Complete nucleotide sequence of a P2 family lysogenic bacteriophage, ! MhaA1-PHL101, from Mannheimia haemolytica serotype A1

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Virology 350 (2006) 79 – 89 www.elsevier.com/locate/yviro

Complete nucleotide sequence of a P2 family lysogenic bacteriophage, ϕMhaA1-PHL101, from Mannheimia haemolytica serotype A1 Sarah K. Highlander a,c,⁎, Sarah Weissenberger a , Laura E. Alvarez a , George M. Weinstock a,b,c , Peter B. Berget d a

Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA b Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA c Human Genome Sequencing Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA d Department of Biological Sciences, Carnegie Mellon University, 4400 5th Avenue, Pittsburgh, PA 15213, USA Received 29 November 2005; returned to author for revision 11 March 2006; accepted 15 March 2006 Available online 2 May 2006

Abstract The 34,525 nucleotide sequence of a double-stranded DNA bacteriophage (ϕMhaA1-PHL101) from Mannheimia haemolytica serotype A1 has been determined. The phage encodes 50 open reading frames. Twenty-three of the proteins are similar to proteins of the P2 family of phages. Other protein sequences are most similar to possible prophage sequences from the draft genome of Histophilus somni 2336. Fourteen open reading frames encode proteins with no known homolog. The P2 orthologues are collinear in ϕMhaA1-PHL101, with the exception of the phage tail protein gene T, which maps in a unique location between the S and V genes. The phage ORFs can be arranged into 17 possible transcriptional units and many of the genes are predicted to be translationally coupled. Southern blot analysis revealed ϕMhaA1-PHL101 sequences in other A1 isolates as well as in serotype A5, A6, A9, and A12 strains of M. haemolytica, but not in the related organisms, Mannheimia glucosida or Pasteurella trehalosi. © 2006 Elsevier Inc. All rights reserved. Keywords: Bacteriophage; Genomic sequence; Mannheimia haemolytica

Introduction The family Pasteurellaceae includes the Haemophilus, Actinobacillus, Pasteurella, and Mannheimia genera of bacteria, which cause a variety of diseases in humans and animals. Bacteriophages have been reported in Haemophilus influenzae (Bendler and Goodgal, 1968; Harm and Rupert, 1963; Hendrix et al., 1990; Samuels and Clarke, 1969; Stuy, 1978; Williams et al., 2002), Actinobacillus actinomycetemcomitans (Loftus and Delisle, 1995; Stevens et al., 1982), Pasteurella multocida (Ackermann and Karaivanov, 1984; Gadberry and Miller, 1978; Pullinger et al., 2004), and Mannheimia haemolytica (Rifkind and Pickett, 1954; Saxena and Hoerlein, 1959) and bacteriophage-like sequences have been identified in Histophilus somni ⁎ Corresponding author. Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, MS280 Houston, TX 77030-3411, USA. Fax: +1 713 798 7375. E-mail address: [email protected] (S.K. Highlander). 0042-6822/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2006.03.024

(previously Haemophilus somnus) (Pontarollo et al., 1997). The genomes of two H. influenzae phages, HP1 and HP2, have been sequenced and both are members of the P2 family of temperate bacteriophage (Esposito et al., 1996; Williams et al., 2002). Recently, the complete genome of a lambdoid temperate bacteriophage from A. actinomycetemcomitans was reported (Resch et al., 2004). No other complete bacteriophage genome sequence has been reported for the Pasteurellaceae, though a Mu-like prophage, ϕflu, was identified within the genome of H. influenzae Rd (Fleischmann et al., 1995; Morgan et al., 2002) and HP1-like sequences were reported in H. somni (Pontarollo et al., 1997). M. haemolytica is the primary bacterial pathogen in bovine respiratory disease complex (Whiteley et al., 1992), yet is also a commensal in ruminants including sheep and goats. Bacteriophage was first isolated from M. haemolytica in the 1950s (Rifkind and Pickett, 1954; Saxena and Hoerlein, 1959). In 1985, Richards, Renshaw and Sneed isolated an icosahedral bacteriophage from UV-treated isolates of M. haemolytica

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biotype A, serotype 1 (A1) (Richards et al., 1985). The phage, called ϕPhaA1, was classified as a member of Bradley's group A (Bradley, 1967) and had a tail with a contractile sheath. ϕPhaA1 was unable to form plaques on any of the A1 strains tested leading the authors to speculate that all strains harbored the same lysogenic bacteriophage (Richards et al., 1985). The genome was not characterized but was assumed to be composed of double-stranded DNA. Froshauer et al. (1996) also showed that danofloxacin could induce a prophage in a serotype A1 isolate of M. haemolytica. Here we report the complete DNA sequence of the doublestranded DNA bacteriophage, ϕMhaA1-PHL101, isolated from a bovine pneumonic isolate of M. haemolytica, A1, strain PHL101. This may be related to the bacteriophage originally isolated by Richards. Results and discussion Genome organization The PHL101 ϕMhaA1-PHL101 genome is 34,525 bp in length and has a 7 bp repeated sequence on each end. These repeats were identified by direct sequencing off the ends of the phage DNA. The terminal repeats form the cos or cohesive end site and have the sequence 5′-TGCGGGC. Fifty open reading frames were predicted from the sequence (Fig. 1 and Table 1). These were categorized as regulatory, structural, and morphogenic (assembly and packaging) or unknown (Fig. 1). The genes encoding structural and assembly components of ϕMhaA1-PHL101 are clustered on the left end of DNA sequence (orf1-orf30). With the exception of orf15, this arrangement is identical to the organization of morphogenesis genes (gpD to gpQ) of bacteriophage P2 (Christie et al., 1998).

In ϕMhaA1-PHL101, the T-like gene lies between the hypothetical genes orf14 and orf15. This is unlike the P2 genome where the T gene lies between the E and U genes. A tblastx comparison of the ϕMhaA1 and P2 genomes, visualized using the Artemis Comparison Tool (Carver et al., 2005), clearly shows the rearrangement of the T gene (Fig. 2). This organization was verified by repeated sequence reads across the region and by PCR performed using phage or chromosomal DNA and primers specific for the S and V genes (data not shown). The Artemis plot also illustrates protein similarities for the Q, P, O, N, M, L, R, S, V, J, I, H, FI, FII, E U, D, and A proteins. Note that a gene encoding an ortholog of the tail fiber protein G, which should map between orf22 and orf25 (H and FI), is not present in ϕMhaA1-PHL101. The P2 tail protein gene can undergo a frameshift to extend the E protein to a longer E + E' protein with an identical amino terminus. This is thought to occur via translational frameshifting in a “slippery” hexa-T sequence upstream of the first stop codon (Christie et al., 2002). In ϕMhaA1-PHL101, a hexa-T sequence is not observed within the gene. However, using the Programmed Frameshift Finder (chainmail.bio.pitt.edu/∼junxu/webshift.html; Xu et al., 2004a), a GGGAAAG slippery sequence was identified upstream of the stop codon. Addition of a base within this sequence would extend the E protein from 104 to 151 amino acids. Also unlike P2, ϕMhaA1-PHL101 encodes a P1-like signal-arrest-release (SAR) endolysin (Xu et al., 2004b) instead of the typical holin-endolysin pair. Similar SAR endolysins are found in HP1 and HP2 (Esposito et al., 1996; Williams et al., 2002) and in H. somni (Pontarollo et al., 1997). The adjacent predicted holin of ϕMhaA1-PHL101 is only weakly similar (e−05 over less than 80% of the protein) to a predicted holin protein in H. somni. Finally, the products of orf1-3, orf5, orf6, orf8, orf9, and orf43 are also similar to corresponding

Fig. 1. Genome map of phage ϕMhaA1-PHL101. Genes are sequentially numbered from 1 to 50. Potential regulatory genes are indicated with light shading and morphogenesis genes are shown with dark shading. Unshaded arrows indicate hypothetical proteins. Protein designations are indicated by capital letters. Predicted transcriptional units are shown as arrows and terminators by the stem–loop structures. A %GC map, generated using a 100 nt sliding window, is shown below the gene map.

S.K. Highlander et al. / Virology 350 (2006) 79–89

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Table 1 Bacteriophage ϕMhaA1-PHL101 ORF analysis ORF

Range (bp)

Size (aa)

Predicted function

Top BLASTP match (organism)

Reference (gi number)

BLASTP e value

H. somni 2336 ortholog (gi number)

BLASTP e value

Capsid portal protein (H. influenzae R2866) Terminase, ATPase subunit (H. influenzae 86-028NP) Chromosome segregation ATPase (H. influenzae R2886) gp5 (Salmonella bacteriophage PSP3) Terminase, endonuclease subunit (H. influenzae 86-028NP) orf4, phage P2-like head completion protein L (H. somni) Phage P2-like tail protein X (H. somni 2336) Possible holin (H. somni) Lysozyme (H. somni 2336) orf8 (H. somni)

46133084

8e−117

53728326

2e−46

38058394

0e+00

53728325

1e−102

42631022

1e−72

32029055

2e−70

41057357 68058391

4e−101 1e−45

32029056 32029057

3e−91 2e−38

915368

1e−46

53728618

3e−45

32029059

8e−13

32029059

8e−13

915370 a 53728269 915373a

9e−06 9e−46 2e−07

32029060a 53728269 53728620a

9e−06 9e−46 2e−07

DnaK (H. influenzae R2866)

42631014

4e−12

Hypothetical protein (Azotobacter vinelandii) orf11 (H. somni) Hypothetical protein NTHI1870 (H. influenzae 86-028NP) Tail protein (H. influenzae R2866) None Plasmid stabilization protein (A. pleuropneumoniae) Baseplate assembly protein V (H. influenzae R2866) Baseplate assembly protein W (H. influenzae 86-028NP) Baseplate assembly protein (H. somni)

67159457

1e−24

32029063a

5e−24

915376 68058378

8e−42 6e−02

32029064 None

1e−41

46133075

1e−80

2e−46

53728835

5e−10

53728622 None None

46133073

3e−61

32029067

8e−41

68058370a

1e−19

53728624a

2e−19

32029069

8e−102

32029069

8e−102

53728625a 46133071a

7e−52 8e−40

7e−52 1e−38

45737848a

1e−09

53728625a 53728626a None 32028843a

32030804 46133067

1e−103 1e−52

32030804 32030805

1e−103 2e−47

37524045a

1e−11

32030806a

4e−08

46133065 46133064 70726007a

2e−41 1e−107 3e−14

None 46155983 46155984 None

7e−41 6e−105

42630997a

4e−10

None

42630996

2e−34

None None

75236053a 46358672

3e−35 1e−13

46155985a 46155986a

3e−34 1e−09

46155987

6e−18

46155987

6e−18

ORF1

1468–428

346

Capsid portal protein Q

ORF2

3294–1477

605

Terminase, ATPase subunit

ORF3

3429–4256

275

Capsid scaffolding protein O

ORF4 ORF5

4270–5298 5308–5997

342 229

Major capsid protein N Terminase, small subunit

ORF6

6109–6624

171

Head completion protein L

ORF7

6621–6833

70

ORF8 ORF9 ORF10

6839–7045 7038–7604 7601–8056

68 188 151

ORF11

8205–8426

73

ORF12

8423–8908

161

ORF13 ORF14

8901–9359 9688–9410

152 92

ORF15 ORF16 ORF17

9727–12,639 12,704–12,880 12,873–13,160

970 58 95

ORF18

13,289–13,894

201

ORF19

13,894–14,229

111

ORF20

14,226–15,143

305

ORF21 ORF22 ORF23 ORF24

15,130–15,762 15,765–18,044 18,045–18,287 18,392–18,562

210 759 80 857

ORF25 ORF26

18,669–19,850 19,859–20,365

393 168

Tail synthesis protein S Conserved hypothetical protein Tail protein T Hypothetical protein Conserved hypothetical protein Baseplate assembly protein V Baseplate assembly protein W Baseplate assembly protein J Tail formation protein I Variable tail fiber protein H Hypothetical protein Pseudogene of conserved hypothetical protein Tail sheath protein FI Tail tube protein FII

ORF27

20,444–20,758

104

Tail protein E

ORF28 ORF29 ORF30 ORF31

20,962–21,309 21,311–21,748 21,748–22,986 23,169–23,978

115 145 412 269

ORF32

24,010–24,270

86

ORF33 ORF34

24,370–24,699 24,798–25,316

111 172

ORF35 ORF36

25,320–26,006 26,130–26,342

228 70

Hypothetical protein Tail protein U Tail protein D Conserved hypothetical phage protein Conserved hypothetical protein Hypothetical protein Conserved hypothetical protein CI family phage repressor Cro family repressor

ORF37 ORF38

26,326–26,610 26,819–27,091

94 90

Tail synthesis protein X Possible holin Lysozyme Conserved hypothetical phage protein Conserved hypothetical protein Tail completion protein R

Hypothetical protein Conserved hypothetical protein

Tail formation protein (H. somni 2336) Tail fiber protein (H. influenzae R2866) None Hypothetical protein (bacteriophage Aaphi23) Tail sheath protein FI (H. somni 2336) Tail tube protein F11 (H. influenzae R2866) Hypothetical protein (Photorhabdus luminescens) None Phage protein U (H. influenzae R2866) Phage protein D (H. influenzae R2866) Hypothetical protein (Staph. haemolyticus JCSC1435) Hypothetical protein (H. influenzae R2866) None NAD-dependent DNA ligase (H. influenzae R2866) Transcriptional regulator (E. coli F11) Cro (Salmonella typhimurium bacteriophage ST104) None Hypothetical protein (H. somni 2336)

1e−06

(continued on next page)

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S.K. Highlander et al. / Virology 350 (2006) 79–89

Table 1 (continued) ORF

Range (bp)

Size (aa)

Predicted function

Top BLASTP match (organism)

Reference (gi number)

BLASTP e value

H. somni 2336 ortholog (gi number)

BLASTP e value

ORF39

27,174–27,506

110

7e−03

46155988a

7e−03

27,519–27,812 27,965–28,207 28,204–28,536 28,533–30,893 30,906–31,238 31,238–31,690

97 80 110 786 110 150

ORF46

31,770–32,033

87

ORF47 ORF48

32,023–32,292 32,267–32,557

89 96

ORF49

33,009–32,827

60

Chromosome segregation ATPase (H. somni 2336) None None None Hypothetical protein (H. somni 2336) None Single-stranded DNA-binding protein (H. somni 129T) gp62 (Burkholderia cepacia phage Bcep43) None ABC transport system, binding protein (H. somni 129T) Hypothetical protein (H. somni 2336)

46155988a

ORF40 ORF41 ORF42 ORF43 ORF44 ORF45

ORF50

34,285–33,287

332

Conserved hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Replication protein Hypothetical protein Single-stranded DNA binding protein Conserved hypothetical phage protein Hypothetical protein Conserved hypothetical protein Conserved hypothetical protein Integrase/recombinase

Integrase/recombinase (H. influenzae)

a

32030813

1e−101

23467571

2e−34

None None None 32030813 None 32029356

41057713a

2e−10

None

23466938

1e−11

None 46155989

2e−11

32030618

6e−11

32030618

6e−11

1175903

5e−105

46155990a

2e−69

1e−101 4e−334

BLAST hit includes
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