Regulatory role of Rhizobium etli CNPAF512 fnrN during symbiosis

The Rhizobium etli CNPAF512 fnrN gene was identified in the fixABCX rpoN(2) region. The corresponding protein contains the hallmark residues characteristic of proteins belonging to the class IB group of Fnr-related proteins. The expression of R. etli fnrN is highly induced under free-living microaerobic conditions and during symbiosis. This microaerobic and symbiotic induction of fnrN is not controlled by the sigma factor RpoN and the symbiotic regulator nifA or fixLJ, but it is due to positive autoregulation. Inoculation of Phaseolus vulgaris with an R. etli fnrN mutant strain resulted in a severe reduction in the bacteroid nitrogen fixation capacity compared to the wild-type capacity, confirming the importance of FnrN during symbiosis. The expression of the R. etli fixN, fixG, and arcA genes is strictly controlled by fnrN under free-living microaerobic conditions and in bacteroids during symbiosis with the host. However, there is an additional level of regulation of fixN and fixG under symbiotic conditions. A phylogenetic analysis of the available rhizobial FnrN and FixK proteins grouped the proteins in three different clusters.     

Catabolic ornithine transcarbamylase of Halobacterium halobium (salinarium): purification, characterization, sequence determination, and evolution.

Halobacterium halobium (salinarium) is able to grow fermentatively via the arginine deiminase pathway, which is mediated by three enzymes and one membrane-bound arginine-ornithine antiporter. One of the enzymes, catabolic ornithine transcarbamylase (cOTCase), was purified from fermentatively grown cultures by gel filtration and ammonium sulfate-mediated hydrophobic chromatography. It consists of a single type of subunit with an apparent molecular mass of 41 kDa. As is common for proteins of halophilic Archaea, the cOTCase is unstable below 1 M salt. In contrast to the cOTCase from Pseudomonas aeruginosa, the halophilic enzyme exhibits Michaelis-Menten kinetics with both carbamylphosphate and ornithine as substrates with Km values of 0.4 and 8 mM, respectively. The N-terminal sequences of the protein and four peptides were determined, comprising about 30% of the polypeptide. The sequence information was used to clone and sequence the corresponding gene, argB. It codes for a polypeptide of 295 amino acids with a calculated molecular mass of 32 kDa and an amino acid composition which is typical of halophilic proteins. The native molecular mass was determined to be 200 kDa, and therefore the cOTCase is a hexamer of identical subunits. The deduced protein sequence was compared to the cOTCase of P. aeruginosa and 14 anabolic OTCases, and a phylogenetic tree was constructed. The halobacterial cOTCase is more distantly related to the cOTCase than to the anabolic OTCase of P. aeruginosa. It is found in a group with the anabolic OTCases of Bacillus subtilis, P. aeruginosa, and Mycobacterium bovis.     

Pseudomonas aeruginosa mutants affected in anaerobic growth on arginine: evidence for a four-gene cluster encoding the arginine deiminase pathway.

Pseudomonas aeruginosa PAO was able to grow in the absence of exogenous terminal electron acceptors, provided that the medium contained 30 to 40 mM L-arginine and 0.4% yeast extract. Under strictly anaerobic conditions (O2 at less than 1 ppm), growth could be measured as an increase in protein and proceeded in a non-exponential way; arginine was largely converted to ornithine but not entirely consumed at the end of growth. In the GasPak anaerobic jar (Becton Dickinson and Co.), the wild-type strain PAO1 grew on arginine-yeast extract medium in 3 to 5 days; mutants could be isolated that were unable to grow under these conditions. All mutants (except one) were defective in at least one of the three enzymes of the arginine deiminase pathway (arcA, arcB, and arcC mutants) or in a novel function that might be involved in anaerobic arginine uptake (arcD mutants). The mutations arcA (arginine deiminase), arcB (catabolic ornithine carbamoyltransferase), arcC (carbamate kinase), and arcD were highly cotransducible and mapped in the 17-min chromosome region. Some mutations in the arc cluster led to low, noninducible levels of all three arginine deiminase pathway enzymes and thus may affect control elements required for induction of the postulated arc operon. Two fluorescent pseudomonads (P. putida and P. fluorescens) and P. mendocina, as well as one PAO mutant, possessed an inducible arginine deiminase pathway and yet were unable to grow fermentatively on arginine. The ability to use arginine-derived ATP for growth may provide P. aeruginosa with a selective advantage when oxygen and nitrate are scarce.     

Effective symbiosis between Rhizobium etli and Phaseolus vulgaris requires the alarmone ppGpp

The symbiotic interaction between Rhizobium etli and Phaseolus vulgaris, the common bean plant, ultimately results in the formation of nitrogen-fixing nodules. Many aspects of the intermediate and late stages of this interaction are still poorly understood. The R. etli relA gene was identified through a genome-wide screening for R. etli symbiotic mutants. RelA has a pivotal role in cellular physiology, as it catalyzes the synthesis of (p)ppGpp, which mediates the stringent response in bacteria. The synthesis of ppGpp was abolished in an R. etli relA mutant strain under conditions of amino acid starvation. Plants nodulated by an R. etli relA mutant had a strongly reduced nitrogen fixation activity (75% reduction). Also, at the microscopic level, bacteroid morphology was altered, with the size of relA mutant bacteroids being increased compared to that of wild-type bacteroids. The expression of the sigma(N)-dependent nitrogen fixation genes rpoN2 and iscN was considerably reduced in the relA mutant. In addition, the expression of the relA gene was negatively regulated by RpoN2, the symbiosis-specific sigma(N) copy of R. etli. Therefore, an autoregulatory loop controlling the expression of relA and rpoN2 seems operative in bacteroids. The production of long- and short-chain acyl-homoserine-lactones by the cinIR and raiIR systems was decreased in an R. etli relA mutant. Our results suggest that relA may play an important role in the regulation of gene expression in R. etli bacteroids and in the adaptation of bacteroid physiology.     

Mapping of the arginine deiminase gene in Pseudomonas aeruginosa

A mutant of Pseudomonas aeruginosa PAO lacking arginine deiminase activity (arcA) was isolated by screening for a derivative of an arcB mutant (deficient in catabolic ornithine carbamoyltransferase) that did not excrete citrulline under conditions of limited aeration. The arcA mutation was highly cotransducible with arcB.     

The arc operon for anaerobic arginine catabolism in Pseudomonas aeruginosa contains an additional gene, arcD, encoding a membrane protein

The arginine deiminase (ADI) pathway in Pseudomonas aeruginosa serves to generate ATP. The three enzymes involved, ADI, catabolic ornithine carbamoyltransferase and carbamate kinase, are induced by oxygen limitation and encoded by the contiguous arcABC genes. A 1.5-kb region upstream from arcABC was sequenced and found to contain an open reading frame, arcD, coding for a hydrophobic polypeptide of 52 kDa. The content and distribution of hydrophobic amino acids suggest that the arcD gene product may be a transmembrane protein. When arcD was fused to an Escherichia coli promoter, the ArcD protein was synthesized in E. coli maxicells and detected in the membrane fraction. In sodium dodecyl sulfate-polyacrylamide-gel electrophoresis the ArcD protein migrated like a 32-kDa protein; such anomalous electrophoretic mobility is known for other highly hydrophobic proteins. Mutations in arcD rendered the cells unable to utilize extracellular arginine as an energy source. Since anaerobic arginine consumption and ornithine release are coupled in P. aeruginosa, it is proposed that arcD specifies an arginine: ornithine antiporter or a part thereof. Insertions of IS21 or Tn1725 in arcD had a strong polar effect on the expression of the arcAB enzymes, indicating that the arc genes are organized as an arcDABC operon.     

Arginine Deiminase in Staphylococcus epidermidis Functions To Augment Biofilm Maturation through pH Homeostasis

Allelic replacement mutants were constructed within arginine deiminase (arcA1 and arcA2) to assess the function of the arginine deiminase (ADI) pathway in organic acid resistance and biofilm formation of Staphylococcus epidermidis 1457. A growth-dependent acidification assay (pH ∼5.0 to ∼5.2) determined that strain 1457 devoid of arginine deiminase activity (1457 ΔADI) was significantly less viable than the wild type following depletion of glucose and in the presence of arginine. However, no difference in viability was noted for individual 1457 ΔarcA1 (native) or ΔarcA2 (arginine catabolic mobile element [ACME]-derived) mutants, suggesting that the native and ACME-derived ADIs are compensatory in S. epidermidis. Furthermore, flow cytometry and electron paramagnetic resonance spectroscopy results suggested that organic acid stress resulted in oxidative stress that could be partially rescued by the iron chelator dipyridyl. Collectively, these results suggest that formation of hydroxyl radicals is partially responsible for cell death via organic acid stress and that ADI-derived ammonia functions to counteract this acid stress. Finally, static biofilm assays determined that viability, ammonia synthesis, and pH were reduced in strain 1457 ΔADI following 120 h of growth in comparison to strain 1457 and the arcA1 and arcA2 single mutants. It is hypothesized that ammonia synthesis via the ADI pathway is important to reduce pH stress in specific microniches that contain high concentrations of organic acids.     

The Rhizobium etli opt operon is required for symbiosis and stress resistance

Rhizobium etli is a Gram-negative root-colonizing soil bacterium capable of fixing nitrogen while living in symbiosis with its leguminous host Phaseolus vulgaris. A genome-wide screening for R. etli symbiotic mutants revealed a R. etli operon encoding an oligopeptide ABC-transporter (Opt), two redA homologous genes and one redB gene. Expression analysis showed this opt operon to be transcribed both under free-living and symbiotic conditions and expression levels were demonstrated to be growth-phase-dependent. Plants nodulated by R. etli opt mutants showed a reduced symbiotic nitrogen fixation activity (approximately 50% reduction). Growth experiments with opt mutants in the presence of oligopeptides as the sole nitrogen source confirmed the involvement of the opt genes in oligopeptide uptake. Further phenotypic analysis of the opt mutants revealed them to display an enhanced resistance to the oligopeptide antibiotic bacitracin, an increased susceptibility to the beta-lactam antibiotic ampicillin and a decreased osmotolerance. In conclusion, our results demonstrate that the opt operon plays a crucial role during symbiosis and stress resistance.     

Ornithine cycle in Nostoc PCC 73102. Arginase,OCT and arginine deiminase,and the effects of addition of external arginine,ornithine, or citrulline

Arginase, ornithine carbamoyl transferase (OCT) and arginine deiminase activities were found in cell-free extracts of Nostoc PCC 73102, a free-living cyanobacterium originally isolated from the cycad Macrozamia. Addition of either arginine, ornithine or citrulline to the growth medium induced significant changes in their in vitro activities. Moreover, growth in darkness, compared to in light, induced higher in vitro activities. The in vitro activities of arginase and arginine deiminase, two catabolic enzymes primarily involved in the breakdown of arginine, increased substantially by a combination of growth in darkness and addition of either arginine, or ornithine, to the growth medium. The most significant effects on the in vitro OCT activities where observed in cells grown with the addition of ornithine. Cells grown in darkness exhibited about 6% of the in vivo nitrogenase activity observed in cells grown in light. However, addition of external carbon (glucose and fructose) to cells grown in darkness resulted in in vivo nitrogenase activity levels similar to, or even higher than, cells grown in light. Growth with high in vivo nitrogenase activity or in darkness with the addition of external carbon, resulted in repressed levels of in vitro arginase and arginine deiminase activities. It is suggested that nitrogen starvation induces a mobilization of the stored nitrogen, internal release of the amino compound arginine, and an induction of two catabolic enzymes arginase and arginine deiminase. A similar and even more pronunced induction can be observed by addition of external arginine to the growth medium.     

Enhanced expression of Rhizobium etli cbb 3 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.     

Ultrastructural analysis of ineffective alfalfa nodules formed by nif::Tn5 mutants of Rhizobium meliloti.

Ineffective alfalfa nodules formed by Rhizobium meliloti nif::Tn5 mutants were examined by light and electron microscopy. R. meliloti nifH::Tn5 mutants formed nodules that were similar in structure to wild-type nodules except that nifH- bacteroids accumulated a compact, electron-dense body. In contrast to nodules induced by wild type and nifH mutants, nifDK- R. meliloti mutants induced nodules which contained numerous starch grains and prematurely senescent bacteroids. In addition, meristematic activity in nifDK- nodules ceased significantly earlier than in nifH- nodules. All mutant nodules exhibited elevated levels of rough endoplasmic reticulum and Golgi membranes compared to wild-type nodule cells. These elevated levels may reflect either a response to nitrogen starvation in the ineffective nodules or an abnormal synthesis and export of nodule-specific proteins during later developmental stages.     

The arginine deiminase pathway in the wine lactic acid bacterium Lactobacillus hilgardii X1B: structural and functional study of the arcABC genes

The genes implicated in the catabolism of the amino acid arginine by Lactobacillus hilgardii X(1)B were investigated to assess the potential for formation of ethyl carbamate precursors in wine. L. hilgardii X(1)B can use arginine via the arginine deiminase pathway. The complete nucleotide sequence of the arc genes involved in this pathway has been determined. They are clustered in an operon-like structure in the order arcABC. No evidence was found for the presence of a homologue of the arcD gene, coding for the arginine/ornithine antiporter. The arc genes have been expressed in Escherichia coli resulting in arginine deiminase (ArcA), ornithine carbamoyltransfera (ArcB) and carbamate kinase (ArcC) activities. The results indicate the need for caution in the selection of lactic acid bacteria for conducting malolactic fermentation in wine since arginine degradation could result in high amounts of ethyl carbamate.