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Sinorhizobium melilotiis a soil bacterium that establishes a symbiosis within root nodules of legumes (Medicago sativa, for example) where it fixes atmospheric nitrogen into ammonia and obtains in return carbon sources and other nutrients. In this symbiosis,S. melilotiundergoes a drastic cellular change leading to a terminal differentiated form (called bacteroid) characterized by genome endoreduplication, increase of cell size and high membrane permeability. The bacterial cell cycle (mis)regulation is at the heart of this differentiation process. In free-living cells, the master regulator CtrA ensures the progression of cell cycle by activating cell division (controlled by the tubulin-like protein FtsZ) and simultaneously inhibiting supernumerary DNA replication, while on the other hand the downregulation of CtrA and FtsZ is essential for bacteroid differentiation during symbiosis, preventing endosymbiont division and permitting genome endoreduplication. Little is known inS. melilotiabout regulators of CtrA and FtsZ, as well as the processes that control bacteroid development. Here, we combine cell biology, biochemistry and bacterial genetics approaches to understand the function(s) of FcrX, a new factor that controls both CtrA and FtsZ, in free-living growth and in symbiosis. Depletion of the essential genefcrXled to abnormally high levels of FtsZ and CtrA and minicell formation. Using multiple complementary techniques, we showed that FcrX is able to interact physically with FtsZ and CtrA. Moreover, its transcription is controlled by CtrA itself and displays an oscillatory pattern in the cell cycle. We further showed that, despite a weak homology with FliJ-like proteins, only FcrX proteins from closely-related species are able to complementS. meliloti fcrXfunction. Finally, deregulation of FcrX showed abnormal symbiotic behaviors in plants suggesting a putative role of this factor during bacteroid differentiation. In conclusion, FcrX is the first known cell cycle regulator that acts directly on both, CtrA and FtsZ, thereby controlling cell cycle, division and symbiotic differentiation.

Methylation of specific DNA sequences is ubiquitous in bacteria and has known roles in immunity and regulation of cellular processes, such as the cell cycle. Here, we explored DNA methylation in bacteria of the genus Ensifer, including its potential role in regulating terminal differentiation during nitrogen-fixing symbiosis with legumes. Using single-molecule real-time sequencing, six genome-wide methylated motifs were identified across four Ensifer strains, five of which were strain-specific. Only the GANTC motif, recognized by the cell cycle-regulated CcrM methyltransferase, was methylated in all strains. In actively dividing cell cultures, methylation of GANTC motifs increased progressively from the ori to ter regions in each replicon, in agreement with a cell cycle-dependent regulation of CcrM. In contrast, there was near full genome-wide GANTC methylation in the early stage of symbiotic differentiation. This was followed by a moderate decrease in the overall extent of methylation and a progressive decrease in chromosomal GANTC methylation from the ori to ter regions in later stages of differentiation. Based on these observations, we suggest that CcrM activity is dysregulated and constitutive during terminal differentiation, which we hypothesize is a driving factor for endoreduplication of terminally differentiated bacteroids. IMPORTANCE Nitrogen fixation by rhizobia in symbiosis with legumes is economically and ecologically important. The symbiosis can involve a complex bacterial transformation—terminal differentiation—that includes major shifts in the transcriptome and cell cycle. Epigenetic regulation is an important regulatory mechanism in diverse bacteria; however, the roles of DNA methylation in rhizobia and symbiotic nitrogen fixation have been poorly investigated. We show that aside from cell cycle regulation, DNA methyltransferases are unlikely to have conserved roles in the biology of bacteria of the genus Ensifer. However, we present evidence consistent with an interpretation that the cell cycle methyltransferase CcrM is dysregulated during symbiosis, which we hypothesize may be a key factor driving the cell cycle switch in terminal differentiation required for effective symbioses.

International audience; Sinorhizobium meliloti of the Alphaproteobacteria class has a fascinating spectrum of lifestyles, thriving as a free-living soil saprophyte, as an endophyte and as a nitrogen-fixing legume symbiont. In symbiosis, it undergoes a striking cellular differentiation process, which is controlled by the host plant through the activity of NCR peptides. NCRs interfere with the cell cycle of S. meliloti and transform the regular cycle consisting of strict successions of single DNA replication followed by cell division into an endoreduplication cycle of multiple genome duplications without divisions. This cellular differentiation results in giant and polyploid symbiotic bacterial cells that fix atmospheric nitrogen. Here we discuss the regulation of the free-living cell cycle in S. meliloti and present the hypothesis that the master regulator CtrA is the ultimate target of the NCR peptides, provoking the cell cycle switch in symbiosis.

Legumes of the Medicago genus form symbiosis with the bacterium Sinorhizobium meliloti and develop root nodules housing large numbers of the intracellular symbionts. Members of the Nodule-specific Cysteine Rich peptide (NCRs) family induce the endosymbionts into a terminal differentiated state. Individual cationic NCRs are antimicrobial peptides that have the capacity to kill the symbiont but the nodule cell environment prevents killing. Moreover, the bacterial broad-specificity peptide uptake transporter BacA and exopolysaccharides contribute to protect the endosymbionts against the toxic activity of NCRs. Here, we show that other S. meliloti functions participate in the protection of the endosymbionts, including an additional broad-specificity peptide uptake transporter encoded by the yejABEF genes, lipopolysaccharide modifications mediated by lpsB and lpxXL as well as rpoH1, encoding a stress sigma factor. Mutants of these genes show in vitro a strain-specific increased sensitivity profile against a panel of NCRs and form nodules in which bacteroid differentiation is affected. The lpsB mutant nodule bacteria do not differentiate, the lpxXL and rpoH1 mutants form some seemingly fully differentiated bacteroids although most of the nodule bacteria are undifferentiated, while the yejABEF mutants form hypertrophied but nitrogen-fixing bacteroids. The nodule bacteria of all the mutants have a strongly enhanced membrane permeability, which is dependent on the transport of NCRs to the endosymbionts. Our results suggest that S. meliloti relies on a suite of functions including peptide transporters, the bacterial envelope structures and stress response regulators to resist the aggressive assault of NCR peptides in the nodule cells.ImportanceThe nitrogen fixing symbiosis of legumes with rhizobium bacteria has a predominant ecological role in the nitrogen cycle and has the potential to provide the nitrogen required for plant growth in agriculture. The host plants allow the rhizobia to colonize specific symbiotic organs, the nodules, in large numbers in order to produce sufficient reduced nitrogen for the plant needs. Some legumes, including Medicago spp., produce massively antimicrobial peptides to keep this large bacterial population in check. These peptides, known as NCRs, have the potential to kill the rhizobia but in nodules, they rather inhibit the division of the bacteria, which maintain a high nitrogen fixing activity. In this study, we show that the tempering of the antimicrobial activity of the NCR peptides in the Medicago symbiont Sinorhizobium meliloti is multifactorial and requires the YejABEF peptide transporter, the lipopolysaccharide outer membrane composition and the stress response regulator RpoH1.

International audience; The symbiotic nitrogen fixing species Sinorhizobium meliloti represents a remarkable model system for the class Alphaproteobacteria, which includes genera such as Caulobacter, Agrobacterium and Brucella. It is capable of living free in the soil, and is also able to establish a complex symbiosis with leguminous plants, during which its cell cycle program is completely rewired presumably due, at least in part, to the action of peptides secreted by the plant. Here we will discuss how the cell cycle regulation works in S. meliloti and the kinds of molecular mechanisms that take place during the infection. We will focus on the complex regulation of the master regulator of the S. meliloti cell cycle, the response regulator CtrA, discussing its implication in symbiosis.

Funding Information: We gratefully acknowledge the contribution of Marielle Bauzan for the cultures in syntrophy and Yann Denis for transcriptomic facilities and Grant Zane (Judy Wall laboratory) for providing us the GZ3621Orp ::mini‐Tn5 strain. The authors are also grateful to Emilien Etienne and the EPR facilities available at the national EPR network RENARD (IR CNRS 3443) and the Aix‐Marseille University EPR center. We also thank Martine Company for technical assistance, Katia Villion for her help on the project during its school program, Béatrice Py and Bruno Guigliarelli for insightful discussions. The French National Research Agency (ANR) funded this research project (ANR‐12‐ISV8‐0003‐01). The project leading to this publication has received funding from the Excellence Initiative of Aix‐Marseille University—A*MIDEX, a French “Investissements d'Avenir” programme and is part of the Institute of Microbiologies and Biotechnology—IM2B (AMX‐19‐IET‐006). This work was supported by Fundação para a Ciência e Tecnologia (FCT) (grant to SRP, FCT‐ANR/BBB‐MET/0023/2012) and by the Belgian Federal Science Policy Office (Belspo) (IAP7/44, iPROS project). SRP was also supported by the Applied Molecular Biosciences Unit‐UCIBIO financed by national funds from FCT (UIDP/04378/2020 and UIDB/04378/2020). This article is based upon work from COST Action CA15133, supported by COST (European Cooperation in Science and Technology). Made available in DSpace on 2021-11-29T23:39:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-07 publishersversion published

Enhancer binding proteins (EBPs) are key players of σ54‐regulation that control transcription in response to environmental signals. In the anaerobic microorganism Desulfovibrio vulgaris Hildenborough (DvH), orp operons have been previously shown to be coregulated by σ54‐RNA polymerase, the integration host factor IHF and a cognate EBP, OrpR. In this study, ChIP‐seq experiments indicated that the OrpR regulon consists of only the two divergent orp operons. In vivo data revealed that (i) OrpR is absolutely required for orp operons transcription, (ii) under anaerobic conditions, OrpR binds on the two dedicated DNA binding sites and leads to high expression levels of the orp operons, (iii) increasing the redox potential of the medium leads to a drastic down‐regulation of the orp operons expression. Moreover, combining functional and biophysical studies on the anaerobically purified OrpR leads us to propose that OrpR senses redox potential variations via a redox‐sensitive [4Fe–4S]2+ cluster in the sensory PAS domain. Overall, the study herein presents the first characterization of a new Fe–S redox regulator belonging to the σ54‐dependent transcriptional regulator family probably advantageously selected by cells adapted to the anaerobic lifestyle to monitor redox stress conditions., Enhancer Binding Proteins (EBPs) are key players of σ54‐regulation that control transcription of genes expression in response to environmental signals. OrpR is a novel type of EBP that presents an unprecedented Fe–S cluster in the PAS sensory domain and detects the redox potential, not O2. In anaerobes, OrpR regulates the expression of genes crucial for growth in anaerobic conditions as a function of the redox potential of the environment.

International audience; Rhizobia are nitrogen-fixing soil bacteria that can infect legume plants to establish root nodules symbiosis. To do that, a complex exchange of molecular signals occurs between plants and bacteria. Among them, rhizobial Nops (Nodulation outer proteins), secreted by a type III secretion system (T3SS) determine the host-specificity for efficient symbiosis with plant roots. Little is known about the molecular function of secreted Nops (also called effectors (T3E)) and their role in the symbiosis process. We performed the structure-function characterization of NopAA, a T3E from Sinorhizobium fredii by using a combination of X-ray crystallography, biochemical and biophysical approaches. this work displays for the first time a complete structural and biochemical characterization of a symbiotic T3E. Our results showed that NopAA has a catalytic domain with xyloglucanase activity extended by a N-terminal unfolded secretion domain that allows its secretion. We proposed that these original structural properties combined with the specificity of NopAA toward xyloglucan, a key component of root cell wall which is also secreted by roots in the soil, can give NopAA a strategic position to participate in recognition between bacteria and plant roots and to intervene in nodulation process. Many bacterial pathogens use type III secretion systems (T3SS) to inject virulence factors, named effectors, directly into the cytoplasm of target eukaryotic cells. Most of the T3SS apparatus components are conserved among plant and animal pathogens, suggesting a common mechanism of recognition and secretion of effec-tors (for review see 1). Secretion of effectors depends on the presence of secretion signals composed by 20 to 30 non-conserved amino acids at the N-terminus. Most of T3SS effectors (T3E) are predicted to possess a N-terminal intrinsically disordered (ID) region enriched in serine residues and required for secretion 2. Many secreted proteins also depend on the interaction with a cytoplasmic specific T3S chaperone 3 that stabilize ID regions in bacterial cytoplasm prior translocation. For SopB, a T3E of Salmonella it has been proposed that its cognate chaperone SigE may be responsible of the formation of ring like hexamers, conformation that may be required for substrate recognition by the type three apparatus 4. In pathogenic bacteria, T3SS genes coding for the secretion apparatus, effectors and accessory proteins are clustered in pathogenicity islands (PAIs) and organized in operons. Interestingly, this secretion strategy is also used by non-pathogenic organisms contributing to symbiotic interactions with hosts as shown for rhizobia 5,6. Rhizobia are soil bacteria having the ability to establish a specific symbiotic association with leguminous host-plant roots. This interaction leads to the formation of root nodules by the plant, specialized organs in which bacteria differentiate into nitrogen fixing bacteroids able to convert atmospheric nitrogen into ammonia for the usage in plant growth 7. This process starts with the secretion of

It is assumed that intracellular pathogenic bacteria have to cope with DNA alkylating stress within host cells. Here we use single-cell reporter systems to show that the pathogen Brucella abortus does encounter alkylating stress during the first hours of macrophage infection. Genes encoding direct repair and base-excision repair pathways are required by B. abortus to face this stress in vitro and in a mouse infection model. Among these genes, ogt is found to be under the control of the conserved cell-cycle transcription factor GcrA. Our results highlight that the control of DNA repair in B. abortus displays distinct features that are not present in model organisms such as Escherichia coli., SCOPUS: ar.j, info:eu-repo/semantics/published

The bacterial envelope is a remarkable and complex compartment of the prokaryotic cell in which many essential functions take place. The article by Herrou and collaborators (Herrou et al., in press), by a clever combination of structural analysis, genetics and functional characterization in free-living bacterial cells and during infection in animal models, elucidates a new factor, named EipA, that plays a major role in Brucella spp envelope biogenesis and cell division. The authors demonstrate a genetic connection between eipA and lipopolysaccharide synthesis, specifically genes involved in the synthesis of the O-antigen portion of lipopolysaccharide (LPS). Beyond its crucial role in Brucella physiology, the conservation of EipA in the class Alphaproteobacteria urges microbiologists to pursue future investigation of its homologs in other species belonging to this important group of bacteria.

Brucella abortus is a pathogen infecting cattle, able to survive, traffic, and proliferate inside host cells. It belongs to the Alphaproteobacteria, a phylogenetic group comprising bacteria with free living, symbiotic, and pathogenic lifestyles. An essential regulator of cell cycle progression named CtrA was described in the model bacterium Caulobacter crescentus. This regulator is conserved in many alphaproteobacteria, but the evolution of its regulon remains elusive. Here we identified promoters that are CtrA targets using ChIP-seq and we found that CtrA binds to promoters of genes involved in cell cycle progression, in addition to numerous genes encoding outer membrane components involved in export of membrane proteins and synthesis of lipopolysaccharide. Analysis of a conditional B. abortus ctrA loss of function mutant confirmed that CtrA controls cell division. Impairment of cell division generates elongated and branched morphologies, that are also detectable inside HeLa cells. Surprisingly, abnormal bacteria are able to traffic to the endoplasmic reticulum, the usual replication niche of B. abortus in host cells. We also found that CtrA depletion affected outer membrane composition, in particular the abundance and spatial distribution of Omp25. Control of the B. abortus envelope composition by CtrA indicates the plasticity of the CtrA regulon along evolution.

Acanthamoeba-infecting Mimiviridae belong to three clades: Mimiviruses (A), Moumouviruses (B) and Megaviruses (C). The uniquely complex mobilome of these giant viruses includes virophages and linear 7 kb-DNA molecules called “transpovirons”. We recently isolated a virophage (Zamilon vitis) and two transpovirons (maBtv and mvCtv) respectively associated to B-clade and C-clade mimiviruses. We used the capacity of the Zamilon virophage to replicate both on B-clade and C-clade host viruses to investigate the three partite interaction network governing the propagation of transpovirons. We found that the virophage could transfer both types of transpovirons to B-clade and C-clade host viruses provided they were devoid of a resident transpoviron (permissive effect). If not, only the resident transpoviron was replicated and propagated within the virophage progeny (dominance effect). Although B- and C-clade viruses devoid of transpoviron could replicate both maBtv and mvCtv, they did it with a lower efficiency across clades, suggesting an ongoing process of adaptive co-evolution. We performed proteomic comparisons of host viruses and virophage particles carrying or cleared of transpovirons in search of proteins involved in this adaptation process. These experiments revealed that transpoviron-encoded proteins are synthetized during the combined mimiviruses/virophage/transpoviron replication process and some of them are specifically incorporated into the virophage and giant virus particles together with the cognate transpoviron DNA. This study also highlights a unique example of intricate commensalism in the viral world, where the transpoviron uses the virophage to propagate from one host virus to another and where the Zamilon virophage and the transpoviron depend on their host giant virus to replicate, this without affecting the giant virus infectious cycle, at least in laboratory conditions.Author SummaryThe Mimiviridae are giant viruses with dsDNA genome up to 1.5 Mb. They build huge viral factories in the host cytoplasm in which the nuclear-like virus-encoded functions (transcription and replication) take place. They are themselves the target of infections by 20 kb-dsDNA virophages, replicating in the giant virus factories. They can also be found associated with 7kb-DNA episomes, dubbed transpovirons. We investigated the relationship between these three players by combining competition experiments involving the newly isolated Zamilon vitis virophage as a vehicle for transpovirons of different origins with proteomics analyses of virophage and giant virus particles. Our results suggest that relationship of the virophage, the transpoviron, and their host giant virus, extend the concept of commensalism to the viral world.

SummaryBacteria are powerful models for understanding how cells divide and accomplish global regulatory programs. In Caulobacter crescentus, a cascade of essential master regulators supervises the correct and sequential activation of DNA replication, cell division and development of different cell types. Among them, the response regulator CtrA plays a crucial role coordinating all those functions. Here, for the first time we describe the role of a novel factor named CcnA, a cell cycle regulated ncRNA located at the origin of replication, presumably activated by CtrA and responsible for the accumulation of CtrA itself. In addition, CcnA may be also involved in the inhibition of translation of the S-phase regulator, GcrA, by interacting with its 5’ untranslated region (5’-UTR). Performing in vitro experiments and mutagenesis, we propose a mechanism of action of CcnA based on liberation (ctrA) or sequestration (gcrA) of their ribosome-binding site (RBS). Finally, its role may be conserved in other alphaproteobacterial species, such as Sinorhizobium meliloti, representing indeed a potentially conserved process modulating cell cycle in Caulobacterales and Rhizobiales.

Acanthamoeba-infecting Mimiviridae are giant viruses with dsDNA genome up to 1.5 Mb. They build viral factories in the host cytoplasm in which the nuclear-like virus-encoded functions take place. They are themselves the target of infections by 20-kb-dsDNA virophages, replicating in the giant virus factories and can also be found associated with 7-kb-DNA episomes, dubbed transpovirons. Here we isolated a virophage (Zamilon vitis) and two transpovirons respectively associated to B- and C-clade mimiviruses. We found that the virophage could transfer each transpoviron provided the host viruses were devoid of a resident transpoviron (permissive effect). If not, only the resident transpoviron originally isolated from the corresponding virus was replicated and propagated within the virophage progeny (dominance effect). Although B- and C-clade viruses devoid of transpoviron could replicate each transpoviron, they did it with a lower efficiency across clades, suggesting an ongoing process of adaptive co-evolution. We analysed the proteomes of host viruses and virophage particles in search of proteins involved in this adaptation process. This study also highlights a unique example of intricate commensalism in the viral world, where the transpoviron uses the virophage to propagate and where the Zamilon virophage and the transpoviron depend on the giant virus to replicate, without affecting its infectious cycle.

The FrzCD chemoreceptor from the gliding bacterium Myxococcus xanthus forms cytoplasmic clusters that occupy a large central region of the cell body also occupied by the nucleoid. In this work, we show that FrzCD directly binds to the nucleoid with its N-terminal positively charged tail and recruits active signaling complexes at this location. The FrzCD binding to the nucleoid occur in a DNA-sequence independent manner and leads to the formation of multiple distributed clusters that explore constrained areas. This organization might be required for cooperative interactions between clustered receptors as observed in membrane-bound chemosensory arrays., Author summary In this work, we show that the cytoplasmic chemoreceptor of the Frz chemosensory system, FrzCD, does not bind the cytoplasmic membrane like most MCPs but bind the bacterial nucleoid directly, thus forming distributed protein clusters also containing the FrzE kinase. In vitro and in vivo experiments show that DNA-binding is not sequence-specific and is mediated by a basic aminoacid sequence of the FrzCD N-terminal domain. The deletion of this motif abolishes FrzCD DNA-binding and cooperativity in the response to signals. This work shows the importance of the nucleoid in the organization and functioning of cytoplasmic signaling systems in bacteria.

In Caulobacter crescentus , methylation of DNA by CcrM plays an important part in the regulation of cell cycle progression. Thanks to this methyltransferase, the activity of which is cell cycle regulated, the chromosome transitions between a hemimethylated state in the S-phase to a fully methylated condition in the G1 and G2 phases. Any perturbation in CcrM expression, such as depletion or constitutive expression, causes severe developmental defects . Several studies suggest that the role of CcrM is conserved across the Alphaproteobacteria . In the past few years, the importance of methylation on the expression of cell cycle regulated genes has emerged, suggesting that CcrM-dependent methylation can direct the binding of transcription factors to specific methylated sequences and affect the expression of genes depending on the methylation state of their promoters. CcrM activity has recently been linked to GcrA, a cell cycle master regulator that controls the expression of several genes during S-phase. Here, we review recent findings that establish the global role of methylation in cell cycle progression, and also explore the significance of a CcrM–GcrA epigenetic module that has co-evolved in Alphaproteobacteria , including Caulobacter , in controlling several genes involved in cell division, polarity, and motility.

Addressing the functionality of genomes is one of the most important and challenging tasks of today's biology. In particular the ability to link genotypes to corresponding phenotypes is of interest in the reconstruction and biotechnological manipulation of metabolic pathways. Over the last years, the OmniLog™ Phenotype Microarray (PM) technology has been used to address many specific issues related to the metabolic functionality of microorganisms. However, computational tools that could directly link PM data with the gene(s) of interest followed by the extraction of information on gene–phenotype correlation are still missing. Here we present DuctApe, a suite that allows the analysis of both genomic sequences and PM data, to find metabolic differences among PM experiments and to correlate them with KEGG pathways and gene presence/absence patterns. As example, an application of the program to four bacterial datasets is presented. The source code and tutorials are available at http://combogenomics.github.io/DuctApe/.

Many bacterial species, such as the alphaproteobacterium Sinorhizobium meliloti, are characterized by open pangenomes and contain multipartite genomes consisting of a chromosome and other large-sized replicons, such as chromids, megaplasmids, and plasmids. The evolutionary forces in both functional and structural aspects that shape the pangenome of species with multipartite genomes are still poorly understood. Therefore, we sequenced the genomes of 10 new S. meliloti strains, analyzed with four publicly available additional genomic sequences. Results indicated that the three main replicons present in these strains (a chromosome, a chromid, and a megaplasmid) partly show replicon-specific behaviors related to strain differentiation. In particular, the pSymB chromid was shown to be a hot spot for positively selected genes, and, unexpectedly, genes resident in the pSymB chromid were also found to be more widespread in distant taxa than those located in the other replicons. Moreover, through the exploitation of a DNA proximity network, a series of conserved "DNA backbones" were found to shape the evolution of the genome structure, with the rest of the genome experiencing rearrangements. The presented data allow depicting a scenario where the pSymB chromid has a distinctive role in intraspecies differentiation and in evolution through positive selection, whereas the pSymA megaplasmid mostly contributes to structural fluidity and to the emergence of new functions, indicating a specific evolutionary role for each replicon in the pangenome evolution.

Brucella abortus is a pathogen infecting cattle, able to survive, traffic, and proliferate inside host cells. It belongs to the Alphaproteobacteria, a phylogenetic group comprising bacteria with free living, symbiotic, and pathogenic lifestyles. An essential regulator of cell cycle progression named CtrA was described in the model bacterium Caulobacter crescentus. This regulator is conserved in many alphaproteobacteria, but the evolution of its regulon remains elusive. Here we identified promoters that are CtrA targets using ChIP-seq and we found that CtrA binds to promoters of genes involved in cell cycle progression, in addition to numerous genes encoding outer membrane components involved in export of membrane proteins and synthesis of lipopolysaccharide. Analysis of a conditional B. abortus ctrA loss of function mutant confirmed that CtrA controls cell division. Impairment of cell division generates elongated and branched morphologies, that are also detectable inside HeLa cells. Surprisingly, abnormal bacteria are able to traffic to the endoplasmic reticulum, the usual replication niche of B. abortus in host cells. We also found that CtrA depletion affected outer membrane composition, in particular the abundance and spatial distribution of Omp25. Control of the B. abortus envelope composition by CtrA indicates the plasticity of the CtrA regulon along evolution.

International audience; The gram-negative bacterium Caulobacter crescentus is a powerful model organism for studies of bacterial cell cycle regulation. Although the major regulators and their connections in Caulobacter have been identified, it still is a challenge to properly understand the dynamics of its circuitry which accounts for both cell cycle progression and arrest. We show that the key decision module in Caulobacter is built from a limit cycle oscillator which controls the DNA replication program. The effect of an induced cell cycle arrest is demonstrated to be a key feature to classify the underlying dynamics.

Developmental events across the prokaryotic life cycle are highly regulated at the transcriptional and posttranslational levels. Key elements of a few regulatory networks are conserved among phylogenetic groups of bacteria, although the features controlled by these conserved systems are as diverse as the organisms encoding them. In this work, we probed the role of the CtrA regulatory network, conserved throughout the Alphaproteobacteria , in the magnetotactic bacterium Magnetospirillum magneticum strain AMB-1, which possesses unique intracellular organization and compartmentalization. While we have shown that CtrA in AMB-1 is not essential for viability, it is required for motility, and its putative phosphorylation state dictates the ability of CtrA to activate the flagellar biosynthesis gene cascade. Gene expression analysis of strains expressing active and inactive CtrA alleles points to the composition of the extended CtrA regulon, including both direct and indirect targets. These results, combined with a bioinformatic study of the AMB-1 genome, enabled the prediction of an AMB-1-specific CtrA binding site. Further, phylogenetic studies comparing CtrA sequences from alphaproteobacteria in which the role of CtrA has been experimentally examined reveal an ancestral role of CtrA in the regulation of motility and suggest that its essential functions in other alphaproteobacteria were acquired subsequently.

Although horizontal gene transfer mediated by plasmids is important to the generation of the genetic variability of Sinorhizobium strains, the barriers which can reduce horizontal gene transfer between bacteria have not yet been studied in Sinorhizobium. We studied the plasmid transfer by electroporation and its restriction in strains of Sinorhizobium meliloti and S. medicae. After conditions for electroporation were established, three S. meliloti strains (including the sequenced type strain Rm1021) and two S. medicae strains were electroporated with plasmid DNA extracted from strains of both species. The efficiency of transformation was found to be variable among different strains. The acquisition of plasmid DNA was found to be donor-dependent in S. meliloti strain Rm1021 that prefers self-DNA more than the DNA from other Sinorhizobium strains. All other strains tested did not show a preference for self-DNA. In strain Rm1021, the inactivation of the hsdR gene, coding for a putative type-I restriction enzyme, increased the efficiency of transformation and conjugation with non-self DNA; the transformation capability was again reduced in hsdR mutant when the cloned hsdR gene was expressed from a lac promoter. Phylogenetic analysis of the hsdR gene clearly indicated that this gene was horizontally transferred to strain Rm1021, explaining its absence in the other strains tested.

Diversity of biological samples is still partially considered by conventional Differential Interference Contrast (DIC) microscopy approaches. Here we propose a new algorithm (developed as an ImageJ macro), the STA (Shadow Technique Algorithm), whose originality relies in the 3D/4D visualization of a large range of biological objects, from bacteria, vegetal tissues to living cells in culture. This new approach does not need extensive calculations, systems modifications or in-depth knowledge of acquisition optics. STA, providing 3D DIC reconstruction every hundredth of ms, can be applied to dissect various cellular phenomena. In addition, we propose different methods of graphic representations, which unable to enlighten the specificities of each category of questioning. Specifically we here addressed: i) tissue imaging, ii) cell cycle and cell death imaging iii) vesicle tracking.

Legumes form symbiotic relationship with root-nodule, rhizobia. The nitrogen (N2) fixed by legumes is a renewable source and of great importance to agriculture. Symbiotic nitrogen fixation (SNF) is constrained by multiple stresses and alleviating them would improve SNF contribution to agroecosystems. Genetic differences in adaptation tolerance to various stresses are known in both host plant and rhizobium. The discovery and use of promiscuous germplasm in soybean led to the release of high-yielding cultivars in Africa. High N2-fixing soybean cultivars are commercially grown in Australia and some countries in Africa and South America and those of pea in Russia. SNF is a complex trait, governed by multigenes with varying effects. Few major quantitative trait loci (QTL) and candidate genes underlying QTL are reported in grain and model legumes. Nodulating genes in model legumes are cloned and orthologs determined in grain legumes. Single nucleotide polymorphism (SNP) markers from nodulation genes are available in common bean and soybean. Genomes of chickpea, pigeonpea, and soybean; and genomes of several rhizobium species are decoded. Expression studies revealed few genes associated with SNF in model and grain legumes. Advances in host plant and rhizobium genomics are helping identify DNA markers to aid breeding of legume cultivars with high symbiotic efficiency. A paradigm shift is needed by breeding programs to simultaneously improve host plant and rhizobium to harness the strength of positive symbiotic interactions in cultivar development. Computation models based on metabolic reconstruction pathways are providing greater insights to explore genotype–phenotype relationships in SNF. Models to simulate the response of N2 fixation to a range of environmental variables and crop growth are assisting researchers to quantify SNF for efficient and sustainable agricultural production systems. Such knowledge helps identifying bottlenecks in specific legume–rhizobia systems that could be overcome by legume breeding to enhance SNF. This review discusses the recent developments to improve SNF and productivity of grain legumes.

Many bacterial pathogens use type three secretion systems (T3SS) to inject virulence factors, named effectors, directly into the cytoplasm of target eukaryotic cells. Most of the T3SS components are conserved among plant and animal pathogens, suggesting a common mechanism of recognition and secretion of effectors. However, no common motif has yet been identified for effectors allowing T3SS recognition. In this work, we performed a biochemical and structural characterization of the Salmonella SopB/SigE chaperone/effector complex by small-angle X-ray scattering (SAXS). Our results showed that the SopB/SigE complex is assembled in dynamic homohexameric-ring-shaped structures with an internal tunnel. In this ring, the chaperone maintains a disordered N-terminal end of SopB molecules, in a good position to be reached and processed by the T3SS. This ring dimensionally fits the ring-organized molecules of the injectisome, including ATPase hexameric rings; this organization suggests that this structural feature is important for ATPase recognition by T3SS. Our work constitutes the first evidence of the oligomerization of an effector, analogous to the organization of the secretion machinery, obtained in solution. As effectors share neither sequence nor structural identity, the quaternary oligomeric structure could constitute a strategy evolved to promote the specificity and efficiency of T3SS recognition.

A fundamental question in developmental biology is how morphogenesis is coordinated with cell cycle progression. In Caulobacter crescentus, each cell cycle produces morphologically distinct daughter cells, a stalked cell and a flagellated swarmer cell. Construction of both the flagellum and stalk requires the alternative sigma factor RpoN (sigma(54)). Here we report that a sigma(54)-dependent activator, TacA, is required for cell cycle regulated stalk biogenesis by collaborating with RpoN to activate gene expression. We have also identified the first histidine phosphotransferase in C. crescentus, ShpA, and show that it too is required for stalk biogenesis. Using a systematic biochemical technique called phosphotransfer profiling we have identified a multicomponent phosphorelay which leads from the hybrid histidine kinase ShkA to ShpA and finally to TacA. This pathway functions in vivo to phosphorylate and hence, activate TacA. Finally, whole genome microarrays were used to identify candidate members of the TacA regulon, and we show that at least one target gene, staR, regulates stalk length. This is the first example of a general method for identifying the connectivity of a phosphorelay and can be applied to any organism with two-component signal transduction systems.

Serpentine soils are characterized by high levels of heavy metals (Ni, Co, Cr), and low levels of important plant nutrients (P, Ca, N). Because of these inhospitable edaphic conditions, serpentine soils are typically home to a very specialized flora including endemic species as the nickel hyperaccumulator Alyssum bertolonii. Although much is known about the serpentine flora, few researches have investigated the bacterial communities of serpentine areas. In the present study bacterial communities were sampled at various distances from A. bertolonii roots in three different serpentine areas and their genetic diversity was assessed by terminal restriction fragment length polymorphism (T-RFLP) analysis. The obtained results indicated the occurrence of a high genetic diversity and heterogeneity of the bacterial communities present in the different serpentine areas. Moreover, TRFs (terminal restriction fragments) common to all the investigated A. bertolonii rhizosphere samples were found. A new cloning strategy was applied to 27 TRFs that were sequenced and taxonomically interpreted as mainly belonging to Gram-positive and alpha-Proteobacteria representatives. In particular, cloned TRFs which discriminated between rhizosphere and soil samples were mainly interpreted as belonging to Proteobacteria representatives.

Sinorhizobium meliloti natural populations show a high level of genetic polymorphism possibly due to the presence of mobile genetic elements such as insertion sequences (IS), transposons, and bacterial mobile introns. The analysis of the DNA sequence polymorphism of the nod region of S. meliloti p SymA megaplasmid in an Italian isolate led to the discovery of a new insertion sequence, IS Rm31. IS Rm31 is 2,803 bp long and has 22-bp-long terminal inverted repeat sequences, 8-bp direct repeat sequences generated by transposition, and three ORFs (A, B, C) coding for proteins of 124, 115, and 541 amino acids, respectively. ORF A and ORF C are significantly similar to members of the transposase family. Amino acid and nucleotide sequences indicate that IS Rm31 is a member of the IS 66 family. IS Rm31 sequences were found in 30.5% of the Italian strains analyzed, and were also present in several collection strains of the Rhizobiaceae family, including S. meliloti strain 1021. Alignment of targets sites in the genome of strains carrying IS Rm31 suggested that IS Rm31 inserts randomly into S. meliloti genomes. Moreover, analysis of IS Rm31 insertion sites revealed DNA sequences not present in the recently sequenced S. meliloti strain 1021 genome. In fact, IS Rm31 was in some cases linked to DNA fragments homologous to sequences found in other rhizobia species.

Sinorhizobium meliloti and Sinorhizobium medicae are two closely related species of the genus Sinorhizobium showing a similar host range, nodulating leguminous species of the genera Medicago, Melilotus and Trigonella, but their phylogenic relationship has not been elucidated yet. In this paper we report the application of three different molecular markers, (i) RFLP of nodD genes, (ii) 16S-23S rDNA intergenic gene spacer fingerprinting and (iii) amplification fragment length polymorphism to S. meliloti and S. medicae strains isolated from the Caucasian area, which is the region of origin of the host plant Medicago. The analysis of data could suggest the origin of S. medicae strains from an ancestral S. meliloti population.

International audience; The cyclic adenosine monophosphate dependent kinase protein (PKA) controls a variety of cellular processes including cell cycle regulation. Here, we took advantages of genetically encoded FRET-based biosensors, using an AKAR-derived biosensor to characterize PKA activity during mitosis in living HeLa cells using a single-cell approach. We measured PKA activity changes during mitosis. HeLa cells exhibit a substantial increase during mitosis, which ends with telophase. An AKAREV T>A inactive form of the biosensor and H89 inhibitor were used to ascertain for the specificity of the PKA activity measured. On a spatial point of view, high levels of activity near to chromosomal plate during metaphase and anaphase were detected. By using the PKA inhibitor H89, we assessed the role of PKA in the maintenance of a proper division phenotype. While this treatment in our hands did not impaired cell cycle progression in a drastic manner, inhibition of PKA leads to a dramatic increase in chromososme misalignement on the spindle during metaphase that could result in aneuploidies. Our study emphasizes the insights that can be gained with genetically encoded FRET-based biosensors, which enable to overcome the shortcomings of classical methologies and unveil in vivo PKA spatiotemporal profiles in HeLa cells.

Sinorhizobium meliloti is a soil bacterium that invades the root nodules it induces on Medicago sativa, whereupon it undergoes an alteration of its cell cycle and differentiates into nitrogen-fixing, elongated and polyploid bacteroid with higher membrane permeability. In Caulobacter crescentus, a related alphaproteobacterium, the principal cell cycle regulator, CtrA, is inhibited by the phosphorylated response regulator DivK. The phosphorylation of DivK depends on the histidine kinase DivJ, while PleC is the principal phosphatase for DivK. Despite the importance of the DivJ in C. crescentus, the mechanistic role of this kinase has never been elucidated in other Alphaproteobacteria. We show here that the histidine kinases DivJ together with CbrA and PleC participate in a complex phosphorylation system of the essential response regulator DivK in S. meliloti. In particular, DivJ and CbrA are involved in DivK phosphorylation and in turn CtrA inactivation, thereby controlling correct cell cycle progression and the integrity of the cell envelope. In contrast, the essential PleC presumably acts as a phosphatase of DivK. Interestingly, we found that a DivJ mutant is able to elicit nodules and enter plant cells, but fails to establish an effective symbiosis suggesting that proper envelope and/or low CtrA levels are required for symbiosis.

Several regulators are involved in the control of cell cycle progression in the bacterial model system Caulobacter crescentus, which divides asymmetrically into a vegetative G1-phase (swarmer) cell and a replicative S-phase (stalked) cell. Here we report a novel functional interaction between the enigmatic cell cycle regulator GcrA and the N6-adenosine methyltransferase CcrM, both highly conserved proteins among Alphaproteobacteria, that are activated early and at the end of S-phase, respectively. As no direct biochemical and regulatory relationship between GcrA and CcrM were known, we used a combination of ChIP (chromatin-immunoprecipitation), biochemical and biophysical experimentation, and genetics to show that GcrA is a dimeric DNA–binding protein that preferentially targets promoters harbouring CcrM methylation sites. After tracing CcrM-dependent N6-methyl-adenosine promoter marks at a genome-wide scale, we show that these marks recruit GcrA in vitro and in vivo. Moreover, we found that, in the presence of a methylated target, GcrA recruits the RNA polymerase to the promoter, consistent with its role in transcriptional activation. Since methylation-dependent DNA binding is also observed with GcrA orthologs from other Alphaproteobacteria, we conclude that GcrA is the founding member of a new and conserved class of transcriptional regulators that function as molecular effectors of a methylation-dependent (non-heritable) epigenetic switch that regulates gene expression during the cell cycle., Author Summary Methylation of genomic DNA at a specific regulatory site can impact a myriad of processes in eukaryotic cells. In bacteria, methylation at the N6 position of adenosine (m6A) is known to mediate a non-adaptive immunity response to protect cells from foreign DNA. While m6A marks are not known to govern expression of cell cycle genes in Gammaproteobacteria, cell cycle transcription in the model alphaproteobacterium Caulobacter crescentus requires the m6A methyltransferase CcrM that introduces m6A marks at GAnTC sequences and the enigmatic factor GcrA. Investigating if a functional and biochemical relationship exists between CcrM and GcrA, we found that CcrM-dependent m6A marks recruit GcrA to the promoters of cell cycle genes in vitro and in vivo and is required for efficient transcription. GcrA interacts with RNA polymerase, explaining how cell cycle transcription is affected. Importantly, m6A-dependent binding is also seen in GcrA orthologs, indicating that this transcriptional regulatory mechanism by CcrM and GcrA is conserved in Alphaproteobacteria.

Background Plant-associated bacterial communities caught the attention of several investigators which study the relationships between plants and soil and the potential application of selected bacterial species in crop improvement and protection. Medicago sativa L. is a legume crop of high economic importance as forage in temperate areas and one of the most popular model plants for investigations on the symbiosis with nitrogen fixing rhizobia (mainly belonging to the alphaproteobacterial species Sinorhizobium meliloti). However, despite its importance, no studies have been carried out looking at the total bacterial community associated with the plant. In this work we explored for the first time the total bacterial community associated with M. sativa plants grown in mesocosms conditions, looking at a wide taxonomic spectrum, from the class to the single species (S. meliloti) level. Results Results, obtained by using Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis, quantitative PCR and sequencing of 16 S rRNA gene libraries, showed a high taxonomic diversity as well as a dominance by members of the class Alphaproteobacteria in plant tissues. Within Alphaproteobacteria the families Sphingomonadaceae and Methylobacteriaceae were abundant inside plant tissues, while soil Alphaproteobacteria were represented by the families of Hyphomicrobiaceae, Methylocystaceae, Bradyirhizobiaceae and Caulobacteraceae. At the single species level, we were able to detect the presence of S. meliloti populations in aerial tissues, nodules and soil. An analysis of population diversity on nodules and soil showed a relatively low sharing of haplotypes (30-40%) between the two environments and between replicate mesocosms, suggesting drift as main force shaping S. meliloti population at least in this system. Conclusions In this work we shed some light on the bacterial communities associated with M. sativa plants, showing that Alphaproteobacteria may constitute an important part of biodiversity in this system, which includes also the well known symbiont S. meliloti. Interestingly, this last species was also found in plant aerial part, by applying cultivation-independent protocols, and a genetic diversity analysis suggested that population structure could be strongly influenced by random drift.

Background Sinorhizobium meliloti is a model system for the studies of symbiotic nitrogen fixation. An extensive polymorphism at the genetic and phenotypic level is present in natural populations of this species, especially in relation with symbiotic promotion of plant growth. AK83 and BL225C are two nodule-isolated strains with diverse symbiotic phenotypes; BL225C is more efficient in promoting growth of the Medicago sativa plants than strain AK83. In order to investigate the genetic determinants of the phenotypic diversification of S. meliloti strains AK83 and BL225C, we sequenced the complete genomes for these two strains. Results With sizes of 7.14 Mbp and 6.97 Mbp, respectively, the genomes of AK83 and BL225C are larger than the laboratory strain Rm1021. The core genome of Rm1021, AK83, BL225C strains included 5124 orthologous groups, while the accessory genome was composed by 2700 orthologous groups. While Rm1021 and BL225C have only three replicons (Chromosome, pSymA and pSymB), AK83 has also two plasmids, 260 and 70 Kbp long. We found 65 interesting orthologous groups of genes that were present only in the accessory genome, consequently responsible for phenotypic diversity and putatively involved in plant-bacterium interaction. Notably, the symbiosis inefficient AK83 lacked several genes required for microaerophilic growth inside nodules, while several genes for accessory functions related to competition, plant invasion and bacteroid tropism were identified only in AK83 and BL225C strains. Presence and extent of polymorphism in regulons of transcription factors involved in symbiotic interaction were also analyzed. Our results indicate that regulons are flexible, with a large number of accessory genes, suggesting that regulons polymorphism could also be a key determinant in the variability of symbiotic performances among the analyzed strains. Conclusions In conclusions, the extended comparative genomics approach revealed a variable subset of genes and regulons that may contribute to the symbiotic diversity.