Bacterial Gene Set Research Articles

The divergent genome of Scorpion Group 1 (SG1) intracellular bacterium from the venom glands of Vaejovis smithi (Scorpiones: Vaejovidae)

Scorpions were among the first animals on land around 430 million years ago. Like many arachnids, scorpions have evolved complex venoms used to paralyze their prey and for self-defense. Here we sequenced and analyzed the metagenomic DNA from venom glands from Vaejovis smithi scorpions. A metagenome-assembled genome (MAG) of 624,025 bp was obtained corresponding to the previously reported Scorpion Group 1 (SG1). The SG1 genome from venom glands had a low GC content (25.8%) characteristic of reduced genomes, many hypothetical genes and genes from the reported minimal set of bacterial genes. Phylogenomic reconstructions placed the uncultured SG1 distant from other reported bacteria constituting a taxonomic novelty. By PCR we detected SG1 in all tested venom glands from 30 independent individuals. Microscopically, we observed SG1 inside epithelial cells from the venom glands using FISH and its presence in scorpion embryos suggested that SG1 is transferred from mother to offspring.

Genes on the circular code alphabet

The X motifs, motifs from the circular code X, are enriched in the (protein coding) genes of bacteria, archaea, eukaryotes, plasmids and viruses, moreover, in the minimal gene set belonging to the three domains of life, as well as in tRNA and rRNA sequences. They allow to retrieve, maintain and synchronize the reading frame in genes, and contribute to the regulation of gene expression. These results lead here to a theoretical study of genes based on the circular code alphabet. A new occurrence relation of the circular code X under the hypothesis of an equiprobable (balanced) strand pairing is given. Surprisingly, a statistical analysis of a large set of bacterial genes retrieves this relation on the circular code alphabet, but not on the DNA alphabet. Furthermore, the circular code X has the strongest balanced circular code pairing among 216 maximal C3 self-complementary trinucleotide circular codes, a new property of this circular code X. As an application of this theory, different tRNAs studied on the circular code alphabet reveal an unexpected stem structure. Thus, the circular code X would have constructed a coding stem in tRNAs as an outline of the future gene structure and the future DNA double helix.

Regulation Mediated by N-Acyl Homoserine Lactone Quorum Sensing Signals in the Rhizobium-Legume Symbiosis.

Soil-dwelling bacteria collectively referred to as rhizobia synthesize and perceive N-acyl-homoserine lactone (AHL) signals to regulate gene expression in a population density-dependent manner. AHL-mediated signaling in these bacteria regulates several functions which are important for the establishment of nitrogen-fixing symbiosis with legume plants. Moreover, rhizobial AHL act as interkingdom signals triggering plant responses that impact the plant-bacteria interaction. Both the regulatory mechanisms that control AHL synthesis in rhizobia and the set of bacterial genes and associated traits under quorum sensing (QS) control vary greatly among the rhizobial species. In this article, we focus on the well-known QS system of the alfalfa symbiont Sinorhizobium (Ensifer) meliloti. Bacterial genes, environmental factors and transcriptional and posttranscriptional regulatory mechanisms that control AHL production in this Rhizobium, as well as the effects of the signaling molecule on bacterial phenotypes and plant responses will be reviewed. Current knowledge of S. meliloti QS will be compared with that of other rhizobia. Finally, participation of the legume host in QS by interfering with rhizobial AHL perception through the production of molecular mimics will also be addressed.

A novel proposal of a simplified bacterial gene set and the neo-construction of a general minimized metabolic network.

A minimal gene set (MGS) is critical for the assembly of a minimal artificial cell. We have developed a proposal of simplifying bacterial gene set to approximate a bacterial MGS by the following procedure. First, we base our simplified bacterial gene set (SBGS) on experimentally determined essential genes to ensure that the genes included in the SBGS are critical. Second, we introduced a half-retaining strategy to extract persistent essential genes to ensure stability. Third, we constructed a viable metabolic network to supplement SBGS. The proposed SBGS includes 327 genes and required 431 reactions. This report describes an SBGS that preserves both self-replication and self-maintenance systems. In the minimized metabolic network, we identified five novel hub metabolites and confirmed 20 known hubs. Highly essential genes were found to distribute the connecting metabolites into more reactions. Based on our SBGS, we expanded the pool of targets for designing broad-spectrum antibacterial drugs to reduce pathogen resistance. We also suggested a rough semi-de novo strategy to synthesize an artificial cell, with potential applications in industry.

Successful completion of a semi-automated enzyme-free cloning method.

Nowadays, in scientific fields such as Structural Biology or Vaccinology, there is an increasing need of fast, effective and reproducible gene cloning and expression processes. Consequently, the implementation of robotic platforms enabling the automation of protocols is becoming a pressing demand. The main goal of our study was to set up a robotic platform devoted to the high-throughput automation of the polymerase incomplete primer extension cloning method, and to evaluate its efficiency compared to that achieved manually, by selecting a set of bacterial genes that were processed either in the automated platform (330) or manually (94). Here we show that we successfully set up a platform able to complete, with high efficiency, a wide range of molecular biology and biochemical steps. 329 gene targets (99%) were effectively amplified using the automated procedure and 286 (87%) of these PCR products were successfully cloned in expression vectors, with cloning success rates being higher for the automated protocols respect to the manual procedure (93.6 and 74.5%, respectively).

High Genetic Diversity of Microbial Cellulase and Hemicellulase Genes in the Hindgut of Holotrichia parallela Larvae.

In this study, we used a culture-independent method based on library construction and sequencing to analyze the genetic diversity of the cellulase and hemicellulase genes of the bacterial community resident in the hindgut of Holotrichia parallela larvae. The results indicate that there is a large, diverse set of bacterial genes encoding lignocellulose hydrolysis enzymes in the hindgut of H. parallela. The total of 101 distinct gene fragments (similarity <95%) of glycosyl hydrolase families including GH2 (24 genes), GH8 (27 genes), GH10 (19 genes), GH11 (14 genes) and GH36 (17 genes) families was retrieved, and certain sequences of GH2 (10.61%), GH8 (3.33%), and GH11 (18.42%) families had <60% identities with known sequences in GenBank, indicating their novelty. Based on phylogenetic analysis, sequences from hemicellulase families were related to enzymes from Bacteroidetes and Firmicutes. Fragments from cellulase family were most associated with the phylum of Proteobacteria. Furthermore, a full-length endo-xylanase gene was obtained, and the enzyme exhibited activity over a broad range of pH levels. Our results indicate that there are large number of cellulolytic and xylanolytic bacteria in the hindgut of H. parallela larvae, and these symbiotic bacteria play an important role in the degradation of roots and other organic matter for the host insect.

Minimal genome encoding proteins with constrained amino acid repertoire

Minimal bacterial gene set comprises the genetic elements needed for survival of engineered bacterium on a rich medium. This set is estimated to include 300–350 protein-coding genes. One way of simplifying an organism with such a minimal genome even further is to constrain the amino acid content of its proteins. In this study, comparative genomics approaches and the results of gene knockout experiments were used to extrapolate the minimal gene set of mollicutes, and bioinformatics combined with the knowledge-based analysis of the structure-function relationships in these proteins and their orthologs, paralogs and analogs was applied to examine the challenges of completely replacing the rarest residue, cysteine. Among several known functions of cysteine residues, their roles in the active centers of the enzymes responsible for deoxyribonucleoside synthesis and transfer RNA modification appear to be crucial, as no alternative chemistry is known for these reactions. Thus, drastic reduction of the content of the rarest amino acid in a minimal proteome appears to be possible, but its complete elimination is challenging.

Conserved Bacterial RNase YbeY Plays Key Roles in 70S Ribosome Quality Control and 16S rRNA Maturation

Quality control of ribosomes is critical for cellular function since protein mistranslation leads to severe physiological consequences. We report evidence of a previously unrecognized ribosome quality control system in bacteria that operates at the level of 70S to remove defective ribosomes. YbeY, a previously unidentified endoribonuclease, and the exonuclease RNase R act together by a process mediated specifically by the 30S ribosomal subunit, to degrade defective 70S ribosomes but not properly matured 70S ribosomes or individual subunits. Furthermore, there is essentially no fully matured 16S rRNA ina ΔybeY mutant at 45°C, making YbeY the only endoribonuclease to be implicated in the critically important processing of the 16S rRNA 3' terminus. These key roles in ribosome quality control and maturation indicate why YbeY is a member of the minimal bacterial gene set and suggest that it could be a potential target for antibacterial drugs.

Phylogroup and lpfA influence epithelial invasion by mastitis associated Escherichia coli

Escherichia coli infection is one of the most common causes of bovine mastitis in well managed dairies. Although E. coli infections are usually transient, E. coli can also cause persistent intramammary infections. We sought to determine whether E. coli isolates recovered from either transient or persistent intramammary infections differed both genetically and in their ability to invade mammary epithelial cells.E. coli isolates from transient (ECtrans, n=16) and persistent (ECpers, n=12) mastitis cases were compared for differences in overall genotype, virulence genes, serotype, phylogroup (A, B1, B2, D), and invasion of bovine mammary epithelial cells, MAC-T by microarray analysis, suppressive subtractive hybridization, PCR and gentamicin protection assays.ECtrans and ECpers were diverse in overall genotype and serotype, and were predominantly of phylogroups A and B1. Both ECtrans and ECpers contained genes encoding type II, IV and VI secretion systems, long polar fimbriae (lpfA) and iron acquisition, and lacked genes associated with virulence in diarrheagenic E. coli. ECtrans had fewer virulence genes than ECpers (p<0.05), but no individual virulence genes were unique to either group. In phylogroup A, ECpers were more invasive than ECtrans (p<0.05), but no difference was observed between them in phylogroup B1. Enhanced epithelial cell invasion was associated with lpfA (p<0.05).Our findings indicate that a genetically diverse group of E. coli is associated with transient and persistent mastitis. We did not identify a set of bacterial genes to account for phenotypic differences. However, we found that mastitis phenotype, phylogroup and presence of lpfA were associated with the ability to invade cultured bovine mammary epithelial cells.

In the case of transmission of &lt;i&gt;Mycobacterium ulcerans&lt;/i&gt; in Buruli ulcer disease &lt;i&gt;Acanthamoeba &lt;/i&gt;species stand accused

Buruli ulcer disease caused by Mycobacterium ulcerans results in extensive destruction of skin and soft tissue and long-term functional disabilities that ultimately require surgery and rehabilitation. The disease is associated with aquatic and swampy environments with the mycobacterium occurring in biofilms, soil, aquatic insects, fish and wildlife however, the mode of transmission to humans remains an enigma. Current transmission ideas including bites from predatory water bugs and mosquitoes, do not explain satisfactorily the spasmodic disease distribution in human populations. Here we argue that Acanthamoeba species are the natural hosts of M. ulcerans and are mainly responsible for disease transmission because; (i) Acanthamoebae are known natural hosts of several microbial pathogens including M. marinum, M. avium and Legionella pneumophila, (ii) culture of slow-to-grow microbial pathogens hosted in nature by Acanthamoeba spp is enhanced when the media is seeded with the protozoa, (iii) acanthamoebae and M. ulcerans share similar bio-ecological and epidemiological settings, (iv) documented evidence that prior growth of L. pneumophila and M. avium in acanthamoebae influences entry mechanisms, intracellular growth and virulence in human monocytes, (v) Acanthamoeba spp also infect humans and cause diseases via routes of openings including broken skin and sites of trauma similar to M. ulcerans and (vi) M. ulcerans is rather a fastidious intracellular organism as recent analysis of the genome indicate. We argue further that temperature plays a significant role in transmission determining the fate of either the intracellular microbe or the host cells. Also, Acanthamoeba-pathogen association has a long evolutionary history because the same set of bacterial genes and gene products e.g. in L. pneumophila are required for survival in both mammalian and protozoan host cells. We suggest that the involvement of Acanthamoeba in the transmission of M. ulcerans to humans better explains the disease's epidemiology.

Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa

Gut microbial composition depends on different dietary habits just as health depends on microbial metabolism, but the association of microbiota with different diets in human populations has not yet been shown. In this work, we compared the fecal microbiota of European children (EU) and that of children from a rural African village of Burkina Faso (BF), where the diet, high in fiber content, is similar to that of early human settlements at the time of the birth of agriculture. By using high-throughput 16S rDNA sequencing and biochemical analyses, we found significant differences in gut microbiota between the two groups. BF children showed a significant enrichment in Bacteroidetes and depletion in Firmicutes (P < 0.001), with a unique abundance of bacteria from the genus Prevotella and Xylanibacter, known to contain a set of bacterial genes for cellulose and xylan hydrolysis, completely lacking in the EU children. In addition, we found significantly more short-chain fatty acids (P < 0.001) in BF than in EU children. Also, Enterobacteriaceae (Shigella and Escherichia) were significantly underrepresented in BF than in EU children (P < 0.05). We hypothesize that gut microbiota coevolved with the polysaccharide-rich diet of BF individuals, allowing them to maximize energy intake from fibers while also protecting them from inflammations and noninfectious colonic diseases. This study investigates and compares human intestinal microbiota from children characterized by a modern western diet and a rural diet, indicating the importance of preserving this treasure of microbial diversity from ancient rural communities worldwide.

Mechanisms of bacterial virulence in pulmonary infections

To consider the relevance to severe human lung infections of recently discovered virulence mechanisms of Staphylococcus aureus and Francisella tularensis. S. aureus has long been considered an opportunistic pathogen. However, due to the emergence of community-acquired methicillin-resistant S. aureus (CA-MRSA) strains that can readily infect and kill normal hosts, S. aureus must now be considered a potentially virulent pathogen. The evolution of S. aureus from an organism associated with asymptomatic nasopharyngeal colonization to one associated with community-acquired lethal infections may reflect horizontal acquisition of bacterial genes that enable efficient spread, aggressive host invasion, and effective immune evasion. Alleviating the burden of staphylococcal disease will require better understanding of host susceptibility and of staphylococcal virulence and antibiotic resistance. In contrast to the rapidly evolving staphylococcal virulence strategy, recent genomic analysis of F. tularensis has revealed a small set of bacterial genes associated with the marked virulence of its North American subspecies. This suggests that a relatively stable strategy of immune evasion underlies this pathogen's ability to establish serious life-threatening lung infections from a very small inoculum. Understanding bacterial pathogenesis will require additional research into both host susceptibility factors and bacterial virulence mechanisms, including horizontal gene transfer. Refinements in the molecular detection of bacteria in the clinical setting, as well as whole genome analysis of both pathogens and patients, are expected to aid in the understanding of bacterial-induced lung injury.

Lifestyle of Lactobacillus plantarum in the mouse caecum

Lactobacillus plantarum is a common inhabitant of mammalian gastrointestinal tracts. Strains of L. plantarum are also marketed as probiotics intended to confer beneficial health effects upon delivery to the human gut. To understand how L. plantarum adapts to its gut habitat, we used whole genome transcriptional profiling to characterize the transcriptome of strain WCFS1 during colonization of the caeca of adult germ-free C57Bl/6 J mice fed a standard low-fat rodent chow diet rich in complex plant polysaccharides or a prototypic Western diet high in simple sugars and fat. Lactobacillus plantarum colonized the digestive tracts of these animals to high levels, although L. plantarum was found in 10-fold higher amounts in the caeca of mice fed the standard chow. Metabolic reconstructions based on the transcriptional data sets revealed that genes involved in carbohydrate transport and metabolism form the principal functional group that is upregulated in vivo compared with exponential phase cells grown in three different culture media, and that a Western diet provides a more nutritionally restricted, growth limiting milieu for the microbe in the distal gut. A set of bacterial genes encoding cell surface-related functions were differentially regulated in both groups of mice. This set included downregulated genes required for the d-alanylation of lipoteichoic acids, extracellular structures of L. plantarum that mediate interactions with the host immune system. These results, obtained in a reductionist gnotobiotic mouse model of the gut ecosystem, provide insights about the niches (professions) of this lactic acid bacterium, and a context for systematically testing features that affect epithelial and immune cell responses to this organism in the digestive tract.

New avenues for Microbial Biotechnology: the beginning of a golden era

New avenues for <i>Microbial Biotechnology</i>: the beginning of a golden era

Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite

From the standpoints of both basic research and biotechnology, there is considerable interest in reaching a clearer understanding of the diversity of biological mechanisms employed during lignocellulose degradation. Globally, termites are an extremely successful group of wood-degrading organisms and are therefore important both for their roles in carbon turnover in the environment and as potential sources of biochemical catalysts for efforts aimed at converting wood into biofuels. Only recently have data supported any direct role for the symbiotic bacteria in the gut of the termite in cellulose and xylan hydrolysis. Here we use a metagenomic analysis of the bacterial community resident in the hindgut paunch of a wood-feeding 'higher' Nasutitermes species (which do not contain cellulose-fermenting protozoa) to show the presence of a large, diverse set of bacterial genes for cellulose and xylan hydrolysis. Many of these genes were expressed in vivo or had cellulase activity in vitro, and further analyses implicate spirochete and fibrobacter species in gut lignocellulose degradation. New insights into other important symbiotic functions including H2 metabolism, CO2-reductive acetogenesis and N2 fixation are also provided by this first system-wide gene analysis of a microbial community specialized towards plant lignocellulose degradation. Our results underscore how complex even a 1-microl environment can be.

Determination of the core of a minimal bacterial gene set.

The availability of a large number of complete genome sequences raises the question of how many genes are essential for cellular life. Trying to reconstruct the core of the protein-coding gene set for a hypothetical minimal bacterial cell, we have performed a computational comparative analysis of eight bacterial genomes. Six of the analyzed genomes are very small due to a dramatic genome size reduction process, while the other two, corresponding to free-living relatives, are larger. The available data from several systematic experimental approaches to define all the essential genes in some completely sequenced bacterial genomes were also considered, and a reconstruction of a minimal metabolic machinery necessary to sustain life was carried out. The proposed minimal genome contains 206 protein-coding genes with all the genetic information necessary for self-maintenance and reproduction in the presence of a full complement of essential nutrients and in the absence of environmental stress. The main features of such a minimal gene set, as well as the metabolic functions that must be present in the hypothetical minimal cell, are discussed.

Structure-function and biogenesis of the type IV pili.

Type IV pili are adhesins expressed by a number of diverse gram-negative microorganisms. These pili are related through similarities in the primary amino acid sequences of the structural subunits, a conserved assembly machinery, and a similar mechanism of transcriptional regulation. Type IV pilus assembly is preceded by proteolytic processing and N-methylation of the pilin polypeptide. This process is carried out by a novel bifunctional enzyme PilD, first identified in Pseudomonas aeruginosa. Moreover, proteins homologous with type IV pilins have been shown to function in extracellular protein secretion in gram-negative bacteria and in transformation competence in gram-positive microorganisms. Like prepilin, these proteins are also processed and N-methylated by PilD. Transcription of the genes for type IV pilins is carried out by an RNA polymerase with a minor sigma factor, RpoN. In P. aeruginosa two other regulatory elements (PilS and PilR) are required for pilin expression. RpoN, but not PilS and PilR, is required for expression of a diverse set of bacterial genes. Therefore, regulation of synthesis and posttranslational modification and assembly of type IV pili serves as a useful model for a number of diverse biological processes in the bacterial cell.

Specialized transducing phage lambda carrying the genes for coupling factor of oxidative phosphorylation of Escherichia coli: increased synthesis of coupling factor on induction of prophage lambda asn.

Studies were made of the synthesis of the coupling factor complex (F1--F0) of oxidative phosphorylation after prophage induction of a set of Escherichia coli strains lysogenic for defective transducing phage lambda asn, lambda uncA, or lambda bglC. The transducing phages had been isolated from a strain of E. coli carrying prophage lambda cI857 S7 within the bglB gene located near the unc gene cluster [Miki, T., Hiraga, S., Nagata, T. & Yura, T. (1978) Proc. Natl. Acad. Sci. USA 75, 5099--5103]. When lysogenic cells carrying lambda asn and lambda cI857 S7 were induced at high temperature, synthesis of the F1-ATPase portion of the complex increased to severalfold that of the noninduced cells. In contrast, no increase was observed upon thermoinduction of cells carrying lambda uncA or lambda bglC. The number of membrane sites that could bind purified F1-ATPase also increased significantly upon induction by lambda asn but not by lambda uncA or lambda bglC. In addition, F1-depleted membranes prepared from lambda asn-induced bacteria required more dicyclohexylcarbodiimide to seal the proton pathway than did those from noninduced bacteria. These results strongly suggest that lambda asn carries a set of bacterial genes coding for all the F1 polypeptides (the alpha, beta, gamma, delta, and probably the epsilon subunits) and at least some of the genes involved in formation of F0 polypeptides. Although lambda uncA carries the structural gene (uncA) for the alpha subunit of F1-ATPase, it apparently does not carry the whole set of F1--F0 genes.

Evidence for a lambda transducing phage carrying the genes for the beta and beta' subunits of Escherichia coli RNA polymerase.

Complementation tests performed between the rifampicin resistance transducing phage λcI857s7drifd18 and a polar amber mutation in RNA polymerase affecting the synthesis of both the β and β′ subunits, show that this phage carries both of these genes. In contrast, a second rif transducing phage, λcI857s7drifd12, fails to complement the polar mutation, but does complement a non-polar amber mutation affecting the synthesis of only the β subunit. Synthesis of β and β′ in the strain containing λcI857s7drifd18 and the polar amber mutation proceeds at the normal rate, suggesting that this phage also carries the promoter region for this set of bacterial genes. Replication of the same lysogen after heat induction produces a moderate gene dosage effect on subunit synthesis.