IDENTIFICATION AND CHARACTERIZATION OF A BACTEROIDES GENE, CSUF, WHICH ENCODES AN OUTER-MEMBRANE PROTEIN THAT IS ESSENTIAL FOR GROWTH ON CHONDROITIN SULFATE

Bacteroides thetaiotaomicron can utilize a variety of polysaccharides, including charged mucopolysaccharides such as chondroitin sulfate (CS ) and hyaluronic acid (HA). Since the enzymes (chondroitin lyases I an d II) that catalyze the first step in breakdown of CS and HA are locat ed in the periplasm, we had proposed that the first step in utilizatio n of these polysaccharides was binding to one or more outer membrane p roteins followed by translocation into the periplasm, but no such oute r membrane proteins had been shown to play a role in CS or HA utilizat ion. Previously we had isolated a transposon-generated mutant, CS4, wh ich was unable to grow an CS or HA but retained the ability to grow on disaccharide components of CS. This phenotype suggested that the muta tion in CS4 either blocked the transport of the mucopolysaccharides in to the periplasmic space or blocked the depolymerization of the mucopo lysaccharides into disaccharides. We have mapped the CS4 mutation to a single gene, csuF, which is capable of encoding a protein of 1,065 am ino acids and contains a consensus signal sequence. Although CsuF had a predicted molecular weight and pI similar to those of chondroitin ly ases, it did not show significant sequence similarity to the Bacteroid es chondroitin lyase II, a Proteus chondroitin ABC lyase, or two hyalu ronidases from Clostridium perfringens and Streptococcus pyogenes, nor was any CS-degrading enzyme activity associated with csuF expression in Bacteroides species or Escherichia coli. The deduced amino acid seq uence of CsuF exhibited features suggestive of an outer membrane prote in. We obtained antibodies to CsuF and demonstrated that the protein i s located in the outer membrane. This is the first evidence that a non enzymatic outer membrane protein is essential for utilization of CS an d HA.