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...