Identification of a Streptococcus pneumoniae Gene Locus Encoding Proteins of an ABC Phosphate Transporter and a Two-Component Regulatory System

The Escherichia coli Pst system belongs to the family of ABC transporters. It is part of a phosphate (PHO) regulon which is regulated by extracellular phosphate. Under conditions of phosphate limitation, the response regulator PhoB is phosphorylated by the histidine kinase PhoR and binds to promoters that share a consensus PHO box. Under conditions of phosphate excess, PhoR, Pst, and PhoU downregulate the PHO regulon. Screening of a library of pneumococcal mutants with defects in exported proteins revealed a putative two-component regulatory system, PnpR-PnpS, and a downstream ABC transporter, similar to the Pst system in E. coli including a gene encoding a PhoU protein. Similar to E. coli, mutagenesis of the ATP-binding cassette gene, pstB, resulted in decreased uptake of phosphate. The effects of the loss of the pneumococcal Pst system extended to decreased transformation and lysis. Withdrawal of phosphate led to transformation deficiency in the parent strain R6x but not to penicillin tolerance, suggesting that reduced bacterial death was independent of phosphate. None of these phenotypes was observed in the pneumococcal loss-of-function mutant phoU. By using a lacZ reporter construct, it was demonstrated that expression of the two-component regulatory system PnpR-PnpS was not influenced by different concentrations of phosphate. These results suggest a more complex role of the Pst system in pneumococcal physiology than in that of E. coli.     

Expression and characterization of the genes encoding azoreductases from Bacillus subtilis and Geobacillus stearothermophilus

Azoreductases have been characterized as enzymes that can decolorize azo dyes by reducing azo groups. In this study, genes encoding proteins having homology with the azoreductase gene of Bacillus sp. OY1-2 were obtained from Bacillus subtilis ATCC6633, B. subtilis ISW1214, and Geobacillus stearotherophilus IFO13737 by polymerase chain reaction. All three genes encoded proteins with 174 amino acids. The deduced amino acid sequences of azoreductase homologs from B. subtilis ISW1214, B. subtilis ATCC6633, and G. stearotherophilus IFO13737 showed similarity of 53.3, 53.9, and 53.3% respectively to that of Bacillus sp. OY1-2.All three genes were expressed in Escherichia coli, and were characterized as having the decolorizing activity of azo dyes in a beta-NADPH dependent manner. The transformation of several azo dyes into colorless compounds by recombinant enzymes was demonstrated to have distinct substrate specificity from that of azoreductase from Bacillus sp. OY1-2.     

高产耐热脂肪酶嗜热脂肪地芽孢杆菌的选育

采用氮离子注入技术对耐热脂肪酶产生菌嗜热脂肪地芽孢杆菌（Geobacillus stearothermophilus）L4进行诱变,筛选获得酶活力有较大提高且传代稳定的正突变菌株L4-3;再对L4-3进行紫外线诱变,得到脂肪酶活力提高的正突变菌株L4-3-2,其脂肪酶活力达25．71U／mL,较原始菌株M提高511．9％。高产突变株L4-3-2所产脂肪酶的最适作用温度为50℃,70℃保温60min的剩余酶活为82％,最适作用pH为7．0～8．0,为一种耐热碱性脂肪酶。     

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a particularly attractive option for studying membrane proteins, especially in the context of ligand-receptor interactions. The method pioneered by Sligar and colleagues is based on the amphipathic properties of an engineered highly a-helical scaffold protein derived from the apolipoprotein A1. The hydrophobic faces of the scaffold protein interact with the fatty acyl side-chains of the lipid bilayer whereas the polar regions face the aqueous environment. Analyses of membrane proteins in nanodiscs have significant advantages over liposome because the particles are small, homogeneous and water-soluble. In addition, biochemical and biophysical methods normally reserved to soluble proteins can be applied, and from either side of the membrane. In this visual protocol, we present a step-by-step reconstitution of a well characterized bacterial ABC transporter, the MalE-MalFGK₂ complex. The formation of the disc is a self-assembly process that depends on hydrophobic interactions taking place during the progressive removal of the detergent. We describe the essential steps and we highlight the importance of choosing a correct protein-to-lipid ratio in order to limit the formation of aggregates and larger polydisperse liposome-like particles. Simple quality controls such as gel filtration chromatography, native gel electrophoresis and dynamic light scattering spectroscopy ensure that the discs have been properly reconstituted.     

The Rcs Two-Component System Regulates Expression of Lysozyme Inhibitors and Is Induced by Exposure to Lysozyme

The Escherichia coli Rcs regulon is triggered by antibiotic-mediated peptidoglycan stress and encodes two lysozyme inhibitors, Ivy and MliC. We report activation of this pathway by lysozyme and increased lysozyme sensitivity when Rcs induction is genetically blocked. This lysozyme sensitivity could be alleviated by complementation with Ivy and MliC.In gram-negative bacteria, the cell envelope represents an important functional compartment that extends from the cytoplasmic membrane to the outer membrane and supports a number of essential processes, such as solute transport, protein translocation, and respiratory energy generation (15). In addition, the cell envelope accommodates the bacterial peptidoglycan layer, a distinct and structurally vital element of the cell. Most recently, Laubacher and Ades (10) have demonstrated that the Rcs phosphorelay system of Escherichia coli, originally described as regulator of capsule synthesis, is activated by β-lactam antibiotics that inhibit penicillin-binding proteins and consequently interfere with peptidoglycan synthesis. Moreover, mutational activation of the Rcs pathway provided significant protection against these antibiotics, indicating that members of this regulon can prevent or repair the peptidoglycan damage caused by β-lactam antibiotics (10).Interestingly, ivy and ydhA, two genes encoding specific lysozyme inhibitors, were found to reside under this Rcs regulon (8, 10). Ivy (inhibitor of vertebrate lysozyme, formerly known as YkfE) was discovered in 2001 as the first bacterial lysozyme inhibitor (1, 14), while the inhibitory activity of YdhA was only recently revealed by our research group (3). Although Ivy and YdhA are both able to inhibit c-type lysozymes, such as human lysozyme and hen egg white lysozyme (HEWL), they are structurally unrelated (1, 16). Interestingly, YdhA belongs to a group of proteins with a common conserved COG3895 domain that are widely spread among the Proteobacteria (3, 16). Unlike Ivy, which resides in the periplasm, YdhA is a lipoprotein and was therefore renamed MliC (membrane-bound lysozyme inhibitor of c-type lysozyme) (3).Given the elementary observation that the two currently known lysozyme inhibitors of E. coli are both part of the Rcs regulon that can in turn be induced by antibiotic-mediated peptidoglycan stress, we wondered whether Rcs induction could also result from exposure to lysozyme itself. To test this, we introduced a tolA knockout from MG1655 tolA (3) into strain DH300 that is equipped with a genomic rprA-lacZ fusion able to report Rcs activation (12), in order to increase outer membrane permeability for HEWL (Table ​(Table11 lists all strains). A stationary-phase culture of the resulting strain, designated LC100, was diluted 1/100 in 4 ml fresh LB medium with different final concentrations of HEWL (0, 5, 10, 25, and 50 μg/ml), and after 2.5 h of further growth at 37°C, β-galactosidase activity was measured (13). Interestingly, rprA-lacZ was significantly induced at HEWL concentrations of >10 μg/ml, up to 4.4-fold at 50 μg/ml (Fig. ​(Fig.1A).1A). This induction could be completely abolished upon the additional introduction of a knockout of rcsB (strain LC102), the response regulator required to activate gene expression in the Rcs pathway. Moreover, knocking out rcsF (strain LC101), the outer membrane lipoprotein sensor that triggers the Rcs pathway upon antibiotic-mediated peptidoglycan stress (10), also resulted in a loss of lysozyme induction. As a comparison, rprA-lacZ induction in DH300 treated with amdinocillin (Sigma-Aldrich, Bornem, Belgium), as previously described (10), resulted in a 16-fold increase in β-galactosidase activity (Fig. ​(Fig.1B).1B). Please note that the difference in basal β-galactosidase levels between LC100 and DH300 (Fig. 1A and B) is probably due to the tolA mutation in LC100, which is known to result in a higher basal expression of the Rcs pathway (5). These data clearly demonstrate that the Rcs phosphorelay can indeed be activated by exposure to lysozyme and that this induction is mediated by the outer membrane sensor rcsF. This also implies that the Rcs pathway responds to different types of peptidoglycan stress, as β-lactam antibiotics block the formation of peptide side-chain cross-links by binding irreversibly to the transpeptidases, while lysozyme hydrolyzes the heteropolysaccharide backbone.Open in a separate windowFIG. 1.Induction of the Rcs pathway in LC100 (tolA::Kn Rcs⁺) with different HEWL concentrations (0 to 50 μg/ml) (A) and in DH300 (Rcs⁺) with (+) or without (−) amdinocillin treatment (B). Rcs induction is measured as β-galactosidase activity originating from a genomic rprA-lacZ reporter fusion and expressed in Miller units (13). Error bars indicate standard deviations of results from three replicate experiments. The corresponding RcsB⁻ strain (LC102) and the RcsF⁻ strain (LC101) showed rprA-lacZ inductions of <10 Miller units when subjected to lysozyme treatments and are therefore not shown.

TABLE 1.

Bacterial strains and plasmids used in the study

应用响应面法优化嗜热脂肪土芽孢杆菌培养基

目的：筛选并优化嗜热脂肪芽孢杆菌CHB1液体发酵培养基。方法：通过Plackett-Burman试验和响应面分析方法,确定培养基的主要影响因素和最佳浓度。结果：利用SAS软件进行分析,确定对响应值影响最大的3个因素为豆粕、酵母粉、K2HPO4。最佳培养基组成为0.51%、豆粕浓度为0.425%、K2HPO4浓度为0.994%。根据模型预测得到的理论最大菌数为2.94&#215;10^8cfu。在初始条件下实验,菌数为2.40&#215;10^8cfu,在优化的最佳培养基条件下,实际的菌数为3.06&#215;10^8cfu。菌数比优化前提高了27.3%。试验值与预测值的误差为4.08%。结论：实验值与模型预测值拟合良好。     

A Multidrug ABC Transporter with a Taste for Salt

Background

LmrA is a multidrug ATP-binding cassette (ABC) transporter from Lactococcus lactis with no known physiological substrate, which can transport a wide range of chemotherapeutic agents and toxins from the cell. The protein can functionally replace the human homologue ABCB1 (also termed multidrug resistance P-glycoprotein MDR1) in lung fibroblast cells. Even though LmrA mediates ATP-dependent transport, it can use the proton-motive force to transport substrates, such as ethidium bromide, across the membrane by a reversible, H⁺-dependent, secondary-active transport reaction. The mechanism and physiological context of this reaction are not known.

Methodology/Principal Findings

We examined ion transport by LmrA in electrophysiological experiments and in transport studies using radioactive ions and fluorescent ion-selective probes. Here we show that LmrA itself can transport NaCl by a similar secondary-active mechanism as observed for ethidium bromide, by mediating apparent H⁺-Na⁺-Cl⁻ symport. Remarkably, LmrA activity significantly enhances survival of high-salt adapted lactococcal cells during ionic downshift.

Conclusions/Significance

The observations on H⁺-Na⁺-Cl⁻ co-transport substantiate earlier suggestions of H⁺-coupled transport by LmrA, and indicate a novel link between the activity of LmrA and salt stress. Our findings demonstrate the relevance of investigations into the bioenergetics of substrate translocation by ABC transporters for our understanding of fundamental mechanisms in this superfamily. This study represents the first use of electrophysiological techniques to analyze substrate transport by a purified multidrug transporter.     

Engineering of the alpha-amylase from Geobacillus stearothermophilus US100 for detergent incorporation

AmyUS100DeltaIG is a variant of the most thermoactive and thermostable maltohexaose forming alpha-amylase produced by Geobacillus stearothermophilus sp.US100. This enzyme which was designed to improve the thermostability of the wild-type enzyme has acquired a very high resistance to chelator agents. According to modeling structural studies and with the aim of enhancing its resistance towards chemical oxidation, a mutant (AmyUS100DeltaIG/M197A) was created by substituting methionine 197 to alanine. The catalytic proprieties of the resulting mutant show alterations in the specific activity and the profile of starch hydrolysis. Interestingly, AmyUS100DeltaIG/M197A displayed the highest resistance to oxidation compared to the AmyUS100DeltaIG and to Termamyl300, the well-known commercial amylase used in detergent. Further, performance of the engineered alpha-amylase was estimated in the presence of commonly used detergent compounds and a wide range of commercial detergent (liquid and solid). These studies indicated a high compatibility and performance of AmyUS100DeltaIG/M197A, suggesting its potential application in detergent industry.     

A Group I Self-Splicing Intron in the Flagellin Gene of the Thermophilic Bacterium Geobacillus stearothermophilus

A group I intron that can be spliced in vivo and in vitro was identified in the flagellin gene of the thermophilic bacterium Geobacillus stearothermophilus. We also found one or two intervening sequences (IVS) of flagellin genes in five additional bacterial species. Furthermore, we report the presence of these sequences in two sites of a highly conserved region in the flagellin gene.     

Molecular basis of S-layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus

The Gram-positive bacterium Geobacillus stearothermophilus NRS 2004/3a possesses a cell wall containing an oblique surface layer (S-layer) composed of glycoprotein subunits. O-Glycans with the structure [-->2)-alpha-L-Rhap-(1-->3)-beta-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->](n) (= 13-18), a2-O-methyl group capping the terminal repeating unit at the nonreducing end and a -->2)-alpha-L-Rhap-[(1-->3)-alpha-L-Rhap](n) (= 1-2)(1-->3)- adaptor are linked via a beta-D-Galp residue to distinct sites of the S-layer protein SgsE. S-layer glycan biosynthesis is encoded by a polycistronic slg (surface layer glycosylation) gene cluster. Four assigned glycosyltransferases named WsaC-WsaF, were investigated by a combined biochemical and NMR approach, starting from synthetic octyl-linked saccharide precursors. We demonstrate that three of the enzymes are rhamnosyltransferases that are responsible for the transfer of L-rhamnose from a dTDP-beta-L-Rha precursor to the nascent S-layer glycan, catalyzing the formation of the alpha1,3- (WsaC and WsaD) and beta1,2-linkages (WsaF) present in the adaptor saccharide and in the repeating units of the mature S-layer glycan, respectively. These enzymes work in concert with a multifunctional methylrhamnosyltransferase (WsaE). The N-terminal portion of WsaE is responsible for the S-adenosylmethionine-dependent methylation reaction of the terminal alpha1,3-linked L-rhamnose residue, and the central and C-terminal portions are involved in the transfer of L-rhamnose from dTDP-beta-L-rhamnose to the adaptor saccharide to form the alpha1,2- and alpha1,3-linkages during S-layer glycan chain elongation, with the methylation and the glycosylation reactions occurring independently. Characterization of these enzymes thus reveals the complete molecular basis for S-layer glycan biosynthesis.