Genome Sequences of Bacteria Isolated from the International Space Station Water Systems.

We report draft genomes of five bacteria recovered from the U.S. and Russian water systems onboard the International Space Station. The five genera include Ralstonia, Burkholderia, Cupriavidus, Methylobacterium, and Pseudomonas. These sequences will help further the understanding of water reclamation and environmental control and life support systems in space.

The catabolite control protein CcpA binds to Pmga and influences expression of the virulence regulator Mga in the Group A streptococcus.

Carbon catabolite repression (CCR) allows bacteria to alter metabolism in response to the availability of specific sugar sources, and increasing evidence suggests that CCR is involved in regulating virulence gene expression in many pathogens. A scan of the M1 SF370 group A streptococcus (GAS) genome using a Bacillus subtilis consensus identified a number of potential catabolite-responsive elements (cre) important for binding by the catabolite control protein A (CcpA), a mediator of CCR in gram-positive bacteria. Intriguingly, a putative cre was identified in the promoter region of mga upstream of its distal P1 start of transcription. Electrophoretic mobility shift assays showed that a His-CcpA fusion protein was capable of binding specifically to the cre in Pmga in vitro. Deletion analysis of Pmga using single-copy Pmga-gusA reporter strains found that Pmga P1 and its upstream cre were not required for normal autoregulated mga expression from Pmga P2 as long as Mga was produced from its native locus. In fact, the Pmga P1 region appeared to show a negative influence on Pmga P2 in these studies. However, deletion of the cre at the native Pmga resulted in a reduction of total mga transcripts as determined by real-time reverse transcription-PCR, supporting a role for CcpA in initial expression. Furthermore, normal transcriptional initiation from the Pmga P1 start site alone was dependent on the presence of the cre. Importantly, inactivation of ccpA in the M6 GAS strain JRS4 resulted in a reduction in Pmga expression and Mga protein levels in late-logarithmic-phase cell growth. These data support a role for CcpA in the early activation of the mga promoter and establish a link between CCR and Mga regulation in the GAS.

Mga is sufficient to activate transcription in vitro of sof-sfbX and other Mga-regulated virulence genes in the group A Streptococcus.

The group A streptococcus (GAS), or Streptococcus pyogenes, is a strict human pathogen of medical significance, causing infections ranging from pharyngitis (strep throat) to necrotizing fasciitis (flesh-eating disease). Several virulence genes that encode factors important for colonization, internalization, and immune evasion are under the control of the multiple gene regulator of the GAS, or Mga. Mga functions as a DNA-binding protein that interacts with sites both proximal (Pemm and PscpA) and distal (PsclA) to the start of transcription for the genes that it regulates. The genes encoding serum opacity factor, sof, and a novel fibronectin-binding protein, sfbX, are cotranscribed and represent two uncharacterized Mga-regulated virulence genes in the GAS. Analysis of the promoter region of sof-sfbX identified a putative Mga-binding site 278 bp upstream of the regulated start of transcription as determined by primer extension. Electrophoretic mobility shift assays demonstrated that Mga is able to bind specifically to the single distal site in a fashion similar to the previously characterized PsclA. In order to better understand the events that take place at this and other Mga-regulated promoters, an in vitro transcription assay was established. Using this assay, we showed that Mga is sufficient to activate transcription in vitro for Mga-regulated promoters containing both proximal (Pemm) and distal (PsclA and Psof-sfbX) binding sites. These results indicate that additional factors are not required for Mga-specific activation at diverse promoters in vitro, although they do not rule out the potential influence of other components on the Mga virulence regulon in vivo.

Transcriptional activation of sclA by Mga requires a distal binding site in Streptococcus pyogenes.

Streptococcus pyogenes (the group A streptococcus [GAS]) is a medically significant pathogen of humans, causing a range of diseases from pharyngitis to necrotizing fasciitis. Several important GAS virulence genes are under the control of a pleiotropic regulator called Mga, or the multiple gene regulator of GAS, including the gene encoding the streptococcal collagen-like protein, or sclA. Analysis of the genome sequence upstream of sclA revealed two potential Mga-binding sites with homology to the published Mga-binding element, which were called PsclA-I (distal) and PsclA-II (proximal) based on their location relative to a predicted start of transcription. Primer extension was used to confirm that the Mga-dependent transcriptional start site for sclA was located adjacent to the proximal PsclA-II binding site. By using overlapping PsclA promoter probes and purified Mga-His fusion protein, it was shown by electrophoretic mobility shift assays that, unlike other Mga-regulated promoters, Mga binds only to a distal DNA-binding site (PsclA-I). Binding of Mga to PsclA-I could be competed with cold probes corresponding to known Mga-regulated promoters (Pemm, PscpA, and Pmga) but not with a nonspecific probe or the proximal PsclA-II fragment. With the use of a plasmid-based green fluorescent protein transcriptional reporter system, the full-length PsclA was not sufficient to reproduce normal Mga-regulated activation. However, studies using a single-copy gusA transcriptional reporter system integrated at the native sclA chromosomal locus clearly demonstrated that the distal PsclA-I binding site is required for Mga regulation. Therefore, PsclA represents a new class of Mga-regulated promoters that requires a single distal binding site for activation.