scholarly journals Temporal Regulation of Genes Encoding the Flagellar Proximal Rod in Caulobacter crescentus

2001 ◽  
Vol 183 (2) ◽  
pp. 725-735 ◽  
Author(s):  
Charles H. Boyd ◽  
James W. Gober
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Developmental Stages ◽  
Caulobacter Crescentus ◽  
Response Regulator ◽  
Class Ii ◽  
Class Iii ◽  
Swarmer Cell ◽  
Sequencing Project

ABSTRACT The gram-negative bacterium Caulobacter crescentus has a life cycle that includes two distinct and separable developmental stages, a motile swarmer phase and a sessile stalked phase. The cell cycle-controlled biogenesis of the single polar flagellum of the swarmer cell is the best-studied aspect of this developmental program. The flagellar regulon is arranged into a rigid trans-acting hierarchy of gene expression in which successful expression of early genes is required for the expression of genes that are later in the hierarchy and in which the order of gene expression mirrors the order of assembly of gene products into the completed flagellum. TheflgBC-fliE genes were identified as a result of the C. crescentus genome sequencing project and encode the homologues of two flagellar proximal rod proteins, FlgB and FlgC, and one conserved protein, FliE, that is of unknown function. Footprint assays on a DNA fragment containing the operon promoter as well as in vivo mutant suppressor analysis of promoter mutations indicate that this operon is controlled by the cell cycle response regulator CtrA, which with ς70 is responsible for regulating transcription of other early flagellar genes in C. crescentus. Promoter analysis, timing of expression, and epistasis experiments place these genes outside of the flagellar regulatory hierarchy; they are expressed in class II mutants, andflgB deletions do not prevent class III gene expression. This operon is also unusual in that it is expressed from a promoter that is divergent from the class II operon containing fliP, which encodes a member of the flagellum-specific protein export apparatus.

scholarly journals The ChvG–ChvI and NtrY–NtrX two-component systems coordinately regulate growth of Caulobacter crescentus

2021 ◽  
Author(s):  
Benjamin J. Stein ◽  
Aretha Fiebig ◽  
Sean Crosson
Keyword(s):  
Gene Expression ◽  
Phosphatase Activity ◽  
Environmental Changes ◽  
Caulobacter Crescentus ◽  
Genetic Interaction ◽  
Response Regulator ◽  
Cell Envelope ◽  
Signaling Systems ◽  
Two Component

Two-component signaling systems (TCSs) are comprised of a sensory histidine kinase and a response regulator protein. In response to environmental changes, sensor kinases directly phosphorylate their cognate response regulator to affect gene expression. Bacteria typically express multiple TCSs that are insulated from one another and regulate distinct physiological processes. There are certainly examples of cross-regulation between TCSs, but this phenomenon remains relatively unexplored. We have identified regulatory links between the ChvG–ChvI (ChvGI) and NtrY–NtrX (NtrYX) TCSs, which control important and often overlapping processes in α-proteobacteria, including maintenance of the cell envelope. Deletion of chvG and chvI in Caulobacter crescentus limited growth in defined medium and a selection for genetic suppressors of this growth phenotype uncovered interactions among chvGI , ntrYX , and ntrZ , which encodes a previously uncharacterized periplasmic protein. Significant overlap in the experimentally-defined ChvI and NtrX transcriptional regulons provided support for the observed genetic connections between ntrYX and chvGI . Moreover, we present evidence that the growth defect of strains lacking chvGI is influenced by the phosphorylation state of NtrX and, to some extent, by levels of the TonB-dependent receptor ChvT. Measurements of NtrX phosphorylation in vivo indicated that NtrZ is an upstream regulator of NtrY, and that NtrY primarily functions as an NtrX phosphatase. We propose a model in which NtrZ functions in the periplasm to inhibit NtrY phosphatase activity; regulation of phosphorylated NtrX levels by NtrZ and NtrY provides a mechanism to modulate and balance expression of the NtrX and ChvI regulons under different growth conditions. Importance Two-component signaling systems (TCSs) enable bacteria to regulate gene expression in response to physiochemical changes in their environment. The ChvGI and NtrYX TCSs regulate diverse pathways associated with pathogenesis, growth, and cell envelope function in many α-proteobacteria. We used Caulobacter crescentus as a model to investigate regulatory connections between ChvGI and NtrYX. Our work defined the ChvI transcriptional regulon in C. crescentus and revealed a genetic interaction between ChvGI and NtrYX, whereby modulation of NtrYX signaling affects the survival of cells lacking ChvGI. In addition, we identified NtrZ as a periplasmic inhibitor of NtrY phosphatase activity in vivo . Our work establishes C. crescentus as an excellent model to investigate multi-level regulatory connections between ChvGI and NtrYX in α-proteobacteria.

scholarly journals Cell cycle progression inCaulobacterrequires a nucleoid-associated protein with high AT sequence recognition

2016 ◽  
Vol 113 (40) ◽  
pp. E5952-E5961 ◽  
Author(s):  
Dante P. Ricci ◽  
Michael D. Melfi ◽  
Keren Lasker ◽  
David L. Dill ◽  
Harley H. McAdams ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Binding Sites ◽  
Cell Cycle Progression ◽  
Target Genes ◽  
Caulobacter Crescentus ◽  
Regulation Of Gene Expression ◽  
Cycle Progression ◽  
Swarmer Cell ◽  
Sequence Recognition

Faithful cell cycle progression in the dimorphic bacteriumCaulobacter crescentusrequires spatiotemporal regulation of gene expression and cell pole differentiation. We discovered an essential DNA-associated protein, GapR, that is required forCaulobactergrowth and asymmetric division. GapR interacts with adenine and thymine (AT)-rich chromosomal loci, associates with the promoter regions of cell cycle-regulated genes, and shares hundreds of recognition sites in common with known master regulators of cell cycle-dependent gene expression. GapR target loci are especially enriched in binding sites for the transcription factors GcrA and CtrA and overlap with nearly all of the binding sites for MucR1, a regulator that controls the establishment of swarmer cell fate. Despite constitutive synthesis, GapR accumulates preferentially in the swarmer compartment of the predivisional cell. Homologs of GapR, which are ubiquitous among the α-proteobacteria and are encoded on multiple bacteriophage genomes, also accumulate in the predivisional cell swarmer compartment when expressed inCaulobacter. TheEscherichia colinucleoid-associated protein H-NS, like GapR, selectively associates with AT-rich DNA, yet it does not localize preferentially to the swarmer compartment when expressed exogenously inCaulobacter, suggesting that recognition of AT-rich DNA is not sufficient for the asymmetric accumulation of GapR. Further, GapR does not silence the expression of H-NS target genes when expressed inE. coli, suggesting that GapR and H-NS have distinct functions. We propose thatCaulobacterhas co-opted a nucleoid-associated protein with high AT recognition to serve as a mediator of cell cycle progression.

scholarly journals A New Class of Caulobacter crescentusFlagellar Genes

1998 ◽  
Vol 180 (19) ◽  
pp. 5010-5019 ◽  
Author(s):  
Guy Leclerc ◽  
Shui Ping Wang ◽  
Bert Ely
Keyword(s):  
Posttranslational Modification ◽  
Caulobacter Crescentus ◽  
Response Regulator ◽  
Class Ii ◽  
Class Iii ◽  
New Class ◽  
Regulator Protein ◽  
Flagellar Filament ◽  
Response Regulator Protein ◽  
Flagellar Genes

ABSTRACT Eight Caulobacter crescentus flagellar genes,flmA, flmB, flmC, flmD,flmE, flmF, flmG, andflmH, have been cloned and characterized. These eight genes are clustered in pairs (flmAB, flmCD,flmEF, and flmGH) that appear to be structurally organized as operons. Homology comparisons suggest that the proteins encoded by the flm genes may be involved in posttranslational modification of flagellins or proteins that interact with flagellin monomers prior to their assembly into a flagellar filament. Expression of the flmAB, flmEF, andflmGH operons was shown to occur primarily in predivisional cells. In contrast, the flmCD operon was expressed throughout the cell cycle, with only a twofold increase in predivisional cells. The expression of the three temporally regulated operons was subject to positive regulation by the CtrA response regulator protein. Mutations in class II and III flagellar genes had no significant effect on the expression of the flm genes. Furthermore, the flm genes did not affect the expression of class II or class III flagellar genes. However, mutations in theflm genes did result in reduced synthesis of the class IV flagellin proteins. Taken together, these data indicate that theflm operons belong to a new class of flagellar genes.

scholarly journals Multilayered control of chromosome replication in Caulobacter crescentus

2019 ◽  
Vol 47 (1) ◽  
pp. 187-196 ◽  
Author(s):  
Antonio Frandi ◽  
Justine Collier
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Caulobacter Crescentus ◽  
Response Regulator ◽  
Classical Model ◽  
Optimal Growth ◽  
Chromosome Replication ◽  
S Phase ◽  
Circular Chromosome ◽  
Swarmer Cell

Abstract The environmental Alphaproteobacterium Caulobacter crescentus is a classical model to study the regulation of the bacterial cell cycle. It divides asymmetrically, giving a stalked cell that immediately enters S phase and a swarmer cell that stays in the G1 phase until it differentiates into a stalked cell. Its genome consists in a single circular chromosome whose replication is tightly regulated so that it happens only in stalked cells and only once per cell cycle. Imbalances in chromosomal copy numbers are the most often highly deleterious, if not lethal. This review highlights recent discoveries on pathways that control chromosome replication when Caulobacter is exposed to optimal or less optimal growth conditions. Most of these pathways target two proteins that bind directly onto the chromosomal origin: the highly conserved DnaA initiator of DNA replication and the CtrA response regulator that is found in most Alphaproteobacteria. The concerted inactivation and proteolysis of CtrA during the swarmer-to-stalked cell transition license cells to enter S phase, while a replisome-associated Regulated Inactivation and proteolysis of DnaA (RIDA) process ensures that initiation starts only once per cell cycle. When Caulobacter is stressed, it turns on control systems that delay the G1-to-S phase transition or the elongation of DNA replication, most probably increasing its fitness and adaptation capacities.

scholarly journals CtrA, a Global Response Regulator, Uses a Distinct Second Category of Weak DNA Binding Sites for Cell Cycle Transcription Control in Caulobacter crescentus

2009 ◽  
Vol 191 (17) ◽  
pp. 5458-5470 ◽  
Author(s):  
William Spencer ◽  
Rania Siam ◽  
Marie-Claude Ouimet ◽  
D. Patrick Bastedo ◽  
Gregory T. Marczynski
Keyword(s):  
Cell Cycle ◽  
Binding Sites ◽  
Caulobacter Crescentus ◽  
Response Regulator ◽  
Cell Cycle Control ◽  
Transcription Control ◽  
Mobility Shift ◽  
Dna Binding Sites ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT CtrA controls cell cycle programs of chromosome replication and genetic transcription. Phosphorylated CtrA∼P exhibits high affinity (dissociation constant [Kd ], <10 nM) for consensus TTAA-N7-TTAA binding sites with “typical” (N = 7) spacing. We show here that ctrA promoters P1 and P2 use low-affinity (Kd , >500 nM) CtrA binding sites with “atypical” (N ≠ 7) spacing. Footprints demonstrated that phosphorylated CtrA∼P does not exhibit increased affinity for “atypical” sites, as it does for sites in the replication origin. Instead, high levels of CtrA (>10 μM) accumulate, which can drive CtrA binding to “atypical” sites. In vivo cross-linking showed that when the stable CtrAΔ3 protein persists during the cell cycle, the “atypical” sites at ctrA and motB are persistently bound. Interestingly, the cell cycle timing of ctrA P1 and P2 transcription is not altered by persistent CtrAΔ3 binding. Therefore, operator DNA occupancy is not sufficient for regulation, and it is the cell cycle variation of CtrA∼P phosphorylation that provides the dominant “activation” signal. Protein dimerization is one potential means of “activation.” The glutathione S-transferase (GST) protein dimerizes, and fusion with CtrA (GST-CtrA) creates a stable dimer with enhanced affinity for TTAA motifs. Electrophoretic mobility shift assays with GST-CtrA revealed cooperative modes of binding that further distinguish the “atypical” sites. GST-CtrA also binds a single TTAA motif in ctrA P1 aided by DNA in the extended TTAACCAT motif. We discuss how “atypical” sites are a common yet distinct category of CtrA regulatory sites and new implications for the working and evolution of cell cycle control networks.

scholarly journals FlbT Couples Flagellum Assembly to Gene Expression in Caulobacter crescentus

1999 ◽  
Vol 181 (19) ◽  
pp. 6160-6170 ◽  
Author(s):  
Erin K. Mangan ◽  
Jaleh Malakooti ◽  
Anthony Caballero ◽  
Paul Anderson ◽  
Bert Ely ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Caulobacter Crescentus ◽  
Class Iii ◽  
Wild Type ◽  
Temporal Expression ◽  
Flagellar Filament ◽  
Class Iii Genes ◽  
The Stability ◽  
Secretory System

ABSTRACT The biogenesis of the polar flagellum of Caulobacter crescentus is regulated by the cell cycle as well as by atrans-acting regulatory hierarchy that functions to couple flagellum assembly to gene expression. The assembly of early flagellar structures (MS ring, switch, and flagellum-specific secretory system) is required for the transcription of class III genes, which encode the remainder of the basal body and the external hook structure. Similarly, the assembly of class III gene-encoded structures is required for the expression of the class IV flagellins, which are incorporated into the flagellar filament. Here, we demonstrate that mutations inflbT, a flagellar gene of unknown function, can restore flagellin protein synthesis and the expression offljK::lacZ (25-kDa flagellin) protein fusions in class III flagellar mutants. These results suggest that FlbT functions to negatively regulate flagellin expression in the absence of flagellum assembly. Deletion analysis shows that sequences within the 5′ untranslated region of the fljK transcript are sufficient for FlbT regulation. To determine the mechanism of FlbT-mediated regulation, we assayed the stability of fljKmRNA. The half-life (t 1/2) of fljKmRNA in wild-type cells was approximately 11 min and was reduced to less than 1.5 min in a flgE (hook) mutant. A flgE flbT double mutant exhibited an mRNA t 1/2of greater than 30 min. This suggests that the primary effect of FlbT regulation is an increased turnover of flagellin mRNA. The increasedt 1/2 of fljK mRNA in aflbT mutant has consequences for the temporal expression offljK. In contrast to the case for wild-type cells,fljK::lacZ protein fusions in the mutant are expressed almost continuously throughout the C. crescentus cell cycle, suggesting that coupling of flagellin gene expression to assembly has a critical influence on regulating cell cycle expression.

scholarly journals A Membrane-Associated Protein, FliX, Is Required for an Early Step in Caulobacter Flagellar Assembly

1998 ◽  
Vol 180 (8) ◽  
pp. 2175-2185 ◽  
Author(s):  
Christian D. Mohr ◽  
Joanna K. MacKichan ◽  
Lucy Shapiro
Keyword(s):  
Cell Cycle ◽  
Caulobacter Crescentus ◽  
Signal Sequence ◽  
Early Stage ◽  
Class Ii ◽  
Flagellar Gene ◽  
Class Iii ◽  
Isolation And Characterization ◽  
Flagellar Assembly ◽  
Flagellar Genes

ABSTRACT The ordered assembly of the Caulobacter crescentusflagellum is accomplished in part through the organization of the flagellar structural genes in a regulatory hierarachy of four classes. Class II genes are the earliest to be expressed and are activated at a specific time in the cell cycle by the CtrA response regulator. In order to identify gene products required for early events in flagellar assembly, we used the known phenotypes of class II mutants to identify new class II flagellar genes. In this report we describe the isolation and characterization of a flagellar gene, fliX. AfliX null mutant is nonmotile, lacks a flagellum, and exhibits a marked cell division defect. Epistasis experiments placedfliX within class II of the flagellar regulatory hierarchy, suggesting that FliX functions at an early stage in flagellar assembly. The fliX gene encodes a 15-kDa protein with a putative N-terminal signal sequence. Expression of fliX is under cell cycle control, with transcription beginning relatively early in the cell cycle and peaking in Caulobacter predivisional cells. Full expression of fliX was found to be dependent onctrA, and DNase I footprinting analysis demonstrated a direct interaction between CtrA and the fliX promoter. ThefliX gene is located upstream and is divergently transcribed from the class III flagellar gene flgI, which encodes the basal body P-ring monomer. Analysis of thefliX-flgI intergenic region revealed an arrangement ofcis-acting elements similar to that of another set ofCaulobacter class II and class III flagellar genes,fliL-flgF, that is also divergently transcribed. In parallel with the FliL protein, FliX copurifies with the membrane fraction, and although its expression is cell cycle controlled, the protein is present throughout the cell cycle.

scholarly journals Loss of the Response Regulator CtrA Causes Pleiotropic Effects on Gene Expression but Does Not Affect Growth Phase Regulation in Rhodobacter capsulatus

2010 ◽  
Vol 192 (11) ◽  
pp. 2701-2710 ◽  
Author(s):  
Ryan G. Mercer ◽  
Stephen J. Callister ◽  
Mary S. Lipton ◽  
Ljiljana Pasa-Tolic ◽  
Hynek Strnad ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Rhodobacter Capsulatus ◽  
Caulobacter Crescentus ◽  
Response Regulator ◽  
Sequence Similarity ◽  
Stationary Phases ◽  
Photosynthetic Bacterium ◽  
Pleiotropic Effects ◽  
Metabolic Versatility

ABSTRACT The purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus has been extensively studied for its metabolic versatility as well as for production of a gene transfer agent called RcGTA. Production of RcGTA is highest in the stationary phase of growth and requires the response regulator protein CtrA. The CtrA protein in Caulobacter crescentus has been thoroughly studied for its role as an essential, master regulator of the cell cycle. Although the CtrA protein in R. capsulatus shares a high degree of sequence similarity with the C. crescentus protein, it is nonessential and clearly plays a different role in this bacterium. We have used transcriptomic and proteomic analyses of wild-type and ctrA mutant cultures to identify the genes dysregulated by the loss of CtrA in R. capsulatus. We have also characterized gene expression differences between the logarithmic and stationary phases of growth. Loss of CtrA has pleiotropic effects, with dysregulation of expression of ∼6% of genes in the R. capsulatus genome. This includes all flagellar motility genes and a number of other putative regulatory proteins but does not appear to include any genes involved in the cell cycle. Quantitative proteomic data supported 88% of the CtrA transcriptome results. Phylogenetic analysis of CtrA sequences supports the hypothesis of an ancestral ctrA gene within the alphaproteobacteria, with subsequent diversification of function in the major alphaproteobacterial lineages.

scholarly journals Differential Expression of the CO2 Fixation Operons of Rhodobacter sphaeroides by the Prr/Reg Two-Component System during Chemoautotrophic Growth

2002 ◽  
Vol 184 (23) ◽  
pp. 6654-6664 ◽  
Author(s):  
Janet L. Gibson ◽  
James M. Dubbs ◽  
F. Robert Tabita
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Cytochrome Oxidase ◽  
Rhodobacter Sphaeroides ◽  
Rhodobacter Capsulatus ◽  
Response Regulator ◽  
Signal Transduction Pathway ◽  
Pentose Phosphate ◽  
Bisphosphate Carboxylase

ABSTRACT In Rhodobacter sphaeroides, the two cbb operons encoding duplicated Calvin-Benson Bassham (CBB) CO2 fixation reductive pentose phosphate cycle structural genes are differentially controlled. In attempts to define the molecular basis for the differential regulation, the effects of mutations in genes encoding a subunit of Cbb3 cytochrome oxidase, ccoP, and a global response regulator, prrA (regA), were characterized with respect to CO2 fixation (cbb) gene expression by using translational lac fusions to the R. sphaeroides cbb I and cbbII promoters. Inactivation of the ccoP gene resulted in derepression of both promoters during chemoheterotophic growth, where cbb expression is normally repressed; expression was also enhanced over normal levels during phototrophic growth. The prrA mutation effected reduced expression of cbbI and cbbII promoters during chemoheterotrophic growth, whereas intermediate levels of expression were observed in a double ccoP prrA mutant. PrrA and ccoP1 prrA strains cannot grow phototrophically, so it is impossible to examine cbb expression in these backgrounds under this growth mode. In this study, however, we found that PrrA mutants of R. sphaeroides were capable of chemoautotrophic growth, allowing, for the first time, an opportunity to directly examine the requirement of PrrA for cbb gene expression in vivo under growth conditions where the CBB cycle and CO2 fixation are required. Expression from the cbbII promoter was severely reduced in the PrrA mutants during chemoautotrophic growth, whereas cbbI expression was either unaffected or enhanced. Mutations in ccoQ had no effect on expression from either promoter. These observations suggest that the Prr signal transduction pathway is not always directly linked to Cbb3 cytochrome oxidase activity, at least with respect to cbb gene expression. In addition, lac fusions containing various lengths of the cbbI promoter demonstrated distinct sequences involved in positive regulation during photoautotrophic versus chemoautotrophic growth, suggesting that different regulatory proteins may be involved. In Rhodobacter capsulatus, ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) expression was not affected by cco mutations during photoheterotrophic growth, suggesting that differences exist in signal transduction pathways regulating cbb genes in the related organisms.

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