Transcription Of katA Research Articles

ArcR contributes to tolerance to fluoroquinolone antibiotics by regulating katA in Staphylococcus aureus.

Staphylococcus aureus is an opportunistic pathogen that shows a unique ability to quickly respond to a variety of antibiotics. The Crp/Fnr family transcriptional regulator ArcR controls expression of arginine deiminase pathway genes arcABDC, which enable the utilization of arginine as an energy source for cell growth under anaerobic conditions. However, ArcR shares low overall similarity with other Crp/Fnr family proteins, suggesting that they differ in the response to environmental stress. In this study, MIC and survival assays were performed to determine the role of ArcR in antibiotic resistance and tolerance. The results showed that deletion of arcR reduced tolerance of S.aureus to fluoroquinolone antibiotics, mainly through a defect in the response to oxidative stress. In ΔarcR mutant, the expression of the major catalase gene katA was downregulated, and katA overexpression restored bacterial resistance to oxidative stress and antibiotics. We showed that ArcR directly regulated katA transcription by binding to the promoter region of katA. Therefore, our results revealed the contribution of ArcR in bacterial tolerance to oxidative stress and subsequently to fluoroquinolones antibiotics. This study added our understanding on the role of Crp/Fnr family in bacterial susceptibility to antibiotics.

Lactobacilli postbiotics reduce biofilm formation and alter transcription of virulence genes of Aggregatibacter actinomycetemcomitans.

Periodontitis is characterized by a dysbiotic microbial community and treatment strategies include the reestablishment of symbiosis by reducing pathogens abundance. Aggregatibacter actinomycetemcomitans (Aa) is frequently associated with rapidly progressing periodontitis. Since the oral ecosystem may be affected by metabolic end-products of bacteria, we evaluated the effect of soluble compounds released by probiotic lactobacilli, known as postbiotics, on Aa biofilm and expression of virulence-associated genes. Cell-free pH-neutralized supernatants (CFS) of Lactobacillus rhamnosus Lr32, L. rhamnosus HN001, Lactobacillus acidophilus LA5, and L. acidophilus NCFM were tested against a fimbriated clinical isolate of Aa JP2 genotype (1×107 CFU/well) on biofilm formation for 24hr, and early and mature preformed biofilms (2 and 24hr). Lactobacilli CFS partially reduced Aa viable counts and biofilms biomass, but did not affect the number of viable non-adherent bacteria, except for LA5 CFS. Furthermore, LA5 CFS and, in a lesser extent HN001 CFS, influenced Aa preformed biofilms. Lactobacilli postbiotics altered expression profile of Aa in a strain-specific fashion. Transcription of cytolethal distending toxin (cdtB) and leukotoxin (ltxA) was downregulated by CFS of LA5 and LR32 CFS. Although all probiotics produced detectable peroxide, transcription of katA was downregulated by lactobacilli CFS. Transcription of dspB was abrogated by LR32 and NCFM CFS, but increased by HN001, whereas expression of pgA was not affected by any postbiotic. Our data indicated the potential of postbiotics from lactobacilli, especially LA5, to reduce colonization levels of Aa and to modulate the expression of virulence factors implicated in evasion of host defenses.

Inactivation of the Agrobacterium tumefaciens ActSR system affects resistance to multiple stresses with increased H2O2 sensitivity due to reduced expression of hemH.

The Agrobacterium tumefaciens ActSR two-component regulatory system is a member of a homologous group of global redox-responsive regulatory systems that adjust the expression of energy-consuming and energy-supplying metabolic pathways in order to maintain cellular redox balance. In this study, the transcriptional organization of the hrpB-actSR locus was determined and the effect of actSR system inactivation on stress resistance was investigated. It was found that hrpB is transcribed as a monocistronic mRNA and actS is transcribed along with actR as a bicistronic mRNA, while actR is also transcribed as a monocistronic message. Each message is initiated from a separate promoter. Inactivation of actR resulted in decreased resistance to membrane stress (sodium dodecyl sulfate), acid stress (pH 5.5), iron starvation (bipyridyl) and iron excess (FeCl3), and antibiotic stress (tetracycline and ciprofloxacin). Resistance to oxidative stress in the form of organic peroxide (cumene hydroperoxide) increased, while resistance to inorganic peroxide (H2O2) decreased. An actR insertion mutant displayed reduced catalase activity, even though transcription of katA and catE remained unchanged. Complementation of the actR inactivation mutant with plasmid-encoded actR or overexpression of hemH, encoding ferrochelatase, restored wild-type catalase activity and H2O2 resistance levels. Gel mobility shift and hemH promoter-lacZ fusion results indicated that ActR is a positive regulator of hemH that binds directly to the hemH promoter region. Thus, inactivation of the A. tumefaciens ActSR system affects resistance to multiple stresses, including reduced resistance to H2O2 resulting from a reduction in catalase activity due to reduced expression of hemH.

The osnR gene of Corynebacterium glutamicum plays a negative regulatory role in oxidative stress responses.

Among the Corynebacterium glutamicum ORFs that have been implicated in stress responses, we chose ORF cg3230, designated osnR, and analyzed it further. Unlike the osnR-deleted strain (ΔosnR), the osnR-overexpressing strain (P180-osnR) developed growth defects and increased sensitivity to various oxidants including H2O2. Transcription in the P180-osnR strain of genes such as sodA (superoxide dismutase), ftn (ferritin biosynthesis), and ahpD (alkyl hydroperoxide reductase; cg2674), which are involved in the detoxification of reactive oxygen species, was only 40% that of the wild type. However, transcription of katA, encoding H2O2-detoxifying catalase, was unchanged in this strain. Genes such as trxB (thioredoxin reductase) and mtr (mycothiol disulfide reductase), which play roles in redox homeostasis, also showed decreased transcription in the strain. 2D-PAGE analysis indicated that genes involved in redox reactions were considerably affected by osnR overexpression. The NADPH/NADP+ ratio of the P180-osnR strain (1.35) was higher than that of the wild-type stain (0.78). Collectively, the phenotypes of the ΔosnR and P180-osnR strains suggest a global regulatory role as well as a negative role for the gene in stress responses, particularly in katA-independent oxidative stress responses.

The ahpD gene of Corynebacterium glutamicum plays an important role in hydrogen peroxide-induced oxidative stress response.

In this study, we analysed the ahpD gene from Corynebacterium glutamicum, which may function in a H2O2-mediated stress responses. Cells overexpressing C. glutamicum ahpD (P180-ahpD) showed increased sensitivity to H2O2 when exposed to the latter in concentrations of 8 mM or greater while showing reduced expression of katA, which encodes catalase. On the other hand, cells that lack ahpD (ΔahpD) displayed increased sensitivity when exposed to low levels of H2O2 while showing katA transcription that was comparable to the level in the wild-type strain. Accordingly, transcription of ahpD and katA was stimulated by low and high concentration of H2O2, respectively. Further, the NAD+/NADH ratio was severely reduced in the ΔahpD (3.03) and P180-ahpD (0.47) strains as compared with that in the wild-type (4.55) strain. Transcriptional analysis indicated that ahpD and upstream genes such as cg2675, cg2676, cg2677 and cg2678, which were annotated as ABC-type transporter, were organized into an operon. Collectively, these findings indicate that C. glutamicum possesses bi-level defence pathways against hydrogen peroxide, involving katA and ahpD. Further, ahpD, along with cg2675-cg2678 genes, may play a novel role in cellular activities against oxidative stress.

Catalase (KatA) Plays a Role in Protection against Anaerobic Nitric Oxide in Pseudomonas aeruginosa

Pseudomonas aeruginosa (PA) is a common bacterial pathogen, responsible for a high incidence of nosocomial and respiratory infections. KatA is the major catalase of PA that detoxifies hydrogen peroxide (H2O2), a reactive oxygen intermediate generated during aerobic respiration. Paradoxically, PA displays elevated KatA activity under anaerobic growth conditions where the substrate of KatA, H2O2, is not produced. The aim of the present study is to elucidate the mechanism underlying this phenomenon and define the role of KatA in PA during anaerobiosis using genetic, biochemical and biophysical approaches. We demonstrated that anaerobic wild-type PAO1 cells yielded higher levels of katA transcription and expression than aerobic cells, whereas a nitrite reductase mutant ΔnirS produced ∼50% the KatA activity of PAO1, suggesting that a basal NO level was required for the increased KatA activity. We also found that transcription of the katA gene was controlled, in part, by the master anaerobic regulator, ANR. A ΔkatA mutant and a mucoid mucA22 ΔkatA bacteria demonstrated increased sensitivity to acidified nitrite (an NO generator) in anaerobic planktonic and biofilm cultures. EPR spectra of anaerobic bacteria showed that levels of dinitrosyl iron complexes (DNIC), indicators of NO stress, were increased significantly in the ΔkatA mutant, and dramatically in a ΔnorCB mutant compared to basal levels of DNIC in PAO1 and ΔnirS mutant. Expression of KatA dramatically reduced the DNIC levels in ΔnorCB mutant. We further revealed direct NO-KatA interactions in vitro using EPR, optical spectroscopy and X-ray crystallography. KatA has a 5-coordinate high spin ferric heme that binds NO without prior reduction of the heme iron (K d ∼6 μM). Collectively, we conclude that KatA is expressed to protect PA against NO generated during anaerobic respiration. We proposed that such protective effects of KatA may involve buffering of free NO when potentially toxic concentrations of NO are approached.

Transcriptional Regulation of the CmeABC Multidrug Efflux Pump and the KatA Catalase by CosR in Campylobacter jejuni

CosR is an essential response regulator in Campylobacter jejuni, a major food-borne pathogen causing enteritis worldwide. A transcriptomic analysis performed in this study discovered 93 genes whose transcriptional levels were changed >2-fold due to the repression of CosR expression by antisense peptide nucleic acid. The identified CosR-regulated genes are involved in various cellular functions, such as energy production, protein synthesis and folding, flagellum biogenesis, and lipid metabolism. Interestingly, 17 of the 93 CosR-regulated genes (18.3%) are predicted essential genes, indicating that CosR may participate in the regulation of vital biological processes in C. jejuni. In particular, CosR knockdown increased the transcriptional levels of cmeA, cmeB, and cmeC genes, whose protein product (CmeABC) is an important determinant conferring multidrug resistance in Campylobacter. Negative regulation of cmeABC by CosR was verified by quantitative real-time PCR (qRT-PCR) and P(cmeABC)::lacZ assay. The results of electrophoretic mobility shift assays (EMSAs) and DNase I footprinting assays demonstrated that CosR directly binds to the cmeABC promoter. Another notable finding is that CosR regulates the transcription of katA, the sole catalase gene in C. jejuni. Further characterization with qRT-PCR, the catalase enzyme assay, EMSA, and DNase I footprinting assays successfully demonstrated that CosR affects the katA transcription and the catalase activity by direct interactions with the katA promoter. The findings in this study clearly demonstrated that CosR regulates resistance mechanisms in C. jejuni by controlling the expression of genes involved in oxidative stress defense and extrusion of toxic compounds out of the cell.

PerR controls peroxide- and iron-responsive expression of oxidative stress defense genes inHelicobacter hepaticus

Chronic intestinal and hepatic colonization with the microaerophilic murine pathogen Helicobacter hepaticus can lead to a range of inflammatory diseases of the lower digestive tract. Colonization is associated with an active cellular immune response and production of oxygen radicals. During colonization, H. hepaticus needs to cope with and respond to oxidative stress, and here we report on the role of the H. hepaticus PerR-regulator (HH0942) in the expression of the peroxidase-encoding katA (HH0043) and ahpC (HH1564) genes. Transcription of katA and ahpC was induced by hydrogen peroxide, and by iron restriction of growth media. This iron- and hydrogen peroxide-responsive regulation of katA and ahpC was mediated at the transcriptional level, from promoters directly upstream of the genes. Inactivation of the perR gene resulted in constitutive, iron-independent high-level expression of the katA and ahpC transcripts and corresponding proteins. Finally, inactivation of the katA gene resulted in increased sensitivity of H. hepaticus to hydrogen peroxide and reduced aerotolerance. In H. hepaticus, iron metabolism and oxidative stress defense are intimately connected via the PerR regulatory protein. This regulatory pattern resembles that observed in the enteric pathogen Campylobacter jejuni, but contrasts with the pattern observed in the closely related human gastric pathogen Helicobacter pylori.

Phosphate limitation induces catalase expression inSinorhizobium meliloti,Pseudomonas aeruginosaandAgrobacterium tumefaciens

Growth of Sinorhizobium meliloti under Pi-limiting conditions induced expression of the major H2O2-inducible catalase (HPII) gene (katA) in this organism. This transcription required the PhoB transcriptional regulator and initiated from a promoter that was distinct from the OxyR-dependent promoter which activates katA transcription in response to addition of H2O2. In N2-fixing root nodules, katA was transcribed from the OxyR- and not the PhoB-dependent promoter. This is consistent with the accumulation of reactive oxygen species (ROS) in nodules and also indicates that bacteroids within nodules are not Pi-limited. Pi-limited growth also induced expression of catalase genes in Agrobacterium tumefaciens (HPI) and Pseudomonas aeruginosa (PA4236-HPI) suggesting that this may be a widespread phenomenon. The response is not a general stress response as in both S. meliloti and P. aeruginosa increased transcription is mediated by the phosphate responsive transcriptional activator PhoB. The phenotypic consequences of this response were demonstrated in S. meliloti by the dramatic increase in H2O2 resistance of wild type but not phoB mutant cells upon growth in Pi-limiting media. Our data indicate that in S. meliloti, katA and other genes whose products are involved in protection from oxidative stress are induced upon Pi-limitation. These observations suggest that as part of the response to Pi-limitation, S. meliloti, P. aeruginosa and A. tumefaciens have evolved a capacity to increase their resistance to oxidative stress. Whether this capacity evolved because Pi-starved cells generate more ROS or whether the physiological changes that occur in the cells in response to Pi-starvation render them more sensitive to ROS remains to be established.

Characterisation of the katA gene encoding a catalase and evidence for at least a second catalase activity in Staphylococcus xylosus, bacteria used in food fermentation

The catalase gene katA of Staphylococcus xylosus was cloned. It encodes a protein of 494 amino acids with a molecular mass of 56.9 kDa, closely related to monofunctional catalases. A katA mutant still showed a relatively high catalase activity demonstrating that S. xylosus possesses more than one enzyme. By Southern blot analysis using a katA probe, a second genetic locus distinct from katA was detected that probably contained the additional catalase gene. To analyse katA expression, a transcriptional fusion of the katA promoter region to a promoterless β-galactosidase gene was integrated into the genome of S. xylosus. katA expression is induced upon entry into stationary phase, by oxygen and hydrogen peroxide. Iron and manganese depletion induced katA transcription. Comparing the resistance of S. xylosus wild-type and the katA mutant strain to hydrogen peroxide clearly showed that KatA is essential for S. xylosus to cope with hydrogen peroxide stress. Therefore, S. xylosus has at least two differentially expressed catalases.

In Staphylococcus aureus, fur is an interactive regulator with PerR, contributes to virulence, and Is necessary for oxidative stress resistance through positive regulation of catalase and iron homeostasis.

The Staphylococcus aureus genome encodes three ferric uptake repressor (Fur) homologues: Fur, PerR, and Zur. To determine the exact role of Fur in S. aureus, we inactivated the fur gene by allelic replacement using a tetracycline resistance cassette, creating strain MJH010 (fur). The mutant had a growth defect in rich medium, and this defect was exacerbated in metal-depleted CL medium. This growth defect was partially suppressed by manganous ion, a metal ion with known antioxidant properties. This suggests that the fur mutation leads to an oxidative stress condition. Indeed, MJH010 (fur) has reduced levels of catalase activity resulting from decreased katA transcription. Using a katA-lacZ fusion we have determined that Fur functions, either directly or indirectly, as an iron-dependent positive regulator of katA expression. Transcription of katA is coregulated by Fur and PerR, since in MJH010 (fur) transcription was still repressed by manganese while transcription in MJH201 (fur perR) was unresponsive to the presence of iron or manganese. Siderophore biosynthesis was repressed by iron in 8325-4 (wild-type) but in MJH010 (fur) was constitutive. A number of putative Fur-regulated genes were identified in the incomplete genome databases using known S. aureus Fur box sequences. Of those tested, the sstABCD and sirABC operons and the fhuD2 and orf4 genes were found to have Fur-regulated expression. MJH010 (fur) was attenuated (P<0.04) in a murine skin abscess model of infection, as was double-mutant MJH201 (fur perR) (P<0.03). This demonstrates the importance in vivo of iron homeostasis and oxidative stress resistance regulation in S. aureus.

An investigation of the molecular basis of the spontaneous occurrence of a catalase-negative phenotype in Helicobacter pylori.

The discovery of a highly active catalase in Helicobacter pylori that in some strains may lose its activity has generated strong scientific interest. We have characterized a spontaneous catalase-negative isolate of H. pylori (UNSW-RU1) and sequenced katA in the parent strain and the promoters of both phenotypes as a prelude to understanding the genetic processes leading to the failure to express catalase. Protein extracts from both phenotypes were examined for catalase on 2D-PAGE and analyzed by Western blot-based immuno-analysis. Presence of catalase mRNA was detected by Northern blot. Hi-Fidelity PCR was used to sequence the katA promoter while katA was sequenced using cycle-sequencing. The transcription start site was located by primer extension. Catalase protein was absent in UNSW-RU1 (KatA-) by 2D-PAGE and Western blot, as was catalase mRNA by Northern blot, indicating that the cause of the KatA- phenotype was at the level of transcription. No mutations were found in the promoter region of the KatA- isolate. The transcription start site was identified 55 bp upstream of the ATG site and putative RNA polymerase binding sites were mapped at "-10" and "-35". A Fur box was identified 181 bp upstream of the transcription start site. The sequences of an 876 bp ORF and a 366 bp Escherichia coli phnA homologue were identified. The UNSW-RU1 (KatA-) phenotype does not express KatA or transcribe katA. The absence of defects in its promoter and a large part of its ORF indicates that loss of activity may be due to a mutation in an accessory gene essential for catalase expression, or to the binding of a repressor preventing katA transcription.

Molecular characterization of catalase from Bordetella pertussis: identification of the katA promoter in an upstream insertion sequence.

In this report we evaluate the role of catalase in the survival of Bordetella pertussis within human polymorphonuclear leukocytes (PMNs). Crude extracts of B. pertussis exhibited a single catalase activity when subjected to non-denaturing polyacrylamide gel electrophoresis and assayed for catalase activity. A plasmid containing B. pertussis katA was identified by complementation of UM255, a catalase-deficient strain of Escherichia coli. The nucleotide sequence of katA predicts a 55 kDa protein that shares homology with a class of haem-containing catalases found in both eubacteria and eukaryotes. Analysis of the nucleotide sequence upstream of katA revealed the presence of a copy of IS481, a B. pertussis-specific insertion sequence. The start site of transcription of katA was mapped to a T residue in IS481 by primer extension analysis performed with B. pertussis RNA and a katA-specific primer. A catalase-deficient strain of B. pertussis, DD900, was constructed by gene replacement. DD900 was more sensitive to killing by 1 and 5 mM H2O2 than the parental strain, BP339. However, there was no difference in the ability of DD900 and BP339 to survive for 2 h in human PMNs. This suggests that catalase plays no significant role in the survival of B. pertussis within PMNs.