Isolation and characterization of Rhizobium etli mutants altered in degradation of asparagine

Abstract

Rhizobium etli mutants unable to grow on asparagine as the nitrogen and carbon source were isolated. Two kinds of mutants were obtained: AHZ1, with very low levels of aspartase activity, and AHZ7, with low levels of asparaginase and very low levels of aspartase compared to the wild-type strain. R. etli had two asparaginases differentiated by their thermostabilities, electrophoretic mobilities, and modes of regulation. The AHZ mutants nodulated as did the wild-type strain and had nitrogenase levels similar to that of the wild-type strain.Amino acid synthesis has been studied in various microorganisms; amino acid degradation has been less studied, despite the fact that the intracellular amino acid concentration must be the result of its synthesis and degradation. In Neurospora crassa and Rhizobium etli, it has been shown that the intracellular glutamine concentration is the result not only of the regulation of the synthesis but also of the degradation of this amino acid (4, 6-8, 15, 16, 31). To determine if the intracellular concentrations of other amino acids are regulated in the same way as that of glutamine, we decided to study the degradation of asparagine (an amino acid very similar to glutamine, with four carbon skeletons instead of five) in R. etli, a bacterium that establishes symbiosis with Phaseolus vulgaris (39).In R. etli, L-asparagine can be utilized as the sole carbon and nitrogen source through the action of two enzymes (20). The first enzyme, asparaginase (EC 3.5.1.1), catalyzes the hydrolysis of L-asparagine to L-aspartate and ammonium. The second enzyme, L-aspartase (EC 4.3.1.1), catalyzes the reversible deamination of L-aspartate to yield fumarate and ammonium. R. etli asparaginase is found to be positively regulated by its substrate asparagine and negatively regulated by the carbon source; it is not regulated by the amount of oxygen dissolved in the growth medium or by nitrogen catabolite repression, and some asparaginase activity is detected when R. etli is grown on ammonium as a nitrogen source and succinate as a carbon source (20). Asparaginase has been studied in other gramnegative bacteria such as Escherichia coli (10,12,22,24,25,37,47), Salmonella enterica (23), Erwinia chrysanthemi (19), and Vibrio proteus (41) and in gram-positive bacteria such as Bacillus subtilis (1, 21, 43) and Staphylococcus aureus (35,42).R. etli aspartase was found to be positively regulated by asparagine, negatively regulated by the carbon source and by ammonium, and not regulated by the amount of oxygen dissolved in the growth medium (20). L-Aspartase has been studied in other bacteria such as E. coli (25,27,33,36), B. subtilis (43), Pseudomonas fluorescens (30, 45), and Serratia marcescens (44).In this report, we describe the isolation and characterization of R. etli mutants altered in the degradation of asparagine, present evidence that R. etli has two asparaginases, and investigate the regulation of these isoenzymes. The cloning of an R. etli DNA fragment that complements the isolated mutants is also reported...

Lipogenesis and Redox Balance in Nitrogen-Fixing Pea Bacteroids

“…Furthermore, free asparagine is not made by Rlv3841, which, from analysis of its genome, uses the GatCAB pathway to insert asparagine into proteins by charging asparaginyl-tRNA with aspartate and then transamidating aspartate to asparagine (43). In addition, catabolism of asparagine and homoserine is not upregulated in bacteroids (44), nor do catabolic mutants show reduced N 2 fixation rates (45,46), consistent with minor roles in symbiosis.…”

Diminished Redundancy of Outer Membrane Factor Proteins in Rhizobiales: A <i>nodT</i> Homolog Is Essential for Free-Living <i>Rhizobium etli</i>

“…To isolate and functionally characterize these nodT genes, a R. etli genomic library [Huerta-Zepeda et al, 1997] was screened using the R . leg .…”

Identification of Functional mob Regions in Rhizobium etli : Evidence for Self-Transmissibility of the Symbiotic Plasmid pRetCFN42d

“…Merodiploid populations of R. etli strains CE3 and CFNX182, a CE3 derivative cured of plasmid p42a (see Table 1), were obtained after introducing by electroporation cosmids from a R. etli CE3 gene library made in vector pLAFR1 (25) into each strain. Each merodiploid population was used as donor en masse in matings with R. etli CFNX218Rif or E. coli HB101 as recipients.…”