Biomedical subjects

A Newton

Publications and source records attributed to A Newton.

At least 37 records · Page 2Linked to original sources

PubMed1997

Requirement of topoisomerase IV parC and parE genes for cell cycle progression and developmental regulation in Caulobacter crescentus.

We have identified the parC and parE genes encoding DNA topoisomerase IV (Topo IV) in Caulobacter crescentus. We have also characterized the effect of conditional Topo IV mutations on cell division and morphology. Topo IV mutants of C. crescentus are unlike mutants of Escherichia coli and S. typhimurium, which form long filamentous cells that are defective in nucleoid segregation and divide frequently to produce anucleate cells. Topo IV mutants of C. crescentus are highly pinched at multiple sites (cell separation phenotype) and they do not divide to produce cells lacking DNA. These results suggest unique regulatory mechanisms coupling nucleoid partitioning and cell division in this aquatic bacterium. In addition, distinctive nucleoid-partitioning defects are not apparent in C. crescentus Topo IV mutants as they are in E. coli and S. typhimurium. However, abnormal nucleoid segregation in parE mutant cells could be demonstrated in a genetic background containing a conditional mutation in the C. crescentus ftsA gene, an early cell division gene that is epistatic to parE for cell division and growth. We discuss these results in connection with the possible roles of C. crescentus Topo IV in the regulation of cell division, chromosome partitioning, and late events in polar morphogenesis. Although the ParC and ParE subunits of Topo IV are very similar in sequence to the GyrA and GyrB subunits of DNA gyrase, we have used DNA sequence analysis to identify a highly conserved 'GyrA box' sequence that is unique to the GyrA proteins and may serve as a hallmark of the GyrA protein family.

Alleles↗

PubMed1997

Roles of the histidine protein kinase pleC in Caulobacter crescentus motility and chemotaxis.

The Caulobacter crescentus histidine kinase genes pleC and divJ have been implicated in the regulation of polar morphogenesis and cell division, respectively. Mutations in pleC also potentiate the cell division phenotype of divJ mutations. To investigate the involvement of the PleC kinase in motility and cell cycle regulation, we carried out a pseudoreversion analysis of the divJ332 allele, which confers a temperature-sensitive motility (Mot-) phenotype. All cold-sensitive pseudorevertants with a Mot+ phenotype at 37 degrees C and a cold-sensitive swarm phenotype in soft agar at 24 degrees C contained extragenic suppressors that were null mutations mapping to pleC. Instead of a cell division defect at the nonpermissive temperature, however, revertants displayed a cold-sensitive defect in chemotaxis (Che-). In addition, the mutant cells were also supermotile, a phenotype previously associated only with mutations in the response regulator gene pleD that block the loss of motility. We also found that the Mot- defect of pleC mutants is suppressed by a pleD301/pleD+ merodiploid and results in a similar, supermotile, cold-sensitive Che- phenotype. These results implicate signal transduction pathways mediated by PleC-DivK and DivJ-PleD in the regulation of chemotaxis as well as motility. We discuss these findings and the observation that although the PleC kinase does not play an indispensable role in cell division, a temperature-sensitive allele of pleC (pleC319) has severely reduced viability under stringent growth conditions.

Bacterial Proteins↗

PubMed1997

The Caulobacter heat shock sigma factor gene rpoH is positively autoregulated from a sigma32-dependent promoter.

Sigma factor sigma32, encoded by rpoH, is required for the recognition of heat shock genes during normal growth conditions and in response to heat shock and other stresses. Unlike the well-studied Escherichia coli rpoH gene, which is transcribed from four promoters recognized by either a sigma70 (sigmaD)- or sigma24 (sigmaE)-containing RNA polymerase, the Caulobacter crescentus rpoH gene is transcribed from two promoters, P1 and P2. In this study, we have examined the structure and expression of these promoters and shown that the rpoH P2 promoter is sigma32 dependent. We present evidence here that P2 is specifically recognized and transcribed by the reconstituted C. crescentus Esigma32 RNA polymerase holoenzyme. We show that site-directed mutations within either the -10 or the -35 regions of P2 have substantial effects on the levels of transcription by the Esigma32 polymerase predicted from the sigma32 promoter consensus sequence. The mutations have similar effects in vivo as assayed with rpoH-lacZ transcription fusions. Analysis of the rpoH P1 promoter provided evidence that it is sigma70 dependent. S1 nuclease protection assays of rpoH P1- and P2-specific expression after heat shock at 42 or 50 degrees C and during synchronous cell division cycles under normal growth conditions showed that the two promoters are differentially regulated. Mutations within the rpoH P2 promoter consensus sequences abolished the response to heat shock induction in C. crescentus. We conclude from these results that, unlike rpoH genes studied previously in other bacteria, the major transcriptional response of the C. crescentus rpoH gene to heat shock depends on positive autoregulation of the sigma32-dependent promoter.

Bacterial Proteins↗

PubMed1997

Identification, characterization, and chromosomal organization of cell division cycle genes in Caulobacter crescentus.

We report a detailed characterization of cell division cycle (cdc) genes in the differentiating gram-negative bacterium Caulobacter crescentus. A large set of temperature-sensitive cdc mutations was isolated after treatment with the chemical mutagen N-methyl-N'-nitro-N-nitrosoguanidine. Analysis of independently isolated mutants at the nonpermissive temperature identified a variety of well-defined terminal phenotypes, including long filamentous cells blocked at various stages of the cell division cycle and two unusual classes of mutants with defects in both cell growth and division. The latter strains are uniformly arrested as either short bagel-shaped coils or large predivisional cells. The polar morphology of these cdc mutants supports the hypothesis that normal cell cycle progression is directly responsible for developmental regulation in C. crescentus. Genetic and physical mapping of the conditional cdc mutations and the previously characterized dna and div mutations identified at least 21 genes that are required for normal cell cycle progression. Although most of these genes are widely scattered, the genetically linked divA, divB, and divE genes were shown by genetic complementation and physical mapping to be organized in one gene cluster at 3200 units on the chromosome. DNA sequence analysis and marker rescue experiments demonstrated that divE is the C. crescentus ftsA homolog and that the ftsZ gene maps immediately adjacent to ftsA. On the basis of these results, we suggest that the C. crescentus divA-divB-divE(ftsA)-ftsZ gene cluster corresponds to the 2-min fts gene cluster of Escherichia coli.

Amino Acid Sequence↗

PubMed1997

Posttranscriptional regulation of Caulobacter flagellin genes by a late flagellum assembly checkpoint.

Flagellum formation in Caulobacter crescentus requires ca. 50 flagellar genes, most of which belong to one of three classes (II, III, or IV). Epistasis experiments suggest that flagellar gene expression is coordinated with flagellum biosynthesis by two assembly checkpoints. Completion of the M/S ring-switch complex is required for the transition from class II to class III gene expression, and completion of the basal body-hook structure is required for the transition from class III to class IV gene expression. In studies focused on regulation of the class IV flagellin genes, we have examined fljK and fljL expression in a large number of flagellar mutants by using transcription and translation fusions to lacZ, nuclease S1 assays, and measurements of protein stability. The fljK-lacZ and fljL-lacZ transcription fusions were expressed in all class III flagellar mutants, although these strains do not make detectable 25- or 27-kDa flagellins. The finding that the fljK-lacZ translation fusion was not expressed in the same collection of class III mutants confirmed that fljK is regulated posttranscriptionally. The requirement of multiple class III genes for expression of the fljK-lacZ fusion suggests that completion of the basal body-hook is an assembly checkpoint for the posttranscriptional regulation of this flagellin gene. Deletion analysis within the 5' untranslated region of fljK identified a sequence between +24 and +38 required for regulation of the fljK-lacZ fusion by class III genes, which implicates an imperfect 14-bp direct repeat in the posttranscriptional regulation of fljK. Our results show that fljL is also regulated posttranscriptionally by class III and unclassified flagellar genes, apparently by a mechanism different from the one regulating fljK.

Bacterial Proteins↗

PubMed1996

Signal transduction in the cell cycle regulation of Caulobacter differentiation.

Caulobacter crescentus differentiates to form a new cell type during asymmetric cell division. Recent results indicate that signal transduction pathways mediated by protein kinases and essential response regulators play a central role in the regulation of development and cell division in response to cell cycle checkpoints.

Caulobacter crescentus↗

PubMed1996

Isolation, identification, and transcriptional specificity of the heat shock sigma factor sigma32 from Caulobacter crescentus.

We report the identification of the Caulobacter crescentus heat shock factor sigma32 as a 34-kDa protein that copurifies with the RNA polymerase holoenzyme. The N-terminal amino acid sequence of this protein was determined and used to design a degenerate oligonucleotide as a probe to identify the corresponding gene, rpoH, which encodes a predicted protein with a molecular mass of 33,659 Da. The amino acid sequence of this protein is similar to those of known bacterial heat shock sigma factors of Escherichia coli (41% identity), Pseudomonas aeruginosa (40% identity), and Citrobacter freundii (38% identity). The isolated C. crescentus gene complements the growth defect of an E. coli rpoH deletion strain at 37 degrees C, and Western blot (immunoblot) analysis confirmed that the gene product is related to the E. coli sigma32 protein. The purified RpoH protein in the presence of RNA polymerase core enzyme specifically recognizes the heat shock-regulated promoter P1 of the C. crescentus dnaK gene, and base pair substitutions in either the -10 or -35 region of this promoter abolish transcription. S1 nuclease mapping indicates that rpoH transcripts originate from two promoters, P1 and P2, under the normal growth conditions. The P2 promoter is similar to the sigma32 promoter consensus, and the P2-specific transcript increases dramatically during heat shock, while the P1-specific transcript remains relatively constant. These results suggest that although the structure and function of C. crescentus sigma32 appear to be very similar to those of its E. coli counterpart, the C. crescentus rpoH gene contains a novel promoter structure and may be positively autoregulated in response to environmental stress.

Amino Acid Sequence↗

PubMed1995

An essential single domain response regulator required for normal cell division and differentiation in Caulobacter crescentus.

Signal transduction pathways mediated by sensor histidine kinases and cognate response regulators control a variety of physiological processes in response to environmental conditions. Here we show that in Caulobacter crescentus these systems also play essential roles in the regulation of polar morphogenesis and cell division. Previous studies have implicated histidine kinase genes pleC and divJ in the regulation of these developmental events. We now report that divK encodes an essential, cell cycle-regulated homolog of the CheY/Spo0F subfamily and present evidence that this protein is a cognate response regulator of the histidine kinase PleC. The purified kinase domain of PleC, like that of DivJ, can serve as an efficient phosphodonor to DivK and as a phospho-DivK phosphatase. Based on these and earlier genetic results we propose that PleC and DivK are members of a signal transduction pathway that couples motility and stalk formation to completion of a late cell division cycle event. Gene disruption experiments and the filamentous phenotype of the conditional divK341 mutant reveal that DivK also functions in an essential signal transduction pathway required for cell division, apparently in response to another histidine kinase. We suggest that phosphotransfer mediated by these two-component signal transduction systems may represent a general mechanism regulating cell differentiation and cell division in response to successive cell cycle checkpoints.

Amino Acid Sequence↗

PubMed1995

Regulation of the Caulobacter crescentus rpoN gene and function of the purified sigma 54 in flagellar gene transcription.

The sequential transcription of flagellar (fla) genes in the Caulobacter crescentus cell cycle is controlled by the organization of these genes in a regulatory hierarchy of four levels (I-IV). Level III and level IV genes at the bottom of the hierarchy are dependent on level II genes and are transcribed late in the cell cycle from sigma 54-dependent promoters. To study the regulation of genes at levels III and IV, we have isolated and sequenced the rpoN gene in order to analyze its expression, purified the rpoN gene product, and examined the role of the RpoN protein in initiation of transcription from sigma 54-dependent promoters. We report here epistasis experiments that show rpoN is required for transcription of level III genes, but that the expression of the rpoN gene itself is not dependent on any of the fla genes examined; these results place rpoN at level II near the top of the hierarchy. Consistent with this conclusion were nuclease S1 assays that mapped the rpoN transcription start site and identified a sequence centered at -24, GTTA/TACCA/TT, which is similar to the core consensus sequence of the level IIB fliF, fliL, and fliQ promoters. We purified the full-length rpoN gene product to near homogeneity and demonstrated that the RpoN protein is required for transcription from the well-characterized sigma 54-dependent glnAp2 promoter of Escherichia coli and specifically recognizes the level III flbG gene promoter of C. crescentus. These last results confirm that rpoN encodes the C. crescentus sigma 54 factor and opens the way for the biochemical analysis of transcriptional regulation of level III and IV fla genes.

Amino Acid Sequence↗

PubMed1995

Global regulation of a sigma 54-dependent flagellar gene family in Caulobacter crescentus by the transcriptional activator FlbD.

Biosynthesis of the Caulobacter crescentus polar flagellum requires the expression of a large number of flagellar (fla) genes that are organized in a regulatory hierarchy of four classes (I to IV). The timing of fla gene expression in the cell cycle is determined by specialized forms of RNA polymerase and the appearance and/or activation of regulatory proteins. Here we report an investigation of the role of the C. crescentus transcriptional regulatory protein FlbD in the activation of sigma 54-dependent class III and class IV fla genes of the hierarchy by reconstituting transcription from these promoters in vitro. Our results demonstrate that transcription from promoters of the class III genes flbG, flgF, and flgI and the class IV gene fliK by Escherichia coli E sigma 54 is activated by FlbD or the mutant protein FlbDS140F (where S140F denotes an S-to-F mutation at position 140), which we show here has a higher potential for transcriptional activation. In vitro studies of the flbG promoter have shown previously that transcriptional activation by the FlbD protein requires ftr (ftr for flagellar transcription regulation) sequence elements. We have now identified multiple ftr sequences that are conserved in both sequence and spatial architecture in all known class III and class IV promoters. These newly identified ftr elements are positioned ca. 100 bp from the transcription start sites of each sigma 54-dependent fla gene promoter, and our studies indicate that they play an important role in controlling the levels of transcription from different class III and class IV promoters. We have also used mutational analysis to show that the ftr sequences are required for full activation by the FlbD protein both in vitro and in vivo. Thus, our results suggest that FlbD, which is encoded by the class II flbD gene, is a global regulator that activates the cell cycle-regulated transcription from all identified sigma 54-dependent promoters in the C. crescentus fla gene hierarchy.

Bacterial Proteins↗

PubMed1995

Identification of a novel response regulator required for the swarmer-to-stalked-cell transition in Caulobacter crescentus.

The onset of motility late in the Caulobacter crescentus cell cycle depends on a signal transduction pathway mediated by the histidine kinase PleC and response regulator DivK. We now show that pleD, whose function is required for the subsequent loss of motility and stalk formation by the motile swarmer cell, encodes a 454-residue protein with tandem N-terminal response regulator domains D1 and D2 and a novel C-terminal GGDEF domain. The identification of pleD301, a semidominant suppressor of the pleC Mot phenotype, as a mutation predicted to result in a D-53-->G change in the D1 domain supports a role for phosphorylation in the PleD regulator. Disruptions constructed in the pleD open reading frame demonstrated that the gene is not essential and that the pleC phenotype can also be suppressed by a recessive, loss-of-function mutation. These results suggest that PleD is part of a signal transduction pathway controlling stalked-cell differentiation early in the C. crescentus cell cycle.

Alleles↗

PubMed1994

The Caulobacter crescentus FlbD protein acts at ftr sequence elements both to activate and to repress transcription of cell cycle-regulated flagellar genes.

The flagellar genes (fla genes) in Caulobacter crescentus are organized into a regulatory hierarchy of four levels, I-IV, in which transcription of the class III and class IV genes late in the cell cycle from sigma 54-dependent promoters depends on expression of the class II genes above them. Timing of fla gene expression has been attributed to sequential activation and repression by specific transcription factors. Here we report that purified FlbD activates transcription in vitro from the sigma 54-dependent class III flbG promoter and repress transcription from the class II fliF promoter by binding to ftr (flagellar transcription regulator) sequence elements required for their transcriptional regulation in vivo. The FlbD protein makes symmetrical base-specific contacts at three highly conserved guanine nucleotides in each half site of ftr1 and ftr1* at flbG and the single ftr4 site at fliF. The dual function of FlbD in activation of class III genes and repression of the class II fliF promoter is consistent with a central role of FlbD as a switch protein mediating the transition from level II to level III fla gene expression.

Bacterial Proteins↗

PubMed1994

Information essential for cell-cycle-dependent secretion of the 591-residue Caulobacter hook protein is confined to a 21-amino-acid sequence near the N-terminus.

Recent findings suggest that axial flagellar proteins and virulence proteins of Gram-negative bacteria are exported from the cytoplasm via conserved translocation systems. To identify residues essential for secretion of flagellar axial proteins we examined the 591-residue Caulobacter crescentus flagellar hook protein. Western blot assays of the culture media of strains producing mutant hook proteins show that only residues 38-58 are essential for its secretion to the cell surface. We discuss the observation that this unprocessed 21-residue sequence is not conserved in other axial proteins and does not correspond to the SGL-, ANNLAN- and heptad repeat motifs that are located just upstream of the essential secretion information in the hook protein and are conserved near the N-termini of other axial proteins. These motifs, for which an essential role in export or assembly has been proposed, are required for motility. However, we also demonstrate that hook protein can only be secreted when the flagellar basal body is present in the cell envelope. The cell-cycle regulation of hook protein secretion confirms the specificity of the assay used in these studies and suggests that the basal body itself may serve as a secretion channel for the hook protein.

Amino Acid Sequence↗

PubMed1994

Multiple structural proteins are required for both transcriptional activation and negative autoregulation of Caulobacter crescentus flagellar genes.

The periodic and sequential expression of flagellar (fla) genes in the Caulobacter crescentus cell cycle depends on their organization into levels I to IV of a regulatory hierarchy in which genes at the top of the hierarchy are expressed early in the cell cycle and are required for the later expression of genes below them. In these studies, we have examined the regulatory role of level II fliF operon, which is located near the top of the hierarchy. The last gene in the fliF operon, flbD, encodes a transcriptional factor required for activation of sigma 54-dependent promoters at levels III and IV and negative autoregulation of the level II fliF promoter. We have physically mapped the fliF operon, identified four new genes in the transcription unit, and determined that the organization of these genes is 5'-fliF-fliG-flbE-fliN-flbD-3'. Three of the genes encode homologs of the MS ring protein (FliF) and two switch proteins (FliG and FliN) of enteric bacteria, and the fourth encodes a predicted protein (FlbE) without obvious similarities to known bacterial proteins. We have introduced nonpolar mutations in each of the open reading frames and shown that all of the newly identified genes (fliF, fliG, flbE, and fliN) are required in addition to flbD for activation of the sigma 54-dependent flgK and flbG promoters at level III. In contrast, fliF, fliG, and flbE, but not fliN, are required in addition to flbD for negative autoregulation of the level II fliF promoter. The simplest interpretation of these results is that the requirements of FlbD in transcriptional activation and repression are not identical, and we speculate that FlbD function is subject to dual or overlapping controls. We also discuss the requirement of multiple structural genes for regulation of levels II and III genes and suggest that fla gene expression in C. crescentus may be coupled to two checkpoints in flagellum assembly.

Amino Acid Sequence↗

PubMed1994

Call selection for the Helicopter Emergency Medical Service: implications for ambulance control.

The increasing sophistication of pre-hospital care, with paramedics and many types of 'rapid response' units, requires the use of advanced systems of ambulance control. The introduction of call selection by a paramedic in the ambulance control room significantly improved the tasking of the Helicopter Emergency Medical Service. This paper illustrates the need for a system to grade 999 calls, so that the appropriate pre-hospital response can be directed to each patient.

Air Ambulances↗

PubMed1993

Identification of the promoter and a negative regulatory element, ftr4, that is needed for cell cycle timing of fliF operon expression in Caulobacter crescentus.

The fliF operon of Caulobacter crescentus, which was previously designated the flaO locus, is near the top of the flagellar-gene regulatory hierarchy, and it is one of the earliest transcription units to be expressed in the cell cycle. In this report, we have identified two cis-acting sequences that are required for cell cycle regulation of fliF transcription. The first sequence was defined by the effects of three 2-bp deletions and five point mutations, each of which greatly reduced the level of fliF operon transcript in vivo. These eight mutations lie between -37 and -22 within an 18-bp sequence that matches, at 11 nucleotides, sequences in the 5' regions of the flaQR (flaS locus) and fliLM operons, which are also expressed early and occupy a high level in the regulatory hierarchy (A. Dingwall, A. Zhuang, K. Quon, and L. Shapiro, J. Bacteriol. 174:1760-1768, 1992). We propose that this 18-bp sequence contains all or part of the fliF promoter. We have also identified a second sequence, 17 bp long and centered at -8, which we have provisionally designated ftr4 because of its similarity to the enhancer-like ftr sequences required for regulation of sigma 54 promoters flaN and flbG (D. A. Mullin and A. Newton, J. Bacteriol. 171:3218-3227, 1989). Six of the seven mutations in ftr4 examined resulted in a large increase in fliF operon transcript levels, suggesting a role for ftr4 in negative regulation. A 2-bp deletion at -12 and -13 in ftr4 altered the cell cycle pattern of fliF operon transcription; the transcript was still expressed periodically, but the period of its synthesis was extended significantly. We suggest that the ftr4 sequence may form part of a developmental switch which is required to turn off fliF operon transcription at the correct time in the cell cycle.

Base Sequence↗

PubMed1993

A sigma 54 promoter and downstream sequence elements ftr2 and ftr3 are required for regulated expression of divergent transcription units flaN and flbG in Caulobacter crescentus.

In this study, we investigated the cis-acting sequences required for transcription of the divergent, cell cycle-regulated flaN and flbG operons of Caulobacter crescentus. Previous work showed that transcription of flbG in vivo depends on a sigma 54 promoter and a sequence element called ftr1 that is located about 100 bp upstream from the transcription start site (D. A. Mullin and A. Newton, J. Bacteriol. 171:3218-3227, 1989). We now show that regulation of flaN transcription in vivo depends on a sigma 54 promoter and two ftr elements located downstream of the transcription start site at +86 (ftr2) and +120 (ftr3). Mutations in or between the conserved elements at -24 and -12 in this sigma 54 promoter reduced or abolished flaN transcription, and one mutation that eliminated flaN expression led to an increased level of flbG transcript. Mutations in ftr2 resulted in greatly reduced levels of flaN transcript but had no noticeable effect on flbG transcript levels. All three mutations constructed in ftr3 resulted in elevated flaN and flbG transcript levels. We conclude that ftr2 is required for positive regulation of flaN, whereas ftr3 appears to play a negative regulatory role in flaN and flbG expression. To explain the coordinated positive activation and negative autoregulation of these two transcription units and the effect of mutations on gene expression, we propose a model in which the flaN and flbG promoters interact through alternative DNA looping to form structures that are transcriptionally active or inactive.

Bacterial Proteins↗

PubMed1992

A histidine protein kinase homologue required for regulation of bacterial cell division and differentiation.

Differentiation in the dimorphic bacterium Caulobacter crescentus results from a sequence of discontinuous, stage-specific events that leads to the production of a stalked cell and a new motile swarmer cell after each asymmetric cell division. As reported previously, pseudoreversion analysis of mutations in the pleiotropic developmental gene pleC identified three cell division genes: divJ, divK, and divL. We show here that one of these genes, divJ, encodes a predicted protein of 596 residues with an extensive hydrophobic N-terminal region and a C-terminal domain containing all of the invariant residues found in the family of bacterial histidine protein kinases. Our results also show that divJ is discontinuously transcribed early in the swarmer cell cycle during a period that coincides with the G1 to S transition. We propose that the DivJ protein is one member of a signal transduction pathway regulating the cell cycle and differentiation in Caulobacter and that protein modification by phosphorylation may play a central role in coupling developmental events to progress through the cell division cycle.

Amino Acid Sequence↗