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PubMed2003

Mechanistic diversity of fosfomycin resistance in pathogenic microorganisms.

Microbial resistance to the antibiotic fosfomycin [(1R,2S)-epoxypropylphosphonic acid, 1] is known to be mediated by thiol transferase enzymes FosA and FosB, which catalyze the addition of glutathione and l-cysteine to C1 of the oxirane, respectively. A probe of the microbial genome database reveals a related group of enzymes (FosX). The genes mlr3345 from Mesorhizobium loti and lmo1702 from Listeria monocytogenes were cloned and the proteins expressed. This heretofore unrecognized group of enzymes is shown to catalyze the Mn(II)-dependent addition of water to C1 of the oxirane. The ability of each enzyme to confer resistance in Escherichia coli is correlated with their catalytic efficiency, such that the M. loti protein confers low resistance while the Listeria enzyme confers very robust resistance. The crystal structure of the FosX from M. loti was solved at a resolution of 1.83 A. The structure reveals an active-site carboxylate (E44) located about 5 A from the expected position of the substrate that appears to be poised to participate in catalysis. Single turnover experiments in H218O and kinetic analysis of the E44G mutant of the FosX enzymes indicate that the carboxylate of E44 acts as a general base in the direct addition of water to 1. The FosX from M. loti also catalyzes the addition of glutathione to the antibiotic. The catalytic promiscuity and low efficiency of the M. loti protein suggest that it may be an intermediate in the evolution of clinically relevant fosfomycin resistance proteins such as the FosX from Listeria monocytogenese.

Bacterial Proteins↗
PubMed2004

Challenges in antimicrobial drug discovery and the potential of nucleoside antibiotics.

Antimicrobial resistance in hospital and community settings is growing at an alarming rate and has been attributed to such organisms as methicillin-resistant staphylococcus aureus, staphylococci with decreased susceptibility to vancomycin, vancomycin-resistant enterococci, multi-drug resistant pseudomonas spp., klebsiella spp., enterobacter spp, and acinetobacter spp., as well as Streptococcus pneumoniae with decreased susceptibility to penicillin and other antibacterials. To address the need for new therapies to combat resistant organisms, drug companies are refocusing their discovery efforts on developing novel agents with new mechanisms of action. The hope is that rapidly emerging technologies including combinatorial chemistry, high throughput screening, proteomics and microbial genomics will have a positive impact on antimicrobial drug discovery. These technologies should aid in the identification of novel drug targets and compounds with unique mechanisms of action other than those currently provided by the traditional antibiotics. Nucleosides are one class of compounds worthy of further investigation as antibacterials since some derivatives have shown moderate to good activity against specific bacterial strains. For example, 5'-peptidyl nucleoside derivatives can inhibit peptide deformylase, an enzyme essential for bacterial survival that is not vital to human cells. This review also includes a list of miscellaneous nucleosides that have been synthesized as potential antibacterials. More detailed investigations on structure, as it relates to the antimicrobial activity of the various classes of nucleosides, need to be conducted in order to maximize the potential of developing a potent nucleoside for the treatment of bacterial infections. This review begins with an introduction to terms followed by discussions regarding the general background and relevance for developing novel antimicrobial agents. Challenges facing the antimicrobial drug discovery process are discussed along with relevant drug targets. An overview of nucleoside chemistry as it relates to antimicrobial activity is presented, followed by a discussion of the evidence which supports the potential of this class of compounds to yield the novel antimicrobial therapies needed in the new millennium.

Animals↗
PubMed1995

Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium.

A protein map of the smallest known self-replicating organism, Mycoplasma genitalium (Class: Mollicutes), revealed a high proportion of acidic proteins. Amino acid composition was used to putatively identify, or provide unique parameters, for 50 gene products separated by two-dimensional gel electrophoresis. A further 19 proteins were subjected to peptide-mass fingerprinting using matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry and 4 were subjected to N-terminal Edman degradation. The majority of M. genitalium proteins remain uncharacterised. However, the combined approach of amino acid analysis and peptide-mass fingerprinting allowed gene products to be linked to homologous genes in a variety of organisms. This has allowed proteins to be identified prior to detection of their respective genes via the M. genitalium sequencing initiative. The principle of 'hierarchical' analysis for the mass screening of proteins and the analysis of microbial genomes via their protein complement or 'proteome' is detailed. Here, characterisation of gene products depends upon the quickest and most economical technologies being employed initially, so as to determine if a large number of proteins are already present in both homologous and heterologous species databases. Initial screening, which lends itself to automation and robotics, can then be followed by more time and cost intensive procedures, when necessary.

Amino Acids↗
PubMed2001

Computational method to assign microbial genes to pathways.

We present techniques that mine fully sequenced microbial genomes for functional relationships between genes. We show that genes related by one of four techniques are more likely to belong to the same cellular pathways. Furthermore, we demonstrate that the pathway of an uncharacterized gene may be inferred from those of its functionally related partners. Therefore, we are now able to assign most of the genes within bacteria to cellular pathways.

Computational Biology↗
PubMed2002

Structure and mechanism of action of a cofactor-dependent phosphoglycerate mutase homolog from Bacillus stearothermophilus with broad specificity phosphatase activity.

The crystal structure of Bacillus stearothermophilus PhoE (originally termed YhfR), a broad specificity monomeric phosphatase with a molecular mass of approximately 24 kDa, has been solved at 2.3 A resolution in order to investigate its structure and function. PhoE, already identified as a homolog of a cofactor-dependent phosphoglycerate mutase, shares with the latter an alpha/beta/alpha sandwich structure spanning, as a structural excursion, a smaller subdomain composed of two alpha-helices and one short beta-strand. The active site contains residues from both the alpha/beta/alpha sandwich and the sub-domain. With the exception of the hydrophilic catalytic machinery conserved throughout the cofactor-dependent phosphoglycerate mutase family, the active-site cleft is strikingly hydrophobic. Docking studies with two diverse, favored substrates show that 3-phosphoglycerate may bind to the catalytic core, while alpha-napthylphosphate binding also involves the hydrophobic portion of the active-site cleft. Combining a highly favorable phospho group binding site common to these substrate binding modes and data from related enzymes, a catalytic mechanism can be proposed that involves formation of a phosphohistidine intermediate on His10 and likely acid-base behavior of Glu83. Other structural factors contributing to the broad substrate specificity of PhoE can be identified. The dynamic independence of the subdomain may enable the active-site cleft to accommodate substrates of different sizes, although similar motions are present in simulations of cofactor-dependent phosphoglycerate mutases, perhaps favoring a more general functional role. A significant number of entries in protein sequence databases, particularly from unfinished microbial genomes, are more similar to PhoE than to cofactor-dependent phosphoglycerate mutases or to fructose-2,6-bisphosphatases. This PhoE structure will therefore serve as a valuable basis for inference of structural and functional characteristics of these proteins.

Amino Acid Sequence↗
PubMed1999

An attempt to pinpoint the phylogenetic introduction of glutaminyl-tRNA synthetase among bacteria.

Until recently it was believed that most Bacteria form Gln-tRNA(GLN) by the amidation of Glu-tRNA(GLN), only a few members of the gamma subdivision of Proteobacteria being able to charge tRNA(GLN) directly. We undertook a phylogenetic study in an attempt to determine at what point the changeover to the direct system may have occurred. To this end, we selected a number of representative Proteobacteria to see if we could find a division point. We constructed degenerate primers and conducted PCR analysis to identify which Bacteria had Gln-tRNA synthetase, on the one hand, and which had the amidotransferase system, on the other. At the same time, we surveyed data banks of completely sequenced microbial genomes, as well as those for genomes in the process of being sequenced. These combined efforts revealed four Proteobacteria in a phylogenetically intermediate position which have the genetic potential for both mechanisms. Perplexingly, however, three distantly related bacteria were also found to have both enzymes.

Amino Acid Sequence↗
PubMed2006

Bacterial type III polyketide synthases: phylogenetic analysis and potential for the production of novel secondary metabolites by heterologous expression in pseudomonads.

Type III polyketide synthases (PKS) were regarded as typical for plant secondary metabolism before they were found in microorganisms recently. Due to microbial genome sequencing efforts, more and more type III PKS are found, most of which of unknown function. In this manuscript, we report a comprehensive analysis of the phylogeny of bacterial type III PKS and report the expression of a type III PKS from the myxobacterium Sorangium cellulosum in pseudomonads. There is no precedent of a secondary metabolite that might be biosynthetically correlated to a type III PKS from any myxobacterium. Additionally, an inactivation mutant of the S. cellulosum gene shows no physiological difference compared to the wild-type strain which is why these type III PKS are assumed to be "silent" under the laboratory conditions administered. One type III PKS (SoceCHS1) was expressed in different Pseudomonas sp. after the heterologous expression in Escherichia coli failed. Cultures of recombinant Pseudomonas sp. harbouring SoceCHS1 turned red upon incubation and the diffusible pigment formed was identified as 2,5,7-trihydroxy-1,4-naphthoquinone, the autooxidation product of 1,3,6,8-tetrahydroxynaphthalene. The successful heterologous production of a secondary metabolite using a gene not expressed under administered laboratory conditions provides evidence for the usefulness of our approach to activate such secondary metabolite genes for the production of novel metabolites.

Acyltransferases↗
PubMed2006

Comparison of methods for total community DNA extraction and purification from compost.

The differences on DNA yield and purity of three different DNA extraction protocols were compared with regard to the use for PCR and other molecular analyses. Total DNA was extracted from compost by the three protocols, and then was purified by spin-bind cartridges after being precipitated by PEG8000. The detection performed on a nucleic acid and protein analyzer showed that all three methods produced high DNA yields. The agarose gel electrophoresis showed that the fragments of crude and purified DNA had a length of about 23 kb. A eubacterial 16S rRNA gene-targeted primer pair was used for PCR amplification, and full length 16S rDNAs were amplified from all the purified DNA samples. After being digested by restriction endonucleases, the restriction map of amplified rDNA showed identical genetic diversity. The products of PCR using primer pair GC341F and 907R were also used for denaturing gradient gel electrophoresis analysis. The results indicated that high-quality DNA was extracted from compost by the three protocols, and each of the protocols is adapted to extract microbial genome DNA from compost expediently and cheaply.

DNA↗
PubMed2004

Intestinal epithelial tight junctions as targets for enteric bacteria-derived toxins.

The application of a multidisciplinary approach to study bacterial pathogenesis, along with the recent sequencing of entire microbial genomes have made possible discoveries that are changing the way scientists view the bacterium-host interaction. Today, research on the molecular basis of the pathogenesis of infectious diarrheal diseases of necessity transcends established boundaries between microbiology, cell biology, intestinal pathophysiology, and immunology. Novel multidisciplinary approaches led to the discovery of new bacteria-host cell interactions involving signals regulating intestinal permeability through the modulation of cell cytoskeleton and intercellular tight junctions (TJ). A century ago, TJ were conceptualized as a secreted extracellular cement forming an absolute and unregulated barrier within the paracellular space. Biological studies of the past several decades have shown that TJ are dynamic structures subjected to structural changes that dictate their functional status under a variety of developmental, physiological, and pathological circumstances. To meet the many diverse physiological challenges to which the intestinal epithelial barrier is subjected, TJ must be capable of rapid and coordinated responses. This requires the presence of a complex regulatory system that orchestrates the state of assembly of the TJ multiprotein network. Many pathogenic bacteria exploit this system to accomplish their pathogenic strategies by ultimately modulating intestinal permeability.

Animals↗
PubMed2005

Multiple Rieske proteins in prokaryotes: where and why?

Many microbial genomes have been sequenced in the recent years. Multiple genes encoding Rieske iron-sulfur proteins, which are subunits of cytochrome bc-type complexes or oxygenases, have been detected in many pro- and eukaryotic genomes. The diversity of substrates, co-substrates and reactions offers obvious explanations for the diversity of the low potential Rieske proteins associated with oxygenases, but the physiological significance of the multiple genes encoding high potential Rieske proteins associated with the cytochrome bc-type complexes remains elusive. For some organisms, investigations into the function of the later group of genes have been initiated. Here, we summarize recent finding on the characteristics and physiological functions of multiple high potential Rieske proteins in prokaryotes. We suggest that the existence of multiple high potential Rieske proteins in prokaryotes could be one way of allowing an organism to adapt their electron transfer chains to changing environmental conditions.

Animals↗
PubMed2005

Alkaline pH homeostasis in bacteria: new insights.

The capacity of bacteria to survive and grow at alkaline pH values is of widespread importance in the epidemiology of pathogenic bacteria, in remediation and industrial settings, as well as in marine, plant-associated and extremely alkaline ecological niches. Alkali-tolerance and alkaliphily, in turn, strongly depend upon mechanisms for alkaline pH homeostasis, as shown in pH shift experiments and growth experiments in chemostats at different external pH values. Transcriptome and proteome analyses have recently complemented physiological and genetic studies, revealing numerous adaptations that contribute to alkaline pH homeostasis. These include elevated levels of transporters and enzymes that promote proton capture and retention (e.g., the ATP synthase and monovalent cation/proton antiporters), metabolic changes that lead to increased acid production, and changes in the cell surface layers that contribute to cytoplasmic proton retention. Targeted studies over the past decade have followed up the long-recognized importance of monovalent cations in active pH homeostasis. These studies show the centrality of monovalent cation/proton antiporters in this process while microbial genomics provides information about the constellation of such antiporters in individual strains. A comprehensive phylogenetic analysis of both eukaryotic and prokaryotic genome databases has identified orthologs from bacteria to humans that allow better understanding of the specific functions and physiological roles of the antiporters. Detailed information about the properties of multiple antiporters in individual strains is starting to explain how specific monovalent cation/proton antiporters play dominant roles in alkaline pH homeostasis in cells that have several additional antiporters catalyzing ostensibly similar reactions. New insights into the pH-dependent Na(+)/H(+) antiporter NhaA that plays an important role in Escherichia coli have recently emerged from the determination of the structure of NhaA. This review highlights the approaches, major findings and unresolved problems in alkaline pH homeostasis, focusing on the small number of well-characterized alkali-tolerant and extremely alkaliphilic bacteria.

Alkalies↗
PubMed2006

Crystallizing new approaches for antimicrobial drug discovery.

Over the past decade, the sequences of microbial genomes have accumulated, changing the strategies for the discovery of novel anti-infective agents. Targets have become plentiful, yet new antimicrobial agents have been slow to emerge from this effort. In part, this reflects the long discovery and development times needed to bring new drugs to market. In addition, bottlenecks have been revealed in the antimicrobial drug discovery process at the steps of identifying good leads, and optimizing those leads into drug candidates. The fruit of structural genomics may provide opportunities to overcome these bottlenecks and fill the antimicrobial pipeline, by using the tools of structure guided drug discovery (SGDD).

Anti-Bacterial Agents↗
PubMed2003

How to find "missing" genes.

Assigning function to "new" proteins is frequently the rate-determining step for deciphering metabolic pathways and regulatory networks. Osterman and Begley break down this barrier by demonstrating that comparative analyses of microbial genomes is a powerful strategy for identifying pathway components.

Bacteria↗
PubMed2006

Oxygen binding and NO scavenging properties of truncated hemoglobin, HbN, of Mycobacterium smegmatis.

Unraveling of microbial genome data has indicated that two distantly related truncated hemoglobins (trHbs), HbN and HbO, might occur in many species of slow-growing pathogenic mycobacteria. Involvement of HbN in bacterial defense against NO toxicity and nitrosative stress has been proposed. A gene, encoding a putative HbN homolog with conserved features of typical trHbs, has been identified within the genome sequence of fast-growing mycobacterium, Mycobacterium smegmatis. Sequence analysis of M. smegmatis HbN indicated that it is relatively smaller in size and lacks N-terminal pre-A region, carrying 12-residue polar sequence motif that is present in HbN of M. tuberculosis. HbN encoding gene of M. smegmatis was expressed in E. coli as a 12.8kD homodimeric heme protein that binds oxygen reversibly with high affinity (P50 approximately 0.081 mm Hg) and autooxidizes faster than M. tuberculosis HbN. The circular dichroism spectra indicate that HbN of M. smegmatis and M. tuberculosis are structurally similar. Interestingly, an hmp mutant of E. coli, unable to metabolize nitric oxide, exhibited very low NO uptake activity in the presence of M. smegmatis HbN as compared to HbN of M. tuberculosis. On the basis of cellular heme content, specific nitric oxide dioxygenase (NOD) activity of M. smegmatis HbN was nearly one-third of that from M. tuberculosis. Additionally, the hmp mutant of E. coli, carrying M. smegmatis HbN, exhibited nearly 10-fold lower cell survival under nitrosative stress and nitrite derived reactive nitrogen species as compared to the isogenic strain harboring HbN of M. tuberculosis. Taken together, these results suggest that NO metabolizing activity and protection provided by M. smegmatis HbN against toxicity of NO and reactive nitrogen is significantly lower than HbN of M. tuberculosis. The lower efficiency of M. smegmatis HbN for NO detoxification as compared to M. tuberculosis HbN might be related to different level of NO exposure and nitrosative stress faced by these mycobacteria during their cellular metabolism.

Amino Acid Motifs↗
PubMed2004

Mercuric resistance genes in gram-positive oral bacteria.

Mercury-resistant bacteria isolated from the oral cavities of children carried one of two types of merA gene that appear to have evolved from a common ancestor. Streptococcus oralis, Streptococcus mitis and a few other species had merA genes that were very similar to merA of Bacillus cereus strain RC607. Unlike the B. cereus RC607 merA gene, however, the streptococcal merA genes were not carried on Tn5084-like transposons. Instead, comparisons with microbial genomic sequences suggest the merA gene is located on a novel type II transposon. Coagulase-negative staphylococci and Streptococcus parasanguis had identical merA genes that represent a new merA variant.

Adolescent↗
PubMed2003

Towards in silico prediction of immunogenic epitopes.

As torrents of new data now emerge from microbial genomics, bioinformatic prediction of immunogenic epitopes remains challenging but vital. In silico methods often produce paradoxically inconsistent results: good prediction rates on certain test sets but not others. The inherent complexity of immune presentation and recognition processes complicates epitope prediction. Two encouraging developments - data driven artificial intelligence sequence-based methods for epitope prediction and molecular modeling methods based on three-dimensional protein structures - offer hope for the future.

Animals↗
PubMed2005

Detection of bacterial pathogens in municipal wastewater using an oligonucleotide microarray and real-time quantitative PCR.

As a first step toward building a comprehensive microarray, two low density DNA microarrays were constructed and evaluated for the accurate detection of wastewater pathogens. The first one involved the direct hybridization of wastewater microbial genomic DNA to the functional gene probes while the second involved PCR amplification of 23S ribosomal DNA. The genomic DNA microarray employed 10 functional genes as detection targets. Sensitivity of the microarray was determined to be approximately 1.0 microg of Esherichia coli genomic DNA, or 2 x 10(8) copies of the target gene, and only E. coli DNA was detected with the microarray assay using municipal raw sewage. Sensitivity of the microarray was enhanced approximately by 6 orders of magnitude when the target 23S rRNA gene sequences were PCR amplified with a novel universal primer set and allowed hybridization to 24 species-specific oligonucleotide probes. The minimum detection limit was estimated to be about 100 fg of E. coli genomic DNA or 1.4 x 10(2) copies of the 23S rRNA gene. The PCR amplified DNA microarray successfully detected multiple bacterial pathogens in wastewater. As a parallel study to verify efficiency of the DNA microarray, a real-time quantitative PCR assay was also developed based on the fluorescent TaqMan probes (Applied Biosystems).

Bacteria↗