The genus Caulobacter and its role in plant microbiomes.

Recent omics approaches have revealed the prevalent microbial taxa that constitute the microbiome of various plant species. Across global scales and environmental conditions, strains belonging to the bacterial genus Caulobacter have consistently been found in association with various plant species. Aligned with agroecological relevance and biotechnological advances, many scientific communications have demonstrated that several Caulobacter strains (spanning several Caulobacter species) harbor the potential to enhance plant biomass for various plant species ranging from Arabidopsis to Citrullus and Zea mays. In the past several years, co-occurrence data have driven mechanistically resolved communications about select Caulobacter-plant interactions. Given the long-standing history of Caulobacter as a model organism for cell cycle regulation, genetic studies, and the prevalence of Caulobacter species in various plant microbiomes, the genus Caulobacter offers researchers a unique opportunity to leverage for investigating plant-microbe interactions and realizing targeted biotechnological applications. In this review, recent developments regarding Caulobacter-plant interactions are presented in terms of model utility for future biotechnological investigations.

Following the terrestrial tracks of Caulobacter - redefining the ecology of a reputed aquatic oligotroph.

For the past 60 years Caulobacter spp. have been commonly attributed an aquatic and oligotrophic lifestyle yet are not uncommon in nutrient-rich or soil environments. This study evaluates the environmental and ecological associations of Caulobacter to reconcile past evidence, largely limited to culturing and microscopy, with currently available metagenomic and genomic data. The distribution of Caulobacter species and their characteristic adhesion-conferring genes, holdfast (hfaAB), were determined using collections of 10,641 16S rRNA gene libraries (196 studies) and 2625 shotgun metagenomes (190 studies) from a range of terrestrial and aquatic environments. Evidence for ecotypic variation was tested in 26 genomes sourced from soil, rhizosphere, plant, groundwater, and water. Caulobacter were, on average, fourfold more relatively abundant in soil than in aquatic environments, and abundant in decomposing wood, compost, and particulate matter (in air and water). Caulobacter holdfast genes were 35-fold more abundant in soils than aquatic environments. Ecotypic differences between soil and aquatic Caulobacter were evident in the environmental associations of several species and differences in genome size and content among isolates. However, most abundant species were common to both environments, suggesting populations exist in a continuum that was evident in the re-analysis of studies on the temporal dynamics of, and sources of bacterioplankton to, lakes and rivers. This study provides a new perspective on the ecological profile of Caulobacter, demonstrating that members of this genus are predominantly soil-borne, possess an overlooked role in plant matter decomposition and a dependency on water-mediated dispersal.

Dynamic translation regulation in Caulobacter cell cycle control.

Progression of the Caulobacter cell cycle requires temporal and spatial control of gene expression, culminating in an asymmetric cell division yielding distinct daughter cells. To explore the contribution of translational control, RNA-seq and ribosome profiling were used to assay global transcription and translation levels of individual genes at six times over the cell cycle. Translational efficiency (TE) was used as a metric for the relative rate of protein production from each mRNA. TE profiles with similar cell cycle patterns were found across multiple clusters of genes, including those in operons or in subsets of operons. Collections of genes associated with central cell cycle functional modules (e.g., biosynthesis of stalk, flagellum, or chemotaxis machinery) have consistent but different TE temporal patterns, independent of their operon organization. Differential translation of operon-encoded genes facilitates precise cell cycle-timing for the dynamic assembly of multiprotein complexes, such as the flagellum and the stalk and the correct positioning of regulatory proteins to specific cell poles. The cell cycle-regulatory pathways that produce specific temporal TE patterns are separate from-but highly coordinated with-the transcriptional cell cycle circuitry, suggesting that the scheduling of translational regulation is organized by the same cyclical regulatory circuit that directs the transcriptional control of the Caulobacter cell cycle.

CspC regulates the expression of the glyoxylate cycle genes at stationary phase in Caulobacter.

BACKGROUND: The Cold Shock proteins are RNA binding proteins involved in various cellular processes, including adaptation to low temperature, nutritional stress, cell growth and stationary phase. They may have an impact on gene expression by interfering with RNA stability and acting as transcription antiterminators. Caulobacter crescentus cspC is an essential gene encoding a stationary phase-induced protein of the Cold Shock Protein family and this work had as goal investigating the basis for the requirement of this gene for survival at this phase. In this work we investigate the role of CspC in C. crescentus stationary phase and discuss the molecular mechanisms that could be involved. RESULTS: The expression of cspC increased significantly at stationary phase in complex media and in glucose depletion, indicating a putative role in responding to carbon starvation. Global transcriptional profiling experiments comparing cspC and the wild type strain both at exponential and stationary phases as well as comparing exponential and stationary phase in wild type strain were carried out by DNA microarray analysis. The results showed that the absence of cspC affected the transcription of 11 genes at exponential phase and 60 genes at stationary phase. Among the differentially expressed genes it is worth noting those encoding respiratory enzymes and genes for sulfur metabolism, which were upregulated, and those encoding enzymes of the glyoxylate cycle, which were severely downregulated in the mutant at stationary phase. mRNA decay experiments showed that the aceA mRNA, encoding isocitrate lyase, was less stable in the cspC mutant, indicating that this effect was at least partially due to posttranscriptional regulation. These observations were supported by the observed arrested growth phenotype of the cspC strain when grown in acetate as the sole carbon source, and by the upregulation of genes for assimilatory sulfate reduction and methionine biosynthesis. CONCLUSIONS: The stationary phase-induced RNA binding protein CspC has an important role in gene expression at this phase, and is necessary for maximal expression of the glyoxylate cycle genes. In the case of aceA, its downregulation may be attributed to the shorter half-life of the mRNA in the cspC mutant, indicating that one of the possible regulatory mechanisms is via altering RNA stabilization.

Genome sequence and phenotypic characterization of Caulobacter segnis.

Caulobacter segnis is a unique species of Caulobacter that was initially deemed Mycoplana segnis because it was isolated from soil and appeared to share a number of features with other Mycoplana. After a 16S rDNA analysis showed that it was closely related to Caulobacter crescentus, it was reclassified C. segnis. Because the C. segnis genome sequence available in GenBank contained 126 pseudogenes, we compared the original sequencing data to the GenBank sequence and determined that many of the pseudogenes were due to sequence errors in the GenBank sequence. Consequently, we used multiple approaches to correct and reannotate the C. segnis genome sequence. In total, we deleted 247 bp, added 14 bp, and changed 8 bp resulting in 233 fewer bases in our corrected sequence. The corrected sequence contains only 15 pseudogenes compared to 126 in the original annotation. Furthermore, we found that unlike Mycoplana, C. segnis divides by fission, producing swarmer cells that have a single, polar flagellum.

Cell cycle transition from S-phase to G1 in Caulobacter is mediated by ancestral virulence regulators.

Zinc-finger domain transcriptional regulators regulate a myriad of functions in eukaryotes. Interestingly, ancestral versions (MucR) from Alpha-proteobacteria control bacterial virulence/symbiosis. Whether virulence regulators can also control cell cycle transcription is unknown. Here we report that MucR proteins implement a hitherto elusive primordial S→G1 transcriptional switch. After charting G1-specific promoters in the cell cycle model Caulobacter crescentus by comparative ChIP-seq, we use one such promoter as genetic proxy to unearth two MucR paralogs, MucR1/2, as constituents of a quadripartite and homeostatic regulatory module directing the S→G1 transcriptional switch. Surprisingly, MucR orthologues that regulate virulence and symbiosis gene transcription in Brucella, Agrobacterium or Sinorhizobium support this S→G1 switch in Caulobacter. Pan-genomic ChIP-seq analyses in Sinorhizobium and Caulobacter show that this module indeed targets orthologous genes. We propose that MucR proteins and possibly other virulence regulators primarily control bacterial cell cycle (G1-phase) transcription, rendering expression of target (virulence) genes periodic and in tune with the cell cycle.

BapE DNA endonuclease induces an apoptotic-like response to DNA damage in Caulobacter.

In the presence of extensive DNA damage, eukaryotes activate endonucleases to fragment their chromosomes and induce apoptotic cell death. Apoptotic-like responses have recently been described in bacteria, but primarily in specialized mutant backgrounds, and the factors responsible for DNA damage-induced chromosome fragmentation and death have not been identified. Here we find that wild-type Caulobacter cells induce apoptotic-like cell death in response to extensive DNA damage. The bacterial apoptosis endonuclease (BapE) protein is induced by damage but not involved in DNA repair itself, and mediates this cell fate decision. BapE fragments chromosomes by cleaving supercoiled DNA in a sequence-nonspecific manner, thereby perturbing chromosome integrity both in vivo and in vitro. This damage-induced chromosome fragmentation pathway resembles that of eukaryotic apoptosis. We propose that damage-induced programmed cell death can be a primary stress response for some bacterial species, providing isogenic bacterial communities with advantages similar to those that apoptosis provides to multicellular organisms.

Cell orientation of swimming bacteria: from theoretical simulation to experimental evaluation.

The motion of small bacteria consists of two phases: relatively long runs alternate with intermittent stops, back-ups, or tumbles, depending on the species. In polar monotrichous bacteria, the flagellum is anchored at the cell pole inherited from the parent generation (old pole) and is surrounded by a chemoreceptor cluster. During forward swimming, the leading pole is always the pole recently formed in cell division (new pole). The flagella of the peritrichous bacterium Escherichia coli often form a bundle behind the old pole. Its cell orientation and receptor positioning during runs generally mimic that of monotrichous bacteria. When encountering a solid surface, peritrichous bacteria exhibit a circular motion with the leading pole dipping downward. Some polar monotrichous bacteria also perform circular motion near solid boundaries, but during back-ups. In this case, the leading pole points upward. Very little is known about behavior near milieu-air interfaces. Biophysical simulations have revealed some of the mechanisms underlying these phenomena, but leave many questions unanswered. Combining biophysics with molecular techniques will certainly advance our understanding of bacterial locomotion.

The architecture and conservation pattern of whole-cell control circuitry.

The control circuitry that directs and paces Caulobacter cell cycle progression involves the entire cell operating as an integrated system. This control circuitry monitors the environment and the internal state of the cell, including the cell topology, as it orchestrates orderly activation of cell cycle subsystems and Caulobacter's asymmetric cell division. The proteins of the Caulobacter cell cycle control system and its internal organization are co-conserved across many alphaproteobacteria species, but there are great differences in the regulatory apparatus' functionality and peripheral connectivity to other cellular subsystems from species to species. This pattern is similar to that observed for the "kernels" of the regulatory networks that regulate development of metazoan body plans. The Caulobacter cell cycle control system has been exquisitely optimized as a total system for robust operation in the face of internal stochastic noise and environmental uncertainty. When sufficient details accumulate, as for Caulobacter cell cycle regulation, the system design has been found to be eminently rational and indeed consistent with good design practices for human-designed asynchronous control systems.

Phylogeny by a polyphasic approach of the order Caulobacterales, proposal of Caulobacter mirabilis sp. nov., Phenylobacterium haematophilum sp. nov. and Phenylobacterium conjunctum sp. nov., and emendation of the genus Phenylobacterium.

Three strains of Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from fresh water and human blood. As determined by analyses of 16S rRNA gene sequences, the prosthecate strain FWC 38T was affiliated to the alphaproteobacterial genus Caulobacter, with Caulobacter henricii (96.8 %) and Caulobacter fusiformis (96.8 %) as its closest relatives. The non-prosthecate strain LMG 11050T and the prosthecate strain FWC 21T both belonged to the genus Phenylobacterium with Phenylobacterium koreense (96.9 %) and Phenylobacterium immobile (96.3 %) as the closest relatives. This affiliation was supported by chemotaxonomic data (polar lipids and cellular fatty acids). Physiological and biochemical tests allowed genotypic and phenotypic differentiation of the novel strains from all hitherto recognized species of the genera Caulobacter and Phenylobacterium. The strains therefore represent novel species, for which the names Caulobacter mirabilis sp. nov. (type strain FWC 38T=LMG 24261T=CCUG 55073T), Phenylobacterium conjunctum (type strain FWC 21T=LMG 24262T=CCUG 55074T), the first described prosthecate Phenylobacterium species, and Phenylobacterium haematophilum sp. nov. (type strain LMG 11050T=CCUG 26751T) are proposed. Marker nucleotides within the 16S rRNA genes were determined for the genera Asticcacaulis, Brevundimonas, Caulobacter and Phenylobacterium and the description of the genus Phenylobacterium is emended.

Systems biology of Caulobacter.

The dynamic range of a bacterial species' natural environment is reflected in the complexity of its systems that control cell cycle progression and its range of adaptive responses. We discuss the genetic network and integrated three-dimensional sensor/response systems that regulate the cell cycle and asymmetric cell division in the bacterium Caulobacter crescentus. The cell cycle control circuitry is tied closely to chromosome replication and morphogenesis by multiple feedback pathways from the modular functions that implement the cell cycle. The sophistication of the genetic regulatory circuits and the elegant integration of temporally controlled transcription and protein synthesis with spatially dynamic phosphosignaling and proteolysis pathways, and epigenetic regulatory mechanisms, form a remarkably robust living system.

[A psychrotolerant Caulobacter sp. from Russian polar tundra soil].

Strain Z-0024, a psychrotolerant aerobic heterotrophic representative of the prosthecate bacteria of the genus Caulobacter, was isolated from a methanotrophic enrichment obtained from Russian polar tundra soil. The cells of the new isolate are vibrios (0.5-0.6 x 1.3-1.8 microm) with a polar stalk. The organism grows in a temperature range from 5 to 36 degrees C, with an optimum at 20 degrees C. The pH range for growth is from 4.5 to 7.0 with an optimum at pH 6.0. Strain Z-0024 utilizes a wide range of organic compounds: sugars, amino acids, volatile fatty acids, and primary alcohols. It tolerates a NaCl concentration in the medium of up to 15 g/l. The G + C content of DNA is 66.6 mol %. The 16S rRNA gene sequence analysis revealed that strain Z-0024 belongs to the cluster of Caulobacter species, showing a 98.8-99.2% sequence similarity to them. DNA-DNA hybridization revealed a low level of homology (24%) between strain Z-0024 and C. vibrioides ATCC 15252. The new isolate is described as Caulobacter sp. Z-0024.

A membrane metalloprotease participates in the sequential degradation of a Caulobacter polarity determinant.

Caulobacter crescentus assembles many of its cellular machines at distinct times and locations during the cell cycle. PodJ provides the spatial cues for the biogenesis of several polar organelles, including the pili, adhesive holdfast and chemotactic apparatus, by recruiting structural and regulatory proteins, such as CpaE and PleC, to a specific cell pole. PodJ is a protein with a single transmembrane domain that exists in two forms, full-length (PodJL) and truncated (PodJS), each appearing during a specific time period of the cell cycle to control different aspects of polar organelle development. PodJL is synthesized in the early predivisional cell and is later proteolytically converted to PodJS. During the swarmer-to-stalked transition, PodJS must be degraded to preserve asymmetry in the next cell cycle. We found that MmpA facilitates the degradation of PodJS. MmpA belongs to the site-2 protease (S2P) family of membrane-embedded zinc metalloproteases, which includes SpoIVFB and YluC of Bacillus subtilis and YaeL of Escherichia coli. MmpA appears to cleave within or near the transmembrane segment of PodJS, releasing it into the cytoplasm for complete proteolysis. While PodJS has a specific temporal and spatial address, MmpA is present throughout the cell cycle; furthermore, periplasmic fusion to mRFP1 suggested that MmpA is uniformly distributed around the cell. We also determined that mmpA and yaeL can complement each other in C. crescentus and E. coli, indicating functional conservation. Thus, the sequential degradation of PodJ appears to involve regulated intramembrane proteolysis (Rip) by MmpA.

Spatial complexity of mechanisms controlling a bacterial cell cycle.

Cell cycle progression in Caulobacter is governed by a multilayered regulatory network linking chromosome replication with polar morphogenesis and cell division. Temporal and spatial regulation have emerged as the central themes, with the abundance, activity and subcellular location of key structural and regulatory proteins changing over the course of the cell cycle. An additional layer of complexity was recently uncovered, showing that each segment of the chromosome is located at a specific cellular position both during and after the completion of DNA replication, raising the possibility that this positioning contributes to temporal and spatial control of gene expression.

Cytokinesis monitoring during development; rapid pole-to-pole shuttling of a signaling protein by localized kinase and phosphatase in Caulobacter.

For successful generation of different cell types by asymmetric cell division, cell differentiation should be initiated only after completion of division. Here, we describe a control mechanism by which Caulobacter couples the initiation of a developmental program to the completion of cytokinesis. Genetic evidence indicates that localization of the signaling protein DivK at the flagellated pole prevents premature initiation of development. Photobleaching and FRET experiments show that polar localization of DivK is dynamic with rapid pole-to-pole shuttling of diffusible DivK generated by the localized activities of PleC phosphatase and DivJ kinase at opposite poles. This shuttling is interrupted upon completion of cytokinesis by the segregation of PleC and DivJ to different daughter cells, resulting in disruption of DivK localization at the flagellated pole and subsequent initiation of development in the flagellated progeny. Thus, dynamic polar localization of a diffusible protein provides a control mechanism that monitors cytokinesis to regulate development.

The Karyote physico-chemical genomic, proteomic, metabolic cell modeling system.

Modeling approaches to the dynamics of a living cell are presented that are strongly based on its underlying physical and chemical processes and its hierarchical spatio-temporal organization. Through the inclusion of a broad spectrum of processes and a rigorous analysis of the multiple scale nature of cellular dynamics, we are attempting to advance cell modeling and its applications. The presentation focuses on our cell modeling system, which integrates data archiving and quantitative physico-chemical modeling and information theory to provide a seamless approach to the modeling/data analysis endeavor. Thereby the rapidly growing mess of genomic, proteomic, metabolic, and cell physiological data can be automatically used to develop and calibrate a predictive cell model. The discussion focuses on the Karyote cell modeling system and an introduction to the CellX and VirusX models. The Karyote software system integrates three elements: (1) a model-building and data archiving module that allows one to define a cell type to be modeled through its reaction network, structure, and transport processes as well as to choose the surrounding medium and other parameters of the phenomenon to be modeled; (2) a genomic, proteomic, metabolic cell simulator that solves the equations of metabolic reaction, transcription/translation polymerization and the exchange of molecules between parts of the cell and with the surrounding medium; and (3) an information theory module (ITM) that automates model calibration and development, and integrates a variety of data types with the cell dynamic computations. In Karyote, reactions may be fast (equilibrated) or slow (finite rate), and the special effects of enzymes and other minority species yielding steady-state cycles of arbitrary complexities are accounted for. These features of the dynamics are handled via rigorous multiple scale analysis. A user interface allows for an automated generation and solution of the equations of multiple timescale, compartmented dynamics. Karyote is based on a fixed intracellular structure. However, cell response to changes in the host medium, damage, development or transformation to abnormality can involve dramatic changes in intracellular structure. As this changes the nature of the cellular dynamics, a new model, CellX, is being developed based on the spatial distribution of concentration and other variables. This allows CellX to capture the self-organizing character of cellular behavior. The self-assembly of organelles, viruses, and other subcellular bodies is being addressed in a second new model, VirusX, that integrates molecular mechanics and continuum theory. VirusX is designed to study the influence of a host medium on viral self-assembly, structural stability, infection of a single cell, and transmission of disease.

A bacterial cell-cycle regulatory network operating in time and space.

Transcriptional regulatory circuits provide only a fraction of the signaling pathways and regulatory mechanisms that control the bacterial cell cycle. The CtrA regulatory network, important in control of the Caulobacter cell cycle, illustrates the critical role of nontranscriptional pathways and temporally and spatially localized regulatory proteins. The system architecture of Caulobacter cell-cycle control involves top-down control of modular functions by a small number of master regulatory proteins with cross-module signaling coordinating the overall process. Modeling the cell cycle probably requires a top-down modeling approach and a hybrid control system modeling paradigm to treat its combined discrete and continuous characteristics.

The Caulobacter cell cycle: timing, spatial organization and checkpoints.

In Caulobacter crescentus, a complex regulatory network integrates temporal and spatial information to control the ordered progression of the cell cycle, and to synchronize cell proliferation with development. Periodicity includes the timed synthesis, activation or destruction of key regulatory proteins, which activate a large number of genes at the appropriate time of the cell cycle. Checkpoints serve to couple cell division and polar development to the replication and segregation state of the chromosome. Asymmetrically positioned regulatory components are involved in the sequential positioning of polar organelles. New work sheds light on the spatial organization of cellular components involved in cell cycle progression and polar differentiation, and starts to define the molecular nature of checkpoints involved in cell cycle control and development.

A signal transduction protein cues proteolytic events critical to Caulobacter cell cycle progression.

Temporally controlled proteolysis of the essential response regulator, CtrA, is critical for cell cycle progression in Caulobacter crescentus. CtrA binds to and silences the origin of replication in swarmer cells. The initiation of replication depends on the proteolysis of CtrA. We present evidence that DivK, an essential single-domain response regulator, contributes to the control of the G(1)-S transition by signaling the temporally controlled proteolysis of CtrA. In a divK-cs mutant at the restrictive temperature, the initiation of DNA replication is blocked because of the retention of CtrA. A shift of cells from restrictive to permissive temperature results in rapid degradation of CtrA, initiation of DNA replication, and the resumption of cell cycle progression, including the ordered expression of genes involved in chromosome replication and polar organelle biogenesis. CtrA binds to and regulates the promoters of two genes critical to its temporally controlled proteolysis, divK and clpP, providing a transcriptional feedback loop for the control of cell cycle progression.

Genes directly controlled by CtrA, a master regulator of the Caulobacter cell cycle.

Studies of the genetic network that controls the Caulobacter cell cycle have identified a response regulator, CtrA, that controls, directly or indirectly, one-quarter of the 553 cell cycle-regulated genes. We have performed in vivo genomic binding site analysis of the CtrA protein to identify which of these genes have regulatory regions bound directly by CtrA. By combining these data with previous global analysis of cell cycle transcription patterns and gene expression profiles of mutant ctrA strains, we have determined that CtrA directly regulates at least 95 genes. The total group of CtrA-regulated genes includes those involved in polar morphogenesis, DNA replication initiation, DNA methylation, cell division, and cell wall metabolism. Also among the genes in this notably large regulon are 14 that encode regulatory proteins, including 10 two-component signal transduction regulatory proteins. Identification of additional regulatory genes activated by CtrA will serve to directly connect new regulatory modules to the network controlling cell cycle progression.