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Most members of the Streptococcus mutans group of microorganisms specify a major cell surface-associated protein, SpaA, that is defined by its antigenic properties. The region of the spaA gene from Streptococcus sobrinus 6715 encoding the immunodominant determinant of the major antigenic component (antigen I) of the SpaA protein has recently been characterized. This study examined whether recognition of the immunodominant determinant is independent of the immunized animal host and whether antibodies elicited by the immunodominant determinant cross-react with cell surface proteins from S. mutans of various serotypes. Mouse and rabbit antisera to the undenatured SpaA protein reacted similarly both with the immunodominant determinant and with other antigenic structures of the protein in Western immunoblots with SpaA polypeptides that were specified by spaA gene fragments expressed in recombinant Escherichia coli. This suggests that the antibody responses of inbred and outbred animals were similar. Furthermore, antibodies raised against both the S. sobrinus SpaA immunodominant determinant expressed by recombinant E. coli and the purified protein from S. sobrinus displayed similar strain specificities and protein band profiles towards cells surface proteins from S. mutans of various serotypes in immunodot and Western blot analyses, respectively. This suggests that for S. sobrinus serotype g, the immune response against the SpaA protein is governed by the immunodominant determinant of antigen I. In addition, it indicates that the SpaA protein domain containing the immunodominant determinant overlaps the domain conferring cross-reactivity to cell surface proteins of S. mutans of various serotypes.

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Bacillus thuringiensis is an important entomopathogenic bacterium belongs to the Bacillus cereus group, which also includes B. anthracis and B. cereus. Several genomes of phages originating from this group had been sequenced, but no genome of Siphoviridae phage from B. thuringiensis has been reported. We recently sequenced and analyzed the genome of a novel phage, BtCS33, from a B. thuringiensis strain, subsp. kurstaki CS33, and compared the gneome of this phage to other phages of the B. cereus group. BtCS33 was the first Siphoviridae phage among the sequenced B. thuringiensis phages. It produced small, turbid plaques on bacterial plates and had a narrow host range. BtCS33 possessed a linear, double-stranded DNA genome of 41,992 bp with 57 putative open reading frames (ORFs). It had a typical genome structure consisting of three modules: the “late” region, the “lysogeny-lysis” region and the “early” region. BtCS33 exhibited high similarity with several phages, B. cereus phage Wβ and some variants of Wβ, in genome organization and the amino acid sequences of structural proteins. There were two ORFs, ORF22 and ORF35, in the genome of BtCS33 that were also found in the genomes of B. cereus phage Wβ and may be involved in regulating sporulation of the host cell. Based on these observations and analysis of phylogenetic trees, we deduced that B. thuringiensis phage BtCS33 and B. cereus phage Wβ may have a common distant ancestor.

The complete nucleotide sequence of the gene for a cell surface protein antigen (SpaA) of Streptococcus sobrinus MT3791 (serotype g) was determined. The spaA gene consisted of 4,698 bp and coded for a protein of 170,202 Da. A putative signal peptide was found in the amino-terminal end of the protein. A potential promoter sequence and a putative Shine-Dalgarno sequence preceded the open reading frame. Two internal repeating amino acid sequences were present in SpaA. One repeating region, located in the amino-terminal region, was rich in alanine, and the other, located in the central region, was rich in proline. The molecular structure of SpaA was very similar to that of the surface protein antigen of Streptococcus mutans.

Surface protein antigen A (SpaA), also called antigen B, antigen I/II, or antigen P1, is an abundant cell envelope protein that is the major antigenic determinant of Streptococcus sobrinus and other members of the Streptococcus mutans group of cariogenic bacteria. This laboratory has previously reported the cloning and expression in Escherichia coli of a BamHI restriction fragment of S. sobrinus DNA containing most of the spaA gene (pYA726) and encoding antigen I. Regions of spaA encoding immunodeterminants of antigen I were analyzed by either deletion mapping or expressing selected restriction fragments from the trc promoter. SpaA proteins produced by mutants harboring nested deletions, constructed by BAL 31 exonuclease treatment at a unique SstI site located towards the 3' end of the gene, were examined by Western immunoblot with rabbit serum against SpaA from S. sobrinus. Only SpaA polypeptides larger than 56 kilodaltons reacted with anti-SpaA serum. Various restriction fragments of the region of spaA encoding the antigenic determinants were cloned into an expression vector. The immunoreactive properties of the polypeptides encoded by those fragments indicated that expression of the immunodominant determinant required topographically assembled residues specified by noncontiguous regions located within 0.48-kilobase PvuII-to-SstI and 1.2-kilobase SstI-to-HindIII fragments which were adjacent on the spaA map.

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We identified and characterized a recombinant Escherichia coli containing the entire gene for surface protein antigen A (spaA) of Streptococcus sobrinus 6715. The recombinant E. coli was isolated from a cosmid gene bank of size-fractionated S. sobrinus DNA fragments, and recombinants expressing the SpaA protein were detected immunologically. Subcloning experiments showed that the DNA sequences encoding the SpaA protein could be isolated on two contiguous EcoRI fragments, 3.7 and 3.3 kilobases (kb) in size, both contained on a 16.2-kb BglII fragment. Southern blot hybridization experiments using the EcoRI fragments to probe genomic DNAs from various serotypes of the mutans group of streptococci revealed DNA sequence homology not only to S. sobrinus 6715 (serotype g) chromosomal DNA but also to S. sobrinus serotype d DNA. Weak hybridization signals to Streptococcus mutans serotypes c, e, and f and to Streptococcus cricetus serotype a were observed with the 3.3-kb EcoRI fragment. These results suggest that the coding sequence for the spaA gene is probably conserved in S. sobrinus strains. Plasmid-encoded polypeptides made in E. coli minicells revealed that transcription of the spaA gene was initiated on the 3.7-kb EcoRI fragment and that its product size was about 210 kilodaltons. The cloned SpaA protein was purified from the periplasmic protein of E. coli, and monospecific antiserum against the cloned product was prepared in rabbits. The data obtained from various physiochemical and immunological procedures allowed us to conclude that the coding sequence for the entire spaA gene of S. sobrinus 6715 had been successfully cloned in E. coli and that faithful expression of the cloned product could be obtained.

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Hydrophobic Actinobacteria are commonly associated with the stabilization of foams in activated sludge systems. One possible attractive approach to control these foam-stabilizing organisms is the use of specific bacteriophages. We describe the genome characterization of a novel polyvalent DNA phage, GTE2, isolated from activated sludge. This phage is lytic for Gordonia terrae, Rhodococcus globerulus, Rhodococcus erythropolis, Rhodococcus erythropolis, Nocardia otitidiscaviarum, and Nocardia brasiliensis. Phage GTE2 belongs to the family Siphoviridae, possessing a characteristic icosahedral head encapsulating a double-stranded DNA linear genome (45,530 bp) having 10-bp 3′-protruding cohesive ends. The genome sequence is 98% unique at the DNA level and contains 57 putative genes. The genome can be divided into two components, where the first is modular and encodes phage structural proteins and lysis genes. The second is not modular, and the genes harbored there are involved in DNA replication, repair, and metabolism. Some have no known function. GTE2 shows promising results in controlling stable foam production by its host bacteria under laboratory conditions, suggesting that it may prove useful in the field as a biocontrol agent.

The gene encoding a protective protein antigen of the gram-positive bacterium Erysipelothrix rhusiopathiae, an important veterinary pathogen responsible for erysipelas in swine and a variety of diseases in animals, was cloned and sequenced. The gene encodes a polypeptide of 597 amino acids plus a putative signal sequence of 29 amino acids, resulting in a mature protein with a molecular mass of 69,017 Da. Sequence analysis of the gene product revealed a C-terminal region composed of nine tandem repeats of 20 amino acids and a total sequence that is nearly identical to that of the 64-kDa cell surface protein (SpaA) of the bacterium. Because of this similarity, the protein was designated SpaA.1. In this study, we examined whether the SpaA.1 protein could induce protective antibodies and whether we could identify the region involved in protective immunity. Both the mature SpaA.1 protein and its C-terminal repeat region, but not the N-terminal segment, were expressed in Escherichia coli and purified as a histidine-tagged fusion recombinant protein. Rabbit antiserum raised against the mature SpaA.1 protein passively protected mice from lethal challenge with a virulent homologous strain, Fujisawa-SmR, suggesting that protection is mediated by humoral antibodies. To determine which domain of the SpaA.1 protein is responsible for the observed protection, mice were actively immunized with either the mature SpaA.1 protein or the C-terminal repeat region and then challenged with Fujisawa-SmR. The result showed that mice immunized with the mature SpaA.1 protein, but not the C-terminal repeat region, were protected, suggesting that the protection-eliciting epitope(s) is located within the N-terminal two-thirds of the SpaA.1 molecule. This was confirmed by passive immunization experiments in which the protective activity of rabbit antiserum, raised against mature SpaA.1 protein, was not abolished by absorption with the purified recombinant C-terminal repeat region. In addition, antibodies specific for the C-terminal repeat region were unable to protect mice from lethal challenge. These results show that the N-terminal two-thirds of the SpaA.1 molecule may constitute a good vaccine candidate against erysipelas.

Different surface organelles contribute to specific interactions of a pathogen with host tissues or infectious partners. Multiple pilus gene clusters potentially encoding different surface structures have been identified in several gram-positive bacterial genomes sequenced to date, including actinomycetales, clostridia, corynebacteria, and streptococci. Corynebacterium diphtheriae has been shown to assemble a pilus structure, with sortase SrtA essential for the assembly of a major subunit SpaA and two minor proteins, SpaB and SpaC. We report here the characterization of a second pilus consisting of SpaD, SpaE, and SpaF, of which SpaD and SpaE form the pilus shaft and SpaF may be located at the pilus tip. The structure of the SpaDEF pilus contains no SpaABC pilins as detected by immunoelectron microscopy. Neither deletion of spaA nor sortase srtA abolishes SpaDEF pilus formation. The assembly of the SpaDEF pilus requires specific sortases located within the SpaDEF pilus gene cluster. Although either sortase SrtB or SrtC is sufficient to polymerize SpaDF, the incorporation of SpaE into the SpaD pili requires sortase SrtB. In addition, an alanine in place of the lysine of the SpaD pilin motif abrogates pilus polymerization. Thus, SpaD, SpaE, and SpaF constitute a different pilus structure that is independently assembled and morphologically distinct from the SpaABC pili and possibly other pili of C. diphtheriae.

In this study, we used a vaccine strain of Salmonella typhimurium to express antigenic determinants of the SpaA antigen of Streptococcus sobrinus, which is involved in the caries-forming process. We cloned either a single repeat (pYA2901) or three tandem repeats (pYA2905) of the 0.48-kb fragment of the spaA gene, which codes for an important component of the SpaA protein, plus a 1.2-kb minor antigenic determinant and measured the resulting immune responses to SpaA in orally immunized BALB/c mice. The single or triple repeat of the spaA gene fragment was inserted into the Asd+ vector pYA292 and was transformed into the S. typhimurium delta cya delta crp vaccine strain chi 4072 containing delta asd in the chromosome. Female BALB/c mice were then orally immunized with two doses of the S. typhimurium containing either of the two SpaA constructs, and the immune responses to the expressed SpaA protein were assessed. Significant serum immunoglobulin G (IgG) anti-SpaA titers were detected in mice immunized with chi 4072(pYA2905) but not chi 4072(pYA2901). Salivary anti-SpaA IgA titers were minimal and were only detected in mice immunized with S. typhimurium expressing the SpaA encoded by pYA2905. Intestinal anti-SpaA IgA titers, however, were detected in both groups of mice, particularly in mice immunized with chi 4072(pYA2905). An oral booster 26 weeks after the initial series of immunizations resulted in increased serum IgG titers in both chi 4072(pYA2901)- and chi 4072(pYA2905)-immunized animals, particularly in the chi 4072(pYA2905)-immunized animals. No anamnestic IgA response was detected in the saliva following the booster immunization.

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Lactobacillus rhamnosus GG is a well-established Gram-positive probiotic strain, whose health-benefiting properties are dependent in part on prolonged residence in the gastrointestinal tract and are likely dictated by adherence to the intestinal mucosa. Previously, we identified two pilus gene clusters (spaCBA and spaFED) in the genome of this probiotic bacterium, each of which contained the predicted genes for three pilin subunits and a single sortase. We also confirmed the presence of SpaCBA pili on the cell surface and attributed an intestinal mucus-binding capacity to one of the pilin subunits (SpaC). Here, we report cloning of the remaining pilin genes (spaA, spaB, spaD, spaE, and spaF) in Escherichia coli, production and purification of the recombinant proteins, and assessment of the adherence of these proteins to human intestinal mucus. Our findings indicate that the SpaB and SpaF pilin subunits also exhibit substantial binding to mucus, which can be inhibited competitively in a dose-related manner. Moreover, the binding between the SpaB pilin subunit and the mucosal substrate appears to operate through electrostatic contacts and is not related to a recognized mucus-binding domain. We conclude from these results that it is conceivable that two pilin subunits (SpaB and SpaC) in the SpaCBA pilus fiber play a role in binding to intestinal mucus, but for the uncharacterized and putative SpaFED pilus fiber only a single pilin subunit (SpaF) is potentially responsible for adhesion to mucus.

Temperate bacteriophages play an important role in the pathogenicity of Staphylococcus aureus, for instance, by mediating the horizontal gene transfer of virulence factors. Here we established a classification scheme for staphylococcal prophages of the major Siphoviridae family based on integrase gene polymorphism. Seventy-one published genome sequences of staphylococcal phages were clustered into distinct integrase groups which were related to the chromosomal integration site and to the encoded virulence gene content. Analysis of three marker modules (lysogeny, tail, and lysis) for phage functional units revealed that these phages exhibit different degrees of genome mosaicism. The prevalence of prophages in a representative S. aureus strain collection consisting of 386 isolates of diverse origin was determined. By linking the phage content to dominant S. aureus clonal complexes we could show that the distribution of bacteriophages varied remarkably between lineages, indicating restriction-based barriers. A comparison of colonizing and invasive S. aureus strain populations revealed that hlb-converting phages were significantly more frequent in colonizing strains.

Two temperate viruses, φ3626 and φ8533, have been isolated from lysogenic Clostridium perfringens strains. Phage φ3626 was chosen for detailed analysis and was inspected by electron microscopy, protein profiling, and host range determination. For the first time, the nucleotide sequence of a bacteriophage infecting Clostridium species was determined. The virus belongs to the Siphoviridae family of the tailed phages, the order Caudovirales. Its genome consists of a linear double-stranded DNA molecule of 33,507 nucleotides, with invariable 3′-protruding cohesive ends of nine residues. Fifty open reading frames were identified, which are organized in three major life cycle-specific gene clusters. The genes required for lytic development show an opposite orientation and arrangement compared to the lysogeny control region. A function could be assigned to 19 gene products, based upon bioinformatic analyses, N-terminal amino acid sequencing, or experimental evidence. These include DNA-packaging proteins, structural components, a dual lysis system, a putative lysogeny switch, and proteins that are involved in replication, recombination, and modification of phage DNA. The presence of genes encoding a putative sigma factor related to sporulation-dependent sigma factors and a putative sporulation-dependent transcription regulator suggests a possible interaction of φ3626 with onset of sporulation in C. perfringens. We found that the φ3626 attachment site attP lies in a noncoding region immediately downstream of int. Integration of the viral genome occurs into the bacterial attachment site attB, which is located within the 3′ end of a guaA homologue. This essential housekeeping gene is functionally independent of the integration status, due to reconstitution of its terminal codons by phage sequence.

Streptococcal antigen I/II or the surface protein antigen A (SpaA) of Streptococcus sobrinus is an adhesin which mediates binding of the organism to tooth surfaces. The complete sequence of the gene which encodes SpaA has been determined. The gene consists of 4,584 bp and encodes a protein of 1,528 amino acid residues. The deduced amino acid sequence shows extensive homology with those of the cell surface adhesins from Streptococcus mutans serotypes c and f and from Streptococcus sanguis. Structural analysis of the N-terminal region (residues 50 to 550), which is rich in alanine and includes four tandem repeats of an 82-residue sequence, suggests that it adopts an alpha-helical coiled-coil conformation. Cell surface hydrophobicity may be associated with this region. The C-terminal region is more conserved and includes two tandem repeats of a 39-residue proline-rich sequence. A further proline-rich sequence in this region is predicted to span the cell wall. Although a hydrophobic sequence is present in the C-terminal region, it appears to be too short to span the cell membrane. Anchoring of SpaA in the cell membrane may therefore require some form of posttranslational modification or association with another membrane protein.

Background

Endolysins produced by bacteriophages lyse bacteria, and are thus considered a novel type of antimicrobial agent. Several endolysins from Bacillus phages or prophages have previously been characterized and used to target Bacillus strains that cause disease in animals and humans. B. thuringiensis phage BtCS33 is a Siphoviridae family phage and its genome has been sequenced and analyzed. In the BtCS33 genome, orf18 was found to encode an endolysin protein (PlyBt33).

Results

Bioinformatic analyses showed that endolysin PlyBt33 was composed of two functional domains, the N-terminal catalytic domain and the C-terminal cell wall binding domain. In this study, the entire endolysin PlyBt33, and both the N- and C-termini,were expressed in Escherichia coli and then purified. The lytic activities of PlyBt33 and its N-terminus were tested on bacteria. Both regions exhibited lytic activity, although PlyBt33 showed a higher lytic activity than the N-terminus. PlyBt33 exhibited activity against all Bacillus strains tested from five different species, but was not active against Gram-negative bacteria. Optimal conditions for PlyBt33 reactivity were pH 9.0 and 50°C. PlyBt33 showed high thermostability, with 40% of initial activity remaining following 1 h of treatment at 60°C. The C-terminus of PlyBt33 bound to B. thuringiensis strain HD-73 and Bacillus subtilis strain 168. This cell wall binding domain might be novel, as its amino acid sequence showed little similarity to previously reported endolysins.

Conclusions

PlyBt33 showed potential as a novel antimicrobial agent at a relatively high temperature and had a broad lytic spectrum within the Bacillus genus. The C-terminus of PlyBt33 might be a novel kind of cell wall binding domain.

The surface protective antigen (Spa) protein of Erysipelothrix rhusiopathiae has been shown to be highly immunogenic and is a potential candidate for a new vaccine against erysipelas. In this study, we cloned and sequenced spa genes from all E. rhusiopathiae serovar reference strains as well as from a serovar 18 strain which was not classified as any species in the genus Erysipelothrix. Sequence analysis revealed that the Spa proteins could be classified into three molecular species, including SpaA, which was previously found in serovars 1a and 2, and the newly designated SpaB and SpaC proteins. The SpaA protein is produced by E. rhusiopathiae serovars 1a, 1b, 2, 5, 8, 9, 12, 15, 16, 17, and N, the SpaB protein is produced by E. rhusiopathiae serovars 4, 6, 11, 19, and 21, and the SpaC protein is produced only by serovar 18. The amino acid sequence similarity was high among members of each Spa type (96 to 99%) but low between different Spa types (∼60%). The greatest diversity in Spa proteins was found in the N-terminal half of the molecule (50 to 57% similarity), which was shown to be involved in immunoprotection. Coinciding with this, immunoblot analysis revealed that rabbit antisera specific to each Spa reacted strongly with the homologous Spa protein but weakly with heterologous Spa proteins. A mouse cross-protection study showed that the three recombinant Spa (rSpa) proteins elicited complete protection against challenge with homologous strains but that the level of protection against challenge with heterologous strains varied depending on the rSpa protein used for immunization. Our study is the first to demonstrate sequence and antigenic diversity in Spa proteins and to indicate that rSpaC may be the most promising antigen for use as a vaccine component because of its broad cross-protectiveness.

Erysipelothrix rhusiopathiae is a causal agent of swine erysipelas, which is of economic importance in the swine industry by virtue of causing acute septicemia, chronic arthritis, and endocarditis. However, little is known about the genetic properties of its protective antigens. Recently, a surface protective antigen (SpaA) gene was identified from serotype 2 in a mouse model. We cloned spaA from virulent strain Fujisawa (serotype 1a) and determined that the N-terminal 342 amino acids without C-terminal repeats of 20 amino acids have the ability to elicit protection in mice. Fusions of 342 amino acids of Fujisawa SpaA and histidine hexamer (HisSpa1.0) protected pigs against challenge with both serotype 1 and serotype 2, the most important serotypes in the swine industry. Pigs immunized with HisSpa1.0 reacted well with both HisSpa1.0 and intact SpaA by enzyme-linked immunosorbent assay and immunoblotting. Serum collected at the time of challenge from a pig immunized with HisSpa1.0 markedly enhanced the in vitro phagocytic and killing activity of pig neutrophils against the bacteria. DNA sequences of protective regions of spaA genes from five strains of serotypes 1 and 2 were almost identical. The full DNA sequences also seemed to be conserved among strains of all 12 serotype reference strains harboring the spaA gene by restriction fragment length polymorphism analysis of PCR products. These results indicates that SpaA is a common protective antigen of serotypes 1 and 2 of E. rhusiopathiae in swine and will be a useful tool for development of new types of vaccines and diagnostic tools for effective control of the disease.

The Shiga-like toxin 1-converting bacteriophage H-19B was recently shown to carry the structural genes for the toxin and was shown to have DNA sequence homology with phage lambda. We present evidence that the linear genome of bacteriophage H-19B has cohesive termini which become covalently associated during prophage integration. Integration occurs through a site on a 4-kilobase-pair EcoRI fragment located near the center of the bacteriophage chromosome. The relationship between bacteriophages H-19B and lambda was examined by Southern hybridization. Homologous regions were mapped on the respective chromosomes which corresponded to the regions of the J gene, the int-xis area, and the O and P genes of phage lambda. The H-19B tox genes were mapped to the right of the O and P gene homology, which was far away from the phage attachment site. We concluded that H-19B is a lambdoid bacteriophage. Unlike other toxin-converting bacteriophages, the toxin genes were not located adjacent to the phage attachment site. It appeared that the Shiga-like toxin 1 genes were not picked up by a simple imprecise prophage excision. H-19B could, however, have acquired chromosomally located toxin genes by a series of events involving deletion and duplication followed by aberrant excision.

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The Streptococcus sobrinus SpaA protein and the Streptococcus mutans P1 protein share 66% sequence homology at the amino acid level. To determine if the SpaA protein can be expressed in S. mutans and functionally replace the P1 protein, the spaA gene of S. sobrinus 6715 was isolated from plasmid pX1303 and inserted into the Escherichia coli-Streptococcus shuttle vector pVA838. The resulting plasmid pX1600 was transformed into the P1-deficient strain S. mutans 834 that has defects in saliva-mediated aggregation and in the ability to adhere to saliva-coated hydroxyapatite surfaces. Western blot (immunoblot) analysis of cellular protein fractions of S. mutans 834 (pX1600) detected in mutanolysin-solubilized cell walls a major protein of 210 kDa with an electrophoretic mobility similar to that of S. sobrinus SpaA protein and a minor 210-kDa protein and a major 64-kDa protein in the extracellular protein fraction. Analysis of virulence traits showed that expression of SpaA protein by S. mutans 834(pX1600) cells had restored the ability of the S. mutans 834 cells to aggregate in the presence of saliva or salivary agglutinin but not to adhere to saliva-coated hydroxyapatite. This cell aggregation was inhibited specifically by antisera to S. sobrinus SpaA protein. These results indicate that SpaA plays a role in the virulence of S. sobrinus by specifically interacting with fluid-phase salivary agglutinin to mediate cell aggregation.

Background

The presence of prophages has been an important variable in genetic exchange and divergence in most bacteria. This study reports the determination of the genomic sequence of Salmonella phage ε34, a temperate bacteriophage that was important in the early study of prophages that modify their hosts' cell surface and is of a type (P22-like) that is common in Salmonella genomes.

Results

The sequence shows that ε34 is a mosaically related member of the P22 branch of the lambdoid phages. Its sequence is compared with the known P22-like phages and several related but previously unanalyzed prophage sequences in reported bacterial genome sequences.

Conclusion

These comparisons indicate that there has been little if any genetic exchange within the procapsid assembly gene cluster with P22-like E. coli/Shigella phages that are have orthologous but divergent genes in this region. Presumably this observation reflects the fact that virion assembly proteins interact intimately and divergent proteins can no longer interact. On the other hand, non-assembly genes in the "ant moron" appear to be in a state of rapid flux, and regulatory genes outside the assembly gene cluster have clearly enjoyed numerous and recent horizontal exchanges with phages outside the P22-like group. The present analysis also shows that ε34 harbors a gtrABC gene cluster which should encode the enzymatic machinery to chemically modify the host O antigen polysaccharide, thus explaining its ability to alter its host's serotype. A comprehensive comparative analysis of the known phage gtrABC gene clusters shows that they are highly mobile, having been exchanged even between phage types, and that most "bacterial" gtrABC genes lie in prophages that vary from being largely intact to highly degraded. Clearly, temperate phages are very major contributors to the O-antigen serotype of their Salmonella hosts.

Summary

Adherence to host tissues mediated by pili is pivotal in the establishment of infection by many bacterial pathogens. Corynebacterium diphtheriae assembles on its surface three distinct pilus structures. The function and the mechanism of how various pili mediate adherence, however, have remained poorly understood. Here we show that the SpaA-type pilus is sufficient for the specific adherence of corynebacteria to human pharyngeal epithelial cells. The deletion of the spaA gene, which encodes the major pilin forming the pilus shaft, abolishes pilus assembly but not adherence to pharyngeal cells. In contrast, adherence is greatly diminished when either minor pilin SpaB or SpaC is absent. Antibodies directed against either SpaB or SpaC block bacterial adherence. Consistent with a direct role of the minor pilins, latex beads coated with SpaB or SpaC protein bind specifically to pharyngeal cells. Therefore, tissue tropism of corynebacteria for pharyngeal cells is governed by specific minor pilins. Importantly, immunoelectron microscopy and immunofluorescence studies reveal clusters of minor pilins that are anchored to cell surface in the absence of a pilus shaft. Thus, the minor pilins may also be cell wall anchored in addition to their incorporation into pilus structures that could facilitate tight binding to host cells during bacterial infection.

Lactobacillus acidophilus ADH is lysogenic and harbors an inducible prophage, phi adh. Bacteriophage were detected in cell lysates induced by treatment with mitomycin C or UV light. Electron microscopy of lysates revealed phage particles with a hexagonal head (62 nm) and a long, noncontractile, flexible tail (398 nm) ending in at last five short fibers. Phage phi adh was classified within Bradley's B1 phage group and the Siphoviridae family. The phi adh genome is a linear double-stranded DNA molecule of 41.7 kilobase pairs with cohesive ends: a physical map of the phi adh genome was constructed. A prophage-cured derivative of strain ADH, designated NCK102, was isolated from cells that survived UV exposure. NCK102 did not exhibit mitomycin C-induced lysis, but broth cultures lysed upon addition of phage. Phage phi adh produced clear plaques on NCK102 in media containing 10 mM CaCl2 at pH values between 5.2 and 5.5. A relysogenized derivative (NCK103) of NCK102 was isolated that exhibited mitomycin C-induced lysis and superinfection immunity to phage phi adh. Hybridization experiments showed that the phi adh genome was present in the ADH and NCK103 chromosomes, but absent in NCK102. These results demonstrated classic lytic and lysogenic cycles of replication for the temperate phage phi adh induced from L. acidophilus ADH. Phage phi adh also mediates transduction of plasmid DNA. Transductants of strain ADH containing pC194, pGK12, pGB354, and pVA797 were detected at frequencies in the range of 3.6 x 10(-8) to 8.3 x 10(-10) per PFU. Rearrangements or deletions were not detected in these plasmids as a consequence of transduction. This is the first description of plasmid transduction in the genus Lactobacillus.

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Bacteriophages are considered to be the most abundant biological entities on the planet. The Siphoviridae are the most commonly encountered tailed phages and contain double-stranded DNA with an average genome size of ∼50 kb. This paper describes the isolation from four different activated sludge plants of the phage RRH1, which is polyvalent, lysing five Rhodococcus species. It has a capsid diameter of only ∼43 nm. Whole-genome sequencing of RRH1 revealed a novel circularly permuted DNA sequence (14,270 bp) carrying 20 putative open reading frames. The genome has a modular arrangement, as reported for those of most Siphoviridae phages, but appears to encode only structural proteins and carry a single lysis gene. All genes are transcribed in the same direction. RRH1 has the smallest genome yet of any described functional Siphoviridae phage. We demonstrate that lytic phage can be recovered from transforming naked DNA into its host bacterium, thus making it a potentially useful model for studying gene function in phages.

Prophage Lrm1 was induced with mitomycin C from an industrial Lactobacillus rhamnosus starter culture, M1. Electron microscopy of the lysate revealed relatively few intact bacteriophage particles among empty heads and disassociated tails. The defective Siphoviridae phage had an isometric head of approximately 55 nm and noncontractile tail of about 275 nm with a small baseplate. In repeated attempts, the prophage could not be cured from L. rhamnosus M1, nor could a sensitive host be identified. Sequencing of the phage Lrm1 DNA revealed a genome of 39,989 bp and a G+C content of 45.5%. A similar genomic organization and mosaic pattern of identities align Lrm1 among the closely related Lactobacillus casei temperate phages A2, ΦAT3, and LcaI and with L. rhamnosus virulent phage Lu-Nu. Of the 54 open reading frames (ORFs) identified, all but 8 shared homology with other phages of this group. Five unknown ORFs were identified that had no homologies in the databases nor predicted functions. Notably, Lrm1 encodes a putative endonuclease and a putative DNA methylase with homology to a methylase in Lactococcus lactis phage Tuc2009. Possibly, the DNA methylase, endonuclease, or other Lrm1 genes provide a function crucial to L. rhamnosus M1 survival, resulting in the stability of the defective prophage in its lysogenic state. The presence of a defective prophage in an industrial strain could provide superinfection immunity to the host but could also contribute DNA in recombination events to produce new phages potentially infective for the host strain in a large-scale fermentation environment.

Pilus assembly in gram-positive bacteria occurs by a two-step mechanism, whereby pilins are polymerized and then covalently anchored to the cell wall. In Corynebacterium diphtheriae, the pilin-specific sortase SrtA catalyzes polymerization of the SpaA-type pilus, consisting of the shaft pilin SpaA, tip pilin SpaC and minor pilin SpaB. Cell wall anchoring of the SpaA polymers is triggered when SrtA incorporates SpaB into the pilus base via lysine-mediated transpeptidation; anchoring to the cell wall peptidoglycan is subsequently catalyzed by the housekeeping sortase SrtF. Here we show that SpaB and SpaC formed a heterodimer independent from SpaA polymerization. SrtA was absolutely required for the formation of the SpaBC heterodimer, while SrtF facilitated the optimal cell wall anchoring of this heterodimer. Alanine substitution of the SpaB lysine residue K139 or truncation of the SpaB cell wall sorting signal (CWSS) abolished assembly of the SpaBC heterodimer, hence underscoring SpaB function in transpeptidation and cell wall linkage. Importantly, sortase specificity for the cell wall anchoring step was found to be dependent on the LAFTG motif within the SpaB CWSS. Thus, C. diphtheriae employs a common sortase-catalyzed mechanism involving lysine-mediated transpeptidation to generate both adhesive pilus and simple heterodimeric structures on the bacterial the cell wall.

Since 1999, a new, epidemic, methicillin-resistant Staphylococcus aureus (MRSA) strain, named the “Cordobes clone,” has emerged in Argentina and coexists with the pandemic Brazilian clone. The purpose of this study was to determine the stability over time of the new clone and to investigate its evolutionary relationship with epidemic international MRSA lineages and with other MRSA and methicillin-susceptible S. aureus (MSSA) major clones distributed in this region. One hundred three MRSA isolates recovered in 2001 from Cordoba, Argentina, hospitals and 31 MSSA strains collected from 1999 to 2002 were analyzed by their antibiotic resistance patterns, phage typing, and pulsed-field gel electrophoresis. Additionally, representative members of most MRSA defined genotypes (A, B, C, E, K, and I) were characterized by multilocus sequence typing (MLST) and spaA and SCCmec typing. The most prevalent MSSA pulsotypes were also analyzed by MLST. Our results support the displacement of the Brazilian clone (sequence type [ST] 239, spaA type WGKAOMQ, SCCmec type IIIA) by the Cordobes clone (ST5, spaA type TIMEMDMGMGMK, SCCmec type I) in the hospital environment. MRSA and MSSA isolates shared only ST5. The data support the origin of the Cordobes clone as a member of a lineage that includes the pediatric and New York/Japan international clones and that is genetically related to the British EMRSA-3 strain. Interestingly, the pediatric clone, isolated from most community-acquired infections in Cordoba, was characterized by ST100, a single-locus variant of ST5 and a new variant of SCCmec type related to SCCmec type IVc.