Miguel A. de Pedro

In Situ Probing of Newly Synthesized Peptidoglycan in Live Bacteria with Fluorescent D‐Amino Acids

Tracking a bugs life: Peptidoglycan (PG) of diverse bacteria is labeled by exploiting the tolerance of cells for incorporating different non-natural D-amino acids. These nontoxic D-amino acids preferably label the sites of active PG synthesis, thereby enabling fine spatiotemporal tracking of cell-wall dynamics in phylogenetically and morphologically diverse bacteria. HCC = 7-hydroxycoumarin, NBD = 7-nitrobenzofurazan, TAMRA = carboxytetramethylrhodamine.

Emerging knowledge of regulatory roles of d-amino acids in bacteria

The d-enantiomers of amino acids have been thought to have relatively minor functions in biological processes. While l-amino acids clearly predominate in nature, d-amino acids are sometimes found in proteins that are not synthesized by ribosomes, and d-Ala and d-Glu are routinely found in the peptidoglycan cell wall of bacteria. Here, we review recent findings showing that d-amino acids have previously unappreciated regulatory roles in the bacterial kingdom. Many diverse bacterial phyla synthesize and release d-amino acids, including d-Met and d-Leu, which were not previously known to be made. These noncanonical d-amino acids regulate cell wall remodeling in stationary phase and cause biofilm dispersal in aging bacterial communities. Elucidating the mechanisms by which d-amino acids govern cell wall remodeling and biofilm disassembly will undoubtedly reveal new paradigms for understanding how extracytoplasmic processes are regulated as well as lead to development of novel therapeutics.

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.

Molecular weight-determination of biosynthetically modified monomeric and oligomeric muropeptides from Escherichia coli by plasma desorption-mass spectrometry

The presence of certain d‐amino acids in the growth media of Escherichia coli results in the accumulation of 2 major and 3–5 minor new muropeptides in the murein sacculus. Preliminary data suggested that the major muropeptides correspond to a monomer and a cross‐linked dimer with one residue of d‐amino acid per molecule. We have analyzed several d‐amino acid‐modified muropeptides by plasma desorption‐mass spectrometry. Our results confirmed that the general structures of the major modified muropeptides are: GlucNAc‐MurNAc‐l‐Ala‐d‐Glu‐m‐A2pm‐d‐X, and GlucNAc‐MurNAc‐l‐Ala‐d‐Glu‐m‐A2pm‐d‐Ala; GlucNAc‐MurNAc‐l‐Ala‐d‐Glu‐m‐A2pm‐d‐X, being X a residue of the d‐amino acid. These results corroborate the usefulness of this technique for the structural analysis of muropeptides.

High-throughput, Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography.

Background: HPLC enables quantification of bacterial cell-wall composition, yet systematic studies across strains, species, and chemical perturbations are lacking. Results: UPLC coupled to computational modeling enables submicroliter injection volumes, and was applied to systematic analysis of several Gram-negative species. Conclusion: Composition is largely decoupled from morphology, although large interspecies differences were evident. Significance: UPLC and automated analysis accelerate discovery regarding peptidoglycan and physiology. The bacterial cell wall is a network of glycan strands cross-linked by short peptides (peptidoglycan); it is responsible for the mechanical integrity of the cell and shape determination. Liquid chromatography can be used to measure the abundance of the muropeptide subunits composing the cell wall. Characteristics such as the degree of cross-linking and average glycan strand length are known to vary across species. However, a systematic comparison among strains of a given species has yet to be undertaken, making it difficult to assess the origins of variability in peptidoglycan composition. We present a protocol for muropeptide analysis using ultra performance liquid chromatography (UPLC) and demonstrate that UPLC achieves resolution comparable with that of HPLC while requiring orders of magnitude less injection volume and a fraction of the elution time. We also developed a software platform to automate the identification and quantification of chromatographic peaks, which we demonstrate has improved accuracy relative to other software. This combined experimental and computational methodology revealed that peptidoglycan composition was approximately maintained across strains from three Gram-negative species despite taxonomical and morphological differences. Peptidoglycan composition and density were maintained after we systematically altered cell size in Escherichia coli using the antibiotic A22, indicating that cell shape is largely decoupled from the biochemistry of peptidoglycan synthesis. High-throughput, sensitive UPLC combined with our automated software for chromatographic analysis will accelerate the discovery of peptidoglycan composition and the molecular mechanisms of cell wall structure determination.

Localized synthesis of the outer envelope from Thermus thermophilus

In agreement with its distinct phylogenetic origin, the envelope of Thermus thermophilus consists of a complex pattern of layers with properties intermediate between those of Gram positives and Proteobacteria. Its cell wall of Gram positive composition is surrounded by an outer envelope that includes a crystalline layer scaffold built up by the SlpA protein, lipids and polysaccharides. The synthesis of this outer envelope has been studied by confocal microscopy. Available amino groups from the cell surface, mainly belonging to the SlpA protein, were covalently labelled in vivo with fluorescent dyes. Stained cells were able to grow without any apparent loss of viability, allowing the localization of the regions of new synthesis as dark nonfluorescent spots. Our results demonstrate that the outer envelope of T. thermophilus is synthesized from a central point in the cells, likely following a helical pattern. Cell poles and subpolar regions are basically inert and retain their label for generations.

Ultra-Sensitive, High-Resolution Liquid Chromatography Methods for the High-Throughput Quantitative Analysis of Bacterial Cell Wall Chemistry and Structure.

High-performance liquid chromatography (HPLC) analysis has been critical for determining the structural and chemical complexity of the cell wall. However this method is very time consuming in terms of sample preparation and chromatographic separation. Here we describe (1) optimized methods for peptidoglycan isolation from both Gram-negative and Gram-positive bacteria that dramatically reduce the sample preparation time, and (2) the application of the fast and highly efficient ultra-performance liquid chromatography (UPLC) technology to muropeptide separation and quantification. The advances in both analytical instrumentation and stationary-phase chemistry have allowed for evolved protocols which cut run time from hours (2-3 h) to minutes (10-20 min), and sample demands by at least one order of magnitude. Furthermore, development of methods based on organic solvents permits in-line mass spectrometry (MS) of the UPLC-resolved muropeptides. Application of these technologies to high-throughput analysis will expedite the better understanding of the cell wall biology.

Complete genome sequence of Hirschia baltica type strain (IFAM 1418 T )

The family Hyphomonadaceae within the Alphaproteobacteria is largely comprised of bacteria isolated from marine environments with striking morphologies and an unusual mode of cell growth. Here, we report the complete genome sequence Hirschia baltica, which is only the second a member of the Hyphomonadaceae with a published genome sequence. H. baltica is of special interest because it has a dimorphic life cycle and is a stalked, budding bacterium. The 3,455,622 bp long chromosome and 84,492 bp plasmid with a total of 3,222 protein-coding and 44 RNA genes were sequenced as part of the DOE Joint Genome Institute Program CSP 2008.

Amino Acids as Useful Tools in the Study of Murein Metabolism in Escherichia coli

The murein sacculus can be envisaged as a bacterial exoskeleton fulfilling mechanical and morphogenetic functions. Metabolism of the murein sacculus is an essential process that compares in complexity with chromosome replication and protein synthesis in bacteria (Holtje and Schwarz, 1985; Holtje and Tuomanen, 1991).

Investigations on Structure and Biosynthesis of Cyanelle Murein from Cyanophora paradoxa

The endosymbiotic origin of plastids and mitochondria from prokaryotic invaders into originally heterotrophic and anaerobic protoeukaryotic host cells is now generally accepted (Margulis, 1981). However, examples for intermediary or side line forms in this long-lasting and presumably still ongoing process that e. g. combine the function and genetic complexity of an organelle with the distinct prokaryotic structural feature of the peptidoglycan wall could be found to date only for the evolutionary line leading to plastids. The cyanelles from the photoautotrophic protist Cyanophora paradoxa have originally been considered as endosymbiotic cyanobacteria since they resemble them in morphology, thylakoid structure (Giddings et al., 1983), the presence of carboxysomes (Mangeney and Gibbs, 1987) and of a lysozyme-sensitive murein sacculus between their inner and outer envelope membranes (Schenk, 1970).