scholarly journals Novel Na+/H+ antiporter (NapA) regulates the motility in Helicobacter pylori

2021 ◽  
Vol 8 (3) ◽  
pp. 027-035
Author(s):  
Masaaki Minami ◽  
Shin-nosuke Hashikawa ◽  
Takafumi Ando ◽  
Hiroshi Kobayashi ◽  
Hidemi Goto ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Pcr Analysis ◽  
Wild Type ◽  
Cellular Homeostasis ◽  
In The Wild ◽  
Flagellar Proteins ◽  
H Pylori

Na+/H+ antiporter plays an important role in maintaining cellular homeostasis by regulating osmotic pressure and intracellular pH. It plays an important role in maintaining cellular homeostasis. In Helicobacter pylori, whole genome sequencing has revealed the presence of two types of Na+/H+ antiporter. A gene (nhaA) homologous to the Na+/H+ antiporter of Escherichia coli has been investigated and its function has been analyzed. However, another gene homologous to the Na+/H+ antiporter of Enterococcus hirae (napA) is not yet known in detail. In this study, we investigated the function of this gene (napA in H. pylori). First, to confirm the genetic presence of napA in 20 H. pylori clinical isolates, PCR analysis was performed, and the napA gene was confirmed in all strains. The amount of Na+ extrusion was measured by atomic absorption spectroscopy. The results showed that the Na+ concentration was decreased in the wild-type strain compared to the napA mutant strain. In addition, there was a significant dose-dependent difference in CFU of Na+ concentration in the napA mutant strain compared to the wild-type strain. We examined whether the napA gene is related to motility using both wild-type and napA mutant strains. As a result, in the motility agar test, the bacterial motility observed in the wild-type strain was not observed in the napA mutant strain. However, no difference in flagellar proteins was observed by SDS-PAGE analysis. These results suggest that the napA gene of H. pylori may regulate homeostasis by extruding Na+ and may also regulate motility.

scholarly journals Involvement of CO2 generated by urease in multiplication of Helicobacter pylori

2021 ◽  
Vol 7 (3) ◽  
pp. 045-053
Author(s):  
Masaaki Minami ◽  
Shin-nosuke Hashikawa ◽  
Takafumi Ando ◽  
Hiroshi Kobayashi ◽  
Hidemi Goto ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Neutral Medium ◽  
Wild Type ◽  
In The Wild ◽  
H Pylori ◽  
Carbon Dioxide Co2 ◽  
The Relationship

Helicobacter pylori (H. pylori) urease generates both ammonia (NH3) and carbon dioxide (CO2) from urea. NH3 helps H. pylori to survive in the stomach in part by neutralizing gastric acid. However, the relationship between CO2 and H. pylori is not completed cleared. We examined the effect of CO2 generated by urease on multiplication of H. pylori by using isogenic ureB mutant and ureB complemented strain from H. pylori strain JP26. Wild-type strain survived in the medium supplement with 1mM urea in room air, however, the urease negative strain did not. To discern whether CO2 was incorporated into H. pylori, 14C in bacillus was counted after 6 hours incubation with 14C urea in both acidic and neutral medium. Significant more 14C uptake was detected in wild-type strain compared to ureB mutant strain and this uptake in the wild-type strain was more under acidic condition compared to under neutral condition, but no difference was identified in the mutant strain. These results suggest that CO2 generated by urease plays a role in multiplication of H. pylori.

scholarly journals An NADPH Quinone Reductase of Helicobacter pylori Plays an Important Role in Oxidative Stress Resistance and Host Colonization

2004 ◽  
Vol 72 (3) ◽  
pp. 1391-1396 ◽  
Author(s):  
Ge Wang ◽  
Robert J. Maier
Keyword(s):  
Oxidative Stress ◽  
Helicobacter Pylori ◽  
Mutant Strain ◽  
Stress Resistance ◽  
Type Strain ◽  
Wild Type Strain ◽  
Quinone Reductase ◽  
Wild Type ◽  
Oxidative Stress Resistance ◽  
H Pylori

ABSTRACT Oxidative stress resistance is one of the key properties that enable pathogenic bacteria to survive the toxic reactive oxygen species released by the host. In a previous study characterizing oxidative stress resistance mutants of Helicobacter pylori, a novel potential antioxidant protein (MdaB) was identified by the observation that the expression of this protein was significantly upregulated to compensate for the loss of other major antioxidant components. In this study, we characterized an H. pylori mdaB mutant and the MdaB protein. While the wild-type strain can tolerate 10% oxygen for growth, the growth of the mdaB mutant was significantly inhibited by this oxygen condition. The mdaB mutant is also more sensitive to H2O2, organic hydroperoxides, and the superoxide-generating agent paraquat. Although the wild-type strain can survive more than 10 h of air exposure, exposure of the mutant strain to air for 8 h resulted in recovery of no viable cells. The oxidative stress sensitivity of the mdaB mutant resulted in a deficiency in the ability to colonize mouse stomachs. H. pylori was recovered from 10 of 11 mouse stomachs inoculated with the wild-type strain, with about 5,000 to 45,000 CFU/g of stomach. However, only 3 of 12 mice that were inoculated with the mdaB mutant strain were found to harbor any H. pylori, and these 3 contained less than 2,000 CFU/g of stomach. A His-tagged MdaB protein was purified and characterized. It was shown to be a flavoprotein that catalyzes two-electron transfer from NAD(P)H to quinones. It reduces both ubiquinones and menaquinones with similar efficiencies and preferably uses NADPH as an electron donor. We propose that the physiological function of the H. pylori MdaB protein is that of an NADPH quinone reductase that plays an important role in managing oxidative stress and contributes to successful colonization of the host.

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 Involvement of the XpsN Protein in Formation of the XpsL-XpsM Complex in Xanthomonas campestris pv. campestris Type II Secretion Apparatus

2001 ◽  
Vol 183 (2) ◽  
pp. 528-535 ◽  
Author(s):  
Hsien-Ming Lee ◽  
Shiaw-Wei Tyan ◽  
Wei-Ming Leu ◽  
Ling-Yun Chen ◽  
David Chanhen Chen ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Mutant Strain ◽  
Type Strain ◽  
Xanthomonas Campestris ◽  
Wild Type Strain ◽  
Type Ii ◽  
Wild Type ◽  
In The Wild ◽  
Steady State Levels ◽  
Type Gene

ABSTRACT The xps gene cluster is required for the second step of type II protein secretion in Xanthomonas campestrispv. campestris. Deletion of the entire gene cluster caused accumulation of secreted proteins in the periplasm. By analyzing protein abundance in the chromosomal mutant strains, we observed mutual dependence for normal steady-state levels between the XpsL and the XpsM proteins. The XpsL protein was undetectable in total lysate prepared from thexpsM mutant strain, and vice versa. Introduction of the wild-type xpsM gene carried on a plasmid into thexpsM mutant strain was sufficient for reappearance of the XpsL protein, and vice versa. Moreover, both XpsL and XpsM proteins were undetectable in the xpsN mutant strain. They were recovered either by reintroducing the wild-type xpsNgene or by introducing extra copies of wild-type xpsL orxpsM individually. Overproduction of wild-type XpsL and -M proteins simultaneously, but not separately, in the wild-type strain of X. campestris pv. campestris caused inhibition of secretion. Complementation of an xpsL orxpsM mutant strain with a plasmid-borne wild-type gene was inhibited by coexpression of XpsL and XpsM. The presence of the xpsN gene on the plasmid along with thexpsL and the xpsM genes caused more severe inhibition in both cases. Furthermore, complementation of thexpsN mutant strain was also inhibited. In both the wild-type strain and a strain with the xps gene cluster deleted (XC17433), carrying pCPP-LMN, which encodes all three proteins, each protein coprecipitated with the other two upon immunoprecipitation. Expression of pairwise combinations of the three proteins in XC17433 revealed that the XpsL-XpsM and XpsM-XpsN pairs still coprecipitated, whereas the XpsL-XpsN pair no longer coprecipitated.

scholarly journals Regulation of ppk Expression and In Vivo Function of Ppk in Streptomyces lividans TK24

2006 ◽  
Vol 188 (17) ◽  
pp. 6269-6276 ◽  
Author(s):  
Sofiane Ghorbel ◽  
Aleksey Smirnov ◽  
Hichem Chouayekh ◽  
Brice Sperandio ◽  
Catherine Esnault ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Sufficient Conditions ◽  
Streptomyces Lividans ◽  
Antibiotic Biosynthesis ◽  
Wild Type ◽  
In The Wild

ABSTRACT The ppk gene of Streptomyces lividans encodes an enzyme catalyzing, in vitro, the reversible polymerization of the γ phosphate of ATP into polyphosphate and was previously shown to play a negative role in the control of antibiotic biosynthesis (H. Chouayekh and M. J. Virolle, Mol. Microbiol. 43:919-930, 2002). In the present work, some regulatory features of the expression of ppk were established and the polyphosphate content of S. lividans TK24 and the ppk mutant was determined. In Pi sufficiency, the expression of ppk was shown to be low but detectable. DNA gel shift experiments suggested that ppk expression might be controlled by a repressor using ATP as a corepressor. Under these conditions, short acid-soluble polyphosphates accumulated upon entry into the stationary phase in the wild-type strain but not in the ppk mutant strain. The expression of ppk under Pi-limiting conditions was shown to be much higher than that under Pi-sufficient conditions and was under positive control of the two-component system PhoR/PhoP. Under these conditions, the polyphosphate content of the cell was low and polyphosphates were reproducibly found to be longer and more abundant in the ppk mutant strain than in the wild-type strain, suggesting that Ppk might act as a nucleoside diphosphate kinase. In light of our results, a novel view of the role of this enzyme in the regulation of antibiotic biosynthesis in S. lividans TK24 is proposed.

scholarly journals Insights into an alternative pathway for glycerol metabolism in a glycerol kinase deficientPseudomonas putidaKT2440

2019 ◽  
Author(s):  
Meg Walsh ◽  
William Casey ◽  
Shane T. Kenny ◽  
Tanja Narancic ◽  
Lars M. Blank ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Alternative Pathway ◽  
Glycerol Kinase ◽  
Glycerol Metabolism ◽  
Wild Type ◽  
Short Chain Length ◽  
Genes Encoding ◽  
In The Wild

AbstractPseudomonas putidaKT2440 is known to metabolise glycerol via glycerol-3-phosphate using glycerol kinase an enzyme previously described as critical for glycerol metabolism (1). However, when glycerol kinase was knocked out inP. putidaKT2440 it retained the ability to use glycerol as the sole carbon source, albeit with a much-extended lag period and 2 fold lower final biomass compared to the wild type strain. A metabolomic study identified glycerate as a major and the most abundant intermediate in glycerol metabolism in this mutated strain with levels 21-fold higher than wild type. Erythrose-4-phosphate was detected in the mutant strain, but not in the wild type strain. Glyceraldehyde and glycraldehyde-3-phosphate were detected at similar levels in the mutant strain and the wild type. Transcriptomic studies identified 191 genes that were more than 2-fold upregulated in the mutant compared to the wild type and 175 that were down regulated. The genes involved in short chain length fatty acid metabolism were highly upregulated in the mutant strain. The genes encoding 3-hydroxybutyrate dehydrogenase were 5.8-fold upregulated and thus the gene was cloned, expressed and purified to reveal it can act on glyceraldehyde but not glycerol as a substrate.

Role of glutathione in the growth of Bradyrhizobium sp. (peanut microsymbiont) under different environmental stresses and in symbiosis with the host plant

2006 ◽  
Vol 52 (7) ◽  
pp. 609-616 ◽  
Author(s):  
Luciano Sobrevals ◽  
Peter Müller ◽  
Adriana Fabra ◽  
Stella Castro
Keyword(s):  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Environmental Stresses ◽  
Wild Type ◽  
Glutamylcysteine Synthetase ◽  
Bradyrhizobium Sp ◽  
In The Wild ◽  
Saline Medium

Glutathione (GSH) plays an important role in the defence of microorganisms and plants against different environmental stresses. To determine the role of GSH under different stresses, such as acid pH, saline shock, and oxidative shock, a GSH-deficient mutant (Bradyrhizobium sp. 6144-S7Z) was obtained by disruption of the gshA gene, which encodes the enzyme γ-glutamylcysteine synthetase. Growth of the mutant strain was significantly reduced in liquid minimal saline medium, and the GSH content was very low, about 4% of the wild-type level. The defect, caused by disruption of the gshA gene in the growth of mutant strain, cannot be reversed by the addition of GSH (up to 100 µmol/L) to the liquid minimal saline medium, and the endogenous GSH level was approximately the same as that observed without the addition of GSH. In contrast, the wild-type strain increased the GSH content under these conditions. However, the growth of the mutant strain in a rich medium (yeast extract – mannitol) increased, suggesting that at least some but not all of the functions of GSH could be provided by peptides and (or) amino acids. The symbiotic properties of the mutant were similar to those found in the wild-type strain, indicating that the mutation does not affect the ability of the mutant to form effective nodules.Key words: glutathione, γ-glutamylcysteine synthetase, Bradyrhizobium sp., peanut.

scholarly journals Screen for Leukotoxin Mutants in Aggregatibacter actinomycetemcomitans: Genes of the Phosphotransferase System Are Required for Leukotoxin Biosynthesis

2008 ◽  
Vol 76 (8) ◽  
pp. 3561-3568 ◽  
Author(s):  
Maria P. Isaza ◽  
Matthew S. Duncan ◽  
Jeffrey B. Kaplan ◽  
Scott C. Kachlany
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Aggressive Periodontitis ◽  
Pcr Analysis ◽  
Growth Media ◽  
Wild Type ◽  
Camp Production ◽  
Phosphotransferase System ◽  
In The Wild ◽  
Transposon Insertions

ABSTRACT Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans is a pathogen that causes localized aggressive periodontitis and extraoral infections including infective endocarditis. Recently, we reported that A. actinomycetemcomitans is beta-hemolytic on certain growth media due to the production of leukotoxin (LtxA). Based on this observation and our ability to generate random transposon insertions in A. actinomycetemcomitans, we developed and carried out a rapid screen for LtxA mutants. Using PCR, we mapped several of the mutations to genes that are known or predicted to be required for LtxA production, including ltxA, ltxB, ltxD, and tdeA. In addition, we identified an insertion in a gene previously not recognized to be involved in LtxA biosynthesis, ptsH. ptsH encodes the protein HPr, a phosphocarrier protein that is part of the sugar phosphotransferase system. HPr results in the phosphorylation of other proteins and ultimately in the activation of adenylate cyclase and cyclic AMP (cAMP) production. The ptsH mutant showed only partial hemolysis on blood agar and did not produce LtxA. The phenotype was complemented by supplying wild-type ptsH in trans, and real-time PCR analysis showed that the ptsH mutant produced approximately 10-fold less ltxA mRNA than the wild-type strain. The levels of cAMP in the ptsH mutant were significantly lower than in the wild-type strain, and LtxA production could be restored by adding exogenous cAMP to the culture.

scholarly journals Characterization of γ-Butyrolactone Autoregulatory Signaling Gene Homologs in the Angucyclinone Polyketide WS5995B Producer Streptomyces acidiscabies

2009 ◽  
Vol 191 (15) ◽  
pp. 4786-4797 ◽  
Author(s):  
Frank G. Healy ◽  
Kevin P. Eaton ◽  
Prajit Limsirichai ◽  
Joel F. Aldrich ◽  
Alaina K. Plowman ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Type Strain ◽  
Wild Type Strain ◽  
Morphological Differentiation ◽  
Pcr Analysis ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Morphological Defects ◽  
In The Wild ◽  
Streptomyces Acidiscabies

ABSTRACT Organisms belonging to the genus Streptomyces produce numerous important secondary metabolites and undergo a sophisticated morphological differentiation program. In many instances these processes are under the control of γ-butyrolactone (GBL) autoregulatory systems. Streptomyces acidiscabies strain 84.104 produces the secondary metabolite aromatic angucyclinone polyketide WS5995B. In order to explore the role of GBL regulatory circuitry in WS5995B production and morphogenesis in S. acidiscabies, a gene cluster encoding GBL autoregulatory signaling homologs was identified and characterized. Two GBL receptor homologs, sabR and sabS, were found flanking a GBL synthase homolog sabA. Strains carrying mutations in sabS produced elevated levels of WS5995B and displayed conditional morphological defects reminiscent of defects seen in Streptomyces bldA mutants. Notably, sabS possesses a TTA codon predicted to be recognized by tRNAleu. sabA mutants produced higher levels of WS5995B than the wild-type strain but to a lesser extent than the levels of WS5995B seen in sabS mutants. Purified recombinant SabR and SabS were tested for their abilities to bind predicted AT-rich autoregulatory element (ARE) boxes within the sabRAS region. SabS did not bind any DNA sequences in this region, while SabR bound an ARE box in the region upstream of sabS. Quantitative reverse transcription-PCR analysis revealed higher levels of sabS transcript in sabR mutants than in the wild-type strain, suggesting that sabS expression is repressed by SabR. Based on these data, we propose that the S. acidiscabies sabRAS genes encode components of a signaling pathway which participates in the regulation of WS5995B production and morphogenesis.

scholarly journals Requirement of Histidine Kinases HP0165 and HP1364 for Acid Resistance in Helicobacter pylori

2006 ◽  
Vol 74 (5) ◽  
pp. 3052-3059 ◽  
Author(s):  
John T. Loh ◽  
Timothy L. Cover
Keyword(s):  
Helicobacter Pylori ◽  
Type Strain ◽  
Wild Type Strain ◽  
Acid Resistance ◽  
Wild Type ◽  
Histidine Kinases ◽  
Mutant Strains ◽  
Two Component Signal Transduction ◽  
Reporter Assays ◽  
H Pylori

ABSTRACT In this study, we investigated a potential requirement of two-component signal transduction systems for acid resistance in Helicobacter pylori. In comparison to a wild-type strain, isogenic strains with null mutations in either HP0165 or HP1364 histidine kinases were impaired in their ability to grow at pH 5.0. The growth of complemented mutant strains was similar to that of the wild-type strain. H. pylori DNA array analyses and transcriptional reporter assays indicated that acid-responsive gene transcription was altered in the HP0165 and HP1364 null mutant strains compared to the parental wild-type strain. These results indicate that intact HP0165 and HP1364 histidine kinases are required for acid resistance in H. pylori.

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