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Degradation of Lon in Caulobacter crescentus.

ABSTRACT: 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.

SUBMITTER: Barros BB

PROVIDER: S-EPMC7723953 | biostudies-literature |

REPOSITORIES: biostudies-literature

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ClpAP is an auxiliary protease for DnaA degradation in Caulobacter crescentus.

Project description:The Clp family of proteases is responsible for controlling both stress responses and normal growth. In Caulobacter crescentus, the ClpXP protease is essential and drives cell cycle progression through adaptor-mediated degradation. By contrast, the physiological role for the ClpAP protease is less well understood with only minor growth defects previously reported for ?clpA cells. Here, we show that ClpAP plays an important role in controlling chromosome content and cell fitness during extended growth. Cells lacking ClpA accumulate aberrant numbers of chromosomes upon prolonged growth suggesting a defect in replication control. Levels of the replication initiator DnaA are elevated in ?clpA cells and degradation of DnaA is more rapid in cells lacking the ClpA inhibitor ClpS. Consistent with this observation, ClpAP degrades DnaA in vitro while ClpS inhibits this degradation. In cells lacking Lon, the protease previously shown to degrade DnaA in Caulobacter, ClpA overexpression rescues defects in fitness and restores degradation of DnaA. Finally, we show that cells lacking ClpA are particularly sensitive to inappropriate increases in DnaA activity. Our work demonstrates an unexpected effect of ClpAP in directly regulating replication through degradation of DnaA and expands the functional role of ClpAP in Caulobacter.

| S-EPMC5161677 | biostudies-literature

The Aerotactic Response of Caulobacter crescentus.

Project description:Many motile microorganisms are able to detect chemical gradients in their surroundings to bias their motion toward more favorable conditions. In this study, we observe the swimming patterns of Caulobacter crescentus, a uniflagellated bacterium, in a linear oxygen gradient produced by a three-channel microfluidic device. Using low-magnification dark-field microscopy, individual cells are tracked over a large field of view and their positions within the oxygen gradient are recorded over time. Motor switching events are identified so that swimming trajectories are deconstructed into a series of forward and backward swimming runs. Using these data, we show that C. crescentus displays aerotactic behavior by extending the average duration of forward swimming runs while moving up an oxygen gradient, resulting in directed motility toward oxygen sources. Additionally, the motor switching response is sensitive both to the steepness of the gradient experienced and to background oxygen levels, exhibiting a logarithmic response.

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Complete genome sequence of Caulobacter crescentus.

Project description:The complete genome sequence of Caulobacter crescentus was determined to be 4,016,942 base pairs in a single circular chromosome encoding 3,767 genes. This organism, which grows in a dilute aquatic environment, coordinates the cell division cycle and multiple cell differentiation events. With the annotated genome sequence, a full description of the genetic network that controls bacterial differentiation, cell growth, and cell cycle progression is within reach. Two-component signal transduction proteins are known to play a significant role in cell cycle progression. Genome analysis revealed that the C. crescentus genome encodes a significantly higher number of these signaling proteins (105) than any bacterial genome sequenced thus far. Another regulatory mechanism involved in cell cycle progression is DNA methylation. The occurrence of the recognition sequence for an essential DNA methylating enzyme that is required for cell cycle regulation is severely limited and shows a bias to intergenic regions. The genome contains multiple clusters of genes encoding proteins essential for survival in a nutrient poor habitat. Included are those involved in chemotaxis, outer membrane channel function, degradation of aromatic ring compounds, and the breakdown of plant-derived carbon sources, in addition to many extracytoplasmic function sigma factors, providing the organism with the ability to respond to a wide range of environmental fluctuations. C. crescentus is, to our knowledge, the first free-living alpha-class proteobacterium to be sequenced and will serve as a foundation for exploring the biology of this group of bacteria, which includes the obligate endosymbiont and human pathogen Rickettsia prowazekii, the plant pathogen Agrobacterium tumefaciens, and the bovine and human pathogen Brucella abortus.

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Small non-coding RNAs in Caulobacter crescentus.

Project description:Small non-coding RNAs (sRNAs) are active in many bacterial cell functions, including regulation of the cell's response to environmental challenges. We describe the identification of 27 novel Caulobacter crescentus sRNAs by analysis of RNA expression levels assayed using a tiled Caulobacter microarray and a protocol optimized for detection of sRNAs. The principal analysis method involved identification of sets of adjacent probes with unusually high correlation between the individual intergenic probes within the set, suggesting presence of a sRNA. Among the validated sRNAs, two are candidate transposase gene antisense RNAs. The expression of 10 of the sRNAs is regulated by either entry into stationary phase, carbon starvation, or rich versus minimal media. The expression of four of the novel sRNAs changes as the cell cycle progresses. One of these shares a promoter motif with several genes expressed at the swarmer-to-stalked cell transition; while another appears to be controlled by the CtrA global transcriptional regulator. The probe correlation analysis approach reported here is of general use for large-scale sRNA identification for any sequenced microbial genome.

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An RNA degradosome assembly in Caulobacter crescentus.

Project description:In many bacterial species, the multi-enzyme RNA degradosome assembly makes key contributions to RNA metabolism. Powering the turnover of RNA and the processing of structural precursors, the RNA degradosome has differential activities on a spectrum of transcripts and contributes to gene regulation at a global level. Here, we report the isolation and characterization of an RNA degradosome assembly from the ?-proteobacterium Caulobacter crescentus, which is a model organism for studying morphological development and cell-cycle progression. The principal components of the C. crescentus degradosome are the endoribonuclease RNase E, the exoribonuclease polynucleotide phosphorylase (PNPase), a DEAD-box RNA helicase and the Krebs cycle enzyme aconitase. PNPase and aconitase associate with specific segments in the C-terminal domain of RNase E that are predicted to have structural propensity. These recognition 'microdomains' punctuate structurally an extensive region that is otherwise predicted to be natively disordered. Finally, we observe that the abundance of RNase E varies through the cell cycle, with maxima at morphological differentiation and cell division. This variation may contribute to the program of gene expression during cell division.

| S-EPMC3045602 | biostudies-literature

Expression analysis of Caulobacter crescentus NA1000 Plac::CCNA_00382 (ccrM) vs Caulobacter crescentus

Project description:Investigation of whole genome gene expression level changes in a Caulonacter crescentus NA1000 Plac::CCNA_00382 (ccrM) mutant, compared to the wild-type strain. The mutations engineered into this strain cause the CcrM DNA methyltransferase to be overexpressed and the chromosome to be constitutively methylated at the adenine at GANTC motifs. References of strains: CcrMOE: Collier, J. and Shapiro, L. (2009) Feedback control of DnaA-mediated replication initiation by replisome-associated HdaA protein in Caulobacter. J Bacteriol, 191, 5706-5716. WT: Marks, M.E., Castro-Rojas, C.M., Teiling, C., Du, L., Kapatral, V., Walunas, T.L. and Crosson, S. (2010) The genetic basis of laboratory adaptation in Caulobacter crescentus. J Bacteriol, 192, 3678-3688; Collier, J. and Shapiro, L. (2009) Feedback control of DnaA-mediated replication initiation by replisome-associated HdaA protein in Caulobacter. J Bacteriol, 191, 5706-5716. A six chip study using total RNA recovered from three separate wild-type cultures of Caulonacter crescentus NA1000 and three separate cultures of a triple mutant strain, Caulonacter crescentus NA1000 Plac::CCNA_00382 (ccrM), in which the ccrM gene coding for a DNA methyltransferase methylating the adenine in GANTC motifs is truncated and its product inactive. Each chip measures the expression level of 3933 genes from Caulobacter crescentus NA1000 with 3 probes per gene and with three-fold technical redundancy.

2014-01-08 | E-GEOD-52722 | biostudies-arrayexpress

CauloBrowser: A systems biology resource for Caulobacter crescentus.

Project description:Caulobacter crescentus is a premier model organism for studying the molecular basis of cellular asymmetry. The Caulobacter community has generated a wealth of high-throughput spatiotemporal databases including data from gene expression profiling experiments (microarrays, RNA-seq, ChIP-seq, ribosome profiling, LC-ms proteomics), gene essentiality studies (Tn-seq), genome wide protein localization studies, and global chromosome methylation analyses (SMRT sequencing). A major challenge involves the integration of these diverse data sets into one comprehensive community resource. To address this need, we have generated CauloBrowser (www.caulobrowser.org), an online resource for Caulobacter studies. This site provides a user-friendly interface for quickly searching genes of interest and downloading genome-wide results. Search results about individual genes are displayed as tables, graphs of time resolved expression profiles, and schematics of protein localization throughout the cell cycle. In addition, the site provides a genome viewer that enables customizable visualization of all published high-throughput genomic data. The depth and diversity of data sets collected by the Caulobacter community makes CauloBrowser a unique and valuable systems biology resource.

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Project description:Podophage Percy infects Caulobacter crescentus, a Gram-negative bacterium that divides asymmetrically and is a commonly used model organism to study the cell cycle, asymmetric cell division, and cell differentiation. Here, we announce the sequence and annotated complete genome of the phiKMV-like podophage Percy and note its prominent features.

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Complete genome sequence of Caulobacter crescentus bacteriophage ?CbK.

Project description:?CbK is a B3 morphotype bacteriophage of the Siphoviridae family that infects Caulobacter crescentus, the preeminent model system for bacterial cell cycle studies. The last 4 decades of research with ?CbK as a genetic and cytological tool to study the biology of the host warrant an investigation of the phage genome composition. Herein, we report the complete genome sequence of ?CbK and highlight unusual features that emerged from its annotation. The complete genome analysis of the ?CbK phage provides new insight into its characteristics and potential interactions with its Caulobacter crescentus host, setting the stage for future functional studies with ?CbK.

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