Roles of water quality and disinfectant application on inactivation of fish pathogenic Streptococcus agalactiae with povidone iodine, quaternary ammonium compounds and glutaraldehyde.

Streptococcosis is an important bacterial disease in Nile tilapia causing severe economic losses to tilapia aquaculture worldwide. The effects of water quality (low- [LS] and high-level [HS] soiling, to mimic clean or dirty surface conditions and temperatures) and disinfectant application (diluted concentrations and exposure time) were characterized on the inactivation of Streptococcus agalactiae isolated from diseased tilapia. Five isolates were tested against three commercial disinfectant products with the main ingredients being povidone iodine (Anidine 100™; AD), benzalkonium chloride (Better BKC 80%™; BKC 80), and a mixture of quaternary ammonium compounds and glutaraldehyde (Chloraldehyde™; CR). CR demonstrated highest efficacy to S. agalactiae inactivation, followed by BKC 80 and AD, respectively. Higher-level soiling, low temperature, diluted concentrations and short exposure time all decreased the disinfectant efficacy. CR and BKC 80 provided more than 5-log inactivation at 1-min exposure at 20°C under HS conditions, and also with ten-fold-diluted concentrations at 60-min exposure time at 30°C. However, AD required 10-min exposure to effectively remove bacteria under LS conditions at 30°C. The results could facilitate aquaculture management planning that leads to operating cost reductions and improvements in biosecurity.

The capability of non-native strains of Bacteroides bacteria to detect bacteriophages as faecal indicators in a tropical area.

AIMS: To evaluate the use of nonlocal, already-available strains of phages to indicate faecal contamination in Thailand waters. METHODS AND RESULTS: Phages of Bacteroides fragilis strains ATCC 700786 (RYC2056PH) and ATCC 51477 (HSP40PH) were measured in 71 human and animal wastewater samples in Thailand using a double-layer agar assay. Bacteriophage RYC2056PH was detected at concentrations comparable to representative human and animal wastewater samples from European and Mediterranean countries, with 61·7 and 33·3% above the threshold value of 100 PFU 100 ml(-1) in wastewater samples of human and animal origins, respectively. On the other hand, HSP40PH was detected at low concentrations in both human- and animal-polluted wastewaters. Moreover, RYC2056PH was found in 12 canal waters with human-influenced pollution and was not detected in 6 nonpolluted river waters being tested in this study. CONCLUSIONS: The presence of RYC2056PH could indicate nonsource-specific faecal contamination in Thailand. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided the first evidence that bacteriophages of the European-isolated B. fragilis strain RYC2056 could be used as nonsource-specific faecal indicators in the Southeast Asian region. The results of this study support the worldwide use of Bacteroides phages as faecal indicators.

Effects of exposure times on the toxic response of ammonia oxidizing mixed culture (AOMC) to phenol and chlorinated phenols.

This study investigated the effect of exposure times on the response of ammonia oxidizing mixed culture (AOMC) to phenolic compounds while having the future goal to develop a biosensor using AOMC for toxicity monitoring. AOMC was used instead of purified nitrifying culture because of the ease of culture development. The oxygen utilization rate (OUR) was measured during three exposure periods; 0-15, 25-40 and 50-65 min. It was found that phenolic compounds have a strong inhibitory effect on AOMC. The percentage of OUR reduction increased with higher concentrations and the extended exposure times improved the toxic response of AOMC, especially to the lower concentrations (0.25, 0.50, 1.0 mg/L). Further, AOMC detoxifying mechanisms might result in the reduction of toxic response when the longest exposure time was applied. However, at the higher concentrations (2.5 and 5.0 mg/L), the extended exposure times did not have a critical effect on the response pattern of AOMC, especially for phenol and mono-chlorinated phenols. It was illustrated that AOMC is very sensitive to phenolic compounds and its sensitivity is high enough for the detection of phenolic compounds at the level of effluent standard in Thailand and Japan with a rapid response time of 15 min. To improve the sensitivity of AOMC to low phenolic compound concentrations, an extended exposure time of 25-40 min would be recommended.

Characterization of Xanthomonas oryzae pv. oryzae recX, a gene that is required for high-level expression of recA.

Analysis of the nucleotide sequence downstream from the Xanthomonas oryzae pv. oryzae recA gene reveals two orfs designated orfX and recX. The former has the potential to code for a 5.6 kDa protein of unknown function while the latter encodes for a putative 14.6 kDa protein with homology to RecX from various bacteria. Northern blot analysis and RT-PCR results show that recA-orfX-recX are co-regulated and arranged in an operon. A recX mutant was constructed. The mutant has no obvious growth defects or stress response defects, except that it cannot support high-level expression of recA from an expression vector. Introduction of the plasmid containing recA into the recX mutant resulted in reduced transformation efficiency and all transformants tested had mutations with reduced RecA levels. Moreover, the recX mutant has reduced basal levels of RecA. This has not been observed in other bacteria. When inactivated recX was complemented in trans, both changes were reversed. recX mutation has no effect on the regulation of the recA promoter, suggesting that its effect on the RecA level could be post-transcriptional.

Complex regulation of the organic hydroperoxide resistance gene (ohr) from Xanthomonas involves OhrR, a novel organic peroxide-inducible negative regulator, and posttranscriptional modifications.

Analysis of the sequence immediate upstream of ohr revealed an open reading frame, designated ohrR, with the potential to encode a 17-kDa peptide with moderate amino acid sequence homology to the MarR family of negative regulators of gene expression. ohrR was transcribed as bicistronic mRNA with ohr, while ohr mRNA was found to be 95% monocistronic and 5% bicistronic with ohrR. Expression of both genes was induced by tert-butyl hydroperoxide (tBOOH) treatment. High-level expression of ohrR negatively regulated ohr expression. This repression could be overcome by tBOOH treatment. In vivo promoter analysis showed that the ohrR promoter (P1) has organic peroxide-inducible, strong activity, while the ohr promoter (P2) has constitutive, weak activity. Only P1 is autoregulated by OhrR. ohr primer extension results revealed three major primer extension products corresponding to the 5' ends of ohr mRNA, and their levels were strongly induced by tBOOH treatment. Sequence analysis of regions upstream of these sites showed no typical Xanthomonas promoter. Instead, the regions can form a stem-loop secondary structure with the 5' ends of ohr mRNA located in the loop section. The secondary structure resembles the structure recognized and processed by RNase III enzyme. These findings suggest that the P1 promoter is responsible for tBOOH-induced expression of the ohrR-ohr operon. The bicistronic mRNA is then processed by RNase III-like enzymes to give high levels of ohr mRNA, while ohrR mRNA is rapidly degraded.

Unusual adaptive, cross protection responses and growth phase resistance against peroxide killing in a bacterial shrimp pathogen, Vibrio harveyi.

Oxidant induced protection against peroxide killing was investigated in a prawn bacterial pathogen, Vibrio harveyi. Exposure to 250 microM H(2)O(2) induced adaptive protection against subsequent exposure to killing concentrations of H(2)O(2). In addition, 200 microM t-butyl hydroperoxide (tBOOH) induced cross protection to H(2)O(2) killing. On the other hand, peroxide pretreatment did not induce protection against tBOOH killing. Peroxide induced adaptive and cross protection responses required new protein synthesis and were abolished by addition of a protein synthesis inhibitor. Pretreatments of V. harveyi with 250 microM H(2)O(2) and 200 microM tBOOH induced an increase in peroxide scavenging enzymes, catalase and alkyl hydroperoxide reductase subunit C. In addition, stationary phase cells of V. harveyi were more resistant to H(2)O(2) and iodoacetamide killing but highly susceptible to tBOOH killing compared to exponential phase cells. Many aspects of the oxidative stress response of V. harveyi are different from those of other bacteria and these factors may be important for bacterial survival in the environment and during interactions with host shrimp.

OhrR is a repressor of ohrA, a key organic hydroperoxide resistance determinant in Bacillus subtilis.

Bacillus subtilis displays a complex adaptive response to the presence of reactive oxygen species. To date, most proteins that protect against reactive oxygen species are members of the peroxide-inducible PerR and sigma(B) regulons. We investigated the function of two B. subtilis homologs of the Xanthomonas campestris organic hydroperoxide resistance (ohr) gene. Mutational analyses indicate that both ohrA and ohrB contribute to organic peroxide resistance in B. subtilis, with the OhrA protein playing the more important role in growing cells. Expression of ohrA, but not ohrB, is strongly and specifically induced by organic peroxides. Regulation of ohrA requires the convergently transcribed gene, ohrR, which encodes a member of the MarR family of transcriptional repressors. In an ohrR mutant, ohrA expression is constitutive, whereas expression of the neighboring ohrB gene is unaffected. Selection for mutant strains that are derepressed for ohrA transcription identifies a perfect inverted repeat sequence that is required for OhrR-mediated regulation and likely defines an OhrR binding site. Thus, B. subtilis contains at least three regulons (sigma(B), PerR, and OhrR) that contribute to peroxide stress responses.

Xanthomonas oryzae pv. oryzae recA is transcribed and regulated from multiple promoters.

Transcription regulation of Xanthomonas oryzae pv. oryzae recA was characterized. Primer extension experiments showed that recA is transcribed from three promoters designated P1, P2 and P3. The sequences of -10 and -35 regions of these promoters have moderate homology to the proposed consensus sequence for a Xanthomonas promoter. Putative SOS boxes were identified in the vicinity of P1 and P2 promoters. Deletion analysis and in vivo monitoring of promoter activity of these promoters revealed that the three promoters have different characteristics. P1 and P2 show stress-inducible high and low promoter strengths respectively. P3 is a non-inducible moderate promoter strength. These promoters are regulated by two SOS boxes. The multiplicity of promoters and SOS boxes provides back-up systems to ensure proper regulation of recA.

A Xanthomonas alkyl hydroperoxide reductase subunit C (ahpC) mutant showed an altered peroxide stress response and complex regulation of the compensatory response of peroxide detoxification enzymes.

Alkyl hydroperoxide reductase subunit C (AhpC) is the catalytic subunit responsible for alkyl peroxide metabolism. A Xanthomonas ahpC mutant was constructed. The mutant had increased sensitivity to organic peroxide killing, but was unexpectedly hyperresistant to H(2)O(2) killing. Analysis of peroxide detoxification enzymes in this mutant revealed differential alteration in catalase activities in that its bifunctional catalase-peroxidase enzyme and major monofunctional catalase (Kat1) increased severalfold, while levels of its third growth-phase-regulated catalase (KatE) did not change. The increase in catalase activities was a compensatory response to lack of AhpC, and the phenotype was complemented by expression of a functional ahpC gene. Regulation of the catalase compensatory response was complex. The Kat1 compensatory response increase in activity was mediated by OxyR, since it was abolished in an oxyR mutant. In contrast, the compensatory response increase in activity for the bifunctional catalase-peroxidase enzyme was mediated by an unknown regulator, independent of OxyR. Moreover, the mutation in ahpC appeared to convert OxyR from a reduced form to an oxidized form that activated genes in the OxyR regulon in uninduced cells. This complex regulation of the peroxide stress response in Xanthomonas differed from that in other bacteria.

Molecular and physiological analysis of an OxyR-regulated ahpC promoter in Xanthomonas campestris pv. phaseoli.

In Xanthomonas campestris pv. phaseoli, a gene for the alkyl hydroperoxide reductase subunit C (ahpC) had unique patterns of regulation by various forms of OxyR. Reduced OxyR repressed expression of the gene, whereas oxidized OxyR activated its expression. This dual regulation of ahpC is unique and unlike all other OxyR-regulated genes. The ahpC transcription start site was determined. Analysis of the region upstream of the site revealed promoter sequences that had high homology to the Xanthomonas consensus promoter sequence. Data from gel shift experiments indicated that both reduced and oxidized OxyR could bind to the ahpC regulatory region. Moreover, the reduced and the oxidized forms of OxyR gave different DNase I footprint patterns, indicating that they bound to different sites. The oxidized OxyR binding site overlapped the -35 region of the ahpC promoter by a few bases. This position is consistent with the role of the protein in activating transcription of the gene. Binding of reduced OxyR to the ahpC promoter showed an extended DNase I footprint and DNase I hypersensitive sites, suggesting that binding of the protein caused a shift in the binding site and bending of the target DNA. In addition, binding of reduced OxyR completely blocked the -35 region of the ahpC promoter and prevented binding of RNA polymerase, leading to repression of the gene. Monitoring of the ahpC promoter activity in vivo confirmed the location of the oxidized OxyR binding site required for activation of the promoter. A mutant that separated OxyR regulation from basal ahpC promoter activity was constructed. The mutant was unable to respond to oxidants by increasing ahpC expression. Physiologically, it had a slower aerobic growth rate and was more sensitive to organic peroxide killing. This indicated that oxidant induction of ahpC has important physiological roles in normal growth and during oxidative stress.

Characterization and mutagenesis of fur gene from Burkholderia pseudomallei.

A homolog of the ferric uptake regulator gene (fur) was isolated from Burkholderia pseudomallei (Bp) by a reverse genetic technique. Sequencing of a 2.2kb DNA fragment revealed an open reading frame with extensive homology to bacterial Fur proteins. The cloned gene encodes a 16kDa protein that cross-reacts with a polyclonal anti-Escherichia coli Fur serum. The transcription start site was determined by the primer extension technique. Expression analysis of fur showed no increased fur mRNA levels in response to various stresses and iron conditions. A positive selection procedure involving the isolation of manganese-resistant mutants was used to isolate mutants that produce altered Fur proteins. Sequencing of a fur mutant revealed a nucleotide change (G to A) converting a conserved amino acid arginine-69 to histidine. The fur missense mutant produced an elevated level of siderophore that could be complemented by a multicopy plasmid carrying the Bp fur. Interestingly, Fur was found to play roles as a positive regulator of FeSOD and peroxidase. The mutant showed a decreased activity of FeSOD and peroxidase, which could be important in its pathogenicity and survival in macrophages.

Exposure of phytopathogenic Xanthomonas spp. to lethal concentrations of multiple oxidants affects bacterial survival in a complex manner.

During plant-microbe interactions and in the environment, Xanthomonas campestris pv. phaseoli is likely to be exposed to high concentrations of multiple oxidants. Here, we show that simultaneous exposures of the bacteria to multiple oxidants affects cell survival in a complex manner. A superoxide generator (menadione) enhanced the lethal effect of an organic peroxide (tert-butyl hydroperoxide) by 1, 000-fold; conversely, treatment of cells with menadione plus H(2)O(2) resulted in 100-fold protection compared to that for cells treated with the individual oxidants. Treatment of X. campestris with a combination of H(2)O(2) and tert-butyl hydroperoxide elicited no additive or protective effect. High levels of catalase alone are sufficient to protect cells against the lethal effect of menadione plus H(2)O(2) and tert-butyl hydroperoxide plus H(2)O(2). These data suggest that H(2)O(2) is the lethal agent responsible for killing the bacteria as a result of these treatments. However, increased expression of individual genes for peroxide (alkyl hydroperoxide reductase, catalase)- and superoxide (superoxide dismutase)-scavenging enzymes or concerted induction of oxidative stress-protective genes by menadione gave no protection against killing by a combination of menadione plus tert-butyl hydroperoxide. However, X. campestris cells in the stationary phase and a spontaneous H(2)O(2)-resistant mutant (X. campestris pv. phaseoli HR) were more resistant to killing by menadione plus tert-butyl hydroperoxide. These findings give new insight into oxidant killing of Xanthomonas spp. that could be generally applied to other bacteria.

Mutations in oxyR resulting in peroxide resistance in Xanthomonas campestris.

A spontaneous Xanthomonas campestris pv. phaseoli H(2)O(2)-resistant mutant emerged upon selection with 1 mM H(2)O(2). In this report, we show that growth of this mutant under noninducing conditions gave high levels of catalase, alkyl hydroperoxide reductase (AhpC and AhpF), and OxyR. The H(2)O(2) resistance phenotype was abolished in oxyR-minus derivatives of the mutant, suggesting that elevated levels and mutations in oxyR were responsible for the phenotype. Nucleotide sequence analysis of the oxyR mutant showed three nucleotide changes. These changes resulted in one silent mutation and two amino acid changes, one at a highly conserved location (G197 to D197) and the other at a nonconserved location (L301 to R301) in OxyR. Furthermore, these mutations in oxyR affected expression of genes in the oxyR regulon. Expression of an oxyR-regulated gene, ahpC, was used to monitor the redox state of OxyR. In the parental strain, a high level of wild-type OxyR repressed ahpC expression. By contrast, expression of oxyR5 from the X. campestris pv. phaseoli H(2)O(2)-resistant mutant and its derivative oxyR5G197D with a single-amino-acid change on expression vectors activated ahpC expression in the absence of inducer. The other single-amino-acid mutant derivative of oxyR5L301R had effects on ahpC expression similar to those of the wild-type oxyR. However, when the two single mutations were combined, as in oxyR5, these mutations had an additive effect on activation of ahpC expression.

Expression analysis and characterization of the mutant of a growth-phase- and starvation-regulated monofunctional catalase gene from Xanthomonas campestris pv. phaseoli.

Analysis of the Xanthomonas campestris pv. phaseoli (Xp) catalase profile using an activity gel revealed at least two distinct monofunctional catalase isozymes denoted Kat1 and Kat2. Kat1 was expressed throughout growth, whereas Kat2 was expressed only during the stationary phase of growth. The nucleotide sequence of a previously isolated monofunctional catalase gene, Xp katE, was determined. The deduced amino acid sequence of Xp KatE showed a high percentage identity to an atypical group of monofunctional catalases that includes the well-characterized E. coli katE. Expression of Xp katE was growth phase-dependent but was not inducible by oxidants. In addition, growth of Xp in a carbon-starvation medium induced expression of the gene. An Xp katE mutant was constructed, and analysis of its catalase enzyme pattern showed that Xp katE coded for the Kat2 isozyme. Xp katE mutant had resistance levels similar to the parental strain against peroxide and superoxide killing at both exponential and stationary phases of growth. Interestingly, the level of total catalase activity in the mutant was similar to that of the parental strain even in stationary phase. These results suggest the existence of a novel compensatory mechanism for the activity of Xp catalase isozymes.

Construction and characterization of regulated L-arabinose-inducible broad host range expression vectors in Xanthomonas.

Several versions of broad host range (BHR), L-arabinose-inducible expression vectors were constructed. These expression vectors were based on a high copy number BHR pBBR1MCS-4 replicon that could replicate in both enteric and non-enteric Gram-negative bacteria. Two versions of expression cassettes containing multiple cloning sites either with or without a ribosome binding site were placed under transcriptional control of the Escherichia coli BAD promoter and araC gene. Three versions of vectors containing ampicillin or kanamycin or tetracycline resistance genes as selectable markers were constructed. In all six new L-arabinose-inducible BHR expression vectors containing many unique cloning sites, selectable markers were made to facilitate cloning and expression of genes in various Gram-negative bacteria. A Tn9 chloramphenicol acetyl transferase (cat) gene was cloned into an expression vector, resulting in pBBad18Acat that was used to establish optimal expression conditions (addition of 0.02% L-arabinose to mid-exponential phase cells for at least 1 h) in a Xanthomonas campestris pv. phaseoli. Comparison of the Cat enzyme activities between uninduced and a 180-min L-arabinose-induced culture showed a greater than 150-fold increased Cat specific activity. In addition, L-arabinose induction of exponential phase cells harboring pBBad18Acat gave a higher amount of Cat than similarly treated stationary phase cells. The usefulness of the expression vector was also demonstrated in both enteric and non-enteric Gram-negative bacteria.

Characterization of a ferric uptake regulator (fur) gene from Xanthomonas campestris pv. phaseoli with unusual primary structure, genome organization, and expression patterns.

A 1.5kb DNA fragment from Xanthomonas campestris pv. phaseoli containing fur was characterized. fur is a single copy gene that is transcribed as a monocistronic mRNA. The predicted amino acid sequence of Xp Fur showed extensive identity to other Fur proteins. However, Xp Fur has many distinct features, particularly a lack of cysteine residues in the conserved metal-binding motifs and unusual modifications in the carboxy-terminus region. The nucleotide sequences of fur genes from four other Xanthomonas spp. were determined. Deduced amino acid sequences all showed the distinct features of Xp Fur. Functionally, Xp Fur partially repressed a Fur-regulated promoter in E. coli. Expression analysis of fur showed increased fur mRNA levels in response to a low iron growth condition. The fur transcription start site was identified by primer extension.

Construction and physiological analysis of a Xanthomonas oryzae pv. oryzae recA mutant.

A Xoo recA insertion inactivation mutant was constructed. The mutant, lacking RecA, showed increased sensitivity towards mutagen killing. This phenotype could be complemented by a cloned, functional recA. Unlike other bacteria, both the recA mutant and the parental strain had similar level of resistance to H2O2 killing and peroxide-induced mutagenesis.

Construction and physiological analysis of a Xanthomonas mutant to examine the role of the oxyR gene in oxidant-induced protection against peroxide killing.

We constructed and characterized a Xanthomonas campestris pv. phaseoli oxyR mutant. The mutant was hypersensitive to H2O2 and menadione killing and had reduced aerobic plating efficiency. The oxidants' induction of the catalase and ahpC genes was also abolished in the mutant. Analysis of the adaptive responses showed that hydrogen peroxide-induced protection against hydrogen peroxide was lost, while menadione-induced protection against hydrogen peroxide was retained in the oxyR mutant. These results show that X. campestris pv. phaseoli oxyR is essential to peroxide adaptation and revealed the existence of a novel superoxide-inducible peroxide protection system that is independent of OxyR.

Identification and characterization of a new organic hydroperoxide resistance (ohr) gene with a novel pattern of oxidative stress regulation from Xanthomonas campestris pv. phaseoli.

We have isolated a new organic hydroperoxide resistance (ohr) gene from Xanthomonas campestris pv. phaseoli. This was done by complementation of an Escherichia coli alkyl hydroperoxide reductase mutant with an organic hydroperoxide-hypersensitive phenotype. ohr encodes a 14.5-kDa protein. Its amino acid sequence shows high homology with several proteins of unknown function. An ohr mutant was subsequently constructed, and it showed increased sensitivity to both growth-inhibitory and killing concentrations of organic hydroperoxides but not to either H2O2 or superoxide generators. No alterations in sensitivity to other oxidants or stresses were observed in the mutant. ohr had interesting expression patterns in response to low concentrations of oxidants. It was highly induced by organic hydroperoxides, weakly induced by H2O2, and not induced at all by a superoxide generator. The novel regulation pattern of ohr suggests the existence of a second organic hydroperoxide-inducible system that differs from the global peroxide regulator system, OxyR. Expression of ohr in various bacteria tested conferred increased resistance to tert-butyl hydroperoxide killing, but this was not so for wild-type Xanthomonas strains. The organic hydroperoxide hypersensitivity of ohr mutants could be fully complemented by expression of ohr or a combination of ahpC and ahpF and could be partially complemented by expression ahpC alone. The data suggested that Ohr was a new type of organic hydroperoxide detoxification protein.

Characterization and expression analysis of a Xanthomonas oryzae pv. oryzae recA.

Nucleotide sequence of Xanthomonas oryzae pv. oryzae (Xoo) DNA from pSM-A1 was determined and sequence analysis revealed an ORF with high homology to RecA proteins. Expression analysis using an anti-RecA antibody demonstrates that MMS treatment induces recA in Xanthomonas strains but not in an Escherichia coli harbouring cloned Xoo recA. This indicates the existence of a recA regulatory mechanism in Xanthomonas that is not function in E. coli. In Xoo, recA was highly induced by treatments with chemical mutagens, UV and peroxides, while superoxides, a thiol agent, a heavy metal and heat shock were not inducers. The increased amount of RecA induced by H2O2 or MMS treatments were due to increased transcription of recA. recA showed no growth phase or starvation regulation. The pattern of recA regulation in Xoo could play important roles in stress survival in the environment and during plant-microbe interactions.