No abstract

We show that inverted membrane vesicles from Corynebacterium gfutamicum, a Gram-positive bacterium, are able to generate and maintain an electrochemical gradient of protons in response to the addition of NADH. This result indicates that the respiratory chain is intact and that the vesicles are reasonably impermeable to protons. These membrane vesicles may be the starting point for in vitro translocation studies of proteins in Gram-positive bacteria.Key words: Gram-positive bacterium; Membrane vesicle; Membrane potential; Protein translocation IntrodllctIonIn vitro translocation systems have been of great use in the elucidation of the molecular mechanism of protein transport across membranes in eucaryotes and procaryotes. In the case of E. coli, a Gram-negative bacterium, the system consists of inverted vesicles obtained by physical treatment (high pressure) of spheroplasts. The precursor form of proteins normally translocated, in vivo, across the cyctoplasmic membrane, may be imported, in vitro, into these vesicles. Although only a small number of proteins are efficiently translocated in vitro, the use of such systems has greatly helped in determining the energy requirement for the translocation (ATP and the proton electrochemical potential) [l-3] and the role of the various components involved in the translocation machinery (set proteins) [4,51 .Our knowledge regarding the secretion of proteins in Grampositive bacteria is limited. Most studies have been performed with Bacillus subtih. It has been shown that secreted proteins are synthesized in the form of precursors with a signal sequence reminiscent of that of periplasmic proteins in Gram-negative bacteria [6]. Moreover, some Gram-positive bacterial genes, homologous to set genes in E. coli, have been cloned and shown to be able to complement set mutations in this strain [7-l 11. All this suggests that a common mechanism may exist for the secretion of proteins in Gram-positive bacteria and for the translocation of proteins across the cytoplasmic membrane in Gramnegative bacteria. This, however, remains to be proven.The elucidation of the molecular mechanism of protein secretion in Gram-positive bacteria is hampered by the difficulty in obtaining secretion mutants and by the absence, despite much effort, of an in vitro translocation system. Recently an in vitro processing assay was developed for proteins secreted by Bacillus subtilk Unfortunately this system is ineffective for translocation [12]. The central part of an in vitro system is inverted membrane vesicles derived from the cytoplasmic membrane. *Corresponding author. Fax: (33) 69 85 37 15.Abbreviations: BHI, brain heart infusion; CCCP, carbonyl cyanide m-chlorophenyl-hydraone; DCCD, dicyclo-hexylcarbo-diimide; PMS, phenaxine methosulfate.The vesicles should be impermeable to protons and be able to generate and maintain an electrochemical gradient of protons (energetically functional vesicles). We have shown recently that Corynebacterium glutamicum, a Gram-positive bacterium, secretes large amo...