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The type IX secretion system (T9SS) is a multiprotein machine distributed in Bacteroidota and responsible for the secretion of various proteins across the outer membrane. Secreted effectors can be either delivered into the medium or anchored to the cell surface. The T9SS is composed of a transenvelope complex consisting of proton-motive force-dependent motors connected to a membrane-associated ring and outer membrane translocons, and a cell-surface anchoring complex that processes effectors once translocated. The T9SS is involved in pathogenesis, metal acquisition, carbohydrate degradation, S-layer biogenesis and gliding motility. The broad spectrum of functions is linked to a highly versatile repertoire of effectors including metallophores, enzymes, toxins and adhesins, that all possess specific signatures to be recruited and transported by the apparatus. This review summarizes the current knowledge on T9SS substrate secretion signals, transport, processing and activities.

Significance The newly described type IX secretion systems (T9SSs) translocate virulence factors and can mediate specialized gliding motility among bacterial pathogens of the Fibrobacteres–Chlorobi–Bacterioidetes superphylum. We visualized the spatial organization of the T9SS in its native context in the Porphorymonas gingivalis cell by cryoelectron tomography. The T9SS exhibits distinct symmetries across the bacterial cell envelope: a cytoplasmic complex requiring PorL and PorM for assembly exhibits 12-fold symmetry; a periplasmic complex composed of PorM exhibits 18-fold symmetry and attaches to a PorKN ring near the outer membrane; and eight Sov translocons are arranged with 8-fold symmetry at the cell surface. The T9SS is the largest of the known bacterial secretion systems and evidently arranges as multiple, independently functioning translocation motors.

Bacteria require membrane fission for both cell division and endospore formation. In Bacillus subtilis, sporulation initiates with an asymmetric division that generates a large mother cell and a smaller forespore that contains only a quarter of its genome. As the mother cell membranes engulf the forespore, a DNA translocase pumps the rest of the chromosome into the small forespore compartment, inflating it due to increased turgor. When the engulfing membrane undergoes fission, the forespore is released into the mother cell cytoplasm. The B. subtilis protein FisB catalyzes membrane fission during sporulation, but the molecular basis is unclear. Here, we show that forespore inflation and FisB accumulation are both required for an efficient membrane fission. Forespore inflation leads to higher membrane tension in the engulfment membrane than in the mother cell membrane, causing the membrane to flow through the neck connecting the two membrane compartments. Thus, the mother cell supplies some of the membrane required for the growth of the membranes surrounding the forespore. The oligomerization of FisB at the membrane neck slows the equilibration of membrane tension by impeding the membrane flow. This leads to a further increase in the tension of the engulfment membrane, promoting its fission through lysis. Collectively, our data indicate that DNA translocation has a previously unappreciated second function in energizing the FisB-mediated membrane fission under energy-limited conditions.\ud \ud

Motile bacteria usually rely on external apparatus like flagella for swimming or pili for twitching. By contrast, gliding bacteria do not rely on obvious surface appendages to move on solid surfaces.Flavobacterium johnsoniaeand other bacteria in the Bacteroidetes phylum use adhesins whose movement on the cell surface supports motility. InF. johnsoniae, secretion and helicoidal motion of the main adhesin SprB are intimately linked and depend on the type IX secretion system (T9SS). Both processes necessitate the proton motive force (PMF), which is thought to fuel a molecular motor that comprises the GldL and GldM cytoplasmic membrane proteins. Here we show thatF. johnsoniaegliding motility is powered by the pH gradient component of the PMF. We further delineate the interaction network between the GldLM transmembrane helices (TMH) and show that conserved glutamate residues in GldL TMH are essential for gliding motility, although having distinct roles in SprB secretion and motion. We then demonstrate that the PMF and GldL trigger conformational changes in the GldM periplasmic domain. We finally show that multiple GldLM complexes are distributed in the membrane suggesting that a network of motors may be present to move SprB along a helical path on the cell surface. Altogether, our results provide evidence that GldL and GldM assemble dynamic membrane channels that use the proton gradient to power both T9SS-dependent secretion of SprB and its motion at the cell surface.

Bacteria require membrane fission for cell division and endospore formation. FisB catalyzes membrane fission during sporulation, but the molecular basis is unclear as it cannot remodel membranes by itself. Sporulation initiates with an asymmetric division that generates a large mother cell and a smaller forespore that contains only 1/4 of its complete genome. As the mother cell membranes engulf the forespore, a DNA translocase pumps the rest of the chromosome into the small forespore compartment, inflating it due to increased turgor. When the engulfing membranes undergo fission, the forespore is released into the mother cell cytoplasm. Here we show that forespore inflation and FisB accumulation are both required for efficient membrane fission. We suggest that high membrane tension in the engulfment membrane caused by forespore inflation drives FisB-catalyzed membrane fission. Collectively our data indicate that DNA-translocation has a previously unappreciated second function in energizing FisB-mediated membrane fission under energy-limited conditions. HIGHLIGHTS- Membrane fission during endospore formation requires rapid forespore inflation by ATP-driven DNA translocation. - Forespore inflation increases the tension of the engulfment and forespore membranes to near lysis tensions. - FisB catalyzes membrane fission, but only if the membrane tension is high. - Membrane fission utilizes chemical energy transduced to mechanical energy during DNA-packing into the forespore.

Penicillin-binding proteins (PBPs) are crucial enzymes of peptidoglycan assembly and targets of β-lactam antibiotics. However, little is known about their regulation. Recently, membrane proteins were shown to regulate the bifunctional transpeptidases/glycosyltransferases aPBPs in some bacteria. However, up to now, regulators of monofunctional transpeptidases bPBPs have yet to be revealed. Here, we propose that TseB could be such a PBP regulator. This membrane protein was previously found to suppress tetracycline sensitivity of a Bacillus subtilis strain deleted for ezrA, a gene encoding a regulator of septation ring formation. In this study, we show that TseB is required for B. subtilis normal cell shape, tseB mutant cells being shorter and wider than wild-type cells. We observed that TseB interacts with PBP2A, a monofunctional transpeptidase. While TseB is not required for PBP2A activity, stability, and localization, we show that the overproduction of PBP2A is deleterious in the absence of TseB. In addition, we showed that TseB is necessary not only for efficient cell wall elongation during exponential phase but also during spore outgrowth, as it was also observed for PBP2A. Altogether, our results suggest that TseB is a new member of the elongasome that regulates PBP2A function during cell elongation and spore germination.

Little is known about mechanisms of membrane fission in bacteria despite their requirement for cytokinesis. The only known dedicated membrane fission machinery in bacteria, FisB, is expressed during sporulation in Bacillus subtilis and is required to release the developing spore into the mother cell cytoplasm. Here we characterized the requirements for FisB-mediated membrane fission. FisB forms mobile clusters of ∼12 molecules that give way to an immobile cluster at the engulfment pole containing ∼40 proteins at the time of membrane fission. Analysis of FisB mutants revealed that binding to acidic lipids and homo-oligomerization are both critical for targeting FisB to the engulfment pole and membrane fission. Experiments using artificial membranes and filamentous cells suggest FisB does not have an intrinsic ability to sense or induce membrane curvature but can bridge membranes. Finally, modeling suggests homo-oligomerization and trans interactions with membranes are sufficient to explain FisB accumulation at the membrane neck that connects the engulfment membrane to the rest of the mother cell membrane during late stages of engulfment. Together, our results show that FisB is a robust and unusual membrane fission protein that relies on homo-oligomerization, lipid-binding and the unique membrane topology generated during engulfment for localization and membrane scission, but surprisingly, not on lipid microdomains, negative-curvature lipids, or curvature-sensing.

Posté dans BioRxiv le 9 mai 2020; We previously linked TSHZ3 haploinsufficiency to autism spectrum disorder (ASD) and showed that embryonic or postnatal Tshz3 deletion in mice results in behavioral traits relevant to the two core domains of ASD, namely social interaction deficits and repetitive behaviors. Here, we provide evidence that cortical projection neurons (CPNs) and striatal cholinergic interneurons (SCINs) are two main and complementary players in the TSHZ3-linked ASD syndrome. We show that in the cerebral cortex, TSHZ3 is expressed in CPNs and in a proportion of GABA interneurons, while not in cholinergic interneurons or glial cells. TSHZ3-expressing cells, which are predominantly SCINs in the striatum, represent a low proportion of neurons in the ascending cholinergic projection system. We then characterized two new conditional knockout (cKO) models generated by crossing Tshz3 flox/flox with Emx1-Cre ( Emx1-cKO ) or Chat-Cre ( Chat-cKO ) mice to decipher the respective role of CPNs and SCINs. Emx1-cKO mice show altered excitatory synaptic transmission onto CPNs and plasticity at corticostriatal synapses, with neither cortical neuron loss nor impaired layer distribution. These animals present social interaction deficits but no repetitive patterns of behavior. Chat-cKO mice exhibit no loss of SCINs but changes in the electrophysiological properties of these interneurons, associated with repetitive patterns of behavior without social interaction deficits. Therefore, dysfunction in either CPNs or SCINs segregates with a distinct ASD behavioral trait. These findings provide novel insights onto the implication of the corticostriatal circuitry in ASD by revealing an unexpected neuronal dichotomy in the biological background of the two core behavioral domains of this disorder.

Magnesium homeostasis is essential for life and depends on magnesium transporters, whose activity and ion selectivity need to be tightly controlled. Rhomboid intramembrane proteases pervade the prokaryotic kingdom, but their functions are largely elusive. Using proteomics, we find that Bacillus subtilis rhomboid protease YqgP interacts with the membrane‐bound ATP‐dependent processive metalloprotease FtsH and cleaves MgtE, the major high‐affinity magnesium transporter in B. subtilis. MgtE cleavage by YqgP is potentiated in conditions of low magnesium and high manganese or zinc, thereby protecting B. subtilis from Mn2+/Zn2+ toxicity. The N‐terminal cytosolic domain of YqgP binds Mn2+ and Zn2+ ions and facilitates MgtE cleavage. Independently of its intrinsic protease activity, YqgP acts as a substrate adaptor for FtsH, a function that is necessary for degradation of MgtE. YqgP thus unites protease and pseudoprotease function, hinting at the evolutionary origin of rhomboid pseudoproteases such as Derlins that are intimately involved in eukaryotic ER‐associated degradation (ERAD). Conceptually, the YqgP‐FtsH system we describe here is analogous to a primordial form of “ERAD” in bacteria and exemplifies an ancestral function of rhomboid‐superfamily proteins., The unprecedented pseudoprotease function of a prokaryotic rhomboid family in degradation of a magnesium transporter member reveals striking analogies to eukaryotic ERAD quality control systems.

International audience; The type VI secretion system (T6SS) is a multiprotein apparatus that injects protein effectors into target cells, hence playing a critical role in pathogenesis and in microbial communities [1, 2, 3, 4]. The T6SS belongs to the broad family of ontractile injection systems (CISs), such as Myoviridae bacteriophages and R-pyocins, that use a spring-like tail to propel a needle loaded with effectors [5, 6]. The T6SS tail comprises an assembly baseplate on which polymerizes a needle, made of stacked Hcp hexamers, tipped by the VgrG-PAAR spike complex and wrapped by the contractile sheath made of TssB and TssC [7, 8, 9, 10, 11, 12, 13]. The T6SS tail is anchored to the cell envelope by a membrane complex that also serves as channel for the passage of the needle upon sheath contraction [14, 15, 16]. In most CISs, the length of the tail sheath is invariable and is usually ensured by a dedicated protein called tape measure protein (TMP) [17, 18, 19, 20, 21, 22]. Here, we show that the length of the T6SS tail is constant in enteroaggregative Escherichia coli cells, suggesting that it is strictly controlled. By overproducing T6SS tail subunits, we demonstrate that component stoichiometry does not participate to the regulation of tail length. The observation of longer T6SS tails when the apparatus is relocalized at the cell pole further shows that tail length is not controlled by a TMP. Finally, we show that tail stops its elongation when in contact with the opposite membrane and thus that T6SS tail length is determined by the cell width.

The type VI secretion system (T6SS) is an injection apparatus that uses a springlike mechanism for effector delivery. The contractile tail is composed of a needle tipped by a sharpened spike and wrapped by the sheath that polymerizes in an extended conformation on the assembly platform, or baseplate. Contraction of the sheath propels the needle and effectors associated with it into target cells. The passage of the needle through the cell envelope of the attacker is ensured by a dedicated trans-envelope channel complex. This membrane complex (MC) comprises the TssJ lipoprotein and the TssL and TssM inner membrane proteins. MC assembly is a hierarchized mechanism in which the different subunits are recruited in a specific order: TssJ, TssM, and then TssL. Once assembled, the MC serves as a docking station for the baseplate. In enteroaggregative Escherichia coli, the MC is accessorized by TagL, a peptidoglycan-binding (PGB) inner membrane-anchored protein. Here, we show that the PGB domain is the only functional domain of TagL and that the N-terminal transmembrane region mediates contact with the TssL transmembrane helix. Finally, we conduct fluorescence microscopy experiments to position TagL in the T6SS biogenesis pathway, demonstrating that TagL is recruited to the membrane complex downstream of TssL and is not required for baseplate docking. IMPORTANCE Bacteria use weapons to deliver effectors into target cells. One of these weapons, called the type VI secretion system (T6SS), could be compared to a nano-spear gun using a springlike mechanism for effector injection. By targeting bacteria and eukaryotic cells, the T6SS reshapes bacterial communities and hijacks host cell defenses. In enteroaggregative Escherichia coli, the T6SS is a multiprotein machine that comprises a cytoplasmic tail and a peptidoglycan-anchored trans-envelope channel. In this work, we show that TagL comprises an N-terminal domain that mediates contact with the channel and a peptidoglycan-binding domain that binds the cell wall. We then determine at which stage of T6SS biogenesis TagL is recruited and how TagL absence impacts the assembly pathway.

Little is known about mechanisms of membrane fission in bacteria despite their requirement for cytokinesis. The only known dedicated membrane fission machinery in bacteria, FisB, is expressed during sporulation in Bacillus subtilis and is required to release the developing spore into the mother cell cytoplasm. Here we characterized the requirements for FisB-mediated membrane fission. FisB forms mobile clusters of ~12 molecules that give way to an immobile cluster at the engulfment pole containing ~40 proteins at the time of membrane fission. Function mutants revealed that binding to acidic lipids and homo-oligomerization are both critical for targeting FisB to the engulfment pole and membrane fission. Our results suggest that FisB is a robust and unusual membrane fission protein that relies on homo-oligomerization, lipid-binding and likely the unique membrane topology generated during engulfment for localization and membrane scission, but surprisingly, not on lipid microdomains or negative-curvature lipids.

International audience; Bacterial division is intimately linked to synthesis and remodeling of the peptidoglycan, a cage-like polymer that surrounds the bacterial cell, providing shape and mechanical resistance. The bacterial division machinery, which is scaffolded by the cytoskeleton protein FtsZ, includes proteins with enzymatic, structural or regulatory functions. These proteins establish a complex network of transient functional and/or physical interactions which preserve cell shape and cell integrity. Cell wall hydrolases required for peptidoglycan remodeling are major contributors to this mechanism. Consistent with this, their deletion or depletion often results in morphological and/or division defects. However, the exact function of most of them remains elusive. In this work, we show that the putative lysozyme activity of the cell wall hydrolase Pmp23 is important for proper morphology and cell division in the opportunistic human pathogen Streptococcus pneumoniae. Our data indicate that active Pmp23 is required for proper localization of the Z-ring and the FtsZ-positioning protein MapZ. In addition, Pmp23 localizes to the division site and interacts directly with the essential peptidoglycan synthase PBP2x. Altogether, our data reveal a new regulatory function for peptidoglycan hydrolases.

The type VI secretion system (T6SS) is a multiprotein weapon used by bacteria to destroy competitor cells. The T6SS contractile sheath wraps an effector-loaded syringe that is injected into the target cell. This tail structure assembles onto the baseplate that is docked to the membrane complex. In enteroaggregative Escherichia coli, TssA plays a central role at each stage of the T6SS assembly pathway by stabilizing the baseplate and coordinating the polymerization of the tail. Here we adapted an assay based on APEX2-dependent biotinylation to identify the proximity partners of TssA in vivo. By using stage-blocking mutations, we define the temporal contacts of TssA during T6SS biogenesis. This proteomic mapping approach also revealed an additional partner of TssA, TagA. We show that TagA is a cytosolic protein tightly associated with the membrane. Analyses of sheath dynamics further demonstrate that TagA captures the distal end of the sheath to stop its polymerization and to maintain it under the extended conformation.

International audience; The central canal along the spinal cord (SC.) and medulla is characterized by the presence of a specific population of neurons that contacts the cerebrospinal fluid (CSF). These medullo-spinal CSF-contacting neurons (CSF-cNs) are identified by the selective expression of the polycystin kidney disease 2-like 1 ionic channel (PKD2L1 or polycystin-L). In adult, they have been shown to express doublecortin (DCX) and Nkx6.1, two markers of juvenile neu-rons along with the neuron-specific nuclear protein (NeuN) typically expressed in mature neurons. They were therefore suggested to remain in a rather incomplete maturation state. The aim of this study was to assess whether such juvenile state is stable in postnatal animals or whether CSF-cNs may reach maturity at older stages than neurons in the par-enchyma. We show, in the cervical SC. and the brainstem that, in relation to age, CSF-cN density declines and that their cell bodies become more distant from the cc, except in its ventral part. Moreover, in adults (from 1 month) by comparison with neonatal mice, we show that CSF-cNs have evolved to a more mature state, as indicated by the increase in the percentage of cells positive for NeuN and of its level of expression. In parallel, CSF-cNs exhibit, in adult, lower DCX immunoreactivity and do not express PSA-NCAM and TUC4, two neurogenic markers. Nevertheless, CSF-cNs still share in adult characteristics of juvenile neurons such as the presence of phospho-CREB and DCX while NeuN expression remained low. This phenotype persists in 12-month-old animals. Thus, despite a pursuit of neuronal maturation during the postnatal period, CSF-cNs retain a durable low differentiated state.

The transcriptional regulator CcpN of Bacillus subtilis has been recently characterized as a repressor of two gluconeogenic genes, gapB and pckA , and of a small noncoding regulatory RNA, sr1 , involved in arginine catabolism. Deletion of ccpN impairs growth on glucose and strongly alters the distribution of intracellular fluxes, rerouting the main glucose catabolism from glycolysis to the pentose phosphate (PP) pathway. Using transcriptome analysis, we show that during growth on glucose, gapB and pckA are the only protein-coding genes directly repressed by CcpN. By quantifying intracellular fluxes in deletion mutants, we demonstrate that derepression of pckA under glycolytic condition causes the growth defect observed in the ccpN mutant due to extensive futile cycling through the pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and pyruvate kinase. Beyond ATP dissipation via this cycle, PckA activity causes a drain on tricarboxylic acid cycle intermediates, which we show to be the main reason for the reduced growth of a ccpN mutant. The high flux through the PP pathway in the ccpN mutant is modulated by the flux through the alternative glyceraldehyde-3-phosphate dehydrogenases, GapA and GapB. Strongly increased concentrations of intermediates in upper glycolysis indicate that GapB overexpression causes a metabolic jamming of this pathway and, consequently, increases the relative flux through the PP pathway. In contrast, derepression of sr1 , the third known target of CcpN, plays only a marginal role in ccpN mutant phenotypes.

CggR belongs to the SorC family of bacterial transcriptional regulators which control the expression of genes and operons involved in carbohydrate catabolism. CggR was first identified in Bacillus subtilis where it represses the gapA operon encoding the five enzymes that catalyze the central part of glycolysis. Here we present a structure/function study demonstrating that the C-terminal region of CggR regulates the DNA binding activity of this repressor in response to binding of a phosphorylated sugar. Molecular modeling of CggR revealed a winged-helix DNA-binding motif followed by a C-terminal domain presenting weak but significant homology with glucosamine-6-phosphate deaminases from the NagB family. In silico ligand screening suggested that the CggR C-terminal domain would bind preferentially bi-phosphorylated compounds, in agreement with previous studies that proposed fructuose-1,6-biphosphate (FBP) as the inducer metabolite. In vitro, FBP was the only sugar compound capable of interfering with CggR cooperative binding to DNA. FBP was also found to protect CggR against trypsin degradation at two arginine residues predicted to reside in a mobile loop forming the active site lid of the NagB enzymes. Replacement of residues predicted to interact with FBP led to mutant CggR with altered repressor activity in vivo but retaining their structural integrity and DNA binding activity in vitro. Interestingly, some of the mutant repressors responded with different specificity towards mono- and di-phospho-fructosides. Based on these results, we propose that the activity of the CggR-like repressors is controlled by a phospho-sugar binding (PSB) domain presenting structural and functional homology with NagB enzymes.

The central glycolytic genes repressor (CggR) controls the transcription of the gapA operon encoding five key glycolytic enzymes in Bacillus subtilis. CggR recognizes a unique DNA target sequence comprising two direct repeats and fructose-1,6-bisphosphate (FBP) is the inducer that negatively controls this interaction. We present here analytical ultracentrifugation and fluorescence anisotropy experiments that demonstrate that CggR binds as a tetramer to the full-length operator DNA in a highly cooperative manner. We also show that CggR binds as a dimer to each direct repeat, the affinity being approximately 100-fold higher for the 3' repeat. In addition, our studies reveal a bimodal effect of FBP on the repressor/operator interaction. At micromolar concentrations, FBP leads to a change in the conformational dynamics of the complex. In the millimolar range, without altering the stoichiometry, FBP leads to a drastic reduction in the affinity and cooperativity of the complex. This bimodal response suggests the existence of two sugar-binding sites in the repressor, a high affinity site at which FBP acts as a structural co-factor and a low affinity site underlying the molecular mechanism of gapA induction.

Summary During sporulation in Bacillus subtilis, the mother cell membranes migrate around the forespore in a phagocytic-like process called engulfment. Developmental gene expression requires the successful completion of this key morphological event. Here we show that perturbations to engulfment block the accumulation of proteins secreted into the space between the mother cell and forespore membranes. Our data support a model in which engulfment defects cause the proteolytic clearance of these secreted proteins. Importantly, we show that this degradative response is reversible; once proper engulfment is restored, secreted proteins again accumulate. In particular, we have found that the forespore signalling protein SpoIVB fails to accumulate when engulfment is impaired and, as a result, late mother cell gene expression under the control of σK is blocked. If engulfment is restored, SpoIVB accumulates and cell–cell signalling resumes. Thus, this degradative pathway functions like a developmental checkpoint ensuring that mother cell gene expression does not commence unless morphogenesis proceeds normally.

The Bacillus subtilis genome contains several sets of paralogs. An extreme case is the four putative malic enzyme genes maeA , malS , ytsJ , and mleA. maeA was demonstrated to encode malic enzyme activity, to be inducible by malate, but also to be dispensable for growth on malate. We report systematic experiments to test whether these four genes ensure backup or cover different functions. Analysis of single- and multiple-mutant strains demonstrated that ytsJ has a major physiological role in malate utilization for which none of the other three genes could compensate. In contrast, maeA , malS , and mleA had distinct roles in malate utilization for which they could compensate one another. The four proteins exhibited malic enzyme activity; MalS, MleA, and MaeA exhibited 4- to 90-fold higher activities with NAD + than with NADP + . YtsJ activity, in contrast, was 70-fold higher with NADP + than with NAD + , with K m values of 0.055 and 2.8 mM, respectively. lacZ fusions revealed strong transcription of ytsJ , twofold higher in malate than in glucose medium, but weak transcription of malS and mleA . In contrast, mleA was strongly transcribed in complex medium. Metabolic flux analysis confirmed the major role of YtsJ in malate-to-pyruvate interconversion. While overexpression of the NADP-dependent Escherichia coli malic enzyme MaeB did not suppress the growth defect of a ytsJ mutant on malate, overexpression of the transhydrogenase UdhA from E. coli partially suppressed it. These results suggest an additional physiological role of YtsJ beyond that of malate-to-pyruvate conversion.

During the process of spore formation in Bacillus subtilis many membrane proteins localize to the sporulation septum where they play key roles in morphogenesis and cell-cell signalling. However, the mechanism by which these proteins are anchored at this site is not understood. In this report we have defined the localization requirements for the mother-cell membrane protein SpoIVFA, which anchors a signalling complex in the septal membrane on the mother cell side. We have identified five proteins (SpoIID, SpoIIP, SpoIIM, BofA and SpoIIIAH) synthesized in the mother cell under the control of sigma(E) and one protein (SpoIIQ) synthesized in the forespore under the control of sigma(F) that are all required for the proper localization of SpoIVFA. Surprisingly, these proteins appear to have complementary and overlapping anchoring roles suggesting that SpoIVFA is localized in the septal membrane through a web of protein interactions. Furthermore, we demonstrate a direct biochemical interaction between the extracellular domains of two of the proteins required to anchor SpoIVFA: the forespore protein SpoIIQ and the mother-cell protein SpoIIIAH. This result supports the idea that the web of interactions that anchors SpoIVFA is itself held in the septal membrane through a zipper-like interaction across the sporulation septum. Importantly, our results suggest that a second mechanism independent of forespore proteins participates in anchoring SpoIVFA. Finally, we show that the dynamic localization of SpoIIQ in the forespore is impaired in the absence of SpoIVFA but not SpoIIIAH. Thus, a complex web of interactions among mother cell and forespore proteins is responsible for static and dynamic protein localization in both compartments of the sporangium. We envision that this proposed network is involved in anchoring other sporulation proteins in the septum and that protein networks with overlapping anchoring capacity is a feature of protein localization in all bacteria.

Summary Glycolysis is one of the best and widely conserved general metabolic pathways. Bacillus subtilis enzymes catalysing the central part of glycolysis, gathering the steps of interconversion of the triose phosphates from dihydroxyacetone-phosphate to phosphoenolpyruvate, are encoded by five genes, gapA , pgk , tpi , pgm and eno . They are transcribed in a hexacistronic operon together with cggR , the first cistron, encoding the repressor of this gapA operon. Using deletion analysis, we have localized the CggR operator between the promoter and the first gene of the operon. CggR was purified and used in gel mobility shift assays and DNase I footprinting experiments to delimit its target sequence. Site-directed mutagenesis and in vivo tests demonstrated that it consists of two direct-repeats (CGGGACN 6 TGTCN 4 CGGGACN 6 TG TC). Sequence analysis and transcriptome comparison of a wild-type and a cggR mutant strain strongly suggested that CggR regulates only the gapA operon. The presence of glycolytic carbon sources induces expression of the gapA operon. Genetic experiments allowed us to identify the metabolic steps required for the formation of the CggR effector. In vitro experiments with the suggested candidates allowed us to demonstrate that fructose-1,6-biphosphate (FBP) acts as an inhibitor of CggR DNA-binding activity (10 mM for full inhibition). FBP is thus the major signal for both CcpA-dependent catabolite repression (or activation) and activation of the central glycolytic genes. Genomic sequence comparisons suggest that these results can apply to numerous low-G + C, Grampositive bacterial species.

This chapter talks about the two cells that comprise the sporangium during spore formation in Bacillus subtilis. Spore formation in B. subtilis has served as an important model for cell-cell signaling in bacteria. The conversation between the forespore and mother cell provides insight into how and why cells communicate and highlights the diversity of ways in which organisms transduce information across their membranes. It is clear that there are three signal transduction pathways between the mother cell and forespore that ensure that gene expression in one compartment is linked to gene expression in the other throughout the sporulation process. The first signal transduction pathway between the forespore and mother cell has as input σF activity in the forespore and as output the activation of σE in the mother cell. Dissection of the molecular mechanisms of R-mediated GA activation and GA-mediated pro-σE processing promises to reveal general principles of how information can be transduced across a lipid bilayer. The second signal transduction pathway, the activation of σG in the forespore under the control of σE in the mother cell, has been the most refractory to genetic and molecular dissection. The third and final signal transduction pathway, the activation of σK in the mother cell under the control of σG in the forespore, is the most well understood of the three. All three of these signaling pathways have served as powerful models for studying cell-cell signaling in bacteria.

13 páginas, 9 figuras, 3 tablas -- PAGS nros. 14996-15008, CggR is the transcriptional repressor of the gapA operon encoding central glycolytic enzymes in Bacillus subtilis. Recently, a detailed mechanistic characterization of gapA induction revealed that the binding of fructose-1,6-bisphosphate (FBP) to a low affinity site on CggR (Kd > 100 μM) is responsible for repressor release from the DNA. In addition, this prior work demonstrated that FBP binds to a second high affinity site on the repressor, causing a conformational change in the CggR/DNA complexes, but with no consequence on CggR affinity for its operator DNA. In the present study we have thoroughly analyzed the structural and thermodynamic consequences of FBP binding to CggR. Results of fluorescence anisotropy titrations, calorimetry and limited proteolysis confirm the existence in CggR of a high affinity site for FBP, with a Kd of around 6 μM. Using analytical size-exclusion chromatography, ultracentrifugation as well as fluorescence correlation spectroscopy (FCS) and pressure perturbation, we show that FBP binding at this site reduces the size of the CggR oligomers and induces conformational changes that stabilize the dimer against denaturation. Hence, FBP has a dual role on CggR structure and regulatory function. In addition to acting as an inducer of transcription at the low affinity site, FBP bound to the high affinity site acts as a structural cofactor for the repressor, with profound effects on its quaternary structure as well as on its conformational dynamics and stability. This high affinity FBP site apparently evolved from the sugar substrate binding site of homologous enzymes

The peptidoglycan (PG) layers surrounding bacterial cells play an important role in determining cell shape. The machinery controlling when and where new PG is made is not understood, but is proposed to involve interactions between bacterial actin homologs such as Mbl, which forms helical cables within cells, and extracellular multiprotein complexes that include penicillin-binding proteins. It has been suggested that labeled antibiotics that bind to PG precursors may be useful for imaging PG to help determine the genes that control the biosynthesis of this polymer. Here, we compare the staining patterns observed in Bacillus subtilis using fluorescent derivatives of two PG-binding antibiotics, vancomycin and ramoplanin. The staining patterns for both probes exhibit a strong dependence on probe concentration, suggesting antibiotic-induced perturbations in PG synthesis. Ramoplanin probes may be better imaging agents than vancomycin probes because they yield clear staining patterns at concentrations well below their minimum inhibitory concentrations. Under some conditions, both ramoplanin and vancomycin probes produce helicoid staining patterns along the cylindrical walls of B. subtilis cells. This sidewall staining is observed in the absence of the cytoskeletal protein Mbl. Although Mbl plays an important role in cell shape determination, our data indicate that other proteins control the spatial localization of the biosynthetic complexes responsible for new PG synthesis along the walls of B. subtilis cells.

During the process of spore formation in Bacillus subtilis many membrane proteins localize to the sporulation septum where they play key roles in morphogenesis and cell-cell signalling. However, the mechanism by which these proteins are anchored at this site is not understood. In this report we have defined the localization requirements for the mother-cell membrane protein SpoIVFA, which anchors a signalling complex in the septal membrane on the mother cell side. We have identified five proteins (SpoIID, SpoIIP, SpoIIM, BofA and SpoIIIAH) synthesized in the mother cell under the control of sigma(E) and one protein (SpoIIQ) synthesized in the forespore under the control of sigma(F) that are all required for the proper localization of SpoIVFA. Surprisingly, these proteins appear to have complementary and overlapping anchoring roles suggesting that SpoIVFA is localized in the septal membrane through a web of protein interactions. Furthermore, we demonstrate a direct biochemical interaction between the extracellular domains of two of the proteins required to anchor SpoIVFA: the forespore protein SpoIIQ and the mother-cell protein SpoIIIAH. This result supports the idea that the web of interactions that anchors SpoIVFA is itself held in the septal membrane through a zipper-like interaction across the sporulation septum. Importantly, our results suggest that a second mechanism independent of forespore proteins participates in anchoring SpoIVFA. Finally, we show that the dynamic localization of SpoIIQ in the forespore is impaired in the absence of SpoIVFA but not SpoIIIAH. Thus, a complex web of interactions among mother cell and forespore proteins is responsible for static and dynamic protein localization in both compartments of the sporangium. We envision that this proposed network is involved in anchoring other sporulation proteins in the septum and that protein networks with overlapping anchoring capacity is a feature of protein localization in all bacteria.

A transcriptome comparison of a wild-typeBacillus subtilisstrain growing under glycolytic or gluconeogenic conditions was performed. In particular, it revealed that theywkAgene, one of the four paralogues putatively encoding a malic enzyme, was more transcribed during gluconeogenesis. Using alacZreporter fusion to theywkApromoter, it was shown thatywkAwas specifically induced by external malate and not subject to glucose catabolite repression. Northern analysis confirmed this expression pattern and demonstrated thatywkAis cotranscribed with the downstreamywkBgene. TheywkAgene product was purified and biochemical studies demonstrated its malic enzyme activity, which was 10-fold higher with NAD than with NADP (kcat/Km102 and 10 s−1 mM−1, respectively). However, physiological tests with single and multiple mutant strains affected inywkAand/or inywkAparalogues showed thatywkAdoes not contribute to efficient utilization of malate for growth. Transposon mutagenesis allowed the identification of the uncharacterized YufL/YufM two-component system as being responsible for the control ofywkAexpression. Genetic analysis andin vitrostudies with purified YufM protein showed that YufM binds just upstream ofywkApromoter and activatesywkAtranscription in response to the presence of malate in the extracellular medium, transmitted by YufL.ywkAandyufL/yufMcould thus be renamedmaeAformalicenzyme andmalK/malRformalatekinase sensor/malate responseregulator, respectively.

Glycolysis is one of the best and widely conserved general metabolic pathways. Bacillus subtilis enzymes catalysing the central part of glycolysis, gathering the steps of interconversion of the triose phosphates from dihydroxyacetone-phosphate to phosphoenolpyruvate, are encoded by five genes, gapA, pgk, tpi, pgm and eno. They are transcribed in a hexacistronic operon together with cggR, the first cistron, encoding the repressor of this gapA operon. Using deletion analysis, we have localized the CggR operator between the promoter and the first gene of the operon. CggR was purified and used in gel mobility shift assays and DNase I footprinting experiments to delimit its target sequence. Site-directed mutagenesis and in vivo tests demonstrated that it consists of two direct-repeats (CGGGACN6TGTCN4CGGGACN6TG TC). Sequence analysis and transcriptome comparison of a wild-type and a cggR mutant strain strongly suggested that CggR regulates only the gapA operon. The presence of glycolytic carbon sources induces expression of the gapA operon. Genetic experiments allowed us to identify the metabolic steps required for the formation of the CggR effector. In vitro experiments with the suggested candidates allowed us to demonstrate that fructose-1,6-biphosphate (FBP) acts as an inhibitor of CggR DNA-binding activity (10 mM for full inhibition). FBP is thus the major signal for both CcpA-dependent catabolite repression (or activation) and activation of the central glycolytic genes. Genomic sequence comparisons suggest that these results can apply to numerous low-G+C, Gram-positive bacterial species.

Super-resolution and fluctuation microscopy in a model DNA-segregation system reveal the architecture and assembly mechanism of the motor responsible for DNA translocation during bacterial cell division., ATP-fuelled molecular motors are responsible for rapid and specific transfer of double-stranded DNA during several fundamental processes, such as cell division, sporulation, bacterial conjugation, and viral DNA transport. A dramatic example of intercompartmental DNA transfer occurs during sporulation in Bacillus subtilis, in which two-thirds of a chromosome is transported across a division septum by the SpoIIIE ATPase. Here, we use photo-activated localization microscopy, structured illumination microscopy, and fluorescence fluctuation microscopy to investigate the mechanism of recruitment and assembly of the SpoIIIE pump and the molecular architecture of the DNA translocation complex. We find that SpoIIIE assembles into ∼45 nm complexes that are recruited to nascent sites of septation, and are subsequently escorted by the constriction machinery to the center of sporulation and division septa. SpoIIIE complexes contain 47±20 SpoIIIE molecules, a majority of which are assembled into hexamers. Finally, we show that directional DNA translocation leads to the establishment of a compartment-specific, asymmetric complex that exports DNA. Our data are inconsistent with the notion that SpoIIIE forms paired DNA conducting channels across fused membranes. Rather, our results support a model in which DNA translocation occurs through an aqueous DNA-conducting pore that could be structurally maintained by the divisional machinery, with SpoIIIE acting as a checkpoint preventing membrane fusion until completion of chromosome segregation. Our findings and proposed mechanism, and our unique combination of innovating methodologies, are relevant to the understanding of bacterial cell division, and may illuminate the mechanisms of other complex machineries involved in DNA conjugation and protein transport across membranes., Author Summary Molecular motors are implicated in myriad cellular processes, notably in the transcription, replication, and segregation of DNA. Segregation or packaging of DNA is essential for production of viable viral particles, proper division of bacterial cells, and production of spores. A dramatic example of this process occurs during sporulation in Bacillus subtilis, in which a large proportion of the chromosome is actively transferred across a division septum by the SpoIIIE motor protein. Here, we use advanced microscopy methods to study the mechanism of recruitment and assembly of the SpoIIIE pump and the architecture of its complex with DNA. We found that SpoIIIE complexes are recruited before the beginning of cell division, and are subsequently escorted by the constriction machinery to the center of the septum. We show that the directionality of DNA transport by SpoIIIE results in the establishment of an asymmetric complex that exports DNA into the nascent spore. Our data are inconsistent with previous models that predicted the formation of a membrane-spanning DNA-conducting channel. Instead, the greater resolution afforded by our approach supports a model in which DNA movement occurs through an open pore structurally maintained by the division apparatus, with SpoIIIE working as a checkpoint preventing membrane fusion until completion of chromosome segregation. Our findings and proposed model may illuminate the mechanisms of other complex machineries involved in DNA conjugation and protein transport across membranes.

In Bacillus subtilis, the transcription of the gapA operon, including the genes of the central part of glycolysis, is stimulated during growth on glycolytic carbon sources. Our in vivo and in vitro studies of CggR, the repressor that controls this stimulation, have demonstrated on one hand that it is able to bind a long single DNA sequence made of two directly repeated motifs (CGGGACN6TGTCN4CGGGACN6TGTC) and located between the promoter and the translation codon of the cggr-gapA operon and on the other hand that its activity is modulated by fructose-1,6-bisphosphate. Sequence analysis and array hybridization indicated that CggR, which is highly conserved in Gram positive bacteria and defines a subfamily of the SorC/DeoR family of transcriptional regulators, is dedicated to the control of the glycolytic genes. A collaboration with structural biologists (CBS, Montpellier) has been engaged to further study this prototype of a not well known sub-family of transcriptional regulators. Two groups of paralogous genes have been detected on Bacillus subtilis chromosome (ywkA et malS, ytsJ and mleA) which products are homologous to malic enzymes. Global transcriptome analysis of a wild-type strain in response to growth in the presence of glucose or malate as sole carbon source has revealed that ywkA expression is induced in the presence of malate. Our experiments have demonstrated that ywkA encodes a NAD-dependent malic enzyme and that its expression is specifically induced by malate and is not subjected to carbon catabolite repression. Moreover, the YufL/YufM two component system has been demonstrated to directly activate ywkA transcription in response to the presence of malate. However, YwkA is not required for efficient growth on malate as sole carbon source. Actually, transcriptome study using macroarray technique is available for our laboratory. Hence, we have started the global comparison of genes expression profile in response to different carbon sources., Chez Bacillus subtilis, la transcription de l'opéron gapA, comprenant les gènes de la partie centrale de la glycolyse, est stimulée en présence de sources de carbone glycolytiques. Nos études in vivo et in vitro de CggR, le répresseur qui contrôle cette stimulation, ont démontré d'une part que celui-ci a la capacité de se lier à une séquence d'ADN inhabituellement longue, consistant en une répétition directe de deux motifs (CGGGACN6TGTCN4CGGGACN6TGTC) et située entre le promoteur et le codon d'initiation de l'opéron cggR-gapA, et d'autre part que son activité est inhibée par le fructose-1,6-biphosphate. Des analyses de séquence et des expériences de transcriptome ont indiqué que CggR, qui est très conservé chez les bactéries à Gram positif et qui définit une sous-famille de la famille de régulateurs transcriptionnels SorC/DeoR, est spécialisé dans le contrôle des gènes de la glycolyse. Ainsi, une collaboration a été engagée avec des structuralistes (CBS, Montpellier) pour aller plus loin dans la connaissance de ce prototype d'une famille encore peu connue de régulateurs. Deux paires de gènes paralogues ont été détectés dans le génome (ywkA et malS, ytsJ et mleA) dont les produits sont homologues à des enzymes maliques. L'analyse transcriptomique globale d'une souche sauvage cultivée en présence de glucose ou de malate comme seule source de carbone montre que l'expression d'ywkA est induite en présence de malate. En collaboration avec l'équipe de Y. Fujita, nous avons montré qu'ywkA codait bien une enzyme malique NADdépendante dont l'expression est spécifiquement induite par le malate extracellulaire et insensible à la répression catabolique. De plus, nous avons montré que le système à deux composants YufL-YufM active directement la transcription d'ywkA en présence de malate. Cependant, YwkA n'est pas requis pour la croissance en présence de malate comme seule source de carbone. La technique d'analyse du transcriptome au moyen de membranes à haute densité est maintenant acquise au laboratoire. Nous avons commencé à mettre à profit cet outil pour une étude globale de l'expression génique en fonction de différentes sources de carbone.