Exploitation of a “hockey-puck” phenotype to identify pilus and biofilm regulators in Serratia marcescens through genetic analysis

Abstract

Pili are essential adhesive determinants for many bacterial pathogens. A suppressor mutation screen that takes advantage of a pilus-mediated self-aggregative “hockey-puck” colony phenotype was designed to identify novel regulators of type I pili in Serratia marcescens. Mutations that decreased pilus biosynthesis mapped to the fimABCD operon; to the genes alaT, fkpA, and oxyR; upstream of the flagellar master regulator operon flhDC; and to an uncharacterized gene encoding a predicted DUF1401 domain. biofilm formation and pilus-dependent agglutination assays were used to characterize the relative importance of the identified genes in pilus biosynthesis. Additional mutagenic or complementation analysis was used to verify the role of candidate genes in pilus biosynthesis. Presented data support a model that CRP negatively regulates pilus biosynthesis through increased expression of flhDC and decreased expression of oxyR. Further studies are warranted to determine the mechanism by which these genes mediate pilus biosynthesis or function.

Repurposing anti-diabetic drug “Sitagliptin” as a novel virulence attenuating agent in Serratia marcescens

“…Fimbria or pili are adhesive organelles of bacteria and are important in establishment of infections. S. marcescens pilus production has been reported and linked to biofilm formation [37]. Genetic analysis showed that the type I pili which encoded by the operon fimABCD are essential for S. marcescens biofilm formation in early stages [38].…”

Serralysin family metalloproteases protects Serratia marcescens from predation by the predatory bacteria Micavibrio aeruginosavorus

“…Deletion of fimbria, which plays a role in cell-cell and cell-surface association 23 – 25 , did not impact predation. Thus, it could be concluded that fimbria does not serve as an attachment site nor could it interfere in the ability of the predator to reach any putative prey binding sites.…”

“… Strain Description Source or reference WT-K904 Clinical contact lens-associated keratitis strain 49 K904 fliC ::pMQ192 K904- Flagella mutant with fliC insertion mutation. 25 K904 fimC ::pMQ167 K904- Fimbriae mutant with fimC insertion mutation. 25 K904 phlA ::pMQ215 K904- Phospholipase-A with phlA insertion mutation.…”

“… 25 K904 fimC ::pMQ167 K904- Fimbriae mutant with fimC insertion mutation. 25 K904 phlA ::pMQ215 K904- Phospholipase-A with phlA insertion mutation. 50 K904 ∆ slaA K904- S-layer mutant with slaA deletion mutation.…”

“…To measure fimbriae production by both the wild type and triple metalloprotease mutant (∆ prtS ∆ slpB slpE ), yeast agglutination was measured as previously described 25 . Bacteria were grown overnight in LB and resuspended in HEPES.…”