Juan A. Ayala

CTX-M-12 β-Lactamase in a Klebsiella pneumoniae Clinical Isolate in Colombia

ABSTRACT We describe the detection of the CTX-M-12 β-lactamase from a clinical isolate of Klebsiella pneumoniae in Colombia. Screening of nosocomial Klebsiella spp. and Escherichia coli isolates from a network of teaching hospitals revealed the presence of CTX-M enzymes in multiple cities. This is the first description of CTX-M in Colombia.

Genetic Dissection of the Type VI Secretion System in Acinetobacter and Identification of a Novel Peptidoglycan Hydrolase, TagX, Required for Its Biogenesis

ABSTRACT The type VI secretion system (T6SS) is a widespread secretory apparatus produced by Gram-negative bacteria that has emerged as a potent mediator of antibacterial activity during interbacterial interactions. Most Acinetobacter species produce a genetically conserved T6SS, although the expression and functionality of this system vary among different strains. Some pathogenic Acinetobacter baumannii strains activate this secretion system via the spontaneous loss of a plasmid carrying T6SS repressors. In this work, we compared the expression of T6SS-related genes via transcriptome sequencing and differential proteomics in cells with and without the plasmid. This approach, together with the mutational analysis of the T6SS clusters, led to the determination of the genetic components required to elaborate a functional T6SS in the nosocomial pathogen A. baumannii and the nonpathogen A. baylyi. By constructing a comprehensive combination of mutants with changes in the T6SS-associated vgrG genes, we delineated their relative contributions to T6SS function. We further determined the importance of two effectors, including an effector-immunity pair, for antibacterial activity. Our genetic analysis led to the identification of an essential membrane-associated structural component named TagX, which we have characterized as a peptidoglycan hydrolase possessing l,d-endopeptidase activity. TagX shows homology to known bacteriophage l,d-endopeptidases and is conserved in the T6SS clusters of several bacterial species. We propose that TagX is the first identified enzyme that fulfills the important role of enabling the transit of T6SS machinery across the peptidoglycan layer of the T6SS-producing bacterium. IMPORTANCE Acinetobacter baumannii is one of the most troublesome and least investigated multidrug-resistant bacterial pathogens. We have previously shown that A. baumannii employs a T6SS to eliminate competing bacteria. Here we provide a comprehensive analysis of the components of the T6SS of Acinetobacter, and our results provide genetic and functional insights into the Acinetobacter T6SS. Through this analysis, we identified a novel peptidoglycan hydrolase, TagX, that is required for biogenesis of the T6SS apparatus. This is the first peptidoglycanase specialized in T6SS function identified in any species. We propose that this enzyme is required for the spatially and temporally regulated digestion of peptidoglycan to allow assembly of the T6SS machinery. Acinetobacter baumannii is one of the most troublesome and least investigated multidrug-resistant bacterial pathogens. We have previously shown that A. baumannii employs a T6SS to eliminate competing bacteria. Here we provide a comprehensive analysis of the components of the T6SS of Acinetobacter, and our results provide genetic and functional insights into the Acinetobacter T6SS. Through this analysis, we identified a novel peptidoglycan hydrolase, TagX, that is required for biogenesis of the T6SS apparatus. This is the first peptidoglycanase specialized in T6SS function identified in any species. We propose that this enzyme is required for the spatially and temporally regulated digestion of peptidoglycan to allow assembly of the T6SS machinery.

Chapter 5 Molecular biology of bacterial septation

Publisher Summary This chapter discusses the molecular biology of bacterial septation. Bacterial division has been studied for many years. Three conclusions are sufficient to introduce the topic of this chapter: (1) bacterial division is very well regulated in time and in space; (2) division is a discontinuous event in topography and chronology; and (3) bacterial cells divide as a consequence of continuous growth. Many advances in the knowledge of division controls are derived from studies on unicellular organisms as yeasts. Cell division in eukaryotic cells includes the partition of two structures, the nucleus and the cytoplasm. In bacteria, the nucleoid divides after replication by segregation, a simpler mechanism. Morphological events during the cell division cycle in prokaryotes and eukaryotes are similar. Growth induces a discontinuous event that leads to the initiation of DNA replication; this is one of the several transitions which regulate the exit from one cell cycle stage and the entrance into the next one. The molecular mechanisms which signal some transition points are well described for the cell cycles of bakers and fission yeasts.

The identification and characterization of IbpA, a novel α-crystallin-type heat shock protein from mycoplasma.

Abstractα-Crystallin-type small heat shock proteins (sHsps) are expressed in many bacteria, animals, plants, and archaea. Among mycoplasmas (Mollicutes), predicted sHsp homologues so far were found only in the Acholeplasmataceae family. In this report, we describe the cloning and functional characterization of a novel sHsp orthologue, IbpA protein, present in Acholeplasma laidlawii. Importantly, similar to the endogenously expressed sHsp proteins, the recombinant IbpA protein was able to spontaneously generate oligomers in vitro and to rescue chemically denatured bovine insulin from irreversible denaturation and aggregation. Collectively, these data suggest that IbpA is a bona fide member of the sHsps family. The immune-electron microscopy data using specific antibodies against IbpA have revealed different intracellular localization of this protein in A. laidlawii cells upon heat shock, which suggests that IbpA not only may participate in the stabilization of individual polypeptides, but may also play a protective role in the maintenance of various cellular structures upon temperature stress.

Application of a charge/size two-dimensional gel electrophoresis system to the analysis of the penicillin-binding proteins of Escherichia coli

nor received Penlcdlm-bmdmg protein NEPHGE SDS-PAGE (E. coli)

Activation parameters and molecular changes induced by substrate hydrolysis of the adenosine triphosphatase of Micrococcus lysodeikticus. A comparison of three different soluble forms of the enzyme

SummaryThe Arrhenius plots for the active and low activity soluble forms of the ATPase purified from the membranes ofMicrococcus lysodeikticus grown at 30°C presented discontinuities at 30 and 33°C, respectively. Their activation parameters differed, being highest for the low activity form of the enzyme.Both forms underwent changes in their molecular properties as a consequence of being enzymically active, i.e., upon incubation with substrates at an adequate temperature. These changes consisted of a decrease in the relative mobilities of some of their subunits in dodecyl sulphate polyacrylamide gel electrophoresis, and the temperature at which they occurred depended on the energy of activation of the particular form of the ATPase used. The low activity form required an incubation temperature of 50°C, whereas for an active form 37°C was sufficient.

Variations in the Metabolism of Peptidoglycan Prior to Polymerization

The biosynthesis of bacterial cell wall peptidoglycan is a complex process which comprises two major stages (Rogers et al., 1980). The first one concerns the sequential formation of a series of cytoplasmic and membrane precursors. The second one concerns the polymerisation reactions with the insertion of the new peptidoglycan material and its maturation. The first stage of peptidoglycan synthesis is considered to begin with UDP-N-acetylglucosamine (UDP-GlcNAc) and to end with N-acetylglucosaminyl-N-acetylmuramyl (pentapeptide) -pyrophosphate undecaprenol (lipid II in Fig.1) which is the substrate for the polymerization reactions. The different steps prior to polymerization have long been identified and certain studied to some extent (Rogers et al., 1980). However little attention had been paid to their in vivo functioning.