Identification of Genes of VSH-1, a Prophage-Like Gene Transfer

Identiﬁcation of Genes of VSH-1, a Prophage-Like Gene Transfer Agent of Brachyspira hyodysenteriae Eric G. Matson,2 M. Greg Thompson,1 Samuel B. Humphrey,1 Richard L. Zuerner,3 and Thad B. Stanton1,3* Enteric Diseases and Food Safety Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, Iowa 500101; Department of Microbiology, Iowa State University, Ames, Iowa 500102; and Zoonotic Diseases Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, Iowa 500103

Received 5 April 2005/Accepted 10 June 2005 Downloaded from VSH-1 is a mitomycin C-inducible prophage of the anaerobic spirochete Brachyspira hyodysenteriae. Puriﬁed VSH-1 virions are noninfectious, contain random 7.5-kb fragments of the bacterial genome, and mediate generalized transduction of B. hyodysenteriae cells. In order to identify and sequence genes of this novel gene transfer agent (GTA), proteins associated either with VSH-1 capsids or with tails were puriﬁed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of 11 proteins were determined. Degenerate PCR primers were designed from the amino acid sequences and used to amplify jb.asm.org several VSH-1 genes from B. hyodysenteriae strain B204 DNA. A ␭ clone library of B. hyodysenteriae B204 DNA was subsequently screened by Southern hybridization methods and used to identify and sequence overlapping DNA inserts containing additional VSH-1 genes. VSH-1 genes spanned 16.3 kb of the B. hyodysenteriae

chromosome and were flanked by bacterial genes. VSH-1 identified genes and unidentified, intervening open at DigiTop -- Digital Desktop Library for USDA on May 31, 2007 reading frames were consecutively organized in head (seven genes), tail (seven genes), and lysis (four genes) clusters in the same transcriptional direction. Putative lysis genes encoding endolysin (Lys) and holin proteins were identified from sequence and structural similarities of their translated protein products with GenBank bacteriophage proteins. Recombinant Lys protein hydrolyzed peptidoglycan purified from B. hyodysenteriae cells. The identified VSH-1 genes exceed the DNA capacity of VSH-1 virions and do not encode traditional bacteriophage early functions involved in DNA replication. These genome properties explain the noninfectious nature of VSH-1 virions and further confirm its resemblance to known prophage-like, GTAs of other bacterial species, such as the GTA from Rhodobacter capsulatus. The identification of VSH-1 genes will enable analysis of the regulation of this GTA and should facilitate investigations of VSH-1-like prophages from other Brach- yspira species.

Brachyspira hyodysenteriae is an anaerobic spirochete and the VSH-1 is the first natural gene transfer mechanism to be de- etiologic agent of swine dysentery, a severe, mucohemorrhagic scribed for a spirochete. intestinal disease afflicting animals in the postweaning period The ability to recognize VSH-1-specific genes would be a of growth (20). B. hyodysenteriae cells contain a defective pro- major advance for investigating intracellular and intercellular phage, VSH-1, which is induced when mitomycin C is added to activities of VSH-1 and for evaluating its ecological signifi- cultures of growing cells (27). This prophage has been purified cance to its host bacterium. To overcome the inability to iden- and characterized previously (28). VSH-1 particles resemble ␭ tify distinct VSH-1 genes carried by virions, structural protein virions in morphology but, with a head diameter of 45 nm and genes were identified from the sequences of VSH-1 head and a tail length of 64 nm, are substantially smaller. tail proteins. Genes for endolysin and holin proteins, proteins VSH-1 virions contain random 7.5-kb fragments of host enabling the release of bacteriophage from bacterial cells, were genomic DNA (28), making it difficult to identify VSH-1 genes. discovered during nucleotide sequence analyses. Recombinant Purified virions are “noninfectious,” that is, they do not lyse endolysin was shown to degrade peptidoglycan. Based on its spirochete cells when added to cultures of B. hyodysenteriae or genome and biological properties, VSH-1 resembles prophage- of other Brachyspira species. VSH-1 transfers bacterial genes like gene transfer agents (GTA) reported for other bacteria (3, between B. hyodysenteriae cells (28, 52) and has likely contrib- 32, 37, 46, 56). uted to the recombinant population structure of B. hyodysenteriae (55). This generalized transduction activity of VSH-1 is useful for constructing B. hyodysenteriae mutant strains (35). MATERIALS AND METHODS Separation of VSH-1 whole virions and tailless heads. VSH-1 particles were harvested from B. hyodysenteriae strain B204 cultures (400 ml) in NT broth 7 to 8 h after mitomycin C treatment (28). Following polyethylene glycol precipita- * Corresponding author. Mailing address: Enteric Diseases and tion, VSH-1 particles from five cultures were combined, extracted once with an

Food Safety Research Unit, National Animal Disease Center, Agri- equal volume of CHCl3, and harvested by CsCl gradient ultracentrifugation (28). cultural Research Service, United States Department of Agriculture, Two bands, one containing intact VSH-1 particles and the other containing 2300 Dayton Road, Ames, IA 50010. Phone: (515) 663-7495. Fax: tailless heads, were collected from the gradient. The virions and heads were (515) 663-7458. E-mail: [email protected]. concentrated by ultraﬁltration (Microcon-100), resuspended in 200 ␮lofSM

5885 5886 MATSON ET AL. J. BACTERIOL. buffer (48), examined by electron microscopy (28), and analyzed by sodium Coomassie blue staining and by Western immunoblot analysis targeting the His dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). tag, following instructions of the manufacturer (Invitrogen).

SDS-PAGE and sequencing of VSH-1 proteins. VSH-1 whole-virion proteins E. coli cells from a 1-liter culture were treated with CHCl3 to remove their (80 ␮g of total protein) were separated by SDS-PAGE. The acrylamide concen- outer membranes (5). The chloroform was removed, and the cells were washed trations of the resolving gels ranged from 12 to 15% depending on the molecular twice with 50 mM Tris-HCl buffer (pH 7.0), resuspended at an optical density at masses of the proteins targeted for separation. Proteins within gels were stained 600 nm of 1.0 in Tris-HCl buffer with 0.1% Triton X-100, and held on ice. with Coomassie blue and electrotransferred to polyvinylidene difluoride mem- Purified recombinant Lys protein was added (0.2 ␮g/ml suspension), and lysis of branes. Selected protein bands were cut from the membranes, and their N- the CHCl3-treated bacteria was monitored as a reduction in suspension turbidity terminal amino acid sequences were determined by Edman degradation and (optical density at 600 nm). high-pressure liquid chromatography analysis at the Iowa State University Pro- To assess the muralytic ability of recombinant Lys, peptidoglycan was purified tein Facility (Ames, Iowa). Reagents, techniques, and equipment for electro- from B. hyodysenteriae B204 cells cultured in a 12-liter fermentor (29, 51). phoresis, electroblotting, and sequencing of proteins have been described previ- Approximately 70 mg of peptidoglycan was obtained from 25 g (wet weight) of ously (28, 41, 51, 54). spirochete cells. Peptidoglycan was also obtained from E. coli cells (2). Samples Throughout this article, VSH-1 virion (structural) proteins are designated Hvp (0.5 mg) of B. hyodysenteriae peptidoglycan, E. coli peptidoglycan, and crab shell (hyodysenteriae viral protein), followed by their molecular mass as estimated chitin (Sigma) were incubated with VSH-1 endolysin (20 ␮g/ml) at 37°C in 50 ␮l from electrophoretic migration. For example, Hvp38 is the VSH-1 major capsid of 50 mM sodium phosphate buffer (pH 6.5). After incubation, the reaction protein with an apparent molecular mass of 38 kDa and hvp38 is the gene mixtures were centrifuged at 20,000 ϫ g, and the supernatants were lyophilized

␮ Downloaded from encoding Hvp38. and dissolved in 20 lof1%K2B7O4. Reduced N-acetylamino sugars were VSH-1 genome sequencing. A 1.1-kb region of VSH-1 DNA was ampliﬁed detected at 585 nm by using the Morgan-Elson reaction (19). Mutanolysin from B. hyodysenteriae B204 DNA by using degenerate PCR primers. The for- (Sigma), with acetylmuramidase activity, and Serratia marcescens chitinase ward primer, 5Ј-AAAAT(T/A)AC(T/A)GAAAAAAA(T/C)AT was designed (Sigma) were used in parallel control assays. from the N-terminal sequence of Hvp19, and the reverse primer, 5Ј-TGAAT(T/ Computer analysis of VSH-1 DNA sequences. Sequence data were assembled A)CC(T/A)GCTTT(T/A)AT(T/A)AT, was based on the N-terminal sequence of and analyzed by using DNASIS (v. 7.11; Hitachi of America) and Vector NTI Hvp13. Based on the 1.1-kb sequence of the amplicon, PCR primers were suite 8 (InforMax). PCR primers were designed by using Oligo version 6.0 designed and used to amplify ﬂanking DNA by the inverse-PCR method (44). B. (Molecular Biology Insights). Gene sequences were compared to GenBank se- jb.asm.org hyodysenteriae B204 DNA was digested with AseI (New England Biolabs). DNA quences via BLASTX (NCBI website, National Library of Medicine). The fragments were circularized by ligation, and samples (0.2 to 1.5 ␮g) were used as BLASTP and PSI-BLAST programs were used to compare proteins with Gen- PCR templates, yielding a 3.6-kb PCR amplicon containing genes for the Hvp19, Bank sequences and to identify proteins from their conserved domains (1, 36).

Hvp13, and Hvp38 proteins. The remaining VSH-1 genes and B. hyodysenteriae VSH-1 holin was analyzed for intramembrane domains by TMpred (26) and at DigiTop -- Digital Desktop Library for USDA on May 31, 2007 flanking genes were determined by a “chromosome walking” strategy. DNA SOSUI (25), accessed through EMBnet (http://www.ch.embnet.org/index.html) probes complementary to sequenced DNA were used to screen by filter hybrid- and the Tokyo University of Agriculture and Technology (http://sosui.proteome ization (48) a clone library of sheared B. hyodysenteriae B204 DNA prepared in .bio.tuat.ac.jp/sosuiframe0.html), respectively. ␭ ZAPII (Stratagene). The DNA from purified plaques was amplified in PCRs by Nucleotide sequence accession number. The sequences of the VSH-1 genes using the hybridization probe as one primer and a second primer targeting the T3 and flanking B. hyodysenteriae genes have been deposited in GenBank under or T7 sequence flanking the cloning site of the ␭ ZAP II vector. Purified PCR accession number AY971355 (1 April 2005, release date). amplification products were sequenced (18) at the ISU Nucleic Acid Facility, Ames, IA. Each nucleotide base was determined at least twice from sequences of both DNA strands. Additionally, VSH-1 gene order and linkages were confirmed RESULTS by PCR amplification, from B. hyodysenteriae DNA, of intergenic regions of both VSH-1 and B. hyodysenteriae genes. Identification and sequencing of VSH-1 virion proteins. PCR conditions. For most amplifications of B. hyodysenteriae B204 DNA, When polyethylene glycol-precipitated VSH-1 particles from AmpliTaq Gold DNA polymerase and GeneAmp reagents (Perkin-Elmer, Ap- mitomycin C-treated B. hyodysenteriae cultures were purified plied Biosystems) were used according to the manufacturer’s recommendations, except that 0.2 ␮M PCR primers (0.4 ␮M for degenerate primers) and 2.5 mM by CsCl density gradient ultracentrifugation, two bands, ap-

MgCl2 were used. Annealing temperatures varied between 45 and 55°C, depend- proximately 1 cm apart, appeared in the gradient. By electron ing on the melting temperature of the primer. Reaction mixes (100 ␮l) contain- microscopy, the lower, prominent band contained VSH-1 ing 100 to 200 ng of template DNA were incubated through 36 cycles in an whole virions (Fig. 1A), whereas the upper, faint band con- UNO-Thermoblock thermal cycler (Biometra). Cloning the VSH-1 endolysin gene (lys). The VSH-1 endolysin gene (lys) was tained tailless VSH-1 heads (Fig. 1B). Most of the tailless amplified from B. hyodysenteriae B204 genomic DNA (50 ng) by using a high- heads appeared somewhat electron dense and were similar in fidelity proofreading polymerase, PfuTurbo (Stratagene, La Jolla, CA). RF2F diameter to whole-virion heads, suggesting they are mature (5Ј-CACCAATCAAGGAGTTTAATAATATGAT) and RF1R (5Ј-ACCTTGTA capsids separated from their tails. ATATTTTAAGAATAAT) were used as primers. The RF2F primer contained Proteins of intact virions were separated by electrophoresis a5Ј sequence (underlined) complementary to the plasmid vector, a putative Shine-Dalgarno sequence (bold), and the lys start codon (ATG in italics). Am- and extracted from gels by electroblotting, and their N-termi- plification conditions consisted of 95°C for 2 min, followed by 35 cycles of 95°C nal amino acid sequences were determined. Unambiguous se- for 30 s, 48°C for 30 s, and 72°C for 1 min and a final 10-min extension at 72°C. quences of 22 to 25 amino acids were obtained for 11 proteins, The lys gene amplicon was ligated into the plasmid expression vector pBAD102/ nine of which are labeled in Fig. 2. Although not apparent in D-TOPO (Invitrogen) and used to transform Escherichia coli One Shot Top10 chemically competent cells (Invitrogen). A recombinant strain, designated BAD/ Fig. 2, two weakly staining proteins, Hvp24 and Hvp28, were lys, was purified by colony subculture. The pBAD/lys plasmid in this strain was detected under different electrophoresis and staining condi- confirmed by sequence analysis to carry an authentic copy of the lys gene. E. coli tions. Top10 cells were also transformed with plasmid pBAD102/D/lacZ, which carries VSH-1 Hvp45, -38, -24, -22, -19, and -13 were present in ␤ a gene for -galactosidase. E. coli strain BAD/lacZ was isolated and used as a both whole virions (Fig. 2, lane WV) and heads (Fig. 2, lane H) positive control for protein expression experiments and as a negative control in peptidoglycan degradation assays. and were presumptive head proteins. By contrast, Hvp101, -60, Identifying VSH-1 endolysin activity. Cells of E. coli strain BAD/lys and strain -53, -32, and -28 either were not detected among the head BAD/lacZ were cultured at 37°C in 1 liter of LB broth containing ampicillin (50 proteins or were reduced in staining intensity. For this reason, ␮g/ml). Expression of His-tagged Lys and LacZ proteins was induced by adding they were considered potential tail proteins. The sequences of L-arabinose (0.003%, wt/vol, final concentration). Cells were disrupted in a these 11 proteins were used to identify VSH-1 genes. French press and centrifuged at 48,000 ϫ g for2hat4°C. Fusion proteins were purified from the supernates by affinity column chromatography (His-Select Identification and sequencing of VSH-1 head and tail genes. Nickel; Sigma). The purity of recombinant Lys was assessed by SDS-PAGE with It was assumed that VSH-1 genes would be contiguous within VOL. 187, 2005 VSH-1 GENOME 5887 Downloaded from

FIG. 1. Transmission electron micrographs of (A) VSH-1 virions and (B) VSH-1 heads. Preparations were negatively stained with 2% (wt/vol) phosphotungstic acid (pH 7.0). Bar ϭ 0.1 ␮m.

the bacterial host chromosome, similar to other prophages. ing VSH-1 genes and ﬂanking B. hyodysenteriae B204 genes, jb.asm.org Consequently, N-terminal amino acid sequences of VSH-1 was sequenced and analyzed (Fig. 3). In subsequent experi- proteins were used to design both forward and reverse degen- ments, the arrangement and orientation of VSH-1 genes and

erate PCR primers for amplification of adjacent VSH-1 genes. B. hyodysenteriae genes, depicted in Fig. 3, were confirmed by at DigiTop -- Digital Desktop Library for USDA on May 31, 2007 Of different primer combinations tested, a forward primer PCR amplification of intergenic regions from B. hyodysenteriae targeting the 5Ј end of the hvp19 gene and a reverse primer genomic DNA. targeting the 5Ј end of hvp13 yielded a 1.1-kb product. The Genes hvp45, hvp19, hvp23, hvp13, hvp38, and hvp24, encod- amplicon contained the predicted coding sequences for both ing head-associated (capsid) proteins, were arranged in a clus- Hvp19 and Hvp22 proteins (hvp19/22 gene), as discussed be- ter (Fig. 3). Two unidentified open reading frames (ORF), low. orfA and orfB, were contained within this cluster. Tail-associ- A “genome walking” strategy using inverse PCR and over- ated genes hvp53, hvp32, hvp101, and hvp28, along with orfC, lapping B. hyodysenteriae chromosomal DNA fragments cloned orfD, and orfE, formed a second cluster. A gene encoding the in ␭ ZAPII enabled DNA sequences upstream and down- Hvp60 protein was not located within the sequenced chromo- stream of the 1.1-kb region to be determined. In total, a somal region (Table 1), suggesting hvp60 is located elsewhere 29.1-kb segment of the B. hyodysenteriae genome, encompass- in the genome. Alternatively, the 60-kDa protein could be a B. hyodysenteriae protein. All of the identified VSH-1 structural genes, and the lysis genes described below, were in the same transcriptional orientation. The staining intensities of Hvp38 and Hvp53 relative to those of other proteins (Fig. 2) suggest that Hvp38 and Hvp53 are, respectively, major head and tail proteins of VSH-1 virions. Two VSH-1 proteins, Hvp19 and Hvp22, appeared as products of posttranslational modification (that is, proteolytic cleavage) of a primary gene product. Hvp19 was encoded by the 5Ј end of hvp19/22, and Hvp22 (N-terminal sequence, MQ KLKNVLEKLISEEKEIEKQAR) was encoded by the 3Ј end (bp 468 to 951). One other protein, Hvp28, displayed the electrophoretic mobility of a protein with a mass greater than that predicted (21.5 kDa) from the hvp28 sequence. The dif- ference may signify posttranslational modification of the hvp28 gene product, although there is no direct evidence for this modification. Identification of VSH-1 lysis genes. None of the translated VSH-1 head and tail proteins had significant sequence similarity with GenBank sequences. Neither did any putative proteins encoded by orfAtoorfF interspersed among these structural protein genes. Downstream of the hvp28 tail gene (Fig. FIG. 2. SDS-PAGE of proteins from VSH-1 whole virions (WV) and heads (H). Molecular mass standards (kDa) are indicated at the 3), however, two ORF, lys and hol, were identified as genes right of the figure, and protein designations are indicated on the left. likely to encode proteins important for the escape of VSH-1 Each lane contained 5 ␮g of protein. virions from B. hyodysenteriae cells. 5888 MATSON ET AL. J. BACTERIOL. Downloaded from

FIG. 3. Map of genes identiﬁed in these studies. VSH-1 genes (ﬁlled arrows), B. hyodysenteriae genes (striped arrows), and ORF greater than 240 bp with no GenBank homolog (open arrows) are oriented according to their direction of transcription.

The gene lys was predicted to encode a 22.9-kDa protein amino acids with a putative endolysin, a peptidoglycan-degrad-

(197 amino acids) with conserved domains typical of glycoside ing enzyme, of enterobacterial phage epsilon15. As described jb.asm.org hydrolase/chitinase enzymes (CD00325; PSI-BLAST, NCBI below, the recombinant Lys protein was found to degrade Entrez Database). Lys shares 38% sequence identity over 191 peptidoglycan. at DigiTop -- Digital Desktop Library for USDA on May 31, 2007 TABLE 1. Genes and proteins of VSH-1 and B. hyodysenteriae identiﬁed in these studiesa

b Protein mass (kDa) Closest protein match Gene designation Protein identity Identiﬁcation basisc Estimated Predicted (GenBank accession no.) VSH-1 hvp45 45 47.9 Head Protein-gene match None hvp19/22 19 36.5 Head Protein-gene match None hvp19/22 22 36.5 Head Protein-gene match None hvp13 13 12.7 Head Protein-gene match None hvp38 38 39.4 Head Protein-gene match None orfA 11.6 None orfB 15.1 None hvp24 24 20.9 Head Protein-gene match None hvp53 53 52.7 Tail Protein-gene match None orfC 23.6 None orfD 10.7 None orfE 14.1 None hvp32 32 31.1 Tail Protein-gene match None hvp101 101 97.2 Tail Protein-gene match None hvp28 28 21.5 Tail Protein-gene match None lys 23.2 Endolysin Enzyme activity Putative endolysin of Salmonella phage epsilon15 (AAO06088); E ϭ 5 ϫ 10Ϫ29 orfF 33.9 None hol 9.6 Holin Predicted properties None orfG 14.2 None

B. hyodysenteriae trep 107.6 Unknown T. denticola hyp. proteind (AAS12502); E ϭ 6 ϫ 10Ϫ30 glt 41.8 Amino acid transport Conserved domains F. nucleatum hyp. Ser/Thr-Naϩ symporter (AAL95344); E ϭ 8 ϫ 10Ϫ93 oxd 37.3 Fe-S oxidoreductase Conserved domains C. tetani hyp. Fe-S oxidoreductase (AAO34830); E ϭ 3 ϫ 10Ϫ38 mcpB 69.2 Methyl-accepting Conserved domains B. hyodysenteriae hyp. McpA protein sensory protein (AAP58978); E ϭ 4 ϫ 10Ϫ94 mcpC 65.5 Methyl-accepting Conserved domains B. hyodysenteriae hyp. McpA protein sensory protein (AAP58978); E ϭ 3 ϫ 10Ϫ89

a A gene encoding Hvp60 (NЈ-M_K_MPYHFLRNKIYKLPPAPYINF . . .) was not among the identified genes. b Estimated masses are based on gel electrophoretic migration (Fig. 2). Predicted masses are based on translated protein sequences. c “Protein-gene match” signifies that 22 N-terminal amino acids of the purified protein were identical to those of the protein predicted from the gene sequence. d hyp., hypothetical. VOL. 187, 2005 VSH-1 GENOME 5889 Downloaded from

FIG. 4. Lysis of CHCl -treated E. coli cells by VSH-1 Lys protein. FIG. 5. Peptidoglycan hydrolysis by VSH-1 Lys protein. Puriﬁed B. 3 ␮ E. coli cells treated with CHCl3 to remove their outer membranes were hyodysenteriae peptidoglycan (0.5 mg) was incubated at 37°C with 1 g incubated at 25°C with 0.2 ␮g/ml recombinant Lys (F), 0.2 ␮g/ml of recombinant Lys (F), 1 ␮g mutanolysin (Œ), or no enzyme (■). ␤-galactosidase (Œ), or no enzyme (■). Amino sugars (reducing sugars) released from peptidoglycan were detected at 585 nm by the Morgan-Elson reaction. jb.asm.org

The gene hol was predicted to encode a protein with characteristics of bacteriophage class II holin proteins (57). It is hyodysenteriae cell suspensions, indicating that the outer mem- small (9.6 kDa, 85 amino acids) and has a hydrophilic C- branes of these bacteria are a barrier to the Lys protein. By at DigiTop -- Digital Desktop Library for USDA on May 31, 2007 terminal end and two hydropathic, predictably transmem- contrast, E. coli cells treated with CHCl3 to remove their outer brane, domains (amino acids 15 to 37 and 49 to 71). membranes were rapidly disrupted by purified Lys (Fig. 4). In Identification of B. hyodysenteriae genes. Flanking the separate experiments (data not shown), EDTA treatment of E. VSH-1 gene clusters were genes homologous to bacterial genes coli cells, which produces holes in the outer membrane without (Fig. 3). Predicted proteins encoded by mcpB and mcpC genes killing the bacteria (39, 40), made the cells sensitive to lysis by have conserved domains of bacterial methyl-accepting sensory both VSH-1 Lys and egg white lysozyme. proteins (smart00283; E values of 6 ϫ 10Ϫ46 and 3 ϫ 10Ϫ41, Purified Lys protein hydrolyzed B. hyodysenteriae peptidogly- respectively). The McpB and McpC proteins share 50% se- can, yielding N-acetylamino sugars detected by the Morgan- quence identity, and they have 34 to 35% identity with the Elson reaction (Fig. 5). Similar activity was observed when B. putative McpA protein of B. hyodysenteriae. hyodysenteriae peptidoglycan was digested with mutanolysin Upstream of VSH-1 genes, bacterial genes were oriented in (Fig. 5). In separate assays, Lys did not hydrolyze crab chitin. the opposite transcriptional direction (Fig. 3). The predicted These results indicated that recombinant VSH-1 Lys is a mu- Glt protein had conserved domains of monovalent cation/di- ralytic enzyme that cleaves the polysaccharide chains of B. carboxylate symporters (COG1301; E value of 7 ϫ 10Ϫ59) and hyodysenteriae peptidoglycan (19). high overall sequence similarity with putative amino acid trans- porters from various bacterial genera (closest match, 52% DISCUSSION identity over 386 amino acids with the serine/threonine-Naϩ symporter of Fusobacterium nucleatum). The translated prod- The findings of these investigations indicate that VSH-1 has uct of the oxd gene had conserved domains predicted for Fe-S features of commonly known bacteriophages but clearly is a oxidoreductase enzymes (COG1242; E value of 7 ϫ 10Ϫ59) and different type of prophage. VSH-1 genes, hvp45 through orfG high overall sequence similarity with oxidoreductases from var- (Fig. 3), span a 16.3-kb region of the B. hyodysenteriae B204 ious bacteria (closest match, 33% identity over 315 amino acids genome. Identified genes and predicted ORF are oriented in with a predicted Fe-S oxidoreductase of Clostridium tetani). the same transcriptional direction and organized in head The Trep protein shared sequence similarity with conserved (seven genes), tail (seven genes), and lysis (four genes) mod- putative proteins with unknown function from Treponema spp. ules. The clustering of similar-function genes and the serial (closest match, 25% identity over 663 amino acids with a hy- head-tail-lysis arrangement of the clusters are typical of late pothetical protein of Treponema denticola; NP 972591; E value operons of various temperate prophages (4, 7, 11, 21, 32). of 6 ϫ 10Ϫ30). VSH-1 genes have an average G ϩ C content of 28 mol% Peptidoglycan-degrading activity of VSH-1 Lys. Recombi- (range of 20 to 33 mol%). nant Lys was purified by affinity chromatography from the VSH-1 genes identified by matching amino acid and nucle- soluble fraction of E. coli BAD/lys cells that had been treated otide sequences include genes encoding capsid proteins with L-arabinose to induce Lys production. Approximately 1 Hvp45, -38, -24, -22, -19, and -13 and tail proteins Hvp101, -53, mg of the recombinant protein was routinely purified from a -32, and -28 (Table 1). Based on their staining intensities (Fig. 1-liter culture. 2), Hvp38 and Hvp53 are predominant VSH-1 structural pro- Neither cell lysis nor loss of viability was observed when Lys teins and are likely to be, respectively, major capsid and tail was added (100 ␮g/ml, final concentration) to E. coli or to B. proteins. Capsid proteins Hvp19 and Hvp22 appear as deriva- 5890 MATSON ET AL. J. BACTERIOL.

TABLE 2. Characteristics of Brachyspira hyodysenteriae VSH-1 and Rhodobacter capsulatus GTA prophagesa

Comparison of:

B. hyodysenteriae VSH-1 R. capsulatus GTA Virions carry random 7.5-kb fragments of host genome (28) Virions carry random 4.5-kb fragments of host genome (49, 59)

Noninfectious; generalized transduction ability (28, 35, 52) Noninfectious; generalized transduction ability (37, 50, 56)

18 genes/ORF (16.3 kb DNA) for head, tail, lysis functions (this study) 19 ORF (15 kb DNA) for putative head and tail functions (33, 34)

Released by host cell lysis; endolysin and putative holin genes identiﬁed Escape mechanism unknown (37); lysis genes not (this study) identiﬁed (33)

Spontaneously produced by growing cells (52) Maximum production by bacteria in stationary growth Downloaded from phase (31, 33, 50)

Induced by mitomycin C (27, 28) Not induced by mitomycin C (37, 49, 59)

Prophage induction presumably regulated by RecA-associated SOS Prophage induction regulated by two-component signal response transduction system (31) jb.asm.org VSH-1-like agents distributed widely among Brachyspira species (6, 53) GTA-like agents distributed widely among R. capsulatus strains (37, 56); GTA genes detected in other species of Rhodobacter and other ␣-

proteobacteria (34) at DigiTop -- Digital Desktop Library for USDA on May 31, 2007

Speciﬁc integration site in B. hyodysenteriae B78T and Brachyspira Speciﬁc integration site in R. capsulatus chromosome pilosicoli P43/6/78T chromosomes (53, 60) (33)

GenBank accession no. AY971355 GenBank accession no. AF181080

a Numbers in parentheses correspond to reference numbers.

tives of a truncated primary gene product involved in VSH-1 B. hyodysenteriae protein associated with VSH-1 particles. The capsid assembly. Posttranslational modifications of bacterio- sequence and chromosome location of this gene will likely be phage gene products, notably proteolytic cleavage of capsid identified when the current B. hyodysenteriae genome sequenc- precursor proteins, commonly occur during virion assembly ing project is completed (T. La and D. Hampson, unpublished (22, 38, 43). data). Significant similarities between VSH-1 structural proteins Consistent with the lysis-mediated release of VSH-1 virions and those of other bacteriophages were not detected during (27), the VSH-1 lys gene encodes an endolysin that was iden- analyses of GenBank sequences by us or during targeted tified from its conserved glycoside hydrolase domains, ability to searches of phage-specific databases (S. Casjens and R. W. lyse CHCl3-treated E. coli cells (Fig. 4), and peptidoglycan- Hendrix, personal communications). Thus, proteins conserved degrading activity (Fig. 5). The VSH-1 hol gene likely encodes among double-stranded DNA tailed phages, such as terminase, a holin protein. The hol gene is strategically positioned near head portal protein, tail tape measure protein, and tail fiber lys. The Hol protein has predicted biochemical features, nota- protein (7, 8, 23, 24), could not be identified. A tail assembly bly two transmembrane domains, typical of phage class II ho- protein may be encoded by orfE, based on a potential transla- lins (57). Holins enable endolysins to gain access to peptidogly- tional frameshift site (TTTTTTG) within the gene (R. W. can and are important in timing the release of virions (57). Hendrix, personal communication) and the previous finding of Direct evidence for its holin identity would come from dem- such sites in tail assembly genes of double-stranded DNA bac- onstrations that the Hol protein forms oligomers, enters mem- teriophages of phylogenetically diverse bacteria, including a branes, and complements heterologous endolysin in a ␭ holin- prophage of the spirochete Borrelia burgdorferi (58). deficient mutant (9, 57). An inability to recognize VSH-1 capsid and tail homologs of What is VSH-1? Although VSH-1 resembles bacterial vi- other phage proteins is not surprising. Characterized phage ruses of the Siphoviridae family of tailed phages (28), both proteins in computer databases are still few, relative to the biological and genomic properties distinguish this agent from diversity of bacteriophages in nature, and are biased towards traditional bacteriophages. VSH-1 preparations are noninfec- certain bacterial taxonomic groups (7, 17). It is also possible tious and a single virion is incapable of self-propagation, based that additional VSH-1 genes are located elsewhere in the B. on indirect and direct evidence as follows. VSH-1 identified hyodysenteriae genome. A gene encoding Hvp60 (Fig. 2; Table structural and lysis genes comprise 16.3 kb of DNA. These 1) was not found among the VSH-1 genes. While the gene late-function genes generally represent about 50 to 60% of the could encode a VSH-1 protein, alternatively, it might encode a genome of self-replicating prophages, such as ␭ (4). Thus, an VOL. 187, 2005 VSH-1 GENOME 5891

entire complement of VSH-1 early- and late-function genes thank Tom Casey and Shirley Halling for helpful, in-depth reviews of would predictably span 30 kb of DNA. A VSH-1 virion which the manuscript. Product names are necessary to report factually on available data; packages 7.5-kb DNA fragments (28) would need to carry however, the USDA neither guarantees nor warrants the standard of multiple DNA fragments for self-propagation. Potential early the product, and the use of the name by USDA implies no approval of genes were not among the identified VSH-1 genes (Fig. 3). the product to the exclusion of others that may also be suitable. Finally, we have not detected increases in VSH-1 gene copy number, a sign of phage gene replication, during mitomycin C ADDENDUM IN PROOF induction of VSH-1 (unpublished data). These considerations support the classification of VSH-1 as a GTA-like defective The genome sequence of prophage LE1 from the spirochete prophage (7). Our use of the term “defective” is intended to Leptospira biflexa was recently described by Bourhy et al. (J. indicate that VSH-1 functions in a different manner from tra- Bacteriol. 187:3931–3940, 2005). We found no significant sim- ditional prophages and to distinguish it from those phages. ilarities among VSH-1 predicted protein sequences and those VSH-1 appears quite effective in its relationship with B. hyo- of phage LE1. dysenteriae.

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