DNA microarray and proteomic analyses of the RpoS regulon in Geobacter sulfurreducens

Abstract

The regulon of the sigma factor RpoS was defined in Geobacter sulfurreducens by using a combination of DNA microarray expression profiles and proteomics. An rpoS mutant was examined under steady-state conditions with acetate as an electron donor and fumarate as an electron acceptor and with additional transcriptional profiling using Fe(III) as an electron acceptor. Expression analysis revealed that RpoS acts as both a positive and negative regulator. Many of the RpoS-dependent genes determined play roles in energy metabolism, including the tricarboxylic acid cycle, signal transduction, transport, protein synthesis and degradation, and amino acid metabolism and transport. As expected, RpoS activated genes involved in oxidative stress resistance and adaptation to nutrient limitation. Transcription of the cytochrome c oxidase operon, necessary for G. sulfurreducens growth using oxygen as an electron acceptor, and expression of at least 13 c-type cytochromes, including one previously shown to participate in Fe(III) reduction (MacA), were RpoS dependent. Analysis of a subset of the rpoS mutant proteome indicated that 15 major protein species showed reproducible differences in abundance relative to those of the wild-type strain. Protein identification using mass spectrometry indicated that the expression of seven of these proteins correlated with the microarray data. Collectively, these results indicate that RpoS exerts global effects on G. sulfurreducens physiology and that RpoS is vital to G. sulfurreducens survival under conditions typically encountered in its native subsurface environments.

FIG. 1.

2DE patterns of soluble-fraction proteins isolated from the G. sulfurreducens DL1 wild-type strain (A) and its rpoS mutant derivative DLCN16 (B) grown with fumarate as electron acceptor. Protein spots showing significant quantitative differences between the two strains are indicated by arrows and numbers. The gel images are oriented with the isoelectric focusing dimension horizontal and the SDS-PAGE dimension vertical. The approximate positions of the SDS-PAGE molecular mass (MW) standards are presented along the vertical axis.

FIG. 2.

Global view of RpoS-regulated genes from fumarate-respiring cells. The genes were identified as either activated or repressed by RpoS with the use of the comparison of gene expression profiles in DL1 versus DLCN16 (ΔrpoS::Km) grown as described in Materials and Methods.

FIG. 3.

Growth of G. sulfurreducens on oxygen. Growth of strain DL1 (circles) and its mutant derivative rpoS (squares) following the introduction of 5% oxygen into the headspaces of cultures pregrown on 5 mM fumarate is shown. Arrows indicate the times at which 5% oxygen was added. Data shown are the means from two replicate cultures.