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  • 1
    ISSN: 1432-072X
    Keywords: Methanobacterium thermoautotrophicum ; Bacteriophage ; Methanogenic bacteria ; Plasmid pME2001
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Bacteriophage ψM1, a virulent, oxygen resistant phage of Methanobacterium thermoautotrophicum strain Marburg, was isolated from an anaerobic sludge digester operated at 55°C to 60°C. A reproducible plaque assay and an enrichment procedure for the preparation of high-titer lysates (2x1010 PFU/ml) were established. One-step growth experiments at 62°C showed that the latent period was 4 h and the burst size was 5–6 infective particles per cell. The phage infected Methanobacterium thermoautotrophicum Marburg but none of three other thermophilic representatives of the genus Methanobacterium that were tested. Electron micrographs showed that phage ψM1 has a polyhedral head of 55 nm diameter and a tail of 210 nm in lenght. The ψM1 genome consists of linear double-stranded DNA with a size of 30.4±1.0 kb. Restriction and hybridization analysis of DNA extracted from phage particles revealed two types of linear molecules with the size of the phage genome. About 85% of the DNA molecules in such preparations were genomes of ψM1 whereas approximately 15% were multimers of the cryptic 4.5-kb plasmid pME2001 of the host. ψM1 DNA did not hybridize with chromosomal DNA of Methanobacterium thermoautotrophicum but it exhibited definite homology to total DNA of Methanobacterium wolfei.
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  • 2
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: 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.
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science, Ltd
    Molecular microbiology 47 (2003), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Several members of the two-component signal transduction family have been implicated in the control of  polar development in Caulobacter crescentus: PleC and DivJ, two polarly localized histidine sensor kinases; and the response regulators DivK and PleD. The PleD protein was shown previously to be required during the swarmer-to-stalked cell transition for flagellar ejection and efficient stalk biogenesis. Here, we present data indicating that PleD also controls the onset of motility and a cell density switch immediately preceding cell division. Constitutively active alleles of pleD or wspR, an orthologue from Pseudomonas fluorescens, almost completely suppressed C. crescentus motility and inhibited the increase in swarmer cell density during cell differentiation. The observation that these alleles also had a dominant-negative effect on motility in a pleC divJ and a pleC divK mutant background indicated that PleD is located downstream of the other components in the signal transduction cascade, which controls the activity of the flagellar motor. In addition, the presence of a constitutive pleD or wspR allele resulted in a doubling of the average stalk length. Together, this is consistent with a model in which the active form of PleD, PleD∼P, negatively controls aspects of differentiation in the late predivisional cell, whereas it acts positively on polar development during the swarmer-to-stalked cell transition. In agreement with such a model, we found that DivJ, which localizes to the stalked pole during cell differentiation, positively controlled the in vivo phosphorylation status of PleD, and the swarmer pole-specific PleC kinase modulated this status in a negative manner. Furthermore, domain switch experiments demonstrated that the WspR GGDEF output domain from P. fluorescens is active in C. crescentus, favouring a more general function for this novel signalling domain over a specific role such as DNA or protein interaction. Possible roles for PleD and its C-terminal output domain in modulating the polar cell surface of C. crescentus are discussed.
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  • 4
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology reviews 24 (2000), S. 0 
    ISSN: 1574-6976
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: 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.
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  • 5
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science Ltd.
    Molecular microbiology 44 (2002), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The ftsH gene of Caulobacter crescentus has been isolated and identified as a component of the general stress response of this organism. In C. crescentus, ftsH expression is transiently induced after temperature upshift and in stationary phase. Consistent with this, mutants deprived of the FtsH protease are viable at normal growth conditions, but are highly sensitive to elevated temperature, increased salt concentration or the presence of antibiotics. Overexpression of ftsH resulted in an increased salt but not thermotolerance, emphasizing the importance of the FtsH protease in stress response. Mutants lacking FtsH were unable to undergo morphological and physiological adaptation in stationary phase and, upon starvation, experienced a more pronounced loss of viability than cells containing FtsH. In addition, cells lacking FtsH had an increased cellular concentration of the heat shock sigma factor σ32, indicating that, as in Escherichia coli, the FtsH protease is involved in the control of the C. crescentus heat shock response. In agreement with this, transcription of the heat-induced σ32-dependent gene dnaK was derepressed at normal temperature when FtsH was absent. In contrast, the groEL gene, which is controlled in response to heat stress by both σ32 and a HcrA/CIRCE mechanism, was not derepressed in an ftsH mutant. Finally, FtsH is involved in C. crescentus development and cell cycle control. ftsH mutants were unable to synthesize stalks efficiently and had a severe cell division phenotype. In the absence of FtsH, swarmer cells differentiated into stalked cells faster than when FtsH was present, even though the entire cell cycle was longer under these conditions. Thus, directly or indirectly, the FtsH protease is involved in the inherent biological clock mechanism, which controls the timing of cell differentiation in C. crescentus.
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  • 6
    Electronic Resource
    Electronic Resource
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 32 (1999), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: 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.
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  • 7
    Publication Date: 2020-07-01
    Description: The bacterial second messenger cyclic diguanylate (c-di-GMP) regulates a wide range of cellular functions from biofilm formation to growth and survival. Targeting a second-messenger network is challenging because the system involves a multitude of components with often overlapping functions. Here, we present a strategy to intercept c-di-GMP signaling pathways by directly targeting the second messenger. For this, we developed a c-di-GMP–sequestering peptide (CSP) that was derived from a CheY-like c-di-GMP effector protein. CSP binds c-di-GMP with submicromolar affinity. The elucidation of the CSP⋅c-di-GMP complex structure by NMR identified a linear c-di-GMP–binding motif, in which a self-intercalated c-di-GMP dimer is tightly bound by a network of H bonds and π-stacking interactions involving arginine and aromatic residues. Structure-based mutagenesis yielded a variant with considerably higher, low-nanomolar affinity, which subsequently was shortened to 19 residues with almost uncompromised affinity. We demonstrate that endogenously expressed CSP intercepts c-di-GMP signaling and effectively inhibits biofilm formation inPseudomonas aeruginosa, the most widely used model for serious biofilm-associated medical implications.
    Print ISSN: 0027-8424
    Electronic ISSN: 1091-6490
    Topics: Biology , Medicine , Natural Sciences in General
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  • 8
    Publication Date: 2019-12-27
    Description: Cytosolic hybrid histidine kinases (HHKs) constitute major signaling nodes that control various biological processes, but their input signals and how these are processed are largely unknown. In Caulobacter crescentus, the HHK ShkA is essential for accurate timing of the G1-S cell cycle transition and is regulated by the corresponding increase in the level of the second messenger c-di-GMP. Here, we use a combination of X-ray crystallography, NMR spectroscopy, functional analyses, and kinetic modeling to reveal the regulatory mechanism of ShkA. In the absence of c-di-GMP, ShkA predominantly adopts a compact domain arrangement that is catalytically inactive. C-di-GMP binds to the dedicated pseudoreceiver domain Rec1, thereby liberating the canonical Rec2 domain from its central position where it obstructs the large-scale motions required for catalysis. Thus, c-di-GMP cannot only stabilize domain interactions, but also engage in domain dissociation to allosterically invoke a downstream effect. Enzyme kinetics data are consistent with conformational selection of the ensemble of active domain constellations by the ligand and show that autophosphorylation is a reversible process.
    Print ISSN: 0027-8424
    Electronic ISSN: 1091-6490
    Topics: Biology , Medicine , Natural Sciences in General
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  • 9
  • 10
    Publication Date: 1989-07-01
    Print ISSN: 0302-8933
    Electronic ISSN: 1432-072X
    Topics: Biology
    Published by Springer
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