Evidence of Localized Prophage-Host Recombination in the lytA Gene, Encoding the Major Pneumococcal Autolysin

Abstract

According to a highly polymorphic region in the lytA gene, encoding the major autolysin of Streptococcus pneumoniae, two different families of alleles can be differentiated by PCR and restriction digestion. Here, we provide evidence that this polymorphic region arose from recombination events with homologous genes of pneumococcal temperate phages.

Streptococcus pneumoniae (pneumococcus) is currently the main cause of acute otitis media, sinusitis, pneumonia requiring hospitalization in adults, and meningitis. These infections cause substantial morbidity and mortality worldwide (31). The three standard tests used in clinical laboratories to differentiate pneumococcus from other alpha-hemolytic streptococci are optochin (Opt) sensitivity, immunological reaction with type-specific antisera (capsular reaction or quellung test), and bile or deoxycholate (Doc) solubility (38). The property of bile solubility was first reported by Neufeld in 1900 (40) and is attributed to triggering of the major pneumococcal autolysin, LytA, an N-acetylmuramoyl-l-alanine amidase (NAM-amidase; EC 3.5.1.28) (30). This amidase is considered an important virulence factor of S. pneumoniae (6, 28, 41, 45).

Pneumococcal LytA (here designated LytA_Spn) exhibits a modular organization whereby the amino (N)-terminal domain acts as the enzyme's catalytic region and the carboxy (C)-terminal domain (choline-binding domain; ChBD) features a tandem of 7 repeated motifs involved in recognizing the choline residues present in the cell wall teichoic acids (for reviews, see references 36 and 37). LytA enzymes belong to the amidase_2 family of proteins (PF01510) (http://pfam.sanger.ac.uk/).

It is now well known that Streptococcus pseudopneumoniae and other streptococci of the mitis group (SMG) also synthesize a LytA-like NAM-amidase. However, and in sharp contrast with LytA_Spn, the LytA_SMG enzymes were inhibited by 1% Doc (43). As a consequence of this inhibition, SMG isolates are unable to lyse with Doc (Doc⁻ phenotype), although they may still harbor a lytA-like gene (4, 12, 20, 34, 43, 55). These Doc⁻ isolates are frequently nontypeable and/or resistant to optochin. Moreover, while lytA_Spn alleles are 957 bp long, SMG lytA alleles (951 bp) show a characteristic 6-bp deletion (ACAGGC) between nucleotide positions 868 and 873, which in the sixth repeated motif of LytA_Spn amidase codes for Thr₂₉₀-Gly₂₉₁ (43). Altogether, the main difference between the S. pneumoniae and SMG lytA alleles resides in 102 nucleotide positions (including the 6 bp missing from the SMG alleles). These nucleotides are perfectly conserved in all the S. pneumoniae alleles examined, as are the corresponding nucleotides in the SMG alleles (34).

Prophages seem to have an important role in S. pneumoniae biology, since it is now clear that more than half of all clinical pneumococcal isolates are lysogenic (47, 50). Especially important are the enzymes (endolysins) involved in cell lysis that are encoded by these phages. Interestingly, all pneumococcal prophages reported to date code for a 318-amino-acid-long NAM-amidase (LytA_PPH) that closely resembles the bacterial LytA enzyme. This characteristic was first reported for the temperate phages HB-3 (48), MM1 (42), and VO1 (44), and recently, 11 more prophages have been added to the list (23, 49). However, all previously reported lytic phages infecting S. pneumoniae carry genes that code for lysozymes (as do the so-called Cp family of phages) or for NAM-amidases (phage Dp-1), but in Dp-1, the endolysin belongs to a protein family (amidase_5; PF05382) different from that of S. pneumoniae (amidase_2) (25). Two temperate bacteriophages of Streptococcus mitis, φB6 and φHER, also code for a LytA_Spn-like endolysin of 318 amino acid residues (51), whereas the EJ-1 inducible prophage isolated from S. mitis 101 harbors a gene (ejl) with the characteristic deletion shown by lytA_SMG alleles (11).

The most significant characteristics of the bacterial strains and temperate phages analyzed here are listed in Table 1. We identified 112 entries in the databases (corresponding to 30 different sequence types [STs]) that fulfilled the requirements established, i.e., at least 900 nucleotides of the lytA gene available and no indeterminate nucleotides. Accessible lytA sequences corresponded to 58 pneumococcal isolates (26 lytA_Spn alleles), 28 SMG (23 lytA_SMG alleles), 23 pneumococcal prophages (19 lytA_PPH alleles), and 3 SMG prophages (3 lytA_SPH alleles) (last date accessed, 20 January 2010). The relatedness between STs was shown as a dendrogram, constructed by the unweighted pair group cluster method with arithmetic averages from the matrix of allelic mismatches between the STs. Multiple sequence alignments of the lytA alleles were generated with the Clustal W 2.0 program (33). Pairwise evolutionary distances (PED) (estimated number of substitutions per 100 bases) were determined with the Distances program (10) with the correction adequate to each case. Tree construction methods and bootstrap analysis were implemented using MEGA version 4.0 (52).

TABLE 1.

lytA alleles analyzed in this study

Gene/family (protein) and allele	Strain/phage designation (accession no.)	Type/groupa	Sequence type (allelic profile)	Length (nt)b	Reference(s)
Pneumococcus
1_Spn/B (1)	Rst7 (M13812)	Nc	128 (7,5,1,5,10,7,15,18)	957	24
	R800 (Z34303)	Nc	128	957	39
	R6 (AE008540)	Nc	128	957	29
	D39 (CP000410)	2	128	957	32
2_Spn/B (2)	TIGR4 (AE007483)	4	205 (10,5,4,5,13,10,18)	957	53
	CDC1873-00 (NZ_ABFS01000011)	6A	376 (6,11,1,1,15,72,77)	957	Unpublished
	SP6-BS73 (NZ_ABAA01000016)	6A	460 (5,7,4,10,10,1,27)	957	27
	CDC0288-04 (NZ_ABGF01000008)	12F	220 (10,20,14,1,9,1,29)	957	Unpublished
	CDC3059-06 (NZ_ABGG01000012)	19A	199 (8,13,14,4,17,4,14)	957	Unpublished
	JJA (CP000919)	14	66 (2,8,2,4,6,1,1)	957	Unpublished
	150 (AF345845)	12		940	Unpublished
3_Spn/B (2)	147 (AF345844)	29		937	Unpublished
4_Spn/A (3)	494 (AJ243399)	3		906	54
5_Spn/A (10)	670c	6B	90 (5,6,1,2,6,3,4)	957	Unpublished
	670 (AJ243400)	6B		906	54
6_Spn/B (9)	INV104Bd	1	227 (12,5,13,5,17,4,20)	957	Unpublished
7_Spn/B (2)	PN58 (AJ243403)	19A		906	54
8_Spn/A (12)	VA1 (AJ243404)	19		906	54
9_Spn/B (13)	CL18 (AJ243405)	10		906	54
10_Spn/A (3)	1012 (AJ243406)	35		906	54
11_Spn/A (3)	PN15 (AJ243407)	12		906	54
12_Spn/A (11)	234 (AJ243408)	23F		906	54
	233 (AJ243409)	23F		906	54
13_Spn/A (3)	949 (AJ490805)	23F	81 (4,4,2,4,4,1,1)	957	44
	ATCC 700669 (FM211187)	23F	81	957	9
	Sp18-BS74 (NZ_ABAE01000010)	6B	New (7,6,1,2,6,15,new)	957	27
	MLV-016 (NZ_ABGH01000008)	11A	62 (2,5,29,12,16,3,14)	957	Unpublished
	TCH8431/19A (NZ_ACJP01000181)	19A	320 (4,16,19,15,6,20,1)	957	Unpublished
	Taiwan19F-14 (CP000921)	19F	236 (15,16,19,15,6,20,26)	957	Unpublished
	PN8 (AJ2431413)	23		906	54
	7751 (AJ243414)	6		906	54
	Canada MDR_19A (ACNU01000124)	19A	320 (4,16,19,15,6,20,1)	957	46
	Canada MDR_19F (ACNV01000155)	19F	320	957	46
14_Spn/A (8)	8249 (AJ490328)	19A	68 (2,8,9,5,12,1,13)	957	44
15_Spn/Ae	S3 (FM865975)	23F	81	956	Unpublished
16_Spn/B (4)	ST344 (AM113493)	NT	344 (8,37,9,29,2,12,53)	957	5, 34
17_Spn/B (6)	ST942 (AM113494)	NT	942 (8,10,15,27,2,28,4)	957	5, 34
18_Spn/A (3)	OXC141d	3	180 (7,15,2,10,6,1,22)	957	Unpublished
	Sp3-BS71 (NZ_AAZZ01000010)	3	180	957	27
19_Spn/A (5)	CDC1087-00 (NZ_ABFT01000024)	7F	191 (8,9,2,1,6,1,17)	957	Unpublished
	219 (AF345846)	7F		940	Unpublished
20_Spn/A (2)	SP195 (NZ_ABGE01000012)	9V	156 (7,11,10,1,6,8,1)	957	Unpublished
	INV200d	14	9 (1,5,4,5,5,1,8)	957	Unpublished
	CGSP14 (CP001033)	14	124 (7,5,1,8,14,11,14)	957	13
	SP9-BS68 (NZ_ABAB01000008)	9V	1269 (7,11,10,1,6,76,14)	957	27
	Sp19-BS75 (NZ_ABAF01000008)	19F	485 (1,5,1,1,1,1,8)	957	27
	Tupelo (FM865976)	14	13 (1,5,4,5,5,27,8)	957	Unpublished
	472 (AJ243410)	3		906	54
	860 (AJ243411)	NK		906	54
	29044 (AJ243412)	14		906	54
21_Spn/A (7)	Sp11-BS70 (NZ_ABAC01000008)	11D	62 (2,5,29,12,16,3,14)	957	27
22_Spn/B (2)	Sp14-BS69 (NZ_ABAD01000012)	14	124 (7,5,1,8,14,11,14)	957	27
	CCRI 1974 (NZ_ABZC01000110)	14	124	957	19
23_Spn/A (2)	Hungary19A-6 (CP000936)	19A	268 (7,13,42,6,10,6,56)	957	Unpublished
	G54 (CP001015)	19F	63 (2,5,36,12,17,21,14)	957	15
24_Spn/B (2)	Sp23-BS72 (NZ_ABAG01000011)	23F	37 (1,8,6,2,6,4,6)	957	27
25_Spn/A (3)	P1031 (CP000920)	1	303 (10,5,4,1,7,19,9)	957	Unpublished
26_Spn/B (2)	70585 (CP000918)	5	289 (16,12,9,1,41,33,33)	957	Unpublished
SMG
1_SMG	101/87 (S43511)	NT		951	12
2_SMG	COL17 (AJ252190)	NT		900	55
3_SMG	COL16 (AJ252192)	NT		900	55
4_SMG	COL20 (AJ252194)	NT		900	55
5_SMG	CCUG 49455T (AM113495)	NT		951	3, 34
	CCUG 48465 (AM113496)	NT		951	3, 34
	COL26 (AJ252195)	NT		900	55
6_SMG	COL27 (AJ252196)	NT		900	55
7_SMG	1508/92 (AJ419973)	NT		951	43
8_SMG	11923/1992 (AJ419974)	NT		951	43
9_SMG	8224/1994 (AJ419975)	NT		951	43
10_SMG	10546/1994 (AJ419976)	19		951	43
11_SMG	782/1996 (AJ419977)	NT		951	43
12_SMG	1230/1996 (AJ419978)	19		951	43
13_SMG	1283/1996 (AJ419979)	NT		951	43
14_SMG	1338/1996 (AJ419980)	NT		951	43
15_SMG	1078/1997 (AJ419981)	NT		951	43
16_SMG	1383/1997 (AJ419982)	19		951	43
17_SMG	1629/1997 (AJ419983)	19		951	43
18_SMG	578 (AM113497)	NT		951	34
19_SMG	1504 (AM113500)	NT		951	34
	1956 (AM113501)	NT		951	4
20_SMG	3072 (AM113504)	NT		951	34
	3198 (AM113505)	NT		951	4
21_SMG	3137 (AM113502)	NT		951	4
22_SMG	2410 (AM113503)	NT		951	4
23_SMG	1237 (AM113498)	NT		951	4
	2859 (AM113499)	NT		951	4
Pneumococcal prophage (host strain)
1_PPH (NK)f	HB-3 (M34652)			957	48
2_PPH (949)	MM1 (AJ302074)			957	42
(DCC1808)	MM1-1998 (DQ113772)			957	35
(ATCC 700669)	MM1-2008 (FM211187)			957	49
3_PPH (8249)	VO1 (AJ490329)			957	44
4_PPH (OXC141)	φSpn_OXCc			957	49
(SP3-BS71)	φSpn-3 (NZ_AAZZ01000017)			957	49
5_PPH (Hungary19A-6)	φSpn_H_1 (CP000936)			957	49
6_PPH (SP195)	φSpn_195_1 (NZ_ABGE01000002)			957	49
7_PPH (SP195)	φSpn_195_2 (NZ_ABGE01000013)			957	49
8_PPH (CDC1873-00)	φSpn_1873 (NZ_ABSF01000005)			957	49
(SVMC28)	SV1 (FJ765451)			957	23
9_PPH (SP11-BS70)	φSpn_11 (NZ_ABAC01000001)			957	49
10_PPH (CDC3059-06)	φSpn_3059 (NZ_ABGG01000014)			957	49
11_PPH (SP19-BS75)	φSpn_19 (NZ_ABAF01000004)			957	49
12_PPH (SP6-BS73)	φSpn_6 (NZ_ABAA01000020)			957	49
13_PPH (SP23-BS72)	φSpn_23 (NZ_ABAG01000010)			957	49
14_PPH (SP14-BS69)	φSpn_14 (NZ_ABAD01000023)			957	49
15_PPH (670)	φSpn_670_1c			957	Unpublished
16_PPH (670)	φSpn_670_2c			957	Unpublished
17_PPH (JJA)	φSpn_JJA (CP000919)			957	Unpublished
18_PPH (P1031)	φSpn_1031 (CP000920)			957	Unpublished
19_PPH (70585)	φSpn_70585 (CP000918)			957	Unpublished
SMG prophage (host strain)
1_SPH (101)	EJ-1 (AJ609634)			951	11
2_SPH (HER 1055)	φHER (AJ617815)			957	51
3_SPH (B6)	φB6 (AJ617816)			957	51

^aNc, nonencapsulated; NT, nontypeable.

^bIndicates the number of nucleotides (nt) sequenced.

^cPreliminary sequence data (ftp://ftp.tigr.org/private/ufmg/s_pneumoniae_spuser_str670/s_pneumoniae-strain670).

^dPreliminary sequence data (http://www.sanger.ac.uk/Projects/S_pneumoniae).

^eThis allele encodes a truncated protein.

^fNK, not known.

Identification of two families of lytA alleles in S. pneumoniae.

Figure 1 shows a diagram of lytA_Spn polymorphism. When full-length lytA_Spn (or LytA_Spn) sequences were compared (17 alleles), they differed at only 44 nucleotide and 13 amino acid positions. Although two peaks around nucleotide positions 767 and 923 were detected, the most remarkably polymorphic region occurs near the middle of the gene (around position 453). Thus, polymorphism between nucleotide positions 421 and 480 accounts for an estimated PED of 18.9%, which contrasts greatly with a PED value of less than 3.5% characteristic of the full lytA_Spn allele length (data not shown). Notably, nucleotide polymorphism is not mirrored in the primary structure of the protein, in which perfect amino acid sequence conservation was observed (Fig. 1B). Upon closer examination of the nucleotide sequence of this polymorphic region, we observed that the 26 different lytA_Spn alleles examined here could be divided into two families (here designated Fam_A and Fam_B) depending on their precise sequence (Fig. 1C). Hence, Fam_A and Fam_B lytA alleles, respectively, include a BamHI or HincII restriction sequence. This feature can be used for rapid discrimination between the two types of alleles (unpublished observations). Fam_B lytA alleles form an independent and consistent clade (Fig. 1D).

FIG. 1.

Polymorphic sites of lytA_Spn and the LytA_Spn NAM-amidase alleles. (A and B) Only full-length (957-bp) lytA_Spn alleles (17 alleles) and the corresponding 318-amino-acid-long LytA_Spn NAM-amidases were aligned using Clustal W 2. Polymorphism within a window of 31 nucleotides (or 11 amino acid residues), while advancing through the sequence, was calculated from a multiple sequence alignment. Overlapping windows were displaced from each other by one nucleotide (or residue). The sum of polymorphic positions found was plotted at the middle position of the window. The blackened bar or the stippled boxes, respectively, represent the N-terminal domain or the C-terminal repeats of NAM-amidase. (C) Nucleotide sequence around position 453 (taking as position 1 the first nucleotide of the ATG initiation codon) that identifies two different lytA_Spn families. The deduced amino acid sequence and its corresponding positions are boldfaced and italicized. Asterisks indicate nucleotides conserved in all lytA_Spn alleles described in Table 1. Nucleotides distinctive of each family of lytA_Spn alleles are highlighted on a gray (Fam_A) or black (Fam_B) background. The BamHI and HincII restriction sites characteristic of Fam_A and Fam_B lytA alleles, respectively, are boxed. (D) Phylogenetic tree of the S. pneumoniae lytA alleles. The neighbor-joining phylogenetic tree (phylogram) shows evolutionary relationships among lytA alleles. Sequences were trimmed so that the length of the alignment was adjusted to 906 nucleotides. The statistical significance of the tree branches was assessed by bootstrap analysis involving the construction of 1,000 trees from resampled data. Bootstrap values of >50% are indicated. The gray box indicates the positions of Fam_B lytA alleles. The scale bar represents the number of nucleotide substitutions per site.

Distribution of polymorphic sites in nonpneumococcal lytA genes.

To determine the possible evolutionary origin of the lytA_Spn families, complete lytA alleles from SMG or pneumococcal prophages were aligned and polymorphic site distributions were compared to that of lytA_Spn. Figure 2 shows that the lytA alleles from SMG or temperate phages are much more polymorphic than those from S. pneumoniae, the variable sites appearing along the whole gene (or protein), in contrast to that noted in the lytA_Spn alleles. The lytA_SMG alleles differed at 138 positions (35 amino acid residues), whereas 158 polymorphic nucleotide positions (31 amino acid residues) were detected in the lytA_PPH alignments. Since phage genes evolve faster than their bacterial counterparts (reviewed in references 1 and 2), it was not surprising to find that the phage alleles were so diverse. In contrast, the reduced polymorphism observed for the lytA_Spn alleles relative to that exhibited by lytA_SMG alleles suggests that the former alleles were acquired fairly recently in evolutionary terms and/or that S. pneumoniae strains are a much more closely related group than SMG, as recently demonstrated (7, 14, 26). Further, in a multiple alignment of the 71 lytA alleles examined here around nucleotide position 453, it was noted that only two phage alleles, lytA_{15_PPH} (φSpn_670_1) and lytA_{2_SPH} (φHER), have sequences identical to those characteristic of Fam_A and Fam_B lytA alleles, respectively (Fig. 3). Quite unexpectedly, signatures identical to either lytA family were absent among the lytA_SMG alleles.

FIG. 2.

Distributions of polymorphic sites in bacterial and phage lytA alleles and their encoded NAM-amidases. Only full-length lytA alleles were analyzed. Polymorphic sites were calculated as indicated in Fig. 1. Nucleotide (nt) and amino acid (aa) positions appear as black or red symbols, respectively. (A) Pneumococcal temperate phages. (B) S. pneumoniae alleles. (C) Streptococci of the mitis group (SMG). A hatched bar indicates the position of the 6-nucleotide deletion distinctive of lytA_SMG alleles.

FIG. 3.

Multiple alignments of pneumococcal, phage, and SMG lytA alleles. Nucleotides distinctive of the Fam_A and Fam_B lytA alleles are shown on a red or on a blue background, respectively. Other polymorphic nucleotides are shown on a yellow background. Asterisks indicate nucleotides identical in all the sequences. The hyphen corresponds to a 1-bp deletion. Nucleotide (and amino acid) positions (taking 1 as the first nucleotide of the ATG initiation codon or the first residue) are shown. Amino acid polymorphisms are indicated.

Prophage-host recombination in the lytA gene.

Recent recombinational events are apparent by visual inspection of sequence alignments, particularly if the replacements are short enough that breakpoints can be defined within the sequenced region (17), as is the case for the mosaicism in the genes of S. pneumoniae coding for the penicillin-binding proteins (16), rpoB genes of rifampin-resistant strains (21), or DNA topoisomerase genes of fluoroquinolone-resistant isolates (22). Descendants from a particular clone that differ at one of the seven loci defined by multilocus sequence typing (MLST) are named single locus variants (SLVs). Variant alleles in SLVs are therefore assigned as having arisen by a point mutation if they differ from the ancestral allele at a single nucleotide site. Variant alleles that differ at multiple sites (plus those that differ at a single site but are present in an unrelated lineage) are assigned as having arisen by recombination (for a review, see reference 17). Following this criterion, evidence strongly suggesting the existence of recombination events in the lytA gene between host and prophages was found, particularly in the case of serotype 14 strains CGSP14 and SP14-BS69, which belong to ST124. Whereas strain CGSP14 harbors a Fam_A allele (lytA_{20_Spn}), SP14-BS69 possesses a Fam_B allele (lytA2_{22_Spn}) (Table 1 and Fig. 4). A nucleotide alignment (not shown) illustrated that the two alleles differ at 13 positions, 10 of them located between positions 441 and 465 (i.e., the signature characteristic of Fam_A/Fam_B lytA alleles), and only 3 more changes elsewhere in the lytA gene (at positions 264, 282, and 567). Moreover, the data presented in Fig. 4 indicate that recombination events between bacteria and phage lytA genes have occurred more than once, because the same alleles are present in isolates that are not closely related. For example, lytA_{20_Spn} (Fam_A) appears in strains of ST9, ST13, ST485, ST156, and ST1269, whereas lytA_{2_Spn} (Fam_B) is present in isolates of ST66, ST199, ST205, ST220, ST376, and ST460.

FIG. 4.

Dendrogram of genetic relationships among 109 strains belonging to 30 different STs based on multilocus sequence typing data (http://www.mlst.net/). Linkage distance is indicated by a scale at the bottom. The lytA_Spn allele present in pneumococcal strains of each ST is shown. Fam_A and Fam_B alleles are shown on a red or on a blue background, respectively.

Out of all the lytA_Spn alleles whose complete sequence is known, the lytA gene of prophage φSpn_670_1 shows the greatest similarity (92% identity; PED, 7.10%) with the lytA gene (lytA_{5_Spn}) of its host strain, i.e., strain 670, a member of the globally distributed, antibiotic-resistant clone Spain^6B-2. In effect, bacterial and phage lytA genes were identical from positions 343 to 495 and from 497 to the gene end (data not shown). In contrast, only 85% identity (817 out of 957 nucleotides) (PED, 16.33%) was seen when alleles lytA_{5_Spn} (from strain 670) and lytA_{16_PPH} (from φSpn_670_2, the second prophage harbored by strain 670) were aligned (not shown). Besides, the lytA gene of the S. mitis prophage φHER (lytA_{2_SPH}) showed the highest similarity (82% identity; PED, 20.12%) with the NAM-amidase gene (lytA_{16_Spn}) of the nontypeable pneumococcal isolate ST344. Although the implication of this finding is unclear, it suggests that temperate phages from SMG play a role in dissemination of the pneumococcal lytA gene among alpha-hemolytic streptococci sharing the same habitat (50).

Genetic transformation is envisaged as the main mechanism of horizontal gene transfer among SMG, whereas the contribution of phages, either lytic or temperate, to the dissemination of clinically relevant genes had not been so well established. Almost 20 years ago, Romero et al. provided direct evidence of recombination between the lytA gene of a pneumococcal prophage and that of the host under laboratory conditions (48). These authors observed remarkable nucleotide similarity (87%) between the lytA gene and the hbl gene. The hbl gene, encoding the NAM-amidase of the temperate phage HB-3 (designated allele 1_PPH in Table 1), transformed NAM-amidase-deficient strains of S. pneumoniae into the wild-type phenotype. In 1999, the possibility of in vivo localized recombination events between phage and bacterial lytA genes was proposed (54). Nevertheless, since only a reduced number of lytA_Spn alleles and only one temperate S. pneumoniae phage (HB-3) and another S. mitis prophage (EJ-1; allele 1_SPH) were known at that time, only a preliminary conclusion could be reached. Our data confirm and extend these past findings and strongly suggest that phage genes could play a significant role in the evolution of bacterial genes involved in disease processes, as is the case for LytA NAM-amidase. The remarkable similarity between the lytA genes either from bacteria or from phages, as well as their capacity to recombine, allows us to properly use the term “homologous.” This homology makes it likely that the phage-encoded NAM-amidase derived directly from the corresponding S. pneumoniae gene, although we cannot exclude the possibility that the host genes are descended from related phage genes, as previously suggested (8).