Model for bacteriophage T4 development in Escherichia coli.
Mathematical relations for the number of mature T4 bacteriophages, both inside and after lysis of an Escherichia coli cell, as a function of time after infection by a single phage were obtained, with the following five parameters: delay time until the first T4 is completed inside the bacterium (eclipse period, nu) and its standard deviation (sigma), the rate at which the number of ripe T4 increases inside the bacterium during the rise period (alpha), and the time when the bacterium bursts (mu) and its standard deviation (beta). Burst size [B = alpha(mu - nu)], the number of phages released from an infected bacterium, is thus a dependent parameter. A least-squares program was used to derive the values of the parameters for a variety of experimental results obtained with wild-type T4 in E. coli B/r under different growth conditions and manipulations (H. Hadas, M. Einav, I. Fishov, and A. Zaritsky, Microbiology 143:179-185, 1997). A "destruction parameter" (zeta) was added to take care of the adverse effect of chloroform on phage survival. The overall agreement between the model and the experiment is quite good. The dependence of the derived parameters on growth conditions can be used to predict phage development under other experimental manipulations. (+info)
Control of corynebacteriophage reproduction by heteroimmune repression.
Corynebacteriophages beta and gamma are closely related but heteroimmune; hence, gamma reproduces in C7(beta). A series of gamma mutants, designated gamma-bin (beta-inhibited), has been isolated. They reproduce in only 2 to 14% of infected C7(beta) cells, and, as a result, plaque with an efficiency of 10(-4) to 10(-5) on this strain. The proportion of C7(beta) cells in which gamma-bin phage can replicate is increased to 30 to 80% when immunity is lifted by UV induction of C7(beta) or by heat induction of C7(beta-tsr3). The gamma-bin mutants carry out a normal vegetative or lysogenic cycle in strain C7 and thus do not appear to be defective in any essential phage function. Infection of C7(beta) by gamma-bin results in cell killing whether the infection is productive or nonproductive. The data support the hypothesis that inhibition of gamma-bin is due to the direct or indirect action of a beta prophage gene. The simplest hypothesis is that gamma-bin phages have sustained mutations in an operator site and that beta repressor now combines with the mutated operator to inhibit normal replication in a significant proportion of infected cells. (+info)
Correlated genetic and EcoRI cleavage map of Bacillus subtilis bacteriophage phi105 DNA.
The seven previously identified EcoRI cleavage fragments of phi 105 DNA were ordered with respect to their sites of origin on the phage genome by marker rescue. One fragment, H, did not carry any determinants essential for replication. This fragment was totally missing in a deletion mutant which exhibited a lysogenization-defective phenotype. There is a nonessential region on the phi 105 genome which begins in fragment B, spans fragment H, and ends in fragment F. The size of the nonessential region, as estimated by alterations observed in the fragmentation patterns of deletion mutant DNAs, is approximately 2.7 X 10(6) daltons. Two new EcoRI cleavage fragments with molecular weights of approximately 0.2 X 10(6) were detected by autoradiography of 32P-labeled DNA. These small fragments were not located on the cleavage map. (+info)
A complex control circuit. Regulation of immunity in temperate bacteriophages.
Temperate bacteriophages can display in a stable way two essentially different behaviours. In the immune state, a gene (cI) produces a repressor which prevents expression of all the other viral genes; in the non-immune state the typically viral functions are expressed. The choice between the two pathways and the establishment of one of them have much in common with cell determination and differentiation. This choice depends on a complex control system, in fact one of the most intricate nets of regulation known in some detail. Our paper provides a formal description and partial analysis of this regulatory net. It is shown that even for relatively simple known models, this kind of analysis uncovers predictions which had previously remained hidden. Some of these predictions were checked experimentally. The experimental part chiefly deals with the efficiency of lysogenization by thermoinducible lambda phage carrying mutations in one or more of the regulatory genes, N, cro and cII. Although N- mutations are widely known for preventing efficient integration, and both N- and cII mutations for preventing efficient establishment of immunity, it is shown that, as predicted by a simple model, both N- and cII- phage efficiently lysogenize at low temperature if they are in addition cro-. In contrast with lambda N- cro+, lambda N- cro- is not propagated as a plasmid at low temperature, precisely because it establishes immunity too efficiently. Genetic control circuits are described in terms of sets of logic equations, which relate the state of expression of genes or of chemical reactions (functions) to input (genetic and environmental) variables and to the presence of gene and reaction products (internal, or memorization varibles). From the set of equations, one derives a matrix which shows the stable stationary states (if any) of the system, and from which one can derive the pathways (temporal sequences of states) consistent with the model. This kind of analysis is complementary to the more widely used analysis based on differential equations; it allows one to analyze in less detail more complex systems. The language might be used as well, mutatis mutandis, in fields very different from genetics. The last part of the discussion deals with the role of positive feedback loops in our specific problem (establishment and maintenance of immunity in temperate bacteriophages) and in developmental genetics in general. As a generalization of an old idea, it is suggested that cell determination (for a given character) depends on a set of genes whose interaction constitutes a positive feedback loop. Such a system has two stable stationary states: which one is chosen will usually depend on additional controls grafted on the loop. (+info)
Cooperative interaction of CI protein regulates lysogeny of Lactobacillus casei by bacteriophage A2.
The temperate bacteriophage A2 forms stable lysogens in Lactobacillus casei. The A2-encoded cI product (CI), which is responsible for maintaining the A2 prophage in the lysogenic state, has been purified. The CI protein, which is a monomer of 25.3 kDa in solution, specifically binds to a 153-bp DNA fragment that contains two divergent promoters, PL and PR. These promoters mediate transcription from cI and a putative cro, respectively. Three similar, although not identical, 20-bp inverted repeated DNA segments (operator sites O1, O2, and O3) were found in this segment. CI selectively interacts with O1, which is placed downstream from the transcription start point of the cro gene, and with O2 and O3, which overlap with the -35 region of the two promoters. Using a heterologous RNA polymerase, we have determined the transcription start points of PL and PR. CI exerts a negative effect on the in vitro transcription of PR by repositioning the RNA polymerase in a concentration-dependent manner. CI, when bound to O1 and O2, enhances the positioning of the RNA polymerase with the PL promoter. Our data indicate that the CI protein regulates the lytic and lysogenic pathways of the A2 phage. (+info)
Site-specific integration of corynephage phi16: construction of an integration vector.
Phi16, a temperate phage induced from Corynebacterium glutamicum ATCC 21792, lysogenizes its host via site-specific recombination. The phage attachment site, attP, was located to a 6.5 kb BamHI fragment of the phi16 genome. This fragment also contained phi16 integrative functions. The minimal phage DNA fragment required for integration was defined. This 1630 bp region contained a large open reading frame, int, encoding a protein of 416 amino acids with similarity in its carboxyl-terminal domain to tyrosine recombinases and particularly to the Xer recombinases. The comparison of the nucleotide sequences of attB, attL, attR, and attP identified a common 29 bp sequence, the core sequence. It lies 11 bp downstream of the 3' end of the integrase gene. phi16 integrase was shown to catalyse site-specific integration in trans to attP with an efficiency of 5x10(3) integrants per microg DNA. The integrating fragment catalysed integration in several Corynebacterium strains that are not infected by phi16, thus enlarging the host spectrum of integrating vectors derived from phi16. In these strains, the phi16 attB site was located in a conserved intergenic region and lies downstream of a clp gene. (+info)
Characterization of the major control region of Vibrio cholerae bacteriophage K139: immunity, exclusion, and integration.
The temperate bacteriophage K139 is highly associated with pathogenic O1 Vibrio cholerae strains. The nucleotide sequence of the major control region of K139 was determined. The sequences of four (cox, cII, cI, and int) of the six deduced open reading frames and their gene order indicated that K139 is related to the P2 bacteriophage family. Two genes of the lysogenic transcript from the mapped promoter PL encode homologs to the proteins CI and Int, with deduced functions in prophage formation and maintenance. Between the cI and int genes, two additional genes were identified: orf2, which has no significant similarity to any other gene, and the formerly characterized gene glo. Further analysis revealed that Orf2 is involved in preventing superinfection. In a previous report, we described that mutations in glo cause an attenuation effect in the cholera mouse model (J. Reidl and J. J. Mekalanos, Mol. Microbiol. 18:685-701, 1995). In this report, we present strong evidence that Glo participates in phage exclusion. Glo was characterized to encode a 13.6-kDa periplasmic protein which inhibits phage infection at an early step, hence preventing reinfection of vibriophage K139 into K139 lysogenic cells. Immediately downstream of gene int, the attP site was identified. Upon analysis of the corresponding attB site within the V. cholerae chromosome, it became evident that phage K139 is integrated between the flagellin genes flaA and flaC of O1 El Tor and O139 V. cholerae lysogenic strains. (+info)
New insertion sequences and a novel repeated sequence in the genome of Mycobacterium tuberculosis H37Rv.
The genome sequence of Mycobacterium tuberculosis H37Rv was found to contain 56 loci with homology to insertion sequences (ISs). As well as the previously described IS6110, IS1081, IS1547 and IS-like elements, new ISs belonging to the IS3, IS5, IS21, IS30, IS110, IS256 and ISL3 families were identified. In addition, six ISs created a grouping of their own to form a new family (the IS1535 family). Elements with similarity to ISs in other actinomycetes were identified, suggesting the movement of ISs between related genera. The location of ISs on the chromosome revealed that an approximately 600 kb region close to the origin of replication lacks ISs, pointing to the possible detrimental effect of insertions in this area. Analysis of the distribution of ISs through the tubercle strains Mycobacterium africanum, M. microti, M. bovis, M. bovis BCG Pasteur, M. tuberculosis H37Ra, M. tuberculosis CSU#93 and 29 clinical isolates revealed that only IS1532, IS1533, 1S1534, and IS1561' were absent from some of the strains tested. A novel repeated sequence, the REP13E12 family, is described that is present in seven copies on the M. tuberculosis H37Rv chromosome and which contains a probable phage attachment site. This study therefore offers an insight into the possible role of ISs and repetitive elements in the evolution of the M. tuberculosis genome, as well as identifying genetic markers that may be useful for phylogenetic and epidemiological analysis of the tubercle complex. (+info)