Molecular analysis of the in situ growth rates of subsurface Geobacter species

Abstract

Molecular tools that can provide an estimate of the in situ growth rate of Geobacter species could improve understanding of dissimilatory metal reduction in a diversity of environments. Whole-genome microarray analyses of a subsurface isolate of Geobacter uraniireducens, grown under a variety of conditions, identified a number of genes that are differentially expressed at different specific growth rates. Expression of two genes encoding ribosomal proteins, rpsC and rplL, was further evaluated with quantitative reverse transcription-PCR (qRT-PCR) in cells with doubling times ranging from 6.56 h to 89.28 h. Transcript abundance of rpsC correlated best (r(2) = 0.90) with specific growth rates. Therefore, expression patterns of rpsC were used to estimate specific growth rates of Geobacter species during an in situ uranium bioremediation field experiment in which acetate was added to the groundwater to promote dissimilatory metal reduction. Initially, increased availability of acetate in the groundwater resulted in higher expression of Geobacter rpsC, and the increase in the number of Geobacter cells estimated with fluorescent in situ hybridization compared well with specific growth rates estimated from levels of in situ rpsC expression. However, in later phases, cell number increases were substantially lower than predicted from rpsC transcript abundance. This change coincided with a bloom of protozoa and increased attachment of Geobacter species to solid phases. These results suggest that monitoring rpsC expression may better reflect the actual rate that Geobacter species are metabolizing and growing during in situ uranium bioremediation than changes in cell abundance.

Fig 1

Microarray results from experiments comparing three different experimental conditions (G. uraniireducens cells grown with Rifle sediment, Fe(III) oxide, or Mn(IV) oxide provided as the electron acceptor) to the control condition (G. uraniireducens cells grown with fumarate provided as the electron acceptor) (P value cutoff of ≤0.01). Transcript levels for both rpsC and rplL were significantly downregulated in all three microarray experiments (indicated by negative fold change in the figure). Triplicate biological and duplicate technical replicates were done for all three microarray experiments.

Fig 2

The number of rpsC (A) and rplL (B) mRNA transcripts normalized against the number of proC mRNA transcripts expressed by G. uraniireducens at a wide range of growth rates. Triplicate biological and technical replicates were done for all specific growth rates.

Fig 3

(A) The number of G. uraniireducens cells in cultures with acetate (5 mM) provided as the electron donor and fumarate (40 mM) provided as the electron acceptor under nitrogen-limiting (no NH₄⁺) or non-nitrogen-limiting (4.67 mM NH₄⁺) conditions; (B) the number of rpsC mRNA transcripts normalized against the number of proC mRNA transcripts expressed by G. uraniireducens cells under nitrogen-limiting or non-nitrogen-limiting conditions. Triplicate biological and technical replicates were done for all samples.

Fig 4

Growth of Geobacteraceae in response to acetate availability during the uranium bioremediation field experiment conducted at Rifle in 2010. (A) Acetate and Fe(II) concentrations in groundwater collected during uranium bioremediation; (B) number of Geobacteraceae in the groundwater estimated by direct counts of cells labeled with Geobacteraceae-specific FISH probes and total bacterial cells estimated with general DAPI staining; (C) proportion of Geobacteraceae in the groundwater estimated by comparing direct counts of cells labeled with Geobacteraceae-specific FISH probes with general DAPI staining of all cells versus proportion of Geobacteraceae found in 16S rRNA gene clone libraries; (D) number of Geobacteraceae rpsC mRNA transcripts normalized against the number of proC mRNA transcripts plotted against the number of Geobacteraceae cells/ml estimated by FISH in the groundwater. Each point is an average of triplicate determinations, and error bars represent standard deviations. Growth rate constants (μ) were calculated from the following equation: y = 511x − 5.3.