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.