Identification of a penicillin-binding protein 3 homolog, PBP3x, in Pseudomonas aeruginosa: gene cloning and growth phase-dependent expression.

A homolog of Pseudomonas aeruginosa penicillin-binding protein 3 (PBP3), named PBP3x in this study, was identified by using degenerate primers based on conserved amino acid motifs in the high-molecular-weight PBPs. Analysis of the translated sequence of the pbpC gene encoding this PBP3x revealed that 41 and 48% of its amino acids were identical to those of Escherichia coli and P. aeruginosa PBP3s, respectively. The downstream sequence of pbpC encoded convergently transcribed homologs of the E. coli soxR gene and the Mycobacterium bovis adh gene. The pbpC gene product was expressed from the T7 promoter in E. coli and was exported to the cytoplasmic membrane of E. coli cells and could bind [3H] penicillin. By using a broad-host-range vector, pUCP27, the pbpC gene was expressed in P. aeruginosa PAO4089. [3H]penicillin-binding competition assays indicated that the pbpC gene product had lower affinities for several PBP3-targeted beta-lactam antibiotics than P. aeruginosa PBP3 did, and overexpression of the pbpC gene product had no effect on the susceptibility to the PBP3-targeted antibiotics tested. By gene replacement, a PBP3x-defective interposon mutant (strain HC132) was obtained and confirmed by Southern blot analysis. Inactivation of PBP3x caused no changes in the cell morphology or growth rate of exponentially growing cells, suggesting that pbpC was not required for cell viability under normal laboratory growth conditions. However, the upstream sequence of pbpC contained a potential sigma(s) recognition site, and pbpC gene expression appeared to be growth rate regulated. [3H]penicillin-binding assays indicated that PBP3 was mainly produced during exponential growth whereas PBP3x was produced in the stationary phase of growth.     

Identification of a Novel Penicillin-Binding Protein from Helicobacter pylori

The Helicobacter pylori genome encodes four penicillin-binding proteins (PBPs). PBPs 1, 2, and 3 exhibit similarities to known PBPs. The sequence of PBP 4 is unique in that it displays a novel combination of two highly conserved PBP motifs and an absence of a third motif. Expression of PBP 4, but not PBP 1, 2, or 3, is significantly increased during mid- to late-log-phase growth.     

Crystal structure of the Bacillus subtilis penicillin-binding protein 4a, and its complex with a peptidoglycan mimetic peptide

The genome of Bacillus subtilis encodes 16 penicillin-binding proteins (PBPs) involved in the synthesis and/or remodelling of the peptidoglycan during the complex life cycle of this sporulating Gram-positive rod-shaped bacterium. PBP4a (encoded by the dacC gene) is a low-molecular mass PBP clearly exhibiting in vitro DD-carboxypeptidase activity. We have solved the crystal structure of this protein alone and in complex with a peptide (D-alpha-aminopymelyl-epsilon-D-alanyl-D-alanine) that mimics the C-terminal end of the Bacillus peptidoglycan stem peptide. PBP4a is composed of three domains: the penicillin-binding domain with a fold similar to the class A beta-lactamase structure and two domains inserted between the conserved motifs 1 and 2 characteristic of the penicillin-recognizing enzymes. The soaking of PBP4a in a solution of D-alpha-aminopymelyl-epsilon-D-alanyl-D-alanine resulted in an adduct between PBP4a and a D-alpha-aminopimelyl-epsilon-D-alanine dipeptide and an unbound D-alanine, i.e. the products of acylation of PBP4a by D-alpha-aminopymelyl-epsilon-D-alanyl-D-alanine with the release of a D-alanine. The adduct also reveals a binding pocket specific to the diaminopimelic acid, the third residue of the peptidoglycan stem pentapeptide of B. subtilis. This pocket is specific for this class of PBPs.     

Site-directed mutagenesis of the mecA gene from a methicillin-resistant strain of Staphylococcus aureus.

The mecA-27r gene from Staphylococcus aureus 27r encodes penicillin-binding protein 2a (PBP2a-27r), which causes this strain to be methicillin resistant. Removal or replacement of the N-terminal transmembrane domain had no effect on binding of penicillin, but removal of portions of the putative transglycosylase domain (144, 245, or 341 amino acids after the transmembrane region) destroyed penicillin-binding activity. The SXXK, SXN, and KSG motifs, present in all penicillin-interacting enzymes, were found in the expected linear spatial arrangement within the putative transpeptidase region of PBP2a-27r. Alterations of amino acids in all three of these motifs resulted in elimination of penicillin-binding activity, confirming their roles in the interaction with penicillin.     

Role of penicillin-binding protein 2 (PBP2) in the antibiotic susceptibility and cell wall cross-linking of Staphylococcus aureus: evidence for the cooperative functioning of PBP2, PBP4, and PBP2A

Ceftizoxime, a beta-lactam antibiotic with high selective affinity for penicillin-binding protein 2 (PBP2) of Staphylococcus aureus, was used to select a spontaneous resistant mutant of S. aureus strain 27s. The stable resistant mutant ZOX3 had an increased ceftizoxime MIC and a decreased affinity of its PBP2 for ceftizoxime and produced peptidoglycan in which the proportion of highly cross-linked muropeptides was reduced. The pbpB gene of ZOX3 carried a single C-to-T nucleotide substitution at nucleotide 1373, causing replacement of a proline with a leucine at amino acid residue 458 of the transpeptidase domain of the protein, close to the SFN conserved motif. Experimental proof that this point mutation was responsible for the drug-resistant phenotype, and also for the decreased PBP2 affinity and reduced cell wall cross-linking, was provided by allelic replacement experiments and site-directed mutagenesis. Disruption of pbpD, the structural gene of PBP4, in either the parental strain or the mutant caused a large decrease in the highly cross-linked muropeptide components of the cell wall and in the mutant caused a massive accumulation of muropeptide monomers as well. Disruption of pbpD also caused increased sensitivity to ceftizoxime in both the parental cells and the ZOX3 mutant, while introduction of the plasmid-borne mecA gene, the genetic determinant of the beta-lactam resistance protein PBP2A, had the opposite effects. The findings provide evidence for the cooperative functioning of two native S. aureus transpeptidases (PBP2 and PBP4) and an acquired transpeptidase (PBP2A) in staphylococcal cell wall biosynthesis and susceptibility to antimicrobial agents.     

Differential functionalities of amphiphilic peptide segments of the cell-septation penicillin-binding protein 3 of Escherichia coli

The class B M1-V577 penicillin-binding protein (PBP) 3 of Escherichia coli consists of a M1-L39 membrane anchor (bearing a cytosolic tail) that is linked via a G40-S70 intervening peptide to an R71-I236 non-catalytic module (containing the conserved motifs 1-3) itself linked via motif 4 to a D237-V577 catalytic module (containing the conserved motifs 5-7 of the penicilloyl serine transferases superfamily). It has been proposed that during cell septation the peptidoglycan crosslinking activity of the acyl transferase module of PBP3 is regulated by the associated M1-I236 polypeptide itself in interaction with other components of the divisome. The fold adopted by the R71-V577 polypeptide of PBP3 has been modelled by reference to the corresponding R76-S634 polypeptide of the class B Streptococcus pneumoniae PBP2x. Based on these data and the results of site-directed mutagenesis of motifs 1-3 and of peptide segments of high amphiphilicity (identified from hydrophobic moment plots), the M1-I236 polypeptide of PBP3 appears to be precisely designed to work in the way proposed. The membrane anchor and the G40-S70 sequence (containing the G57-Q66 peptide segment) upstream from the non-catalytic module have the information ensuring that PBP3 undergoes proper insertion within the divisome at the cell septation site. Motif 1 and the I74-L82 overlapping peptide segment, motif 2 and the H160-G172 overlapping peptide segment, and the G188-D197 motif 3 are located at or close to the intermodule junction. They contain the information ensuring that PBP3 folds correctly and the acyl transferase catalytic centre adopts the active configuration. The E206-V217 peptide segment is exposed at the surface of the non-catalytic module. It has the information ensuring that PBP3 fulfils its cell septation activity within the fully complemented divisome.     

Molecular analysis of a beta-lactam resistance gene encoded within the cephamycin gene cluster of Streptomyces clavuligerus.

A Streptomyces clavuligerus gene (designated pcbR) which is located immediately downstream from the gene encoding isopenicillin N synthase in the cephamycin gene cluster was characterized. Nucleotide sequence analysis and database searching of PcbR identified a significant similarity between PcbR and proteins belonging to the family of high-molecular-weight group B penicillin-binding proteins (PBPs). Eight of nine boxes (motifs) conserved within this family of proteins are present in the PcbR protein sequence in the same order and with approximately the same spacing between them. When a mutant disrupted in pcbR was constructed by gene replacement, the resulting pcbR mutant exhibited a significant decrease in its resistance to benzylpenicillin and cephalosporins, indicating that pcbR is involved in beta-lactam resistance in this organism. Western blot (immunoblot) analysis of S. clavuligerus cell membranes using PcbR-specific antibodies suggested that PcbR is a membrane protein. PcbR was also present in cell membranes when expressed in Escherichia coli and was able to bind radioactive penicillin in a PBP assay, suggesting that PcbR is a PBP. When genomic DNAs from several actinomycetes were probed with pcbR, hybridization was observed to some but not all beta-lactam-producing actinomycetes.     

Characterization of HMW-PBPs from the rod-shaped actinomycete Corynebacterium glutamicum: peptidoglycan synthesis in cells lacking actin-like cytoskeletal structures

Analysis of the complete genome sequence of Corynebacterium glutamicum indicated that, in addition to ftsI, there are eight proteins with sequence motifs that are strongly conserved in penicillin binding proteins (PBPs): four genes that code for high-molecular-weight (HMW)-PBPs (PBP1a, PBP1b, PBP2a and PBP2b), two genes encoding low-molecular-weight PBPs (PBP4 and PBP4b) and two probable beta-lactamases (PBP5 and PBP6). Here, the function of the four HMW-PBPs in C. glutamicum was investigated using a combination of genetic knockouts, enhanced green fluorescent protein 2 (EGFP2) fusions and penicillin staining of membrane preparations. The four HMW-PBPs were expressed in a growing culture of C. glutamicum, but none of four pbp genes was individually essential for the growth of the bacterium, and only the simultaneous disruption of both pbp1b and pbp2b was lethal. The fused EGFP2-PBP proteins were functional in vivo, which allowed correct determination of their cellular localization. EGFP2 fusions to PBP1a, PBP1b and PBP2b localized at the poles and at the septum, whereas EGFP2-PBP2a was predominantly found at the septum. Cefsulodin treatment specifically delocalized PBP1a and PBP1b (class A HMW-PBPs), whereas mecillinam caused the specific delocalization of PBP2b and PBP2a (class B HMW-PBPs). The results provide new insight into the mechanisms involved in the synthesis of the cell wall in this bacterial species, which lacks a known actin-like cytoskeletal structure.     

An evolutionary basis for scrapie disease: identification of a fish prion mRNA

Infectious prion proteins cause neurodegenerative disease in mammals owing to the acquisition of an aberrant conformation. We cloned a Fugu rubripes gene that encodes a structurally conserved prion protein, and found rapid rates of molecular divergence among prions from different vertebrate classes, along with molecular stasis within each class. We propose that a directional trend in the evolution of prion sequence motifs associated with pathogenesis and infectivity could account for the origin of scrapie in mammals.     

耐甲氧西林金黄色葡萄球菌PBP2a转肽酶区的克隆表达及纯化鉴定

目的:对编码耐甲氧西林金黄色葡萄球菌(MRSA)青霉素结合蛋白2a(PBP2a)转肽酶区的mecA基因片段进行克隆、表达、纯化及鉴定。方法:根据基因文库登录的mecA基因的编码序列,设计合成了一对寡核苷酸引物,应用PCR技术从MRSA基因组DNA中扩增获得编码PBP2a转肽酶区的DNA片段,将此目的基因片段克隆至pET-His载体,经酶切鉴定、测序正确后,转化E.coliBL21(DE3)plysS;用IPTG进行诱导表达后,利用Ni2 亲和层析技术从表达蛋白中纯化目的蛋白;对表达的蛋白以MRSA胶乳凝集试剂盒进行鉴定。结果:成功构建了PBP2a转肽酶区原核表达载体,并获得了高效表达,制备了高纯度的目的蛋白。结论:获得了高纯度的PBP2a转肽酶区蛋白,为其进一步研究奠定了基础。     

A novel member of murine Polycomb-group proteins, Sex comb on midleg homolog protein, is highly conserved, and interacts with RAE28/mph1 in vitro

The Polycomb group of (PcG) genes were originally described in Drosophila, but many PcG genes have mammalian homologs. Genetic studies in flies and mice show that mutations in PcG genes cause posterior transformations caused by failure to maintain repression of homeotic loci, suggesting that PcG proteins have conserved functions. The Drosophila gene Sex comb on midleg (Scm) encodes an unusual PcG protein that shares motifs with the PcG protein polyhomeotic, and with a Drosophila tumor suppressor, lethal(3)malignant brain tumor (l(3)mbt). Expressed sequence tag (EST) databases were searched to recover putative mammalian Scm homologs, which were used to screen murine cDNA libraries. The recovered cDNA encodes two mbt repeats and the SPM domain that characterize Scm, but lacks the cysteine clusters and the serine/threonine-rich region found at the amino terminus of Scm. Accordingly, we have named the gene Sex comb on midleg homolog 1 (Scmh1). Like their Drosophila counterparts, Scmh1 and the mammalian polyhomeotic homolog RAE28/mph1 interact in vitro via their SPM domains. We analyzed the expression of Scmh1 and rae28/mph1 using northern analysis of embryos and adult tissues, and in situ hybridization to embryos. The expression of Scmh1 and rae28/mph1 is well correlated in most tissues of embryos. However, in adults, Scmh1 expression was detected in most tissues, whereas mph1/rae28 expression was restricted to the gonads. Scmh1 is strongly induced by retinoic acid in F9 and P19 embryonal carcinoma cells. Scmh1 maps to 4D1-D2.1 in mice. These data suggest that Scmh1 will have an important role in regulation of homeotic genes in embryogenesis and that the interaction with RAE28/mph1 is important in vivo.     

A mother cell-specific class B penicillin-binding protein, PBP4b, in Bacillus subtilis

The Bacillus subtilis genome encodes 16 penicillin-binding proteins (PBPs), some of which are involved in synthesis of the spore peptidoglycan. The pbpI (yrrR) gene encodes a class B PBP, PBP4b, and is transcribed in the mother cell by RNA polymerase containing sigma(E). Loss of PBP4b, alone and in combination with other sporulation-specific PBPs, had no effect on spore peptidoglycan structure.     

Importance of the conserved residues in the peptidoglycan glycosyltransferase module of the class A penicillin-binding protein 1b of Escherichia coli

The peptidoglycan glycosyltransferase (GT) module of class A penicillin-binding proteins (PBPs) and monofunctional GTs catalyze glycan chain elongation of the bacterial cell wall. These enzymes belong to the GT51 family, are characterized by five conserved motifs, and have some fold similarity with the phage lambda lysozyme. In this work, we have systematically modified all the conserved amino acid residues of the GT module of Escherichia coli class A PBP1b by site-directed mutagenesis and determined their importance for the in vivo and in vitro activity and the thermostability of the protein. To get an insight into the GT active site of this paradigm enzyme, a model of PBP1b GT domain was constructed based on the available crystal structures (PDB codes 2OLV and 2OLU). The data show that in addition to the essential glutamate residues Glu233 of motif 1 and Glu290 of motif 3, the residues Phe237 and His240 of motif 1 and Gly264, Thr267, Gln271, and Lys274 of motif 2, all located in the catalytic cavity of the GT domain, are essential for the in vitro enzymatic activity of the PBP1b and for its in vivo functioning. Thus, the first three conserved motifs contain most of the residues that are required for the GT activity of the PBP1b. The residues Asp234, Phe237, His240, Thr267, and Gln271 are proposed to maintain the structure of the active site and the positioning of the catalytic Glu233.     

Molecular identification of wheat endoxylanase inhibitor TAXI-I1, member of a new class of plant proteins

Triticum aestivum endoxylanase inhibitors (TAXIs) are wheat proteins that inhibit family 11 endoxylanases commonly used in different (bio)technological processes. Here, we report on the identification of the TAXI-I gene which encodes a mature protein of 381 amino acids with a calculated molecular mass of 38.8 kDa. When expressed in Escherichia coli, the recombinant protein had the specificity and inhibitory activity of natural TAXI-I, providing conclusive evidence that the isolated gene encodes an endoxylanase inhibitor. Bioinformatical analysis indicated that no conserved domains nor motifs common to other known proteins are present. Sequence analysis revealed similarity with a glycoprotein of carrot and with gene families in Arabidopsis thaliana and rice, all with unknown functions. Our data indicate that TAXI-I belongs to a newly identified class of plant proteins for which a molecular function as glycoside hydrolase inhibitor can now be suggested.     

Choristoneura fumiferana Granulovirus p74 protein, a highly conserved baculoviral envelope protein

A gene that encodes a homologue to baculoviral p74, an envelope-associated viral structural protein, has been identified and sequenced on the genome of Choristoneura fumiferana granulovirus (ChfuGV). A part of the ChfuGV p74 gene was located on an 8.9 kb BamHI subgenomic fragment using different sets of degenerated primers. These were designed using the results of the protein sequencing of a major 74 kDa structural protein that is associated with the occlusion-derived virus (ODV). The gene has a 1992 nucleotide (nt) open-reading frame (ORF) that encodes a protein with 663 amino acids with a predicted molecular mass of 74,812 Da. Comparative studies revealed the presence of two major conserved regions in the ChfuGV p74 protein. This study also shows that all of the p74 proteins contain two putative transmembrane domains at their C-terminal segments. At the nucleotide sequence level, two late promoter motifs (TAAG and GTAAG) were located upstream of the first ATG of the p74 gene. The gene contained a canonical poly(A) signal, AATAAA, at its 3 non-translated region. A phylogenetic tree for baculoviral p74 was constructed using a maximum parsimony analysis. The phylogenetic estimation demonstrated that ChfuGV p74 is related the closest to those of Cydia pomonella granulovirus (CpGV) and Phthorimaea operculella granulovirus (PhopGV).