Control of the bacterial cell cycle.

New insights into the control of DNA replication through growth, hemimethylated DNA and DnaA protein have been described. Fundamental shifts in thinking have resulted in the identification of new cell cycle genes with potential roles in initiation of DNA replication, chromosomal segregation and division. Excitingly, this trend may also narrow the apparent differences between the prokaryotic and eukaryotic cell cycles.

Intracellular routing of GLcNAc-bearing molecules in thyrocytes: selective recycling through the Golgi apparatus.

Previous experiments led us to speculate that thyrocytes contain a recycling system for GlcNAc-bearing immature thyroglobulin molecules which prevents these molecules from lysosomal degradation (Miquelis, R., C. Alquier, and M. Monsigny. 1987. J. Biol. Chem. 262:15291-15298). To confirm this hypothesis, the fate of GlcNAc-bearing proteins after internalization by thyrocytes was monitored and compared to that of fluid phase markers. Kinetic internalization studies were performed using 125I-GlcNAc-BSA and 131I-Man-BSA. We observed that the apparent intake rate as well as the amount of hydrolyzed GlcNAc-BSA are smaller than the corresponding values for Man-BSA. These differences were reduced by GlcNAc competitors (thyroglobulin and ovomucoid) or a weak base (chloroquine). Part of the internalized GlcNAc-BSA was released into the extracellular milieu at a higher rate and shorter half life (t1/2 = approximately 30 min) than the Man-BSA (t1/2 = approximately 8 h). Subcellular homing was first studied by cell fractionation after internalization using 125I-ovomucoid and 131I-BSA. During Percoll density gradient fractionation, endogenous thyroperoxidase was used to separate subsets of organelles involved in the biosynthetic exocytotic pathway. Incubation of the cell homogenate in the presence of DAB and H2O2 before cell fractionation give rise to a shift in the density of organelles containing 3.5 times more ovomucoid than BSA. Discontinuous sucrose gradient showed that: (a) thyroperoxidase was colocalized with galactosyltransferase-contraining organelles in Golgi-rich subfractions; and (b) that at every time studied from 10 to 100 min, the ovomucoid/BSA ratio was higher in these organelles than in other subfractions. Finally we also observed that: (a) ovomucoid sequestered in the Golgi-rich subfraction incorporated [3H]galactose; and (b) that part of internalized ovomucoid was localized on the Golgi stacks as well as elements of the trans-Golgi, as revealed by immunogold labeling on ultrathin cryosections. These data prove that in thyrocytes GlcNAc accessible sugar moieties on soluble internalized molecules are sufficient to trigger their recycling via the Golgi apparatus.

Verapamil, a Ca2+ channel inhibitor acts as a local anesthetic and induces the sigma E dependent extra-cytoplasmic stress response in E. coli.

Verapamil is used clinically as a Ca(2+) channel inhibitor for the treatment of various disorders such as angina, hypertension and cardiac arrhythmia. Here we study the effect of verapamil on the bacterium Escherichia coli. The drug was shown to inhibit cell division at growth sub inhibitory concentrations, independently of the SOS response. We show verapamil is a membrane active drug, with similar effects to dibucaine, a local anesthetic. Thus, both verapamil and dibucaine abolish the proton motive force and decrease the intracellular ATP concentration. This is accompanied by induction of degP expression, as a result of the activation of the RpoE (SigmaE) extra-cytoplasmic stress response, and activation of the psp operon. Such effects of verapamil, as a membrane active compound, could explain its general toxicity in eukaryotic cells.

Cloning and analysis of the entire Escherichia coli ams gene. ams is identical to hmp1 and encodes a 114 kDa protein that migrates as a 180 kDa protein.

We have used an antibody to a previously identified 180 kDa (Hmp1) protein in Escherichia coli to clone the corresponding gene, which encodes a polypeptide of 114 kDa that has a mobility equivalent to 180 kDa in SDS/PAGE. We have demonstrated that the 180 kDa polypeptide is the primary gene product and not due to aggregation with other molecules. Moreover, our data indicate that the highly charged C-terminal region of the protein is responsible for its anomalous behaviour when analysed by SDS/PAGE. The hmp1 gene is in fact identical to ams (abnormal mRNA stability), also designated rne (RnaseE), and reported to have an ORF of 91 kDa. This discrepancy with the data in this paper can be ascribed to the omission of two bases in the previously reported sequence, generating an apparent stop codon. We previously demonstrated that the 180 kDa Hmp1/Ams protein cross reacted with both a polyclonal antibody and a monoclonal antibody raised against a yeast heavy chain myosin. However, we could detect no homology with myosin genes in the ams/hmp1 sequence. From the DNA sequence data, we identified a putative nucleotide binding site and a transmembrane domain in the N-terminal half of the molecule. In the C-terminal half, which appears to constitute a separate domain dominated by proline and charged amino acids, we also identified a region homologous to the highly conserved 70 kDa snRNP protein, involved in RNA splicing in eukaryotes. This feature would be consistent with reports that ams encodes RNaseE, an enzyme required for the processing of several stable RNAs in E. coli.

An assessment of the role of intracellular free Ca2+ in E. coli.

We have previously proposed that fluctuations in Ca(2+) levels should play an important role in bacteria as in eukaryotes in regulating cell cycle events (Norris et al., J. Theor. Biol. 134 (1998) 341-350). This proposal implied the presence of Ca(2+) uptake systems in bacteria, cell cycle mutants simultaneously defective in Ca(2+)-homeostasis, and perturbation of cell cycle processes when cellular Ca(2+) levels are depleted. We review the properties of new cell cycle mutants in E. coli and B. subtilis resistant to inhibitors of calmodulin, PKC or Ca(2+)-channels; the evidence for Ca(2+)-binding proteins including Acp and FtsZ; and Ca(2+)-transporters. In addition, the effects of EGTA and verapamil (a Ca(2+) channel inhibitor) on growth, protein synthesis and cell cycle events in E. coli are described. We also describe new measurements of free Ca(2+)-levels, using aequorin, in E. coli. Several new cell cycle mutants were obtained using this approach, affecting either initiation of DNA replication or in particular cell division at non-permissive temperature. Several of the mutants were also hypersensitive to EGTA and or Ca(2+). However, none of the mutants apparently involved direct alteration of a drug target and surprisingly in some cases involved specific tRNAs or a tRNA synthetase. The results also indicate that the expression of several genes in E. coli may be regulated by Ca(2+). Cell division in particular appears very sensitive to the level of cell Ca(2+), with the frequency of division clearly reduced by EGTA and by verapamil. However, whilst the effect of EGTA was clearly correlated with depletion of cellular Ca(2+) including free Ca(2+), this was not the case with verapamil which appears to change membrane fluidity and the consequent activity of membrane proteins. Measurement of free Ca(2+) in living cells indicated levels of 200-300 nM, tightly regulated in wild type cells in exponential phase, somewhat less so in stationary phase, with apparently La(2+)-sensitive PHB-polyphosphate complexes involved in Ca(2+) influx. The evidence reviewed increasingly supports a role for Ca(2+) in cellular processes in bacteria, however, any direct link to the control of cell cycle events remains to be established.

Analysis of a myosin-like protein and the role of calcium in the E. coli cell cycle.

For a number of years now, we have argued that current models for the control of initiation of DNA synthesis, chromosomal partitioning and septum formation in Escherichia coli are unsatisfactory. Indeed, we could argue that despite considerable efforts, with the possible exception of dnaA and ftsZ, no genes specifically implicated in these control processes have been identified. In the cases of DnaA and FtsZ, no evidence has appeared to indicate how such molecules might be regulated to act once per cycle. In 1988, we formulated a specific proposal that the timing of cell cycle events in E. coli might be determined by a Ca++ flux, mediated by calcium-binding proteins and protein kinases and culminating, in the case of chromosome segregation and division, in the action of force-generating proteins such as myosin (Norris et al., 1988). In formulating this proposal, we took the view that the fundamental elements of cell cycle regulation are likely to be highly conserved across all species including prokaryotes. In this presentation, we shall describe the approaches we have been taking in order to test this hypothesis and to summarize the data obtained, in particular in relation to new genes identified which may play a role in the E. coli cell cycle. We shall also briefly indicate recent data from other laboratories consistent with our general hypothesis.

A DNA-binding protein specific for the early replicated region of the chromosome obtained from Escherichia coli membrane fractions.

After extensive dialysis of Escherichia coli membranes treated with Triton X-100, three membrane proteins (A, B and B') with an affinity for DNA have been isolated and purified. They bind to either double-stranded or single-stranded DNA. A and B' proteins preferentially attach to DNA even in the presence of poly(uridylic acid). Only protein B' can recognize some base sequence of DNA because pulse-labelled DNA made at the initiation of replication in a synchronized dnaC mutant has been selectively retained by the protein.

A novel DnaJ-like protein in Escherichia coli inserts into the cytoplasmic membrane with a type III topology.

We describe a novel Escherichia coli protein, DjlA, containing a highly conserved J-region motif, which is present in the DnaJ protein chaperone family and required for interaction with DnaK. Remarkably, DjlA is shown to be a membrane protein, localized to the inner membrane with the unusual Type III topology (N-out, C-in). Thus, DjlA appears to present an extremely short N-terminus to the periplasm and has a single transmembrane domain (TMD) and a large cytoplasmic domain containing the C-terminal J-region. Analysis of the TMD of DjlA and recently identified homologues in Coxiella burnetti and Haemophilus influenzae revealed a striking pattern of conserved glycines (or rarely alanine), with a four-residue spacing. This motif, predicted to form a spiral groove in the TMD, is more marked than a repeating glycine motif, implicated in the dimerization of TMDs of some eukaryotic proteins. This feature of DjlA could represent a promiscuous docking mechanism for interaction with a variety of membrane proteins. DjlA null mutants can be isolated but these appear rapidly to accumulate suppressors to correct envelope and growth defects. Moderate (10-fold) overproduction of DjlA suppresses a mutation in FtsZ but markedly perturbs cell division and cell-envelope growth in minimal medium. We propose that DjlA plays a role in the correct assembly, activity and/or maintenance of a number of membrane proteins, including two-component signal-transduction systems.

Recognition sites for a membrane-derived DNA binding protein preparation in the E. coli replication origin.

The DNA binding protein B' preparation, isolated from the membrane of E. coli, recognizes two sites, one of which is located in the minimum oriC (35-270 bp) and the other between base pairs 417 and 488. Recognition is only possible when restriction fragments containing these sites are in single-stranded state. At the first site the strand reading 3'OH-5'P in the direction of the E. coli genetic map is recognized, at the second site the 5'P-3'OH strand.

Point mutations in the transmembrane domain of DjlA, a membrane-linked DnaJ-like protein, abolish its function in promoting colanic acid production via the Rcs signal transduction pathway.

DjIA is a novel DnaJ-like protein localized to the inner membrane of Escherichia coli through the single transmembrane domain (TMD) found at the N-terminus. The overproduction of DjIA activates expression of the cps operon, controlling synthesis and export of the extracellular polysaccharide colanic acid via the Rcs/B two-component signal transduction pathway. We now show that both the TMD and the J-region are essential for the induction of cps expression observed with the overproduction of DjIA. Furthermore, we describe the isolation and characterization of different point mutations in the TMD that completely or partially block the induction of cps expression associated with overproduction of DjIA. These mutations were shown not to affect the localization, stability or topology of the mutant DjIA proteins. We propose that these mutations are affecting specific interactions between the TMD of DjIA and its substrate protein(s), for example RcsC, the membrane sensor kinase partner of the Rcs/B signal transduction pathway.

The secD locus of E.coli codes for two membrane proteins required for protein export.

Cold-sensitive mutations in the secD locus of Escherichia coli result in severe defects in protein export at the non-permissive temperature of 23 degrees C. DNA sequence of a cloned fragment that includes the secD locus reveals open reading frames for seven polypeptide chains. Both deletions and TnphoA insertions in this clone have been used in maxicell and complementation studies to define the secD locus and its products. The secD mutations fall into two complementation groups, defining genes we have named secD and secF. These two genes comprise an operon, the first case of two genes involved in the export process being co-transcribed. The DNA sequence of the two genes along with alkaline phosphatase fusion analysis indicates that they code for integral proteins of the cytoplasmic membrane. We suggest that these two proteins may form a complex in the membrane which acts at late steps in the export process.

Purification and characterization of a low-molecular-weight membrane protein with affinity for the Escherichia coli origin of replication.

A purification procedure was devised for a low-molecular-mass (about 10-kilodalton) membrane protein from Escherichia coli that was shown to bind specifically to the chromosomal replication origin region (oriC). Nitrocellulose membrane retention assays showed the binding site to be adjacent to the right boundary of the oriC minimal sequence. We determined the amino acid sequence of the N-terminal and C-terminal regions as well as the global amino acid composition of this membrane protein. Specific antibodies against the protein were produced and used to confirm the cell membrane location of the protein. These results demonstrate that this is a new membrane protein, different from the previously described B' protein, with specific binding activity for the oriC region. We propose that this protein be called membrane oriC-binding protein 2 (MOB2 protein).

Purification and characterisation of a 12 K dalton peptide with an affinity for oriC containing DNA fragments from Escherichia coli K12 membrane fractions.

A DNA binding protein capable of interacting specifically with oriC containing DNA was purified to near homogeneity from E. coli membrane fractions. It has a molecular weight of 12 KD in denaturing conditions. The specific binding to oriC DNA is more resistant to the presence of salt than the non specific binding. Neither the DNA binding activity nor the specificity for oriC DNA was destroyed by heating at 100 degrees C for 2 min.

Genetic analysis of the RcsC sensor kinase from Escherichia coli K-12.

The Rcs two-component pathway is involved in the regulation of capsule production in Escherichia coli. RcsC is predicted to be the sensor component of this two-component pathway, and in this study we present the first genetic data that support the role of RcsC as a hybrid sensor kinase.

EGTA induces the synthesis in Escherichia coli of three proteins that cross-react with calmodulin antibodies.

Escherichia coli mutants, (verA, dilA) specifically resistant to the Ca2+ channel inhibitors verapamil and diltiazem, respectively, are hypersensitive to EGTA and BAPTA. We have shown, using 1-D and 2-D gel electrophoresis, that the synthesis of at least 25 polypeptides in the mutants was enhanced by treatment with Ca2+ chelators and the synthesis of at least 11 polypeptides was repressed. This pattern of induction was not observed in heat- or SDS-treated cells and therefore does not appear to be a general stress response. The majority of the induced proteins are low molecular weight, extremely heat stable and acidic, characteristic properties of calmodulin. Moreover, of the major induced species, three with apparent molecular masses of 12, 18, and 34 kDa all cross-reacted with polyclonal and monoclonal antibodies to eukaryote calmodulins or calerythrin, a heat-resistant Ca(2+)-binding protein from Saccharopolyspora erythraea. The verA, dilA mutants, in being hypersensitive to EGTA and to the Ca2+ ionophore A23187 + Ca2+, may be defective in the regulation of the level of free intracellular Ca2+.

Some properties of HU are modified after the infection of Escherichia coli by bacteriophage T4.

Escherichia coli HU, an abundant, nucleoid-associated, DNA-binding protein, plays a role in several biological processes including DNA replication. Many other bacteria have well-conserved HU homologs, and there are several more-distantly related members of the family, including TF1, encoded by Bacillus subtilis phage SPO1. We have asked whether coliphage T4, like SPO1, encodes an HU homolog or whether it alters the properties of host HU. We have been unable to detect a T4-specified HU homolog, but we have shown that E. coli HU extracted from phage-infected cells differs in some properties from that extracted from uninfected cells. First, HU from uninfected cells inhibits a reconstituted T4 DNA replication system, whereas HU from infected cells does not. Second, HU from infected cells appears to bind a T4-encoded polypeptide, as shown by coimmunoprecipitation. We propose that such binding alters HU function in T4-infected cells.

Increased sensitivity of E. coli to novobiocin, EDTA and the anticalmodulin drug W7 following overproduction of DjlA requires a functional transmembrane domain.

In earlier studies we found that E. coli is sensitive to anticalmodulin drugs such as W7. Mutants that are resistant to this drug were isolated, including wseA1. In an attempt to clone the wseA gene, we isolated a clone that restored sensitivity to the drug in the mutant. We found that this clone in fact suppresses W7 resistance through expression of djlA, which encodes a novel DnaJ-like protein. It was found previously that overproduction of DjlA could induce capsule synthesis via activation of the two-component regulatory pathway RcsC/B. In addition to suppression of wseA1, djlA overexpression increases the sensitivity of cells to EDTA and novobiocin, but not to other drugs tested. Although overexpression of a form of the protein carrying a mutation in, or lacking, the J-region of DjlA also led to increased sensitivity, indicating that the chaperone activity of this protein was not strictly required. the full-length, wild-type protein had a more pronounced effect. In contrast, a point mutation which affects the function of the transmembrane domain but not the localisation or stability of DjlA abolished the effects of DjlA overproduction.

The secE gene encodes an integral membrane protein required for protein export in Escherichia coli.

Genetic screening and selection procedures employing a secA-lacZ fusion strain repeatedly have yielded mutations in four genes affecting the protein export pathway of Escherichia coli. These genes are secA, secD, prlA/secY, and secE. We discuss the significance of the failure to find new sec genes after extensive use of this approach. One of the genes, secE, has been characterized in some detail. From the DNA sequence of the gene and analysis of alkaline phosphatase fusions to the SecE protein, we propose that it is a 13,600-dalton integral cytoplasmic membrane protein. The data presented here and in the accompanying paper strongly suggest that secE has an important role in E. coli protein export.

The transmembrane domain of the DnaJ-like protein DjlA is a dimerisation domain.

DjlA is a bitopic inner membrane protein, which belongs to the DnaJ co-chaperone family in Escherichia coli. Overproduction of DjlA leads to the synthesis of colanic acid, resulting in mucoidy, via the activation of the two-component regulatory system RcsC/B that controls the cps (capsular polysaccharide) operon. This induction requires both the co-chaperone activity of DjlA, in cooperation with DnaK and GrpE, and its unique transmembrane (TM) domain. Here, we show that the TM segment of DjlA acts as a dimerisation domain: when fused to the N-terminal DNA-binding domain of the lambda cI repressor protein, it can substitute for the native C-terminal dimerisation domain of cI, thus generating an active cI repressor. Replacing the TM domain of DjlA by other TM domains, with or without dimerising capacity, revealed that dimerisation is not sufficient for the induction of cps expression, indicating an additional sequence- or structurally specific role for the TM domain. Finally, the conserved glycines present in the TM domain of DjlA are essential for the induction of mucoidy, but not for dimerisation.