Faculty Bibliography

Plasmid pSA5700 from Staphylococcus aureus coding for erythromycin (EmR) and chloramphenicol (CmR) resistance was transformed into Streptococcus pneumoniae. High-copy-number and EmR constitutive mutants of this plasmid were isolated. Transformation frequencies in S. pneumoniae as high as 70% were obtained with a constitutive plasmid as donor DNA, into a recipient cell containing a resident, inducible, high-copy-number plasmid. With the aid of these high frequencies, the site of constitutive mutations could be mapped via a simple marker rescue technique that uses purified restriction endonuclease-generated fragments. One of the EmR constitutive mutants, pFB9, a plasmid originating from a Gram-positive host, was shown to replicate and express EmR and CmR in a Gram-negative organism, Escherichia coli. Four derivatives of pFB9 containing large (0.6-0.9 megadalton) insertion sequences that arose spontaneously in E. coli demonstrated unusual transforming activity, as well as enhanced EmR, in E. coli. The inserted elements mapped to the region in front of the EmR gene. Three of these inserted elements had the size and restriction patterns of insertion sequence IS1, IS2, and IS5. Plasmid pFB9 and derivatives are useful for isolation of new insertion sequences and for comparison of gene expression and illegitimate recombination between Gram-positive and Gram-negative species.

Derivatives of filamentous phage, f1, fd, and M13, useful as cloning vectors are listed, and procedures for their use are reviewed. Methods for growing phage, preparing single- and double-stranded DNA, and cloning are given in the "cook-book" form. These procedures minimize the practical problem often associated with filamentous-phage cloning, i.e., deletion of inserts.

Gene III protein of bacteriophage f1 is inserted into the host cell membrane where it is assembled into phage particles. A truncated form of gene III protein, encoded by a recombinant plasmid and lacking the carboxyl terminus, does not remain in the membrane but instead appears to slip through it. Fusion of a hydrophobic "membrane anchor" from another membrane protein, the gene VIII protein, to the truncated gene III protein (by manipulation of the recombinant plasmid) restores membrane anchoring. A model for the relationship of gene III protein with the Escherichia coli membrane is discussed.

Plasmids which encode bacteriophage f1 coat protein genes VIII and III are responsible for a number of unusual properties suggesting that they have a drastic effect on the bacterial outer membrane. Analysis of several such recombinant plasmids and selection of mutant plasmids unable to cause this effect established that the properties were caused by gene III protein or its amino-terminal fragment.

The assembly of filamentous bacteriophages has been studied in cells infected by wild-type and mutant phage; host mutants defective in bacteriophage assembly have also been isolated. Phage assembly takes place at the membrane, and requires insertion of the viral major coat protein. We present data on the physiology of this process and on the effects of amino acid sequence variations near to the coat protein amino terminus on membrane insertion, processing, and phage assembly.

Simple methods for introducing one or two extra codons of genetic information into the f1 genome in vitro have been devised. The methods use various combinations of enzymes to insert three or six base-pairs into the RF1 DNA of the bacteriophage. Since such insertions do not cause frameshifts in coding regions, a number of these mutants are viable. Several such mutants were mapped and characterized. The methods described and variations of them can be applied to other circular DNA genomes.