The formation of bacterial biofilms requires an extracellular matrix to facilitate adherence of bacteria to the surface they colonize. Carbohydrate polymers, known as exopolysaccharides, form a key component of most biofilm matrices. A wide variety of medically-important biofilm forming bacterial strains, including S. epidermidis, S. aureus, E. coli, B. pertussis, and Y. pestis generate the same β-1,6-N-acetyl glucosamine (PNAG) homopolymer as a key biofilm matrix exopolysaccharide. In E. coli, as well as in the other bacterial strains, the PNAG undergoes partial enzymatic de-N-acetylation, which is essential for surface attachment and subsequent biofilm formation. In vivo studies implied that the enzyme responsible for carrying out de-N-acetylation in E. coli is PgaB, an enzyme with sequence homologues in many Gram negative species capable of forming biofilms. In this work, the first biochemical characterization of PgaB is presented. We confirmed the activity of PgaB on β-1,6-GlcNAc oligosaccharides. The activity of PgaB is specific for the β-1,6 linkage and no de-N-acetylation of β-1,4-GlcNAc oligosaccharides was detected. Enzyme activity is dependent on the degree of substrate polymerization, as the second order rate constant for pentasaccharide substrate was determined to be four times higher than that of the tetrasaccharide substrate. Oligosaccharide sequencing studies indicate that there may be a pattern in the de-N-acetylation of substrates by PgaB. The central residue is modified in mono-de-N-acetylated pentasaccharide substrate, while di-de-N-acetylated hexasaccharide substrate shows modification mainly at the third and fifth residues from the non-reducing terminus of the substrate. Activity studies revealed that PgaB is activated by Ni2+ as well as by Fe2+, which is uncommon for deacetylase enzymes. Metal coordination to active site residues His184 and His189 was confirmed by mutagenesis studies, which also indicated that the metal likely plays a catalytic role. The results of these metal dependence studies support the observed binding of nickel and iron to the active site in PgaB crystal structures. The characterization studies presented in this thesis allow us to gain a better understanding of the de-N-acetylation aspect of the PNAG biosynthetic process and will serve as a basis for enzyme inhibitor design.

The formation of bacterial biofilms requires an extracellular matrix to facilitate adherence of bacteria to the surface they colonize. Carbohydrate polymers, known as exopolysaccharides, form a key component of most biofilm matrices. A wide variety of medically-important biofilm forming bacterial strains, including S. epidermidis, S. aureus, E. coli, B. pertussis, and Y. pestis generate the same β-1,6-N-acetyl glucosamine (PNAG) homopolymer as a key biofilm matrix exopolysaccharide. In E. coli, as well as in the other bacterial strains, the PNAG undergoes partial enzymatic de-N-acetylation, which is essential for surface attachment and subsequent biofilm formation. In vivo studies implied that the enzyme responsible for carrying out de-N-acetylation in E. coli is PgaB, an enzyme with sequence homologues in many Gram negative species capable of forming biofilms. In this work, the first biochemical characterization of PgaB is presented. We confirmed the activity of PgaB on β-1,6-GlcNAc oligosaccharides. The activity of PgaB is specific for the β-1,6 linkage and no de-N-acetylation of β-1,4-GlcNAc oligosaccharides was detected. Enzyme activity is dependent on the degree of substrate polymerization, as the second order rate constant for pentasaccharide substrate was determined to be four times higher than that of the tetrasaccharide substrate. Oligosaccharide sequencing studies indicate that there may be a pattern in the de-N-acetylation of substrates by PgaB. The central residue is modified in mono-de-N-acetylated pentasaccharide substrate, while di-de-N-acetylated hexasaccharide substrate shows modification mainly at the third and fifth residues from the non-reducing terminus of the substrate. Activity studies revealed that PgaB is activated by Ni2+ as well as by Fe2+, which is uncommon for deacetylase enzymes. Metal coordination to active site residues His184 and His189 was confirmed by mutagenesis studies, which also indicated that the metal likely plays a catalytic role. The results of these metal dependence studies support the observed binding of nickel and iron to the active site in PgaB crystal structures. The characterization studies presented in this thesis allow us to gain a better understanding of the de-N-acetylation aspect of the PNAG biosynthetic process and will serve as a basis for enzyme inhibitor design.

Poly-N-acetylglucosamine (PNAG) is an important Staphylococcus aureus virulence factor. It is a major component of the extracellular polymeric matrix in biofilms, and contributes to resistance to the innate immune response. The proteins encoded in the icaADBC (intercellular adhesin) operon are responsible for PNAG production. Here, we present evidence of a new mechanism of phase variation for switching off PNAG production that involves slipped strand mutagenesis, along with further insight into the regulation and expression of the icaADBC operon. S. aureus strain MN8m (mucoid) is a spontaneous mutant of strain MN8 (non-mucoid). It is characterized by constitutive overproduction of PNAG, resulting from a 5 bp deletion in the icaADBC promoter region. First, we explored the effect of this mutation on the binding of the positive regulator SarA and its regulation of icaADBC in MN8m; and demonstrated that while binding is not inhibited by the MN8m promoter mutation, it appears that SarA exhibits a negative influence on transcriptional activation in this strain background. Second, we found that non-mucoid mutants were frequently detected in MN8m cultures. The PNAG mutants still exhibited elevated icaADBC transcript levels and the 5 bp deletion was still present, but they were PNAG-negative. The most frequent mutation responsible for the phenotype was a slipped strand mispairing in the icaC gene. We found that, in vitro, PNAG-negative mutants had a growth advantage and increased fitness relative to the mucoid, PNAG-overproducing strain. Therefore, the slipped strand mutants in the PNAG-off state had a competitive growth advantage and eventually predominated the culture. Reversion back to mucoid occurred but revertants were isolated infrequently, due to their compromised fitness. The region of slipped strand mispairing was found to vary in several clinical S. aureus isolates, indicating in vivo relevance of this new mechanism. Third, a non-mucoid variant was isolated with the same phenotype and growth properties that instead had a nonsense mutation in icaB, which has demonstrated that there is a polar translational relationship between icaB and icaC. Together, these studies have given us insight into the regulation and expression of icaADBC expression and have revealed a growth disadvantage associated with PNAG overproduction.