Role of a highly conserved bacterial protein in outer membrane protein assembly

After transport across the cytoplasmic membrane, bacterial outer membrane proteins are assembled into the outer membrane. Meningococcal Omp85 is a highly conserved protein in Gram-negative bacteria, and its homolog Toc75 is a component of the chloroplast protein-import machinery. Omp85 appeared to be essential for viability, and unassembled forms of various outer membrane proteins accumulated upon Omp85 depletion. Immunofluorescence microscopy revealed decreased surface exposure of outer membrane proteins, which was particularly apparent at the cell-division planes. Thus, Omp85 is likely to play a role in outer membrane protein assembly.     

Role of 4.5S RNA in assembly of the bacterial signal recognition particle with its receptor

The mechanism by which a signal recognition particle (SRP) and its receptor mediate protein targeting to the endoplasmic reticulum or to the bacterial plasma membrane is evolutionarily conserved. In Escherichia coli, this reaction is mediated by the Ffh/4.5S RNA ribonucleoprotein complex (Ffh/4.5S RNP; the SRP) and the FtsY protein (the SRP receptor). We have quantified the effects of 4.5S RNA on Ffh-FtsY complex formation by monitoring changes in tryptophan fluorescence. Surprisingly, 4.5S RNA facilitates both assembly and disassembly of the Ffh-FtsY complex to a similar extent. These results provide an example of an RNA molecule facilitating protein-protein interactions in a catalytic fashion.     

鼠伤寒沙门氏菌鞭毛蛋白FliC的原核表达及纯化

对鼠伤寒沙门氏菌的鞭毛蛋白FliC的基因进行克隆、鉴定和原核表达,为鞭毛蛋白佐剂作用的研究奠定基础。以鼠伤寒沙门氏菌8014菌株基因组DNA为模板,PCR扩增Flic基因,产物与T载体连接,经测序鉴定正确后与表达载体pQE30(+)连接构建重组表达质粒Flic-pQE30,将此重组质粒转化入表达宿主E.coli XL1-Blue菌株内抽提质粒,酶切鉴定正确后对转化菌株以IPTG进行诱导后,表达产物经镍离子亲和层析柱纯化后,进行SDS-PAGE和Western blot分析。测序结果显示,FliC为完整的编码区基因1 485 bp,编码495个氨基酸残基,蛋白相对分子质量约为56 kDa。经SDS-PAGE分析表明,以37℃、1 mM的IPTG诱导5 h,表达效果最好,诱导产物是与理论值相符的56 kDa的融合蛋白。经western-blotting检测,表达的重组蛋白FliC可与小鼠抗鼠伤寒沙门氏菌全菌体多抗血清反应得到清晰的目的条带,表明表达的重组蛋白具有良好的反应原性。成功进行了鼠伤寒沙门氏菌鞭毛蛋白Flic基因的克隆表达,为进一步研究其免疫佐剂作用奠定了基础。     

鼠伤寒沙门氏茵鞭毛蛋白FIiC的原核表达及纯化

对鼠伤寒沙门氏菌的鞭毛蛋白FliC的基因进行克隆、鉴定和原核表达,为鞭毛蛋白佐剂作用的研究奠定基础。以鼠伤寒沙门氏菌8014菌株基因组DNA为模板,PCR扩增Flic基因,产物与T载体连接,经测序鉴定正确后与表达载体pQE30（＋）连接构建重组表达质粒Flic—pQE30,将此重组质粒转化人表达宿主E．coliXLI—Blue菌株内抽提质粒,酶切鉴定正确后对转化菌株以1PTG进行诱导后,表达产物经镍离子亲和层析柱纯化后,进行SDS—PAGE和Westernblot分析。测序结果显示,FliC为完整的编码区基因1485bp,编码495个氨基酸残基,蛋白相对分子质量约为56kDa。经SDS—PAGE分析表明,以37℃、1mM的IPTG诱导5h,表达效果最好,诱导产物是与理论值相符的56kDa的融合蛋白。经western—blotting检测,表达的重组蛋白FliC可与小鼠抗鼠伤寒沙门氏菌全菌体多抗血清反应得到清晰的目的条带,表明表达的重组蛋白具有良好的反应原性。成功进行了鼠伤寒沙门氏菌鞭毛蛋白nic基因的克隆表达,为进一步研究其免疫佐剂作用奠定了基础。     

Crystallization of a sulfate-binding protein (permease) from Salmonella typhimurium

Crystallization is reported of a protein that is a component of an active transport system for sulfate into Salmonella typhimurium. This appears to be the component that is specific for binding the substrate.     

Role of inorganic polyphosphate in promoting ribosomal protein degradation by the Lon protease in E. coli

Inorganic polyphosphate (polyP), a polymer of hundreds of phosphate (Pi) residues, accumulates in Escherichia coli in response to stresses, including amino acid starvation. Here we show that the adenosine 5'-triphosphate-dependent protease Lon formed a complex with polyP and degraded most of the ribosomal proteins, including S2, L9, and L13. Purified S2 also bound to polyP and formed a complex with Lon in the presence of polyP. Thus, polyP may promote ribosomal protein degradation by the Lon protease, thereby supplying the amino acids needed to respond to starvation.     

Role of C. elegans TAT-1 protein in maintaining plasma membrane phosphatidylserine asymmetry

The asymmetrical distribution of phospholipids on the plasma membrane is critical for maintaining cell integrity and physiology and for regulating intracellular signaling and important cellular events such as clearance of apoptotic cells. How phospholipid asymmetry is established and maintained is not fully understood. We report that the Caenorhabditis elegans P-type adenosine triphosphatase homolog, TAT-1, is critical for maintaining cell surface asymmetry of phosphatidylserine (PS). In animals deficient in tat-1, PS is abnormally exposed on the cell surface, and normally living cells are randomly lost through a mechanism dependent on PSR-1, a PS-recognizing phagocyte receptor, and CED-1, which contributes to recognition and engulfment of apoptotic cells. Thus, tat-1 appears to function in preventing appearance of PS in the outer leaflet of plasma membrane, and ectopic exposure of PS on the cell surface may result in removal of living cells by neighboring phagocytes.     

A yeast actin-binding protein is encoded by SAC6, a gene found by suppression of an actin mutation

The protein encoded by SAC6, a gene that can mutate to suppress a temperature-sensitive defect in the yeast actin gene, has been identified as a 67-kilodalton actin-binding protein (ABP 67) that associates with all identifiable actin structures. This finding demonstrates the in vivo functional importance of the actin-ABP 67 interaction previously established in vitro and illustrates the use of suppressor analysis to identify physically interacting proteins.     

Distribution of protein and RNA in the 30S ribosomal subunit

In Escherichia coli, the small ribosomal subunit has a sedimentation coefficient of 30S, and consists of a 16S RNA molecule of 1541 nucleotides complexed with 21 proteins. Over the last few years, a controversy has emerged regarding the spatial distribution of RNA and protein in the 30S subunit. Contrast variation with neutron scattering was used to suggest that the RNA was located in a central core of the subunit and the proteins mainly in the periphery, with virtually no separation between the centers of mass of protein and RNA. However, these findings are incompatible with the results of efforts to locate individual ribosomal proteins by immune electron microscopy and triangulation with interprotein distance measurements. The conflict between these two views is resolved in this report of small-angle neutron scattering measurements on 30S subunits with and without protein S1, and on subunits reconstituted from deuterated 16S RNA and unlabeled proteins. The results show that (i) the proteins and RNA are intermingled, with neither component dominating at the core or the periphery, and (ii) the spatial distribution of protein and RNA is asymmetrical, with a separation between their centers of mass of about 25 angstroms.     

A sorting platform determines the order of protein secretion in bacterial type III systems

Bacterial type III protein secretion systems deliver effector proteins into eukaryotic cells in order to modulate cellular processes. Central to the function of these protein-delivery machines is their ability to recognize and secrete substrates in a defined order. Here, we describe a mechanism by which a type III secretion system from the bacterial enteropathogen Salmonella enterica serovar Typhimurium can sort its substrates before secretion. This mechanism involves a cytoplasmic sorting platform that is sequentially loaded with the appropriate secreted proteins. The sequential loading of this platform, facilitated by customized chaperones, ensures the hierarchy in type III protein secretion. Given the presence of these machines in many important pathogens, these findings can serve as the bases for the development of novel antimicrobial strategies.     

GFI LANguard N.S.S在网络漏洞扫描中的应用

漏洞扫描技术从一个新的角度解决了网络安全问题。GFI LANguard Network Security Scanner作为一个网络漏洞扫描系统,能够扫描、检测、评估和修复网络中的任何安全漏洞,使用GFI LANguard N.S.S可以更有效地解决漏洞管理的主要问题。     

A bacterial protein enhances the release and efficacy of liposomal cancer drugs

Clostridium novyi-NT is an anaerobic bacterium that can infect hypoxic regions within experimental tumors. Because C. novyi-NT lyses red blood cells, we hypothesized that its membrane-disrupting properties could be exploited to enhance the release of liposome-encapsulated drugs within tumors. Here, we show that treatment of mice bearing large, established tumors with C. novyi-NT plus a single dose of liposomal doxorubicin often led to eradication of the tumors. The bacterial factor responsible for the enhanced drug release was identified as a previously unrecognized protein termed liposomase. This protein could potentially be incorporated into diverse experimental approaches for the specific delivery of chemotherapeutic agents to tumors.     

Regulated pole-to-pole oscillations of a bacterial gliding motility protein

Little is known about directed motility of bacteria that move by type IV pilus-mediated (twitching) motility. Here, we found that during periodic cell reversals of Myxoccocus xanthus, type IV pili were disassembled at one pole and reassembled at the other pole. Accompanying these reversals, FrzS, a protein required for directed motility, moved in an oscillatory pattern between the cell poles. The frequency of the oscillations was controlled by the Frz chemosensory system, which is essential for directed motility. Pole-to-pole migration of FrzS appeared to involve movement along a filament running the length of the cell. FrzS dynamics may thus regulate cell polarity during directed motility.