A c-type cytochrome and a transcriptional regulator responsible for enhanced extracellular electron transfer in Geobacter sulfurreducens revealed by adaptive evolution.
The stimulation of subsurface microbial metabolism often associated with engineered bioremediation of groundwater contaminants presents subsurface microorganisms, which are adapted for slow growth and metabolism in the subsurface, with new selective pressures. In order to better understand how Geobacter species might adapt to selective pressure for faster metal reduction in the subsurface, Geobacter sulfurreducens was put under selective pressure for rapid Fe(III) oxide reduction. The genomes of two resultant strains with rates of Fe(III) oxide reduction that were 10-fold higher than those of the parent strain were resequenced. Both strains contain either a single base-pair change or a 1 nucleotide insertion in a GEMM riboswitch upstream of GSU1761, a gene coding for the periplasmic c-type cytochrome designated PgcA. GSU1771, a gene coding for a SARP regulator, was also mutated in both strains. Introduction of either of the GEMM riboswitch mutations upstream of pgcA in the wild-type increased the abundance of pgcA transcripts, consistent with increased expression of pgcA in the adapted strains. One of the mutations doubled the rate of Fe(III) oxide reduction. Interruption of GSU1771 doubled the Fe(III) oxide reduction rate. This was associated with an increased in expression of pilA, the gene encoding the structural protein for the pili thought to function as microbial nanowires. The combination of the GSU1771 interruption with either of the pgcA mutations resulted in a strain that reduced Fe(III) as fast as the comparable adapted strain. These results suggest that the accumulation of a small number of beneficial mutations under selective pressure, similar to that potentially present during bioremediation, can greatly enhance the capacity for Fe(III) oxide reduction in G. sulfurreducens. Furthermore, the results emphasize the importance of the c-type cytochrome PgcA and pili in Fe(III) oxide reduction and demonstrate how adaptive evolution studies can aid in the elucidation of complex mechanisms, such as extracellular electron transfer.
Tremblay
Pier-Luc
PL
Department of Microbiology, University of Massachusetts, Amherst, MA, USACenter for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC, USADepartment of Bioengineering, University of California, San Diego, La Jolla, CA, USA.
Summers
Zarath M
ZM
Department of Microbiology, University of Massachusetts, Amherst, MA, USACenter for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC, USADepartment of Bioengineering, University of California, San Diego, La Jolla, CA, USA.
Glaven
Richard H
RH
Department of Microbiology, University of Massachusetts, Amherst, MA, USACenter for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC, USADepartment of Bioengineering, University of California, San Diego, La Jolla, CA, USA.
Nevin
Kelly P
KP
Department of Microbiology, University of Massachusetts, Amherst, MA, USACenter for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC, USADepartment of Bioengineering, University of California, San Diego, La Jolla, CA, USA.
Zengler
Karsten
K
Department of Microbiology, University of Massachusetts, Amherst, MA, USACenter for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC, USADepartment of Bioengineering, University of California, San Diego, La Jolla, CA, USA.
Barrett
Christian L
CL
Department of Microbiology, University of Massachusetts, Amherst, MA, USACenter for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC, USADepartment of Bioengineering, University of California, San Diego, La Jolla, CA, USA.
Qiu
Yu
Y
Department of Microbiology, University of Massachusetts, Amherst, MA, USACenter for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC, USADepartment of Bioengineering, University of California, San Diego, La Jolla, CA, USA.
Palsson
Bernhard O
BO
Department of Microbiology, University of Massachusetts, Amherst, MA, USACenter for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC, USADepartment of Bioengineering, University of California, San Diego, La Jolla, CA, USA.
Lovley
Derek R
DR
Department of Microbiology, University of Massachusetts, Amherst, MA, USACenter for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC, USADepartment of Bioengineering, University of California, San Diego, La Jolla, CA, USA.
D000222:Adaptation, Physiological / Q000235:genetics*; D001673:Biodegradation, Environmental; D003574:Cytochrome c Group / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics; D004579:Electron Transport; D019143:Evolution, Molecular; D005290:Ferric Compounds / Q000378:metabolism*; D020869:Gene Expression Profiling; D005798:Genes, Bacterial; D016680:Genome, Bacterial; D045302:Geobacter / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D016254:Mutagenesis, Insertional; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D010084:Oxidation-Reduction; D058928:Riboswitch; D017422:Sequence Analysis, DNA
D003574:Cytochrome c Group; D004269:DNA, Bacterial; D005290:Ferric Compounds; D058928:Riboswitch; C000499:ferric oxide