Lack of levofloxacin and moxyfloxacin efficacy in experimental plague of albino mice infected with nalidixic acid resistant pathogen (Nal(r))

The efficacy of levofloxacin and moxyfloxacin vs. the previously tested fluoroquinolones was studied on albino mice with experimental plague due to the Nal(r) mutants of Yersinia pestis 231 and 231 FI-. The plague microbe mutants resistant to nalidixic acid (Nal(r)) generated at a frequency of 10(-10)-10(-9). The resistance to nalidixic acid was not accompanied by the strains loss of the virulence. The Nal(r) mutants were cross resistant to fluoroquinolones (ciprofloxacin, moxyfloxacin). The LD50 for the nontreated animals did not differ from that for the mice treated with nalidixic acid and the fluoroquinolones (when the animals were infected with Nal(r) mutants). The results showed that the criteria of the plague microbe susceptibility/resistance to fluoroquinolones should be revised.     

Unusual association of a plasmid with nalidixic acid resistance in an epidemic strain of Shigella dysenteriae type 1 from Asia

The association of a 20-MDa plasmid with nalidixic acid resistant (Nalr) strains of Shigella dysenteriae 1 has been examined. The plasmid, which is readily transferable, does not itself code for nalidixic acid resistance but offers a survival advantage to its host under nalidixic acid stress. The plasmid-containing cultures of S. dynsenteriae 1 produced Nalr mutants in vitro at a frequency 1000-fold higher than their plasmidless parent strains, after two exposures to nalidixic acid. Using a similar procedure, mutants resistant to other antibiotics such as chloramphenicol, tetracycline, or ciprofloxacin could not be isolated. The genome of S. dysenteriae 1 appears to carry a heavy load of the insertion sequence IS1. The propensity of the plasmid-containing strains to readily mutate to nalidixic acid resistance and its possible relevance to the observed association of the plasmid with Nalr clinical isolates is discussed.     

Formation of virulent antigen-modified mutants (Fra-, Fra-Tox-) of plague bacteria resistant to rifampicin and quinolones]

Experiments were performed with two strains of plague bacteria--231 (isolated from marmot) and 358 (isolated from human) and their isogenic variants with Fra- and Fra-Tox- phenotype. Mutants resistant to rifampicin (Rifr) and nalidixic acid (Nalr) appeared independently of pathogen phenotype and genotype with frequency n.10(-8)-n.10(-9), subsequently. Rifr mutation influenced on virulence manifestation at albino mice and antigendeficient variants with Fra- and Fra-Tox- phenotype. In every group of strains highly virulent subcultures were registered. Resistance to nalidixic acid mainly was not associated with virulence loss. Nalr mutants of parent and antigenmodified mutants were cross resistant to fluoroqinolones (ciprofloxacin, ofloxacin, pefloxacin, lomefloxacin). LD50 for untreated albino mice did not differ from LD50, for mice treated with rifampicin (when mice were infected with strain resistant to rifampicin) or with nalidixic acid and fluoroquinolones (when animals were infected with Nalr mutants). Antigenmodified strains of plague bacteria and their Rifr, Nalr mutants were able to overcome specific immune reaction. The drugs should be used in synergic combinations (with aminoglycosides or cephalosporines of III generation) to prevent appearance of virulent strains resistant to rifampicin and fluroquinolones.     

Chromosomal resistance of plague agent to quinolones]

The nature of increasing chromosomal resistance to quinolones was studied in a model of the plague microbe. Five virulent strains of the natural plague microbe (Y. pestis) were used in the experiment: 363 (1/1479), 231, 2385, 2442 and 2444. The one-stage procedure for isolation of the mutants was applied. It was shown that the frequency of the one-stage mutants resistant to oxalinic acid, pefloxacin and ciprofloxacin amounted to 10(-9)-10(-11) and was 2 to 3 orders of magnitude lower than that of the mutants resistant to nalidixic acid. Two types of the plague microbe mutants resistant to the quinolones were detected: those resistant to the quinolones to the generations (Nalr-phenotype) and those resistant to the representatives of the 3rd generation quinolones (Nals-phenotype). The quinolones were not efficient in the treatment of albino mice with experimental plague caused by the quinolone-resistant forms of the plague microbe.     

Properties of adenyl cyclase and cyclic adenosine 3'',5''-monophosphate receptor protein-deficient mutants of Escherichia coli.

Several spontaneous cya and crp mutants of Escherichia coli have been selected as clones simultaneously resistant to phage lambda and nalidixic acid and characterized. Both cya and crp mutants have been found to grow as cocci with increased doubling times. They have increased resistance to some mutagens (methylmethanesulfonate, ultraviolet light, gamma rays), antibiotics (nalidixic acid, ampicillin), phages (lambda, T6), sublethal heat and hypotonic shock, and decreased resistance to neutral detergents (sodium dodecyl sulfate, sodium deoxycholate), a protein synthesis inhibitor (streptomycin), and a respiratory inhibitor (sodium azide). The nature of changes in cell parameters indicate fundamental alterations in the envelope structure of the cya and crp mutant cells. The new cya and crp mutants have been found to be multiply carbohydrate negative and nonmotile in conformity with similar previously isolated mutants. Studies of revertants and phi80 cya+ and phi80 cya transductants indicated that the pleiotropic phenotype is related to a single mutational event at the cya or the crp locus in the mutants.     

Involvement of host DNA gyrase in growth of bacteriophage T5.

Bacteriophage T5 did not grow at the nonpermissive temperature of 42 degrees C in Escherichia coli carrying a temperature-sensitive mutation in gyrB [gyrB(Ts)], but it did grow in gyrA(Ts) mutants at 42 degrees C. These findings indicate that the A subunit of host DNA gyrase is unnecessary, whereas the B subunit is necessary for growth of T5. The necessity for the B subunit was confirmed by a strong inhibition of T5 growth by novobiocin and coumermycin A1, which interfere specifically with the function of the B subunit of host DNA gyrase. However, T5 growth was also strongly inhibited by nalidixic acid, which interferes specifically with the function of the A subunit. This inhibition was due to the interaction of nalidixic acid with the A subunit and not just to its binding to DNA, because appropriate mutations in the gyrA gene of the host conferred nalidixic acid resistance to the host and resistance to T5 growth in such a host. The inhibition by nalidixic acid was also not due to a cell poison formed between nalidixic acid and the A subunit (K. N. Kreuzer and N. R. Cozzarelli, J. Bacteriol. 140:424-435, 1979) because nalidixic acid inhibited growth of T5 in a gyrA(Ts) mutant (KNK453) at 42 degrees C. We suggest that T5 grows in KNK453 at 42 degrees C because its gyrA(Ts) mutation is leaky for T5. Inhibition of T5 growth due to inactivation of host DNA gyrase was caused mainly by inhibition of T5 DNA replication. In addition, however, late T5 genes were barely expressed when host DNA gyrase was inactivated.     

Isolation and Characterization of an Escherichia coli ruv Mutant Which Forms Nonseptate Filaments After Low Doses of Ultraviolet Light Irradiation

Two ultraviolet light (UV)-sensitive mutants have been isolated from Escherichia coli K-12. These mutants, designated RuvA(-) and RuvB(-), were controlled by a gene located close to the his gene on the chromosome map. They were sensitive to UV (10- to 20-fold increase) and slightly sensitive to gamma rays (3-fold increase). Host cell reactivation, UV reactivation and genetic recombination were normal in these mutants. Irradiation of the mutants with UV resulted in the production of single-strand breaks in deoxyribonucleic acid, which was repaired upon incubation in a growth medium. After UV irradiation, these mutants resumed deoxyribonucleic acid synthesis at a normal rate, as did the parent wild-type bacteria, and formed nonseptate, multinucleate filaments. From these results we concluded that the mutants have some defect in cell division after low doses of UV irradiation, similar to the lon(-) or fil(+) mutant of E. coli. The ruv locus was divided further into ruvA and ruvB with respect to nalidixic acid sensitivity and the effect of minimal agar or pantoyl lactone on survival of the UV-irradiated cell. The ruvB(-)mutant was more sensitive to nalidixic acid than were ruvA(-) and the parent strain. There was a great increase in the surviving fraction of the UV-irradiated ruvB(-) mutant when it was plated on minimal agar or L agar containing pantoyl lactone. No such increase in survival was observed in the ruvA(-) mutant.     

The role of RamA on the development of ciprofloxacin resistance in Salmonella enterica serovar Typhimurium

Active efflux pump is a primary fluoroquinolone resistant mechanism of clinical isolates of Salmonella enterica serovar Typhimurium. RamA is an essential element in producing multidrug resistant (MDR) S. enterica serovar Typhimurium. The aim of the present study was to elucidate the roles of RamA on the development of ciprofloxacin, the first choice for the treatment of salmonellosis, resistance in S. enterica serovar Typhimurium. Spontaneous mutants were selected via several passages of S. enterica serovar Typhimurium CVCC541 susceptible strain (ST) on M-H agar with increasing concentrations of ciprofloxacin (CIP). Accumulation of ciprofloxacin was tested by the modified fluorometric method. The expression levels of MDR efflux pumps were determined by real time RT-PCR. In ST and its spontaneous mutants, the ramA gene was inactivated by insertion of the kan gene and compensated on a recombinant plasmid pGEXΦ(gst-ramA). The mutant prevention concentration (MPC) and mutant frequencies of ciprofloxacin against ST and a spontaneous mutant in the presence, absence and overexpression of RamA were tested. Four spontaneous mutants (SI1-SI4) were obtained. The SI1 (CIP MICs, 0.1 mg/L) without any target site mutation in its quinolone resistant determining regions (QRDRs) and SI3 (CIP MICs, 16 mg/L) harboring the Ser83→Phe mutation in its QRDR of GyrA strains exhibited reduced susceptibility and resistance to multidrugs, respectively. In SI1, RamA was the main factor that controlled the susceptibility to ciprofloxacin by activating MdtK as well as increasing the expression level of acrAB. In SI3, RamA played predominant role in ciprofloxacin resistance via increasing the expression level of acrAB. Likewise, the deficiency of RamA decreased the MPCs and mutant frequencies of ST and SI2 to ciprofloxacin. In conclusion, the expression of RamA promoted the development of ciprofloxacin resistant mutants of S. enterica serovar Typhimurium. The inhibition of RamA could decrease the appearance of the ciprofloxacin resistant mutants.     

Global analysis of the genes involved in the thermotolerance mechanism of thermotolerant Acetobacter tropicalis SKU1100

Acetobacter tropicalis SKU1100 is a thermotolerant acetic acid bacterium that grows even at 42 °C, a much higher temperature than the limit for the growth of mesophilic strains. To elucidate the mechanism underlying the thermotolerance of this strain, we attempted to identify the genes essential for growth at high temperature by transposon (Tn10) mutagenesis followed by gene or genome analysis. Among the 4,000 Tn10-inserted mutants obtained, 32 exhibited a growth phenotype comparable to that of the parent strain at 30 °C but not at higher temperatures. We identified the insertion site of Tn10 on the chromosomes of all the mutant strains by TAIL (Thermal Asymmetric Interlaced)-PCR, and found 24 genes responsible for thermotolerance. The results also revealed a partial overlap between the genes required for thermotolerance and those required for acetic acid resistance. In addition, the origin and role of these thermotolerant genes are discussed.     

Clonal expansion may account for high levels of quinolone resistance in Salmonella enterica serovar enteritidis

We have observed a high incidence of isolated nalidixic acid resistance in Salmonella enterica serovar Enteritidis isolates in Ireland, particularly isolates of phage type 1 (PT1). A group of nalidixic acid-resistant (n = 22) and nalidixic acid-susceptible (n = 28) isolates of serovar Enteritidis from multiple sites in Ireland were selected. Isolates were typed by pulsed-field gel electrophoresis (PFGE) with XbaI, and the MICs for nalidixic acid and ciprofloxacin were determined. Mutations associated with nalidixic acid resistance in clinical isolates and laboratory mutants of serovar Enteritidis and 32 nalidixic acid-resistant isolates of 15 other salmonella serovars were identified. PFGE had limited discriminatory power. A specific point mutation (G246T) associated with amino acid substitution Asp87Tyr in the quinolone resistance determining region of the gyrA gene accounted for 95% of all mutations in serovar Enteritidis and for all mutations in PT1 isolates. Greater diversity of mutations was observed among all non-Enteritidis salmonella serovars studied. Rates of nalidixic acid resistance in serovar Enteritidis may predominantly reflect clonal expansion after infrequent mutation or selection events.     

Selection for citrate synthase deficiency in icd mutants of Escherichia coli.

Cultures of isocitrate dehydrogenase-deficient (icd) mutants were overgrown by double mutants (icd glt) lacking citrate synthase activity also. The icd mutants grew more slowly than wild-type cells or the double mutants because they accumulated an inhibitory metabolite (possibly citrate). Intracellular citrate levels were several hundred-fold higher in icd cells than in wild-type or icd glt cells. Final growth yields of the wild type and the icd mutant on limiting glucose were equivalent and greater than the growth yield of icd glt double mutants. The icd gene mapped between 60 and 74 min. icd mutants were resistant to nalidixic acid, but glt and icd glt mutants and wild-type cells were sensitive, indicating that resistance results from accumulation of isocitrate, citrate, or a derivative of these compounds.     

Temporal sequence of events during the initiation process in Escherichia coli deoxyribonucleic acid replication: roles of the dnaA and dnaC gene products and ribonucleic acid polymerase.

Three thermosensitive deoxyribonucleic acid (DNA) initiation mutants of Escherichia coli exposed to the restrictive temperature for one to two generations were examined for the ability to reinitiate DNA replication after returning to the permissive temperature in the presence of rifampin, chloramphenicol, or nalidixic acid. Reinitiation in the dnaA mutant was inhibited by rifampin but not by chloramphenicol, whereas renitiation was not inhibited by rifampin but not by chloramphenicol, whereas reinitiation was not inhibited in two dnaC mutants by either rifampin or chloramphenicol. To observe the rifampin inhibition, the antibiotic must be added at least 10 min before return to the permissive temperature. The rifampin inhibition of reinitiation was not observed when a rifampin-resistant ribonucleic acid ((RNA) polymerase gene was introduced into the dnaA mutant, demonstrating that RNA polymerase synthesizes one or more RNA species required for the initation of DNA replication (origin-RNA). Reinitiation at 30 degrees C was not inhibited by streptolydigin in a stretolydigin-sensitive dnaA muntant. Incubation in the presence of nalidixic acid prevented subsequent reinitiation in the dnaC28 mutant but did not inhibit reinitiation in the dnaA5 muntant. These results demonstrate that the dnaA gene product acts before or during the synthesis of an origin-RNA, RNA polymerase synthesizes this origin RNA, and the dnaC gene product is involved in a step after this RNA synthesis event. Furthermore, these results suggest that the dnaC gene product is involved in the first deoxyribounucleotide polymerization event wheareas the dnaA gene product acts prior to this event. A model is presented describing the temporal sequence of events that occur during initiation of a round of DNA replication, based on results in this and the accompanying paper.     

WQ-3810 exerts high inhibitory effect on quinolone-resistant DNA gyrase of Salmonella Typhimurium

ABSTRACT

The inhibitory effect of WQ-3810 on DNA gyrase was assayed to evaluate the potential of WQ-3810 as a candidate drug for the treatment of quinolone resistant Salmonella Typhymurium infection. The inhibitory effect of WQ-3810, ciprofloxacin and nalidixic acid was compared by accessing the drug concentration that halves the enzyme activity (IC₅₀) of purified S. Typhimurium wildtype and mutant DNA gyrase with amino acid substitution at position 83 or/and 87 in subunit A (GyrA) causing quinolone resistance. As a result, WQ-3810 reduced the enzyme activity of both wildtype and mutant DNA gyrase at a lower concentration than ciprofloxacin and nalidixic acid. Remarkably, WQ-3810 showed a higher inhibitory effect on DNA gyrase with amino acid substitutions at position 87 than with that at position 83 in GyrA. This study revealed that WQ-3810 could be an effective therapeutic agent, especially against quinolone resistant Salmonella enterica having amino acid substitution at position 87.     

Characterization of the Arabidopsis thermosensitive mutant atts02 reveals an important role for galactolipids in thermotolerance

Plants are constantly challenged with various abiotic stresses in their natural environment. Elevated temperatures have a detrimental impact on overall plant growth and productivity. Many plants increase their tolerance to high temperatures through an adaptation response known as acquired thermotolerance. To identify the various mechanisms that plants have evolved to cope with high temperature stress, we have isolated a series of Arabidopsis mutants that are defective in the acquisition of thermotolerance after an exposure to 38 degrees C, a treatment that induces acquired thermotolerance in wild-type plants. One of these mutants, atts02, was not only defective in acquiring thermotolerance after the treatment, but also displayed a reduced level of basal thermotolerance in a 30 degrees C growth assay. The affected gene in atts02 was identified by positional cloning and encodes digalactosyldiacylglycerol synthase 1 (DGD1) (the atts02 mutant was, at that point, renamed dgd1-2). An additional dgd1 allele, dgd1-3, was identified in two other mutant lines displaying altered acquired thermotolerance, atts100 and atts104. Expression patterns of several heat shock proteins (HSPs) in heat-treated dgd1-2 homozygous plants were similar to those from identically treated wild-type plants, suggesting that the thermosensitivity in the dgd1-2 mutant was not caused by a defect in HSP induction. Lipid analysis of wild-type and mutant plants indicated a close correlation between the ability to acquire thermotolerance and the increases in digalactosyldiacylglycerol (DGDG) level and in the ratio of DGDG to monogalactosyldiacylglycerol (MGDG). Thermosensitivity in dgd1-2 and dgd1-3 was associated with (1) a decreased DGDG level and (2) an inability to increase the ratio of DGDG to MGDG upon exposure to a 38 degrees C sublethal temperature treatment. Our results suggest that the DGDG level and/or the ratio of DGDG to MGDG may play an important role in basal as well as acquired thermotolerance in Arabidopsis.     

Conditional lethal mutants of bacteriophage T4 unable to grow on a streptomycin resistant mutant of Escherichia coli.

Sixteen conditional lethal mutants of bacteriophage T4D have been isolated which grow on Escherichia coli CR63 (a su+ streptomycin-sensitive K12 strain) but are restricted by CR/s (a streptomycin-resistant derivative of CR63). These mutants have been given the prefix str. Four of these mutants are amber and 12 appear to be missense. Eleven of the 12 missense mutants appear to be "pseudo-amber" (i.e. they are restricted by a su- E. coli B strain but not by a su- K12 strain); the other missense mutant was not restricted by either B or K12. The str mutations mapped in 12 different genes. Most were clustered in a region of early genes (gene 56 to gene 47). Fifty-eight amber and 10 "pseudo-amber" mutants isolated previously for their inability to grow on E. coli B were tested for restriction by CR/s. All the amber mutants grew normally on CR/s, whereas all 10 "pseudo-amber" mutants were restricted by CR/s. This implies that the phenotype of the "pseudo-amber" mutants is the result of a ribosomal difference between the permissive host CR63 and the restrictive hosts B and CR/s. These str mutants should prove to be useful alternatives to amber mutants for genetic and biochemical studies of bacteriophage T4 and for studies of the E. coli ribosome. It should be possible ot isolate similar mutants in other bacteriophages provided that streptomycin resistant hosts are available.     

Novel mutations in β-tubulin gene in Trichoderma harzianum mutants resistant to methyl benzimidazol-2-YL carbamate

Twelve low resistant (LR) mutants of Trichoderma harzianum with the capability of grow fast at 0.8 μg/mL methyl benzimidazol-2-yl carbamate (MBC) were obtained using UV mutagenesis. MR and HR mutants which could grow fast at 10 and 100 μg/mL MBC, respectively, were isolated by step-up selection protocols in which UV-treated mutants were induced and mycelial sector screening was made in plates with growth medium. Subsequently, β-tubulin genes of 14 mutants were cloned to describe the molecular lesion likely to be responsible for MBC resistance. Comparison of the β-tubulin sequences of the mutant and sensitive strains of T. harzianum revealed 2 new MBC-binding sites differed from those in other plant pathogens. A single mutation at amino acid 168, having Phe (TTC) instead of Ser (TCC), was demonstrated for the HR mutant; a double mutation in amino acid 13 resulting in the substitution of Gly (GGC) by Val (GTG) was observed in β-tubulin gene of MR mutant. On the other hand, no substitutions were identified in the β-tubulin gene and its 5’-flanking regions in 12 LR mutants of T. harzianum.     

Clonal Expansion May Account for High Levels of Quinolone Resistance in Salmonella enterica Serovar Enteritidis

We have observed a high incidence of isolated nalidixic acid resistance in Salmonella enterica serovar Enteritidis isolates in Ireland, particularly isolates of phage type 1 (PT1). A group of nalidixic acid-resistant (n = 22) and nalidixic acid-susceptible (n = 28) isolates of serovar Enteritidis from multiple sites in Ireland were selected. Isolates were typed by pulsed-field gel electrophoresis (PFGE) with XbaI, and the MICs for nalidixic acid and ciprofloxacin were determined. Mutations associated with nalidixic acid resistance in clinical isolates and laboratory mutants of serovar Enteritidis and 32 nalidixic acid-resistant isolates of 15 other salmonella serovars were identified. PFGE had limited discriminatory power. A specific point mutation (G246T) associated with amino acid substitution Asp87Tyr in the quinolone resistance determining region of the gyrA gene accounted for 95% of all mutations in serovar Enteritidis and for all mutations in PT1 isolates. Greater diversity of mutations was observed among all non-Enteritidis salmonella serovars studied. Rates of nalidixic acid resistance in serovar Enteritidis may predominantly reflect clonal expansion after infrequent mutation or selection events.     

Efficacy of Ciprofloxacin for Treatment of Cholera Associated with Diminished Susceptibility to Ciprofloxacin to Vibrio cholerae O1

Objective

We identified a poor clinical response to treatment of cholera with a single 1 g dose of ciprofloxacin, a standard treatment for cholera.

Methods

To determine reasons for the poor response and better therapeutic approaches we examined the minimal inhibitor concentration (MIC, n = 275) and disc-diffusion zone sizes (n = 205) for ciprofloxacin and nalidixic acid of V. cholerae O1 strains isolated in Bangladesh from 1994 to 2012, and reexamined data from 161patients infected with Vibrio cholerae O1 recruited in four clinical trials who received single- or multiple-dose ciprofloxacin for treatment of cholera and compared their clinical response to the V. cholerae O1 susceptibility.

Results

Although all 275 isolates of V. cholerae O1 remained susceptible to ciprofloxacin using standard MIC and disc-diffusion thresholds, the MIC⁹⁰ to ciprofloxacin increased from 0.010 in 1994 to 0.475 μgm/ml in 2012. Isolates became frankly resistant to nalidixic with the MIC⁹⁰ increasing from 21 μgm/ml in 1994 to >256 μgm/ml and 166 of 205 isolates from 1994 to 2005 being frankly resistant using disc-diffusion testing. Isolates resistant to nalidixic acid by disc-diffusion testing had a median ciprofloxacin MIC of 0.190 μgm/ml (10^th-90^th centiles 0.022 to 0.380); nalidixic acid-susceptible isolates had a median ciprofloxacin MIC of 0.002 (0.002 to 0.012).The rate of clinical success with single-dose ciprofloxacin treatment for nalidixic acid-susceptible strains was 94% (61 of 65 patients) and bacteriologic success 97% (63/65) compared to 18% (12/67) and 8% (5/67) respectively with nalidixic acid-resistant strains (P<0.001 for both comparisons). Multiple-dose treatment with ciprofloxacin had 86% and 100% clinical and bacteriologic success rates respectively in patients infected with nalidixic acid-susceptible strains of V. cholerae O1 compared to clinical success 67% and bacteriologic success 60% with nalidixic acid-resistant strains.

Conclusions

Single-dose ciprofloxacin is not effective for treating cholera caused by V. cholerae O1 with diminished susceptibility to ciprofloxacin, and nalidixic acid disc-diffusion testing effectively screens for such isolates.     

Characterization of the nirK gene encoding the respiratory, Cu-containing nitrite reductase of Bradyrhizobium japonicum.

The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from Bradyrhizobium japonicum USDA110 has been isolated and sequenced. The deduced amino acid sequence exhibited a high degree of similarity to other Cu-containing nitrite reductases from various sources. The full-length protein included a signal peptide for protein export. Analysis of the sequence upstream from the structural nirK gene revealed the presence of an anaerobox located 83 base pairs from the putative translational start codon. Cells of strain GRK308, a nitrite reductase-deficient derivative of strain USDA110, were unable to grow when cultured under microaerobic conditions (1% O(2)) in the presence of either nitrate or nitrite. Maximal expression of a nirK-lacZ fusion in strain USDA110 required simultaneously both low level oxygen conditions and the presence of nitrate. Expression of beta-galactosidase activity was not detected in the B. japonicum fixL 7403, fixJ 7360 and fixK(2) 9043 mutants transformed with the nirK-lacZ fusion after incubation of the cells under oxygen-limiting conditions either with or without nitrate. Complementation of B. japonicum 9043 with the fixK(2) gene restored beta-galactosidase activity to levels similar to those found in the parental strain. These results suggest that nirK expression depends on the low-oxygen-responsive two-component regulatory system FixLJ and on the Fnr/FixK-like DNA binding protein FixK(2).