Search

Here, we describe a simple, non-time consuming and inexpensive method for monitoring of Calcofluor white M2R-binding exopolysaccharides in individual bacterial cells. This method was demonstrated by time-lapse microscopy of succinoglycan-producing cells of the plant-symbiotic alpha-proteobacterium Sinorhizobium meliloti . The method is most likely applicable to other bacteria producing β-(1 → 3) and β-(1 → 4) linked polysaccharides. [ABSTRACT FROM AUTHOR]

The plant growth-promoting rhizobacterium, Ochrobactrum sp. 11a displays a high intrinsic salinity tolerance and has been used in this work to study the molecular basis of bacterial responses to high concentrations of NaCl. A collection of Ochrobactrum sp. 11a mutants was generated by Tn 5-B21 mutagenesis and screened for sensitivity to salinity. One clone, designated PBP and unable to grow on glutamate mannitol salt agar medium supplemented with 300 mM NaCl was selected and further characterized. The PBP mutant carries a single transposon insertion in a gene showing a high degree of identity to the serine-typed-alanyl-d-alanine carboxypeptidase gene of Ochrobactrum anthropi. Interestingly, the expression of this gene was shown to be upregulated by salt in the PBP mutant. Moreover, evidence is presented for the requirement of the gene product for adaptation to high-salt conditions as well as to overcome the toxicity of LiCl, KCl, sucrose, polyethylene glycol (PEG), AlCl3, CuSO4, and ZnSO4. In addition to the altered tolerance to both ionic and osmotic stresses, the PBP mutant exhibited changes in colony and cell morphology, exopolysaccharide production, and an increased sensitivity to detergents. [ABSTRACT FROM AUTHOR]

Bacteria of the genus Azospirillum are free-living nitrogen-fixing, rhizobacteria that are found in close association with plant roots, where they exert beneficial effects on plant growth and yield in many crops of agronomic importance. Unlike other bacteria, little is known about the genetics and biochemistry of exopolysaccharides in Azospirillum brasilense. In an attempt to characterize genes associated with exopolysaccharides production, we generated an A. brasilense Cd Tn 5 mutant that showed exopolysaccharides overproduction, decreased tolerance to saline conditions, altered cell morphology, and increased sensitivity to detergents. Genetic characterization showed that the Tn 5 was inserted within a ddlB gene encoding for ad-alanine–d-alanine ligase, and located upstream of the ftsQAZ gene cluster responsible for cell division in different bacteria. Heterologous complementation of the ddlB Tn 5 mutant restored the exopolysaccharides production to wild-type levels and the ability to grow in the presence of detergents, but not the morphology and growth characteristics of the wild-type bacteria, suggesting a polar effect of Tn 5 on the fts genes. This result and the construction of a nonpolar ddlB mutant provide solid evidence of the presence of transcriptional coupling between a gene associated with peptidoglycan biosynthesis and the fts genes required to control cell division. [ABSTRACT FROM AUTHOR]

The interaction between Azospirillum brasilense and plants is not fully understood, although several bacterial surface components like exopolysaccharides (EPS), flagella, and capsular polysaccharides are required for attachment and colonization. While in other plant–bacteria associations (Rhizobium–legume, Pseudomonas–potato), lipopolysaccharides (LPS) play a key role in the establishment of an effective association, their role in the root colonization by Azospirillum had not been determined. In this study, we isolated a Tn5 mutant of A. brasilense Cd (EJ1) with an apparently modified LPS core structure, non-mucoid colony morphology, increased EPS production, and affected in maize root colonization. A 3790-bp region revealed the presence of three complete open reading frames designated rmlC, rmlB and rmlD. The beginning of a fourth open reading frame was found and designated rmlA. These genes are organized in a cluster which shows homology to the cluster involved in the synthesis of dTDP-rhamnose in other bacteria. Additionally, the analysis of the monosaccharide composition of LPSs showed a diminution of rhamnose compared to the wild-type strain. [Copyright &y& Elsevier]

The role of the sesquiterpene botrydial in the interaction of the phytopathogenic fungus Botrytis cinerea and plant-associated bacteria was analyzed. From a collection of soil and phyllospheric bacteria, nine strains sensitive to growth-inhibition by B. cinerea were identified. B. cinerea mutants unable to produce botrydial caused no bacterial inhibition, thus demonstrating the inhibitory role of botrydial. A taxonomic analysis showed that these bacteria corresponded to different Bacillus species (six strains), Pseudomonas yamanorum (two strains) and Erwinia aphidicola (one strain). Inoculation of WT and botrydial non-producing mutants of B. cinerea along with B acillus amyloliquefaciens strain MEP 2 18 in soil demonstrated that both microorganisms exert reciprocal inhibitory effects; the inhibition caused by B. cinerea being dependent on botrydial production. Moreover, botrydial production was modulated by the presence of B. amyloliquefaciens MEP 2 18 in confrontation assays in vitro. Purified botrydial in turn, inhibited growth of Bacillus strains in vitro and cyclic lipopeptide (surfactin) production by B. amyloliquefaciens MEP 2 18. As a whole, results demonstrate that botrydial confers B. cinerea the ability to inhibit potential biocontrol bacteria of the genus Bacillus. We propose that resistance to botrydial could be used as an additional criterion for the selection of biocontrol agents of plant diseases caused by B. cinerea. Image 1 • The sesquiterpene botrydial produced by Botrytis cinerea inhibits bacteria associated to plants. • Botrydial lets B. cinerea antagonize Bacillus amyloliquefaciens in soil. • Botrydial lets B. cinerea resist the antagonistic effects of B. amyloliquefaciens. • Botrydial inhibits surfactin production by antagonistic B. amyloliquefaciens. • The ecological role of botrydial is wider than its phytotoxic effects on plant hosts of B. cinerea. [ABSTRACT FROM AUTHOR]

Bacillus sp. MEP218, a soil bacterium with high potential as a source of bioactive molecules, produces mostly C16–C17 fengycin and other cyclic lipopeptides (CLP) when growing under previously optimized culture conditions. This work addressed the elucidation of the genome sequence of MEP218 and its taxonomic classification. The genome comprises 3,944,892 bp, with a total of 3474 coding sequences and a G + C content of 46.59%. Our phylogenetic analysis to determine the taxonomic position demonstrated that the assignment of the MEP218 strain to Bacillus velezensis species provides insights into its evolutionary context and potential functional attributes. The in silico genome analysis revealed eleven gene clusters involved in the synthesis of secondary metabolites, including non-ribosomal CLP (fengycins and surfactin), polyketides, terpenes, and bacteriocins. Furthermore, genes encoding phytase, involved in the release of phytic phosphate for plant and animal nutrition, or other enzymes such as cellulase, xylanase, and alpha 1–4 glucanase were detected. In vitro antagonistic assays against Salmonella typhimurium, Acinetobacter baumanii, Escherichia coli, among others, demonstrated a broad spectrum of C16–C17 fengycin produced by MEP218. MEP218 genome sequence analysis expanded our understanding of the diversity and genetic relationships within the Bacillus genus and updated the Bacillus databases with its unique trait to produce antibacterial fengycins and its potential as a resource of biotechnologically useful enzymes. [ABSTRACT FROM AUTHOR]

The Fusarium head blight of grain cereals is a significant disease worldwide. In Argentina, high levels of contamination with Fusarium proliferatum have been found in crops. Many strains of the Pseudomonas genus antagonize the growth of fungi by different mechanisms, such as the production of antibiotics, siderophores, volatiles, and extracellular enzymes. In this work, we have designed a new system for studying the growth inhibition of F. proliferatum-namely by volatile compounds produced by Pseudomonas fluorescens MGR12. In both rich and minimal media, the bacterium released volatiles that negatively affected the mycelial growth of that phytopathogenic fungus. These bacterial compounds were analyzed by gas chromatography-mass spectrometry, but only a few could be identified by comparing their mass spectra with the libraries of the National Institutes of Standards and Technology MS search. [ABSTRACT FROM AUTHOR]

A growing body of evidence has reinforced the central role of microbiomes in the life of sound multicellular eukaryotes, thus more properly described as true holobionts. Though soil was considered a main source of plant microbiomes, seeds have been shown to be endophytically colonized by microorganisms thus representing natural carriers of a selected microbial inoculum to the young seedlings. In this work we have investigated the type of culturable endophytic bacteria that are carried within surface-sterilized alfalfa seeds. MALDI-TOF analysis revealed the presence of bacteria that belonged to 40 separate genera, distributed within four taxa ( Proteobacteria , Actinobacteria , Firmicutes , and Bacteroidetes ). Nonsymbiotic members of the Rhizobiaceae family were also found. The evaluation of nine different in-vitro biochemical activities demonstrated isolates with complex combinations of traits that, upon a Principal-Component-Analysis, could be classified into four phenotypic groups. That isolates from nearly half of the genera identified had been able to colonize alfalfa plants grown under axenic conditions was remarkable. Further analyses should be addressed to investigating the colonization mechanisms of the alfalfa seeds, the evolutionary significance of the alfalfa-seed endophytes, and also how after germination the seed microbiome competes with spermospheric and rhizospheric soil bacteria to colonize newly emerging seedlings. [ABSTRACT FROM AUTHOR]

Bacillus amyloliquefaciens MEP 2 18 is a native isolate with a broad spectrum of antifungal activity against plant pathogenic fungi. The ability of strain MEP 2 18 to antagonize phytopathogens is due to the production of cyclic lipopeptides (CLPs). In this work, different carbon (C) and nitrogen (N) sources and C to N ratios were evaluated in order to improve both, biomass and CLPs production by strain MEP 2 18. Among the C and N sources and C to N ratios tested, glucose and NH 4 NO 3 at the C to N ratio of 10:1 enhanced significantly biomass and CLPs yield. Moreover, CLPs produced in this medium exhibited higher antibacterial activity against Xanthomonas axonopodis pv. vesicatoria ( Xav ) than those obtained in the recommended medium for CLPs production. Interestingly, CLPs addition influenced the development of Xav biofilm on biotic and abiotic surfaces. A comparison of HPLC-chromatograms of CLPs obtained in the optimized medium versus the ones obtained in the recommended medium showed a notable increase of surfactin in the CLPs obtained in the optimized medium. Furthermore, two peaks with antibacterial activity against Xav , identified by mass spectrometry analysis as fengycins A and B, were detected in the CLPs from the strain MEP 2 18, grown in the optimized culture medium. The results obtained in this work suggest that changes in C and N sources and C to N ratios affect the yield and type of CLPs produced by B. amyloliquefaciens MEP 2 18. To the best of our knowledge, this is the first study to report the finding of fengycins with antibacterial activity. CLPs produced by the strain MEP 2 18 are potential candidates for controlling bacterial spot disease in tomato and pepper. [ABSTRACT FROM AUTHOR]

In this work, we surveyed the genome of P. protegens CHA0 in order to identify novel mRNAs possibly under the control of the Gac-Rsm cascade that might, for their part, serve to elucidate as-yet-unknown functions involved in the biocontrol of plant pathogens and/or in cellular processes required for fitness in natural environments. In view of the experimental evidence from former studies on the Gac-Rsm cascade, we developed a computational screen supported by a combination of sequence, structural and evolutionary constraints that led to a dataset of 43 potential novel mRNA targets. We then confirmed several mRNA targets experimentally and next focused on two of the respective genes that are physically linked to the orfamide biosynthetic gene cluster and whose predicted open-reading frames resembled cognate LuxR-type transcriptional regulators of cyclic lipopeptide clusters in related pseudomonads. In this report, we demonstrate that in strain CHA0, orfamide production is stringently dependent on a functional Gac-Rsm cascade and that both mRNAs encoding transcriptional regulatory proteins are under direct translational control of the RsmA/E proteins. Our results have thus revealed a hierarchical control over the expression of orfamide biosynthetic genes with the final transcriptional control subordinated to the global Gac-Rsm post-transcriptional regulatory system. [ABSTRACT FROM AUTHOR]

Azospirillum brasilense is a soil bacterium capable of promoting plant growth. Several surface components were previously reported to be involved in the attachment of A . brasilense to root plants. Among these components are the exopolysaccharide (EPS), lipopolysaccharide (LPS) and the polar flagellum. Flagellin from polar flagellum is glycosylated and it was suggested that genes involved in such a posttranslational modification are the same ones involved in the biosynthesis of sugars present in the O -antigen of the LPS. In this work, we report on the characterization of two homologs present in A. brasilense Cd, to the well characterized flagellin modification genes, flmA and flmB , from Aeromonas caviae . We show that mutations in either flmA or flmB genes of A. brasilense resulted in non-motile cells due to alterations in the polar flagellum assembly. Moreover, these mutations also affected the capability of A. brasilense cells to adsorb to maize roots and to produce LPS and EPS. By generating a mutant containing the polar flagellum affected in their rotation, we show the importance of the bacterial motility for the early colonization of maize roots. [ABSTRACT FROM AUTHOR]

Sinorhizobium meliloti associates with Medicago and Melilotus species to develop nitrogen-fixing symbioses. The agricultural relevance of these associations, the worldwide distribution of acid soils, and the remarkable acid sensitivity of the microsymbiont have all stimulated research on the responses of the symbionts to acid environments. We show here that an adaptive acid-tolerance response (ATR) can be induced in S. meliloti, as shown previously for Sinorhizobium medicae, when the bacteria are grown in batch cultures at the slightly acid pH of 6.1. In marked contrast, no increased tolerance to hydrogen ions is obtained if rhizobia are grown in a chemostat under continuous cultivation at the same pH. The adaptive ATR appears as a complex process triggered by an increased hydrogen-ion concentration, but operative only if other – as yet unknown – concomitant factors that depend on the culture conditions are present (although not provided under continuous cultivation). Although the stability of the ATR and its influence on acid tolerance has been characterized in rhizobia, no data have been available on the effect of the adapted state on symbiosis. Coinoculation experiments showed that acid-adapted indicator rhizobia (ATR+) were present in >90% of the nodules when nodulation was performed at pH 5.6, representing a >30% increase in occupancy compared with a control test. We show that the ATR represents a clear advantage in competing for nodulation at low pH. It is not yet clear whether such an effect results from an improved performance in the acid environment during preinfection, an enhanced ability to initiate infections, or both conditions. The practical use of ATR+ rhizobia will depend on validation experiments with soil microcosms and on field testing, as well as on the possibility of preserving the physiology of ATR+ bacteria in inoculant formulations. [ABSTRACT FROM AUTHOR]

Background: The bacterial Hfq protein is able to interact with diverse RNA molecules, including regulatory small non- coding RNAs (sRNAs), and thus it is recognized as a global post-transcriptional regulator of gene expression. Loss of Hfq has an extensive impact in bacterial physiology which in several animal pathogens influences virulence. Sinorhizobium meliloti is a model soil bacterium known for its ability to establish a beneficial nitrogen-fixing intracellular symbiosis with alfalfa. Despite the predicted general involvement of Hfq in the establishment of successful bacteria-eukaryote interactions, its function in S. meliloti has remained unexplored. Results:Two independentS. melilotimutants, 201 1-3.4and lO2lΔhfq,were obtained bydisruption and deletion ofthe hfq gene in the wild-type strains 2011 and 1021, respectively, both exhibiting similar growth defects as free-living bacteria. Transcriptomic profiling of 1 O2lAhfq revealed a general down-regulation of genes of sugar transporters and some enzymes of the central carbon metabolism, whereas transcripts specifying the uptake and metabolism of nitrogen sources (mainly amino acids) were more abundant than in the wild-type strain. Proteomic analysis of the 2011-3.4 mutant independently confirmed these observations. Symbiotic tests showed that lack of Hfq led to a delayed nodulation, severely compromised bacterial competitiveness on alfalfa roots and impaired normal plant growth. Furthermore, a large proportion of nodules (55%-64%) elicited by the 102 lAhfq mutant were non-fixing, with scarce content in bacteroids and signs of premature senescence of endosymbiotic bacteria. RT-PCR experiments on RNA from bacteria grown under aerobic and microoxic conditions revealed that Hfq contributes to regulation of nifA and fixKl/K2, the genes controlling nitrogen fixation, although the Hfq-mediated regulation of fixKis only aerobiosis dependent. Finally, we found that some of the recently identified 5. meliloti sRNAs co-inmunoprecipitate with a FLAG- epitope tagged Hfq protein. Conclusions: Our results support that the 5. meliloti RNA chaperone Hfq contributes to the control of central metabolic pathways in free-living bacteria and influences rhizospheric competence, survival of the microsymbiont within the nodule cells and nitrogen fixation during the symbiotic interaction with its legume host alfalfa. The identified 5. meliloti Hfq-binding sRNAs are predicted to participate in the Hfq regulatory network. [ABSTRACT FROM AUTHOR]

A bacterial collection of approximately one thousand native strains, isolated from saline soils of Cordoba province (Argentina), was established. From this collection, a screening to identify those strains showing plant growth promotion and biocontrol activities, as well as salt tolerance, was performed. Eight native strains tolerant to 1 M NaCl and displaying plant growth promotion and/or biocontrol features were selected for further characterization. Strains MEP2 18, MRP2 26, MEP2 11a, MEP3 1, and MEP3 3b significantly increased the growth of maize seedlings under normal and saline conditions, whereas isolates ARP2 3, AEP1 5, and ARP2 6 were able to increase the root dry weight of agropyre under saline conditions. On the other hand, strains MEP2 18 and ARP2 3 showed antagonistic activity against phytopathogenic fungi belonging to Sclerotinia and Fusarium genus. Antifungal activity was found in cell-free supernatants, and it was heat and protease resistant. Strains MEP218 and ARP23 were identified as Bacillus sp. and strains MEP211a and MEP33b as Ochrobactrum sp. according to the sequence analysis of 16S rRNA gene. [ABSTRACT FROM AUTHOR]

A correction to the article "Biocontrol and PGPR Features in Native Strains Isolated from Saline Soils of Argentina" that was published in the 2007 issue is presented.