Recombination and chromosome rearrangements

This project was initiated in an attempt to better understand the structure of the folded bacterial chromosome and the selective forces that have conserved the genetic map over the past 100 million years or so, since the divergence of Salmonella and E. coli. We have found that duplications are frequent, well-tolerated and can be selectively valuable while inversions are rare and sometimes deleterious. This has suggested that the recombination system of bacteria may be structured to minimize exchanges between inverse-order repeats.

In the course of this work, we have realized that rearrangements provide a very useful system for studying the process of recombination. Repeated chromosomal sequences provide substrates that lack the double stranded ends provided by sexual assays and thus may more accurately reflect the role functions of recombination. Duplications can be generated by half-exchanges while inversions require full (reciprocal) exchanges. In addition, using inverse-order sequences we can recover both products of a recombination event, which is impossible in standard sexual assays. These assays have provided some new ways of studying recombination.

We have also been using P22-mediated transduction to develop new recombination assays. It is our general belief that there is still a role for genetic analysis in the study of recombination, and in assessing the biological role of known recombination proteins.

Synthesis and significance of cobalamin (B12)

This cofactor is unevenly distributed among living things, suggesting that it might be associated with particular lifestyles or growth conditions. We discovered that Salmonella dedicates about 1% of its genome to B12 production and import, suggesting frequent use of the cofactor under natural conditions. Under standard laboratory conditions, there is no growth phenotype associated with B12 deficiency. We are using genetics to approach the synthetic pathway of B12 and it's regulation. We are also trying to understand the importance of B12 to natural populations. In doing this we are working on two B12-dependent processes -- breakdown of ethanolamine and propanediol. Operons for both of these processes are being intensively studied.

Recycling of NAD

While NAD is well known as an electron carrier, it also energizes DNA ligation. We are trying to understand the synthesis and recycling of NAD and how it relates to energy metabolism, DNA repair and oxidative stress. The large NAD pool is subject to breakdown to NMN which is recycled to NAD through two pathways. The ligase-dependent NAD breakdown is only about 20% of the NMN production under aerobic conditions. The rest of the cycle is eliminated during anaerobic growth by unknown reactions. In pursuit of this major metabolic cycle we have isolated mutants for almost every known step of NAD synthesis and metabolism. We have discovered two NAD kinases (producing NADP) which have different cofactor requirements and we have found that a deficit in NAD kinase leads to oxygen-sensitivity. We have recently discovered a recycling activity (NMN deamidase) that depends on DNA ligase and acts only in the presence of oxygen. Thus oxygen appears to be a major player in control and significance of the recycling pathway.

Adaptive mutation

We become involved in this controversial but very interesting area as a spinoff of an older notion of ours about mutagenic response to stress, namely that randomly formed local duplications and subsequently expanded arrays of genes may provide sufficient residual activity to allow growth, which in turn would allow for selection of useful mutations within a target set much larger than normally found in wild type. In testing the idea, we discovered that the dominant system used by other laboratories to demonstrate "adaptive mutation" requires that the gene under selection be located on an F' plasmid and subject to transfer replication. We are proceeding to look for evidence of "adaptive mutability" under less specialized conditions so we can more definitively test our model.

Last Update: Monday July 29 2019
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Eric Kofoid eckofoid at