Genomic Degeneration and Reduction in the Fish Pathogen Mycobacterium shottsii.

Mycobacterium shottsii is a dysgonic, nonpigmented mycobacterium originally isolated from diseased striped bass (Morone saxatilis) in the Chesapeake Bay, USA. Genomic analysis reveals that M. shottsii is a Mycobacterium ulcerans/Mycobacterium marinum clade (MuMC) member, but unlike the superficially similar M. pseudoshottsii, also isolated from striped bass, it is not an M. ulcerans ecovar, instead belonging to a transitional group of strains basal to proposed "Aronson" and "M" lineages. Although phylogenetically distinct from the human pathogen M. ulcerans, the M. shottsii genome shows parallel but nonhomologous genomic degeneration, including massive accumulation of pseudogenes accompanied by proliferation of unique insertion sequences (ISMysh01, ISMysh03), large-scale deletions, and genomic reorganization relative to typical M. marinum strains. Coupled with its observed ecological characteristics and loss of chromogenicity, the genomic structure of M. shottsii is suggestive of evolution toward a state of obligate pathogenicity, as observed for other Mycobacterium spp., including M. ulcerans, M. tuberculosis, and M. leprae. IMPORTANCE Morone saxatilis (striped bass) is an ecologically and economically important finfish species on the United States east coast. Mycobacterium shottsii and Mycobacterium pseudoshottsii were originally described in the early 2000s as novel species from outbreaks of visceral and dermal mycobacteriosis in this species. Biochemical and genetic characterization place these species within the Mycobacterium ulcerans/M. marinum clade (MuMC), and M. pseudoshottsii has been proposed as an ecovar of M. ulcerans. Here, we describe the complete genome of M. shottsii, demonstrating that it is clearly not an M. ulcerans ecovar; however, it has undergone parallel genomic modification suggestive of a transition to obligate pathogenicity. As in M. ulcerans, the M. shottsii genome demonstrates widespread pseudogene formation driven by proliferation of insertion sequences, as well as genomic reorganization. This work clarifies the phylogenetic position of M. shottsii relative to other MuMC members and provides insight into processes shaping its genomic structure.

Transcription properties of RNA polymerase holoenzymes isolated from the purple nonsulfur bacterium Rhodobacter sphaeroides.

We have been characterizing RNA polymerase holoenzymes from Rhodobacter sphaeroides. RNA polymerase purified from R. sphaeroides transcribed from promoters recognized by Escherichia coli E sigma 32 or E sigma 70 holoenzyme. Antisera to E. coli sigma 32 or sigma 70 indicated that related polypeptides of approximately 37 kDa (sigma 37) and 93 kDa (sigma 93), respectively, are present in this preparation. Transcription of sigma 32-dependent promoters was observed in a further fractionated R. sphaeroides holoenzyme containing the sigma 37 polypeptide, while a preparation enriched in sigma 93 transcribed sigma 70-dependent promoters. To demonstrate further that the sigma 93 polypeptide functions like E. coli sigma 70, we obtained an R. sphaeroides E sigma 93 holoenzyme capable of transcription from sigma 70-dependent promoters by combining sigma 93 with (i) an E sigma 37 fraction with diminished sigma 93 polypeptide content or (ii) E. coli core RNA polymerase. The generation of analogous DNase I footprints on the lacUV5 promoter by R. sphaeroides E sigma 93 and by E. coli E sigma 70 suggests that the overall structures of these two holoenzymes are similar. However, some differences in promoter specificity between R. sphaeroides E sigma 93 and E. coli E sigma 70 exist because transcription of an R. sphaeroides rRNA promoter was detected only with E sigma 93.

In vitro transcription of pathogenesis-related genes by purified RNA polymerase from Staphylococcus aureus.

The RNA polymerase (RNAP) holoenzyme of Staphylococcus aureus was purified by DNA affinity, gel filtration, and ion-exchange chromatography. This RNAP contained four major subunits with apparent molecular masses of 165, 130, 60, and 47 kDa. All four subunits of the RNAP were serologically related to the subunits of Escherichia coli E sigma 70 holoenzyme by Western immunoblot analysis. The 60-kDa subunit was subsequently isolated and found to react with a monoclonal antibody specific to the E. coli sigma 70 subunit. This sigma 70-related protein allowed E. coli core RNAP promoter-specific initiation and increased transcription by S. aureus RNAP that is unsaturated with sigma. We therefore suggest that this 60-kDa protein is a sigma factor. Purified S. aureus RNAP transcribed from the promoters of several important S. aureus virulence genes (sea, sec, hla, and agr P2) in vitro. The in vitro transcription start sites of the sea, sec, and agr P2 promoters, mapped by primer extension, were similar to those identified in vivo. The putative promoter hexamers of these three genes showed strong sequence similarity to the E. coli sigma 70 consensus promoter, and transcription by E sigma 70 from some of these promoters has been observed. Conversely, S. aureus RNAP does not transcribe from all E. coli sigma 70-dependent promoters. Taken together, our results indicate that the promoter sequences recognized by purified S. aureus RNAP are similar but not identical to those recognized by E. coli E sigma 70.

Activation of the cycA P2 promoter for the Rhodobacter sphaeroides cytochrome c2 gene by the photosynthesis response regulator.

The Rhodobacter sphaeroides photosynthesis response regulator, PrrA, positively regulates cycA P2 expression. Deletion analysis has identified sequences within 73 bp upstream of the transcription initiation site that are required for the activation of cycA P2 by PrrA. A mutant form of the Rhodobacter capsulatus PrrA homologue, whose activity is independent of phosphorylation (RegA*), protects an approximately 26 bp region of cycA P2 that is centred at approximately -50 from DNase digestion, and activates transcription of a mutant -14T promoter with increased activity when using either R. sphaeroides RNA polymerase or Escherichia coli Esigma70. A 4 bp target site mutation that eliminated DNA binding and transcription activation by RegA* in vitro also abolished PrrA activation of cycA P2 transcription in vivo, indicating that this region contains a PrrA binding site. By analysing the behaviour of the -14T mutant cycA P2 promoter in vivo, we also found that PrrA uses the same target site to activate expression in both the presence and the absence of O2. However, the extent of transcription activation by PrrA at cycA P2 in vivo is greater under anaerobic conditions.

Transcription of the Rhodobacter sphaeroides cycA P1 promoter by alternate RNA polymerase holoenzymes.

These experiments sought to identify what form of RNA polymerase transcribes the P1 promoter for the Rhodobacter sphaeroides cytochrome c2 gene (cycA). In vitro, cycA P1 was recognized by an RNA polymerase holoenzyme fraction that transcribes several well-characterized Escherichia coli heat shock (sigma32) promoters. The in vivo effects of mutations flanking the transcription initiation site (+1) also suggested that cycA P1 was recognized by an RNA polymerase similar to E. coli Esigma32. Function of cycA P1 was not altered by mutations more than 35 bp upstream of position +1 or by alterations downstream of -7. A point mutation at position -34 that is towards the E. coli Esigma32 -35 consensus sequence (G34T) increased cycA P1 activity approximately 20-fold, while several mutations that reduced or abolished promoter function changed highly conserved bases in presumed -10 or -35 elements. In addition, cycA P1 function was retained in mutant promoters with a spacer region as short as 14 nucleotides. When either wild-type or G34T promoters were incubated with reconstituted RNA polymerase holoenzymes, cycA P1 transcription was observed only with samples containing either a 37-kDa subunit that is a member of the heat shock sigma factor family (Esigma37) or a 38-kDa subunit that also allows core RNA polymerase to recognize E. coli heat shock promoters (Esigma38). (R. K. Karls, J. Brooks, P. Rossmeissl, J. Luedke, and T. J. Donohue, J. Bacteriol. 180:10-19, 1998).

Metabolic roles of a Rhodobacter sphaeroides member of the sigma32 family.

We report the role of a gene (rpoH) from the facultative phototroph Rhodobacter sphaeroides that encodes a protein (sigma37) similar to Escherichia coli sigma32 and other members of the heat shock family of eubacterial sigma factors. R. sphaeroides sigma37 controls genes that function during environmental stress, since an R. sphaeroides deltaRpoH mutant is approximately 30-fold more sensitive to the toxic oxyanion tellurite than wild-type cells. However, the deltaRpoH mutant lacks several phenotypes characteristic of E. coli cells lacking sigma32. For example, an R. sphaeroides deltaRpoH mutant is not generally defective in phage morphogenesis, since it plates the lytic virus RS1, as well as its wild-type parent. In characterizing the response of R. sphaeroides to heat, we found that its growth temperature profile is different when cells generate energy by aerobic respiration, anaerobic respiration, or photosynthesis. However, growth of the deltaRpoH mutant is comparable to that of a wild-type strain under each of these conditions. The deltaRpoH mutant mounted a heat shock response when aerobically grown cells were shifted from 30 to 42 degrees C, but it exhibited altered induction kinetics of approximately 120-, 85-, 75-, and 65-kDa proteins. There was also reduced accumulation of several presumed heat shock transcripts (rpoD P(HS), groESL1, etc.) when aerobically grown deltaRpoH cells were placed at 42 degrees C. Under aerobic conditions, it appears that another sigma factor enables the deltaRpoH mutant to mount a heat shock response, since either RNA polymerase preparations from an deltaRpoH mutant, reconstituted Esigma37, or a holoenzyme containing a 38-kDa protein (sigma38) each transcribed E. coli Esigma32-dependent promoters. The lower growth temperature profile of photosynthetic cells is correlated with a difference in heat-inducible gene expression, since neither wild-type cells or the deltaRpoH mutant mount a typical heat shock response after such cultures were shifted from 30 to 37 degrees C.