Identification, phylogenetic analysis, and genome mining of the tetracycline-resistant Bacillus thuringiensis strain m401 reveal its potential for biotechnological and biocontrol applications.

Bacillus thuringiensis is an entomopathogen belonging to the Bacillus cereus clade. We isolated a tetracycline-resistant strain called m401, recovered it from honey, and identified it as Bacillus thuringiensis sv. kumamotoensis based on the average nucleotide identity calculations (ANIb) comparison and the analysis of the gyrB gene sequences of different B. thuringiensis serovars. Sequences with homology to virulence factors [cytK, nheA, nheB, nheC, hblA, hblB, hblC, hblD, entFM, and inhA] and tetracycline resistance genes [tet(45), tet(V), and tet(M)/tet(W)/tet(O)/tet(S) family] were identified in the bacterial chromosome. The prediction of plasmid-coding regions revealed homolog sequences to the MarR and TetR/AcrR family of transcriptional regulators, toxins, and lantipeptides. The genome mining analysis revealed 12 regions of biosynthetic gene clusters responsible for synthesizing secondary metabolites. We identified biosynthetic gene clusters coding for bacteriocins, siderophores, ribosomally synthesized post-translationally modified peptide products, and non-ribosomal peptide synthetase clusters that provide evidence for the possible use of Bt m401 as a biocontrol agent. Furthermore, Bt m401 showed high inhibition against all Paenibacillus larvae genotypes tested in vitro. In conclusion, Bt m401 owns various genes involved in different biological processes, such as transductional regulators associated with antibiotic resistance, toxins, and antimicrobial peptides with potential biotechnological and biocontrol applications.

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.

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.

Global transcriptome analysis of Rhizobium favelukesii LPU83 in response to acid stress.

Acidic environments naturally occur worldwide and inappropriate agricultural management may also cause acidification of soils. Low soil pH values are an important barrier in the plant-rhizobia interaction. Acidic conditions disturb the establishment of the efficient rhizobia usually used as biofertilizer. This negative effect on the rhizobia-legume symbiosis is mainly due to the low acid tolerance of the bacteria. Here, we describe the identification of relevant factors in the acid tolerance of Rhizobium favelukesii using transcriptome sequencing. A total of 1924 genes were differentially expressed under acidic conditions, with ∼60% underexpressed. Rhizobium favelukesii acid response mainly includes changes in the energy metabolism and protein turnover, as well as a combination of mechanisms that may contribute to this phenotype, including GABA and histidine metabolism, cell envelope modifications and reverse proton efflux. We confirmed the acid-sensitive phenotype of a mutant in the braD gene, which showed higher expression under acid stress. Remarkably, 60% of the coding sequences encoded in the symbiotic plasmid were underexpressed and we evidenced that a strain cured for this plasmid featured an improved performance under acidic conditions. Hence, this work provides relevant information in the characterization of genes associated with tolerance or adaptation to acidic stress of R. favelukesii.

Role of plant compounds in the modulation of the conjugative transfer of pRet42a.

One of the most studied mechanisms involved in bacterial evolution and diversification is conjugative transfer (CT) of plasmids. Plasmids able to transfer by CT often encode beneficial traits for bacterial survival under specific environmental conditions. Rhizobium etli CFN42 is a Gram-negative bacterium of agricultural relevance due to its symbiotic association with Phaseolus vulgaris through the formation of Nitrogen-fixing nodules. The genome of R. etli CFN42 consists of one chromosome and six large plasmids. Among these, pRet42a has been identified as a conjugative plasmid. The expression of the transfer genes is regulated by a quorum sensing (QS) system that includes a traI gene, which encodes an acyl-homoserine lactone (AHL) synthase and two transcriptional regulators (TraR and CinR). Recently, we have shown that pRet42a can perform CT on the root surface and inside nodules. The aim of this work was to determine the role of plant-related compounds in the CT of pRet42a. We found that bean root exudates or root and nodule extracts induce the CT of pRet42a in the plant rhizosphere. One possibility is that these compounds are used as nutrients, allowing the bacteria to increase their growth rate and reach the population density leading to the activation of the QS system in a shorter time. We tested if P. vulgaris compounds could substitute the bacterial AHL synthesized by TraI, to activate the conjugation machinery. The results showed that the transfer of pRet42a in the presence of the plant is dependent on the bacterial QS system, which cannot be substituted by plant compounds. Additionally, individual compounds of the plant exudates were evaluated; among these, some increased and others decreased the CT. With these results, we suggest that the plant could participate at different levels to modulate the CT, and that some compounds could be activating genes in the conjugation machinery.

Polyphasic characterization and identification of the bioremediation agent Bacillus sp. SFC 500-1E.

Bacillus sp. SFC 500-1E is used for the effective treatment of tannery effluents since it consistently removes hexavalent chromium from diverse contaminated matrices. The aim of the present study was to complete identification of the strain through a polyphasic characterization, which included the pattern of carbohydrate utilization, fatty acids profile, multilocus sequence analysis, multiplex PCR profile and the analysis of the complete genome sequence. Morpho-physiological and biochemical characterization results and analysis of 16S rRNA sequences were not conclusive. The strain formed a monophyletic clade with B. toyonensis BCT-7112, B. thuringiensis MC28 and B. cereus Rock 1-3. However, genomic comparisons with type strains of B. cereus and B. thuringiensis showed that the isolated belonged to a different species. Results of this study highlight the relevance of the genome sequence of this strain, identified as Bacillus toyonensis SFC 500-1E, to expand knowledge of its bioremediation potential and to explore unknown decontamination activities.

Proteomic Analysis of Rhizobium favelukesii LPU83 in Response to Acid Stress.

Acid soils constitute a severe problem for leguminous crops mainly through a disturbance in rhizobium-legume interactions. Rhizobium favelukesii-an acid-tolerant rhizobium able to nodulate alfalfa-is highly competitive for nodule occupation under acid conditions but inefficient for biologic nitrogen fixation. In this work, we obtained a general description of the acid-stress response of R. favelukesii LPU83 by means of proteomics by comparing the total proteome profiles in the presence or absence of acid stress by nanoflow ultrahigh-performance liquid chromatography coupled to mass spectrometry. Thus, a total of 336 proteins were identified with a significant differential expression, 136 of which species were significantly overexpressed and 200 underexpressed in acidity. An in silico functional characterization with those respective proteins revealed a complex and pleiotropic response by these rhizobia involving components of oxidative phosphorylation, glutamate metabolism, and peptidoglycan biosynthesis, among other pathways. Furthermore, a lower permeability was evidenced in the acid-stressed cells along with several overexpressed proteins related to γ-aminobutyric acid metabolism, such as the gene product of livK, which gene was mutated. This mutant exhibited an acid-sensitive phenotype in agreement with the proteomics results. We conclude that both the γ-aminobutyric acid metabolism and a modified cellular envelope could be relevant to acid tolerance in R. favelukesii.

Genome Sequence of the Symbiotic Type Strain Mesorhizobium helmanticense CSLC115N Isolated from Lotus corniculatus Nodules.

Mesorhizobium helmanticense is a novel species that was isolated from root nodules of Lotus corniculatus grown in an alfisol soil from Carbajosa de la Sagrada, a Mediterranean region in the province of Salamanca in northwest Spain. The whole-genome sequence of the type strain M. helmanticense CSLC115N is reported in this study.

rptA, a novel gene from Ensifer (Sinorhizobium) meliloti involved in conjugal transfer.

We approached the identification of Ensifer (Sinorhizobium) meliloti conjugal functions by random Tn5-B13 mutagenesis of the pSmeLPU88a plasmid of E. meliloti strain LPU88 and the subsequent selection of those mutants that had lost the ability to mobilize the small plasmid pSmeLPU88b. The Tn5-B13-insertion site of one of the mutants was cloned as an EcoRI-restricted DNA fragment that after subsequent isolation and sequencing demonstrated that a small open reading frame of 522 bp (designated rptA, for rhizobium plasmid transfer A) had been disrupted. The predicted gene product encoded by the rptA sequence shows a significant similarity to two hypothetical proteins of the plasmid pSmed03 of Ensifer medicae WSM419 and other rhizobia plasmids. No significant similarity was found to any protein sequence of known function registered in the databases. Although the rptA gene was required for pSmeLPU88b-plasmid mobilization in the strain 2011 background, it was not required in the original strain LPU88 background.

Cultural conditions required for the induction of an adaptive acid-tolerance response (ATR) in Sinorhizobium meliloti and the question as to whether or not the ATR helps rhizobia improve their symbiosis with alfalfa at low pH.

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.