katG Promoter Research Articles

Establishment and evaluation of an overlap extension polymerase chain reaction technique for rapid and efficient detection of drug-resistance in Mycobacterium tuberculosis

BackgroundRapid and accurate detection of drug resistance in Mycobacterium tuberculosis is critical for effective control of tuberculosis (TB). Herein, we established a novel, low cost strategy having high accuracy and speed for the detection of M. tuberculosis drug resistance, using gene splicing by overlap extension PCR (SOE PCR).MethodsThe SOE PCR assay and Sanger sequencing are designed and constructed to detect mutations of rpoB, embB, katG, and inhA promoter, which have been considered as the major contributors to rifampicin (RFP), isoniazid (INH), and ethambutol (EMB) resistance in M. tuberculosis. One hundred and eight M. tuberculosis isolates came from mycobacterial cultures of TB cases at Chongqing Public Health Medical Center in China from December 2018 to April 2019, of which 56 isolates were tested with the GeneXpert MTB/RIF assay. Performance evaluation of the SOE PCR technique was compared with traditional mycobacterial culture and drug susceptibility testing (DST) or GeneXpert MTB/RIF among these isolates. Kappa identity test was used to analyze the consistency of the different diagnostic methods.ResultsWe found that the mutations of S531L, S315T and M306V were most prevalent for RFP, INH and EMB resistance, respectively, in the 108 M. tuberculosis isolates. Compared with phenotypic DST, the sensitivity and specificity of the SOE PCR assay for resistance detection were 100.00% and 88.00% for RFP, 94.64% and 94.23% for INH, and 68.97% and 79.75% for EMB, respectively. Compared with the GeneXpert MTB/RIF, the SOE PCR method was completely consistent with results of the GeneXpert MTB/RIF, with a concordance of 100% for resistance to RFP.ConclusionsIn present study, a novel SOE PCR diagnostic method was successfully developed for the accurate detection of M. tuberculosis drug resistance. Our results using this method have a high consistency with that of traditional phenotypic DST or GeneXpert MTB/RIF, and SOE PCR testing in clinical isolates can also be conducted rapidly and simultaneously for detection of drug resistance to RFP, EMB, and INH.Graphical

Characterization of rifampicin-resistant Mycobacterium tuberculosis in Khyber Pakhtunkhwa, Pakistan

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is endemic in Pakistan. Resistance to both firstline rifampicin and isoniazid drugs (multidrug-resistant TB; MDR-TB) is hampering disease control. Rifampicin resistance is attributed to rpoB gene mutations, but rpoA and rpoC loci may also be involved. To characterise underlying rifampicin resistance mutations in the TB endemic province of Khyber Pakhtunkhwa, we sequenced 51 M. tuberculosis isolates collected between 2016 and 2019; predominantly, MDR-TB (n = 44; 86.3%) and lineage 3 (n = 30, 58.8%) strains. We found that known mutations in rpoB (e.g. S405L), katG (e.g. S315T), or inhA promoter loci explain the MDR-TB. There were 24 unique mutations in rpoA, rpoB, and rpoC genes, including four previously unreported. Five instances of within-host resistance diversity were observed, where two were a mixture of MDR-TB strains containing mutations in rpoB, katG, and the inhA promoter region, as well as compensatory mutations in rpoC. Heteroresistance was observed in two isolates with a single lineage. Such complexity may reflect the high transmission nature of the Khyber Pakhtunkhwa setting. Our study reinforces the need to apply sequencing approaches to capture the full-extent of MDR-TB genetic diversity, to understand transmission, and to inform TB control activities in the highly endemic setting of Pakistan.

Effect of Deuterium on the Expression of Inducible Genes in Escherichia coli

It has been shown for the first time that preliminary incubation of bacteria in a medium containing deuterium oxide (D2O) at concentrations of 2.5 to 10% leads to an increase in recA expression induced by hydrogen peroxide at concentrations of 2.2–8.8 mmol/L. The induction of recA in deuterated and non-deuterated (control) cultures was compared using a biosensor based on the E. coli strain K12 MG1655 (pRecA-lux), where luminescence occurs as a result of recA promoter activation in response to DNA damage caused by H2O2. The most effective D2O concentrations were 5.0 and 7.5%. To explain the phenomenon, expression of the catalase gene was studied in deuterated and non-deuterated cultures of E. coli K12 MG1655 (pKatG-lux) biosensor. The luminescence of this biosensor results from activation of the katG promoter in response to an increase in the concentration of H2O2 in the cell. It was found that D2O downregulated katG expression, which can lead to H2O2 accumulation, and, as a consequence, to an increase in the level of DNA damage as seen by an increase in recA expression.

Role of the hepcidin-ferroportin axis in controlling the iron content of the cytosol and Salmonella-containing vacuoles in infected macrophages

Abstract Iron plays a dual-role in bacterial infection: First, it is a critical micronutrient required for the proliferation of infecting bacteria and second, it acts as a cofactor in the generation of bactericidal free radicals. Macrophages provide a major source of serum iron by releasing cytoplasmic iron via the membrane bound iron export protein, Ferroportin (FPN), degradation of which is triggered by hepcidin produced by hepatocytes upon bacterial infection. Salmonella typhimurium is an intracellular pathogen capable of invading macrophages and proliferating in the membranous Salmonella-Containing Vacuole (SCV). In this study, we first demonstrate that FPN is localized on the SCV and plays a role in iron transport into the SCV. To measure iron content in the SCV, a biosensor was constructed by fusing the iron responsive iroB promoter of Salmonella to a mutant GFP with a short half-life (gfpOVA). Using this construct, we estimated the iron levels in macrophages in animals as well as in in vitro cultured macrophages in the presence and absence of hepcidin. In contrast to the generally accepted opinion, the iron level in the SCV in the presence of the iron transporter (FPN) was higher than in its absence (+hepcidin). In general, host defense against pathogens relies on the generation of Reactive Oxygen Species (ROS) in phagocytic cells, especially during the early stage of infection. Thus, we examined for the generation of bactericidal ROS in the SCV using another biosensor composed of the ROS-responsive katG promoter of Salmonella fused to gfpOVA. To our surprise, ROS generation in the SCV was higher in the presence of FPN than in its absence. The relatively high level of iron in the SCV increased the generation of bactericidal ROS, which in turn decreased the number of intra-macrophage Salmonella and extended infected animal survival. Thus, this study reveals the mechanism via which a block in the hepcidin-FPN circuit controls intra-macrophage Salmonella infection.

Early detection of multidrug- and pre-extensively drug-resistant tuberculosis from smear-positive sputum by direct sequencing

BackgroundEmergence of multidrug- and extensively drug-resistant tuberculosis (M/XDR-TB) is a major hurdle for TB control programs especially in developing countries like China. Resistance to fluoroquinolones is high among MDR-TB patients. Early diagnosis of MDR/pre-XDR-TB is essential for lowering transmission of drug-resistant TB and adjusting the treatment regimen.MethodsSmear-positive sputum specimens (n = 186) were collected from Wuhan Institute for Tuberculosis Control. The DNA was extracted from the specimens and run through a Sanger sequencing assay to detect mutations associated with MDR/pre-XDR-TB including the rpoB core region for rifampicin (RIF) resistance; katG and inhA promoter for isoniazid (INH) resistance; and gyrA for fluoroquinolone (FQ) resistance. Sequencing data were compared to phenotypic Lowenstein-Jensen (L-J) proportion method drug susceptibility testing (DST) results for performance analysis.ResultsBy comparing the mutation data with phenotypic results, the detection rates of MDR-TB and pre-XDR-TB were 84.31% (43/51) and 83.33% (20/24), respectively. The sequencing assay illustrated good sensitivity for the detection of resistance to RIF (96.92%), INH (86.89%), FQ (77.50%). The specificities of the assay were 98.35% for RIF, 99.20% for INH, and 97.26% for FQ.ConclusionsThe sequencing assay is an efficient, accurate method for detection of MDR-TB and pre-XDR-TB from clinical smear-positive sputum specimens, should be considered as a supplemental method for obtaining early DST results before the availability of phenotypic DST results. This could be of benefit to early diagnosis, adjusting the treatment regimen and controlling transmission of drug-resistant TB.

The Mycobacterial LysR-Type Regulator OxyS Responds to Oxidative Stress and Negatively Regulates Expression of the Catalase-Peroxidase Gene

Protection against oxidative stress is one of the primary defense mechanisms contributing to the survival of Mycobacterium tuberculosis in the host. In this study, we provide evidence that OxyS, a LysR-type transcriptional regulator functions as an oxidative stress response regulator in mycobacteria. Overexpression of OxyS lowers expression of the catalase-peroxidase (KatG) gene in M. smegmatis. OxyS binds directly with the katG promoter region and a conserved, GC-rich T-N11-A motif for OxyS binding was successfully characterized in the core binding site. Interestingly, the DNA-binding activity of OxyS was inhibited by H2O2, but not by dithiothreitol. Cys25, which is situated at the DNA-binding domain of OxyS, was found to have a regulatory role for the DNA-binding ability of OxyS in response to oxidative stress. In contrast, the other three cysteine residues in OxyS do not appear to have this function. Furthermore, the mycobacterial strain over-expressing OxyS had a higher sensitivity to H2O2.Thus, OxyS responds to oxidative stress through a unique cysteine residue situated in its DNA-binding domain and negatively regulates expression of the katG gene. These findings uncover a specific regulatory mechanism for mycobacterial adaptation to oxidative stress.

Molecular diversity of katG genes in the soil bacteria Comamonas

Three complete katG genes coding for bifunctional catalase-peroxidases (KatGs) from the beta-proteobacterium Comamonas terrigena and two related strains of Comamonas testosteroni have been cloned and sequenced. Catalase-peroxidases are unique bifunctional enzymes known to be expressed in these soil bacteria in response to environmental and/or oxidative stress. The evolutionary and structural diversity of these enzymes is investigated based on multiple sequence alignment and comprehensive phylogenetic analysis. The reconstructed phylogenetic tree and well-known structure-function relationships were applied to inspect the conservation of essential residues. Observed diversity is discussed with respect to the fact that KatGs are distinctive gene-duplicated peroxidases comprising a N-terminal (enzymatically active) and a C-terminal (heme-less) domain. The unique promoter motifs regulating katG transcription in four strains of Comamonas were detected and compared with E. coli katG promoter. The relationship between the promoter sequences and the corresponding expression levels was analyzed. A significant difference in heat shock-inducible catalatic and peroxidatic activities between E. coli K12 and Comamonas terrigena & testosteroni strains was observed. The peculiar variability in gene-coding sequences appears to be more significant for such activity output among Comamonas strains than differences in their promoter regions. The functional role of observed increased diversity in the C-terminal domain is discussed with respect to potential modification of catalytic features at the N-terminal domain that could be relevant for these soil bacteria to cope with stressors. The online version of this article (doi:10.1007/s00203-009-0541-4) contains supplementary material, which is available to authorized users.

DpsA protects the human pathogen Burkholderia pseudomallei against organic hydroperoxide.

The human pathogen, Burkholderia pseudomalle, is able to survive and multiply in hostile environments such as within macrophages. In an attempt to understand its strategy to cope with oxidative stress, the physiological role and gene regulation of a nonspecific DNA-binding protein (DpsA) was investigated. Expression of dpsA increases in response to oxidative stress through increased transcription from the upstream katG (catalase-peroxidase) promoter, which is OxyR dependent. dpsA is also transcribed from its own promoter, which is activated by osmotic stress in an OxyR-independent manner. DpsA-deficient mutants are hypersensitive to tert-butyl hydroperoxide, while overexpression of DpsA leads to increased resistance to organic oxidants. B. pseudomallei DpsA can also protect Escherichia coli against organic hydroperoxide toxicity. The mechanism of DpsA-mediated resistance to organic hydroperoxides was shown to differ from that of alkyl hydroperoxide reductase.

Enhancement of the multi-channel continuous monitoring system through the use of Xenorhabdus luminescens lux fusions

The enhancement of the multi-channel continuous toxicity monitoring system developed previously was studied. To achieve better and more stable results from the system, the use of thermo- lux fusion strains that express the luxCDABE genes from Xenorhabdus luminescens was evaluated. A total of six recombinant Escherichia coli strains with the promoters from three oxidative-stress responsive genes, i.e. the katG, sodA and pqi-5 genes, fused to either the lux genes from Vibrio fischeri or X. luminescens were characterized and their responses to different chemicals compared. It was found that the basal level bioluminescence (BL) from the thermo- lux fusion strains was always higher while that of the V. fischeri lux strains were always near or below the lower limit of detection of the system. For example, the katG::V. fischeri lux strain, DPD2511, gave no discernible response due to its low level expression while a fusion of the katG promoter with the X. luminescens lux operon was clearly responsive and capable of detecting hydrogen peroxide down to about 1 ppm. The use of the thermo- lux strains found them to be as sensitive as the V. fischeri lux strains while providing a brighter, more stable basal level bioluminescence, making the analysis and monitoring of water-borne toxicity more reliable.

An Escherichia coli biosensor capable of detecting both genotoxic and oxidative damage.

A two-plasmid dual reporter Escherichia coli biosensor was developed using the genes for bacterial bioluminescence and a mutant of the green fluorescent protein, GFPuv4. To achieve this, the two plasmids, which were derivatives of pBR322 and pACYC184, had compatible origins of replication and different antibiotic selection markers: ampicillin and tetracycline. The parent strains DK1 and ACRG43, each carrying a single plasmid with one of the fusion genes (strain DK1 harboring a fusion of the katG promoter to the lux operon while in ACRG43, the recA promoter was fused with the GFP gene), were responsive to oxidative and DNA damage, respectively, resulting in higher bioluminescence or fluorescence under the relevant toxic conditions. The responses of the dual sensor strain, DUAL22, to various toxicants, e.g., mitomycin C, N-methyl-N-nitro--nitrosoguanidine, hydrogen peroxide and cadmium chloride, were characterized and compared with the responses of the parent strains to the same chemicals. Finally, several chemical mixtures that cause various stress responses were tested to demonstrate the ability of this biosensor to detect specific stress responses within a multiple toxicity environment.

Regulation of the katG-dpsA operon and the importance of KatG in survival of Burkholderia pseudomallei exposed to oxidative stress

Homologues of the catalase-peroxidase gene katG and the gene for the non-specific DNA binding protein dpsA were identified downstream of oxyR in Burkholderia pseudomallei. Northern experiments revealed that both katG and dpsA are co-transcribed during oxidative stress. Under conditions where the katG promoter is not highly induced, dpsA is transcribed from a second promoter located within the katG-dpsA intergenic region. A katG insertion mutant was found to be hypersensitive to various oxidants. Analysis of katG expression in the oxyR mutant indicates that OxyR is a dual function regulator that represses the expression of katG during normal growth and activates katG during exposure to oxidative stress. Both reduced and oxidized OxyR were shown to bind to the katG promoter.

The Mycobacterium tuberculosis mysB gene product is a functional equivalent of the Escherichia coli sigma factor, KatF.

Mycobacterium tuberculosis, the causative agent of tuberculosis, may remain dormant within its host for many years. The nature of this dormant or latent state is not known, but it may be a specialized form of the stationary growth phase. In Escherichia coli, KatF (or RpoS) is the major stationary phase sigma factor regulating an array of genes expressed in this phase of growth. A potential M. tuberculosis katF homologue was cloned using a fragment of the E. coli katF gene as a probe. DNA sequence analysis of a resultant clone showed 100% identity to a fragment of DNA encoding the M. tuberculosis mysA and mysB genes. Overexpression of mysB in M. bovis BCG resulted in an increase in katG mRNA and catalase and peroxidase activity, and an increase in sensitivity of the cells to isoniazid. An increase in katG promoter activity from a reporter vector was demonstrated when mysB was overexpressed from the same plasmid, indicating a direct relationship between MysB and katG expression.

The Mycobacterium tuberculosis katG promoter region contains a novel upstream activator.

An Escherichia coli-mycobacterial shuttle vector, pJCluc, containing a luciferase reporter gene, was constructed and used to analyse the Mycobacterium tuberculosis katG promoter. A 1.9 kb region immediately upstream of katG promoted expression of the luciferase gene in E. coli and Mycobacterium smegmatis. A smaller promoter fragment (559 bp) promoted expression with equal efficiency, and was used in all further studies. Two transcription start sites were mapped by primer extension analysis to 47 and 56 bp upstream of the GTG initiation codon. Putative promoters associated with these show similarity to previously identified mycobacterial promoters. Deletions in the promoter fragment, introduced with BAL-31 nuclease and restriction endonucleases, revealed that a region between 559 and 448 bp upstream of the translation initiation codon, designated the upstream activator region (UAR), is essential for promoter activity in E. coli, and is required for optimal activity in M. smegmatis. The katG UAR was also able to increase expression from the Mycobacterium paratuberculosis P(AN) promoter 15-fold in E. coli and 12-fold in M. smegmatis. An alternative promoter is active in deletion constructs in which either the UAR or the katG promoters identified here are absent. Expression from the katG promoter peaks during late exponential phase, and declines during stationary phase. The promoter is induced by ascorbic acid, and is repressed by oxygen limitation and growth at elevated temperatures. The promoter constructs exhibited similar activities in Mycobacterium bovis BCG as they did in M. smegmatis.

Oxidative stress detection with Escherichia coli harboring a katG'::lux fusion.

A plasmid containing a transcriptional fusion of the Escherichia coli katG promoter to a truncated Vibrio fischeri lux operon (luxCDABE) was constructed. An E. coli strain bearing this plasmid (strain DPD2511) exhibited low basal levels of luminescence, which increased up to 1,000-fold in the presence of hydrogen peroxide, organic peroxides, redox-cycling agents (methyl viologen and menadione), a hydrogen peroxide-producing enzyme system (xanthine and xanthine oxidase), and cigarette smoke. An oxyR deletion abolished hydrogen peroxide-dependent induction, confirming that oxyR controlled katG'::lux luminescence. Light emission was also induced by ethanol by an unexplained mechanism. A marked synergistic response was observed when cells were exposed to both ethanol and hydrogen peroxide; the level of luminescence measured in the presence of both inducers was much higher than the sum of the level of luminescence observed with ethanol and the level of luminescence observed with hydrogen peroxide. It is suggested that this construction or similar constructions may be used as a tool for assaying oxidant and antioxidant properties of chemicals, as a biosensor for environmental monitoring and as a tool for studying cellular responses to oxidative hazards.

Mapping of the OxyR protein contact site in the C-terminal region of RNA polymerase alpha subunit.

The Escherichia coli OxyR protein requires the C-terminal contact site I region of the RNA polymerase alpha subunit for cooperative interaction with and transcription activation at OxyR-dependent promoters, suggesting direct protein-protein contact between OxyR and the C-terminal region of the alpha subunit. To determine the precise location of the OxyR protein contact site(s) in this region, we carried out mutational analysis of the 3' half of E. coli rpoA, the gene encoding the alpha subunit of RNA polymerase. We isolated a number of rpoA mutants defective in oxyR-dependent transcription activation at the E. coli katG promoter. Nucleotide sequence analysis of the rpoA gene from these mutants revealed that the mutations showing clear phenotypes are all clustered at two narrow regions (amino acid residues 265 to 269 and 293 to 300) within the C terminus of the alpha subunit. Reconstituted RNA polymerases containing the mutant alpha subunits were unable to respond to transcription activation in vitro at the katG, ahpC, and oxyX promoters by OxyR. These results suggest that these two regions comprise the contact surfaces on the alpha subunit for OxyR.

Involvement of the RNA polymerase α subunit C‐terminal region in co‐operative interaction and transcriptional activation with OxyR protein

The role of the alpha subunit of Escherichia coli RNA polymerase in transcription activation by the OxyR protein was investigated using in-vitro-reconstituted RNA polymerase containing alpha subunits carrying C-terminal truncations or an amino acid substitution. Mutant RNA polymerases failed to respond to transcription activation of the E. coli OxyR-dependent promoters. DNase I footprinting analysis indicates that the OxyR protein exerts a co-operative effect on the binding of wild-type RNA polymerase, but not the mutant RNA polymerases, to the katG promoter. Together, these results suggest that direct protein-protein contact between the OxyR protein and the C-terminal contact site I region of the RNA polymerase alpha subunit plays an essential role in transcription activation at the OxyR-dependent promoters.

Identification and molecular analysis of oxyR-regulated promoters important for the bacterial adaptation to oxidative stress

The oxyR-encoded regulatory protein, OxyR, acts to induce the synthesis of a family of hydrogen peroxide-inducible proteins in Salmonella typhimurium and Escherichia coli. To further define the mechanism by which oxyR regulates the production of these proteins, we identified, mapped, and characterized oxyR-regulated promoters upstream from the S. typhimurium ahp genes (encoding an alkyl hydroperoxide reductase) and the E. coli katG gene (encoding catalase). A set of ahpC promoter deletions was constructed in vitro and analysis of these deletions revealed the location of sequences that are involved in oxyR-mediated induction of the ahpC gene product. DNase I protection studies of the ahpC promoter region revealed an oxyR-dependent footprint that overlapped the sequences found to be important for oxyR control. E. coli strains containing transcriptional fusions between the katG promoter and the lacZ gene showed strongly increased synthesis of β-galactosidase in response to hydrogen peroxide treatment. This stimulation was found to be oxyR-dependent. DNase I protection studies of the katG promoter region revealed an oxyR-dependent footprint in the same location relative to the basal promoter elements as was observed with the ahpC promoter. Although both the ahpC and katG promoters were shown to bind the same factor, no strong sequence similarities were found between the two, or between the two and a third oxyR-dependent binding site upstream from the E. coli oxyR gene itself.