Capsular and holdfast extracellular polymeric substances of Hyphomonas strain VP-6 mediate adhesion to solid substrata

The formation of biofilms is a serious problem for the medical, sanitation and shipping industries and the U.S. Navy and Coast Guard, causing billions of dollars worth of damage worldwide each year. The formation of biofilms begins with the attachment of bacteria to a solid surface and continues as new microorganisms settle, replicate and secrete viscous exopolymers. The process by which primary fouling bacteria attach to surfaces is incompletely understood. Two strains of the genus Hyphomonas, VP-6 and MHS-3, were used as model organisms to investigate this process. Specific chemical and biological probes including lectins, dyes and monoclonal antibodies directed toward the extracellular polymeric substances (EPS) of both strains revealed that they secreted EPS capsules to adhere to surfaces. Both strains adhered to hydrophilic and hydrophobic surfaces, as well as those precoated with proteins. Hyphomonas VP-6 synthesized two separate adhesive EPS; one that is produced polarly and temporally and a second that surrounds the entire cell at all morphogenic stages. Scanning electron microscopy revealed a direct correlation between the location of EPS production on both strains and the orientation of cell adhesion to surfaces. VP-6 used polar EPS, termed holdfast, for initial adhesion to surfaces and the capsular polysaccharide for permanent adhesion. Analysis of the capsular EPS has revealed that it is a negatively charged polymer with a molecular weight of {dollar}sim{dollar}550 kDa. Lectins specific for holdfast and capsular EPS revealed that both polysaccharides possess N-acetylated amino sugars. A spontaneous, non-biofilm forming mutant of Hyphomonas VP-6 termed reduced adhesion (RAD) initially adhered to surfaces like the wild type but formed biofilms of decreased mass. Thus, RAD mutants are capable of initial adhesion but are deficient in the accumulation phase of biofilm formation. Monoclonal antibodies to a VP-6 capsular EPS-associated protein, found in WT but not RAD strains, blocked adhesion. It is concluded that EPS is essential for attachment of both VP-6 and MHS-3 and that VP-6 also synthesizes an EPS associated protein for EPS cohesion and consequently, biofilm accretion.