Folding LacZ in the periplasm of Escherichia coli

Abstract

Targeted, translational LacZ fusions provided the initial support for the signal sequence hypothesis in prokaryotes and allowed for selection of the mutations that identified the Sec translocon. Many of these selections relied on the fact that expression of targeted, translational lacZ fusions like malE-lacZ and lamB-lacZ42-1 causes lethal toxicity as folded LacZ jams the translocation pore. However, there is another class of targeted LacZ fusions that do not jam the translocon. These targeted, nonjamming fusions also show toxic phenotypes that may be useful for selecting mutations in genes involved in posttranslocational protein folding and targeting; however, they have not been investigated to the same extent as their jamming counterparts. In fact, it is still unclear whether LacZ can be fully translocated in these fusions. It may be that they simply partition into the inner membrane where they can no longer participate in folding or assembly. In the present study, we systematically characterize the nonjamming fusions and determine their ultimate localization. We report that LacZ can be fully translocated into the periplasm, where it is toxic. We show that this toxicity is likely due to LacZ misfolding and that, in the absence of the periplasmic disulfide bond catalyst DsbA, LacZ folds in the periplasm. Using the novel phenotype of periplasmic β-galactosidase activity, we show that the periplasmic chaperone FkpA contributes to LacZ folding in this nonnative compartment. We propose that targeted, nonjamming LacZ fusions may be used to further study folding and targeting in the periplasm of Escherichia coli.

FIG 1

Construction of JCM944. (A, B, D, and F) Note that the λ insertion is λp1 (209). (A) CWB299. CWB299 is an H*lamB merodiploid, containing a copy of H*lamB in addition to the H*lamB-lacZ fusion. (B) JCM911. Construction of a haploid H*lamB-lacZ strain began with a strain containing the Δ60lamB-lacZ fusion and pKD46. (C to E) JCM911 was recombineered with the linear DNA fragment depicted in panel C. The fragment was obtained by PCR amplification of the Kan^r cassette present on pKD4 with primers having 5′ homology to malK and lamB. The primers were designed to generate a Kan^r insertion-deletion removing 27 nucleotides upstream of the lamB open reading frame and 200 nucleotides downstream (Δ230::Kan^r). The result of the recombineering reaction is JCM932, depicted in panel D. The Δ230::Kan^r insertion-deletion is polar on the lac operon, preventing expression of lacY. As a result, JCM932 cannot grow on minimal melibiose at 42°C. JCM932 was then recombineered with the linear DNA fragment depicted in panel E. The fragment was obtained by PCR amplification of malK and H*lamB from pCWB36. Recombinants that replaced the Δ230::Kan^r insertion-deletion and restored lacY expression were selected on minimal melibiose at 42°C and subsequently screened for Kan^s. (F) JCM944 was identified as a Mel⁺ Kan^s recombinant.

FIG 2

Effect of DsbA on whole-cell β-galactosidase activity. Overnight cultures were washed and subcultured 1:50 into liquid LB cultures. Cultures were incubated with aeration at 37°C for 2 to 3 h until reaching mid-log phase. β-Galactosidase activity was determined from V_max of whole-cell lysates in a kinetic Miller assay. Δ60 indicates strains JCM913 (dsbA⁺) and RSD117 (dsbA::Kan^r). 42-1 indicates strains JCM912 (dsbA⁺) and RSD118 (dsbA::Kan^r). H* indicates strains JCM944 (dsbA⁺) and RSD120 (dsbA::Kan^r), and 102 indicates strains RSD06 (dsbA⁺) and RSD121 (dsbA::Kan^r). Error bars show standard deviations calculated from three independent biological replicates.

FIG 3

Effect of DsbA on periplasmic β-galactosidase activity. (A and B) Error bars show standard deviations calculated from three independent biological replicates. (A) Bypassing lacY. Overnight cultures were washed and subcultured in minimal medium containing glycerol or lactose as the sole carbon source. Δ60 indicates strains containing the Δ60lamB-lacZ fusion in a wild-type background (JCM913, white), a lacY::Tn9 background (RSD122, gray), or a lacY::Tn9 dsbA::Kan^r background (RSD124, black). H* indicates strains containing the H*lamB-lacZ fusion in a wild-type background (JCM944, white), a lacY::Tn9 background (RSD123, gray), or a lacY::Tn9 dsbA::Kan^r background (RSD125, black). 102 indicates strains containing the malF-lacZ102 fusion in a wild-type background without lacY (RSD06, gray) or a dsbA::Kan^r background (RSD121, black). (B and C) Overnight cultures were washed and subcultured 1:50 into liquid LB cultures. Cultures were incubated with aeration at 37°C for 2 to 3 h until reaching mid-log phase. Δ60 refers to RSD117 (dsbA::Kan^r), and H* refers to RSD120 (dsbA::Kan^r). (B) Releasing soluble periplasmic β-galactosidase activity. β-Galactosidase activity was determined from V_max of whole-cell lysates or filtered spheroplast supernatants in a kinetic Miller assay. Periplasmic β-galactosidase activity is reported as a fraction of whole-cell β-galactosidase activity. (C) Spheroplasting lysis control. Whole-cell lysates or filtered spheroplast supernatants were subjected to SDS-PAGE and Western blotting. MBP is used as a marker for soluble periplasmic proteins. CpxR is used as a marker of soluble cytoplasmic proteins.

FIG 4

Effect of periplasmic chaperones on periplasmic β-galactosidase activity. Overnight cultures were washed and subcultured 1:50 into liquid LB cultures. Cultures were incubated with aeration at 30°C for 2 to 3 h until reaching mid-log phase. β-Galactosidase activity was determined from V_max of whole-cell lysates in a kinetic Miller assay. β-Galactosidase activity is normalized to RSD121. Error bars show standard deviations calculated from three independent biological replicates. 102 indicates RSD121. Δskp indicates RSD126. fkpA::Cm^r indicates RSD127.