Overcoming the anaerobic hurdle in phenotypic microarrays: Generation and visualization of growth curve data for Desulfovibrio vulgaris Hildenborough

Abstract

1Growing anaerobic microorganisms in phenotypic microarrays (PM) and 96-well microtiter 2 plates is an emerging technology that allows a high throughput survey of the growth and 3 physiology and/or phenotype of cultivable microorganisms. For non-model bacteria, a swift 4 method for phenotypic analysis is invaluable, not only to serve as a starting point for further 5 evaluation, but also to provide a broad understanding of the physiology of an uncharacterized 6 wild-type organism or the physiology/phenotype of a newly created mutant of that organism. 7Given recent advances in genetic characterization and targeted mutations to elucidate genetic 8 networks and metabolic pathways, high-throughput methods for determining phenotypic 9 differences are essential. Here we outline challenges presented in studying the physiology 10 and phenotype of a sulfate-reducing anaerobic delta proteobacterium, Desulfovibrio vulgaris 11Hildenborough. Modifications of the commercially available OmniLog TM system (Hayward, 12 CA) for experimental setup, and configuration, as well as considerations in PM data analysis 13 are presented. Also highlighted here is data viewing software that enables users to view and 14 compare multiple PM data sets. The PM method promises to be a valuable strategy in our 15 systems biology approach to D. vulgaris studies and is readily applicable to other anaerobic 16 and aerobic bacteria. 17 18 Introduction 1Desulfovibrio vulgaris Hildenborough, a sulfate-reducing anaerobic, delta 2 proteobacterium, has been identified as a model organism in many types of sulfate-reducing 3 environments, especially those related to metal-contaminated sites (Caumette 1993; Telang, 4 Voordouw et al. 1994;Noguera, Brusseau et al. 1998;Cottrell and Cary 1999; Wind, Stubner 5 et al. 1999; Heidelberg, Seshadri et al. 2004). Detailed studies on growth and metabolism of 6 this organism are necessary to understand metal reduction processes under a variety of 7 environmental conditions. Conducting a growth curve to determine lag time, growth rate, and 8 maximum cell densities often is necessary to understand the physiology or phenotype and the 9 specific effect of environmental stressors on the organism. Culturing in batch cultures has the 10 disadvantage of large amounts of media with single or multiple component differences, of 11 large numbers of tubes or flasks, and of the inconvenience of manually monitoring optical 12 densities of large numbers of cultures. In addition, growth times often are difficult to predict 13 and key experimental data may be lost if sampling is not sufficiently frequent (Sani, Peyton et 14 al. 2003). Increased convenience and quantity of the data can be obtained by automation of 15 the growth curve measurements in 96-well microtiter plates (hereafter referred to as plates). 16After preparation, plates can be incubated and growth monitored by recording opacity changes 17 by automation at discrete intervals, continuously over several days. Microtiter plate readers can rapidly measure tur...

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