Unintended Laboratory-Driven Evolution Reveals Genetic Requirements for Biofilm Formation by Desulfovibrio vulgaris Hildenborough

Abstract

Biofilms of sulfate-reducing bacteria (SRB) are of particular interest as members of this group are culprits in corrosion of industrial metal and concrete pipelines as well as being key players in subsurface metal cycling. Yet the mechanism of biofilm formation by these bacteria has not been determined. Here we show that two supposedly identical wild-type cultures of the SRB Desulfovibrio vulgaris Hildenborough maintained in different laboratories have diverged in biofilm formation. From genome resequencing and subsequent mutant analyses, we discovered that a single nucleotide change within DVU1017, the ABC transporter of a type I secretion system (T1SS), was sufficient to eliminate biofilm formation in D. vulgaris Hildenborough. Two T1SS cargo proteins were identified as likely biofilm structural proteins, and the presence of at least one (with either being sufficient) was shown to be required for biofilm formation. Antibodies specific to these biofilm structural proteins confirmed that DVU1017, and thus the T1SS, is essential for localization of these adhesion proteins on the cell surface. We propose that DVU1017 is a member of the lapB category of microbial surface proteins because of its phenotypic similarity to the adhesin export system described for biofilm formation in the environmental pseudomonads. These findings have led to the identification of two functions required for biofilm formation in D. vulgaris Hildenborough and focus attention on the importance of monitoring laboratory-driven evolution, as phenotypes as fundamental as biofilm formation can be altered.

An N-Terminal Retention Module Anchors the Giant Adhesin LapA of Pseudomonas fluorescens at the Cell Surface: a Novel Subfamily of Type I Secretion Systems

“…Although bacteria initiate surface contact and biofilm formation through a variety of strategies, many microbes need cell surface-associated protein adhesins to bind a surface. A family of giant type I secreted repeats-in-toxin (RTX)-containing adhesins have been shown to be critical for biofilm formation or surface binding by a variety of organisms, including Pseudomonas, Salmonella, Bordetella, Legionella, Vibrio, Shewanella, Desulfovibrio, and Marinomonas species (3)(4)(5)(6)(7)(8)(9)(10). Cell surface display of these adhesins, which promotes biofilm formation, typically coincides with high intracellular cyclic di-GMP (c-di-GMP) levels.…”

Novel Mode of Molybdate Inhibition of Desulfovibrio vulgaris Hildenborough

“…Plasmids ( Supplementary Table 1 ) were constructed by sequence and ligation independent cloning, SLIC ( Li and Elledge, 2007 ), with methods described previously ( De León et al, 2017 ). In short, the plasmids were designed to contain the pUC origin of replication, the spectinomycin-resistance gene ( aadAI ), a PCR amplicon of the region upstream of the gene(s) to be deleted (upstream), a PCR amplicon of the region downstream of the gene(s) to be deleted (downstream).…”

“…Sequencing of the molybdate-resistant culture was performed at the University of Missouri DNA Core facilities. Genomic DNA was isolated and prepared as described previously ( De León et al, 2017 ). Raw sequences were mapped to the D. vulgaris Hildenborough genome (NCBI reference accession no.…”

Large-scale Genetic Characterization of a Model Sulfate-Reducing Bacterium

“…Other differences in gene essentiality between JW710 and wild-type DvH in our study, or between gene essentiality in wild-type DvH in our study or in Fels et al (Fels et al, 2013), could be affected by sequence differences between these strains. Laboratory-acquired mutations in DvH can lead to large phenotypic differences between closely-related strains (De León et al, 2017). To identify genetic variants, we sequenced the JW710 genome and compared these data to our transposon mapping data for our wild-type strain (Materials and Methods).…”