Inoculation of alfalfa plants with either incompatible Rhizobium or a Rhizobium mutant blocked in Nod factor synthesis led to an accumulation of salicylic acid in roots, in contrast to plants inoculated with a wild-type (compatible) R. meliloti strain. When salicylic acid was exogenously applied prior to inoculation of alfalfa plants with either purified Nod factor or compatible Rhizobium strains, a significant inhibition of nodule primordia formation and a reduction of the number of emerging nodules, respectively, as well as a delay in nodule visualization, were observed. These results suggest an involvement of Rhizobium-synthesized Nod factors in the inhibition of salicylic acid-mediated defense in legumes.

Bacterial retrons consist of a reverse transcriptase (RT) and a contiguous non-coding RNA (ncRNA) gene. One third of annotated retrons carry additional open reading frames (ORFs), the contribution and significance of which in retron biology remains to be determined. In this study we developed a computational pipeline for the systematic prediction of genes specifically associated with retron RTs based on a previously reported large dataset representative of the diversity of prokaryotic RTs. We found that retrons generally comprise a tripartite system composed of the ncRNA, the RT and an additional protein or RT-fused domain with diverse enzymatic functions. These retron systems are highly modular, and their components have coevolved to different extents. Based on the additional module, we classified retrons into 13 types, some of which include additional variants. Our findings provide a basis for future studies on the biological function of retrons and for expanding their biotechnological applications.

SummaryGroup II introns are large catalytic RNA molecules that act as mobile genetic elements. They were initially identified in the organelle genomes of lower eukaryotes and plants, and it has been suggested that they are the progenitors of nuclear spliceosomal introns. Group II self-splicing introns were shown to be present in bacteria in 1993, since when the various bacterial genome sequencing projects have led to a significant increase in the number of group II intron sequences present in databases. However, few of these introns have been characterized, and most were identified on the basis of their intron-encoded protein (IEP), with little data available concerning their ribozyme/RNA structure. Their frequency in prokaryotes is also unknown. We attempt here to provide a first comprehensive review of bacterial group II introns based on recent genome sequencing data and mechanistic studies.

Sinorhizobium meliloti RmInt1 is an efficient mobile group II intron that uses an unknown reverse transcriptase priming mechanism as the intron ribonucleoprotein complex can reverse splice into DNA target substrates but cannot carry out site-specific second strand cleavage due to the lack of a C-terminal DNA endonuclease domain. We show here that, like other mobile group II introns, RmInt1 moves around by an efficient RNA-based retrohoming mechanism. We found evidence of two distinct RmInt1 retrohoming pathways for mobility depending on the orientation of the target site relative to the direction of DNA replication. The preferred retrohoming pathway is consistent with reverse splicing of the intron RNA into single-stranded DNA at a replication fork, using a nascent lagging DNA strand as the primer for reverse transcription. This strand bias is the opposite of that reported for mobility of the lactococcal Ll.ltrB intron in the absence of second strand cleavage. The mobility mechanism found here for RmInt1 may be used for dissemination by many bacterial group II introns encoding proteins lacking the DNA endonuclease domain.

No abstract

RmInt1 is a functional group II intron found in Sinorhizobium meliloti where it interrupts a group of IS elements of the IS630‐Tc1 family. In contrast to many other group II introns, the intron‐encoded protein (IEP) of RmInt1 lacks the characteristic conserved part of the Zn domain associated with the IEP endonuclease activity. Nevertheless, in this study, we show that RmInt1 is capable of inserting into a vector containing the DNA spanning the RmInt1 target site from the genome of S. meliloti. Efficient homing was also observed in the absence of homologous recombination (RecA− strains). In addition, it is shown that RmInt1 is able to move to its target in a heterologous host (S. medicae). Homing of RmInt1 occurs very efficiently upon DNA target uptake (conjugation/electroporation) by the host cell resulting in a proportion of invaded target of 11–30%. Afterwards, the remaining intronless target DNA is protected from intron invasion.

SummaryBy sequence analysis of Sinorhizobium meliloti strain GR4 plasmid pRmeGR4b, we have identified a group II intron named RmInt1 inserted within the insertion sequence ISRm2011-2 of the IS630-Tc1/IS3 retroposon superfamily. Like some other group II introns, RmInt1 possesses, in addition to the structurally conserved ribozyme core, an open reading frame (ORF) with homology to reverse transcriptases. Using a T7 expression system in Escherichia coli, we show that the intron is active in splicing in vivo and that splicing efficiency requires the intron-encoded ORF, which suggests that the putative intron encoded protein has a maturase function. DNA hybridization studies indicate that intron RmInt1 is widespread within S. meliloti native populations and appears to be mostly located within this IS element. Nevertheless, some S. meliloti strains harbour one copy of RmInt1 at a different location. DNA sequence analysis of the 5Ј exon of one of these heterologous intron insertion sites revealed the presence of a putative IS element closely related to insertion sequence ISRm2011-2. The intron-binding sites (IBS1 and IBS2 motifs) are conserved, although a transition of a G→A in the IBS1 has occurred. Our results demonstrate an association of intron RmInt1 with particular insertion sequences of the IS630-Tc1/ IS3 retroposon superfamily that may have ensured the spread and maintenance of this group II intron in S. meliloti.

The soil microbial community is highly complex and contains a high density of antibiotic-producing bacteria, making it a likely source of diverse antibiotic resistance determinants. We used functional metagenomics to search for antibiotic resistance genes in libraries generated from three different soil samples, containing 3.6 Gb of DNA in total. We identified 11 new antibiotic resistance genes: 3 conferring resistance to ampicillin, 2 to gentamicin, 2 to chloramphenicol and 4 to trimethoprim. One of the clones identified was a new trimethoprim resistance gene encoding a 26.8 kDa protein closely resembling unassigned reductases of the dihydrofolate reductase group. This protein, Tm8-3, conferred trimethoprim resistance in Escherichia coli and Sinorhizobium meliloti (γ- and α-proteobacteria respectively). We demonstrated that this gene encoded an enzyme with dihydrofolate reductase activity, with kinetic constants similar to other type I and II dihydrofolate reductases (K(m) of 8.9 µM for NADPH and 3.7 µM for dihydrofolate and IC(50) of 20 µM for trimethoprim). This is the first description of a new type of reductase conferring resistance to trimethoprim. Our results indicate that soil bacteria display a high level of genetic diversity and are a reservoir of antibiotic resistance genes, supporting the use of this approach for the discovery of novel enzymes with unexpected activities unpredictable from their amino acid sequences.