ATP-Driven Separation of Liquid Phase Condensates in Bacteria.

Liquid-liquid phase-separated (LLPS) states are key to compartmentalizing components in the absence of membranes; however, it is unclear whether LLPS condensates are actively and specifically organized in the subcellular space and by which mechanisms. Here, we address this question by focusing on the ParABS DNA segregation system, composed of a centromeric-like sequence (parS), a DNA-binding protein (ParB), and a motor (ParA). We show that parS and ParB associate to form nanometer-sized, round condensates. ParB molecules diffuse rapidly within the nucleoid volume but display confined motions when trapped inside ParB condensates. Single ParB molecules are able to rapidly diffuse between different condensates, and nucleation is strongly favored by parS. Notably, the ParA motor is required to prevent the fusion of ParB condensates. These results describe a novel active mechanism that splits, segregates, and localizes non-canonical LLPS condensates in the subcellular space.

NsrA, a Predicted ß-Barrel Outer Membrane Protein Involved in Plant Signal Perception and the Control of Secondary Infection in Sinorhizobium meliloti.

An ongoing signal exchange fine-tunes the symbiotic interactions between rhizobia and legumes, ensuring the establishment and maintenance of mutualism. In a recently identified regulatory loop, endosymbiotic Sinorhizobium meliloti exerts negative feedback on root infection in response to unknown plant cues. Upon signal perception, three bacterial adenylate cyclases (ACs) of the inner membrane, namely, CyaD1, CyaD2, and CyaK, synthesize the second messenger cAMP, which, together with the cAMP-dependent Clr transcriptional activator, activates the expression of genes involved in root infection control. The pathway that links signal perception at the surface of the cell to cytoplasmic cAMP production by ACs was thus far unknown. Here we first show that CyaK is the cognate AC for the plant signal, called signal 1, that was observed previously in mature nodule and shoot extracts. We also show that inactivation of the gene immediately upstream of cyaK, nsrA (smb20775), which encodes a ß-barrel protein of the outer membrane, abolished signal 1 perception ex planta, whereas nsrA overexpression increased signal 1 responsiveness. Inactivation of the nsrA gene abolished all Clr-dependent gene expression in nodules and led to a marked hyperinfection phenotype on plants, similar to that of a cyaD1 cyaD2 cyaK triple mutant. We suggest that the NsrA protein acts as the (co)receptor for two signal molecules, signal 1 and a hypothetical signal 1', in mature and young nodules that cooperate in controlling secondary infection in S. meliloti-Medicago symbiosis. The predicted topology and domain composition of the NsrA protein hint at a mechanism of transmembrane signaling.IMPORTANCE Symbiotic interactions, especially mutualistic ones, rely on a continuous signal exchange between the symbionts. Here we report advances regarding a recently discovered signal transduction pathway that fine-tunes the symbiotic interaction between S. meliloti and its Medicago host plant. We have identified an outer membrane protein of S. meliloti, called NsrA, that transduces Medicago plant signals to adenylate cyclases in the inner membrane, thereby triggering a cAMP signaling cascade that controls infection. Besides their relevance for the rhizobium-legume symbiosis, these findings shed light on the mechanisms of signal perception and transduction by adenylate cyclases and transmembrane signaling in bacteria.

Transcriptomic Insight in the Control of Legume Root Secondary Infection by the Sinorhizobium meliloti Transcriptional Regulator Clr.

The cAMP-dependent transcriptional regulator Clr of Sinorhizobium meliloti regulates the overall number of infection events on Medicago roots by a so-far unknown mechanism requiring smc02178, a Clr-target gene of unknown function. In order to shed light on the mode of action of Clr on infection and potentially reveal additional biological functions for Clr, we inventoried genomic Clr target genes by transcriptome profiling. We have found that Clr positively controls the synthesis of cAMP-dependent succinoglycan as well as the expression of genes involved in the synthesis of a so-far unknown polysaccharide compound. In addition, Clr activated expression of 24 genes of unknown function in addition to smc02178. Genes negatively controlled by Clr were mainly involved in swimming motility and chemotaxis. Functional characterization of two novel Clr-activated genes of unknown function, smb20495 and smc02177, showed that their expression was activated by the same plant signal as smc02178 ex planta. In planta, however, symbiotic expression of smc02177 proved independent of clr. Both smc02177 and smb20495 genes were strictly required for the control of secondary infection on M. sativa. None of the three smc02177, smc02178 and smb20495 genes were needed for plant signal perception. Altogether this work provides a refined view of the cAMP-dependent Clr regulon of S. meliloti. We specifically discuss the possible roles of smc02177, smc02178, smb20495 genes and other Clr-controlled genes in the control of secondary infection of Medicago roots.

Bacterial partition complexes segregate within the volume of the nucleoid.

Precise and rapid DNA segregation is required for proper inheritance of genetic material. In most bacteria and archaea, this process is assured by a broadly conserved mitotic-like apparatus in which a NTPase (ParA) displaces the partition complex. Competing observations and models imply starkly different 3D localization patterns of the components of the partition machinery during segregation. Here we use super-resolution microscopies to localize in 3D each component of the segregation apparatus with respect to the bacterial chromosome. We show that Par proteins locate within the nucleoid volume and reveal that proper volumetric localization and segregation of partition complexes requires ATPase and DNA-binding activities of ParA. Finally, we find that the localization patterns of the different components of the partition system highly correlate with dense chromosomal regions. We propose a new mechanism in which the nucleoid provides a scaffold to guide the proper segregation of partition complexes.

Biochemical and functional characterization of SpdA, a 2', 3'cyclic nucleotide phosphodiesterase from Sinorhizobium meliloti.

BACKGROUND: 3', 5'cAMP signaling in Sinorhizobium meliloti was recently shown to contribute to the autoregulation of legume infection. In planta, three adenylate cyclases CyaD1, CyaD2 and CyaK, synthesizing 3', 5'cAMP, together with the Crp-like transcriptional regulator Clr and smc02178, a gene of unknown function, are involved in controlling plant infection. RESULTS: Here we report on the characterization of a gene (smc02179, spdA) at the cyaD1 locus that we predicted to encode a class III cytoplasmic phosphodiesterase.First, we have shown that spdA had a similar pattern of expression as smc02178 in planta but did not require clr nor 3', 5'cAMP for expression.Second, biochemical characterization of the purified SpdA protein showed that, contrary to expectation, it had no detectable activity against 3', 5'cAMP and, instead, high activity against the positional isomers 2', 3'cAMP and 2', 3'cGMP.Third, we provide direct experimental evidence that the purified Clr protein was able to bind both 2', 3'cAMP and 3', 5'cAMP in vitro at high concentration. We further showed that Clr is a 3', 5'cAMP-dependent DNA-binding protein and identified a DNA-binding motif to which Clr binds. In contrast, 2', 3'cAMP was unable to promote Clr specific-binding to DNA and activate smc02178 target gene expression ex planta.Fourth, we have shown a negative impact of exogenous 2', 3'cAMP on 3', 5'cAMP-mediated signaling in vivo. A spdA null mutant was also partially affected in 3', 5'cAMP signaling. CONCLUSIONS: SpdA is a nodule-expressed 2', 3' specific phosphodiesterase whose biological function remains elusive. Circumstantial evidence suggests that SpdA may contribute insulating 3', 5'cAMP-based signaling from 2', 3' cyclic nucleotides of metabolic origin.

Plant-activated bacterial receptor adenylate cyclases modulate epidermal infection in the Sinorhizobium meliloti-Medicago symbiosis.

Legumes and soil bacteria called rhizobia have coevolved a facultative nitrogen-fixing symbiosis. Establishment of the symbiosis requires bacterial entry via root hair infection threads and, in parallel, organogenesis of nodules that subsequently are invaded by bacteria. Tight control of nodulation and infection is required to maintain the mutualistic character of the interaction. Available evidence supports a passive bacterial role in nodulation and infection after the microsymbiont has triggered the symbiotic plant developmental program. Here we identify in Sinorhizobium meliloti, the Medicago symbiont, a cAMP-signaling regulatory cascade consisting of three receptor-like adenylate cyclases, a Crp-like regulator, and a target gene of unknown function. The cascade is activated specifically by a plant signal during nodule organogenesis. Cascade inactivation results in a hyperinfection phenotype consisting of abortive epidermal infection events uncoupled from nodulation. These findings show that, in response to a plant signal, rhizobia play an active role in the control of infection. We suggest that rhizobia may modulate the plant's susceptibility to infection. This regulatory loop likely aims at optimizing legume infection.