Tight coupling of cell width to nucleoid structure in Escherichia coli.

Cell dimensions of rod-shaped bacteria such as Escherichia coli are connected to mass growth and chromosome replication. During their interdivision cycle (τ min), cells enlarge by elongation only, but at faster growth in richer media, they are also wider. Changes in width W upon nutritional shift-up (shortening τ) occur during the division process. The elusive signal directing the mechanism for W determination is likely related to the tightly linked duplications of the nucleoid (DNA) and the sacculus (peptidoglycan), the only two structures (macromolecules) existing in a single copy that are coupled, temporally and spatially. Six known parameters related to the nucleoid structure and replication are reasonable candidates to convey such a signal, all simple functions of the key number of replication positions n(=C/τ), the ratio between the rates of growth (τ-1) and of replication (C-1). The current analysis of available literature-recorded data discovered that, of these, nucleoid complexity NC[=(2n-1)/(n×ln2)] is by far the most likely parameter affecting cell width W. The exceedingly high correlations found between these two seemingly unrelated measures (NC and W) indicate that coupling between them is of major importance to the species' survival. As an exciting corollary, to the best of our knowledge, a new, indirect approach to estimate DNA replication rate is revealed. Potential involvement of DNA topoisomerases in W determination is also proposed and discussed.

Transient enhanced cell division by blocking DNA synthesis in Escherichia coli.

Duplication of the bacterial nucleoid is necessary for cell division hence specific arrest of DNA replication inhibits divisions culminating in filamentation, nucleoid dispersion and appearance of a-nucleated cells. It is demonstrated here that during the first 10 min however, Escherichia coli enhanced residual divisions: the proportion of constricted cells doubled (to 40%), nucleoids contracted and cells remodelled dimensions: length decreased and width increased. The preliminary data provides further support to the existence of temporal and spatial couplings between the nucleoid/replisome and the sacculus/divisome, and is consistent with the idea that bacillary bacteria modulate width during the division process exclusively.

Cell-shape homeostasis in Escherichia coli is driven by growth, division, and nucleoid complexity.

Analysis of recently published high-throughput measurements of wild-type Escherichia coli cells growing at a wide range of rates demonstrates that cell width W, which is constant at any particular growth rate, is related (with a CV = 2.4%) to the level of nucleoid complexity, expressed as the amount of DNA in genome equivalents that is associated with chromosome terminus (G/terC). The relatively constant (CV = 7.3%) aspect ratio of newborn cells (Lb/W) in populations growing at different rates indicates existence of cell-shape homeostasis. Enlarged W of thymine-limited thyA mutants growing at identical rates support the hypothesis that nucleoid complexity actively affects W. Nucleoid dynamics is proposed to transmit a primary signal to the peptidoglycan-synthesizing system through the transertion mechanism, i.e., coupled transcription/translation of genes encoding membrane proteins and inserting these proteins into the membrane.

Maximizing yields of virulent phage: the T4/Escherichia coli system as a test case.

A hybrid mathematical model was devised to obtain optimal values for bacterial doubling time and initial phage/bacteria multiplicity of infection for the purpose of reaching the highest possible phage titers in steady-state exponentially growing cultures. The computational model consists of an initial probabilistic stage, followed by a second one processed by a system of delayed differential equations. The model's approach can be used in any phage/bacteria system for which the relevant parameters have been measured. Results of a specific case, based on the detailed, known information about the interactions between virulent T4 phage and its host bacterium Escherichia coli, display a range of possible such values along a highlighted strip of parameter values in the relevant parameter plane. In addition, times to achieve these maxima and gains in phage concentrations are evaluated.

Phospho-regulation of kinesin-5 during anaphase spindle elongation.

The kinesin-5 Saccharomyces cerevisiae homologue Cin8 is shown here to be differentially phosphorylated during late anaphase at Cdk1-specific sites located in its motor domain. Wild-type Cin8 binds to the early-anaphase spindles and detaches from the spindles at late anaphase, whereas the phosphorylation-deficient Cin8-3A mutant protein remains attached to a larger region of the spindle and spindle poles for prolonged periods. This localization of Cin8-3A causes faster spindle elongation and longer anaphase spindles, which have aberrant morphology. By contrast, the phospho-mimic Cin8-3D mutant exhibits reduced binding to the spindles. In the absence of the kinesin-5 homologue Kip1, cells expressing Cin8-3D exhibit spindle assembly defects and are not viable at 37°C as a result of spindle collapse. We propose that dephosphorylation of Cin8 promotes its binding to the spindle microtubules before the onset of anaphase. In mid to late anaphase, phosphorylation of Cin8 causes its detachment from the spindles, which reduces the spindle elongation rate and aids in maintaining spindle morphology.

Extending Validity of the Bacterial Cell Cycle Model through Thymine Limitation: A Personal View.

The contemporary view of bacterial physiology was established in 1958 at the "Copenhagen School", culminating a decade later in a detailed description of the cell cycle based on four parameters. This model has been subsequently supported by numerous studies, nicknamed BCD (The Bacterial Cell-Cycle Dogma). It readily explains, quantitatively, the coupling between chromosome replication and cell division, size and DNA content. An important derivative is the number of replication positions n, the ratio between the time C to complete a round of replication and the cell mass doubling time τ; the former is constant at any temperature and the latter is determined by the medium composition. Changes in cell width W are highly correlated to n through the equation for so-called nucleoid complexity NC (=(2n - 1)/(ln2 × n)), the amount of DNA per terC (i.e., chromosome) in genome equivalents. The narrow range of potential n can be dramatically extended using the method of thymine limitation of thymine-requiring mutants, which allows a more rigorous testing of the hypothesis that the nucleoid structure is the primary source of the signal that determines W during cell division. How this putative signal is relayed from the nucleoid to the divisome is still highly enigmatic. The aim of this Opinion article is to suggest the possibility of a new signaling function for nucleoid DNA.

Novel Principles and Methods in Bacterial Cell Cycle Physiology: Celebrating the Charles E. Helmstetter Prize in 2022.

This Special Issue celebrates the creation of the Charles E [...].

Instructive simulation of the bacterial cell division cycle.

The coupling between chromosome replication and cell division includes temporal and spatial elements. In bacteria, these have globally been resolved during the last 40 years, but their full details and action mechanisms are still under intensive study. The physiology of growth and the cell cycle are reviewed in the light of an established dogma that has formed a framework for development of new ideas, as exemplified here, using the Cell Cycle Simulation (CCSim) program. CCSim, described here in detail for the first time, employs four parameters related to time (replication, division and inter-division) and size (cell mass at replication initiation) that together are sufficient to describe bacterial cells under various conditions and states, which can be manipulated environmentally and genetically. Testing the predictions of CCSim by analysis of time-lapse micrographs of Escherichia coli during designed manipulations of the rate of DNA replication identified aspects of both coupling elements. Enhanced frequencies of cell division were observed following an interval of reduced DNA replication rate, consistent with the prediction of a minimum possible distance between successive replisomes (an eclipse). As a corollary, the notion that cell poles are not always inert was confirmed by observed placement of division planes at perpendicular planes in monstrous and cuboidal cells containing multiple, segregating nucleoids.

Expression in Escherichia coli of the native cyt1Aa from Bacillus thuringiensis subsp. israelensis.

The gene cyt1Aa is one of the genes in the complex determining the mosquito larvicidity of Bacillus thuringiensis subsp. israelensis. Previous cloning in Escherichia coli resulted in a 48-bp addition upstream, encoding a chimera. Here, cyt1Aa was recloned without the artifact, and its toxicity against Aedes aegypti larvae and host E. coli cells was retested.

Kinetics of repeat propagation in the microgene polymerization reaction.

Repetitive DNA is a periodic copolymer with the intrinsic property of exponential propagation to longer repeats. Microgene polymerization reaction (MPR) is a model system in which a short nonrepetitive homo-duplex DNA evolves to multiple repetitive products during heat-cool cycles. The mechanism underlying this process involves staggered annealing of complementary DNA strands of variable lengths and polymerase-mediated filling-in of the generated overhangs. MPR is considered here as a process sharing common features with two polymerization types, chain-growth and step-growth, and significant distinctions from both types were highlighted. The involved reaction stages were formulated and a kinetic model was derived and tested experimentally. The model can quantitatively explain MPR propagation and be used as a good approximation for this phenomenon.

Site-specific recombination in the cyanobacterium Anabaena sp. strain PCC 7120 catalyzed by the integrase of coliphage HK022.

The integrase (Int) of the lambda-like coliphage HK022 catalyzes the site-specific integration and excision of the phage DNA into and from the chromosome of its host, Escherichia coli. Int recognizes two different pairs of recombining sites attP x attB and attL x attR for integration and excision, respectively. This system was adapted to the cyanobacterium Anabaena sp. strain PCC 7120 as a potential tool for site-specific gene manipulations in the cyanobacterium. Two plasmids were consecutively cointroduced by conjugation into Anabaena cells, one plasmid that expresses HK022 Int recombinase and the other plasmid that carries the excision substrate P(glnA)-attL-T1/T2-attR-lacZ, where T1/T2 are the strong transcription terminators of rrnB, to prevent expression of the lacZ reporter under the constitutive promoter P(glnA). The Int-catalyzed site-specific recombination reaction was monitored by the expression of lacZ emanating as a result of T1/T2 excision. Int catalyzed the site-specific excision reaction in Anabaena cells when its substrate was located either on the plasmid or on the chromosome with no need to supply an accessory protein, such as integration host factor and excisionase (Xis), which are indispensable for this reaction in its host, E. coli.

Does the Nucleoid Determine Cell Dimensions in Escherichia coli?

Bacillary, Gram-negative bacteria grow by elongation with no discernible change in width, but during faster growth in richer media the cells are also wider. The mechanism regulating the change in cell width W during transitions from slow to fast growth is a fundamental, unanswered question in molecular biology. The value of W that changes in the divisome and during the division process only, is related to the nucleoid complexity, determined by the rates of growth and of chromosome replication; the former is manipulated by nutritional conditions and the latter-by thymine limitation of thyA mutants. Such spatio-temporal regulation is supported by existence of a minimal possible distance between successive replisomes, so-called eclipse that limits the number of replisomes to a maximum. Breaching this limit by slowing replication in fast growing cells results in maximal nucleoid complexity that is associated with maximum cell width, supporting the notion of Nucleoid-to-Divisome signal transmission. Physical signal(s) may be delivered from the nucleoid to assemble the divisome and to fix the value of W in the nascent cell pole.

Diversity of Bacterial Biota in Capnodis tenebrionis (Coleoptera: Buprestidae) Larvae.

The bacterial biota in larvae of Capnodis tenebrionis, a serious pest of cultivated stone-fruit trees in the West Palearctic, was revealed for the first time using the MiSeq platform. The core bacterial community remained the same in neonates whether upon hatching or grown on peach plants or an artificial diet, suggesting that C. tenebrionis larvae acquire much of their bacterial biome from the parent adult. Reads affiliated with class levels Gammaproteobacteria and Alphaproteobacteria (phylum Proteobacteria ca. 86%), and Actinobacteria (ca. 14%) were highly abundant. Most diverse reads belong to the families Xanthomonadaceae (50%), Methylobacteriaceae (20%), Hyphomicrobiaceae (9%), Micrococcaceae (7%) and Geodermatophilaceae (4.5%). About two-thirds of the reads are affiliated with the genera Lysobacter, Microvirga, Methylobacterium, and Arthrobacter, which encompass species displaying cellulolytic and lipolytic activities. This study provides a foundation for future studies to elucidate the roles of bacterial biota in C. tenebrionis.

Variations in the mosquito larvicidal activities of toxins from Bacillus thuringiensis ssp. israelensis.

Comparing activities of purified toxins from Bacillus thuringiensis ssp. israelensis against larvae of seven mosquito species (vectors of tropical diseases) that belong to three genera, gleaned from the literature, disclosed highly significant variations in the levels of LC(50) as well as in the hierarchy of susceptibilities. Similar toxicity comparisons were performed between nine transgenic Gram-negative species, four of which are cyanobacterial, expressing various combinations of cry genes, cyt1Aa and p20, against larvae of four mosquito species as potential agents for biological control. Reasons for inconsistencies are listed and discussed. Standard conditions for toxin isolation and presentation to larvae are sought. A set of lyophilized powders prepared identically from six Escherichia coli clones expressing combinations of four genes displayed toxicities against larvae of three mosquito species, with levels that differed between them but with identical hierarchy.

Initiation of the microgene polymerization reaction with non-repetitive homo-duplexes.

Microgene Polymerization Reaction (MPR) is used as an experimental system to artificially simulate evolution of short, non-repetitive homo-duplex DNA into multiply-repetitive products that can code for functional proteins. Blunt-end ligation by DNA polymerase is crucial in expansion of homo-duplexes (HDs) into head-to-tail multiple repeats in MPR. The propagation mechanism is known, but formation of the initial doublet (ID) by juxtaposing two HDs and polymerization through the gap has been ambiguous. Initiation events with pairs of HDs using Real-Time PCR were more frequent at higher HD concentrations and slightly below the melting temperature. A process molecularity of about 3.1, calculated from the amplification efficiency and the difference in PCR cycles at which propagation was detected at varying HD concentrations, led to a simple mechanism for ID formation: the gap between two HDs is bridged by a third. Considering thermodynamic aspects of the presumed intermediate "nucleation complex" can predict relative propensity for the process with other HDs.

Thermodynamics of unstable DNA structures from the kinetics of the microgene PCR.

The microgene polymerization reaction (MPR) generates head-to-tail tandem repeats from homoduplexes (HDs). In MPR initiation, one HD putatively aligns two others in the proximity required to form a nucleation complex, thus allowing the DNA polymerase to skip the intertemplate gap and generate an initial doublet (ID) prone to repeat propagation. The current investigation refines this stage by additional thermodynamic considerations and elucidates the fundamental mechanism underlying propagation. Four different HD types were designed to extend the range of melting temperatures and to simultaneously modify the stabilities of their secondary structures. Following the propagation kinetics with these, using real-time PCR at different temperatures revealed a new stage in the MPR, amplification of an ID by an original HD, and enabled us to decipher the biphasic kinetics of the process. This amplification merges with the propagation stage if the lifetime of the staggered conformation of the ID is sufficiently long for DNA polymerase to fill in the overhangs. The observed increase with temperature of thermodynamically unfavorable conformations of singlet and doublet HDs that underlies, respectively, MPR initiation and propagation is well correlated with simulations by UNAFold.

Exposing cryptic antibacterial activity in Cyt1Ca from Bacillus thuringiensis israelensis by genetic manipulations.

In an attempt to endow Cyt1Ca with Cyt1Aa-like antibacterial activity, both derived from Bacillus thuringiensis subsp. israelensis, two amino acids were replaced, E117V and N125A, so as to raise the hydrophobicity of the corresponding region, considered to be the membrane-active motif. The clones obtained included multiple repeats of VIEVLKSLLGIALA, corresponding to head-to-tail polymerization of the primer, translated in frame with Cyt1Ca. These versions of Cyt1Ca caused instant arrest in biomass growth and decreased viability upon expression in Escherichia coli. Multiple insertions of the non-mutated motif VIEELKSLLGINLA into the polypeptide were also lethal. To expose toxicity of the latter motif in the original Cyt1Ca, cyt1Ca was appropriately truncated.

Does the eclipse limit bacterial nucleoid complexity and cell width?

Cell size of bacteria M is related to 3 temporal parameters: chromosome replication time C, period from replication-termination to subsequent division D, and doubling time τ. Steady-state, bacillary cells grow exponentially by extending length L only, but their constant width W is larger at shorter τ's or longer C's, in proportion to the number of chromosome replication positions n (= C/τ), at least in Escherichia coli and Salmonella typhimurium. Extending C by thymine limitation of fast-growing thyA mutants result in continuous increase of M, associated with rising W, up to a limit before branching. A set of such puzzling observations is qualitatively consistent with the view that the actual cell mass (or volume) at the time of replication-initiation Mi (or Vi), usually relatively constant in growth at varying τ's, rises with time under thymine limitation of fast-growing, thymine-requiring E. coli strains. The hypothesis will be tested that presumes existence of a minimal distance lmin between successive moving replisomes, translated into the time needed for a replisome to reach lmin before a new replication-initiation at oriC is allowed, termed Eclipse E. Preliminary analysis of currently available data is inconsistent with a constant E under all conditions, hence other explanations and ways to test them are proposed in an attempt to elucidate these and other results. The complex hypothesis takes into account much of what is currently known about Bacterial Physiology: the relationships between cell dimensions, growth and cycle parameters, particularly nucleoid structure, replication and position, and the mode of peptidoglycan biosynthesis. Further experiments are mentioned that are necessary to test the discussed ideas and hypotheses.

Chromosome replication, cell growth, division and shape: a personal perspective.

The origins of Molecular Biology and Bacterial Physiology are reviewed, from our personal standpoints, emphasizing the coupling between bacterial growth, chromosome replication and cell division, dimensions and shape. Current knowledge is discussed with historical perspective, summarizing past and present achievements and enlightening ideas for future studies. An interactive simulation program of the bacterial cell division cycle (BCD), described as "The Central Dogma in Bacteriology," is briefly represented. The coupled process of transcription/translation of genes encoding membrane proteins and insertion into the membrane (so-called transertion) is invoked as the functional relationship between the only two unique macromolecules in the cell, DNA and peptidoglycan embodying the nucleoid and the sacculus respectively. We envision that the total amount of DNA associated with the replication terminus, so called "nucleoid complexity," is directly related to cell size and shape through the transertion process. Accordingly, the primary signal for cell division transmitted by DNA dynamics (replication, transcription and segregation) to the peptidoglycan biosynthetic machinery is of a physico-chemical nature, e.g., stress in the plasma membrane, relieving nucleoid occlusion in the cell's center hence enabling the divisome to assemble and function between segregated daughter nucleoids.

Localizing cell division in spherical Escherichia coli by nucleoid occlusion.

The spatial relationship between FtsZ localization and nucleoid segregation was followed in Escherichia coli thyA cells, made spheroidal by brief exposure to mecillinam and after manipulating chromosome replication time using changes ('steps') in thymine concentration [Zaritsky et al., Microbiology 145 (1999) 1015-1022]. In such cells, fluorescent FtsZ-GFP arcs did not overlap the DAPI-stained nucleoids. It is concluded that FtsZ rings are deposited between segregating nucleoids, consistent with the nucleoid occlusion model [Woldringh et al., J. Bacteriol. 176 (1994) 6030-6038].