Comparative genomics and informational content analysis uncovered internal regions of the core genes rpoD, pepN and gltX for an MLSA with genome-level resolving power within the genus Pseudomonas.

In the field of prokaryotic taxonomy, there has been a recent transition towards phylogenomics as the gold standard approach. However, genome-based phylogenetics is still restrictive for its cost when managing large amounts of isolates. Fast, cheap, and taxonomically competent alternatives, like multilocus sequence analysis (MLSA) are thus recommendable. Nevertheless, the criteria for selecting the conserved genes for MLSA have not been explicit for different bacterial taxa, including the broadly diverse Pseudomonas genus. Here, we have carried out an unbiased and rational workflow to select internal sequence regions of Pseudomonas core genes (CG) for a MLSA with the best phylogenetic power, and with a resolution comparable to the genome-based ANI approach. A computational workflow was established to inspect 126 complete genomes of representatives from over 60 Pseudomonas species and subspecies, in order to identify the most informative CG internal regions and determine which combinations in sets of three partial CG sequences have comparable phylogenetic resolution to that of the current ANI standard. We found that the rpoD346-1196-pepN1711-2571-gltX86-909 concatenated sequences were the best performing in terms of phylogenetic robustness and resulted highly sensitive and specific when contrasted with ANI. The rpoD-pepN-gltX MLSA was validated in silico and in vitro. Altogether, the results presented here supports the proposal of the rpoD-pepN-gltX MLSA as a fast, affordable, and robust phylogenetic tool for members of the Pseudomonas genus.

Azospirillum baldaniorum Sp245 exploits Pseudomonas fluorescens A506 biofilm to overgrow in dual-species macrocolonies.

Biofilms are essential for plant-associated bacteria to colonize their host. In this work, we analysed the interaction of Azospirillum baldaniorum Sp245 and Pseudomonas fluorescens A506 in mixed macrocolony biofilms. We identified certain culture conditions where A. baldaniorum Sp245 exploits P. fluorescens A506 to boost its growth. Azospirillum growth increased proportionally to the initial number of pseudomonads building the biofilm, which in turn were negatively affected in their growth. Physical contact with P. fluorescens A506 was essential for A. baldaniorum Sp245 growth increase. Biofilm ultrastructure analysis revealed that Pseudomonas produces a thick structure that hosts Azospirillum cells in its interior. Additional experimentation demonstrated that Azospirillum growth boost is compromised when interacting with biofilm-deficient Pseudomonas mutants, and that a low oxygen concentration strongly induce A. baldaniorum Sp245 growth, overriding Pseudomonas stimulation. In this line, we used a microaerophilia reporter strain of A. baldaniorum Sp245 to confirm that dual-species macrocolonies contain a higher number of cells under microaerophilic conditions. Taking all the results into consideration, we propose that A. baldaniorum Sp245 can benefit from P. fluorescens A506 partnership in mixed biofilms by taking advantage of the low oxygen concentration and scaffold made up of Pseudomonas-derived matrix, to expand its growth.

7-hydroxytropolone is the main metabolite responsible for the fungal antagonism of Pseudomonas donghuensis strain SVBP6.

Pseudomonas donghuensis strain SVBP6, an isolate from an agricultural plot in Argentina, displays a broad-spectrum and diffusible antifungal activity, which requires a functional gacS gene but could not be ascribed yet to known secondary metabolites typical of Pseudomonas biocontrol species. Here, we report that Tn5 mutagenesis allowed the identification of a gene cluster involved in both the fungal antagonism and the production of a soluble tropolonoid compound. The ethyl acetate extract from culture supernatant showed a dose-dependent inhibitory effect against the phytopathogenic fungus Macrophomina phaseolina. The main compound present in the organic extract was identified by spectroscopic and X-ray analyses as 7-hydroxytropolone (7HT). Its structure and tautomerism was confirmed by preparing the two key derivatives 2,3-dimethoxy- and 2,7-dimethoxy-tropone. 7HT, but not 2,3- or 2,7-dimethoxy-tropone, mimicked the fungal inhibitory activity of the ethyl acetate extract from culture supernatant. The activity of 7HT, as well as its production, was barely affected by the presence of up to 50 µM added iron (Fe+2 ). To summarize, P. donghuensis SVBP6 produces 7HT under the positive control of the Gac-Rsm cascade and is the main active metabolite responsible for the broad-spectrum inhibition of different phytopathogenic fungi.

Expression of the small regulatory RNA gene mmgR is regulated negatively by AniA and positively by NtrC in Sinorhizobium meliloti 2011.

In the N2-fixing symbiont of alfalfa root nodules, Sinorhizobium meliloti 2011, the mmgR gene encodes a 77 nt small untranslated RNA (sRNA) that negatively regulates the accumulation of polyhydroxybutyrate (PHB) when the bacterium is grown under conditions of surplus carbon (C) in relation to nitrogen (N). We previously showed that the expression of mmgR is primarily controlled at the transcriptional level and that it depends on the cellular N status, although the regulatory mechanism and the factors involved were unknown. In this study, we provide experimental data supporting that: (a) mmgR is induced upon N limitation with the maximum expression found at the highest tested C/N molar ratio in the growth medium; (b) a conserved heptamer TTGTGCA located between the -35 and -10 mmgR promoter elements is necessary and sufficient for induction by N limitation; (c) induction of mmgR requires the N-status regulator NtrC; (d) under C limitation, mmgR transcription is repressed by AniA, a global regulator of C flow; (e) the mmgR promoter contains a conserved dyadic motif (TGC[N3]GCA) partially overlapping the heptamer TTGTGCA, which was also found in the promoters of the PHB-related genes phaP1, phaP2, phaZ and phaR (aniA) of S. meliloti and other alpha-proteobacteria. Taken together, these results suggest that the mmgR promoter would integrate signals from the metabolism of C and N through - at least - the global regulators NtrC and AniA, to provide an optimal level of the MmgR sRNA to fine-tune gene expression post-transcriptionally according to varying C and N availability.

Regulation of Polyhydroxybutyrate Accumulation in Sinorhizobium meliloti by the Trans-Encoded Small RNA MmgR.

Riboregulation has a major role in the fine-tuning of multiple bacterial processes. Among the RNA players, trans-encoded untranslated small RNAs (sRNAs) regulate complex metabolic networks by tuning expression from multiple target genes in response to numerous signals. In Sinorhizobium meliloti, over 400 sRNAs are expressed under different stimuli. The sRNA MmgR (standing for Makes more granules Regulator) has been of particular interest to us since its sequence and structure are highly conserved among the alphaproteobacteria and its expression is regulated by the amount and quality of the bacterium's available nitrogen source. In this work, we explored the biological role of MmgR in S. meliloti 2011 by characterizing the effect of a deletion of the internal conserved core of mmgR (mmgRΔ33-51). This mutation resulted in larger amounts of polyhydroxybutyrate (PHB) distributed into more intracellular granules than are found in the wild-type strain. This phenotype was expressed upon cessation of balanced growth owing to nitrogen depletion in the presence of surplus carbon (i.e., at a carbon/nitrogen molar ratio greater than 10). The normal PHB accumulation was complemented with a wild-type mmgR copy but not with unrelated sRNA genes. Furthermore, the expression of mmgR limited PHB accumulation in the wild type, regardless of the magnitude of the C surplus. Quantitative proteomic profiling and quantitative reverse transcription-PCR (qRT-PCR) revealed that the absence of MmgR results in a posttranscriptional overexpression of both PHB phasin proteins (PhaP1 and PhaP2). Together, our results indicate that the widely conserved alphaproteobacterial MmgR sRNA fine-tunes the regulation of PHB storage in S. melilotiIMPORTANCE High-throughput RNA sequencing has recently uncovered an overwhelming number of trans-encoded small RNAs (sRNAs) in diverse prokaryotes. In the nitrogen-fixing alphaproteobacterial symbiont of alfalfa root nodules Sinorhizobium meliloti, only four out of hundreds of identified sRNA genes have been functionally characterized. Thus, uncovering the biological role of sRNAs currently represents a major issue and one that is particularly challenging because of the usually subtle quantitative regulation contributed by most characterized sRNAs. Here, we have characterized the function of the broadly conserved alphaproteobacterial sRNA gene mmgR in S. meliloti Our results strongly suggest that mmgR encodes a negative regulator of the accumulation of polyhydroxybutyrate, the major carbon and reducing power storage polymer in S. meliloti cells growing under conditions of C/N overbalance.

The Irr and RirA Proteins Participate in a Complex Regulatory Circuit and Act in Concert To Modulate Bacterioferritin Expression in Ensifer meliloti 1021.

In this work we found that the bfr gene of the rhizobial species Ensifer meliloti, encoding a bacterioferritin iron storage protein, is involved in iron homeostasis and the oxidative stress response. This gene is located downstream of and overlapping the smc03787 open reading frame (ORF). No well-predicted RirA or Irr boxes were found in the region immediately upstream of the bfr gene although two presumptive RirA boxes and one presumptive Irr box were present in the putative promoter of smc03787 We demonstrate that bfr gene expression is enhanced under iron-sufficient conditions and that Irr and RirA modulate this expression. The pattern of bfr gene expression as well as the response to Irr and RirA is inversely correlated to that of smc03787 Moreover, our results suggest that the small RNA SmelC759 participates in RirA- and Irr-mediated regulation of bfr expression and that additional unknown factors are involved in iron-dependent regulation.IMPORTANCEE. meliloti belongs to the Alphaproteobacteria, a group of bacteria that includes several species able to associate with eukaryotic hosts, from mammals to plants, in a symbiotic or pathogenic manner. Regulation of iron homeostasis in this group of bacteria differs from that found in the well-studied Gammaproteobacteria In this work we analyzed the effect of rirA and irr mutations on bfr gene expression. We demonstrate the effect of an irr mutation on iron homeostasis in this bacterial genus. Moreover, results obtained indicate a complex regulatory circuit where multiple regulators, including RirA, Irr, the small RNA SmelC759, and still unknown factors, act in concert to balance bfr gene expression.

Major cereal crops benefit from biological nitrogen fixation when inoculated with the nitrogen-fixing bacterium Pseudomonas protegens Pf-5 X940.

A main goal of biological nitrogen fixation research has been to expand the nitrogen-fixing ability to major cereal crops. In this work, we demonstrate the use of the efficient nitrogen-fixing rhizobacterium Pseudomonas protegens Pf-5 X940 as a chassis to engineer the transfer of nitrogen fixed by BNF to maize and wheat under non-gnotobiotic conditions. Inoculation of maize and wheat with Pf-5 X940 largely improved nitrogen content and biomass accumulation in both vegetative and reproductive tissues, and this beneficial effect was positively associated with high nitrogen fixation rates in roots. 15 N isotope dilution analysis showed that maize and wheat plants obtained substantial amounts of fixed nitrogen from the atmosphere. Pf-5 X940-GFP-tagged cells were always reisolated from the maize and wheat root surface but never from the inner root tissues. Confocal laser scanning microscopy confirmed root surface colonization of Pf-5 X940-GFP in wheat plants, and microcolonies were mostly visualized at the junctions between epidermal root cells. Genetic analysis using biofilm formation-related Pseudomonas mutants confirmed the relevance of bacterial root adhesion in the increase in nitrogen content, biomass accumulation and nitrogen fixation rates in wheat roots. To our knowledge, this is the first report of robust BNF in major cereal crops.

Phylogenetic distribution and evolutionary pattern of an α-proteobacterial small RNA gene that controls polyhydroxybutyrate accumulation in Sinorhizobium meliloti.

It has become clear that sRNAs play relevant regulatory functions in bacteria. However, a comprehensive understanding of their biological roles considering evolutionary aspects has not been achieved for most of them. Thus, we have characterized the evolutionary and phylogenetic aspects of the Sinorhizobium meliloti mmgR gene encoding the small RNA MmgR, which has been recently reported to be involved in the regulation of polyhydroxybutyrate accumulation in this bacterium. We constructed a covariance model from a multiple sequence and structure alignment of mmgR close homologs that allowed us to extend the search and to detect further remote homologs of the sRNA gene. From our results, mmgR seemed to evolve from a common ancestor of the α-proteobacteria that diverged from the order of Rickettsiales. We have found mmgR homologs in most current species of α-proteobacteria, with a few exceptions in which genomic reduction events or gene rearrangements seem to explain its absence. Furthermore, a strong microsyntenic relationship was found between a large set of mmgR homologs and homologs of a gene encoding a putative N-formyl glutamate amidohydrolase (NFGAH) that allowed us to trace back the evolutionary path of this group of mmgR orthologs. Among them, structure and sequence traits have been completely conserved throughout evolution, namely a Rho-independent terminator and a 10-mer (5'-UUUCCUCCCU-3') that is predicted to remain in a single-stranded region of the sRNA. We thus propose the definition of the new family of α-proteobacterial sRNAs αr8, as well as the subfamily αr8s1 which encompass S. meliloti mmgR orthologs physically linked with the downstream open reading frame encoding a putative NFGAH. So far, mmgR is the trans-encoded small RNA with the widest phylogenetic distribution of well recognized orthologs among α-proteobacteria. Expression of the expected MmgR transcript in rhizobiales other than S. meliloti (Sinorhizobium fredii, Rhizobium leguminosarum and Rhizobium etli) was confirmed by Northern blot. These findings will contribute to the understanding of the biological role(s) of mmgR in the α-proteobacteria.

[Selective enrichment of Pseudomonas spp. in the rhizoplane of different plant species]. / Enriquecimiento diferencial de Pseudomonas spp. en el rizoplano de distintas especies cultivadas.

In contrast to rhizobia-legume symbiosis, the specificity for root colonization by pseudomonads seems to be less strict. However, several studies about bacterial diversity in the rhizosphere highlight the influence of plant species on the selective enrichment of certain microorganisms from the bulk soil community. In order to evaluate the effect that different crops have on the structure of pseudomonad community on the root surface, we performed plant trap experiments, using surface-disinfected maize, wheat or soybean seeds that were sown in pots containing the same pristine soil as substrate. Rhizoplane suspensions were plated on a selective medium for Pseudomonas, and pooled colonies served as DNA source to carry out PCR-RFLP community structure analysis of the pseudomonads-specific marker genes oprF and gacA. PCR-RFLP profiles were grouped by plant species, and were distinguished from those of bulk soil samples. Partial sequencing of 16S rDNA genes of some representative colonies of Pseudomonas confirmed the selective enrichment of distinctive genotypes in the rhizoplane of each plant species. These results support the idea that the root systems of agricultural crops such as soybean, maize and wheat, select differential sets of pseudomonads from the native microbial repertoire inhabiting the bulk soil.

An Integrated Affinity Chromatography-Based Approach to Unravel the sRNA Interactome in Nitrogen-Fixing Rhizobia.

The activity mechanism and function of bacterial base-pairing small non-coding RNA regulators (sRNAs) are largely shaped by their main interacting cellular partners, i.e., proteins and mRNAs. We describe here an MS2 affinity chromatography-based procedure adapted to unravel the sRNA interactome in nitrogen-fixing legume endosymbiotic bacteria. The method consists of tagging of the bait sRNA at its 5'-end with the MS2 aptamer followed by pulse overexpression and immobilization of the chimeric transcript from cell lysates by an MS2-MBP fusion protein conjugated to an amylose resin. The sRNA-binding proteins and target mRNAs are further profiled by mass spectrometry and RNAseq, respectively.

Insights into the noncoding RNome of nitrogen-fixing endosymbiotic α-proteobacteria.

Symbiotic chronic infection of legumes by rhizobia involves transition of invading bacteria from a free-living environment in soil to an intracellular state as differentiated nitrogen-fixing bacteroids within the nodules elicited in the host plant. The adaptive flexibility demanded by this complex lifestyle is likely facilitated by the large set of regulatory proteins encoded by rhizobial genomes. However, proteins are not the only relevant players in the regulation of gene expression in bacteria. Large-scale high-throughput analysis of prokaryotic genomes is evidencing the expression of an unexpected plethora of small untranslated transcripts (sRNAs) with housekeeping or regulatory roles. sRNAs mostly act in response to environmental cues as post-transcriptional regulators of gene expression through protein-assisted base-pairing interactions with target mRNAs. Riboregulation contributes to fine-tune a wide range of bacterial processes which, in intracellular animal pathogens, largely compromise virulence traits. Here, we summarize the incipient knowledge about the noncoding RNome structure of nitrogen-fixing endosymbiotic bacteria as inferred from genome-wide searches for sRNA genes in the alfalfa partner Sinorhizobium meliloti and further comparative genomics analysis. The biology of relevant S. meliloti RNA chaperones (e.g., Hfq) is also reviewed as a first global indicator of the impact of riboregulation in the establishment of the symbiotic interaction.

A CsrA/RsmA translational regulator gene encoded in the replication region of a Sinorhizobium meliloti cryptic plasmid complements Pseudomonas fluorescens rsmA/E mutants.

Members of the CsrA/RsmA family are global regulatory proteins that bind to mRNAs, usually at the ribosome-binding site, to control mRNA translation and stability. Their activity is counteracted by small non-coding RNAs (sRNAs), which offer several binding sites to compete with mRNA binding. The csrA/rsmA genes are widespread in prokaryotic chromosomes, although certain phylogenetic groups such as Alphaproteobacteria lack this type of global regulator. Interestingly, a csrA/rsmA-like sequence was identified in the replication region of plasmid pMBA19a from the alphaproteobacterium Sinorhizobium meliloti. This rsmA-like allele (rsmA(Sm)) is 58 % identical to Xanthomonas axonopodis pv. citri chromosomal rsmA and bears an unusual C-terminal extension that may fold into an extra α-helix. Homology-based modelling of RsmA(Sm) suggests that all key mRNA-binding residues are conserved and correctly positioned in the RNA-binding pocket. In fact, a 1.6 kb fragment from pMBA19a encompassing the rsmA(Sm) locus restored rsmA/E-dependent phenotypes of rsmA/E gacS Pseudomonas fluorescens mutants. The functionality of RsmA(Sm) was confirmed by the gain of control over target aprA'-'lacZ and hcnA'-'lacZ translational fusions in the same mutant background. The RsmA(Sm) activity correlated with Western blot detection of the polypeptide. Phenotype and translational fusion data from rsmA/E P. fluorescens mutants expressing RsmX/Y/Z RNAs indicated that RsmA(Sm) is able to bind these antagonistic sRNAs. In agreement with the latter observation, it was also found that the sRNA RsmY was stabilized by RsmA(Sm). Deletion of the C-terminal extra α-helix of RsmA(Sm) affected its cellular concentration, but increased its relative RNA-binding activity. This is believed to be the first report of the presence and characterization of a functional csrA/rsmA homologue in a mobile genetic element.

The bacterial protein Hfq: much more than a mere RNA-binding factor.

Most of the sequenced bacterial genomes contain a gene encoding a protein known as Hfq that resembles the eukaryotic RNA-binding proteins of the LSm family. It was originally identified in Escherichia coli as a host factor required for replication of the Qß RNA phage. In this review, we present a comprehensive summary of 40 years of investigation to learn that Hfq is an influential, though not essential, global regulator of gene expression in bacteria and that this feature is undoubtedly linked to Hfq's RNA-binding properties. This protein intervenes in different RNA transactions, notably the promotion of antisense interactions between messenger RNAs and small regulatory RNAs. Yet, several aspects of its molecular mechanism remain not understood. In addition, mechanistic studies have been exclusively carried out in enterobacterial models, highlighting the need to expand the research on Hfq function to other taxons. Upon reviewing the genetic, structural, biochemical, and biological aspects of this extraordinary protein, we discuss recent findings on interactions with macromolecules other than RNA suggesting a broader participation of Hfq in major steps in the flow of genetic information. We show that, although significant progress has been achieved to elucidate Hfq role at the molecular level, many open questions remain.

Agronomic efficiency and genome mining analysis of the wheat-biostimulant rhizospheric bacterium Pseudomonas pergaminensis sp. nov. strain 1008T.

Pseudomonas sp. strain 1008 was isolated from the rhizosphere of field grown wheat plants at the tillering stage in an agricultural plot near Pergamino city, Argentina. Based on its in vitro phosphate solubilizing capacity and the production of IAA, strain 1008 was formulated as an inoculant for bacterization of wheat seeds and subjected to multiple field assays within the period 2010-2017. Pseudomonas sp. strain 1008 showed a robust positive impact on the grain yield (+8% on average) across a number of campaigns, soil properties, seed genotypes, and with no significant influence of the simultaneous seed treatment with a fungicide, strongly supporting the use of this biostimulant bacterium as an agricultural input for promoting the yield of wheat. Full genome sequencing revealed that strain 1008 has the capacity to access a number of sources of inorganic and organic phosphorus, to compete for iron scavenging, to produce auxin, 2,3-butanediol and acetoin, and to metabolize GABA. Additionally, the genome of strain 1008 harbors several loci related to rhizosphere competitiveness, but it is devoid of biosynthetic gene clusters for production of typical secondary metabolites of biocontrol representatives of the Pseudomonas genus. Finally, the phylogenomic, phenotypic, and chemotaxonomic comparative analysis of strain 1008 with related taxa strongly suggests that this wheat rhizospheric biostimulant isolate is a representative of a novel species within the genus Pseudomonas, for which the name Pseudomonas pergaminensis sp. nov. (type strain 1008T = DSM 113453T = ATCC TSD-287T) is proposed.

Circadian variation in Pseudomonas fluorescens (CHA0)-mediated paralysis of Caenorhabditis elegans.

Abiotic and biotic environmental stressors play a key role in the ecophysiology of most organisms. As the presence and activity of stress-inducing agents vary along the day, organisms that are able to predict these periodic changes are better fit to survive. Caenorhabditis elegans, a soil-dwelling nematode, is subjected to daily changes in its natural environment, and its tolerance to osmotic and oxidative stress varies along the day. Pseudomonas fluorescens strain CHA0 is a soil bacterium that produces a set of secondary metabolites that antagonize phytopathogenic fungi and therefore promote healthy growth of several plant species. Here we show that strain CHA0 is able to affect C. elegans either under growth limiting conditions (i.e., slow-killing) or by rapid paralysis in nutrient replete conditions (fast-killing). Both types of toxicity require the post-transcriptional Gac/Rsm regulatory cascade, and the fast paralytic killing depends strongly on hydrogen cyanide production. The response observed in C. elegans nematodes to fast paralytic killing varies along the day and its sensitivity is higher during the night, at Zeitgeber Time (ZT) 12 (lights off). This behavior correlates well with HCN tolerance, which is higher during the day, at ZT0 (lights on). The innate immune response to P. fluorescens CHA0 might depend on the stress response pathway of C. elegans. The fact that the tolerance varies daily gives further proof of an underlying clock that governs cyclic behavior in C. elegans.

Evidences of autoregulation of hfq expression in Sinorhizobium meliloti strain 2011.

Riboregulation comprises gene expression regulatory mechanisms that rely upon the activity of small non-coding RNAs (sRNAs) and in most cases RNA binding proteins. In γ-proteobacteria, the Sm-like protein Hfq is a key player in riboregulatory processes, because it promotes sRNA-mRNA interactions and influences mRNA polyadenylation or translation. In the α-proteobacterium Sinorhizobium meliloti, the large number of detected small RNA transcripts and the pleiotropic effects of hfq mutations lead to the hypothesis that riboregulatory mechanisms are important in this soil microorganism to adjust gene expression both in free-living conditions and as a nitrogen-fixing endosymbiont within legume root nodules. In this study, homology modeling of S. meliloti Hfq protein and cross-complementation experiments of S. meliloti and Escherichia coli mutants indicates that hfq ( Sm ) encodes an RNA chaperone that can be functionally exchanged by its homolog from E. coli. A transcriptional and translational analysis of S. meliloti hfq expression by means of lacZ reporter fusions strongly suggests that the S. meliloti Hfq protein autocontrols its expression at the translational level, a phenomenon that was evident in the natural host S. meliloti as well as in the heterologous host E. coli.

Structural studies on the O-specific polysaccharide of the lipopolysaccharide from Pseudomonas donghuensis strain SVBP6, with antifungal activity against the phytopathogenic fungus Macrophomina phaseolina.

An O-specific polysaccharide (OPS) was isolated from the lipopolysaccharide (LPS) of Pseudomonas donghuensis SVBP6, a bacterium with a broad-spectrum antifungal activity in vitro, particularly that against Macrophomina phaseolina. This latter is one of the most virulent and dangerous pathogens of plants, including soybean which is an economically important crop in Argentina today. The OPS was studied by sugar analysis and spectroscopy (1D and 2D 1H and 13C NMR) showing the following trisaccharide repeating unit: →6)-ɑ-D-ManpNAc-(1 â†’ 3)-ß-l-Rhap-(1 â†’ 4)-ß-D-Glcp-(1→. The crude LPS, the purified LPS and the O-chain were assayed for their antifungal activity against M. phaseolina at 25, 50, 100, and 200 µg plug-1. The results showed that the crude LPS best inhibition was at 200 µg plug-1, able to inhibit the fungus growth by about 45%, while purified LPS and the corresponding OPS, in the same condition, reduced fungus growth by 65%, and 75%, respectively. Furthermore, the purified LPS and OPS significantly reduced the growth of M. phaseolina already at 100 µg plug-1 compared to the crude LPS. The structure of the O-chain is unique among the bacterial LPS and this is the first time that both the antifungal activity of a bacterial LPS and its corresponding O-chain were described.

Comparative Genomics and Evolutionary Analysis of RNA-Binding Proteins of the CsrA Family in the Genus Pseudomonas.

Gene expression is adjusted according to cellular needs through a combination of mechanisms acting at different layers of the flow of genetic information. At the posttranscriptional level, RNA-binding proteins are key factors controlling the fate of nascent and mature mRNAs. Among them, the members of the CsrA family are small dimeric proteins with heterogeneous distribution across the bacterial tree of life, that act as global regulators of gene expression because they recognize characteristic sequence/structural motifs (short hairpins with GGA triplets in the loop) present in hundreds of mRNAs. The regulatory output of CsrA binding to mRNAs is counteracted in most cases by molecular mimic, non-protein coding RNAs that titrate the CsrA dimers away from the target mRNAs. In γ-proteobacteria, the regulatory modules composed by CsrA homologs and the corresponding antagonistic sRNAs, are mastered by two-component systems of the GacS-GacA type, which control the transcription and the abundance of the sRNAs, thus constituting the rather linear cascade Gac-Rsm that responds to environmental or cellular signals to adjust and coordinate the expression of a set of target genes posttranscriptionally. Within the γ-proteobacteria, the genus Pseudomonas has been shown to contain species with different number of active CsrA (RsmA) homologs and of molecular mimic sRNAs. Here, with the help of the increasing availability of genomic data we provide a comprehensive state-of-the-art picture of the remarkable multiplicity of CsrA lineages, including novel yet uncharacterized paralogues, and discuss evolutionary aspects of the CsrA subfamilies of the genus Pseudomonas, and implications of the striking presence of csrA alleles in natural mobile genetic elements (phages and plasmids).

Biocontrol Traits Correlate With Resistance to Predation by Protists in Soil Pseudomonads.

Root-colonizing bacteria can support plant growth and help fend off pathogens. It is clear that such bacteria benefit from plant-derived carbon, but it remains ambiguous why they invest in plant-beneficial traits. We suggest that selection via protist predation contributes to recruitment of plant-beneficial traits in rhizosphere bacteria. To this end, we examined the extent to which bacterial traits associated with pathogen inhibition coincide with resistance to protist predation. We investigated the resistance to predation of a collection of Pseudomonas spp. against a range of representative soil protists covering three eukaryotic supergroups. We then examined whether patterns of resistance to predation could be explained by functional traits related to plant growth promotion, disease suppression and root colonization success. We observed a strong correlation between resistance to predation and phytopathogen inhibition. In addition, our analysis highlighted an important contribution of lytic enzymes and motility traits to resist predation by protists. We conclude that the widespread occurrence of plant-protective traits in the rhizosphere microbiome may be driven by the evolutionary pressure for resistance against predation by protists. Protists may therefore act as microbiome regulators promoting native bacteria involved in plant protection against diseases.