Biosynthesis of the glycolipid anchor in lipoteichoic acid of Staphylococcus aureus RN4220: role of YpfP, the diglucosyldiacylglycerol synthase

In Staphylococcus aureus RN4220, lipoteichoic acid (LTA) is anchored in the membrane by a diglucosyldiacylglycerol moiety. The gene (ypfP) which encodes diglucosyldiacylglycerol synthase was recently cloned from Bacillus subtilis and expressed in Escherichia coli (P. Jorasch, F. P. Wolter, U. Zahringer, and E. Heinz, Mol. Microbiol. 29:419-430, 1998). To define the role of ypfP in this strain of S. aureus, a fragment of ypfP truncated from both ends was cloned into the thermosensitive replicon pVE6007 and used to inactivate ypfP. Chloramphenicol-resistant (ypfP::cat) clones did not synthesize the glycolipids monoglucosyldiacylglycerol and diglucosyldiacylglycerol. Thus, YpfP would appear to be the only diglucosyldiacylglycerol synthase in S. aureus providing glycolipid for LTA assembly. In LTA from the mutant, the glycolipid anchor is replaced by diacylglycerol. Although the doubling time of the mutant was identical to that of the wild type in Luria-Bertani (LB) medium, growth of the mutant in LB medium containing 1% glycine was not observed. This inhibition was antagonized by either L- or D-alanine. Moreover, viability of the mutant at 37 degrees C in 0.05 M phosphate (pH 7.2)-saline for 12 h was reduced to <0.1%. Addition of 0.1% D-glucose to the phosphate-saline ensured viability under these conditions. The autolysis of the ypfP::cat mutant in the presence of 0.05% Triton X-100 was 1.8-fold faster than that of the parental strain. Electron microscopy of the mutant revealed not only a small increase in cell size but also the presence of pleomorphic cells. Each of these phenotypes may be correlated with either (or both) a deficiency of free glycolipid in the membrane or the replacement of the usual glycolipid anchor of LTA with diacylglycerol.     

trans-acting factors affecting carbon catabolite repression of the hut operon in Bacillus subtilis

In Bacillus subtilis, CcpA-dependent carbon catabolite repression (CCR) mediated at several cis-acting carbon repression elements (cre) requires the seryl-phosphorylated form of both the HPr (ptsH) and Crh (crh) proteins. During growth in minimal medium, the ptsH1 mutation, which prevents seryl phosphorylation of HPr, partially relieves CCR of several genes regulated by CCR. Examination of the CCR of the histidine utilization (hut) enzymes in cells grown in minimal medium showed that neither the ptsH1 nor the crh mutation individually had any affect on hut CCR but that hut CCR was abolished in a ptsH1 crh double mutant. In contrast, the ptsH1 mutation completely relieved hut CCR in cells grown in Luria-Bertani medium. The ptsH1 crh double mutant exhibited several growth defects in glucose minimal medium, including reduced rates of growth and growth inhibition by high levels of glycerol or histidine. CCR is partially relieved in B. subtilis mutants which synthesize low levels of active glutamine synthetase (glnA). In addition, these glnA mutants grow more slowly than wild-type cells in glucose minimal medium. The defects in growth and CCR seen in these mutants are suppressed by mutational inactivation of TnrA, a global nitrogen regulatory protein. The inappropriate expression of TnrA-regulated genes in this class of glnA mutants may deplete intracellular pools of carbon metabolites and thereby result in the reduction of the growth rate and partial relief of CCR.     

The ftsQ1p gearbox promoter of Escherichia coli is a major sigma S-dependent promoter in the ddlB–ftsA region

We have isolated the ypfP gene (accession number P54166) from genomic DNA of Bacillus subtilis Marburg strain 60015 ( Freese and Fortnagel, 1967 ) using PCR. After cloning and expression in E. coli , SDS–PAGE showed strong expression of a protein that had the predicted size of 43.6 kDa. Chromatographic analysis of the lipids extracted from the transformed E. coli revealed several new glycolipids. These glycolipids were isolated and their structures determined by nuclear magnetic resonance (NMR) and mass spectrometry. They were identified as 3-[ O -β- D -glucopyranosyl-(1→6)- O -β- D -glucopyranosyl]-1,2-diacylglycerol, 3-[ O -β- D -glucopyranosyl-(1→6)- O -β- D -glucopyranosyl-(1→6)- O -β- D -glucopyranosyl]-1,2-diacylglycerol and 3-[ O -β- D -glucopyranosyl-(1→6)- O -β- D -glucopyranosyl-(1→6)- O -β- D -glucopyranosyl-(1→6)- O -β- D -glucopyranosyl]-1,2-diacylglycerol. The enzymatic activity expected to catalyse the synthesis of these compounds was confirmed by in vitro assays with radioactive substrates. In these assays, one additional glycolipid was formed and tentatively identified as 3-[ O -β- D -glucopyranosyl]-1,2-diacylglycerol, which was not detected in the lipid extract of transformed cells. Experiments with some of the above-described glycolipids as ¹⁴C-labelled sugar acceptors and unlabelled UDP-glucose as glucose donor suggest that the ypfP gene codes for a new processive UDP-glucose: 1,2-diacylglycerol-3-β- D -glucosyl transferase. This glucosyltransferase can use diacylglycerol, monoglucosyl-diacylglycerol, diglucosyldiacylglycerol or triglucosyldiacylglycerol as sugar acceptor, which, apart from the first member, are formed by repetitive addition of a glucopyranosyl residue in β (1→6) linkage to the product of the preceding reaction.     

Identification of a putative Bacillus subtilis rho gene.

Transposon Tn917 mutagenesis of Bacillus subtilis BD99 followed by selection for protonophore resistance led to the isolation of strain MS119, which contained a single Tn917 insertion in an open reading frame whose deduced amino acid sequence was 56.6% identical to that of the Escherichia coli rho gene product. The insertional site was near the beginning of the open reading frame, which was located in a region of the B. subtilis chromosome near the spoOF gene; new sequence data for several open reading frames surrounding the putative rho gene are presented. The predicted B. subtilis Rho protein would have 427 amino acids and a molecular weight of 48,628. The growth of the mutant strain was less than that of the wild type on defined medium at 30 degrees C. On yeast extract-supplemented medium, the growth of MS119 was comparable to that of the wild type on defined medium at 30 degrees C. On yeast extract-supplemented medium, the growth of MS119 was comparable to that of the wild type at 30 degrees C but was much slower at lower temperatures; sporulation occurred and competence was developed in cells of the mutant grown at 30 degrees C. To determine whether the protonophore resistance and sensitivity to low growth temperature resulted from the insertion, a chloramphenicol resistance cassette was inserted into the wild-type B. subtilis rho gene of strain BD170; the resulting derivative displayed the same phenotype as MS119.     

Deletion of the yhhP gene results in filamentous cell morphology in Escherichia coli

The Escherichia coli yhhP gene was predicted to encode a small hypothetical protein of 81 amino acids, the cellular function of which is not known. To gain insight into the function of this uncharacterized YhhP protein, genetic and biochemical studies were done. We first tried to express and purify the YhhP protein to prepare an anti-YhhP antiserum. Western blotting showed that the hypothetical yhhP gene is indeed transcribed and translated as a minor cytoplasmic protein. YhhP-deficient (delta yhhP) cells formed colonies poorly on a rich medium (e.g., Luria-Bertani medium) containing a relatively low concentration of NaCl, while they can grow normally either in LB containing 3% NaCl or in a synthetic medium (e.g., M9-glucose). During exponential growth in rich medium, an early step of cell division was inhibited in delta yhhP cells, forming filaments. For the YhhP-deficient filamentous cells, the FtsZ-ring formation was analyzed with immunofluorescence microscopy. The FtsZ-ring formation did not occur normally in the delta yhhP filaments, although the filamentous cells contained the FtsZ protein at a certain level comparable to that in the wild-type cells. The ftsZ gene was found to function as a multicopy suppressor of the delta yhhP mutant. Another multicopy suppressor gene was identified as the dksA gene. Provided that either the ftsZ or dksA gene was introduced into the mutant cells with its multicopy state, the resulting transformants were capable of growing in rich medium, formed wild-type short rods. These results are discussed with regard to the presumed function of this ubiquitous protein.     

Integration of hybrid plasmids-derivatives of staphylococcal plasmid pE3692 and pBR322 vector with Bacillus subtilis chromosome

Bacillus subtilis is a nonpathogenic microorganism which certainly for also that feature is widely used in several biotechnological processes. Certainly, because of that an elaboration of gene cloning methods in Bacillus subtilis cells is of great significance both from scientific and practical point of view. It was found hybrid plasmids constructed by ligation of pE3692 and pE3692-9 staphylococcal plasmids with pBR322 vector can be integrated with B. subtilis chromosome. Selection of cells in the presence of erythromycin at 50 degrees C allows to isolate clones containing exclusively integrated plasmids. Growth rate of cells containing these plasmids in the selective medium is the same or slightly higher at 50 degrees C than at 37 degrees C. It seems that hybrid plasmid tested pBE4 and pBE91 after appropriate reconstruction in vitro could serve as vectors for B. subtilis gene cloning.     

Fusion of pro region of subtilisin to staphylococcal protein A and its secretion by Bacillus subtilis

Subtilisin is synthesized as a preproenzyme in Bacillus subtilis. We fused that region of the subtilisin gene, (apr[BamP]), which encodes the signal sequence and pro region, to the mature gene sequence (spa) for a heterologous protein (staphylococcal protein A). B. subtilis cells harboring this gene fusion synthesized a fusion protein consisting of the signal and pro sequence of subtilisin fused to the protein A; the signal sequence was processed and a fusion protein (pro + protein A) was secreted into the growth medium.     

Chromosome DNA fragmentation and excretion caused by defective prophage gene expression in the early-exponential-phase culture of Bacillus subtilis

Bacillus subtilis 168 and its major autolysin mutant, AN8, were shown to excrete two size classes of DNA when cultured in Luria-Bertani medium. Pulsed-field gel electrophoresis of DNA harvested from the cell surface demonstrated the presence of 13-kb-long and circa 50-kb-long strands. Restriction digestion of both sizes of DNA resulted in a smearing pattern, as observed by agarose gel electrophoresis. Shotgun sequencing of DNase I partial digests of 50-kb DNA fragments revealed that the strands originate from various sites on the chromosome. SDS-PAGE analysis of cell surface fractions and culture supernatants demonstrated the presence of several proteins that were thought to be associated with the DNA. Of these, three major proteins were identified, i.e., XkdG, XkdK, and XkdM, by tandem mass spectrometry, all of which were proteins of a defective prophage PBSX residing in the Bacillus subtilis chromosome. Disruption of these PBSX genes resulted in a reduction of 13-kb fragment generation and excretion and also a great reduction of 50-kb fragment excretion. Electron microscopy showed that a few mature phages and numerous membrane vesicle-like particles existed in the cell surface fractions of strain 168. The present findings suggest that the spontaneous generation and excretion of chromosome DNA fragments in Bacillus subtilis are both closely related to the expression of defective prophage genes.     

Cloning the alpha-amylase gene of Streptococcus bovis and its expression in Bacillus subtilis cells

The gene coding for alpha-amylase from the ruminant bacterium Streptococcus bovis was cloned on the plasmid pMX39 in Bacillus subtilis cells. An alpha-amylase positive colony was isolated in the initial screening of 3900 colonies on the medium containing insoluble starch. The size of the insert was approximately 2.8 kb. The recombinant plasmid was stably maintained in Bacillus subtilis cells under the nonselective conditions.     

磷脂酰丝氨酸合成酶基因的克隆及在枯草芽孢杆菌中的表达

应用 PCR技术从 Escherichia coli K12 Sgal- (ExPASy P23830) 中扩增到大小为 1350bp编码磷脂酰丝氨酸合成酶的 DNA片段,将其插入枯草芽孢杆菌诱导型表达载体 pBES,获得重组质粒 pBES-pss后转化 Bacillus subtilis DB104。经蔗糖诱导后,该磷脂酰丝氨酸合成酶在 Bacillus subtilis DB104 (pBES-pss)中获得胞外分泌表达,SDS-PAGE分析发现目的蛋白分子量约为 52kDa,酶联比色法检测酶活力为 1.50U/mL,提高了磷脂酰丝氨酸合成酶的表达产量,为工业化发酵生产磷脂酰丝氨酸合成酶奠定了良好的基础。     

枯草杆菌碱性蛋白酶基因诱导表达载体的构建

以PCR方法扩增sacB基因的启动子-信号肽序列(称为sacR),将其与枯草芽孢杆菌碱性蛋白酶的前肽-成熟酶基因连接后克隆入载体pUBH,构建了含碱性蛋白酶基因的分泌型诱导表达载体pUBS,将其转化枯草芽孢杆菌DB403后,获得基因工程菌DB403(pUBS)。碱性蛋白酶基因在sacR的调控和蔗糖的诱导下实现了表达分泌,获得了具生物学活性的碱性蛋白酶。     

Comparative analysis of the development of swarming communities of Bacillus subtilis 168 and a natural wild type: critical effects of surfactin and the composition of the medium

The natural wild-type Bacillus subtilis strain 3610 swarms rapidly on the synthetic B medium in symmetrical concentric waves of branched dendritic patterns. In a comparison of the behavior of the laboratory strain 168 (trp) on different media with that of 3610, strain 168 (trp), which does not produce surfactin, displayed less swarming activity, both qualitatively (pattern formation) and in speed of colonization. On E and B media, 168 failed to swarm; however, with the latter, swarming was arrested at an early stage of development, with filamentous cells and rafts of cells (characteristic of dendrites of 3610) associated with bud-like structures surrounding the central inoculum. In contrast, strain 168 apparently swarmed efficiently on Luria-Bertani (LB) agar, colonizing the entire plate in 24 h. However, analysis of the intermediate stages of development of swarms on LB medium demonstrated that, in comparison with strain 3610, initiation of swarming of 168 (trp) was delayed and the greatly reduced rate of expansion of the swarm was uncoordinated, with some regions advancing faster than others. Moreover, while early stages of swarming in 3610 are accompanied by the formation of large numbers of dendrites whose rapid advance involves packs of cells at the tips, strain 168 advanced more slowly as a continuous front. When sfp+ was inserted into the chromosome of 168 (trp) to reestablish surfactin production, many features observed with 3610 on LB medium were now visible with 168. However, swarming of 168 (sfp+) still showed some reduced speed and a distinctive pattern compared to swarming of 3610. The results are discussed in terms of the possible role of surfactin in the swarming process and the different modes of swarming on LB medium.