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1.
Two genes, pbpA (orf18) and pbp2 (orf19) located on the downstream of clavulanic acid (CA) gene cluster of Streptomyces clavuligerus were cloned into pET-28a(+), and confirmed to encode a family of high molecular-weight penicillin-binding proteins (PBPs). Both genes were amplified from genomic DNA by PCR and expressed in E. coli BL21 (DE3). Hydropathy plots of the proteins revealed a single stretch of hydrophobic amino acids indicating them to be transmembrane proteins. Pbp2 had lower affinity to penicillin G compared to PbpA, and was essential to the cell growth in contrast to PbpA. Revisions requested 3 November 2005; Revisions received 13 December 2005  相似文献   

2.
A genomic analysis of putative penicillin-binding proteins (PBPs) that are involved in the synthesis of the peptidoglycan layer of the cell wall and are encoded in 12 cyanobacterial genomes was performed in order to help elucidate the role(s) of these proteins in peptidoglycan synthesis, especially during cyanobacterial cellular differentiation. The analysis suggested that the minimum set of PBPs needed to assemble the peptidoglycan layer in cyanobacteria probably does not exceed one bifunctional transpeptidase–transglycosylase Class A high-molecular-weight PBP; two Class B high-molecular-weight PBPs, one of them probably involved in cellular elongation and the other in septum formation; and one low-molecular-weight PBP. The low-molecular-weight PBPs of all of the cyanobacteria analyzed are putative endopeptidases and are encoded by fewer genes than in Escherichia coli. We show that in Anabaena sp. strain PCC 7120, predicted proteins All2981 and Alr4579, like Alr5101, are Class A high-molecular-weight PBPs that are required for the functional differentiation of aerobically diazotrophic heterocysts, indicating that some members of this class of PBPs are required specifically for cellular developmental processes.  相似文献   

3.
The regimen conferring competence for uptake of transforming DNA is shown to render Escherichia coli osmolabile. Three different K-12 strains were exposed to the standard procedure of competence induction, i.e. incubation in the presence of 0.1 M Ca2+ or Mg2+ for 50 min at 0°C, interrupted by a heat shock for 5 min at 37°C. Upon osmotic challenge of competent cells formation of protoplasts was observed in approximately 2% of the treated cells. Incubation of competent cells of strain W1485 in phosphate-buffered saline for 1, 2, and 3 h reduced the viable counts to 67, 58, and 41%, respectively. Competence induction with divalent cations altered the affinity of penicillin-binding proteins (PBPs) for [125I]ampicillin. In isolated cell envelopes the presence of Ca2+ and Mg2+ stimulated the binding of [125I]ampicillin to PBPs 1, 3, 4, 5, and 6, whereas the binding to PBP 2 remained unchanged. The binding to PBP 1 C was inhibited by 0.23 M Ca2+. In living cells the binding to PBPs 1, 3, and 4 was enhanced, while the binding to PBP 8 was inhibited. Newly [125I]ampicillin-labelled proteins of M r 55,000 and 45,000 were apparent, especially after competence induction with Ca2+. Interaction of divalent cations with PBPs is suggested to contribute to osmolability of competent cells. Disintegration of the cell wall may be necessary for uptake of transforming DNA.Abbreviations PBP(s) penicillin-binding protein(s) - PBS phosphate-buffered saline - k kilodaltons - SDS sodium dodecyl sulfate  相似文献   

4.
Spheroplasts of the unstable l-form of Proteus mirabilis with fragile, shape defective cell walls grown in medium containing 120 mg/l penicillin G and then killed and permeabilized by ether treatment, were capable of in vitro synthesis of peptidoglycan from the precursors UDP-GlcNAc and UDP-MurNAc-l-Ala-d-Glu(ms-A2pm-d-Ala-d-Ala). The in vitro peptidoglycan was extensively peptide-crosslinked, indicating a continuing function of peptidoglycan transpeptidase in the spheroplasts. The seven penicillin-binding proteins (PBPs) of P. mirabilis with their functions as multiple peptidoglycan transpeptidases were shown to be saturated in the spheroplasts and thereby functionally inactivated by the penicillin of the growth medium to a very different degree. Complete or almost complete saturation occurred with the PBPs 1A, 1B, and 3, for which functions as indispensible transpeptidases in Escherichia coli have been postulated. In contrast, PBPs 5 and 6 were not saturated in the l-form spheroplasts. Transpeptidase function has been described previously in PBP 5 of P. mirabilis. The working hypothesis is proposed that synthesis of the functionally defective peptidoglycan of l-form spheroplasts in the presence of penicillin takes place with transpeptidase function of PBP 5.Dedicated to Professor Dr. H.-G. Schlegel on the occasion of his 60th birthday  相似文献   

5.
The aim of this study was to analyse the chemical composition of peptidoglycan and the state of some of the enzymes involved in its metabolism in Escherichia coli KN126 in the viable but nonculturable (VBNC) state which is a survival strategy adopted by bacteria (including those of medical interest) when exposed to environmental stresses. When entering the VBNC state, E. coli cells miniaturised and became coccus-shaped. Analysis of peptidoglycan chemical composition, by separation in HPLC of muropeptides released by muramidase digestion of purified peptidoglycan, indicated a high degree of cross-linking, a threefold increase in unusual DAP–DAP cross-linking, an increase in muropeptides bearing covalently bound lipoprotein, and a shortening of the average length of glycan strands in comparison with dividing cells. Analysis of penicillin-binding proteins (PBPs), enzymes involved in the terminal stage of peptidoglycan assembly showed the disappearance of high-molecular-weight PBPs 1A, 1B, 2, and 3 in VBNC cells. Finally, VBNC cells displayed an autolytic capability which was far higher than that of exponentially growing cells. It is suggested that part of these alterations of peptidoglycan may be connected with the VBNC state. Received: 20 March 2001 / Accepted: 7 June 2001  相似文献   

6.
The penicillin-binding proteins of 11 pathogenic Escherichia coli strains, including enteropathogenic, enterotoxigenic, enteroinvasive, enteroaggregative, and enterohemorrhagic E. coli, were detected in gels following the labeling of isolated cell envelopes with [3H]benzylpenicillin. The electrophoretic profiles, sensitivities to and morphological changes induced by β-lactam antibiotics showed that the penicillin-binding proteins of most pathogenic E. coli possess structural and physiological functions similar to those of E. coli K12.  相似文献   

7.
Transposon mutagenesis of Anabaena sp. strain PCC7120 led to the isolation of a mutant strain, SNa1, which is unable to fix nitrogen aerobically but is perfectly able to grow with combined nitrogen (i.e., nitrate). Reconstruction of the transposon mutation of SNa1 in the wild-type strain reproduced the phenotype of the original mutant. The transposon had inserted within an open reading frame whose translation product shows significant homology with a family of proteins known as high-molecular-weight penicillin-binding proteins (PBPs), which are involved in the synthesis of the peptidoglycan layer of the cell wall. A sequence similarity search allowed us to identify at least 12 putative PBPs in the recently sequenced Anabaena sp. strain PCC7120 genome, which we have named and organized according to predicted molecular size and the Escherichia coli nomenclature for PBPs; based on this nomenclature, we have denoted the gene interrupted in SNal as pbpB and its product as PBP2. The wild-type form of pbpB on a shuttle vector successfully complemented the mutation in SNa1. In vivo expression studies indicated that PBP2 is probably present when both sources of nitrogen, nitrate and N2, are used. When nitrate is used, the function of PBP2 either is dispensable or may be substituted by other PBPs; however, under nitrogen deprivation, where the differentiation of the heterocyst takes place, the role of PBP2 in the formation and/or maintenance of the peptidoglycan layer is essential.  相似文献   

8.
Aquaporin Z (AqpZ), a typical orthodox aquaporin with six transmembrane domains, was expressed as a fusion protein with TrxA in E. coli in our previous work. In the present study, three fusion partners (DsbA, GST and MBP) were employed to improve the expression level of this channel protein in E. coli. The result showed that, compared with the expression level of TrxA-AqpZ, five- to 40-fold increase in the productivity of AqpZ with fusion proteins was achieved by employing these different fusion partners, and MBP was the most efficient fusion partner to increase the expression level. By using E. coli C43 (DE3)/pMAL-AqpZ, the effects of different expression conditions were investigated systematically to improve the expression level of MBP-AqpZ in E. coli. The high productivity of MBP-AqpZ (200 mg/l) was achieved under optimized conditions. The present work provides a novel approach to improve the expression level of membrane proteins in E. coli.  相似文献   

9.
TLHS1 is a class I low molecular weight heat-shock protein (LMW HSP) of tobacco (Nicotiana tabacum). For a functional study of TLHS1, a recombinant DNA coding for TLHS1 with a hexahistidine tag at the aminoterminus was constructed and expressed in Escherichia coli. An expressed fusion protein, H6TLHS1, was purified using a Ni2+ affinity column and a Sephacryl S400 HR column. A polyclonal antibody against H6TLHS1 was produced to follow the fate of H6TLHS1 in E. coli. The fusion protein in E. coli maintained its solubility at a temperature of up to 90°C and most of the proteins in the E. coli cell lysate with H6TLHS1 were prevented from thermally induced aggregation at up to 90°C. We compared the viability of E. coli cells expressing H6TLHS1 to the E. coli cells without H6TLHS1 at a temperature of 50°C. After 8 h of high temperature treatment, E. coli cells with H6TLHS1 survived about three thousand times more than the bacterial cells without H6TLHS1. These results showed that a plant class I LMW HSP, TLHS1, can protect proteins of E. coli from heat denaturation, which could lead to a higher survival rate of the bacterial cells at high temperature.  相似文献   

10.
The penicillin-binding protein (PBP) profiles of 33Clostridium perfringens and sixClostridium species isolated from clinically significant infections were analyzed. Three new PBPs—PBPs 2B, 4B, and 5B (84, 70, and 49 kDa respectively)—and a high-molecular-weight PBP 6 (45 kDa) were demonstrated in theC. perfringens isolates. In addition to PBPs 1 and 2, PBPs 2B and 4B were seen to show low binding affinities for penicillin, although further studies are required to determine their possible roles in the development of penicillin resistance. The PBP profiles of theC. perfringens isolates were complex. Variations in apparent molecular weights (M r s) of all PBPs, with the exception of PBP 5 and the presence or absence of PBPs 2, 3, and 4B, gave rise to nine different PBP patterns. The high-M rPBPs 5 and 6, which exhibited high-penicillin-binding affinities, were with only one exception consistent within theC. perfringens isolates. These PBPs 5 and 6 of theC. perfringens isolates and independent PBPs found in the otherClostridium species studied indicate that PBP analysis may assist in the differentiation ofClostridium spacies.  相似文献   

11.
Random screening for inhibitors of chromosome partitioning in Escherichia coli was done by the anucleate cell blue assay. A novel S-benzylisothiourea derivative, S-(3,4-dichlorobenzyl)isothiourea, tentatively named A22, was found to induce spherical cells and spherical anucleate cells in E. coli. Mecillinam, a specific inhibitor of penicillin-binding protein 2, which induces spherical cells in E. coli, also caused anucleate cell production. Spherical cells induced by treatment with either A22 or mecillinam varied in size, and anucleate cells seemed to be more frequent among the smaller cells. These results suggest that loss of the rod shape in E. coli leads to asymmetric cell division that results in production of anucleate cells. No competition was observed even in the presence of a 10-fold excess A22 in an in vitro assay of 14C-penicillin G binding, but mecillinam specifically inhibited binding of 14C-penicillin G to penicillin-binding protein 2. Simultaneous treatment with mecillinam and cephalexin, a specific inhibitor of penicillin-binding protein 3, induced lysis of E. coli cells, but a combination of A22 and cephalexin did not. These results suggest that the target molecule(s) of A22 was not penicillin-binding protein 2. A22 may act on a rod-shape-determining protein(s) other than penicillin-binding protein 2, such as RodA or MreB.  相似文献   

12.
Gram-negative bacteria possess stress responses to maintain the integrity of the cell envelope. Stress sensors monitor outer membrane permeability, envelope protein folding, and energization of the inner membrane. The systems used by gram-negative bacteria to sense and combat stress resulting from disruption of the peptidoglycan layer are not well characterized. The peptidoglycan layer is a single molecule that completely surrounds the cell and ensures its structural integrity. During cell growth, new peptidoglycan subunits are incorporated into the peptidoglycan layer by a series of enzymes called the penicillin-binding proteins (PBPs). To explore how gram-negative bacteria respond to peptidoglycan stress, global gene expression analysis was used to identify Escherichia coli stress responses activated following inhibition of specific PBPs by the β-lactam antibiotics amdinocillin (mecillinam) and cefsulodin. Inhibition of PBPs with different roles in peptidoglycan synthesis has different consequences for cell morphology and viability, suggesting that not all perturbations to the peptidoglycan layer generate equivalent stresses. We demonstrate that inhibition of different PBPs resulted in both shared and unique stress responses. The regulation of capsular synthesis (Rcs) phosphorelay was activated by inhibition of all PBPs tested. Furthermore, we show that activation of the Rcs phosphorelay increased survival in the presence of these antibiotics, independently of capsule synthesis. Both activation of the phosphorelay and survival required signal transduction via the outer membrane lipoprotein RcsF and the response regulator RcsB. We propose that the Rcs pathway responds to peptidoglycan damage and contributes to the intrinsic resistance of E. coli to β-lactam antibiotics.  相似文献   

13.
Two kinds of spherical mutants, mrdA and mrdB mutants, have been isolated from Escherichia coli strain K12. The mrdA mutants have thermosensitive penicillin-binding protein 2, while the mrdB mutants have normal penicillin-binding proteins. Both kinds of mutants form spherical cells at 42°C and are resistant to the amidinopenicillin, mecillinam, at the same temperature. The two mutations have been mapped very close to lip at 14.2 min (revised chromosome linkage map, 1980) on the E. coli chromosome. Both mutations cause supersensitivities of cell growth to various β-lactam antibiotics, such as ampicillin, cephalexin, cefoxitin and nocardicin A at 42°C.  相似文献   

14.
Genetic identification of exported proteins in Streptococcus pneumoniae   总被引:18,自引:3,他引:15  
A strategy was developed to mutate and genetically identify exported proteins in Streptococcus pneumoniae. Vectors were created and used to screen pneumococcal DNA in Escherichia coli and S. pneumoniae for translational gene fusions to alkaline phosphatase (PhoA), Twenty five PhoA+ pneumococcal mutants were isolated and the loci from eight of these mutants showed similarity to known exported or membrane-associated proteins. Homologues were found to: (i) protein-dependent peptide permeases, (ii) penicillin-binding proteins, (iii) Cip proteases, (iv) two-component sensor regulators, (v) the phospho-enolpyruvate:carbohydrate phosphotransferase permeases, (vi) membrane-associated dehydrogenases, (vii) P-type (E1E2-type) cation transport ATPases, (viii) ABC transporters responsible for the translocation of the RTX class of bacterial toxins. Unexpectedly one PhoA+ mutant contained a fusion to a member of the DEAD protein family of ATP-dependent RNA helicases suggesting export of these proteins.  相似文献   

15.

Background  

Bacterial penicillin-binding proteins (PBPs) can be visualized by their ability to bind radiolabeled or fluorescent β-lactam derivatives both whole cells and membrane/cell enriched fractions. Analysis of the Listeria monocytogenes genome sequence predicted ten genes coding for putative PBPs, but not all of their products have been detected in studies using radiolabeled antibiotics, thus hindering their characterization. Here we report the positive identification of the full set of L. monocytogenes PBPs and the characteristics of the hitherto undescribed PBPD2 (Lmo2812).  相似文献   

16.
Overproduction of membrane proteins can be a cumbersome task, particularly if high yields are desirable. NADH:quinone oxidoreductase (Complex I) contains several very large membrane‐spanning protein subunits that hitherto have been impossible to express individually in any appreciable amounts in Escherichia coli. The polypeptides contain no prosthetic groups and are poorly antigenic, making optimization of protein production a challenging task. In this work, the C‐terminal ends of the Complex I subunits NuoH, NuoL, NuoM, and NuoN from E. coli Complex I and the bona fide antiporters MrpA and MrpD were genetically fused to the cytochrome c domain of Bacillus subtilis cytochrome c550. Compared with other available fusion‐protein tagging systems, the cytochrome c has several advantages. The heme is covalently bound, renders the proteins visible by optical spectroscopy, and can be used to monitor, quantify, and determine the orientation of the polypeptides in a plethora of experiments. For the antiporter‐like subunits NuoL, NuoM, and NuoN and the real antiporters MrpA and MrpD, unprecedented amounts of holo‐cytochrome fusion proteins could be obtained in E. coli. The NuoHcyt polypeptide was also efficiently produced, but heme insertion was less effective in this construct. The cytochrome c550 domain in all the fusion proteins exhibited normal spectra and redox properties, with an Em of about +170 mV. The MrpA and MrpD antiporters remained functional after being fused to the cytochrome c‐tag. Finally, a his‐tag could be added to the cytochrome domain, without any perturbations to the cytochrome properties, allowing efficient purification of the overexpressed fusion proteins.  相似文献   

17.
Strains isolated from rabbit, chicken, and rat feces and from sewage and fermented milk products, all identified asBifidobacterium animalis, were found to show phase variations in colony appearance and in cellular morphology. The rate of transition in a switching system from opaque to transparent colonies and vice versa was determined. Differences in protein components and in penicillin-binding proteins (PBPs) of the cells from different colony types are shown.  相似文献   

18.
The genes for the bacteriocins enterocin A and B were isolated from Enterococcus faecium ATB 197a. Using the pET37b(+) vector, the enterocin genes were fused to an Escherichia coli specific export signal sequence, a cellulose-binding domain (CBDcenA) and a S-tag under the control of a T7lac promotor. The constructs were subsequently cloned into E. coli host cells. The expression of the recombinant enterocins had different effects on both the host cells and other Gram-positive bacteria. The expression of entA in Esc. coli led to the synthesis and secretion of functional active enterocin A fusion proteins, which were active against some Gram-positive indicator bacteria, but did not influence the viability of the host cells. In contrast, the expression of enterocin B fusion proteins led to a reduced viability of the host cells, indicating a misfolding of the protein or interference with the cellular metabolism of Esc. coli. Indicator strains of Gram-positive bacteria were not inhibited by purified enterocin B fusion proteins. However, recombinant enterocin B displayed inhibitory activity after the proteolytic cleavage of the fused peptides.  相似文献   

19.
Periplasmic binding proteins (PBPs) are essential components of bacterial transport systems, necessary for bacterial growth and survival. The two‐domain structures of PBPs are topologically classified into three groups based on the number of crossovers or hinges between the globular domains: group I PBPs have three connections, group II have two, and group III have only one. Although a large number of structures for group I or II PBPs are known, fewer group III PBPs have been structurally characterized. Group I and II PBPs exhibit significant domain motions during transition from the unbound to ligand‐bound form, however, no large conformational changes have been observed to date in group III PBPs. We have solved the crystal structure of a periplasmic binding protein FitE, part of an iron transport system, fit, recently identified in a clinical E. coli isolate. The structure, determined at 1.8 Å resolution, shows that FitE is a group III PBP containing a single α‐helix bridging the two domains. Among the individual FitE molecules present in two crystal forms we observed three different conformations (open, closed, intermediate). Our crystallographic and molecular dynamics results strongly support the notion that group III PBPs also adopt the same Venus flytrap mechanism as do groups I and II PBPs. Unlike other group III PBPs, FitE forms dimers both in solution and in the crystals. The putative siderophore binding pocket is lined with arginine residues, suggesting an anionic nature of the iron‐containing siderophore. Proteins 2009. © 2008 Wiley‐Liss, Inc.  相似文献   

20.
Combined multidimensional liquid chromatography and electrospray ionisation tandem mass spectrometry was employed to analyse platinated tryptic peptides from Escherichia coli cells treated with the anticancer drug cis-[PtCl2(NH3)2] at pH 7.0. Prerequisites for the LC/LC/MS/MS analysis of protein targets that are fulfilled by cisplatin are (a) that the original protein binding sites have a high kinetic stability over the range 2.3 < pH < 8.5, and (b) that the metal fragment remains coordinated to a significant number of b+ and y+ peptide ions under MS/MS fragmentation conditions. Matching the MS/MS spectra of the platinated tryptic peptides to sequences of proteins in the E. coli database enabled the identification of 31 protein targets for cisplatin. Whereas six of these are high-abundance enzymes and ribosomal proteins in E. coli cells, five low-abundance DNA-binding proteins were also identified as specific targets. These include the DNA mismatch repair protein mutS, the DNA helicase II (uvrD) and topoisomerase I (top1). Two efflux proteins (acrD, mdtA), the redox regulator thioredoxin 1 (thiO) and the external filament-like type-1 fimbrial protein A chain (fimA1) were also characterised as specific cisplatin-binding proteins. Kinetically favoured carboxylate (D, E) and hydroxy (S, T, Y) O atoms were identified as the Pt coordination sites in 18 proteins and methionyl S atoms in 9 proteins.  相似文献   

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