Mariano Pistorio

Response of alfalfa (Medicago sativa L.) to single and mixed inoculation with phosphate-solubilizing bacteria and Sinorhizobium meliloti

The objectives of this work were to phenotypically and genetically characterize alfalfa rhizosphere bacteria and to evaluate the effect of single or mixed inoculation upon nodulation and biological nitrogen fixation. Thirty-two strains showed tricalcium phosphate solubilization ability, and two of them caused bigger or equal solubilization halos than the control strain P. putida SP22. The comparison of the 16S ribosomal DNA sequences indicated that these strains are phylogenetically related to Bacillus spp. and Pseudomonas spp. A beneficial effect of both isolates on alfalfa growth was observed in coinoculation assays. Pseudomonas sp. FM7d caused a significant increase in root and shoot dry weight, length, and surface area of roots, number, and symbiotic properties of alfalfa plants. The plants coinoculated with Sinorhizobium meliloti B399 and the Bacillus sp. M7c showed significant increases in the measured parameters. Our results indicating that strains Pseudomonas sp. FM7d and Bacillus sp. M7c can be considered for the formulation of new inoculants.

Identification of a transmissible plasmid from an Argentine Sinorhizobium meliloti strain which can be mobilised by conjugative helper functions of the European strain S. meliloti GR4

We describe in this work the identification and the conjugal properties of two cryptic plasmids present in the strain Sinorhizobium meliloti LPU88 isolated from an Argentine soil. One of the plasmids, pSmeLPU88b (22 kb), could be mobilised from different S. meliloti strains to other bacteria by conjugation only if the other plasmid, pSmeLPU88a (139 kb), was present. This latter plasmid, however, could not be transferred via conjugation (frequency <10(-9) transconjugants per recipient) contrasting with the conjugal system from the previously described strain GR4, where one plasmid is mobilisable and a second one (helper) is self-transmissible. Despite the differences between the two systems, the conjugative helper functions present in the cryptic plasmids of strain GR4 were active in the mobilisation of plasmid pSmeLPU88b from strain LPU88. Contrasting with this, plasmid pSmeLPU88b was not mobilised by the helper functions of the broad-host-range plasmid RP4. Eckhardt gel analysis showed that none of the plasmids from strain GR4 were excluded in the presence of plasmid pSmeLPU88b suggesting that they all belong to different incompatibility groups for replication. The small plasmid from strain LPU88, pSmeLPU88b, was only able to replicate in members of the Rhizobiaceae family such as Rhizobium leguminosarum, Rhizobium tropici and Agrobacterium tumefaciens, but not in Escherichia coli or Pseudomonas fluorescens. The observation suggests that most likely plasmid pSmeLPU88b was not received from a phylogenetically distant bacterium.

Conjugal properties of the Sinorhizobium meliloti plasmid mobilome

The biology and biochemistry of plasmid transfer in soil bacteria is currently under active investigation because of its central role in prokaryote adaptation and evolution. In this work, we examined the conjugal properties of the cryptic plasmids present in a collection of the N(2)-fixing legume-symbiont Sinorhizobium meliloti. The study was performed on 65 S. meliloti isolates recovered from 25 humic soils of Argentina, which were grouped into 22 plasmid-profile types [i.e. plasmid operational taxonomic units (OTUs)]. The cumulative Shannon index calculated for the observed plasmid profiles showed a clear saturation plateau, thus indicating an adequate representation of the S. meliloti plasmid-profile types in the isolates studied. The results show that isolates of nearly 14% of the plasmid OTUs hosted transmissible plasmids and that isolates of 29% of the plasmid OTUs were able to retransfer the previously characterized mobilizable-cryptic plasmid pSmeLPU88b to a third recipient strain. It is noteworthy that isolates belonging to 14% of the plasmid OTUs proved to be refractory to the entrance of the model plasmid pSmeLPU88b, suggesting either the presence of surface exclusion phenomena or the occurrence of restriction incompatibility with the incoming replicon. Incompatibility for replication between resident plasmids and plasmid pSmeLPU88b was observed in c. 20% of the OTUs. The results reported here reveal a widespread compatibility among the conjugal functions of the cryptic plasmids in S. meliloti, and this fact, together with the observed high proportion of existing donor genotypes, points to the extrachromosomal compartment of the species as being an extremely active plasmid mobilome.

Identification and Characterization of a nodH Ortholog from the Alfalfa-Nodulating Or191-Like Rhizobia

Nodulation of Medicago sativa (alfalfa) is known to be restricted to Sinorhizobium meliloti and a few other rhizobia that include the poorly characterized isolates related to Rhizobium sp. strain Or191. Distinctive features of the symbiosis between alfalfa and S. meliloti are the marked specificity from the plant to the bacteria and the strict requirement for the presence of sulfated lipochitooligosaccharides (Nod factors [NFs]) at its reducing end. Here, we present evidence of the presence of a functional nodH-encoded NF sulfotransferase in the Or191-like rhizobia. The nodH gene, present in single copy, maps to a high molecular weight megaplasmid. As in S. meliloti, a nodF homolog was identified immediately upstream of nodH that was transcribed in the opposite direction (local synteny). This novel nodH ortholog was cloned and shown to restore both NF sulfation and the Nif+Fix+ phenotypes when introduced into an S. meliloti nodH mutant. Unexpectedly, however, nodH disruption in the Or191-like bacteria did not abolish their ability to nodulate alfalfa, resulting instead in a severely delayed nodulation. In agreement with evidence from other authors, the nodH sequence analysis strongly supports the idea that the Or191-like rhizobia most likely represent a genetic mosaic resulting from the horizontal transfer of symbiotic genes from a sinorhizobial megaplasmid to a not yet clearly identified ancestor.

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.

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.

Rhizobial plasmid pLPU83a is able to switch between different transfer machineries depending on its genomic background

Plasmids have played a major role in bacterial evolution, mainly by their capacity to perform horizontal gene transfer (HGT). Their conjugative transfer (CT) properties are usually described in terms of the plasmid itself. In this work, we analyzed structural and functional aspects of the CT of pLPU83a, an accessory replicon from Rhizobium sp. LPU83, able to transfer from its parental strain, from Ensifer meliloti, or from Rhizobium etli. pLPU83a contains a complete set of transfer genes, featuring a particular organization, shared with only two other rhizobial plasmids. These plasmids contain a TraR quorum-sensing (QS) transcriptional regulator, but lack an acyl-homoserine lactone (AHL) synthase gene. We also determined that the ability of pLPU83a to transfer from R. etli CFN42 genomic background was mainly achieved through mobilization, employing the machinery of the endogenous plasmid pRetCFN42a, falling under control of the QS regulators from pRetCFN42a. In contrast, from its native or from the E. meliloti background, pLPU83a utilized its own machinery for conjugation, requiring the plasmid-encoded traR. Activation of TraR seemed to be AHL independent. The results obtained indicate that the CT phenotype of a plasmid is dictated not only by the genes it carries, but by their interaction with its genomic context.

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.

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.

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.