金黄色葡萄球菌hmp基因缺失突变株的构建及抗一氧化氮能力分析

摘要：【目的】:构建金黄色葡萄球菌RN6390黄素血红蛋白(flavohaemoglobin, HMP)基因缺失突变株,研究其抗一氧化氮(Nitric Oxide, NO) 能力及其在细菌生物被膜形成中的作用。【方法】：根据同源重组技术的原理,利用PCR扩增RN6390的hmp基因上下游同源臂,经过抗生素和温度交替培养筛选hmp基因缺失突变株,利用基因组PCR、定量PCR对突变菌株进行鉴定。以硝普钠（SNP）为NO供体,检测了hmp基因缺失菌株的抗NO能力,并初步研究了hmp基因在生物被膜形成中的作用。【结果】：成功构建了RN6390的hmp基因缺失突变株,外源NO能够诱导菌株hmp基因的表达,hmp基因缺失菌株抗NO能力明显下降,但其生物被膜形成能力有明显提高。【结论】：获得了RN6390的hmp基因缺失突变株,该突变株的获得为进一步研究hmp基因的生物功能,以及细菌内源性NO的作用奠定了良好的技术平台。     

Role of signal peptides in targeting of proteins in cyanobacteria.

Proteins of cyanobacteria may be transported across one of two membrane systems: the typical eubacterial cell envelope (consisting of an inner membrane, periplasmic space, and an outer membrane) and the photosynthetic thylakoids. To investigate the role of signal peptides in targeting in cyanobacteria, Synechococcus sp. strain PCC 7942 was transformed with vectors carrying the chloramphenicol acetyltransferase reporter gene fused to coding sequences for one of four different signal peptides. These included signal peptides of two proteins of periplasmic space origin (one from Escherichia coli and the other from Synechococcus sp. strain PCC 7942) and two other signal peptides of proteins located in the thylakoid lumen (one from a cyanobacterium and the other from a higher plant). The location of the gene fusion products expressed in Synechococcus sp. strain PCC 7942 was determined by a chloramphenicol acetyltransferase enzyme-linked immunosorbent assay of subcellular fractions. The distribution pattern for gene fusions with periplasmic signal peptides was different from that of gene fusions with thylakoid lumen signal peptides. Primary sequence analysis revealed conserved features in the thylakoid lumen signal peptides that were absent from the periplasmic signal peptides. These results suggest the importance of the signal peptide in protein targeting in cyanobacteria and point to the presence of signal peptide features conserved between chloroplasts and cyanobacteria for targeting of proteins to the thylakoid lumen.     

Nitric oxide formation by Escherichia coli. Dependence on nitrite reductase,the NO-sensing regulator Fnr,and flavohemoglobin Hmp

Nitric oxide (NO) is a key signaling and defense molecule in biological systems. The bactericidal effects of NO produced, for example, by macrophages are resisted by various bacterial NO-detoxifying enzymes, the best understood being the flavohemoglobins exemplified by Escherichia coli Hmp. However, many bacteria, including E. coli, are reported to produce NO by processes that are independent of denitrification in which NO is an obligatory intermediate. We demonstrate using an NO-specific electrode that E. coli cells, grown anaerobically with nitrate as terminal electron acceptor, generate significant NO on adding nitrite. The periplasmic cytochrome c nitrite reductase (Nrf) is shown, by comparing Nrf+ and Nrf- mutants, to be largely responsible for NO generation. Surprisingly, an hmp mutant did not accumulate more NO but, rather, failed to produce detectable NO. Anaerobic growth of the hmp mutant was not stimulated by nitrate, and the mutant failed to produce periplasmic cytochrome(s) c, leading to the hypothesis that accumulating NO in the absence of Hmp inactivates the global anaerobic regulator Fnr by reaction with the [4Fe-4S]2+ cluster (Cruz-Ramos, H., Crack, J., Wu, G., Hughes, M. N., Scott, C., Thomson, A. J., Green, J., and Poole, R. K. (2002) EMBO J. 21, 3235-3244). Fnr thus failed to up-regulate nitrite reductase. The model is supported by the inability of an fnr mutant to generate NO and by the restoration of NO accumulation to hmp mutants upon introducing a plasmid encoding Fnr* (D154A) known to confer activity in the presence of oxygen. A cytochrome bd-deficient mutant retained NO-generating activity. The present study reveals a critical balance between NO-generating and -detoxifying activities during anaerobic growth.     

Characterization of an Escherichia coli rotA mutant, affected in periplasmic peptidyl-prolyl cis/trans isomerase

The rotA gene of Escherichia coli encodes a peptidyl-prolyl cis/trans isomerase (PPlase), which is supposed to catalyse protein folding in the periplasm. To investigate the importance of the enzyme, the rotA gene was cloned and a chromosomal deletion mutant was created. The rotA mutant was normally viable. No residual PPlase activity could be detected in the periplasmic fraction of the mutant. Comparison of the patterns of periplasmic and outer membrane proteins by SDS-PAGE revealed no differences in protein composition between the rotA mutant and its parental strain. Similarly, the kinetics of periplasmic protein folding and outer membrane protein assembly appeared unaffected by the rotA mutation. Our results show that the periplasmic PPlase of E. coli is not essential and that the protein does not play an important role in protein folding.     

Localisation of Helicobacter pylori catalase in both the periplasm and cytoplasm, and its dependence on the twin-arginine target protein, KapA, for activity

Helicobacter pylori induces a severe inflammatory response in the gastric mucosa. It is able to withstand the inflammatory response by producing proteins such as KatA and KapA. The C-terminus of KatA possesses a unique tetra-lysine motif not found in other catalases or other known protein sequences. Mutants deficient in this motif were constructed by site-directed mutagenesis. Cytoplasmic and periplasmic catalase activities were measured for the parental strain, a truncated KatA mutant (deficient in the unique C-terminal tetra-lysine motif) and a previously constructed KapA-deficient mutant (confirming previous observations regarding the possible periplasmic localisation of KatA). No differences were observed in the cytoplasmic catalase activities, however, the KapA-deficient mutant had approximately 5.5 times less catalase activity in the periplasmic extract when compared to the periplasmic preparations of either parental strain or KatA truncated mutant. N-terminal sequencing of KatA revealed no cleaved N-terminal signal peptide, indicating Sec-independent transport. These findings support previous reports that there is some form of interaction between KatA and KapA of H. pylori, an interaction which still needs to be characterised.     

Identification of major and minor chaperone proteins involved in the export of 987P fimbriae.

The 987P fimbriae of Escherichia coli consist mainly of the major subunit, FasA, and two minor subunits, FasF and FasG. In addition to the previously characterized outer membrane or usher protein FasD, the FasB, FasC, and FasE proteins are required for fimbriation. To better understand the roles of these minor proteins, their genes were sequenced and the predicted polypeptides were shown to be most similar to periplasmic chaperone proteins of fimbrial systems. Western blot (immunoblot) analysis and immunoprecipitation of various fas mutants with specific antibody probes identified both the subcellular localizations and associations of these minor components. FasB was shown to be a periplasmic chaperone for the major fimbrial subunit, FasA. A novel periplasmic chaperone, FasC, which stabilizes and specifically interacts with the adhesin, FasG, was identified. FasE, a chaperone-like protein, is also located in the periplasm and is required for optimal export of FasG and possibly other subunits. The use of different chaperone proteins for various 987P subunits is a novel observation for fimbrial biogenesis in bacteria. Whether other fimbrial systems use a similar tactic remains to be discovered.     

Septal Localization of FtsQ, an Essential Cell Division Protein in Escherichia coli

Septation in Escherichia coli requires several gene products. One of these, FtsQ, is a simple bitopic membrane protein with a short cytoplasmic N terminus, a membrane-spanning segment, and a periplasmic domain. We have constructed a merodiploid strain that expresses both FtsQ and the fusion protein green fluorescent protein (GFP)-FtsQ from single-copy chromosomal genes. The gfp-ftsQ gene complements a null mutation in ftsQ. Fluorescence microscopy revealed that GFP-FtsQ localizes to the division site. Replacing the cytoplasmic and transmembrane domains of FtsQ with alternative membrane anchors did not prevent the localization of the GFP fusion protein, while replacing the periplasmic domain did, suggesting that the periplasmic domain is necessary and sufficient for septal targeting. GFP-FtsQ localization to the septum depended on the cell division proteins FtsZ and FtsA, which are cytoplasmic, but not on FtsL and FtsI, which are bitopic membrane proteins with comparatively large periplasmic domains. In addition, the septal localization of ZipA apparently did not require functional FtsQ. Our results indicate that FtsQ is an intermediate recruit to the division site.     

Sterol carrier protein 2 (non-specific lipid transfer protein) is localized in membranous fractions of Leydig cells and Sertoli cells but not in germ cells.

The cellular and subcellular distribution of sterol carrier protein 2 (SCP2; nsL-TP) was reinvestigated in rat testicular cells by Western blotting and immunocytochemistry, using the affinity purified antibody against rat liver SCP2. Western blot analysis revealed high levels of the protein in the somatic cells of the testis, e.g., Leydig and Sertoli cells whereas it could not be detected in germ cells. This cellular localization of SCP2 was confirmed by Northern blotting. Immunocytochemical techniques revealed that in Leydig cells, immunoreactive proteins were concentrated in peroxisomes. Although SCP2 was also detected in Sertoli cells, a specific subcellular localization could not be shown. SCP2 was absent from germ cells. Analysis of subcellular fractions of Leydig cells showed that SCP2 is membrane bound without detectable amounts in the cytosolic fraction. These results are at variance with data published previously which suggested that in Leydig cells a substantial amount of SCP2 was present in the cytosol and that the distribution between membranes and cytosol was regulated by luteinizing hormone. The present data raise the question in what way SCP2 is involved in cholesterol transport between membranes in steroidogenic cells but also in non-steroidogenic cells.     

Topology and subcellular localization of FtsH protein in Escherichia coli.

FtsH protein in Escherichia coli is an essential protein of 70.7 kDa (644 amino acid residues) with a putative ATP-binding sequence. Western blots (immunoblots) of proteins from fractionated cell extracts and immunoelectron microscopy of the FtsH-overproducing strain showed exclusive localization of the FtsH protein in the cytoplasmic membrane. Most of the FtsH-specific labeling with gold particles was observed in the cytoplasmic membrane and the adjacent cytoplasm; much less was observed in the outer membrane and in the bulk cytoplasm. Genetic analysis by TnphoA insertions into ftsH revealed that the 25- to 95-amino-acid region, which is flanked by two hydrophobic stretchs, protrudes into the periplasmic space. From these results, we concluded that FtsH protein is an integral cytoplasmic membrane protein spanning the membrane twice and that it has a large cytoplasmic carboxy-terminal part with a putative ATP-binding domain. The average number of FtsH molecules per cell was estimated to be approximately 400.     

Flavohemoglobin Hmp,but not its individual domains,confers protection from respiratory inhibition by nitric oxide in Escherichia coli

Escherichia coli possesses a two-domain flavohemoglobin, Hmp, implicated in nitric oxide (NO) detoxification. To determine the contribution of each domain of Hmp toward NO detoxification, we genetically engineered the Hmp protein and separately expressed the heme (HD) and the flavin (FD) domains in a defined hmp mutant. Expression of each domain was confirmed by Western blot analysis. CO-difference spectra showed that the HD of Hmp can bind CO, but the CO adduct showed a slightly blue-shifted peak. Overexpression of the HD resulted in an improvement of growth to a similar extent to that observed with the Vitreoscilla hemeonly globin Vgb, whereas the FD alone did not improve growth. Viability of the hmp mutant in the presence of lethal concentrations of sodium nitroprusside was increased (to 30% survival after 2 h in 5 mM sodium nitroprusside) by overexpressing Vgb or the HD. However, maximal protection was provided only by holo-Hmp (75% survival under the same conditions). Cellular respiration of the hmp mutant was instantaneously inhibited in the presence of 13.5 microM NO but remained insensitive to NO inhibition when these cells overexpressed Hmp. When HD or FD was expressed separately, no significant protection was observed. By contrast, overexpression of Vgb provided partial protection from NO respiratory inhibition. Our results suggest that, despite the homology between the HD from Hmp and Vgb (45% identity), their roles seem to be quite distinct.     

Molecular construction of a multidrug exporter system, AcrAB: molecular interaction between AcrA and AcrB, and cleavage of the N-terminal signal sequence of AcrA

The AcrAB system of Escherichia coli is an intrinsic efflux protein with a broad substrate specificity. AcrA was thought to be localized in the periplasmic space, and to be linked to AcrB and TolC. The AcrAB-TolC system directly exports diverse substrates from the cell interior to the medium. In this study, we have determined the cellular localization of AcrA. By using the osmotic shock method, sucrose density gradient centrifugation, urea washing and Western blotting analysis, we reveal that AcrA is a peripheral inner membrane protein. A mutant plasmid encoding both the AcrA-TetBCt fusion protein and the AcrB-His fusion protein was constructed. Membrane vesicles prepared from cells expressing these fusion proteins were solubilized and AcrB-His was immunoprecipitated with an anti-polyhistidine antibody. After SDS-PAGE, Western blotting was performed with anti-TetBCt antiserum, resulting in the appearance of a 40 kDa band, indicating that AcrA co-precipitated with AcrB. Next we performed site-directed chemical labeling of Cys-introduced mutants of AcrA with [(14)C]N-ethylmaleimide. As judged from the labeling pattern and the molecular mass shift, the N-terminus of AcrA was removed and the mature protein is on the periplasmic surface. On the other hand, C25A mutants retained the N-terminal signal sequence on the cytoplasmic side of the membrane. We conclude that AcrA exists as a complex with AcrB on the periplasmic surface of the inner membrane after removal of the signal sequence.     

The nitrosative stress response of Staphylococcus aureus is required for resistance to innate immunity

Staphylococcus aureus is a highly virulent human pathogen with an extensive array of strategies to subvert the innate immune response. An important aspect of innate immunity is the production of the nitrogen monoxide radical (Nitric Oxide, NO.). Here we describe an adaptive response to nitrosative stress that allows S. aureus to replicate at high concentrations of NO.. Microarray analysis revealed 84 staphylococcal genes with significantly altered expression following NO. exposure. Of these, 30 are involved with iron-homeostasis, potentially under the control of the Fur regulator. Another seven induced genes are involved in hypoxic/fermentative metabolism, including the flavohaemoprotein, Hmp. The SrrAB two-component system has been shown to regulate the expression of many of the NO.-induced metabolic genes. Indeed, inactivation of hmp, srrAB and fur resulted in heightened NO. sensitivity. Hmp was responsible for c. 90% of measurable staphylococcal NO. consumption and therefore critical for efficient NO. detoxification. While SrrAB was required for maximal hmp expression, srrAB mutants still exhibited significant NO. scavenging and NO.-dependent induction of hmp. Yet S. aureus lacking SrrAB were more sensitive to nitrosative stress than hmp mutants, indicating that the contribution of SrrAB to NO. resistance extends beyond the regulation of hmp expression. Both Hmp and SrrAB were required for full virulence in a murine sepsis model, however, only the attenuation of the hmp mutant was restored by the abrogation of host NO. production. Thus, the S. aureus Hmp protein has evolved to serve as an iNOS-dependent virulence determinant.     

Molecular cloning of the gene which encodes beta-N-acetylglucosaminidase from a marine bacterium, Alteromonas sp. strain O-7.

The gene encoding the periplasmic beta-N-acetylglucosaminidase (GlcNAcase B) from a marine Alteromonas sp. strain, O-7, was cloned and sequenced. The protein sequence of GlcNAcase B revealed a highly significant homology with Vibrio GlcNAcase and alpha- and beta-chains of human beta-hexosaminidase.     

Plant-type and bacterial-type ferredoxins in a nitrogen-fixing cyanobacterium: Nostoc sp. strain PCC 73102

Abstract The cyanobacterium Nostoc sp. strain PCC 73102, cultured under nitrogen-fixing conditions, was investigated for the occurrence of ferrodoxins by SDS-PAGE/Western immunoblots using antisera directed against both a major plant-type and a bacterial-type ferredoxin purified from Anabaena variabilis . Immunocytological labelling and transmission electron microscopy were used to study the distribution of both types of ferredoxins in the Nostoc cells. SDS-PAGE/Western immunoblots revealed two proteins/polypeptides in the Nostoc strain, immunologically related to two soluble ferredoxins purified from Anabaena variabilis : the major plant-type ferredoxin (Fd I) and a bacterial-type ferredoxin (Fd III). Immunolocalization showed a uniform distribution of the plant-type and the bacterial-type ferredoxin in both the photosynthetic vegetative cells and in the nitrogen-fixing heterocysts, with no specific association with any subcellular inclusions. Using the particle analysis of an image processor, the labelling associated with the vegetative cells, expressed as number of gold particles per cell area, was found to be only slightly higher (1.2x) or almost twice as high (1.9x) compared to the heterocysts for the major plant-type and the bacterial-type ferredoxin, respectively.     

Localization of hydrogenase in Desulfovibrio gigas cells

The localization of hydrogenase protein in Desulfovibrio gigas cells grown either in lactate-sulfate or hydrogen-sulfate media, has been investigated by subcellular fractionation with immunoblotting and by electron microscopic immunocytochemistry. Subcellular fractionation experiments suggest that no integral membrane-bound hydrogenase is present in D. gigas. About 40% of the hydrogenase activity could be extracted by treatment of D. gigas cells with Tris-EDTA buffer. The rest of the soluble hydrogenase activity (50%) was found in the soluble fraction which was obtained after disruption of Tris-EDTA extracted cells and high speed centrifugation. Both soluble hydrogenase fractions purified to homogeneity showed identical molecular properties including the N-terminal aminoacid sequences of their large and small subunits. Polyacrylamide gel electrophoresis of the proteins of the subcellular fractions revealed a single band of hydrogenase activity exhibiting the same mobility as purified D. gigas hydrogenase. Western blotting carried out on these subcellular fractions revealed crossreactivity with the antibodies raised against (NiFe) hydrogenase. The lack of crossreactivity with antibodies against (FE) or (NiFeSe) hydrogenases, indicated that only (NiFe) type hydrogenase is present in D. gigas.Immunocytolocalization in ultrathin frozen sections of D. gigas cells grown either in lactate-sulfate, pyruvate-sulfate or hydrogen-sulfate media showed only a (NiFe) hydrogenase located in the periplasmic space. The bioenergetics of D. gigas are discussed in the light of these findings.     

The subcellular distribution of protein kinase Calpha, -epsilon, and -zeta isoforms during cardiac cell differentiation.

There is little information on the molecular events that control the subcellular distribution of protein kinase C during cardiac cell differentiation. We examined protein kinase C activity and the subcellular distribution of representatives of the "classical," "novel," and "atypical" protein kinase C's in P19 murine teratoma cells induced to undergo differentiation into cardiac myocytes by the addition of dimethylsulfoxide to the medium (Grepin et al., Development 124, 2387-2395, 1997). Differentiation was assessed by the presence of striated myosin, a morphological marker for cardiac cells. Addition of dimethyl sulfoxide to the medium resulted in the appearance of striated myosin by 10 days postincubation. Immunolocalization and Western blot studies revealed that a significant proportion of protein kinase Calpha, -epsilon, and -zeta were associated with the particulate fraction in P19 cells prior to differentiation. Differentiation into cardiac cells resulted in a translocation of protein kinase C activity from the particulate fraction to cytosol and localization of most of protein kinase Calpha, -epsilon, and -zeta to the cytoplasmic compartment. The total cellular protein kinase C activity was unaltered during differentiation. The translocation of protein kinase C activity during differentiation of P19 cells into cardiac myocytes was associated with a decrease in the levels of cellular 1, 2-diacyl-sn-glycerol. The cellular levels of phosphatidylserine and phosphatidylinositol did not change during differentiation. Addition of 1,2-dioctanoyl-sn-glycerol, a cell-permeant 1, 2-diacyl-sn-glycerol analog, reversed the differentiation-induced switch in the relative distribution of protein kinase C activity and dramatically increased the association of protein kinase Calpha with the particulate fraction. Addition of 1,2-dioctanoyl-sn-glycerol did not reverse the pattern of distribution for protein kinase Cepsilon or -zeta. The results indicate that protein kinase C activity and protein kinase Calpha, -epsilon and -zeta isoforms are redistributed from the particulate to the cytosolic fraction during differentiation of P19 cells into cardiomyocytes. The mechanism for the redistribution of protein kinase Calpha may be related to the reduction in the cellular 1,2-diacyl-sn-glycerol levels that accompany differentiation.     

HMP19 is a new metastasis suppressor in epithelial ovarian cancer

HMP19 is a neuron-specific gene; its expression product belongs to a family of neuronal proteins which can be found in numerous kinds of human cancers. However, the clinicopathological significance of HMP19 expression in epithelial ovarian cancer (EOC) is as yet unknown. In this study, protein expression levels of HMP19 in cancerous tissues were determined by tissue microarray immunohistochemistry analysis (TMA-IHC) (n = 117). HMP19 protein levels in cancer tissues were associated with clinical characteristics and overall survival rates of patients with EOC. It was found that both mRNA and protein levels of HMP19 were significantly lower in EOC than those in normal ovary or fallopian tube tissues (P<0.05). The protein expression level of HMP19 was significantly associated with a lower FIGO stage, a lower level of CA-125 and a lower presence of metastasis. Consistent with related adverse clinical pathological features, the overall survival (OS) rate of patients with low or non HMP19-expressing tumors was inferior compared to those with high HMP19-expressing tumors. This is in accordance with further studies that found high HMP19 protein level to be an independent prognostic factor for OS in EOC. Multivariate analysis demonstrated that tumor patients with low HMP19 expression had an exceedingly poor OS. HMP19 plays a role in metastasis/tumor suppression and offers a prognostic value for EOC. HMP19, as a new inhibitor, strongly inhibits metastasis and partially attenuates tumor growth in EOC.     

A species-specific periplasmic flagellar protein of Serpulina (Treponema) hyodysenteriae.

We have previously reported that a 46-kDa protein present in an outer membrane protein preparation seemed to be a species-specific antigen of Serpulina hyodysenteriae (Z. S. Li, N. S. Jensen, M. Bélanger, M.-C. L'Espérance, and M. Jacques, J. Clin. Microbiol. 30:2941-2947, 1992). The objective of this study was to further characterize this antigen. A Western blot (immunoblot) analysis and immunogold labeling with a monospecific antiserum against this protein confirmed that the protein was present in all S. hyodysenteriae reference strains but not in the nonpathogenic organism Serpulina innocens. The immunogold labeling results also indicated that the protein was associated with the periplasmic flagella of S. hyodysenteriae. N-terminal amino acid sequencing confirmed that the protein was in fact a periplasmic flagellar sheath protein. The molecular mass of this protein, first estimated to be 46 kDa by Western blotting, was determined to be 44 kDa when the protein was evaluated more precisely by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the protein was glycosylated, as determined by glycoprotein staining and also by N-glycosidase F treatment. Five other periplasmic flagellar proteins of S. hyodysenteriae, which may have been the core proteins and had molecular masses of 39, 35, 32, 30, and 29 kDa, were antigenically related and cross-reacted with the periplasmic flagellar proteins of S. innocens. Finally, serum from a pig experimentally infected with S. hyodysenteriae recognized the 44-kDa periplasmic flagellar sheath protein. Our results suggest that the 44-kDa periplasmic flagellar sheath protein of S. hyodysenteriae is a species-specific glycoprotein antigen.