Chlorate Resistance Research Articles

Mechanisms of chlorate toxicity and resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that often encounters hypoxic/anoxic environments within the host, which increases its tolerance to many conventional antibiotics. Toward identifying novel treatments, we explored the therapeutic potential of chlorate, a pro-drug that kills hypoxic/anoxic, antibiotic-tolerant P. aeruginosa populations. While chlorate itself is relatively nontoxic, it is enzymatically reduced to the toxic oxidizing agent, chlorite, by hypoxically induced nitrate reductase. To better assess chlorate's therapeutic potential, we investigated mechanisms of chlorate toxicity and resistance in P. aeruginosa. We used transposon mutagenesis to identify genes that alter P. aeruginosa fitness during chlorate treatment, finding that methionine sulfoxide reductases (Msr), which repair oxidized methionine residues, support survival during chlorate stress. Chlorate treatment leads to proteome-wide methionine oxidation, which is exacerbated in a ∆msrA∆msrB strain. In response to chlorate, P. aeruginosa upregulates proteins involved in a wide range of functions, including metabolism, DNA replication/repair, protein repair, transcription, and translation, and these newly synthesized proteins are particularly vulnerable to methionine oxidation. The addition of exogenous methionine partially rescues P. aeruginosa survival during chlorate treatment, suggesting that widespread methionine oxidation contributes to death. Finally, we found that mutations that decrease nitrate reductase activity are a common mechanism of chlorate resistance.

Identification of a Novel QTL for Chlorate Resistance in Rice (Oryza sativa L.)

Chlorate resistance analysis is an effective approach commonly used to distinguish the genetic variation between Oryza sativa L. ssp. indica and japonica, and predict the nitrogen use efficiency (NUE). This study aimed at investigating the response of a doubled haploid (DH) population derived from anther culture of 93-11 × Milyang352 exposed to 0.1% potassium chlorate (KClO3) at the seedling stage. The results revealed that the parental rice lines 93-11 (indica) and Milyang352 (japonica) showed distinctive phenotypic responses. The parental line 93-11 scored highly sensitive (0% survival) and Milyang352 scored resistant (66.7% survival) 7 days after treatment. The DH lines reflected the differential phenotypic response observed in parental lines. Interestingly, we identified a novel quantitative trait locus (QTL) for chlorate resistance on chromosome 3 (qCHR-3, 136 cM, logarithm of the odds—LOD: 4.1) using Kompetitive Allele-Specific PCR (KASP) markers. The additive effect (−11.97) and phenotypic variation explained (PVE; 14.9%) indicated that the allele from Milyang352 explained the observed phenotypic variation. In addition, shoot growth showed a significant difference between parental lines, but not root growth. Moreover, in silico analysis identified candidate genes with diverse and interesting molecular and physiological functions. Therefore, this study suggested that the QTL qCHR-3 harbors promising candidate genes that could play a role in the regulation of nitrogen metabolism in rice.

Heterokaryon Formation from Color and Nutrient Mutants Resistant to Chlorate and Selenite in Fungus Alternaria alternate

The frequency of spontaneous mutants resistant to both chlorate and selenite was compared with the frequency of mutations induced by ultraviolet rays. The results showed that, the frequencies of chlorate resistance strains which isolated from two different parenting strains, AA1 and SW1, were higher than 27 and 23 times from spontaneous frequencies respectively, so as for selenite resistant results, the induced and isolating mutants of two different parenting strains, AA1 and SW3, were about 5 and 8 times higher than their spontaneous frequency, respectively. The mutant strains (Chl5-Chl9) and (Chl13-Chl27) were all resistant to chlorate-nitrate auxotrophic but they were distributed between spontaneous and induced as well as white or black conidia, while the mutants sel1-sel5 resistance to the selenite are color mutants (white conidia) and sulfates auxotrophic at the same time. Heterokaryon was also formed in Alternaria alternata between the two black Chl10 strains, nitrate auxotrophic, have the genotype (nit10, w2 +, s2 +) and the white Sel2 strain, have the genotype (nit10 +, w2s2) which represent important Preliminary event at the beginning of a para-sexual analysis in this plant pathogen fungus

Identification of QTLs Associated withindica-japonicaDifferentiation-Related Traits in Rice (Oryza sativaL.)

Understanding the morphological and physiological characteristics which are attributable to determine indica and japonica subspecies of Asian rice (Oryza sativa L.) is important to conceive breeding strategy. However, information of quantitative trait loci (QTLs) associated with targeted traits related to subspecies domestication and differentiation-related traits remains limited. Two reciprocal F2 mapping populations generated from a cross between Ilpumbyeo (japonica) and Dasanbyeo (indica) were developed and used for constructing genetic maps to identify QTLs responsible for subspecies specific-related traits using 107 sequence-tagged site (STS) markers. A total of 26 main-effect QTLs for subspecies-related traits such as grain length, grain width, grain length-width ratio, apiculus hair length, and potassium chlorate resistance, were detected on seven chromosomes (1, 2, 3, 4, 5, 6, and 8). Of the detected QTLs, 14 QTLs corresponding to the previously reported QTLs suggesting the functional conservation of QTLs across populations and the remaining 12 QTLs appeared to be novel QTLs. A total of 29 significant epistatic QTLs were also detected in present study demonstrating an important genetic basis of epistatic interactions for complex subspecies-related traits. These results suggest that the subspecies-related traits and detected QTLs should be taken into consideration to incorporate with marker-assisted selection strategy in rice breeding program.

Characterization of novel nitrate reductase-deficient mutants for transgenic Dunaliella salina systems.

The aim of the present study was to isolate and characterize novel nitrate reductase (NR)-deficient mutants, which may be useful for the transgenic manipulation of Dunaliella salina. Three NR-deficient mutants of D. salina, J-1, J-2, and J-3, were successfully isolated by screening for chlorate resistance after chemical mutagenesis with ethylnitrosourea. NR activity was not detected in the mutants and the expression of NR mRNA was significantly decreased. Growth analysis of D. salina strains grown in media containing different nitrogen sources revealed that these mutants were capable of utilizing nitrite and urea, but not nitrate as a nitrogen source, indicating that these mutants are indeed NR-deficient. Mutation analysis of NR cDNA sequences revealed that there were 11 point mutations shared by the J-1, J-2, and J-3 mutants. Furthermore, the results of the functional complementation experiment showed that NR activity of transformant T-1 derived from J-1 was recovered to 48.1 % of that of the wild-type D. salina. The findings of the present study indicate that nitrate may be used as a selective agent rather than antibiotics or herbicides for the isolated NR-deficient mutants in future transgenic D. salina systems.

Development of Transformation System of Verticillium lecanii (Lecanicillium spp.) (Deuteromycotina: Hyphomycetes) Based on Nitrate Reductase Gene of Aspergillus nidulans

A heterologous transformation system was developed for V. lecanii based on the complementation of a nitrate reductase mutant. Nitrate reductase mutants were obtained by resistance to chlorate in a rate of 23.24% when compared to other mutations that lead to the chlorate resistance. Mutant no. 01 and 04 was chosen for the transformation experiments. Plasmid pBT was used as transformation vector containing the Aspergillus nidulans nitrate reductase gene. A frequency of approximately 3 transformants/μg DNA was obtained using the circular vector pBT. The establishment of a transformation system for V. lecanii is fundamental for genetic manipulation of this microorganism.

Effect of Translesion DNA Polymerases, Endonucleases and RpoS on Mutation Rates inSalmonella typhimurium

It has been suggested that bacteria have evolved mechanisms to increase their mutation rate in response to various stresses and that the translesion DNA polymerase Pol IV under control of the LexA regulon and the alternative sigma factor RpoS are involved in regulating this mutagenesis. Here we examined in Salmonella enterica serovar Typhimurium LT2 the rates for four different types of mutations (rifampicin, nalidixic acid, and chlorate resistance and Lac(+) reversion) during various growth conditions and with different levels of four translesion DNA polymerases (Pol II, Pol IV, Pol V, and SamAB) and RpoS. Constitutive derepression of the LexA regulon by a lexA(def) mutation had no effect on Lac(+) reversion rates but increased the other three mutation rates up to 11-fold, and the contribution of the translesion DNA polymerases to this mutagenesis varied with the type of mutation examined. The increase in mutation rates in the lexA(def) mutant required the presence of the LexA-controlled UvrB protein and endonucleases UvrC and Cho. With regard to the potential involvement of RpoS in mutagenesis, neither an increase in RpoS levels conferred by artificial overexpression from a plasmid nor long-term stationary phase incubation or slow growth caused an increase in any of the four mutation rates measured, alone or in combination with overexpression of the translesion DNA polymerases. In conclusion, mutation rates are remarkably robust and no combination of growth conditions, induction of translesion DNA polymerases by inactivation of LexA, or increased RpoS expression could confer an increase in mutation rates higher than the moderate increase caused by derepression of the LexA regulon alone.

Effect of chlorate, molybdate, and shikimic acid on Salmonella enterica serovar Typhimurium in aerobic and anaerobic cultures

Experiments were conducted to determine factors that affect sensitivity of Salmonella enterica serovar Typhimurium to sodium chlorate (5 mM). In our first experiment, cultures grown without chlorate grew more rapidly than those with chlorate. An extended lag before logarithmic growth was observed in anaerobic but not aerobic cultures containing chlorate. Chlorate inhibition of growth during aerobic culture began later than that observed in anaerobic cultures but persisted once inhibition was apparent. Conversely, anaerobic cultures appeared to adapt to chlorate after approximately 10 h of incubation, exhibiting rapid compensatory growth. In anaerobic chlorate-containing cultures, 20% of total viable counts were resistant to chlorate by 6 h and had propagated to 100% resistance (>10 9 CFU mL −1) by 24 h. In the aerobic chlorate-containing cultures, 12.9% of colonies had detectable resistance to chlorate by 6 h, but only 1% retained detectable resistance at 24 h, likely because these cultures had opportunity to respire on oxygen and were thus not enriched via the selective pressure of chlorate. In another study, treatment with shikimic acid (0.34 mM), molybdate (1 mM) or their combination had little effect on aerobic or anaerobic growth of Salmonella in the absence of added chlorate. As observed in our earlier study, chlorate resistance was not detected in any cultures without added chlorate. Chlorate resistant Salmonella were recovered at equivalent numbers regardless of treatment after 8 h of aerobic or anaerobic culture with added chlorate; however, by 24 h incubation chlorate sensitivity was completely restored to aerobic but not anaerobic cultures treated with shikimic acid or molybdate but not their combination. Results indicate that anaerobic adaptation of S. Typhimurium to sodium chlorate during pure culture is likely due to the selective propagation of low numbers of cells exhibiting spontaneous resistance to chlorate and this resistance is not reversible by molybdenum supplementation.

Substrate Specificity of RdgB Protein, a Deoxyribonucleoside Triphosphate Pyrophosphohydrolase

We have previously reported the identification of a DNA repair system in Escherichia coli for the prevention of the stable incorporation of noncanonical purine dNTPs into DNA. We hypothesized that the RdgB protein is active on 2'-deoxy-N-6-hydroxylaminopurine triphosphate (dHAPTP) as well as deoxyinosine triphosphate. Here we show that RdgB protein and RdgB homologs from Saccharomyces cerevisiae, mouse, and human all possess deoxyribonucleoside triphosphate pyrophosphohydrolase activity and that all four RdgB homologs have high specificity for dHAPTP and deoxyinosine triphosphate compared with the four canonical dNTPs and several other noncanonical (d)NTPs. Kinetic analysis reveals that the major source of the substrate specificity lies in changes in K(m) for the various substrates. The expression of these enzymes in E. coli complements defects that are caused by the incorporation of HAP and an endogenous noncanonical purine into DNA. Our data support a preemptive role for the RdgB homologs in excluding endogenous and exogenous modified purine dNTPs from incorporation into DNA.

QTLs and candidate genes for chlorate resistance in rice (Oryzasativa L.)

Chlorate resistance is one of the reliable characters in Indica/Japonica classifica- tion. To understand the genetic basis of chlorate resistance is very important for revealing the evolutionary mechanism of Indica/Japonica dif- ferentiation. In this study, a doubled haploid (DH) population derived from anther culture of ZYQ8/JX17, a typical Indica and Japonica hybrid, was used as the genetic material to investigate chlorate sensitivity of the parents and DH lines. The quantitative trait loci (QTLs) of chlorate resistance were analyzed based on the molecular linkage map of this population. Total of 3 QTLs (qCHR-2, qCHR-8 and qCHR-10) for chlorate resistance were detected on chromosomes 2, 8 and 10, respectively. A

Development of a transformation system for Penicillium brevicompactum based on the Fusarium oxysporum nitrate reductase gene

Penicillium brevicompactum is a filamentous fungus that presents a potential for industrial use due its efficient pectinase production. A heterologous transformation system was developed for P. brevicompactum based on the complementation of a nitrate reductase mutant. Nitrate reductase mutants were obtained by resistance to chlorate in a rate of 23.24% when compared to other mutations that lead to the chlorate resistance. One mutant named 4457-18X was chosen for the transformation experiments with the pNH24 vector containing de Fusarium oxysporum nitrate reductase gene. A frequency of approximately 3 transformants/µg DNA was obtained using the circular vector pNH24. This frequency was multiplied about 10 fold using the linearized vector with the Xba I restriction enzyme. Southern analysis of the transformants showed a tendency of the linearized vector to diminish the number of integrations compared to the use of the circular vector. The integration was random and stable in the analyzed transformants. The establishment of a transformation system for P. brevicompactum is fundamental for genetic manipulation of this microorganism.

Effects of Sodium Chlorate on Antibiotic Resistance inEscherichia coliO157:H7

The spread of antibiotic resistance from farm animals to human pathogens has become a matter of great public health significance, and methods to reduce foodborne pathogenic bacteria without utilizing traditional antibiotics have been sought. Chlorate kills Escherichia coli O157:H7 in vitro and in vivo and has been proposed as a feed additive to be included in food animal rations immediately prior to slaughter to reduce E. coli O157:H7 populations in the animal gastrointestinal tract. This study was designed to examine the effect that the development of chlorate resistance in E. coli O157:H7 has on resistance to 32 medically important antibiotics. Chlorate resistance numerically increased antibiotic resistance in E. coli O157:H7 strain 6058 and strain 933 to six and four antibiotics, respectively, but chlorate resistance affected resistance to only two antibiotics (cefoxotin and sulfadimethoxine) in both strains examined. Based on the fact that chlorate resistant mutants do not appear to compete well in vivo and based on the results of the current study, it appears unlikely that chlorate treatment will result in a dissemination of antibiotic resistance. However, further research is needed to confirm these results.

Chlorate resistance as a tool to study the effect of nitrate reductase antisense gene in wheat

The herbicide chlorate has been used extensively for isolation of mutants or for selection of transgenic plants defective in nitrate reduction. Chlorate, acting as an analogue of nitrate, is substrate of the enzyme nitrate reductase (NR), which reduces chlorate to the toxic chlorite. Plants being lack of NR function are resistant to chlorate. In this work chlorate resistance was used to control the efficiency of gene transfer cnxl antisense in wheat. Cnxl was transferred with cotransformation of pAHC25 containing bar and gus into immature embryo-derived calli of a spring wheat genotype (CY-45) by microprojectile bombardment The nitrate - chlorate interaction was investigated via nitrate reductase activity (NRA) assay and necrotic symptoms of chlorate toxicity. The optimal conditions of chlorate resistance test signalling the decrease of NRA were determined, and found to be 0.4 mM nitrate for NR induction, and 7 mM chlorate content for evoking greatest differences in toxic symptoms. We succeeded to regenerate of 10 plantlets resistant to phosphinothricine (PPT) indicating the expression of bar, and 6 of them were fertile producing 6 PPT resistant transgenic lines. Among the T 1 progenies of PPT transgenics, two (designated as 117 and 124) showed a significant reduction of NRA. Though T 2 progenies had a less decreased NRA in the mean of the population, but a higher chlorate resistance as compared to that of the control was still experienced.

Escherichia coli O157:H7 becomes resistant to sodium chlorate in pure culture, but not in mixed culture or in vivo.

Chlorate kills Escherichia coli O157:H7 and may be an effective feed additive for use in food animals. This study was designed to determine if development of chlorate-resistant E. coli O157:H7 strains was likely. Escherichia coli O157:H7 was chlorate-sensitive, but became chlorate-resistant in pure batch culture and in sterilized faecal fluid; it was killed in mixed culture and did not become resistant. Addition of chlorate to continuous pure cultures caused chlorate resistance, but chlorate addition to mixed continuous culture eliminated E. coli O157:H7 and no resistance occurred. Piglets challenged with E. coli O157:H7 were treated with chlorate; populations were reduced and colonies were always chlorate-sensitive. Chlorate-resistant E. coli O157:H7 can be selected in pure, but not mixed culture, and results suggest that terminal chlorate feeding will not select for chlorate-resistance in vivo. Chlorate can reduce food-borne pathogens prior to harvest, but development of resistance does not appear likely in vivo.

Molecular mapping of the cnx2 locus involved in molybdenum cofactor biosynthesis in rice (Oryza sativa L.)

Molybdenum cofactor (Moco) is essential for nitrate reductase (NR), xanthine dehydrogenase (XDH), and aldehyde oxidase to perform their catalytic functions in plants. Moco biosynthesis is a complex process involving many genes. Little is known about the genetics and molecular aspects of Moco biosynthesis in plants and other eukaryotes. In rice, we previously isolated a Moco mutant C25 with a mutation in the CNX2 gene from a mutagenized indica cultivar IR30 and characterized its biochemical properties. This mutant was crossed with a japonica cultivar, Norin 8, to investigate the linkage of cnx2 to restriction fragment length polymorphism (RFLP) and cleaved amplified polymorphic sequence (CAPS) markers. Chlorate resistance was used to trace the cnx2 mutation because of its cosegregation with the loss of NR and XDH activities observed earlier. RFLP and CAPS analyses show the location of the cnx2 locus on the long arm of chromosome 4. It is mapped between RFLP markers C513 and C377 with a distance of 9.5 and 13.1 cM, respectively. It is also linked with CAPS marker RA0738 at a distance of 30.3 cM.

Isolation and characterization of chlorate resistant mutants from nitrate-nonutilizing fungus Phycomyces blakesleeanus

Chlorate resistant mutants, which were first isolated in the zygomycetous fungusPhycomyces blakesleeanus, were found to be resistant up to a concentration of at least 300 mM of potassium chlorate. The dose-response relationship showed that although the mutants could be divided into two groups based on chlorate resistance in the mycelial elongation assay on the solid minimal medium, this was not observed in the assay using liquid culture. Genetic analysis of heterokaryons revealed the mutant alleles to be dominant. Enzymatic activities of three nitrate reductases and chlorate reductase were deficient in both the parent strain and the mutants. Intracellular incorporation of chlorate ion varied from strain to strain; however, the variation could not explain the mechanism of chlorate resistance. One unexpected characteristic of the mutants was that the intracellular sulfate ion concentration was 3.5 to 5.5 times higher than in the parent strain. We designated this mutant genotypecrw, chlorate resistant mutant from nitrate-nonutilizing wild type.

A Chlorate Resistant Mutant of Rice(Oryza sativa L.) with Normal Nitrate Uptake and Nitrate Reductase Activity.

Most mutants lacking nitrate reductase (NR) activity show a chlorate resistance, but a mutant line M1004, screened based on chlorate resistance, exhibited the same level of NR activity as the wild type Norin 8. Here, we characterized a mutant line M1004 and compared it with a NR deficient mutant line M819 and original cultivar Norin 8. M1004 showed a higher chlorate resistance than M819 at a higher chlorate concentration (1 mM). The same level of NO-3 and ClO-3 uptake as well as NRmRNA expression was observed in the mutant lines and wild type. NR-mRNA expression was induced in KClO3 solution although the NR activity was inhibited. M1004 exhibited higher a deactivation velocity of NR than the wild type. The results suggested that chlorate resistance in M1004 was caused by the rapid deactivation of NR. This chlorate resistance was transmitted by a single recessive nuclear gene.

CHL1 encodes a component of the low-affinity nitrate uptake system in Arabidopsis and shows cell type-specific expression in roots.

The Arabidopsis CHL1 (AtNRT1) gene confers sensitivity to the herbicide chlorate and encodes a nitrate-regulated nitrate transporter. However, how CHL1 participates in nitrate uptake in plants is not yet clear. In this study, we examined the in vivo function of CHL1 with in vivo uptake measurements and in situ hybridization experiments. Under most conditions tested, the amount of nitrate uptake by a chl1 deletion mutant was found to be significantly less than that of the wild type. This uptake deficiency was reversed when a CHL1 cDNA clone driven by the cauliflower mosaic virus 35S promoter was expressed in transgenic chl1 plants. Furthermore, tissue-specific expression patterns showed that near the root tip, CHL1 mRNA is found primarily in the epidermis, but further from the root tip, the mRNA is found in the cortex or endodermis. These results are consistent with the involvement of CHL1 in nitrate uptake at different stages of root cell development. A functional analysis in Xenopus oocytes indicated that CHL1 is a low-affinity nitrate transporter with a K(m) value of approximately 8.5 mM for nitrate. This finding is consistent with the chlorate resistance phenotype of chl1 mutants. However, these results do not fit the current model of a single, constitutive component for the low-affinity uptake system. To reconcile this discrepancy and the complex uptake behavior observed, we propose a "two-gene" model for the low-affinity nitrate uptake system of Arabidopsis.

Selection for mutants with low nitrate uptake ability in rice (Oryza sativa)

Screening for mutants deficient in the high affinity system of nitrate uptake was performed using mutagenized M2 population of rice (Oryza sativa, cv. Nipponbare or Kinmaze). For selecting mutants, M2 seedlings were transferred individually to 10 ml solution containing 250 μM potassium nitrate and 500 μM calcium sulphate at 20 or 28°C. After 6 or 24 h, nitrate concentration of the solution was determined with a nitrate selective electrode and the seedlings showing impaired nitrate uptake were selected as nitrate uptake deficient variants. Of 74 variants, three were confirmed to be mutants with low nitrate uptake ability in the M3 generation. Potassium uptake ability also decreased in the mutants. Three mutants were divided into two groups based on the analysis of nitrate reductase (NR, EC 1.6.6.1) activity and chlorate resistance. Two, NUE13 and NUE36, had a lower level of NR activity than the original cultivar and were not resistant to chlorate, while the seedlings of NUE50 had the same level of NR activity as the original cultivar and were more resistant to chlorate than the original cultivar. All mutants were resistant to cesium, a toxic ion analogue for potassium, suggesting that the decreased levels of both nitrate and potassium uptake were coupled to the change of plasma membrane H+‐ATPase activity.

Relationship Between the Shape of Silica Body from Motor Cells and Morphological and Physiological Characters of Grain for the Discriminations of indica and japonica Rice in China.

Methods of discrimination of indica and japonica rice based on the shape of silica body from motor cells in the leaf blade and on morphological and physiological characters of grain were compared, and their relationship was investigated. Fifty Chinese native and im-proved rice varieties (25 Hsien and 25 Keng) were used. Four morphological characters of silica body from the motor cells (vertical length, VL; horizontal length, HL; Iateral length, LL and shape coefficient b/a) (Fig. 1), grain shape (length, L; width, W and their ratio L/W), apiculus hair length (AH), phenol reaction of the hull (PR) and potassium chlorate (KCI03) resist-ance of seedlings (KC) based on injury scores (O to 3) were analysed. Based on the above characters of indica and japonica cultivars defined by Sato's discriminant function (Z (SD) =PR+1.313KC-0.82AH-1.251)