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PubMed2004

G-PRIMER: greedy algorithm for selecting minimal primer set.

UNLABELLED: G-PRIMER, a web-based primer design program, has been developed to compute a minimal primer set specifically annealed to all the open reading frames in a given microbial genome. This program has been successfully used in the microarray experiment for analyzing the expression of genes in the Xanthomonas campestris genome. AVAILABILITY: It is available at http://mammoth.bii.a-star.edu.sg/gprimer/. Its source code is available upon request.

Algorithms↗
PubMed2004

LabArray: real-time imaging and analytical tool for microarrays.

UNLABELLED: Microarrays have been used to perform high-throughput genetic analyses such as single-nucleotide polymorphisms detection and microbial genome analysis. Some of these analyses require real-time monitoring of the hybridization signals with respect to a varying experimental condition, such as temperature. However, current microarray imaging and analysis packages typically do not possess such real-time capabilities. Therefore, microarray image analyses are often time-consuming and labour-intensive. LabArray was developed to expedite such processes by enabling real-time monitoring of microarray signals. AVAILABILITY: LabArray is available at http://www.eng.nus.edu.sg/civil/Labarray/labarray.htm CONTACT: cveliuwt@nus.edu.sg SUPPLEMENTARY INFORMATION: Screenshots and instructions for use are available at the above website.

Algorithms↗
PubMed2002

New approaches towards the identification of antibiotic and vaccine targets in Streptococcus pneumoniae.

Streptococcus pneumoniae causes more than one million deaths every year, mostly of young children in developing countries, due to pneumonia, bacteremia and meningitis. The emergence and dissemination of drug-resistant pneumococcal strains, coupled to changing patterns of virulence and the inadequacy of available vaccines, calls for an aggressive search for novel targets for antibiotic and vaccine development. Microbial genomics techniques allow genetic and biochemical tools to be employed to tackle discovery, design and development of new anti-infective agents based on the identification of hundreds of new targets. In this review, novel approaches employed to identify potential antibiotic and vaccine targets in S. pneumoniae are highlighted. Recently identified virulence factors, as well as molecules essential for bacterial viability, cell wall integrity and infectivity, are discussed.

Anti-Bacterial Agents↗
PubMed2026

Identification and Classification of Expressed Orphan Genes, Spurious Orphan Genes, and Conserved Genes in the Human Gut Microbiome.

Orphan genes (OGs)-genes lacking detectable homologs outside a species-are widespread in microbial genomes and are thought to contribute to their adaptation and molecular innovation. However, not all predicted OGs may represent novel functional coding sequences. False positive OGs, also called spurious OGs, can arise from gene prediction errors. We reason that OGs lacking detectable expression are more likely to be spurious. To test this, we combined large-scale metatranscriptomic profiling of the human gut microbiome with machine learning to distinguish expressed OGs from spurious ones and compare them with conserved genes (CGs) found in multiple species. Using nearly 5,000 metatranscriptome libraries, we identified ∼218,000 OGs supported by expression evidence, while ∼330,000 predicted OGs lacked detectable expression and were classified as spurious. We extracted 154 features for sequence, structural, and evolutionary properties for each gene and trained XGBoost classifiers while accounting for genomic representation. The models achieved an area under the receiver operating characteristic curve (AUC) of 0.82 in distinguishing expressed OGs from spurious OGs and an AUC of 0.93 in distinguishing expressed OGs from CGs. Interpretation based on SHAP (SHapley Additive exPlanations) revealed clear biological signals. Particularly, expressed orphans were present in more genomes than spurious ones, and expressed OGs were shorter than CGs. This work improves OG discovery and suggests that expressed OGs differ systematically from CGs and spurious OGs in sequence composition, structural constraints, and evolutionary signals.

Humans↗
PubMed2002

Predicted structure and phyletic distribution of the RNA-binding protein Hfq.

Hfq, a bacterial RNA-binding protein, was recently shown to contain the Sm1 motif, a characteristic of Sm and LSm proteins that function in RNA processing events in archaea and eukaryotes. In this report, comparative structural modeling was used to predict a three-dimensional structure of the Hfq core sequence. The predicted structure aligns with most major features of the Methanobacterium thermoautotrophicum LSm protein structure. Conserved residues in Hfq are positioned at the same structural locations responsible for subunit assembly and RNA interaction in Sm proteins. A highly conserved portion of Hfq assumes a structural fold similar to the Sm2 motif of Sm proteins. The evolution of the Hfq protein was explored by conducting a BLAST search of microbial genomes followed by phylogenetic analysis. Approximately half of the 140 complete or nearly complete genomes examined contain at least one gene coding for Hfq. The presence or absence of Hfq closely followed major bacterial clades. It is absent from high-level clades and present in the ancient Thermotogales-Aquificales clade and all proteobacteria except for those that have undergone major reduction in genome size. Residues at three positions in Hfq form signatures for the beta/gamma proteobacteria, alpha proteobacteria and low GC Gram-positive bacteria groups.

Amino Acid Sequence↗
PubMed2002

A one-step method for in vitro production of tRNA transcripts.

Sequencing of a large number of microbial genomes has led to the discovery of new enzymes involved in tRNA biosynthesis and tRNA function. Preparation of a great variety of RNA molecules is, therefore, of major interest for biochemical characterization of these proteins. We describe a fast, cost-effective and efficient method for in vitro production of tRNA transcripts. T7 RNA polymerase requires a double-stranded DNA promoter in order to initiate transcription; however, elongation does not require a double-stranded DNA template. A partially double-stranded transcription template formed by annealing of a short oligonucleotide, complementary to the T7 promoter, to a larger oligonucleotide is shown to be a good substrate for in vitro transcription. This method allows rapid production of a variety of tRNA transcripts which can be aminoacylated well. This eliminates the need for cloning of tRNA genes, large-scale plasmid preparation and enzymatic digestion.

DNA↗
PubMed2025

Opsins are Phospholipid Scramblases in All Domains of Life.

Opsins are highly abundant retinal proteins in the membranes of photoheterotrophic bacteria. However, some microbial genomes encode an opsin but lack the gene for the final enzyme in retinal synthesis. To account for this paradox, we hypothesized that bacterial opsins play a role in membrane structure and/or biogenesis independent from their potential for light-driven signaling or proton pumping. After purifying actinorhodopsin from a cell-free expression system and from E. coli membranes upon overexpression, we demonstrated both in vitro and in silico that actinorhodopsin from Nanopelagicus ca. is a phospholipid scramblase, serving in its pentameric state as a retinal-independent phospholipid diffusion channel. Phospholipid headgroups move along a transbilayer path between actinorhodopsin protomers, to equilibrate lipid content in the inner and outer leaflets. Two profound activities, membrane biosynthesis and capture of light energy, are thus facilitated by one ancient bacterial polypeptide. Light-dependent activity and light-independent phospholipid scrambling are shared functions of eukaryotic, archaeal, and bacterial rhodopsins.

ActR↗
PubMed2004

High-throughput mutation detection underlying adaptive evolution of Escherichia coli-K12.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis of base-specific cleavage products is an efficient, highly accurate tool for the detection of single base sequence variations. We describe the first application of this comparative sequencing strategy for automated high-throughput mutation detection in microbial genomes. The method was applied to identify DNA sequence changes that occurred in Escherichia coli K-12 MG1655 during laboratory adaptive evolution to new optimal growth phenotypes. Experiments were based on a genome-scale in silico model of E. coli metabolism and growth. This model computes several phenotypic functions and predicts optimal growth rates. To identify mutations underlying a 40-d adaptive laboratory evolution on glycerol, we resequenced 4.4% of the E. coli-K12 MG1655 genome in several clones picked at the end of the evolutionary process. The 1.54-Mb screen was completed in 13.5 h. This resequencing study is the largest reported by MALDI-TOF mass spectrometry to date. Ten mutations in 40 clones and three deviations from the reference sequence were detected. Mutations were predominantly found within the glycerol kinase gene. Functional characterization of the most prominent mutation shows its metabolic impact on the process of adaptive evolution. All sequence changes were independently confirmed by genotyping and Sanger-sequencing. We demonstrate that comparative sequencing by base-specific cleavage and MALDI-TOF mass spectrometry is an automated, fast, and highly accurate alternative to capillary sequencing.

Adaptation, Biological↗
PubMed2000

Crystallization and preliminary crystallographic study of b0220, an 'ORFan' protein of unknown function from Escherichia coli.

Newly sequenced microbial genomes continue to reveal up to 50% functionally uncharacterized 'anonymous' genes. A significant fraction of these anonymous ORFs does not exhibit any sequence similarity to any protein in the databases and constitutes a set of unique sequences, denoted 'ORFans'. The structure determination of ORFan proteins is both of evolutionary and functional interest. Here, the first crystallization of an Escherichia coli ORFan gene product, the 157 amino-acid b0220 protein, is reported. The crystals belong to the trigonal space group P3 or P3(1), with unit-cell parameters a = b = 47.2, c = 88.4 A. There are two molecules in the asymetric unit. Frozen crystals diffract to 1.6 A resolution using synchrotron radiation. Phasing was performed using multiwavelength anomalous dispersion (MAD) on the selenomethionine-substituted b0220 protein.

Bacterial Proteins↗
PubMed2005

Chemokines: key players in innate and adaptive immunity.

Healthy individuals initiate an immediate immune response to microbes by using a set of germline-encoded receptors that recognize common molecular patterns found on the surface of pathogens that are distinct from self-antigens. This innate immune response is the first line of defense against microorganisms in vertebrates, and constitutes the only immune response in plants and invertebrates. The innate immune system includes cellular components, as well as a host of soluble products (antimicrobial peptides, complement fragments, cytokines, and chemokines). The adaptive immune response, which provides long-lasting protection, takes days to develop and requires somatic mutations leading to the development of antigen-specific T cell receptors (cell-mediated immunity) and immunoglobulins (humoral immunity). Members of the chemokine superfamily are crucially involved in both innate and adaptive responses. We review the biological actions of the chemokine superfamily, focusing on several functions that are relevant for both immune responses, such as cell recruitment, microbicidal activity, cell activation, polarization of CD4+ T cells, and effects on structural cells. In particular, we will illustrate the central role that chemokines play in host defense, best demonstrated by the tremendous number of chemokine and chemokine receptor homologs found in microbial genomes, which deflect the immune response of the host.

Animals↗
PubMed2005

Bacterial degradation of xenobiotic compounds: evolution and distribution of novel enzyme activities.

Bacterial dehalogenases catalyse the cleavage of carbon-halogen bonds, which is a key step in aerobic mineralization pathways of many halogenated compounds that occur as environmental pollutants. There is a broad range of dehalogenases, which can be classified in different protein superfamilies and have fundamentally different catalytic mechanisms. Identical dehalogenases have repeatedly been detected in organisms that were isolated at different geographical locations, indicating that only a restricted number of sequences are used for a certain dehalogenation reaction in organohalogen-utilizing organisms. At the same time, massive random sequencing of environmental DNA, and microbial genome sequencing projects have shown that there is a large diversity of dehalogenase sequences that is not employed by known catabolic pathways. The corresponding proteins may have novel functions and selectivities that could be valuable for biotransformations in the future. Apparently, traditional enrichment and metagenome approaches explore different segments of sequence space. This is also observed with alkane hydroxylases, a category of proteins that can be detected on basis of conserved sequence motifs and for which a large number of sequences has been found in isolated bacterial cultures and genomic databases. It is likely that ongoing genetic adaptation, with the recruitment of silent sequences into functional catabolic routes and evolution of substrate range by mutations in structural genes, will further enhance the catabolic potential of bacteria toward synthetic organohalogens and ultimately contribute to cleansing the environment of these toxic and recalcitrant chemicals.

Amino Acid Sequence↗
PubMed2001

Classification and phylogeny of hydrogenases.

Hydrogenases (H2ases) catalyze the reversible oxidation of molecular hydrogen and play a central role in microbial energy metabolism. Most of these enzymes are found in Archaea and Bacteria, but a few are present in Eucarya as well. They can be distributed into three classes: the [Fe]-H2ases, the [NiFe]-H2ases, and the metal-free H2ases. The vast majority of known H2ases belong to the first two classes, and over 100 of these enzymes have been characterized genetically and/or biochemically. Compelling evidence from sequences and structures indicates that the [NiFe]- and [Fe]-H2ases are phylogenetically distinct classes of proteins. The catalytic core of the [NiFe]-H2ases is a heterodimeric protein, although additional subunits are present in many of these enzymes. Functional classes of [NiFe]-H2ases have been defined, and they are consistent with categories defined by sequence similarity of the catalytic subunits. The catalytic core of the [Fe]-H2ases is a ca. 350-residue domain that accommodates the active site (H-cluster). A few monomeric [Fe]-H2ases are barely larger than the H-cluster domain. Many others are monomeric as well, but possess additional domains that contain redox centers, mostly iron-sulfur. Some [Fe]-H2ases are oligomeric. The modular structure of H2ases is strikingly illustrated in recently unveiled sequences and structures. It is also remarkable that most of the accessory domains and subunits of H2ases have counterparts in other redox complexes, in particular NADH-ubiquinone oxidoreductase (Complex I) of respiratory chains. Microbial genome sequences are bringing forth a significant body of additional H2ase sequence data and contribute to the understanding of H2ase distribution and evolution. Altogether, the available data suggest that [Fe]-H2ases are restricted to Bacteria and Eucarya, while [NiFe]-H2ases, with one possible exception, seem to be present only in Archaea and Bacteria. H2ase processing and maturation involve the products of several genes which have been identified and are currently being characterized in the case of the [NiFe]-H2ases. In contrast, near to nothing is known regarding the maturation of the [Fe]-H2ases. Inspection of the currently available genome sequences suggests that the [NiFe]-H2ase maturation proteins have no similar counterparts in the genomes of organisms possessing [Fe]-H2ases only. This observation, if confirmed, would be consistent with the phylogenetic distinctiveness of the two classes of H2ases. Sequence alignments of catalytic subunits of H2ases have been implemented to construct phylogenetic trees that were found to be consistent, in the main, with trees derived from other data. On the basis of the comparisons performed and discussed here, proposals are made to simplify and rationalize the nomenclature of H2ase-encoding genes.

Amino Acid Sequence↗
PubMed2006

Microbial biochemistry, physiology, and biotechnology of hyperthermophilic Thermotoga species.

High-throughput sequencing of microbial genomes has allowed the application of functional genomics methods to species lacking well-developed genetic systems. For the model hyperthermophile Thermotoga maritima, microarrays have been used in comparative genomic hybridization studies to investigate diversity among Thermotoga species. Transcriptional data have assisted in prediction of pathways for carbohydrate utilization, iron-sulfur cluster synthesis and repair, expolysaccharide formation, and quorum sensing. Structural genomics efforts aimed at the T. maritima proteome have yielded hundreds of high-resolution datasets and predicted functions for uncharacterized proteins. The information gained from genomics studies will be particularly useful for developing new biotechnology applications for T. maritima enzymes.

Bacterial Proteins↗
PubMed2006

Postgenomic scan of metallo-beta-lactamase homologues in rhizobacteria: identification and characterization of BJP-1, a subclass B3 ortholog from Bradyrhizobium japonicum.

The diffusion of metallo-beta-lactamases (MBLs) among clinically important human pathogens represents a therapeutic issue of increasing importance. However, the origin of these resistance determinants is largely unknown, although an important number of proteins belonging to the MBL superfamily have been identified in microbial genomes. In this work, we analyzed the distribution and function of genes encoding MBL-like proteins in the class Rhizobiales. Among 12 released complete genomes of members of the class Rhizobiales, a total of 57 open reading frames (ORFs) were found to have the MBL conserved motif and identity scores with MBLs ranging from 8 to 40%. On the basis of the best identity scores with known MBLs, four ORFs were cloned into Escherichia coli for heterologous expression. Among their products, one (blr6230) encoded by the Bradyrhizobium japonicum USDA110 genome, named BJP-1, hydrolyzed beta-lactams when expressed in E. coli. BJP-1 enzyme is most closely related to the CAU-1 enzyme from Caulobacter vibrioides (40% amino acid sequence identity), a member of subclass B3 MBLs. A kinetic analysis revealed that BJP-1 efficiently hydrolyzed most beta-lactam substrates, except aztreonam, ticarcillin, and temocillin, with the highest catalytic efficiency measured with meropenem. Compared to other MBLs, BJP-1 was less sensitive to inactivation by chelating agents.

Agrobacterium tumefaciens↗
PubMed2003

Intracellular expression of Peptide fusions for demonstration of protein essentiality in bacteria.

We describe a "protein knockout" technique that can be used to identify essential proteins in bacteria. This technique uses phage display to select peptides that bind specifically to purified target proteins. The peptides are expressed intracellularly and cause inhibition of growth when the protein is essential. In this study, peptides that each specifically bind to one of seven essential proteins were identified by phage display and then expressed as fusions to glutathione S-transferase in Escherichia coli. Expression of peptide fusions directed against E. coli DnaN, LpxA, RpoD, ProRS, SecA, GyrA, and Era each dramatically inhibited cell growth. Under the same conditions, a fusion with a randomized peptide sequence did not inhibit cell growth. In growth-inhibited cells, inhibition could be relieved by concurrent overexpression of the relevant target protein but not by coexpression of an irrelevant protein, indicating that growth inhibition was due to a specific interaction of the expressed peptide with its target. The protein knockout technique can be used to assess the essentiality of genes of unknown function emerging from the sequencing of microbial genomes. This technique can also be used to validate proteins as drug targets, and their corresponding peptides as screening tools, for discovery of new antimicrobial agents.

Bacterial Proteins↗
PubMed2006

Novel quantitative biosystem for modeling physiological fluid shear stress on cells.

The response of microbes to changes in the mechanical force of fluid shear has important implications for pathogens, which experience wide fluctuations in fluid shear in vivo during infection. However, the majority of studies have not cultured microbes under physiological fluid shear conditions within a range commonly encountered by microbes during host-pathogen interactions. Here we describe a convenient batch culture biosystem in which (i) the levels of fluid shear force can be varied within physiologically relevant ranges and quantified via mathematical models and (ii) large numbers of cells can be planktonically grown and harvested to examine the effect of fluid shear levels on microbial genomic and phenotypic responses. A quantitative model based on numerical simulations and in situ imaging analysis was developed to calculate the fluid shear imparted by spherical beads of different sizes on bacterial cell cultures grown in a rotating wall vessel (RWV) bioreactor. To demonstrate the application of this model, we subjected cultures of the bacterial pathogen Salmonella enterica serovar Typhimurium to three physiologically-relevant fluid shear ranges during growth in the RVW and demonstrated a progressive relationship between the applied fluid shear and the bacterial genetic and phenotypic responses. By applying this model to different cell types, including other bacterial pathogens, entire classes of genes and proteins involved in cellular interactions may be discovered that have not previously been identified during growth under conventional culture conditions, leading to new targets for vaccine and therapeutic development.

Bacterial Proteins↗
PubMed1999

Identification of the yqhE and yafB genes encoding two 2, 5-diketo-D-gluconate reductases in Escherichia coli.

The identification of a gene (yiaE) encoding 2-ketoaldonate reductase (2KR) in our previous work led to the hypothesis that Escherichia coli has other ketogluconate reductases including 2, 5-diketo-D-gluconate reductase (25DKGR) and to study of the related ketogluconate metabolism. By using the deduced amino acid sequences of 5-diketo-D-gluconate reductase (5KDGR) of Gluconobacter oxydans and 25DKGR of Corynebacterium sp., protein databases were screened to detect homologous proteins. Among the proteins of E. coli, an oxidoreductase encoded by yjgU and having 56% similarity to 5KDGR of G. oxydans and two hypothetical oxidoreductases encoded by yqhE and yafB and having 49.8 and 42% similarity, respectively, to 25DKGR of Corynebacterium sp. were detected. Recently, the yjgU gene was identified as encoding 5KDGR and renamed idnO (C. Bausch, N. Peekhaus, C. Utz, T. Blais, E. Murray, T. Lowary, and T. Conway, J. Bacteriol. 180:3704-3710, 1998). The pathways involved in the metabolism of ketogluconate by E. coli have been predicted by biochemical analysis of purified enzymes and chemical analysis of the pathway intermediates. The gene products of yqhE and yafB were identified as 25DKGR-A, and 25DKGR-B, respectively, catalyzing the reduction of 25KDG to 2-keto-L-gulonate (2KLG). The native 25DKGR-A, 25DKGR-B, and 5KDGR had apparent molecular weights of about 30,000, 30,000, and 54,000, respectively. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, all three enzymes showed protein bands with a molecular weight of about 29,000, which indicated that 25DKGR-A, 25DKGR-B, and 5KDGR may exist as monomeric, monomeric, and dimeric proteins, respectively. The optimum pHs for reduction were 7.5, 7.0, and 8.0, respectively. The 5KDGR was active with NADH, whereas 25DKGR-A and 25DKGR-B were active with NADPH as a preferred electron donor. 25DKG can be converted to 5KDG by 2KR, which is then reduced to D-gluconate by 5KDGR. The pathways were compared with those of Erwinia sp. and Corynebacterium sp. A BLAST search of published and incomplete microbial genome sequences revealed that the ketogluconate reductases and their related metabolism may be widespread in many species.

Base Sequence↗