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.

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.

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.

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.

Rhizobium favelukesii sp. nov., isolated from the root nodules of alfalfa (Medicago sativa L).

Strains LPU83T and Or191 of the genus Rhizobium were isolated from the root nodules of alfalfa, grown in acid soils from Argentina and the USA. These two strains, which shared the same plasmid pattern, lipopolysaccharide profile, insertion-sequence fingerprint, 16S rRNA gene sequence and PCR-fingerprinting pattern, were different from reference strains representing species of the genus Rhizobium with validly published names. On the basis of previously reported data and from new DNA-DNA hybridization results, phenotypic characterization and phylogenetic analyses, strains LPU83T and Or191 can be considered to be representatives of a novel species of the genus Rhizobium, for which the name Rhizobium favelukesii sp. nov. is proposed. The type strain of this species is LPU83T (=CECT 9014T=LMG 29160T), for which an improved draft-genome sequence is available.

Characterization of a collection of plasmid-containing bacteria isolated from an on-farm biopurification system used for pesticide removal.

Biopurification systems (BPS) are complex soil-related and artificially-generated environments usually designed for the removal of toxic compounds from contaminated wastewaters. The present study has been conducted to isolate and characterize a collection of cultivable plasmid-carrying bacterial isolates recovered from a BPS established for the decontamination of wastewater generated in a farmyard. Out of 1400 isolates, a collection of 75 plasmid-containing bacteria was obtained, of which 35 representative isolates comprising in total at least 50 plasmids were chosen for further characterization. Bacterial hosts were taxonomically assigned by 16S ribosomal RNA gene sequencing and phenotypically characterized according to their ability to grow in presence of different antibiotics and heavy metals. The study demonstrated that a high proportion of the isolates was tolerant to antibiotics and/or heavy metals, highlighting the on-farm BPS enrichment in such genetic traits. Several plasmids conferring such resistances in the bacterial collection were detected to be either mobilizable or selftransmissible. Occurrence of broad host range plasmids of the incompatibility groups IncP, IncQ, IncN and IncW was examined with positive results only for the first group. Presence of the IS1071 insertion sequence, frequently associated with xenobiotics degradation genes, was detected in DNA obtained from 24 of these isolates, strongly suggesting the presence of yet-hidden catabolic activities in the collection of isolates. The results showed a remarkable diversity in the plasmid mobilome of cultivable bacteria in the BPS with the presence of abundant resistance markers of different types, thus providing a suitable environment to investigate the genetic structure of the mobile genetic pool in a model on-farm biofilter for wastewater decontamination in intensive agricultural production.

Characterization of an accessory plasmid of Sinorhizobium meliloti and its two replication-modules.

Rhizobia are Gram-negative bacteria known for their ability to fix atmospheric N2 in symbiosis with leguminous plants. Current evidence shows that rhizobia carry in most cases a variable number of plasmids, containing genes necessary for symbiosis or free-living, a common feature being the presence of several plasmid replicons within the same strain. For many years, we have been studying the mobilization properties of pSmeLPU88b from the strain Sinorhizobium meliloti LPU88, an isolate from Argentina. To advance in the characterization of pSmeLPU88b plasmid, the full sequence was obtained. pSmeLPU88b is 35.9 kb in size, had an average GC % of 58.6 and 31 CDS. Two replication modules were identified in silico: one belonging to the repABC type, and the other to the repC. The replication modules presented high DNA identity to the replication modules from plasmid pMBA9a present in an S. meliloti isolate from Canada. In addition, three CDS presenting identity with recombinases and with toxin-antitoxin systems were found downstream of the repABC system. It is noteworthy that these CDS present the same genetic structure in pSmeLPU88b and in other rhizobial plasmids. Moreover, in all cases they are found downstream of the repABC operon. By cloning each replication system in suicide plasmids, we demonstrated that each of them can support plasmid replication in the S. meliloti genetic background, but with different stability behavior. Interestingly, while incompatibility analysis of the cloned rep systems results in the loss of the parental module, both obtained plasmids can coexist together.

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.

Genetic and functional characterization of a yet-unclassified rhizobial Dtr (DNA-transfer-and-replication) region from a ubiquitous plasmid conjugal system present in Sinorhizobium meliloti, in Sinorhizobium medicae, and in other nonrhizobial Gram-negative bacteria.

Rhizobia are Gram-negative bacteria that live in soils and associate with leguminous plants to establish nitrogen-fixing symbioses. The ability of these bacteria to undergo horizontal gene transfer (HGT) is thought to be one of the main features to explain both the origin of their symbiotic life-style and the plasticity and dynamics of their genomes. In our laboratory we have previously characterized at the species level the non-pSym plasmid mobilome in Sinorhizobium meliloti, the symbiont of Medicago spp., and have found a high incidence of conjugal activity in many plasmids (Pistorio et al., 2008). In this work we characterized the Dtr (DNA-transfer-and-replication) region of one of those plasmids, pSmeLPU88b. This mobilization region was found to represent a previously unclassified Dtr type in rhizobia (hereafter type-IV), highly ubiquitous in S. meliloti and found in other genera of Gram-negative bacteria as well; including Agrobacterium, Ochrobactrum, and Chelativorans. The oriT of the type-IV Dtr described here could be located by function within a DNA fragment of 278 bp, between the divergent genes parA and mobC. The phylogenetic analysis of the cognate relaxase MobZ indicated that this protein groups close to the previously defined MOB(P3) and MOB(P4) type of enzymes, but is located in a separate and novel cluster that we have designated MOB(P0). Noteworthy, MOB(P0) and MOB(P4) relaxases were frequently associated with plasmids present in rhizospheric soil bacteria. A comparison of the nod-gene locations with the phylogenetic topology of the rhizobial relaxases revealed that the symbiotic genes are found on diverse plasmids bearing any of the four Dtr types, thus indicating that pSym plasmids are not specifically associated with any particular mobilization system. Finally, we demonstrated that the type-IV Dtr promoted the mobilization of plasmids from S. meliloti to Sinorhizobium medicae as well as from these rhizobia to other bacteria by means of their own helper functions. The results present an as-yet-unclassified and seemingly ubiquitous conjugal system that provides a mechanistic support for the HGT between sympatric rhizobia of Medicago roots, and between other soil and rhizospheric bacteria.

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.

Enterococcus faecalis virulence regulator FsrA binding to target promoters.

The FsrABDC signal transduction system is a major virulence regulator in Enterococcus faecalis. The FsrC sensor histidine kinase, upon activation by the gelatinase biosynthesis-activating pheromone (GBAP) peptide encoded by the fsrBD genes, phosphorylates the FsrA response regulator required for the transcription of the fsrBDC and the gelE-sprE genes from the fsrB promoter and the gelE promoter, respectively. FsrA belongs to the LytTR family of proteins, which includes other virulence regulators, such as AgrA of Staphylococcus aureus, AlgR of Pseudomonas aeruginosa, and VirR of Clostridium perfringens. The LytTR DNA-binding domain that characterizes these proteins generally binds to two imperfect direct repeats separated by a number of bases that place the repeats on the same face of the DNA helix. In this study, we demonstrated that FsrA also binds to two imperfect direct repeats separated by 13 bp, based on the consensus sequence of FsrA, T/AT/CAA/GGGAA/G, which is consistent with the binding characteristics of LytTR domains.

The nodulation of alfalfa by the acid-tolerant Rhizobium sp. strain LPU83 does not require sulfated forms of lipochitooligosaccharide nodulation signals.

The induction of root nodules by the majority of rhizobia has a strict requirement for the secretion of symbiosis-specific lipochitooligosaccharides (nodulation factors [NFs]). The nature of the chemical substitution on the NFs depends on the particular rhizobium and contributes to the host specificity imparted by the NFs. We present here a description of the genetic organization of the nod gene cluster and the characterization of the chemical structure of the NFs associated with the broad-host-range Rhizobium sp. strain LPU83, a bacterium capable of nodulating at least alfalfa, bean, and Leucena leucocephala. The nod gene cluster was located on the plasmid pLPU83b. The organization of the cluster showed synteny with those of the alfalfa-nodulating rhizobia, Sinorhizobium meliloti and Sinorhizobium medicae. Interestingly, the strongest sequence similarity observed was between the partial nod sequences of Rhizobium mongolense USDA 1844 and the corresponding LPU83 nod genes sequences. The phylogenetic analysis of the intergenic region nodEG positions strain LPU83 and the type strain R. mongolense 1844 in the same branch, which indicates that Rhizobium sp. strain LPU83 might represent an early alfalfa-nodulating genotype. The NF chemical structures obtained for the wild-type strain consist of a trimeric, tetrameric, and pentameric chitin backbone that shares some substitutions with both alfalfa- and bean-nodulating rhizobia. Remarkably, while in strain LPU83 most of the NFs were sulfated in their reducing terminal residue, none of the NFs isolated from the nodH mutant LPU83-H were sulfated. The evidence obtained supports the notion that the sulfate decoration of NFs in LPU83 is not necessary for alfalfa nodulation.

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.

Characterization of extrachromosomal replicons present in the extended host range Rhizobium sp. LPU83.

In several rhizobia, bacteria that inhabit the soil in free-living conditions and associate in symbiosis with the root of legumes as nitrogen-fixing organisms, plasmid DNA can constitute a high percentage of the genome. We have characterized acid-tolerant isolates of rhizobia-here represented by the strain Rhizobium sp. LPU83-that have an extended nodulation-host range including alfalfa, the common bean, and Leucena leucocephala. In this study we analyzed the plasmids of R. sp. LPU83 in order to characterize their role in the evolution of Medicago symbionts and their involvement in symbiotic behavior. The pLPU83a plasmid was found to be transmissible with no associated phenotypic traits. The symbiotic plasmid pLPU83b could be transferred at very low frequencies under laboratory conditions only when pLPU83a was present; could restore nodulation to a strain cured of its symbiotic plasmid, S. meliloti A818; but could not restore the full nitrogen fixation associated with alfalfa.

Isolation and characterization of endophytic plant growth-promoting (PGPB) or stress homeostasis-regulating (PSHB) bacteria associated to the halophyte Prosopis strombulifera.

This study was designed to isolate and characterize endophytic bacteria from halophyte Prosopis strombulifera grown under extreme salinity and to evaluate in vitro the bacterial mechanisms related to plant growth promotion or stress homeostasis regulation. Isolates obtained from P. strombulifera were compared genotypically by BOX-polymerase chain reaction, grouped according to similarity, and identified by amplification and partial sequences of 16S DNAr. Isolates were grown until exponential growth phase to evaluate the atmospheric nitrogen fixation, phosphate solubilization, siderophores, and phytohormones, such as indole-3-acetic acid, zeatin, gibberellic acid and abscisic acid production, as well as antifungal, protease, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. A total of 29 endophytic strains were grouped into seven according to similarity. All bacteria were able to grow and to produce some phytohormone in chemically defined medium with or without addition of a nitrogen source. Only one was able to produce siderophores, and none of them solubilized phosphate. ACC deaminase activity was positive for six strains. Antifungal and protease activity were confirmed for two of them. In this work, we discuss the possible implications of these bacterial mechanisms on the plant growth promotion or homeostasis regulation in natural conditions.

Ethanolamine activates a sensor histidine kinase regulating its utilization in Enterococcus faecalis.

Enterococcus faecalis is a gram-positive commensal bacterium of the human intestinal tract. Its opportunistic pathogenicity has been enhanced by the acquisition of multiple antibiotic resistances, making the treatment of enterococcal infections an increasingly difficult problem. The extraordinary capacity of this organism to colonize and survive in a wide variety of ecological niches is attributable, at least in part, to signal transduction pathways mediated by two-component systems (TCS). Here, the ability of E. faecalis to utilize ethanolamine as the sole carbon source is shown to be dependent upon the RR-HK17 (EF1633-EF1632) TCS. Ethanolamine is an abundant compound in the human intestine, and thus, the ability of bacteria to utilize it as a source of carbon and nitrogen may provide an advantage for survival and colonization. Growth of E. faecalis in a synthetic medium with ethanolamine was abolished in the response regulator RR17 mutant strain. Transcription of the response regulator gene was induced by the presence of ethanolamine. Ethanolamine induced a 15-fold increase in the rate of autophosphorylation in vitro of the HK17 sensor histidine kinase, indicating that this is the ligand recognized by the sensor domain of the kinase. These results assign a role to the RR-HK17 TCS as coordinator of the enterococcal response to specific nutritional conditions existing at the site of bacterial invasion, the intestinal tract of an animal host.

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.

Full activation of Enterococcus faecalis gelatinase by a C-terminal proteolytic cleavage.

Enterococci account for nearly 10% of all nosocomial infections and constitute a significant treatment challenge due to their multidrug resistance properties. One of the well-studied virulence factors of Enterococcus faecalis is a secreted bacterial protease, termed gelatinase, which has been shown to contribute to the process of biofilm formation. Gelatinase belongs to the M4 family of bacterial zinc metalloendopeptidases, typified by thermolysin. Gelatinase is synthesized as a preproenzyme consisting of a signal sequence, a putative propeptide, and then the mature enzyme. We determined that the molecular mass of the mature protein isolated from culture supernatant was 33,030 Da, which differed from the predicted molecular mass, 34,570 Da, by over 1,500 Da. Using N-terminal sequencing, we confirmed that the mature protein begins at the previously identified sequence VGSEV, thus suggesting that the 1,500-Da molecular mass difference resulted from a C-terminal processing event. By using mutants with site-directed mutations within a predicted C-terminal processing site and mutants with C-terminal deletions fused to a hexahistidine tag, we determined that the processing site is likely to be between residues D304 and I305 and that it requires the Q306 residue. The results suggest that the E. faecalis gelatinase requires C-terminal processing for full activation of protease activity, making it a unique enzyme among the members of the M4 family of proteases of gram-positive bacteria.

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.

The symbiotic defect in a Sinorhizobium meliloti lipopolysaccharide mutant can be overcome by expression of other surface polysaccharides.

In this work we have examined the extent of functional complementation in symbiosis among different Sinorhizobium meliloti surface polysaccharides including lipopolysaccharide (LPS). We show that a symbiotic deficiency associated with an LPS defect can be reversed by appropriate expression of other surface polysaccharides such as galactoglucan (EPSII) and a particular form of capsular polysaccharide (KdoPS). It is noteworthy that, while succinoglycan EPSI and LPS cannot functionally substitute for each other, they can both be replaced by the same common set of polysaccharides (i.e., EPSII/KdoPS). The complex pattern of functional complementation in symbiosis among S. meliloti surface polysaccharides was shown to be different in Medicago truncatula compared to that previously reported for M. sativa.