Biomedical subjects

James W Gober

Publications and source records attributed to James W Gober.

14 recordsLinked to original sources

PubMed2005

The cell-shape protein MreC interacts with extracytoplasmic proteins including cell wall assembly complexes in Caulobacter crescentus.

The bacterial actin homolog, MreB, forms helical cables within the cell that are required for maintenance of a rod shape. These helical structures are thought to be involved in the spatial organization of cell wall (peptidoglycan) synthesizing complexes of penicillin-binding proteins (PBPs). Here, we examined the role of the MreC cell shape protein in this process in Caulobacter crescentus. Subcellular fractionation experiments showed that MreC is a periplasmic protein and, as assayed by immunofluorescence microscopy, adopted helical or banded patterns along the cell length reminiscent of those formed by MreB and PBP2. The pattern of MreC and PBP2 localization remained when MreB cables were disrupted by treatment with the inhibitor A22. However, long-term absence of MreB led to cell shape changes and an eventual loss of MreC localization, suggesting that an independent structure, perhaps an intact peptidoglycan layer, contributes to the MreC localization pattern. Using affinity chromatography with MreC covalently bound to Sepharose, we isolated several PBPs from cell extracts that eluted from the column as heterogeneous complexes. In this same experiment, using mass spectrometry-based protein identification, we identified several outer membrane proteins, including TonB-dependent receptor transport proteins, that interacted with MreC. Imaging live cells containing fusions of these outer membrane proteins to green fluorescent protein showed that they adopted a subcellular localization pattern that was similar to that of MreC. These results suggest that MreC may function in the spatial organization of PBPs as well as other proteins that lie outside the cytoplasmic membrane.

Bacterial Proteins↗

PubMed2005

Role of core promoter sequences in the mechanism of swarmer cell-specific silencing of gyrB transcription in Caulobacter crescentus.

BACKGROUND: Each Caulobacter crescentus cell division yields two distinct cell types: a flagellated swarmer cell and a non-motile stalked cell. The swarmer cell is further distinguished from the stalked cell by an inability to reinitiate DNA replication, by the physical properties of its nucleoid, and its discrete program of gene expression. Specifically, with regard to the latter feature, many of the genes involved in DNA replication are not transcribed in swarmer cells. RESULTS: We show that for one of these genes involved in DNA replication, gyrB, its pattern of temporal expression depends upon an 80 base pair promoter region with strong resemblance to the Caulobacter crescentus sigma73 consensus promoter sequence; regulation does not appear to be affected by the general strength of the promoter activity, as mutations that increased its conformity with the consensus did not affect its cell-cycle expression pattern. Transcription from the gyrB promoter in vitro required only the presence of the sigma73 RNA polymerase (from E. coli) and the requisite nucleoside triphosphates, although a distinct binding activity, present in crude whole-cell extracts, formed a complex gyrB promoter DNA. We also assayed the effect on gyrB expression in strains containing mutations in either smc or dps, two genes encoding proteins that condense DNA. However we found there was no change in the temporal pattern of gyrB transcription in strains containing deletions in either of these genes. CONCLUSION: These experiments demonstrate that gyrB transcription does not require any auxiliary factors, suggesting that temporal regulation is not dependent upon an activator protein. Swarmer-specific silencing may not be attributable to the observed physical difference in the swarmer cell nucleoid, since mutations in either smc or dps, two genes encoding proteins that condense DNA, did not alter the temporal pattern of gyrB transcription in strains containing deletions in either of these genes. Rather a repressor that specifically recognizes sequences in the gyrB promoter region that are also probably essential for transcription, is likely to be responsible for controlling cell cycle expression.

Base Sequence↗

PubMed2005

The trans-acting flagellar regulatory proteins, FliX and FlbD, play a central role in linking flagellar biogenesis and cytokinesis in Caulobacter crescentus.

The FliX/FlbD-dependent temporal transcription of late flagellar genes in Caulobacter crescentus requires the assembly of an early, class II-encoded flagellar structure. Class II flagellar-mutant strains exhibit a delay in the completion of cell division, with the accumulation of filamentous cells in culture. It is shown here that this cell-division defect is attributable to an arrest in the final stages of cell separation. Normal cell morphology could be restored in class II mutants by gain-of-function alleles of FliX or FlbD, suggesting that the timely completion of cell division requires these trans-acting factors. In synchronized cultures, inhibition of cell division by depleting FtsZ resulted in normal initial expression of the late, FlbD-dependent fliK gene; however, the cell cycle-regulated cessation of transcription was delayed, indicating that cell division may be required to negatively regulate FlbD activity. Interestingly, prolonged depletion of FtsZ resulted in an eventual loss of FlbD activity that could be bypassed by a constitutive mutant of FlbD, but not of FliX, suggesting the possible existence of a second cell cycle-dependent pathway for FlbD activation.

Bacterial Proteins↗

PubMed2005

The conserved flaF gene has a critical role in coupling flagellin translation and assembly in Caulobacter crescentus.

The expression of the flagellin proteins in Caulobacter crescentus is regulated by the progression of flagellar assembly both at the transcriptional and post-transcriptional levels. An early basal body structure is required for the transcription of flagellin genes, whereas the ensuing assembly of a hook structure is required for flagellin protein synthesis. Previous experiments have shown that the negative regulatory protein, FlbT, operates this second post-transcriptional checkpoint by associating with the 5' untranslated region (UTR) of the fljK flagellin transcript, inhibiting translation and destabilizing the mRNA. In this paper we examine the role of flaF in flagellar biogenesis. The flaF gene, which is conserved in several speices of flagellated alpha-proteobacteria, is required for motility and flagellin protein synthesis. A deletion of flbT in a DeltaflaF strain restored flagellin protein expression, but not motility, indicating that FlaF functions in filament assembly. Mutant strains with a deletion in flaF had no detectable fljK mRNA, the levels of which were restored by an additional mutation in flbT. Assay of fljK gene expression using transcription and translation reporter fusions indicated that FlaF was essential for the translation of fljK mRNA. FlaF protein levels were under cell cycle control, peaking during the period of flagellin expression and filament assembly, whereas FlbT was present throughout the cell cycle. These results suggest that FlbT and FlaF activities oppose one another in the regulation of flagellin expression in response to both the progression of flagellar assembly and the cell cycle.

Amino Acid Sequence↗

PubMed2005

Linking structural assembly to gene expression: a novel mechanism for regulating the activity of a sigma54 transcription factor.

In Caulobacter crescentus, the temporal and spatial expression of late flagellar genes is regulated by the sigma54 transcriptional activator, FlbD. Genetic experiments have indicated that the trans-acting factor FliX regulates FlbD in response to the progression of flagellar assembly, repressing FlbD activity until an early flagellar basal body structure is assembled. Following assembly of this structure, FliX is thought to function as an activator of FlbD. Here we have investigated the mechanism of FliX-mediated regulation of FlbD activity. In vitro transcription experiments showed that purified FliX could function as a repressor of FlbD-activated transcription. Transcription activated by a gain-of-function mutant of FlbD (FlbD-1204) that is active in vivo in the absence of an early flagellar structure, was resistant to the repressive effects of FliX. DNA binding studies showed that FliX inhibited the interaction of wild-type FlbD with enhancer DNA but did not effect FlbD-catalysed ATPase activity. DNA binding activity of FlbD-1204 was relatively unaffected by FliX indicating that this mutant protein bypasses the transcriptional requirement for early flagellar assembly by escaping FliX-mediated negative regulation. Gel filtration and co-immunoprecipitation experiments indicated that FliX formed a stable complex with FlbD. These experiments demonstrate that regulation of FlbD activity is unusual among the well-studied sigma54 transcriptional activators, apparently combining a two-component receiver domain with additional control imposed via interaction with a partner protein, FliX.

Adenosine Triphosphatases↗

PubMed2005

Role of integration host factor in the transcriptional activation of flagellar gene expression in Caulobacter crescentus.

In the Caulobacter crescentus predivisional cell, class III and IV flagellar genes, encoding the extracytoplasmic components of the flagellum, are transcribed in the nascent swarmer compartment. This asymmetric expression pattern is attributable to the compartmentalized activity of the sigma54-dependent transcriptional activator FlbD. Additionally, these temporally transcribed flagellar promoters possess a consensus sequence for the DNA-binding protein integration host factor (IHF), located between the upstream FlbD binding site and the promoter sequences. Here, we deleted the C. crescentus gene encoding the beta-subunit of the IHF, ihfB (himD), and examined the effect on flagellar gene expression. The DeltaihfB strain exhibited a mild defect in cell morphology and impaired motility. Using flagellar promoter reporter fusions, we observed that expression levels of a subset of class III flagellar promoters were decreased by the loss of IHF. However, one of these promoters, fliK-lacZ, exhibited a wild-type cell cycle-regulated pattern of expression in the absence of IHF. Thus, IHF is required for maximal transcription of several late flagellar genes. The DeltaihfB strain was found to express significantly reduced amounts of the class IV flagellin, FljL, as a consequence of reduced transcriptional activity. Our results indicate that the motility defect exhibited by the DeltaihfB strain is most likely attributable to its failure to accumulate the class IV-encoded 27-kDa flagellin subunit, FljL.

Bacterial Proteins↗

PubMed2004

Measurements of DNA lengths remaining in a viral capsid after osmotically suppressed partial ejection.

The effect of external osmotic pressure on the extent of DNA ejection from bacteriophage-lambda was recently investigated (Evilevitch et al., 2003). The total length of DNA ejected was measured via the 260-nm absorption by free nucleotides, after opening of the capsids in the presence of varying amounts of polyethylene glycol 8000 and DNase I. As a function of osmolyte concentration, this absorption was shown to decrease progressively, ultimately vanishing completely for a sufficiently high external osmotic pressure. In this work we report the results of both sedimentation and gel analysis of the length of DNA remaining inside the capsids, as a function of osmolyte concentration. It is confirmed in this way that the progressive inhibition of DNA ejection corresponds to partial ejection from all of the capsids.

Absorption↗

PubMed2004

MreB, the cell shape-determining bacterial actin homologue, co-ordinates cell wall morphogenesis in Caulobacter crescentus.

The bacterial actin homologue, MreB, is required for the maintenance of a rod-shaped cell and has been shown to form spirals that traverse along the longitudinal axis of Bacillus subtilis and Escherichia coli cells. The depletion of MreB in Caulobacter crescentus resulted in lemon-shaped cells that possessed defects in the integrity of the cell wall. MreB localization appeared as bands or spirals that encircled the cell along its entire length and switched to a mid-cell location at a time that coincided with the initiation of cell division. The formation of smaller MreB spirals or bands at the mid-cell was dependent on the presence on the cytokinetic protein, FtsZ. Penicillin-binding protein 2 (PBP2) also formed band-like structures perpendicular to the cell periphery that resembled, and depended upon, MreB localization. PBP2 co-immunoprecipitated with several other penicillin-binding proteins, suggesting that these proteins are in association in Caulobacter cells. We hypothesize that MreB filaments function as a cytoskeleton that serves as an organizer or tracking device for the PBP2-peptidoglycan biosynthesis complex.

Actins↗

PubMed2004

Regulation of FlbD activity by flagellum assembly is accomplished through direct interaction with the trans-acting factor, FliX.

The temporal and spatial transcription of late flagellar genes in Caulobacter crescentus is regulated by the sigma54 transcriptional activator, FlbD. One requirement for FlbD activity is the assembly of a structure encoded by early, class II flagellar genes. In this report, we show that the trans-acting factor FliX predominantly functions as a negative regulator of FlbD activity in the absence of the class II-encoded flagellar structure. In contrast, a mutant FliX that bypasses the transcriptional requirement for early flagellar assembly is incapable of repressing FlbD in a class II flagellar mutant. Expression of this mutant allele, fliX1, does not alter the temporal pattern of FlbD-dependent transcription. Remarkably, this mutation confers the correct cell cycle timing of hook operon transcription in a strain that cannot assemble the flagellum, indicating that the progression of flagellar assembly is a minor influence on temporal gene expression. Using a two-hybrid assay, we present evidence that FliX regulates FlbD through a direct interaction, a novel mechanism for this class of sigma54 transcriptional activator. Furthermore, increasing the cellular levels of FliX results in an increase in the concentration of FlbD, and a corresponding increase in FlbD-activated transcription, suggesting that FliX and FlbD form a stable complex in Caulobacter. FliX and FlbD homologues are present in several polar-flagellated bacteria, indicating that these proteins constitute an evolutionarily conserved regulatory pair in organisms where flagellar biogenesis is likely to be under control of the cell division cycle.

Amino Acid Sequence↗

PubMed2003

Productive interaction between the chromosome partitioning proteins, ParA and ParB, is required for the progression of the cell cycle in Caulobacter crescentus.

In Caulobacter crescentus the partitioning proteins ParA and ParB operate a molecular switch that couples chromosome partitioning to cytokinesis. Homologues of these proteins have been shown to be important for the stable inheritance of F-plasmids and the prophage form of bacteriophage P1. In C. crescentus, ParB binds to sequences adjacent to the origin of replication and is required for the initiation of cell division. Additionally, ParB influences the nucleotide-bound state of ParA by acting as a nucleotide exchange factor. Here we have performed a genetic analysis of the chromosome partitioning protein ParB. We show that C. crescentus ParB, like its plasmid homologues, is composed of three domains: a carboxyl-terminal dimerization domain; a central DNA-binding, helix-turn-helix domain; and an amino-terminal domain required for the interaction with ParA. In vivo expression of amino-terminally deleted parB alleles has a dominant lethal effect resulting in the inhibition of cell division. Fluorescent in situ hybridization experiments indicate that this phenotype is not caused by a chromosome partitioning defect, but by the reversal of the amounts of ATP- versus ADP- bound ParA inside the cell. We present evidence suggesting that amino-terminally truncated and full-length, wild-type ParB form heterodimers which fail to interact with ParA, thereby reversing the intracellular ParA-ATP to ParA-ADP ratio. We hypothesize that the amino-terminus of ParB is required to regulate the nucleotide exchange of ParA which, in turn, regulates the initiation of cell division.

Adenosine Diphosphate↗

PubMed2003

Cell shape, division and development: the 2002 American Society for Microbiology (ASM) conference on prokaryotic development.

In the last decade, the use of cytological techniques, together with the analysis of complete genomes, has dramatically advanced our understanding of bacterial development. Work on several well-developed model systems such as Bacillus subtilis, Caulobacter crescentus, Myxococcus xanthus and Streptomyces spp., has provided us with an in-depth understanding of processes such as sporulation, multicellular behaviour and the bacterial cell cycle. At the same time, these studies have revolutionized our view of the bacterial cell and shown it to be a highly complex entity with spatial and temporal organization. The recent American Society for Microbiology (ASM) conference on prokaryotic development demonstrated that several laboratories have now started to connect data obtained through functional genomic analysis with subcellular organization, thereby generating three-dimensional regulatory networks. This meeting report highlights new findings in the field, such as regulation of protein localization during sporulation and the cell cycle, control of cell-cell interaction and the initiation of cell division.

Bacillus subtilis↗

PubMed2002

Bacterial chromosome segregation.

Recent studies have made great strides toward our understanding of the mechanisms of microbial chromosome segregation and partitioning. This review first describes the mechanisms that function to segregate newly replicated chromosomes, generating daughter molecules that are viable substrates for partitioning. Then experiments that address the mechanisms of bulk chromosome movement are summarized. Recent evidence indicates that a stationary DNA replication factory may be responsible for supplying the force necessary to move newly duplicated DNA toward the cell poles. Some factors contributing to the directionality of chromosome movement probably include centromere-like-binding proteins, DNA condensation proteins, and DNA translocation proteins.

Bacterial Proteins↗

PubMed2002

ParB-stimulated nucleotide exchange regulates a switch in functionally distinct ParA activities.

ParA and ParB of Caulobacter crescentus belong to a conserved family of bacterial proteins implicated in chromosome segregation. ParB binds to DNA sequences adjacent to the origin of replication and localizes to opposite cell poles shortly following the initiation of DNA replication. ParA has homology to a conserved and widespread family of ATPases. Here, we show that ParB regulates the ParA ATPase activity by promoting nucleotide exchange in a fashion reminiscent of the exchange factors of eukaryotic G proteins. Furthermore, we demonstrate that ADP-bound ParA binds single-stranded DNA, whereas the ATP-bound form dissociates ParB from its DNA binding sites. Increasing the fraction of ParA-ADP in the cell inhibits cell division, suggesting that this simple nucleotide switch may regulate cytokinesis.

Adenosine Diphosphate↗

PubMed2002

Mutations in FlbD that relieve the dependency on flagellum assembly alter the temporal and spatial pattern of developmental transcription in Caulobacter crescentus.

The transcription factor FlbD regulates the temporal and spatial transcription of flagellar genes in the bacterium Caulobacter crescentus. Activation of FlbD requires cell cycle progression and the assembly of an early (class II) flagellum structure. In this report, we identify 20 independent gain-of-function mutations in flbD that relieve regulation by flagellar assembly. One of these, flbD-1204, contained a mutation in the receiver domain (V17M) and another, flbD-1231, in the DNA binding domain (V451G). Both of these mutations resulted in an aberrant pattern of cell cycle transcription. The presence of the FlbD-1204 allele also resulted in a loss of swarmer-pole-specific transcription. These results indicate that temporal and spatial transcription is influenced by the assembly of the nascent flagellar structure. The trans-acting positive and negative regulatory factor, FliX, couples flagellar assembly to the activation of FlbD and, as we show here, also influences temporal transcription. Furthermore, we show that FliX can suppress the activity of FlbD mutants that cannot be phosphorylated, and that FliX is required for FlbD stability, and vice versa. These results indicate that FliX may interact directly with FlbD to regulate its activity.

Amino Acid Sequence↗