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.
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Amino Acid Sequence↗
Biomedical subjects
V Norris
Publications and source records attributed to V Norris.
Immunogold localization of GyrA and GyrB proteins in Escherichia coli.
Immunogold preparations of Escherichia coli, using anti-GyrA and anti-GyrB antibodies to the subunits of DNA gyrase, showed clear labelling with both secondary antibody and protein A-gold conjugates. Both proteins were located mainly in the cytoplasm, with typically less than 10% in the nucleoid. This partitioning of gyrase proteins between nucleoid and cytoplasm was nonrandom and was consistently observed for a range of different cell preparations. Total gold particle counts were highly variable but suggested levels of at least 1000-3000 molecules per cell for both GyrA and GyrB. Sequential treatment with both anti-GyrA and anti-GyrB monoclonal antibodies resulted in simultaneous labelling of both proteins and revealed no clear association between the two groups of molecules. Treatment of cells with chloramphenicol caused marked changes in nucleoid conformation, but no reduction in cytoplasmic labelling of gyrase proteins. On the assumption that gyrase complexes within the nucleoid are not differentially masked from the monoclonal antibodies, the results obtained in this study suggest that most of the gyrase proteins are not associated with either central nucleoid DNA or cytoplasmic loops of peripheral single-stranded DNA, but are distributed randomly throughout the cytoplasm.
Antibodies, Monoclonal↗
Sequestration of origins of chromosome replication in Escherichia coli by lipid compartments: the pocket hypothesis.
After initiation of chromosome replication in Escherichia coli, the newly replicated, hemi-methylated origins of replication are exempt from both re-initiation and methylation for up to a third of the generation time. In the model proposed here, the exemption from methylation is attributed to the phospholipid preferences of proteins binding hemi-methylated origins to membrane; these proteins associate with unsaturated phosphatidylethanolamine which forms structures with small radii of curvature; hence, a lipid compartment or "pocket" forms around the origin making it inaccessible to cytoplasmic enzymes such as Dam methyltransferase. Since autolysins and peptidoglycan-synthesizing enzymes are sensitive to lipid composition, peptidoglycan synthesis is stimulated around this compartment and creates structures obtained in membrane fractionation that specifically bind hemi-methylated origins.
Bacterial Proteins↗
Designer antiviruses for HIV.
Potentially, antiviruses that interfere with HIV propagation could be used as AIDS therapy. If problems associated with HIV recombination and the dynamics of the interactions between HIV and antivirus can be resolved by an appropriate design, an antivirus might defer or prevent the development of AIDS, and might benefit AIDS patients.
Acquired Immunodeficiency Syndrome↗
Deformations in the cytoplasmic membrane of Escherichia coli direct the synthesis of peptidoglycan. The hernia model.
To explain the growth of the Gram-negative envelope and in particular how it could be strengthened where it is weakest, we propose in the hernia model that local weakening of the peptidoglycan sacculus allows turgor pressure to cause the envelope to bulge outwards in a hernia; the consequent local alteration in the radius of curvature of the cytoplasmic membrane causes local alterations in phospholipid structure and composition that determine both the synthesis and hydrolysis of peptidoglycan. This proposal is supported by evidence that phospholipid composition determines the activity of phospho-N-acetylmuramic acid pentapeptide translocase, UDP-N-acetylglucosamine:N-acetylmuramic acid-(pentapeptide)-P-P-bactoprenyl-N-acetylglucosamine transferase, bactoprenyl phosphate phosphokinase, and N-acetylmuramyl-L-alanine amidase. We also propose that the shape of Escherichia coli is maintained by contractile proteins acting at the hernia. Given the universal importance of membranes, these proposals have implications for the determination of shape in eukaryotic cells.
Bacterial Proteins↗
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.
Amino Acid Sequence↗
Phospholipid domains determine the spatial organization of the Escherichia coli cell cycle: the membrane tectonics model.
Escherichia coli normally divides at its equator between segregated nucleoids. Such division is inhibited during perturbations of chromosome replication (even in the absence of inducible division inhibitors); eventually, division resumes at sites which are not at this equator. Escherichia coli will also divide at its poles to generate minicells following overproduction of the FtsZ or MinE proteins. The mechanisms underlying the division inhibition and the positioning of the division sites are unknown. In the membrane tectonics model, I propose that the formation of phospholipid domains within the cytoplasmic membrane positions division sites. The particular phospholipid composition of a domain attracts particular proteins and determines their activity; conversely, particular proteins change the composition of domains. Principally via such proteins, the interaction of the chromosome with the membrane creates a chromosomal domain. The development of chromosomal domains during replication and nucleoid formation contributes to the formation and positioning of a septal domain between them. During septation (cell division), this septal domain matures into a polar domain. Each domain attracts and activates different enzymes. The septal domain attracts and activates enzymes necessary for septation. Preventing the formation of the septal domain by preventing chromosome replication prevents normal division. Altering the composition of the polar domain may allow septation enzymes to function there and generate minicells. A corollary of the model explains how the formation of an origin domain by the attachment of hemi-methylated origin DNA to the membrane may underlie the creation and migration of structures within the envelope, the periseptal annuli.
Animals↗
A single base change in the acceptor stem of tRNA(3Leu) confers resistance upon Escherichia coli to the calmodulin inhibitor, 48/80.
We have isolated several classes of spontaneous mutants resistant to the calmodulin inhibitor 48/80 which inhibits cell division in Escherichia coli K12. Several mutants were also temperature sensitive for growth and this property was exploited to clone a DNA fragment from an E. coli gene library restoring growth at 42 degrees C and drug sensitivity at 30 degrees C in one such mutant. Physical and genetic mapping confirmed that both the mutation and the cloned DNA were located at 15.5 min on the E. coli chromosome at a locus designated feeB. By subcloning, complementation analysis and sequencing, the feeB locus was identified as identical to the tRNA(CUALEU) gene. When the mutant locus was isolated and sequenced, the mutation was confirmed as a single base change, C to A, at position 77 in the acceptor stem of this rare Leu tRNA. In other studies we obtained evidence that this mutant tRNA, recognizing the rare Leu codon, CUA, was defective in translation at both permissive and non-permissive temperatures. The feeB1 mutant is defective in division and shows a reduced growth rate at non-permissive temperature. We discuss the possibility that the mutant tRNA(3Leu) is limiting for the synthesis of a polypeptide(s), requiring several CUA codons for translation which in turn regulates in some way the level or activity of the drug target, a putative cell cycle protein.
Base Sequence↗
Calcium in bacteria: a solution to which problem?
Calcium and calcium-binding proteins including those resembling calmodulin are implicated in numerous diverse processes in bacteria. These processes include chemotaxis, sporulation, virulence, the transport of sugars and proteins, phosphorylation, heat shock, the initiation of DNA replication, septation, nucleoid structure, nuclease activity and recombination, the stability of the envelope, and phospholipid synthesis and configuration. That such varied processes should have a common factor, calcium, suggests major underlying principles of calcium metabolism which have yet to be discovered.
Bacteria↗
A protein kinase C-like activity in Escherichia coli.
The protein kinase C (PKC) family comprises calcium- and phospholipid-dependent kinases whose activity is stimulated by diacylglycerol and tumour-promoting phorbol esters such as 12-tetradecanoyl phorbol-13-acetate (TPA). In the Gram-negative bacterium Escherichia coli, functional similarity to PKC was demonstrated in crude extracts by calcium and phospholipid-dependent, TPA-stimulated phosphorylation of a small number of endogenous substrates. Activity was reduced by sphingosine, a known inhibitor of eukaryotic PKC. Structural similarity to PKC was demonstrated in crude and partially purified bacterial extracts by cross-reactivity with several monoclonal antibodies. This revealed isozyme-specific homology between a protein(s) of relative molecular mass 80-85,000 in E. coli and the alpha- and gamma-isozymes, but probably not the beta-isozyme, of eukaryotic PKC.
Antibodies, Monoclonal↗
DNA replication in Escherichia coli is initiated by membrane detachment of oriC. A model.
An adequate model for the initiation of chromosome replication in Escherichia coli should explain why the introduction of multiple copies of the chromosomal origin of replication, oriC, does not perturb cells seriously and why such multiple origins are replicated synchronously; it should explain why the key initiator protein, DnaA, is activated in vitro by binding specifically to acidic phospholipids and why the Dam methyltransferase is essential for the correct timing of initiation; it should explain why phospholipid synthesis and fluidity are necessary for initiation. In the detachment model, presented here, cyclical changes in the phospholipid composition of the cytoplasmic membrane activate initiator proteins such as DnaA protein and cause origins to detach; this detachment allows torsional stresses to open 13mer sequences in oriC; DnaA assists in the serial opening of these sequences and guides the entry of the helicase to form a pre-priming complex and trigger initiation; the greater affinity of hemi-methylated origin for membrane is re-interpreted as a mechanism for preventing re-initiation.
Cell Cycle↗
Cytoskeletal elements and calcium: do they play a role in the Escherichia coli cell cycle?
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Bacterial Proteins↗
Identification of a 180 kD protein in Escherichia coli related to a yeast heavy-chain myosin.
A high molecular-weight protein from Escherichia coli sharing structural homology at the protein level with a yeast heavy-chain myosin encoded by the MYO1 gene is described. This 180 kD protein (180-HMP) can be enriched in cell fractions following the procedure normally utilized for the purification of non-muscle myosins. In Western blots this protein cross-reacts with a monoclonal antibody against yeast heavy-chain myosin. Moreover, antibodies raised against the 180 kD protein cross-react with the yeast myosin and with a myosin heavy chain from chicken. Recognition by anti-180-HMP antibodies of an overexpressed fragment of yeast myosin encoded by MYO1 allows the localization of one of the shared epitopes to a specific region around the ATP binding site of the yeast myosin heavy chain. The existence of a high molecular-weight protein with structural similarity to myosin in E. coli raises the possibility that such a protein might generate the force required for movement in processes such as nucleoid segregation and cell division.
Antigens, Bacterial↗
Phospholipid flip-out controls the cell cycle of Escherichia coli.
Phospholipids are the principal constituents of biological membranes. In Escherichia coli, phospholipids are involved in the metabolism of other envelope constituents such as lipoprotein, lipopolysaccharide, certain envelope proteins and peptidoglycan. They are also involved in the regulation of the cell cycle. DNAA, the key protein in the initiation of chromosome replication, is activated by acidic phospholipids only when these are in fluid bilayers, whilst interruptions of phospholipid synthesis inhibit both the initiation of chromosome replication and cell division. The transmembrane movement or flip-flop of phospholipids from one monolayer to the other requires the passage of the polar head group through the hydrophobic core of the bilayer. Hence, in many systems, flip-flop is a slow process with half-time of days. Flip-flop accompanies the formation of non-bilayer structure. Such structures form under certain conditions of packing density and composition and have been observed both in vitro and in vivo. In bacteria, flip-flop appears to be extremely rapid, with half-times as fast as 3 min being observed. However, such rapid flip-flop may not be characteristic of all phospholipids. The asymmetrical distribution of phosphatidylethanolamine in the plasma membrane of Bacillus megaterium has been attributed to the existence of two classes of this phospholipid. In E. coli, studies of the metabolic turnover of phosphatidylserine, phosphatidylglycerol and phosphatidic acid also reveal the existence of distinct classes of these phospholipids. In this article I propose that, in E. coli, a class of phospholipids does indeed escape the rapid flip-flop mechanism; this class probably includes a subpopulation of the acidic phospholipids. Therefore during the cell cycle these phospholipids accumulate in the inner monolayer of the cytoplasmic membrane and so cause an increase in its packing density; at a critical density, phospholipids "flip out" from the inner to the outer monolayer. This flip-out occurs once per cycle and initiates cell cycle events.
Cell Cycle↗
A single calcium flux triggers chromosome replication, segregation and septation in bacteria: a model.
Abrupt changes in the concentration of intracellular calcium, through the mediation of calmodulin, is presumed to play an essential role in many molecular processes in eukaryotes including triggering cell cycle events. Although early studies failed to establish any role for calcium in the growth of bacteria, recent studies have demonstrated that bacteria have several calcium transport systems, and an intracellular concentration of free calcium identical to that of higher organisms, which appears to fluctuate during the cell cycle. Moreover, calmodulin-like proteins have been reported in bacteria, and the growth of E. coli is sensitive to calmodulin inhibitors. In this article we propose that a single flux of calcium, abruptly raising the intracellular concentration of free calcium, is responsible for the triggering in bacteria of the major cell cycle events, initiation of DNA replication, chromosome partition and cell division. We predict that major roles in this process will involve a bacterial calmodulin-like protein and a primitive cytoskeleton. The mechanism of triggering different cell cycle events by a single calcium flux is discussed.
Bacteria↗
SOS-independent coupling between DNA replication and cell division in Escherichia coli.
Inhibition of DNA synthesis in Escherichia coli mutants in which the SOS-dependent division inhibitors SfiA and SfiC were unable to operate led to a partial arrest of cell division. This SOS-independent mechanism coupling DNA replication and cell division was characterized with respect to residual division, particle number, and DNA content. Whether DNA replication was blocked in the initiation or the elongation step, numerous normal-sized anucleate cells were produced (not minicells or filaments). Their production was used to evaluate the efficiency of this coupling mechanism, which seems to involve the cell division protein FtsZ (SulB), also known to be the target of the division inhibitors SfiA and SfiC. In the absence of DNA synthesis, the efficiency of coupling was modulated by the cyclic-AMP-cyclic-AMP receptor protein complex, which was required for anucleate cell production.
Adenylyl Cyclases↗
DNA replication termination in Escherichia coli parB (a dnaG allele), parA, and gyrB mutants affected in DNA distribution.
We investigated the Escherichia coli mutants carrying the parB, parA, and gyrB mutations, all of which display faulty chromosome partitioning at the nonpermissive temperature, to see whether their phenotype reflected a defect in the termination of DNA replication. In the parB strain DNA synthesis slowed down at 42 degrees C and the SOS response was induced, whereas in the parA strain DNA synthesis continued normally for 120 min and there was no SOS induction. To see whether replication forks accumulated in the vicinity of terC at the nonpermissive temperature, the mutants were incubated for 60 min at 42 degrees C and then returned to low temperature and pulse-labeled with [3H]thymidine. In all cases the restriction pattern of the labeled DNA was incompatible with that of the terC region, suggesting that replication termination was normal. In the parA mutant no DNA sequences were preferentially labeled, whereas in the parB and gyrB strains there was specific labeling of sequences whose restriction pattern resembled that of oriC. In the case of parB this was confirmed by DNA-DNA hybridization with appropriate probes. This test further revealed that the parB mutant over initiates at oriC after the return to the permissive temperature. Like dna(Ts) strains, the parB mutant formed filaments at 42 degrees C in the absence of SOS-associated division inhibition, accompanied by the appearance of anucleate cells of nearly normal size (28% of the population after 3 h), as revealed by autoradiography. The DNA in the filaments was either centrally located or distributed throughout. The parB mutation lies at 67 min, and the ParB- phenotype is corrected by a cloned dnaG gene or by a plasmid primase, strongly suggesting that parB is an allele of dnaG, the structural gene of the E. coli primase. It is thus likely that the parB mutant possesses an altered primase which does not affect replication termination but causes a partial defect in replication initiation and elongation and in chromosome distribution.
Alleles↗
Restriction map of Tn7.
Tn7, a transposon of 14 kb, encodes resistance to trimethoprim (Tp) and streptomycin (Sm). A cleavage site map of this transposon for twenty-two different restriction enzymes as determined by comparison of restriction enzyme cleavage patterns of the plasmids ColE1 and ColE1::Tn7 is presented. The precise localization of these sites was facilitated by the use of two deletion derivatives of ColE1::Tn7: pGB2 and ColE1::Tn7 delta 6, and by the use of pOB14 and pOB15 which contain a part of Tn7 cloned into the plasmid pBR322. This map should aid in the study of the structural and genetic organization of this transposon.
Chromosome Mapping↗