Aeromonas hydrophila secretes protein toxins via the type II pathway, involving the products of at least two operons, exeAB (gspAB) and exeC-N (gspC-N). In the studies reported here, aerolysin secretion was restored to C5.84, an exeA::Tn5-751 mutant, by overexpression of exeD alone in trans. Expression studies indicated that these results did not reflect a role of ExeAB in the regulation of the exeC-N operon. Instead, immunoblot analysis showed that ExeD did not multimerize in C5.84, and fractionation of the membranes showed that the monomeric ExeD remained in the inner membrane. Expression of ExeAB, but not either protein alone, from a plasmid in C5.84 resulted in increases in the amount of multimeric ExeD, which correlated with increases in aerolysin secretion. Pulse-chase analysis also suggested that the induction of ExeAB allowed multimerization of previously accumulated monomer ExeD. In C5.84 cells overproducing ExeD, it multimerized even in the absence of ExeAB and, although most remained in the inner membrane, an amount similar to that in wild-type outer membranes fractionated with the outer membrane of the overproducing cells. These results indicate that the secretion defect of exeAB mutants is a result of an inability to assemble the ExeD secretin in the outer membrane. The localization and multimerization of overproduced ExeD in these mutants further suggests that the ExeAB complex plays either a direct or indirect role in the transport of ExeD into the outer membrane.

SummaryAeromonas hydrophila transports extracellular protein toxins via the type II secretion system, an export mechanism comprised of numerous proteins that spans both the inner and outer membranes. Two components of this secretion system, ExeA and ExeB, form a complex in the inner membrane that functions to locate and/or assemble the ExeD secretin in the outer membrane. In the studies reported here, twocodon insertion mutagenesis of exeA revealed that an insertion at amino acid 495 in the C-terminal region of ExeA did not alter ExeAB complex formation yet completely abrogated its involvement in ExeD secretin assembly and thus rendered the bacteria secretion negative. In silico analysis of protein motifs with similar amino acid profiles revealed that this amino acid is located within a putative peptidoglycan (PG) binding motif in the periplasmic domain of ExeA. Substitution mutations of three highly conserved amino acids in the motif were constructed. In cells expressing each of these mutants, the ability to assemble the ExeD secretin or secrete aerolysin was lost, while ExeA retained the ability to form a complex with ExeB. In in vivo cross-linking experiments, wild-type ExeA could be cross-linked to PG, whereas the three substitution mutants of ExeA could not. These data indicate that PG binding and/or remodelling plays a role in the function of the ExeAB complex during assembly of the ExeD secretin.

DegP is a periplasmic protease that is a member of both the E and Cpx extracytoplasmic stress regulons of Escherichia coli and is essential for viability at temperatures above 42°C. [U-14 C]acetate labeling experiments demonstrated that phospholipids were degraded in degP mutants at elevated temperatures. In addition, chloramphenicol acetyltransferase, ␤-lactamase, and ␤-galactosidase assays as well as sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis indicated that large amounts of cellular proteins are released from degP cells at the nonpermissive temperature. A mutation in pldA, which encodes outer membrane phospholipase A (OMPLA), was found to rescue degP cells from the temperature-sensitive phenotype. pldA degP mutants had a normal plating efficiency at 42°C, displayed increased viability at 44°C, showed no degradation of phospholipids, and released far lower amounts of cellular protein to culture supernatants. degP and pldA degP mutants containing chromosomal lacZ fusions to Cpx and E regulon promoters indicated that both regulons were activated in the pldA mutants. The overexpression of the envelope lipoprotein, NlpE, which induces the Cpx regulon, was also found to suppress the temperature-sensitive phenotype of degP mutants but did not prevent the degradation of phospholipids. These results suggest that the absence of OMPLA corrects the degP temperature-sensitive phenotype by inducing the Cpx and E regulons rather than by inactivating the phospholipase per se.Extracytoplasmic stress, such as that caused by heat shock or the overproduction of outer membrane proteins in Escherichia coli, is believed to be caused by the accumulation and aggregation of denatured and misfolded proteins in the membranes and periplasm. Under these conditions the Cpx two-component signal transduction pathway and the alternative sigma factor E direct the synthesis of several proteins that are involved in the degradation and refolding of these denatured and misfolded periplasmic proteins, leading to alleviation of the stress (for a review, see reference 50).The rpoE gene encoding E is essential for the viability of cells at all temperatures (22).E is known to direct the transcription of degP (htrA), fkpA, rpoE, rpoH, and many others (12,14,15,26,34). DegP is a protease/chaperone that digests abnormal proteins in the periplasm and has been demonstrated to be necessary for cell viability at temperatures of 42°C and above (33,35,55,57,58,59), and FkpA is a peptidyl prolyl cis/trans isomerase (28, 41). RseA, RseB, and RseC are involved in regulating the transcription of genes in the E regulon (21, 42). Under heat shock conditions or upon overexpression of outer membrane proteins, denatured and misfolded proteins in the periplasm are sensed by RseA and/or RseB (38). E is then released by the cytoplasmic domain, allowing it to direct transcription of the genes in the E regulon.

Inactivation of the gene encoding the periplasmic protease DegP confers a high-temperature-sensitive phenotype in Escherichia coli. We have previously demonstrated that a degP mutant of E. coli strain CBM (W3110 pldA1) is not temperature sensitive and showed that this was most likely due to constitutive activation of the sigma E and Cpx extracytoplasmic stress regulons in the parent strain. In this study, further characterization of this strain revealed a previously unknown cryptic mutation that rescued the degP temperaturesensitive phenotype by inducing the extracytoplasmic stress regulons. We identified the cryptic mutation as an 11-bp deletion of nucleotides 1884 to 1894 of the adenylate cyclase-encoding cyaA gene (cyaA⌬11). The mechanism in which cyaA⌬11 induces the sigma E and Cpx regulons involves decreased activity of the mutant adenylate cyclase. Addition of exogenous cyclic AMP (cAMP) to the growth medium of a cyaA⌬11 mutant strain that contains a Cpx-and sigma E-inducible degP-lacZ reporter fusion decreased ␤-galactosidase expression to levels observed in a cyaA ؉ strain. We also found that a cyaA null mutant displayed even higher levels of extracytoplasmic stress regulon activation compared to a cyaA⌬11 mutant. Thus, we conclude that the lowered concentration of cAMP in cyaA mutants induces both sigma E and Cpx extracytoplasmic stress regulons and thereby rescues the degP temperature-sensitive phenotype.Gram-negative bacteria such as Escherichia coli have developed a set of response regulons designed to maintain cell viability under a variety of stressful environmental conditions. Under conditions that increase the amount of misfolded or aggregated proteins in the periplasm, such as those generated under conditions of heat shock or the overproduction of outer membrane porins, a pair of regulons known as sigma E and Cpx are induced. These regulons prevent cell death by inducing the expression of genes that encode chaperones and proteases that function to refold or degrade misfolded proteins in the periplasm (36).The sigma E extracytoplasmic stress response regulon initially described by Erickson and Gross (16) utilizes a regulated intramembrane proteolytic pathway (5) to transduce a stress signal from the periplasm to the cytoplasm, whereupon genes in the sigma E regulon are induced. A cascade of events initiating with detection of aberrant proteins in the periplasm results in the release of sigma E normally sequestered by the inner-membrane-spanning protein RseA to the cytoplasm. Release allows members of the sigma E regulon to be upregulated, including; rpoE, rseA, rseB, and rseC (14). Other sigma E-controlled genes include degP, which encodes the protease/ chaperone DegP (46); fkpA, which encodes the peptidyl/prolyl isomerase FkpA (31); rpoH, which encodes H of the cytoplasmic heat shock response regulon (16); and many others involved in performing basic cellular functions such as metabolism and phospholipid biosynthesis (37).The Cpx regulon is a three-component regulatory system composed of an inne...