Identification and analysis of bacterial virulence genes in vivo

Signature-tagged mutagenesis is a mutation-based screening method for the identification of virulence genes of microbial pathogens. Genes isolated by this approach fall into three classes: those with known biochemical function, those of suspected function and some whose functions cannot be predicted from database searches. A variety of in vitro and in vivo methods are available to elucidate the function of genes of the second and third classes. We describe the use of some of these approaches to study the function of the Salmonella pathogenicity island 2 type III secretion system of Salmonella typhimurium. This virulence determinant is required for intracellular survival. Secretion by this system is induced by an acidic pH, and its function may be to alter trafficking of the Salmonella-containing vacuole. Use of a temperature-sensitive non-replicating plasmid and competitive index tests with other genes show that in vivo phenotypes do not always correspond to those predicted from in vitro studies.     

Gene fusion in <Emphasis Type="Italic">Helicobacter pylori</Emphasis>: making the ends meet

Fusion genes have been reported as a means of enabling the development of novel or enhanced functions. In this report, we analyzed fusion genes in the genomes of two Helicobacter pylori strains (26695 and J99) and identified 32 fusion genes that are present as neighbours in one strain (components) and are fused in the second (composite), and vice-versa. The mechanism for each case of gene fusion is explored. 28 out of 32 genes identified as fusion products in this analysis were reported as essential genes in the previously documented transposon mutagenesis of H. pylori strain G27. This observation suggests the potential of the products of fusion genes as putative microbial drug targets. These results underscore the utility of bacterial genomic sequence comparisons for understanding gene evolution and for in silico drug target identification in the post-genomic era. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Unraveling the Secret Lives of Bacteria: Use of In Vivo Expression Technology and Differential Fluorescence Induction Promoter Traps as Tools for Exploring Niche-Specific Gene Expression

A major challenge for microbiologists is to elucidate the strategies deployed by microorganisms to adapt to and thrive in highly complex and dynamic environments. In vitro studies, including those monitoring genomewide changes, have proven their value, but they can, at best, mimic only a subset of the ensemble of abiotic and biotic stimuli that microorganisms experience in their natural habitats. The widely used gene-to-phenotype approach involves the identification of altered niche-related phenotypes on the basis of gene inactivation. However, many traits contributing to ecological performance that, upon inactivation, result in only subtle or difficult to score phenotypic changes are likely to be overlooked by this otherwise powerful approach. Based on the premise that many, if not most, of the corresponding genes will be induced or upregulated in the environment under study, ecologically significant genes can alternatively be traced using the promoter trap techniques differential fluorescence induction and in vivo expression technology (IVET). The potential and limitations are discussed for the different IVET selection strategies and system-specific variants thereof. Based on a compendium of genes that have emerged from these promoter-trapping studies, several functional groups have been distinguished, and their physiological relevance is illustrated with follow-up studies of selected genes. In addition to confirming results from largely complementary approaches such as signature-tagged mutagenesis, some unexpected parallels as well as distinguishing features of microbial phenotypic acclimation in diverse environmental niches have surfaced. On the other hand, by the identification of a large proportion of genes with unknown function, these promoter-trapping studies underscore how little we know about the secret lives of bacteria and other microorganisms.     

Unraveling the secret lives of bacteria: use of in vivo expression technology and differential fluorescence induction promoter traps as tools for exploring niche-specific gene expression.

A major challenge for microbiologists is to elucidate the strategies deployed by microorganisms to adapt to and thrive in highly complex and dynamic environments. In vitro studies, including those monitoring genomewide changes, have proven their value, but they can, at best, mimic only a subset of the ensemble of abiotic and biotic stimuli that microorganisms experience in their natural habitats. The widely used gene-to-phenotype approach involves the identification of altered niche-related phenotypes on the basis of gene inactivation. However, many traits contributing to ecological performance that, upon inactivation, result in only subtle or difficult to score phenotypic changes are likely to be overlooked by this otherwise powerful approach. Based on the premise that many, if not most, of the corresponding genes will be induced or upregulated in the environment under study, ecologically significant genes can alternatively be traced using the promoter trap techniques differential fluorescence induction and in vivo expression technology (IVET). The potential and limitations are discussed for the different IVET selection strategies and system-specific variants thereof. Based on a compendium of genes that have emerged from these promoter-trapping studies, several functional groups have been distinguished, and their physiological relevance is illustrated with follow-up studies of selected genes. In addition to confirming results from largely complementary approaches such as signature-tagged mutagenesis, some unexpected parallels as well as distinguishing features of microbial phenotypic acclimation in diverse environmental niches have surfaced. On the other hand, by the identification of a large proportion of genes with unknown function, these promoter-trapping studies underscore how little we know about the secret lives of bacteria and other microorganisms.     

Cloning and targeted disruption of two lipopolysaccharide biosynthesis genes, kdsA and waaG, of Pseudomonas aeruginosa PAO1 by site-directed mutagenesis

The emergence of antibiotic resistance in bacterial pathogens poses a great challenge to public health and emphasizes the need for new antimicrobial targets. The recent development of microbial genomics and the availability of genome sequences allows for the identification of essential genes which could be novel and potential targets for antibacterial drugs. However, these predicted targets need experimental validation to confirm essentiality. Here, we report on experimental validation of a two potential targets in the lipopolysaccharide (LPS) biosynthesis pathway of the pathogen Pseudomonas aeruginosa PAO1 using insertion duplication. Two genes, kdsA and waaG, from LPS encoding proteins 2-dehydro-3-deoxyphosphooctonate aldolase and UDP-glucose (heptosyl) LPS α-1,3-glucosyltransferase were selected as putative target candidates for the gene disruption experiments using plasmid insertion mutagenesis to determine essentiality. The introduction of a selectable ampicillin and kanamycin resistance marker into the chromosome resulted in lack of recovery of antibiotic-resistant colonies suggesting the essentiality of these genes for the survival of P. aeruginosa. Several molecular analyses were carried out in order to confirm the essentiality of these genes. We propose that the above two validated drug targets are essential and can be screened for functional inhibitors for the discovery of novel therapeutic compounds against antibiotic-resistant opportunistic pathogen P. aeruginosa.     

Signature-tagged mutagenesis in the identification of virulence genes in pathogens

Signature-tagged mutagenesis is a functional genomics technique that identifies microbial genes required for infection within an animal host, or within host cells. The application of this technique to a range of microbial pathogens has resulted in the identification of novel virulence determinants in each screen performed to date, so that cumulatively several hundred genes have been ascribed a role in virulence.     

斑马鱼反转录病毒插入突变技术的发展及其在基因饱和突变与筛选中的应用

用于大规模基因突变与筛选的主要策略有化学诱变、插入突变、基因诱捕。插入突变是一种通过外源DNA整合的方式来获得突变体,并克隆得到对应突变基因的方法。运用反转录病毒介导的插入突变技术,在脊椎动物斑马鱼中已经获得了许多影响胚胎发育和细胞生长过程的突变体,并找到了对应的基因。基因诱捕技术也被运用于反转录病毒载体的构建。这套系统的建立使斑马鱼成为第一个有可能达到基因饱和突变和筛选的脊椎动物。     

转座子相关技术在微生物功能基因组研究中的应用

转座子是DNA插入因子的一种,是指能在基因组间或组内跳跃的DNA片段。转座子作为插入突变剂或分子标签已被广泛地应用于基因的分离和克隆,且因其独特的性质已成为发现新基因和基因功能分析的有效工具。这使得转座子无论是在单基因水平还是全基因组水平,都成为细菌、酵母和其他微生物研究的有力工具。简单而有效的体外转座反应可以对一些以往难以进行分析的顽固微生物进行转座诱变分析。而建立在转座子基础上的信号标签诱变技术和遗传足迹法的应用则发现了一些新的病原微生物毒力因子,从而可以更好地对这些病原微生物的致病机理进行阐述。这些再次说明转座子是微生物功能基因组研究中的有力工具。本文综述了转座子及其衍生载体介导的一些技术,并讨论其在微生物功能基因组研究中的应用。     

Functional genetics in Apicomplexa: potentials and limits

The Apicomplexans are obligate intracellular protozoan parasites and the causative agents of severe diseases in humans and animals. Although complete genome sequences are available since many years and for several parasites, they are replete with putative genes of unassigned function. Forward and reverse genetic approaches are limited only to a few Apicomplexans that can either be propagated in vitro or in a convenient animal model. This review will compare and contrast the most recent strategies developed for the genetic manipulation of Plasmodium falciparum, Plasmodium berghei and Toxoplasma gondii that have taken advantage of the intrinsic features of their respective genomes. Efforts towards the improvement of the transfection efficiencies in malaria parasites, the development of approaches to study essential genes and the elaboration of high-throughput methods for the identification of gene function will be discussed.     

Bioinformatics Annotation of the Hypothetical Proteins Found by Omics Techniques Can Help to Disclose Additional Virulence Factors

The advent of genomics should have facilitated the identification of microbial virulence factors, a key objective for vaccine design. When the bacterial pathogen infects the host it expresses a set of genes, a number of them being virulence factors. Among the genes identified by techniques as microarrays, in vivo expression technology, signature-tagged mutagenesis and differential fluorescence induction there are many related to cellular stress, basal metabolism, etc., which cannot be directly involved in virulence, or at least cannot be considered useful candidates to be deleted for designing a live attenuated vaccine. Among the genes disclosed by these methodologies there are a number of hypothetical or unknown proteins. As they can hide some true virulence factors, we have reannotated all of these hypothetical proteins from several respiratory pathogens by a careful and in-depth analysis of each one. Although some of the re-annotations match with functions that can be related to microbial virulence, the identification of virulence factors remains difficult.     

Insertional mutagenesis in zebrafish: genes for development, genes for disease.

In order to rapidly identify a substantial fraction of the genes with a unique and essential role in vertebrate development, the laboratory of Nancy Hopkins at MIT has performed a large insertional mutagenesis screen in zebrafish using a pseudotyped retroviral vector as the mutagen. We have recovered mutations in about one-quarter of the embryonic essential genes in this organism, and have identified the mutated genes in nearly all of these (333). As the ease of gene identification allowed us to clone the mutated genes for nearly all of the mutants rather than prioritizing based upon the initially observed phenotypes, this has provided an unbiased view of the diversity of genes required for vertebrate development as well as a large collection of mutants to be screened for more specific phenotypes. In collaboration with other labs, we have screened the insertional mutant for the development of a variety of organs and cell types, as well as phenotypes that could represent disease models, such as cystic kidney and hepatomegaly. Furthermore, while all of these mutants are embryonic lethal in their homozygous state, we are investigating the heterozygous adults for additional phenotypes, such as cancer predisposition.     

Bacterial alkaline proteases: molecular approaches and industrial applications

Proteolytic enzymes are ubiquitous in occurrence, being found in all living organisms, and are essential for cell growth and differentiation. The extracellular proteases are of commercial value and find multiple applications in various industrial sectors. Although there are many microbial sources available for producing proteases, only a few are recognized as commercial producers. A good number of bacterial alkaline proteases are commercially available, such as subtilisin Carlsberg, subtilisin BPN' and Savinase, with their major application as detergent enzymes. However, mutations have led to newer protease preparations with improved catalytic efficiency and better stability towards temperature, oxidizing agents and changing wash conditions. Many newer preparations, such as Durazym, Maxapem and Purafect, have been produced, using techniques of site-directed mutagenesis and/or random mutagenesis. Directed evolution has also paved the way to a great variety of subtilisin variants with better specificities and stability. Molecular imprinting through conditional lyophilization is coming up to match molecular approaches in protein engineering. There are many possibilities for modifying biocatalysts through molecular approaches. However, the search for microbial sources of novel alkaline proteases in natural diversity through the "metagenome" approach is targeting a hitherto undiscovered wealth of molecular diversity. This fascinating development will allow the biotechnological exploitation of uncultured microorganisms, which by far outnumber the species accessible by cultivation, regardless of the habitat. In this review, we discuss the types and sources of proteases, protease yield-improvement methods, the use of new methods for developing novel proteases and applications of alkaline proteases in industrial sectors, with an overview on the use of alkaline proteases in the detergent industry.     

Identification and modeling of a drug target for Clostridium perfringens SM101

In the present study, comparative genome analysis between Clostridium perfringens and the human genome was carried out to identify genes that are essential for the pathogen's survival, and non-homologous to the genes of human host, that can be used as potential drug targets. The study resulted in the identification of 426 such genes. The number of these potential drug targets thus identified is significantly lower than the genome's protein coding capacity (2558 protein coding genes). The 426 genes of C. perfringens were further analyzed for overall similarities with the essential genes of 14 different bacterial species present in Database of Essential Genes (DEG). Our results show that there are only 5 essential genes of C. perfringens that exhibit similarity with 12 species of the 14 different bacterial species present in DEG database. Of these, 1 gene was similar in 12 species and 4 genes were similar in 11 species. Thus, the study opens a new avenue for the development of potential drugs against the highly pathogenic bacterium. Further, by selecting these essential genes of C. perfringens, which are common and essential for other pathogenic microbial species, a broad spectrum anti-microbial drug can be developed. As a case study, we have built a homology model of one of the potential drug targets, ABC transporter-ATP binding protein, which can be employed for in silico docking studies by suitable inhibitors.     

Novel integrative genomics strategies to identify genes for complex traits

Forward genetics is a common approach to dissecting complex traits like common human diseases. The ultimate aim of this approach was the identification of genes that are causal for disease or other phenotypes of interest. However, the forward genetics approach is by definition restricted to the identification of genes that have incurred mutations over the course of evolution or that incurred mutations as a result of chemical mutagenesis, and that as a result lead to disease or to variations in other phenotypes of interest. Genes that harbour no such mutations, but that play key roles in parts of the biological network that lead to disease, are systematically missed by this class of approaches. Recently, a class of novel integrative genomics approaches has been devised to elucidate the complexity of common human diseases by intersecting genotypic, molecular profiling, and clinical data in segregating populations. These novel approaches take a more holistic view of biological systems and leverage the vast network of gene–gene interactions, in combination with DNA variation data, to establish causal relationships among molecular profiling traits and Fbetween molecular profiling and disease (or other classic phenotypes). A number of novel genes for disease phenotypes have been identified as a result of these approaches, highlighting the utility of integrating orthogonal sources of data to get at the underlying causes of disease.     

Forward genetics and map-based cloning approaches

Whereas reverse genetics strategies seek to identify and select mutations in a known sequence, forward genetics requires the cloning of sequences underlying a particular mutant phenotype. Map-based cloning is tedious, hampering the quick identification of candidate genes. With the unprecedented progress in the sequencing of whole genomes, and perhaps even more with the development of saturating marker technologies, map-based cloning can now be performed so efficiently that, at least for some plant model systems, it has become feasible to identify some candidate genes within a few months. This, in turn, will boost the use of forward genetics approaches, as applied (for example) to isolating genes involved in natural variation and genes causing phenotypic mutations as derived from (second-site) mutagenesis screens.     

生物信息学在工业生物催化研究中的应用

随着石油等不可再生资源的日益减少以及环境污染问题的日益严重,应用工业生物催化技术改造或取代传统化工工艺已经成为新世纪化学工业可持续发展的研究热点。工业生物催化技术的研究对象是生物催化剂及其催化过程。近来,利用生物信息学技术进行工业生物催化研究已经越来越受到人们的重视。随着工业生物催化的发展,生物信息学将直接指导并加快新型高效生物催化剂的发现及功能改造进程。     

Lethal mutations in the isoprenoid pathway of Salmonella enterica

Essential isoprenoid compounds are synthesized using the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in many gram-negative bacteria, some gram-positive bacteria, some apicomplexan parasites, and plant chloroplasts. The alternative mevalonate pathway is found in archaea and eukaryotes, including cytosolic biosynthesis in plants. The existence of orthogonal essential pathways in eukaryotes and bacteria makes the MEP pathway an attractive target for the development of antimicrobial agents. A system is described for identifying mutations in the MEP pathway of Salmonella enterica serovar Typhimurium. Using this system, point mutations induced by diethyl sulfate were found in the all genes of the essential MEP pathway and also in genes involved in uptake of methylerythritol. Curiously, none of the MEP pathway genes could be identified in the same parent strain by transposon mutagenesis, despite extensive searches. The results complement the biochemical and bioinformatic approaches to the elucidation of the genes involved in the MEP pathway and also identify key residues for activity in the enzymes of the pathway.