Deletion of HAPS_2096 Increases Sensitivity to Cecropin B in Haemophilus parasuis

2015 ◽  
Vol 25 (4) ◽  
pp. 284-291
Author(s):  
Fanjie Chen ◽  
Han Hu ◽  
Zhonghua Li ◽  
Jiacheng Huang ◽  
Xuwang Cai ◽  
...  
Keyword(s):  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Sublethal Concentration ◽  
Haemophilus Parasuis ◽  
Wild Type ◽  
Microscopy Observation ◽  
Cecropin B ◽  
In The Wild ◽  
Substrate Binding Protein

Cecropin B (CB) is a very effective natural antimicrobial peptide that has shown great potential for future antimicrobial drug development. HAPS_2096 is a <i>Haemophilus parasuis</i> gene that encodes the periplasmic substrate-binding protein of an ATP-binding cassette-type amino acid transporter. In this research, we constructed and verified an HAPS_2096 deletion mutant and a complementary HAPS_2096 mutant of <i>H. parasuis</i> JS0135. A bactericidal assay revealed that the HAPS_2096 deletion mutant was significantly more sensitive than the wild-type strain to 0.25-0.5 µg/ml CB. However, the gene complementation alleviated the CB sensitivity of the mutant. Immunoelectron microscopy observation following a 30-min treatment with a sublethal concentration of CB (0.25 μg/ml) revealed more extensive morphological damage in the mutant strain than in the wild-type strain. Hence, our results suggest that the HAPS_2096 gene contributes to <i>H. parasuis</i> resistance to CB.

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 Expression and function of the cdgD gene, encoding a CHASE–PAS-DGC-EAL domain protein, in Azospirillum brasilense

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
José Francisco Cruz-Pérez ◽  
Roxana Lara-Oueilhe ◽  
Cynthia Marcos-Jiménez ◽  
Ricardo Cuatlayotl-Olarte ◽  
María Luisa Xiqui-Vázquez ◽  
...  
Keyword(s):  
Azospirillum Brasilense ◽  
Type Strain ◽  
Wild Type Strain ◽  
Soil Conditions ◽  
Wild Type ◽  
Gene Encoding ◽  
Wheat Roots ◽  
Genes Encoding ◽  
In The Wild ◽  
Plant Growth Promoting

AbstractThe plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

scholarly journals Activation of 2,4-Diaminoquinazoline in Mycobacterium tuberculosis by Rv3161c, a Putative Dioxygenase

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Eduard Melief ◽  
Shilah A. Bonnett ◽  
Edison S. Zuniga ◽  
Tanya Parish
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Type A ◽  
Wild Type ◽  
Metabolite Analysis ◽  
Content Type ◽  
Data Support ◽  
In The Wild

ABSTRACT The diaminoquinazoline series has good potency against Mycobacterium tuberculosis. Resistant isolates have mutations in Rv3161c, a putative dioxygenase. We carried out metabolite analysis on a wild-type strain and an Rv3161c mutant strain after exposure to a diaminoquinazoline. The parental compound was found in intracellular extracts from the mutant but not the wild type. A metabolite consistent with a monohydroxylated form was identified in the wild type. These data support the hypothesis that Rv3161c metabolizes diaminoquinazolines in M. tuberculosis.

scholarly journals Disruption of the RAD52 gene alters the spectrum of spontaneous SUP4-o mutations in Saccharomyces cerevisiae.

Genetics ◽  
1989 ◽  
Vol 122 (3) ◽  
pp. 535-542 ◽  
Author(s):  
B A Kunz ◽  
M G Peters ◽  
S E Kohalmi ◽  
J D Armstrong ◽  
M Glattke ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Base Pair ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mutator Phenotype ◽  
In The Wild ◽  
Single Base Pair ◽  
Almost All

Abstract Defects in the RAD52 gene of the yeast Saccharomyces cerevisiae confer a mutator phenotype. To characterize this effect in detail, a collection of 238 spontaneous SUP4-o mutations arising in a strain having a disrupted RAD52 gene was analyzed by DNA sequencing. The resulting mutational spectrum was compared to that derived from an examination of 222 spontaneous mutations selected in a nearisogenic wild-type (RAD52) strain. This comparison revealed that the mutator phenotype was associated with an increase in the frequency of base-pair substitutions. All possible types of substitution were detected but there was a reduction in the relative fraction of A.T----G.C transitions and an increase in the proportion of G.C----C.G transversions. These changes were sufficient to cause a twofold greater preference for substitutions at G.C sites in the rad52 strain despite a decrease in the fraction of G.C----T.A transversions. There were also considerable differences between the distributions of substitutions within the SUP4-o gene. Base-pair changes occurred at fewer sites in the rad52 strain but the mutated sites included several that were not detected in the RAD52 background. Only two of the four sites that were mutated most frequently in the rad52 strain were also prominent in the wild-type strain and mutation frequencies at almost all sites common to both strains were greater for the rad52 derivative. Although single base-pair deletions occurred in the two strains with similar frequencies, several classes of mutation that were recovered in the wild-type background including multiple base-pair deletions, insertions of the yeast transposable element Ty, and more complex changes, were not detected in the rad52 strain.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals SdiA Aids Enterohemorrhagic Escherichia coli Carriage by Cattle Fed a Forage or Grain Diet

2013 ◽  
Vol 81 (9) ◽  
pp. 3472-3478 ◽  
Author(s):  
Haiqing Sheng ◽  
Y. N. Nguyen ◽  
Carolyn J. Hovde ◽  
Vanessa Sperandio
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Rumen Fluid ◽  
Acid Resistance ◽  
Enterohemorrhagic Escherichia Coli ◽  
Dependent Manner ◽  
Bacterial Survival ◽  
Wild Type

ABSTRACTEnterohemorrhagicEscherichia coli(EHEC) causes hemorrhagic colitis and life-threatening complications. The main reservoirs for EHEC are healthy ruminants. We reported that SdiA senses acyl homoserine lactones (AHLs) in the bovine rumen to activate expression of the glutamate acid resistance (gad) genes priming EHEC's acid resistance before they pass into the acidic abomasum. Conversely, SdiA represses expression of the locus of enterocyte effacement (LEE) genes, whose expression is not required for bacterial survival in the rumen but is necessary for efficient colonization at the rectoanal junction (RAJ) mucosa. Our previous studies show that SdiA-dependent regulation was necessary for efficient EHEC colonization of cattle fed a grain diet. Here, we compared the SdiA role in EHEC colonization of cattle fed a forage hay diet. We detected AHLs in the rumen of cattle fed a hay diet, and these AHLs activatedgadgene expression in an SdiA-dependent manner. The rumen fluid and fecal samples from hay-fed cattle were near neutrality, while the same digesta samples from grain-fed animals were acidic. Cattle fed either grain or hay and challenged with EHEC orally carried the bacteria similarly. EHEC was cleared from the rumen within days and from the RAJ mucosa after approximately one month. In competition trials, where animals were challenged with both wild-type and SdiA deletion mutant bacteria, diet did not affect the outcome that the wild-type strain was better able to persist and colonize. However, the wild-type strain had a greater advantage over the SdiA deletion mutant at the RAJ mucosa among cattle fed the grain diet.

scholarly journals Plasmid DNA Production in Proteome-Reduced Escherichia coli

2020 ◽  
Vol 8 (9) ◽  
pp. 1444
Author(s):  
Mitzi de la Cruz ◽  
Elisa A. Ramírez ◽  
Juan-Carlos Sigala ◽  
José Utrilla ◽  
Alvaro R. Lara
Keyword(s):  
Escherichia Coli ◽  
Plasmid Dna ◽  
Type Strain ◽  
Wild Type Strain ◽  
The Novel ◽  
Wild Type ◽  
Batch Mode ◽  
Cell Factories ◽  
Starting Point ◽  
In The Wild

The design of optimal cell factories requires engineering resource allocation for maximizing product synthesis. A recently developed method to maximize the saving in cell resources released 0.5% of the proteome of Escherichia coli by deleting only three transcription factors. We assessed the capacity for plasmid DNA (pDNA) production in the proteome-reduced strain in a mineral medium, lysogeny, and terrific broths. In all three cases, the pDNA yield from biomass was between 33 and 53% higher in the proteome-reduced than in its wild type strain. When cultured in fed-batch mode in shake-flask, the proteome-reduced strain produced 74.8 mg L−1 pDNA, which was four times greater than its wild-type strain. Nevertheless, the pDNA supercoiled fraction was less than 60% in all cases. Deletion of recA increased the pDNA yields in the wild type, but not in the proteome-reduced strain. Furthermore, recA mutants produced a higher fraction of supercoiled pDNA, compared to their parents. These results show that the novel proteome reduction approach is a promising starting point for the design of improved pDNA production hosts.

scholarly journals Group III Histidine Kinase Is a Positive Regulator of Hog1-Type Mitogen-Activated Protein Kinase in Filamentous Fungi

2005 ◽  
Vol 4 (11) ◽  
pp. 1820-1828 ◽  
Author(s):  
Akira Yoshimi ◽  
Kaihei Kojima ◽  
Yoshitaka Takano ◽  
Chihiro Tanaka
Keyword(s):  
Osmotic Stress ◽  
Filamentous Fungi ◽  
Histidine Kinase ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Group Iii ◽  
Positive Regulator ◽  
Mitogen Activated Protein ◽  
In The Wild

ABSTRACT We previously reported that the group III histidine kinase Dic1p in the maize pathogen Cochliobolus heterostrophus is involved in resistance to dicarboximide and phenylpyrrole fungicides and in osmotic adaptation. In addition, exposure to the phenylpyrrole fungicide fludioxonil led to improper activation of Hog1-type mitogen-activated protein kinases (MAPKs) in some phytopathogenic fungi, including C. heterostrophus. Here we report, for the first time, the relationship between the group III histidine kinase and Hog1-related MAPK: group III histidine kinase is a positive regulator of Hog1-related MAPK in filamentous fungi. The phosphorylation pattern of C. heterostrophus BmHog1p (Hog1-type MAPK) was analyzed in wild-type and dic1-deficient strains by Western blotting. In the wild-type strain, phosphorylated BmHog1p was detected after exposure to both iprodione and fludioxonil at a concentration of 1 μg/ml. In the dic1-deficient strains, phosphorylated BmHog1p was not detected after exposure to 10 μg/ml of the fungicides. In response to osmotic stress (0.4 M KCl), a trace of phosphorylated BmHog1p was found in the dic1-deficient strains, whereas the band representing active BmHog1p was clearly detected in the wild-type strain. Similar results were obtained for Neurospora crassa Os-2p MAPK phosphorylation in the mutant of the group III histidine kinase gene os-1. These results indicate that group III histidine kinase positively regulates the activation of Hog1-type MAPKs in filamentous fungi. Notably, the Hog1-type MAPKs were activated at high fungicide (100 μg/ml) and osmotic stress (0.8 M KCl) levels in the histidine kinase mutants of both fungi, suggesting that another signaling pathway activates Hog1-type MAPKs in these conditions.

scholarly journals Mutations in Four Regulatory Genes Have Interrelated Effects on Heterocyst Maturation in Anabaena sp. Strain PCC 7120

2006 ◽  
Vol 188 (21) ◽  
pp. 7387-7395 ◽  
Author(s):  
Sigal Lechno-Yossef ◽  
Qing Fan ◽  
Shigeki Ehira ◽  
Naoki Sato ◽  
C. Peter Wolk
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Response Regulator ◽  
Regulatory System ◽  
Transcript Abundance ◽  
Regulatory Genes ◽  
Wild Type ◽  
Serine Threonine Kinase ◽  
In The Wild ◽  
Pcc 7120

ABSTRACT Regulatory genes hepK, hepN, henR, and hepS are required for heterocyst maturation in Anabaena sp. strain PCC 7120. They presumptively encode two histidine kinases, a response regulator, and a serine/threonine kinase, respectively. To identify relationships between those genes, we compared global patterns of gene expression, at 14 h after nitrogen step-down, in corresponding mutants and in the wild-type strain. Heterocyst envelopes of mutants affected in any of those genes lack a homogeneous, polysaccharide layer. Those of a henR mutant also lack a glycolipid layer. patA, which encodes a positive effector of heterocyst differentiation, was up-regulated in all mutants except the hepK mutant, suggesting that patA expression may be inhibited by products related to heterocyst development. hepS and hepK were up-regulated if mutated and so appear to be negatively autoregulated. HepS and HenR regulated a common set of genes and so appear to belong to one regulatory system. Some nontranscriptional mechanism may account for the observation that henR mutants lack, and hepS mutants possess, a glycolipid layer, even though both mutations down-regulated genes involved in formation of the glycolipid layer. HepK and HepN also affected transcription of a common set of genes and therefore appear to share a regulatory pathway. However, the transcript abundance of other genes differed very significantly from expression in the wild-type strain in either the hepK or hepN mutant while differing very little from wild-type expression in the other of those two mutants. Therefore, hepK and hepN appear to participate also in separate pathways.

scholarly journals Glycerol Utilization Gene Cluster in Streptomyces clavuligerus

2009 ◽  
Vol 75 (9) ◽  
pp. 2991-2995 ◽  
Author(s):  
Sonia Baños ◽  
Rosario Pérez-Redondo ◽  
Bert Koekman ◽  
Paloma Liras
Keyword(s):  
Clavulanic Acid ◽  
Gene Cluster ◽  
Acid Production ◽  
Type Strain ◽  
Wild Type Strain ◽  
Streptomyces Clavuligerus ◽  
Wild Type ◽  
Glycerol Utilization ◽  
In The Wild

ABSTRACT The Streptomyces clavuligerus ATCC 27064 glycerol cluster gylR-glpF1K1D1 is induced by glycerol but is not affected by glucose. S. clavuligerus growth and clavulanic acid production are stimulated by glycerol, but this does not occur in a glpK1-deleted mutant. Amplification of glpK1D1 results in transformants yielding larger amounts of clavulanic acid in the wild-type strain and in overproducer S. clavuligerus Gap15-7-30 or S. clavuligerus ΔrelA strains.

scholarly journals C-5(6) Sterol Desaturase from Tetrahymena thermophila: Gene Identification and Knockout, Sequence Analysis, and Comparison to Other C-5(6) Sterol Desaturases

2009 ◽  
Vol 8 (8) ◽  
pp. 1287-1297 ◽  
Author(s):  
Alejandro D. Nusblat ◽  
Sebastián R. Najle ◽  
Mariela L. Tomazic ◽  
Antonio D. Uttaro ◽  
Clara B. Nudel
Keyword(s):  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Tetrahymena Thermophila ◽  
Gene Replacement ◽  
Gene Identification ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Gene Coding ◽  
Transmembrane Regions

ABSTRACT The gene coding for a C-5(6) sterol desaturase in Tetrahymena thermophila, DES5A, has been identified by the knockout of the TTHERM_01194720 sequence. Macronucleus transformation was achieved by biolistic bombardment and gene replacement through phenotypic assortment, using paromomycin as the selective agent. A knockout cell line (KO270) showed a phenotype consistent with that of the DES5A deletion mutant. KO270 converted only 6% of the added sterol into the C-5 unsaturated derivative, while the wild type accumulated 10-fold larger amounts under similar conditions. The decreased desaturation activity is specific for the C-5(6) position of lathosterol and cholestanol; other desaturations, namely C-7(8) and C-22(23), were not affected. Analysis by reverse transcription-PCR reveals that DES5A is transcribed both in the presence and absence of cholestanol in wild-type cells, whereas the transcribed gene was not detected in KO270. The growth of KO270 was undistinguishable from that of the wild-type strain. Des5Ap resembles known C-5(6) sterol desaturases, displaying the three typical histidine motifs, four hydrophobic transmembrane regions, and two other highly conserved domains of unknown function. A phylogenetic analysis placed T. thermophila's enzyme and Paramecium orthologues in a cluster together with functionally characterized C-5 sterol desaturases from vertebrates, fungi, and plants, although in a different branch.

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