Construction and properties of a chimeric bacteriophage lysis gene

Abstract The genomes of DNA phage ΦX174 and of RNA phage MS2 each encode a single lysis protein, E protein and L protein, respectively. Based on the known DNA and protein sequences, and with the aid of structural predictions of the proteins, a chimeric gene was constructed. The resulting protein was composed of the N-terminal sequence of E protein and the C-terminal sequence of L protein. The truncated E and L polypeptides used in this construction were functionally inactive. However, the chimeric gene product had very high lysis-inducing activity. This construction is an example which could be extended to the engineering of other lysis proteins designed with specific biotechnological processes in mind.     

The cis-acting DNA sequences required in vivo for bacteriophage Mu helper-mediated transposition and packaging

The 37,000 bp double-stranded DNA genome of bacteriophage Mu behaves as a plaque-forming transposable element of Escherichia coli. We have defined the cis-acting DNA sequences required in vivo for transposition and packaging of the viral genome by monitoring the transposition and maturation of Mu DNA-containing pSC101 and pBR322 plasmids with an induced helper Mu prophage to provide the trans-acting functions. We found that nucleotides 1 to 54 of the Mu left end define an essential domain for transposition, and that sequences between nucleotides 126 and 203, and between 203 and 1,699, define two auxiliary domains that stimulate transposition in vivo. At the right extremity, the essential sequences for transposition require not more than the first 62 base pairs (bp), although the presence of sequences between 63 and 117 bp from the right end increases the transposition frequency about 15-fold in our system. Finally, we have delineated the pac recognition site for DNA maturation to nucleotides 32 to 54 of the Mu left end which reside inside of the first transposase binding site (L1) located between nucleotides 1–30. Thus, the transposase binding site and packaging domains of bacteriophage Mu DNA can be separated into two well-defined regions which do not appear to overlap.Abbreviations attL attachment site left - attR attachment site right - bp base pairs - Kb kilobase pair - nt nucleotide - Pu Purine - Py pyrimidine - Tn transposable element State University of New York, Downstate Medical Center, Brooklyn, NY 11204 USA     

Bacteriophage lambda DNA packaging: A mutant terminase that is independent of integration host factor

Summary ⁺ is able to grow in Escherichia coli cells lacking integration host factor (IHF), producing a burst of approximately 25% that produced in IHF⁺ cells. In vitro, however, we find that the DNA packaging enzyme terminase is strongly dependent on IHF in both cos cleavage reactions and DNA packaging reactions. The cos59 mutation renders dependent on IHF in vivo. The cos59 mutation is a deletion of 3 base pairs at the XmnI site in the cohesive end site (cos) of . Variants of cos59 that were able to grow in the absence of IHF were isolated and found to carry a mutation, called ms1, in the Nu1 gene, which codes for the small subunit of terminase. The Nu1ms1 mutation results in a change of the 40th amino acid of the Nu1 gene product from leucine to phenylalanine. The Nu1ms1 terminase was independent of IHF in packaging reactions in vitro. The results indicate that the mutation either renders terminase: (1) able to utilize some host protein other than IHF, or (2) totally independent of host factors.     

A selection cartridge for rapid detection and analysis of spontaneous mutations including insertions of transposable elements in Enterobacteriaceae

Summary We present a method that allows positive selection and rapid analysis of mutations in Enterobacteriaceae. Mutations are detected in a 2630 bp selection cartridge inserted in two different bacterial mutlicopy plasmid vectors. Spontaneous mutations in Escherichia coli, Enterobacter cloacae and Citrobacter freundii include insertions, deletions and point mutations. The small size of the target sequence facilitates rapid analysis of DNA rearrangements by cleavage with restriction enzymes and of any type of mutation by DNA sequence analysis. While in E. coli insertions of the mobile elements IS1, IS2 and IS5 were readily found, insertions of putative new transposable elements were detected in Enterobacter cloacae. The selection cartridge can thus serve as a tool for studying the spectrum of insertion mutations in Enterobacteriaceae and probably other Gramnegative bacteria, and the dependency of this spectrum on physiological and environmental factors and the host's genetic background can be investigated.     

Phage resistance and altered growth habit in a strain of Streptococcus bovis

Bacteriophage (phi Sb01) of Streptococcus bovis, isolated from pooled rumen fluid of cattle, was a small siphovirus of morphotype B1. It contained double-stranded DNA of length 30.9 kb, which was digested by the restriction endonucleases, EcoRI, HindIII, and PvuII. Bacteria which survived phi Sb01 infection (strain 2BAr) grew in long chains (100-200 cells), ultimately forming large clumps of cells. This growth habit was in distinct contrast to that of the parent host strain which grew predominantly in the form of single cells or diplococci. Strain 2BAr was genetically stable, resistant to phi Sb01 attack, and the observed differences in the growth characteristics of the parent strain and 2BAr indicated that cells of 2BAr were more adherent. In the rumen ecosystem, the selection of phage-resistant bacteria with altered growth characteristics may be a factor in modifying bacterial phenotypes, and thus increasing variability among bacteria which are closely related genetically.     

青枯假单胞杆菌噬菌体ZP-2和ZP-3的生物学性状

噬菌体可作为检测和区分植物病原细菌的一种手段。有关青枯假单胞杆菌(Pseudomo-n~(as solanacearum) 噬菌体的研究工作报道不多,姜瘟菌株上的噬菌体则还未见报道。本文所报道的关于分离自土壤的青枯假单胞杆菌姜瘟菌株(ZO-12)的噬菌体ZP-2和ZP-3生物学性状的研究结果,可为这些噬菌体的利用提供参考依据。     

Virulence of Salmonella enteritidis phage type 4 is related to the possession of a 38 MDa plasmid

Nine strains of Salmonella enteritidis phage type 4 were examined for virulence in BALB/c mice. The possession of a 38 MDa plasmid was necessary for full virulence. Strains carrying this plasmid had LD50 values of less than 20 bacteria whilst plasmid-free strains had LD50 values of greater than 10(6) bacteria when challenged intraperitoneally. Pathogenesis of disease involved the widespread distribution of bacteria throughout the tissues. Possession of the 38 MDa plasmid could not be linked with the ability of strains to express novel outer membrane proteins, to produce toxins affecting Vero, Y1, HeLa, Henle or HEp-2 cells, or to invade HEp-2 cells. Furthermore, the 38 MDa plasmid did not encode an aerobactin-mediated iron uptake system or the production of a haemolysin. Strains of S. enteritidis PT4 isolated in 1967, 1978 or 1979 and possessing the 38 MDa plasmid showed the same virulence properties as the current plasmid-carrying strains. This suggests that the enhanced virulence of the current strains for poultry is unlikely to be the result of changes in the 38 MDa plasmid.