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Nutrients availability is the sinews of the war for single microbial cells, driving growth and cell cycle progression. Therefore, coordinating cellular processes with nutrients availability is crucial, not only to survive upon famine or fluctuating conditions but also to rapidly thrive and colonize plentiful environments. While metabolism is traditionally seen as a set of chemical reactions taking place in cells to extract energy and produce building blocks from available nutrients, numerous connections between metabolic pathways and cell cycle phases have been documented. The few regulatory systems described at the molecular levels show that regulation is mediated either by a second messenger molecule or by a metabolite and/or a metabolic enzyme. In the latter case, a secondary moonlighting regulatory function evolved independently of the primary catalytic function of the enzyme. In this review, we summarize our current understanding of the complex cross-talks between metabolism and cell cycle in bacteria.

Stringent factors orchestrate bacterial cell reprogramming through increasing the level of the alarmones (p)ppGpp. In Beta- and Gammaproteobacteria, SpoT hydrolyzes (p)ppGpp to counteract the synthetase activity of RelA. However, structural information about how SpoT controls the levels of (p)ppGpp is missing. Here we present the crystal structure of the hydrolase-only SpoT from Acinetobacter baumannii and uncover the mechanism of intramolecular regulation of ‘long’-stringent factors. In contrast to ribosome-associated Rel/RelA that adopt an elongated structure, SpoT assumes a compact τ-shaped structure in which the regulatory domains wrap around a Core subdomain that controls the conformational state of the enzyme. The Core is key to the specialization of long RelA-SpoT homologs toward either synthesis or hydrolysis: the short and structured Core of SpoT stabilizes the τ-state priming the hydrolase domain for (p)ppGpp hydrolysis, whereas the longer, more dynamic Core domain of RelA destabilizes the τ-state priming the monofunctional RelA for efficient (p)ppGpp synthesis. [Figure not available: see fulltext.]. We are grateful to the Protein Expertise Platform at Umeå University for constructing plasmids.

In the model organism Caulobacter crescentus, a network of two-component systems involving the response regulators CtrA, DivK and PleD coordinate cell cycle progression with differentiation. Active phosphorylated CtrA prevents chromosome replication in G1 cells while simultaneously regulating expression of genes required for morphogenesis and development. At the G1-S transition, phosphorylated DivK (DivK~P) and PleD (PleD~P) accumulate to indirectly inactivate CtrA, which triggers DNA replication initiation and concomitant cellular differentiation. The phosphatase PleC plays a pivotal role in this developmental program by keeping DivK and PleD phosphorylation levels low during G1, thereby preventing premature CtrA inactivation. Here, we describe CckN as a second phosphatase akin to PleC that dephosphorylates DivK~P and PleD~P in G1 cells. However, in contrast to PleC, we do not detect kinase activity with CckN. The effects of CckN inactivation are largely masked when PleC is present, but become evident when PleC and DivJ, the major kinase for DivK and PleD, are absent. Accordingly, mild overexpression of cckN restores most phenotypic defects of a pleC null mutant. We also show that CckN and PleC are proteolytically degraded in a ClpXP-dependent way well before the onset of the S phase. Surprisingly, known ClpX adaptors are dispensable for PleC and CckN proteolysis, suggesting the existence of adaptors specifically involved in proteolytic removal of cell cycle regulators. Since cckN expression is induced in stationary phase, depending on the stress alarmone (p)ppGpp, we propose that CckN acts as an auxiliary factor responding to environmental stimuli to modulate CtrA activity under suboptimal conditions.ImportanceTwo-component signal transduction systems are widely used by bacteria to sense environmental signals and respond accordingly by modulating various cellular processes, such as cell cycle progression. In Caulobacter crescentus, PleC acts as a phosphatase that indirectly protects the response regulator CtrA from premature inactivation during the G1 phase of the cell cycle. Here, we provide genetic and biochemical evidence that PleC is seconded by another phosphatase, CckN. The activity of PleC and CckN phosphatases is restricted to G1 phase since both proteins are timely degraded by proteolysis just before the G1-S transition. This degradation requires new proteolytic adaptors as well as an unsuspected N-terminal motif for CckN. Our work illustrates a typical example of redundant functions between two-component proteins.

Bacteria use dedicated mechanisms to respond adequately to fluctuating environments and to optimize their chances of survival in harsh conditions. One of the major stress responses used by virtually all bacteria relies on the sharp accumulation of an alarmone, the guanosine penta- or tetra-phosphate commonly referred to as (p)ppGpp. Under stressful conditions, essentially nutrient starvation, these second messengers completely reshape the metabolism and physiology by coordinately modulating growth, transcription, translation and cell cycle. As a central regulator of bacterial stress response, the alarmone is also involved in biofilm formation, virulence, antibiotics tolerance and resistance in many pathogenic bacteria. Intracellular concentrations of (p)ppGpp are determined by a highly conserved and widely distributed family of proteins called RelA-SpoT Homologs (RSH). Recently, several studies uncovering mechanisms that regulate RSH activities have renewed a strong interest in this field. In this review, we outline the diversity of the RSH protein family as well as the molecular devices used by bacteria to integrate and transform environmental cues into intracellular (p)ppGpp levels., The bacterial alarmone (p)ppGpp used to face stressful conditions is synthesized and hydrolysed by a highly conserved and widely distributed family of enzymes (RSH) whose activity is regulated by stress-sensitive mechanisms.

The class Alphaproteobacteria includes Gram-negative free-living, symbiotic and obligate intracellular bacteria, as well as important plant, animal and human pathogens. Recent work has established the key antagonistic roles that phosphorylated guanosines, cyclic-di-GMP (c-di-GMP) and the alarmones guanosine tetraphosphate and guanosine pentaphosphate (collectively referred to as (p)ppGpp), have in the regulation of the cell cycle in these bacteria. In this Review, we discuss the insights that have been gained into the regulation of the initiation of DNA replication and cytokinesis by these second messengers, with a particular focus on the cell cycle of Caulobacter crescentus. We explore how the fluctuating levels of c-di-GMP and (p)ppGpp during the progression of the cell cycle and under conditions of stress control the synthesis and proteolysis of key regulators of the cell cycle. As these signals also promote bacterial interactions with host cells, the enzymes that control (p)ppGpp and c-di-GMP are attractive antibacterial targets.

Sensory and regulatory domains allow bacteria to adequately respond to environmental changes. The regulatory ACT (Aspartokinase, Chorismate mutase and TyrA) domains are mainly found in metabolicrelated proteins as well as in long (p)pp Gpp synthetase/hydrolase enzymes. Here, we investigate the functional role of the ACT domain of SpoT, the only (p)pp Gpp synthetase/hydrolase of Caulobacter crescentus. We show that SpoT requires the ACT domain to efficiently hydrolyze (p)pp Gpp. In addition, our in vivo and in vitro data show that the phosphorylated version of EIIANtr (EIIANtr∼P) interacts directly with the ACT and inhibits the hydrolase activity of SpoT. Finally, we highlight the conservation of the ACT-dependent interaction between EIIANtr∼P and SpoT/Rel along with the phosphotransferase system (PTSNtr)-dependent regulation of (p)pp Gpp accumulation upon nitrogen starvation in Sinorhizobium meliloti, a plant-associated α-proteobacterium. Thus, thiswork suggests that α-proteobacteria might have inherited from a common ancestor, a PTSNtr dedicated to modulate (p)pp Gpp levels in response to nitrogen availability., SCOPUS: ar.j, info:eu-repo/semantics/published

Afin d’optimiser les chances de survie d’une cellule dans son environnement, le métabolisme et le cycle cellulaire doivent nécessairement être interconnectés. En effet, les cellules possèdent des mécanismes leur permettant, d’une part, de vérifier leur état métabolique avant d’initier l’une ou l’autre étape du cycle cellulaire souvent énergivore et, d’autre part, d’achever certaines étapes du cycle cellulaire avant d’éventuellement modifier leur métabolisme. Parce que les bactéries ne dérogent pas à cette règle, un nombre croissant d’exemples de connexions entre le métabolisme et le cycle cellulaire a émergé ces dernières années. L’identification d’enzymes métaboliques comme messagers coordonnant métabolisme et cycle cellulaire, permet d’ajouter une nouvelle dimension aux cartes métaboliques. La très grande conservation de ces mêmes cartes métaboliques, des bactéries jusqu’à l’homme, permet également d’imaginer des recherches sur les cellules tumorales qui ont un cycle cellulaire débridé et un appétit vorace., To increase chances for a cell to survive in its natural environment, metabolism and cell cycle necessarily have to be interconnected. Indeed, cells need, on the one hand, to check their metabolic status before initiating a cell cycle step often energy-consuming, and, on the other hand, to complete some cell cycle steps before modifying their metabolism. Because bacteria do not differ from the rules, an increasing number of examples of connection between metabolism and cell cycle emerged these last years. Identifying metabolic enzymes as messengers coordinating metabolism and cell cycle allows the addition of another dimension to metabolic maps. Likewise, the very high conservation of these metabolic maps, from bacteria to human, allows inspiring research on tumor cells that are known to have an unrestrained cell cycle and a voracious appetite.

Sensory and regulatory domains allow bacteria to adequately respond to environmental changes. The regulatory ACT domains are mainly found in metabolic-related proteins as well as in long (p)ppGpp synthetase/hydrolase (SD/HD) enzymes. Here, we investigate the functional role of the ACT domain of SpoT, the only (p)ppGpp SD/HD ofCaulobacter crescentus. We show that SpoT requires the ACT domain to hydrolyse ppGpp in an efficient way. In addition, ourin vivoandin vitrodata show that the phosphorylated version of EIIANtr(EIIANtr~P) interacts directly with the ACT to inhibit the hydrolase activity of SpoT. Finally, we highlight the conservation of the ACT-dependent interaction between EIIANtr~P and SpoT/Rel along with the PTSNtr-dependent regulation of (p)ppGpp accumulation upon nitrogen starvation inSinorhizobium meliloti, a plant-associated α-proteobacterium. Thus, this work suggests that α-proteobacteria might have inherited from a common ancestor, a PTSNtrdedicated to modulate (p)ppGpp levels.

Type II toxin-antitoxin (TA) systems are generally composed of two genes organized in an operon, encoding a labile antitoxin and a stable toxin. They were first discovered on plasmids where they contribute to plasmid stability by a phenomenon denoted as 'addiction', and subsequently in bacterial chromosomes. To discover novel families of antitoxins and toxins, we developed a bioinformatics approach based on the 'guilt by association' principle. Extensive experimental validation in Escherichia coli of predicted antitoxins and toxins increased significantly the number of validated systems and defined novel toxin and antitoxin families. Our data suggest that toxin families as well as antitoxin families originate from distinct ancestors that were assembled multiple times during evolution. Toxin and antitoxin families found on plasmids tend to be promiscuous and widespread, indicating that TA systems move through horizontal gene transfer. We propose that due to their addictive properties, TA systems are likely to be maintained in chromosomes even though they do not necessarily confer an advantage to their bacterial hosts. Therefore, addiction might play a major role in the evolutionary success of TA systems both on mobile genetic elements and in bacterial chromosomes., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

Type II toxin-antitoxin (TA) systems are considered as protein pairs in which a specific toxin is associated with a specific antitoxin. We have identified a novel antitoxin family (paaA) that is associated with parE toxins. The paaA-parE gene pairs form an operon with a third component (paaR) encoding a transcriptional regulator. Two paralogous paaR-paaA-parE systems are found in E. coli O157:H7. Deletions of the paaA-parE pairs in O157:H7 allowed us to show that these systems are expressed in their natural host and that PaaA antitoxins specifically counteract toxicity of their associated ParE toxin. For the paaR2-paaA2-parE2 system, PaaR2 and Paa2-ParE2 complex are able to regulate the operon expression and both are necessary to ensure complete repression. The paaR2-paaA2-parE2 system mediates ClpXP-dependent post-segregational killing. The PaaR2 regulator appears to be essential for this function, most likely by maintaining an appropriate antitoxin : toxin ratio in steady-state conditions. Ectopic overexpression of ParE2 is bactericidal and is not resuscitated by PaaA2 expression. ParE2 colocalizes with the nucleoid, while it is diffusely distributed in the cytoplasm when PaaA2 is coexpressed. This indicates that ParE2 interacts with DNA-gyrase cycling on DNA and that coexpression of PaaA2 antitoxin sequesters ParE2 away from its target by protein-protein complex formation.

Prior to initiate energy-consuming processes, such as DNA replication or cell division, cells need to evaluate their metabolic status. We have recently identified and characterized a new connection between metabolism and cell division in the α-proteobacterium Caulobacter crescentus. We showed that an NAD-dependent glutamate dehydrogenase (GdhZ) coordinates growth with cell division according to its enzymatic activity. Here we report the conserved role of GdhZ in controlling cell division in another α-proteobacterium, the facultative intracellular pathogen Brucella abortus. We also discuss the importance of amino acids as a main carbon source for α-proteobacteria.

Many organisms use polar localization of signalling proteins to control developmental events in response to completion of asymmetric cell division. Asymmetric division was recently reported for Brucella abortus, a class III facultative intracellular pathogen generating two sibling cells of slightly different size. Here we characterize PdhS, a cytoplasmic histidine kinase essential for B. abortus viability and homologous to the asymmetrically distributed PleC and DivJ histidine kinases from Caulobacter crescentus. PdhS is localized at the old pole of the large cell, and after division and growth, the small cell acquires PdhS at its old pole. PdhS may therefore be considered as a differentiation marker as it labels the old pole of the large cell. Moreover, PdhS colocalizes with its paired response regulator DivK. Finally, PdhS is able to localize at one pole in other alpha-proteobacteria, suggesting that a polar structure associating PdhS with one pole is conserved in these bacteria. We propose that a differentiation event takes place after the completion of cytokinesis in asymmetrically dividing alpha-proteobacteria. Altogether, these data suggest that prokaryotic differentiation may be much more widespread than expected.

Coupling cell cycle with nutrient availability is a crucial process for all living cells. But how bacteria control cell division according to metabolic supplies remains poorly understood. Here, we describe a molecular mechanism that coordinates central metabolism with cell division in the α-proteobacterium Caulobacter crescentus. This mechanism involves the NAD-dependent glutamate dehydrogenase GdhZ and the oxidoreductase-like KidO. While enzymatically active GdhZ directly interferes with FtsZ polymerization by stimulating its GTPase activity, KidO bound to NADH destabilizes lateral interactions between FtsZ protofilaments. Both GdhZ and KidO share the same regulatory network to concomitantly stimulate the rapid disassembly of the Z-ring, necessary for the subsequent release of progeny cells. Thus, this mechanism illustrates how proteins initially dedicated to metabolism coordinate cell cycle progression with nutrient availability. Synopsis GdhZ and KidO are metabolic regulators of cell division in Caulobacter crescentus. Once bound to their substrates, both proteins synergistically stimulate cytokinesis by triggering Z-ring disassembly by two complementary mechanisms in late predivisional cells (G2). Caulobacter crescentus modulates cell division according to its metabolic activity. Catalytically active GdhZ stimulates GTPase activity of FtsZ. KidO bound to NADH inhibits lateral interactions between FtsZ protofilaments. KidO and GdhZ cooperate to disassemble the Z-ring. GdhZ and KidO are complementary negative regulators of FtsZ that connect metabolic conditions to cell division in Caulobacter crescentus. GdhZ is an inhibitor of FtsZ polymerization while KidO prevents FtsZ filament bundling in response to nutrient availability.

B. melitensis 16M genome analysis revealed the presence of six putative sigma factor-encoding genes: rpoD , rpoH1 , rpoH2 , rpoE1 , rpoE2 , and rpoN . We mutated all these genes except rpoD . Phenotypic analysis of the mutants reveals that a strain carrying an rpoH2 null mutation (Δ rpoH2 ) is impaired for growth at 21 and 42°C and shows increased sensitivity to hydrogen peroxide. Compared to the wild-type strain, the Δ rpoH2 mutant is attenuated in all virulence models tested. Three other null mutants (Δ rpoH1 , Δ rpoE1 , and Δ rpoE2 mutants) are also defective for survival in mice at 4 weeks postinfection. We also demonstrated that rpoH2 deletion strongly reduces the expression of two major virulence factors in B. melitensis , the type IV secretion system and the flagellum.

The bacteria of the Brucella genus are responsible for a worldwide zoonosis called brucellosis. They belong to the α-proteobacteria group, as many other bacteria that live in close association with a eukaryotic host. Importantly, the Brucellae are mainly intracellular pathogens, and the molecular mechanisms of their virulence are still poorly understood. Using the complete genome sequence of Brucella melitensis, we generated a database of protein-coding open reading frames (ORFs) and constructed an ORFeome library of 3091 Gateway Entry clones, each containing a defined ORF. This first version of the Brucella ORFeome (v1.1) provides the coding sequences in a user-friendly format amenable to high-throughput functional genomic and proteomic experiments, as the ORFs are conveniently transferable from the Entry clones to various Expression vectors by recombinational cloning. The cloning of the Brucella ORFeome v1.1 should help to provide a better understanding of the molecular mechanisms of virulence, including the identification of bacterial protein-protein interactions, but also interactions between bacterial effectors and their host's targets.

Type II toxin-antitoxin (TA) systems are considered as protein pairs in which a specific toxin is associated with a specific antitoxin. We have identified a novel antitoxin family (paaA) that is associated with parE toxins. The paaA-parE gene pairs form an operon with a third component (paaR) encoding a transcriptional regulator. Two paralogous paaR-paaA-parE systems are found in E. coli O157:H7. Deletions of the paaA-parE pairs in O157:H7 allowed us to show that these systems are expressed in their natural host and that PaaA antitoxins specifically counteract toxicity of their associated ParE toxin. For the paaR2-paaA2-parE2 system, PaaR2 and Paa2-ParE2 complex are able to regulate the operon expression and both are necessary to ensure complete repression. The paaR2-paaA2-parE2 system mediates ClpXP-dependent post-segregational killing. The PaaR2 regulator appears to be essential for this function, most likely by maintaining an appropriate antitoxin : toxin ratio in steady-state conditions. Ectopic overexpression of ParE2 is bactericidal and is not resuscitated by PaaA2 expression. ParE2 colocalizes with the nucleoid, while it is diffusely distributed in the cytoplasm when PaaA2 is coexpressed. This indicates that ParE2 interacts with DNA-gyrase cycling on DNA and that coexpression of PaaA2 antitoxin sequesters ParE2 away from its target by protein-protein complex formation.

Twenty Gateway-compatible destination vectors were constructed. The vectors comprise fluorescent and epitope fusion tags, various drug markers, and replication origins that should make them useful for exploring existing microbial ORFeomes. In an attempt to validate several of these vectors, we observed polar and oscillating localization of MinD in Brucella abortus .

Summary Physiological adaptation of intracellular bacteria is critical for timely interaction with eukaryotic host cells. One mechanism of adaptation, the stringent response, is induced by nutrient stress via its effector molecule (p)ppGpp, synthesized by the action of RelA/SpoT homologues. The intracellular pathogen Brucella spp., causative agent of brucellosis, pos- sesses a gene homologous to relA / spoT , named rsh , encoding a (p)ppGpp synthetase as confirmed by het- erologous complementation of a relA mutant of Sinorhizobium meliloti . The Rsh deletion mutants in Brucella suis and Brucella melitensis were character- ized by altered morphology, and by reduced survival under starvation conditions and in cellular and murine models of infection. Most interestingly, we evi- denced that expression of virB , encoding the type IV secretion system, a major virulence factor of Brucella , was Rsh-dependent. All mutant phenotypes, includ- ing lack of VirB proteins, were complemented with the rsh gene of Brucella . In addition, RelA of S. meliloti functionally replaced Brucella Rsh, describing the capacity of a gene from a plant symbiont to restore virulence in a mammalian pathogen. We therefore concluded that in the intramacrophagic environment encountered by Brucella , Rsh might participate in the adaptation of the pathogen to low-nutrient environ- ments, and indirectly in the VirB-mediated formation of the final replicative niche.

The release of an increasing number of complete bacterial genomic sequences allows the evolutionary analysis of processes such as regulatory networks. CtrA is a response regulator of the OmpR subfamily, belonging to a complex regulatory network in the dimorphic bacterium Caulobacter crescentus. It coordinates the cell cycle with an asymmetric division, which is part of the adaptation of Caulobacter to poor-nutrient environments. CtrA is only found in alpha-proteobacteria, a group of bacteria encompassing genera with very distinct lifestyles, including host-associated bacteria. Analyses of CtrA regulatory networks and morphological examinations of some alpha-proteobacteria are presented. Our observations suggest that the core of the CtrA regulation network is conserved and that alpha-proteobacteria divide asymmetrically. We propose that the two daughter cells might be differentiated bacteria, each one displaying specific functions.