Caulobacter Research Articles

Article of Significant Interest in This Issue

ϕCbK infects Caulobacter crescentus first by interactions with flagellar filaments. Notably, there are six distinct flagellin genes in C. crescentus that presumably offer a complex landscape for ϕCbK infection. Montemayor et al. (e00399-20) show that no single flagellin is essential for infection by ϕCbK, although flagellin FljK appears to generate filaments that are optimal substrates for infection. High-resolution cryo-electron microscopy reconstructions of simplified C. crescentus flagellar filaments show posttranslational modifications on the periphery of filaments, at loci that are well conserved among the six flagellin paralogs. As such, ϕCbK likely recognizes a common epitope along the body of assembled filaments.

Pseudomonas taiwanensis biofilms for continuous conversion of cyclohexanone in drip flow and rotating bed reactors.

In this study, the biocatalytic performance of a Baeyer‐Villiger monooxygenase (BVMO) catalyzing the reaction of cyclohexanone to ε‐caprolactone was investigated in Pseudomonas biofilms. Biofilm growth and development of two Pseudomonas taiwanensis VLB120 variants, Ps_BVMO and Ps_BVMO_DGC, were evaluated in drip flow reactors (DFRs) and rotating bed reactors (RBRs). Engineering a hyperactive diguanylate cyclase (DGC) from Caulobacter crescentus into Ps_BVMO resulted in faster biofilm growth compared to the control Ps_BVMO strain in the DFRs. The maximum product formation rates of 92 and 87 g m–2 d–1 were observed for mature Ps_BVMO and Ps_ BVMO_DGC biofilms, respectively. The application of the engineered variants in the RBR was challenged by low biofilm surface coverage (50–60%) of rotating bed cassettes, side‐products formation, oxygen limitation, and a severe drop in production rates with time. By implementing an active oxygen supply mode and a twin capillary spray feed, the biofilm surface coverage was maximized to 70–80%. BVMO activity was severely inhibited by cyclohexanol formation, resulting in a decrease in product formation rates. By controlling the cyclohexanone feed concentration at 4 mM, a stable product formation rate of 14 g m–2 d–1 and a substrate conversion of 60% was achieved in the RBR.

Using Molecular Dynamics Simulations to Elucidate a Role for Bacterial Ceramides

Sphingolipids synthesis was thought to be rare in Gram-negative bacteria, previously only found in a handful of taxa. We recently discovered ceramides in Caulobacter crescentus and demonstrated that these lipids play an important role in antibiotic and phage sensitivity. However, the mechanism by which ceramides affect resistance to antimicrobials, as well as their effects on the integrity of the cell membrane are not yet clear. In this study, a coarse-grained molecular dynamics simulation of a prototypical bacterial outer membrane is used to observe changes in the conformation of the outer membrane lipids in the presence of ceramides.

Recombinant cellulase of Caulobacter crescentus: potential applications for biofuels and textile industries

Caulobacter crescentus (C. crescentus) has a single celA gene encoding the bacterial cellulase (EC 3.2.1.4) of the GH9 family that is induced at transcriptional levels (q-PCR) by agroindustrial residues. Corn straw (1%, w/v) was more efficient in inducing bacterial mRNA-celA expression than corn cob (1%, w/v) and a parallel protein expression profile was observed, since enzyme activity was also higher in presence of corn straw in the bacterium. In order to evaluate the biotechnological role of cellulase from C. crescentus (NA1000), the celA gene of 1,878 bp (CCNA_02310) was cloned in the pTrCHisA vector and successfully expressed in Escherichia coli. The recombinant cellulase CelA purified showed 73 kDa, specificity to CMC, optimal pH of 5.5 and pH stability of 6, optimal temperature at 40 °C and thermostability at 35 °C, KM = 0.66 mg/mL and VMax = 2.41 U/mg/min. CelA was strongly activated by ions Mn2+ (70%), Sn2+ (49%) and Co2+ (46%) and was efficient in the hydrolysis of corn straw and corn cob (1%; w/v) releasing 2.62 µmol/mL and 1.02 µmol/mL of reducing sugar, respectively, being an alternative for the production of chemicals and biofuels. CelA was applied directly to the jeans fiber and scanning electron micrographs (SEM) generated showed a clean and smooth surface of the fibers compared to the control, indicating the application of enzyme for the biopolishing of denim jeans. This work supports a set of biotechnological capabilities of cellulase from C. crescentus suggested by the bacterial genomic sequence that has not been described yet.

Projection Microstereolithographic Microbial Bioprinting for Engineered Biofilms.

Microbes are critical drivers of all ecosystems and many biogeochemical processes, yet little is known about how the three-dimensional (3D) organization of these dynamic organisms contributes to their overall function. To probe how biofilm structure affects microbial activity, we developed a technique for patterning microbes in 3D geometries using projection stereolithography to bioprint microbes within hydrogel architectures. Bacteria were printed and monitored for biomass accumulation, demonstrating postprint viability of cells using this technique. We verified our ability to integrate biological and geometric complexity by fabricating a printed biofilm with two E. coli strains expressing different fluorescence. Finally, we examined the target application of microbial absorption of metal ions to investigate geometric effects on both the metal sequestration efficiency and the uranium sensing capability of patterned engineered Caulobacter crescentus strains. This work represents the first demonstration of the stereolithographic printing of microbials and presents opportunities for future work of engineered biofilms and other complex 3D structured cultures.

Z-Ring-Associated Proteins Regulate Clustering of the Replication Terminus-Binding Protein ZapT in Caulobacter crescentus.

Regulated organization of the chromosome is essential for faithful propagation of genetic information. In the model bacterium Caulobacter crescentus, the replication terminus of the chromosome is spatially arranged in close proximity to the cytokinetic Z-ring during the cell cycle. Although the Z-ring-associated proteins ZapA and ZauP interact with the terminus recognition protein ZapT, the molecular functions of the complex that physically links the terminus and the Z-ring remain obscure. In this study, we found that the physical linkage helps to organize the terminus DNA into a clustered structure. Neither ZapA nor ZauP was required for ZapT binding to the terminus DNA, but clustering of the ZapT-DNA complexes over the Z-ring was severely compromised in cells lacking ZapA or ZauP. Biochemical characterization revealed that ZapT, ZauP, and ZapA interacted directly to form a highly ordered ternary complex. Moreover, multiple ZapT molecules were sequestered by each ZauP oligomer. Investigation of the functional structure of ZapT revealed that the C terminus of ZapT specifically interacted with ZauP and was essential for timely positioning of the Z-ring in vivo Based on these findings, we propose that ZauP-dependent oligomerization of ZapT-DNA complexes plays a distinct role in organizing the replication terminus and the Z-ring. The C termini of ZapT homologs share similar chemical properties, implying a common mechanism for the physical linkage between the terminus and the Z-ring in bacteria.IMPORTANCE Rapidly growing bacteria experience dynamic changes in chromosome architecture during chromosome replication and segregation, reflecting the importance of mechanisms that organize the chromosome globally and locally within a cell to maintain faithful transmission of genetic material across generations. During cell division in the model bacterium Caulobacter crescentus, the replication terminus of the chromosome is physically linked to the cytokinetic Z-ring at midcell. However, the functions of this physical linkage are not fully understood. We adopted biochemical and cell-biological techniques to characterize the linkage, including the terminus-binding protein ZapT and the Z-ring-associated protein ZauP. We obtained evidence that the Z-ring organizes the chromosome terminus into a compact structure at midcell via specific interaction between ZapT and ZauP oligomers. Because these proteins are conserved in diverse Gram-negative bacteria, our findings highlight a novel and conserved role for the linker complex in regulated organization of the chromosome terminus.

Characterizing the portability of phage-encoded homologous recombination proteins.

Efficient genome editing methods are essential for biotechnology and fundamental research. Homologous recombination (HR) is the most versatile method of genome editing, but techniques that rely on host RecA-mediated pathways are inefficient and laborious. Phage-encoded ssDNA annealing proteins (SSAPs) improve HR 1000-fold above endogenous levels; however, they are not broadly functional. Using Escherichia coli, Lactococcus lactis, Mycobacterium smegmatis, Lactobacillus rhamnosus, and Caulobacter crescentus we investigated the limited portability of SSAPs. We find that these proteins specifically recognize the C-terminal tail of the host’s single-stranded DNA-binding protein (SSB), and are portable between species if compatibility with this host domain is maintained. Furthermore, we find that co-expressing SSAPs with a paired SSB can significantly improve activity, in some species enabling SSAP functionality even without host-compatibility. Finally, we find that high-efficiency HR far surpasses the mutational capacity of commonly used random mutagenesis methods, generating exceptional phenotypes inaccessible through sequential nucleotide conversions.

Mechanical feedback promotes bacterial adaptation to antibiotics

To maximize their fitness, cells must be able to respond effectively to stresses. This demands making trade-offs between processes that conserve resources to promote survival, and processes that use resources to promote growth and division. Understanding the nature of these trade-offs and the physics underlying them remains an outstanding challenge. Here we combine single-cell experiments and theoretical modelling to propose a mechanism for antibiotic adaptation through mechanical feedback between cell growth and morphology. Under long-term exposure to sublethal doses of ribosome-targeting antibiotics, we find that Caulobacter crescentus cells can recover their pre-stimulus growth rates and undergo dramatic changes in cell shape. Upon antibiotic removal, cells recover their original forms over multiple generations. These phenomena are explained by a physical theory of bacterial growth, which demonstrates that an increase in cell width and curvature promotes faster growth under protein synthesis inhibition. Shape changes thereby make bacteria more adaptive to surviving antibiotics. Certain bacteria cells respond to the stress of long-term exposure to antibiotics by changing their shape. Single-cell experiments and modelling cast this as a mechanical feedback strategy that makes bacteria more adaptive to surviving antibiotics.

AFM force-clamp spectroscopy captures the nanomechanics of the Tad pilus retraction.

Motorization of bacterial pili is key to generate traction forces to achieve cellular function. The Tad (or Type IVc) pilus from Caulobacter crescentus is a widespread motorized nanomachine crucial for bacterial survival, evolution and virulence. An unusual bifunctional ATPase motor drives Tad pilus retraction, which helps the bacteria to land on target surfaces. Here, we use a novel platform combining a fluorescence-based screening of piliated bacteria and atomic force microscopy (AFM) force-clamp spectroscopy, to monitor over time (30 s) the nanomechanics and dynamics of the Tad nanofilament retraction under a high constant tension (300 pN). We observe striking transient variations of force and height originating from two phenomena: active pilus retraction and passive hydrophobic interactions between the pilus and the hydrophobic substrate. That the Tad pilus is able to retract under high tensile loading - at a velocity of ∼150 nm s-1 - indicates that this nanomachine is stronger than previously anticipated. Our findings show that pilus retraction and hydrophobic interactions work together to mediate bacterial cell landing and surface adhesion. The motorized pilus retraction actively triggers the cell to approach the substrate. At short distances, passive hydrophobic interactions accelerate the approach phenomenon and promote strong cell-substrate adhesion. This mechanism could provide a strategy to save ATP-based energy by the retraction ATPase. Our force-clamp AFM methodology offers promise to decipher the physics of bacterial nanomotors with high sensitivity and temporal resolution.

Crosstalking second messengers.

By competing for the same binding site on SmbA, a regulatory nucleotide-binding protein, the bacterial second messengers c-di-GMP and (p)ppGpp inversely balance cell cycle re-entry of growth-arrested swarmer cells of Caulobacter crescentus.

Discovery of oscillations in rotational speed of body-tetheredCaulobacter crescentus

Swarmer cells of Caulobacter crescentus have been found to tether to glass at a point on the cell body. The rolling of the freely rotating flagellum near the glass surface causes the cell body to rotate. We describe the discovery of damped oscillations in the rotational speed of these cell bodies. We show that the damped oscillations are robust over multiple cells and that they depend more on the cell's accumulated rotation angle than on time. We also find that their phase is determined by the moment the flagellar motor changes the direction of its rotation. The oscillations occur only for one direction of cell rotation, when the flagellum is in pulling mode. We discuss possible explanations for these oscillations, including fluctuations in flagellar motor torque and periodic changes in flagellar orientation, and illustrate both of these cases using simplified computer models. Finally, we present the hypothesis that the oscillations are the result of fluctuations in the proton motive force, initiated by a sudden change in proton current that occurs when the motor switches rotation direction.

Article of Significant Interest in This Issue

In bacteria, the Lon protease destroys poor-quality polypeptides to prevent the toxic accumulation of misfolded proteins. However, Lon also degrades essential regulatory proteins, such as DnaA in Caulobacter crescentus; hence, activity must be carefully monitored. Barros et al. (e00344-20) show that the Lon protease is itself degraded in vivo. This process requires active Lon proteases and requires carboxy-terminal residues also found in various Lon orthologs. In Caulobacter, expression of stabilized Lon drastically reduces DnaA levels, leading to cell death. They suggest that this posttranslational control balances the requirement for Lon with the toxicity associated with unconstrained proteolysis.

Degradation of Lon in Caulobacter crescentus.

Protein degradation is an essential process in all organisms. This process is irreversible and energetically costly; therefore, protein destruction must be tightly controlled. While environmental stresses often lead to upregulation of proteases at the transcriptional level, little is known about posttranslational control of these critical machines. In this study, we show that in Caulobacter crescentus levels of the Lon protease are controlled through proteolysis. Lon turnover requires active Lon and ClpAP proteases. We show that specific determinants dictate Lon stability with a key carboxy-terminal histidine residue driving recognition. Expression of stabilized Lon variants results in toxic levels of protease that deplete normal Lon substrates, such as the replication initiator DnaA, to lethally low levels. Taken together, results of this work demonstrate a feedback mechanism in which ClpAP and Lon collaborate to tune Lon proteolytic capacity for the cell.IMPORTANCE Proteases are essential, but unrestrained activity can also kill cells by degrading essential proteins. The quality-control protease Lon must degrade many misfolded and native substrates. We show that Lon is itself controlled through proteolysis and that bypassing this control results in toxic consequences for the cell.

Antibiotic binding releases autoinhibition of the TipA multidrug-resistance transcriptional regulator

Investigations of bacterial resistance strategies can aid in the development of new antimicrobial drugs as a countermeasure to the increasing worldwide prevalence of bacterial antibiotic resistance. One such strategy involves the TipA class of transcription factors, which constitute minimal autoregulated multidrug resistance (MDR) systems against diverse antibiotics. However, we have insufficient information regarding how antibiotic binding induces transcriptional activation to design molecules that could interfere with this process. To learn more, we determined the crystal structure of SkgA from Caulobacter crescentus as a representative TipA protein. We identified an unexpected spatial orientation and location of the antibiotic-binding TipAS effector domain in the apo state. We observed that the α6–α7 region of the TipAS domain, which is canonically responsible for forming the lid of antibiotic-binding cleft to tightly enclose the bound antibiotic, is involved in the dimeric interface and stabilized via interaction with the DNA-binding domain in the apo state. Further structural and biochemical analyses demonstrated that the unliganded TipAS domain sterically hinders promoter DNA binding but undergoes a remarkable conformational shift upon antibiotic binding to release this autoinhibition via a switch of its α6–α7 region. Hence, the promoters for MDR genes including tipA and RNA polymerases become available for transcription, enabling efficient antibiotic resistance. These insights into the molecular mechanism of activation of TipA proteins advance our understanding of TipA proteins, as well as bacterial MDR systems, and may provide important clues to block bacterial resistance.

Spatial organization enhances versatility and specificity in cyclic di-GMP signaling.

The second messenger cyclic di-GMP regulates a variety of processes in bacteria, many of which are centered around the decision whether to adopt a sessile or a motile life style. Regulatory circuits include pathogenicity, biofilm formation, and motility in a wide variety of bacteria, and play a key role in cell cycle progression in Caulobacter crescentus. Interestingly, multiple, seemingly independent c-di-GMP pathways have been found in several species, where deletions of individual c-di-GMP synthetases (DGCs) or hydrolases (PDEs) have resulted indistinct phenotypes that would not be expected based on a freely diffusible second messenger. Several recent studies have shown that individual signaling nodes exist,and additionally, that protein/protein interactions between DGCs, PDEs and c-di-GMP receptors play an important role in signaling specificity. Additionally, subcellular clustering has been shown to be employed by bacteria to likely generate local signaling of second messenger, and/or to increase signaling specificity. This review highlights recent findings that reveal how bacteria employ spatial cues to increase the versatility of second messenger signaling.

Protein Pulldown Assays to Monitor the Composition of the Bacterial RNA Degradosome.

The method of co-immunoprecipitation (co-IP or pulldown) enables the identification of proteins interacting in macromolecular assemblies, through the purification of a key protein by affinity chromatography using specific antibodies immobilized on a matrix. The advantages of using epitope-tagged proteins include the ability to use commercially available antibodies for affinity purifications, and typically they do not disrupt the structure of the protein complexes. Here we describe the utilization of an epitope-tagged version of Caulobacter crescentus RNase E in order to determine the composition of the RNA degradosome under different growth conditions. Several proteins that interact with the RNA degradosome were identified.

Searching Hit Potential Antimicrobials in Natural Compounds Space against Biofilm Formation.

Biofilms are communities of microorganisms that can colonize biotic and abiotic surfaces and thus play a significant role in the persistence of bacterial infection and resistance to antimicrobial. About 65% and 80% of microbial and chronic infections are associated with biofilm formation, respectively. The increase in infections by multi-resistant bacteria instigates the need for the discovery of novel natural-based drugs that act as inhibitory molecules. The inhibition of diguanylate cyclases (DGCs), the enzyme implicated in the synthesis of the second messenger, cyclic diguanylate (c-di-GMP), involved in the biofilm formation, represents a potential approach for preventing the biofilm development. It has been extensively studied using PleD protein as a model of DGC for in silico studies as virtual screening and as a model for in vitro studies in biofilms formation. This study aimed to search for natural products capable of inhibiting the Caulobacter crescentus enzyme PleD. For this purpose, 224,205 molecules from the natural products ZINC15 database, have been evaluated through molecular docking and molecular dynamic simulation. Our results suggest trans-Aconitic acid (TAA) as a possible starting point for hit-to-lead methodologies to obtain new inhibitors of the PleD protein and hence blocking the biofilm formation.

Reciprocal growth control by competitive binding of nucleotide second messengers to a metabolic switch in Caulobacter crescentus.

Bacteria use small signalling molecules such as (p)ppGpp or c-di-GMP to tune their physiology in response to environmental changes. It remains unclear whether these regulatory networks operate independently or whether they interact to optimize bacterial growth and survival. We report that (p)ppGpp and c-di-GMP reciprocally regulate the growth of Caulobacter crescentus by converging on a single small-molecule-binding protein, SmbA. While c-di-GMP binding inhibits SmbA, (p)ppGpp competes for the same binding site to sustain SmbA activity. We demonstrate that (p)ppGpp specifically promotes Caulobacter growth on glucose, whereas c-di-GMP inhibits glucose consumption. We find that SmbA contributes to this metabolic switch and promotes growth on glucose by quenching the associated redox stress. The identification of an effector protein that acts as a central regulatory hub for two global second messengers opens up future studies on specific crosstalk between small-molecule-based regulatory networks.

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.

Specificity in glycosylation of multiple flagellins by the modular and cell cycle regulated glycosyltransferase FlmG.

How specificity is programmed into post-translational modification of proteins by glycosylation is poorly understood, especially for O-linked glycosylation systems. Here we reconstitute and dissect the substrate specificity underpinning the cytoplasmic O-glycosylation pathway that modifies all six flagellins, five structural and one regulatory paralog, in Caulobacter crescentus, a monopolarly flagellated alpha-proteobacterium. We characterize the biosynthetic pathway for the sialic acid-like sugar pseudaminic acid and show its requirement for flagellation, flagellin modification and efficient export. The cognate NeuB enzyme that condenses phosphoenolpyruvate with a hexose into pseudaminic acid is functionally interchangeable with other pseudaminic acid synthases. The previously unknown and cell cycle-regulated FlmG protein, a defining member of a new class of cytoplasmic O-glycosyltransferases, is required and sufficient for flagellin modification. The substrate specificity of FlmG is conferred by its N-terminal flagellin-binding domain. FlmG accumulates before the FlaF secretion chaperone, potentially timing flagellin modification, export, and assembly during the cell division cycle.