Unlocking the secrets of chitinase secretion

T o secrete proteins into their extra-cellular environment, gram-negative bacteria must transport them across both the inner and outer cell membranes , as well as the intervening peptido-glycan-rich periplasm. To date, researchers have identifi ed six different pathways for protein secretion (1). Some of them take secretory proteins directly from the cyto-plasm to the cell exterior, whereas others involve a two-legged crossing, with a brief layover in the periplasm en route. Hamilton et al. now uncover a seventh pathway, which the pathogenic bacterium Serratia marcescens uses to secrete enzymes that break down the polysaccharide chitin (2). S. marcescens, a major cause of health-care-acquired infections, secretes large amounts of three chitinases (ChiA, ChiB, and ChiC) and a chitin-binding protein, Cbp21. These proteins allow S. marcescens to use chitin as a food source, but they may also facilitate the bacterium's attachment to host cell membranes (3, 4). Frank Sargent, a biotechnologist from the University of Dundee in the UK, was interested in using S. marcescens chitinases to transform chitin, which is highly abundant throughout nature, into biofuel. First, however, he told graduate student Jaeger Hamilton to look up how the enzymes are secreted so that the whole chitinolytic machinery could be transferred into a more convenient host, such as E. coli. " He came back and said there was nothing known about how these chitinases were secreted, " Sargent recalls. " S. marcescens doesn't even have half of the secretion systems written about in the literature. " Hamilton et al. therefore carried out a genetic screen to identify S. marcescens mutants unable to break down chitin (2). The screen revealed that bacteria lacking a gene that the researchers named chiW were unable to release the chitinolytic machinery from the cell. The chitinases ChiA and ChiC, for example, got trapped in the periplasm in ChiW's absence. chiW encodes a member of the holin family of membrane proteins, which bacteriophages often express at high levels to form membrane pores and induce host cell lysis. Indeed, chiW is located in an operon containing several other genes encoding homologues of viral lysis proteins. Hamilton et al. found that one of these proteins, an l-alanyl-d-glutamate endopeptidase called ChiX, was also required for chitinase secretion. Sargent and colleagues hypothesized that, as an inner membrane holin-like protein, ChiW might translocate ChiX to the periplasm, allowing the endopeptidase to clear a path through the peptido-glycan layer for the chitin-olytic machinery …