Biomedical subjects

J Errington

Publications and source records attributed to J Errington.

At least 19 recordsLinked to original sources

PubMed2001

Division site selection protein DivIVA of Bacillus subtilis has a second distinct function in chromosome segregation during sporulation.

DivIVA is a coiled-coil, tropomyosin-like protein of Gram-positive bacteria. Previous work showed that this protein is targeted to division sites and retained at the cell poles after division. In vegetative cells, DivIVA sequesters the MinCD division inhibitor to the cell poles, thereby helping to direct cell division to the correct midcell site. We now show that DivIVA has a second, quite separate role in sporulating cells of Bacillus subtilis. It again acts at the cell pole but in this case interacts with the chromosome segregation machinery to help position the oriC region of the chromosome at the cell pole, in preparation for polar division. We isolated mutations in divIVA that separate the protein's role in sporulation from its vegetative function in cell division. DivIVA therefore appears to be a bifunctional protein with distinct roles in division-site selection and chromosome segregation.

Adenosine Triphosphatases↗

PubMed2001

Control of cell shape in bacteria: helical, actin-like filaments in Bacillus subtilis.

In the absence of an overt cytoskeleton, the external cell wall of bacteria has traditionally been assumed to be the primary determinant of cell shape. In the Gram-positive bacterium Bacillus subtilis, two related genes, mreB and mbl, were shown to be required for different aspects of cell morphogenesis. Subcellular localization of the MreB and Mbl proteins revealed that each forms a distinct kind of filamentous helical structure lying close to the cell surface. The distribution of the proteins in different species of bacteria, and the similarity of their sequence to eukaryotic actins, suggest that the MreB-like proteins have a cytoskeletal, actin-like role in bacterial cell morphogenesis.

Actins↗

PubMed2001

DNA transport in bacteria.

DNA transport is important in various biological contexts--particularly chromosome segregation and intercellular gene transfer. Recently, progress has been made in understanding the function of a family of bacterial proteins involved in DNA transfer, and we focus here on one of the best-understood members, SpoIIIE. Studies of SpoIIIE-like proteins show that they might couple DNA transport to processes such as cell division, conjugation (mating) and the resolution of chromosome dimers.

Bacteria↗

PubMed2001

Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli.

The twin-arginine translocation (Tat) system targets cofactor-containing proteins across the Escherichia coli cytoplasmic membrane via distinct signal peptides bearing a twin-arginine motif. In this study, we have analysed the mechanism and capabilities of the E. coli Tat system using green fluorescent protein (GFP) fused to the twin-arginine signal peptide of TMAO reductase (TorA). Fractionation studies and fluorescence measurements demonstrate that GFP is exported to the periplasm where it is fully active. Export is almost totally blocked in tat deletion mutants, indicating that the observed export in wild-type cells occurs predominantly, if not exclusively, by the Tat pathway. Imaging studies reveal a halo of fluorescence in wild-type cells corresponding to the exported periplasmic form; the GFP is distributed uniformly throughout the cytoplasm in a tat mutant. Because previous work has shown GFP to be incapable of folding in the periplasm, we propose that GFP is exported in a fully folded, active state. These data also show for the first time that heterologous proteins can be exported in an active form by the Tat pathway.

Amino Acid Sequence↗

PubMed2001

Cytological and biochemical characterization of the FtsA cell division protein of Bacillus subtilis.

The actin-like protein FtsA is present in many eubacteria, and genetic experiments have shown that it plays an important, sometimes essential, role in cell division. Here, we show that Bacillus subtilis FtsA is targeted to division sites in both vegetative and sporulating cells. As in other organisms FtsA is probably recruited immediately after FtsZ. In sporulating cells of B. subtilis FtsZ is recruited to potential division sites at both poles of the cell, but asymmetric division occurs at only one pole. We have now found that FtsA is recruited to only one cell pole, suggesting that it may play an important role in the generation of asymmetry in this system. FtsA is present in much higher quantities in B. subtilis than in Escherichia coli, with approximately one molecule of FtsA for five of FtsZ. This means that there is sufficient FtsA to form a complete circumferential ring at the division site. Therefore, FtsA may have a direct structural role in cell division. We have purified FtsA and shown that it behaves as a dimer and that it has both ATP-binding and ATP-hydrolysis activities. This suggests that ATP hydrolysis by FtsA is required, together with GTP hydrolysis by FtsZ, for cell division in B. subtilis (and possibly in most eubacteria).

Adenosine Triphosphatases↗

PubMed2001

The impact of p53 status on cellular sensitivity to antifolate drugs.

The impact of p53 status on cellular sensitivity to antifolate drugs has been examined in seven human cell lines (A549, MCF7, T-47D, CCRF-CEM, COR-L23, A2780, and HCT-116) and p53 nonfunctional counterparts of two of the cell lines (HCT-116/N7 and A2780/CP70). p53 status was determined by sequencing and functional assays. The sensitivities of the cell lines to growth inhibition (sulphorhodamine B assay) produced by four antifolate drugs (Alimta, methotrexate, raltitrexed, and lometrexol) were studied. There was no clear relationship between functional p53 status and sensitivity to methotrexate or lometrexol, whereas a functional p53 status was possibly associated with resistance to Alimta- and raltitrexed-induced growth inhibition. In contrast, in the two pairs of related human tumor cell lines (HCT-116 and HCT-116/N7 and A2780 and A2780/CP70) cells with functional p53 were more sensitive to Alimta- and raltitrexed-induced growth inhibition (P = 0.002). Detailed studies were performed with the A2780 cell lines, and in the parental cells sensitivity to Alimta- and raltitrexed-induced cytotoxicity (clonogenic assay) was similar to the sensitivity determined in the sulphorhodamine B assay. However, in A2780/CP70 cells, 1 microM of drug resulted in only 40-60% growth inhibition yet > or = 85% cytotoxicity. After Alimta and raltitrexed exposure for < or = 72 h, there were no differences between the A2780 and A278/CP70 cell lines in cell cycle phase distribution, absolute cell number, or the induction of apoptosis. However, the cellular protein content of the A2780/CP70 cells was 3-6-fold higher than in A2780 cells after Alimta and raltitrexed treatment, suggesting that cells without functional p53 can maintain protein synthesis in the absence of cell division (unbalanced cell growth). In conclusion, the apparent impact of functional p53 status on sensitivity to antifolate drugs may depend upon the phenotypic/genotypic background as well as the assay used to measure cellular sensitivity.

Apoptosis↗

PubMed2000

Role of Bacillus subtilis SpoIIIE in DNA transport across the mother cell-prespore division septum.

The SpoIIIE protein of Bacillus subtilis is required for chromosome segregation during spore formation. The COOH-terminal cytoplasmic part of SpoIIIE was shown to be a DNA-dependent adenosine triphosphatase (ATPase) capable of tracking along DNA in the presence of ATP, and the NH(2)-terminal part of the protein was found to mediate its localization to the division septum. Thus, during sporulation, SpoIIIE appears to act as a DNA pump that actively moves one of the replicated pair of chromosomes into the prespore. The presence of SpoIIIE homologs in a broad range of bacteria suggests that this mechanism for active transport of DNA may be widespread.

Adenosine Triphosphatases↗

PubMed2000

Dynamic relocalization of phage phi 29 DNA during replication and the role of the viral protein p16.7.

We have examined the localization of DNA replication of the Bacillus subtilis phage phi 29 by immunofluorescence. To determine where phage replication was localized within infected cells, we examined the distribution of phage replication proteins and the sites of incorporation of nucleotide analogues into phage DNA. On initiation of replication, the phage DNA localized to a single focus within the cell, nearly always towards one end of the host cell nucleoid. At later stages of the infection cycle, phage replication was found to have redistributed to multiple sites around the periphery of the nucleoid, just under the cell membrane. Towards the end of the cycle, phage DNA was once again redistributed to become located within the bulk of the nucleoid. Efficient redistribution of replicating phage DNA from the initial replication site to various sites surrounding the nucleoid was found to be dependent on the phage protein p16.7.

Bacillus Phages↗

PubMed2000

Promiscuous targeting of Bacillus subtilis cell division protein DivIVA to division sites in Escherichia coli and fission yeast.

The Bacillus subtilis divIVA gene encodes a coiled-coil protein that shows weak similarity to eukaryotic tropomyosins. The protein is targeted to the sites of cell division and mature cell poles where, in B.subtilis, it controls the site specificity of cell division. Although clear homologues of DivIVA are present only in Gram-positive bacteria, and its role in division site selection is not conserved in the Gram-negative bacterium, Escherichia coli, a DivIVA-green fluorescent protein (GFP) fusion was targeted accurately to division sites and retained at the cell pole in this organism. Remarkably, the same fusion protein was also targeted to nascent division sites and growth zones in the fission yeast Schizosaccharomyces pombe, mimicking the localization of the endogenous tropomyosin-like cell division protein Cdc8p, and F-actin. The results show that a targeting signal for division sites is conserved across the eukaryote-prokaryote divide.

Actins↗

PubMed2000

Direct interaction between the cell division protein FtsZ and the cell differentiation protein SpoIIE.

SpoIIE is a bifunctional protein with two critical roles in the establishment of cell fate in Bacillus subtilis. First, SpoIIE is needed for the normal formation of the asymmetrically positioned septum that forms early in sporulation and separates the mother cell from the prespore compartment. Secondly, SpoIIE is essential for the activation of the first compartment-specific transcription factor sigma(F) in the prespore. After initiation of sporulation, SpoIIE localizes to the potential asymmetric cell division sites near one or both cell poles. Localization of SpoIIE was shown to be dependent on the essential cell division protein FtsZ. To understand how SpoIIE is targeted to the asymmetric septum we have now analysed its interaction with FtsZ in vitro. Using the yeast two-hybrid system and purified FtsZ, and full-length and truncated SpoIIE proteins, we demonstrate that the two proteins interact directly and that domain II and possibly domain I of SpoIIE are required for the interaction. Moreover, we show that SpoIIE interacts with itself and suggest that this self-interaction plays a role in assembly of SpoIIE into the division machinery.

Bacillus subtilis↗

PubMed2000

Compartmentalization of transcription and translation in Bacillus subtilis.

Using fusions of green fluorescent protein to subunits of RNA polymerase (RNAP) and ribosomes, we have investigated the subcellular localization of the transcriptional and translational machinery in the bacterium Bacillus subtilis. Unexpectedly, we found that RNAP resides principally within the nucleoid. Conversely, ribosomes localized almost exclusively outside the nucleoid, concentrating particularly towards sites of cell division. This zonal localization was not dependent on cell division and is probably due, at least in part, to exclusion from the nucleoid. Dual labelling of RNAP and ribosomes was used to confirm the spatial separation of the two processes. We conclude that, even in the absence of a nuclear membrane, transcription and translation occur predominantly in separate functional domains. At higher growth rates, concentrations of RNAP developed, probably representing the sites of rRNA synthesis. These may represent a further spatial specialization, possibly equivalent to the eukaryotic nucleolus.

Bacillus subtilis↗

PubMed2000

Role of penicillin-binding protein PBP 2B in assembly and functioning of the division machinery of Bacillus subtilis.

We have characterized the role of the penicillin-binding protein PBP 2B in cell division of Bacillus subtilis. We have shown that depletion of the protein results in an arrest in division, but that this arrest is slow, probably because the protein is relatively stable. PBP 2B-depleted filaments contained, at about their mid-points, structures resembling partially formed septa, into which most, if not all, of the division proteins had assembled. Although clearly deficient in wall material, membrane invagination seemed to continue, indicating that membrane and wall ingrowth can be uncoupled. At other potential division sites along the filaments, no visible ingrowths were observed, although FtsZ rings assembled at regular intervals. Thus, PBP 2B is apparently required for both the initiation of division and continued septal ingrowth. Immunofluorescence microscopy showed that the protein is recruited to the division site. The pattern of localization suggested that this recruitment occurs continually during septal ingrowth. During sporulation, PBP 2B was present transiently in the asymmetrical septum of sporulating cells, and its availability may play a role in the regulation of sporulation septation.

Bacillus subtilis↗

PubMed2000

Intrinsic instability of the essential cell division protein FtsL of Bacillus subtilis and a role for DivIB protein in FtsL turnover.

Cell division in most eubacteria is driven by an assembly of about eight conserved division proteins. These proteins form a ring structure that constricts in parallel with the formation of the division septum. Here, we show that one of the division proteins, FtsL, is highly unstable. We also show that the protein is targeted to the ring structure and that targeting occurs in concert with the recruitment of several other membrane-associated division proteins. FtsL stability is further reduced in the absence of DivIB protein (probably homologous to E. coli FtsQ) at high temperature, suggesting that DivIB is involved in the control of FtsL turnover. The reduced stability of FtsL may explain the temperature dependence of divIB mutants, because their phenotype can be suppressed by overexpression of FtsL. The results provide new insights into the roles of the FtsL and DivIB proteins in bacterial cell division.

Bacillus subtilis↗

PubMed2000

The Bacillus subtilis cell division protein FtsL localizes to sites of septation and interacts with DivIC.

FtsL is a small bitopic membrane protein required for vegetative cell division and sporulation in Bacillus subtilis. We investigated its localization by fluorescence microscopy using a green fluorescent protein (GFP) fusion. GFP-FtsL was localized at mid-cell in vegetative cells and at the asymmetric septum in sporulating cells. We also show that FtsL forms a ring-like structure at the division site and that it remains localized at mid-cell during the whole septation process. By yeast two-hybrid analysis and non-denaturing polyacrylamide gel electrophoresis (PAGE) with purified proteins, FtsL was found to interact with another membrane-bound division protein, the FtsL-like DivIC protein.

Animals↗

PubMed2000

Analysis of the essential cell division gene ftsL of Bacillus subtilis by mutagenesis and heterologous complementation.

The ftsL gene is required for the initiation of cell division in a broad range of bacteria. Bacillus subtilis ftsL encodes a 13-kDa protein with a membrane-spanning domain near its N terminus. The external C-terminal domain has features of an alpha-helical leucine zipper, which is likely to be involved in the heterodimerization with another division protein, DivIC. To determine what residues are important for FtsL function, we used both random and site-directed mutagenesis. Unexpectedly, all chemically induced mutations fell into two clear classes, those either weakening the ribosome-binding site or producing a stop codon. It appears that the random mutagenesis was efficient, so many missense mutations must have been generated but with no phenotypic effect. Substitutions affecting hydrophobic residues in the putative coiled-coil domain, introduced by site-directed mutagenesis, also gave no observable phenotype except for insertion of a helix-breaking proline residue, which destroyed FtsL function. ftsL homologues cloned from three diverse Bacillus species, Bacillus licheniformis, Bacillus badius, and Bacillus circulans, could complement an ftsL null mutation in B. subtilis, even though up to 66% of the amino acid residues of the predicted proteins were different from B. subtilis FtsL. However, the ftsL gene from Staphylococcus aureus (whose product has 73% of its amino acids different from those of the B. subtilis ftsL product) was not functional. We conclude that FtsL is a highly malleable protein that can accommodate a large number of sequence changes without loss of function.

Alleles↗

PubMed2000

Identification and characterization of a new prespore-specific regulatory gene, rsfA, of Bacillus subtilis.

Differential gene expression during Bacillus subtilis sporulation is controlled by sigma factors and other regulatory effectors. The first compartmentalized sigma factor, sigma(F), is active specifically in the prespore compartment. During our screening for new chromosome segregation mutants using a sigma(F)-dependent gpr-lacZ reporter as a probe, we identified a new gene (ywfN) required for maximal expression of the reporter and named it rsfA. The product of rsfA has features of gene regulatory proteins, and the protein colocalizes with DNA. The expression of rsfA is under the control of both sigma(F) and sigma(G). Null mutations in rsfA have different effects on the expression of sigma(F)-dependent genes, suggesting that the RsfA protein is a regulator of transcription that fine-tunes gene expression in the prespore.

Amino Acid Sequence↗

PubMed2000

Cellular ATP depletion by LY309887 as a predictor of growth inhibition in human tumor cell lines.

The antifolate LY309887 is a specific glycinamide ribonucleotide formyltransferase inhibitor that blocks de novo purine synthesis and produces a depletion of purine nucleotides. The activity of LY309887 in six human tumor cell lines has been examined by growth inhibition and clonogenic assay after continuous exposure for three cell doubling times and by ATP depletion at 24 h. Three cell lines (CCRF-CEM, MCF7, and GC3) were sensitive to LY309887-induced growth inhibition (IC50: 5.6-8.1 nM), whereas the other cell lines (COR-L23, T-47D, and A549) were comparatively resistant (IC50: 36-55 nM). Sensitivity to LY309887 cytotoxicity was consistent with sensitivity to growth inhibition in four of five cell lines tested (MCF7/GC3: 0.01% survival and COR-L23/T-47D: 1-5% survival at 100 nM LY309887). LY309887-induced ATP depletion was measured by luciferase-based ATP assay and confirmed by high performance liquid chromatography measurements. There was a linear relationship between ATP depletion and growth inhibition when data were analyzed for all six cell lines (r2 = 0.93; P < 0.0001). Depletion of 24-h cellular ATP concentrations to < 1 mM was associated with both cell growth inhibition and cytotoxicity in all cell lines studied. In conclusion, cellular ATP depletion induced by LY309887 can be used to predict growth inhibition and cytotoxicity in human tumor cells.

Adenosine Triphosphate↗

PubMed1999

Upheaval in the bacterial nucleoid. An active chromosome segregation mechanism.

Recent advances have completely overturned the classical view of chromosome segregation in bacteria. Far from being a passive process involving gradual separation of the chromosomes, an active, possibly mitotic-like machinery is now known to exist. Soon after the initiation of DNA replication, the newly replicated copies of the oriC region, behaving rather like eukaryotic centromeres, move rapidly apart towards opposite poles of the cell. They then determine the positions that will be taken up by the newly formed sister nucleoids when DNA replication has been completed. Thus, the gradual expansion of the diffuse nucleoid camouflages an underlying active mechanism. Several genes involved in chromosome segregation in bacteria have now been defined; their possible functions are discussed.

Anaphase↗