Helicobacter pylori Physiology Predicted from Genomic Comparison of Two Strains

Abstract

Helicobacter pylori is a gram-negative bacteria which colonizes the gastric mucosa of humans and is implicated in a wide range of gastroduodenal diseases. This paper reviews the physiology of this bacterium as predicted from the sequenced genomes of two unrelated strains and reconciles these predictions with the literature. In general, the predicted capabilities are in good agreement with reported experimental observations. H. pylori is limited in carbohydrate utilization and will use amino acids, for which it has transporter systems, as sources of carbon. Energy can be generated by fermentation, and the bacterium possesses components necessary for both aerobic and anaerobic respiration. Sulfur metabolism is limited, whereas nitrogen metabolism is extensive. There is active uptake of DNA via transformation and ample restriction-modification activities. The cell contains numerous outer membrane proteins, some of which are porins or involved in iron uptake. Some of these outer membrane proteins and the lipopolysaccharide may be regulated by a slipped-strand repair mechanism which probably results in phase variation and plays a role in colonization. In contrast to a commonly held belief that H. pylori is a very diverse species, few differences were predicted in the physiology of these two unrelated strains, indicating that host and environmental factors probably play a significant role in the outocme of H. pylori-related disease.

Helicobacter pylori is a gram-negative bacterium which colonizes the gastric mucosa of humans, causes gastritis and peptic ulcer disease, and is associated with certain types of gastric cancer (27, 65, 87). Once colonized, the host can be chronically infected for life unless antimicrobial therapy is administered. The ability to colonize and persist in the human stomach for many years indicates that H. pylori is specifically adapted to occupy only this niche, and such adaptation should be reflected in a unique complement of physiological capabilities. Furthermore, physiological differences resulting from the apparent genomic variation among strains have been suggested to be responsible for the diversity of diseases associated with H. pylori infection (16, 95).

Bacterial genomics, the identification and annotation of the entire coding potential of a bacterium, allows a more complete understanding of bacterial physiology and pathogenesis. The recent analysis of the complete genomic sequence of two unrelated, pathogenic H. pylori strains (J99 and 26695) demonstrated that even though the chromosomes are organized differently in a limited number of discrete regions, the genome size, genetic content, and gene order of these two strains are remarkably similar (6). We have used the data resulting from this comparative sequence analysis as the starting point to examine, from a functional perspective, the genes that are common and unique to the two strains. The presence or absence of orthologous genes or metabolic pathways in both unrelated H. pylori strains implies that these genes or pathways are present or absent, respectively, in this species. Our comparison has defined the set of common H. pylori metabolic capabilities as well as a small number that are strain specific.

ANALYSES OF GENETIC AND FUNCTIONAL CONSERVATION

The predicted genes from both H. pylori genomes (6) were assigned a likely function, if their predicted amino acid sequence exhibited similarity to a protein of known function, and categorized as shown in Table 1. Function was annotated conservatively; e.g., proteins which showed sequence similarity to transporters, the majority of which did not have the same substrate specificity, were assigned to the general category of transporters pending experimental evidence for their specificity. The two H. pylori genomes are highly conserved with respect to gene content (1,495 and 1,552 open reading frames [ORFs] in J99 and 26695, respectively [6]), functional categorization (Table 2), and gene order (Fig. 1). In both strains, approximately 58% of the gene products were assigned a putative function based upon their having significant sequence similarity to a protein of known function; nearly 18% were conserved in other species but had no known function; and about 23% were specific to H. pylori (Table 2). Eighty-nine genes were specific to strain J99, and 117 were specific to strain 26695; 26 of these genes in each of the strains J99 and 26695 had an assigned function. Preliminary analysis of the Campylobacter jejuni genome (based on analysis of the recently completed genome by the Sanger Centre) indicated that approximately 90 of the H. pylori specific genes will have an orthologue in this closely related species. This will reduce the proportion of H. pylori-specific genes to approximately 17%.

TABLE 1.

List of H. pylori J99 genes and corresponding 26695 orthologs with putative functional assignments^a

Gene no. in:		Gene name	Function
J99	26695
Amino acid biosynthesis
General
206	0220		Aminotransferase
568	0624		Aminotransferase
632	0696		Hydantoin utilization
633	0695		Hydantoin utilization
673	0736		Aminotransferase
976	0405		Aminotransferase
Aromatic amino acid family
122	0134	aroF	Phospho-2-dehydro-3-deoxyheptonate aldolase
145	0157	aroK	Shikamate kinase I
268	0283	aroB	3-Dehydroquinate synthase
386	1038	aroD	3-Dehydroquinate dehydratase
608	0663	aroC	Chorismate synthase
980	0401	aroA	3-Phosphoshikimate 1-carboxyvinyl transferase
1170	1249	aroE	Shikimate 5-dehydrogenase
1198	1277	trpA	Tryptophan synthase alpha chain
1199	1278	trpB	Tryptophan synthase beta chain
1200	1279	trpC	Indole-3-glycerol phosphate synthase
1201	1280	trpD	Anthranilate phosphoribosyltransferase
1202	1281	trpG	Anthranilate synthase component II
1203	1282	trpE	Anthranilate synthase component I
1294	1380	tyrA	Prephenate dehydrogenase
Aspartate family
90	0098	thrC	Threonine synthase
98	0106	metB	Cystathionine gamma-synthase
198	0212	dapE	Succinyl-diaminopimelate desuccinylase
275	0290	lysA	Diaminopimelate decarboxylase
375	1050	thrB	Homoserine kinase
410	1013	dapA	Dihydrodipicolinate synthase
460	0510	dapB	Dihydrodipicolinate reductase
513	0566	dapF	Diaminopimelate epimerase
570	0626	dapD	2,3,4,5-Tetrahydropyridine-2-carboxylate-N-succinyltransferase
594	0649	aspA	Aspartate ammonia-lyase
615	0672	aspB	Aspartate aminotransferase
761	0822	hom	Homoserine dehydrogenase
1114	1189	asd	Aspartate-semialdehyde dehydrogenase
1150	1229	lysC	Aspartokinase 2 alpha and beta subunits
Glutamate family
461	0512	glnA	Glutamine synthetase
1001	0380	gdhA	Glutamate dehydrogenase
1085	1158	proC	Pyrroline-5-carboxylate reductase
Pyruvate family
313	0330	ilvC	Ketol-acid reductoisomerase
1361	1468	ilvE	Branched-chain amino acid aminotransferase
Serine family
99	0107	cysK	Cysteine synthase
171	0183	glyA	Serine hydroxymethyltransferase
597	0652	serB	Phosphoserine phosphatase
984	0397	serA	Phosphoglycerate dehydrogenase
1133	1210	cysE	Serine acetyltransferase
Biosynthesis of cofactors, prosthetic groups, and carriers
Biotin
25	0029	bioD	Dethiobiotin synthetase
545	0598	bioF	8-Amino-7-oxononanoate synthase
910	0976	bioA	Adenosylmethionine-8-amino-7-oxononanoate aminotransferase
1068	1140		Biotin activation protein
1298	1406	bioB	Biotin synthetase
Folic acid
278	0293	pabB	p-Aminobenzoate synthetase
388	1036	folK	7,8-Dihydro-6-hydroxymethylpterin-pyrophosphokinase
524	0577	folD	Methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase
862	0928	folE	GTP cyclohydrolase I
863	0928	folE	GTP cyclohydrolase I
1153	1232	folP	Dihydropteroate synthase
1403	1510	folB	Dihydroneopterin aldolase
1454	1545	folC	Folylpolyglutamate synthase
Heme and porphyrin
150	0163	hemB	δ-Aminolevulinic acid dehydratase
222	0237	hemC	Porphobilinogen deaminase
224	0239	hemA	Glutamyl-tRNA reductase
291	0306	hemL	Glutamate-1-semialdehyde-2,1-aminomutase
551	0604	hemE	Uroporphyrinogen decarboxylase
610	0665	hemN	Oxygen-independent coproporphyrinogen III oxidase
1000	0381	hemG	Protoporphyrinogen oxidase
1005	0376	hemH	Ferrochelatase
1145	1224	hemD	Uroporphyrinogen III synthase
1147	1226	hemN	Oxygen-independent coproporphyrinogen III oxidase
Menaquinone and ubiquinone
225	0240	ispB	Octaprenyl-diphosphate synthase
864	0929	ispA	Geranyltransferase
1278	1360	ubiA	4-Hydroxybenzoate octaprenyltransferase
1369	1476	ubiD	Octaprenyl-4-hydroxybenzoate carboxy-lyase
1376	1483	ubiE	Ubiquinone/menaquinone biosynthesis methyltransferase
Molybdopterin
158	0172	moeA	Molybdopterin biosynthesis protein
705	0768	moaA	Molybdopterin cofactor biosynthetic protein
706	0769	mobA	Molybdopterin-guanine dinucleotide biosynthesis protein A
734	0798	moaC	Molybdenum cofactor biosynthesis protein C
735	0799	mog	Molybdopterin biosynthesis protein
736	0800	moaE	Molybdopterin-converting factor, subunit 2
737	0801	moaD	Molybdopterin-converting factor, subunit 1
750	0814	moeB	Molybdopterin-synthase sulfurylase
Pantothenate
6	0006	panC	Pantoate-β-alanine ligase
30	0034	panD	Aspartate-1-decarboxylase
367	1058	panB	3-Methyl-2-oxobutanoate hydroxymethyltransferase
779	0841	dfp	Pantothenate metabolism flavoprotein
Pyridoxine
328	0354	dxs	1-Deoxyxylulose-5-phosphate synthase
1489	1582	pdxJ	Pyridoxal phosphate synthetase
1490	1583	pdxA	Pyridoxal phosphate biosynthetic protein A
Riboflavin
2	0002	ribE	Riboflavin synthase beta chain
338	1087	ribF	Riboflavin kinase
738	0802	ribA	GTP cyclohydrolase II
740	0804	ribBA	GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate synthase
1398	1505	ribD	Riboflavin-specific deaminase
1482	1574	ribC	Riboflavin synthase alpha chain
Thioredoxin, glutaredoxin, and glutathione
763	0824	trxA	Thioredoxin
764	0825	trxB	Thioredoxin reductase
1046	1118	ggt	Gamma-glutamyl transpeptidase
1091	1164	trxB	Thioredoxin reductase
1351	1458		Thioredoxin
Thiamine
781	0843	thiE	Thiamine phosphate pyrophosphorylase
782	0844	thiD	Phosphomethylpyrimidine kinase
783	0845	thiM	Hydroxyethylthiazole kinase
Pyridine nucleotides
312	0329	nadE	NH3-dependent NAD+ synthetase
1273	1355	nadC	Nicotinate-nucleotide pyrophosphorylase
1274	1356	nadA	Quinolinate synthetase
Cell envelope
Membranes and porins
7	0009		Outer membrane protein
21	0025		Outer membrane protein
73	0078		Outer membrane protein
	0079
117	0127		Outer membrane protein
195	0209		Outer membrane protein
212	0227		Outer membrane protein
214	0229	hopA	Outer membrane protein—porin
237	0252		Outer membrane protein
238	0253, 0254		Outer membrane protein
307	0324		Outer membrane protein
342	1083		Outer membrane protein
359	1066		Outer membrane protein
424	0472		Outer membrane protein
429	0477		Outer membrane protein
438	0486		Outer membrane protein
439	0487		Outer membrane protein
456	0506		Outer membrane protein
514	0567		Inner membrane protein
581	0638		Outer membrane protein
600	0655		Outer membrane protein
614	0671		Outer membrane protein
634	0694		Outer membrane protein
645	0706	hopE	Outer membrane protein—porin
649	0710		Outer membrane protein
659	0722		Outer membrane protein
662	0725		Outer membrane protein
663	0726		Outer membrane protein
719	0782		Outer membrane protein
725	0788		Outer membrane protein
732	0796		Outer membrane protein
777	0839		Outer membrane protein
810	0876	frpB	Iron-regulated outer membrane protein
833	1243	babB	Outer membrane protein—adhesin
848	0912	hopC	Outer membrane protein—porin
849	0913	hopB	Outer membrane protein—porin
850	0914		Outer membrane protein
851	0915, 0916	frpB	Iron-regulated outer membrane protein
857	0923		Outer membrane protein
870			Outer membrane protein
1008	0373		Outer membrane protein
1022	0358		Outer membrane protein
1034	1107		Outer membrane protein
1040	1113		Outer membrane protein
1054	1125		Outer membrane protein
1083	1156		Outer membrane protein
1084	1157		Outer membrane protein
1094	1167		Outer membrane protein
1103	1177		Outer membrane function
1138	1215, 1216	imp	Role in outer membrane permeability
1164	0896	babA	Outer membrane protein—adhesin
1261	1342		Outer membrane protein
1343	1450		Inner membrane protein
1346	1453		Outer membrane protein
1349	1456	lpp20	Conserved lipoprotein
1360	1467		Outer membrane protein
1362	1469		Outer membrane protein
1394	1501		Outer membrane protein
1405	1512	frpB	Iron-regulated outer membrane protein
1432	1395		Outer membrane protein
1472	1564		Outer membrane protein
1479	1571		Outer membrane protein
Murein sacculus and peptidoglycan
445	0493	mraY	Phospho-N-acetylmuramoyl-pentapeptide-transferase
446	0494	murD	UDP-N-acetylmuramoylalanine-d-glutamate ligase
496	0549	murI	Glutamate racemase
544	0597		Penicillin-binding protein
567	0623	murC	UDP-N-acetylmuramate-alanine ligase
590	0645		Lytic murein transglycosylase
593	0648	murA	UDP-N-acetylglucosamine enolpyruvyltransferase
675	0738	ddl	d-Alanine–d-alanine ligase
677	0740	murF	d-Alanyl–d-alanine-adding enzyme
709	0772	amiA	Probable N-acetylmuramoyl-l-alanine amidase
876	0941	alr	Alanine racemase, biosynthetic
1082	1155	murG	UDP-N-acetylglucosamine-N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenoln-acetylglucosaminetransferase
1313	1418	murB	UDP-N-acetylenolpyruvoyl glucosamine reductase
1387	1494	murE	UDP-N-acetylmuramyl-tripeptide synthetase
1464	1556		Penicillin-binding protein
1473	1565		Penicillin-binding protein
Surface polysaccharides, lipopolysaccharides, and antigens
3	0003	kdsA	3-Deoxy-d-manno-octulosonic acid 8-phosphate synthase
37	0043	manA/manC	Phosphomannose isomerase/GDP-mannose pyrophosphorylase
38	0044	Gmd	GDP-d-mannose dehydratase
39	0045		Sugar nucleotide biosynthesis
70	0075	glmM	Phosphoglucosamine mutase
86	0093, 0094		α-(1,2)-Fucosyltransferase
147	0159		Lipopolysaccharide biosynthesis protein
166	0178	neuB	Sialic acid synthase
182	0196	lpxD	UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acetyltransferase
194	0208		Lipopolysaccharide biosynthesis protein
215	0230	kdsB	3-Deoxy-manno-octulosonate cytidylyltransferase
264	0279	waaC	Lipopolysaccharide heptosyltransferase-1
265	0280	waaM	Lipid A biosynthesis acyltransferase
309	0326	neuA	Acylneuraminate cytidylyltransferase
311	0328	lpxK	Tetraacyldisaccharide-1-p 4′-kinase
373	1052	lpxC	UDP-3-O-[3-hydroxymyristoyl]-N-acetylglucosamine deacetylase
562			Lipopolysaccharide biosynthesis protein
563	0619		Lipopolysaccharide biosynthesis protein
596	0651	fucT	α-(1,3)-Fucosyltransferase
620	0679		Lipopolysaccharide biosynthesis protein
741	0805		Lipopolysaccharide biosynthesis protein
765	0826		Lipopolysaccharide biosynthesis protein
778	0840		Sugar nucleotide biosynthesis protein
791	0857	gmhA	Phosphoheptose isomerase
792	0858	waaE	ADP-d-glycero-d-mannoheptose synthase
793	0859	gmhD	ADP-l-glycero-d-mannoheptose-6-epimerase
801	0867	lpxB	Lipid-A-disaccharide synthase
820			Lipopolysaccharide biosynthesis protein
891	0957	waaA	3-Deoxy-d-manno-octulosonic-acid transferase
963	0421		Polysaccharide biosynthesis protein
1002	0379	fucU	α-(1,3)-Fucosyltransferase
1015	0366		Sugar nucleotide biosynthesis
1020	0360	galE	UDP-glucose 4-epimerase
1031	1105		Lipopolysaccharide biosynthesis protein
1032			Lipopolysaccharide biosynthesis protein
1116	1191	waaF	ADP-heptose-lipopolysaccharide heptosyltransferase II
1196	1275	manB	Phosphomannomutase
1289	1375	lpxA	UDP-N-acetylglucosamine acyltransferase
1311	1416		Lipopolysaccharide biosynthesis protein
1368	1475	kdtB	Lipopolysaccharide core biosynthesis protein
1488	1581	wecA	Undecaprenyl-phosphate-α-N-acetylglucosaminyltransferase
Surface structures
107	0115	flaB	Flagellin B
159	0173	fliR	Flagellar biosynthesis protein
217	0232		Motility protein
231	0246	flgI	Flagellar P-ring protein
280	0295	flgL	Flagellar hook-associated protein 3 (hap3)
308	0325	flgH	Flagellar L-ring protein precursor (basal body L-ring protein)
310	0327	flaG	Flagellar biosynthesis protein
325	0351	fliF	Flagellar M-ring protein
326	0352	fliG	Flagellar motor switch protein
327	0353	fliH	Flagellar export apparatus
333	1092	flgG	Flagellar basal-body rod protein
383	1041	flhA	Flagellar biosynthesis protein
389	1035	flhF	Flagellar biosynthesis protein
393	1031	fliM	Flagellar motor switch protein
394	1030		Flagellar motor switch protein
444	0492		Paralogue of hpaA
531	0584	fliN	Flagellar motor switch protein
548	0601	flaA	Flagellin A
625	0684, 0685	fliP	Flagellar biosynthesis protein
688	0751		Flagellin protein
689	0752	fliD	Flagellar hook-associated protein 2 (hap2)
690	0753	fliS	Flagellar protein
707	0770	flhB	Flagellar biosynthesis protein
733	0797	hpaA	Neuraminyllactose-binding hemagglutinin precursor
745	0809	fliL	Flagellar biosynthesis protein
751	0815	motA	Chemotaxis protein (motility protein a)
752	0816	motB	Flagellar motor protein
804	0870	flgE	Flagellar hook protein
843	0907		Flagellar biosynthesis protein
844	0908		Flagellar basal-body/rod/hook protein
971	0410		Paralogue of hpaA
1047	1119	flgK	Flagellar hook-associated protein 1 (Hap1)
1117	1192		Motility protein
1195	1274	pflA	Flagellar functional protein
1314	1419	fliQ	Flagellar biosynthesis protein
1315	1420	fliI	Flagellum-specific ATP synthase
1355	1462		Motility protein
1465	1557	fliE	Flagellar hook–basal-body complex protein
1466	1558	flgC	Flagellar basal-body rod protein
1467	1559	flgB	Flagellar basal-body rod protein
1483	1575	flhB	Flagellar biosynthesis protein
1492	1585	flgG	Flagellar basal-body rod protein (distal rod protein)
Cellular processes
General
4	0004	icfA	Carbonic anhydrase
161	0175		Peptidyl-prolyl cis-trans isomerase
183	0197	metK	S-Adenosylmethionine synthetase
228	0243	napA	Neutrophil-activating protein A
466	0517	era	GTP-binding protein
678	0741		HIT family protein
865	0930	surE	Stationary-phase protein
977	0404		HIT family protein
1112	1186		Carbonic anhydrase
Cell division
314	0331	minD	Cell division inhibitor
315	0332	minE	Cell division topological specificity factor
335	1090	ftsK	Septum formation protein
680	0743	rodA	Rod shape-determining protein
912	0978	ftsA	Septum formation protein
913	0979	ftsZ	GTPase in circumferential ring formation
1086	1159	fic	cAMP-induced cell filamentation protein
1287	1373	mreB	Rod shape-determining protein
1468	1560	rodA	Rod shape-determining protein
Cell killing
274	0289		Vacuolating cytotoxin (VacA) paralogue
339	1086	hlyA	Hemolysin
556	0609, 0610		Vacuolating cytotoxin (VacA) paralogue
819	0887	vacA	Vacuolating cytotoxin
856	0922		Vacuolating cytotoxin (VacA) paralog
Cag island proteins and transposable elements
15	0017	virB4a	DNA transfer protein
36	0041, 0042	virB10	DNA transfer protein
469	0520	orf6	Cag island protein
470	0521	orf7	Cag island protein
471	0522	orf8	Cag island protein
472	0523	orf9	Cag island protein
473	0524	virD4	Cag island protein, DNA transfer protein
474	0525	virB11a	Cag island protein, DNA transfer protein
475	0526	orf12	Cag island protein
476	0527	orf13/14	Cag island protein
477	0528	orf15	Cag island protein
478	0529	orf16	Cag island protein
479	0530	orf17	Cag island protein
480	0531	orf18	Cag island protein
481	0532	cagT	Cag island protein
482	0534	cagS	Cag island protein
483	0535	cagQ	Cag island protein
484	0536	cagP	Cag island protein
485	0537	cagM	Cag island protein
486	0538	cagN	Cag island protein
487	0539	cagL	Cag island protein
488	0540	cagI	Cag island protein
489	0541	cagH	Cag island protein
490	0542	cagG	Cag island protein
491	0543	cagF	Cag island protein
492	0544	cagE	DNA transfer protein (Agrobacterium VirB4 homologue)
493	0545	cagD	Cag island protein
494	0546	cagC	Cag island protein
495	0547	cagA	Cag island protein, cytotoxicity-associated immunodominant antigen
826		tnpB	IS606 transposase
827		tnpA	IS606 transposase
917		virB4b	DNA transfer protein
918		virB4c	DNA transfer protein
1279	1361	comEC	DNA transfer protein
1316	1421	virB11b	DNA transfer protein
Chaperones
8	0010	groEL	60-kDa chaperone
9	0011	groES	10-kDa chaperone
101	0109	dnaK	70-kDa chaperone
102	0110	grpE	24-kDa chaperone
196	0210	htpG	90-kDa chaperone
400	1024	dnaJ2	Cochaperone with DnaK
861	0927	htpX	Stress protein
1252	1332	dnaJ1	Cochaperone with DnaK
Detoxification
809	0875	katA	Catalase
991	0390	tpx	Thiol peroxidase
992	0389	sodF	Iron-dependent superoxide dismutase
1471	1563	tsaA	Peroxidase
Protein and peptide secretion
69	0074	lspA	Lipoprotein signal peptidase
168	0180	lnt	Apolipoprotein N-acyltransferase
329	0355	lepA	GTP-binding protein
523	0576	lepB	Signal peptidase I
700	0763	ftsY	Functional homolog of srp receptor
723	0786	secA	Preprotein translocase subunit
731	0795	tig	Trigger factor
889	0955	lgt	Prolipoprotein diacylglyceryl transferase
1079	1152	ffh	Signal recognition particle protein
1126A	1203A	secE	Preprotein translocase subunit
1176	1255	secG	Protein export membrane protein
1220	1300	secY	Preprotein translocase subunit
1449	1550	secD	Protein export membrane protein
1450	1549	secF	Protein export membrane protein
Phosphorus compounds
413	1010	ppk	Polyphosphate kinase
564	0620	ppa	Inorganic pyrophosphatase
Polyamine biosynthesis
18	0020	nspC	Carboxynorspermidine decarboxylase
771	0832	speE	Spermidine synthase
962	0422	speA	Arginine decarboxylase
Urea
62	0067	ureH	Urease accessory protein
63	0068	ureG	Urease accessory protein
64	0069	ureF	Urease accessory protein
65	0070	ureE	Urease accessory protein
67	0072	ureB	Urease beta subunit
68	0073	ureA	Urease alpha subunit
DNA replication
10	0012	dnaG	DNA primase
108	0116	topA	DNA topoisomerase I
199	0213	gidA	Glucose-inhibited division protein A
362	1063	gidB	Glucose-inhibited division protein B
452	0500	dnaN	DNA polymerase III, beta chain
453	0501	gyrB	DNA gyrase subunit B
558	0615	lig	DNA ligase
641	0701	gyrA	DNA gyrase subunit A
655	0717	dnaX	DNA polymerase III subunits gamma and tau
847	0911		ATP-dependent helicase
919		topA	Topoisomerase I
931		topA	Topoisomerase I
994	0387	priA	Primosomal protein n′ (replication factor y)
1152	1231	holB	DNA polymerase III subunit delta′
1166	1245	ssb	Single-strand binding protein
1280	1362	dnaB	Replicative DNA helicase
1353	1460	dnaE	DNA polymerase III, alpha chain
1363	1470	polA	DNA polymerase I
1371	1478	rep	ATP-dependent DNA helicase
1412	1523	recG	ATP-dependent DNA helicase
1417	1529	dnaA	Chromosomal replication initiator protein
1438	1387		DNA polymerase III
1446	1553	pcrA	ATP-dependent helicase
DNA restriction, modification, recombination, and repair
43	0050		Type II DNA modification enzyme (methyltransferase)
44			Type II DNA modification enzyme (methyltransferase)
45			Type II DNA modification enzyme (methyltransferase)
46			Type II restriction enzyme
84	0091		Type II restriction enzyme
85	0092		Type II DNA modification enzyme (methyltransferase)
130	0142	mutY	A/G-specific adenine glycosylase
141	0153	recA	Recombination protein
164			Restriction enzyme
209	0223	radA	DNA repair protein
243	0259	xseA	Exodeoxyribonuclease large subunit
244	0260		Type II DNA modification enzyme (methyltransferase)
248	0263		Type II DNA modification enzyme (methyltransferase)
306	0323		Endonuclease
322	0348	recJ	Single-stranded-DNA-specific exonuclease
366	1059	ruvB	Holliday junction DNA helicase
414		hsdS1	Type I restriction enzyme (specificity subunit)
415	0463	hsdM1	Type I restriction enzyme (modification subunit)
416	0464	hsdR1	Type I restriction enzyme (restriction subunit)
430	0478		Type II DNA modification enzyme (methyltransferase)
433	0481		Type II DNA modification enzyme (methyltransferase)
435	0483		Type II DNA modification enzyme (methyltransferase)
532	0585	nth	Endonuclease III
549	0602		Endonuclease III
565	0621	mutS	DNA mismatch repair protein
606	0661	rnhA	RNase HI
617	0675		Integrase-recombinase protein (xerCD family)
618	0676	ogt	Methylated-DNA–protein-cysteine methyltransferase
629			Type II DNA modification enzyme (methyltransferase)
630			Type II restriction enzyme
644	0705	uvrA	Excinuclease ABC subunit A
726		hsdS	Type I restriction enzyme (specificity subunit)
756			Type II DNA modification enzyme (methyltransferase)
760	0821	uvrC	Excinuclease ABC subunit C
784	0846	hsdR2	Type I restriction enzyme (restriction subunit)
785	0848, 0849	hsdS2	Type I restriction enzyme (specificity subunit)
786	0850	hsdM2	Type I restriction enzyme (modification subunit)
811	0877	ruvC	Crossover junction endodeoxyribonuclease
815	0883	ruvA	Holliday junction DNA helicase
846	0910		Type II DNA modification enzyme (methyltransferase)
859	0925	recR	Recombination protein
941	0995		Integrase/recombinase (xerCD family)
951			Integrase/recombinase (xerCD family)
1012	0369		Type II DNA modification enzyme (methyltransferase)
1041	1114	uvrB	Excinuclease ABC subunit B
1050	1121		Type II DNA modification enzyme (methyltransferase)
1131	1208	M.HpyI	Type II DNA modification enzyme (methyltransferase)
1149	1228	mutT	dGTP pyrophosphohydrolase
1243	1323	rnhB	RNase HII
1266	1347	ung	Uracil-DNA glycosylase
1271	1352		Type II DNA modification enzyme (methyltransferase)
1284			Type II DNA modification enzyme (methyltransferase)
1295	1382		Endonuclease
1296		mod1	Type III DNA modification enzyme (methyltransferase)
1297		res1	Type III restriction enzyme
1364	1471		Type II restriction enzyme
1365	1472		Type II DNA modification enzyme (methyltransferase)
1409			Type II DNA modification enzyme (methyltransferase)
1410	1521	res2	Type III restriction enzyme
1411	1522	mod2	Type III DNA modification enzyme (methyltransferase)
1415	1526	exoA	Exodeoxyribonuclease
1422		hsdS3	Type I restriction enzyme (specificity subunit)
1423	1403	hsdM3	Type I restriction enzyme (modification subunit)
1424	1402	hsdR3	Type I restriction enzyme (restriction subunit)
1434	1393	recN	DNA repair protein
1442	1366		Type II restriction enzyme
Energy metabolism
Amino acids and amines
120	0132	sdaB	l-Serine/l-Threonine deaminase
279	0294	aimE	Aliphatic amidase
585			3-Hydroxyacid dehydrogenase
661	0723	ansB	l-Asparaginase II
1159	1238		Aliphatic amidase
1427	1399	rocF	Arginase
1428	1398	ald	l-Alanine dehydrogenase
ATP-proton motive force interconversion
767	0828	atpB	ATP synthase F0, subunit a
1059	1131	atpC	ATP synthase F1, subunit epsilon
1060	1132	atpD	ATP synthase F1, subunit beta
1061	1133	atpG	ATP synthase F1, subunit gamma
1062	1134	atpA	ATP synthase F1, subunit alpha
1063	1135	atpH	ATP synthase F1, subunit delta
1064	1136	atpF	ATP synthase F0, subunit b
1065	1137	atpX	ATP synthase B′
1135	1212	atpE	ATP synthase F0, subunit c
Electron transport
40	0047	hypE	Hydrogenase expression/formation protein
48	0056	putA	Proline/pyrroline-5-carboxylate dehydrogenase
132	0144	fixN	Cytochrome oxidase (CBB3-TYPE)
133	0145	fixO	Cytochrome oxidase (CBB3-TYPE)
134	0146	fixQ	Cytochrome oxidase (CBB3-TYPE)
135	0147	fixP	Cytochrome oxidase (CBB3-TYPE)
177	0191	frdB	Fumarate reductase
178	0192	frdA	Fumarate reductase
179	0193	frdC	Fumarate reductase
250	0265	ccdA	Cytochrome c biogenesis protein
262	0277		Ferredoxin
459	0509	glcD	Glycolate oxidase
574	0631	hyaA	Hydrogenase, small subunit
575	0632	hyaB	Hydrogenase, large subunit
576	0633	hyaC	Hydrogenase, cytochrome subunit
577	0634	hyaD	Hydrogenase expression/formation protein
611	0666	glpC	Glycerol-3-phosphate dehydrogenase
803	0869	hypA	Hydrogenase expression/formation protein
835	0898	hypD	Hydrogenase expression/formation protein
836	0899	hypC	Hydrogenase expression/formation protein
837	0900	hypB	Hydrogenase expression/formation protein
878	0943	dadA	d-Amino acid dehydrogenase
895	0961	gpsA	Glycerol-3-phosphate dehydrogenase (NAD+)
974	0407		S/N-oxide reductase
1003	0378		Cytochrome c biogenesis protein
1035	1108	porG	Pyruvate ferrodoxin oxidoreductase
1036	1109	porD	Pyruvate ferrodoxin oxidoreductase
1037	1110	porA	Pyruvate ferrodoxin oxidoreductase
1038	1111	porB	Pyruvate ferrodoxin oxidoreductase
1088	1161	fldA	Flavodoxin
1090	1163	fixS	Component of cation transport for cbb3-type oxidase
1143	1222	dld	d-Lactate dehydrogenase
1148	1227		Periplasmic cytochrome c-553
1181	1260	nuoA	NADH oxidoreductase I
1182	1261	nuoB	NADH oxidoreductase I
1183	1262	nuoC	NADH oxidoreductase I
1184	1263	nuoD	NADH oxidoreductase I
1185	1264	nuoE	NADH oxidoreductase I
1186	1265	nuoF	NADH oxidoreductase I
1187	1266	nuoG	NADH oxidoreductase I
1188	1267	nuoH	NADH oxidoreductase I
1189	1268	nuoI	NADH oxidoreductase I
1190	1269	nuoJ	NADH oxidoreductase I
1191	1270	nuoK	NADH oxidoreductase I
1192	1271	nuoL	NADH oxidoreductase I
1193	1272	nuoM	NADH oxidoreductase I
1194	1273	nuoN	NADH oxidoreductase I
1354	1461		Cytochrome c peroxidase
1401	1508	fixG	Component of cation transport for cbb3-type oxidase
1459	1540	petA	Ubiquinol cytochrome c oxidoreductase, 2Fe-2S subunit
1460	1539	petB	Ubiquinol cytochrome c oxidoreductase, cytochrome b subunit
1461	1538	petC	Ubiquinol cytochrome c oxidoreductase, cytochrome c1 subunit
Entner-Doudoroff pathway
1025	1099	eda	2-Keto-3-deoxy-6-phosphogluconate aldolase
1026	1100	edd	Phosphogluconate dehydratase
Fermentation
840	0903	ackA	Acetate kinase
841	0904, 0905	pta	Phosphotransacetylase
1030	1104		Zinc-dependent alcohol dehydrogenase
1429			Zinc-dependent alcohol dehydrogenase
Gluconeogenesis
111	0121	ppsA	Phosphoenolpyruvate synthase
142	0154	eno	Enolase
162	0176	fba	Fructose-bisphosphate aldolase
180	0194	tpi	Triose-phosphate isomerase
855	0921	gap	Glyceraldehyde-3-phosphate dehydrogenase
908	0974	pgm	2,3-Bisphosphoglycerate-independent phosphoglycerate mutase
1093	1166	pgi	Glucose-6-phosphate isomerase
1264	1345	pgk	Phosphoglycerate kinase
1265	1346	gap	Glyceraldehyde 3-phosphate dehydrogenase
1440	1385	fbp	Fructose-1,6-bisphosphatase
Phosphopentose pathway
337	1088	tktA	Transketolase
521	0574	rpi	Ribose 5-phosphate isomerase
1027	1101	zwf	Glucose-6-phosphate-1-dehydrogenase
1388	1495	tal	Transaldolase
1439	1386	rpe	Ribulose-phosphate-3-epimerase
Sugars
1029	1103	glk	Glucokinase
Tricarboxylic acid cycle
22	0026	gltA	Citrate synthase
23	0027	icd	Isocitrate dehydrogenase
536	0588	oorD	Subunit of 2-oxoglutarate oxidoreductase
537	0589	oorA	Subunit of 2-oxoglutarate oxidoreductase
538	0590	oorB	Subunit of 2-oxoglutarate oxidoreductase
539	0591	oorC	Subunit of 2-oxoglutarate oxidoreductase
716	0779	acnB	Aconitate hydratase
1245	1325	fumC	Fumarase
Other
88	0096		Keto-acid dehydrogenase
586	0642		Oxidoreductase
888	0954		Aldehyde dehydrogenase
1023	0357		Oxidoreductase
1028	1102		Dehydrogenase
1345	1452	thdF	Thiophene/furan oxidation protein
Fatty acid and phospholipid metabolism
83	0090	fabD	Malonyl-CoA-ACP transacylase
176	0190		Cardiolipin synthase
181	0195	fabI	Enoyl-ACP reductase
187	0201	plsX	Fatty acid/phospholipid synthesis protein
188	0202	fabH	β-Ketoacyl-ACP synthase III
201	0215	cdsA	CDP-diacylglycerol synthase
354	1071	pssA	Phosphatidylserine synthase
407	1016	pgsA	Phosphatidylglycerophosphate synthase
409	1014		Short-chain dehydrogenase
451	0499	pldA	Phospholipase A1
504	0557	accA	Acetyl-CoAa carboxylase subunit A
505	0558	fabB	β-Ketoacyl-ACP synthase I
506	0559	acpP	ACP
508	0561	fabG	Acetyl-CoA carboxylase subunit A
636	0692	scoB	3-Oxoacid CoA-transferase, subunit B
637	0691	scoA	3-Oxoacid CoA-transferase, subunit A
638	0690	thl	Acetyl-CoA acetyltransferase
640	0700	dgkA	Diacylglycerol kinase
674	0737	pgpA	Phosphatidylglycerophosphatase A
744	0808	acpS	Holo-ACP synthase
805	0871	cdh	CDP-diacylglycerol pyrophosphatase
884	0950	accD	Acetyl-CoA carboxylase subunit B
968	0416	cfa	Cyclopocyclopropane fatty acid synthase
1010	0371	accB	Biotin carboxyl carrier protein
1011	0370	accC	Biotin carboxylase
1267	1348	plsC	1-Acyl-SN-glycerol-3-phosphate acyltransferase
1275	1357	psd	Phosphatidylserine decarboxylase
1290	1376	fabZ	Hydroxymyristoyl-ACP dehydratase
Purines, pyrimidines, nucleosides and nucleotides
2′-Deoxyribonucleotide metabolism
621	0680	nrdA	Ribonucleoside-diphosphate reductase 1 alpha chain
799	0865	dut	Deoxyuridine 5′-triphosphate nucleotidohydrolase
1009	0372	dcd	Deoxycytidine triphosphate deaminase
1016	0364	nrdB	Ribonucleoside-diphosphate reductase 1 beta chain
Purine ribonucleotide biosynthesis
1039	1112	purB	Adenylosuccinate lyase
1140	1218	purD	Glycinamide ribonucleotide synthetase
1327	1434	purU	Formyltetrahydrofolate hydrolase
Pyrimidine ribonucleotide biosynthesis
5	0005	pyrF	Orotidine 5′-phosphate decarboxylase
184	0198	ndk	Nucleoside diphosphate kinase
251	0266	pyrC1	Dihydroorotase
323	0349	pyrG	CTP synthase
341	1084	pyrB	Aspartate carbamoyltransferase catalytic chain
412	1011	pyrD	Dihydroorotate dehydrogenase
528	0581	pyrC2	Dihydroorotase
714	0777	pyrH	Uridylate kinase
853	0919	pyrA1	Carbamoyl-phosphate synthase large chain
1158	1237	pyrA2	Carbamoyl-phosphate synthase small chain
1178	1257	pyrE	Orotate phosphoribosyltransferase
Salvage and interconversion of nucleosides and nucleotides
96	0104	cpdB	2′,3′-Cyclic nucleotide 2′-phosphodiesterase
239	0255	purA	Adenylosuccinate synthetase
304	0321	gmk	Guanylate kinase
519	0572	apt	Adenine phosphoribosyltransferase
561	0618	adk	Adenylate kinase
672	0735	gpt	Xanthine-guanine phosphoribosyltransferase
679	0742	prsA	Phosphoribosyl pyrophosphate synthetase
768	0829	guaB	Inosine-5′-monophosphate dehydrogenase
790	0854	guaC	GMP reductase
972	0409	guaA	GMP synthetase
1104	1178	deoD	Purine nucleoside phosphorylase
1105	1179	deoB	Phosphopentomutase
1367	1474	tmk	Thymidylate kinase
Sugar-nucleotide biosynthesis and conversions
591	0646	galU	UTP-glucose-1-phosphate uridylyltransferase
624	0683	glmU	UDP-N-acetylglucosamine pyrophosphorylase
1420	1532	glmS	Glutamine fructose-6-phosphate minotransferase
Regulatory functions
General
41	0048		Transcriptional regulator
81	0088	rpoD	RNA polymerase sigma 70 factor
151	0164, 0165		Histidine kinase sensor protein
152	0166		Transcriptional regulator
229	0244		Histidine kinase sensor protein
263	0278	gppA	Guanosine-5′-triphosphate,3′-diphosphate pyrophosphatase
381	1043		Transcriptional regulator
392	1032	fliA	RNA polymerase sigma 28 factor
397	1027	fur	Ferric uptake regulation protein
399	1025		Transcriptional regulator
403	1021		Transcriptional regulator
643	0703		Transcriptional regulator
652	0714	rpoN	RNA polymerase sigma-54 factor
664	0727		Transcriptional regulator
712	0775	spoT	Guanosine-3′,5′-bis(diphosphate)-3′-pyrophosphohydrolase
981	0400	lytB	Lysis tolerance protein
1207	1287		Transcriptional regulator
1282	1364		Histidine kinase sensor protein
1283	1365		Transcriptional regulator
1335	1442	csrA	Carbon storage regulator
1443	1365		Transcriptional regulator
1480	1572	dniR	Regulatory protein
Chemotaxis and motility
17	0019	cheV1	Chemotaxis protein
75	0082		MCP
91	0099		MCP
95	0103		MCP
358	1067	cheY	Response regulator
546	0599		MCP
559	0616	cheV2	Chemotaxis protein
988	0393	cheV3	Chemotaxis protein
989	0392	cheA	Histidine kinase
990	0391	cheW	Histidine kinase-MCP coupling protein
Transcription
Degradation of RNA
1136	1213	pnp	Polyribonucleotide nucleotidyltransferase
1169	1248	vacB	RNase II family protein
1299	1407	rbn	RNase N
DNA-dependent RNA polymerase
1121	1198	rpoB	DNA-directed RNA polymerase, beta subunit
1213	1293	rpoA	DNA-directed RNA polymerase, alpha subunit
1458	1541	mfd	Transcription-repair coupling factor
Transcription factors
1	0001	nusB	Transcription termination
497	0550	rho	Transcription termination factor
800	0866	greA	Transcription elongation factor (transcript cleavage factor)
1126	1203	nusG	Transcription antitermination protein
1407	1514	nusA	N utilization substance protein A
RNA processing
583	0640	pcnB	Polynucleotide adenylyltransferase
607	0662	rnc	RNase III
Translation
Aminoacyl-tRNA synthetases
113	0123	thrS	Threonyl-tRNA synthetase
170	0182	lysS	Lysyl-tRNA synthetase
223	0238	proS	Prolyl-tRNA synthetase
302	0319	argS	Arginyl-tRNA synthetase
428	0476	gltX	Glutamyl-tRNA synthetase
560	0617	aspS	Aspartyl-tRNA synthetase
588	0643	gltX	Glutamyl-tRNA synthetase
711	0774	tyrS	Tyrosyl-tRNA synthetase
818	0886	cysS	Cysteinyl-tRNA synthetase
894	0960	glyQ	Glycyl-tRNA synthetase alpha chain
906	0972	glyS	Glycyl-tRNA synthetase beta chain
967	0417	metG	Methionyl-tRNA synthetase
978	0403	pheS	Phenylalanyl-tRNA synthetase alpha chain
979	0402	pheT	Phenylalanyl-tRNA synthetase beta chain
1080	1153	valS	Valyl-tRNA synthetase
1115	1190	hisS	Histidyl-tRNA synthetase
1162	1241	alaS	Alanyl-tRNA synthetase
1174	1253	trpS	Tryptophanyl-tRNA synthetase
1317	1422	ileS	Isoleucyl-tRNA synthetase
1373	1480	serS	Seryl-tRNA synthetase
1452	1547	leuS	Leucyl-tRNA synthetase
Degradation of proteins, peptides and glycopeptides
29	0033	clpA	ATP-dependent protease, ATP-binding subunit
155	0169		Protease
249	0264	clpB	Heat shock protein
271	0286	ftsH	ATP-dependent Zn metallopeptidase
356	1069	ftsH	ATP-dependent Zn metallopeptidase
387	1037	pepQ	Proline peptidase
405	1019	htrA	Protease DO
411	1012		Zn protease
422	0470	pepF	Oligopeptidase
464	0515	hslV	Heat shock protein
465	0516	hslU	Heat shock protein
517	0570	pepA	Aminopeptidase
602	0657		Processing protease
603	0658	gatB	Glu-tRNA amidotransferase, subunit B
730	0794	clpP	ATP-dependent protease, proteolytic subunit
769	0830	gatA	Glu-tRNA amidotransferase, subunit A
909	0975	gatC	Glu-tRNA amidotransferase, subunit C
999	0382		Zn-metallo protease
1269	1350	prc	Carboxyl-terminal protease
1288	1374	clpX	ATP-dependent protease, ATP-binding subunit
1293	1379	lon	ATP-dependent protease 1a
1328	1435	sppA	Protease
1491	1584	ydiE	O-Sialoglycoprotein endopeptidase
Nucleoproteins
774	0835		DNA-binding protein HU
1052	1123	slyD	FKBP-type peptidyl-prolyl cis-trans isomerase
Protein modification
210	0224		Peptide methionine sulfoxide reductase
729	0793	def	Polypeptide deformylase
1017	0363	pcm	Protein-l-isoaspartate O-methyltransferase
1219	1299	map	Methionine aminopeptidase
1334	1441	ppiA	Peptidyl-prolyl cis-trans isomerase
Ribosomal proteins, synthesis and modification
71	0076	rpsT	30S ribosomal protein S20
76	0083	rpsI	30S ribosomal protein S9
77	0084	rplM	50S ribosomal protein L13
115	0125	rpmI	50S ribosomal protein L35
116	0126	rplT	50S ribosomal protein L20
186	0200	rpmF	50S ribosomal protein L32
281	0296	rplU	50S ribosomal protein L21
282	0297	rpmA	50S ribosomal protein L27
357	1068	prmA	Ribosomal protein L11 methyltransferase
378	1047	rbfA	Ribosome-binding factor A
384	1040	rpsO	30S ribosomal protein S15
443	0491	rpmB	50S ribosomal protein L28
463	0514	rplI	50S ribosomal protein L9
498	0551	rpmE	50S ribosomal protein L31
509	0562	rpsU	30S ribosomal protein S21
982	0399	rpsA	30S ribosomal protein S1
1074	1147	rplS	50S ribosomal protein L19
1078	1151	rpsP	30S ribosomal protein S16
1119	1196	rpsG	30S ribosomal protein S7
1120	1197	rpsL	30S ribosomal protein S12
1122	1199	rplL	50S ribosomal protein L7/L12
1123	1200	rplJ	50S ribosomal protein L10
1124	1201	rplA	50S ribosomal protein L1
1125	1202	rplK	50S ribosomal protein L11
1127	1204	rpmG	50S ribosomal protein L33
1165	1244	rpsR	30S ribosomal protein S18
1167	1246	rpsF	30S ribosomal protein S6
1212	1292	rplQ	50S ribosomal protein L17
1214	1294	rpsD	30S ribosomal protein S4
1215	1295	rpsK	30S ribosomal protein S11
1217	1296	rpsM	30S ribosomal protein S13
1217	1297	rpmJ	50S ribosomal protein L36
1221	1301	rplO	50S ribosomal protein L15
1222	1302	rpsE	30S ribosomal protein S5
1223	1303	rplR	50S ribosomal protein L18
1224	1304	rplF	50S ribosomal protein L6
1225	1305	rpsH	30S ribosomal protein S8
1226	1306	rpsN	30S ribosomal protein S14
1227	1307	rplE	50S ribosomal protein L5
1228	1308	rplX	50S ribosomal protein L24
1229	1309	rplN	50S ribosomal protein L14
1230	1310	rpsQ	30S ribosomal protein S17
1231	1311	rpmC	50S ribosomal protein L29
1232	1312	rplP	50S ribosomal protein L16
1233	1313	rpsC	30S ribosomal protein S3
1234	1314	rplV	50S ribosomal protein L22
1235	1315	rpsS	30S ribosomal protein S19
1236	1316	rplB	50S ribosomal protein L2
1237	1317	rplW	50S ribosomal protein L23
1238	1318	rplD	50S ribosomal protein L4
1239	1319	rplC	50S ribosomal protein L3
1240	1320	rpsJ	30S ribosomal protein S10
1340	1447	rpmH	50S ribosomal protein L34
1389	1496	rplY	50S ribosomal protein L25
1445	1554	rpsB	30S ribosomal protein S2
tRNA modification
266	0281	tgt	Queuine-tRNA-ribosyltransferase
363	1062	queA	S-Adenosylmethionine tRNA ribosyltransferase-isomerase
1019	0361	truA	Pseudouridylate synthase I
1069	1141	fmt	Methionyl-tRNA formyltransferase
1075	1148	trmD	tRNA (guanine-n1)-methyltransferase
1254	1335	trmU	tRNA(5-methylaminomethyl-2-thiouridylate)-methyltransferase
1310	1415	miaA	tRNA delta(2)-isopentenylpyrophosphate transferase
1341	1448	rnpA	RNase protein component
1390	1497	pth	Peptidyl-tRNA hydrolase
1406	1513	selA	l-Seryl-tRNA selenium transferase
Translation factors
72	0077	prfA	Peptide chain release factor 1
114	0124	infC	Translation initiation factor IF-3
157	0171	prfB	Peptide chain release factor 2 (RF-2)
163	0177	efp	Elongation factor P (EF-P)
232	0247	deaD	ATP-dependent RNA helicase dead
377	1048	infB	Translation initiation factor IF-2
1118	1195	fusA	Elongation factor G (EF-G)
1128	1205	tufA	Elongation factor TU (EF-TU)
1177	1256	frr	Ribosome recycling factor (ribosome-releasing factor [RRF])
1218	1298	infA	Translation initiation factor IF-1
1322	1431	ksgA	Dimethyladenosine transferase
1444	1555	tfs	Elongation factor TS (EF-TS)
Transport and binding proteins
General
66	0071	ureI	Urea transporter
167	0179		ABC transporter, ATP-binding protein
200	0214		Transporter
235	0250		ABC transporter, ATP-binding protein
236	0251		ABC transporter, permease
300	0613		ABC transporter, ATP-binding protein
343	1082	msbA	Multidrug resistance protein
449	0497		Transporter
450	0498		Transporter
547	0600		Secretion/efflux ABC transporter, ATP-binding protein
553	0606		Efflux transporter
554	0607		Efflux transporter
653	0715		ABC transporter, ATP-binding protein
685	0748		ABC transporter, ATP-binding protein
754	0818		Osmoprotection binding protein
757	0818		Osmoprotection binding protein
758	0819		Osmoprotection ATP-binding protein
789	0853		ABC transporter, ATP-binding protein
806	0872	phnA	Alkylphosphonate uptake protein
871	0936	proP	Proline/betaine transporter
1055	1126	tolB	tonB-independent protein-uptake protein
1057	1129	exbD1	Biopolymer transport protein
1058	1130	exbB1	Biopolymer transport protein
1107	1181		Transporter
1129	1206		ABC transporter, ATP-binding protein
1141	1220		ABC transporter, ATP-binding protein
1320	1427		Histidine-rich metal-binding protein
1321	1432		Histidine- and glutamine-rich metal-binding protein
1338	1445	exbB3	Biopolymer transport protein
1339	1446	exbD3	Biopolymer transport protein
1484	1576		ABC transporter, ATP-binding protein
1485	1577		ABC transporter, permease
Amino acids, peptides, and amines
47	0055	putP	Sodium/proline symporter
121	0133	sdaC	l-Serine transporter
283	0298	dppA	Periplasmic dipeptide transport substrate-binding protein
284	0299	dppB	Dipeptide transport system permease protein
285	0300	dppC	Dipeptide transport system permease protein
286	0301	dppD	Dipeptide transport system ATP-binding protein
287	0302	dppF	Dipeptide transport system ATP-binding protein
406	1017		Amino acid permease
874	0939		Amino acid ABC transporter, permease protein
875	0940		Amino acid ABC transporter, binding protein precursor
877	0942		Sodium/alanine symporter
1096	1169		Amino acid ABC transporter, permease protein
1097	1170		Amino acid ABC transporter, permease protein
1098	1171		Amino acid ABC transporter, ATP-binding protein
1099	1172		Amino acid ABC transporter, binding protein precursor
1172	1251		Peptide ABC transporter, ATP-binding protein
1358	1465		Amino acid ABC transporter, ATP-binding protein
1399	1506	gltS	Sodium/glutamate symporter
Anions
425	0473	modA	Molybdenum ABC transporter, periplasmic binding protein
426	0474	modB	Molybdenum ABC transporter, permease
427	0475	modC	Molybdenum ABC transporter, ATP-binding protein
1384	1491		Phosphate permease
Carbohydrates, organic alcohols, and acids
128	0140	lldP	l-Lactate permease
129	0141	lldP	l-Lactate permease
334	1091	kgtP	α-Ketoglutarate permease
635	0693	atoE	Short-chain fatty acids transporter
660	0724	dcuA	Anaerobic C4-dicarboxylate membrane transporter
1101	1174	gluP	Glucose/galactose transporter
Cations
124	0136	bcp	Bacterioferritin comigratory protein
348	1077	nixA	High-affinity nickel transport protein
352	1073	copP	Copper-associated protein
353	1072	copA	Copper-transporting P-type ATPase
423	0471	kefB	Glutathione-regulated potassium efflux system protein
442	0490		Putative potassium channel protein
529	0582	tonB1	Siderophore-mediated iron transport protein
598	0653	pfr	Nonheme iron-containing ferritin
626	0686	fecA1	Iron(III) dicitrate transport protein
627	0687	feoB	Ferrous iron transport protein B
727	0791	hmcT	Heavy-metal cation-transporting P-type ATPase
743	0807	fecA2	Iron(III) dicitrate transport protein
821	0888	fecE	Iron(III) dicitrate transport system ATP-binding protein
822	0889	fecD	Iron(III) dicitrate transport system permease protein
903	0969	czcA1	Cation efflux system protein
904	0970	czcB1	Cation efflux system protein
1109	1183		Na+/H+ antiporter
1248	1328	czcB2	Cation efflux system protein
1249	1329	czcA2	Cation efflux system protein
1258	1339	exbB2	Biopolymer transport protein
1259	1340	exbD2	Biopolymer transport protein
1260	1341	tonB2	Siderophore-mediated iron transport protein
1263	1344	corA	Magnesium and cobalt transport protein
1396	1503	fixI	Component of cation transport for cbb3-type oxidase
1426	1400	fecA3	Iron(III) dicitrate transport protein
1447	1552	nhaA	Na+/H+ antiporter I
Nucleosides, purines, and pyrimidines
1106	1180		Nucleoside transporter
1210	1290	pnuC	Nicotinamide mononucleotide transporter

^aGene numbers correspond to those in Fig. 1. 

TABLE 2.

Annotation and classification of genes from H. pylori J99 and 26695

Annotation category	No. of genes in:
	H. pylori J99	H. pylori 26695	Both strainsa
Functionally classified	877	898
Conserved with no known function	275	290
H. pylori specific	343	364
Total	1,495	1,552
Amino acid biosynthesis	44	44	44
Biosynthesis of cofactors, etc.	60b	59	59
Cell envelope	160	164	156cde
Cellular processes	96	113	92cde
DNA replication	23	23	21ce
DNA restriction-modification, etc.	66	68	51cde
Energy metabolism	104	104	102cde
Fatty acid and phospholipid metabolism	28	29	28e
Purine and pyrimidine biosynthesis	34	34	34
Regulatory functions	32	32	31df
Transcription	13	13	13
Translation	128	128	128
Transport and binding proteins	88g	87	87
Conserved with no known function	275	290	267cde
H. pylori specific	343	364	288cde
Total	1,495	1,552	1,401h

^aUsing J99 genes as the basis for counting. 

^bIncludes the partial duplication of folE (JHP862). 

^cCategories which include H. pylori J99-specific genes (see the text for details). 

^dThese numbers include the “split” genes based on the H. pylori J99 definition. There are 6 J99 genes which constitute 12 26695 genes in the cell envelope; 2 J99 genes which constitute 4 26695 genes in cellular processes; 1 J99 gene which constitutes 2 26695 genes in each of DNA restriction-modification, energy metabolism, and Regulatory functions; 5 J99 genes which constitute 7 26695 genes in conserved with no known function; and 24 J99 genes which constitute 33 26695 genes in H. pylori specific. 

^eCategories which include H. pylori 26695-specific genes. 

^fDoes not include the duplication of the response regulator (JHP1283 and JHP1443). 

^gIncludes the partial duplication of proX (JHP754). 

^hThe remaining 94 genes represent the 89 J99-specific genes, tnpA/B from IS606, and the partial or complete duplications of three genes. 

FIG. 1.

Linear representation of the H. pylori J99 chromosome, illustrating the location of each predicted protein-coding region, rRNA gene, tRNA gene, IS605 or IS606 element and related fragment, and NotI endonuclease site. The predicted protein-coding regions are color coded based on functional classification (see the bottom of the figure for the code), with the direction of transcription indicated by an arrowhead. H. pylori J99 ORFs are numbered sequentially in red, and the corresponding homologous gene, if it exists in strain 26695, is numbered in black. The positions of the NotI endonuclease sites in J99 are indicated with the number of conserved nucleotides in the recognition sequence (x/8) at the corresponding position in strain 26695. The numbers associated with the tRNA symbols (inverted triangles) represent the number of tRNA genes at a specific locus. Vertical hash marks, below the linear chromosome, are located every 20 kb.

Comparison of orthologous genes and their encoded products showed a high degree of conservation. Sequence variation between the two strains was significantly greater at the nucleotide level than at the amino acid level. Because the nucleotide variation occurred most commonly in the third position of a coding triplet, the primary sequence of the encoded protein was highly conserved (Table 3). The fact that many of the nucleotide differences are silent with respect to the protein sequence suggests that there is a strong selective pressure for functional conservation at the protein level.

TABLE 3.

Nucleotide and amino acid identity between genes common to H. pylori J99 and 26695

% Identity	No. (%) of predicted ORFsa
	Nucleotide	Amino acid
100	0 (0)	41 (2.9)
98.0–99.9	8 (0.6)	269 (19.3)
96.0–97.9	249 (17.8)	359 (25.7)
94.0–95.9	566 (40.5)	279 (20.0)
92.0–93.9	306 (21.9)	169 (12.1)
90.0–91.9	89 (6.4)	86 (6.2)
85.0–89.9	81 (5.8)	77 (5.5)
<85	97 (7.0)	116 (8.3)
Total	1,396 (100)	1,396 (100)

^aGenes that appear “split” by putative frameshifts in either strain have been classed as the larger ORF in the above analysis. 

The nucleotide drift in the third position of a coding triplet is probably responsible for the majority of the DNA-based “diversity” reported for H. pylori (3, 4, 12, 54, 75, 77, 148). For example, pulsed-field gel electrophoresis mapping data have been interpreted to mean that the gene order and physical arrangement of the chromosome are highly variable from strain to strain (75, 148). By using this technique, strain J99 and strain 26695 would appear to be highly divergent in both the number of NotI fragments and gene location (6). This apparent genetic diversity is easily explained by two inversions in combination with the silent nucleotide drift, which is responsible for six of the seven additional NotI sites found in strain J99 compared to strain 26695 (6). Although the genomic content and the resulting physiological capabilities of the two strains are almost identical, these few differences in gene arrangement would have classified these strains as diverse. This example reveals the limitations of DNA-based methods when used to examine strain diversity.

NUTRITIONAL REQUIREMENTS

Amino Acids and Polyamines

Both sequenced strains of H. pylori have homologues to all the genes that would be needed to synthesize eight amino acids from central intermediary metabolites (Table 4). Studies of the growth requirements for several strains of H. pylori have shown an absolute need for arginine, histidine, leucine, isoleucine, valine, methionine, and phenylalanine (118, 132), a finding consistent with the genomic sequence information.

TABLE 4.

Predicted biosynthetic abilities and auxotrophies of H. pylori

Category and Compound	No. of genes presenta	Predicted synthetic abilityb
Amino acids
Aspartic acid	2 (2)	Y
Cysteine	2 (3)c	Y
Glutamic acid	1 (1)	Y
Glutamine	1 (1)	Y
Glycine	1 (1)	Y
Lysine	6 (7)d	Y
Threonine	5 (5)	Y
Tryptophan	5 (5)	Y
Alanine	1 (2)	N
Arginine	0 (9)	N
Histidine	0 (8)	N
Isoleucine	2 (9)	N
Leucine	1 (6)	N
Methionine	1 (7)	N
Phenylalanine	1 (3)	N
Proline	1 (3)	N
Valine	2 (9)	N
Asparagine	0 (2)e	?
Serine	2 (3)	?
Tyrosine	1 (3)	?
Cofactors and vitamins
Biotin	4 (4)	Y
CoA	0 (5)d	Y
Folate	7 (10)d	Y
Molybdopterin	8 (9)c	Y
Panothenate	4 (4)	Y
Protoheme	10 (10)	Y
Pyridine nucleotides	3 (5)d	Y
Pyridoxial phosphate	3 (?)d	Y
Riboflavin	6 (7)c	Y
Thiamine	3 (9)cd	Y
Thioredoxin	2 (2)	Y
B12
Glutathione	0 (2)	N
Siroheme	0 (1)	N
Ubiquinone	3 (8)	?
Menaquinone	0 (6)	?f
Polyamines
Agmatine	1 (1)	Y
Putrescine	1 (3)	N
Spermidine	3 (5)	?g
Pyrimidines	11 (10)h	Y
Purines	3 (10)	Ni

^aNumber of genes assigned in E. coli shown in parentheses. Data from reference 119. 

^bY, yes; N, no; ?, not clear. 

^cH. pylori has a gene for each step in the biosynthetic pathway. The number of genes in E. coli is larger due to redundancy. 

^dNot all genes in this pathway have been identified in E. coli. 

^eSynthesis may occur via tRNA (see the text). 

^fThe presence of menaquinones in H. pylori has been demonstrated (see the text). 

^gSpermidine synthesis may be mediated by NapC. 

^hTwo copies of pyrC were found in H. pylori. 

ⁱSalvage pathway is present (see the text). 

Although no homologues to the genes involved in the amidation of aspartate were detected in H. pylori, several strains have been reported to grow in the absence of asparagine (132). It is possible that asparagine is synthesized by an aspartyl-tRNA-asparagine amidotransferase, similar to what has been observed with glutaminyl-tRNA biosynthesis in Bacillus subtilis (see “Transcription and translation” below) (29).

Both the serine and tyrosine biosynthetic pathways were complete except for a homologue to their respective specific transaminase. However, each of these reactions may be catalyzed by one of the several identified transaminases with undetermined substrate specificity (JHP206/HP220, JHP568/HP624, JHP673/HP736, and JHP976/HP405). Such an enzymatic activity would allow the de novo synthesis of these amino acids, as observed in some strains of H. pylori (132). Regardless of whether H. pylori can synthesize serine, it possesses a specific transporter which allows the acquisition of this amino acid from the environment. sdaC, which encodes the serine transporter, is contiguous with sdaB, whose protein product in Escherichia coli converts l-serine to pyruvate. Similarly, putP, which encodes a proline transporter, is adjacent to putA, which encodes a bifunctional enzyme that oxidizes proline to l-glutamate in E. coli. The alanine transporter gene (JHP877/HP0942) is clustered with two other genes (alr and dadA) which are involved in alanine metabolism (Fig. 2). A positive regulator of the dad operon is thought to be upstream of this gene cluster in E. coli. In H. pylori, a putative regulatory gene (JHP879/HP0944) has also been identified upstream of the dad gene cluster. This putative regulatory gene does not have homology to the putative E. coli regulator, a finding which may indicate that the regulation of alanine catabolism is different in these two species. The H. pylori gene clusters described above would allow for the uptake and utilization of serine, proline, and alanine as carbon and nitrogen sources. In addition, H. pylori has a transporter for the uptake of glutamate (JHP1399/HP1506), an amino acid abundant in gastric juice (82).

FIG. 2.

Central metabolic pathways of H. pylori. Boxed compounds are key central intermediates. Black lines are reactions predicted to occur from the genomic analysis. Blue lines represent reactions that have been reported in the literature and are consistent with genomic analysis. Green lines represent a predicted reaction occurring only in strain 26695. Yellow lines represent a predicted reaction occurring only in strain J99. Broken magenta lines represent reactions reported in the literature but for which no homologue to the enzyme has been identified in either genome. Red lines represent key steps regulating gluconeogenesis/glycolysis.

H. pylori also encodes homologues for four other amino acid uptake systems with unknown specificity. One of these systems consists of a gene cluster encoding a multisubunit periplasmic permease (JHP1096–1099/HP1169–1172). The putative operon encodes two permeases, an ATP-binding protein and a periplasmic binding protein. Based on sequence similarity, the ligand for this high-affinity transporter may be either glutamine, histidine, or arginine. H. pylori is unable to synthesize the last two amino acids and therefore requires transport systems for them. Furthermore, the apparent inability of H. pylori to synthesize phenylalanine, methionine, and the branched-chain amino acids necessitates specific transport of these amino acids. No such specific transport systems were identified, but they may be encoded by any of the transport systems with unassigned ligand specificity.

In addition to transporting amino acids, H. pylori may have the ability to transport the abundant peptides which are found in the stomach. Homologues to a dipeptide transport system are present and its five genes (JHP283–287/HP0298–0302) are arranged contiguously, similar to the organization of the dpp operon in E. coli (1). There is also a single gene (JHP1172/HP1251) which displays significant sequence similarity to an oligopeptide transporter.

The genomic sequence provides little information on the composition of polyamines in H. pylori, which are needed for optimal growth in most cells. The homologue of SpeA allows the conversion of arginine to agmatine in H. pylori. Although H. pylori has a homologue to speE, which encodes spermidine synthetase, it is unlikely that this enzyme can catalyze spermidine biosynthesis since no homologues for the enzymes that provide precursors for SpeE (SpeB to SpeD) were detected. However, H. pylori may be able to synthesize spermidine through nspC. The product of this gene can synthesize spermidine by decarboxylating carboxyspermidine (117). It is also possible that H. pylori uses nspC for the synthesis of norspermidine, a polyamine found in Vibrio alginolyticus.

Cofactors and Vitamins

Both sequenced strains of H. pylori have all the identified genes needed for the biosynthesis of biotin, folate, heme, molybdopterin, pantothenate, pyridoxial phosphate, riboflavin, and thioredoxin (Table 4). H. pylori has all the genes necessary for the synthesis of NAD with the exception of nadB, which encodes the aspartate oxidase subunit of quinolate synthetase in E. coli (138). This polypeptide is the oxygen-utilizing subunit of an enzyme which converts l-aspartate to iminoaspartate. The absence of NadB is not unexpected since the mechanism by which anaerobic or microaerophilic bacteria, such as H. pylori, synthesize iminoaspartate is unknown and is not likely to be oxygen dependent. In addition, nicotinamide mononucleotide transporter (JHP1210/HP1290) was identified. No homologues to enzymes involved in vitamin B₁₂ and coenzyme A biosynthesis were identified. Vitamin B₁₂ is an important cofactor for certain enzymes involved in anaerobic metabolism such as methionine synthase. H. pylori may not need to synthesize this vitamin, since homologues to B₁₂-requiring enzymes were not identified. Bacteria synthesize coenzyme A de novo from pantothenate. However, no homologues to known enzymes involved in its biosynthesis were identified in H. pylori, making this pathway unique with respect to those previously reported. The pathway for thiamine biosynthesis has not been completely defined. Some of the genes believed to be involved in thiamine synthesis were found, suggesting that H. pylori can make this vitamin. However, it has been reported that H. pylori requires thiamine for growth (118).

H. pylori has homologues to all of the genes necessary to produce riboflavin. A single gene in H. pylori, homologous to both ribB and ribA (JHP740/HP0804), encodes a bifunctional enzyme with both GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone 4-phosphate synthase activities (158). In addition, H. pylori has a separate GTP cyclohydrolase II (RibA) homologue (JHP738/HP802) downstream from the bifunctional RibAB. Worst et al. (158) have shown that expression of RibAB but not RibA is regulated by iron limitation in H. pylori. The significance of this enzymatic duplication and differential gene regulation is unknown.

Purine and Pyrimidine Biosynthesis, Salvage, and Interconversion

Enzymes for the de novo biosynthesis of purines are largely absent in both sequenced strains of H. pylori, which implies that this bacterium cannot synthesize purine nucleotides from formate, glycine, or serine. Genes that encode homologues for all of the purine salvage and interconversion enzymes are present (Fig. 3A). No homologues to a purine transporter were identified, although there is biochemical evidence for the transport of purine bases in H. pylori (107, 118). Based on similarity, it is likely that the putative transporter (JHP1106/HP1180) is specific for nucleosides, which would allow H. pylori to obtain purines via the salvage and interconversion pathways.

FIG. 3.

Pyrimidine salvage and interconversion (A) and purine salvage pathways (B). A, adenosine; G, guanine; HX, hypoxanthine; X, xanthine; R, ribonucleoside; dR, deoxyribonucleoside. Adapted from reference 119.

H. pylori possesses homologues to all of the genes necessary for the de novo synthesis of UTP and CTP, consistent with experimental results that show that radiolabeled pyrimidine nucleotide precursors are incorporated into DNA (108). Unlike E. coli, H. pylori possesses a second pyrC homologue encoding dihydroorotase, raising the possibility that the H. pylori enzyme exists as a heterodimer rather than as a homodimer as reported in E. coli. One of the two homologues (JHP528/HP0581) is more closely related to a PyrC in gram-negative organisms, whereas the other homologue (JHP251/HP0266) is more closely related to a PyrC in gram-positive organisms. Furthermore, H. pylori lacks a homologue for the regulatory chain of the aspartate transcarbamoylase (PyrI), suggesting that either a paralogous gene serves this function or PyrB functions in the absence of a regulatory subunit.

H. pylori has homologues for all of the enzymes used for the interconversion of pyrimidine deoxyribonucleotides (Fig. 3B) with the exception of thymidylate synthase (ThyA), which is required for the interconversion of dUMP to dTMP. In addition, homologues for all of the enzymes associated with the pyrimidine salvage pathway are absent except for DeoB. This absence is consistent with poor utilization of uracil and uridine and with the failure to detect incorporation of added thymine, cytosine, or deoxycytidine into DNA by H. pylori (108). Although thymidine kinase activity has been found in crude extracts of H. pylori (108), no gene encoding a homologue of thymidine kinase (tdk) was identified in H. pylori.

Inorganic Elements and Heavy Metals

Phosphorus is an essential element in bacteria. H. pylori possesses homologues to both polyphosphate kinase and inorganic pyrophosphatase. These enzymes confer the ability to synthesize and hydrolyze polyphosphate, in agreement with experimental evidence (17). H. pylori also possesses a phosphate transporter (JHP1384/HP1491).

Sulfur assimilation is restricted in H. pylori compared to E. coli. Homologues to the genes necessary for the assimilation of sulfide and cysteine (cysE and cysK) are present in both sequenced strains, whereas those for the assimilation of sulfate (cysA, cysC, cysD, cysH, and cysN), an energy-consuming process, are not. The absence of an identifiable sulfate permease supports the apparent inability of H. pylori to use sulfate. Whereas H. pylori can utilize only sulfide as a source of inorganic sulfur, the closely related bacterium Campylobacter jejuni has homologues to the genes necessary to assimilate sulfate, sulfite, and sulfide (based on analysis of the recently completed genome by the Sanger Centre). This difference in sulfur assimilation between H. pylori and Campylobacter spp. is also seen in sulfur dissimilation. Unlike many Campylobacter spp., H. pylori does not have the homologues necessary for the respiration of many sulfur compounds. The absence of these sulfur assimilatory and dissimilatory genes in H. pylori probably reflects the evolved physiology resulting from its unique gastric niche.

In E. coli and Salmonella typhimurium, nitrogen is derived mainly from the primary products of ammonia assimilation, i.e., glutamate and glutamine. Glutamine synthetase (GlnA) catalyzes the formation of glutamine, while either glutamate dehydrogenase (GdhA) or glutamate synthase (GltBD) catalyzes the formation of glutamate (131). Homologues to glnA and gdhA were identified, and the gene product of glnA has been characterized in H. pylori (45). Glutamate dehydrogenase and glutamine synthetase allow H. pylori to incorporate nitrogen from urea into amino acids, presumably via ammonia, as demonstrated previously (157). No homologue of GltBD was identified. In other bacteria, the absence of this enzyme results in the inability to grow in a medium with low levels of free ammonia (18, 32, 126). Presumably, H. pylori does not need GltBD because sufficient levels of free ammonia are generated by enzymes such as urease.

Homologues of genes belonging to several iron uptake systems were identified in both sequenced strains, indicating the importance of iron metabolism in H. pylori is similar to that in other pathogenic bacteria. H. pylori possesses homologues to some of the genes involved in the ferric citrate (Fec) transport system, but no identifiable homologues to FecB, a periplasmic protein, or FecC, a component of the cytoplasmic membrane channel, were found. Three homologues to genes encoding the outer membrane receptor FecA are present in H. pylori, one of which may be involved in iron uptake via the ferric citrate system. One of the three fecA homologues is adjacent to the feoB homologue, a gene encoding a cytoplasmic ferrous iron permease. These two genes may be involved in ferrous, rather than ferric, uptake. Another fecA homologue may be part of the TonB-dependent iron uptake system (55, 80). The tonB homologue is adjacent to homologues of the remaining two genes (exbB and exbD) comprising the TonB-dependent iron uptake system. In addition, two other sets of exbB and exbD genes were identified but are probably involved in other transport processes (13). H. pylori may also acquire iron through FrpB, for which four homologues were identified. In Neisseria spp., FrpB paralogs are outer membrane proteins that are induced at low iron concentrations, have homology to lactoferrin- and heme-binding proteins, and are postulated to be involved in iron acquisition. Under iron limitation, many bacteria use siderophores to acquire iron. Whether H. pylori produces siderophores is controversial (67, 70). No homologues to genes involved in siderophore biosynthesis were found. H. pylori may not need siderophores, because the amount of free inorganic iron present in the stomach should be sufficient to support bacterial growth.

In addition to iron transport, H. pylori possesses homologues to several other heavy-metal transporters, including NixA, which is responsible for Ni²⁺ uptake, a function necessary for urease activity (114), and CopAP, which is responsible for Cu²⁺ uptake (47). H. pylori also possesses homologues to a high-affinity, multisubunit molybdate uptake system, which is needed for the biosynthesis of molybdopterin.

Carbohydrates

Genomic analyses indicate that H. pylori has a limited capability to acquire sugars from the environment, which is in agreement with experimental findings (102). Only a homologue for a phosphoenolpyruvate-independent glucose and galactose transporter was identified, consistent with previous metabolic studies (98, 102). The apparent absence of other sugar uptake systems suggests a limited ability for sugar catabolism in H. pylori (see below). Several homologues to organic acid transporters were identified: two l-lactate permeases, a ketoglutarate permease, and a C₄-dicarboxylate transporter that is used under anaerobic conditions in other bacteria. The presence of these transporters suggests that organic acids serve as important sources of carbon for H. pylori (see below).

In summary, based on the analysis of the sequence from both strains, H. pylori is capable of synthesizing the cofactors necessary for growth and acquiring important inorganic elements, although it is limited in its ability to use sulfur. H. pylori would be auxotrophic for at least nine amino acids and purines. Interestingly, no complex sugar transport or degradation homologues were found, suggesting that the bacterium does not acquire sugars from the environment and uses them as sources of energy or as sugar precursors.

CENTRAL INTERMEDIARY AND ENERGY METABOLISM

Central Intermediary Metabolism

Glycolysis and gluconeogenesis.

Genes involved in the metabolism of saccharides to simple sugars were not identified. This finding is consistent with metabolic studies which suggest that complex sugars are not a major energy source for H. pylori (102). Homologues for all enzymes, which carry out reversible steps in the pathway, are present in both sequenced strains of H. pylori. Since homologues of the two nonreversible gluconeogenic enzymes (fructose-1,6-bisphosphatase and pyruvate dikinase) are present and since homologues of the two nonreversible glycolytic enzymes (phosphofructose kinase and pyruvate kinase) were not identified, it appears that H. pylori uses the enzymes of the glycolytic/gluconeogenic pathway for anabolic biosynthesis rather than for catabolic energy production. The experimental evidence supports this hypothesis (59).

Entner-Doudoroff and phosphopentose pathways.

The genomic sequences show that H. pylori possesses homologues of all the genes involved in the Entner-Doudoroff pathway, suggesting that glucose can be used as a source of energy, which is consistent with published data (20, 103, 104). H. pylori has homologues of genes that encode all the enzymes in the phosphopentose shunt except gluconate-6-phosphate dehydrogenase. The presence of this enzymatic activity in crude extracts of H. pylori has been suggested (59, 100). However, the conversion of gluconate-6-phosphate to ribulose-5-phosphate could occur indirectly via the Entner-Doudoroff and the phosphopentose pathways (Fig. 2). The phosphopentose pathway enzymes that were identified in H. pylori allow for the generation of all the intermediates normally produced by this pathway.

Pyruvate metabolism.

The genomic analyses suggest that glucose or malate is not the primary source for production of pyruvate in H. pylori but, rather, that lactate, l-alanine, l-serine, and d-amino acids are the primary sources, which is consistent with the literature (106, 143). H. pylori appears only to convert pyruvate to acetyl coenzyme Δ (acetyl-6A) by pyruvate oxidoreductase (63, 64), lacking homologues for pyruvate dehydrogenase, pyruvate formate-lyase, and pyruvate oxidase. H. pylori dissimilates pyruvate to produce acetate, formate, succinate, and lactate (99, 106), an experimental observation consistent with the genomic analyses.

Fermentation.

H. pylori ferments pyruvate to acetate. Genetic analysis indicates that strain J99 can carry out this fermentation but that strain 26695 cannot do so due to a frameshift in its phosphotransacetylase gene. This mutation also implies that strain 26695 cannot convert acetate to acetyl-CoA by running the fermentative pathway in the reverse direction. However, in H. pylori 26695 the single, identifiable strain-specific gene involved in energy metabolism is an acetyl-CoA synthetase (HP1045) (149), which allows the direct conversion of acetate to acetyl-CoA.

The presence of alcohol dehydrogenase activity in H. pylori suggests that this bacterium can ferment pyruvate to ethanol (135, 136). Indeed, a homologue of this enzyme was identified in both strains. In addition, the single, identifiable strain-specific gene involved in energy metabolism in strain J99 (JHP1429) is a second alcohol dehydrogenase homologue. The role of alcohol dehydrogenase in H. pylori is unclear, because this organism apparently cannot ferment pyruvate to ethanol due to the absence of an identifiable acetaldehyde dehydrogenase homologue. The absence of such a homologue is consistent with biochemical studies (135, 136). The ability of H. pylori to produce acetaldehyde from ethanol (133) suggests that this bacterium is very sensitive to alcohols since it cannot detoxify the resulting acetaldehyde.

Tricarboxylic acid cycle.

The tricarboxylic acid (TCA) cycle of H. pylori in both sequenced strains is similar to the branched anaerobic TCA pathway used by E. coli (Fig. 2) with the following exceptions. Succinyl-CoA is generated from 2-oxoglutarate rather than from succinate (64). Furthermore, in H. pylori, fumarate is generated in the TCA pathway from aspartate rather than from malate. In contrast, genomic analysis suggests that in the closely related bacterium C. jejuni, succinyl-CoA is synthesized from succinate and that fumarate is synthesized from malate (based on analysis of the recently completed genome by the Sanger Centre). H. pylori possesses a homologue of fumarase, explaining the conversion of malate to fumarate observed in crude extracts (101). Unlike C. jejuni, no other malate-utilizing enzymes, such as malate dehydrogenase, malate synthase, or malate oxidoreductase, were identified in H. pylori.

Fatty acid degradation.

Both sequenced strains of H. pylori contain the genes necessary for C₂ or short-chain fatty acid atabolism (25) and for a short-chain fatty acid transporter. No identifiable homologues were found to the genes involved in long-chain fatty acid β-oxidation. Together, these observations indicate that H. pylori may utilize acetoacetate and not acetobutyrate as a source for short chain fatty acid catabolites.

Electron Transport Chain

Electron donors.

The initial transfer of electrons during the oxidation of d-lactate and NADH may be performed by homologues to d-lactate dehydrogenase (dld), NADH dehydrogenase I (nuoA-nouN), and hydrogenase (hyaA to hyaDD). The presence of these genes in both sequenced strains is consistent with measured activities in H. pylori cell membranes (22, 93, 101, 105). Although homologues to NuoE and NuoF were not identified in H. pylori, the nuo cluster contained two ORFs in an identical location and of similar size to the two E. coli genes. Thus, it is likely that these two H. pylori ORFs encode proteins with orthologous functions to NuoE and NuoF. In addition, homologues for the following electron-transferring systems were found: pyruvate ferredoxin oxidoreductase (porA, porB, porG, and porD), glycerol-3-phosphate dehydrogenase (glpC), and proline dehydrogenase (putA).

No genes encoding a succinate dehydrogenase homologue were identified, although such an activity has been observed in extracts from several H. pylori strains, including J99 (34, 59, 120). This apparent discrepancy can be explained by the observation that fumarate reductase can convert succinate to fumarate in vitro. The importance of fumarate reductase in respiration depends on environmental conditions. Under microaerophilic conditions, fumarate reductase (46) is not essential in H. pylori, which explains why high concentrations of fumarate reductase-specific antimicrobials are required to inhibit the growth of and kill H. pylori in vitro (105). Under these conditions, oxygenic respiration may be used by the bacterium and hence reduces the importance of fumarate reductase in metabolism. In the absence of oxygen, this enzyme may be essential. In the presence of oxygen and fumarate, H. pylori, like members of the related genus Wolinella, may prefer fumarate as a terminal electron acceptor over performing oxygenic respiration (61). The role fumarate reductase plays in respiration cannot be assessed until the microenvironment of H. pylori is better defined.

Quinones and cytochromes.

Analysis of the genomic sequence provides no clear indication to the composition of the H. pylori quinone pool. No homologues to genes involved in the biosynthesis of menaquinones, elements of anaerobic respiration, were identified despite the reported presence of menaquinone-6 and menaquinone-1 in the cell membranes of H. pylori (52, 94). The absence of identifiable homologues implies that H. pylori obtains menaquinones either by synthesis with genes that have yet to be identified or by uptake from its environment. H. pylori does contain homologues for three ubiquinone-biosynthetic enzymes (UbiA, UbiD, and UbiE), but no significant homologues for UbiB, UbiC, UbiF, UbiG, or UbiH were identified. UbiB, UbiF, and UbiH are oxygen-utilizing enzymes, and their apparent absence in H. pylori might be the result of the microaerophilic metabolism of the bacterium.

The biosynthesis of cytochromes by H. pylori has been reviewed in detail recently (51, 125). H. pylori uses a type II system for such biosynthesis, which is similar to that in many gram-positive bacteria and some members of the β-proteobacteria. Both strains possess homologues to all of the components needed for this biosynthetic system.

Terminal electron acceptors.

There appear to be three putative terminal electron acceptor systems in both sequenced strains of H. pylori, i.e., fumarate reductase, N-oxide reductase, and cytochrome c oxidase, suggesting that H. pylori may be able to use fumarate, N-oxides (i.e., dimethyl sulfoxide and trimethylamine-N-oxide), or oxygen as electron sinks. Whereas the presence of a cytochrome c oxidase in H. pylori would allow aerobic respiration, the presence of both fumarate reductase and an N-oxide reductase suggests that the bacterium may respire anaerobically as well. The terminal oxidase complex is similar to cbb₃-type oxidase complexes and is encoded by a gene cluster composed of homologues to genes encoding the Rhizobium FixN, FixO, FixP, and FixQ subunits. The arrangement of these genes is identical to that found in Rhizobium spp. (130), and the presence of such a terminal oxidase in H. pylori is consistent with previous findings (116).

ATP-Proton Motive Force Conversion

The bacterial ATP synthase, a multisubunit enzyme, is composed of the F₀ complex, which consists of three subunits that form a proton channel, and the F₁ complex, which consists of five subunits that constitute the catalytic site for ATP synthesis. In E. coli, all eight subunits are encoded within the atp operon (76). All five subunits of the F₁ complex and the F₀ b subunit are contiguous on the H. pylori chromosome. The remaining two subunits of the F₀ complex are encoded by genes present in other chromosomal regions. H. pylori has an additional subunit (JHP1065/HP1137), which is homologous to the ATPase b′, a diverged and duplicated form of the b subunit found among plants and photosynthetic bacteria. The gene encoding this homologue is located at one end of the ATP synthase gene cluster. Functionally, the H. pylori ATPase is similar to other bacterial ATPases in that it uses the proton motive force generated by the electron transport chain to synthesize ATP (97).

Detoxification

Organisms that come in contact with oxygen, like the microaerophilic H. pylori, must be able to protect themselves from the toxic products of oxygen metabolism, such as superoxide and hydrogen peroxide. Both sequenced strains of H. pylori possess a superoxide dismutase and a catalase, consistent with previous biochemical findings (57, 122, 140). Thus, H. pylori is a microaerophile not because of an inability to neutralize the toxic products of oxygen metabolism but, more probably, as a consequence of other metabolic limitations. H. pylori has two genes encoding peroxidases (JHP991/HP0390 and JHP1471/HP1563), one of which is located adjacent to the superoxide dismutase gene. The ability to isolate catalase-negative mutants of H. pylori (122, 156) suggests that at least one of the peroxidases can function as a catalase.

In summary, it would appear that H. pylori can use amino acids or simple carbohydrates as a major sources of energy. The bacterium is restricted with respect to pyruvate metabolism. Further, H. pylori possesses an electron transport chain that can use oxygen as a terminal electron acceptor, but homologues to fumarate reductase and N-oxide reductase suggest that the bacterium is capable of at least limited anaerobic metabolism.

MACROMOLECULE BIOSYNTHESIS AND MODIFICATION

DNA Replication, Recombination, and Restriction-Modification

H. pylori contains genes encoding homologues to the DnaE, DnaN, DnaX, DnaQ, and HolB subunits of the DNA polymerase III holoenzyme, which is responsible for DNA replication. While the H. pylori holoenzyme contains fewer than the 10 subunits that comprise DNA polymerase III in E. coli, the total number of subunits is consistent with that found in other bacterial genomes (24, 31, 43, 81, 84, 139). Indeed, the only common subunits among these different species are DnaE, DnaN, and DnaX. H. pylori contains homologues to all genes encoding enzymes involved in initiation and DNA chain elongation, except dnaC, which is also absent in several other bacterial genomes (24, 31, 43, 81, 84, 139).

H. pylori possesses homologues to several nucleases, including UvrABC endonuclease, UvrD, ExoA, and RecJ. Even though H. pylori contains homologues to xseA and mutS, it lacks recognizable homologues encoding the other subunits of exonuclease VII (XseB) and the MutHLS endonuclease repair complex, respectively. Recombinational repair in H. pylori appears to occur in a RecBC-independent manner (92), since the RecBCD exonuclease V is absent and a RecR homologue was identified. Other homologues identified in the recombination system include RecA and RecN, while no homologue to recE encoded exonuclease VIII was found. In addition, homologues of ruvABC and recG, whose products are involved in the branch migration and resolution of Holliday structures, were identified in H. pylori. Both H. pylori genomes also possess several homologues to the DNA topoisomerase I gene (topA), some of which are strain specific (6). There are genes encoding several other ATP-dependent helicases in H. pylori, including a homologue for pcrA and rep. Both sequenced H. pylori strains have numerous genes with similarity to DNA restriction and modification enzymes, many of which are strain specific. This finding suggests that H. pylori strains have their own unique complement of these genes (2, 6). While many of the products of these genes can be classified as type I, II, or III restriction or modification enzymes, their exact specificity remains to be determined.

Both H. pylori genomes appear to have three type I systems. In all three systems, the modification (HsdM) and restriction (HsdR) subunits are highly homologous between the strains but the specificity subunits (HsdS) have limited identity. Domains of HsdS proteins can be shuffled to produce new specificity, and it is this modular nature which allows the type I systems to evolve rapidly to a new DNA specificity.

Some of the type II systems in H. pylori 26695 and J99 appear to be functionally equivalent and may possess the same DNA specificity, while others have identity within the methyltransferase enzyme but differences in the restriction enzyme. There are nine type II modification methylases common to both genomes (6). In addition, H. pylori J99 possesses two unique type II restriction-modification systems. In H. pylori 26695 and several other strains, the M.HpyI gene, encoding a type II modification enzyme (159), flanks iceA, which encodes a putative type II restriction endonuclease (149). The absence of iceA in H. pylori J99, a recent clinical isolate from a patient diagnosed with a duodenal ulcer, suggests that iceA is not required for gastrointestinal disease and may not represent an informative epidemiological marker of pathogenicity and virulence, as previously hypothesized (15, 152).

Both H. pylori 26695 and J99 contain two type III restriction-modification systems, one of which is strain specific. Whereas in the other system (JHP1410/1411, HP1521/1522) the restriction gene product is 93% identical in the two strains, the modification gene product is conserved only at the N and C termini. This difference may result in unique specificity for each modification enzyme. The modification gene appears to be regulated by a slipped-strand repair mechanism and is frameshifted in both J99 and 26695 (6).

Transcription and Translation

Analysis of the H. pylori genomes identified only three sigma factors (RpoD, RpoN, and FliA). The presence of these three sigma factors had been suggested by putative promotors found upstream of H. pylori genes (83, 86, 89, 122, 141, 147). No homologue to the stationary-phase sigma factor (RpoS) or the heat shock-specific sigma factor (RpoH) was identified, implying that H. pylori responds to stress in different fashion from that described in other bacteria. Both sequenced H. pylori strains contain a fusion of rpoB and rpoC, which encode the β and β′ subunits of RNA polymerase (149, 160). H. pylori contains homologues to three termination factors (nusA, nusB, and rho) and lacks identifiable homologues to the tRNA maturation genes rnd, rph, and rnpB. The lack of identifiable transcriptional termination stem-loop structures suggests that in H. pylori termination is largely Rho dependent (6).

All the aminoacyl-tRNA synthetases are present in H. pylori, except glutaminyl- and asparaginyl-tRNA synthetases. Two copies of the gene encoding glutamyl-tRNA synthetase (gltX) are present in both H. pylori strains. It had been suggested that one of these copies may function as a glutaminyl-tRNA synthetase (149). However, the presence of homologues to the gatA, gatB, and gatC genes, which have been demonstrated to replace glutaminyl-tRNA synthetase activity in Bacillus subtilis (29, 146), makes it more likely that glutaminyl-tRNA synthetase activity in H. pylori is encoded by the gatABC homologues rather than gltX (6). Thus, the role of the second glutamyl-tRNA synthetase in H. pylori is unclear. A similar transamidation reaction, encoded by these three homologues or other genes, may function in H. pylori to synthesize asparaginyl-tRNA from aspartyl-tRNA, explaining the ability of H. pylori to grow without added asparagine despite its apparent inability to synthesize this amino acid.

Fatty Acid and Phospholipid

Identifiable homologues were found to many of the genes required for initiation and elongation of fatty acid biosynthesis. Both H. pylori strains contain the characteristically small acyl carrier protein (ACP) (78 amino acids) (JHP744/HP0808). H. pylori 26695 but not J99 possesses a second, significantly larger (153-amino-acid) homologue with an extended N-terminal domain (HP962). Genomic analysis indicates that H. pylori has a homologue to cyclopropane fatty acid synthase (JHP969/HP0416), consistent with the experimental evidence that H. pylori has a preponderance of C_19:0 cyclopropane chains (73). No homologue to β-hydroxydecanoyl-ACP dehydrase, which catalyzes the formation of cis-3-decenoyl-ACP, an important intermediate in the biosynthesis of unsaturated fatty acids in E. coli, was found. The absence of this homologue is surprising, since unsaturated fatty acids are present in H. pylori (73).

The phospholipid composition of H. pylori consists mainly of phosphatidylethanolamine, cardiolipin, and phosphotidylglycerol, with smaller quantities of phosphatidylserine and phosphatidylcholine (73). The genome appears to encode all of the proteins necessary for the synthesis of these phospholipids except for cardiolipin synthase (cls), which catalyzes the final step of cardiolipin synthesis in E. coli. The H. pylori genome encodes at least two of the three enzymes necessary for phosphatidic acid synthesis (JHP895/HP0961 and JHP1267/HP1348). A homologue to the glycerol-3-phosphate acyltransferase was not identified in the H. pylori genome.

A characteristic feature of the lipid profile of H. pylori is the presence of cholesterol glucosides, which account for about 25% of the total lipid of the bacterium (56, 58). H. pylori does not appear to encode known enzymes for the synthesis of cholesterol and presumably scavenges this molecule from the environment. Little is known about glucoside-modified cholesterol synthesis, but the enzymes responsible are expected to be found among the species-specific genes with unknown function.

Peptidoglycan

The cytoplasmic synthesis of UDP-activated precursors of bacterial peptidoglycan assembly is well understood. The H. pylori genome encodes homologues to all of the enzymes in this pathway, beginning with the synthesis of UDP-N-acetylmuramic acid and ending with UDP-disaccharide pentapeptide linked to an undecaprenol lipid carrier (Fig. 4).

FIG. 4.

Peptidoglycan synthesis and recycling. GlcNAc, N-acetylglucosamine; MurNAc, N-acetylmuramic acid; Ala, alanine; Glu, glutamate, Dap, diaminopimelic acid. Adapted from reference 62.

After transport through the cytoplasmic membrane, peptidoglycan precursors are incorporated into the existing peptidoglycan layer by the penicillin-binding proteins (PBPs) (50). The genome of H. pylori encodes three proteins which show homology to PBPs, in agreement with a published gel electrophoresis study (68). The precise metabolic function of each PBP is uncertain. However, one of the PBPs (JHP544/HP597) has a transglycosylase motif. Since no additional genes with a similar motif were detected, this PBP may be the only enzyme in H. pylori that is involved in lengthening the glycan chain.

From the analysis of the genomes, it is uncertain if peptidoglycan fragments can be recycled by H. pylori. Although this bacterium has homologues to genes encoding a lytic amidase (JHP709/HP0772) and an N-acetylmuranoyl-l-alanine transglycosylase (JHP590/HP0645), it does not appear to have the other genes required for this recycling system (ampG, ampD, mpl) (127).

Outer Membrane

Approximately 4% of the coding capacity of both strains is devoted to outer membrane proteins. This amount is significantly larger than that of any other bacterial genome sequenced to date. The majority of these proteins belong to three paralogous families, the largest having 20 and 21 members in J99 and 26695, respectively (6). Several members of the largest paralogous family are porins (33, 41) or adhesins specific for the Lewis B carbohydrate moiety found on host cells (72). The sequence identity of orthologous members of this large family is high (greater than 95%), suggesting that strain-specific sequence differences play only a limited role in antigenic variation. Five orthologous pairs of this outer membrane protein family, including BabB (Lewis B adhesin), contain dinucleotide (CT) repeats in their signal sequences. Slipped-strand repair has been proposed to regulate the expression of these proteins (6, 149). Significantly, the predicted expression status (in frame or out of frame) of these five pairs is identical despite the presence of a different number of dinucleotide repeats in the two strains in each case (6).

Lipopolysaccharide

H. pylori has homologues to all enzymes required for 2-keto-3-deoxyoctulosonic acid (KDO)-lipid A biosynthesis. Compared to lipid A in E. coli, this moiety in H. pylori is underacylated, has longer fatty acid chains (C₁₆ and C₁₈), and lacks a phosphate group at the 4′-hydroxyl position on the nonreducing glucosamine of the disaccharide (115). Taken together, these findings suggest that in H. pylori lipid A is assembled as an acylated and diphosphorylated disaccharide, which is then modified by an unidentified phosphatase and esterase enzyme(s).

The chemical structures of the lipopolysaccharide cores from two H. pylori strains were shown to be identical heptasaccharides (9, 10). Synthesis of such a structure requires several glycosyltransferases. There are seven ORFs encoding these putative glycosyltransferases (JHP147/HP0159, JHP194/HP0208, JHP563/HP0619, JHP620/HP0679, JHP741/HP0805, JHP765/HP0826, and JHP1031/HP1105), which are common to both strains, three strain-specific ORFs in J99 (JHP562, JHP820, and JHP1032), and one strain-specific ORF in 26695 (HP1578). However, none of these ORFs can be assigned a substrate specificity. Whether the presence of these strain-specific glycosyltransferases in J99 and 26695 results in a different lipopolysaccharide core structure remains to be determined.

The O chain from lipopolysaccharides of H. pylori is composed of Lewis acids [Le^x and Le^y; mono- and difucosylated repeating disaccharides of β-(1,4)-linked galactose and N-acetylglucosamine, respectively] (9, 10). These carbohydrate moieties, which are identical to those found on host tissues, have been implicated in colonization and persistence and may also play a role in autoimmunity (7, 8). The biosynthetic pathway of these O chains has not been determined. One enzyme, α-(1,3)-fucosyltransferase (JHP596/HP0651), is to be involved in this pathway (21, 96). Each genome has two α-(1,3)-fucosyltransferases which differ in the number of a 7-amino-acid sequence repeat (YDDLRVN). Regulation of these genes appears to occur through slipped-strand repair at two distinct polynucleotide repeats (6).

Flagella

Flagellar biosynthesis in gram-negative bacteria has been extensively studied. The assembly of a functional flagellum requires numerous proteins and is a highly regulated process. Homologues to all of the required genes involved in flagellar biosynthesis were identified in both sequenced strains. In other gram-negative bacteria, including C. jejuni, inactivation of the biosynthetic pathway disrupts the expression of the system as a whole. However, in H. pylori, inactivation of the hook protein does not result in suppression of flagellin expression (124), indicating that flagellar biosynthesis is not as highly regulated as in other bacteria. This difference can be explained by the absence of FlgM in H. pylori, a protein that in other bacteria controls feedback regulation of the flagellar biosynthetic cascade.

The flagellar filament of H. pylori is composed of two flagellin subunits, FlaA and FlaB, and genes encoding both are present. Despite reports suggesting that the genes for these flagellin subunits exhibit strain variation (66, 123), in strains J99 and 26695 the FlaA protein sequences are identical and the FlaB protein sequences differ by only a single amino acid. In addition, the protein sequences reported for FlaA and FlaB from another strain are nearly identical to those from strains J99 and 26695 (89).

The flagellar filament of H. pylori is encased within a sheath that is continuous with the outer membrane (90, 91, 128). The sheath, whose composition has yet to be defined, probably protects the polymeric filament from dissociation in the low pH of the stomach. Likewise, other polymeric structures, such as fimbriae, would also be subject to dissociation at low pH, thus explaining why adhesion to the host epithelium by H. pylori appears to be mediated by integral outer membrane proteins (71, 72).

In summary, H. pylori has the necessary homologues for DNA, RNA, and protein synthesis.

CELLULAR PROCESSES

Protein Secretion

H. pylori has homologues to two leader peptidases, enzymes required for protein secretion. The type I leader peptidase, LepB, is responsible for cleavage of the signal sequence from most periplasmic and outer membrane proteins. The lspA (previously called ureD) gene product is the leader peptidase responsible for processing prelipoproteins.

The secretion of proteins through the cytoplasmic membrane utilizes specific machinery which consists of several sec gene products. H. pylori contains all the known Sec proteins except SecB and SecG. Mutation of secB, which encodes a chaperone, affects the secretion of only a limited number of proteins in E. coli, and other preproteins may utilize alternative chaperones. Indeed, it has been demonstrated that groESL mutants in E. coli have significant effects on the secretion of some proteins (85). Thus, it is likely that in H. pylori, other cytoplasmic chaperones are used to usher proteins, in conjunction with SecA, to the cytoplasmic membrane in preparation for secretion. SecE is proposed to be integral to the translocation machinery (121). Initial analysis of both genomes did not identify a SecE orthology. However, a region between JHP1126/HP1203 and JHP1127/HP1204 contains a 59-amino-acid ORF that has similarity to the functionally important region of E. coli SecE.

Cag Pathogenicity Island

H. pylori strains associated with clinically severe gastric disease (peptic ulcers) more commonly possess an approximately 40-kb pathogenicity island containing cytotoxin-associated genes (Cag pathogenicity island [cagPAI]) than do strains isolated from patients with uncomplicated gastritis (26). Type I strains have been defined as H. pylori isolates which have the entire cagPAI, express the cytotoxicity-associated immunodominant antigen (CagA) and an active vacuolating cytotoxin (VacA), and induce interleukin-8 (IL-8) secretion by gastric epithelial cells. In addition to cagA, it has been demonstrated by mutational analysis that several of the genes in the cagPAI are required for wild-type induction of IL-8 secretion by gastric epithelial cells. In contrast, Type II strains do not express CagA, have no vacuolating activity even though a truncated VacA may be produced, and do not induce IL-8 secretion at levels comparable to that induced by type I strains.

The initial cagPAI sequence was produced in two parts by two different laboratories, each of which sequenced clones from the same ordered cosmid library that had been constructed from H. pylori NCTC 11638 (2, 19). These groups showed that the cagPAI was separated into the cagI and cagII segments by an intervening stretch of DNA that was itself bordered on each side by a newly identified insertion sequence (IS605) element. In contrast, the cagPAI in H. pylori J99 and 26695, both type I strains, consists of the cagI and cagII segments fused as a single unit without the stretch of intervening DNA flanked on each side by IS605. In all three strains, the cagI and cagII segments are essentially the same and the cagPAI is found in the same relative location.

Many of the genes in the cagPAI are required for specific host cell responses to infection by H. pylori, including induction of (i) IL-8 secretion by gastric epithelial cells (2, 19, 137), (ii) tyrosine phosphorylation in host proteins (137), and (iii) cytoskeletal rearrangements during actin pedestal formation at the host cell surface (137). The manner in which cagPAI genes cause these host cell responses is unknown. However, five ORFs show significant sequence similarity to genes encoding the VirB protein family, which is responsible for DNA transfer in Agrobacterium tumefaciens (type IV secretion system) (23). These genes also have sequence similarity to genes involved in conjugative transfer of plasmids in E. coli and protein export in Bordetella pertussis (155). It is unknown how these components of conjugative and protein transport systems function in H. pylori. It has been suggested that the cagPAI encodes a type of contact-mediated secretion apparatus analogous to the type III secretion systems identified and characterized in several enteric pathogens such as Yersinia, Salmonella, Shigella, and pathogenic E. coli (88). It is therefore likely that the virB homologues of the cagPAI region encode components of the secretion apparatus which may deliver effector molecules (DNA or protein) directly to the host cell to elicit the responses mentioned above.

Insertion Elements

The IS element IS605 contains genes encoding two previously identified transposases flanked by a short nucleotide sequence with dyadic symmetry and a common central core sequence (19). IS605 transposes as a single unit in E. coli, suggesting that it could also be functional in H. pylori (79). One of the transposase genes (tnpA) is related to IS200 found in gram-negative bacteria, and the other (tnpB) is related to IS1341 found in a gram-positive thermophylic bacterium. It is unusual to find an insertion element with transposases from apparently two different origins. The IS605 element was first described within the cagPAI of NCTC 11638. Strain 26695 has five full copies of IS605 and one copy of a related insertion element, IS606, none of which is located within the cagPAI. Strain J99 has no complete copies of IS605 but has one copy of IS606 on its chromosome. The short flanking sequence of IS605, without the transposases (is605), is present on both ends of the cagPAI in strain NCTC 11638. These sequences flanking cagPAI are thought to be remnants of a recent transposition or of another type of recombinational event. The dyadic repeats of IS605 and IS606 are also found within the J99 and 26695 genomes, at both common and distinct locations. IS605 dyadic repeats are coincident, in one or the other genomes, with several of the major organizational differences between the two sequenced strains. H. pylori plasmid pHPM186 (GenBank accession no. AF077006) contains an IS605 element which is adjacent to three genes that flank an IS605 element in the plasticity zone of strain 26695 (6). This finding suggests that plasmid integration plays a role in generating the limited genomic diversity in H. pylori. Thus, insertion elements, such as IS605, may have been involved in the acquisition of cagPAI and the plasticity zone by H. pylori via horizontal transfer, as has also been postulated for other pathogenicity islands found in a wide range of pathogenic bacteria.

Transformation

Many H. pylori strains are naturally competent for DNA transformation, and the efficiency of this process varies from strain to strain. Besides the conjugative DNA transfer/protein export homologues (VirB proteins) encoded by the cagPAI, both strains contain additional members of this family, some of which are strain specific. Recently, some members of the VirB family have been shown to play a role in DNA transformation in H. pylori (60). The existence of strain-specific VirB family homologues may explain the variation in DNA transformation efficiency seen between strains. One additional transformation-associated gene (comEC), which is required for uptake of DNA into B. subtilis, was also identified.

Chemotaxis

Chemotaxis, the sensory adaptation mechanism by which motile bacteria recognize and react to environmental conditions, has been found in H. pylori (110). Three homologues of the chemotaxis pathway in E. coli (CheW, CheA, and CheY), as well as four methyl-accepting chemotaxis proteins (MCPs), which mediate specificity for ligands, were identified in both sequenced strains of H. pylori. Proteins with similarity to MCPs are not necessarily involved in flagellar chemotaxis (5, 30). Neither strain contains identifiable homologues to CheR or CheB, enzymes which, respectively, add methyl groups to or remove methyl groups from the MCPs, precisely modulating the chemotactic response. By contrast, C. jejuni does possess homologues to both CheR and CheB. Therefore, the chemotaxis observed in H. pylori may occur by a CheB- and CheR-independent mechanism, similar to that seen in CheB CheR mutants of E. coli (145). The apparent inability of H. pylori to precisely modulate chemotaxis may reflect its limited but unique gastric niche.

Both H. pylori strains have three homologues to CheV, another chemotaxis protein, which has an N-terminal domain similar to CheW and a C-terminal response regulator domain similar to CheY (44). The CheW protein and the N-terminal domain of CheV are both capable of modulating the CheA-MCP interaction (134). The three CheV orthologous pairs have greater than 97% identity, whereas none of the paralogues have more than 40% identity at the amino acid level, suggesting that each orthologous pair has functional similarity and that each paralogue has a different specific function.

Cell Division and Morphology

Several genes implicated in bacterial cell division were found in both sequenced strains of H. pylori. Among these are ftsZ and ftsA, which are adjacent to each other, as is generally observed (154). H. pylori does not possess an unambiguous homologue to FtsW, but it does possess two homologues to RodA, a protein with significant sequence similarity to FtsW (69). It is possible that one of the RodA homologues actually functions as FtsW. In E. coli, at least one of the peptidoglycan-synthesizing enzymes is specifically involved in cell division (142). H. pylori has three homologues to peptidoglycan-synthesizing enzymes, one of which could act specifically during cell division. Two homologues of the metalloprotease FtsH, phenotypically associated with cell division, were identified. Interestingly, mutagenesis studies have shown that one of the FtsH homologues (JHP356/HP1069) is essential for growth in vitro (48). This suggests that the second homologue (JHP271/HP0286) is unable to functionally replace the first homologue and probably has a different function. In addition, homologues of ftsK and fic were identified.

No homologues to ZipA, which is believed to initiate formation of the FtsZ ring (53), SulA, which is thought to inhibit the formation of the FtsZ ring (14), FtsQ, FtsN, or FtsL were identified. Also, no homologue to MinC, a cell division inhibitor, was found, whereas homologues to the activator (MinD) and cofactor (MinE) of MinC were identified.

Two homologues of cell morphology-determining proteins, RodA and MreB, were found. RodA is required for the catalytic activity of a PBP during elongation in E. coli (74), whereas the function of MreB is unknown.

Virulence Factors

A number of H. pylori proteins have been implicated in pathogenesis (for reviews, see references 36 and 95). All of the reported genes encoding potential virulence factors in H. pylori were identified in both strains, with the exception of iceA, which is missing from strain J99 (see “DNA replication, recombination, and restriction-modification” above).

Bacterial motility has been suggested to be required for colonization of the gastric mucosa, since H. pylori mutants unable to synthesize either of the flagellar subunits (FlaA or FlaB) cannot colonize gnotobiotic piglets (38, 40). Whether motility is needed for the persistence of an infection is not known, but owing to the rapid gastric epithelium and mucous turnover, it would probably be required.

The cytotoxin VacA induces vacuole formation in cultured epithelial cells and may be an important component in the induction of gastric cell lesions by H. pylori (27, 49). Different alleles of the vacA gene have been reported (11, 28). Strains J99 and 26695 possess different vacA alleles (s1b/ml and s1a/ml, respectively) and were isolated from patients with H. pylori-related disease of different severity. Epidemiological studies with humans have correlated the level of expression of the vacA gene with disease outcome (42), and particular vacA genotypes are associated with more severe disease symptoms (109, 144, 151). Interestingly, in gnotobiotic piglets, a mutant with a knockout mutation of vacA had no discernible effect on colonization, epithelial vacuolation, or gastritis, suggesting that VacA is not a virulence factor in this animal model (37).

The urease enzyme of H. pylori has been extensively studied (for reviews, see references 111 to 113) and has been shown to be a colonization factor (39, 78, 150). Urease is found in the cytoplasm as well as on the surface of H. pylori. The mechanism by which this enzyme is translocated is controversial (129, 153).

Other components which have been implicated in virulence, include the cagPAI, lipopolysaccharide, outer membrane proteins, and a number of enzymes, such as phospholipases, catalase, superoxide dismutase, and a mucinase homologue (36). The involvement of these components in pathogenesis remains to be elucidated. Homologues to the genes encoding all these products were present in both strains.

In summary, H. pylori possess homologues to the genes necessary for DNA replication, transcription, translation, and cell division, as well for as the synthesis of other important cellular macromolecules such as lipids and peptidoglycan. Each strain appears to possess a number of unique restriction enzymes that would result in selectivity to transformation with foreign DNA. Further features of this bacterium are the large number of related outer membrane proteins and the presence of a putative pathogenicity island.

CONCLUSION

The genomic analysis suggests that both strains have essentially identical metabolic potential. The strain-specific genes that encode proteins with an assigned function are predicted to have little impact on the physiology of H. pylori. For the most part, the reported experimental data for biochemical activities present or absent in H. pylori are in agreement with the predicted metabolic capabilities. Our genomic analysis reveals nutritional requirements which would restrict the environments in which H. pylori can survive and, in part, explains its limited niche. H. pylori has broad catabolic capabilities. While it can perform oxygenic respiration, anaerobic respiration, and fermentation, H. pylori is limited in what it can use as a carbon source. Carbohydrate utilization is restricted to glucose, via the Entner-Doudoroff pathway, and to sugars with shorter carbon backbones than C₆, for which transport systems are present. In addition, H. pylori possesses numerous transporter systems for the uptake of amino acids, which must be available in the gastric environment. Other than carbohydrates, H. pylori apparently uses amino acids as an important source of carbon which would yield less energy than using hexoses and could result in slow growth as occurs in vitro. Further, the deamination of amino acids, together with the action of urease, requires a mechanism to deal with increased levels of ammonia.

Colonization by H. pylori involves an interaction between the outer membrane of the bacterium and the gastric epithelium of the host. The outer membrane composition of H. pylori is unique in its protein content and lipopolysaccharide structure, which are consistent with the persistence of H. pylori in a restricted niche. Compared to other bacteria, H. pylori devotes a significantly higher percentage of its coding capacity to that of outer membrane proteins, further emphasizing the importance of these proteins. A number of genes involved in determining the composition of the outer membrane are differentially regulated by slipped-strand repair (6). This differential regulation and strain-specific outer membrane-related genes may play a role in the severity of H. pylori-related disease and the ability of H. pylori to persist chronically in its host. The other strain-specific genes may also play a role in these aspects of pathogenesis. None of these strain-specific genes with an assigned function are predicted to have a significant impact on the pathophysiological capabilities of H. pylori, although those with an unassigned function may be important. However, there is evidence that host factors are involved in H. pylori-related diseases (35), and our genomic analyses suggest that these host factors may play a more significant role than was previously appreciated.

The analysis presented here, like previous analyses of other sequenced bacterial genomes, has found several biochemical pathways for which we were unable to identify all of the genes which should be present for that pathway to be functional. Some of these genes may not be present in the genome. In other cases, the biochemical activity may be present within the organism but the gene responsible for this activity may be unidentifiable by current in silico techniques. The apparent incompleteness of the same metabolic pathways in two unrelated strains may suggest that these pathways are functional in H. pylori and that the unidentified genes are different from previously described orthologues. These genes would be among the 40% of the genome to which no function has been assigned.

For the first time, the genomes of two strains from the same bacterial species have been compared (6). This publication provided an opportunity to begin defining the physiology of H. pylori, a globally important pathogen. Future genomic comparisons of multiple strains, carefully correlated with epidemiological data, will identify the minimal genomic complement of this species and the genes required for virulence. Such an approach will be applied to other pathogenic bacteria, and the genes identified from these studies will become candidates for therapeutic intervention.