Interactions between populations ofPseudomonas putida leading to the expression of a cryptic dehalogenase gene (dehll)

In appropriate environments containing 2-monochloropropionic acid (2MCPA), mutations in a population of nondehalogenatingPseudomonas putida, strain PP40-040 (parent population), resulted in the formation of 2mcpa⁺ papillae as a result of the decryptification of adehII gene. Increasing the size of the parent population, for example by increasing the availability of a metabolizable substrate such as succinate or lactate, increased the number of 2mcpa⁺ papillae formed because there were more parent cells available for mutation to the 2mcpa⁺ phenotype. The presence of a dehalogenating population, such asP. putida strain PP3, in close proximity to the non-dehalogenating population, also increased the number of 2mcpa⁺ papillae formed. This was due to the excretion of dehalogenases into the growth medium, which caused localized dehalogenation of the available 2MCPA, yielding a metabolizable substrate. This substrate stimulated the growth of the non-dehalogenating population, in turn increasing the number of 2mcpa⁺ papillae formed. Barriers, such as dialysis membranes, which prevented the excretion of the dehalogenases into the growth medium, prevented the stimulation of 2mcpa⁺ papillae formation by preventing release of metabolizable substrates from 2MCPA breakdown. Cell-free extracts (CFE) from dehalogenase-producing populations had a similar effect for the same reason. CFE without dehalogenase activity or in which the dehalogenase activity had been destroyed by heating failed to stimulate parent population growth and 2mcpa⁺ papillae formation. In the case ofPseudomonas putida strain PP3, which carries an easily transposed dehalogenase-encoding transposon, treatment of CFE with DNAase eliminated an additional factor involved in the formation of 2mcpa⁺ papillae.The authors are with the School of Pure and Applied Biology, University of Wales-Cardiff, P.O. Box 915, Cardiff CF1 3TL, UK     

The crystal structure of DehI reveals a new alpha-haloacid dehalogenase fold and active-site mechanism

Haloacid dehalogenases catalyse the removal of halides from organic haloacids and are of interest for bioremediation and for their potential use in the synthesis of industrial chemicals. We present the crystal structure of the homodimer DehI from Pseudomonas putida strain PP3, the first structure of a group I α-haloacid dehalogenase that can process both l- and d-substrates. The structure shows that the DehI monomer consists of two domains of ∼ 130 amino acids that have ∼ 16% sequence identity yet adopt virtually identical and unique folds that form a pseudo-dimer. Analysis of the active site reveals the likely binding mode of both l- and d-substrates with respect to key catalytic residues. Asp189 is predicted to activate a water molecule for nucleophilic attack of the substrate chiral centre resulting in an inversion of configuration of either l- or d-substrates in contrast to d-only enzymes. These details will assist with future bioengineering of dehalogenases.     

Cloning and nucleotide sequence of a D,L-haloalkanoic acid dehalogenase encoding gene from Alcaligenes xylosoxidans ssp. denitrificans ABIV

We have cloned DNA fragments of plasmid pFL40 from Alcaligenes xylosoxidans ssp. denitrificans ABIV encoding a D,L-2-haloalkanoic acid halidohydrolase (DhlIV). A 6.5-kb EcoRI/SalI-fragment with inducible expression of the halidohydrolase was cloned in Pseudomonas fluorescens and Escherichia coli. A 1.9-kb HindII-fragment demonstrated expression of the dehalogenase only due to the presence of the promoter from the pUC vector in Escherichia coli. The nucleotide sequence of this DNA-fragment was determined. It had an open reading frame coding for 296 amino acid residues (molecular weight of 32783 D). The dhlIV gene showed sequence homology to a short segment of a D-specific dehalogenase (hadD) from Pseudomonas putida AJ1, but not to any other known DNA sequences. Restriction enzyme patterns indicated similarity between dhlIV and the D,L-isomer specific dehI dehalogenase gene from Pseudomonas putida PP3. There are some indications from restriction enzyme patterns and initial sequencing data, that a gene encoding a ⁵⁴ activator protein, similar to the dehR _Iregulatory gene from Pseudomonas putida PP3 is located upstream of dhlIV. In contrast to DehI, dehalogenation of D-or L-chloropropionic acid by the DhlIV-protein leads to lactic acid of inverted configuration.     

Potential of biotechnological conversion of lignocellulose hydrolyzates by Pseudomonas putida KT2440 as a model organism for a bio‐based economy

Lignocellulose‐derived hydrolyzates typically display a high degree of variation depending on applied biomass source material as well as process conditions. Consequently, this typically results in variable composition such as different sugar concentrations as well as degree and the presence of inhibitors formed during hydrolysis. These key obstacles commonly limit its efficient use as a carbon source for biotechnological conversion. The gram‐negative soil bacterium Pseudomonas putida KT2440 is a promising candidate for a future lignocellulose‐based biotechnology process due to its robustness and versatile metabolism. Recently, P. putida KT2440_xylAB which was able to metabolize the hemicellulose (HC) sugars, xylose and arabinose, was developed and characterized. Building on this, the intent of the study was to evaluate different lignocellulose hydrolyzates as platform substrates for P. putida KT2440 as a model organism for a bio‐based economy. Firstly, hydrolyzates of different origins were evaluated as potential carbon sources by cultivation experiments and determination of cell growth and sugar consumption. Secondly, the content of major toxic substances in cellulose and HC hydrolyzates was determined and their inhibitory effect on bacterial growth was characterized. Thirdly, fed‐batch bioreactor cultivations with hydrolyzate as the carbon source were characterized and a diauxic‐like growth behavior with regard to different sugars was revealed. In this context, a feeding strategy to overcome the diauxic‐like growth behavior preventing accumulation of sugars is proposed and presented. Results obtained in this study represent a first step and proof‐of‐concept toward establishing lignocellulose hydrolyzates as platform substrates for a bio‐based economy.     

Pseudomonas putida NBRIC19 dihydrolipoamide succinyltransferase (SucB) gene controls degradation of toxic allelochemicals produced by Parthenium hysterophorus

Aims: The aim of this study was to identify the gene responsible for degradation of toxic allelochemicals of Parthenium by generating Tn5‐induced mutant of Pseudomonas putida NBRIC19. Furthermore, the study characterizes the mutant at physiological, biochemical and molecular level that helped in understanding the mechanisms of reducing the allelopathic inhibition of Parthenium by Ps. putida NBRIC19. Methods and Results: Tn5 mutant S‐74.3 showing inability to degrade toxic allelochemicals was selected after screening 22 000 transconjugants. Tn5 flanking SucB gene (dihydrolipoamide succinyltransferase) of Ps. putida NBRIC19 was found to be responsible for the degradation of toxic allelochemicals that also affected biofilm formation, chemotaxis and alginate production under toxic environment of allelochemicals. Phenotypic microarray data revealed that the respiratory activity of Ps. putida NBRIC19 and S‐74.3 differed on 47 substrates including amino acids, carboxylic acids, peptides and some chemical inhibitors. Conclusions: Study revealed that SucB gene regulates processes either directly or indirectly in Ps. putida NBRIC19, which on inactivation made the mutant less compatible for tolerating stress. Significance and Impact of the Study: This work provides the first evidence for a functional role of Ps. putida SucB gene in degradation of toxic allelochemicals of Parthenium that lead to reversal of plant growth inhibition by these toxic allelochemicals. The investigation also revealed interesting features about the involvement of microbes in plant–plant allelopathic interactions.     

Cr(III) Oxidation and Cr Toxicity in Cultures of the Manganese(II)-Oxidizing Pseudomonas putida Strain GB-1

Abstract

Mn oxides have long been considered the primary environmental oxidant of Cr(III), however, since most of the reactive Mn oxides in the environment are believed to be of biological origin, microorganisms may indirectly mediate Cr(III) oxidation and accelerate the rate over that seen in purely abiotic systems. In this study, we examined the ability of the Mn(II)-oxidizing bacterium, Pseudomonas putida strain GB-1, to oxidize Cr(III). Our results show that GB-1 cannot oxidize Cr(III) directly, but that in the presence of Mn(II), Cr(III) can be rapidly and completely oxidized. Growth studies suggest that in growth medium with few organics the resulting Cr(VI) may be less toxic to P. putida GB-1 than Cr(III), which is generally considered less hazardous. In addition, Cr(III) present during the growth of P. putida GB-1 appeared to cause iron stress as determined by the production of the fluorescent siderophore pyoverdine. When stressed by Fe limitation or Cr(III) toxicity, Mn(II) oxidation by GB-1 is inhibited.     

Comparative effectiveness of <Emphasis Type="Italic">Pseudomonas</Emphasis> and <Emphasis Type="Italic">Serratia</Emphasis> sp. containing ACC-deaminase for improving growth and yield of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) under salt-stressed conditions

Ethylene synthesis is accelerated in response to various environmental stresses like salinity. Ten rhizobacterial strains isolated from wheat rhizosphere taken from different salt affected areas were screened for growth promotion of wheat under axenic conditions at 1, 5, 10 and 15 dS m⁻¹. Three strains, i.e., Pseudomonas putida (N21), Pseudomonas aeruginosa (N39) and Serratia proteamaculans (M35) showing promising performance under axenic conditions were selected for a pot trial at 1.63 (original), 5, 10 and 15 dS m⁻¹. Results showed that inoculation was effective even in the presence of higher salinity levels. P. putida was the most efficient strain compared to the other strains and significantly increased the plant height, root length, grain yield, 100-grain weight and straw yield up to 52, 60, 76, 19 and 67%, respectively, over uninoculated control at 15 dS m⁻¹. Similarly, chlorophyll content and K⁺/Na⁺ of leaves also increased by P. putida over control. It is highly likely that under salinity stress, 1-aminocyclopropane-1-carboxylic acid-deaminase activity of these microbial strains might have caused reduction in the synthesis of stress (salt)-induced inhibitory levels of ethylene. The results suggested that these strains could be employed for salinity tolerance in wheat; however, P. putida may have better prospects in stress alleviation/reduction.     

Solubilization of organic and inorganic phosphates by three highly efficient soil bacterial isolates

Screening soil samples collected from a diverse range of slightly alkaline soil types, we have isolated 22 competent phosphate solubilizing bacteria (PSB). Three isolates identified as Pantoea agglomerans strain P5, Microbacterium laevaniformans strain P7 and Pseudomonas putida strain P13 hydrolyzed inorganic and organic phosphate compounds effectively. Bacterial growth rates and phosphate solubilization activities were measured quantitatively under various environmental conditions. In general, a close association was evident between phosphate solubilizing ability and growth rate which is an indicator of active metabolism. All three PSB were able to withstand temperature as high as 42°C, high concentration of NaCl upto 5% and a wide range of initial pH from 5 to 11 while hydrolyzing phosphate compounds actively. Such criteria make these isolates superior candidates for biofertilizers that are capable of utilizing both organic and mineral phosphate substrates to release absorbable phosphate ion for plants.     

Microbial production of <Emphasis Type="Italic">cis</Emphasis>-1,2-dihydroxy-cyclohexa-3,5-diene-1-carboxylate by genetically modified <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

Arene cis-diols are interesting chemicals because of their chiral structures and great potentials in industrial synthesis of useful chiral chemical products. Pseudomonas putida KT2442 was genetically modified to transform benzoic acid (benzoate) to 1,2-dihydroxy-cyclohexa-3,5-diene-1-carboxylic acid (DHCDC) or named benzoate cis-diol. BenD gene encoding cis-diol dehydrogenase was deleted to generate a mutant named P. putida KTSY01. Genes benABC encoding benzoate dioxygenase were cloned into plasmid pSYM01 and overexpressed in P. putida KTSY01. The recombinant bacteria P. putida KTSY01 (pSYM01) showed strong ability to transform benzoate to DHCDC. DHCDC of 2.3 g/L was obtained with a yield of 73% after 24 h of cultivation in shake flasks incubated under optimized growth conditions. Transformation of benzoate carried out in a 6-L fermentor using a benzoate fed-batch process produced over 17 g/L DHCDC after 48 h of fermentation. The average DHCDC production rate was 0.356 g L⁻¹ h⁻¹. DHCDC purified from the fermentation broth showed a purity of more than 95%, and its chemical structure was confirmed by nuclear magnetic resonance.     

Cosubstrate effects in reductive dehalogenation byPseudomonas putida G786 expressing cytochrome P-450CAM

Cytochrome P-450_CAM was shown to be the primary catalyst mediating reductive dehalogenation of polychlorinated ethanes byPseudomonas putida G786. Under anaerobic conditions, the enzyme catalyzed reductive elimination reactionsin vivo with the substrates hexachloroethane, pentachloroethane, and 1,1,1,2-tetrachloroethane; the products were tetrachloroethylene, trichloroethylene, and 1,1-dichloroethylene, respectively.In vivo reaction rates were determined. No reaction was observed with 1,1,2,2-tetrachloroethane or 1,1,1-trichloroethane. Purified cytochrome P-450_CAM was used to measure dissociation constants of polychlorinated ethanes for the enzyme active site. Observed rates and dissociation constants were used to predict the course of a reaction with the three substrates simultaneously. Data obtained from experiments withP. putida G786 generally followed the simulated reaction curves. Oxygen suppressed the reductive dechlorination reactions and, in the case of 1,1,1,2-tetrachloroethane, 2,2,2-trichloroacetaldehyde was formed. Significant rates of reductive dechlorination were observed at 5% oxygen suggesting that these reactions could occur under partially aerobic conditions. These studies highlight the potential to use an aerobic bacterium,P. putida G786, under a range of oxygen tensions to reductively dehalogenate mixed wastes which are only degraded at very low rates by obligately anaerobic bacteria.Abbreviations GC/MS Gas chromatography/mass spectrometry - P-450_CAM Cytochrome m of the camphor oxidizing system ofP. putida - pca Polychlorinated ethane     

Microbial transformation of chlorinated benzoates

Chlorinated benzoates enter the environment through their use as herbicides or as metabolites of other halogenated compounds. Ample evidence is available indicating biodegradation of chlorinated benzoates to CO₂ and chloride in the environment under aerobic as well as anaerobic conditions. Under aerobic conditions, lower chlorinated benzoates can serve as sole electron and carbon sources supporting growth of a large list of taxonomically diverse bacterial strains. These bacteria utilize a variety of pathways ranging from those involving an initial degradative attack by dioxygenases to those initiated by hydrolytic dehalogenases. In addition to monochlorinated benzoates, several bacterial strains have been isolated that can grow on dichloro-, and trichloro- isomers of chlorobenzoates. Some aerobic bacteria are capable of cometabolizing chlorinated benzoates with simple primary substrates such as benzoate. Under anaerobic conditions, chlorinated benzoates are subject to reductive dechlorination when suitable electron-donating substrates are available. Several halorespiring bacteria are known which can use chlorobenzoates as electron acceptors to support growth. For example, Desulfomonile tiedjei catalyzes the reductive dechlorination of 3-chlorobenzoate to benzoate. The benzoate skeleton is mineralized by other microorganisms in the anaerobic environment. Various dichloro- and trichlorobenzoates are also known to be dechlorinated in anaerobic sediments.     

Haloacetic acid-degrading bacterial communities in drinking water systems as determined by cultivation and by terminal restriction fragment length polymorphism of PCR-amplified haloacid dehalogenase gene fragments

Aims: To characterize the HAA‐degrading bacteria in drinking water systems. Methods and Results: Haloacetic acid (HAA)‐degrading bacteria were analysed in drinking water systems by cultivation and by a novel application of terminal restriction fragment length polymorphism (tRFLP). Substantial similarities were observed among the tRFLP patterns of dehI and dehII gene fragments in drinking water samples obtained from three different cities (Minneapolis, MN; St Paul, MN; Bucharest, Romania) and from one biologically active granular activated carbon filter (Hershey, PA). The dominant fragment in the tRFLP profiles of dehI genes from the drinking water samples matched the pattern from an Afipia sp. that was previously isolated from drinking water. In contrast, the dominant fragment in the tRFLP profiles of dehII genes did not match any previously characterized dehII gene fragment. PCR cloning was used to characterize this gene fragment, which had <65% nucleotide sequence identity with any previously characterized dehII gene. Conclusions: Afipia spp. are an appropriate model organism for studying the biodegradation of HAAs in drinking water distribution systems as encoded by dehI genes; the organism that harbours the most prominent dehII gene in drinking water has yet to be cultivated and identified. Significance and Impact of the Study: The development of a novel application of tRFLP targeting dehI and dehII genes could be broadly useful in understanding HAA‐degrading bacteria in numerous environments.     

Toxic effects of toluene on the growth of activated sludge bacteria

Two bacteria were isolated from the activated sludge sample of a wastewater treatment plant in Dublin by enrichment culture technique with toluene as the sole source of carbon and energy. They were identified as Aeromonas caviae (To-4) and Pseudomonas putida (To-5). The growth of these bacteria depended on the manner in which toluene was supplied. In general, growth was better when toluene was supplied in the vapour phase. When toluene was added directly to the growth medium it was found to be toxic to the organisms but the toxic effect could be alleviated in the presence of other carbon sources and by the acclimation of the cells. The growth of To-4 on toluene has never been previously reported.     

Toluene-Degrading Bacteria Are Chemotactic towards the Environmental Pollutants Benzene, Toluene, and Trichloroethylene

The bioremediation of polluted groundwater and toxic waste sites requires that bacteria come into close physical contact with pollutants. This can be accomplished by chemotaxis. Five motile strains of bacteria that use five different pathways to degrade toluene were tested for their ability to detect and swim towards this pollutant. Three of the five strains (Pseudomonas putida F1, Ralstonia pickettii PKO1, and Burkholderia cepacia G4) were attracted to toluene. In each case, the response was dependent on induction by growth with toluene. Pseudomonas mendocina KR1 and P. putida PaW15 did not show a convincing response. The chemotactic responses of P. putida F1 to a variety of toxic aromatic hydrocarbons and chlorinated aliphatic compounds were examined. Compounds that are growth substrates for P. putida F1, including benzene and ethylbenzene, were chemoattractants. P. putida F1 was also attracted to trichloroethylene (TCE), which is not a growth substrate but is dechlorinated and detoxified by P. putida F1. Mutant strains of P. putida F1 that do not oxidize toluene were attracted to toluene, indicating that toluene itself and not a metabolite was the compound detected. The two-component response regulator pair TodS and TodT, which control expression of the toluene degradation genes in P. putida F1, were required for the response. This demonstration that soil bacteria can sense and swim towards the toxic compounds toluene, benzene, TCE, and related chemicals suggests that the introduction of chemotactic bacteria into selected polluted sites may accelerate bioremediation processes.     

C<Subscript>1</Subscript> compounds as auxiliary substrate for engineered <Emphasis Type="Italic">Pseudomonas putida</Emphasis> S12

The solvent-tolerant bacterium Pseudomonas putida S12 was engineered to efficiently utilize the C₁ compounds methanol and formaldehyde as auxiliary substrate. The hps and phi genes of Bacillus brevis, encoding two key steps of the ribulose monophosphate (RuMP) pathway, were introduced to construct a pathway for the metabolism of the toxic methanol oxidation intermediate formaldehyde. This approach resulted in a remarkably increased biomass yield on the primary substrate glucose when cultured in C-limited chemostats fed with a mixture of glucose and formaldehyde. With increasing relative formaldehyde feed concentrations, the biomass yield increased from 35% (C-mol biomass/C-mol glucose) without formaldehyde to 91% at 60% relative formaldehyde concentration. The RuMP-pathway expressing strain was also capable of growing to higher relative formaldehyde concentrations than the control strain. The presence of an endogenous methanol oxidizing enzyme activity in P. putida S12 allowed the replacement of formaldehyde with the less toxic methanol, resulting in an 84% (C-mol/C-mol) biomass yield. Thus, by introducing two enzymes of the RuMP pathway, co-utilization of the cheap and renewable substrate methanol was achieved, making an important contribution to the efficient use of P. putida S12 as a bioconversion platform host.     

The use of transgenic canola (Brassica napus) and plant growth-promoting bacteria to enhance plant biomass at a nickel-contaminated field site

The applicability of transgenic plants and plant growth-promoting bacteria to improve plant biomass accumulation as a phytoremediation strategy at a nickel (Ni)-contaminated field site was examined. Two crops of 4-day old non-transformed and transgenic canola (Brassica napus) seedlings in the presence and absence of Pseudomonas putida strain UW4 (crop #1) or P. putida strain HS-2 (crop #1 and 2) were transplanted at a Ni-contaminated field site in 2005. Overall, transgenic canola had increased growth but decreased shoot Ni concentrations compared to non-transformed canola, resulting in similar total Ni per plant. Under optimal growth conditions (crop #2), the addition of P. putida HS-2 significantly enhanced growth for non-transformed canola. Canola with P. putida HS-2 had trends of higher total Ni per plant than canola without P. putida HS-2, indicating the potential usefulness of this bacterium in phytoremediation strategies. Modifications to the planting methods may be required to increase plant Ni uptake.     

Fate of genetically modified microorganisms in the corn rhizosphere

The fates of genetically modified (GM)Escherichia coli andPseudomonas putida in the corn rhizosphere were investigated. Under hydrophonic and sterile conditions, both bacteria grew well in the presence of root exudates used as a sole carbon source. The growth patterns of wild types and genetically modified strains ofE. coli andP. putida were similar under the conditions tested.The presence of rhizospheric microorganisms affected the survival pattern ofE. coli. In the presence of corn roots and rhizospheric microorganisms,E. coli numbers increased during the first 3 days but were later drastically reduced, probably as a result of competition with rhizospheric microorganisms for the carbon source. However, in the presence of rhizospheric microorganisms,P. putida survived better thanE. coli in the simulated corn rhizosphere.     

The solvent efflux system of Pseudomonas putida S12 is not involved in antibiotic resistance

The active efflux system contributing to the solvent tolerance of Pseudomonas putida S12 was characterized physiologically. The mutant P. putida JK1, which lacks the active efflux system, was compared with the wild-type organism. None of 20 known substrates of common multi-drug-resistant pumps had a stronger growth-inhibiting effect on the mutant than on the wild type. The amount of [¹⁴C]toluene accumulating in P. putida S12 increased in the presence of the solvent xylene and in the presence of uncouplers. The effect of uncouplers confirms the proton dependency of the efflux system in P. putida S12. Other compounds, potential substrates for the solvent pump, did not affect the accumulation of [¹⁴C]toluene. These results show that the efflux system in P. putida S12 is specific for organic solvents and does not export antibiotics or other known substrates of multi-drug-resistant pumps. Received: 15 February 2000 / Received revision: 16 June 2000 / Accepted: 18 June 2000     

Biocontrol of <Emphasis Type="Italic">Meloidogyne javanica</Emphasis> by <Emphasis Type="Italic">Rhizobium</Emphasis> and plant growth-promoting rhizobacteria on lentil

Biocontrol of the root-knot nematode Meloidogyne javanica was studied on lentil using plant growth-promoting rhizobacteria (PGPR) namely Pseudomonas putida, P. alcaligenes, Paenibacillus polymyxa and Bacillus pumilus and root nodule bacterium Rhizobium sp. Pseudomonas putida caused greater inhibitory effect on the hatching and penetration of M. javanica followed by P. alcaligenes, P. polymyxa and B. pumilus. Inoculation of any PGPR species alone or together with Rhizobium increased plant growth both in M. javanica-inoculated and -uninoculated plants. Inoculation of Rhizobum caused greater increase in plant growth than caused by any species of plant growth-promoting rhizobacteria in nematode-inoculated plants. Among PGPR, P. putida caused greater increase in plant growth and higher reduction in galling and nematode multiplication followed by P. alcaligenes, P. polymyxa and B. pumilus. Combined use of Rhizobium with any species of PGPR caused higher reduction in galling and nematode multiplication than their individual inoculation. Use of Rhizobium plus P. putida caused maximum reduction in galling and nematode multiplication followed by Rhizobium plus P. alcaligens. Pseudomonas putida caused greater root colonization and siderophore production followed by P. alcaligenes, P. polymyxa and B. pumilus. Analysis of the protein bands of these four species by SDS-PAGE revealed that P. putida had a different protein band profile compared to the protein profiles of P. alcaligenes, P. polymyxa and B. pumilus. However, the protein profiles of P. acaligenes, P. polymyxa and B. pumilus were similar.     

Isolation and characterisation of a strain of Pseudomonas putida that can express a periplasmic nitrate reductase

A strain of Pseudomonas putida that can express a nitrate reductase that is located in the periplasmic compartment was isolated from freshwater. The enzyme was active in vivo during arginine fermentation and at the onset of oxygen limitation in batch cultures. The activity of the enzyme increased the yield of bacteria following fermentative growth under anoxic conditions with arginine, but nitrate reduction did not support growth on nonfermentable carbon substrates under anoxic conditions. Cells expressing the periplasmic nitrate reductase were capable of reducing nitrate in the presence of oxygen. Nitrate reduction under oxic conditions was clearly coupled to a respiratory electron transport chain because: (1) the process was sensitive to the respiratory inhibitors rotenone and 2-n-heptyl-4-hydroxyquinoline N-oxide, and (2) membrane-bound and periplasmic cytochromes were involved. This is the first report of the presence of a periplasmic nitrate reductase in a member of the proteobacteria.