Proteome investigation of the global regulatory role of sigma 54 in response to gentisate induction in Pseudomonas alcaligenes NCIMB 9867

Pseudomonas alcaligenes NCIMB 9867 (strain P25X) utilizes the gentisate pathway for the degradation of aromatic hydrocarbons. The gene encoding the alternative sigma (sigma) factor sigma(54), rpoN, was cloned from strain P25X and a rpoN knock-out strain, designated G54, was constructed by insertional inactivation with a kanamycin resistance gene cassette. The role of sigma(54) in the physiological response of P. alcaligenes P25X to gentisate induction was assessed by comparing the global protein expression profiles of the wild-type P25X with the rpoN mutant strain G54. Analysis of two-dimensional polyacrylamide gel electrophoresis gels showed that 39 out of 355 prominent protein spots exhibited differential expression as a result of the insertional inactivation of rpoN. Identification of the protein spots by matrix-assisted laser desorption/ionization-time of flight/time of flight revealed a wide diversity of proteins that are affected by the sigma(54) mutation, the largest group being proteins that are involved in carbon metabolism. The strictly inducible gentisate 1,2-dioxygenase, one of two isofunctional copies of the key enzyme in the gentisate pathway, and enzymes of the TCA cycle, pyruvate metabolism and gluconeogenesis were part of this group. Other proteins that are part of the sigma(54) regulon include enzymes implicated in nitrogen metabolism, transport proteins, stress-response proteins and proteins involved in cell motility. The results of this study showed that sigma(54) plays a global regulatory role in the expression of a wide variety of genes in P. alcaligenes, including the wild-type response to the presence of the aromatic inducer, gentisate.     

Polyphosphate-Mediated Inhibition of Tartrate-Resistant Acid Phosphatase and Suppression of Bone Resorption of Osteoclasts

Inorganic polyphosphate (poly(P)) has recently been found to play an important role in bone formation. In this study, we found that tartrate-resistant acid phosphatase (TRAP), which is abundantly expressed in osteoclasts, has polyphosphatase activity that degrades poly(P) and yields Pi as well as shorter poly(P) chains. Since the TRAP protein that coprecipitated with anti-TRAP monoclonal antibodies exhibited both polyphosphatase and the original phosphatase activity, poly(P) degradation activity is dependent on TRAP and not on other contaminating enzymes. The ferrous chelator α, α’-bipyridyl, which inhibits the TRAP-mediated production of reactive oxygen species (ROS), had no effect on such poly(P) degradation, suggesting that the degradation is not dependent on ROS. In addition, shorter chain length poly(P) molecules were better substrates than longer chains for TRAP, and poly(P) inhibited the phosphatase activity of TRAP depending on its chain length. The IC50 of poly(P) against the original phosphatase activity of TRAP was 9.8 µM with an average chain length more than 300 phosphate residues, whereas the IC50 of poly(P) with a shorter average chain length of 15 phosphate residues was 8.3 mM. Finally, the pit formation activity of cultured rat osteoclasts differentiated by RANKL and M-CSF were markedly inhibited by poly(P), while no obvious decrease in cell number or differentiation efficiency was observed for poly(P). In particular, the inhibition of pit formation by long chain poly(P) with 300 phosphate residues was stronger than that of shorter chain poly(P). Thus, poly(P) may play an important regulatory role in osteoclastic bone resorption by inhibiting TRAP activity, which is dependent on its chain length.     

Analysis of the role of RsbV, RsbW, and RsbY in regulating {sigma}B activity in Bacillus cereus

The alternative sigma factor sigma(B) is an important regulator of the stress response of Bacillus cereus. Here, the role of the regulatory proteins RsbV, RsbW, and RsbY in regulating sigma(B) activity in B. cereus is analyzed. Functional characterization of RsbV and RsbW showed that they act as an anti-sigma factor antagonist and an anti-sigma factor, respectively. RsbW can also act as a kinase on RsbV. These data are in line with earlier functional characterizations of RsbV and RsbW homologs in B. subtilis. The rsbY gene is unique to B. cereus and its closest relatives and is predicted to encode a protein with an N-terminal CheY domain and a C-terminal PP2C domain. In an rsbY deletion mutant, the sigma(B) response upon stress exposure was almost completely abolished, but the response could be restored by complementation with full-length rsbY. Expression analysis showed that rsbY is transcribed from both a sigma(A)-dependent promoter and a sigma(B)-dependent promoter. The central role of RsbY in regulating the activity of sigma(B) indicates that in B. cereus, the sigma(B) activation pathway is markedly different from that in other gram-positive bacteria.     

degS (hhoB) is an essential Escherichia coli gene whose indispensable function is to provide σE activity

DegS (HhoB), a putative serine protease related to DegP/HtrA, regulates the basal and induced activity of the essential Escherichia coli sigma factor sigma (E), which is involved in the cellular response to extracytoplasmic stress. DegS promotes the destabilization of the sigma (E)-specific anti-sigma factor RseA, thereby releasing sigma (E) to direct gene expression. We demonstrate that degS is an essential E. coli gene and show that the essential function of DegS is to provide the cell with sigma (E) activity. We also show that the putative active site of DegS is periplasmic and that DegS requires its N-terminal transmembrane domain for its sigma (E)-related function.