Spatial organization of bacterial sphingolipid synthesis enzymes.

Sphingolipids are produced by nearly all eukaryotes where they play significant roles in cellular processes such as cell growth, division, programmed cell death, angiogenesis, and inflammation. While it was previously believed that sphingolipids were quite rare among bacteria, bioinformatic analysis of the recently identified bacterial sphingolipid synthesis genes suggests that these lipids are likely to be produced by a wide range of microbial species. The sphingolipid synthesis pathway consists of three critical enzymes. Serine palmitoyltransferase catalyzes the condensation of serine with palmitoyl-CoA (or palmitoyl-acyl carrier protein), ceramide synthase adds the second acyl chain, and a reductase reduces the ketone present on the long-chain base. While there is general agreement regarding the identity of these bacterial enzymes, the precise mechanism and order of chemical reactions for microbial sphingolipid synthesis is more ambiguous. Two mechanisms have been proposed. First, the synthesis pathway may follow the well characterized eukaryotic pathway in which the long-chain base is reduced prior to the addition of the second acyl chain. Alternatively, our previous work suggests that addition of the second acyl chain precedes the reduction of the long-chain base. To distinguish between these two models, we investigated the subcellular localization of these three key enzymes. We found that serine palmitoyltransferase and ceramide synthase are localized to the cytoplasm, whereas the ceramide reductase is in the periplasmic space. This is consistent with our previously proposed model wherein the second acyl chain is added in the cytoplasm prior to export to the periplasm where the lipid molecule is reduced.

Characterization of an evolutionarily distinct bacterial ceramide kinase from Caulobacter crescentus.

A common feature among nearly all gram-negative bacteria is the requirement for lipopolysaccharide (LPS) in the outer leaflet of the outer membrane. LPS provides structural integrity to the bacterial membrane, which aids bacteria in maintaining their shape and acts as a barrier from environmental stress and harmful substances such as detergents and antibiotics. Recent work has demonstrated that Caulobacter crescentus can survive without LPS due to the presence of the anionic sphingolipid ceramide-phosphoglycerate (CPG). Based on genetic evidence, we predicted that protein CpgB functions as a ceramide kinase and performs the first step in generating the phosphoglycerate head group. Here, we characterized the kinase activity of recombinantly expressed CpgB and demonstrated that it can phosphorylate ceramide to form ceramide 1-phosphate. The pH optimum for CpgB was 7.5, and the enzyme required Mg2+ as a cofactor. Mn2+, but no other divalent cations, could substitute for Mg2+. Under these conditions, the enzyme exhibited typical Michaelis-Menten kinetics with respect to NBD C6-ceramide (Km,app = 19.2 ± 5.5 µM; Vmax,app = 2590 ± 230 pmol/min/mg enzyme) and ATP (Km,app = 0.29 ± 0.07 mM; Vmax,app = 10,100 ± 996 pmol/min/mg enzyme). Phylogenetic analysis of CpgB revealed that CpgB belongs to a new class of ceramide kinases, which is distinct from its eukaryotic counterpart; furthermore, the pharmacological inhibitor of human ceramide kinase (NVP-231) had no effect on CpgB. The characterization of a new bacterial ceramide kinase opens avenues for understanding the structure and function of the various microbial phosphorylated sphingolipids.

Compartmentalization of RNA Degradosomes in Bacteria Controls Accessibility to Substrates and Ensures Concerted Degradation of mRNA to Nucleotides.

RNA degradosomes are multienzyme complexes composed of ribonucleases, RNA helicases, and metabolic enzymes. RNase E-based degradosomes are widespread in Proteobacteria. The Escherichia coli RNA degradosome is sequestered from transcription in the nucleoid and translation in the cytoplasm by localization to the inner cytoplasmic membrane, where it forms short-lived clusters that are proposed to be sites of mRNA degradation. In Caulobacter crescentus, RNA degradosomes localize to ribonucleoprotein condensates in the interior of the cell [bacterial ribonucleoprotein-bodies (BR-bodies)], which have been proposed to drive the concerted degradation of mRNA to nucleotides. The turnover of mRNA in growing cells is important for maintaining pools of nucleotides for transcription and DNA replication.Membrane attachment of the E. coli RNA degradosome is necessary to avoid wasteful degradation of intermediates in ribosome assembly. Sequestering RNA degradosomes to C. crescentus BR-bodies, which exclude structured RNA, could have a similar role in protecting intermediates in ribosome assembly from degradation.

Regulation of the activity of the bacterial histidine kinase PleC by the scaffolding protein PodJ.

Scaffolding proteins can customize the response of signaling networks to support cell development and behaviors. PleC is a bifunctional histidine kinase whose signaling activity coordinates asymmetric cell division to yield a motile swarmer cell and a stalked cell in the gram-negative bacterium Caulobacter crescentus. Past studies have shown that PleC's switch in activity from kinase to phosphatase correlates with a change in its subcellular localization pattern from diffuse to localized at the new cell pole. Here we investigated how the bacterial scaffolding protein PodJ regulates the subcellular positioning and activity of PleC. We reconstituted the PleC-PodJ signaling complex through both heterologous expressions in Escherichia coli and in vitro studies. In vitro, PodJ phase separates as a biomolecular condensate that recruits PleC and inhibits its kinase activity. We also constructed an in vivo PleC-CcaS chimeric histidine kinase reporter assay and demonstrated using this method that PodJ leverages its intrinsically disordered region to bind to PleC's PAS sensory domain and regulate PleC-CcaS signaling. Regulation of the PleC-CcaS was most robust when PodJ was concentrated at the cell poles and was dependent on the allosteric coupling between PleC-CcaS's PAS sensory domain and its downstream histidine kinase domain. In conclusion, our in vitro biochemical studies suggest that PodJ phase separation may be coupled to changes in PleC enzymatic function. We propose that this coupling of phase separation and allosteric regulation may be a generalizable phenomenon among enzymes associated with biomolecular condensates.

Three-dimensional structure of xylonolactonase from Caulobacter crescentus: A mononuclear iron enzyme of the 6-bladed ß-propeller hydrolase family.

Xylonolactonase Cc XylC from Caulobacter crescentus catalyzes the hydrolysis of the intramolecular ester bond of d-xylonolactone. We have determined crystal structures of Cc XylC in complex with d-xylonolactone isomer analogues d-xylopyranose and (r)-(+)-4-hydroxy-2-pyrrolidinone at high resolution. Cc XylC has a 6-bladed ß-propeller architecture, which contains a central open channel having the active site at one end. According to our previous native mass spectrometry studies, Cc XylC is able to specifically bind Fe2+ . The crystal structures, presented here, revealed an active site bound metal ion with an octahedral binding geometry. The side chains of three amino acid residues, Glu18, Asn146, and Asp196, which participate in binding of metal ion are located in the same plane. The solved complex structures allowed suggesting a reaction mechanism for intramolecular ester bond hydrolysis in which the major contribution for catalysis arises from the carbonyl oxygen coordination of the xylonolactone substrate to the Fe2+ . The structure of Cc XylC was compared with eight other ester hydrolases of the ß-propeller hydrolase family. The previously published crystal structures of other ß-propeller hydrolases contain either Ca2+ , Mg2+ , or Zn2+ and show clear similarities in ligand and metal ion binding geometries to that of Cc XylC. It would be interesting to reinvestigate the metal binding specificity of these enzymes and clarify whether they are also able to use Fe2+ as a catalytic metal. This could further expand our understanding of utilization of Fe2+ not only in oxidative enzymes but also in hydrolases.

Xylonolactonase from Caulobacter crescentus Is a Mononuclear Nonheme Iron Hydrolase.

Caulobacter crescentus xylonolactonase (Cc XylC, EC 3.1.1.68) catalyzes an intramolecular ester bond hydrolysis over a nonenzymatic acid/base catalysis. Cc XylC is a member of the SMP30 protein family, whose members have previously been reported to be active in the presence of bivalent metal ions, such as Ca2+, Zn2+, and Mg2+. By native mass spectrometry, we studied the binding of several bivalent metal ions to Cc XylC and observed that it binds only one of them, namely, the Fe2+ cation, specifically and with a high affinity (Kd = 0.5 µM), pointing out that Cc XylC is a mononuclear iron protein. We propose that bivalent metal cations also promote the reaction nonenzymatically by stabilizing a short-lived bicyclic intermediate on the lactone isomerization reaction. An analysis of the reaction kinetics showed that Cc XylC complexed with Fe2+ can speed up the hydrolysis of d-xylono-1,4-lactone by 100-fold and that of d-glucono-1,5-lactone by 10-fold as compared to the nonenzymatic reaction. To our knowledge, this is the first discovery of a nonheme mononuclear iron-binding enzyme that catalyzes an ester bond hydrolysis reaction.

Coordination between nucleotide excision repair and specialized polymerase DnaE2 action enables DNA damage survival in non-replicating bacteria.

Translesion synthesis (TLS) is a highly conserved mutagenic DNA lesion tolerance pathway, which employs specialized, low-fidelity DNA polymerases to synthesize across lesions. Current models suggest that activity of these polymerases is predominantly associated with ongoing replication, functioning either at or behind the replication fork. Here we provide evidence for DNA damage-dependent function of a specialized polymerase, DnaE2, in replication-independent conditions. We develop an assay to follow lesion repair in non-replicating Caulobacter and observe that components of the replication machinery localize on DNA in response to damage. These localizations persist in the absence of DnaE2 or if catalytic activity of this polymerase is mutated. Single-stranded DNA gaps for SSB binding and low-fidelity polymerase-mediated synthesis are generated by nucleotide excision repair (NER), as replisome components fail to localize in the absence of NER. This mechanism of gap-filling facilitates cell cycle restoration when cells are released into replication-permissive conditions. Thus, such cross-talk (between activity of NER and specialized polymerases in subsequent gap-filling) helps preserve genome integrity and enhances survival in a replication-independent manner.

The Cleavage Profile of Protein Substrates by ClpXP Reveals Deliberate Starts and Pauses.

Cells rely on protein degradation by AAA+ proteases. A well-known example is the hexameric ClpX unfoldase, which captures ATP hydrolysis to feed substrates into the oligomeric ClpP peptidase. Recent studies show that an asymmetric ClpX spiral cycles protein translocation upon ATP hydrolysis. However, how this cycle affects peptide products is less explored in part because ClpP cleavage is thought to be solely defined by sequence constraints. Here, we comprehensively characterize peptides from Caulobacter crescentus ClpXP degradation of three different substrates using high-resolution mass spectrometry and find that cleavage of translocated substrates is driven by factors other than sequence. We report that defined locations in a translocated protein are especially sensitive to cleavage spaced on average every 10-13 residues. These sites are not exclusively controlled by sequence and are independent of bulk changes in catalytic peptidase sites, ATP hydrolysis, or the efficiency of initial recognition. These results fit a model in which processive translocation through ClpX starts at a specific location in a polypeptide and pauses during reset of the ClpX hexamer after a cycle of translocation. Our work suggests that defined peptides, which could be used as signaling molecules, can be generated from a given substrate by a nonspecific peptidase.

Cell cycle regulated DNA methyltransferase: fluorescent tracking of a DNA strand-separation mechanism and identification of the responsible protein motif.

DNA adenine methylation by Caulobacter crescentus Cell Cycle Regulated Methyltransferase (CcrM) is an important epigenetic regulator of gene expression. The recent CcrM-DNA cocrystal structure shows the CcrM dimer disrupts four of the five base pairs of the (5'-GANTC-3') recognition site. We developed a fluorescence-based assay by which Pyrrolo-dC tracks the strand separation event. Placement of Pyrrolo-dC within the DNA recognition site results in a fluorescence increase when CcrM binds. Non-cognate sequences display little to no fluorescence changes, showing that strand separation is a specificity determinant. Conserved residues in the C-terminal segment interact with the phospho-sugar backbone of the non-target strand. Replacement of these residues with alanine results in decreased methylation activity and changes in strand separation. The DNA recognition mechanism appears to occur with the Type II M.HinfI DNA methyltransferase and an ortholog of CcrM, BabI, but not with DNA methyltransferases that lack the conserved C-terminal segment. The C-terminal segment is found broadly in N4/N6-adenine DNA methyltransferases, some of which are human pathogens, across three Proteobacteria classes, three other phyla and in Thermoplasma acidophilum, an Archaea. This Pyrrolo-dC strand separation assay should be useful for the study of other enzymes which likely rely on a strand separation mechanism.

The Lon Protease Links Nucleotide Metabolism with Proteotoxic Stress.

During proteotoxic stress, bacteria maintain critical processes like DNA replication while removing misfolded proteins, which are degraded by the Lon protease. Here, we show that in Caulobacter crescentus Lon controls deoxyribonucleoside triphosphate (dNTP) pools during stress through degradation of the transcription factor CcrM. Elevated dNTP/nucleotide triphosphate (NTP) ratios in Δlon cells protects them from deletion of otherwise essential deoxythymidine triphosphate (dTTP)-producing pathways and shields them from hydroxyurea-induced loss of dNTPs. Increased dNTP production in Δlon results from higher expression of ribonucleotide reductase driven by increased CcrM. We show that misfolded proteins can stabilize CcrM by competing for limited protease and that Lon-dependent control of dNTPs improves fitness during protein misfolding conditions. We propose that linking dNTP production with availability of Lon allows Caulobacter to maintain replication capacity when misfolded protein burden increases, such as during rapid growth. Because Lon recognizes misfolded proteins regardless of the stress, this mechanism allows for response to a variety of unanticipated conditions.

Beta class amino methyltransferases from bacteria to humans: evolution and structural consequences.

S-adenosyl-l-methionine dependent methyltransferases catalyze methyl transfers onto a wide variety of target molecules, including DNA and RNA. We discuss a family of methyltransferases, those that act on the amino groups of adenine or cytosine in DNA, have conserved motifs in a particular order in their amino acid sequence, and are referred to as class beta MTases. Members of this class include M.EcoGII and M.EcoP15I from Escherichia coli, Caulobacter crescentus cell cycle-regulated DNA methyltransferase (CcrM), the MTA1-MTA9 complex from the ciliate Oxytricha, and the mammalian MettL3-MettL14 complex. These methyltransferases all generate N6-methyladenine in DNA, with some members having activity on single-stranded DNA as well as RNA. The beta class of methyltransferases has a unique multimeric feature, forming either homo- or hetero-dimers, allowing the enzyme to use division of labor between two subunits in terms of substrate recognition and methylation. We suggest that M.EcoGII may represent an ancestral form of these enzymes, as its activity is independent of the nucleic acid type (RNA or DNA), its strandedness (single or double), and its sequence (aside from the target adenine).

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 control by citrate synthase independent of enzymatic activity.

Proliferating cells must coordinate central metabolism with the cell cycle. How central energy metabolism regulates bacterial cell cycle functions is not well understood. Our forward genetic selection unearthed the Krebs cycle enzyme citrate synthase (CitA) as a checkpoint regulator controlling the G1→S transition in the polarized alpha-proteobacterium Caulobacter crescentus, a model for cell cycle regulation and asymmetric cell division. We find that loss of CitA promotes the accumulation of active CtrA, an essential cell cycle transcriptional regulator that maintains cells in G1-phase, provided that the (p)ppGpp alarmone is present. The enzymatic activity of CitA is dispensable for CtrA control, and functional citrate synthase paralogs cannot replace CitA in promoting S-phase entry. Our evidence suggests that CitA was appropriated specifically to function as a moonlighting enzyme to link central energy metabolism with S-phase entry. Control of the G1-phase by a central metabolic enzyme may be a common mechanism of cellular regulation.

hipBA toxin-antitoxin systems mediate persistence in Caulobacter crescentus.

Antibiotic persistence is a transient phenotypic state during which a bacterium can withstand otherwise lethal antibiotic exposure or environmental stresses. In Escherichia coli, persistence is promoted by the HipBA toxin-antitoxin system. The HipA toxin functions as a serine/threonine kinase that inhibits cell growth, while the HipB antitoxin neutralizes the toxin. E. coli HipA inactivates the glutamyl-tRNA synthetase GltX, which inhibits translation and triggers the highly conserved stringent response. Although hipBA operons are widespread in bacterial genomes, it is unknown if this mechanism is conserved in other species. Here we describe the functions of three hipBA modules in the alpha-proteobacterium Caulobacter crescentus. The HipA toxins have different effects on growth and macromolecular syntheses, and they phosphorylate distinct substrates. HipA1 and HipA2 contribute to antibiotic persistence during stationary phase by phosphorylating the aminoacyl-tRNA synthetases GltX and TrpS. The stringent response regulator SpoT is required for HipA-mediated antibiotic persistence, but persister cells can form in the absence of all hipBA operons or spoT, indicating that multiple pathways lead to persister cell formation in C. crescentus.

A combined experimental and modelling approach for the Weimberg pathway optimisation.

The oxidative Weimberg pathway for the five-step pentose degradation to α-ketoglutarate is a key route for sustainable bioconversion of lignocellulosic biomass to added-value products and biofuels. The oxidative pathway from Caulobacter crescentus has been employed in in-vivo metabolic engineering with intact cells and in in-vitro enzyme cascades. The performance of such engineering approaches is often hampered by systems complexity, caused by non-linear kinetics and allosteric regulatory mechanisms. Here we report an iterative approach to construct and validate a quantitative model for the Weimberg pathway. Two sensitive points in pathway performance have been identified as follows: (1) product inhibition of the dehydrogenases (particularly in the absence of an efficient NAD+ recycling mechanism) and (2) balancing the activities of the dehydratases. The resulting model is utilized to design enzyme cascades for optimized conversion and to analyse pathway performance in C. cresensus cell-free extracts.

Selective sequestration of signalling proteins in a membraneless organelle reinforces the spatial regulation of asymmetry in Caulobacter crescentus.

Selective recruitment and concentration of signalling proteins within membraneless compartments is a ubiquitous mechanism for subcellular organization1-3. The dynamic flow of molecules into and out of these compartments occurs on faster timescales than for membrane-enclosed organelles, presenting a possible mechanism to control spatial patterning within cells. Here, we combine single-molecule tracking and super-resolution microscopy, light-induced subcellular localization, reaction-diffusion modelling and a spatially resolved promoter activation assay to study signal exchange in and out of the 200 nm cytoplasmic pole-organizing protein popZ (PopZ) microdomain at the cell pole of the asymmetrically dividing bacterium Caulobacter crescentus4-8. Two phospho-signalling proteins, the transmembrane histidine kinase CckA and the cytoplasmic phosphotransferase ChpT, provide the only phosphate source for the cell fate-determining transcription factor CtrA9-18. We find that all three proteins exhibit restricted rates of entry into and escape from the microdomain as well as enhanced phospho-signalling within, leading to a submicron gradient of activated CtrA-P19 that is stable and sublinear. Entry into the microdomain is selective for cytosolic proteins and requires a binding pathway to PopZ. Our work demonstrates how nanoscale protein assemblies can modulate signal propagation with fine spatial resolution, and that in Caulobacter, this modulation serves to reinforce asymmetry and differential cell fate of the two daughter cells.

The cell cycle-regulated DNA adenine methyltransferase CcrM opens a bubble at its DNA recognition site.

The Caulobacter crescentus cell cycle-regulated DNA methyltransferase (CcrM) methylates the adenine of hemimethylated GANTC after replication. Here we present the structure of CcrM in complex with double-stranded DNA containing the recognition sequence. CcrM contains an N-terminal methyltransferase domain and a C-terminal nonspecific DNA-binding domain. CcrM is a dimer, with each monomer contacting primarily one DNA strand: the methyltransferase domain of one molecule binds the target strand, recognizes the target sequence, and catalyzes methyl transfer, while the C-terminal domain of the second molecule binds the non-target strand. The DNA contacts at the 5-base pair recognition site results in dramatic DNA distortions including bending, unwinding and base flipping. The two DNA strands are pulled apart, creating a bubble comprising four recognized base pairs. The five bases of the target strand are recognized meticulously by stacking contacts, van der Waals interactions and specific Watson-Crick polar hydrogen bonds to ensure high enzymatic specificity.

Lon recognition of the replication initiator DnaA requires a bipartite degron.

DnaA initiates chromosome replication in bacteria. In Caulobacter crescentus, the Lon protease degrades DnaA to coordinate replication with nutrient availability and to halt the cell cycle during acute stress. Here, we characterize the mechanism of DnaA recognition by Lon. We find that the folded state of DnaA appears crucial for its degradation, in contrast to the well-known role of Lon in degrading misfolded proteins. We fail to identify a single degradation motif (degron) sufficient for DnaA degradation, rather we show that both the ATPase domain and a species-specific N-terminal motif are important for productive Lon degradation of full-length DnaA. Mutations in either of these determinants disrupt DnaA degradation in vitro and in vivo. However, analysis of truncation products reveals that appending other extensions to the ATPase domain is sufficient to trigger degradation, suggesting plasticity in Lon recognition. Our final working model is that Lon engages DnaA through at least two elements, one of which anchors DnaA to Lon and the other acting as an initiation site for degradation.

Regulation of (p)ppGpp hydrolysis by a conserved archetypal regulatory domain.

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 metabolic-related proteins as well as in long (p)ppGpp synthetase/hydrolase enzymes. Here, we investigate the functional role of the ACT domain of SpoT, the only (p)ppGpp synthetase/hydrolase of Caulobacter crescentus. We show that SpoT requires the ACT domain to efficiently hydrolyze (p)ppGpp. 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)ppGpp accumulation upon nitrogen starvation in Sinorhizobium meliloti, a plant-associated α-proteobacterium. Thus, this work suggests that α-proteobacteria might have inherited from a common ancestor, a PTSNtr dedicated to modulate (p)ppGpp levels in response to nitrogen availability.

Identification of Small-Molecule Modulators of Diguanylate Cyclase by FRET-Based High-Throughput Screening.

The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) is a key regulator of cellular motility, the cell cycle, and biofilm formation with its resultant antibiotic tolerance, which can make chronic infections difficult to treat. Therefore, diguanylate cyclases, which regulate the spatiotemporal production of c-di-GMP, might be attractive drug targets for control of biofilm formation that is part of chronic infections. We present a FRET-based biochemical high-throughput screening approach coupled with detailed structure-activity studies to identify synthetic small-molecule modulators of the diguanylate cyclase DgcA from Caulobacter crescentus. We identified a set of seven small molecules that regulate DgcA enzymatic activity in the low-micromolar range. Subsequent structure-activity studies on selected scaffolds revealed a remarkable diversity of modulatory behavior, including slight chemical substitutions that reverse the effects from allosteric enzyme inhibition to activation. The compounds identified represent new chemotypes and are potentially developable into chemical genetic tools for the dissection of c-di-GMP signaling networks and alteration of c-di-GMP-associated phenotypes. In sum, our studies underline the importance of detailed mechanism-of-action studies for inhibitors of c-di-GMP signaling and demonstrate the complex interplay between synthetic small molecules and the regulatory mechanisms that control the activity of diguanylate cyclases.