The genetic and phylogenetic relationships for strains of Mesorhizobium tianshanense and its relatives were compared by an analysis of the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins, DNA-DNA hybridization, and full 16s rRNA gene sequencing. The strains of M. tianshanense formed a cluster which was distinct from those of other rhizobium species in the clustering analysis of SDS-PAGE. DNA-DNA relatedness between A-1BS (type strain of M. tianshanense) and the type or reference strains for Mesorhizobium loti, M. huakuii, M. ciceri, M. mediterraneum, and cluster U, an unnamed rhizobial group, ranged from 4.4 to 43.8%. The phylogenetic analysis based on the 16s rRNA gene sequences showed that M. tianshunense was closely related to the Mesorhizobium phylogenetic branch and could be distinguished from the other four species in this branch. These results further confirmed that these bacteria constitute a distinct rhizobial species. ~ ~~The genetic approaches now widely applied to the taxonomy of root nodule bacteria have opened the possibility to infer their phylogenies and to correctly define the species and genera of these bacteria. The improved methods for identifying bacteria and a growing interest in characterization of new rhizobial isolates have brought about many changes in the taxonomy of rhizobia since 1984; a revised taxonomic system for these bacteria was proposed in "Bergey's Manual of Systematic Bacteriology" (16). Several reviews on the development of rhizobial taxonomy and phylogeny have been published recently (20,33). The main developments include the descriptions of the genera Azorhizobium (S), Sinorhizobium (4, 6 ) , and Mesorhizobium (14, IS), as well as many new species. Up to now, six distinct phylogenetic branches, Azorhizobium, Bradyrhizobium, the Rhizobium-Agrobacterium rhizogenes branch, Mesorhizobium (14), the Rhizobium galegae-Agrobacterium branch, and Sinorhizobium, have been identified, and all of them were located in the alpha subclass of Proteobacteria (6, 31-33). Mesorhizobium has been proposed recently by Jarvis et al. (14), and five species, M. loti (15), M. huakuii (3), M. ciceri (23), M. mediterraneum (22), and M. tianshanense (2) were included on the basis of the data from full sequences of 16s rRNA genes. A group of rhizobia named cluster U has been classified in this genus by 16s ribosomal DNA (rDNA) sequencing (6). Some isolates from nodules of Amolpha fruticosa also belong to it, as indicated by the PCR-based restriction fragment length polymorphism patterns of their 16s rDNA (unpublished data). M. tianshanense was described in our previous paper dealing with a group of rhizobia isolated from saline and arid soils in the Xinjiang region of China (2). Some of the strains in this species grow as slowly as Bradyrhizobium spp. Other strains grow faster than Bradyrhizobium but slower than Rhizobium leguminosarum. According to the data from the partial 16s rDNA sequence, this species belongs to the M. loti-M. huakuii branch ...

Summary Phospholipids are the membrane‐forming constituents in all living organisms. In addition to phosphorus‐containing lipids, the membranes of numerous bacteria contain significant amounts of phosphorus‐free polar lipids, often derived from amino acids. Although lipids derived from the amino acid ornithine are widespread among bacteria, their biosynthesis is unknown. Here, we describe the isolation of mutants of Sinorhizobium meliloti deficient in the biosynthesis of ornithine‐derived lipids (OL). Complementation of such mutants with a sinorhi‐zobial cosmid gene bank, subcloning of the complementing fragment and sequencing of the subclone led to the identification of a gene (olsA) coding for a presumptive acyltransferase. Amplification of this gene and its expression in OL‐deficient mutant backgrounds of S. meliloti demonstrates that it is required for OL biosynthesis. An OL‐deficient mutant of S. meliloti disrupted in olsA shows wild type‐like growth behaviour and is capable of inducing nitrogen‐fixing nodules on legume hosts. A lyso‐ornithine lipid‐dependent acyltransferase activity forming OL requires acyl‐AcpP as the acyl donor and expression of the olsA gene.

Rhizobia are Gram-negative soil bacteria able to establish nitrogenfixing root nodules with their respective legume host plants. Besides phosphatidylglycerol, cardiolipin, and phosphatidylethanolamine, rhizobial membranes contain phosphatidylcholine (PC) as a major membrane lipid. Under phosphate-limiting conditions of growth, some bacteria replace their membrane phospholipids with lipids lacking phosphorus. In Sinorhizobium meliloti, these phosphorus-free lipids are sulfoquinovosyl diacylglycerol, ornithinecontaining lipid, and diacylglyceryl trimethylhomoserine (DGTS). Pulse-chase experiments suggest that the zwitterionic phospholipids phosphatidylethanolamine and PC act as biosynthetic precursors of DGTS under phosphorus-limiting conditions. A S. meliloti mutant, deficient in the predicted phosphatase SMc00171 was unable to degrade PC or to form DGTS in a similar way as the wild type. Cell-free extracts of Escherichia coli, in which SMc00171 had been expressed, convert PC to phosphocholine and diacylglycerol, showing that SMc00171 functions as a phospholipase C. Diacylglycerol , in turn, is the lipid anchor from which biosynthesis is initiated during the formation of the phosphorus-free membrane lipid DGTS. Inorganic phosphate can be liberated from phosphocholine. These data suggest that, in S. meliloti under phosphate-limiting conditions, membrane phospholipids provide a pool for metabolizable inorganic phosphate, which can be used for the synthesis of other essential phosphorus-containing biomolecules. This is an example of an intracellular phospholipase C in a bacterial system; however, the ability to degrade endogenous preexisting membrane phospholipids as a source of phosphorus may be a general property of Gram-negative soil bacteria.nimal cells have access to relatively abundant sources of phosphorus for the formation of biomolecules such as membrane phospholipids and nucleic acids. The characteristic lipid composition for a particular animal cell membrane is thought to result from a steady state between formation and turnover of the lipids. In contrast, plants and many environmental microbes often live in environments where available phosphorus is a growth-limiting factor. The strategies employed by organisms to deal with phosphorus limitation include: (i) increased solubilization of phosphorus-containing compounds; (ii) more efficient uptake into cells; and (iii) less phosphorus use when synthesizing their biomolecules (1). The replacement of phospholipids by galacto-and sulfolipids in plant membranes constitutes an important adaptive process for growth on phosphate-limited soils. In Arabidopsis thaliana, several phospholipases D and C (2-5) are induced under phosphate-limiting conditions, and they degrade membrane phospholipids to phosphatidic acid or diacylglycerol (DAG), respectively. DAG then serves as the initial substrate for the formation of galacto-and sulfolipids, which lack phosphorus.In some bacteria, the membrane phospholipids are partially replaced during phosphate limitation by phosphoru...

SummaryUnder phosphate-limiting conditions, some bacteria replace their membrane phospholipids by lipids not containing any phosphorus. One of these phosphorus-free lipids is an ornithine-containing lipid (OL) that is widespread among eubacteria. In earlier work, we had identified a gene ( olsA ) required for OL biosynthesis that probably encodes an O -acyltransferase using acyl-acyl carrier protein (acyl-AcpP) as an acyl donor and that converts lyso-ornithine lipid into OL. We now report on a second gene ( olsB ) required for OL biosynthesis that is needed for the incorporation of radiolabelled ornithine into OL. Overexpression of OlsB in an olsA -deficient mutant of Sinorhizobium (Rhizobium) meliloti leads to the transient accumulation of lyso-ornithine lipid, the biosynthetic intermediate of OL biosynthesis. Overexpression of OlsB in Escherichia coli is sufficient to cause the in vivo formation of lyso-ornithine lipid in this organism and is the cause for a 3-hydroxyacylAcpP-dependent acyltransferase activity forming lyso-ornithine lipid from ornithine. These results demonstrate that OlsB is required for the first step of OL biosynthesis, in which ornithine is N -acylated with a 3-hydroxy-fatty acyl residue in order to obtain lysoornithine lipid. OL formation in a wild-type S. meliloti is increased upon growth under phosphate-limiting conditions. Expression of OlsB from a broad host range vector leads to the constitutive formation of relatively high amounts of OL (12-14% of total membrane lipids) independently of whether strains are grown in the presence of low or high concentrations of phosphate, suggesting that in S. meliloti the formation of OlsB is usually limiting for the amount of OL formed in this organism. Open reading frames homologous to OlsA and OlsB were identified in many eubacteria and although in S. meliloti the olsB and olsA gene are 14 kb apart, in numerous other bacteria they form an operon.

Paenibacillus polymyxa is a plant growth-promoting rhizobacterium that has immense potential to be used as an environmentally friendly replacement of chemical fertilizers and pesticides. In the present study, Paenibacillus polymyxa SK1 was isolated from bulbs of Lilium lancifolium. The isolated endophytic strain showed antifungal activities against important plant pathogens like Botryosphaeria dothidea, Fusarium oxysporum, Botrytis cinerea, and Fusarium fujikuroi. The highest percentage of growth inhibition, i.e., 66.67 ± 2.23%, was observed for SK1 against Botryosphaeria dothidea followed by 61.19 ± 3.12%, 60.71 ± 3.53%, and 55.54 ± 2.89% against Botrytis cinerea, Fusarium fujikuroi, and Fusarium oxysporum, respectively. The metabolite profiling of ethyl acetate fraction was assessed through the UHPLC-LTQ-IT-MS/MS analysis, and putative identification was done with the aid of the GNPS molecular networking workflow. A total of 29 compounds were putatively identified which included dipeptides, tripeptides, cyclopeptides (cyclo-(Leu-Leu), cyclo(Pro-Phe)), 2-heptyl-3-hydroxy 4-quinolone, 6-oxocativic acid, anhydrobrazilic acid, 1-(5-methoxy-1H-indol-3-yl)-2-piperidin-1-ylethane-1,2-dione, octadecenoic acid, pyochelin, 15-hydroxy-5Z,8Z,11Z, 13E-eicosatetraenoic acid, (Z)-7-[(2R,3S)-3-[(2Z,5E)-Undeca-2,5-dienyl]oxiran-2-yl]hept-5-enoic acid, arginylasparagine, cholic acid, sphinganine, elaidic acid, gossypin, L-carnosine, tetrodotoxin, and ursodiol. The high antifungal activity of SK1 might be attributed to the presence of these bioactive compounds. The isolated strain SK1 showed plant growth-promoting traits such as the production of organic acids, ACC deaminase, indole-3-acetic acid (IAA), siderophores, nitrogen fixation, and phosphate solubilization. IAA production was strongly correlated with the application of exogenous tryptophan concentrations in the medium. Furthermore, inoculation of SK1 enhanced plant growth of two Lilium varieties, Tresor and White Heaven, under greenhouse condition. In the light of these findings, the P. polymyxa SK1 may be utilized as a source of plant growth promotion and disease control in sustainable agriculture.

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A fast-growing rhizobial group isolated from leguminous plants in Hainan Province, a tropical region of China, is proposed as a new Rhizobium species on the basis of 16s rRNA gene sequencing, DNA-DNA hybridization, and phenotypic characterization. This new species belongs to the phylogenetic branch which includes Rhizobium leguminosarum. We propose the name Rhizobium hainanense sp. nov. for this species. The strain CCBAU 57015 (166) is the type strain; it has been deposited in the culture collection of Beijing Agricultural University, People's Republic of China.Diversity in rhizobia nodulating tropical legumes has been revealed by many studies, and these bacteria belong to several bacterial genera and species. Many slow-growing strains isolated from tropical legumes belong to the genus Bradyrhizobium (11,15). A unique group of strains inducing stem and root nodules on Sesbania rostrata, a tropical legume of Africa, constitute the genus Azorhizobium (8). Rhizobium tropici (18) and Rhizobium etli (22) were proposed for the rhizobia isolated from the common bean (Phaseolus vulgaris) and Leucaena leucocephala in the tropical region. Recently, Sinorhizobium saheli and Sinorhizobium teranga were proposed for some isolates from Acacia and Sesbania (6). Some other groups nodulating plants of the genera Acacia and Leucaena were identified by numerical taxonomy (33) and genetic analysis (13). In our previous research, some rhizobia were isolated from nodules of various species of leguminous plants, including trees, herbs, and vines growing in Hainan province, a tropical region of China, and were characterized by using numerical taxonomy and DNA-DNA hybridization (1 1). All slow-growing rhizobia among them were classified as Bradyrhizobium japonicum strains, while the fast-growing rhizobia from Hainan were diverse both in phenotypic and genetic aspects. Some strains belonged to previously described Rhizobium species; the others formed unique subgroups. Among them subgroup IV was distinguished from all previously described Rhizobium species by numerical taxonomy and by an analysis of DNA hybridization data. The 13 strains in this subgroup were isolated from 12 leguminous species classified in nine different genera (11). A partial 16s rRNA gene sequence from strain 166, the reference strain of subgroup IV, showed a close relationship with those of Rhizobium species but could not be used to separate subgroup IV from R. tropici B, because the partial 16s rRNA gene sequence was identical to that of R. tropici B CIAT 899T (25). In order to determine the exact taxonomic position of subgroup IV, some further studies were performed, including full 16s rRNA gene (ribosomal DNA [rDNA]) sequencing, analysis of symbiotic performance with hosts for this subgroup and with selected hosts according to the guidance in the "Proposed Minimal Standards for the Description of New Genera and Species of Root-and Stem-Nodulating Bacteria" (12), and additional research on DNA relatedness between this group and R. tropici, R. etli, and Agrobacterium...

In the present study, a new strain of Bacillus stratosphericus LW-03 was isolated from the bulbs of Lilium wardii. The isolated endophytic strain LW-03 exhibited excellent antifungal activity against common plant pathogens, such as Fusarium oxysporum, Botryosphaeria dothidea, Botrytis cinerea, and Fusarium fujikuroi. The growth inhibition percentage of Botryosphaeria dothidea was 74.56 ± 2.35%, which was the highest, followed by Botrytis cinerea, Fusarium fujikuroi, and Fusarium oxysporum were 71.91 ± 2.87%, 69.54 ± 2.73%, and 65.13 ± 1.91%, respectively. The ethyl acetate fraction revealed a number of bioactive compounds and several of which were putatively identified as antimicrobial agents, such as 4-hydroxy-2-nonenylquinoline N-oxide, sphingosine ceramides like cer(d18:0/16:0(2OH)), cer(d18:0/16:0), and cer(d18:1/0:0), di-peptides, tri-peptide, cyclopeptides [cyclo(D-Trp-L-Pro)], [cyclo (Pro-Phe)], dehydroabietylamine, oxazepam, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine like compound (PC(0:0/20:4), phosphatidylethanolamine (PE(18:1/0:0)), 3-Hydroxyoctadecanoic acid, 7.alpha.,27-Dihydroxycholesterol, N-Acetyl-d-mannosamine, p-Hydroxyphenyllactic acid, Phytomonic acid, and 2-undecenyl-quinoloin-4 (1H). The LW-03 strain exhibits multiple plant growth-promoting traits, including the production of organic acids, ACC deaminase, indole-3-acetic acid (IAA), siderophores, and nitrogen fixation activity. The beneficial effects of the endophytic strain LW-03 on the growth of two lily varieties were further evaluated under greenhouse conditions. Our results revealed plant growth-promoting activity in inoculated plants relative to non-inoculated control plants. The broad-spectrum antifungal activity and multiple plant growth-promoting properties of Bacillus stratosphericus LW-03 make it an important player in the development of biological fertilizers and sustainable agricultural biological control strategies.