scholarly journals Identification of an Exopolysaccharide Biosynthesis Gene in Bradyrhizobium diazoefficiens USDA110

2021 ◽  
Vol 9 (12) ◽  
pp. 2490
Author(s):  
Chunxia Xu ◽  
Huaqin Ruan ◽  
Wenjie Cai ◽  
Christian Staehelin ◽  
Weijun Dai
Keyword(s):  
Insertion Site ◽  
Phenotypic Screening ◽  
Biosynthesis Pathway ◽  
Plant Interactions ◽  
Symbiotic Interaction ◽  
Transposon Insertion ◽  
Biosynthesis Gene ◽  
Exopolysaccharide Biosynthesis ◽  
Soybean Plants ◽  
Insertion Mutants

Exopolysaccharides (EPS) play critical roles in rhizobium-plant interactions. However, the EPS biosynthesis pathway in Bradyrhizobium diazoefficiens USDA110 remains elusive. Here we used transposon (Tn) mutagenesis with the aim to identify genetic elements required for EPS biosynthesis in B. diazoefficiens USDA110. Phenotypic screening of Tn5 insertion mutants grown on agar plates led to the identification of a mutant with a transposon insertion site in the blr2358 gene. This gene is predicted to encode a phosphor-glycosyltransferase that transfers a phosphosugar onto a polyprenol phosphate substrate. The disruption of the blr2358 gene resulted in defective EPS synthesis. Accordingly, the blr2358 mutant showed a reduced capacity to induce nodules and stimulate the growth of soybean plants. Glycosyltransferase genes related to blr2358 were found to be well conserved and widely distributed among strains of the Bradyrhizobium genus. In conclusion, our study resulted in identification of a gene involved in EPS biosynthesis and highlights the importance of EPS in the symbiotic interaction between USDA110 and soybeans.

scholarly journals AcrAB Multidrug Efflux Pump Is Associated with Reduced Levels of Susceptibility to Tigecycline (GAR-936) in Proteus mirabilis

2003 ◽  
Vol 47 (2) ◽  
pp. 665-669 ◽  
Author(s):  
Melissa A. Visalli ◽  
Ellen Murphy ◽  
Steven J. Projan ◽  
Patricia A. Bradford
Keyword(s):  
Proteus Mirabilis ◽  
Efflux Pump ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Transposon Insertion ◽  
Spectrum Activity ◽  
Insertion Mutants ◽  
Broad Spectrum Activity ◽  
Increased Susceptibility

ABSTRACT Tigecycline has good broad-spectrum activity against many gram-positive and gram-negative pathogens with the notable exception of the Proteeae. A study was performed to identify the mechanism responsible for the reduced susceptibility to tigecycline in Proteus mirabilis. Two independent transposon insertion mutants of P. mirabilis that had 16-fold-increased susceptibility to tigecycline were mapped to the acrB gene homolog of the Escherichia coli AcrRAB efflux system. Wild-type levels of decreased susceptibility to tigecycline were restored to the insertion mutants by complementation with a clone containing a PCR-derived fragment from the parental wild-type acrRAB efflux gene cluster. The AcrAB transport system appears to be associated with the intrinsic reduced susceptibility to tigecycline in P. mirabilis.

scholarly journals Multiple Elements Controlling Adherence of Enterohemorrhagic Escherichia coli O157:H7 to HeLa Cells

2003 ◽  
Vol 71 (9) ◽  
pp. 4985-4995 ◽  
Author(s):  
Alfredo G. Torres ◽  
James B. Kaper
Keyword(s):  
Escherichia Coli ◽  
Hela Cells ◽  
Culture Conditions ◽  
Enterohemorrhagic Escherichia Coli ◽  
Differentially Expressed ◽  
Wild Type ◽  
Transposon Insertion ◽  
Reduced Adherence ◽  
Insertion Mutants

ABSTRACT Adherence of enterohemorrhagic Escherichia coli (EHEC) to the intestinal epithelium is essential for initiation of infection. Intimin is the only factor demonstrated to play a role in intestinal colonization by EHEC O157:H7. Other attempts to identify additional adhesion factors in vitro have been unsuccessful, suggesting that expression of these factors is under tight regulation. We sought to identify genes involved in the control of adherence of EHEC O157:H7 to cultured epithelial cells. A total of 5,000 independent transposon insertion mutants were screened for their ability to adhere to HeLa cells, and 7 mutants were isolated with a markedly enhanced adherence. The mutants adhered at levels 113 to 170% that of the wild-type strain, and analysis of the protein profiles of these mutants revealed several proteins differentially expressed under in vitro culture conditions. We determined the sequence of the differentially expressed proteins and further investigated the function of OmpA, whose expression was increased in a mutant with an insertionally inactivated tcdA gene. An isogenic ompA mutant showed reduced adherence compared to the parent strain. Disruption of the ompA gene in the tdcA mutant strain abolished the hyperadherent phenotype, and anti-OmpA serum inhibited adhesion of wild-type and tdcA mutant strains to HeLa cells. Enhanced adhesion mediated by OmpA was also observed with Caco-2 cells, and anti-OmpA serum blocked adherence to HeLa cells of other EHEC O157:H7 strains. Our results indicate that multiple elements control adherence and OmpA acts as an adhesin in EHEC O157:H7.

scholarly journals Identification of gmhA, a Yersinia pestis Gene Required for Flea Blockage, by Using a Caenorhabditis elegans Biofilm System

2005 ◽  
Vol 73 (11) ◽  
pp. 7236-7242 ◽  
Author(s):  
Creg Darby ◽  
Sandya L. Ananth ◽  
Li Tan ◽  
B. Joseph Hinnebusch
Keyword(s):  
Caenorhabditis Elegans ◽  
Yersinia Pestis ◽  
Biofilm Formation ◽  
Yersinia Pseudotuberculosis ◽  
C Elegans ◽  
Transposon Insertion ◽  
Insertion Mutants ◽  
Random Screening

ABSTRACT Yersinia pestis, the cause of bubonic plague, blocks feeding by its vector, the flea. Recent evidence indicates that blockage is mediated by an in vivo biofilm. Y. pestis and the closely related Yersinia pseudotuberculosis also make biofilms on the cuticle of the nematode Caenorhabditis elegans, which block this laboratory animal's feeding. Random screening of Y. pseudotuberculosis transposon insertion mutants with a C. elegans biofilm assay identified gmhA as a gene required for normal biofilms. gmhA encodes phosphoheptose isomerase, an enzyme required for synthesis of heptose, a conserved component of lipopolysaccharide and lipooligosaccharide. A Y. pestis gmhA mutant was constructed and was severely defective for C. elegans biofilm formation and for flea blockage but only moderately defective in an in vitro biofilm assay. These results validate use of the C. elegans biofilm system to identify genes and pathways involved in Y. pestis flea blockage.

scholarly journals A whole-genome screen identifies Salmonella enterica serovar Typhi genes involved in fluoroquinolone susceptibility

2020 ◽  
Vol 75 (9) ◽  
pp. 2516-2525
Author(s):  
A Keith Turner ◽  
Sabine E Eckert ◽  
Daniel J Turner ◽  
Muhammud Yasir ◽  
Mark A Webber ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Insertion Site ◽  
Whole Genome ◽  
Genome Screen ◽  
Polysaccharide Biosynthesis ◽  
Salmonella Enterica Serovar Typhi ◽  
Transposon Insertion ◽  
Associated Functions ◽  
The Impact ◽  
Insertion Mutations

Abstract Objectives A whole-genome screen at sub-gene resolution was performed to identify candidate loci that contribute to enhanced or diminished ciprofloxacin susceptibility in Salmonella enterica serovar Typhi. Methods A pool of over 1 million transposon insertion mutants of an S. Typhi Ty2 derivative were grown in a sub-MIC concentration of ciprofloxacin, or without ciprofloxacin. Transposon-directed insertion site sequencing (TraDIS) identified relative differences between the mutants that grew following the ciprofloxacin treatment compared with the untreated mutant pool, thereby indicating which mutations contribute to gain or loss of ciprofloxacin susceptibility. Results Approximately 88% of the S. Typhi strain’s 4895 annotated genes were assayed, and at least 116 were identified as contributing to gain or loss of ciprofloxacin susceptibility. Many of the identified genes are known to influence susceptibility to ciprofloxacin, thereby providing method validation. Genes were identified that were not known previously to be involved in susceptibility, and some of these had no previously known phenotype. Susceptibility to ciprofloxacin was enhanced by insertion mutations in genes coding for efflux, other surface-associated functions, DNA repair and expression regulation, including phoP, barA and marA. Insertion mutations that diminished susceptibility were predominantly in genes coding for surface polysaccharide biosynthesis and regulatory genes, including slyA, emrR, envZ and cpxR. Conclusions A genomics approach has identified novel contributors to gain or loss of ciprofloxacin susceptibility in S. Typhi, expanding our understanding of the impact of fluoroquinolones on bacteria and of mechanisms that may contribute to resistance. The data also demonstrate the power of the TraDIS technology for antibacterial research.

scholarly journals Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

10.3791/57298 ◽  
2018 ◽  
Author(s):  
Eliana Drenkard ◽  
Rhianna M. Hibbler ◽  
D. Alina Gutu ◽  
Alexander D. Eaton ◽  
Amy L. Silverio ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transposon Insertion ◽  
Insertion Mutants

scholarly journals A Diverse Repertoire of Exopolysaccharide Biosynthesis Gene Clusters in Lactobacillus Revealed by Comparative Analysis in 106 Sequenced Genomes

2019 ◽  
Vol 7 (10) ◽  
pp. 444 ◽  
Author(s):  
Dipti Deo ◽  
Dimple Davray ◽  
Ram Kulkarni
Keyword(s):  
Biofilm Formation ◽  
Gene Clusters ◽  
High Variability ◽  
Protein Families ◽  
Free Living ◽  
Biosynthesis Gene ◽  
Exopolysaccharide Biosynthesis ◽  
Pharmaceutical Industries ◽  
Living Group ◽  
Core Genes

Production of exopolysaccharides (EPS) is one of the unique features of Lactobacillus genus. EPS not only have many physiological roles such as in stress tolerance, quorum sensing and biofilm formation, but also have numerous applications in the food and pharmaceutical industries. In this study, we identified and compared EPS biosynthesis gene clusters in 106 sequenced Lactobacillus genomes representing 27 species. Of the 146 identified clusters, only 41 showed the typical generic organization of genes as reported earlier. Hierarchical clustering showed highly varied nature of the clusters in terms of the gene composition; nonetheless, habitat-wise grouping was observed for the gene clusters from host-adapted and nomadic strains. Of the core genes required for EPS biosynthesis, epsA, B, C, D and E showed higher conservation, whereas gt, wzx and wzy showed high variability in terms of the number and composition of the protein families. Analysis of the distribution pattern of the protein families indicated a higher proportion of mutually exclusive families in clusters from host-adapted and nomadic strains, whereas those from the free-living group had very few unique families. Taken together, this analysis highlights high variability in the EPS gene clusters amongst Lactobacillus with some of their properties correlated to the habitats.

scholarly journals Two C—P Lyase Operons in Pseudomonas stutzeri and Their Roles in the Oxidation of Phosphonates, Phosphite, and Hypophosphite

2004 ◽  
Vol 186 (14) ◽  
pp. 4730-4739 ◽  
Author(s):  
Andrea K. White ◽  
William W. Metcalf
Keyword(s):  
Insertion Site ◽  
Pseudomonas Stutzeri ◽  
Gene Organization ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
E Coli ◽  
Reverse Transcription Pcr ◽  
Transposon Insertion ◽  
Intergenic Sequences ◽  
Reading Frames

ABSTRACT DNA sequencing and analysis of two distinct C—P lyase operons in Pseudomonas stutzeri WM88 were completed. The htxABCDEFGHIJKLMN operon encodes a hypophosphite-2-oxoglutarate dioxygenase (HtxA), whereas the predicted amino acid sequences of HtxB to HtxN are each homologous to the components of the Escherichia coli phn operon, which encodes C—P lyase, although homologs of E. coli phnF and phnO are absent. The genes in the htx operon are cotranscribed based on gene organization, and the presence of the intergenic sequences is verified by reverse transcription-PCR with total RNA. Deletion of the htx locus does not affect the ability of P. stutzeri to grow on phosphonates, indicating the presence of an additional C—P lyase pathway in this organism. To identify the genes comprising this pathway, a Δhtx strain was mutagenized and one mutant lacking the ability to grow on methylphosphonate as the sole P source was isolated. A ca.-10.6-kbp region surrounding the transposon insertion site of this mutant was sequenced, revealing 13 open reading frames, designated phnCDEFGHIJKLMNP, which were homologous to the E. coli phn genes. Deletion of both the htx and phn operons of P. stutzeri abolishes all growth on methylphosphonate and aminoethylphosphonate. Both operons individually support growth on methylphosphonate; however, the phn operon supports growth on aminoethylphosphonate and phosphite, as well. The substrate ranges of both C—P lyases are limited, as growth on other phosphonate compounds, including glyphosate and phenylphosphonate, was not observed.

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