An Adaptor Hierarchy Regulates Proteolysis during a Bacterial Cell Cycle.

Regulated protein degradation is essential. The timed destruction of crucial proteins by the ClpXP protease drives cell-cycle progression in the bacterium Caulobacter crescentus. Although ClpXP is active alone, additional factors are inexplicably required for cell-cycle-dependent proteolysis. Here, we show that these factors constitute an adaptor hierarchy wherein different substrates are destroyed based on the degree of adaptor assembly. The hierarchy builds upon priming of ClpXP by the adaptor CpdR, which promotes degradation of one class of substrates and also recruits the adaptor RcdA to degrade a second class of substrates. Adding the PopA adaptor promotes destruction of a third class of substrates and inhibits degradation of the second class. We dissect RcdA to generate bespoke adaptors, identifying critical substrate elements needed for RcdA recognition and uncovering additional cell-cycle-dependent ClpXP substrates. Our work reveals how hierarchical adaptors and primed proteases orchestrate regulated proteolysis during bacterial cell-cycle progression.

The CTPase activity of ParB determines the size and dynamics of prokaryotic DNA partition complexes.

ParB-like CTPases mediate the segregation of bacterial chromosomes and low-copy number plasmids. They act as DNA-sliding clamps that are loaded at parS motifs in the centromere of target DNA molecules and spread laterally to form large nucleoprotein complexes serving as docking points for the DNA segregation machinery. Here, we solve crystal structures of ParB in the pre- and post-hydrolysis state and illuminate the catalytic mechanism of nucleotide hydrolysis. Moreover, we identify conformational changes that underlie the CTP- and parS-dependent closure of ParB clamps. The study of CTPase-deficient ParB variants reveals that CTP hydrolysis serves to limit the sliding time of ParB clamps and thus drives the establishment of a well-defined ParB diffusion gradient across the centromere whose dynamics are critical for DNA segregation. These findings clarify the role of the ParB CTPase cycle in partition complex assembly and function and thus advance our understanding of this prototypic CTP-dependent molecular switch.

Stereoisomer-specific reprogramming of a bacterial flagellin sialyltransferase.

Glycosylation of surface structures diversifies cells chemically and physically. Nucleotide-activated sialic acids commonly serve as glycosyl donors, particularly pseudaminic acid (Pse) and its stereoisomer legionaminic acid (Leg), which decorate eubacterial and archaeal surface layers or protein appendages. FlmG, a recently identified protein sialyltransferase, O-glycosylates flagellins, the subunits of the flagellar filament. We show that flagellin glycosylation and motility in Caulobacter crescentus and Brevundimonas subvibrioides is conferred by functionally insulated Pse and Leg biosynthesis pathways, respectively, and by specialized FlmG orthologs. We established a genetic glyco-profiling platform for the classification of Pse or Leg biosynthesis pathways, discovered a signature determinant of eubacterial and archaeal Leg biosynthesis, and validated it by reconstitution experiments in a heterologous host. Finally, by rewiring FlmG glycosylation using chimeras, we defined two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that either donates Leg or Pse and a specialized flagellin-binding domain that identifies the acceptor.

Adaptive ß-lactam resistance from an inducible efflux pump that is post-translationally regulated by the DjlA co-chaperone.

The acquisition of multidrug resistance (MDR) determinants jeopardizes treatment of bacterial infections with antibiotics. The tripartite efflux pump AcrAB-NodT confers adaptive MDR in the polarized α-proteobacterium Caulobacter crescentus via transcriptional induction by first-generation quinolone antibiotics. We discovered that overexpression of AcrAB-NodT by mutation or exogenous inducers confers resistance to cephalosporin and penicillin (ß-lactam) antibiotics. Combining 2-step mutagenesis-sequencing (Mut-Seq) and cephalosporin-resistant point mutants, we dissected how TipR uses a common operator of the divergent tipR and acrAB-nodT promoter for adaptive and/or potentiated AcrAB-NodT-directed efflux. Chemical screening identified diverse compounds that interfere with DNA binding by TipR or induce its dependent proteolytic turnover. We found that long-term induction of AcrAB-NodT deforms the envelope and that homeostatic control by TipR includes co-induction of the DnaJ-like co-chaperone DjlA, boosting pump assembly and/or capacity in anticipation of envelope stress. Thus, the adaptive MDR regulatory circuitry reconciles drug efflux with co-chaperone function for trans-envelope assemblies and maintenance.

The DUF1013 protein TrcR tracks with RNA polymerase to control the bacterial cell cycle and protect against antibiotics.

How DNA-dependent RNA polymerase (RNAP) acts on bacterial cell cycle progression during transcription elongation is poorly investigated. A forward genetic selection for Caulobacter crescentus cell cycle mutants unearthed the uncharacterized DUF1013 protein (TrcR, transcriptional cell cycle regulator). TrcR promotes the accumulation of the essential cell cycle transcriptional activator CtrA in late S-phase but also affects transcription at a global level to protect cells from the quinolone antibiotic nalidixic acid that induces a multidrug efflux pump and from the RNAP inhibitor rifampicin that blocks transcription elongation. We show that TrcR associates with promoters and coding sequences in vivo in a rifampicin-dependent manner and that it interacts physically and genetically with RNAP. We show that TrcR function and its RNAP-dependent chromatin recruitment are conserved in symbiotic Sinorhizobium sp. and pathogenic Brucella spp Thus, TrcR represents a hitherto unknown antibiotic target and the founding member of the DUF1013 family, an uncharacterized class of transcriptional regulators that track with RNAP during the elongation phase to promote transcription during the cell cycle.

Phenolic Substitution in Fidaxomicin: A Semisynthetic Approach to Antibiotic Activity Across Species.

Fidaxomicin (Fdx) is a natural product antibiotic with potent activity against Clostridioides difficile and other Gram-positive bacteria such as Mycobacterium tuberculosis. Only a few Fdx derivatives have been synthesized and examined for their biological activity in the 50 years since its discovery. Fdx has a well-studied mechanism of action, namely inhibition of the bacterial RNA polymerase. Yet, the targeted organisms harbor different target protein sequences, which poses a challenge for the rational development of new semisynthetic Fdx derivatives. We introduced substituents on the two phenolic hydroxy groups of Fdx and evaluated the resulting trends in antibiotic activity against M. tuberculosis, C. difficile, and the Gram-negative model organism Caulobacter crescentus. As suggested by the target protein structures, we identified the preferable derivatisation site for each organism. The derivative ortho-methyl Fdx also exhibited activity against the Gram-negative C. crescentus wild type, a first for fidaxomicin antibiotics. These insights will guide the synthesis of next-generation fidaxomicin antibiotics.

Integrative and quantitative view of the CtrA regulatory network in a stalked budding bacterium.

The Alphaproteobacteria show a remarkable diversity of cell cycle-dependent developmental patterns, which are governed by the conserved CtrA pathway. Its central component CtrA is a DNA-binding response regulator that is controlled by a complex two-component signaling network, mediating distinct transcriptional programs in the two offspring. The CtrA pathway has been studied intensively and was shown to consist of an upstream part that reads out the developmental state of the cell and a downstream part that integrates the upstream signals and mediates CtrA phosphorylation. However, the role of this circuitry in bacterial diversification remains incompletely understood. We have therefore investigated CtrA regulation in the morphologically complex stalked budding alphaproteobacterium Hyphomonas neptunium. Compared to relatives dividing by binary fission, H. neptunium shows distinct changes in the role and regulation of various pathway components. Most notably, the response regulator DivK, which normally links the upstream and downstream parts of the CtrA pathway, is dispensable, while downstream components such as the pseudokinase DivL, the histidine kinase CckA, the phosphotransferase ChpT and CtrA are essential. Moreover, CckA is compartmentalized to the nascent bud without forming distinct polar complexes and CtrA is not regulated at the level of protein abundance. We show that the downstream pathway controls critical functions such as replication initiation, cell division and motility. Quantification of the signal flow through different nodes of the regulatory cascade revealed that the CtrA pathway is a leaky pipeline and must involve thus-far unidentified factors. Collectively, the quantitative system-level analysis of CtrA regulation in H. neptunium points to a considerable evolutionary plasticity of cell cycle regulation in alphaproteobacteria and leads to hypotheses that may also hold in well-established model organisms such as Caulobacter crescentus.

The conserved transcriptional regulator CdnL is required for metabolic homeostasis and morphogenesis in Caulobacter.

Bacterial growth and division require regulated synthesis of the macromolecules used to expand and replicate components of the cell. Transcription of housekeeping genes required for metabolic homeostasis and cell proliferation is guided by the sigma factor σ70. The conserved CarD-like transcriptional regulator, CdnL, associates with promoter regions where σ70 localizes and stabilizes the open promoter complex. However, the contributions of CdnL to metabolic homeostasis and bacterial physiology are not well understood. Here, we show that Caulobacter crescentus cells lacking CdnL have severe morphological and growth defects. Specifically, ΔcdnL cells grow slowly in both rich and defined media, and are wider, more curved, and have shorter stalks than WT cells. These defects arise from transcriptional downregulation of most major classes of biosynthetic genes, leading to significant decreases in the levels of critical metabolites, including pyruvate, α-ketoglutarate, ATP, NAD+, UDP-N-acetyl-glucosamine, lipid II, and purine and pyrimidine precursors. Notably, we find that ΔcdnL cells are glutamate auxotrophs, and ΔcdnL is synthetic lethal with other genetic perturbations that limit glutamate synthesis and lipid II production. Our findings implicate CdnL as a direct and indirect regulator of genes required for metabolic homeostasis that impacts morphogenesis through availability of lipid II and other metabolites.

Cryptosporulation in Kurthia spp. forces a rethinking of asporogenesis in Firmicutes.

Endosporulation is a complex morphophysiological process resulting in a more resistant cellular structure that is produced within the mother cell and is called endospore. Endosporulation evolved in the common ancestor of Firmicutes, but it is lost in descendant lineages classified as asporogenic. While Kurthia spp. is considered to comprise only asporogenic species, we show here that strain 11kri321, which was isolated from an oligotrophic geothermal reservoir, produces phase-bright spore-like structures. Phylogenomics of strain 11kri321 and other Kurthia strains reveals little similarity to genetic determinants of sporulation known from endosporulating Bacilli. However, morphological hallmarks of endosporulation were observed in two of the four Kurthia strains tested, resulting in spore-like structures (cryptospores). In contrast to classic endospores, these cryptospores did not protect against heat or UV damage and successive sub-culturing led to the loss of the cryptosporulating phenotype. Our findings imply that a cryptosporulation phenotype may have been prevalent and subsequently lost by laboratory culturing in other Firmicutes currently considered as asporogenic. Cryptosporulation might thus represent an ancestral but unstable and adaptive developmental state in Firmicutes that is under selection under harsh environmental conditions.

Insights into the global effect on Staphylococcus aureus growth arrest by induction of the endoribonuclease MazF toxin.

A crucial bacterial strategy to avoid killing by antibiotics is to enter a growth arrested state, yet the molecular mechanisms behind this process remain elusive. The conditional overexpression of mazF, the endoribonuclease toxin of the MazEF toxin-antitoxin system in Staphylococcus aureus, is one approach to induce bacterial growth arrest, but its targets remain largely unknown. We used overexpression of mazF and high-throughput sequence analysis following the exact mapping of non-phosphorylated transcriptome ends (nEMOTE) technique to reveal in vivo toxin cleavage sites on a global scale. We obtained a catalogue of MazF cleavage sites and unearthed an extended MazF cleavage specificity that goes beyond the previously reported one. We correlated transcript cleavage and abundance in a global transcriptomic profiling during mazF overexpression. We observed that MazF affects RNA molecules involved in ribosome biogenesis, cell wall synthesis, cell division and RNA turnover and thus deliver a plausible explanation for how mazF overexpression induces stasis. We hypothesize that autoregulation of MazF occurs by directly modulating the MazEF operon, such as the rsbUVW genes that regulate the sigma factor SigB, including an observed cleavage site on the MazF mRNA that would ultimately play a role in entry and exit from bacterial stasis.

Molecular architecture of the DNA-binding sites of the P-loop ATPases MipZ and ParA from Caulobacter crescentus.

The spatiotemporal regulation of chromosome segregation and cell division in Caulobacter crescentus is mediated by two different P-loop ATPases, ParA and MipZ. Both of these proteins form dynamic concentration gradients that control the positioning of regulatory targets within the cell. Their proper localization depends on their nucleotide-dependent cycling between a monomeric and a dimeric state and on the ability of the dimeric species to associate with the nucleoid. In this study, we use a combination of genetic screening, biochemical analysis and hydrogen/deuterium exchange mass spectrometry to comprehensively map the residues mediating the interactions of MipZ and ParA with DNA. We show that MipZ has non-specific DNA-binding activity that relies on an array of positively charged and hydrophobic residues lining both sides of the dimer interface. Extending our analysis to ParA, we find that the MipZ and ParA DNA-binding sites differ markedly in composition, although their relative positions on the dimer surface and their mode of DNA binding are conserved. In line with previous experimental work, bioinformatic analysis suggests that the same principles may apply to other members of the P-loop ATPase family. P-loop ATPases thus share common mechanistic features, although their functions have diverged considerably during the course of evolution.

Bacterial cell cycle and growth phase switch by the essential transcriptional regulator CtrA.

Many bacteria acquire dissemination and virulence traits in G1-phase. CtrA, an essential and conserved cell cycle transcriptional regulator identified in the dimorphic alpha-proteobacterium Caulobacter crescentus, first activates promoters in late S-phase and then mysteriously switches to different target promoters in G1-phase. We uncovered a highly conserved determinant in the DNA-binding domain (DBD) of CtrA uncoupling this promoter switch. We also show that it reprograms CtrA occupancy in stationary cells inducing a (p)ppGpp alarmone signal perceived by the RNA polymerase beta subunit. A simple side chain modification in a critical residue within the core DBD imposes opposing developmental phenotypes and transcriptional activities of CtrA and a proximal residue can direct CtrA towards activation of the dispersal (G1-phase) program. Hence, we propose that this conserved determinant in the CtrA primary structure dictates promoter reprogramming during the growth transition in other alpha-proteobacteria that differentiate from replicative cells into dispersal cells.

De- and repolarization mechanism of flagellar morphogenesis during a bacterial cell cycle.

Eukaryotic morphogenesis is seeded with the establishment and subsequent amplification of polarity cues at key times during the cell cycle, often using (cyclic) nucleotide signals. We discovered that flagellum de- and repolarization in the model prokaryote Caulobacter crescentus is precisely orchestrated through at least three spatiotemporal mechanisms integrated at TipF. We show that TipF is a cell cycle-regulated receptor for the second messenger--bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP)--that perceives and transduces this signal through the degenerate c-di-GMP phosphodiesterase (EAL) domain to nucleate polar flagellum biogenesis. Once c-di-GMP levels rise at the G1 → S transition, TipF is activated, stabilized, and polarized, enabling the recruitment of downstream effectors, including flagellar switch proteins and the PflI positioning factor, at a preselected pole harboring the TipN landmark. These c-di-GMP-dependent events are coordinated with the onset of tipF transcription in early S phase and together enable the correct establishment and robust amplification of TipF-dependent polarization early in the cell cycle. Importantly, these mechanisms also govern the timely removal of TipF at cell division coincident with the drop in c-di-GMP levels, thereby resetting the flagellar polarization state in the next cell cycle after a preprogrammed period during which motility must be suspended.

Peptidoglycan in obligate intracellular bacteria.

Peptidoglycan is the predominant stress-bearing structure in the cell envelope of most bacteria, and also a potent stimulator of the eukaryotic immune system. Obligate intracellular bacteria replicate exclusively within the interior of living cells, an osmotically protected niche. Under these conditions peptidoglycan is not necessarily needed to maintain the integrity of the bacterial cell. Moreover, the presence of peptidoglycan puts bacteria at risk of detection and destruction by host peptidoglycan recognition factors and downstream effectors. This has resulted in a selective pressure and opportunity to reduce the levels of peptidoglycan. In this review we have analysed the occurrence of genes involved in peptidoglycan metabolism across the major obligate intracellular bacterial species. From this comparative analysis, we have identified a group of predicted 'peptidoglycan-intermediate' organisms that includes the Chlamydiae, Orientia tsutsugamushi, Wolbachia and Anaplasma marginale. This grouping is likely to reflect biological differences in their infection cycle compared with peptidoglycan-negative obligate intracellular bacteria such as Ehrlichia and Anaplasma phagocytophilum, as well as obligate intracellular bacteria with classical peptidoglycan such as Coxiella, Buchnera and members of the Rickettsia genus. The signature gene set of the peptidoglycan-intermediate group reveals insights into minimal enzymatic requirements for building a peptidoglycan-like sacculus and/or division septum.

Deconstructing the Chlamydial Cell Wall.

The evolutionary separated Gram-negative Chlamydiales show a biphasic life cycle and replicate exclusively within eukaryotic host cells. Members of the genus Chlamydia are responsible for many acute and chronic diseases in humans, and Chlamydia-related bacteria are emerging pathogens. We revisit past efforts to detect cell wall material in Chlamydia and Chlamydia-related bacteria in the context of recent breakthroughs in elucidating the underlying cellular and molecular mechanisms of the chlamydial cell wall biosynthesis. In this review, we also discuss the role of cell wall biosynthesis in chlamydial FtsZ-independent cell division and immune modulation. In the past, penicillin susceptibility of an invisible wall was referred to as the "chlamydial anomaly." In light of new mechanistic insights, chlamydiae may now emerge as model systems to understand how a minimal and modified cell wall biosynthetic machine supports bacterial cell division and how cell wall-targeting beta-lactam antibiotics can also act bacteriostatically rather than bactericidal. On the heels of these discussions, we also delve into the effects of other cell wall antibiotics in individual chlamydial lineages.

A novel nucleoid-associated protein coordinates chromosome replication and chromosome partition.

We searched for regulators of chromosome replication in the cell cycle model Caulobacter crescentus and found a novel DNA-binding protein (GapR) that selectively aids the initiation of chromosome replication and the initial steps of chromosome partitioning. The protein binds the chromosome origin of replication (Cori) and has higher-affinity binding to mutated Cori-DNA that increases Cori-plasmid replication in vivo. gapR gene expression is essential for normal rapid growth and sufficient GapR levels are required for the correct timing of chromosome replication. Whole genome ChIP-seq identified dynamic DNA-binding distributions for GapR, with the strongest associations at the partitioning (parABS) locus near Cori. Using molecular-genetic and fluorescence microscopy experiments, we showed that GapR also promotes the first steps of chromosome partitioning, the initial separation of the duplicated parS loci following replication from Cori. This separation occurs before the parABS-dependent partitioning phase. Therefore, this early separation, whose mechanisms is not known, coincides with the poorly defined mechanism(s) that establishes chromosome asymmetry: C. crescentus chromosomes are partitioned to distinct cell-poles which develop into replicating and non-replicating cell-types. We propose that GapR coordinates chromosome replication with asymmetry-establishing chromosome separation, noting that both roles are consistent with the phylogenetic restriction of GapR to asymmetrically dividing bacteria.

In-phase oscillation of global regulons is orchestrated by a pole-specific organizer.

Cell fate determination in the asymmetric bacterium Caulobacter crescentus (Caulobacter) is triggered by the localization of the developmental regulator SpmX to the old (stalked) cell pole during the G1→S transition. Although SpmX is required to localize and activate the cell fate-determining kinase DivJ at the stalked pole in Caulobacter, in cousins such as Asticcacaulis, SpmX directs organelle (stalk) positioning and possibly other functions. We define the conserved σ54-dependent transcriptional activator TacA as a global regulator in Caulobacter whose activation by phosphorylation is indirectly down-regulated by SpmX. Using a combination of forward genetics and cytological screening, we uncover a previously uncharacterized and polarized component (SpmY) of the TacA phosphorylation control system, and we show that SpmY function and localization are conserved. Thus, SpmX organizes a site-specific, ancestral, and multifunctional regulatory hub integrating the in-phase oscillation of two global transcriptional regulators, CtrA (the master cell cycle transcriptional regulator A) and TacA, that perform important cell cycle functions.

Cell Cycle Constraints and Environmental Control of Local DNA Hypomethylation in α-Proteobacteria.

Heritable DNA methylation imprints are ubiquitous and underlie genetic variability from bacteria to humans. In microbial genomes, DNA methylation has been implicated in gene transcription, DNA replication and repair, nucleoid segregation, transposition and virulence of pathogenic strains. Despite the importance of local (hypo)methylation at specific loci, how and when these patterns are established during the cell cycle remains poorly characterized. Taking advantage of the small genomes and the synchronizability of α-proteobacteria, we discovered that conserved determinants of the cell cycle transcriptional circuitry establish specific hypomethylation patterns in the cell cycle model system Caulobacter crescentus. We used genome-wide methyl-N6-adenine (m6A-) analyses by restriction-enzyme-cleavage sequencing (REC-Seq) and single-molecule real-time (SMRT) sequencing to show that MucR, a transcriptional regulator that represses virulence and cell cycle genes in S-phase but no longer in G1-phase, occludes 5'-GANTC-3' sequence motifs that are methylated by the DNA adenine methyltransferase CcrM. Constitutive expression of CcrM or heterologous methylases in at least two different α-proteobacteria homogenizes m6A patterns even when MucR is present and affects promoter activity. Environmental stress (phosphate limitation) can override and reconfigure local hypomethylation patterns imposed by the cell cycle circuitry that dictate when and where local hypomethylation is instated.

Cell Cycle Control by the Master Regulator CtrA in Sinorhizobium meliloti.

In all domains of life, proper regulation of the cell cycle is critical to coordinate genome replication, segregation and cell division. In some groups of bacteria, e.g. Alphaproteobacteria, tight regulation of the cell cycle is also necessary for the morphological and functional differentiation of cells. Sinorhizobium meliloti is an alphaproteobacterium that forms an economically and ecologically important nitrogen-fixing symbiosis with specific legume hosts. During this symbiosis S. meliloti undergoes an elaborate cellular differentiation within host root cells. The differentiation of S. meliloti results in massive amplification of the genome, cell branching and/or elongation, and loss of reproductive capacity. In Caulobacter crescentus, cellular differentiation is tightly linked to the cell cycle via the activity of the master regulator CtrA, and recent research in S. meliloti suggests that CtrA might also be key to cellular differentiation during symbiosis. However, the regulatory circuit driving cell cycle progression in S. meliloti is not well characterized in both the free-living and symbiotic state. Here, we investigated the regulation and function of CtrA in S. meliloti. We demonstrated that depletion of CtrA cause cell elongation, branching and genome amplification, similar to that observed in nitrogen-fixing bacteroids. We also showed that the cell cycle regulated proteolytic degradation of CtrA is essential in S. meliloti, suggesting a possible mechanism of CtrA depletion in differentiated bacteroids. Using a combination of ChIP-Seq and gene expression microarray analysis we found that although S. meliloti CtrA regulates similar processes as C. crescentus CtrA, it does so through different target genes. For example, our data suggest that CtrA does not control the expression of the Fts complex to control the timing of cell division during the cell cycle, but instead it negatively regulates the septum-inhibiting Min system. Our findings provide valuable insight into how highly conserved genetic networks can evolve, possibly to fit the diverse lifestyles of different bacteria.

A cell cycle and nutritional checkpoint controlling bacterial surface adhesion.

In natural environments, bacteria often adhere to surfaces where they form complex multicellular communities. Surface adherence is determined by the biochemical composition of the cell envelope. We describe a novel regulatory mechanism by which the bacterium, Caulobacter crescentus, integrates cell cycle and nutritional signals to control development of an adhesive envelope structure known as the holdfast. Specifically, we have discovered a 68-residue protein inhibitor of holdfast development (HfiA) that directly targets a conserved glycolipid glycosyltransferase required for holdfast production (HfsJ). Multiple cell cycle regulators associate with the hfiA and hfsJ promoters and control their expression, temporally constraining holdfast development to the late stages of G1. HfiA further functions as part of a 'nutritional override' system that decouples holdfast development from the cell cycle in response to nutritional cues. This control mechanism can limit surface adhesion in nutritionally sub-optimal environments without affecting cell cycle progression. We conclude that post-translational regulation of cell envelope enzymes by small proteins like HfiA may provide a general means to modulate the surface properties of bacterial cells.