Putative Penicillin-binding Protein Research Articles

In silico analyses of penicillin binding proteins in Burkholderia pseudomallei uncovers SNPs with utility for phylogeography, species differentiation, and sequence typing

Burkholderia pseudomallei causes melioidosis. Sequence typing this pathogen can reveal geographical origin and uncover epidemiological associations. Here, we describe B. pseudomallei genes encoding putative penicillin binding proteins (PBPs) and investigate their utility for determining phylogeography and differentiating closely related species. We performed in silico analysis to characterize 10 PBP homologs in B. pseudomallei 1026b. As PBP active site mutations can confer β-lactam resistance in Gram-negative bacteria, PBP sequences in two resistant B. pseudomallei strains were examined for similar alterations. Sequence alignments revealed single amino acid polymorphisms (SAAPs) unique to the multidrug resistant strain Bp1651 in the transpeptidase domains of two PBPs, but not directly within the active sites. Using BLASTn analyses of complete assembled genomes in the NCBI database, we determined genes encoding PBPs were conserved among B. pseudomallei (n = 101) and Burkholderia mallei (n = 26) strains. Within these genes, single nucleotide polymorphisms (SNPs) useful for predicting geographic origin of B. pseudomallei were uncovered. SNPs unique to B. mallei were also identified. Based on 11 SNPs identified in two genes encoding predicted PBP-3s, a dual-locus sequence typing (DLST) scheme was developed. The robustness of this typing scheme was assessed using 1,523 RefSeq genomes from B. pseudomallei (n = 1,442) and B. mallei (n = 81) strains, resulting in 32 sequence types (STs). Compared to multi-locus sequence typing (MLST), the DLST scheme demonstrated less resolution to support the continental separation of Australian B. pseudomallei strains. However, several STs were unique to strains originating from a specific country or region. The phylogeography of Western Hemisphere B. pseudomallei strains was more highly resolved by DLST compared to internal transcribed spacer (ITS) typing, and all B. mallei strains formed a single ST. Conserved genes encoding PBPs in B. pseudomallei are useful for strain typing, can enhance predictions of geographic origin, and differentiate strains of closely related Burkholderia species.

Resistance to β-lactam antibiotics conferred by point mutations in penicillin-binding proteins PBP3, PBP4 and PBP6 in Salmonella enterica.

Penicillin-binding proteins (PBPs) are enzymes responsible for the polymerization of the glycan strand and the cross-linking between glycan chains as well as the target proteins for β-lactam antibiotics. Mutational alterations in PBPs can confer resistance either by reducing binding of the antibiotic to the active site or by evolving a β-lactamase activity that degrades the antibiotic. As no systematic studies have been performed to examine the potential of all PBPs present in one bacterial species to evolve increased resistance against β-lactam antibiotics, we explored the ability of fifteen different defined or putative PBPs in Salmonella enterica to acquire increased resistance against penicillin G. We could after mutagenesis and selection in presence of penicillin G isolate mutants with amino-acid substitutions in the PBPs, FtsI, DacB and DacC (corresponding to PBP3, PBP4 and PBP6) with increased resistance against β-lactam antibiotics. Our results suggest that: (i) most evolved PBPs became ‘generalists” with increased resistance against several different classes of β-lactam antibiotics, (ii) synergistic interactions between mutations conferring antibiotic resistance are common and (iii) the mechanism of resistance of these mutants could be to make the active site more accessible for water allowing hydrolysis or less binding to β-lactam antibiotics.

Inactivation of mrcA gene derepresses the basal-level expression of L1 and L2 -lactamases in Stenotrophomonas maltophilia

To characterize the relationship between inactivation of the mrcA gene and β-lactamase expression and β-lactams resistance in Stenotrophomonas maltophilia KJ and to investigate the involvement of ampR, ampN-ampG, ampD(I) and creBC in this. The mrcA deletion mutant KJΔmrcA was constructed to investigate the role of this putative penicillin-binding protein 1a (PBP1a) in β-lactamase expression and β-lactam resistance. The ΔampR, ΔampNG, ΔampDI and ΔcreBC alleles were introduced into KJΔmrcA, and KJΔDIΔBC and KJΔDIΔmrcAΔBC were also constructed for comparison. All the mutants and their corresponding parent strains were assayed for β-lactamase activities and MICs of β-lactams. Inactivation of mrcA caused basal L1/L2 β-lactamase production to increase by ∼100-fold, but made little difference to cefuroxime-induced β-lactamase activity and the MICs of β-lactams. The ΔmrcA-derived basal β-lactamase hyperproduction was ampR and ampN-ampG dependent. Simultaneous inactivation of ampD(I) and mrcA did not augment β-lactamase production over and above that seen in an ampD(I) mutant alone. Furthermore, we could find no evidence for a role of the creBC two-component regulatory system in β-lactamase hyperproduction in a ΔampD(I) or ΔmrcA background. Inactivation of mrcA, predicted to encode PBP1a, causes basal L1/L2 β-lactamase hyperproduction in S. maltophilia.

Purification, crystallization and preliminary X-ray crystallographic analysis of Lmo0540 fromListeria monocytogenes

Penicillin-binding proteins catalyze the biosynthesis of the peptidoglycan chains of the bacterial cell wall, which protects cells from osmotic pressure. Although Lmo0540 has been identified as a putative penicillin-binding protein that contributes to the virulence of Listeria monocytogenes, the biochemical role of Lmo0540 remains unclear. To provide insights into its biochemical function, Lmo0540 was overexpressed, purified and crystallized by the sitting-drop vapour-diffusion method. Diffraction data were collected to 1.5 Å resolution using synchrotron radiation. The crystal belonged to the C-centred monoclinic space group C2, with unit-cell parameters a = 82.5, b = 75.7, c = 75.9 Å, α = γ = 90, β = 121.8°. A full structural determination is under way in order to elucidate the structure-function relationship of this protein.

Expression and Characterization of Penicillin-Binding Proteins in Burkholderia cenocepacia

Putative penicillin-binding proteins (PBPs) were identified in the genome of the Burkholderia cenocepacia strain J2315 based on homology to E. coli PBPs. The three sequences identified as homologs of E. coli PBP1a, BCAL2021, BCAL0274, and BCAM2632, were cloned and expressed as His(6)-tagged fusion proteins in E. coli. The fusion proteins were isolated and shown to bind beta-lactams, indicating these putative PBPs have penicillin-binding activity.

The putative penicillin-binding proteins 1 and 2 are important for viability, growth and cell morphology of Brucella melitensis

The penicillin-binding proteins (PBPs) are enzymes that regulate the assembly of the peptidoglycan layer of the bacterial cell wall. The genome of Brucella melitensis strain 16 M possesses seven pbp genes: three in pbp-1 family (designated as 1A, 1B, and 1C); one in pbp-2 family; and three in pbp-6 family (designated as 6A, 6B, and 6C). We investigated the importance of pbp-1 and pbp-2 genes to viability, cell morphology and infectivity of B. melitensis. A recombinant B. melitensis strain (designated 16 MΔ pbp1C) was generated by disrupting the pbp-1C of strain 16 M by allelic exchange. This strain produced nearly 20% smaller colonies on trypticase soy agar plates, and grew slower in trypticase soy broth compared to the strain 16 M. Electron microscopy revealed that strain 16 M exhibited native cocco-bacillus morphology, while 16 MΔ pbp1C possessed a spherical morphology. Strain 16 MΔ pbp1C did not differ from strain 16 M in terms of recovery from infected mouse macrophage cell line J774.1, or recovery from spleens of infected BALB/c mice, suggesting that pbp-1C is dispensable for intracellular persistence of B. melitensis. Expression of mRNA of fixR, the gene downstream of pbp-1C was similar between the strains 16 M and 16 MΔ pbp1C suggesting that disruption of pbp-1C did not induce any polar effects. Multiple attempts to mutate pbp-1A, pbp-1B, or pbp-2 genes failed, most probably because these genes are indispensable for viability of B. melitensis. Our findings suggest that pbp-1C regulates in vitro growth and cell morphology, whereas pbp-1A, pbp-1B, and pbp-2 are essential for viability of B. melitensis.

Wide variation in the cyanobacterial complement of presumptive penicillin-binding proteins

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.

Identification of Group A Streptococcus Antigenic Determinants Upregulated In Vivo

Group A Streptococcus (GAS) causes a range of diseases in humans, from mild noninvasive infections to severe invasive infections. The molecular basis for the varying severity of disease remains unclear. We identified genes expressed during invasive disease using in vivo-induced antigen technology (IVIAT), applied for the first time in a gram-positive organism. Convalescent-phase sera from patients with invasive disease were pooled, adsorbed against antigens derived from in vitro-grown GAS, and used to screen a GAS genomic expression library. A murine model of invasive GAS disease was included as an additional source of sera for screening. Sequencing DNA inserts from clones reactive with both human and mouse sera indicated 16 open reading frames with homology to genes involved in metabolic activity to genes of unknown function. Of these, seven genes were assessed for their differential expression by quantitative real-time PCR both in vivo, utilizing a murine model of invasive GAS disease, and in vitro at different time points of growth. Three gene products-a putative penicillin-binding protein 1A, a putative lipoprotein, and a conserved hypothetical protein homologous to a putative translation initiation inhibitor in Vibrio vulnificus-were upregulated in vivo, suggesting that these genes play a role during invasive disease.

Comparative study of the protein composition of three strains of Streptococcus thermophilus grown either in M17 medium or in milk

Streptococcus thermophilus is a lactic acid bacterium widely used for manufacturing milk products. The protein composition of three strains of S. thermophilus grown in either M17 me- dium or milk was investigated. These conditions were chosen on the basis of the observed technologi- cal differences between these strains. Protein extracts prepared from cells harvested at the same pH (5.2 ± 0.1), were analyzed by two-dimensional gel electrophoresis. Gel comparison revealed qualita- tive and quantitative differences between the two growth media and between the strains. Some pro- teins with quantitative variation were partially identified. The most significant medium-related differences concerned the number of spots detected and the heat shock protein (GroEL, HSP70) levels which were higher in milk than in M17. On the contrary, the levels of enzymes (proton translocating ATPase, pyruvate kinase, tributyrin esterase, phospho-glycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, fructose diphosphate aldolase, superoxide dismutase) and of a putative penicillin binding protein were lower in milk than in M17. Strain-related differences were accentuated in milk for heat shock proteins, tributryrine esterase and dinitrogenase reductase. Streptococcus thermophilus / milk / proteome / 2D-PAGE electrophoresis

Spontaneous nisin-resistant Listeria monocytogenes mutants with increased expression of a putative penicillin-binding protein and their sensitivity to various antibiotics.

A concern regarding the use of bacteriocins, as for example the lantibiotic nisin, for biopreservation of certain food products is the possibility of resistance development and potential cross-resistance to antibiotics in the target organism. The genetic basis for nisin resistance development is as yet unknown. We analyzed changes in gene expression following nisin resistance development in Listeria monocytogenes 412 by restriction fragment differential display. The mutant had increased expression of a protein with strong homology to the glycosyltransferase domain of high-molecular-weight penicillin-binding proteins (PBPs), a histidine protein kinase, a protein of unknown function, and ClpB (putative functions from homology). The three former proteins had increased expression in a total of six out of 10 independent mutants originating from five different wild-type strains, indicating a prevalent nisin resistance mechanism under the employed isolation conditions. Increased expression of the putative PBP may affect the cell wall composition and thereby alter the sensitivity to cell wall-targeting compounds. The mutants had an isolate-specific increase in sensitivity to different beta-lactams and a slight decrease in sensitivity to another lantibiotic, mersacidin. A model incorporating these observations is proposed based on current knowledge of nisin's mode of action.

Molecular analysis of tlrB, an antibiotic-resistance gene from tylosin-producing Streptomyces fradiae, and discovery of a novel resistance mechanism.

The tlrB gene, which confers inducible resistance to a range of macrolide antibiotics including biosynthetic precursors of tylosin, was isolated and sequenced. In the genome of Streptomyces fradiae, it lies between pbp, which encodes a putative penicillin-binding protein, and tylN, encoding a glycosyltransferase involved in tylosin biosynthesis. The TlrB protein was produced in E. coli as a fusion to MalE. The fusion protein, but not MalE alone, inactivates macrolides in the presence of S-adenosyl-methionine (SAM) but the modified product(s) has not been characterised.