Constant peptidoglycan density in the sacculus of escherichia coli B/r growing at different rates

The determination and maintenance of bacterial cell shape are problems still far from being understood (reviewed [ 1,2]). Several models have been advanced during the last decade, for mechanisms governing th

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

Studies on DNA replication and cell division in bacteria.

Eleven independent methods for estimating the replication time (C) of the bacterial chromosome are described and discussed. Four of these methods are used to demonstrate that the replication velocity (which is inversely proportional to C) in two non-related thymine-requiring strains of Escherichia coli is a function of the thymine concentration present in their growth medium. By varying the concentration of thymine C can be varied over a two- to three-fold rang, without causing any significant change in the doubling time of the cells. This demonstrates the non-coupling between replication velocity of the chrome-somes and growth rate of the culture. The best absolute values of C obtained in this study are derived from measurement of the rate-stimulation-factor. This is the factor by which the rate of DNA synthesis in the culture is stimulated after a period of thymine starvation equivalent to one mass doubling. However, the most reliable estimates of the relative replication velocities are obtained from the relative DNA/mass ratios in steady-state exponentially-growing cultures. The rate of change of C with the thymine concentration ia similar in glycerol-grown cells to that in glucose-grown cultures. Average cell size in glucose-grown cultures of both strains (but not in glycerol-grown cultures) increases steadily with time. A partial analysis of this phenomenon lad to the hypothesis that an irreversible event, which interferes with call division occurs with a probability that is inversely proportional to the thymine concentration. Evidence is presented suggesting that the time between completion of a round of chromosome replication and the subsequent cell division is coupled in some unknown way to C

Partial Restoration of Antibacterial Activity of the Protein Encoded by a Cryptic Open Reading Frame (cyt1Ca) from Bacillus thuringiensis subsp. israelensis by Site-Directed Mutagenesis

Insecticidal crystal proteins of Bacillus thuringiensis belong to two unrelated toxin families: receptor-specific Cry toxins against insects and Cyt toxins that lyse a broad range of cells, including bacteria, via direct binding to phospholipids. A new cyt-like open reading frame (cyt1Ca) encoding a 60-kDa protein, has recently been discovered (C. Berry et al., Appl. Environ. Microbiol. 68:5082-5095, 2002). Cyt1Ca displays the structure of a two-domain fusion protein: the N-terminal moiety resembles the full-length Cyt toxins, and the C-terminal moiety is similar to the receptor-binding domains of several ricin-like toxins, such as Mtx1. Neither the larvicidal activity of cyt1Ca expressed in Escherichia coli nor the hemolytic effect of His-tagged purified Cyt1Ca has been observed (R. Manasherob et al., unpublished). This was attributed to five amino acid differences between the sequences of its N-terminal moiety and Cyt1Aa. The 3′ end of cyt1Ca was truncated (removing the ricin-binding domain of Cyt1Ca), and six single bases were appropriately changed by site-directed mutagenesis, sequentially replacing the noncharged amino acids by charged ones, according to Cyt1Aa, to form several versions. Expression of these mutated cyt1Ca versions caused loss of the colony-forming ability of the corresponding E. coli cells to different extents compared with the original gene. In some mutants this antibacterial effect was associated by significant distortion of cell morphology and in others by generation of multiple inclusion bodies spread along the cell envelope. The described deleterious effects of mutated cyt1Ca versions against E. coli may reflect an evolutionary relationship between Cyt1Aa and Cyt1Ca

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

The Bacterial Cell: Coupling between Growth, Nucleoid Replication, Cell Division and Shape

Bacterial Physiology was inaugurated as a discipline by the seminal research of Maaløe, Schaechter and Kjeldgaard published in 1958. Their work clarified the relationship between cell composition and growth rate and led to unravel the temporal coupling between chromosome replication and the subsequent cell division by Helmstetter et al. a decade later. Now, after half a century this field has become a major research direction that attracts interest of many scientists from different disciplines. The outstanding question how the most basic cellular processes - mass growth, chromosome replication and cell division - are inter-coordinated in both space and time is still unresolved at the molecular level. Several particularly pertinent questions that are intensively studied follow: (a) what is the primary signal to place the Z-ring precisely between the two replicating and segregating nucleoids? (b) Is this coupling related to the structure and position of the nucleoid itself? (c) How does a bacterium determine and maintain its shape and dimensions? Possible answers include gene expression-based mechanisms, self-organization of protein assemblies and physical principles such as micro-phase separations by excluded volume interactions, diffusion ratchets and membrane stress or curvature. The relationships between biochemical reactions and physical forces are yet to be conceived and discovered. This e-book discusses the above mentioned and related questions. The book also serves as an important depository for state-of-the-art technologies, methods, theoretical simulations and innovative ideas and hypotheses for future testing. Integrating the information gained from various angles will likely help decipher how a relatively simple cell such as a bacterium incorporates its multitude of pathways and processes into a highly efficient self-organized system. The knowledge may be helpful in the ambition to artificially reconstruct a simple living system and to develop new antibacterial drugs