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PubMed2006

An AFLP-based database of Shigella flexneri and Shigella sonnei isolates and its use for the identification of untypable Shigella strains.

Amplified fragment length polymorphism (AFLP) can be used to assess the genetic diversity of closely related microbial genomes. In this study, the first of its kind for identification of Shigella, the high discriminatory power of AFLP has been used to determine the genetic relatedness of 230 isolates of Shigella flexneri and Shigella sonnei strains. An AFLP database was generated to demonstrate its utility in the discrimination of closely related strains. Based on AFLP, S. flexneri strains could be grouped into separate clusters according to their serotypes. Within each serotype, strains demonstrated 80-100% similarity indicating that identical strains and closely related strains could be distinguished by this technique. S. flexneri 6 formed a distinct cluster with 55% similarity to the rest of the S. flexneri strains showing significant divergence from the rest of the S. flexneri strains. Significantly, S. sonnei isolates formed a distinct group and showed approximately the same level of genetic linkage to S. flexneri as Escherichia coli strains. Untypable isolates that showed conflicting agglutination reactions with conventional typing sera were identifiable by AFLP. Thus AFLP can be used for genetic fingerprinting of Shigella strains and aid in the identification of variant untypable isolates.

Bacterial Typing Techniques↗
PubMed2004

Scanning the Escherichia coli chromosome by random transposon mutagenesis and multiple phenotypic screening.

Analysis of the complete DNA sequences of many microbial genomes available reveals a fair number of putative ORFs without an identified function. A systematic scan of the Escherichia coli chromosome was achieved by random transposition with a newly developed Tn5 minitransposon derivative carrying the arabinose-inducible araP(BAD) promoter oriented outward at one end (Tn5-araP(BAD)). The transposon insertion mutants obtained were assayed for conditional lethal phenotypes (arabinose dependence or sensitivity), for growth at two temperatures (37 and 15 degrees C) and in different media (rich and minimal medium). The Tn5-araP(BAD)-tagged genes were identified by sequencing the transposon insertion points. In this way we found a new essential gene cluster (yhbN-yhbG), produced conditional lethal (arabinose-dependent) mutations in already known essential genes (folD, frr, plsC, thiL, serS, thrS, and trpS) and provided a new phenotype (cold sensitivity) to other known genes (holD, ahpC, and tolA). Moreover, we identified eight putative ORFs (kch, ycaM, ycbQ, yddA, yddB, ydeK, ydeX, and yliF) that appear to be required in optimum growth conditions (rich medium at 37 degrees C) but not in the cold and in minimal medium.

Chromosomes, Bacterial↗
PubMed2005

Do different surrogate methods detect lateral genetic transfer events of different relative ages?

Non-tree-based ("surrogate") methods have been used to identify instances of lateral genetic transfer in microbial genomes but agreement among predictions of different methods can be poor. It has been proposed that this disagreement arises because different surrogate methods are biased towards the detection of certain types of transfer events. This conjecture is supported by a rigorous phylogenetic analysis of 3776 proteins in Escherichia coli K12 MG1655 to map the ages of transfer events relative to one another.

Base Composition↗
PubMed2005

A comparative categorization of gene flux in diverse microbial species.

Microbial genomes harbor genomic islands (GIs), genes presumably acquired via horizontal gene transfer (HGT). We compared GIs of hyperthermophilic, thermophilic, mesophilic, and pathogenic/nonpathogenic species and of small and large genomes. The COG database was used to characterize gene-encoded functions. Putative donors were determined to quantify gene flux between superkingdoms. In hyperthermophiles, more than 10% of the genes were on average acquired across the superkingdom border. For thermophiles and particularly mesophiles, we identified a nearly unidirectional export from bacteria to archaea. Additionally, we analyzed GI composition for Escherichia, and pairs of Listeria, Rhizobiales, Methanosarcinaceae, and Thermus thermophilus/Deinococcus radiodurans. For Escherichia and Listeria, the composition of GIs in pathogenic and nonpathogenic species did not differ significantly with respect to encoded COG classes. The analysis of related genomes showed that the composition of GIs cannot be explained with trends of gene content known to depend on genome size.

Archaea↗
PubMed2003

Unexpected catalytic site variation in phosphoprotein phosphatase homologues of cofactor-dependent phosphoglycerate mutase.

The cofactor-dependent phosphoglycerate mutase (dPGM) superfamily contains, besides mutases, a variety of phosphatases, both broadly and narrowly substrate-specific. Distant dPGM homologues, conspicuously abundant in microbial genomes, represent a challenge for functional annotation based on sequence comparison alone. Here we carry out sequence analysis and molecular modelling of two families of bacterial dPGM homologues, one the SixA phosphoprotein phosphatases, the other containing various proteins of no known molecular function. The models show how SixA proteins have adapted to phosphoprotein substrate and suggest that the second family may also encode phosphoprotein phosphatases. Unexpected variation in catalytic and substrate-binding residues is observed in the models.

Amino Acid Sequence↗
PubMed2003

The diversity of globin-coupled sensors.

The recently discovered globin-coupled sensors (GCSs) are heme-containing two-domain transducers distinct from the PAS domain superfamily. We have identified an additional 22 GCSs with varying multi-domain C-terminal transmitters through a search of the complete and incomplete microbial genome datasets. The GCS superfamily is composed of two major subfamilies: the aerotactic and gene regulators. We postulate the existence of protoglobin in Archaea as the predecessor to the chimeric GCS.

Amino Acid Sequence↗
PubMed2003

Computational approach for prediction of domain organization and substrate specificity of modular polyketide synthases.

Modular polyketide synthases (PKSs) are large multi-enzymatic, multi-domain megasynthases, which are involved in the biosynthesis of a class of pharmaceutically important natural products, namely polyketides. These enzymes harbor a set of repetitive active sites termed modules and the domains present in each module dictate the chemical moiety that would add to a growing polyketide chain. This modular logic of biosynthesis has been exploited with reasonable success to produce several novel compounds by genetic manipulation. However, for harnessing their vast potential of combinatorial biosynthesis, it is essential to develop knowledge based in silico approaches for correlating the sequence and domain organization of PKSs to their polyketide products. In this work, we have carried out extensive sequence analysis of experimentally characterized PKS clusters to develop an automated computational protocol for unambiguous identification of various PKS domains in a polypeptide sequence. A structure based approach has been used to identify the putative active site residues of acyltransferase (AT) domains, which control the specificities for various starter and extender units during polyketide biosynthesis. On the basis of the analysis of the active site residues and molecular modelling of substrates in the active site of representative AT domains, we have identified a crucial residue that is likely to play a major role in discriminating between malonate and methylmalonate during selection of extender groups by this domain. Structural modelling has also explained the experimentally observed chiral preference of AT domain in substrate selection. This computational protocol has been used to predict the domain organization and substrate specificity for PKS clusters from various microbial genomes. The results of our analysis as well as the computational tools for prediction of domain organization and substrate specificity have been organized in the form of a searchable computerized database (PKSDB). PKSDB would serve as a valuable tool for identification of polyketide products biosynthesized by uncharacterized PKS clusters. This database can also provide guidelines for rational design of experiments to engineer novel polyketides.

Amino Acid Sequence↗
PubMed2005

Microbial dimethylsulfoxide and trimethylamine-N-oxide respiration.

Over the last two decades, the biochemistry and genetics of dimethylsulfoxide (DMSO) and trimethylamine-N-oxide (TMAO) respiration has been characterised, particularly in Escherichia coli marine bacteria of the genus Shewanella and the purple phototrophic bacteria, Rhodobacter sphaeroides and R. capsulatus. All of the enzymes (or catalytic subunits) involved the final step in DMSO and TMAO respiration contain a pterin molybdenum cofactor and are members of the DMSO reductase family of molybdoenzymes. In E. coli, the dimethylsulfoxide reductase (DmsABC) can be purified from membranes as a complex, which exhibits quinol-DMSO oxidoreductase activity. The enzyme is anchored to the membrane via the DmsC subunit and its catalytic subunit DmsA is now considered to face the periplasm. Electron transfer to DmsA involves the DmsB subunit, which is a polyferredoxin related to subunits found in other molybdoenzymes such as nitrate reductase and formate dehydrogenase. A characteristic of the DmsAB-type DMSO reductase is its ability to reduce a variety of S- and N-oxides. E. coli contains a trimethylamine-N-oxide reductase (TorA) that is highly specific for N-oxides. This enzyme is located in the periplasm and is connected to the quinone pool via a membrane-bound penta-haem cytochrome (TorC). DorCA in purple phototrophic bacteria of the genus Rhodobacter is very similar to TorCA with the critical difference that DorA catalyses reduction of both DMSO and TMAO. It is known as a DMSO reductase because the S-oxide is the best substrate. Crystal structures of DorA and TorA have revealed critical differences at the Mo active site that may explain the differences between substrate specificity between the two enzymes. DmsA, TorA and DorA possess a "twin arginine" N-terminal signal sequence consistent with their secretion via the TAT secretory system and not the Sec system. The enzymes are secreted with their bound prosthetic groups: this take place in the cytoplasm and the biogenesis involves a chaperone protein, which is cognate for each enzyme. Expression of the DMSO and TMAO respiratory operons is induced in response to a fall in oxygen tension. dmsABC expression is positively controlled by the oxygen-responsive transcription factor, Fnr and ModE, a transcription factor that binds molybdate. In contrast, torCAD expression is not under Fnr- or ModE-control but is dependent upon a sensor histidine kinase-response regulator pair, TorSR, which activate gene expression under conditions of low oxygen tension in the presence of N- or S-oxide. Regulation of dorCDA expression is similar to that seen for torCAD but it appears that the expression of the sensor histidine kinase-response regulator pair, DorSR is regulated by Fnr and there is an additional tier of regulation involving the ModE-homologue MopB, molybdate and the transcription factor DorX. Analysis of microbial genomes has revealed the presence of dms and tor operons in a wide variety of bacteria and in some archaea and duplicate dms and tor operons have been identified in E. coli. Challenges ahead will include the determination of the significance of the presence of the dms operon in bacterial pathogens and the determination of the significance of DMSO respiration in the global turnover of marine organo-sulfur compounds.

Dimethyl Sulfoxide↗
PubMed1999

Mechanisms and applications of immune stimulatory CpG oligodeoxynucleotides.

Immune stimulation has been widely recognized as an undesirable side effect of certain antisense oligodeoxynucleotides (ODN) which can interfere with their therapeutic application. It is now clear that these dose-dependent immune stimulatory effects primarily result from the presence of an unmethylated CpG dinucleotide in particular base contexts ('CpG motif). The sequence-specific immune activation is not just an experimental artifact, but is actually a highly evolved immune defense mechanism whose actual 'goal' is the detection of microbial nucleic acids. In contrast to vertebrate DNA, in which CpG dinucleotides are 'suppressed' and are highly methylated, microbial genomes do not generally feature CpG suppression or methylation [1]. Immune effector cells such as B cells, macrophages, dendritic cells, and natural killer cells appear to have evolved pattern recognition receptors (PRR) that by binding the microbe-restricted structure of CpG motifs, trigger protective immune responses. Although the specific immune activation appears to have a variety of potential therapeutic applications, it is generally undesirable in antisense ODN. Immune stimulation may be avoided in antisense oligos by the selection of CpG-free target sequences, by the use of ODN backbones that do not support immune stimulation, or by selective modifications of the cytosine in any CpG dinucleotides.

Adjuvants, Immunologic↗
PubMed2002

A putative iron-regulated TonB-dependent receptor of Mannheimia (Pasteurella) haemolytica A1: possible mechanism for phase variation.

A recombinant plasmid that codes for a novel iron receptor protein (Irp) of Mannheimia (Pasteurella) haemolytica A1 was isolated by the partial complementation of an Escherichia coli fur mutant. The deduced amino acid sequence of Irp exhibited characteristics typical of TonB-dependent receptors. These include: a TonB-box at the N-terminal; a 50 amino acid region homologous to the "plug" domain of the E. coli FhuA and FepA receptors; and a C-terminal TonB-dependent signature which likely functions as an outer membrane anchoring domain. Previously uncharacterized Irp homologues were detected by BLAST analysis of available databases and incomplete microbial genomes. When the irp homologues from Neisseria gonorrhoeae and N. meningitidis were cloned by PCR and expressed in E. coli, novel proteins of the predicted size (84kDa) were detected in cell lysates, demonstrating that these are functional genes. The M. haemolytica A1 irp gene undergoes phase variation at a nucleotide region which contain the sequence AAAAAAATTAAAA (7A-2T-4A) flanked by a short inverted repeat. Site-specific mutagenesis of the 7A-2T-4A sequence as well as replacement of the inverted repeats resulted in a stable construct that expressed the Irp protein without phase variation. The expression of irp in M. haemolytica A1 was regulated by iron concentrations and most likely a Fur homologue, consistent with the proposed function of Irp in iron metabolism. The irp genes may represent contingency loci that play a role in iron acquisition during infection.

Amino Acid Sequence↗
PubMed2003

CD8 T cell immunome analysis of Listeria monocytogenes.

The identification of T cell epitopes is crucial for the understanding of the host response during infections with pathogenic microorganisms. Generally, the identification of relevant T cell responses is based on the analysis of T cell lines propagated in vitro. We used an ex vivo approach for the analysis of the CD8 T cell response against Listeria monocytogenes that is based upon the fractionation of naturally processed antigenic peptides and subsequent analysis with T cells in an enzyme-linked immunospot (ELISPOT) assay. Our data indicate that the direct ex vivo ELISPOT analysis of peptides extracted from infected tissues represents a versatile and potent test system for the analysis of the CD8 T cell immunome of microorganisms that furthermore requires neither the knowledge of the microbial genome nor of the specificity of responding T cells.

Animals↗
PubMed1997

Developments in metabolic engineering.

The complete sequencing of several microbial genomes has resulted in the increased availability of genes for metabolic engineering. The number of databases and computational tools to deal with this information has also increased. This development has stimulated, and will continue to stimulate, advances in metabolic engineering. Specific recent advances include improvement of pathways for aromatic metabolites, the development of a more complete understanding of the effect of bacterial hemoglobin on cell performance, the development of NMR-based methods for the monitoring of intracellular metabolites and metabolic flux, and the application of metabolic control analysis and metabolic flux analysis to a variety of systems.

Journal Article↗
PubMed2003

Bacterial "competence" genes: signatures of active transformation, or only remnants?

An exhaustive review published ten years ago reported natural genetic transformation, a potential mechanism for intra- and interspecies gene transfer, in approximately 40 species belonging to different taxonomic and trophic groups. Since then, considerable progress has been made in characterizing DNA-uptake machineries and regulatory circuits controlling their expression in cells competent for genetic transformation. In this article, in light of the recent description of a Group A streptococcal isolate capable of DNA transfer in mixed cultures, we discuss whether the detection in completely sequenced microbial genomes of intact homologues of key competence-regulatory and/or DNA-uptake proteins enables the prediction of new transformable species.

Bacteria↗
PubMed1999

Metabolic symbiosis at the origin of eukaryotes.

Thirty years after Margulis revived the endosymbiosis theory for the origin of mitochondria and chloroplasts, two novel symbiosis hypotheses for the origin of eukaryotes have been put forward. Both propose that eukaryotes arose through metabolic symbiosis (syntrophy) between eubacteria and methanogenic Archaea. They also propose that this was mediated by interspecies hydrogen transfer and that, initially, mitochondria were anaerobic. These hypotheses explain the mosaic character of eukaryotes (i.e. an archaeal-like genetic machinery and a eubacterial-like metabolism), as well as distinct eukaryotic characteristics (which are proposed to be products of symbiosis). Combined data from comparative genomics, microbial ecology and the fossil record should help to test their validity.

Archaea↗
PubMed2001

Phage display for target-based antibacterial drug discovery.

Increasing bacterial drug resistance and hard-to-eradicate opportunistic infections have created a need for new antibiotics. Sequencing of microbial genomes has yielded many new potential targets for antibacterial drug discovery. However, little is known about the biochemical activities of many of these targets, making it difficult to develop HTS assays for them. Peptides isolated by phage display can be used as 'surrogate ligands' in competition assays for screening of targets of unknown function with small-molecule libraries. These screening assays can be adapted into a variety of high-throughput formats, including those based on radioactive, luminescence or fluorescence detection.

Journal Article↗
PubMed2003

Mining the microbial metabolome: a new frontier for natural product lead discovery.

Traditionally, natural products have been important sources of new leads for the pharmaceutical industry, but with discovery rates of novel structural classes in decline, the need to bioprospect alternate sources of chemical diversity is evident. Microbial genome sequencing projects have revealed the presence of 'silent' biosynthetic gene clusters where there is no current detectable product. Likewise, culture-independent techniques have provided access to the collective genomes of environmental microflora. Both sources of molecular diversity could encode potentially valuable metabolites. The ability to measure the entire complement of metabolites within microorganisms that are used as surrogate hosts to express such gene clusters will be crucial to the exploitation of these yet untapped reservoirs of metabolic diversity for future natural product drug discovery.

Biological Factors↗
PubMed2000

Monitoring gene expression using DNA microarrays.

The concurrent development of high-density array technologies and the complete sequencing of a number of microbial genomes is providing the opportunity to comprehensively and efficiently survey the transcription profile of microorganisms under different conditions and well-defined genotypes. Microarray-based studies are uncovering broad patterns of genetic activity, providing new understanding of gene functions and, in some cases, generating unexpected insight into transcriptional processes and biological mechanisms. One topic that has come to the forefront is how best to effectively manage and interpret the large data sets being generated. Although progress has been made, this remains a challenging opportunity for functional genomics research.

Gene Expression Regulation, Bacterial↗
PubMed2003

Regulation of the FecI-type ECF sigma factor by transmembrane signalling.

Induction of the ferric citrate transport genes of Escherichia coli K-12 involves a signalling cascade that starts at the cell surface and proceeds to the cytoplasm. Three specific proteins are involved: FecA in the outer membrane, FecR in the cytoplasmic membrane, and FecI in the cytoplasm. The binding of dinuclear ferric citrate to FecA causes substantial structural changes in FecA, triggering the signal cascade. The amino-proximal end of FecA interacts with the carboxy-proximal end of FecR in the periplasm. FecR then transmits the signal across the cytoplasmic membrane into the cytoplasm and activates the FecI sigma factor, which binds to the RNA polymerase core enzyme and directs the RNA polymerase to the promoter upstream of the fecABCDE transport genes to initiate transcription. Transcription of the fecIR regulatory genes and the fec transport genes is repressed by the Fur protein loaded with Fe(2+). Therefore, transcription of the fec transport genes is subjected to double control: cells first detect iron deficiency and respond by synthesis of the regulatory proteins FecI and FecR, which initiate transcription of the fec transport genes, provided ferric citrate is available. FecI belongs to the extracytoplasmic function sigma factors, which are widespread among bacteria. With the recent sequencing of complete microbial genomes, it has become apparent that the FecIRA cascade is now a paradigm for the regulatory control of FecI family sigmas in Gram-negative bacteria.

Amino Acid Sequence↗