scholarly journals Characterization of a Second Rhodococcus erythropolis SQ1 3-Ketosteroid 9α-Hydroxylase Activity Comprising a Terminal Oxygenase Homologue, KshA2, Active with Oxygenase-Reductase Component KshB

2008 ◽  
Vol 74 (23) ◽  
pp. 7197-7203 ◽  
Author(s):  
R. van der Geize ◽  
G. I. Hessels ◽  
M. Nienhuis-Kuiper ◽  
L. Dijkhuizen
Keyword(s):  
Mutant Strain ◽  
Parent Strain ◽  
Gene Deletion ◽  
Amino Acid Level ◽  
Rhodococcus Erythropolis ◽  
Pcr Primers ◽  
Transcriptional Analysis ◽  
Reporter Protein ◽  
Steroid Substrate ◽  
Key Enzyme

ABSTRACT Previously we have characterized 3-ketosteroid 9α-hydroxylase (KSH), a key enzyme in microbial steroid degradation in Rhodococcus erythropolis strain SQ1, as a two-component iron-sulfur monooxygenase, comprised of the terminal oxygenase component KshA1 and the oxygenase-reductase component KshB. Deletion of the kshA1 gene resulted in the loss of the ability of mutant strain RG2 to grow on the steroid substrate 4-androstene-3,17-dione (AD). Here we report characteristics of a close KshA1 homologue, KshA2 of strain SQ1, sharing 60% identity at the amino acid level. Expression of the kshA2 gene in mutant strain RG2 restored growth on AD and ADD, indicating that kshA2 also encodes KSH activity. The functional complementation was shown to be dependent on the presence of kshB. Transcriptional analysis showed that expression of kshA2 is induced in parent strain R. erythropolis SQ1 in the presence of AD. However, promoter activity studies, using β-lactamase of Escherichia coli as a convenient transcription reporter protein for Rhodococcus, revealed that the kshA2 promoter in fact is highly induced in the presence of 9α-hydroxy-4-androstene-3,17-dione (9OHAD) or a metabolite thereof. Inactivation of kshA2 in parent strain SQ1 by unmarked gene deletion did not affect growth on 9OHAD, cholesterol, or cholic acid. We speculate that KshA2 plays a role in preventing accumulation of toxic intracellular concentrations of ADD during steroid catabolism. A third kshA homologue was additionally identified in a kshA1 kshA2 double gene deletion mutant strain of R. erythropolis SQ1. The developed degenerate PCR primers for kshA may be useful for isolation of kshA homologues from other (actino) bacteria.

scholarly journals New Insights into the Genetic Organization of the FK228 Biosynthetic Gene Cluster inChromobacterium violaceumNo. 968

2010 ◽  
Vol 77 (4) ◽  
pp. 1508-1511 ◽  
Author(s):  
Vishwakanth Y. Potharla ◽  
Shane R. Wesener ◽  
Yi-Qiang Cheng
Keyword(s):  
Natural Product ◽  
Gene Cluster ◽  
Gene Deletion ◽  
Regulatory Gene ◽  
Biosynthetic Gene Cluster ◽  
Transcriptional Analysis ◽  
Biosynthetic Gene ◽  
Genetic Organization

ABSTRACTThe biosynthetic gene cluster of FK228, an FDA-approved anticancer natural product, was identified and sequenced previously. The genetic organization of this gene cluster has now been delineated through systematic gene deletion and transcriptional analysis. As a result, the gene cluster is redefined to contain 12 genes:depAthroughdepJ,depM, and a newly identified pathway regulatory gene,depR.

scholarly journals Transposition of Insertion Sequences was Triggered by Oxidative Stress in Radiation-Resistant Bacterium Deinococcus geothermalis

2019 ◽  
Vol 7 (10) ◽  
pp. 446 ◽  
Author(s):  
Chanjae Lee ◽  
Nakjun Choi ◽  
Min K. Bae ◽  
Kyungsil Choo ◽  
Sung-Jae Lee
Keyword(s):  
Oxidative Stress ◽  
Family Member ◽  
Mutant Strain ◽  
Mutant Cell ◽  
Carotenoid Biosynthesis ◽  
Wild Type ◽  
Is Element ◽  
Deinococcus Geothermalis ◽  
Key Enzyme ◽  
Radiation Resistant

During an oxidative stress-response assay on a putative Dps-like gene-disrupted Δdgeo_0257 mutant strain of radiation-resistant bacterium Deinococcus geothermalis, a non-pigmented colony was observed among the normal reddish color colonies. This non-pigmented mutant cell subsequently displayed higher sensitivity to H2O2. While carotenoid has a role in protecting as scavenger of reactive oxygen species the reddish wild-type strain from radiation and oxidative stresses, it is hypothesized that the carotenoid biosynthesis pathway has been disrupted in the mutant D. geothermalis cell. Here, we show that, in the non-pigmented mutant cell of interest, phytoene desaturase (Dgeo_0524, crtI), a key enzyme in carotenoid biosynthesis, was interrupted by transposition of an ISDge7 family member insertion sequence (IS) element. RNA-Seq analysis between wild-type and Δdgeo_0257 mutant strains revealed that the expression level of ISDge5 family transposases, but not ISDge7 family members, were substantially up-regulated in the Δdgeo_0257 mutant strain. We revealed that the non-pigmented strain resulted from the genomic integration of ISDge7 family member IS elements, which were also highly up-regulated, particularly following oxidative stress. The transposition path for both transposases is a replicative mode. When exposed to oxidative stress in the absence of the putative DNA binding protein Dgeo_0257, a reddish D. geothermalis strain became non-pigmented. This transformation was facilitated by transposition of an ISDge7 family IS element into a gene encoding a key enzyme of carotenoid biosynthesis. Further, we present evidence of additional active transposition by the ISDge5 family IS elements, a gene that was up-regulated during the stationary phase regardless of the presence of oxidative stress.

scholarly journals PCR Detection of Genes Encoding Nitrite Reductase in Denitrifying Bacteria

1999 ◽  
Vol 65 (4) ◽  
pp. 1652-1657 ◽  
Author(s):  
Sara Hallin ◽  
Per-Eric Lindgren
Keyword(s):  
Wastewater Treatment ◽  
Activated Sludge ◽  
Nitrite Reductase ◽  
Wastewater Treatment Plants ◽  
Paracoccus Denitrificans ◽  
Pcr Primers ◽  
Genes Encoding ◽  
Pcr Products ◽  
Key Enzyme ◽  
Sequence Relationships

ABSTRACT Using consensus regions in gene sequences encoding the two forms of nitrite reductase (Nir), a key enzyme in the denitrification pathway, we designed two sets of PCR primers to amplifycd 1- and Cu-nir. The primers were evaluated by screening defined denitrifying strains, denitrifying isolates from wastewater treatment plants, and extracts from activated sludge. Sequence relationships ofnir genes were also established. Thecd 1 primers were designed to amplify a 778 to 799-bp region of cd1-nir in the six published sequences. Likewise, the Cu primers amplified a 473-bp region in seven of the eight published Cu-nir sequences. Together, the two sets of PCR primers amplified nir genes in nine species within four genera, as well as in four of the seven sludge isolates. The primers did not amplify genes of nondenitrifying strains. The Cu primers amplified the expected fragment in all 13 sludge samples, but cd1-nir fragments were only obtained in five samples. PCR products of the expected sizes were verified as nir genes after hybridization to DNA probes, except in one case. The sequenced nir fragments were related to other nir sequences, demonstrating that the primers amplified the correct gene. The selected primer sites for Cu-nir were conserved, while broad-range primers targeting conserved regions of cd1-nir seem to be difficult to find. We also report on the existence of Cu-nir in Paracoccus denitrificans Pd1222.

scholarly journals Metabolism of pyrithiamine by the pyrithiamine-requiring mutant of Staphylococcus aureus

1968 ◽  
Vol 107 (2) ◽  
pp. 165-169 ◽  
Author(s):  
Asru K. Sinha ◽  
G. C. Chatterjee
Keyword(s):  
Staphylococcus Aureus ◽  
Mutant Strain ◽  
Parent Strain ◽  
Culture Broth ◽  
Exponential Phase ◽  
Optimum Growth ◽  
Solvent Fractionation ◽  
Prolonged Incubation ◽  
Inhibition Index

1. A mutant strain of Staphylococcus aureus that requires pyrithiamine for its optimum growth was found to utilize pyrithiamine during the exponential phase of growth. 2. Pyrithiamine was deaminated by the organism to form oxypyrithiamine, the reaction being enzymic with no cofactor requirement. 3. On prolonged incubation of S. aureus A cultures, the concentration of deaminating enzyme increased in the culture broth, from which pyrithiamine-deaminating enzyme could be isolated by solvent fractionation. 4. Oxypyrithiamine is not a competitive analogue of thiamine although it inhibited the growth of the parent strain of S. aureus; the inhibition index of this compound, however, was lower than that of pyrithiamine.

scholarly journals Loss of Catabolite Repression Function of HPr, the Phosphocarrier Protein of the Bacterial Phosphotransferase System, Affects Expression of the cry4A Toxin Gene in Bacillus thuringiensis subsp. israelensis

2002 ◽  
Vol 184 (19) ◽  
pp. 5410-5417 ◽  
Author(s):  
Sharik R. Khan ◽  
Nirupama Banerjee-Bhatnagar
Keyword(s):  
Bacillus Thuringiensis ◽  
Mutant Strain ◽  
Catabolite Repression ◽  
Parent Strain ◽  
Inhibitory Effect ◽  
Toxin Gene ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Gene Transcripts ◽  
Bacterial Phosphotransferase System

ABSTRACT HPr, the phosphocarrier protein of the bacterial phosphotransferase system, mediates catabolite repression of a number of operons in gram-positive bacteria. In order to participate in the regulatory process, HPr is activated by phosphorylation of a conserved serine-46 residue. To study the potential role of HPr in the regulation of Cry4A protoxin synthesis in Bacillus thuringiensis subsp. israelensis, we produced a catabolite repression-negative mutant by replacing the wild-type copy of the ptsH gene with a mutated copy in which the conserved serine residue of HPr was replaced with an alanine. HPr isolated from the mutant strain was not phosphorylated at Ser-45 by HPr kinase, but phosphorylation at His-14 was found to occur normally. The enzyme I and HPr kinase activities of the mutant were not affected. Analysis of the B. thuringiensis subsp. israelensis mutant harboring ptsH-S45A in the chromosome showed that cry4A expression was derepressed from the inhibitory effect of glucose. The mutant strain produced both cry4A and σ35 gene transcripts 4 h ahead of the parent strain, but there was no effect on σ28 synthesis. In wild-type B. thuringiensis subsp. israelensis cells, cry4A mRNA was observed from 12 h onwards, while in the mutant it appeared at 8 h and was produced for a longer period. The total amount of cry4A transcripts produced by the mutant was higher than by the parent strain. There was a 60 to 70% reduction in the sporulation efficiency of the mutant B. thuringiensis subsp. israelensis strain compared to the wild-type strain.

scholarly journals Oxalate-Degrading Activity in Bifidobacterium animalis subsp. lactis: Impact of Acidic Conditions on the Transcriptional Levels of the Oxalyl Coenzyme A (CoA) Decarboxylase and Formyl-CoA Transferase Genes

2010 ◽  
Vol 76 (16) ◽  
pp. 5609-5620 ◽  
Author(s):  
Silvia Turroni ◽  
Claudia Bendazzoli ◽  
Samuele C. F. Dipalo ◽  
Marco Candela ◽  
Beatrice Vitali ◽  
...  
Keyword(s):  
Coenzyme A ◽  
Genomic Library ◽  
Open Reading Frames ◽  
Transcriptional Analysis ◽  
Bifidobacterium Animalis ◽  
Reverse Transcription Pcr ◽  
Coa Transferase ◽  
Degrading Bacteria ◽  
Key Enzyme ◽  
Acidic Conditions

ABSTRACT Oxalic acid occurs extensively in nature and plays diverse roles, especially in pathological processes. Due to its highly oxidizing effects, hyperabsorption or abnormal synthesis of oxalate can cause serious acute disorders in mammals and can be lethal in extreme cases. Intestinal oxalate-degrading bacteria could therefore be pivotal in maintaining oxalate homeostasis and reducing the risk of kidney stone development. In this study, the oxalate-degrading activities of 14 bifidobacterial strains were measured by a capillary electrophoresis technique. The oxc gene, encoding oxalyl-coenzyme A (CoA) decarboxylase, a key enzyme in oxalate catabolism, was isolated by probing a genomic library of Bifidobacterium animalis subsp. lactis BI07, which was one of the most active strains in the preliminary screening. The genetic and transcriptional organization of oxc flanking regions was determined, unraveling the presence of two other independently transcribed open reading frames, potentially responsible for the ability of B. animalis subsp. lactis to degrade oxalate. pH-controlled batch fermentations revealed that acidic conditions were a prerequisite for a significant oxalate degradation rate, which dramatically increased in cells first adapted to subinhibitory concentrations of oxalate and then exposed to pH 4.5. Oxalate-preadapted cells also showed a strong induction of the genes potentially involved in oxalate catabolism, as demonstrated by a transcriptional analysis using quantitative real-time reverse transcription-PCR. These findings provide new insights into the characterization of oxalate-degrading probiotic bacteria and may support the use of B. animalis subsp. lactis as a promising adjunct for the prophylaxis and management of oxalate-related kidney disease.

scholarly journals Degradation of 2,4,6-Trinitrotoluene (TNT): Involvement of Protocatechuate 3,4-Dioxygenase (P34O) in Buttiauxella sp. S19-1

Toxics ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 231
Author(s):  
Miao Xu ◽  
Dong Liu ◽  
Ping Sun ◽  
Yunuo Li ◽  
Ming Wu ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Degradation Pathway ◽  
Degradation Efficiency ◽  
Bacterial Degradation ◽  
Contaminated Sites ◽  
Gas Chromatography Mass Spectrometry ◽  
Severe Damage ◽  
Gene Encoding ◽  
Key Enzyme ◽  
Intermediate Metabolite

Extensive use and disposal of 2,4,6-trinitrotoluene (TNT), a primary constituent of explosives, pollutes the environment and causes severe damage to human health. Complete mineralization of TNT via bacterial degradation has recently gained research interest as an effective method for the restoration of contaminated sites. Here, screening for TNT degradation by six selected bacteria revealed that Buttiauxella sp. S19-1, possesses the strongest degrading ability. Moreover, BuP34O (a gene encoding for protocatechuate 3,4-dioxygenase—P34O, a key enzyme in the β-ketoadipate pathway) was upregulated during TNT degradation. A knockout of BuP34O in S19-1 to generate S-M1 mutant strain caused a marked reduction in TNT degradation efficiency compared to S19-1. Additionally, the EM1 mutant strain (Escherichia coli DH5α transfected with BuP34O) showed higher degradation efficiency than DH5α. Gas chromatography mass spectrometry (GC-MS) analysis of TNT degradation by S19-1 revealed 4-amino-2,6-dinitrotolune (ADNT) as the intermediate metabolite of TNT. Furthermore, the recombinant protein P34O (rP34O) expressed the activity of 2.46 µmol/min·mg. Our findings present the first report on the involvement of P34O in bacterial degradation of TNT and its metabolites, suggesting that P34O could catalyze downstream reactions in the TNT degradation pathway. In addition, the TNT-degrading ability of S19-1, a Gram-negative marine-derived bacterium, presents enormous potential for restoration of TNT-contaminated seas.

scholarly journals Efficacy of trimethoprim in murine experimental infection with a thymidine kinase-deficient mutant of Escherichia coli.

1997 ◽  
Vol 41 (5) ◽  
pp. 1042-1045 ◽  
Author(s):  
T Tokunaga ◽  
K Oka ◽  
A Takemoto ◽  
Y Ohtsubo ◽  
N Gotoh ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Activity ◽  
Thymidine Kinase ◽  
Mutant Strain ◽  
Experimental Infection ◽  
Therapeutic Efficacy ◽  
Parent Strain ◽  
E Coli

The antimicrobial activity of trimethoprim is antagonized by thymidine in in vitro susceptibility tests. The purpose of this investigation was to determine whether this antagonism also occurred during experimental infection in mice, which have high serum thymidine concentrations. We derived a mutant strain of Escherichia coli, TT-48, incapable of utilizing exogenous thymidine from parent strain E. coli KC-14 and then investigated the in vitro and in vivo antimicrobial activities of trimethoprim, sulfamethoxazole, cefdinir, and ofloxacin against these strains. E. coli TT-48 lacked the activity of thymidine kinase, which catalyzes the conversion of thymidine to thymidylate, but its growth curve remained close to that of the parent strain. The MICs of all of the antimicrobial agents tested, except cefdinir, for the mutant strain were slightly inferior to those for the parent strain. The bactericidal effect of trimethoprim against the parent strain was antagonized by thymidine at concentrations of more than 1 microg/ml, while that against the mutant strain was not affected by thymidine even at the highest concentration (10 microg/ml). The therapeutic efficacy of trimethoprim in experimental murine infections was significantly higher when the mutant rather than the parent strain was used, whereas the therapeutic efficacy of cefdinir or ofloxacin, whose antimicrobial action is independent of folic acid synthesis, was the same with both strains. Unexpectedly, sulfamethoxazole also had similar efficacy against both strains. Thus, high thymidine concentrations antagonized the antimicrobial activity of trimethoprim in vitro and in vivo.

scholarly journals Pathogenicity and Immunogenicity of a Mutant Strain ofListeria monocytogenesin the Chicken Infection Model

2011 ◽  
Vol 18 (3) ◽  
pp. 500-505 ◽  
Author(s):  
Yuelan Yin ◽  
Debin Tian ◽  
Hongmei Jiao ◽  
Chenju Zhang ◽  
Zhiming Pan ◽  
...  
Keyword(s):  
Immune Response ◽  
Mutant Strain ◽  
Parent Strain ◽  
Lethal Dose ◽  
Oral Route ◽  
Infection Model ◽  
Listeriolysin O ◽  
Wild Type ◽  
Strong Immune Response ◽  
Booster Immunization

ABSTRACTListeria monocytogeneshas been exploited as a vaccine carrier based upon its ability to induce a strong cell-mediated immune response. At present, the safety of live, attenuatedL. monocytogenesvaccines in patients is being studied in clinical trials.L. monocytogenesis also an attractive vaccine vector for use in poultry; however, the pathogenicity and immunogenicity of this organism in poultry remain to be fully elucidated. In this study, we investigated the pathogenicity and immunogenicity of anactA- andplcB-deficientL. monocytogenesstrain, yzuLM4ΔactA/plcB, and its wild-type parent strain, yzuLM4, in an avian infection model. The results showed that the wild-type strain could infect ISA brown chickens, causing serious tissue disruptions, including various degrees of degeneration, necrotic lesions, and inflammatory cell infiltration in the liver, spleen, heart, and kidney. However, the mutant strain showed reduced virulence in embryonated eggs compared with that of the parent strain (the 50% lethal dose [LD50] was 3 logs higher). The mutant strain also showed low virulence in chickens and was rapidly eliminated by the host. There were no obvious pathological changes in tissue sections, but the mutant strain still retained the ability to stimulate high levels of antibody against the protein listeriolysin O (LLO). Booster immunization with the mutant strain led to rapid bacterial clearance from the livers and spleens of chickens challenged by the intramuscular route or the oral route. Collectively, our data suggest that the wild-type serotype 1/2aL. monocytogenesstrain can cause serious disease in chickens but the mutant strain with a deletion of theactAandplcBgenes is less virulent but induces a strong immune response. This mutant strain ofL. monocytogenesis therefore a promising candidate as a safe and effective vector for the delivery of heterologous antigens to prevent zoonosis and infectious disease in poultry.

scholarly journals Biological Role of Xanthomonadin Pigments inXanthomonas campestris pv. Campestris

2000 ◽  
Vol 66 (12) ◽  
pp. 5123-5127 ◽  
Author(s):  
A. R. Poplawsky ◽  
S. C. Urban ◽  
W. Chun
Keyword(s):  
Mutant Strain ◽  
Parent Strain ◽  
Xanthomonas Campestris ◽  
Uv Light ◽  
High Light Intensity ◽  
Biological Role ◽  
Insertion Mutant ◽  
Mutant Strains ◽  
Epiphytic Survival

ABSTRACT Previous studies have indicated that the yellow pigments (xanthomonadins) produced by phytopathogenic Xanthomonasbacteria are unimportant during pathogenesis but may be important for protection against photobiological damage. We used a Xanthomonas campestris pv. campestris parent strain, single-site transposon insertion mutant strains, and chromosomally restored mutant strains to define the biological role of xanthomonadins. Although xanthomonadin mutant strains were comparable to the parent strain for survival when exposed to UV light; after their exposure to the photosensitizer toluidine blue and visible light, survival was greatly reduced. Chromosomally restored mutant strains were completely restored for survival in these conditions. Likewise, epiphytic survival of a xanthomonadin mutant strain was greatly reduced in conditions of high light intensity, whereas a chromosomally restored mutant strain was comparable to the parent strain for epiphytic survival. These results are discussed with respect to previous results, and a model for epiphytic survival of X. campestris pv. campestris is presented.

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