To determine the phospholipid requirement of the preprotein translocase in vitro, the Escherichia coli SecYEG complex was purified in a delipidated form using the detergent dodecyl maltoside. SecYEG was reconstituted into liposomes composed of defined synthetic phospholipids, and proteoliposomes were analyzed for their preprotein translocation and SecA translocation ATPase activity. The activity strictly required the presence of anionic phospholipids, whereas the non-bilayer lipid phosphatidylethanolamine was found stimulatory. The latter effect could also be induced by dioleoylglycerol, a lipid that adopts a non-bilayer conformation. Phosphatidylethanolamine derivatives that prefer the bilayer state were unable to stimulate translocation. In the absence of SecG, activity was reduced, but the phospholipid requirement was unaltered. Remarkably, nonbilayer lipids were found essential for the activity of the Bacillus subtilis SecYEG complex. Optimal activity required a mixture of anionic and non-bilayer lipids at concentrations that correspond to concentrations found in the natural membrane.

In contrast to Gram-negative bacteria, secretory proteins of Gram-positive bacteria only need to traverse a single membrane to enter the extracellular environment. For this reason, Gram-positive bacteria (e.g. various Bacillus species) are often used in industry for the commercial production of extracellular proteins that can be produced in yields of several grams per liter culture medium. The central components of the main protein translocation system (Sec system) of Gram-negative and Gram-positive bacteria show a high degree of conservation, suggesting similar functions and working mechanisms. Despite this fact, several differences can be identified such as the absence of a clear homolog of the secretion-specific chaperone SecB in Gram-positive bacteria. The now available detailed insight into the organization of the Gram-positive protein secretion system and how it differs from the well-characterized system of Escherichia coli may in the future facilitate the exploitation of these organisms in the high level production of heterologous proteins which, so far, is sometimes very inefficient due to one or more bottlenecks in the secretion pathway. In this review, we summarize the current knowledge on the various steps of the protein secretion pathway of Gram-positive bacteria with emphasis on Bacillus subtilis, which during the last decade, has arisen as a model system for the study of protein secretion in this industrially important class of microorganisms.

Three Xanthobacter Py2 mutants (M3, M8 and M10) lacking epoxyalkanedegrading activity were isolated and characterized. All mutants were able to grow on acetone, the degradation product of 1,2-epoxypropane conversions. Furthermore, they contained the unidentified 'low molecular mass fraction (LMF) necessary for epoxyalkane-degrading activity. Three cosmids from a gene bank complemented the mutation in M I 0 and M8 but not in mutant M3. Epoxyalkane-degrading activity in crude extracts of 1,2-epoxypropane-grown complemented mutants was similar t o the wild-type activity. Surprisingly, M10 transformed with complementing cosmid PEP9 showed a constitutively expressed epoxyal kane-degrading activity, which was not observed in the wild-type strain. The cosmid PEP9 was conjugated into Xanthobacter autotrophicus GJIO, which is not able to degrade 1,2-epoxypropane. In crude extracts of X. autotrophicus GJ1 O(pEP9), epoxyalkane-degrading activity was demonstrated, but only after the addition of the LMF from Xanthobacter Py2. Hybridization experiments demonstrated an overlap on complementing cosmids pEP1, pEP3 and pEP9. Subcloning revealed a 4.8 kb EcoRI-Hindlll fragment t o be necessary for complementing the mutant M10. In the sequence of this fragment four different ORFs were found.Keywords : Xanthobacter Py2, epoxyalkane degradation, complementation INTRODUCTIONSeveral methods have been described to produce optically pure epoxides by biological methods (Weijers et al., 198813; Leak e t a/., 1992;De Bont, 1993). One such method is the enantioselective degradation of racemic epoxides; however, such a process yields a maximum of 50% product. Xantbobacter Py2 was isolated on propene as sole carbon and energy source (Van Ginkel & De Bont, 1986). This strain is able to degrade the (2s)-forms of trans-2,3-epoxybutane and 2,3-epoxypentane, resulting in optically pure (2R)-2,3-epoxyalkanes ; the C,-C, 1,2-epoxyalkanes are degraded completely (Weijers et al., 1988a The EMBL accession number for the sequence reported in this paper is X79863.Ketones have been identified as degradation products of epoxyalkane metabolism in crude extracts of propenegrown cells (Fig. 1). It has been demonstrated that both N A D and an unidentified ' low molecular mass fraction ' (LMF) are involved in the reaction. In a reaction mechanism proposed by Weijers et al. (1994) it was suggested that the LMF is involved in the reduction of the epoxyalkanes to secondary alcohols since the LMF can be replaced by reducing compounds like DTT and other dithiol compounds. The presence of N A D is necessary for the oxidation of the alcohols to the corresponding ketones.Attempts to purify the epoxyalkane-degrading enzyme have not been successful. Consequently, we have now investigated the genetics of epoxyalkane degradation in Xantbobacter Py2. Eventually, we hope to be able to obtain the enzyme to study further its reaction mechanism.In this study we report the selection and characterization of mutants devoid of epoxyalkane-degrading activity, the complementation o...

Epoxide degradation in cell extracts of Xanthobacter strain Py2 has been reported to be dependent on NAD ؉ and dithiols. This multicomponent system has now been fractionated. A key protein encoded by a DNA fragment complementing a Xanthobacter strain Py2 mutant unable to degrade epoxides was purified and analyzed. This NADP-dependent protein, a novel type of pyridine nucleotide-disulfide oxidoreductase, is essential for epoxide degradation. NADPH, acting as the physiological cofactor, replaced the dithiols in epoxide conversion.Propene-grown Xanthobacter strain Py2 (15) contains an enzyme system capable of degrading epoxyalkanes, which are metabolites arising from alkenes by the action of alkene monooxygenase. Recently, both the monooxygenase (23) and the epoxide-degrading system of the organism have received considerable attention. Initially, this interest was based on applied aspects because the organism may be used in the degradation of chlorinated alkenes and epoxides (3, 4, 7, 10) and in the production of optically pure epoxides (18). As it turns out, the epoxide-degrading enzyme system has very intriguing properties and was therefore investigated in detail.Initially, epoxide degradation was studied at the whole-cell level (9, 18). Recently, Weijers et al. (19) were able to demonstrate enzyme activity in extracts if both NAD ϩ and a lowmolecular-mass fraction were included in the assay system. Furthermore, they showed that the low-molecular-mass fraction can be replaced by a range of artificial dithiol compounds, such as dithiothreitol (DTT). Ketones were the product formed under their assay conditions. Allen and Ensign (1) also studied the fate of epoxides in extracts. They included carbonate in their assay system and proposed that the enzyme system of Xanthobacter strain Py2 carboxylated 1,2-epoxyalkanes to form ␤-keto acids. In their view, ketones are a dead-end product which is formed only when carbonate is limiting. The formation of either product from an epoxide is redox neutral. The requirement for both NAD ϩ and a dithiol therefore suggests that reduction of the epoxide is followed by oxidation or vice versa.The first report of a successful fractionation of the epoxidedegrading enzyme system was by Leak and coworkers (Imperial College, London, United Kingdom). They were able to devise a method resulting in two fractions, both of which were required to reconstitute an active epoxide-degrading system (16).Swaving et al. (13) reported the cloning of a 4.8-kb DNA fragment required for complementation of mutants of Xanthobacter strain Py2 defective in epoxide degradation. From the deduced amino acid sequences of the four open reading frames of this fragment no clear information on how the degradation of epoxides proceeds could be gained. However, the protein encoded by ORF3 was of great interest because of its homology to the family of pyridine nucleotide-disulfide oxidoreductases (22). The homology was of special interest because most members of this family use dithiols as a substrate, whereas dithiol...

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CsaA from the Gram-positive bacterium Bacillus subtilis has been identified previously as a suppressor of the growth and protein-export defect of Escherichia coli secA(Ts) mutants. CsaA has chaperone-like activities in vivo and in vitro. To examine the role of CsaA in protein export in B. subtilis, expression of the csaA gene was repressed. While export of most proteins remained unaffected, export of at least two proteins was significantly reduced upon CsaA depletion. CsaA co-immunoprecipitates and co-purifies with the SecA proteins of E. coli and B. subtilis, and binds the B. subtilis preprotein prePhoB. Purified CsaA stimulates the translocation of prePhoB into E. coli membrane vesicles bearing the B. subtilis translocase, whereas it interferes with the SecB-mediated translocation of proOmpA into membrane vesicles of E. coli. The specific interaction with the SecA translocation ATPase and preproteins suggests that CsaA acts as a chaperone that promotes the export of a subset of preproteins in B. subtilis.

Bacilli vigorously secrete proteins into the extracellular environment, and are therefore used in industry for the bulk production of enzymes such as proteinases and amylases. Studies on the mechanism of protein translocation in these Gram-positive bacteria have been hampered by the lack of an in vitro system. To establish such a system for Bacillus subtilis, everted membranes were isolated from a strain deficient in the alkaline and neutral protease. Translocation-competent membrane vesicles were obtained only when a broad range proteinase-inhibitor cocktail was used during membrane isolation. This method efficiently prevented proteolysis of SecY, one of the core integral membrane components of the preprotein translocase. Translocation of the urea-denatured precursor of the Bacillus licheniformis A-amylase, preAmyL, and B. subtilis alkaline phosphatase, prePhoB, into the B. subtilis membrane vesicles require the B. subtilis SecA protein and are driven by ATP hydrolysis and the proton-motive force. These studies establish an authentic in vitro translocation system for protein secretion in B. subtilis.