Impact of L-arginine on Streptococcus gordonii gene expression and biofilm formation

Abstract

Streptococcus gordonii is an oral commensal bacterium, and an early coloniser of the acquired salivary pellicle that coats tooth surfaces. As such, it is a key organism in the establishment of dental plaque biofilms. The amino acid L-arginine has been previously shown to play a role in biofilm formation in other oral species, and depletion of L-arginine has a significant impact upon S. gordonii growth and gene regulation. Three L-arginine-dependent transcription regulators have been identified in S. gordonii, but it is currently not clear how these co-ordinate to sense and respond to changes in the exogenous L-arginine concentration. Therefore, the major aims of this work were to (i) further elucidate the impacts of L-arginine on S. gordonii growth and biofilm formation, (ii) investigate the roles of three putative arginine-dependent regulators in modulating arginine-responsive gene regulation, and (iii) assess the effects of L-arginine-dependent gene regulators on S. gordonii biofilm formation. Initial growth experiments revealed that high concentrations (≥500 mM) of L-arginine retard S. gordonii planktonic growth in a chemically defined medium, resulting in lower growth yields than intermediate (0.5 mM) L-arginine. However, 500 mM L-arginine was not toxic to S. gordonii cells incubated in natural human saliva. S. gordonii has previously been shown to be conditionally auxotrophic for L-arginine, since it can biosynthesise L-arginine under strictly anaerobic conditions or during the gradual depletion of extracellular L-arginine, but it cannot grow following a rapid shift to medium lacking L-arginine. A similar lack of growth was also found following rapid depletion of L-histidine and the branched-chain amino acids. S. gordonii is predicted to encode all genes required for L-histidine and branched chain amino acids, and it is possible that this organism is conditionally auxotrophic for multiple amino acids. Rapid depletion of L-arginine was shown previously to result in a change in expression of >20% of the S. gordonii genome. By comparing expression levels of some of the most strongly arginine-regulated genes when cells were challenged with depletion of L-histidine or branched chain amino acids, it was shown that some of the genes (for example, argC, SGO_1686, asp5) were specifically regulated by arginine depletion, whereas others (bfbF, SGO_1699) were similarly regulated following depletion of all amino acids. Therefore, it appears that depletion of L-arginine results in both an arginine-specific response and a more generalised stress response, presumably associated with growth arrest in this medium. iv Investigation of the roles of three arginine-dependent regulators (ArcR, ArgR and AhrC) by gene expression microarrays identified a number of genes that were arginine-responsive and were differentially-regulated in the wild-type compared with the isogenic mutants ΔarcR, ΔargR or ΔahrC. There was extensive overlap between the genes regulated by the ArgR and AhrC regulators, suggesting that these regulators perform similar and interdependent roles in S. gordonii. Regulatory responses following arcR disruption were distinct from those seen in the argR and ahrC mutants. In addition to three loci that have previously been described, one particular gene, SGO_0846, encoding a hypothetical protein, was highly up-regulated in response to arcR deletion. This thesis is the first holistic study of the three arginine-dependent regulators in S. gordonii, and shows that each one plays a key role in arginine-dependent gene regulation. Finally, previous unpublished work from our group had demonstrated that S. gordonii ΔarcR displayed a defective biofilm phenotype, whereas the deletion strains of the other two regulators showed no such phenotype. To determine whether up-regulation of SGO_0846 is responsible for the biofilm attenuation in S. gordonii ΔarcR, a deletion mutant of SGO_0846 was constructed in both the wild-type and ΔarcR background. Disruption of the SGO_0846 gene showed no significant differences in biofilm formation levels in comparison to the wild-type background, and showed no effect on the biofilm defective phenotype of the ΔarcR mutant. This suggests that changes in expression of SGO_0846 are not responsible for the biofilm defects seen in the ΔarcR knockout, and that the ArcR regulator is affecting biofilm formation via another mechanism. In conclusion, this thesis provides evidence that arginine has a clear impact on gene expression and biofilm formation in S. gordonii, and furthermore, that the ArcR regulator is critical for optimal biofilm formation. It is possible that in the future, this could be used as a target for controlling S. gordonii biofilm formation, and subsequent dental plaque development.