Vol. 176, No. 22 JOURNAL OF BACTERIOLOGY, Nov. 1994, p. 6819-6826 0021-9193/94/$04.00+0 Copyright © 1994, American Society for Microbiology Isolation and Characterization of a Second exe Operon Required for Extracellular Protein Secretion in Aeromonas hydrophila RAVINDRA JAHAGIRDAR AND S. PETER HOWARD* University of Regina, Regina, Saskatchewan, Canada S4S OA2 Received 18 July 1994/Returned for modification 26 August 1994/Accepted 12 September 1994 Strain C5.84 is a TnS-751 insertion mutant of Aeromonas hydrophila which is unable to secrete extracellular proteins, instead accumulating them in the periplasm (B. Jiang and S. P. Howard, J. Bacteriol. 173:1241-1249, 1991). A 3.5-kb Bglll fragment which complements this mutation was isolated from the chromosome of the parent strain. Analysis of this fragment revealed an operon-like structure with two complete genes, exeA and exeB, a functional promoter 5' to the exeA gene, and a 13-bp inverted repeat immediately 3' to the exeB gene. Although the transposon had inserted in exeA, provision of a wild-type copy of this gene alone in trans did not restore competence for export to C5.84. Complementation required the presence of both exeA and exeB, and marker exchange mutagenesis confirmed the requirement for both gene products for secretion. In vitro expression as well as analysis of the deduced amino acid sequence of ExeA indicated that it is a hydrophilic 60-kDa protein with a consensus ATP binding site. ExeB is a 25-kDa basic protein which shares limited homology with PulB, a protein of unknown function associated with the maltose regulon of Kiebsiella oxytoca, and OutB, a protein which has been shown to be required for efficient secretion in Erwinia chrysanthemi. The hydrophilic character of these proteins and preliminary localization studies suggested that they are anchored to the inner membrane. These results demonstrate the involvement of a second operon encoding a putative ATP-binding protein in the secretion of extracellular proteins from gram-negative bacteria and further suggest that the cytoplasmic compartment may play a greater role in protein translocation across the outer membrane from the periplasm than previously thought. A number of distinct modes of extracellular protein secre- tion exist in gram-negative bacteria (24, 29). One of the better-understood pathways involves secretion of proteins across both inner and outer membranes in a single step, possibly through membrane adhesion sites, thereby bypassing the periplasm, as exemplified by the hemolysin of Escherichia coli (9). It is more common, however, for secretion to involve translocation first across the inner membrane and then, in a separate step, across the outer membrane. Numerous genetic and biochemical studies have characterized the translocation of proteins across the inner membrane; this complex machin- ery is termed the Sec-dependent or general export pathway (reviewed in reference 24). The general export pathway con- sists of the translocase SecA, SecY, SecE, SecD, and SecF proteins and the chaperone SecB (41). More recently, secre- tion across the outer membrane has become the subject of intensive study and has been shown in a number of cases to involve a host of additional proteins comprising a secretion machinery which is known as the general secretion pathway. The components of this pathway have been best characterized in Kiebsiella oxytoca, in which they are involved in the secretion of pullulanase, and the pathway has also been identified in a growing number of other families of gram-negative bacteria (24, 25). Since the proteins exported by this pathway contain typical amino-terminal signal sequences and accumulate in the periplasmic space in mutants, it is generally considered to be an extension of the sec gene pathway for translocation across the inner membrane and to function only after the proteins * Corresponding author. Mailing address: Department of Biology, University of Regina, Regina, Saskatchewan, Canada S4S OA2. Phone: (306) 585-5223. Fax: (306) 585 4894. Electronic mail address: howardsp @meena.cc.uregina.ca. have arrived at the periplasm via this route. This is despite the fact that one of the proteins encoded by this operon (PulE and its homologs) contains a consensus ATP binding site which is essential for its function (23, 35). During our earlier studies into the mechanism of extracel- lular secretion in Aeromonas hydrophila, we isolated transpo- son TnS-751 insertion mutants which were defective in secre- tion of extracellular proteins (14). One of these, L1.97, led to the isolation of the 12-gene operon exeC-N, which is highly homologous to the pul operon and which is required for secretion of a large group of toxins by this bacterium (13). It is also required, however, for correct assembly of the outer membrane, since mutations in any one of three different genes of the operon were shown to cause a marked reduction in the quantity of the OmpF homolog protein II in the outer mem- brane, an effect which renders the bacteria osmotically fragile (13, 14). In the same mutagenesis experiment, we isolated another mutant, C5.84, which like L1.97 produces all of the extracellular proteins assayed but accumulates them in the periplasmic space. Southern hybridization experiments dem- onstrated that in this mutant the transposon had not inserted in the exeC-N operon. Furthermore, these mutants differ in possessing an outer membrane protein profile identical to that of the wild type (14). Together, these results suggested that the mutation in C5.84 had affected an aspect or step in extracel- lular secretion which is separate from that controlled by the e.xeC-N operon. In the studies reported here, we have isolated and se- quenced a 3.5-kb BglII fragment which complemented the mutation in C5.84. By subcloning, complementation, analysis of expression in vitro, and marker exchange mutagenesis, we show that this locus consists of an operon composed of two genes, each of which is required for extracellular secretion. 6819