Screening of a Haloferax volcanii Transposon Library Reveals Novel Motility and Adhesion Mutants

Abstract

Archaea, like bacteria, use type IV pili to facilitate surface adhesion. Moreover, archaeal flagella—structures required for motility—share a common ancestry with type IV pili. While the characterization of archaeal homologs of bacterial type IV pilus biosynthesis components has revealed important aspects of flagellum and pilus biosynthesis and the mechanisms regulating motility and adhesion in archaea, many questions remain. Therefore, we screened a Haloferax volcanii transposon insertion library for motility mutants using motility plates and adhesion mutants, using an adapted air–liquid interface assay. Here, we identify 20 genes, previously unknown to affect motility or adhesion. These genes include potential novel regulatory genes that will help to unravel the mechanisms underpinning these processes. Both screens also identified distinct insertions within the genomic region lying between two chemotaxis genes, suggesting that chemotaxis not only plays a role in archaeal motility, but also in adhesion. Studying these genes, as well as hypothetical genes hvo_2512 and hvo_2876—also critical for both motility and adhesion—will likely elucidate how these two systems interact. Furthermore, this study underscores the usefulness of the transposon library to screen other archaeal cellular processes for specific phenotypic defects.

Molecular Factors of Hypochlorite Tolerance in the Hypersaline Archaeon Haloferax volcanii

“…H. volcanii H26 (a trpA + derivative of H295) was included as a control. The H. volcanii H295 transposon library was multiplied as previously described [ 18 ], stored in 20% glycerol at −80 °C, and thawed upon use. The cell mixture (10 μL) was diluted with 990 μL GMM (+uracil).…”

Haloferax volcanii immersed liquid biofilms develop independently of known biofilm machineries and exhibit rapid honeycomb pattern formation

“…Since a screen of an H. volcanii transposon insertion library for motility or adhesion-defective H. volcanii mutants revealed two mutant strains having insertions in the intergenic regions between chemotaxis genes cheB (hvo_1224) and cheW1 (hvo_1225) (42) and one mutant strain with a transposon insertion within cheF (hvo_1221) (data not shown) that have severe motility and adhesion defects, chemotaxis likely plays an important role in adhesion as a prerequisite to biofilm formation in H. volcanii. However, immersed liquid biofilm formation comparable to that of H. volcanii wild-type cultures was observed in the cheB::tn as well as cheF::tn mutant strains ( Table 2 and Fig.…”

“…Strains lacking the genes encoding the adhesion pilins, the prepilin peptidase, or components of the pilus biosynthesis pathway (∆pilA1-6, ∆pibD, and ∆pilB1C1 or ∆pilB3C3, respectively) are impaired in adhesion to coverslips at the ALI (36,(38)(39)(40)(41). While biofilm formation in H. volcanii presumably also requires the chemotaxis machinery, as transposon insertions between the cheB and cheW1 genes result in a mutant having an adhesion defect, H. volcanii biofilm formation is not impaired in a non-motile mutant lacking the archaellins arlA1 and arlA2 (38,42).…”

Archaeal cell surface biogenesis

“…In fact, screens for adhesion mutants in a transposon insertion library recently led to the identification of mutants having transposon insertions in chemotaxis genes, indicating that chemotaxis is important for the proper function of type IV pili (Legerme et al. 2016 ).…”