The legume symbiont Sinorhizobium meliloti expresses a plethora of small noncoding RNAs (sRNAs) whose function is mostly unknown. Here, we have functionally characterized two tandemly encoded S. meliloti Rm1021 sRNAs that are similar in sequence and structure. Homologous sRNAs (designated AbcR1 and AbcR2) have been shown to regulate several ABC transporters in the related α-proteobacteria Agrobacterium tumefaciens and Brucella abortus. In Rm1021, AbcR1 and AbcR2 exhibit divergent unlinked regulation and are stabilized by the RNA chaperone Hfq. AbcR1 is transcribed in actively dividing bacteria, either in culture, rhizosphere or within the invasion zone of mature alfalfa nodules. Conversely, AbcR2 expression is induced upon entry into stationary phase and under abiotic stress. Only deletion of AbcR1 resulted into a discrete growth delay in rich medium, but both are dispensable for symbiosis. Periplasmic proteome profiling revealed down-regulation of the branched-chain amino acid binding protein LivK by AbcR1, but not by AbcR2. A double-plasmid reporter assay confirmed the predicted specific targeting of the 5′-untranslated region of the livK mRNA by AbcR1 in vivo. Our findings provide evidences of independent regulatory functions of these sRNAs, probably to fine-tune nutrient uptake in free-living and undifferentiated symbiotic rhizobia.
Much less is known about reverse transcriptases (RTs) in prokaryotes than in eukaryotes, with most prokaryotic enzymes still uncharacterized. Two surveys involving BLAST searches for RT genes in prokaryotic genomes revealed the presence of large numbers of diverse, uncharacterized RTs and RT-like sequences. Here, using consistent annotation across all sequenced bacterial species from GenBank and other sources via RAST, available from the PATRIC (Pathogenic Resource Integration Center) platform, we have compiled the data for currently annotated reverse transcriptases from completely sequenced bacterial genomes. RT sequences are broadly distributed across bacterial phyla, but green sulfur bacteria and cyanobacteria have the highest levels of RT sequence diversity (≤85% identity) per genome. By contrast, phylum Actinobacteria, for which a large number of genomes have been sequenced, was found to have a low RT sequence diversity. Phylogenetic analyses revealed that bacterial RTs could be classified into 17 main groups: group II introns, retrons/retron-like RTs, diversity-generating retroelements (DGRs), Abi-like RTs, CRISPR-Cas-associated RTs, group II-like RTs (G2L), and 11 other groups of RTs of unknown function. Proteobacteria had the highest potential functional diversity, as they possessed most of the RT groups. Group II introns and DGRs were the most widely distributed RTs in bacterial phyla. Our results provide insights into bacterial RT phylogeny and the basis for an update of annotation systems based on sequence/domain homology.
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.
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