Endophytic bacteria reside within plant tissues and have often been found to promote plant growth. Fourteen strains of putative endophytic bacteria, not including endosymbiotic Bradyrhizobium strains, were isolated from surface-sterilized soybean (Glycine max. (L.) Merr.) root nodules. These isolates were designated as non-Bradyrhizobium endophytic bacteria (NEB). Three isolates (NEB4, NEB5, and NEB17) were found to increase soybean weight when plants were co-inoculated with one of the isolates and Bradyrhizobium japonicum under nitrogen-free conditions, compared with plants inoculated with B. japonicum alone. In the absence of B. japonicum, these isolates neither nodulated soybean, nor did they affect soybean growth. All three isolates were Gram-positive spore-forming rods. While Biolog tests indicated that the three isolates belonged to the genus Bacillus, it was not possible to determine the species. Phylogenetic analysis of 16S rRNA gene hypervariant region sequences demonstrated that both NEB4 and NEB5 are Bacillus subtilis strains, and that NEB17 is a Bacillus thuringiensis strain.

SummaryMalate dehydrogenase (MDH) catalyzes the readily reversible reaction of oxaloacetate ≤ malate using either NADH or NADPH as a reductant. In plants, the enzyme is important in providing malate for C 4 metabolism, pH balance, stomatal and pulvinal movement, respiration, β-oxidation of fatty acids, and legume root nodule functioning. Due to its diverse roles the enzyme occurs as numerous isozymes in various organelles. While antibodies have been produced and cDNAs characterized for plant mitochondrial, glyoxysomal, and chloroplast forms of MDH, little is known of other forms. Here we report the cloning and characterization of cDNAs encoding five different forms of alfalfa MDH, including a plant cytosolic MDH (cMDH) and a unique novel nodule-enhanced MDH (neMDH). Phylogenetic analyses show that neMDH is related to mitochondrial and glyoxysomal MDHs, but diverge from these forms early in land plant evolution. Four of the five forms could effectively complement an E. coli Mdh -mutant. RNA and protein blots show that neMDH is most highly expressed in effective root nodules. Immunoprecipitation experiments show that antibodies produced to cMDH and neMDH are immunologically distinct and that the neMDH form comprises the major form of total MDH activity and protein in root nodules. Kinetic analysis showed that neMDH has a turnover rate and specificity constant that can account for the extraordinarily high synthesis of malate in nodules.

Aims:  The aim of this study was to identify and characterize a compound produced by the plant growth promoting bacterium, Bacillus thuringiensis non‐Bradyrhizobium Endophytic Bacterium 17. Methods and Results:  The bacterial peptide was analysed and purified via HPLC. Using the disk diffusion assay this peptide inhibited the growth of 16/19 B. thuringiensis strains, 4/4 Bacillus cereus strains, among others, as well as a Gram‐negative strain Escherichia coli MM294 (pBS42). Both bactericidal and bacteristatic effects were observed on B. cereus ATCC 14579 and bactericidal effects were observed on B. thuringiensis ssp. thuringiensis Bt1267. The molecular weight of the peptide was estimated via SDS‐PAGE and confirmed with Matrix Assisted Laser Desorption Ionization Quadrapole Time of Flight mass spectrometry; its weight is 3162 Da. The peptide is biologically active after exposure to 100°C for 15 min, and within the pH range 1·00–9·25. Its activity disappeared when treated with proteinase K and protease, but not with α‐amylase or catalase. Conclusions:  We conclude that this is the first report of a bacteriocin produced by a plant growth promoting rhizobacteria (B. thuringiensis) species and have named the bacteriocin thuricin 17. Significance and Impact of the Study:  Our work has characterized a bacteriocin produced by a plant growth promoting bacterium. This strain is previously reported to increase soya bean nodulation.

DEAE-cellulose chromatography of extracts of free-living Rhizobium meliloti cells revealed separate NAD(+)-dependent and NADP(+)-dependent malic enzyme activities. The NAD+ malic enzyme exhibited more activity with NAD+ as cofactor, but also showed some activity with NADP+. The NADP+ malic enzyme only showed activity when NADP+ was supplied as cofactor. Three independent transposon-induced mutants of R. meliloti which lacked NAD+ malic enzyme activity (dme-) but retained NADP+ malic enzyme activity were isolated. In an otherwise wild-type background, the dme mutations did not alter the carbon utilization phenotype; however, nodules induced by these mutants failed to fix N2. Structurally, these nodules appeared to develop like wild-type nodules up to the stage where N2-fixation would normally begin. These results support the proposal that NAD+ malic enzyme, together with pyruvate dehydrogenase, functions in the generation of acetyl-CoA required for TCA cycle function in N2-fixing bacteroids which metabolize C4-dicarboxylic acids supplied by the plant.

The bacterium Rhizobium meliloti, which forms N 2 -fixing root nodules on alfalfa, has two distinct malic enzymes; one is NADP ؉ dependent, while a second has maximal activity when NAD ؉ is the coenzyme. The diphosphopyridine nucleotide (NAD ؉ )-dependent malic enzyme (DME) is required for symbiotic N 2 fixation, likely as part of a pathway for the conversion of C 4 -dicarboxylic acids to acetyl coenzyme A in N 2 -fixing bacteroids. Here, we report the cloning and localization of the tme gene (encoding the triphosphopyridine nucleotide [NADP ؉ ]-dependent malic enzyme) to a 3.7-kb region. We constructed strains carrying insertions within the tme gene region and showed that the NADP ؉ -dependent malic enzyme activity peak was absent when extracts from these strains were eluted from a DEAE-cellulose chromatography column. We found that NADP ؉ -dependent malic enzyme activity was not required for N 2 fixation, as tme mutants induced N 2 -fixing root nodules on alfalfa. Moreover, the apparent NADP ؉ -dependent malic enzyme activity detected in wild-type (N 2 -fixing) bacteroids was only 20% of the level detected in free-living cells. Much of that residual bacteroid activity appeared to be due to utilization of NADP ؉ by DME. The functions of DME and the NADP ؉ -dependent malic enzyme are discussed in light of the above results and the growth phenotypes of various tme and dme mutants.Malic enzymes convert malate to pyruvate and CO 2 with the simultaneous reduction of NAD -dependent malic enzymes (DME and TME, respectively) in Escherichia coli (25,44), and Hansen and Juni (21) have isolated mutants of E. coli which lack either DME or both DME and TME. Kobayashi et al. (27) have reported the properties of TME together with the nucleotide sequence of the corresponding gene from Bacillus stearothermophilus.A TME has been partially purified from Bradyrhizobium japonicum bacteroids (26), which have both DME and TME activities (7,28). Both malic enzyme activities have also been reported in Rhizobium sp. strain NGR234 free-living cells (39) and in both free-living cells and bacteroids of Rhizobium leguminosarum (30).We have been studying the role(s) of the bacterial malic enzymes in symbiotic nitrogen fixation within the alfalfa-Rhizobium meliloti symbiosis (9, 10). C 4 -dicarboxylic acids appear to be the principal source of carbon and energy supplied by the plant to N 2 -fixing bacteria (bacteroids) within root nodules (1,17,41). Bacteroids appear to metabolize C 4 -dicarboxylic acids directly via the citric acid cycle (31,47,48). In bacteroids, acetyl coenzyme A appears to be synthesized via the DME and pyruvate dehydrogenase (9, 30), and R. meliloti dme mutants (lacking DME) induce root nodules which contain bacteria but which fail to fix nitrogen (9).To isolate R. meliloti dme mutants, we constructed a strain within which dme mutations generated a succinate-negative growth phenotype. R. meliloti pckA mutants lack phosphoenolpyruvate carboxykinase (PCK) activity and grow poorly on minimal media with succinate or tricar...

Biofilm communities cultivated in rotating annular bioreactors using water from the South Saskatchewan River were assessed for the effects of seasonal variations and nutrient (C, N, and P) additions. Confocal laser microscopy revealed that while control biofilms were consistently dominated by bacterial biomass, the addition of nutrients shifted biofilms of summer and fall water samples to phototrophic-dominated communities. In nutrient-amended biofilms, similar patterns of nitrification, denitrification, and hexadecane mineralization rates were observed for winter and spring biofilms; fall biofilms had the highest rates of nitrification and hexadecane mineralization, and summer biofilms had the highest rates of denitrification. Very low rates of all measured activities were detected in control biofilms (without nutrient addition) regardless of season. Nutrient addition caused large increases in hexadecane mineralization and denitrification rates but only modest increases, if any, in nitrification rates, depending upon the season. Generally, both alkB and nirK were more readily PCR amplified from nutrient-amended biofilms. Both genes were amplified from all samples except for nirK from the fall control biofilm. It appears that bacterial production in the South Saskatchewan River water is limited by the availability of nutrients and that biofilm activities and composition vary with nutrient availability and time of year.

The pckA gene of Rhizobium meliloti, encoding phosphoenolpyruvate carboxykinase, was isolated from a genomic cosmid library by complementation of the succinate growth phenotype of a Pck ؊ mutant. The gene region was mapped by subcloning and Tn5 insertion mutagenesis. The DNA sequence for a 2-kb region containing the structural gene and its promoter was determined. The pckA gene encodes a 536-amino-acid protein that shows homology with other ATP-dependent Pck enzymes. The promoter was identified following primer extension analysis and is similar to 70 -like promoters. Expression analysis with a pckA::lacZ gene fusion indicated that the pckA gene was strongly induced at the onset of stationary phase in complex medium. When defined carbon sources were tested, the expression level of the pckA gene was found to be high when cells were grown in minimal media with succinate or arabinose as the sole carbon source but almost absent when glucose, sucrose, or glycerol was the sole carbon source. Glucose and sucrose were not found to strongly repress pckA induction by succinate.Phosphoenolpyruvate carboxykinase (EC 4.1.1.49) (Pck) catalyzes the decarboxylation and phosphorylation of oxaloacetate to phosphoenolpyruvate. This reaction is the first step in the gluconeogenic pathway in which tricarboxylic acid (TCA) cycle intermediates are converted to hexose sugars. With the exceptions of phosphoenolpyruvate carboxykinase and fructose bisphosphatase, all of the enzymes employed in the gluconeogenic pathway are also used in the glycolytic pathway.With N 2 -fixing root nodules induced by bacteria such as the alfalfa symbiont Rhizobium meliloti, there is much evidence to suggest that the plant supplies the C 4 -dicarboxylic acids succinate and malate to the N 2 -fixing bacteria (bacteroids) within nodules (5,9,17,42,47,51). Pck is required for growth and metabolism of C 4 -dicarboxylates and other TCA cycle intermediates in free-living cells of R. meliloti, Rhizobium leguminosarum, and Rhizobium sp. strain NGR234. The symbiotic importance of Pck during nodule infection and N 2 fixation requires clarification. While Pck Ϫ mutants of R. meliloti show a reduced level of nitrogen fixation, Pck activity is not detected in wild-type bacteroids (15). R. leguminosarum Pck Ϫ mutants have no apparent symbiotic phenotype, yet Pck activity was detected at low levels in wild-type bacteroids (33). A Pck Ϫ mutant of Rhizobium sp. strain NGR234, a broad-host-range fast-growing rhizobium, exhibited a host-dependent symbiotic phenotype. N 2 fixation was reduced to 60 and 20% of the wild-type level in Leucaena leucocephala and Macroptilium atropurpureum, respectively, whereas on Vigna unguiculata, this mutant induced completely Fix Ϫ nodules (39). The structural analysis of these nodules showed defects in the nodule development process and the occurrence of early senescence of bacteroids. These results suggest that the importance of Pck in symbiosis is dependent on the plant host metabolites available to the bacteria during the infection process an...

The wild-type NAD+-dependent malic enzyme (dme) gene of Rhizobium (now Sinorhizobium) meliloti was cloned and localized to a 3.1 kb region isolated on the cosmid pTH69. This cosmid complemented the symbiotic nitrogen fixation (Fix') phenotype of R. meliloti dme mutants. The dme gene was mapped by conjugation to between the cys-I7 and leu-53 markers on the R, meliloti chromosome. 8-Galactosidase activities measured in bacterial strains carrying either dme-lacZ or tme-lac2 gene fusions (the tme gene encodes NADP+-dependent malic enzyme) indicated that the dme gene was expressed constitutively in free-living cells and in N,-fixing bacteroids whereas expression of the tme gene was repressed in bacteroids. The R. meliloti dme gene product (DME) was overexpressed in and partially purified from Escherichia coli. The properties of this enzyme, together with those of the NADP+-dependent malic enzyme (TME) partially purified from R. meliloti dme mutants, were determined. Acetyl-CoA inhibited DME but not TME activity. This result supports the hypothesis that DME, together with pyruvate dehydrogenase, forms a pathway in which malate is converted to acetyl-CoA.