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The flagellar cytoplasmic ring (C ring), which consists of three proteins, FliG, FliM, and FliN, is located on the cytoplasmic side of the flagellum. The C ring is a multifunctional structure necessary for flagellar protein secretion, torque generation, and switching of the rotational direction of the motor. The deletion of any one of the fliG, fliM, and fliN genes results in a [Fla.sup.-] phenotype. Here, we show that the overproduction of the flagellum-specific ATPase FliI overcomes the inability of basal bodies with partial C-ring structures to produce complete flagella. Flagella made upon FliI overproduction were paralyzed, indicating that an intact C ring is essential for motor function. In FliN-or FliM-deficient mutants, flagellum production was about 10% of the wild-type level, while it was only a few percent in FliG-deficient mutants, suggesting that the size of partial C rings affects the extent of flagellation. For flagella made in C-ring mutants, the hook length varied considerably, with many being markedly shorter or longer than that of the wild type. The broad distribution of hook lengths suggests that defective C rings cannot control the hook length as tightly as the wild type even though FliK and FlhB are both intact. doi: 10.1128/JB.00601-09

We constructed a Bacillus subtilis Marburg strain that harbors deletion mutations in all seven extracytoplasmic function (ECF) sigma genes. The strain shows wild-type growth at 37[degrees]C both in a complex and in a synthetic medium and exhibits wild-type sporulation. ECF sigma genes of B. subtilis are dispensable as long as no stress is imposed, although they seem to be required for quick response to stresses.

Penicillin-binding protein 1B (PBP1B) of Escherichia coil is a bifunctional murein synthase containing both a transpeptidase domain and a transglycosylase domain. The protein is present in three forms ([alpha], [beta], and [gamma]) which differ in the length of their N-terminal cytoplasmic region. Expression piilasmids allowing the production of native PBP1B or of PBP1B variants with an inactive transpeptidase or transglycosylase domain or both were constructed. The inactive domains contained a single amino acid exchange in an essential active-site residue. Overproduction of the inactive PBP1B variants, but not of the active proteins, caused lysis of wild-type cells. The cells became tolerant to lysis by inactive PBP1B at a pH of 5.0, which is similar to the known tolerance for penicillin-induced lysis under acid pH conditions. Lysis was also reduced in mutant strains lacking several murein hydrolases. In particular, a strain devoid of activity of all known lytic transglycosylases was virtually tolerant, indicating that mainly the lyric transglycosylases are responsible for the observed lysis effect. A possible structural interaction between PBP1B and murein hydrolases in vivo by the formation of a muitienzyme complex is discussed.

The Bacillus subtilis extracytoplasmic function sigma factor [[sigma].sup.Y] is of unknown function. We demonstrate that the sigY operon is expressed from an autoregulatory promoter site, [P.sub.Y]. We selected for transposon-induced mutations that upregulate [P.sub.Y] transcription in an attempt to identify genes involved in [sigma].sup.Y] regulation. The resulting insertions disrupted yxlC, the gene immediately downstream of sigY. However, the phenotype of the yxlC::Tn10 insertion was due to polarity on the downstream genes of the sigY operon; a nonpolar insertion in yxlC did not lead to derepression of [P.sub.y] Further analyses revealed that both yxlD and yxlE encoded proteins important for the negative regulation of [[sigma].sup.Y] activity. A comparison of the transcriptomes of wild-type and yxlC::Tn10 mutant strains revealed elevated expression of several operons. However, only one additional gene, ybgB, was unambiguously identified as a direct target for [[sigma].sup.y] This was supported by analysis of direct targets for [[sigma].sup.Y] transcription with whole-genome runoff transcription followed by macroarray analysis.

The HAMP linker, a predicted structural element observed in many sensor kinases and methyl-accepting chemotaxis proteins, transmits signals between sensory input modules and output modules. HAMP linkers are located immediately inside the cytoplasmic membrane and are predicted to form two short amphipathic [alpha]-helices (AS-1 and AS-2) joined by an unstructured connector. HAMP linkers are found in the Escherichia coli nitrate- and nitrite-responsive sensor kinases NarX and NarQ (which respond to ligand by increasing kinase activity) and the sensor kinase CpxA (which responds to ligand by decreasing kinase activity). We constructed a series of hybrids with fusion points throughout the HAMP linker, in which the sensory modules of NarX or NarQ are fused to the transmitter modules of NarX, NarQ, or CpxA. A hybrid of the NarX sensor module and the CpxA HAMP linker and transmitter module (NarX-CpxA. 1) responded to nitrate by decreasing kinase activity, whereas a hybrid in which the HAMP linker of NarX was replaced by that of CpxA (NarX-CpxA-NarX-1) responded to nitrate by increasing kinase activity. However, sequence variations between HAMP linkers do not allow free exchange of HAMP linkers or their components. Certain deletions in the NarX HAMP linker resulted in characteristic abnormal responses to ligand; similar deletions in the NarQ and NarX-CpxA-1 HAMP linkers resulted in responses to ligand generally similar to those seen in NarX. We conclude that the structure and action of the HAMP linker are conserved and that the HAMP linker transmits a signal to the output domain that ligand is bound.

Proteins of the Tol-Pal (Tol-OprL) system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria. Here we describe an additional role for this system in the transport of various carbon sources across the cytoplasmic membrane. Growth of Pseudomonas putida tol-oprL mutant strains in minimal medium with glycerol, fructose, or arginine was impaired, and the growth rate with succinate, proline, or sucrose as the carbon source was lower than the growth rate of the parental strain. Assays with radiolabeled substrates revealed that the rates of uptake of these compounds by mutant cells were lower than the rates of uptake by the wild-type strain. The pattern and amount of outer membrane protein in the P. putida tol-oprL mutants were not changed, suggesting that the transport defect was not in the outer membrane. Consistently, the uptake of radiolabeled glucose and glycerol in spheroplasts was defective in the P. putida tol-oprL mutant strains, suggesting that there was a defect at the cytoplasmic membrane level. Generation of a proton motive force appeared to be unaffected in these mutants. To rule out the possibility that the uptake defect was due to a lack of specific transporter proteins, the PutP symporter was overproduced, but this overproduction did not enhance proline uptake in the tol-oprL mutants. These results suggest that the Tol-OprL system is necessary for appropriate functioning of certain uptake systems at the level of the cytoplasmic membrane.

Cadmium and zinc are removed from cells of Ralstonia metallidurans by the CzcCBA efflux pump and by two soft-metal-transporting P-type ATPases, CadA and ZntA. The czcCBA genes are located on plasmid pMOL30, and the cadA and zntA genes are on the bacterial chromosome. Expression of zntA from R. metallidurans in Escherichia coli predominantly mediated resistance to zinc, and expression of cadA predominantly mediated resistance to cadmium. Both transporters decreased the cellular content of zinc or cadmium in this host. In the plasmid-free R. metallidurans strain AE104, single gene deletions of cadA or zntA had only a moderate effect on cadmium and zinc resistance, but zinc resistance decreased 6-fold and cadmium resistance decreased 350-fold in double deletion strains. Neither single nor double gene deletions affected zinc resistance in the presence of czcCBA. In contrast, cadmium resistance of the cadA zntA double mutant could be elevated only partially by the presence of CzcCBA. lacZ reporter gene fusions indicated that expression of cadA was induced by cadmium but not by zinc in R. metallidurans strain AE104. In the absence of the zntA gene, expression of cadA occurred at lower cadmium concentrations and zinc now served as an inducer. In contrast, expression of zntA was induced by both zinc and cadmium, and the induction pattern did not change in the presence or absence of CadA. However, expression of both genes, zntA and cadA, was diminished in the presence of CzcCBA. This indicated that CzcCBA efficiently decreased cytoplasmic cadmium and zinc concentrations. It is discussed whether these data favor a model in which the cations are removed either from the cytoplasm or the periplasm by CzcCBA.

Research has been conducted on Bacillus subtilis bcrC bacitracin resistance gene. Results demonstrate that this gene is important for bacitracin resistance, is controlled by sigma (super)x and sigma (super)m and is inducible by bacitracin.

Research has been conducted on the nature of the glycolytic flux control. Results indicate that the expression of the F (sub)1 subunits from the H (super)+ -ATP synthase leads to the ATPase activity in Escherichia coli cytoplasm and that the added ATPase activity dramatically increases glycolysis rate.

Research has been conducted on the Escherichia coli ferric citrate transport genes. The transcriptional regulation of these genes initiated by ferric citrate binding to the outer membrane protein FecA and the interaction between transmembrane protein FecR and sigma factor FecI has been investigated and discussed.

Research has been conducted on the xanthomonadin, genus Xanthomonas' aryl-polyene pigments. The characterization of Xanthomonas oryzae pv. oryzae genetic locus containing open reading frames has been carried out and the results indicate that the putative cytoplasmic membrane protein encoded in this locus is required for the outer membrane localization of xanthomonadin in this bacterium.

Research has been conducted on the Aeromonas salmonicida that contains the cloned gene for the proaerolysin. Results indicate that the alteration of the region near the aerA's beginning has led to the increase in the proaerolysin production.

In gram-negative bacteria, coexpression of single- and multicomponent efflux pumps may result in multiplicative enhancement of the level of resistance against cytoplasmically acting antibiotics. Here, a simple model is presented to show that this cooperative effect can be accounted for only if substrate capture by the multicomponent efflux transporter occurs in the periplasm but not the cytosol.

Research has been conducted on sigma factor sigma (super)E which responds to stress in Escherichia coli extracytoplasmic compartment. The authors report that sigma (super)E activity increases after stress response is initiated by a shift to elevated temperature and remains at the same level during the stress.

Research has been conducted on pssT gene involved in exopolysaccharides' polymerization, biosynthesis and transport in Rhizobium leguminosarum bv. trifolii strain TA1. Resuslts suggest that PssT topological model consists of transmembrane segments, and that amino and carboxyl termini are located in the cytoplasm.

Research has been conducted on Bordetella pertussis. The results of functional and proteomic analyses have demonstrated that genes encoding extracytoplasmic solute receptors are expressed in B. pertussis.

Research has been conducted on Bacillus subtilis K (super)+ uptake systems. Results demonstrate that K (super)+ uptake carried out via KtrAB and KtrCD is important in the cellular defense of B. subtilis against osmotic stress.

Research has been conducted on Pseudomonas aeruginosa. The ability of p yoverdine to control FpvA production in P. aeruginosa has been investigated, and the details are presented.

Research has been conducted on Bacillus subtilis heat shock genes htpG. Results demonstrate that the heat induction factor depends on the absolute temperature and that nonative proteins within the cytoplasm cannot induce htpG.

Research has been conducted on histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein and phosphatase (HAMP). Results demonstrate that HAMP domain's features are amphipathic alpha-helices.