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.

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 [...].

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.

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.

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.

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.

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.

Richness and diversity in dust stormborne biomes at the southeast mediterranean.

Dust storms include particulate matter that is transported over land and sea with biota that could impact downwind ecosystems. In addition to the physico-chemical compositions, organismal diversities of dust from two storm events in southern Israel, December 2012 (Ev12) and January 2013 (Ev13), were determined by pyro-sequencing using primers universal to 16S and 18S rRNA genes and compared. The bio-assemblages in the collected dust samples were affiliated with scores of different taxa. Distinct patterns of richness and diversity of the two events were influenced by the origins of the air masses: Ev13 was rich with reads affiliated to Betaproteobacteria and Embryophyta, consistent with a European origin. Ev12, originated in north-Africa, contained significantly more of the Actinobacteria and fungi, without conifers. The abundance of bacterial and eukaryotic reads demonstrates dissemination of biological material in dust that may impose health hazards of pathogens and allergens, and influence vegetation migration throughout the world.

Growth and development of Aedes aegypti larvae at limiting food concentrations.

Mosquitoes have a complex life-cycle with dramatic changes in shape, function, and habitat. Aedes aegypti was studied by growing individual larvae at different concentrations of a defined rich food source. At higher food concentrations, rate of larval growth was faster, but the time required for 4th instar larvae to molt into the pupal stage was unexpectedly extended. These opposite tendencies resulted in constant times from hatching to pupation and up to adult eclosion at permissive food concentrations. The results demonstrate that nutritional conditions of 4th instar larvae impact initiation of the first metamorphic molt.

The basis for rootstock resilient to Capnodis species: screening for genes encoding δ-endotoxins from Bacillus thuringiensis.

BACKGROUND: Conventional methods often fail to control the flatheaded borers Capnodis spp., major pests of stone fruit trees; the larvae are protected from insecticides and predation because they feed deep in the roots. A potential solution is transgenic trees producing in their roots toxic compounds such as Cry proteins of Bacillus thuringiensis (Bt). RESULTS: Toxicities against Capnodis larvae were demonstrated by exploiting a recently designed artificial larval diet and an available collection of field isolated Bt. An isolate of Bt tenebrionis (Btt) from commercial bioinsecticide (Novodor) displayed LC50 and LC95 values of 3.2 and 164 mg g(-1) , respectively, against neonates of Capnodis tenebrionis, whereas values of the most toxic field isolate K-7 were 1.9 and 25.6 mg g(-1) respectively. Weights of surviving larvae after 1 month on diets containing low concentrations of K-7 (0.1-1.0 mg g(-1) ) were lower than on Btt or untreated larvae. K-7 was also toxic against larvae of C. cariosa and C. miliaris and found to harbour genes encoding Cry9Ea-like and Cry23Aa/Cry37Aa binary toxins. CONCLUSION: Larvae of Capnodis spp. are susceptible to Bt Cry toxins. Expressing cry genes active against these pests thus seems a feasible solution towards production of transgenic rootstock trees resilient to the pest.

Cyt1Aa toxin: crystal structure reveals implications for its membrane-perforating function.

During sporulation, Bacillus thuringiensis subsp. israelensis produces a mosquito larvicidal protein complex containing several crystalline and cytolytic (Cyt) toxins. Here, the activated monomeric form of Cyt1Aa, the most toxic Cyt family member, was isolated and crystallized, and its structure was determined for the first time at 2.2 Å resolution. Cyt1Aa adopts a typical cytolysin fold containing a ß-sheet held by two surrounding α-helical layers. The absence of a ß-strand (between residues V26 and I37) in the dimeric structure of Cyt2Aa led us to deduce that this is the only essential segment for dimer formation and that activation of the toxin occurs by proteolytic processing of its N-terminus. Based on the Cyt1Aa structure, we suggest that the toxicity of Cyt1Aa and other nonrelated proteins, all sharing a cytolysin fold, is correlated with their ability to undergo conformational changes that are necessary prior to their membrane insertion and perforation. This fold allows the α-helical layers to swing away, exposing the ß-sheet to insert into the membrane. The identification of a putative lipid binding pocket between the ß-sheet and the helical layer of Cyt1Aa supports this mechanism. Sequence-based structural analysis of Cyt1Aa revealed that the lack of activity of Cyt1Ca may be related to the latter's inability to undergo this conformational change due to its lack of flexibility. The pattern of the hemolytic activity of Cyt1Aa presented here (resembling that of pore-forming agents), while differing from that imposed by ionic and nonionic detergents, further supports the pore-forming model by which conformational changes occur prior to membrane insertion and perforation.

Tandem repeats in a new toxin gene from Bacillus thuringiensis and in other cry11-like genes.

A new gene, cry11Bb2 from a field isolate of Bacillus thuringiensis, was cloned for expression in Escherichia coli. The encoded protein, with a deduced molecular mass of 89.5 kDa, exhibits 97 and 79% identities with the overlap regions of Cry11Bb1 from B. thuringiensis ssp. medellin and Cry11Ba1 from ssp. jegathesan, respectively. It is however longer than Cry11Bb1 by 42 amino acids in its carboxy-terminus, of which 32 comprise 2 tandem repeats additional to the 5 existing in the latter polypeptide. Possible roles for this recurrent motif among Cry toxins and their accessory proteins, and for their encoding genes are proposed.

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.

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.

Modular columns to study depth-dependence behavior of mosquito larvae and toxicity of Bacillus thuringiensis subsp. israelensis.

Modular transparent column system was designed to study depth-dependence behavior of mosquito larvae. The system was used in preliminary experiments to evaluate the effect of water depth on the larvicidal activity of Bacillus thuringiensis subsp. israelensis de Barjac against bottom feeder larvae of Aedes aegypti (Linn.) (Diptera: Culicidae), and suggestions for increasing the efficiency of the device are discussed.