Clinical strains of Streptococcus agalactiae carry two different variants of pathogenicity island XII.

Streptococcus agalactiae or Group B streptococci (GBS) are a common cause of serious diseases of newborns and adults. GBS pathogenicity largely depends on genes located on the accessory genome including several pathogenicity islands (PAI). The present paper is focused on the structure and molecular epidemiological analysis of one of the GBS pathogenicity islands-the pathogenicity island PAI XII (Glaser et al. Mol Microbiol 45(6):1499-1513, 2002). This PAI was found to be composed of three different mobile genetic elements: a composite transposon (PAI-C), a genomic islet (PAI-B), and a pathogenicity island associated with gene sspB1 (PAI-A). PAI-A in GBS has a homolog--PAI-A1 with similar, but a different genetic constellation. PCR-based analysis of GBS collections from different countries revealed that a strains lineage with PAI-A is less common than PAI-A1 and was determined to be present only among the strains obtained from Russia. Our results suggest that PAI-A and PAI-A1 have the same progenitor, which evolved independently and appeared in the GBS genome as separate genetic events. Results of this study reflect specific geographical distribution of the GBS strains with the mobile genetic element under study.

Probing oral microbial functionality--expression of spxB in plaque samples.

The Human Oral Microbiome Database (HOMD) provides an extensive collection of genome sequences from oral bacteria. The sequence information is a static snapshot of the microbial potential of the so far sequenced species. A major challenge is to connect the microbial potential encoded in the metagenome to an actual function in the in vivo oral biofilm. In the present study we took a reductionist approach and identified a considerably conserved metabolic gene, spxB to be encoded by a majority of oral streptococci using the HOMD metagenome information. spxB encodes the pyruvate oxidase responsible for the production of growth inhibiting amounts of hydrogen peroxide (H2O2) and has previously been shown as important in the interspecies competition in the oral biofilm. Here we demonstrate a strong correlation of H2O2 production and the presence of the spxB gene in dental plaque. Using Real-Time RT PCR we show that spxB is expressed in freshly isolated human plaque samples from several donors and that the expression is relative constant when followed over time in one individual. This is the first demonstration of an oral community encoded gene expressed in vivo suggesting a functional role of spxB in oral biofilm physiology. This also demonstrates a possible strategy to connect the microbial potential of the metagenome to its functionality in future studies by identifying similar highly conserved genes in the oral microbial community.

Multiple roles of RNase Y in Streptococcus pyogenes mRNA processing and degradation.

Control over mRNA stability is an essential part of gene regulation that involves both endo- and exoribonucleases. RNase Y is a recently identified endoribonuclease in Gram-positive bacteria, and an RNase Y ortholog has been identified in Streptococcus pyogenes (group A streptococcus [GAS]). In this study, we used microarray and Northern blot analyses to determine the S. pyogenes mRNA half-life of the transcriptome and to understand the role of RNase Y in global mRNA degradation and processing. We demonstrated that S. pyogenes has an unusually high mRNA turnover rate, with median and mean half-lives of 0.88 min and 1.26 min, respectively. A mutation of the RNase Y-encoding gene (rny) led to a 2-fold increase in overall mRNA stability. RNase Y was also found to play a significant role in the mRNA processing of virulence-associated genes as well as in the rapid degradation of rnpB read-through transcripts. From these results, we conclude that RNase Y is a pleiotropic regulator required for mRNA stability, mRNA processing, and removal of read-through transcripts in S. pyogenes.

Dynamics of speB mRNA transcripts in Streptococcus pyogenes.

Streptococcus pyogenes (group A streptococcus [GAS]) is a human-specific pathogen that causes a variety of diseases ranging from superficial infections to life-threatening diseases. SpeB, a potent extracellular cysteine proteinase, plays an important role in the pathogenesis of GAS infections. Previous studies show that SpeB expression and activity are controlled at the transcriptional and posttranslational levels, though it had been unclear whether speB was also regulated at the posttranscriptional level. In this study, we examined the growth phase-dependent speB mRNA level and decay using quantitative reverse transcription-PCR (qRT-PCR) and Northern blot analyses. We observed that speB mRNA accumulated rapidly during exponential growth, which occurred concomitantly with an increase in speB mRNA stability. A closer observation revealed that the increased speB mRNA stability was mainly due to progressive acidification. Inactivation of RNase Y, a recently identified endoribonuclease, revealed a role in processing and degradation of speB mRNA. We conclude that the increased speB mRNA stability contributes to the rapid accumulation of speB transcript during growth.

Counteractive balancing of transcriptome expression involving CodY and CovRS in Streptococcus pyogenes.

Streptococcus pyogenes (group A streptococcus [GAS]) responds to environmental changes in a manner that results in an adaptive regulation of the transcriptome. The objective of the present study was to understand how two global transcriptional regulators, CodY and CovRS, coordinate the transcriptional network in S. pyogenes. Results from expression microarray data and quantitative reverse transcription-PCR (qRT-PCR) showed that the global regulator CodY controls the expression of about 250 genes, or about 17% of the genome of strain NZ131. Additionally, the codY gene was shown to be negatively autoregulated, with its protein binding directly to the promoter region with a CodY binding site. In further studies, the influence of codY, covRS, and codY-covRS mutations on gene expression was analyzed in growth phase-dependent conditions using C medium, reported to mimic nutritional abundance and famine conditions similar to those found during host GAS infection. Additional biological experiments of several virulence phenotypes, including pilin production, biofilm formation, and NAD glycohydrolase activity, demonstrated the role that both CodY and CovRS play in their regulation. Correlation analysis of the overall data revealed that, in exponentially growing cells, CodY and CovRS act in opposite directions, with CodY stimulating and CovRS repressing a substantial fraction of the core genome, including many virulence factors. This is the first report of counteractive balancing of transcriptome expression by global transcription regulators and provides important insight into how GAS modulates gene expression by integrating important extracellular and intracellular information.

Genomic island TnSmu2 of Streptococcus mutans harbors a nonribosomal peptide synthetase-polyketide synthase gene cluster responsible for the biosynthesis of pigments involved in oxygen and H2O2 tolerance.

The oral biofilm community consists of >800 microbial species, among which Streptococcus mutans is considered a primary pathogen for dental caries. The genomic island TnSmu2 of S. mutans comprises >2% of the genome. In this study, we demonstrate that TnSmu2 harbors a gene cluster encoding nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and accessory proteins and regulators involved in nonribosomal peptide (NRP) and polyketide (PK) biosynthesis. Interestingly, the sequences of these genes and their genomic organizations and locations are highly divergent among different S. mutans strains, yet each TnSmu2 region encodes NRPS/PKS and accessory proteins. Mutagenesis of the structural genes and putative regulatory genes in strains UA159, UA140, and MT4653 resulted in colonies that were devoid of their yellow pigmentation (for strains UA140 and MT4653). In addition, these mutant strains also displayed retarded growth under aerobic conditions and in the presence of H(2)O(2). High-performance liquid chromatography profiling of cell surface extracts identified unique peaks that were missing in the mutant strains, and partial characterization of the purified product from UA159 demonstrated that it is indeed a hybrid NRP/PK, as predicted. A genomic survey of 94 clinical S. mutans isolates suggests that the TnSmu2 gene cluster may be more prevalent than previously recognized.

Bacteriophage content of M49 strains of Streptococcus pyogenes.

Bacteriophages are common autonomous migrating mobile genetic elements in group A Streptococcus (GAS) and are often associated with the carriage of various virulence genes, including toxins, mitogens and enzymes. Two collections of GAS type M49 strains isolated from invasive (22 strains) and noninvasive (16 strains) clinical cases have been studied for the presence of phage and phage-associated virulence genes. All the GAS strains carried from at least two to six phage genomes as determined by the number of known phage integrase genes found. A sampling of the invasive M49 strains showed that they belonged to the same multilocus sequence typing type, carried two specific integrase genes (int5 and int7), and contained the toxin genes speA, speH and speI. Other invasive strains lacking this gene profile carried the prophage integrating in mutL-mutS region and inducing the 'mutator' phenotype. We suggest that this specific phage-related virulence gene constellation might be an important factor increasing M49 GAS pathogenicity.

Genome sequence of a nephritogenic and highly transformable M49 strain of Streptococcus pyogenes.

The 1,815,783-bp genome of a serotype M49 strain of Streptococcus pyogenes (group A streptococcus [GAS]), strain NZ131, has been determined. This GAS strain (FCT type 3; emm pattern E), originally isolated from a case of acute post-streptococcal glomerulonephritis, is unusually competent for electrotransformation and has been used extensively as a model organism for both basic genetic and pathogenesis investigations. As with the previously sequenced S. pyogenes genomes, three unique prophages are a major source of genetic diversity. Two clustered regularly interspaced short palindromic repeat (CRISPR) regions were present in the genome, providing genetic information on previous prophage encounters. A unique cluster of genes was found in the pathogenicity island-like emm region that included a novel Nudix hydrolase, and, further, this cluster appears to be specific for serotype M49 and M82 strains. Nudix hydrolases eliminate potentially hazardous materials or prevent the unbalanced accumulation of normal metabolites; in bacteria, these enzymes may play a role in host cell invasion. Since M49 S. pyogenes strains have been known to be associated with skin infections, the Nudix hydrolase and its associated genes may have a role in facilitating survival in an environment that is more variable and unpredictable than the uniform warmth and moisture of the throat. The genome of NZ131 continues to shed light upon the evolutionary history of this human pathogen. Apparent horizontal transfer of genetic material has led to the existence of highly variable virulence-associated regions that are marked by multiple rearrangements and genetic diversification while other regions, even those associated with virulence, vary little between genomes. The genome regions that encode surface gene products that will interact with host targets or aid in immune avoidance are the ones that display the most sequence diversity. Thus, while natural selection favors stability in much of the genome, it favors diversity in these regions.

CodY-affected transcriptional gene expression of Streptococcus pyogenes during growth in human blood.

In an attempt to expand the available knowledge of pathogen-host interactions during ex vivo growth of Streptococcus pyogenes (GAS) in nonimmune whole human blood, the extents to which the expression of 51 genes including regulators with known targets, established virulence factors, physiologically important transporters and metabolic enzyme genes was differentially affected in the presence or absence of a functional codY gene were determined. The results obtained by quantitative real-time PCR using the M49 strain NZ131 showed that CodY influenced GAS gene activity in a dynamic fashion, with differential responses detected for 26 genes and occasionally characterized by discordance in the blood environment compared to laboratory medium. Degenerate derivatives of the recently discovered CodY box potentially serving as a cis-regulatory element for CodY action were identified in the upstream regions of 15 genes of the NZ131 genome, and these genes featured sequence motifs identical to the NZ131 CodY box in all completely sequenced S. pyogenes genomes. As none of these genes represented a genuine virulence factor, it seems likely, therefore, that the observed differential transcription of the majority of virulence genes was caused by indirect actions of CodY as part of a regulatory network.

Linking the nutritional status of Streptococcus pyogenes to alteration of transcriptional gene expression: the action of CodY and RelA.

In this investigation, we identify the CodY protein from Streptococcus pyogenes as a pleiotropic transcription regulator with global features. The notion that acquisition of nutrients by this polyauxotrophic organism is the primary event occurring during the establishment of infection and that virulence expression is a result of this quest, led us to study the action of codY and relA genes on transcriptional gene expression under different nutritional conditions using complex and chemically defined media. Real-time reverse transcription PCR was used to determine the extent to which inactivation of codY and relA affects the mRNA levels of selected transcription factors, virulence genes, transporters, and genes encoding metabolic enzymes. The results show that CodY and RelA did not affect the expression of each other but that both exhibited strong negative autoregulatory properties. Genes negatively controlled by the relA-directed stringent response to amino acid starvation included, besides relA itself, transporters, metabolic enzymes, and at least two virulence genes (graB and speH). The expression of many genes of all four groups studied proved to be subject to direct or indirect control by CodY, often in a nutritional status-dependent fashion. One of the most important results implicates CodY in growth phase-dependent positive transcriptional regulation of pel/sagA and mga, loci that themselves positively affect the expression of numerous virulence factors. Increasing the cellular activity of nicotinamidase in both a codY mutant and wild-type background induced extensive transcriptional reprogramming, altering, among others, the growth phase-dependent transcription pattern of the genes for cysteine protease (speB) and several transporters. Inasmuch as CodY influenced the expression of other regulators (pel/sagA, mga, covRS, ropB, pyrR), its action is amplified and expands the complex regulatory network that governs gene expression in S. pyogenes.

Comparative genomics of streptococcal species.

Microbial genome sequencing has produced an unprecedented amount of new information and insights into an organism's metabolic activities, virulence properties, and evolution. The complete genome sequence has been reported for four different species of streptococci, including Streptococcus pyogenes, S. agalactiae, S. pneumoniae and S. mutans. Comparative genome analysis among organisms of the same species not only shows a high degree of similarity in gene content and organization, but also a high degree of sequence heterogeneity as evidenced by the large number of single nucleotide polymorphisms present. Considerable differences were also observed in the number of mobile genetic elements found in each organism, including complete and partial bacteriophage genomes, IS elements, transposons, and plasmids. S. pyogenes was the only species to contain complete bacteriophage genomes in its genome, while only S. pneumoniae and S. mutans contained the full complement of competence genes essential for natural transformation. Comparative genome analysis between the species showed that S. pyogenes was more closely related to S. agalactiae than with S. pneumoniae or S. mutans.

Construction of a GBS-GAS DNA subtraction library allows discovery of previously unidentified GBS genes and rapid location of unique regions on the GBS chromosome.

A subtraction library of group B streptococcus (GBS) strain O9OR with GAS chromosomal DNA (strain SF370) was constructed and more than 100 plasmid clones sequenced. DNA sequences of the plasmid inserts were analyzed using the BLAST gene search. Most inserts had little or no homology to GAS chromosomal DNA and 26 clones from the library had no gene homologues in the gene bank. The majority of genes discovered represented house keeping GBS genes, but several could be considered as possible virulence factors. Inserts from 21 clones were labeled and used as probes for hybridization with GBS DNA fragments separated by pulsed field electrophoresis. A genetic map of GBS strain O9OR was constructed.

Novel genomic rearrangement that affects expression of the Streptococcus pyogenes streptolysin O (slo) gene.

A RecA-independent chromosomal rearrangement in the upstream region of the streptolysin O (slo) gene of Streptococcus pyogenes which affects slo expression was identified. PCR analysis was used to demonstrate that this kind of rearrangement was found in several strains of different lineages. Chromosomal loci involved in the recombination were found to be 746 kb apart on the 1.85-Mb-long chromosome. The primary structure of the splicing region, the reproducibility of the rearrangement, and the fact that reconstructed recombinant molecules fused to erm and lacZ reporter genes affected their expression indicate that this event is not accidental but may play a role in the expression of the slo gene. In addition, the product of the recombining DNAs, including the splicing site, does not follow any example of a known recombination mechanism. The implications of this rearrangement for slo expression are discussed.

Genome analysis of an inducible prophage and prophage remnants integrated in the Streptococcus pyogenes strain SF370.

The mitomycin C inducible prophage SF370.1 from the highly pathogenic M1 serotype Streptococcus pyogenes isolate SF370 showed a 41-kb-long genome whose genetic organization resembled that of SF11-like pac-site Siphoviridae. Its closest relative was prophage NIH1.1 from an M3 serotype S. pyogenes strain, followed by S. pneumoniae phage MM1 and Lactobacillus phage phig1e, Listeria phage A118, and Bacillus phage SPP1 in a gradient of relatedness. Sequence similarity with the previously described prophages SF370.2 and SF370.3 from the same polylysogenic SF370 strain were mainly limited to the tail fiber genes. As in these two other prophages, SF370.1 encoded likely lysogenic conversion genes between the phage lysin and the right attachment site. The genes encoded the pyrogenic exotoxin C of S. pyogenes and a protein sharing sequence similarity with both DNases and mitogenic factors. The screening of the SF370 genome revealed further prophage-like elements. A 13-kb-long phage remnant SF370.4 encoded lysogeny and DNA replication genes. A closely related prophage remnant was identified in S. pyogenes strain Manfredo at a corresponding genome position. The two prophages differed by internal indels and gene replacements. Four phage-like integrases were detected; three were still accompanied by likely repressor genes. All prophage elements were integrated into coding sequences. The phage sequences complemented the coding sequences in all cases. The DNA repair genes mutL and mutS were separated by the prophage remnant SF370.4; prophage SF370.1 and S. pneumoniae phage MM1 integrated into homologous chromosomal locations. The prophage sequences were interpreted with a hypothesis that predicts elements of cooperation and an arms race between phage and host genomes.

Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen.

Streptococcus mutans is the leading cause of dental caries (tooth decay) worldwide and is considered to be the most cariogenic of all of the oral streptococci. The genome of S. mutans UA159, a serotype c strain, has been completely sequenced and is composed of 2,030,936 base pairs. It contains 1,963 ORFs, 63% of which have been assigned putative functions. The genome analysis provides further insight into how S. mutans has adapted to surviving the oral environment through resource acquisition, defense against host factors, and use of gene products that maintain its niche against microbial competitors. S. mutans metabolizes a wide variety of carbohydrates via nonoxidative pathways, and all of these pathways have been identified, along with the associated transport systems whose genes account for almost 15% of the genome. Virulence genes associated with extracellular adherent glucan production, adhesins, acid tolerance, proteases, and putative hemolysins have been identified. Strain UA159 is naturally competent and contains all of the genes essential for competence and quorum sensing. Mobile genetic elements in the form of IS elements and transposons are prominent in the genome and include a previously uncharacterized conjugative transposon and a composite transposon containing genes for the synthesis of antibiotics of the gramicidin/bacitracin family; however, no bacteriophage genomes are present.

Autonomous expression of the slo gene of the bicistronic nga-slo operon of Streptococcus pyogenes.

A recent model for cytolysin-mediated translocation in Streptococcus pyogenes proposes that NAD-glycohydrolase is translocated through streptolysin O-generated pores into a host cell (J. Madden, N. Ruiz, and M. Caparon, Cell 104:143-152, 2001). This model also assumes that the NAD-glycohydrolase (nga) and streptolysin O (slo) genes that code for these products are organized in an operon-like structure expressed from a single promoter only (nga). We expand this model by showing that slo possesses its own autonomous promoter, which is located 155 bp upstream of the slo gene. Under experimental conditions in which S. pyogenes is grown in THY medium, the strength of the slo promoter, as measured by the activity of a lacZ reporter gene, resulted in low but highly reproducible values. Finally, we demonstrated that sloR, a S. pyogenes gene that closely resembles the Clostridium perfringens pfoR gene, exerts a negative effect on the expression of the slo gene.