Michael Malamy

My training projects in biochemistry with Horecker, and bacterial genetics and physiology with Monod and then Pardee, were based in E. coli. After establishing my own laboratory at Tufts, I continued working with E. coli studying inorganic phosphate transport, regulation and functions of the F- transfer factor, the role of the F-factor in inhibiting bacteriophage development, and in the identification and characterization of insertion sequences (IS elements) which caused highly polar mutations in the lactose operon of E. coli. I continued to study the pif region of the F-factor and characterized the pif operon and the pifC regulator protein. Just adjacent to the pif region on F, we discovered and characterized the reactions at the rfs site at which site-specific recombination could occur to fuse or resolve plasmids containing this site. In the mid 1980's I began a collaboration with Dr. Francis Tally to study virulence factors in the opportunistic pathogen Bacteroides fragilis. Our first challenge was to test whether outbreaks of clindamycin resistant Bacteroides strains in a clinical setting represented dissemination of a resistance determinant by horizontal transfer. At that time, there were few, if any genetic tools that could be used to study gene transfer in B. fragilis. I adapted the approach that Lederberg used when he discovered vectorial transfer of DNA between E. coli donor and recipient cells, for use in Bacteroides. We quickly established that the clindamycin determinant could be transferred by conjugation and we described pBFTM10, as the plasmid in B. fragilis that contained the clindamycin resistance gene and the ability to transfer it to recipients. At about that time Macrina's lab described another plasmid in B. fragilis, pBF4, that was larger than pBFTM10 and also transferable. Our labs collaborated to show that the clindamycin resistance determinant on both plasmids was the same. This lead to the characterization of Tn4400 in B. fragilis, a transposon capable of "hopping" from plasmid to plasmid or to the chromosome. In order to take advantage of the genetic tools available in E.coli but not yet established in B. fragilis, we developed a series of shuttle plasmids that could be passed from E. coli to B. fragilis; pJST61 and 63 could replicate in E.coli and B. fragilis because they contained both an E.coli replicon (derived from pBR322) and a B. fragilis replicon (derived from pBFTM10). This allowed us to clone, mutagenize, and manipulate B. fragilis sequences in E.coli then reintroduce them into B. fragilis by conjugation. Another shuttle plasmid, pJST55, is a suicide plasmid that can be transferred to B. fragilis but which is incapable of replication in B. fragilis but it contains a drug resistance markers that allowed for its selection in B. fragilis. This plasmid became the basis for introducing mutations into the B. fragilis chromosome, and delivering new transposons into B. fragilis to create Tn mutant libraries, etc. We have sent these plasmids, and derivatives containing our transposons to most of the labs involved with Bacteroides work in the US and abroad. Our recent work has focused on the response of B. fragilis to oxygen and resulted in the discovery that B. fragilis could actually grow and benefit from the presence of nanomolar concentrations of oxygen- thus B. fragilis is a nanaerobe. As a result of this work we characterized the cytochrome bd oxidase in B. fragilis. We had previously demonstrated, contrary to the prevailing view, that B. fragilis contains a complete TCA cycle; we have also published our work on the fumarate reductase, succinic dehydrogenase, and the aconitase enzymes. More recently we discovered that mutants of B. fragilis could be isolated that were able to grow in rich media in oxygen concentrations up to 2%, and in rich or defined media when incubated anaerobically. Remarkably, all the enabling mutations occurred in one gene that we named oxe (oxygen enabled). Alt