New Flagellin-Specifying Genes in Some Escherichia coli Strains

Abstract

Data for further development of the flagellar antigen genetics of the species Escherichia coli are reported. Two new flagellin genes named fllA and flmA were found in E. coli 781-55, E2987-73, and E223-69, the test strains for E. coli flagellar antigens H44, H55, and H54, respectively (collection of the International Escherichia and Klebsiella Centre of the World Health Organization, Copenhagen, Denmark). Two alleles of fllA were identified that encode flagellar antigens H44 (fllA₄₄) and H55 (fllA₅₅), and the only flmA allele found (flmA₅₄) encodes antigen H54. The sites of their integration in the E. coli K-12 chromosome after P1-mediated transduction were approximately determined and found to be separate from each other and from the known regions of flagellar genes of E. coli and salmonellae. The region of flm₅₄ was found to repress the expression of some alleles of the flagellin gene fliC. In addition, cryptic genes encoding antigens H4 and H38 were found in phenotypically monophasic test strains 781-55 and E2987-73, respectively.

Escherichia coli K-12, the E. coli strain genetically studied in the greatest detail, possesses a single flagellin gene and is therefore monophasic (2, 14). The gene was initially designated hag and recently renamed fliC (10). For a long time, this was believed to describe the phenotypic behavior and genetics of flagellar antigens in E. coli strains in general; the different flagellar antigen serotypes would correspond to different alleles of fliC (hag).

However, some natural isolates of E. coli were then found to contain two alternatively expressed, not cotransducible flagellin genes, hagA and hagB; the former is an allele of the fliC gene of E. coli K-12, while the latter is located at a distance from it (20, 23). It was also shown that the region of hagB is a distinct one integrating in the E. coli K-12 chromosome outside its hitherto known three flagellar regions, as well as far from the site of the integration of the salmonella phase 2 flagellin region. In conformity with the new nomenclature (10), it therefore seems expedient to rename the hagB gene flkA. (The designations of flagellin genes are discussed in the Discussion.) The alteration of the activity of these genes was found to be usually nonreversible (unilateral): fliC^off, flkA^on → fliC^on, flkA^off. The on or off state of fliC correlates with the off or on state of flkA, respectively, because the flk region produces a special (phase-specific) repressor activity similar to that of the product of salmonella repressor gene fljA, acting upon some (sensitive) alleles of fliC; therefore, the existence of a gene similar to fljA that is coexpressed with flkA was postulated (20, 21, 23). New data reported here show an even larger variety in the genetics of flagellar antigens in the natural population of E. coli.

MATERIALS AND METHODS

Strains.

All of the E. coli strains used are listed in Table 1.

TABLE 1.

E. coli strains used in this study

Straina (H anti- gen expressed)	Flagellar genotypeb	Other relevant markers	Origin (reference[s])c
781-55 (H44)	fliC4offfllA44on		A (19)
E2987-73 (H55)	fliC38offfllA55on		A (19)
E223-69 (H54)	flmA54on		A (19)
EJ34 (NM)	fliCiah-1		E. coli K-12 derivative (7)
EJ262 (e,n,x)	fliCiah-1 hin+ fljBe,n,xfljA+		Salmonella phase 2 flagellin region transduced into EJ34 (6)
PM298 (H48)	fliC48on	Strr RifrproA23 trp-30 his-51	E. coli K-12 derivative (23)
PM304 (H44)	fliC4offfllA44on	Strr RifrproA23 trp-30	A; PM298 with flagellin genes received from F′ variant of strain 781-55
PM315 (H3)	fliCiah-1 flkA3on		EJ34 with flkA3 introduced (23)
PM335 (H48)	fliC48on	Nalr Rifrstr-104 leuB6 hisG1 argG6 metB1 malT1 xyl-7 mtlA2	E. coli K-12 derivative (23)
PM336 (H16)	fliC16on	Same as in PM335	E. coli K-12 derivative (23)
PM342 (H16)	fliC16on	str-3 hisG1 purF pheA2 argH mtlA2 malT1 xyl-7	E. coli K-12 derivative (23)
PM470 (H44)	fliCiah-1 fllA44on		A; EJ34 with fllA44 transduced from PM304
PM471 (H55)	fliCiah-1 fllA55on		A; EJ34 with fllA55 transduced from E2987-69
PM472 (H54)	fliCiah-1 flmA54on		A; EJ34 with flmA transduced from E223-69
PM476F′ (H55)	fliCiah-1 fllA55on		A; PM471 with sex factor F′ Lac+ Kmr introduced
PM479a (H54)	fliC16offflmA54on	Δ(pro-lac) thi-1	A; Hfr-donord
PM479b (H54)	fliC16offflmA54on	Δ(pro-lac) thi-1	A; Hfr-donord
B99-2 (H6)	fliC6on		(20, 21)

^aIn strain EJ34, native E. coli K-12 flagellin gene fliC₄₈ was replaced with salmonella phase 1 flagellin gene fliC_i (previously named H1) altered by the mutation ah-1 and unable to produce flagellin; therefore, the strain was nonflagellated and NM, although all of the other flagellar genes were intact (7). 

^bSuperscripts: off, latent state; on, expressed state of a flagellin gene. 

^cA, flagellar genotype determined during this study. 

^dHfr donors PM479a and PM479b were similar but produced separately during this study from E. coli K-12 Hfr strain KL191 (13) by substituting its fliC₄₈ (repressor-insensitive allele) for fliC₁₆ (repressor-sensitive allele) and then introducing flmA₅₄. 

Culture media.

Solid, semisolid, and liquid rich media (20) and minimal glucose salt agar (16) were used. Antibacterial drugs, amino acids, nucleic acid bases and/or H antisera were added when appropriate (23).

Crosses.

Transductional experiments were carried out by means of temperature-inducible phage P1clr100Km^r-3 (8, 20). Conjugal crosses were carried out in broth (16) for 100 min with Hfr or F′ donors containing F′ Lac⁺ Km^r (23).

Selection of transductants and exconjugants.

Flagellar transductants were selected for motility on semisolid medium supplemented with appropriate anti-H serum to counterselect (by immobilization) bacteria retaining the recipient’s flagellar antigen, but no antiserum was used when nonmotile (NM), flagellin-nonproducing strain EJ34 served as the recipient. For the selection of exconjugants by their nutrition requirements, minimal agar with necessary additions was used. H-antigen specificities were determined by means of an agglutination test (20).

Selection of alternative flagellar phase.

Three to five transductants of each class were grown on semisolid medium containing antiserum to the expressed H antigen, thus providing strict conditions for testing of their ability to produce an alternative flagellar antigen phase (20, 22).

RESULTS

Identification of a new flagellin locus in the standard test strains for H55 (E2987-73) and H44 (781-55).

It was easy to propagate the phage used (P1clr100Km^r-3) on strain E2987-73 but not on strain 781-55. Therefore, fertile bacteria produced from the latter by introduction of plasmid F′ Lac⁺ Km^r were mated with strain PM298, selecting for Str^r Rif^r His⁺ recombinants; one that was motile on semisolid agar containing antiserum H48 and exhibited flagellar antigen H44 was designated PM304 (Table 1) and used for following genetic analysis.

Transduction experiments were carried out with donor strains PM304 (H44) and E2987-73 (H55) and several recipients, either NM (EJ34) or motile, representing different flagellar antigens (Table 2, experiment A). A large number of transductants were obtained from NM recipient EJ34; they all were either H44 (with PM304 as the donor) or H55 (with E2987-73 as the donor), demonstrating that the genes encoding H44 and H55 were efficiently transferred and could be expressed in E. coli K-12 derivatives. (Two of these transductants, designated PM470 with H44 and PM471 with H55 [Table 1], were used in further experiments.) By contrast (Table 2, experiment A), no transductants were obtained with either donor from recipient strains PM335 (H48), PM336 (H16), and PM315 (H3) when selecting for motility in the presence of antiserum to flagellar antigens expressed by them. Note that all of the recipients were derivatives of E. coli K-12 and, moreover, that PM315 was derived directly from EJ34, which was shown above to be capable of inheriting flagellin genes of the donors. The most probable explanation for the absence of transductants is that the cells inheriting donor genes continued to produce the recipient native H antigen and therefore were immobilized under selective conditions. This indicated that donor genes for H44 and H55 had not been able to replace flagellin genes fliC₄₈, fliC₁₆, and flkA₃ and were therefore not alleles of either fliC or flkA, and if integrated elsewhere, they did not suppress these genes.

TABLE 2.

Transductional analyses of new flagellin gene fllA of strains PM304 and E2987-73

Expt and donor, recipient strains	Counter- selecting antiserum	Appearance of motile clones	Description of motile clones tested
			No.	H antigen expressed	Alternative phasea
Expt A
PM304, EJ34	None	Yes	31	H44 (all)	NT
PM304, PM335	H48	No
PM304, PM336	H16	No
PM304, PM315	H3	No
E2987-73, EJ34	None	Yes	41	H55 (all)	NT
E2987-73, PM335	H48	No
E2987-73, PM336	H16	No
E2987-73, PM315	H3	No
Expt B
EJ262, PM470	H44	No
EJ262, PM471	H55	No
EJ262, PM336	H16	Yes	10	e,n,x (all)	H16
Expt C
PM304, PM471	H55	Yes	22	H44 (all)	NF
E2987-73, PM470	H44	Yes	34	H55 (all)	NF
Expt D
PM304, B99-2	H6	Yes	5	H4 (all)	NF
E2987-73, B99-2	H6	Yes	5	H38 (all)	NF

^aNT, not tested; NF, not found. 

To explore the relationship between the genes controlling antigens H44 and H55, on the one hand, and the salmonella phase 2 flagellin region, on the other hand, strain EJ262 was used as the donor (Table 2, experiment B) and PM470 (H44), PM471 (H55), and PM336 (H16) were used as recipients. The latter served as a control and showed that the phage grown on EJ262 transduced the salmonella phase 2 region containing flagellin-specifying gene fljB_e,n,x and another one (fljA) repressing recipient flagellin gene fliC₁₆. It must be emphasized that EJ262, as well as PM470 and PM471, is a derivative of EJ34 (6, 8), and the salmonella phase 2 flagellin region (the same as that in EJ262) was shown to be easily transduced to and inherited by EJ34 (6). However, no motile (under selective conditions) transductants resulted from recipients PM470 and PM471, thus indicating (in analogy to the above explanation) that, most probably, the sites of integration of the genes for H44 and H55, on the one hand, and of the salmonella phase 2 flagellin region, on the other hand, were different, and the repressor gene of the latter did not repress the gene for H44 or H55.

To determine whether the genes for H44 and H55 are alleles of one locus, transductions were carried out between PM304 (H44) and PM471 (H55), as well as between E2987-73 (H55) and PM470 (H44). They showed (Table 2, experiment C) that these genes are mutually exclusive and thus alleles of the same new flagellin locus, which I named fllA.

To localize approximately the site of the integration of fllA in the E. coli K-12 chromosome, PM471 (H55) was converted to a male PM476F′ by infection with F′ Lac⁺ Km^r and mated with female H16 strains PM336 and PM342 with the different chromosomal markers that allowed selection for their respective donor alleles; the H antigen was then determined as a nonselected character. Table 3 shows the percentages of recombinants for the different markers that inherited fllA₅₅; the closest linkage of fllA₅₅ observed was to pheA2, at map position 57.

TABLE 3.

Percent linkage of new flagellin gene fllA (allele fllA₅₅) with different chromosome markers transferred from donor strain PM476F′ into recipient strains PM336 and PM342 by conjugation

Recipient strain (H antigen expressed)	% (±mp) inheritance of fllA55 by recombinant selected for donor allele of the following markera:
	hisG1 (44)	argG6 (69)	malT1 (75)	xyl-7 (80)	mtlA2 (81)	metB1 (90)	hisG1 (44)	purF (50)	pheA2 (57)	argH (90)
PM336 (H16)	22.0 ± 4.2	19.4 ± 4.0	0	1.0	0	0
PM342 (H16)					0		21.0 ± 4.1	35.0 ± 4.8	63.0 ± 4.8	0

^aNinety-eight Arg⁺ (marker argG6) recombinant clones and 100 recombinant clones of each other class were tested. Counterselection of the donor was done with streptomycin. The map positions of the genes (in parentheses) are according to references 1 and 2. m_p, percent error. 

Four flagellar patterns were found among the recombinants tested, and their proportion depended on a marker used for selection. For instance, the Phe⁺ recombinants consisted of clones expressing (i) only recipient antigen H16 (35%), (ii) only donor antigen H55 (3%), (iii) the H16 H55 mixed phenotype (60%), or (iv) the NM phenotype (2%). NM recombinants produced rare motile variants (usually after several successive passages in broth) with salmonella flagellar antigen i, thus indicating that the immobility was a consequence of replacing recipient fliC₁₆ with the latent donor allele fliC_i with ah-1. Neither H16 nor H55 recombinants produced an alternative flagellar phase, presumably because H16 recombinants possessed only the recipient flagellin gene, while H55 recombinants inherited both donor flagellin loci fliC_i with ah-1 (in place of the native recipient allele fliC₁₆) and fllA₅₅. It is clear that the recombinants expressing two flagellar antigens (H16 and H55) possessed two different flagellin loci not repressing each other. These findings corroborate the supposition inferred from the results achieved by means of transduction.

Cryptic flagellin genes in standard strains 781-55 and E2987-73.

When transductions similar to those in experiment A of Table 2 were carried out with donors PM304 (H44) and E2987-73 (H55) and recipient B99-2 (whose H6 antigen is determined by fliC₆, which is insensitive to phase-specific repression), the results were different: all of the transductants obtained were either H4 (with the donor PM304) or H38 (with the donor E2987-73), and no transductants were found with H44 or H55 (Table 2, experiment D). This indicated, first of all, that the fliC allele, fliC₆, of the recipient B99-2 was not repressed by fllA₄₄ or fllA₅₅ and, furthermore, that the genes encoding flagellar antigens H4 and H38 were present (but not expressed) in the donor strains and then expressed in transductants. Why were they not expressed in the donor strains? Were they under phase-specific repression controlled by the fllA region?

To answer this question, transductants with flagellar antigen H4 or H38 were used as recipients for second-step transduction experiments carried out with the homologous donor strain, PM304 or E2987-73, on semisolid agar containing antiserum to H4 or H38, respectively; no transductants were obtained (data not shown), indicating that all eventual transductants inheriting the fllA region still expressed antigen H4 or H38 and were therefore immobilized by the serum. The reason for the latency of the genes in the H44 and H55 test strains thus remained unknown.

Identification of another new flagellin region in E223-69, the standard test strain for flagellar antigen H54.

H54 transductants were obtained with E223-69 as the donor and strain EJ34 (NM) or PM336 (H16) as the recipient, but attempts to obtain similar transductants from four other recipient strains (PM335 with H48, PM315 with H3, PM304 with H44, and PM471 with H55) failed (Table 4, experiment A), despite the close relationship of three of them to EJ34 or PM336 (PM335 was the immediate ancestor of PM336, and PM315 and PM471 were derived directly from EJ34). The most probable interpretation is that a gene responsible for H54 can be inherited and expressed in strains of the E. coli K-12 line (EJ34 and PM336) but is not able to replace or suppress any of the flagellin genes fliC₄₈, flkA₃, fllA₄₄, and fllA₅₅. On the other hand, the tested H54 transductants arising from recipient strain PM336 (H16) were able to produce an alternative flagellar phase, H16, thus indicating that the expression of allele fliC₁₆ (which is known to be sensitive to phase-specific repression) was governed by the introduced flagellin gene region of the donor. In this respect, the latter resembled the flkA₃ region of E. coli and the salmonella phase 2 flagellin region, and therefore it seemed interesting to look into its interaction with the relevant salmonella flagellin gene. So, transduction was carried out with donor strain EJ262 containing the salmonella phase 2 flagellin region in an active state and recipient strain PM472 (an H54-expressing transductant of EJ34) (Table 4, experiment B). No motile transductants were obtained on semisolid medium containing anti-H54 serum, indicating that, most probably, the gene for H54 could not be replaced with salmonella phase 2 flagellin gene fljB; the control transduction with recipient strain PM336 (H16) yielded the expected transductants expressing salmonella flagellar antigen e,n,x and capable of phase variation between it and H16.

TABLE 4.

Transductional analysis of new flagellin gene flmA of strain E223-69 (H54)

Expt and donor, recipient strains	Counter- selecting antiserum	Appearance of motile clones	Description of motile clones tested
			No.	H antigen expressed	Alternative phase
Expt A
E223-69, EJ34	None	Yes	60	H54 (all)	NFa
E223-69, PM336	H16	Yes	56	H54 (all)	H16
E223-69, PM335	H48	No
E223-69, PM315	H3	No
E223-69, PM304	H44	No
E223-69, PM471	H55	No
Expt B
EJ262, PM336	H16	Yes	21	e,n,x (all)	H16
EJ262, PM472	H54	No
Expt C
Bi7327-41, PM335	H48	Yes	21	H16 (all)	NF
Bi7327-41, PM336	H16	Yes	28	H3 (all)	H16
Bi7327-41, PM472	H54	No

^aNF, not found. 

The presumable inability of the gene encoding H54 (strain PM472) to be replaced with flk₃, on the one hand, and the ability of its region to suppress fliC₁₆, on the other hand, were inferred from the results shown in Table 4 (experiment C): although the phage propagated on strain Bi7327-41 effectively transduced both fliC₁₆ and flk₃ (the results obtained with recipient strains PM335 and PM336), no transductants were found if the recipient was PM472 (despite the fact that its ancestor, EJ34, possessed the ability to easily inherit and express fliC₁₆ and flk₃ of Bi7327-41 [22]).

These data indicated that the gene encoding H54 represented a new E. coli flagellin locus which was designated flmA (flmA₅₄). Mating experiments performed like those shown in Table 3, in which H antigen was the nonselected character, showed a close linkage of flmA₅₄ to the chromosomal marker mtlA2 (Table 5).

TABLE 5.

Percent linkage of flmA₅₄ to different chromosome markers transferred into recipient strain PM336 (H16) by conjugation

Donor strain	% (±mp) inheritance of flmA54 by recombinant selected for donor allele of the following marker:a
	argG6 (69)	malT1 (75)	xyl-7 (80)	mtlA2 (81)	metB1 (90)
PM479a	1.3	7.3 ± 2.1	44.7 ± 4.1	84.0 ± 3.0	31.3 ± 3.8
PM479b	2.0	5.3 ± 1.8	52.0 ± 4.1	88.0 ± 2.7	38.7 ± 4.0

^aOne hundred fifty clones of each selected recombinant class were studied. Streptomycin was used for counterselection of the donor. The map positions of marker genes (in parentheses) are according to references 1 and 2. Recombinants of only two flagellar phenotypes, H16 and H54, were found; the H16 recombinants tested were stable, while those of the H54 phenotype produced an alternative flagellar phase, H16. m_p, percent error. 

DISCUSSION

The experimental data presented here show an unexpected diversity of the flagellin gene pool in the E. coli population in nature. These findings, as well as other published data on the variety of E. coli flagellar antigen genetics, are summarized in Table 6. In this investigation, two new loci, fllA (alleles fllA₄₄ and fllA₅₅, determining antigens H44 and H55 in E. coli standard H-test strains 781-55 and E2987-73, respectively) and flmA (allele flmA₅₄ for antigen H54 in E. coli standard H-test strain E223-69), were discovered and shown, when being transferred in E. coli K-12 derivatives, each to have a different chromosomal localization than any of the previously identified flagellin genes of E. coli: fllA was closely linked to pheA2 (map position 57) and showed no linkage to mtlA2 (map position 81), while flmA was closely linked to the latter; fliC, the single flagellin gene of E. coli K-12 (10), is located at position 43 (2), and flkA (formerly hagB) is very close to argG6 (map position 69) (23). flkA (23), fllA, and flmA are not alleles of salmonella gene fljB because it does not replace them. The insertion site of the latter into the E. coli K-12 chromosome is at map position ca. 55 (6). The results of the conjugational crosses confirmed the proposals and conclusions inferred from the experiments carried out by means of transduction that fliC, flkA, fllA, flmA, and salmonella fljB belong to distinct flagellar regions and therefore, when transduced, are unable to replace one another.

TABLE 6.

Flagellin-specifying genes identified in E. coli strains

Flagellin gene		Approximate map position	Allele	Relationship to phase-specific repressora		Combination of two flagellin genes in some wild strainsb	Reason for latent state of fliCc	Flagellar antigend expressed (latent)	Straine	Refer- ence(s)f
Former name	New name			Sensitivity to repressor	Phase-specific repressor activity
hag, hagA, or hagC	fliC	43	fliC2	Sensitive		fliC2off, flk3on	PSR	H3 (H2)	1-391	21
			fliC6	Insensitive		Not reported		H6 (?)	B-99	20
			fliC16	Sensitive		fliC16off, flk3on	PSR	H3 (H16)	Bi7327-41	20, 23
			fliC21	Sensitive		fliC21off, flk47on	PSR	H47 (H21)	1755-58	1, 21, 23
			fliC38	Insensitive		fliC38off, fll55on	Unknown	H55 (H38)	E2987-73	A
			fliC48	Insensitive		An only gene		H48 (None)	E. coli K-12	6, 20, 23
			fliC4 or fliC′4	Insensitive		fliC4off, fll44on	Unknown	H44 (H4)	781-55	A
			fliC"17	Insensitive		fliC′4off, fliC"17on	Unknown	H17 (H4)	P12b	22
hagB	flkA	Linked to argG6 (position 69)	flkA3		+	fliC16off, flkA3on	PSR	H3 (H16)	Bi7327-41	20, 23
			flkA36		−	An only gene		H36 (none)	5017-53	23
			flkA47		+	fliC21off, flkA47on	PSR	H47 (H21)	1755-58	23
			flkA53		+	An only gene		H53 (none)	5017-53	23
Not known before	fllA	Linked to pheA (position 57)	fllA44		−	fliC4off, fll44on	Unknown	H44 (H4)	781-55	A
			fllA55		−	fliC38off, fll55on	Unknown	H55 (H38)	E2987-73	A
Not known before	flmA	Linked to mtlA2 (position 81)	flmA54		+	An only gene		H54 (none)	E223-69	A

^a+, presence of phase-specific repressor activity (suppressing sensitive alleles of fliC) owing to coexpression of a flagellin gene and a putative repressor gene similar to salmonella gene fljA; −, no phase-specific repressor activity. 

^bAn only gene, no other intact flagellin gene found; off, latent state; on, expressed state of a flagellin-specifying gene. 

^cPSR, phase-specific repressor activity. 

^dNone, absence of a latent flagellar antigen, confirmed by genetic data; ?, very rare alteration of the flagellar antigen was sometimes observed, but its genetic background is unknown. 

^eThe strains listed, with the exception of 1-391, B-99, and E. coli K-12, are all standard H test strains from the collection of the International Escherichia and Klebsiella Centre of the World Health Organization. Some other wild E. coli strains possessing the flkA₃ (hagB₃) gene or the phase-specific repressor-sensitive fliC₂ gene as the only flagellin-specifying locus have been reported (15, 21). 

^fA, this work. 

As to the nature of the integration into the E. coli K-12 chromosome of the flagellin gene regions other than that of fliC, one cannot exclude the possible presence in the relevant sites of the E. coli K-12 chromosome of sequences displaying at least some homology to these flagellar regions (flagellin genes), although the integration might be determined by sequences flanking rather a big P1-transduced piece of the chromosome.

The two newly described flagellar regions differ in the ability to suppress alleles of fliC sensitive to phase-specific repression. That of flmA₅₄ possesses this activity and is thus similar in this respect to the salmonella phase 2 flagellin region and the E. coli flk region. In contrast, the fll region (for both flagellin gene alleles fllA₄₄ and fllA₅₅) lacks such a feature, and particularly in this connection one may expect the existence of E. coli strains that express two flagellin genes. Indeed, I found naturally occurring E. coli strains of serogroup O18 exhibiting two different flagellar antigens, H14 and H55, simultaneously (24).

Concerning the gene nomenclature used, it is worth noting again that of the above-named E. coli flagellin genes, only one (fliC) has been found in E. coli K-12 and that this strain represents a genetically rather rare clone among the naturally occurring E. coli strains (5). Nevertheless, the principle developed for the new unified nomenclature of the E. coli K-12 and Salmonella typhimurium flagellar genes (10) seems to be also applicable for designation of the E. coli flagellar genes not reported to occur in these strains. The principle is based on the fact that the flagellar genes gather in three (E. coli K-12) or four (S. typhimurium) separate chromosome regions (14). Each region is assigned a three-letter symbol starting with fl, while the third letter is determined by the relevant flagellar region and used in alphabetical order starting with g. Therefore, three similar regions in E. coli K-12 and S. typhimurium have been designated flg, flh, and fli and the phase 2 flagellin region of S. typhimurium has been designated flj. Inside a region, the genes (to be distinguished) are given alphabetical (capital letter) extensions according to their genome order (e.g., fljA and fljB); however, exceptions to the latter rule are allowed (10). According to this principle, the designations flk, fll, and flm were used for the three distinct flagellin regions not found in E. coli K-12, with the extension A for the flagellin-specifying genes, and a subsequent subscript extension for an allele indicating the serotype of the flagellin determined by it.

However, sometimes the use of the principle seems not to be so simple regarding wild strains. Some wild strains may possess several (at least two) flagellin genes in the same region. For example, in E. coli P12b, the standard test strain for E. coli antigen H17, two flagellin genes formerly designated hagA₄ (assumed for an allele of hag, i.e., fliC) and hagC₁₇ were reported, and owing to their high cotransducibility (22), both should be considered constituents of the same third flagellar region (fli). The difficulty of naming such flagellin genes arises from the absence of any direct criteria for identifying genuine fliC in strain P12b, as well as in other wild E. coli strains, especially in view of the fact that the genome arrangement, at least in the vicinity near the flagellin gene(s) in the fli region in some wild strains, may be different from that of E. coli K-12. Therefore, the designations fliC′ and fliC" seem to be useful with respect to P12b; on the other hand, if only a single flagellin gene is found in this flagellar region, the gene designation fliC without any specification seems the most appropriate.

Phase-specific repressor sensitivity or resistance determined by salmonella gene fljA or by its analog (e.g., flkB, according to the principle of the new nomenclature) postulated in E. coli (20, 23) is not an intrinsic feature of fliC, as fliC₄₈ of E. coli K-12 is resistant, while fliC_i of S. typhimurium is sensitive. Besides, other sensitive (hagA₂, hagA₁₆, and hagA₂₁) and resistant (hagA₄ and hagA₆) alleles of fliC have been reported in wild E. coli strains (15, 20–23). On the other hand, genes insensitive to such kinds of repression may be cryptic for a different yet enigmatic reason, as was reported for cryptic gene fliC′₄ of strain P12b, which became active when fliC"₁₇ was spontaneously suppressed (22). Therefore, the presence of cryptic genes fliC₄ and fliC₃₈ in strains 781-55 (H44) and E2987-73 (H55), respectively, seems to be not extraordinary. Interestingly, however, the attempts to obtain these alleles in an active state by transducing them into E. coli K-12 derivatives failed (Table 2, experiment A), but it turned out to be possible when strain B99-2 (H6) was used as the recipient (Table 2, experiment D). The inability of H4 and H38 transductants to restore the H6 phenotype indicated that fliC₆ was not repressed but replaced by a donor flagellin gene or a sequence closely linked to and inserted into the recipient chromosome together with it. Therefore, the genes encoding H4 and H38 should be considered constituents of the third flagellar region and designated fliC₄ and fliC₃₈, respectively. It may be that the discrepancy between the results of transduction to different recipients depended on the probable difference between the genome arrangements in the vicinities of fliC₄₈ (E. coli K-12) and fliC₆ (B99-2) and that the silent state of fliC₄ and fliC₃₈ resulted from a very closely linked alteration.

The methods of transduction and conjugation used in this study were fruitful in discovering new phenomena and provided cogent arguments for the above-stated conclusions which gave new knowledge about E. coli flagellar genetics concerning the existence of the new flagellar regions and flagellin genes, as well as the new examples corroborating the natural occurrence of E. coli strains each containing two flagellin genes. Molecular genetic analysis should be used for further investigation of the phenomena, but it is inaccessible to me because of the catastrophic situation of science in my country.

The genetics of bacterial flagellins has recently become more complicated in other taxonomic groups, too (3, 4, 9, 11, 12, 17, 25–27). Although in E. coli the genetics of H-antigen specificity, like that of other features, was earlier focused on K-12, there have been rare attempts to study it in other E. coli wild strains (15, 18). The data presented here, together with those reported earlier (20–24), show that the knowledge of the flagellar genetics of E. coli K-12 only partially reflects the much more complicated and rather diverse flagellar genetics of the bacterial population covered by the taxonomic term E. coli.