Identification and functional analysis of recent IS transposition events in rhizobia.

Rhizobia are alpha- and beta- Proteobacteria that, through the establishment of symbiotic interactions with leguminous plants, are able to fix atmospheric nitrogen as ammonium. The successful establishment of a symbiotic interaction is highly dependent on the availability of nitrogen sources in the soil, and on the specific rhizobia strain. Insertion sequences (ISs) are simple transposable genetic elements that can move to different locations within the host genome and are known to play an important evolutionary role, contributing to genome plasticity by acting as recombination hot-spots, and disrupting coding and regulatory sequences. Disruption of coding sequences may have occurred either in a common ancestor of the species or more recently. By means of ISComapare, we identified Differentially Located ISs (DLISs) in nearly related rhizobial strains of the genera Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium. Our results revealed that recent IS transposition could have a role in adaptation by enabling the activation and inactivation of genes that could dynamically affect the competition and survival of rhizobia in the rhizosphere.

Diversity, Genomics and Symbiotic Characteristics of Sinorhizobia That Nodulate Desmanthus spp. in Northwest Argentina.

Desmanthus spp. are legumes with the ability to associate with diverse α-proteobacteria-a microsymbiont-in order to establish nitrogen-fixing root nodules. A previous investigation from our laboratory revealed that the main bacteria associated with Desmanthus paspalaceus in symbiosis in central Argentina (Province of Santa Fe) were quite diverse and belonged to the genera Rhizobium and Mesorhizobium. To achieve a more extensive view of the local microsymbionts associated with Desmanthus spp., we sampled three different sites in Jujuy and Salta, in northwest Argentina. Matrix-assisted Laser-Desorption-Ionization Time-of-Flight mass spectrometry (MALDI-TOF) typing, 16S-rDNA analysis, and genome sequencing demonstrated that the dominant root-nodule microsymbionts belonged to the genus Sinorhizobium, with some sequenced genomes related to Sinorhizobium mexicanum, Sinorhizobium chiapanecum, and Sinorhizobium psoraleae. An analysis of nodA and nodC markers indicated that, in some of the isolates, horizontal gene transfer appeared to be responsible for the lack of congruence between the phylogenies of the chromosome and of the symbiotic region. These results revealed diverse evolutionary strategies for reaching the current Desmanthus-microsymbiont diversity. What is remarkable beside their observed genetic diversity is that the tolerance profiles of these isolates to abiotic stresses (temperature, salt concentration, pH) were quite coincident with the separation of the sinorhizobia according to place of origin, suggesting possible ecoedaphic adaptations. This observation, together with the higher aerial dry-weight matter that some isolates generated in Desmanthus virgatus cv. Marc when compared to the biomass generated by the commercial strain Sinorhizobium terangae CB3126, distinguish the collected sinorhizobia as constituting valuable germplasm for evaluation in local fields to select for more efficient symbiotic pairs.

Comparative Proteomic Analysis Revealing ActJ-Regulated Proteins in Sinorhizobium meliloti.

To adapt to different environmental conditions, Sinorhizobium meliloti relies on finely tuned regulatory networks, most of which are unexplored to date. We recently demonstrated that deletion of the two-component system ActJK renders an acid-vulnerable phenotype in S. meliloti and negatively impacts bacteroid development and nodule occupancy as well. To fully understand the role of ActJ in acid tolerance, S. meliloti wild-type and S. meliloti ΔactJ proteomes were compared in the presence or absence of acid stress by nanoflow ultrahigh-performance liquid chromatography coupled to mass spectrometry. The analysis demonstrated that proteins involved in the synthesis of exopolysaccharides (EPSs) were notably enriched in ΔactJ cells in acid pH. Total EPS quantification further revealed that although EPS production was augmented at pH 5.6 in both the ΔactJ and the parental strain, the lack of ActJ significantly enhanced this difference. Moreover, several efflux pumps were found to be downregulated in the ΔactJ strain. Promoter fusion assays suggested that ActJ positively modulated its own expression in an acid medium but not at under neutral conditions. The results presented here identify several ActJ-regulated genes in S. meliloti, highlighting key components associated with ActJK regulation that will contribute to a better understanding of rhizobia adaptation to acid stress.

Curtobacterium, A Foliar Pathogen Isolated from Maize in Central Argentina.

Plant pathogens, such as fungi, bacteria, and viruses, can cause serious damage to crops and significantly reduce yield and quality. Bacterial diseases of agronomic crops, however, have been little studied. The present study aims to isolate and identify bacteria recovered from symptomatic maize (Zea mays) leaves collected from field samples in the province of Cordoba, Argentina. Bacterial strains were identified using whole-cell matrix-assisted laser-desorption-ionization-time-off light mass spectrometry and 16S rDNA sequencing. Members of the genera Exiguobacterium and Curtobacterium were dominant in the studied vegetal material. Two strains (RC18-1/2 and RC18-3/1) were selected for further studies. The pathogenicity test showed that plants inoculated with Curtobacterium sp. RC18-1/2 exhibited the same symptoms as those previously detected in the field. To our knowledge, this study provides the first evidence about the isolation of a Curtobacterium pathogenic strain in maize. Effective crop disease management will require the use of integrated strategies, such as resistant cultivars and/or biocontrol agents.

ubiF is involved in acid stress tolerance and symbiotic competitiveness in Rhizobium favelukesii LPU83.

The acidity of soils significantly reduces the productivity of legumes mainly because of the detrimental effects of hydrogen ions on the legume plants, leading to the establishment of an inefficient symbiosis and poor biological nitrogen fixation. We recently reported the analysis of the fully sequenced genome of Rhizobium favelukesii LPU83, an alfalfa-nodulating rhizobium with a remarkable ability to grow, nodulate and compete in acidic conditions. To gain more insight into the genetic mechanisms leading to acid tolerance in R. favelukesii LPU83, we constructed a transposon mutant library and screened for mutants displaying a more acid-sensitive phenotype than the parental strain. We identified mutant Tn833 carrying a single-transposon insertion within LPU83_2531, an uncharacterized short ORF located immediately upstream from ubiF homolog. This gene encodes a protein with an enzymatic activity involved in the biosynthesis of ubiquinone. As the transposon was inserted near the 3' end of LPU83_2531 and these genes are cotranscribed as a part of the same operon, we hypothesized that the phenotype in Tn833 is most likely due to a polar effect on ubiF transcription.We found that a mutant in ubiF was impaired to grow at low pH and other abiotic stresses including 5 mM ascorbate and 0.500 mM Zn2+. Although the ubiF mutant retained the ability to nodulate alfalfa and Phaseolus vulgaris, it was unable to compete with the R. favelukesii LPU83 wild-type strain for nodulation in Medicago sativa and P. vulgaris, suggesting that ubiF is important for competitiveness. Here, we report for the first time an ubiF homolog being essential for nodulation competitiveness and tolerance to specific stresses in rhizobia.

BioF is a novel B2 metallo-ß-lactamase from Pseudomonas sp. isolated from an on-farm biopurification system.

Antimicrobial resistance represents a major global health concern and environmental bacteria are considered a source of resistance genes. Carbapenems are often used as the last antibiotic option to treat multidrug-resistant bacteria. Metallo-ß-lactamases (MBLs) are able to render resistance to almost all ß-lactam antibiotics, including carbapenems. Unfortunately, there are no inhibitors against MBLs for clinical use. Subclass B2 MBLs are the only enzymes working as strict carbapenemases, under-represented, encoded in chromosome genes and only functional as mono-zinc enzymes. Despite current efforts in MBLs inhibitor development, B2 carbapenemase activity is especially difficult to suppress, even in vitro. In this study we characterized BioF, a novel subclass B2 MBL identified in a new environmental Pseudomonas sp. strain isolated from an on-farm biopurification system (BPS). Although blaBioF is most likely a chromosomal gene, it is found in a genomic island and may represent a step previous to the horizontal transmission of B2 genes. The new B2 MBL is active as a mono-zinc enzyme and is a potent carbapenemase with incipient activity against some cephalosporins. BioF activity is not affected by excess zinc and is only inhibited at high metal chelator concentrations. The discovery and characterization of B2 MBL BioF as a potent carbapenemase in a BPS bacterial isolate emphasizes the importance of exploring antibiotic resistances existing in the environmental microbiota under the influence of human activities before they could emerge clinically.

Exopolysaccharide Characterization of Rhizobium favelukesii LPU83 and Its Role in the Symbiosis With Alfalfa.

One of the greatest inputs of available nitrogen into the biosphere occurs through the biological N2-fixation to ammonium as result of the symbiosis between rhizobia and leguminous plants. These interactions allow increased crop yields on nitrogen-poor soils. Exopolysaccharides (EPS) are key components for the establishment of an effective symbiosis between alfalfa and Ensifer meliloti, as bacteria that lack EPS are unable to infect the host plants. Rhizobium favelukesii LPU83 is an acid-tolerant rhizobia strain capable of nodulating alfalfa but inefficient to fix nitrogen. Aiming to identify the molecular determinants that allow R. favelukesii to infect plants, we studied its EPS biosynthesis. LPU83 produces an EPS I identical to the one present in E. meliloti, but the organization of the genes involved in its synthesis is different. The main gene cluster needed for the synthesis of EPS I in E. meliloti, is split into three different sections in R. favelukesii, which probably arose by a recent event of horizontal gene transfer. A R. favelukesii strain devoided of all the genes needed for the synthesis of EPS I is still able to infect and nodulate alfalfa, suggesting that attention should be directed to other molecules involved in the development of the symbiosis.

The two-component system ActJK is involved in acid stress tolerance and symbiosis in Sinorhizobium meliloti.

The nitrogen-fixing α-proteobacterium Sinorhizobium meliloti genome codifies at least 50 response regulator (RR) proteins mediating different and, in many cases, unknown processes. RR-mutant library screening allowed us to identify genes potentially implicated in survival to acid conditions. actJ mutation resulted in a strain with reduced growth rate under mildly acidic conditions as well as a lower capacity to tolerate a sudden shift to lethal acidic conditions compared with the parental strain. Mutation of the downstream gene actK, which encodes for a histidine kinase, showed a similar phenotype in acidic environments suggesting a functional two-component system. Interestingly, even though nodulation kinetics, quantity, and macroscopic morphology of Medicago sativa nodules were not affected in actJ and actK mutants, ActK was required to express the wild-type nitrogen fixation phenotype and ActJK was necessary for full bacteroid development and nodule occupancy. The actJK regulatory system presented here provides insights into an evolutionary process in rhizobium adaptation to acidic environments and suggests that actJK-controlled functions are crucial for optimal symbiosis development.

Identification and Characterization of a Novel Plasmid-Encoded Laccase-Like Multicopper Oxidase from Ochrobactrum sp. BF15 Isolated from an On-Farm Bio-Purification System.

RESEARCH BACKGROUND: In recent decades, laccases (p-diphenol-dioxygen oxidoreductases; EC 1.10.3.2) have attracted the attention of researchers due to their wide range of biotechnological and industrial applications. Laccases can oxidize a variety of organic and inorganic compounds, making them suitable as biocatalysts in biotechnological processes. Even though the most traditionally used laccases in the industry are of fungal origin, bacterial laccases have shown an enormous potential given their ability to act on several substrates and in multiple conditions. The present study aims to characterize a plasmid-encoded laccase-like multicopper oxidase (LMCO) from Ochrobactrum sp. BF15, a bacterial strain previously isolated from polluted soil. EXPERIMENTAL APPROACH: We used in silico profile hidden Markov models to identify novel laccase-like genes in Ochrobactrum sp. BF15. For laccase characterization, we performed heterologous expression in Escherichia coli, purification and activity measurement on typical laccase substrates. RESULTS AND CONCLUSIONS: Profile hidden Markov models allowed us to identify a novel LMCO, named Lac80. In silico analysis of Lac80 revealed the presence of three conserved copper oxidase domains characteristic of three-domain laccases. We successfully expressed Lac80 heterologously in E. coli, allowing us to purify the protein for further activity evaluation. Of thirteen typical laccase substrates tested, Lac80 showed lower activity on 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), pyrocatechol, pyrogallol and vanillic acid, and higher activity on 2,6-dimethoxyphenol. NOVELTY AND SCIENTIFIC CONTRIBUTION: Our results show Lac80 as a promising laccase for use in industrial applications. The present work shows the relevance of bacterial laccases and highlights the importance of environmental plasmids as valuable sources of new genes encoding enzymes with potential use in biotechnological processes.

Genome sequence of Bradyrhizobium yuanmingense strain P10 130, a highly efficient nitrogen-fixing bacterium that could be used for Desmodium incanum inoculation.

Strain P10 130, an isolated Bradyrhizobium strain from Argentina which promotes the growth of the leguminous plant Desmodium incanum by different mechanisms was previously selected as the best candidate for D. incanum inoculation based on broader selective criteria. A close relationship between this strain and B. yuanmingense was determined by MALDI BioTyper identification and 16S rRNA gene phylogenetic analysis. This study aimed to analyse the genome sequence of B. yuanmingense P10 130 in order to deepen our knowledge regarding its plant growth-promoting traits and to establish its phylogenetic relationship with other species of Bradyrhizobium genus. The genome size of strain P10 130 was estimated to be 7.54 Mb that consisted of 65 contigs. Genome Average Nucleotide Identity (ANI) analysis revealed that B. yuanmingense CCBAU 10071 T was the closest strain to P10 130 with ca. 96% identity. Further analysis of the genome of B. yuanmingense P10 130 identified 20 nod/nol/NOE, 14 nif/18 fix, 5 nap/5 nor genes, which may be potentially involved in nodulation, nitrogen fixation, and denitrification process respectively. Genome sequence of B. yuanmingense P10 130 is a valuable source of information to continue the research of its potential industrial production as a biofertilizer of D. incanum.

Codon Usage Optimization in the Prokaryotic Tree of Life: How Synonymous Codons Are Differentially Selected in Sequence Domains with Different Expression Levels and Degrees of Conservation.

Prokaryote genomes exhibit a wide range of GC contents and codon usages, both resulting from an interaction between mutational bias and natural selection. In order to investigate the basis underlying specific codon changes, we performed a comprehensive analysis of 29 different prokaryote families. The analysis of core gene sets with increasing ancestries in each family lineage revealed that the codon usages became progressively more adapted to the tRNA pools. While, as previously reported, highly expressed genes presented the most optimized codon usage, the singletons contained the less selectively favored codons. The results showed that usually codons with the highest translational adaptation were preferentially enriched. In agreement with previous reports, a C bias in 2- to 3-fold pyrimidine-ending codons, and a U bias in 4-fold codons occurred in all families, irrespective of the global genomic GC content. Furthermore, the U biases suggested that U3-mRNA-U34-tRNA interactions were responsible for a prominent codon optimization in both the most ancestral core and the highly expressed genes. A comparative analysis of sequences that encode conserved (cr) or variable (vr) translated products, with each one being under high (HEP) and low (LEP) expression levels, demonstrated that the efficiency was more relevant (by a factor of 2) than accuracy to modeling codon usage. Finally, analysis of the third position of codons (GC3) revealed that in genomes with global GC contents higher than 35 to 40%, selection favored a GC3 increase, whereas in genomes with very low GC contents, a decrease in GC3 occurred. A comprehensive final model is presented in which all patterns of codon usage variations are condensed in four distinct behavioral groups.IMPORTANCE The prokaryotic genomes-the current heritage of the most ancient life forms on earth-are comprised of diverse gene sets, all characterized by varied origins, ancestries, and spatial-temporal expression patterns. Such genetic diversity has for a long time raised the question of how cells shape their coding strategies to optimize protein demands (i.e., product abundance) and accuracy (i.e., translation fidelity) through the use of the same genetic code in genomes with GC contents that range from less than 20 to more than 80%. Here, we present evidence on how codon usage is adjusted in the prokaryotic tree of life and on how specific biases have operated to improve translation. Through the use of proteome data, we characterized conserved and variable sequence domains in genes of either high or low expression level and quantitated the relative weight of efficiency and accuracy-as well as their interaction-in shaping codon usage in prokaryotes.

Guidelines for Inferring and Characterizing a Family of Bacterial trans-Acting Small Noncoding RNAs.

So far, every sequenced bacterial transcriptome encompasses hundreds of small regulatory noncoding RNAs (sRNAs). From those sRNAs that have been already characterized, we learned that their regulatory functions could span over almost every bacterial process, mostly acting at the posttranscriptional control of gene expression (Wagner and Romby, Adv Genet 90:133-208, 2015). Canonical molecular mechanisms of sRNA action have been described to rely on both sequence and/or structural traits of the RNA molecule. As for protein-coding genes, the conservation of sRNAs among species suggests conserved and adjusted functions across evolution. Knowing the phylogenetic distribution of an sRNA gene and how its functional traits have evolved may help to get a broad picture of its biological role in each single species. Here, we present a simple computational workflow to identify close and distant sRNA homologs present in sequenced bacterial genomes, which allows defining novel sRNA families. This strategy is based on the use of Covariance Models (CM) and assumes the conservation of sequence and structure of functional sRNA genes throughout evolution. Moreover, by carefully inspecting the conservation of the close genomic context of every member of the RNA family and how the patterns of microsynteny follow the path of species evolution, it is possible to define subgroups of sRNA orthologs, which in turn enables the definition of RNA subfamilies.

Novel environmental class 1 integrons and cassette arrays recovered from an on-farm bio-purification plant.

Rapid dissemination and emergence of novel antibiotic resistance genes among bacteria are rising problems worldwide. Since their discovery in clinical isolates in the late 1980s, class 1 integrons have been found in a wide range of bacterial genera and have been extensively studied as contributors to dissemination of antibiotic resistance. The present study aimed to investigate the presence and structure of class 1 integrons in plasmid-carrying bacterial isolates obtained from a biopurification system used for decontamination of pesticide-contaminated water as well as their possible role as reservoir of antimicrobial resistance gene cassettes. A total of 35 representative isolates were screened for the presence of class 1 integron integrase encoded by intI1. PCR and DNA sequencing revealed the presence of six class 1 integrons with four variable regions: 5΄CS-aadA1b-3΄CS, 5΄CS-aadA2-3΄CS, 5΄CS-aadA11cΔ-3΄CS and 5΄CS-dfrB3-aadA1di-catB2-aadA6k-3΄CS, the last two being unseen arrays of antimicrobial resistance gene cassettes associated with novel environmental alleles of intI1. These four class 1 integrons were identified as being present in four different genera, including Ochrobactrum, and Variovorax, where class 1 integrons have not been previously reported. The results provide evidence of the biopurification systems as a tank of class 1 integron carrying strains and novel environmental class 1 integron integrases associated with antimicrobial resistance gene cassette arrays.

Isolation, taxonomic analysis, and phenotypic characterization of bacterial endophytes present in alfalfa (Medicago sativa) seeds.

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.

Burkholderia puraquae sp. nov., a novel species of the Burkholderia cepacia complex isolated from hospital settings and agricultural soils.

Bacteria from the Burkholderia cepacia complex (Bcc) are capable of causing severe infections in patients with cystic fibrosis (CF). These opportunistic pathogens are also widely distributed in natural and man-made environments. After a 12-year epidemiological surveillance involving Bcc bacteria from respiratory secretions of Argentinean patients with CF and from hospital settings, we found six isolates of the Bcc with a concatenated species-specific allele sequence that differed by more than 3 % from those of the Bcc with validly published names. According to the multilocus sequence analysis (MLSA), these isolates clustered with the agricultural soil strain, Burkholderia sp. PBP 78, which was already deposited in the PubMLST database. The isolates were examined using a polyphasic approach, which included 16S rRNA, recA, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), DNA base composition, average nucleotide identities (ANIs), fatty acid profiles, and biochemical characterizations. The results of the present study demonstrate that the seven isolates represent a single novel species within the Bcc, for which the name Burkholderia puraquae sp. nov. is proposed. Burkholderia puraquae sp. nov. CAMPA 1040T (=LMG 29660T=DSM 103137T) was designated the type strain of the novel species, which can be differentiated from other species of the Bcc mainly from recA gene sequence analysis, MLSA, ANIb, MALDI-TOF MS analysis, and some biochemical tests, including the ability to grow at 42 °C, aesculin hydrolysis, and lysine decarboxylase and ß-galactosidase activities.

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.

Draft Genome Sequence of Burkholderia puraquae Type Strain CAMPA 1040, Isolated from Hospital Settings in Córdoba, Argentina.

We report here the draft genome sequence of Burkholderia puraquae type strain CAMPA 1040, a member of the Burkholderia cepacia complex. This strain, isolated from a hemodialysis water reservoir, harbors several stress tolerance genes, such as the systems for low oxygen survival, for copper tolerance, and for osmotic stress resistance.

Nitrogen-fixing rhizobial strains isolated from Desmodium incanum DC in Argentina: Phylogeny, biodiversity and symbiotic ability.

Desmodium spp. are leguminous plants belonging to the tribe Desmodieae of the subfamily Papilionoideae. They are widely distributed in temperated and subtropical regions and are used as forage plants, for biological control, and in traditional folk medicine. The genus includes pioneer species that resist the xerothermic environment and grow in arid, barren sites. Desmodium species that form nitrogen-fixing symbiosis with rhizobia play an important role in sustainable agriculture. In Argentina, 23 native species of this genus have been found, including Desmodium incanum. In this study, a total of 64 D. incanum-nodulating rhizobia were obtained from root nodules of four Argentinean plant populations. Rhizobia showed different abiotic-stress tolerances and a remarkable genetic diversity using PCR fingerprinting, with more than 30 different amplification profiles. None of the isolates were found at more than one site, thus indicating a high level of rhizobial diversity associated with D. incanum in Argentinean soils. In selected isolates, 16S rDNA sequencing and whole-cell extract MALDI TOF analysis revealed the presence of isolates related to Bradyrhizobium elkanii, Bradyrhizobium japonicum, Bradyrhizobium yuanmingense, Bradyrhizobium liaoningense, Bradyrhizobium denitrificans and Rhizobium tropici species. In addition, the nodC gene studied in the selected isolates showed different allelic variants. Isolates were phenotypically characterized by assaying their growth under different abiotic stresses. Some of the local isolates were remarkably tolerant to high temperatures, extreme pH and salinity, which are all stressors commonly found in Argentinean soils. One of the isolates showed high tolerance to temperature and extreme pH, and produced higher aerial plant dry weights compared to other inoculated treatments. These results indicated that local isolates could be efficiently used for D. incanum inoculation.

Specificity traits consistent with legume-rhizobia coevolution displayed by Ensifer meliloti rhizosphere colonization.

Rhizobia are α- and ß-proteobacteria that associate with legumes in symbiosis to fix atmospheric nitrogen. The chemical communication between roots and rhizobia begins in the rhizosphere. Using signature-tagged-Tn5 mutagenesis (STM) we performed a genome-wide screening for Ensifer meliloti genes that participate in colonizing the rhizospheres of alfalfa and other legumes. The analysis of ca. 6,000 mutants indicated that genes relevant for rhizosphere colonization account for nearly 2% of the rhizobial genome and that most (ca. 80%) are chromosomally located, pointing to the relevance and ancestral origin of the bacterial ability to colonize plant roots. The identified genes were related to metabolic functions, transcription, signal transduction, and motility/chemotaxis among other categories; with several ORFs of yet-unknown function. Most remarkably, we identified a subset of genes that impacted more severely the colonization of the roots of alfalfa than of pea. Further analyses using other plant species revealed that such early differential phenotype could be extended to other members of the Trifoliae tribe (Trigonella, Trifolium), but not the Fabeae and Phaseoleae tribes. The results suggest that consolidation of E. meliloti into its current symbiotic state should have occurred in a rhizobacterium that had already been adapted to rhizospheres of the Trifoliae tribe.

First time identification of Pandoraea sputorum from a patient with cystic fibrosis in Argentina: a case report.

BACKGROUND: Pandoraea species are considered emerging pathogens in the context of cystic fibrosis (CF) and are difficult to identify by conventional biochemical methods. These multidrug resistant bacteria remain poorly understood particularly in terms of natural resistance, mechanisms of acquired resistance and impact on the prognosis of the disease and the lung function. Among them, Pandoraea sputorum has been previously described in few cases of CF patients from Spain, Australia, France and United States, underlining the need of more clinical data for a better knowledge of its pathogenicity. This is the first report relating to P. sputorum in a CF patient in Argentina. CASE PRESENTATION: Pandoraea sputorum was identified in a nine-year-old cystic fibrosis patient from Argentina, after treatment failure during an exacerbation. The isolates were successfully identified by combining molecular techniques based on 16S rRNA sequencing and mass spectrometry (MS) methods, after reassessing previous misidentified isolates by conventional methods. After first isolation of P. sputorum, patient's clinical condition worsened but later improved after a change in the treatment. Although isolates showed susceptibility to trimethoprim-sulfamethoxazole and imipenem, in our case, the antibiotic treatment failed in the eradication of P. sputorum. CONCLUSIONS: All combined data showed a chronic colonization with P. sputorum associated to a deterioration of lung function. We noted that the presence of P. sputorum can be underestimated in CF patients and MALDI-TOF MS appears to be a promising means of accurate identification of Pandoraea species.