Many virulence factors secreted from pathogenic Gram-negative bacteria are autotransporter proteins. The final step of autotransporter secretion is C 3 N-terminal threading of the passenger domain through the outer membrane (OM), mediated by a cotranslated C-terminal porin domain. The native structure is formed only after this final secretion step, which requires neither ATP nor a proton gradient. Sequence analysis reveals that, despite size, sequence, and functional diversity among autotransporter passenger domains, >97% are predicted to form parallel ␤-helices, indicating this structural topology may be important for secretion. We report the folding behavior of pertactin, an autotransporter passenger domain from Bordetella pertussis. The pertactin ␤-helix folds reversibly in isolation, but folding is much slower than expected based on size and native-state topology. Surprisingly, pertactin is not prone to aggregation during folding, even though folding is extremely slow. Interestingly, equilibrium denaturation results in the formation of a partially folded structure, a stable core comprising the C-terminal half of the protein. Examination of the pertactin crystal structure does not reveal any obvious reason for the enhanced stability of the C terminus. In vivo, slow folding would prevent premature folding of the passenger domain in the periplasm, before OM secretion. Moreover, the extra stability of the C-terminal rungs of the ␤-helix might serve as a template for the formation of native protein during OM secretion; hence, vectorial folding of the ␤-helix could contribute to the energyindependent translocation mechanism. Coupled with the sequence analysis, the results presented here suggest a general mechanism for autotransporter secretion.parallel ␤-sheet ͉ contact order ͉ outer membrane protein ͉ protein structure prediction ͉ virulence factor

SPR3 is one of at least nine genes which are expressed in sporulating Saccharomyces cerevisiae cells at the time of meiosis I. We show below that strains homozygous for null alleles of SPR3 are capable of normal meiosis and the production of viable ascospores. We have also monitored SPR3 expression in a series of strains that are defective in meiotic development, using an SPR3:lacZ fusion carried on a single copy plasmid. beta-Galactosidase activity occurred at wild-type levels in diploid strains homozygous for mutations in spo13, rad50, rad57 and cdc9, but was greatly reduced in strains carrying cdc8 or spo7 defects. We conclude that SPR3 expression is a valid monitor of early meiotic development, even though the gene is inessential for the sporulation process.

The relative transcription rates of three sporulation-regulated genes of yeast (SPR1, SPR2 and SPR3) were determined at intervals during sporulation, using a filter binding assay. The binding of in vivo labeled RNA to the corresponding DNAs increased 3- to 12-fold at the time of meiosis I, in parallel with the accumulation of the SPR transcripts. SPR1 and SPR3 mRNA abundance increased from less than 0.7 to 130 and 90 copies per cell, respectively, between the time of shift to sporulation medium and the initiation of spore formation. This represented a 150-to 200-fold increase in the steady-state levels of these RNAs. Similarly, the levels of beta-galactosidase present in sporulating cells harboring fusions between SPR3 and Escherichia coli lacZ increased at least 700-fold. We conclude that SPR1, SPR2 and SPR3 transcription is modulated during sporulation, possibly in response to earlier events in the process.

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