Transcriptomic and proteomic analyses of Desulfovibrio vulgaris biofilms: carbon and energy flow contribute to the distinct biofilm growth state

Abstract

Background: Desulfovibrio vulgaris Hildenborough is a sulfate-reducing bacterium (SRB) that is intensively studied in the context of metal corrosion and heavy-metal bioremediation, and SRB populations are commonly observed in pipe and subsurface environments as surface-associated populations. In order to elucidate physiological changes associated with biofilm growth at both the transcript and protein level, transcriptomic and proteomic analyses were done on mature biofilm cells and compared to both batch and reactor planktonic populations. The biofilms were cultivated with lactate and sulfate in a continuously fed biofilm reactor, and compared to both batch and reactor planktonic populations.

Results: The functional genomic analysis demonstrated that biofilm cells were different compared to planktonic cells, and the majority of altered abundances for genes and proteins were annotated as hypothetical (unknown function), energy conservation, amino acid metabolism, and signal transduction. Genes and proteins that showed similar trends in detected levels were particularly involved in energy conservation such as increases in an annotated ech hydrogenase, formate dehydrogenase, pyruvate:ferredoxin oxidoreductase, and rnf oxidoreductase, and the biofilm cells had elevated formate dehydrogenase activity. Several other hydrogenases and formate dehydrogenases also showed an increased protein level, while decreased transcript and protein levels were observed for putative coo hydrogenase as well as a lactate permease and hyp hydrogenases for biofilm cells. Genes annotated for amino acid synthesis and nitrogen utilization were also predominant changers within the biofilm state. Ribosomal transcripts and proteins were notably decreased within the biofilm cells compared to exponential-phase cells but were not as low as levels observed in planktonic, stationary-phase cells. Several putative, extracellular proteins (DVU1012, 1545) were also detected in the extracellular fraction from biofilm cells.

Conclusions: Even though both the planktonic and biofilm cells were oxidizing lactate and reducing sulfate, the biofilm cells were physiologically distinct compared to planktonic growth states due to altered abundances of genes/proteins involved in carbon/energy flow and extracellular structures. In addition, average expression values for multiple rRNA transcripts and respiratory activity measurements indicated that biofilm cells were metabolically more similar to exponential-phase cells although biofilm cells are structured differently. The characterization of physiological advantages and constraints of the biofilm growth state for sulfate-reducing bacteria will provide insight into bioremediation applications as well as microbially-induced metal corrosion.

Figure 1

Protein (●,○) and carbohydrate (■,□) levels for biofilm and planktonic cells, respectively from continuous culture with lactate and sulfate.

Figure 2

Hierarchal clustering (a) and principal components analysis (b) of the nine samples from planktonic and biofilm (T2-T4 represent exponential-phase and T5-T9 represent stationary-phase) grown on lactate and sulfate for Desulfovibrio vulgaris. The samples are grouped based upon similarities in the expression patterns of all genes with significant changes.

Figure 3

Correlation matrix for D. vulgaris gene expression that compares biofilm cells to planktonic cells grown with different substrates using a centered Pearson correlation. Cells were grown in a defined S4D medium with sulfate (except for ‘pyruvate only’), and provided with a different carbon and energy source (lactate, pyruvate, hydrogen, or formate). The correlation matrix was generated with MicrobesOnline functional genomics analysis (microbesonline.org).

Figure 4

Cell-wide depiction of significantly up-expressed transcripts for mature D. vulgaris biofilms compared to both batch and chemostat planktonic cells. The following genes were also detected as significantly up-expressed proteins: SodB, KatA, Hsp20 (DVU2441), Hsp20 (DVU2442), UspA (DVU0423).

Figure 5

Transcript levels for 15 ribosomal proteins compared among four (expontial, transition, stationary, and biofilm) different growth stages. The genes are sorted highest to lowest with respect to the levels observed for exponential-phase cells.

Figure 6

Conceptual model (a) of the transcriptional responses in energy metabolism and pyruvate-acetate metabolic node for a mature D. vulgaris biofilm (red is up-expressed and blue is down-expressed) compared to planktonic cells.

Figure 7

Expression levels of representative genes involved in carbon and energy flow that displayed altered expression distinct to biofilm cells when compared to reactor planktonic cells, batch exponential-phase, or batch stationary-phase.

Figure 8

Graphical representation of the biofilm to planktonic comparison via log₂transcript ratios. As an example, Ech hydrogenase genes and cytochrome c-553 components had increased expression in biofilm cells and tryptophan biosynthesis genes were down expressed.