Descripción: In order to isolate very strong promoters from bacteria and bacteriophage a plasmid named pProm was constructed. It possesses an origin (ORI) for replication in Gram-negative bacteria, an ORI for replication in Gram-positive bacteria, a promoterless ampicillin resistance gene with a multiple cloning site (MCS) in the position formerly occupied by the ampicillin promoter, a tetracycline resistance gene for selection in Gram-negative bacteria and a chloramphenicol resistance gene for selection in Gram-positive bacteria. Insertion in the MCS of DNA fragments of Staphylococcus aureus bacteriophages resulted in isolation of several clones very resistant to ampicillin. The DNA fragments inserted in these recombinant plasmids were sequenced and all of them contained putative promoter motifs. Direct measurement of the penicillinase activity indicated that one of the isolated promoters could be included within a group of the stronger known prokaryotic promoters. According to these results pProm is a powerful tool to perform studies on promoter strength and for industrial applications. Copyright (C) 1999 Federation of European Microbiological Societies.

Descripción: Bacteriophage Recombineering of Electroporated DNA (BRED) has been described for construction of gene deletion and point mutations in mycobacteriophages. Using BRED, we inserted a Phsp60-egfp cassette (1143 bp) into the mycobacteriophage D29 genome to construct a new reporter phage, which was used for detection of mycobacterial cells. The cassette was successfully inserted and recombinant mycobacteriophage purified. DNA sequencing of the cassette did not show any mutations even after several phage generations. Mycobacterium smegmatis mc2155 cells were infected with D29::Phsp60-egfp (MOI of 10) and evaluated for EGFP expression by microscopy. Fluorescence was observed at around 2 h after infection, but dissipated in later times because of cell lysis. We attempted to construct a lysis-defective mutant by deleting the lysA gene, although we were unable to purify the mutant to homogeneity even with complementation. These observations demonstrate the ability of BRED to insert c. 1 kbp-sized DNA segments into mycobacteriophage genomes as a strategy for constructing new diagnostic reporter phages. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd.

Descripción: Phages may cause lysis of lactic acid bacteria used in cheese production. Three virulent bacteriophages specific for Streptococcus lactis subsp. lactis C2 were isolated and purified from cheese whey. They showed distinct plaque sizes, and although they had similar morphology by electron microscopy examination, their dimensions were slightly different. The phage heads were elongated and hexagonal in shape, and the flexible tails appeared periodically cross-striated. They were DNA phages based on the acridine orange test. On infection, phage was adsorbed on the bacterial surface by the free end of the tail. After 80 min of incubation at 25°C, the phage heads appeared empty, slightly collapsed, and possessed a visible hollow tube through which the genetic material had been injected.

Descripción: Addition of affinity tags to bacteriophage particles facilitates a variety of applications, including vaccine construction and diagnosis of bacterial infections. Addition of tags to phage capsids is desirable, as modification of the tails can lead to poor adsorption and loss of infectivity. Although tags can readily be included as fusions to head decoration proteins, many phages do not have decoration proteins as virion components. The addition of a small (10-amino-acid) Strep-tag II (STAG II) to the mycobacteriophage TM4 capsid subunit, gp9, was not tolerated as a genetically homogenous recombinant phage but could be incorporated into the head by growth of wild-type phage on a host expressing the capsid-STAG fusion. Particles with capsids composed of wild-type and STAG-tagged subunit mixtures could be grown to high titers, showed good infectivities, and could be used to isolate phage-bacterium complexes. Preparation of a STAG-labeled fluoromycobacteriophage enabled capture of bacterial complexes and identification of infected bacteria by fluorescence. © 2013, American Society for Microbiology.

Descripción: Strains derived from HfrH carrying the arcA2 null mutation exhibit a higher respiratory rate, enhanced glucose consumption, and a more-reduced intracellular redox state than arcA deletion mutants of a different lineage. The phenotype of the arcA2 mutants was due to the presence of a creC constitutive mutation introduced by P1 transduction. Copyright © 2008, American Society for Microbiology. All Rights Reserved.