scholarly journals Targeted Mutagenesis in Pathogenic Leptospira Species: Disruption of the LigB Gene Does Not Affect Virulence in Animal Models of Leptospirosis

2008 ◽  
Vol 76 (12) ◽  
pp. 5826-5833 ◽  
Author(s):  
Julio Croda ◽  
Claudio Pereira Figueira ◽  
Elsio A. Wunder ◽  
Cleiton S. Santos ◽  
Mitermayer G. Reis ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Wild Type Strain ◽  
Cultured Cells ◽  
Immunoblot Analysis ◽  
Targeted Mutagenesis ◽  
Wild Type ◽  
Chromosomal Locus ◽  
Hamster Model ◽  
Pathogenic Leptospira ◽  
Leptospira Species

ABSTRACT The pathogenic mechanisms of Leptospira interrogans, the causal agent of leptospirosis, remain largely unknown. This is mainly due to the lack of tools for genetically manipulating pathogenic Leptospira species. Thus, homologous recombination between introduced DNA and the corresponding chromosomal locus has never been demonstrated for this pathogen. Leptospiral immunoglobulin-like repeat (Lig) proteins were previously identified as putative Leptospira virulence factors. In this study, a ligB mutant was constructed by allelic exchange in L. interrogans; in this mutant a spectinomycin resistance (Spcr) gene replaced a portion of the ligB coding sequence. Gene disruption was confirmed by PCR, immunoblot analysis, and immunofluorescence studies. The ligB mutant did not show decrease virulence compared to the wild-type strain in the hamster model of leptospirosis. In addition, inoculation of rats with the ligB mutant induced persistent colonization of the kidneys. Finally, LigB was not required to mediate bacterial adherence to cultured cells. Taken together, our data provide the first evidence of site-directed homologous recombination in pathogenic Leptospira species. Furthermore, our data suggest that LigB does not play a major role in dissemination of the pathogen in the host and in the development of acute disease manifestations or persistent renal colonization.

scholarly journals The Ser/Thr Kinase PrkC Participates in Cell Wall Homeostasis and Antimicrobial Resistance inClostridium difficile

2019 ◽  
Vol 87 (8) ◽  
Author(s):  
Elodie Cuenot ◽  
Transito Garcia-Garcia ◽  
Thibaut Douche ◽  
Olivier Gorgette ◽  
Pascal Courtin ◽  
...  
Keyword(s):  
Cell Wall ◽  
Type Strain ◽  
Wild Type Strain ◽  
Cell Envelope ◽  
Wild Type ◽  
Hamster Model ◽  
Content Type ◽  
Physiological Processes ◽  
Signal Transduction Networks ◽  
Mean Size

ABSTRACTClostridium difficileis the leading cause of antibiotic-associated diarrhea in adults. During infection,C. difficilemust detect the host environment and induce an appropriate survival strategy. Signal transduction networks involving serine/threonine kinases (STKs) play key roles in adaptation, as they regulate numerous physiological processes. PrkC ofC. difficileis an STK with two PASTA domains. We showed that PrkC is membrane associated and is found at the septum. We observed that deletion ofprkCaffects cell morphology with an increase in mean size, cell length heterogeneity, and presence of abnormal septa. A ΔprkCmutant was able to sporulate and germinate but was less motile and formed more biofilm than the wild-type strain. Moreover, a ΔprkCmutant was more sensitive to antimicrobial compounds that target the cell envelope, such as the secondary bile salt deoxycholate, cephalosporins, cationic antimicrobial peptides, and lysozyme. This increased susceptibility was not associated with differences in peptidoglycan or polysaccharide II composition. However, the ΔprkCmutant had less peptidoglycan and released more polysaccharide II into the supernatant. A proteomic analysis showed that the majority ofC. difficileproteins associated with the cell wall were less abundant in the ΔprkCmutant than the wild-type strain. Finally, in a hamster model of infection, the ΔprkCmutant had a colonization delay that did not significantly affect overall virulence.

Recombination and ligation of transfected DNA in CHO mutant EM9, which has high levels of sister chromatid exchange

1987 ◽  
Vol 7 (5) ◽  
pp. 2007-2011
Author(s):  
C A Hoy ◽  
J C Fuscoe ◽  
L H Thompson
Keyword(s):  
Homologous Recombination ◽  
Sister Chromatid ◽  
Sister Chromatid Exchange ◽  
Type Strain ◽  
Wild Type Strain ◽  
Strand Break ◽  
Wild Type ◽  
Dna Strand Break ◽  
Foreign Dna ◽  
Dna Strand

Transformation frequencies were measured in CHO mutant EM9 after transfection with intact or modified plasmid pSV2-gpt. The mutant and wild-type strain behaved similarly under all conditions except when homologous recombination was required to produce an intact plasmid. Therefore, the defect of the mutant which renders it slow in DNA strand break rejoining and high in sister chromatid exchange induction reduces its ability to recombine foreign DNA molecules.

scholarly journals The chvH Locus ofAgrobacterium Encodes a Homologue of an Elongation Factor Involved in Protein Synthesis

2001 ◽  
Vol 183 (1) ◽  
pp. 36-45 ◽  
Author(s):  
Wen-Tao Peng ◽  
Lois M. Banta ◽  
Trevor C. Charles ◽  
Eugene W. Nester
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Elongation Factor ◽  
Peptide Bond ◽  
Single Copy ◽  
Tumor Formation ◽  
Wild Type ◽  
Chromosomal Locus ◽  
Gene Encoding ◽  
Elongation Factor P

ABSTRACT The virulence of Agrobacterium tumefaciens depends on both chromosome- and Ti plasmid-encoded gene products. In this study, we characterize a chromosomal locus, chvH, previously identified by TnphoA mutagenesis and shown to be required for tumor formation. Through DNA sequencing and comparison of the sequence with identified sequences in the database, we show that this locus encodes a protein similar in sequence to elongation factor P, a protein thought to be involved in peptide bond synthesis inEscherichia coli. The analysis of vir-lacZ andvir-phoA translational fusions as well as Western immunoblotting revealed that the expression of Vir proteins such as VirE2 was significantly reduced in the chvH mutant compared with the wild-type strain. The E. coli efp gene complemented detergent sensitivity, virulence, and expression of VirE2 in the chvH mutant, suggesting that chvH andefp are functionally homologous. As expected, ChvH exerts its activity at the posttranscriptional level. Southern analysis suggests that the gene encoding this elongation factor is present as a single copy in A. tumefaciens. We constructed achvH deletion mutant in which a 445-bp fragment within its coding sequence was deleted and replaced with an omega fragment. On complex medium, this mutant grew more slowly than the wild-type strain, indicating that elongation factor P is important but not essential for the growth of Agrobacterium.

scholarly journals Mutations in Homologous Recombination Genes Rescue top3 Slow Growth in Saccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 162 (2) ◽  
pp. 647-662 ◽  
Author(s):  
Erika Shor ◽  
Serge Gangloff ◽  
Marisa Wagner ◽  
Justin Weinstein ◽  
Gavrielle Price ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Type Strain ◽  
Wild Type Strain ◽  
Slow Growth ◽  
Recq Helicase ◽  
Wild Type ◽  
Growth Defects ◽  
Topoisomerase Iii ◽  
Hyper Sensitivity ◽  
Instability Phenotype

Abstract In budding yeast, loss of topoisomerase III, encoded by the TOP3 gene, leads to a genomic instability phenotype that includes slow growth, hyper-sensitivity to genotoxic agents, mitotic hyper-recombination, increased chromosome missegregation, and meiotic failure. Slow growth and other defects of top3 mutants are suppressed by mutation of SGS1, which encodes the only RecQ helicase in S. cerevisiae. sgs1 is epistatic to top3, suggesting that the two proteins act in the same pathway. To identify other factors that function in the Sgs1-Top3 pathway, we undertook a genetic screen for non-sgs1 suppressors of top3 defects. We found that slow growth and DNA damage sensitivity of top3 mutants are suppressed by mutations in RAD51, RAD54, RAD55, and RAD57. In contrast, top3 mutants show extreme synergistic growth defects with mutations in RAD50, MRE11, XRS2, RDH54, and RAD1. We also analyzed recombination at the SUP4-o region, showing that in a rad51, rad54, rad55, or rad57 background top3Δ does not increase recombination to the same degree as in a wild-type strain. These results suggest that the presence of the Rad51 homologous recombination complex in a top3 background facilitates creation of detrimental intermediates by Sgs1. We present a model wherein Rad51 helps recruit Sgs1-Top3 to sites of replicative damage.

scholarly journals CadA Negatively Regulates Escherichia coli O157:H7 Adherence and Intestinal Colonization

2008 ◽  
Vol 76 (11) ◽  
pp. 5072-5081 ◽  
Author(s):  
Roberto C. Vazquez-Juarez ◽  
Jeeba A. Kuriakose ◽  
David A. Rasko ◽  
Jennifer M. Ritchie ◽  
Melissa M. Kendall ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Cultured Cells ◽  
Expression Patterns ◽  
Gene Array ◽  
Rabbit Model ◽  
Gene Expression Patterns ◽  
Wild Type

ABSTRACT Adherence of pathogenic Escherichia coli strains to intestinal epithelia is essential for infection. For enterohemorrhagic E. coli (EHEC) serotype O157:H7, we have previously demonstrated that multiple factors govern this pathogen's adherence to HeLa cells (A. G. Torres and J. B. Kaper, Infect. Immun. 71:4985-4995, 2003). One of these factors is CadA, a lysine decarboxylase, and this protein has been proposed to negatively regulate virulence in several enteric pathogens. In the case of EHEC strains, CadA modulates expression of the intimin, an outer membrane adhesin involved in pathogenesis. Here, we inactivated cadA in O157:H7 strain 86-24 to investigate the role of this gene in EHEC adhesion to tissue-cultured monolayers, global gene expression patterns, and colonization of the infant rabbit intestine. The cadA mutant did not possess lysine decarboxylation activity and was hyperadherent to tissue-cultured cells. Adherence of the cadA mutant was nearly twofold greater than that of the wild type, and the adherence phenotype was independent of pH, lysine, or cadaverine in the media. Additionally, complementation of the cadA defect reduced adherence back to wild-type levels, and it was found that the mutation affected the expression of the intimin protein. Disruption of the eae gene (intimin-encoding gene) in the cadA mutant significantly reduced its adherence to tissue-cultured cells. However, adherence of the cadA eae double mutant was greater than that of an 86-24 eae mutant, suggesting that the enhanced adherence of the cadA mutant is not entirely attributable to enhanced expression of intimin in this background. Gene array analysis revealed that the cadA mutation significantly altered EHEC gene expression patterns; expression of 1,332 genes was downregulated and that of 132 genes was upregulated in the mutant compared to the wild-type strain. Interestingly, the gene expression variation shows an EHEC-biased gene alteration including intergenic regions. Two putative adhesins, flagella and F9 fimbria, were upregulated in the cadA mutant, suggestive of their association with adherence in the absence of the Cad regulatory mechanism. In the infant rabbit model, the cadA mutant outcompeted the wild-type strain in the ileum but not in the cecum or mid-colon, raising the possibility that CadA negatively regulates EHEC pathogenicity in a tissue-specific fashion.

The Critical Role of Polyketide Synthase Gene On The Swainsonine Biosynthesis in The Fungus Metarhizium Anisopliae

2021 ◽  
Author(s):  
Lu Sun ◽  
Enxia Huang ◽  
Yu Zhang ◽  
Ziyu Guo ◽  
Kexin Wu ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Metarhizium Anisopliae ◽  
Type Strain ◽  
Polyketide Synthase ◽  
Wild Type Strain ◽  
Biosynthesis Pathway ◽  
Wild Type ◽  
Knock Out ◽  
Polyketide Synthase Gene

Abstract Swainsonine (SW) is the principal toxic ingredient of locoweeds, and is produced by fungi including Metarhizium anisopliae, Slafractonia leguminicola, and Alternaria oxytropis. While the SW biosynthesis pathway of fungi and the catalytic enzyme genes that regulate synthesis are not cleanly. In this study, we used homologous recombination (HR) to knock out and interfere with the polyketide synthase gene (pks) of M. anisopliae to determine its effect on the SW biosynthesis pathway. The concentration of SW was measured in the fermentation broth of M. anisopliae at 1 d, 2 d, 3 d, 4 d, 5 d, 6 d or 7 d using LC-MS. The gene for the pks gene was detected by RT-qPCR. Day 5 of M. anisopliae gave the highest content of SW and the highest expression of the pks gene. To determine the role of the pks gene in the SW biosynthesis pathway of M. anisopliae, we used PEG-mediated homologous recombination (HR) to transform a wild-type strain (WT) with a Benomyl (ben)-resistant fragment to knock out the pks gene producing a mutant-type strain (MT) and used PEG-mediated RNAi to transform a wild-type strain (WT) with a Benomyl (ben)-resistant plasmid to interfere with the pks gene. A complemented-type (CT) strain was produced by adding a complementation vector that contains the geneticin (G418) resistance gene as a marker. The content of SW didn’t detected in MT strain, and returned to the original level in the CT strain, while the content of SW was significantly decreased in RNAi strain. We suggest that mutation and RNAi in the pks gene affect the cell wall formation of M. anisopliae, while the colony diameters, phenotypes, and growth rates did not change significantly, and no obvious changes in other cellular organelles were noted. These results indicate that the pks gene plays a crucial role in the SW biosynthesis of M. anisopliae, which provides an important theoretical basis for illuminating the SW biosynthesis and solving locoism in livestock.

scholarly journals Recombination and ligation of transfected DNA in CHO mutant EM9, which has high levels of sister chromatid exchange.

1987 ◽  
Vol 7 (5) ◽  
pp. 2007-2011 ◽  
Author(s):  
C A Hoy ◽  
J C Fuscoe ◽  
L H Thompson
Keyword(s):  
Homologous Recombination ◽  
Sister Chromatid ◽  
Sister Chromatid Exchange ◽  
Type Strain ◽  
Wild Type Strain ◽  
Strand Break ◽  
Wild Type ◽  
Dna Strand Break ◽  
Foreign Dna ◽  
Dna Strand

Transformation frequencies were measured in CHO mutant EM9 after transfection with intact or modified plasmid pSV2-gpt. The mutant and wild-type strain behaved similarly under all conditions except when homologous recombination was required to produce an intact plasmid. Therefore, the defect of the mutant which renders it slow in DNA strand break rejoining and high in sister chromatid exchange induction reduces its ability to recombine foreign DNA molecules.

scholarly journals Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 383-391 ◽  
Author(s):  
Yasumasa Tsukamoto ◽  
Jun-ichi Kato ◽  
Hideo Ikeda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quantitative Analysis ◽  
Structural Analysis ◽  
Recombination Rate ◽  
Type Strain ◽  
Negative Selection ◽  
Wild Type Strain ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

scholarly journals The role of Zur-regulated lipoprotein A in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles in Acinetobacter baumannii

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Nayeong Kim ◽  
Hyo Jeong Kim ◽  
Man Hwan Oh ◽  
Se Yeon Kim ◽  
Mi Hyun Kim ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Mutant Strain ◽  
Antimicrobial Susceptibility ◽  
Type Strain ◽  
Wild Type Strain ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Wild Type ◽  
Bacterial Morphology

Abstract Background Zinc uptake-regulator (Zur)-regulated lipoprotein A (ZrlA) plays a role in bacterial fitness and overcoming antimicrobial exposure in Acinetobacter baumannii. This study further characterized the zrlA gene and its encoded protein and investigated the roles of the zrlA gene in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles (OMVs) in A. baumannii ATCC 17978. Results In silico and polymerase chain reaction analyses showed that the zrlA gene was conserved among A. baumannii strains with 97–100% sequence homology. Recombinant ZrlA protein exhibited a specific enzymatic activity of D-alanine-D-alanine carboxypeptidase. Wild-type A. baumannii exhibited more morphological heterogeneity than a ΔzrlA mutant strain during stationary phase. The ΔzrlA mutant strain was more susceptible to gentamicin than the wild-type strain. Sizes and protein profiles of OMVs were similar between the wild-type and ΔzrlA mutant strains, but the ΔzrlA mutant strain produced 9.7 times more OMV particles than the wild-type strain. OMVs from the ΔzrlA mutant were more cytotoxic in cultured epithelial cells than OMVs from the wild-type strain. Conclusions The present study demonstrated that A. baumannii ZrlA contributes to bacterial morphogenesis and antimicrobial resistance, but its deletion increases OMV production and OMV-mediated host cell cytotoxicity.

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