Oxygen-insensitive synthesis of the photosynthetic membranes of Rhodobacter sphaeroides: a mutant histidine kinase.

Two new loci, prrB and prrC, involved in the positive regulation of photosynthesis gene expression in response to anaerobiosis, have been identified in Rhodobacter sphaeroides. prrB encodes a sensor histidine kinase that is responsive to the removal of oxygen and functions through the response regulator PrrA. Inactivation of prrB results in a substantial reduction of photosynthetic spectral complexes as well as in the inability of cells to grow photosynthetically at low to medium light intensities. Together, prrB and prrA provide the major signal involved in synthesis of the specialized intracytoplasmic membrane (ICM), harboring components essential to the light reactions of photosynthesis. Previously, J. K. Lee and S. Kaplan (J. Bacteriol. 174:1158-1171, 1992) identified a mutant which resulted in high-level expression of the puc operon, encoding the apoproteins giving rise to the B800-850 spectral complex, in the presence of oxygen as well as in the synthesis of the ICM under conditions of high oxygenation. This mutation is shown to reside in prrB, resulting in a leucine-to-proline change at position 78 in mutant PrrB (PRRB78). Measurements of mRNA levels in cells containing the prrB78 mutation support the idea that prrB is a global regulator of photosynthesis gene expression. Two additional mutants, PRRB1 and PRRB2, which make two truncated forms of the PrrB protein, possess substantially reduced amounts of spectral complexes. Although the precise role of prrC remains to be determined, evidence suggests that it too is involved in the regulatory cascade involving prrB and prrA. The genetic organization of the photosynthesis response regulatory (PRR) region is discussed.     

prrA, a putative response regulator involved in oxygen regulation of photosynthesis gene expression in Rhodobacter sphaeroides.

A new locus, prrA, involved in the regulation of photosynthesis gene expression in response to oxygen, has been identified in Rhodobacter sphaeroides. Inactivation of prrA results in the absence of photosynthetic spectral complexes. The prrA gene product has strong homology to response regulators associated with signal transduction in other prokaryotes. When prrA is present in multiple copies, cells produce light-harvesting complexes under aerobic growth conditions, suggesting that prrA affects photosynthesis gene expression positively in response to oxygen deprivation. Analysis of the expression of puc::lacZ fusions in wild-type and PrrA- cells revealed a substantial decrease in LacZ expression in the absence of prrA under all conditions of growth, especially when cells were grown anaerobically in the dark in the presence of dimethyl sulfoxide. Northern (RNA) and slot blot hybridizations confirmed the beta-galactoside results for puc and revealed additional positive regulation of puf, puhA, and cycA by PrrA. The effect of truncated PrrA on photosynthesis gene expression in the presence of low oxygen levels can be explained by assuming that PrrA may be effective as a multimer. PrrA was found to act on the downstream regulatory sequences (J. K. Lee and S. Kaplan, J. Bacteriol. 174:1146-1157, 1992) of the puc operon regulatory region. Finally, two spontaneous prrA mutations that abolish prrA function by changing amino acids in the amino-terminal domain of the protein were isolated.     

A non-RD1 gene cluster is required for Snm secretion in Mycobacterium tuberculosis

The Snm secretion system is a crucial virulence determinant of Mycobacterium tuberculosis. Genes encoding all known components of this alternative secretion pathway are clustered at the same genetic locus, known as RD1. Here, we show that a mutant M. tuberculosis strain containing a transposon insertion in the Rv3615c gene, which is situated outside the RD1 locus, results in loss of Snm secretion. Complementation analysis revealed that both Rv3615c and the downstream gene Rv3614c are required for Snm secretion. Thus, we have renamed the two genes snm9 and snm10 respectively. The snm9::Tn mutant phenocopies bona fide snm mutants, exhibiting attenuation in mice, macrophage growth defects and failure to suppress cytokine induction. Furthermore, yeast two-hybrid analysis revealed a physical interaction between Snm10 and Snm7 (Rv3882c), suggesting that Snm10 may function in complex with other Snm proteins during secretion. Thus, snm9 and snm10 are the first genes located outside the RD1 locus identified as critical components of Snm secretion. These data indicate that Snm secretion consists of an elaborate network of interactions that likely arose from multiple duplication events during the evolution of M. tuberculosis.     

Large Mg(2+)-dependent currents are associated with the increased expression of ALR1 in Saccharomyces cerevisiae

Mycobacteria adapt to a decrease in oxygen tension by entry into a non-replicative persistent phase. It was shown earlier that the two-component system, DevR-DevS, was induced in Mycobacterium tuberculosis and Mycobacterium bovis BCG cultures during hypoxia, suggesting that it may play a regulatory role in their adaptation to oxygen limitation. The presence of a homologous genetic system in Mycobacterium smegmatis was predicted by scanning its unfinished genome sequence with devR and devS genes of M. tuberculosis. Rv3134c, which is cotranscribed with devR-devS in M. tuberculosis, was also present in M. smegmatis at a similar location upstream from devR. The expression of all three genes was induced at the RNA and protein levels in M. smegmatis cultures grown under microaerobic and anaerobic conditions. The M. smegmatis genome also contained the hspX gene, encoding chaperone alpha-crystallin, Acr, that was induced during hypoxia. The similarity in sequences and hypoxia-responsive behaviour of devR-devS, Rv3134c and hspX genes in M. smegmatis and M. tuberculosis suggests that the molecular mechanisms involved in the dormancy response are likely conserved in these two species. M. smegmatis could therefore serve as a useful model for the delineation of the hypoxia response in general and DevR-DevS regulated pathways in particular.     

Differential expression of the CO2 fixation operons of Rhodobacter sphaeroides by the Prr/Reg two-component system during chemoautotrophic growth

In Rhodobacter sphaeroides, the two cbb operons encoding duplicated Calvin-Benson Bassham (CBB) CO2 fixation reductive pentose phosphate cycle structural genes are differentially controlled. In attempts to define the molecular basis for the differential regulation, the effects of mutations in genes encoding a subunit of Cbb3 cytochrome oxidase, ccoP, and a global response regulator, prrA (regA), were characterized with respect to CO2 fixation (cbb) gene expression by using translational lac fusions to the R. sphaeroides cbb(I) and cbb(II) promoters. Inactivation of the ccoP gene resulted in derepression of both promoters during chemoheterotophic growth, where cbb expression is normally repressed; expression was also enhanced over normal levels during phototrophic growth. The prrA mutation effected reduced expression of cbb(I) and cbb(II) promoters during chemoheterotrophic growth, whereas intermediate levels of expression were observed in a double ccoP prrA mutant. PrrA and ccoP1 prrA strains cannot grow phototrophically, so it is impossible to examine cbb expression in these backgrounds under this growth mode. In this study, however, we found that PrrA mutants of R. sphaeroides were capable of chemoautotrophic growth, allowing, for the first time, an opportunity to directly examine the requirement of PrrA for cbb gene expression in vivo under growth conditions where the CBB cycle and CO2 fixation are required. Expression from the cbb(II) promoter was severely reduced in the PrrA mutants during chemoautotrophic growth, whereas cbb(I) expression was either unaffected or enhanced. Mutations in ccoQ had no effect on expression from either promoter. These observations suggest that the Prr signal transduction pathway is not always directly linked to Cbb3 cytochrome oxidase activity, at least with respect to cbb gene expression. In addition, lac fusions containing various lengths of the cbb(I) promoter demonstrated distinct sequences involved in positive regulation during photoautotrophic versus chemoautotrophic growth, suggesting that different regulatory proteins may be involved. In Rhodobacter capsulatus, ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) expression was not affected by cco mutations during photoheterotrophic growth, suggesting that differences exist in signal transduction pathways regulating cbb genes in the related organisms.     

A mycobacterial virulence gene cluster extending RD1 is required for cytolysis, bacterial spreading and ESAT-6 secretion

Initiation and maintenance of infection by mycobacteria in susceptible hosts are not well understood. A screen of Mycobacterium marinum transposon mutant library led to isolation of eight mutants that failed to cause haemolysis, all of which had transposon insertions in genes homologous to a region between Rv3866 and Rv3881c in Mycobacterium tuberculosis, which encompasses RD1 (Rv3871-Rv3879c), a known virulence gene cluster. The M. marinum mutants showed decreased virulence in vivo and failed to secrete ESAT-6, like M. tuberculosis RD1 mutants. M. marinum mutants in genes homologous to Rv3866-Rv3868 also failed to accumulate intracellular ESAT-6, suggesting a possible role for those genes in synthesis or stability of the protein. These transposon mutants and an ESAT-6/CFP-10 deletion mutant all showed reduced cytolysis and cytotoxicity to macrophages and significantly decreased intracellular growth at late stages of the infection only when the cells were infected at low multiplicity of infection, suggesting a defect in spreading. Direct evidence for cell-to-cell spread by wild-type M. marinum was obtained by microscopic detection in macrophage and epithelial monolayers, but the mutants all were defective in this assay. Expression of M. tuberculosis homologues complemented the corresponding M. marinum mutants, emphasizing the functional similarities between M. tuberculosis and M. marinum genes in this region that we designate extRD1 (extended RD1). We suggest that diminished membranolytic activity and defective spreading is a mechanism for the attenuation of the extRD1 mutants. These results extend recent findings on the genomic boundaries and functions of M. tuberculosis RD1 and establish a molecular cellular basis for the role that extRD1 plays in mycobacterial virulence. Disruption of the M. marinum homologue of Rv3881c, not previously implicated in virulence, led to a much more attenuated phenotype in macrophages and in vivo, suggesting that this gene plays additional roles in M. marinum survival in the host.