Vic Norris

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 DNAS replication, septation, nucleoid structure, nuclease activity and recombination, the stability of the envelope, and phospholipids synthesis and configuration. That such varied processes should have a common factor, calcium, suggests major underlying principles of calcium metabolism metabolism which have yet to be discovered.

Hypothesis: chromosome separation in Escherichia coli involves autocatalytic gene expression, transertion and membrane-domain formation

To explain how daughter chromosomes are separated into discrete nucleoids and why chromosomes are partitioned with pole preferences, I propose that differential gene expression occurs during DNA replication in Escherichia coli. This differential gene expression means that the daughter chromosomes have different patterns of gene expression and that cell division is not a simple process of binary fission. Differential gene expression arises from autocatalytic gene expression and creates a separate proteolipid domain around each developing chromosome via the coupled transcription‐translation–insertion of proteins into membranes (transertion). As these domains are immiscible, daughter chromosomes are simultaneously replicated and separated into discrete nucleoids. I also propose that the partitioning relationship between chromosome age and cell age arises because the poles of cells have a proteolipid composition that favours transertion from one nucleoid rather than from the other. This hypothesis forms part of an ensemble of related hypotheses which attempt to explain cell division, differentiation and wall growth in bacteria in terms of the physical properties and interactions of the principal constituents of cells.

The Escherichia coli enzoskeleton

The nature of the structure of the bacterial cell is becoming clearer. The envelope contains periseptal annuli, a discontinuous periplasm and adhesion sites, whilst the cytoplasmic membrane is probably organized into distinct proteolipid domains by the coupled transcription–translation–insertion (transertion) of membrane proteins. The structure of the nucleoid is determined by proteins which self‐associate and by attachment to membrane, which is achieved in part by transertion. Metabolic pathways form multi‐enzyme complexes which channel substrates and which connect membranes and nucleic acids to create the extensive, cross‐linked, intracellular structure we term the ‘enzoskeleton’. This enzoskeleton includes eukaryotic‐like cytoskeletal structures and elements such as the MukB and FtsZ proteins. We propose that the enzoskeleton is regulated by calcium and by protein phosphorylation during adaptation to different environments and during the 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.

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.

Identification of phosphoproteins in Escherichia coli

The substrates of ion‐ and lipld‐stimulated protein kinase activity in extracts of Escherichia coli were purified by chromatography. Subsequent N‐terminal sequencing suggests that these substrates include the following: a novel 80kDa protein co‐purifying with RNA polymerase but partially homologous to elongation factor G; a protein with an apparent molecular weight of 65kDa identified as the ribosomal protein S1; and a 32 kDa protein identified as succinyl CoA synthetase, a key enzyme in the tricarboxylic acid cycle. The phosphorylation of these three proteins was markedly stimulated by the addition of manganese, and occurred on threonine, serine or tyrosine residues as indicated by the stability of the phosphoresidues during acid treatment. In addition, a calcium‐stimulated protein of 70kDa was identified as the heat‐shook protein DnaK, and a 17kDa lipid‐stimulated phosphoprotein as nucleotide diphosphate kinase.

Electrospray ionization mass spectrometric analysis of phospholipids of Escherichia coli.

A range of lipid species (Fig. IA ) . The principal peaks have molecular masses that correspond to those of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) with a PE:PG ratio of 73:27; it should be noted that the apparent dominance of PG species in the spectrum is due to the technique being 10 times less sensitive to PE than PG (data not shown). To confirm identifications, in-source collisionally induced dissociation (CID) was used to simultaneously split all these molecules into their constituent groups, enabling the identification of six fatty acid chains and two ions corresponding to cleavage of the head

Identification of a 180kD protein in Escherichia coli related to a yeast heavy‐chain myosin

A high molecular‐weight protein from Escherichia coli sharing structural ho mo logy at the protein level with a yeast heavy‐chain myosin encoded by the MYO1 gene is described. This 180kD 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 180kD 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.

DNA replication in Escherichia coli is initiated by membrane detachment of oriC

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.

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.