scholarly journals Monocyclic Aromatic Hydrocarbon Degradation by Rhodococcus sp. Strain DK17

2002 ◽  
Vol 68 (7) ◽  
pp. 3270-3278 ◽  
Author(s):  
Dockyu Kim ◽  
Young-Soo Kim ◽  
Seong-Ki Kim ◽  
Si Wouk Kim ◽  
Gerben J. Zylstra ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Proton Nuclear Magnetic Resonance ◽  
Gas Chromatography Mass Spectrometry ◽  
Ring Cleavage ◽  
Oxidation Reactions ◽  
Wild Type ◽  
Initial Oxidation ◽  
In The Wild

ABSTRACT Rhodococcus sp. strain DK17 was isolated from soil and analyzed for the ability to grow on o-xylene as the sole carbon and energy source. Although DK17 cannot grow on m- and p-xylene, it is capable of growth on benzene, phenol, toluene, ethylbenzene, isopropylbenzene, and other alkylbenzene isomers. One UV-generated mutant strain, DK176, simultaneously lost the ability to grow on o-xylene, ethylbenzene, isopropylbenzene, toluene, and benzene, although it could still grow on phenol. The mutant strain was also unable to oxidize indole to indigo following growth in the presence of o-xylene. This observation suggests the loss of an oxygenase that is involved in the initial oxidation of the (alkyl)benzenes tested. Another mutant strain, DK180, isolated for the inability to grow on o-xylene, retained the ability to grow on benzene but was unable to grow on alkylbenzenes due to loss of a meta-cleavage dioxygenase needed for metabolism of methyl-substituted catechols. Further experiments showed that DK180 as well as the wild-type strain DK17 have an ortho-cleavage pathway which is specifically induced by benzene but not by o-xylene. These results indicate that DK17 possesses two different ring-cleavage pathways for the degradation of aromatic compounds, although the initial oxidation reactions may be catalyzed by a common oxygenase. Gas chromatography-mass spectrometry and 300-MHz proton nuclear magnetic resonance spectrometry clearly show that DK180 accumulates 3,4-dimethylcatechol from o-xylene and both 3- and 4-methylcatechol from toluene. This means that there are two initial routes of oxidation of toluene by the strain. Pulsed-field gel electrophoresis analysis demonstrated the presence of two large megaplasmids in the wild-type strain DK17, one of which (pDK2) was lost in the mutant strain DK176. Since several other independently derived mutant strains unable to grow on alkylbenzenes are also missing pDK2, the genes encoding the initial steps in alkylbenzene metabolism (but not phenol metabolism) appear to be present on this approximately 330-kb plasmid.

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.

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.

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 The CcpA Protein Is Necessary for Efficient Sporulation and Enterotoxin Gene (cpe) Regulation in Clostridium perfringens

2004 ◽  
Vol 186 (16) ◽  
pp. 5221-5229 ◽  
Author(s):  
John Varga ◽  
Veronica L. Stirewalt ◽  
Stephen B. Melville
Keyword(s):  
Mutant Strain ◽  
Clostridium Perfringens ◽  
Exponential Growth ◽  
Catabolite Repression ◽  
Type Strain ◽  
Wild Type Strain ◽  
Food Poisoning ◽  
Wild Type ◽  
Antibiotic Associated Diarrhea ◽  
In The Wild

ABSTRACT Clostridium perfringens is the cause of several human diseases, including gas gangrene (clostridial myonecrosis), enteritis necroticans, antibiotic-associated diarrhea, and acute food poisoning. The symptoms of antibiotic-associated diarrhea and acute food poisoning are due to sporulation-dependent production of C. perfringens enterotoxin encoded by the cpe gene. Glucose is a catabolite repressor of sporulation by C. perfringens. In order to identify the mechanism of catabolite repression by glucose, a mutation was introduced into the ccpA gene of C. perfringens by conjugational transfer of a nonreplicating plasmid into C. perfringens, which led to inactivation of the ccpA gene by homologous recombination. CcpA is a transcriptional regulator known to mediate catabolite repression in a number of low-G+C-content gram-positive bacteria, of which C. perfringens is a member. The ccpA mutant strain sporulated at a 60-fold lower efficiency than the wild-type strain in the absence of glucose. In the presence of 5 mM glucose, sporulation was repressed about 2,000-fold in the wild-type strain and 800-fold in the ccpA mutant strain compared to sporulation levels for the same strains grown in the absence of glucose. Therefore, while CcpA is necessary for efficient sporulation in C. perfringens, glucose-mediated catabolite repression of sporulation is not due to the activity of CcpA. Transcription of the cpe gene was measured in the wild-type and ccpA mutant strains grown in sporulation medium by using a cpe-gusA fusion (gusA is an Escherichia coli gene encoding the enzyme β-glucuronidase). In the exponential growth phase, cpe transcription was two times higher in the ccpA mutant strain than in the wild-type strain. Transcription of cpe was highly induced during the entry into stationary phase in wild-type cells but was not induced in the ccpA mutant strain. Glucose repressed cpe transcription in both the wild-type and ccpA mutant strain. Therefore, CcpA appears to act as a repressor of cpe transcription in exponential growth but is required for efficient sporulation and cpe transcription upon entry into stationary phase. CcpA was also required for maximum synthesis of collagenase (kappa toxin) and acted as a repressor of polysaccharide capsule synthesis in the presence of glucose, but it did not regulate synthesis of the phospholipase PLC (alpha toxin).

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 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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