Microbial responses to nitric oxide and nitrosative stress: Growth, "omic," and physiological methods

The study of bacterial responses to nitric oxide (NO), nitrosating agents, and other agents of nitrosative stress has a short history but has rapidly produced important insights into the interactions of these agents with model microbial systems as well as pathogenic species. Several methodological problems arise in attempting to define the global responses to these agents, whether in simply measuring growth or performing "omic" experiments in which the objective is to determine the genome-wide (transcriptomic) or proteome-wide responses. The first problem is the relatively long timescale over which the experiments are conducted-minutes, hours, or days in the case of slow-growing cultures. The second problem is not unique to NO and its congeners but concerns the difficulties encountered when sensitive and comprehensive analytical techniques (such as transcriptomics) are applied to cultures whose growth and physiology are perturbed by an inhibitor. In essence, the problem is "seeing the wood for the trees." This chapter reviews briefly the state of knowledge of NO responses and mechanisms in bacteria, particularly Escherichia coli and Campylobacter jejuni. Continuous culture has several advantages for investigating the consequences of NO exposure, and this approach is outlined with examples of recent results and conclusions. The major advantage of the chemostat is establishment of a reproducible quasi-steady state in growth, in which the growth rate can be controlled and maintained. Contrary to common belief, neither the concept nor the apparatus is difficult. Commercially available and homemade systems are described with practical advice. Establishing continuous cultures paves the way for other "omic" approaches, particularly proteomics and metabolomics, which are not covered here, as their application to the field of NO biology is in its infancy. A key to the literature describing methods suitable for assessing toxicity to microbes of NO and reactive nitrogen species is given.