The mechanism of site-specific recombination; structure/function analysis of recombination proteins
1. The mechanism of site-specific recombination mediated by the Integrase (Int) protein of bacteriophage lambda.
Site-specific recombination reactions are wide-spread in nature, and perform many functions in cells including the control of gene expression and the separation of dimeric chromosomes to allow their proper segregation to daughter cells. Many bacteriophages use site-specific recombination to integrate bacteriophage genomes into host cell chromosomes and to excise prophages to resume the lytic life cycle.
The mechanism of the recombinases that mediate the reactions we study is related to the mechanism of eukaryotic type I topoisomerase enzymes: both enzyme families nick and reseal DNA one strand at a time. Topoisomerases do this fairly randomly, in order to relieve topological stress which accumulates during transcription or replication. The Int enzyme and similar recombinases are much more specific: Int acts at pairs of sequences known as att sites, bringing them together in unique synaptic complexes. Within these complexes, several Int proteins helped by accessory proteins perform two rounds of DNA cleavage, exchange and ligation reactions to rearrange the continuity of both DNA strands of each att substrate.
The synaptic complexes can be very delicate and short-lived, making them difficult to study. We have developed tools to stabilize and isolate these complexes and study their geometry in each pathway of Int-mediated recombination. To do this, we are using imaging methods such as atomic force microscopy (see figure) as well as physical methods such as protein-protein and protein-DNA crosslinking in order to understand the physical relationships of protein and DNA molecules to each other. We have recently identified several peptide inhibitors of the recombination reaction.
Some of these also inhibit the related topoisomerase encoded by the Vaccinia virus, human topoisomerase I, as well as the bacterial type I topoisomerase. We are using these peptide inhibitors to probe the intermediates of recombination, and are testing the action of the peptides in vivo in order to explore their potential use as antibiotics and cancer therapeutics (Funded by NIH RO1 GM52847).
2. Structure of the Salmonella chromosome in vivo.
Several lines of evidence have suggested that bacterial chromosomes may have a specific folded structure. We are investigating this possibility by using Int-mediated site-specific recombination as a probe. We are placing pairs of att sites at different locations in the chromosome. Recombination between them would result in an inversion of the intervening chromosomal segment with respect to the rest of the chromosome. We are thus testing the frequency of recombination between different pairs of att sites to measure the accessibility of different chromosomal regions to each other.
We already have data that shows recombination efficiency changes depending on where in the chromosome the recombination targets are found, and we have found that cell physiology can have a profound effect on recombination efficiency, we think by its effect on chromosome structure. We are also testing the effect of the inversion rearrangement on the physiology of the cell. While these studies are still relatively new, we are hoping to gain an in-depth view of the organization of the chromosome and to identify the genes that control and maintain this structure. Such genes may be exploited as targets for developing antibiotics (Funded by NSF CAREER Award 9733332 and NIH RO1 GM52847).
3. The diversity of marine bacteriophage.
The ocean is the Earth’s largest ecosystem, yet it is one of the least studied. Bacteria process approximately 50% of the carbon in marine environments. Bacterial counts range between 105 and 106/ml of seawater, and bacterial populations are an extremely important part of the food web (Azam, 1999). Marine environments are home to an even larger population of marine bacteriophage which are found at concentration of 107 and 108 particles/ml, and as high as 109/ml. Bacteriophage are expected to profoundly affect bacterial populations by lysis, and should also be a major agent of DNA exchange among marine bacterial species.
The diversity of bacterial populations is only beginning to be explored. A large problem facing investigations of bacterial species is that as many 90% of the species are unculturable in the laboratory. This has been solved to a large extent by molecular detection methods, in particular PCR. Using PCR methods, bacteria can be identified using highly conserved regions of ribosomal RNA. Although PCR would be equally sensitive against bacteriophage, phage do not have any genes which are highly conserved like ribosomal RNAs. Therefore, measuring the diversity of phage has been largely restricted by the ability to find appropriate host bacteria.
Through a collaboration with the laboratory of Dr. Farooq Azam at the Scripps Institution of Oceanography, we are exploring the diversity of marine bacteriophage using molecular, genomic, and bioinformatics tools. One of our approaches has been to sequence the genomes of individual marine bacteriophage with known hosts.
We have already completed one such genome and are in the process of sequencing several others. Once phage genomic sequences have been obtained, we can analyze directly the similarity between marine and terrestrial viruses, compare marine phage to each other to identify possible common genes, and construct specific “probes” that would allow us to identify these viruses in different marine environments around the globe.
Using these probes, we can gauge the effect of geography, climate, nutrients and myriad other factors on the distribution of viruses. This approach was used to show that Roseophage SIO1 was in the water off of Scripps Pier 8 years after the phage was originally isolated (Rohwer et al., 2000). Another approach is to isolate DNA from marine viruses without propagating them in culture and investigate their diversity using molecular methods.
Many bacteriophage carry host genes and serve as an agent of genetic exchange among bacterial species. Thus phage contribute directly to the molecular evolution of their bacterial hosts. By identifying phage-encoded host genes, we should gain insight into modes in which bacteria adapt to changing marine environmental conditions and to major stresses that marine bacteria face.
For example, in sequencing Roseophage SIO1, we found that it encodes a protein very similar to PhoH, a protein induced specifically under conditions of phosphate starvation. This suggests that the phage host might need to grow in such phosphate deprived conditions (Funded by NSF 0221763 Biocomplexity program).