Heat shock regulation in the ftsH null mutant of Escherichia coli: dissection of stability and activity control mechanisms of sigma32 in vivo

The heat shock response of Escherichia coli is regulated by the cellular level and the activity of sigma32, an alternative sigma factor for heat shock promoters. FtsH, a membrane-bound AAA-type metalloprotease, degrades sigma32 and has a central role in the control of the sigma32 level. The ftsH null mutant was isolated, and establishment of the DeltaftsH mutant allowed us to investigate control mechanisms of the stability and the activity of sigma32 separately in vivo. Loss of the FtsH function caused marked stabilization and consequent accumulation of sigma32 ( approximately 20-fold of the wild type), leading to the impaired downregulation of the level of sigma32. Surprisingly, however, DeltaftsH cells express heat shock proteins only two- to threefold higher than wild-type cells, and they also show almost normal heat shock response upon temperature upshift. These results indicate the presence of a control mechanism that downregulates the activity of sigma32 when it is accumulated. Overproduction of DnaK/J reduces the activity of sigma32 in DeltaftsH cells without any detectable changes in the level of sigma32, indicating that the DnaK chaperone system is responsible for the activity control of sigma32 in vivo. In addition, CbpA, an analogue of DnaJ, was demonstrated to have overlapping functions with DnaJ in both the activity and the stability control of sigma32.     

Lambda Xis degradation in vivo by Lon and FtsH

Lambda Xis, which is required for site-specific excision of phage lambda from the bacterial chromosome, has a much shorter functional half-life than Int, which is required for both integration and excision (R. A. Weisberg and M. E. Gottesman, p. 489-500, in A. D. Hershey, ed., The Bacteriophage Lambda, 1971). We found that Xis is degraded in vivo by two ATP-dependent proteases, Lon and FtsH (HflB). Xis was stabilized two- to threefold more than in the wild type in a lon mutant and as much as sixfold more in a lon ftsH double mutant at the nonpermissive temperature for the ftsH mutation. Integration of lambda into the bacterial chromosome was delayed in the lon ftsH background, suggesting that accumulation of Xis in vivo interferes with integration. Overexpression of Xis in wild-type cells from a multicopy plasmid inhibited integration of lambda and promoted curing of established lysogens, confirming that accumulation of Xis interferes with the ability of Int to establish and maintain an integrated prophage.     

Host regulation of lysogenic decision in bacteriophage lambda: transmembrane modulation of FtsH (HflB), the cII degrading protease, by HflKC (HflA)

The cII gene product of bacteriophage lambda is unstable and required for the establishment of lysogenization. Its intracellular amount is important for the decision between lytic growth and lysogenization. Two genetic loci of Escherichia coli are crucial for these commitments of infecting lambda genome. One of them, hflA encodes the HflKC membrane protein complex, which has been believed to be a protease degrading the cII protein. However, both its absence and overproduction stabilized cII in vivo and the proposed serine protease-like sequence motif in HflC was dispensable for the lysogenization control. Moreover, the HflKC protein was found to reside on the periplasmic side of the plasma membrane. In contrast, the other host gene, ftsH (hflB) encoding an integral membrane ATPase/protease, is positively required for degradation of cII, since loss of its function stabilized cII and its overexpression accelerated the cII degradation. In vitro, purified FtsH catalyzed ATP-dependent proteolysis of cII and HflKC antagonized the FtsH action. These results, together with our previous finding that FtsH and HflKC form a complex, suggest that FtsH is the cII degrading protease and HflKC is a modulator of the FtsH function. We propose that this transmembrane modulation differentiates the FtsH actions to different substrate proteins such as the membrane-bound SecY protein and the cytosolic cII protein. This study necessitates a revision of the prevailing view about the host control over lambda lysogenic decision.     

The RNA-binding protein HF-I, known as a host factor for phage Qbeta RNA replication, is essential for rpoS translation in Escherichia coli

The rpoS-encoded sigma(S) subunit of RNA polymerase in Escherichia coli is a global regulatory factor involved in several stress responses. Mainly because of increased rpoS translation and stabilization of sigma(S), which in nonstressed cells is a highly unstable protein, the cellular sigma(S) content increases during entry into stationary phase and in response to hyperosmolarity. Here, we identify the hfq-encoded RNA-binding protein HF-I, which has been known previously only as a host factor for the replication of phage Qbeta RNA, as an essential factor for rpoS translation. An hfq null mutant exhibits strongly reduced sigma(S) levels under all conditions tested and is deficient for growth phase-related and osmotic induction of sigma(S). Using a combination of gene fusion analysis and pulse-chase experiments, we demonstrate that the hfq mutant is specifically impaired in rpoS translation. We also present evidence that the H-NS protein, which has been shown to affect rpoS translation, acts in the same regulatory pathway as HF-I at a position upstream of HF-I or in conjunction with HF-I. In addition, we show that expression and heat induction of the heat shock sigma factor sigma(32) (encoded by rpoH) is not dependent on HF-I, although rpoH and rpoS are both subject to translational regulation probably mediated by changes in mRNA secondary structure. HF-I is the first factor known to be specifically involved in rpoS translation, and this role is the first cellular function to be identified for this abundant ribosome-associated RNA-binding protein in E. coli.     

Casein kinase 2 phosphorylation of recombinant rat osteopontin enhances adhesion of osteoclasts but not osteoblasts

We have analysed the function of a gene of Bacillus subtilis, the product of which shows significant homology with eukaryotic SMC proteins essential for chromosome condensation and segregation. Two mutant strains were constructed; in one, the expression was under the control of the inducible spac promoter (conditional null) and, in the other, the gene was disrupted by insertion (disrupted null). Both could form colonies at 23 degree C but not at 37 degree C in the absence of the expression of the Smc protein, indicating that the B. subtilis smc gene was essential for cell growth at higher temperatures. Microscopic examination revealed the formation of anucleate and elongated cells and diffusion of nucleoids within the elongated cells in the disrupted null mutant grown at 23 degree C and in the conditional null mutant grown in low concentrations of IPTG at 37 degree C. In addition, immunofluorescence microscopy showed that subcellular localization of the SpoOJ partition protein was irregular in the smc disrupted null mutant, compared with bipolar localization in wild-type cells. These results indicate that the B. subtilis smc gene is essential for chromosome partition. The role of B. subtilis Smc protein in chromosome partition is discussed.     

Bacillus subtilis RNase III gene: cloning, function of the gene in Escherichia coli, and construction of Bacillus subtilis strains with altered rnc loci

The rnc gene of Bacillus subtilis, which has 36% amino acid identity with the gene that encodes Escherichia coli RNase III endonuclease, was cloned in E. coli and shown by functional assays to encode B. subtilis RNase III (Bs-RNase III). The cloned B. subtilis rnc gene could complement an E. coli rnc strain that is deficient in rRNA processing, suggesting that Bs-RNase III is involved in rRNA processing in B. subtilis. Attempts to construct a B. subtilis rnc null mutant were unsuccessful, but a strain was constructed in which only a carboxy-terminal truncated version of Bs-RNase III was expressed. The truncated Bs-RNase III showed virtually no activity in vitro but was active in vivo. Analysis of expression of a copy of the rnc gene integrated at the amy locus and transcribed from a p(spac) promoter suggested that expression of the B. subtilis rnc is under regulatory control.     

Expression of a phosphorylation-resistant eukaryotic initiation factor 2 alpha-subunit mitigates heat shock inhibition of protein synthesis

Protein synthesis is dramatically reduced upon exposure of cells to elevated temperature. Concordant with this inhibition, multiple phosphorylation and dephosphorylation reactions occur on specific eukaryotic initiation factors that are required for protein synthesis. Most notably, phosphorylation of the alpha-subunit of eukaryotic initiation factor-2 (eIF-2 alpha) on serine residue 51 occurs. To identify the importance of phosphorylation in control of protein synthesis, we have evaluated the effects of expression of a mutant eIF-2 alpha which is resistant to phosphorylation. Expression of a serine to alanine mutant at residue 51 of eIF-2 alpha partially protected cells from the inhibition of protein synthesis in response to heat treatment. The overexpressed serine to alanine 51 mutant subunit was incorporated into the eIF-2 heterotrimer and was resistant to phosphorylation. These results are consistent with the hypothesis that heat shock inhibition of translation is mediated in part through phosphorylation of eIF-2 alpha. Expression of the wild type or mutant eIF-2 alpha did not affect cell survival or induction of hsp70 mRNA upon heat shock, indicating that although eIF-2 alpha is a heat shock-induced protein, its increased synthesis during heat shock does not alter the heat-shock response.     

Unique regulation of carbohydrate chemotaxis in Bacillus subtilis by the phosphoenolpyruvate-dependent phosphotransferase system and the methyl-accepting chemotaxis protein McpC

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) plays a major role in the ability of Escherichia coli to migrate toward PTS carbohydrates. The present study establishes that chemotaxis toward PTS substrates in Bacillus subtilis is mediated by the PTS as well as by a methyl-accepting chemotaxis protein (MCP). As for E. coli, a B. subtilis ptsH null mutant is severely deficient in chemotaxis toward most PTS carbohydrates. Tethering analysis revealed that this mutant does respond normally to the stepwise addition of a PTS substrate (positive stimulus) but fails to respond normally to the stepwise removal of such a substrate (negative stimulus). An mcpC null mutant showed no response to the stepwise addition or removal of D-glucose or D-mannitol, both of which are PTS substrates. Therefore, in contrast to E. coli PTS carbohydrate chemotaxis, B. subtilis PTS carbohydrate chemotaxis is mediated by both MCPs and the PTS; the response to positive stimulus is primarily McpC mediated, while the duration or magnitude of the response to negative PTS carbohydrate stimulus is greatly influenced by components of the PTS and McpC. In the case of the PTS substrate D-glucose, the response to negative stimulus is also partially mediated by McpA. Finally, we show that B. subtilis EnzymeI-P has the ability to inhibit B. subtilis CheA autophosphorylation in vitro. We hypothesize that chemotaxis in the spatial gradient of the capillary assay may result from a combination of a transient increase in the intracellular concentration of EnzymeI-P and a decrease in the concentration of carbohydrate-associated McpC as the cell moves down the carbohydrate concentration gradient. Both events appear to contribute to inhibition of CheA activity that increases the tendency of the bacteria to tumble. In the case of D-glucose, a decrease in D-glucose-associated McpA may also contribute to the inhibition of CheA. This bias on the otherwise random walk allows net migration, or chemotaxis, to occur.     

Molecular cloning and nucleotide sequence of the superoxide dismutase gene and characterization of its product from Bacillus subtilis

Bacillus subtilis was found to possess one detectable superoxide dismutase (Sod) in both vegetative cells and spores. The Sod activity in vegetative cells was maximal at stationary phase. Manganese was necessary to sustain Sod activity at stationary phase, but paraquat, a superoxide generator, did not induce the expression of Sod. The specific activity of purified Sod was approximately 2, 600 U/mg of protein, and the enzyme was a homodimer protein with a molecular mass of approximately 25,000 per monomer. The gene encoding Sod, designated sodA, was cloned by the combination of several PCR methods and the Southern hybridization method. DNA sequence analysis revealed the presence of one open reading frame consisting of 606 bp. Several putative promoter sites were located in the upstream region of sodA. The deduced amino acid sequence showed high homology with other bacterial manganese Sods. Conserved regions in bacterial manganese Sod could also be seen. The phenotype of double mutant Escherichia coli sodA sodB, which could not grow in minimal medium without supplemental amino acids, was complemented by the expression of B. subtilis sodA.     

A response regulatory protein with the site of phosphorylation blocked by an arginine interaction: crystal structure of Spo0F from Bacillus subtilis

Spo0F is a secondary messenger in the "two-component" system controlling the sporulation of Bacillus subtilis. Spo0F, like the chemotaxis protein CheY, is a single-domain protein homologous to the N-terminal activator domain of the response regulators. We recently reported the crystal structure of a phosphatase-resistant mutant Y13S of Spo0F with Ca2+ bound in the active site. The crystal structure of wild-type Spo0F in the absence of a metal ion is presented here. A comparison of the two structures reveals that the cation induces significant changes in the active site. In the present wild-type structure, the carboxylate of Asp11 points away from the center of the active site, whereas when coordinated to the Ca2+, as in the earlier structure, it points toward the active site. In addition, Asp54, the site of phosphorylation, is blocked by a salt bridge interaction of an Arg side chain from a neighboring molecule. From fluorescence quenching studies with Spo0F Y13W, we found that only the amino acid Arg binds to Spo0F in a saturable manner (Kd = 15 mM). This observation suggests that a small molecule with a shape complementary to the active site and having a guanidinium group might inhibit phosphotransfer between response regulators and their cognate histidine kinases.     

"Repty" scales of strokes. Functional index "Repty" for the evaluation of ADL in hemiplegic patients after cerebral stroke. Part II

The Escherichia coli FtsH protein is a membrane-bound and ATP-dependent protease. In this study, we describe ATP-dependent conformational changes in FtsH as well as a polypeptide binding ability of this protein. A 33 kDa segment of FtsH became trypsin resistant in the presence of ATP. ATP and ATPgammaS prevented self-aggregation of detergent-solubilized FtsH-His6-Myc at 37 degrees C, again suggesting that the binding of ATP induces a conformational change in FtsH. Affinity chromatography showed that FtsH-His6-Myc can associate with denatured alkaline phosphatase (PhoA) but not with the native enzyme. Denatured PhoA also prevented the aggregation of FtsH, and these two proteins co-sedimented through a sucrose gradient. Binding between FtsH-His6-Myc and detergent-solubilized SecY was also demonstrated. Although FtsH-bound SecY was processed further for ATP-dependent proteolysis, FtsH-bound PhoA was not. Thus, FtsH association with denatured PhoA is uncoupled from proteolysis. Overproduction of FtsH significantly increased the cytoplasmic localization of the PhoA moiety of a MalF-PhoA hybrid protein, in which a charged residue had been introduced into a transmembrane segment. Thus, denatured PhoA binding of FtsH may also occur in vivo.     

RpoS-dependent expression of the second lysine decarboxylase gene in Escherichia coli

The second lysine decarboxylase gene (ldc) is at 4.7 min on the Escherichia coli chromosome [Kikuchi et al., J. Baceriol. 179, 4486-4492 (1997)]. This report showes that the expression of ldc as well as cadA was induced at stationary phase in the wild type of E. coli. The ldc was not expressed in a rpoS deletion mutant of E. coli at any growing stage. In contrast, cadA was expressed in the rpoS mutant. Thus, we conclude that the expression of ldc but not cadA at stationary phase is regulated by a RpoS-dependent mechanism (s) in E. coli.     

A novel serine/threonine protein kinase homologue of Pseudomonas aeruginosa is specifically inducible within the host infection site and is required for full virulence in neutropenic mice

A genetic locus of Pseudomonas aeruginosa was identified that is highly and specifically inducible during infection of neutropenic mice. This locus, ppkA, encodes a protein that is highly homologous to eukaryote-type serine/threonine protein kinases. A ppkA null mutant strain shows reduced virulence in neutropenic mice compared to the wild type. Overexpression of the PpkA protein greatly inhibited the growth of Escherichia coli or P. aeruginosa. However, a single amino acid change at the catalytic site of the kinase domain eliminated the toxic effect of PpkA on bacterial cells, suggesting that the kinase domain of PpkA is functional within bacterial cells.