Genome-wide analysis of the RpoN regulon in Geobacter sulfurreducens

Abstract

Background: The role of the RNA polymerase sigma factor RpoN in regulation of gene expression in Geobacter sulfurreducens was investigated to better understand transcriptional regulatory networks as part of an effort to develop regulatory modules for genome-scale in silico models, which can predict the physiological responses of Geobacter species during groundwater bioremediation or electricity production.

Results: An rpoN deletion mutant could not be obtained under all conditions tested. In order to investigate the regulon of the G. sulfurreducens RpoN, an RpoN over-expression strain was made in which an extra copy of the rpoN gene was under the control of a taclac promoter. Combining both the microarray transcriptome analysis and the computational prediction revealed that the G. sulfurreducens RpoN controls genes involved in a wide range of cellular functions. Most importantly, RpoN controls the expression of the dcuB gene encoding the fumarate/succinate exchanger, which is essential for cell growth with fumarate as the terminal electron acceptor in G. sulfurreducens. RpoN also controls genes, which encode enzymes for both pathways of ammonia assimilation that is predicted to be essential under all growth conditions in G. sulfurreducens. Other genes that were identified as part of the RpoN regulon using either the computational prediction or the microarray transcriptome analysis included genes involved in flagella biosynthesis, pili biosynthesis and genes involved in central metabolism enzymes and cytochromes involved in extracellular electron transfer to Fe(III), which are known to be important for growth in subsurface environment or electricity production in microbial fuel cells. The consensus sequence for the predicted RpoN-regulated promoter elements is TTGGCACGGTTTTTGCT.

Conclusion: The G. sulfurreducens RpoN is an essential sigma factor and a global regulator involved in a complex transcriptional network controlling a variety of cellular processes.

Figure 1

The rpoN gene cluster and the mutation schemes. (a) Genes surrounding rpoN are shown as open arrows. HP: conserved hypothetical protein with unknown function; YhbG: ABC transporter, ATP binding protein; YfiA: ribosomal subunit interface-associated sigma-54 modulation protein; HprK: Hpr(Ser) kinase/phosphorylase. Insertion of a kanamycin resistance cassette upstream or downstream of the intergenic region of the rpoN gene resulted in viable mutants (a). (b) Scheme showing attempts of construction of deletion of (i) the 5'-end, (ii) the whole, or (iii) the 3'-end of the rpoN coding region. (c) An extra copy of the rpoN gene was inserted on the chromosome and was under the control of the chloramphenicol resistance cassette promoter. (d) An extra copy of the rpoN gene was introduced in trans under the control of a lac promoter (constitutively expressed) or a taclac promoter (IPTG-inducible). The position of insertion of the antibiotic resistance cassette (kanamycin, Kan or gentamycin, Gm) is indicated with an inverted triangle and a vertical bar. The regions which were attempted to replace with the antibiotic resistance cassette insertion are indicated by dashed line.

Figure 2

RpoN expression. (a) RpoN expression under different growth conditions. 1: NBAF; 2: NBH₂F; 3: NBLF; 4: ammonium-free NBAF; 5: FWAFC; 6: FWH₂FC; 7: FWLFC; 8: FWAF; 9: FWH₂F; 10: FWLF. Media abbreviations were detailed in Methods. (b) RpoN over-expression. Total protein (5 μg) was separated by 10% SDS-PAGE and analyzed by Western blot analysis with the RpoN-specific antiserum. Two biological samples were shown for IPTG-induced WT^V and RpoN⁺ strains. IPTG was added at final concentration 1 mM.

Figure 3

Characterization of the RpoN over-expression strain. Cell growth with fumarate as an electron acceptor was monitored by absorbance at 600 nm (a)(b). (a) acetate as the electron donor and fumarate as the electron acceptor (NBAF medium); (b): ammonia-free NBAF. Growth with Fe(III) as an electron acceptor was monitored by Fe(II) production (c) as well as cell numbers (d). Filled square: the WT^V strain without IPTG; Empty square: the WT^V strain with IPTG. Filled circle: the RpoN⁺ strain without IPTG; empty circle: the RpoN⁺ strain with IPTG. (a)-(d): Data are means ± standard deviations of triplicates. The production of pili was measured by agglutination assays (e). Data are means ± standard deviation of triplicates from two independent experiments (e).

Figure 4

RpoN-dependent gene expression. Representative genes, (a) GSU1836 and GSU2751 (up-regulated in the RpoN⁺ strain), and (b) GSU0420, GSU0777, GSU2806 and GSU3046 (down-regulated in the RpoN⁺ strain) identified by the microarray analysis were further analyzed by primer extension assays. The results of the primer extension assays and their promoter regions are shown. The 5' ends of mRNA are indicated by asterisks. The putative -24/-12 elements and RBS are underlined. Translation start codons are shown in bold and are indicated by Met.

Figure 5

G. sulfurreducens RpoN-dependent promoter elements. (a) Alignment of G. sulfurreducens RpoN-dependent promoters identified by primer extension assays in this study. Conserved nucleotides which are the same to the consensus sequences from (b) are labeled in red. (b) Sequence logo of 110 G. sulfurreducens RpoN-regulated promoters predicted by PromScan in non-coding regions upstream of target protein-coding genes.