scholarly journals A Single-Nucleotide Insertion in a Drug Transporter Gene Induces a Thermotolerance Phenotype inGluconobacter frateuriiby Increasing the NADPH/NADP+Ratio via Metabolic Change

2018 ◽  
Vol 84 (10) ◽  
Author(s):  
Nami Matsumoto ◽  
Hiromi Hattori ◽  
Minenosuke Matsutani ◽  
Chihiro Matayoshi ◽  
Hirohide Toyama ◽  
...  
Keyword(s):  
Acetic Acid ◽  
High Temperatures ◽  
Type Strain ◽  
Wild Type Strain ◽  
Metabolic Change ◽  
Drug Transporter ◽  
Transporter Gene ◽  
Wild Type ◽  
Content Type ◽  
In The Wild

ABSTRACTThermotolerant microorganisms are beneficial to the fermentation industry because they reduce the need for cooling and offer other operational advantages. Previously, we obtained a thermally adaptedGluconobacter frateuriistrain by experimental evolution. In the present study, we found only a single G insertion in the adapted strain, which causes a frameshift in a gene encoding a putative drug transporter. A mutant derivative strain with the single G insertion in the transporter gene (Wild-G) was constructed from the wild-type strain and showed increased thermotolerance. We found that the thermotolerant strains accumulated substantial intracellular trehalose and manifested a defect in sorbose assimilation, suggesting that the transporter is partly involved in trehalose efflux and sorbose uptake and that the defect in the transporter can improve thermotolerance. The ΔotsABstrain, constructed by elimination of the trehalose synthesis gene in the wild type, showed no trehalose production but, unexpectedly, much better growth than the adapted strain at high temperatures. The ΔotsABmutant produced more acetate as the final metabolite than the wild-type strain did. We hypothesized that trehalose does not contribute to thermotolerance directly; rather, a metabolic change including increased carbon flux to the pentose phosphate pathway may be the key factor. The NADPH/NADP+ratio was higher in strain Wild-G, and much higher in the ΔotsABstrain, than in the wild-type strain. Levels of reactive oxygen species (ROS) were lower in the thermotolerant strains. We propose that the defect of the transporter causes the metabolic flux to generate more NADPH, which may enhance thermotolerance inG. frateurii.IMPORTANCEThe biorefinery industry has to ensure that microorganisms are robust and retain their viability and function at high temperatures. Here we show thatGluconobacterfrateurii, an industrially important member of the acetic acid bacteria, exhibited enhanced thermotolerance through the reduction of trehalose excretion after thermal adaptation. Although intracellular trehalose may play a key role in thermotolerance, the molecular mechanisms of action of trehalose in thermotolerance are a matter of debate. Our mutated strain that was defective in trehalose synthase genes, producing no trehalose but a larger amount of acetic acid as the end metabolite instead, unexpectedly showed higher thermotolerance than the wild type. Our adapted and mutated thermotolerant strains showed increased NADPH/NADP+ratios and reductions in ROS levels. We concluded that inG. frateurii, trehalose does not contribute to thermotolerance directly; rather, the metabolic change increases the NADPH/NADP+ratio to enhance thermotolerance.

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 Serratia marcescensarn, a PhoP-Regulated Locus Necessary for Polymyxin B Resistance

2014 ◽  
Vol 58 (9) ◽  
pp. 5181-5190 ◽  
Author(s):  
Quei Yen Lin ◽  
Yi-Lin Tsai ◽  
Ming-Che Liu ◽  
Wei-Cheng Lin ◽  
Po-Ren Hsueh ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Response Regulator ◽  
Challenge Test ◽  
Polymyxin B ◽  
Resistant Bacteria ◽  
Wild Type ◽  
Mobility Shift ◽  
Content Type ◽  
In The Wild

ABSTRACTPolymyxins, which are increasingly being used to treat infections caused by multidrug-resistant bacteria, perform poorly againstSerratia marcescens. To investigate the underlying mechanisms, Tn5mutagenesis was performed and two mutants exhibiting increased polymyxin B (PB) susceptibility were isolated. The mutants were found to have Tn5inserted into thearnBandarnCgenes. In other bacteria,arnBandarnCbelong to the seven-genearnoperon, which is involved in lipopolysaccharide (LPS) modification. LPSs ofarnmutants had greater PB-binding abilities than that of wild-type LPS. Further, we identified PhoP, a bacterial two-component response regulator, as a regulator of PB susceptibility inS. marcescens. By the reporter assay, we found PB- and low-Mg2+-induced expression ofphoPandarnin the wild-type strain but not in thephoPmutant. Complementation of thephoPmutant with the full-lengthphoPgene restored the PB MIC and induction by PB and low Mg2+levels, as in the wild type. An electrophoretic mobility shift assay (EMSA) further demonstrated that PhoP bound directly to thearnpromoter. The PB challenge test confirmed that pretreatment with PB and low Mg2+levels protectedS. marcescensfrom a PB challenge in the wild-type strain but not in thephoPmutant. Real-time reverse transcriptase-PCR also indicated that PB serves as a signal to regulate expression ofugd, a gene required for LPS modification, inS. marcescensthrough a PhoP-dependent pathway. Finally, we found that PB-resistant clinical isolates displayed greater expression ofarnAupon exposure to PB than did susceptible isolates. This is the first report to describe the role ofS. marcescensarnin PB resistance and its modulation by PB and Mg2+through the PhoP protein.

scholarly journals Roles offkbNin Positive Regulation andtcs7in Negative Regulation of FK506 Biosynthesis in Streptomyces sp. Strain KCTC 11604BP

2012 ◽  
Vol 78 (7) ◽  
pp. 2249-2255 ◽  
Author(s):  
SangJoon Mo ◽  
Young Ji Yoo ◽  
Yeon Hee Ban ◽  
Sung-Kwon Lee ◽  
Eunji Kim ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Protein ◽  
Strain Improvement ◽  
Transcriptional Regulators ◽  
Biosynthetic Gene Cluster ◽  
Wild Type ◽  
Biosynthetic Genes ◽  
Content Type ◽  
In The Wild

ABSTRACTFK506 is an important 23-member polyketide macrolide with immunosuppressant activity. Its entire biosynthetic gene cluster was previously cloned fromStreptomycessp. strain KCTC 11604BP, and sequence analysis identified three putative regulatory genes,tcs2,tcs7, andfkbN, which encode proteins with high similarity to the AsnC family transcriptional regulators, LysR-type transcriptional regulators, and LAL family transcriptional regulators, respectively. Overexpression and in-frame deletion oftcs2did not affect the production of FK506 or co-occurring FK520 compared to results for the wild-type strain, suggesting thattcs2is not involved in their biosynthesis.fkbNoverexpression improved the levels of FK506 and FK520 production by approximately 2.0-fold, and a deletion offkbNcaused the complete loss of FK506 and FK520 production. Although the overexpression oftcs7decreased the levels of FK506 and FK520 production slightly, a deletion oftcs7caused 1.9-fold and 1.5-fold increases in FK506 and FK520 production, respectively. Finally,fkbNoverexpression in thetcs7deletion strain resulted in a 4.0-fold (21 mg liter−1) increase in FK506 production compared to that by the wild-type strain. This suggests thatfkbNencodes a positive regulatory protein essential for FK506/FK520 biosynthesis and that the gene product oftcs7negatively regulates their biosynthesis, demonstrating the potential of exploiting this information for strain improvement. Semiquantitative reverse transcription-PCR (RT-PCR) analyses of the transcription levels of the FK506 biosynthetic genes in the wild-type and mutant strains proved that most of the FK506 biosynthetic genes are regulated byfkbNin a positive manner and negatively bytcs7.

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 A Novel CO-Responsive Transcriptional Regulator and Enhanced H2Production by an Engineered Thermococcus onnurineus NA1 Strain

2014 ◽  
Vol 81 (5) ◽  
pp. 1708-1714 ◽  
Author(s):  
Min-Sik Kim ◽  
Ae Ran Choi ◽  
Seong Hyuk Lee ◽  
Hae-Chang Jung ◽  
Seung Seob Bae ◽  
...  
Keyword(s):  
Production Rate ◽  
Gene Cluster ◽  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory System ◽  
Transcriptional Regulator ◽  
Bioreactor Culture ◽  
Wild Type ◽  
Content Type ◽  
Transcriptional Regulatory

ABSTRACTGenome analysis revealed the existence of a putative transcriptional regulatory system governing CO metabolism inThermococcus onnurineusNA1, a carboxydotrophic hydrogenogenic archaeon. The regulatory system is composed of CorQ with a 4-vinyl reductase domain and CorR with a DNA-binding domain of the LysR-type transcriptional regulator family in close proximity to the CO dehydrogenase (CODH) gene cluster. Homologous genes of the CorQR pair were also found in the genomes ofThermococcusspecies and “CandidatusKorarchaeum cryptofilum” OPF8. In-frame deletion of eithercorQorcorRcaused a severe impairment in CO-dependent growth and H2production. WhencorQandcorRdeletion mutants were complemented by introducing thecorQRgenes under the control of a strong promoter, the mRNA and protein levels of the CODH gene were significantly increased in a ΔCorR strain complemented with integratedcorQR(ΔCorR/corQR↑) compared with those in the wild-type strain. In addition, the ΔCorR/corQR↑strain exhibited a much higher H2production rate (5.8-fold) than the wild-type strain in a bioreactor culture. The H2production rate (191.9 mmol liter−1h−1) and the specific H2production rate (249.6 mmol g−1h−1) of this strain were extremely high compared with those of CO-dependent H2-producing prokaryotes reported so far. These results suggest that thecorQRgenes encode a positive regulatory protein pair for the expression of a CODH gene cluster. The study also illustrates that manipulation of the transcriptional regulatory system can improve biological H2production.

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 The Riemerella anatipestiferAS87_01735Gene Encodes Nicotinamidase PncA, an Important Virulence Factor

2016 ◽  
Vol 82 (19) ◽  
pp. 5815-5823 ◽  
Author(s):  
Xiaolan Wang ◽  
Beibei Liu ◽  
Yafeng Dou ◽  
Hongjie Fan ◽  
Shaohui Wang ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Vaccine Candidate ◽  
Wild Type ◽  
Salvage Pathway ◽  
Effective Protection ◽  
Domestic Ducks ◽  
Content Type ◽  
Key Enzyme ◽  
Important Virulence Factor

ABSTRACTRiemerella anatipestiferis a major bacterial pathogen that causes septicemic and exudative diseases in domestic ducks. In our previous study, we found that deletion of theAS87_01735gene significantly decreased the bacterial virulence ofR. anatipestiferstrain Yb2 (mutant RA625). TheAS87_01735gene was predicted to encode a nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, which is an important reaction in the NAD+salvage pathway. In this study, theAS87_01735gene was expressed and identified as the PncA-encoding gene, using an enzymatic assay. Western blot analysis demonstrated thatR. anatipestiferPncA was localized to the cytoplasm. The mutant strain RA625 (named Yb2ΔpncAin this study) showed a similar growth rate but decreased NAD+quantities in both the exponential and stationary phases in tryptic soy broth culture, compared with the wild-type strain Yb2. In addition, Yb2ΔpncA-infected ducks showed much lower bacterial loads in their blood, and no visible histological changes were observed in the heart, liver, and spleen. Furthermore, Yb2ΔpncAimmunization of ducks conferred effective protection against challenge with the virulent wild-type strain Yb2. Our results suggest that theR. anatipestiferAS87_01735gene encodes PncA, which is an important virulence factor, and that the Yb2ΔpncAmutant can be used as a novel live vaccine candidate.IMPORTANCERiemerella anatipestiferis reported worldwide as a cause of septicemic and exudative diseases of domestic ducks. ThepncAgene encodes a nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, which is an important reaction in the NAD+salvage pathway. In this study, we identified and characterized thepncA-homologous geneAS87_01735inR. anatipestiferstrain Yb2.R. anatipestiferPncA is a cytoplasmic protein that possesses similar PncA activity, compared with other organisms. Generation of thepncAmutant Yb2ΔpncAled to a decrease in the NAD+content, which was associated with decreased capacity for invasion and attenuated virulence in ducks. Furthermore, Yb2ΔpncAimmunization of ducks conferred effective protection against challenge with the virulent wild-type strain Yb2. Altogether, these results suggest that PncA contributes to the virulence ofR. anatipestiferand that the Yb2ΔpncAmutant can be used as a novel live vaccine candidate.

scholarly journals Influence of Chemotaxis and Swimming Patterns on the Virulence of the Coral PathogenVibrio coralliilyticus

2018 ◽  
Vol 200 (15) ◽  
Author(s):  
Blake Ushijima ◽  
Claudia C. Häse
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Bacterial Colonization ◽  
Valuable Insight ◽  
Wild Type ◽  
Coral Mucus ◽  
Content Type ◽  
Swimming Pattern ◽  
Vibrio Coralliilyticus ◽  
Insight Into

ABSTRACTChemotaxis, the directed movement toward or away from a chemical signal, can be essential to bacterial pathogens for locating hosts or avoiding hostile environments. The coral pathogenVibrio coralliilyticuschemotaxes toward coral mucus; however, chemotaxis has not been experimentally demonstrated to be important for virulence. To further examine this, in-frame mutations were constructed in genes predicted to be important forV. coralliilyticuschemotaxis. MostVibriogenomes contain multiple homologs of various chemotaxis-related genes, and two paralogs of each forcheB,cheR, andcheAwere identified. Based on single mutant analyses, the paralogscheB2,cheR2, andcheA1were essential for chemotaxis in laboratory assays. As predicted, the ΔcheA1and ΔcheR2strains had a smooth-swimming pattern, while the ΔcheB2strain displayed a zigzag pattern when observed under light microscopy. However, these mutants, unlike the parent strain, were unable to chemotax toward the known attractants coral mucus, dimethylsulfoniopropionate, andN-acetyl-d-glucosamine. The ΔcheB2strain and an aflagellate ΔfliG1strain were avirulent to coral, while the ΔcheA1and ΔcheR2strains were hypervirulent (90 to 100% infection within 14 h on average) compared to the wild-type strain (66% infection within 36 h on average). Additionally, the ΔcheA1and ΔcheR2strains appeared to better colonize coral fragments than the wild-type strain. These results suggest that although chemotaxis may be involved with infection (the ΔcheB2strain was avirulent), a smooth-swimming phenotype is important for bacterial colonization and infection. This study provides valuable insight into understandingV. coralliilyticuspathogenesis and how this pathogen may be transmitted between hosts.IMPORTANCECorals are responsible for creating the immense structures that are essential to reef ecosystems; unfortunately, pathogens like the bacteriumVibrio coralliilyticuscan cause fatal infections of reef-building coral species. However, compared to related human pathogens, the mechanisms by whichV. coralliilyticusinitiates infections and locates new coral hosts are poorly understood. This study investigated the effects of chemotaxis, the directional swimming in response to chemical signals, and bacterial swimming patterns on infection of the coralMontipora capitata. Infection experiments with different mutant strains suggested that a smooth-swimming pattern resulted in hypervirulence. These results demonstrate that the role of chemotaxis in coral infection may not be as straightforward as previously hypothesized and provide valuable insight intoV. coralliilyticuspathogenesis.

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.

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