Abstract
L-arabinose inducible promoters are commonly used in gene expression analysis. However, nutrient source and availability also play a role in biofilm formation; therefore, L-arabinose metabolism could impact biofilm development. In this study we examined the impact of L-arabinose on Salmonella enterica serovar Typhimurium (S. Typhimurium) biofilm formation. Using mutants impaired for the transport and metabolism of L-arabinose, we showed that L-arabinose metabolism negatively impacts S. Typhimurium biofilm formation in vitro. When L-arabinose metabolism is abrogated, biofilm formation returned to baseline levels. However, without the ability to import extracellular L-arabinose, biofilm formation significantly increased. Using RNA-Seq we identified several gene families involved in these different phenotypes including curli expression, amino acid synthesis, and L-arabinose metabolism. Several individual candidate genes were tested for their involvement in the L-arabinose-mediated biofilm phenotypes, but most played no significant role. Interestingly, in the presence of L-arabinose the diguanylate cyclase gene adrA was downregulated in wild type S. Typhimurium. Meanwhile cyaA, encoding an adenylate cyclase, was downregulated in an L-arabinose transport mutant. Using an IPTG-inducible plasmid to deplete c-di-GMP via vieA expression, we were able to abolish the increased biofilm phenotype seen in the transport mutant. However, the mechanism by which the L-arabinose import mutant forms significantly larger biofilms remains to be determined. Regardless, these data suggest that L-arabinose metabolism influences intracellular c-di-GMP levels and therefore biofilm formation. These findings are important when considering the use of an L-arabinose inducible promoter in biofilm conditions.
Highlights
Typhimurium strains containing the L-arabinose inducible pBAD plasmid controlling genes of interest grown in a biofilm with or without the addition of 0.2% L-arabinose, we observed a dramatic decrease in biofilm formation in our control
Typhimurium regained biofilm growth similar to without L-arabinose, suggesting that the metabolism of L-arabinose is involved in the reduction in biofilm formation
These data suggested that while metabolism of L-arabinose via AraA resulted in decreased biofilm formation, the inability to import L-arabinose via arabinose import systems: high-affinity (AraE) increased biofilm formation
Summary
Typhimurium) can grow as either individual planktonic cells or as aggregates adhered to a surface known as a biofilm in response to environmental stimuli (Flemming and Wuertz, 2019). These cells are surrounded by a self-produced extracellular matrix (ECM) comprised of proteins, exopolysaccharides, and nucleic acids. The dinucleotide second messenger cyclic-di-GMP (c-diGMP) has been identified as one of the factors that controls ECM synthesis and adhesion (Caly et al, 2015), regulating the transition between the planktonic and biofilm lifestyles of many bacteria, including S. The diguanylate cyclase, encoded by adrA, synthesizes c-di-GMP which activates cellulose synthase (Zogaj et al, 2001)
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