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U Jenal

Publications and source records attributed to U Jenal.

18 recordsLinked to original sources
PubMed2005

Protease susceptibility of the Caulobacter crescentus flagellar hook-basal body: a possible mechanism of flagellar ejection during cell differentiation.

When motile swarmer cells of Caulobacter crescentus differentiate into sessile stalked cells, the flagellum is ejected. To elucidate the molecular mechanism of the flagellar ejection, flagellar hook-basal body (HBB) complexes from C. crescentus were purified and characterized. The purified HBBs were less stable against acidic pH or protease treatment than HBBs of Salmonella typhimurium, supporting the view that flagellar ejection from C. crescentus is initiated by destruction of the fragile basal structures. In addition, protease treatment of the purified flagella resulted in the specific digestion of the MS ring complex, revealing for the first time the intact structure of the whole rod.

Amino Acid Sequence↗
PubMed2001

Proteomic analysis of the bacterial cell cycle.

A global approach was used to analyze protein synthesis and stability during the cell cycle of the bacterium Caulobacter crescentus. Approximately one-fourth (979) of the estimated C. crescentus gene products were detected by two-dimensional gel electrophoresis, 144 of which showed differential cell cycle expression patterns. Eighty-one of these proteins were identified by mass spectrometry and were assigned to a wide variety of functional groups. Pattern analysis revealed that coexpression groups were functionally clustered. A total of 48 proteins were rapidly degraded in the course of one cell cycle. More than half of these unstable proteins were also found to be synthesized in a cell cycle-dependent manner, establishing a strong correlation between rapid protein turnover and the periodicity of the bacterial cell cycle. This is, to our knowledge, the first evidence for a global role of proteolysis in bacterial cell cycle control.

Caulobacter crescentus↗
PubMed2001

Precise amounts of a novel member of a phosphotransferase superfamily are essential for growth and normal morphology in Caulobacter crescentus.

The Caulobacter crescentus chromosomal clp locus contains the genes encoding the components of ClpXP, a multisubunit protease required for cell cycle progression in this organism. Here, we report the identification and characterization of cicA, a gene located between the clpX and clpP genes on the Caulobacter chromosome. cicA is a novel morphogene in C. crescentus and, like clpX and clpP, is essential for growth. A conditional cicA mutant stopped growth, but retained viability under restrictive conditions. In contrast, an increased concentration of CicA led to an immediate loss of the normal rod shape, an almost 10-fold increase of the cell's volume and a cell division block. In parallel with this drastic morphological change, cells rapidly lost viability. Primary sequence analysis suggested that the cicA gene encodes a member of a large superfamily of phosphotransferases, that include phosphoserine phosphatases, the ATPase domain of P-type ATPases and receiver domains of response regulators. Four conserved motifs of this protein family that have been implicated in the catalysis of phosphotransfer reactions were investigated by site-directed mutagenesis and were found to be critical for in vivo function of CicA. Based on our observations, we postulate that CicA is involved in essential phosphotransferase reactions in Caulobacter and that increased activity of CicA has a deleterious effect on cell wall biosynthesis, morphogenesis and cell division.

Adenosine Triphosphatases↗
PubMed2000

Signal transduction mechanisms in Caulobacter crescentus development and cell cycle control.

The life cycle of the aquatic bacterium Caulobacter crescentus includes an asymmetric cell division and an obligate cell differentiation. Each cell division gives rise to a motile but replication inert swarmer cell and a sessile, replication competent stalked cell. While the stalked progeny immediately reinitiates DNA replication and cell division, the swarmer cell remains motile and chemotactically active for a constant period of the cell cycle before it differentiates into a stalked cell. During this process, the cell looses motility by ejecting the flagellum, synthesizes a stalk and eventually initiates chromosome replication and cell division. The link of morphogenic transitions to the replicative cycle of Caulobacter implies that the developmental programs which determine asymmetry and cell differentiation must be tightly connected with cell cycle control. This has been confirmed by the recent identification of signal transduction mechanisms, which are involved in temporal and spatial control of both development and cell cycle. Interestingly, the cell has recruited two-component signal transduction systems for this internal control, a family of regulatory proteins which usually are involved in the information transfer between the environment and the inside of a cell. The response regulator protein CtrA controls several key cell cycle events like the initiation of DNA replication, DNA methylation, cell division, and flagellar biogenesis. The activity of this master regulator is subject to complex temporal and spatial control during the C. crescentus cell cycle, including regulated transcription, phosphorylation and degradation. Three membrane bound sensor kinases have been proposed to control the phosphorylation status of CtrA. Two of these, CckA and DivJ, exhibit specific subcellular localization and, in the case of CckA, dynamic rearrangement in the course of the cell cycle. These findings support the idea that the developmental program of C. crescentus is controlled at least in part by localized cues that act as checkpoints for the control of morphological changes and cell cycle progression.

Caulobacter crescentus↗
PubMed2000

Regulatory circuits in Caulobacter.

The transcriptional regulator CtrA controls DNA replication, DNA methylation and cell division during the Caulobacter cell cycle. Recent work on this master cell cycle regulator has provided insight into its control mechanisms. Feedback regulation of ctrA transcription together with timed proteolysis of CtrA determine the levels of the regulator during the cell cycle. Multiple phosphorylation pathways regulate CtrA activity by a mechanism that includes dynamic subcellular localization of the corresponding sensor kinases.

Bacterial Proteins↗
PubMed1999

Cell cycle-dependent degradation of a flagellar motor component requires a novel-type response regulator.

The poles of each Caulobacter crescentus cell undergo morphological development as a function of the cell cycle. A single flagellum assembled at one pole during the asymmetric cell division is later ejected and replaced by a newly synthesized stalk when the motile swarmer progeny differentiates into a sessile stalked cell. The removal of the flagellum during the swarmer-to-stalked cell transition coincides with the degradation of the FliF flagellar anchor protein. We report here that the cell cycle-dependent turnover of FliF does not require the structural components of the flagellum itself, arguing that it is the initial event leading to the ejection of the flagellum. Analysis of a polar development mutant, pleD, revealed that the pleD gene was required for efficient removal of FliF and for ejection of the flagellar structure during the swarmer-to-stalked cell transition. The PleD requirement for FliF degradation was also not dependent on the presence of any part of the flagellar structure. In addition, only 25% of the cells were able to synthesize a stalk during cell differentiation when PleD was absent. The pleD gene codes for a member of the response regulator family with a novel C-terminal regulatory domain. Mutational analysis confirmed that a highly conserved motif in the PleD C-terminal domain is essential to promote both FliF degradation and stalk biogenesis during cell differentiation. Signalling through the C-terminal domain of PleD is thus required for C. crescentus polar development. A second gene, fliL, was shown to be required for efficient turnover of FliF, but not for stalk biogenesis. The possible roles of PleD and FliL in C. crescentus polar development are discussed.

Bacterial Proteins↗
PubMed1999

Identification and transcriptional control of the genes encoding the Caulobacter crescentus ClpXP protease.

The region of the Caulobacter crescentus chromosome harboring the genes for the ClpXP protease was isolated and characterized. Comparison of the deduced amino acid sequences of the C. crescentus ClpP and ClpX proteins with those of their homologues from several gram-positive and gram-negative bacteria revealed stronger conservation for the ATPase regulatory subunit (ClpX) than for the peptidase subunit (ClpP). The C. crescentus clpX gene was shown by complementation analysis to be functional in Escherichia coli. However, clpX from E. coli was not able to substitute for the essential nature of the clpX gene in C. crescentus. The clpP and clpX genes are separated on the C. crescentus chromosome by an open reading frame pointing in the opposite direction from the clp genes, and transcription of clpP and clpX was found to be uncoupled. clpP is transcribed as a monocistronic unit with a promoter (PP1) located immediately upstream of the 5' end of the gene and a terminator structure following its 3' end. PP1 is under heat shock control and is induced upon entry of the cells into the stationary phase. At least three promoters for clpX (PX1, PX2, and PX3) were mapped in the clpP-clpX intergenic region. In contrast to PP1, the clpX promoters were found to be downregulated after heat shock but were also subject to growth phase control. In addition, the clpP and clpX promoters showed different activity patterns during the cell cycle. Together, these results demonstrate that the genes coding for the peptidase and the regulatory subunits of the ClpXP protease are under independent transcriptional control in C. crescentus. Determination of the numbers of ClpP and ClpX molecules per cell suggested that ClpX is the limiting component compared with ClpP.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1998

An essential protease involved in bacterial cell-cycle control.

Proteolytic inactivation of key regulatory proteins is essential in eukaryotic cell-cycle control. We have identified a protease in the eubacterium Caulobacter crescentus that is indispensable for viability and cell-cycle progression, indicating that proteolysis is also involved in controlling the bacterial cell cycle. Mutants of Caulobacter that lack the ATP-dependent serine protease ClpXP are arrested in the cell cycle before the initiation of chromosome replication and are blocked in the cell division process. ClpXP is composed of two types of polypeptides, the ClpX ATPase and the ClpP peptidase. Site-directed mutagenesis of the catalytically active serine residue of ClpP confirmed that the proteolytic activity of ClpXP is essential. Analysis of mutants lacking ClpX or ClpP revealed that both proteins are required in vivo for the cell-cycle-dependent degradation of the regulatory protein CtrA. CtrA is a member of the response regulator family of two-component signal transduction systems and controls multiple cell-cycle processes in Caulobacter. In particular, CtrA negatively controls DNA replication and our findings suggest that specific degradation of the CtrA protein by the ClpXP protease contributes to G1-to-S transition in this organism.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1997

Isolation and characterization of a xylose-dependent promoter from Caulobacter crescentus.

An inducible promoter is a useful tool for the controlled expression of a given gene. Accordingly, we identified, cloned, and sequenced a chromosomal locus, xylX, from Caulobacter crescentus which is required for growth on xylose as the sole carbon source and showed that transcription from a single site is dependent on the presence of xylose in the growth medium. P(xylX) promoter activity was determined as a function of the composition of the growth medium both in single copy and on a plasmid using different reporter genes. One hundred micromolar exogenously added xylose was required for maximal induction of P(xylX) in a strain that is unable to metabolize xylose. P(xylX) activity was induced immediately after the addition of xylose and repressed almost completely when xylose was removed from the growth medium. In addition to the strong transcriptional control, the expression of xylX is also regulated on the translational level.

Amino Acid Sequence↗
PubMed1996

Cell cycle-controlled proteolysis of a flagellar motor protein that is asymmetrically distributed in the Caulobacter predivisional cell.

Flagellar biogenesis and release are developmental events tightly coupled to the cell cycle of Caulobacter crescentus. A single flagellum is assembled at the swarmer pole of the predivisional cell and is released later in the cell cycle. Here we show that the MS-ring monomer FliF, a central motor component that anchors the flagellum in the cell membrane, is synthesized only in the predivisional cell and is integrated into the membrane at the incipient swarmer cell pole, where it initiates flagellar assembly. FliF is proteolytically turned over during swarmer-to-stalked cell differentiation, coinciding with the loss of the flagellum, suggesting that its degradation is coupled to flagellar release. The membrane topology of FliF was determined and a region of the cytoplasmic C-terminal domain was shown to be required for the interaction with a component of the motor switch. The very C-terminal end of FliF contains a turnover determinant, required for the cell cycle-dependent degradation of the MS-ring. The cell cycle-dependent proteolysis of FliF and the targeting of FliF to the swarmer pole together contribute to the asymmetric localization of the MS-ring in the predivisional cell.

Amino Acid Sequence↗
PubMed1996

Bacterial differentiation: sizing up sporulation.

New results on Bacillus subtilis sporulation suggest that size differences between the post-septation compartments trigger differential gene expression, which is then coordinated by communication between the nascent mother cell and forespore compartments.

Bacillus subtilis↗
PubMed1996

Flagellar assembly in Caulobacter crescentus: a basal body P-ring null mutation affects stability of the L-ring protein.

The P- and L-rings are structural components of the flagellar basal body that are positioned in the periplasmic space and outer membrane, respectively. In order to explore the mechanism of P- and L-ring assembly, we examined the effect of a null mutation in the gene encoding the P-ring subunit, FlgI, on the expression, stability, and subcellular localization of the L-ring subunit, FlgH, in Caulobacter crescentus. Transcription of the L-ring gene and synthesis of the L-ring protein were both increased in the P-ring null mutant. However, steady-state L-ring protein levels were dramatically reduced compared with those of wild type. This reduction, which was not observed in flagellar hook mutants, was due to a decreased stability of the L-ring protein. The instability of the L-ring protein was apparent throughout the cell cycle of the P-ring mutant and contrasted with the fairly constant level of L-ring protein during the cell cycle of wild-type cells. Low levels of the L-ring protein were detected exclusively in the cell envelope of cells lacking the P-ring, suggesting that, in the absence of P-ring assembly, L-ring monomers are unable to form multimeric rings and are thus subject to proteolysis in the periplasm.

Bacterial Proteins↗
PubMed1994

Caulobacter flagellar function, but not assembly, requires FliL, a non-polarly localized membrane protein present in all cell types.

Caulobacter crescentus has a single polar flagellum, which is assembled in the predivisional cell. Known flagellar genes encode structural and regulatory components that are required for flagellar assembly and function. These genes are organized in several classes which form a transcriptional regulatory hierarchy. A member of the Class II genes, the fliLM operon, encodes homologs of the Escherichia coli flagellar switch protein, FliM, and a protein with a hitherto unknown function, FliL. We report here that flagellar rotation requires the FliL protein. In-frame deletions in the chromosomal copy of the fliL gene result in cells that form a flagellum but are non-motile. The FliL protein was found to be associated with the inner membrane and to be present in all cell types. This is the first report of a Caulobacter crescentus protein that is essential for motility but is not spatially restricted to the region of the flagellar basal body. Although FliL is required for flagellar function, it is not part of the transcriptional hierarchy, supporting the hypothesis that, as is the case for the enterics, the regulatory hierarchy responds to assembly cues rather than directly to the expression of flagellar proteins.

Amino Acid Sequence↗
PubMed1994

Regulation of tryptophan biosynthesis in Methanobacterium thermoautotrophicum Marburg.

A tryptophan-auxotrophic mutant of the archaeon Methanobacterium thermoautotrophicum Marburg was grown with growth-promoting and growth-limiting concentrations of tryptophan. The specific activities of anthranilate synthase (TrpEG) and tryptophan synthase (TrpB) increased 30- to 40-fold in tryptophan-starved cells. Levels of trpE-specific and trpD-specific mRNAs (transcripts of the first and the last genes, respectively, of the M. thermoautotrophicum Marburg trp gene cluster) increased about 10-fold upon starvation for tryptophan. Thus, the expression of the trp genes appears to be regulated primarily at the level of transcription. These data support transcription of trp genes as an operon and support a regulatory model involving a repressor. Anthranilate synthase was feedback inhibited by L-tryptophan, with a Ki of 3.0 microM. In a leucine-auxotrophic mutant starved for L-leucine, the level of alpha-isopropylmalate synthase (LeuA) was 10-fold higher than in cells grown with L-leucine. In addition to the finding of specific regulation of gene expression by the end products of their respective pathways, it was found that the levels of anthranilate synthase and alpha-isopropylmalate synthase were reduced upon growth in the presence of amino acids of other families, such as L-alanine, L-proline, or L-arginine. Conversely, starvation for tryptophan caused a slight elevation of alpha-isopropylmalate synthase and starvation for leucine caused a significant increase of anthranilate synthase and tryptophan synthase specific activities. The latter effect was also observed at the level of trp-specific mRNA and is reminiscent of general amino acid control.

2-Isopropylmalate Synthase↗
PubMed1993

Transcription of the ileS operon in the archaeon Methanobacterium thermoautotrophicum Marburg.

In the thermophilic archaeon Methanobacterium thermoautotrophicum Marburg, the structural gene for isoleucyl-tRNA synthetase (ileS) is flanked upstream by orf401 and downstream by purL. orf401 encodes a 43.5-kDa protein with an unknown function. Northern (RNA) hybridization and S1 nuclease protection experiments showed that the orf401, ileS, and purL genes are cotranscribed from an archael consensus promoter in front of orf401. The corresponding transcript was about eightfold increased in cells that had been exposed to pseudomonic acid A, a specific inhibitor of isoleucyl-tRNA synthetase. Growth inhibition by puromycin, tryptophan starvation, or starvation for hydrogen did not affect the level of this transcript. The level of a trpE transcript, however, was drastically elevated upon tryptophan starvation, while inhibition by pseudomonic acid A had no effect on the level of this transcript. Expression of ileS thus appears to be controlled by a regulatory mechanism which specifically responds to the availability of isoleucyl-tRNA. Extensive decay of the orf401-ileS-purL message was observed. Degradation occurred, presumably by endonucleolytic cleavage, within the orf401 region.

Amino Acid Sequence↗
PubMed1991

Isoleucyl-tRNA synthetase of Methanobacterium thermoautotrophicum Marburg. Cloning of the gene, nucleotide sequence, and localization of a base change conferring resistance to pseudomonic acid.

The ileS gene encoding the isoleucyl-tRNA synthetase of the thermophilic archaebacterium Methanobacterium thermoautotrophicum Marburg was isolated and sequenced. ileS was closely flanked by an unknown open reading frame and by purL and thus is arranged differently from the organizations observed in several eubacteria or in Saccharomyces cerevisiae. The deduced amino acid sequence of isoleucyl-tRNA synthetase was compared with primary sequences of isoleucyl-, valyl-, leucyl-, and methionyl-tRNA synthetases from eubacteria and yeast. The archaebacterial enzyme fitted well into this group of enzymes. It contained the two short consensus sequences observed in class I aminoacyl-tRNA synthetases as well as regions of homology with enzymes of the isoleucine family. Comparison between the isoleucyl-tRNA synthetases of M. thermoautotrophicum yielded 36% amino acid identity with the yeast enzyme and 32% identity with the corresponding enzyme from Escherichia coli. The ileS gene of the pseudomonic acid-resistant M. thermoautotrophicum mutant MBT10 was also sequenced. The mutant enzyme had undergone a glycine to aspartic acid transition at position 590, in a conserved region comprising the KMSKS consensus sequence. The inhibition constants of pseudomonic acid, KiIle and KiATP, for the mutant enzyme were 10-fold higher than those determined for the wild-type enzyme. Both the mutant and the wild-type ileS gene were expressed in E. coli, and their products displayed the expected difference in sensitivity toward pseudomonic acid.

Amino Acid Sequence↗