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Biomedical subjects

A Newton

Publications and source records attributed to A Newton.

At least 55 records · Page 3Linked to original sources
PubMed1992

Cell cycle regulation in bacteria.

Significant progress has been made in the study of ftsZ expression and the topology of FtsZ protein localization in Escherichia coli cells. Exciting results on the identification of new genes required for chromosome resolution and partitioning after the completion of DNA synthesis have also been reported. A recent area of study is asymmetric cell division and its role in differentiation in Bacillus subtilis and Caulobacter crescentus. Biochemical activities of bacterial cell division gene products are also beginning to be addressed.

Bacteria↗
PubMed1991

Pseudoreversion analysis indicates a direct role of cell division genes in polar morphogenesis and differentiation in Caulobacter crescentus.

A pseudoreversion analysis was used to examine the role of cell division genes in polar morphogenesis in Caulobacter crescentus. Extragenic suppressors of temperature sensitive mutations in pleC, a pleiotropic gene required for cell motility, formation of polar phi CbK bacteriophage receptors, and stalk formation, were isolated. These suppressors, which restored motility at 37 degrees C, simultaneously conferred a cold sensitive cell division phenotype and they were mapped to the three new cell division genes divJ, divL and divK. The cold-sensitive mutations in divL, and to a lesser extent divJ, exhibited a relatively narrow range of suppression. The cold-sensitive cell division mutation in divK, by contrast, suppressed all pleC mutations examined and behaved as a classical bypass suppressor. The direct role of this cell division gene in the regulation of motility is suggested by the observation that divK341 mapped to the same locus as pleD301, a pleiotropic mutation that prevents loss of motility and stalk formation. These results provide strong evidence that the cell division and developmental pathways are interconnected and they support our earlier conclusion that cell division is required for the regulation of polar morphogenesis and differentiation in C. crescentus.

Caulobacter↗
PubMed1991

The cell cycle-regulated flagellar gene flbF of Caulobacter crescentus is homologous to a virulence locus (lcrD) of Yersinia pestis.

We have characterized flbF, a key locus located at the top of the flagellar gene hierarchy of Caulobacter crescentus. This gene is required for transcription from sigma 54 promoters of fla genes expressed late in the cell cycle. We have determined the nucleotide sequence of the gene, mapped the 5' end of the flbF RNA, and examined the pattern of expression in the cell cycle. Our results show that flbF is expressed earlier in the cell cycle than other fla genes, that it is expressed at a low level throughout the stalked cell cycle, and that its 5' regulatory region contains sequences that can be aligned with the sigma 28 promoter consensus reported for enteric bacteria. flbF contains an open reading frame of 700 residues with an amino-terminal half rich in hydrophobic residues that could correspond to six to eight transmembrane domains. The translated flbF sequence is very similar to LcrD (low calcium response) encoded by virulence plasmids of pathogenic Yersinia spp. (G. Plano, S. Barve, and S. Straley, J. Bacteriol. 173:7293-7303, 1991). LcrD and FlbF can be aligned over the entire length of the proteins with the greatest degree of sequence identity (45%) in the hydrophobic amino-terminal region. The high degree of sequence homology of proteins derived from widely differing organisms, including Caulobacter and Yersinia species, suggests that FlbF and LcrD may be representatives of a larger family of regulatory proteins with a common sensor mechanism for modifying responses to appropriate stimuli.

Amino Acid Sequence↗
PubMed1991

Timing of flagellar gene expression in the Caulobacter cell cycle is determined by a transcriptional cascade of positive regulatory genes.

The Caulobacter crescentus flagellar (fla) genes are organized in a regulatory hierarchy in which genes at each level are required for expression of those at the next lower level. To determine the role of this hierarchy in the timing of fla gene expression, we have examined the organization and cell cycle regulation of genes located in the hook gene cluster. As shown here, this cluster is organized into four multicistronic transcription units flaN, flbG, flaO, and flbF that contain fla genes plus a fifth transcription unit II.1 of unknown function. Transcription unit II.1 is regulated independently of the fla gene hierarchy, and it is expressed with a unique pattern of periodicity very late in the cell cycle. The flaN, flbG, and flaO operons are all transcribed periodically, and flaO, which is near the top of the hierarchy and required in trans for the activation of flaN and flbG operons, is expressed earlier in the cell cycle than the other two transcription units. We have shown that delaying flaO transcription by fusing it to the II.1 promoter also delayed the subsequent expression of the flbG operon and the 27- and 25-kDa flagellin genes that are at the bottom of the regulatory hierarchy. Thus, the sequence and timing of fla gene expression in the cell cycle are determined in large measure by the positions of these genes in the regulatory hierarchy. These results also suggest that periodic transcription is a general feature of fla gene expression in C. crescentus.

Bacterial Proteins↗
PubMed1990

FlbD of Caulobacter crescentus is a homologue of the NtrC (NRI) protein and activates sigma 54-dependent flagellar gene promoters.

The periodic transcription of flagellar genes in the Caulobacter crescentus cell cycle is controlled, in part, by their organization in a regulatory hierarchy. The flbG (hook operon), flaN, and flagellin gene operons, which are at the lowest levels of the hierarchy and expressed late in the cell cycle, contain Ntr-like promoters. We report that flbD, one of the early genes required in trans for expression of these operons, codes for a 52-kDa protein homologous to the transcriptional activators NtrC (NRI), NifA, DctD, HydG, and XylR. Our results show that in Escherichia coli flbD partially complements glnG (ntrC) mutations and stimulates transcription of the C. crescentus sigma 54 RNA polymerase-dependent flbG gene. Additionally, the sequence predicts that FlbD protein, along with NtrC, DctD, and HydG proteins, is structurally related at the amino-terminal domain to a larger family of response regulators that mediate cellular responses to environmental stimuli. FlbD may be a singular member of this large protein family in that its function is tied to an internal cell-cycle signal. FlbD is also unusual in that its amino-terminal domain contains only one of the three residues conserved in previously described members of this family of response regulators.

Amino Acid Sequence↗
PubMed1990

Identification, distribution, and sequence analysis of new insertion elements in Caulobacter crescentus.

We describe two insertion elements isolated from Caulobacter crescentus that are designated IS298 and IS511. These insertion elements were cloned from spontaneous flagellar (fla) gene mutants SC298 and SC511 derived from the wild-type strain CB15 (ATCC 19089), in which they were originally identified as insertions in the flbG operon of the hook gene cluster (N. Ohta, E. Swanson, B. Ely, and A. Newton, J. Bacteriol. 158:897-904, 1984). IS298 and IS511 were each present in C. crescentus CB2 and CB15 in at least four different positions, but neither was present in strain CB13 or in several Caulobacter species examined, including C. vibrioides, C. leidyia, and C. henricii. Nucleotide sequence analysis across the chromosome-insertion element junctions showed that IS298 is located 152 base pairs (bp) upstream from the ATG translation start of the hook protein gene flaK, where it is bounded by a 4-bp direct repeat derived from the site of insertion, and that IS511 is inserted at codon 186 of the flaK coding sequence, where it is also bounded by a 4-bp direct repeat duplicated from the site of insertion. The ilvB102 mutation in strain SC125 was also shown to result from insertion sequence IS511, but no duplication of the genomic sequence was present at the insertion element junctions. IS298 contains an imperfect terminal inverted repeat 16 bp long, and IS511 contains a 32-bp inverted repeat at the termini. IS298 and IS511 are the first insertion elements described in C. crescentus.

Bacteria↗
PubMed1990

Cloning and cell cycle-dependent expression of DNA replication gene dnaC from Caulobacter crescentus.

Chromosome replication in the asymmetrically dividing bacteria Caulobacter crescentus is discontinuous with the new, motile swarmer cell undergoing an obligatory presynthetic gap period (G1 period) of 60 min before the initiation of DNA synthesis and stalk formation. To examine the regulation of the cell division cycle at the molecular level, we have cloned the DNA chain elongation gene dnaC from a genomic DNA library constructed in cosmid vector pLAFR1-7. To ensure that the cloned sequence corresponded to dnaC, we isolated the gene by genetic complementation of the temperature-sensitive allele dnaC303 on DNA fragment that contained a Tn5 insertion element tightly linked by transduction to dnaC. The size of the dnaC gene was estimated to be 1,500 bp or less based on the pattern of complementation by subcloned restriction and BAL 31 deletion fragments. Nuclease S1 assays were used to map the transcription start site and to determine the pattern of dnaC expression in the cell cycle. Large amounts of the dnaC transcript began to accumulate only in the late G1 period of the swarmer cell and then peaked early during chromosome replication. We confirmed that the gene is periodically transcribed by monitoring the rate of beta-galactosidase synthesis directed by a dnaC promoter-lacZ fusion in a synchronous cell culture. dnaC is the first C. crescentus cell cycle gene whose regulation has been reported, and the discontinuous pattern of its expression suggests that the DNA synthetic period in these dimorphic bacteria is regulated in part by the stage-specific expression of DNA replication genes.

Bacterial Proteins↗
PubMed1989

Genetic switching in the flagellar gene hierarchy of Caulobacter requires negative as well as positive regulation of transcription.

Caulobacter crescentus flagellar (fla, flb, or flg) genes are periodically expressed in the cell cycle and they are organized in a regulatory hierarchy. We have analyzed the genetic interactions required for fla gene expression by determining the effect of mutations in 30 known fla genes on transcription from four operons in the hook gene cluster. These results show that the flaO (transcription unit III) and flbF (transcription unit IV) operons are located at or near the top of the hierarchy. They also reveal an extensive network of negative transcriptional controls that are superimposed on the positive regulatory cascade described previously. The strong negative autoregulation observed for the flaN (transcription unit I), flbG (transcription unit II), and flaO (transcription unit III) promoters provides one possible mechanism for turning off fla gene expression at the end of the respective synthetic periods. We suggest that these positive and negative transcriptional interactions are components of genetic switches that determine the sequence in which fla genes are turned on and off in the C. crescentus cell cycle.

Bacteria↗
PubMed1989

Cyclitic glaucoma.

A series of patients is presented showing the widely variable clinical behaviour of different groups of patients who share the common features of cyclitic glaucoma. These cyclitic glaucomas may be classified: 1. Glaucomatocyclitic crises: unilateral or bilateral. This condition presents acutely, almost always in males with a strong tendency to spontaneous resolution but a liability to subsequent recurrence. Some cases are slow to resolve and may recur frequently. 2. Heterochromic cyclitis: glaucoma infrequently complicates heterochromic cyclitis and then with only a mild rise in intraocular pressure (IOP). 3. Chronic cyclitis: unilateral or bilateral. This is more common in females often presenting with a high rise in IOP which may be difficult to control medically. An insidious form of chronic cyclitis occurs in middle-aged, blue-eyed females, often with sarcoid, and is likely to progress to profound visual loss if unrecognised.

Adolescent↗
PubMed1989

Escherichia coli sigma 54 RNA polymerase recognizes Caulobacter crescentus flbG and flaN flagellar gene promoters in vitro.

A set of the periodically regulated flagellar (fla) genes of Caulobacter crescentus contain conserved promoter sequence elements at -24 and -12 that are very similar to the sequence of the nitrogen assimilation (Ntr) and nitrogen fixation (Nif) promoters of enteric bacteria and Rhizobium spp. Transcription from Ntr and Nif promoters requires RNA polymerase containing sigma 54 instead of the usual sigma 70 and, in the case of the Ntr promoters, is activated by the transcription factors NRI and NRII. We have now demonstrated that the C. crescentus flbG and flaN promoters, which contain the Ntr/Nif type of consensus sequence, are utilized by purified Escherichia coli sigma 54 RNA polymerase (E sigma 54) in the presence of NRI and NRII but not by the purified sigma 70 RNA polymerase (E sigma 70) of E. coli. Oligonucleotide-generated flbG promoter deletions that removed the highly conserved GG dinucleotide at -24 or the GC dinucleotide at -12 or altered the spacing between the -24 and -12 sequence elements prevented utilization of the flbG promoter by the E. coli E sigma 54. Transversions of T to G at positions -26 and -15 also inactivated flbG promoter function in the E. coli cell-free transcription system, while a transition of G to A at position -16 in the nonconserved spacer region had no effect. The C. crescentus flaO and flbF promoters, which do not contain the Ntr/Nif-type promoter consensus sequence, were not utilized by either purified E sigma 54 or E sigma 70 from E. coli. Our results help to define the features of the Ntr/Nif-type consensus sequence required for promoter utilization by purified E. coli E sigma 54 and support the idea that C. crescentus may contain a specialized polymerase with similar promoter specificity required for expression of a set of fla genes.

Bacteria↗
PubMed1989

Turning off flagellum rotation requires the pleiotropic gene pleD: pleA, pleC, and pleD define two morphogenic pathways in Caulobacter crescentus.

We have identified mutations in three pleiotropic genes, pleA, pleC, and pleD, that are required for differentiation in Caulobacter crescentus. pleA and pleC mutants were isolated in an extensive screen for strains defective in both motility and adsorption of polar bacteriophage phi CbK; using temperature-sensitive alleles, we determined the time at which the two genes act. pleA was required for a short period at 0.7 of the swarmer cell cycle for flagellum biosynthesis, whereas pleC was required during an overlapping period from 0.6 to 0.95 of the cell cycle to activate flagellum rotation as well as to enable loss of the flagellum and stalk formation by swarmer cells after division. The third pleiotropic gene, pleD, is described here for the first time. A pleD mutation was identified as a bypass suppressor of a temperature-sensitive pleC allele. Strains containing this mutation were highly motile, did not shed the flagellum or form stalks, and retained motility throughout the cell cycle. Since pleD was required to turn off motility and was a bypass suppressor of pleC, we conclude that it acts after the pleA and pleC gene functions in the cell cycle. No mutants defective in both flagellum biosynthesis and stalk formation were identified. Consequently, we propose that the steps required for formation of swarmer cells and subsequent development into stalked cells are organized into at least two developmental pathways: a pleA-dependent sequence of events, responsible for flagellum biosynthesis in predivisional cells, and a pleC-pleD-dependent sequence, responsible for flagellum activation in predivisional cells and loss of motility and stalk formation in progeny swarmer cells.

Bacteria↗
PubMed1989

Ntr-like promoters and upstream regulatory sequence ftr are required for transcription of a developmentally regulated Caulobacter crescentus flagellar gene.

The flbG (hook operon or transcription unit II) and flaN (transcription unit I) operons of Caulobacter crescentus have a -12, -24 nucleotide sequence motif that is very similar to those of the Nif and Ntr promoters of enteric bacteria and Rhizobium spp. and a conserved ftr (flagellar gene transcription regulation) sequence, previously designated II-1 (D. A. Mullin, S. A. Minnich, L.-S. Chen, and A. Newton, J. Mol. Biol. 195:939-943, 1987) at approximately -100. We have used site-directed mutagenesis to examine the role of these sequences in the transcriptional regulation of these periodically expressed flagellar genes. Mutations in the flbG promoter that removed the conserved GC at -12, -13, the GG at -24, -25, or an AC base pair at -18, -19 in the nonconserved sequence between the -12, -24 elements completely eliminated detectable transcription. Mutations at other positions resulted in either a slight decrease (position 26), no change (position 15), or an elevated level (position -16 or -19) of the flbG transcript. By contrast, most of these flbG promoter mutations resulted in greatly elevated levels of transcription from the opposing flaN operon. Similar experiments were used to confirm the location of the flaN promoter to a -12, -24 Nif and Ntr sequence motif. Deletion of all or part of the ftr element or point mutations in the sequence drastically reduced the level of flbG transcript and resulted in increased levels of the flaN transcript. Thus, the conserved sequences at -12 and -24 in flbG and flaN are required for transcription of these genes in vivo, and the ftr element is required for transcription of flbG. This analysis also suggested that the ftr sequence and sequences in the flbG promoter are required for the negative autoregulation of the flbG and flaN operons. We speculate that the flbG and flaN promoters and the ftr element interact in some way to mediate the negative control of these divergent transcription units.

Base Sequence↗
PubMed1988

Determination of diastereoisomers of bromadiolone, an anticoagulant rodenticide, in animal tissues by high-performance liquid chromatography.

Two components isolated by semi-preparative normal phase high-performance liquid chromatography (HPLC) of bromadiolone reference material were tentatively identified as diastereoisomeric forms. Examination by mass spectroscopy confirmed this identification and supporting evidence was provided by identical UV fluorescence characteristics. The separated isomers were used to examine the chromatographic properties of bromadiolone in ion-pair, ion-suppression and weak ion-exchange HPLC modes. Conditions suitable for the analytical determination of the individual diastereoisomers were established for each mode. The influence of mobile phase pH on the resolution of coumarin-based rodenticides by weak ion-exchange HPLC on an aminopropyl-bonded phase was studied. Clean-up techniques for the determination of residues of bromadiolone in animal tissue extracts were compared. A combined gel permeation and adsorption chromatographic procedure was preferred for sensitive assay; it permitted the use of either fluorescence or UV detection. The lower practical limit of determination of each isomer in animal tissues was 0.005 mg kg-1 using UV detection and 0.0005 mg kg-1 using fluorescence detection.

4-Hydroxycoumarins↗
PubMed1988

Sequential regulation of developmental events during polar morphogenesis in Caulobacter crescentus: assembly of pili on swarmer cells requires cell separation.

Pili, along with the flagellum and DNA bacteriophage receptors, are structural markers for polar morphogenesis in Caulobacter crescentus. Pili act as primary receptors for a number of small, C. crescentus-specific DNA and RNA bacteriophages, and the timing of pilus-dependent adsorption of bacteriophage phiCb5 in synchronized cell populations has led to the general conclusion that pili are formed coordinately with the flagellum and other polar surface structures in the predivisional cell. The use of rotary platinum shadow casting and electron microscopy as a direct assay for formation of flagella and pili in synchronous cell cultures now shows, however, that when expressed as fractions of the swarmer cell cycle, flagella are assembled on the predivisional cells at approximately 0.8 and that pili are assembled on the new swarmer cells at approximately 0.1 of the next cell cycle. Adsorption of pilus-specific bacteriophage phiCb5 prevented the loss of pili from swarmer cells during development, which suggests that these structures are retracted at the time of stalk formation. Examination of temperature-sensitive cell division mutants showed that the assembly of pili depends on completion of cell separation. These results indicate that the stage-specific events required for polar morphogenesis in C. crescentus occur sequentially, rather than coordinately in the cell cycle, and that the timing of these events reflects the order of underlying cell cycle steps.

Bacteria↗
PubMed1988

Identification of two new cell division genes that affect a high-molecular-weight penicillin-binding protein in Caulobacter crescentus.

Penicillin-binding proteins (PBPs) are membrane proteins associated with the synthesis of the bacterial cell wall. We report the characterization of 14 PBPs in Caulobacter crescentus, using in vivo and in vitro penicillin-binding assays and experiments to determine their possible role in cell division. New conditional cell cycle mutants were isolated by selecting cephalosporin-C-resistant mutants of the beta-lactamase strain SC1107 at 30 degrees C that are also defective in cell division at 37 degrees C. They fall into two classes, represented by strains PC8002 and PC8003. Strain PC8002 produced short cells arrested at all stages of cell division at 37 degrees C and was found to contain a high-molecular-weight PBP 1B which was temperature sensitive when assayed in vivo and in vitro. Strain PC8003 was blocked at an early stage of cell division and formed tightly coiled, unpinched filaments. This cephalosporin-C-resistant strain was also defective in PBP 1B, but only when assayed in vivo. PBP 1B behaved like a high-affinity PBP, and in competition assays, beta-lactams that induced filamentation bound preferentially to PBP 1B. These results and the phenotype of mutant PC8002 suggest that PBP 1B is required for cell division, as well as for cell growth, in C. crescentus. The behavior of strain PC8003 suggests that it contains a conditionally defective gene product that interacts in some way with PBP 1B at an early stage of cell division. None of the mutants showed an allele-specific PBP pattern when assayed in vitro at the nonpermissive temperature, but all of them displayed temperature-sensitive PBP 1C (102 kilodaltons) activity. Thus, it appears that PBP 1C is inhibited at 37 degree C as a consequence of filamentous growth.

Anti-Bacterial Agents↗