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Plant growth-promoting rhizobacteria (PGPR) are members of the plant rhizomicrobiome that enhance plant growth and stress resistance by increasing nutrient availability to the plant, producing phytohormones or other secondary metabolites, stimulating plant defense responses against abiotic stresses and pathogens, or fixing nitrogen. The use of PGPR to increase crop yield with minimal environmental impact is a sustainable and readily applicable replacement for a portion of chemical fertilizer and pesticides required for the growth of high-yielding varieties. Increased plant health and productivity have long been gained by applying PGPR as commercial inoculants to crops, although with uneven results. The establishment of plant-PGPR relationships requires the exchange of chemical signals and nutrients between the partners, and polyamines (PAs) are an important class of compounds that act as physiological effectors and signal molecules in plant-microbe interactions. In this review, we focus on the role of PAs in interactions between PGPR and plants. We describe the basic ecology of PGPR and the production and function of PAs in them and the plants with which they interact. We examine the metabolism and the roles of PAs in PGPR and plants individually and during their interaction with one another. Lastly, we describe some directions for future research.

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We previously showed that specific polyamines (PAs) present in the extracellular environment markedly affect extracellular polysaccharide (EPS) production, biofilm formation and motility in Sinorhizobium meliloti Rm8530. We hypothesized that extracellular PA signals were sensed and transduced by the NspS and MbaA proteins, respectively, which are homologs of the PA-sensing, c-di-GMP modulating NspS-MbaA proteins described in Vibrio cholerae. Here we show that the decrease in biofilm formation and EPS production in the quorum-sensing (QS)-deficient S. meliloti wild-type strain 1021 in cultures containing putrescine or spermine did not occur in a 1021 nspS mutant (1021 nspS). The transcriptional expression of nspS in strain 1021 was significantly increased in cultures containing either of these polyamines, but not by exogenous cadaverine, 1,3-diaminopropane (DAP), spermidine (Spd) or norspermidine (NSpd). Cell aggregation in liquid cultures did not differ markedly between strain 1021 and 1021 nspS in the presence or absence of PAs. The S. meliloti QS-proficient Rm8530 wild-type and nspS mutant (Rm8530 nspS) produced similar levels of biofilm under control conditions and 3.2- and 2.2-fold more biofilm, respectively, in cultures with NSpd, but these changes did not correlate with EPS production. Cells of Rm8530 nspS aggregated from two- to several-fold more than the wild-type in cultures without PAs or in those containing Spm. NSpd, Spd and DAP differently affected swimming and swarming motility in strains 1021 and Rm8530 and their respective nspS mutants. nspS transcription in strain Rm8530 was greatly reduced by exogenous Spm. Bioinformatic analysis revealed similar secondary structures and functional domains in the MbaA proteins of S. meliloti and V. cholerae, while their NspS proteins differed in some residues implicated in polyamine recognition in the latter species. NspS-MbaA homologs occur in a small subset of soil and aquatic bacterial species that commonly interact with eukaryotes. We speculate that the S. meliloti NspS-MbaA system modulates biofilm formation, EPS production and motility in response to environmental or host plant-produced PAs.

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Biotin is a key cofactor of metabolic carboxylases, although many rhizobial strains are biotin auxotrophs. When some of these strains were serially subcultured in minimal medium, they showed diminished growth and increased excretion of metabolites. The addition of biotin, or genetic complementation with biotin synthesis genes resulted in full growth of Rhizobium etli CFN42 and Rhizobium phaseoli CIAT652 strains. Half of rhizobial genomes did not show genes for biotin biosynthesis, but three-quarters had genes for biotin transport. Some strains had genes for an avidin homologue (rhizavidin), a protein with high affinity for biotin but an unknown role in bacteria. A CFN42-derived rhizavidin mutant showed a sharper growth decrease in subcultures, revealing a role in biotin storage. In the search of biotin-independent growth of subcultures, CFN42 and CIAT652 strains with excess aeration showed optimal growth, as they also did, unexpectedly, with the addition of aspartic acid analogues α- and N-methyl aspartate. Aspartate analogues can be sensed by the chemotaxis aspartate receptor Tar. A tar homologue was identified and its mutants showed no growth recovery with aspartate analogues, indicating requirement of the Tar receptor in such a phenotype. Additionally, tar mutants did not recover full growth with excess aeration. A Rubisco-like protein was found to be necessary for growth as the corresponding mutants showed no recovery either with high aeration or aspartate analogues; also, diminished carboxylation was observed. Taken together, our results indicate a route of biotin-independent growth in rhizobial strains that included oxygen, a Tar receptor and a previously uncharacterized Rubisco-like protein.

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The metalloenzyme arginase hydrolyzes l-arginine to produce l-ornithine and urea. In bacteria, arginase has important functions in basic nitrogen metabolism and redistribution, production of the key metabolic precursor l-ornithine, stress resistance and pathogenesis. We describe the regulation and specific functions of the arginase pathway as well as summarize key characteristics of related arginine catabolic pathways. The use of arginase-derived ornithine as a precursor molecule is reviewed. We discuss the biochemical and transcriptional regulation of arginine metabolism, including arginase, with the latter topic focusing on the RocR and AhrC transcriptional regulators in the model organism Bacillus subtilis. Finally, we consider similarities and contrasts in the structure and catalytic mechanism of the arginases from Bacillus caldovelox and Helicobacter pylori. The overall aim of this review is to provide a panorama of the diversity of physiological functions, regulation and biochemical features of arginases in a variety of bacterial species.

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In bacteria, l-arginine is a precursor of various metabolites and can serve as a source of carbon and/or nitrogen. Arginine catabolism by arginase, which hydrolyzes arginine to l-ornithine and urea, is common in nature but has not been studied in symbiotic nitrogen-fixing rhizobia. The genome of the alfalfa microsymbiont Sinorhizobium meliloti 1021 has two genes annotated as arginases, argI1 (smc03091) and argI2 (sma1711). Biochemical assays with purified ArgI1 and ArgI2 (as 6His-Sumo-tagged proteins) showed that only ArgI1 had detectable arginase activity. A 1021 argI1 null mutant lacked arginase activity and grew at a drastically reduced rate with arginine as sole nitrogen source. Wild-type growth and arginase activity were restored in the argI1 mutant genetically complemented with a genomically integrated argI1 gene. In the wild-type, arginase activity and argI1 transcription were induced several fold by exogenous arginine. ArgI1 purified as a 6His-Sumo-tagged protein had its highest in vitro enzymatic activity at pH 7.5 with Ni2+ as cofactor. The enzyme was also active with Mn2+ and Co2+, both of which gave the enzyme the highest activities at a more alkaline pH. The 6His-Sumo-ArgI1 comprised three identical subunits based on the migration of the urea-dissociated protein in a native polyacrylamide gel. A Lrp-like regulator (smc03092) divergently transcribed from argI1 was required for arginase induction by arginine or ornithine. This regulator was designated ArgIR. Electrophoretic mobility shift assays showed that purified ArgIR bound to the argI1 promoter in a region preceding the predicted argI1 transcriptional start. Our results indicate that ArgI1 is the sole arginase in S. meliloti, that it contributes substantially to arginine catabolism in vivo and that argI1 induction by arginine is dependent on ArgIR.

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In nitrogen-fixing rhizobia, emerging evidence shows significant roles for polyamines in growth and abiotic stress resistance. In this work we show that a polyamine-deficient ornithine decarboxylase null mutant (odc2) derived from Sinorhizobium meliloti Rm8530 had significant phenotypic differences from the wild-type, including greatly reduced production of exopolysaccharides (EPS; ostensibly both succinoglycan and galactoglucan), increased sensitivity to oxidative stress and decreased swimming motility. The introduction of the odc2 gene borne on a plasmid into the odc2 mutant restored wild-type phenotypes for EPS production, growth under oxidative stress and swimming. The production of calcofluor-binding EPS (succinoglycan) by the odc2 mutant was also completely or mostly restored in the presence of exogenous spermidine (Spd), norspermidine (NSpd) or spermine (Spm). The odc2 mutant formed about 25 % more biofilm than the wild-type, and its ability to form biofilm was significantly inhibited by exogenous Spd, NSpd or Spm. The odc2 mutant formed a less efficient symbiosis with alfalfa, resulting in plants with significantly less biomass and height, more nodules but less nodule biomass, and 25 % less nitrogen-fixing activity. Exogenously supplied Put was not able to revert these phenotypes and caused a similar increase in plant height and dry weight in uninoculated plants and in those inoculated with the wild-type or odc2 mutant. We discuss ways in which polyamines might affect the phenotypes of the odc2 mutant.

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Polyamines are ubiquitous molecules containing two or more amino groups that fulfill varied and often essential physiological and regulatory roles in all organisms. In the symbiotic nitrogen-fixing bacteria known as rhizobia, putrescine and homospermidine are invariably produced while spermidine and norspermidine synthesis appears to be restricted to the alfalfa microsymbiont Sinorhizobium meliloti. Studies with rhizobial mutants deficient in the synthesis of one or more polyamines have shown that these compounds are important for growth, stress resistance, motility, exopolysaccharide production and biofilm formation. In this review, we describe these studies and examine how polyamines are synthesized and regulated in rhizobia.

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Polyamines (PAs) are ubiquitous polycations derived from basic l-amino acids whose physiological roles are still being defined. Their biosynthesis and functions in nitrogen-fixing rhizobia such as Sinorhizobium meliloti have not been extensively investigated. Thin layer chromatographic and mass spectrometric analyses showed that S. meliloti Rm8530 produces the PAs, putrescine (Put), spermidine (Spd) and homospermidine (HSpd), in their free forms and norspermidine (NSpd) in a form bound to macromolecules. The S. meliloti genome encodes two putative ornithine decarboxylases (ODC) for Put synthesis. Activity assays with the purified enzymes showed that ODC2 (SMc02983) decarboxylates both ornithine and lysine. ODC1 (SMa0680) decarboxylates only ornithine. An odc1 mutant was similar to the wild-type in ODC activity, PA production and growth. In comparison to the wild-type, an odc2 mutant had 45 % as much ODC activity and its growth rates were reduced by 42, 14 and 44 % under non-stress, salt stress or acid stress conditions, respectively. The odc2 mutant produced only trace levels of Put, Spd and HSpd. Wild-type phenotypes were restored when the mutant was grown in cultures supplemented with 1 mM Put or Spd or when the odc2 gene was introduced in trans. odc2 gene expression was increased under acid stress and reduced under salt stress and with exogenous Put or Spd. An odc1 odc2 double mutant had phenotypes similar to the odc2 mutant. These results indicate that ODC2 is the major enzyme for Put synthesis in S. meliloti and that PAs are required for normal growth in vitro.

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Flavonoids excreted by legume roots induce the expression of symbiotically essential nodulation (nod) genes in rhizobia, as well as that of specific protein export systems. In the bean microsymbiont Rhizobium etli CE3, nod genes are induced by the flavonoid naringenin. In this study, we identified 693 proteins in the exoproteome of strain CE3 grown in minimal medium with or without naringenin, with 101 and 100 exoproteins being exclusive to these conditions, respectively. Four hundred ninety-two (71%) of the extracellular proteins were found in both cultures. Of the total exoproteins identified, nearly 35% were also present in the intracellular proteome of R. etli bacteroids, 27% had N-terminal signal sequences and a significant number had previously demonstrated or possible novel roles in symbiosis, including bacterial cell surface modification, adhesins, proteins classified as MAMPs (microbe-associated molecular patterns), such as flagellin and EF-Tu, and several normally cytoplasmic proteins as Ndk and glycolytic enzymes, which are known to have extracellular "moonlighting" roles in bacteria that interact with eukaryotic cells. It is noteworthy that the transmembrane ß (1,2) glucan biosynthesis protein NdvB, an essential symbiotic protein in rhizobia, was found in the R. etli naringenin-induced exoproteome. In addition, potential binding sites for two nod-gene transcriptional regulators (NodD) occurred somewhat more frequently in the promoters of genes encoding naringenin-induced exoproteins in comparison to those ofexoproteins found in the control condition.

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The tropical and mycoparasite strain Streptomyces galilaeus CFFSUR-B12 was evaluated as an antagonist of Mycosphaerella fijiensis Morelet, causal agent of the Black Sigatoka Disease (BSD) of banana. On zymograms of CFFSUR-B12 culture supernatants, we detected four chitinases of approximately 32 kDa (Chi32), 20 kDa (Chi20), and two with masses well over 170 kDa (ChiU) that showed little migration during denaturing electrophoresis at different concentrations of polyacrylamide. The thymol-sulphuric acid assay showed that the ChiU were glycosylated chitinases. Moreover, matrix assisted laser desorption ionization time-of-flight MS analysis revealed that the ChiU are the same protein and identical to a family 18 chitinase from Streptomyces sp. S4 (gi|498328075). Chi32 was similar to an extracellular protein from Streptomyces albus J1074 (gi|478687481) and Chi20 was non-significantly similar to chitinases from five different strains of Streptomyces (P > 0.05). Subsequently, Chi32 and Chi20 were partially purified by anion exchange and hydrophobic interaction chromatography and tested against M. fijiensis. Chitinases failed to inhibit ascospore germination, but inhibited up to 35 and 62% of germ tube elongation and mycelial growth, respectively. We found that crude culture supernatant and living cells of S. galilaeus CFFSUR-B12 were the most effective in inhibiting M. fijiensis and are potential biocontrol agents of BSD.

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