Interactions between Viral Regulatory Proteins Ensure an MOI-Independent Probability of Lysogeny during Infection by Bacteriophage P1

Phage P1 has been shown potentially to play an important role in disseminating antibiotic resistance among bacteria during lysogenization, as evidenced by the prevalence of P1 phage-like elements in animal and human pathogens. In contrast to phage λ, a cell fate decision-making paradigm, P1 lysogenization was shown to be independent of MOI.

Virus–Host Interaction Gets Curiouser and Curiouser. PART I: Phage P1vir Enhanced Development in an E. coli DksA-Deficient Cell

Bacteriophage P1 is among the best described bacterial viruses used in molecular biology. Here, we report that deficiency in the host cell DksA protein, an E. coli global transcription regulator, improves P1 lytic development. Using genetic and microbiological approaches, we investigated several aspects of P1vir biology in an attempt to understand the basis of this phenomenon. We found several minor improvements in phage development in the dksA mutant host, including more efficient adsorption to bacterial cell and phage DNA replication. In addition, gene expression of the main repressor of lysogeny C1, the late promoter activator Lpa, and lysozyme are downregulated in the dksA mutant. We also found nucleotide substitutions located in the phage immunity region immI, which may be responsible for permanent virulence of phage P1vir. We suggest that downregulation of C1 may lead to a less effective repression of lysogeny maintaining genes and that P1vir may be balancing between lysis and lysogeny, although finally it is able to enter the lytic pathway only. The mentioned improvements, such as more efficient replication and more “gentle” cell lysis, while considered minor individually, together may account for the phenomenon of a more efficient P1 phage development in a DksA-deficient host.

Genetic updating and marks of cellular lines

Despite great advances in the biology of stem cells, there are still many dark spots. Genetic modification techniques, which can be used to track the lines of different cells, primarily stem cells, help to solve this problem. Various methods of biotechnology research are considered, allowing to evaluate the options of introducing new genes into cells and even whole organisms, as well as methods of controlling their expression in time and space, their activation, differentiation and decrease in functional activity, expression of several target genes. Options with multi-cystron vectors encoding several proteins are described. Options for introducing genes using plasmids, electroportation of their disadvantages and advantages are characterized. The most promising and the safest is a retroviral vector using lentivirus vectors capable of generating additional copies of itself, which is very important in the field of biotechnology security. A line of packing cells, usually 293T cells, is used to produce a viral vector. Prospects for the use of adenovirus and adenoassociated vectors are characterized. The achievement of modern biotechnology methods is the system of short palindrome repetitions located in groups, which is a unique tool for genome editing. At the heart of this system is the process of cutting out sequences of deoxyribonucleic acid, which are permanent and which are supported by cells regardless of subsequent divisions or changes in condition. The system allows geneticists and medical researchers to edit parts of the genome by removing, adding or modifying successive sites of deoxyribonucleic acid. An important problem with biotechnology methods is how to control the expression of transgenes. Today, it is quite effective to control expression with a factor present in the gene delivery vector itself and which is only active in a certain type of cell. Endonuclease bacteriophage P1 is used to regulate transgene expression, which cuts deoxyribonucleic acid only at specific sites. This system is introduced in both eukaryotic and prokaryotic systems.

DNA transduction in Sodalis species: implications for the genetic modification of uncultured endosymbionts of insects

Bacteriophages (phages) are ubiquitous in nature. These viruses play a number of central roles in microbial ecology and evolution by, for instance, promoting horizontal gene transfer (HGT) among bacterial species. The ability of phages to mediate HGT through transduction has been widely exploited as an experimental tool for the genetic study of bacteria. As such, bacteriophage P1 represents a prototypical generalized transducing phage with a broad host range that has been extensively employed in the genetic manipulation of Escherichia coli and a number of other model bacterial species. Here we demonstrate that P1 is capable of infecting, lysogenizing and promoting transduction in members of the bacterial genus Sodalis, including the maternally inherited insect endosymbiont Sodalis glossinidius. While establishing new tools for the genetic study of these bacterial species, our results suggest that P1 may be used to deliver DNA to many Gram negative endosymbionts in their insect host, thereby circumventing a culturing requirement to genetically manipulate these organisms.SummaryA large number of economically important insects maintain intimate associations with maternally inherited endosymbiotic bacteria. Due to the inherit nature of these associations, insect endosymbionts cannot be usually isolated in pure culture nor genetically manipulated. Here we use a broad-host range bacteriophage to deliver exogenous DNA to an insect endosymbiont and a closely related free-living species. Our results suggest that broad host range bacteriophages can be used to genetically alter insect endosymbionts in their insect host and, as a result, bypass a culturing requirement to genetically alter these bacteria.

Application of Recombinase-Based In Vivo Expression Technology to Bifidobacterium longum subsp. longum for Identification of Genes Induced in the Gastrointestinal Tract of Mice

Bifidobacteria are one of the major components in human gut microbiota and well-known as beneficial microbes. However, clarification of commensal mechanisms of bifidobacteria in the intestines is still ongoing, especially in the presence of the gut microbiota. Here, we applied recombinase-based in vivo expression technology (R-IVET) using the bacteriophage P1 Cre/loxP system to Bifidobacterium longum subsp. longum 105-A (B. longum 105-A) to identify genes that are specifically expressed in the gastrointestinal tract of conventionally raised mice. Oral administration of the genomic DNA library of B. longum 105-A to conventionally raised mice resulted in the identification of 73 in vivo-induced genes. Four out of seven tested genes were verified in vivo-specific induction at least in the cecum by quantitative reverse transcription PCR. Although there is still room for improvement of the system, our findings can contribute to expanding our understanding of the commensal behavior of B. longum in the gut ecosystem.

Isolation of Full Size BAC Inserts by DNA Gap Repair inE. coli

DNA polymers can comprise millions of base pairs and encode thousands of structural and regulatory genetic elements. Thus, the precise isolation of specific DNA segments is required for accurate gene dissection. Although polymerase chain reaction (PCR) is a standard tool for this purpose, increasing DNA template size leads to the accumulation of polymerase errors, hindering the precise isolation of large-size DNA fragments. Unlike PCR amplification, DNA gap repair (DGR) is a virtually error-free process. However, the maximal size of bacterial artificial chromosome (BAC) insert isolated so far by recombination-mediated genetic engineering (recombineering) is <90 Kilobase pairs (Kbp) in length. Here, we developed a compact bacteriophage P1 artificial chromosome (PAC) vector, and we used it to retrieve a DNA segment of 203 Kbp in length from a human BAC by DGR inEscherichia coli(E. coli). We analyzed the efficiency of DGR with repressed (recombineering-) and derepressed lambda phageredgenes (recombineering+). We showed that both DGR efficiency and the percentage of PAC clones containing the expected 203 Kbp BAC insert improved with increasing size of homology arms. In recombineering+E. colicells and with an efficiency of electroporation of 8×109/1µg pUC plasmid DNA, DGR efficiency and the percentage of correct PAC clones were about 5×10-6and 1% for 30 bp; 6×10-6and 30% for 40 bp; and 1.5×10-5and 80% for 80 bp homology arms, respectively. These data show that using long homology arms and a newly developed vector, we isolated for the first time nearly a full size BAC insert with a frequency of correct clones not previously reported.