Zdzislaw Markiewicz

Identification of the full set of Listeria monocytogenes penicillin-binding proteins and characterization of PBPD2 (Lmo2812)

BackgroundBacterial 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).ResultsEight L. monocytogenes PBPs were identified by the binding of fluorescent β-lactam antibiotic derivatives Boc-FL, Boc-650 and Amp-Alexa430 to proteins in whole cells or membrane/cell wall extracts. The gene encoding a ninth PBP (Lmo2812) was cloned and expressed in Escherichia coli as a His-tagged protein. The affinity purified recombinant protein had DD-carboxypeptidase activity and preferentially degraded low-molecular-weight substrates. L. monocytogenes mutants lacking the functional Lmo2812 enzyme were constructed and, compared to the wild-type, the cells were longer and slightly curved with bent ends.Protein Lmo1855, previously designated PBPD3, did not bind any of the antibiotic derivatives tested, similarly to the homologous enterococcal protein VanY.ConclusionsNine out of the ten putative L. monocytogenes PBP genes were shown to encode proteins that bind derivatives of β-lactam antibiotics, thus enabling their positive identification. PBPD2 (Lmo2812) was not visualized in whole cell extracts, most probably due to its low abundance, but it was shown to bind Boc-FL after recombinant overexpression and purification. Mutants lacking Lmo2812 and another low molecular mass (LMM) PBP, PBP5 (PBPD1) - both with DD-carboxypeptidase activity - displayed only slight morphological alterations, demonstrating that they are dispensable for cell survival and probably participate in the latter stages of peptidoglycan synthesis. Since Lmo2812 preferentially degrades low-molecular- mass substrates, this may indicate a role in cell wall turnover.

The intrinsic cephalosporin resistome of Listeria monocytogenes in the context of stress response, gene regulation, pathogenesis and therapeutics

Intrinsic resistance to antibiotics is a serious therapeutic problem in the case of many bacterial species. The Gram‐positive human pathogen Listeria monocytogenes is intrinsically resistant to broad spectrum cephalosporin antibiotics, which are commonly used in therapy of bacterial infections. Besides three penicillin‐binding proteins the intrinsic cephalosporin resistome of L. monocytogenes includes multidrug resistance transporter transporters, proteins involved in peptidoglycan biosynthesis and modification, cell envelope proteins with structural or general detoxification function, cytoplasmic proteins with unknown function and regulatory proteins. Analysis of the regulation of the expression of genes involved in the intrinsic resistance of L. monocytogenes to cephalosporins highlights the high complexity of control of the intrinsic resistance phenotype. The regulation of the transcription of the intrinsic resistome determinants involves the activity of eight regulators, namely LisR, CesR, LiaR, VirR, σB, σH, σL and PrfA, of which the most prominent role play LisR, CesR and σB. Furthermore, the vast majority of the intrinsic resistome determinants contribute to the tolerance of different stress conditions and virulence. A study indicates that O‐acetyltransferase OatA is the most promising candidate for co‐drug development since an agent targeting OatA should sensitize L. monocytogenes to certain antibiotics, therefore improving the efficacy of listeriosis treatment as well as food preservation measures.

Re-evaluation of the significance of penicillin binding protein 3 in the susceptibility of Listeria monocytogenes to β-lactam antibiotics

BackgroundPenicillin binding protein 3 (PBP3) of L. monocytogenes has long been thought of as the primary lethal target for β-lactam antibiotics due to the excellent correlation between the MICs of different β-lactams and their affinity for this protein. The gene encoding PBP3 has not yet been directly identified in this gram-positive bacterium, but based on in silico analysis, this protein is likely to be encoded by lmo1438. However, studies examining the effects of mutations in genes encoding known and putative L. monocytogenes PBPs have demonstrated that inactivation of lmo1438 does not affect sensitivity to β-lactams.ResultsIn this study, overexpression of lmo1438 was achieved using an inducible (nisin-controlled) expression system. This permitted the direct demonstration that lmo1438 encodes PBP3. PBP3 overexpression was accompanied by slightly elevated PBP4 expression. The recombinant strain overexpressing PBP3 displayed significant growth retardation and greatly reduced cell length in the stationary phase of growth in culture. In antibiotic susceptibility assays, the strain overexpressing PBP3 displayed increased sensitivity to subinhibitory concentrations of several β-lactams and decreased survival in the presence of a lethal dose of penicillin G. However, the MIC values of the tested β-lactams for this recombinant strain were unchanged compared to the parent strain.ConclusionsThe present study allows a reevaluation of the importance of PBP3 in the susceptibility of L. monocytogenes to β-lactams. It is clear that PBP3 is not the primary lethal target for β-lactams, since neither the absence nor an excess of this protein affect the susceptibility of L. monocytogenes to these antibiotics. The elevated level of PBP4 expression observed in the recombinant strain overexpressing PBP3 demonstrates that the composition of the L. monocytogenes cell wall is subject to tight regulation. The observed changes in the morphology of stationary phase cells in response to PBP3 overexpression suggests the involvement of this protein in cell division during this phase of growth.

Antibiotics and Antibiotics Resistance Genes Dissemination in Soils

This chapter describes the dissemination of antibiotics and antibiotic resistance genes in soil. It starts with an overview of the current knowledge about the natural resistome in soil—mainly bacteria-producing antibiotics—and also the contribution of agriculture, animal husbandry and natural fertilization, and the use of water from the effluent to irrigate crop fields in dissemination of antibiotics in soil. The aspects related to the degradation of antibiotics in the environment and their dependence on environmental conditions are also discussed. Attention has also been paid to the complexity of the soil microbes in the biofilms community and different answers to subinhibitory concentrations of various antibiotics like the SOS response, biofilm formation, or changes in primary metabolism. The next part of this chapter focuses on antibiotic-resistant bacteria in soil and their dissemination. There are numerous examples of intrinsically resistant bacteria and also mechanisms of the acquisition or development of resistance to various antibiotics. Also emphasized is the role of antibiotic pressure leading to higher levels of resistance and the acquisition and exchange of genetic material also that from pathogenic bacteria introduced into the environment from medical settings, municipal wastewater systems, and animal husbandry facilities. The last part is dedicated to antibiotic resistance genes and the mechanism of their transfer and dissemination. This phenomenon is related to horizontal (or lateral) gene transfer through mobile genetic elements. The issue of dissemination of anthropogenically associated antibiotic resistance genes to soil is also discussed. Finally, a holistic model for understanding antibiotic resistance gene dynamics in soil is proposed.

Murein Structure of Three Different Species of Chemolithotrophic Sulfur Bacteria: Thiobacillus tepidarius, Thiobacillus neapolitanus and Thiobacillus versutus

The genus Thiobacillus is an extremely heterogeneous group of bacteria. The only criterion for placing all the species in a single genus is that they all obtain energy for autotrophic growth from the oxidation of inorganic sulfur substrates. Some species are obligately chemolitotrophic and autotrophic (e.g. Thiobacillus neapolitanus and Thiobacillus tepidarius) whereas others are facultatively autotrophic and capable of heterotrophic growth using a wide range of organic substrates (e.g. Thiobacillus versutus). The facultative species are frequently indistinguishable from some of the bacteria grouped as Pseudomonas, except for their capacity for growth with reduced sulfur compounds (Kelly and Harrison, 1989). It has been suggested that the facultative or mixotrophic Thiobacillus species be reassigned to the appropriate genera of chemoorganotrophic bacteria (Friedrich and Mitrenga, 1981).

N-unsubstituted glucosamine residues and other modifications in murein of the obligatory chemolithotroph Thiobacillus neapolitanus

Purified murein from Thiobacillus neapolitanus was poorly digested by lysozyme. Its sensitivity to the enzyme greatly increased after N-acetylation. The murein was found to contain 30 to 35% glucosamine residues lacking N-acetyl groups. It also contained phosphomuramic acid. Further modifications included amidation of diaminopimelic acid in the peptide side chains and a low alanine content. None of these modifications were found in the murein of another sulphur bacterium, Thiobacillus versutus.

Purification and some properties of ATP-dependent deoxyribonuclease of Caulobacter crescentus

An ATP-dependent deoxyribonuclease has been partially purified from extracts of Caulobacter crescentus cells in a procedure involving ion-exchange and affinity chromatography. The enzyme was purified approximately 350-fold and was free of contaminating nucleolytic and ATPase activity. The nuclease hydrolyzes linear, double-stranded DNA with subsequent release of short oligonucleotides, mostly from one to four bases in length. The release of nucleotides is accompanied by hydrolysis of ATP, 7.6 nmol ATP being consumed for each nmol of acid-soluble products of DNA degradation. The enzyme shows an absolute requirement for divalent cations and in most active at pH 7.6 to 8.8. The molecular weight of the nuclease, estimated by gel filtration and sucrose density gradient centrifugation, is 280 000.