葡萄球菌呼吸相关双组分系统SrrAB研究进展

葡萄球菌呼吸相关双组分系统SrrAB能感应外界O2浓度,并将信号传至胞内,调控下游基因的转录,以应对外界环境的变化。有研究表明,金黄色葡萄球菌SrrAB在有氧条件下促进毒力因子的表达,抑制生物膜的形成;在厌氧条件下抑制毒力因子的表达,促进生物膜的形成。另外,在有氧及厌氧条件下,金黄色葡萄球菌SrrAB调控生长代谢的途径也不一致。表皮葡萄球菌中也存在类似的双组分系统SrrAB,且与金黄色葡萄球菌SrrAB具有较高同源性,但目前尚不清楚两者在生长代谢及毒力调控方面的异同。结合课题组研究工作,简要综述葡萄球菌SrrAB的调控机制,着重比较其在有氧及厌氧条件下的调控差异,这对临床诊治葡萄球菌引起的感染具有一定的借鉴意义。     

A genomic analysis of two-component signal transduction in Streptococcus pneumoniae

A genomics-based approach was used to identify the entire gene complement of putative two-component signal transduction systems (TCSTSs) in Streptococcus pneumoniae. A total of 14 open reading frames (ORFs) were identified as putative response regulators, 13 of which were adjacent to genes encoding probable histidine kinases. Both the histidine kinase and response regulator proteins were categorized into subfamilies on the basis of phylogeny. Through a systematic programme of mutagenesis, the importance of each novel TCSTS was determined with respect to viability and pathogenicity. One TCSTS was identified that was essential for the growth of S. pneumoniaeThis locus was highly homologous to the yycFG gene pair encoding the essential response regulator/histidine kinase proteins identified in Bacillus subtilis and Staphylococcus aureus. Separate deletions of eight other loci led in each case to a dramatic attenuation of growth in a mouse respiratory tract infection model, suggesting that these signal transduction systems are important for the in vivo adaptation and pathogenesis of S. pneumoniae. The identification of conserved TCSTSs important for both pathogenicity and viability in a Gram-positive pathogen highlights the potential of two-component signal transduction as a multicomponent target for antibacterial drug discovery.     

Biochemical characterization of the first essential two-component signal transduction system from Staphylococcus aureus and Streptococcus pneumoniae

The YYCFG two-component signal transduction system (TCSTS) has been shown to be essential to the viability of several gram-positive bacteria. However, the function of the gene pair remains unknown. Interestingly, while both components are essential to Staphylococcus aureus and Bacillus subtilis, only the response regulator (YYCF) is essential to Streptococcus pneumoniae. To study this essential TCSTS further, the S. pneumoniae and S. aureus truncated YycG histidine kinase and full-length YycF response regulator proteins were characterized at a biochemical level. The recombinant proteins from both organisms were expressed in Escherichia coli and purified. The YycG autophosphorylation activities were activated by ammonium. The apparent K(m )(ATP) of S. aureus YycG autophosphorylation was 130 microM and S. pneumoniae was 3.0 microM. Each had similar K(cat )values of 0.036 and 0.024 min(-1), respectively. Cognate phosphotransfer was also investigated indicating different levels of the phosphorylated YycG intermediates during the reaction. The S. pneumoniae YycG phosphorylated intermediate was not detectable in the presence of its cognate YycF, while phosphorylated S. aureus YycG and YycF were detected concurrently. In addition, noncognate phosphotransfer was demonstrated between the two species. These studies thoroughly compare the essential YycFG TCSTS from the two species at the biochemical level and also establish methods for assaying the activities of these antibacterial targets.     

Respiratory-competent conditional developmental mutant of Mucor racemosus

A conditional developmental mutant of Mucor racemosus which is capable of oxidative energy metabolism is described. Unlike the wild-type strain the mutant was highly fermentative and exhibited the yeast morphology when grown aerobically in glucose-containing media. The high fermentative activity and yeast morphology under these conditions correlated well with maximal expression of glycolytic enzymes and with expression of some polypeptides characteristic of anaerobic growth. Aerobic growth of the mutant on amino acids as the sole carbon source resulted in growth in the mycelial morphology. The mutant was fully capable of oxidative metabolism as judged by its ability to grow on amino acids, respiratory capacity, and complement of tricarboxylic acid cycle enzymes. The results support the hypothesis that oxygen controls both the expression of glycolytic enzymes and the expression of proteins involved in morphogenesis. Moreover, they suggest that there are common regulatory elements in the control of these two classes of gene products. Abnormally high levels of aconitase and isocitrate dehydrogenase in the mutant are consistent with the proposal that pool sizes of citrate may act as a regulator of genes responsive to environmental oxygen concentration.     

A defect in menadione biosynthesis induces global changes in gene expression in Staphylococcus aureus

Both the high-resolution two-dimensional protein gel electrophoresis technique and full-genome DNA microarrays were used for identification of Staphylococcus aureus genes whose expression was changed by a mutation in menD. Because the electron transport chain is interrupted, the mutant should be unable to use oxygen and nitrate as terminal electron acceptors. Consistent with this, a mutation in menD was found to cause a gene expression pattern typically detected under anaerobic conditions in wild-type cells: proteins involved in glycolytic as well as in fermentation pathways were upregulated, whereas tricarboxylic acid (TCA) cycle enzymes were significantly downregulated. Moreover, the expression of genes encoding enzymes for nitrate respiration and the arginine deiminase pathway was strongly increased in the mutant strain. These results indicate that the menD mutant, just as the site-directed S. aureus hemB mutant, generates ATP from glucose or fructose mainly by substrate phosphorylation and might be defective in utilizing a variety of carbon sources, including TCA cycle intermediates and compounds that generate ATP only via electron transport phosphorylation. Of particular interest is that there are also differences in the gene expression patterns between hemB and menD mutants. While some anaerobically active enzymes were present in equal amounts in both strains (Ldh1, SACOL2535), other classically anaerobic enzymes seem to be present in higher amounts either in the hemB mutant (e.g., PflB, Ald1, IlvA1) or in the menD mutant (arc operon). Only genes involved in nitrate respiration and the ald1 operon seem to be additionally regulated by a depletion of oxygen in the hemB and/or menD mutant.     

Regulation of nitrogen metabolism in Bacillus subtilis: vive la différence!

Nitrogen metabolism genes of Bacillus subtilis are regulated by the availability of rapidly metabolizable nitrogen sources, but not by any mechanism analogous to the two-component Ntr regulatory system found in enteric bacteria. Instead, at least three regulatory proteins independently control the expression of gene products involved in nitrogen metabolism in response to nutrient availability. Genes expressed at high levels during nitrogen-limited growth are controlled by two related proteins, GlnR and TnrA, which bind to similar DNA sequences under different nutritional conditions. The TnrA protein is active only during nitrogen limitation, whereas GlnR-dependent repression occurs in cells growing with excess nitrogen. Although the nitrogen signal regulating the activity of the GlnR and TnrA proteins is not known, the wild-type glutamine synthetase protein is required for the transduction of this signal to the GlnR and TnrA proteins. Examination of GlnR- and TnrA-regulated gene expression suggests that these proteins allow the cell to adapt to growth during nitrogen-limited conditions. A third regulatory protein, CodY, controls the expression of several genes involved in nitrogen metabolism, competence and acetate metabolism in response to growth rate. The highest levels of CodY-dependent repression occur in cells growing rapidly in a medium rich in amino acids, and this regulation is relieved during the transition to nutrient-limited growth. While the synthesis of amino acid degradative enzymes in B. subtilis is substrate inducible, their expression is generally not regulated in response to nitrogen availability by GlnR and TnrA. This pattern of regulation may reflect the fact that the catabolism of amino acids produced by proteolysis during sporulation and germination provides the cell with substrates for energy production and macromolecular synthesis. As a result, expression of amino acid degradative enzymes may be regulated to ensure that high levels of these enzymes are present in sporulating cells and in dormant spores.     

Regulation of narK gene expression in Escherichia coli in response to anaerobiosis, nitrate, iron, and molybdenum.

The regulation of the narK gene in Escherichia coli was studied by constructing narK-lacZ gene and operon fusions and analyzing their expression in various mutant strains in response to changes in cell growth conditions. Expression of narK-lacZ was induced 110-fold by a shift to anaerobic growth and a further 8-fold by the presence of nitrate. The fnr gene product mediates this anaerobic response, while nitrate control is mediated by the narL, narX, and narQ gene products. The narX and narQ gene products were shown to sense nitrate independently of one another and could each activate narK expression in a NarL-dependent manner. We provide the first evidence that NarL and FNR interact to ensure optimal expression of narK. IHF and Fis proteins are also required for full activation of narK expression, and their roles in DNA bending are discussed. Finally, the availability of molybdate and iron ions is necessary for optimal narK expression, whereas the availability of nitrite is not. Although the role of the narK gene product in cell metabolism remains uncertain, the pattern of narK gene expression is consistent with a proposed role of NarK in nitrate uptake by the cell for nitrate-linked electron transport.     

Characterization of genes encoding dimethyl sulfoxide reductase of Rhodobacter sphaeroides 2.4.1T: an essential metabolic gene function encoded on chromosome II.

Rhodobacter sphaeroides 2.4.1T is a purple nonsulfur facultative phototrophic bacterium which exhibits remarkable metabolic diversity as well as genomic complexity. Under anoxic conditions, in the absence of light and the presence of dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO), R. sphaeroides 2.4.1T utilizes DMSO or TMAO as the terminal electron acceptor for anaerobic respiration, which is mediated by the molybdoenzyme DMSO reductase. Sequencing of a 13-kb region of chromosome II revealed the presence of 10 putative open reading frames, of which 5 possess homology to genes encoding the TMAO reductase (the tor system) of Escherichia coli. The dorS and dorR genes encode a sensor-regulator pair of the two-component sensory transduction protein family, homologous to the torS and torR gene products. The dorC gene was shown to encode a 44-kDa DMSO-inducible c-type cytochrome. The dorB gene encodes a membrane protein of unknown function homologous to the torD gene product. The dorA gene encodes DMSO reductase, containing the molybdopterin active site. Mutations were constructed in each of these dor genes, and the resulting mutants were shown to be impaired for DMSO-dependent anaerobic growth in the dark. The mutant strains exhibited negligible levels of DMSO reductase activity compared to the wild-type strain under similar growth conditions. Further, no DorA protein was detected in DorS and DorR mutant strains with anti-DorA antisera, suggesting that the products of these genes are required for the positive regulation of dor expression in response to DMSO. This characterization of the dor gene cluster is the first evidence that genes of chromosome CII encode metabolic functions which are essential under particular growth conditions.     

拟南芥AtrbohD和AtrbohF缺失对叶蛋白质组的影响

拟南芥NADPH氧化酶AtrbohD和AtrbohF在脱落酸（abscisic acid,ABA）抑制主根伸长、ABA诱导气孔关闭以及植物应答干旱、盐及病菌侵染等逆境胁迫反应中发挥重要作用,但这2个蛋白亚基缺失对拟南芥（Arabidopsis thaliana）蛋白质组的影响还未见报道。我们以营养土中生长16 d的野生型及AtrbohD和AtrbohF双基因突变体atrbohD1/F1叶片为材料进行蛋白组学分析,在双向电泳图谱上可分辨出约1 000个蛋白点,且蛋白表达谱存在差异。选取42个显著差异蛋白点进行MALDI-TOF/TOF质谱鉴定,成功鉴定出20个差异蛋白,这些蛋白主要与氧化还原、能量代谢、蛋白代谢、转录和信号传导等相关,还有一些蛋白功能未知。     

The design of oligonucleotide primers for the universal amplification of the N-acetylglucosaminidase gene (nag1) in Chytridiomycetes with emphasis on the anaerobic Neocallimastigales

The common feature of all chytridiomycetous fungi, aerobic as well as anaerobic, is an abundance of chitin in their cell wall. The genes coding for chitinases have therefore been widely used as phylogenetic markers in ascomycetes. As their utility for Chytridiomycetes has not been determined we chose the gene encoding an enzyme involved in chitin degradation and energy metabolism, the beta-(1,4)-N-acetylglucosaminidase (nag1). Primer pair Nag-forward and Nag-reverse was used to create PCR product from 5 strains of anaerobic and 7 strains of aerobic chytrids. However, Blast search of sequenced amplicons showed that these primers are specific only for fungus Emericella nidulans. Amino acid alignment of Nag1 proteins of fungal, protozoal and bacterial origin available in GenBank database was therefore performed. Five amino acid regions were found to be conserved enough to serve as a suitable domain for the design of a set of primers for the universal amplification of the nag1 gene in the Neocallimastigales fungi.