Ilona Dusha

Location of nodulation and nitrogen fixation genes on a high molecular weight plasmid of R. meliloti

SummaryR. meliloti strain 41 (Rm41) was shown to harbour two indigenous plasmids with molecular weights of 140 Mdal (pRmc41a) and more than 300 Mdal (pRme41b), respectively. Using a heat-treatment procedure, derivatives of Rm41 defective in nodulation (Nod-) or nitrogen fixation (Fix-) have been readily obtained. In some Nod- mutants the deletion of a segment of plasmid pRme41b was found.Based on the demonstrated homology between the nitrogen fixation (nif) genes of Klebsiella pneumoniae and of R. meliloti the Rhizobium nif region has been cloned into the cosmid vector pHC79, then recloned into pBR322 and the restriction map of the nif region has been determined. 32P-labelled nick-translated probe prepared from the cloned nif DNA fragment hybridized to pRme41b of Rm41 but for most Nod- mutants this hybridization was not detected. Hybridization of a cosmid containing Rm41 DNA to total DNA digests from the wild-type bacterium and from a series of Nod- mutants revealed that at least a 24 kb DNA fragment including the nif structural genes was missing from most of the Nod- mutants. These results, together with the genetic analyses of these symbiotic mutations suggest that some nod and fix genes are located on pRme41b.

The Rhizobium meliloti early nodulation genes (nodABC) are nitrogen-regulated: Isolation of a mutant strain with efficient nodulation capacity on alfalfa in the presence of ammonium

SummaryThe presence of combined nitrogen in the soil suppresses the formation of nitrogen-fixing root nodules by Rhizobium. We demonstrate that bacterial genes determining early nodulation functions (nodABC) as well as the regulatory gene nodD3 are under nitrogen (NH4+) control. Our results suggest that the gene product of nodD3 has a role in mediating the ammonia regulation of early nod genes. The general nitrogen regulatory (ntr) system as well as a chromosomal locus mutated in Rhizobium meliloti were also found to be involved in the regulation of nod gene expression. A R. meliloti mutant with altered sensitivity to ammonia regulation was isolated, capable of more efficient nodulation of alfalfa than the wild-type strain in the presence of 2 mM ammonium sulfate.

Genes at different regulatory levels are required for the ammonia control of nodulation in Rhizobium meliloti

The expression of the nodulation genes nodABC of Rhizobium meliloti, which determine early response functions to plant host signals, is regulated by the level of ammonia, the primary product of symbiotic nitrogen fixation. We show that the pathway that links the ammonia-induced signal to the transcriptional control of the nodABC genes involves at least two regulatory levels. The fluctuating nitrogen level is sensed and the signal is mediated by the members of the general nitrogen regulatory (ntr) system, then transmitted to the syrM-nodD3 genes representing the nod-specific level of ammonia regulation. At low ammonia concentration, the activator protein NtrC exerts its effect via nodD3. In conditions of nitrogen excess ntrR, involved in the repression of nod genes, may function in coordination with the syrM gene. Finally, the NodD3 protein may relay the nitrogen status signal to the transcriptional control of the nodABC genes.

The ntrPR Operon of Sinorhizobium meliloti Is Organized and Functions as a Toxin-Antitoxin Module

The chromosomal ntrPR operon of Sinorhizobium meliloti encodes a protein pair that forms a toxin-antitoxin (TA) module, the first characterized functional TA system in Rhizobiaceae. Similarly to other bacterial TA systems, the toxin gene ntrR is preceded by and partially overlaps with the antitoxin gene ntrP. Based on protein homologies, the ntrPR operon belongs to the vapBC family of TA systems. The operon is negatively autoregulated by the NtrPNtrR complex. Promoter binding by NtrP is weak; stable complex formation also requires the presence of NtrR. The N-terminal part of NtrP is responsible for the interaction with promoter DNA, whereas the C-terminal part is required for protein-protein interactions. In the promoter region, a direct repeat sequence was identified as the binding site of the NtrPNtrR complex. NtrR expression resulted in the inhibition of cell growth and colony formation; this effect was counteracted by the presence of the antitoxin NtrP. These results and our earlier observations demonstrating a less effective downregulation of a wide range of symbiotic and metabolic functions in the ntrR mutant under microoxic conditions and an increased symbiotic efficiency with the host plant alfalfa suggest that the ntrPR module contributes to adjusting metabolic levels under symbiosis and other stressful conditions.

syrM Is Involved in the Determination of the Amount and Ratio of the Two Forms of the Acidic Exopolysaccharide EPSI in Rhizobium meliloti

For the invasion of root nodule cells of alfalfa by its symbiotic partner Rhizobium meliloti, the bacterial succinoglycan (EPSI) is required. Nitrogen starvation of R. meliloti results in a considerable increase of EPSI production. We have shown that the presence of nitrogen also altered the ratio of high and low molecular weight (HMW and LMW, respectively) forms of EPSI, favoring the production of LMW molecules. The transcription of two exo genes (exoF, exoP) was found to be under the control of combined nitrogen. In contrast, exoH and exoK were expressed independently of the nitrogen level. ntrC and syrM, regulatory genes for the nitrogen control of nod gene expression, were shown to be involved in the regulation of EPSI production. In an syrM mutant both the amount and the ratio of the two main forms of EPSI were altered, compared with the wild-type strain: a reduced level of EPSI and the dominance of the LMW form in nitrogenstarved cultures, while an increased level of EPSI and the accumulation of the HMW fraction in nitrogen-containing samples was observed. An enhanced expression level of the exoK gene and the higher ratio of the LMW form to the total EPSI in nitrogen-free samples of the syrM mutant suggest a regulatory role for syrM in exoK expression and a contribution of exoK to the accumulation of the LMW form of EPSI. During the formation of nitrogen-fixing root nodules, signal molecules are exchanged between the host plant alfalfa (Medicago sativa) and its symbiotic partner Rhizobium meliloti. The signal molecules are required for the control of the highly specific plant-bacterium recognition and for the coordinated interaction of the two organisms. Molecular mechanisms acting at the early steps of nodule formation have been characterized in the R. meliloti‐M. sativa

Wide-range transcriptional modulating effect of ntrR under microaerobiosis in Sinorhizobium meliloti.

A mutation in the second gene in the ntrPR operon results in increased expression of nodulation ( nod) and nitrogen fixation ( nif) genes in Sinorhizobium meliloti. Since this pleiotropic effect is particularly pronounced in the presence of external combined nitrogen, a nitrogen regulatory function has been suggested for NtrR. To identify the complete set of protein-coding genes influenced by loss of ntrR function, microarray hybridizations were carried out to compare transcript levels in the wild type and mutant strains grown under aerobic and microaerobic conditions. Of the 6207 genes examined, representing the entire genome of S. meliloti, 7% exhibited altered expression: 4.5% of the genes are affected under oxic, 2.5% under microoxic conditions. 0.4% of all the genes are affected under both oxygen concentrations. A microoxic environment is required for the induction of genes related to symbiotic functions but results in the down-regulation of other (e.g. metabolic) functions. When the alterations in transcription levels at low oxygen concentration in the mutant strain were compared to those of the wild type, a modulating effect of the ntrR mutation was observed. For example, symbiotic nif/fix genes were induced in both strains, but the level of induction was higher in the ntrR mutant. In contrast, genes related to transcription/translation functions were down-regulated in both strains, and the effect was greater in the wild-type strain than in the ntrR mutant. A relatively wide range of functions was affected by this modulating influence, suggesting that ntrR is not a nitrogen regulatory gene. Since genes encoding various unrelated functions were affected, we propose that NtrR may either interfere with general regulatory mechanisms, such as phosphorylation/dephosphorylation, or may influence RNA stability.

The roles of different regions of the CycH protein in c-type cytochrome biogenesis in Sinorhizobium meliloti

Cytochrome c heme lyases encoded by the Sinorhizobium meliloti cycHJKL operon are responsible for generating the covalent bond between the heme prosthetic group and apocytochromes c. The CycH protein with its presumably membrane-associated N-terminal and periplasmic C-terminal parts is thought to be responsible for binding apocytochrome and presenting it to the heme ligation machinery. We propose that these two modules of CycH play roles in different functions of the protein. The N-terminal 96 amino acids represent an active subdomain of the protein, which is able to complement the protoporphyrin IX (PPIX) accumulation phenotype of the cycH mutant strain AT342, suggesting that it is involved in the final steps of heme C biosynthesis. Furthermore, three tetratricopeptide (TPR) domains have been identified in the C-terminal periplasmic region of the CycH protein. TPR domains are known to mediate protein-protein interactions. Each of these CycH domains is absolutely required for protein function, since plasmid constructs carrying cycH genes with in-frame TPR deletions were not able to complement cycH mutants for their nitrate reductase (Rnr−) and nitrogen-fixing (Fix−) phenotypes. We also found that the 309-amino acid N-terminal portion of the CycH, which includes all the TPR domains, is able to mediate the assembly of the c-type cytochromes required for the Rnr+ phenotype. In contrast, only the full-length protein confers the ability to fix nitrogen.

Purification and properties of tyrosine-sensitive 3-deoxy-D-arabino-heptolosonate-7-phosphate synthetase of Escherichia coli K12.

1. The tyrosine-sensitive allosteric first enzyme of the aromatic amino acid biosynthetic pathway, 3-deoxy-D-arabinoheptulosonate 7-phosphate synthetase (7-Phospho-2-keto-3-deoxy-D-arabino-heptonate D-erythrose 4-phosphate-lyase (pyruvate phosphorylating), EC 4.1.2.15) has been purified from a mutant strain of Escherichia coli. 2. The enzyme activity was inhibited to 50% at 2-10(-5) M tyrosine and to 90% at 2-10(-4) M tyrosine concentration. At tyrosine concentrations lower than 2-10(-5) M a cooperative interaction between tyrosine binding sites was observed. 3. Co2+ increased the enzyme activity about 2-2.5-fold. The presence of Co2+ ions stabilized the enzyme. EDTA inhibited the enzyme activity, and this inhibition was reversed by Co2+. Tyrosine-sensitive DAHP synthetase seems to be a metal containing enzyme. 4. Kinetic experiments were carried out to study the catalytic action. Contrary to earlier suggestions it is concluded, that the reaction mechanism appears to be more complex--with either the ping-pong or sequential type predominating, depending on conditions.

Atypical Transcriptional Regulation and Role of a New Toxin-Antitoxin-Like Module and Its Effect on the Lipid Composition of Bradyrhizobium japonicum

A toxin-antitoxin (TA)-like system (designated as bat/bto genes) was identified in Bradyrhizobium japonicum, based on sequence homology and similarities in organization and size to known TA systems. Deletion of the bat/bto module resulted in pleiotropic alterations in cell morphology and metabolism. The generation time of the mutant was considerably decreased in rich media. Atomic force microscopy revealed the modified shape (shorter and wider) and softness of mutant cells. The synthesis of phosphatidylcholine was completely blocked in the mutant bacteria, and vaccenic acid, the predominant fatty acid of membranes of the wild-type cell, was replaced by palmitic acid in the mutant membranes. The mutant bacteria synthesized incomplete lipopolysaccharide molecules. Remarkable changes in the membrane lipid composition may explain the observed morphological alterations and growth properties of the mutant bacteria. The overlapping promoter region of bat/bto and glpD (coding for the aerobic sn-glycerol-3-phosphate dehydrogenase) genes suggests a complex regulation and the involvement of bat/bto in the control of main metabolic pathways and an important role in the maintenance of a normal physiological state of B. japonicum. These data reveal new aspects of the role of TA systems in bacteria.

A simplified assay of enzymes catalyzing ATP-pyrophosphate exchange reactions

Abstract The ATP-pyrophosphate exchange reaction is a widely used method for determination of the activity of enzymes forming enzyme-adenylate intermediates in the course of their catalytic action (1–4). For separation of the labeled ATP formed in the enzyme-catalyzed reaction from the labeled pyrophosphate, the adsorption of ATP on activated Norit is generally used. This method, however, is rather time consuming (considering the extensive washings required for the removal of pyrophosphate from Norit), and its reproducibility is highly dependent on pretreatment of charcoal with acid, Another method of separation applies anion-exchange paper (5). According to our experience, separation on DEAE-cellulose paper (Whatman DE 81) is not sufficient. The present paper describes a simple and reproducible thin-layer chromatographic method for the separation of ATP and pyrophosphate. The system consists of commercial chromatoplates precoated with strong anion-exchange resin (Ionex-25 SB-Ac or Fixion 2X8) and 0.1 m aqueous sodium pyrophosphate, pH 8, as developing solvent.