A Noise Trimming and Positional Significance of Transposon Insertion System to Identify Essential Genes in Yersinia pestis

This is the final version of the article. Available from Springer Nature via the DOI in this record.Massively parallel sequencing technology coupled with saturation mutagenesis has provided new and global insights into gene functions and roles. At a simplistic level, the frequency of mutations within genes can indicate the degree of essentiality. However, this approach neglects to take account of the positional significance of mutations - the function of a gene is less likely to be disrupted by a mutation close to the distal ends. Therefore, a systematic bioinformatics approach to improve the reliability of essential gene identification is desirable. We report here a parametric model which introduces a novel mutation feature together with a noise trimming approach to predict the biological significance of Tn5 mutations. We show improved performance of essential gene prediction in the bacterium Yersinia pestis, the causative agent of plague. This method would have broad applicability to other organisms and to the identification of genes which are essential for competitiveness or survival under a broad range of stresses.This work was supported by the Defence Science and Technology Laboratory under contract DSTLX-1000060221 (WP1)

An integrated computational-experimental approach reveals Yersinia pestis genes essential across a narrow or a broad range of environmental conditions

This is the final version. Available from BMC via the DOI in this recordAvailability of data and materials: The datasets supporting the conclusions of this article are available at the NCBI GEO website https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE100226.BACKGROUND: The World Health Organization has categorized plague as a re-emerging disease and the potential for Yersinia pestis to also be used as a bioweapon makes the identification of new drug targets against this pathogen a priority. Environmental temperature is a key signal which regulates virulence of the bacterium. The bacterium normally grows outside the human host at 28 °C. Therefore, understanding the mechanisms that the bacterium used to adapt to a mammalian host at 37 °C is central to the development of vaccines or drugs for the prevention or treatment of human disease. RESULTS: Using a library of over 1 million Y. pestis CO92 random mutants and transposon-directed insertion site sequencing, we identified 530 essential genes when the bacteria were cultured at 28 °C. When the library of mutants was subsequently cultured at 37 °C we identified 19 genes that were essential at 37 °C but not at 28 °C, including genes which encode proteins that play a role in enabling functioning of the type III secretion and in DNA replication and maintenance. Using genome-scale metabolic network reconstruction we showed that growth conditions profoundly influence the physiology of the bacterium, and by combining computational and experimental approaches we were able to identify 54 genes that are essential under a broad range of conditions. CONCLUSIONS: Using an integrated computational-experimental approach we identify genes which are required for growth at 37 °C and under a broad range of environments may be the best targets for the development of new interventions to prevent or treat plague in humans.This work was funded by the Defence Science and Technology Laboratory, award DSTLX-1000060221 (WP1)

An integrated computational-experimental approach reveals Yersinia pestis genes essential across a narrow or a broad range of environmental conditions

Background The World Health Organization has categorized plague as a re-emerging disease and the potential for Yersinia pestis to also be used as a bioweapon makes the identification of new drug targets against this pathogen a priority. Environmental temperature is a key signal which regulates virulence of the bacterium. The bacterium normally grows outside the human host at 28 °C. Therefore, understanding the mechanisms that the bacterium used to adapt to a mammalian host at 37 °C is central to the development of vaccines or drugs for the prevention or treatment of human disease. Results Using a library of over 1 million Y. pestis CO92 random mutants and transposon-directed insertion site sequencing, we identified 530 essential genes when the bacteria were cultured at 28 °C. When the library of mutants was subsequently cultured at 37 °C we identified 19 genes that were essential at 37 °C but not at 28 °C, including genes which encode proteins that play a role in enabling functioning of the type III secretion and in DNA replication and maintenance. Using genome-scale metabolic network reconstruction we showed that growth conditions profoundly influence the physiology of the bacterium, and by combining computational and experimental approaches we were able to identify 54 genes that are essential under a broad range of conditions. Conclusions Using an integrated computational-experimental approach we identify genes which are required for growth at 37 °C and under a broad range of environments may be the best targets for the development of new interventions to prevent or treat plague in humans

Global Analysis of Genes Essential for Francisella tularensis Schu S4 Growth In Vitro and for Fitness during Competitive Infection of Fischer 344 Rats

This is the final version. Available from American Society for Microbiology via the DOI in this record The highly virulent intracellular pathogen Francisella tularensis is a Gram-negative bacterium that has a wide host range, including humans, and is the causative agent of tularemia. To identify new therapeutic drug targets and vaccine candidates and investigate the genetic basis of Francisella virulence in the Fischer 344 rat, we have constructed an F. tularensis Schu S4 transposon library. This library consists of more than 300,000 unique transposon mutants and represents a transposon insertion for every 6 bp of the genome. A transposon-directed insertion site sequencing (TraDIS) approach was used to identify 453 genes essential for growth in vitro Many of these essential genes were mapped to key metabolic pathways, including glycolysis/gluconeogenesis, peptidoglycan synthesis, fatty acid biosynthesis, and the tricarboxylic acid (TCA) cycle. Additionally, 163 genes were identified as required for fitness during colonization of the Fischer 344 rat spleen. This in vivo selection screen was validated through the generation of marked deletion mutants that were individually assessed within a competitive index study against the wild-type F. tularensis Schu S4 strain.IMPORTANCE The intracellular bacterial pathogen Francisella tularensis causes a disease in humans characterized by the rapid onset of nonspecific symptoms such as swollen lymph glands, fever, and headaches. F. tularensis is one of the most infectious bacteria known and following pulmonary exposure can have a mortality rate exceeding 50% if left untreated. The low infectious dose of this organism and concerns surrounding its potential as a biological weapon have heightened the need for effective and safe therapies. To expand the repertoire of targets for therapeutic development, we initiated a genome-wide analysis. This study has identified genes that are important for F. tularensis under in vitro and in vivo conditions, providing candidates that can be evaluated for vaccine or antibacterial development.Biotechnology & Biological Sciences Research Council (BBSRC)Defence Science and Technology Laboratory (DSTL

Tyrosine 331 and phenylalanine 334 in Clostridium perfringens α-toxin are essential for cytotoxic activity

AbstractDifferences in the biological properties of the Clostridium perfringens phospholipase C (α-toxin) and the C. bifermentans phospholipase C (Cbp) have been attributed to differences in their carboxy-terminal domains. Three residues in the carboxy-terminal domain of α-toxin, which have been proposed to play a role in membrane recognition (D269, Y331 and F334), are not conserved in Cbp (Y, L and I respectively). We have characterised D269Y, Y331L and F334I variant forms of α-toxin. Variant D269Y had reduced phospholipase C activity towards aggregated egg yolk phospholipid but increased haemolytic and cytotoxic activity. Variants Y331L and F334I showed a reduction in phospholipase C, haemolytic and cytotoxic activities indicating that these substitutions contribute to the reduced haemolytic and cytotoxic activity of Cbp

Role of trehalose biosynthesis in environmental survival and virulence of Salmonella enterica serovar typhimurium.

The otsA and otsB genes, encoding trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase respectively, have been isolated from Salmonella enterica serovar typhimurium and nucleotide-sequenced. Induction of trehalose biosynthesis by exposure of bacteria to high osmotic strength resulted in the intracellular accumulation of trehalose. An otsA mutant of S. enterica serovar typhimurium was more susceptible to killing by heat, and grew poorly under conditions of high osmolarity. The wild-type and otsA mutant strains showed similar abilities to colonise spleen tissues after oral dosing of mice. These findings suggest that the otsBA gene products play a role in environmental survival, but not in virulence, of S. enterica serovar typhimurium

The regulation of the alpha-toxin gene of Clostridium perfringens

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