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In Pseudomonas putida KT2440, cfcR encodes an orphan multidomain response regulator with diguanylate cyclase activity, which is responsible for the synthesis of c-di-GMP, a second messenger key in the transition from planktonic to sessile bacterial lifestyles. When overexpressed, cfcR enhances biofilm formation and causes other phenotype alterations. The cfcA gene, encoding a membrane-anchored multisensory CHASE3/GAF hybrid histidine kinase (HK), is required to develop this pleiotropic phenotype. Here we show autophosphorylation of CfcA through HisKA/HATPase_c domains and then transfer of the phosphoryl group to an internal receiver (REC) domain. CfcA REC domains are nonessential for phosphotransfer from CfcA~P to the REC domain of CfcR. CfcA~P also phosphorylates the REC domain of CfcD, a second HK encoded in the same gene cluster as CfcA, which negatively regulates the CfcA/CfcR pathway. To evaluate the impact of CfcA domains on CfcR activity, a battery of mutants with in-frame domain deletions was generated, whose CfcA protein locations were also examined. CfcA membrane anchorage contributes to protein stability and CfcR activation. Salt enhances c-di-GMP levels through CfcR, a response which is hampered by alteration of a presumed ligand-binding motif in the CHASE3 sensor domain. Thus, in P. putida, c-di-GMP is salt-regulated through the CfcA/CfcR/CfcD system.

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Mobile group II introns are ribozymes and retroelements that probably originate from bacteria. Sinorhizobium meliloti, the nitrogen-fixing endosymbiont of legumes of genus Medicago, harbors a large number of these retroelements. One of these elements, RmInt1, has been particularly successful at colonizing this multipartite genome. Many studies have improved our understanding of RmInt1 and phylogenetically related group II introns, their mobility mechanisms, spread and dynamics within S. meliloti and closely related species. Although RmInt1 conserves the ancient retroelement behavior, its evolutionary history suggests that this group II intron has played a role in the short- and long-term evolution of the S. meliloti genome. We will discuss its proposed role in genome evolution by controlling the spread and coexistence of potentially harmful mobile genetic elements, by ectopic transposition to different genetic loci as a source of early genomic variation and by generating sequence variation after a very slow degradation process, through intron remnants that may have continued to evolve, contributing to bacterial speciation.

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Bacterial group II introns are self-splicing catalytic RNAs and mobile retroelements that have an open reading frame encoding an intron-encoded protein (IEP) with reverse transcriptase (RT) and RNA splicing or maturase activity. Some IEPs carry a DNA endonuclease (En) domain, which is required to cleave the bottom strand downstream from the intron-insertion site for target DNA-primed reverse transcription (TPRT) of the inserted intron RNA. Host factors complete the insertion of the intron. By contrast, the major retrohoming pathway of introns with IEPs naturally lacking endonuclease activity, like the Sinorhizobium meliloti intron RmInt1, is thought to involve insertion of the intron RNA into the template for lagging strand DNA synthesis ahead of the replication fork, with possible use of the nascent strand to prime reverse transcription of the intron RNA. The host factors influencing the retrohoming pathway of such introns have not yet been described. Here, we identify key candidates likely to be involved in early and late steps of RmInt1 retrohoming. Some of these host factors are common to En+ group II intron retrohoming, but some have different functions. Our results also suggest that the retrohoming process of RmInt1 may be less dependent on the intracellular free Mg2+ concentration than those of other group II introns.

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Group II introns are large catalytic RNAs and mobile retroelements that encode a reverse transcriptase. Here, we provide methods for their identification in bacterial genomes and further analysis of their splicing and mobility capacities.

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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.

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We report the complete genome sequence of the RmInt1 group II intronless Sinorhizobium meliloti strain RMO17 isolated from Medicago orbicularis nodules from Spanish soil. The genome consists of 6.73 Mb distributed between a single chromosome and two megaplasmids (the chromid pSymB and pSymA).

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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.

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Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP) in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns.

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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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RmInt1 is a self-splicing and mobile group II intron initially identified in the bacterium Sinorhizobium meliloti, which encodes a reverse transcriptase-maturase (Intron Encoded Protein, IEP) lacking the C-terminal DNA binding (D) and DNA endonuclease domains (En). RmInt1 invades cognate intronless homing sites (ISRm2011-2) by a mechanism known as retrohoming. This work describes how the RmInt1 intron spreads in the S.meliloti genome upon acquisition by conjugation. This process was revealed by using the wild-type intron RmInt1 and engineered intron-donor constructs based on ribozyme coding sequence (DeltaORF)-derivatives with higher homing efficiency than the wild-type intron. The data demonstrate that RmInt1 propagates into the S.meliloti genome primarily by retrohoming with a strand bias related to replication of the chromosome and symbiotic megaplasmids. Moreover, we show that when expressed in trans from a separate plasmid, the IEP is able to mobilize genomic DeltaORF ribozymes that afterward displayed wild-type levels of retrohoming. Our results contribute to get further understanding of how group II introns spread into bacterial genomes in nature.

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