Identification of the endosymbionts from Sulla spinosissima growing in a lead mine tailings in Eastern Morocco as Mesorhizobium camelthorni sv. aridi.

AIMS: To identify the bacteria nodulating Sulla spinosissima growing profusely in a lead and zinc mine tailings in Eastern Morocco. METHODS AND RESULTS: In all, 32 rhizobial cultures, isolated from root nodules of S. spinosissima growing in soils of the mining site, were tolerant to different heavy metals. The ERIC-polymerase chain reaction (PCR) fingerprinting analysis clustered the isolates into seven different groups, and the analysis of the 16S rRNA sequences of four selected representative strains, showed they were related to different species of the genus Mesorhizobium. The atpD, glnII and recA housekeeping genes analysis confirmed the affiliation of the four representative strains to Mesorhizobium camelthorni CCNWXJ40-4T , with similarity percentages varying from 96·30 to 98·30%. The sequences of the nifH gene had 97·33-97·78% similarities with that of M. camelthorni CCNWXJ40-4T ; however, the nodC phylogeny of the four strains diverged from the type and other reference strains of M. camelthorni and formed a separated cluster. The four strains nodulate also Astragalus gombiformis and A. armatus but did not nodulate A. boeticus, Vachellia gummifera, Prosopis chilensis, Cicer arietinum, Lens culinaris, Medicago truncatula, Lupinus luteus or Phaseolus vulgaris. CONCLUSIONS: Based on similarities of the nodC symbiotic gene and differences in the host range, the strains isolated from S. spinosissima growing in soils of the Sidi Boubker mining site may form a different symbiovar within Mesorhizobium for which the name aridi is proposed. SIGNIFICANCE AND IMPACT OF THE STUDY: In this work, we show that strains of M. camelthorni species nodulating S. spinosissima in the arid area of Eastern Morocco constitute a distinct phylogenetic clade of nodulation genes; we named symbiovar aridi, which encompasses also mesorhizobia from other Mediterranean desert legumes.

Microvirga sp. symbiovar mediterranense nodulates Lupinus cosentinii grown wild in Morocco.

AIM: To analyse the diversity of nodule-forming bacteria isolated from Lupinus cosentinii naturally grown in the Maamora cork oak forest (Rabat, Morocco). METHODS AND RESULTS: Of the 31 bacterial strains, four were selected based on their REP-PCR fingerprinting that were studied by sequencing and phylogenetic analysis of their 16S rRNA, gyrB, dnaK, recA and rpoB housekeeping genes as well as the nodC symbiotic gene. The nearly complete 16S rRNA gene sequence of the four representative strains showed that they are related to Tunisian strains of genus Microvirga isolated from L. micranthus with nucleotide identity values ranging from 98·67 to 97·13%. The single and concatenated sequences of the 16S rRNA, gyrB, dnaK, recA and rpoB housekeeping genes indicated that the L. cosentinii-isolated strains had 99·2-99·9% similarities with the Tunisian L. micranthus microsymbionts. The nodC gene phylogeny revealed that the Moroccan strains clustered in the newly described mediterranense symbiovar, and nodulation tests showed that they nodulated not only L. cosentinii but also L. angustifolius, L. luteus and L. albus. CONCLUSIONS: To the best of our knowledge, this is the first report concerning the isolation, molecular identification and phylogenetic diversity of L. cosentinii nodule-forming endosymbionts and of their description as members of the Microvirga genus. SIGNIFICANCE AND IMPACT OF THE STUDY: In this work, we show that Microvirga sp. can be isolated from root nodules of wild-grown L. cosentinii in Northeast Africa, that selected strains also nodulate L. angustifolius, L. luteus and L. albus, and that they belong to symbiovar mediterranense. In addition, our data support that the ability of Microvirga to nodulate lupines could be related to the soil pH, its geographical distribution being more widespread than expected.

Purple corn-associated rhizobacteria with potential for plant growth promotion.

AIMS: Purple corn (Zea mays var. purple amylaceum) is a native variety of the Peruvian Andes, cultivated at 3000 m since the pre-Inca times without N fertilization. We aimed to isolate and identify native plant growth-promoting rhizobacteria (PGPR) for future microbial-based inoculants. METHODS AND RESULTS: Eighteen strains were isolated from the rhizosphere of purple corn plants grown without N fertilization in Ayacucho (Peru). The 16S rRNA gene clustered the 18 strains into nine groups that contained species of Bacillus, Stenotrophomonas, Achromobacter, Paenibacillus, Pseudomonas and Lysinibacillus. A representative strain from each group was selected and assayed for N2 fixation, phosphate solubilization, indole acetic and siderophore production, 1-aminocyclopropane-1-carboxylic acid deaminase activity and biocontrol abilities. Inoculation of purple corn plants with single and combined strains selected after a principal component analysis caused significant increases in root and shoot dry weight, total C and N contents of the plants. CONCLUSIONS: PGPRs can support growth and crop production of purple corn in the Peruvian Andes and constitute the base for microbial-based inoculants. SIGNIFICANCE AND IMPACT OF THE STUDY: This study enlarges our knowledge on plant-microbial interactions in high altitude mountains and provides new applications for PGPR inoculation in purple amylaceum corn, which is part of the staple diet for the native Quechua communities.

Nitrous Oxide Metabolism in Nitrate-Reducing Bacteria: Physiology and Regulatory Mechanisms.

Nitrous oxide (N2O) is an important greenhouse gas (GHG) with substantial global warming potential and also contributes to ozone depletion through photochemical nitric oxide (NO) production in the stratosphere. The negative effects of N2O on climate and stratospheric ozone make N2O mitigation an international challenge. More than 60% of global N2O emissions are emitted from agricultural soils mainly due to the application of synthetic nitrogen-containing fertilizers. Thus, mitigation strategies must be developed which increase (or at least do not negatively impact) on agricultural efficiency whilst decrease the levels of N2O released. This aim is particularly important in the context of the ever expanding population and subsequent increased burden on the food chain. More than two-thirds of N2O emissions from soils can be attributed to bacterial and fungal denitrification and nitrification processes. In ammonia-oxidizing bacteria, N2O is formed through the oxidation of hydroxylamine to nitrite. In denitrifiers, nitrate is reduced to N2 via nitrite, NO and N2O production. In addition to denitrification, respiratory nitrate ammonification (also termed dissimilatory nitrate reduction to ammonium) is another important nitrate-reducing mechanism in soil, responsible for the loss of nitrate and production of N2O from reduction of NO that is formed as a by-product of the reduction process. This review will synthesize our current understanding of the environmental, regulatory and biochemical control of N2O emissions by nitrate-reducing bacteria and point to new solutions for agricultural GHG mitigation.

Role of bacteria in marine barite precipitation: a case study using Mediterranean seawater.

Marine bacteria isolated from natural seawater were used to test their capacity to promote barite precipitation under laboratory conditions. Seawater samples were collected in the western and eastern Mediterranean at 250 m and 200 m depths, respectively, since marine barite formation is thought to occur in the upper water column. The results indicate that Pseudoalteromonas sp., Idiomarina sp. and Alteromonas sp. actually precipitate barite under experimental conditions. Barite precipitates show typical characteristics of microbial precipitation in terms of size, morphology and composition. Initially, a P-rich phase precipitates and subsequently evolves to barite crystals with low P contents. Under laboratory conditions barite formation correlates with extracellular polymeric substances (EPS) production. Barite precipitates are particularly abundant in cultures where EPS production is similarly abundant. Our results further support the idea that bacteria may provide appropriate microenvironments for mineral precipitation in the water column. Therefore, bacterial production in the past ocean should be considered when using Ba proxies for paleoproductivity reconstructions.

Functional analysis of the copy 1 of the fixNOQP operon of Ensifer meliloti under free-living micro-oxic and symbiotic conditions.

AIM: In this work, phenotypic analyses of a Ensifer meliloti fixN1 mutant under free-living and symbiotic conditions have been carried out. METHODS AND RESULTS: Ensifer meliloti fixN1 mutant showed a defect in growth as well as in TMPD-dependent oxidase activity when cells were incubated under micro-oxic conditions. Furthermore, haem c staining analyses of a fixN1 and a fixP1 mutant identified two membrane-bound c-type cytochromes of 27 and 32 kDa, present in microaerobically grown cells and in bacteroids, as the FixO and FixP components of the E. meliloti cbb3 oxidase. Under symbiotic conditions, fixN1 mutant showed a clear nitrogen fixation defect in alfalfa plants that were grown in an N-free nutrient solution during 3 weeks. However, in plants grown for a longer period, fixNOQP1 copy was not indispensable for symbiotic nitrogen fixation. CONCLUSIONS: The copy 1 of the fixNOQP operon is involved in E. meliloti respiration and growth under micro-oxic conditions as well as in the expression of the FixO and FixP components of the cbb3 oxidase present in free-living microaerobic cultures and in bacteroids. This copy is important for nitrogen fixation during the early steps of the symbiosis. SIGNIFICANCE AND IMPACT OF THE STUDY: It is the first time that a functional analysis of the E. meliloti copy 1 of the fixNOQP operon is performed. In this work, the cytochromes c that constitute the cbb3 oxidase operating in free-living micro-oxic cultures and in bacteroids of E. meliloti have been identified.

Enhanced expression of Rhizobium etli cbb3 oxidase improves drought tolerance of common bean symbiotic nitrogen fixation.

To investigate the involvement of Rhizobium etli cbb(3) oxidase in the response of Phaseolus vulgaris to drought, common bean plants were inoculated with the R. etli strain, CFNX713, overexpressing this oxidase in bacteroids (cbb(3)(+)) and subjected to drought conditions. The negative effect of drought on plant and nodule dryweight, nitrogen content, and nodule functionality was more pronounced in plants inoculated with the wild-type (WT) strain than in those inoculated with the cbb(3)(+) strain. Regardless of the plant treatment, bacteroids produced by the cbb(3)(+) strain showed higher respiratory capacity than those produced by the WT strain. Inoculation of plants with the cbb(3)(+) strain alleviated the negative effect of a moderate drought on the respiratory capacity of bacteroids and the energy charge of the nodules. Expression of the FixP and FixO components of the cbb(3) oxidase was higher in bacteroids of the cbb(3)(+) strain than in those of the WT strain under all experimental conditions. The decline in sucrose synthase activity and the decrease in dicarboxylic acids provoked by moderate drought stress were more pronounced in nodules from plants inoculated with the WT strain than in those inoculated with the cbb(3)(+) strain. Taken together, these results suggest that inoculation of plants with a R. etli strain having enhanced expression of cbb(3) oxidase in bacteroids reduces the sensitivity of P. vulgaris-R. etli symbiosis to drought and can modulate carbon metabolism in nodules.

Burkholderia phymatum strains capable of nodulating Phaseolus vulgaris are present in Moroccan soils.

Phylogenetic analysis of 16S rRNA, nodC, and nifH genes of four bacterial strains isolated from root nodules of Phaseolus vulgaris grown in Morocco soils were identified as Burkholderia phymatum. All four strains formed N(2)-fixing nodules on P. vulgaris and Mimosa, Acacia, and Prosopis species and reduced acetylene to ethylene when cultured ex planta.

The Bradyrhizobium japonicum napEDABC genes are controlled by the FixLJ-FixK(2)-NnrR regulatory cascade.

Nitrate respiration by the N(2)-fixing symbiotic bacteria Bradyrhizobium japonicum USDA110 is mediated by a Nap (periplasmic nitrate reductase) encoded by the napEDABC genes. Expression of a transcriptional fusion of the nap promoter region to the reporter gene lacZ, P(napE)-lacZ, was very low in aerobically grown cells of USDA110, but expression was induced approx. 3-fold when the cells were cultured under microaerobic conditions, and 12-fold when nitrate was added to the microaerobic incubation medium. The P(napE)-lacZ fusion was not expressed in the fixL 7403, fixJ 7360 and fixK(2) 9043 mutant strains. Microaerobic induction of the P(napE)-lacZ fusion was retained in the nnrR 8678 mutant, but no increase in beta-galactosidase activity was observed upon nitrate addition. Western-blot and Methyl Viologen-dependent nitrate reductase activity assays showed that synthesis and activity of the catalytic NapA subunit in USDA110 was similar to that in the napC 0906 and nirK GRK308 mutant strains incubated microaerobically with nitrate. These results suggest that nitrate and nitrite, which are not reduced by the napC 0906 and nirK GRK308 mutant cells respectively, induced the synthesis and activity of NapA; conversely, formation of endogenous NO was not required for induction of Nap expression.

The role of Bradyrhizobium japonicum nitric oxide reductase in nitric oxide detoxification in soya bean root nodules.

The identification of nitric oxide-bound leghaemoglobin within soya bean nodules has led to the question of how Bradyrhizobium japonicum bacteroids overcome the toxicity of this nitric oxide. It has previously been shown that one candidate for nitric oxide detoxification, the respiratory nitric oxide reductase, is expressed in soya bean nodules from plants supplied with nitrate. In this paper, the role of this enzyme in nitric oxide detoxification is assessed and discussion is provided on other possible B. japonicum nitric oxide detoxification systems.

Molybdate-dependent expression of the periplasmic nitrate reductase in Bradyrhizobium japonicum.

The napEDABC genes of Bradyrhizobium japonicum encode the periplasmic nitrate reductase, an Mo-containing enzyme which catalyses the reduction of nitrate to nitrite when oxygen concentrations are limiting. In this bacterium, another set of genes, modABC, code for a high affinity ABC-type Mo transport system. A B. japonicum modA mutant has been obtained that is not capable of growing anaerobically with nitrate and lacks nitrate reductase activity. Under nitrate respiring conditions, when Mo concentrations are limiting, the B. japonicum modA mutant lacked both the 90 kDa protein corresponding to the NapA component of the periplasmic nitrate reductase, and the membrane-bound 25 kDa c-type cytochrome NapC. Regulatory studies using a napE-lacZ fusion indicated that napE expression was highly reduced in the modA mutant background when the cells were incubated anaerobically with nitrate under Mo-deficient conditions.

The complete denitrification pathway of the symbiotic, nitrogen-fixing bacterium Bradyrhizobium japonicum.

Denitrification is an alternative form of respiration in which bacteria sequentially reduce nitrate or nitrite to nitrogen gas by the intermediates nitric oxide and nitrous oxide when oxygen concentrations are limiting. In Bradyrhizobium japonicum, the N(2)-fixing microsymbiont of soya beans, denitrification depends on the napEDABC, nirK, norCBQD, and nosRZDFYLX gene clusters encoding nitrate-, nitrite-, nitric oxide- and nitrous oxide-reductase respectively. Mutational analysis of the B. japonicum nap genes has demonstrated that the periplasmic nitrate reductase is the only enzyme responsible for nitrate respiration in this bacterium. Regulatory studies using transcriptional lacZ fusions to the nirK, norCBQD and nosRZDFYLX promoter region indicated that microaerobic induction of these promoters is dependent on the fixLJ and fixK(2) genes whose products form the FixLJ-FixK(2) regulatory cascade. Besides FixK(2), another protein, nitrite and nitric oxide respiratory regulator, has been shown to be required for N-oxide regulation of the B. japonicum nirK and norCBQD genes. Thus nitrite and nitric oxide respiratory regulator adds to the FixLJ-FixK(2) cascade an additional control level which integrates the N-oxide signal that is critical for maximal induction of the B. japonicum denitrification genes. However, the identity of the signalling molecule and the sensing mechanism remains unknown.

Function of the Rhizobium etli CFN42 nirK gene in nitrite metabolism.

Rhizobium etli CFN42 is not capable of growing anaerobically with nitrate but it grows with nitrite as a terminal electron acceptor. This bacterium contains the nirK gene encoding the copper-containing Nir (nitrite reductase), which is located on the cryptic plasmid pCFN42f. Mutational analysis has demonstrated that a nirK deficient mutant was not capable of growing under nitrite-respiring conditions. Moreover, microaerobic growth of this mutant was inhibited by the presence of nitrite. Nir activity and nitrite uptake were highly diminished in a nirK mutant, compared with the wild-type levels after incubation under anaerobic conditions. Our results suggest that the copper-containing Nir may have both a respiratory and a nitrite-detoxifying role in R. etli.

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).

Detection, purification and characterisation of quorum-sensing signal molecules in plant-associated bacteria.

Quorum sensing (also called autoinduction) is a term that describes an environmental sensing system that allows bacteria to monitor their own population density. Autoinduction relies upon the interaction of a small diffusible signal molecule (the autoinducer) with a transcriptional activator protein to couple gene expression with cell population density. These signal molecules diffuse from bacterial cells and accumulate in the environment as a function of cell growth. Once a threshold concentration is reached, these signals serve as co-inducers to regulate the transcription of (a) set(s) of target genes. In Gram-negative bacteria, most autoinducers belong to the family of N-acylhomoserine lactones (AHLs). The detection of AHLs (or AHL-like activities) has been greatly facilitated by the development of sensitive bioassays that allow fast screening of microorganisms for diffusible signal molecules. AHL or diketopiperazine-mediated cell-cell signalling play roles in regulating different bacterial functions, such as antibiotic biosynthesis, production of virulence factors, exopolysaccharide biosynthesis, bacterial swarming, plasmid conjugal transfer and transition into the stationary phase. Several bacterial species that interact with plants produce AHL-like compounds. In this review, we will summarise the current knowledge about the detection, characterisation and purification of quorum-sensing molecules from plant-associated bacteria. We will also discuss some of the future prospects and biotechnological applications of autoinducers.

The nir, nor, and nos denitrification genes are dispersed over the Bradyrhizobium japonicum chromosome.

Cleavage of genomic DNA from Bradyrhizobium japonicum strain 3I1b110 by the restriction enzymes PmeI, PacI, and SwaI has been used together with pulsed-field gel electrophoresis and Southern hybridization to locate the nirK, norCBQD, and nosRZDFYLX denitrification genes on the chromosomal map of B. japonicum strain 110spc4. Mutant strains GRK13, GRC131, and GRZ25 were obtained by insertion of plasmid pUC4-KIXX-aphII-PSP, which carries recognition sites for the enzymes PacI, PmeI and SwaI, into the B. japonicum 3I1b110 nirK, norC and nosZ genes, respectively. Restriction of strain 3I1b110 genomic DNA with PacI, PmeI and SwaI yielded three, five and nine fragments, respectively. Pulsed-field gel electrophoresis of restricted mutant DNAs resulted in an altered fragment pattern that allowed determination of the position of the selected genes. Complementary mapping data were obtained by hybridization using digoxigenin-labeled B. japonicum 3I1b110 nirK, norBQD and nosZD as gene probes. The nirK, norCBQD and nosRZDFYLX genes were located close to the groEL(2), cycH and cycVWX genes, respectively, on the strain 110spc4 genetic map. In contrast to other denitrifiers, B. japonicum 3I1b110 denitrification genes were dispersed over the entire chromosome.

One of two hemN genes in Bradyrhizobium japonicum is functional during anaerobic growth and in symbiosis.

Previously, we screened the symbiotic gene region of the Bradyrhizobium japonicum chromosome for new NifA-dependent genes by competitive DNA-RNA hybridization (A. Nienaber, A. Huber, M. Göttfert, H. Hennecke, and H. M. Fischer, J. Bacteriol. 182:1472-1480, 2000). Here we report more details on one of the genes identified, a hemN-like gene (now called hemN(1)) whose product exhibits significant similarity to oxygen-independent coproporphyrinogen III dehydrogenases involved in heme biosynthesis in facultatively anaerobic bacteria. In the course of these studies, we discovered that B. japonicum possesses a second hemN-like gene (hemN(2)), which was then cloned by using hemN(1) as a probe. The hemN(2) gene maps outside of the symbiotic gene region; it is located 1.5 kb upstream of nirK, the gene for a Cu-containing nitrite reductase. The two deduced HemN proteins are similar in size (445 and 450 amino acids for HemN(1) and HemN(2), respectively) and share 53% identical (68% similar) amino acids. Expression of both hemN genes was monitored with the help of chromosomally integrated translational lacZ fusions. No significant expression of either gene was detected in aerobically grown cells, whereas both genes were strongly induced (> or = 20-fold) under microaerobic or anaerobic conditions. Induction was in both cases dependent on the transcriptional activator protein FixK(2). In addition, maximal anaerobic hemN(1) expression was partially dependent on NifA, which explains why this gene had been identified by the competitive DNA-RNA hybridization approach. Strains were constructed carrying null mutations either in individual hemN genes or simultaneously in both genes. All mutants showed normal growth in rich medium under aerobic conditions. Unlike the hemN(1) mutant, strains lacking a functional hemN(2) gene were unable to grow anaerobically under nitrate-respiring conditions and largely failed to fix nitrogen in symbiosis with the soybean host plant. Moreover, these mutants lacked several c-type cytochromes which are normally detectable by heme staining of proteins from anaerobically grown wild-type cells. Taken together, our results revealed that B. japonicum hemN(2), but not hemN(1), encodes a protein that is functional under the conditions tested, and this conclusion was further corroborated by the successful complementation of a Salmonella enterica serovar Typhimurium hemF hemN mutant with hemN(2) only.

Carbon monoxide dehydrogenase activity in Bradyrhizobium japonicum.

Bradyrhizobium japonicum strain 110spc4 was capable of chemolithoautotrophic growth with carbon monoxide (CO) as a sole energy and carbon source under aerobic conditions. The enzyme carbon monoxide dehydrogenase (CODH; EC 1.2.99.2) has been purified 21-fold, with a yield of 16% and a specific activity of 58 nmol of CO oxidized/min/mg of protein, by a procedure that involved differential ultracentrifugation, anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. The purified enzyme gave a single protein and activity band on nondenaturing polyacrylamide gel electrophoresis and had a molecular mass of 230,000 Da. The 230-kDa enzyme was composed of large (L; 75-kDa), medium (M; 28.4-kDa), and small (S; 17.2-kDa) subunits occurring in heterohexameric (LMS)(2) subunit composition. The 75-kDa polypeptide exhibited immunological cross-reactivity with the large subunit of the CODH of Oligotropha carboxidovorans. The B. japonicum enzyme contained, per mole, 2.29 atoms of Mo, 7.96 atoms of Fe, 7.60 atoms of labile S, and 1.99 mol of flavin. Treatment of the enzyme with iodoacetamide yielded di(carboxamidomethyl)molybdopterin cytosine dinucleotide, identifying molybdopterin cytosine dinucleotide as the organic portion of the B. japonicum CODH molybdenum cofactor. The absorption spectrum of the purified enzyme was characteristic of a molybdenum-containing iron-sulfur flavoprotein.

Characterization of Bradyrhizobium japonicum pcaBDC genes involved in 4-hydroxybenzoate degradation.

The pca structural genes encode enzymes that participate in the conversion of protocatechuate to succinate and acetylcoenzyme A. A 3. 05-kb region of the Bradyrhizobium japonicum strain USDA110 genome has been characterized, which contains the pcaB, pcaD and pcaC genes. The predicted protein sequences of the three genes have extensive homologies with beta-carboxy-cis,cis-muconate cycloisomerase (PcaB), beta-ketodiapate enol-lactone hydrolase (PcaD), and gamma-carboxymuconolactone decarboxylase (PcaC), respectively, from Acinetobacter calcoaceticus and Pseudomonas putida. The DNA sequence revealed that the pca genes are probably arranged in a single transcriptional unit, pcaBDC, similar to that described in P. putida. A pcaB deletion mutant constructed by marker exchange mutagenesis lost the ability to use 4-hydroxybenzoate or protocatechuate as the only carbon source, demonstrating functionality of the characterized genes in catabolism of hydroxyaromatics by B. japonicum. Furthermore, 4-hydroxybenzoate and protocatechuate became toxic for the pcaB mutant, indicating that hydroxyaromatics catabolism serves both nutritional and detoxifying purposes.

Genes involved in the formation and assembly of rhizobial cytochromes and their role in symbiotic nitrogen fixation.

Rhizobia fix nitrogen in a symbiotic association with leguminous plants and this occurs in nodules. A low-oxygen environment is needed for nitrogen fixation, which paradoxically has a requirement for rapid respiration to produce ATP. These conflicting demands are met by control of oxygen flux and production of leghaemoglobin (an oxygen carrier) by the plant, coupled with the expression of a high-affinity oxidase by the nodule bacteria (bacteroids). Many of the bacterial genes encoding cytochrome synthesis and assembly have been identified in a variety of rhizobial strains. Nitrogen-fixing bacteroids use a cytochrome cbb3-type oxidase encoded by the fixNOQP operon; electron transfer to this high-affinity oxidase is via the cytochrome bc1 complex. During free-living growth, electron transport from the cytochrome bc1 complex to cytochrome aa3 occurs via a transmembrane cytochrome c (CycM). In some rhizobia (such as Bradyrhizobium japonicum) there is a second cytochrome oxidase that also requires electron transport via the cytochrome bc1 complex. In parallel with these cytochrome c oxidases there are quinol oxidases that are expressed during free-living growth. A cytochrome bb3 quinol oxidase is thought to be present in B. japonicum; in Rhizobium leguminosarum, Rhizobium etli and Azorhizobium caulinodans cytochrome d-type oxidases have been identified. Spectroscopic data suggest the presence of a cytochrome o-type oxidase in several rhizobia, although the absence of haem O in B. japonicum may indicate that the absorption attributed to cytochrome o could be due to a high-spin cytochrome b in a cytochrome bb3-type oxidase. In some rhizobia, mutation of genes involved in cytochrome c assembly does not strongly affect growth, presumably because the bacteria utilize the cytochrome c-independent quinol oxidases. In this review, we outline the work on various rhizobial mutants affected in different components of the electron transport pathways, and the effects of these mutations on symbiotic nitrogen fixation and free-living growth.