SummaryThe region of temperate bacteriophage T12 responsible for integration into the chromosome of Streptococcus pyogenes has been identified. The integrase gene (int ) and the phage attachment site (attP ) are found immediately upstream of the gene for speA, the latter of which is known to be responsible for the production of erythrogenic toxin A (also known as pyrogenic exotoxin A). The integrase gene has a coding capacity for a protein of 41 457 Da, and the Cterminus of the deduced protein is similar to other conserved C-terminal regions typical of phage integrases. Upstream of int is a second open reading frame, which is capable of encoding an acidic protein of 72 amino acids (8744 Da); the position of this region in relation to int suggests it to be the phage excisionase gene (xis). The arms flanking the integrated prophage (attL and attR ) were identified, allowing determination of the sequences of the phage (attP ) and bacterial (attB ) attachment sites. A fragment containing the integrase gene and attP was cloned into a streptococcal suicide vector; when introduced into S. pyogenes by electrotransformation, this plasmid stably integrated into the bacterial chromosome at attB. The insertion site for the phage into the S. pyogenes chromosome was found to be in the anticodon loop of a putative type II gene for a serine tRNA. attP and attB share a region of identity that is 96 bp in length; this region of identity corresponds to the 3Ј end of the tRNA gene such that the coding sequence remains intact after integration of the prophage. The symmetry of the core region of att may set this region apart from previously described phage attachment sites (Campbell, 1992), and may play a role in the biology of this medically important bacteriophage.
Streptococcus pyogenes Rgg is a regulatory protein that controls the transcription of 588 genes in strain NZ131 during the post-exponential phase of growth, including the virulence-associated genes encoding the extracellular SpeB protease, pullulanase A (PulA), and two extracellular nucleases (SdaB and Spd-3). Rgg binds to DNA proximally to the speB promoter (PspeB) to activate transcription; however, it is not known if Rgg binds to the promoters of other genes to influence expression, or if the perturbation of other global regulons accounts for the genome-wide changes in expression associated with the mutant. To address this issue, chromatin immunoprecipitation followed by DNA microarray analysis (ChIP-chip) was used to identify the DNA binding sites of Rgg. Rgg bound to 65 sites in the chromosome. Thirty-five were within noncoding DNA, and 43% of these were adjacent to genes previously identified as regulated by Rgg. Electrophoretic mobility shift assays were used to assess the binding of Rgg to a subset of sites bound in vivo, including the noncoding DNA upstream of speB, the genes encoding PulA, Spd-3, and a transcriptional regulator (SPY49_1113), and prophage-associated genes encoding a putative integrase (SPY49_0746) and a surface antigen (SPY49_0396). Rgg bound to all target DNAs in vitro, consistent with the in vivo results. Finally, analyses with a transcriptional reporter system showed that the DNA bound by Rgg contained an active promoter that was regulated by Rgg. Overall, the results indicate that Rgg binds specifically to multiple sites in the chromosome, including prophage DNA, to influence gene expression.
Lytic bacteriophage A25, which infects and several related species, has been used to better understand phage-microbe interactions due to its ability to mediate high-efficiency transduction. Most of these studies, however, are decades old and were conducted prior to the advent of next-generation sequencing and bioinformatics. The aim of our study was to gain a better understanding of the mechanism of high-efficiency transduction through analysis of the A25 genome. We show here that phage A25 is related to a family of genome prophages and became a lytic phage following escape from lysogeny. A lambdoid-like residual lysogeny module consisting of an operator site with two promoters and a-like antirepressor gene was identified, but the genes for the cI-like repressor and integrase are missing. Additionally, the genetic organization of the A25 genome was found to be modular in nature and similar to that of many prophages of as well as from other streptococcal species. A study of A25 homology to all annotated prophages within revealed near identity within the remnant lysogeny module of the A25 phage genome to the corresponding regions in resident prophages of genome strains MGAS10270 (M2), MGAS315 (M3), MGAS10570 (M4), and STAB902 (M4). Host range studies of MGAS10270, MGAS315, and MGAS10750 demonstrated that these strains were resistant to A25 infection. The resistance mechanism of superinfection immunity was confirmed experimentally through complementation of the operator region and -like repressor from prophage MGAS10270.2 into susceptible strains SF370, CEM1Δ4 (SF370ΔSpyCIM1), and ATCC 12204, which rendered all three strains resistant to A25 infection. prediction of packaging through homology analysis of the terminase large subunit from bacteriophages within the known packaging mechanism of Gram-positive bacteria as well as the evidence of terminally redundant and/or circularly permuted sequences suggested that A25 grouped with phages employing the less stringent -type packaging mechanisms, which likely explains the characteristic A25 high-efficiency transduction capabilities. Only a few examples of lytic phages appearing following loss of part or all of the lysogeny module have been reported previously, and the genetic mosaicism of A25 suggests that this event may not have been a recent one. However, the discovery that this lytic bacteriophage shares some of the genetic pool of prophages emphasizes the importance of genetic and biological characterization of bacteriophages when selecting phages for therapeutics or disinfectants, as phage-phage and phage-microbe interactions can be complex, requiring more than just assessment of host range and carriage of toxoid or virulence genes. Bacteriophages (bacterial viruses) play an important role in the shaping of bacterial populations as well as the dissemination of bacterial genetic material to new strains, resulting in the spread of virulence factors and antibiotic resistance genes. This study identified the genetic origins of phage A25 and uncovered the molecular mechan...
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