An alternate pathway for the processing of the prolipoprotein signal peptide in Escherichia coli

Previous studies showed that when the signal sequence plus 9 amino acid residues from the amino terminus of the major lipoprotein of Escherichia coli was fused to beta-lactamase, the resulting hybrid protein was modified, proteolytically processed, and assembled into the outer membrane as was the wild-type lipoprotein (Ghrayeb, J., and Inouye, M. (1983) J. Biol. Chem. 259, 463-467). We have constructed several hybrid proteins with mutations at the cleavage site of the prolipoprotein signal peptide. These mutations are known to block the lipid modification of the lipoprotein at the cysteine residue, resulting in the accumulation of unprocessed, unmodified prolipoprotein in the outer membrane. The mutations blocked the lipid modification of the hybrid protein. However, in contrast to the mutant lipoproteins, the cleavage of the signal peptides for the mutant hybrid proteins did occur, although less efficiently than the unaltered prolipo-beta-lactamase. The mutant prolipo-beta-lactamase proteins were cleaved at a site 5 amino acid residues downstream of the prolipoprotein signal peptide cleavage site. This new cleavage between alanine and lysine residues was resistant to globomycin, a specific inhibitor for signal peptidase II. This indicates that signal peptidase II, the signal peptidase which cleaves the unaltered prolipo-beta-lactamase, is not responsible for the new cleavage. The results demonstrate that the cleavage of the signal peptide is a flexible process that can occur by an alternative pathway when the normal processing pathway is blocked.     

A single amino acid determinant of the membrane localization of lipoproteins in E. coli

When beta-lactamase was fused with the signal peptide plus the amino-terminal 9 amino acid residues of the major outer membrane lipoprotein, the resultant lipo-beta-lactamase (LL-1) was shown to be localized to the outer membrane. However, when the 9 residue sequence was replaced with the amino-terminal 12 residue sequence of lipoprotein-28, an inner membrane protein, the resultant lipo-beta-lactamase (LL-2) was found exclusively in the inner membrane. The localization of LL-2 was shifted to the outer membrane simply by substituting the second amino acid residue (Asp) of LL-2 with Ser. Conversely, the alteration of the second residue (Ser) of LL-1 to Asp resulted in the localization of LL-1 to the inner membrane. These results suggest that the second amino acid residue of the lipoproteins plays a crucial role in determining their final locations in the E. coli envelope.     

Inhibition of secretion of a mutant lipoprotein across the cytoplasmic membrane by the wild-type lipoprotein of the Escherichia coli outer membrane.

A globomycin-resistant mutant of Escherichia coli was found to produce a precursor of the major outer membrane lipoprotein (prolipoprotein), in which the glycine residue at position 14 within the signal peptide was replaced by an aspartic acid residue. The same mutation has been reported by Lin et al. (Proc. Natl. Acad. Sci. U.S.A. 175:4891-4895, 1978). The structural gene of the mutant prolipoprotein was inserted into an inducible expression cloning vehicle. When the mutant prolipoprotein was produced in lipoprotein-minus host cells, 82% of the unprocessed protein was found in the membrane fraction, with the remaining 18% localized in the soluble fraction. However, when the production of the mutant prolipoprotein was induced in the wild-type lpp+ host cells, only 31% of the mutant prolipoprotein was found in the membrane fraction, leaving the remaining 69% in the soluble, cytoplasmic fraction. In addition, the assembly of the wild-type lipoprotein in these cells was not affected, whether the mutant prolipoprotein was produced or not. These results suggest that secretions of both mutant and wild-type prolipoproteins utilize the same component(s) responsible for the initial stages of secretion across the cytoplasmic membrane. However, it appears that the wild-type lipoprotein has a higher affinity for these components than does the mutant lipoprotein.     

Localization of the outer membrane subunit OprM of resistance-nodulation-cell division family multicomponent efflux pump in Pseudomonas aeruginosa

The outer membrane subunit OprM of the multicomponent efflux pump of Pseudomonas aeruginosa has been assumed to form a transmembrane xenobiotic exit channel across the outer membrane. We challenged this hypothesis to clarify the underlying ambiguity by manipulating the amino-terminal signal sequence of the OprM protein of the MexAB-OprM efflux pump in P. aeruginosa. [(3)H]Palmitate uptake experiments revealed that OprM is a lipoprotein. The following lines of evidence unequivocally established that the OprM protein functioned at the periplasmic space. (i) The OprM protein, in which a signal sequence including Cys-18 was replaced with that of periplasmic azurin, appeared in the periplasmic space but not in the outer membrane fraction, and the protein fully functioned as the pump subunit. (ii) The hybrid OprM containing the N-terminal transmembrane segment of the inner membrane protein, MexF, appeared exclusively in the inner membrane fraction. The hybrid protein containing 186 or 331 amino acid residues of MexF was fully active for the antibiotic extrusion, but a 42-residue protein was totally inactive. (iii) The mutant OprM, in which the N-terminal cysteine residue was replaced with another amino acid, appeared unmodified with fatty acid and was fractionated in both the periplasmic space and the inner membrane fraction but not in the outer membrane fraction. The Cys-18-modified OprM functioned for the antibiotic extrusion indistinguishably from that in the wild-type strain. We concluded, based on these results, that the OprM protein was anchored in the outer membrane via fatty acid(s) attached to the N-terminal cysteine residue and that the entire polypeptide moiety was exposed to the periplasmic space.     

Structural determinants in addition to the amino-terminal sorting sequence influence membrane localization of Escherichia coli lipoproteins.

The lipid-modified nine-residue amino-terminal sequence of the mature form of the major outer membrane lipoprotein of Escherichia coli contains information that is responsible for sorting to either the inner or outer membrane. Fusion of this sorting sequence to beta-lactamase is sufficient for localization of the resultant lipo-beta-lactamase to the outer membrane (J. Ghrayeb and M. Inouye, J. Biol. Chem. 259:463-467, 1984). Substitution of the serine adjacent to the amino-terminal lipid-modified cysteine residue of the sorting sequence with the negatively charged residue aspartate causes inner membrane localization (K. Yamaguchi, F. Yu, and M. Inouye, Cell 53:423-432, 1988). Fusion of the aspartate-containing nine-residue inner membrane localization signal to the normally outer membrane lipoprotein bacteriocin release protein does cause partial localization to the inner membrane. However, a single replacement of the glutamine adjacent to the amino-terminal lipid-modified cysteine residue of bacteriocin release protein with aspartate causes no inner membrane localization. Therefore, an aspartate residue itself lacks the information necessary for inner membrane sorting when removed from the structural context provided by the additional eight residues of the sorting sequence. Although the aspartate-containing inner membrane sorting sequence causes an almost quantitative localization to the inner membrane when fused to the otherwise soluble protein beta-lactamase, this sequence cannot prevent significant outer membrane localization when fused to proteins (bacteriocin release protein and OmpA) normally found in the outer membrane. Therefore, structural determinants in addition to the amino-terminal sorting sequence influence the membrane localization of lipoproteins.     

Amino acid replacement in a mutant lipoprotein of the Escherichia coli outer membrane.

The primary structure of a mutant lipoprotein of the outer membrane of Escherichia coli was investigated. This mutant was previously described as a mutant that forms a dimer of the lipoprotein by an S-S bridge (H. Suzuki et al., J. Bacteriol. 127:1494-1501, 1976). The amino acid analysis of the mutant lipoprotein revealed that the mutant lipoprotein had an extra cysteine residue, with concomitant loss of an arginine residue. From the analysis of the mutant lipoprotein revealed that the mutant lipoprotein had an extra cysteine residue, with concomitant loss of an arginine residue. From the analysis of tryptic peptides, it was found that the arginine residue at position 57 was replaced with a cysteine residue. The amino terminal structure of the mutant lipoprotein was found to be glycerylcysteine, as in the case of the wild-type lipoprotein. The present results show that the mutation that was previously determined to map at 36.5 min on the E. coli chromosome occurred in the structure gene (lpp) for the lipoprotein. This was further confirmed by the fact that a merodiploid carrying both lpp+ and lpp produces not only the wild-type lipoprotein but also the mutant lipoprotein.     

Lethal mutations in the structural gene of an outer membrane protein (OmpA) of Escherichia coli K12

Summary The gene ompA encodes a major outer membrane protein of Escherichia coli. Localized mutagenesis of the part of the gene corresponding to the 21-residue signal sequence and the first 45 residues of the protein resulted in alterations which caused cell lysis when expressed. DNA sequence analyses revealed that in one mutant type the last CO₂H-terminal residue of the signal sequence, alanine, was replaced by valine. The proteolytic removal of the signal peptide was much delayed and most of the unprocessed precursor protein was fractioned with the outer membrane. However, this precursor was completely soluble in sodium lauryl sarcosinate which does not solubilize the OmpA protein or fragments thereof present in the outer membrane. Synthesis of the mutant protein did not inhibit processing of the OmpA or OmpF proteins. In the other mutant type, multiple mutational alterations had occurred leading to four amino acid substitutions in the signal sequence and two affecting the first two residues of the mature protein. A reduced rate of processing could not be clearly demonstrated. Membrane fractionation suggested that small amounts of this precursor were associated with the plasma membrane but synthesis of this mutant protein also did not inhibit processing of the wild-type OmpA or OmpF proteins. Several lines of evidence left no doubt that the mature, mutant protein is stably incorporated into the outer membrane. It is suggested that the presence, in the outer membrane, of the mutant precursor protein in the former case, or of the mutant protein in the latter case perturbs the membrane architecture enough to cause cell death.     

Lipoprotein from the osmoregulated ABC transport system OpuA of Bacillus subtilis: purification of the glycine betaine binding protein and characterization of a functional lipidless mutant.

The OpuA transport system of Bacillus subtilis functions as a high-affinity uptake system for the osmoprotectant glycine betaine. It is a member of the ABC transporter superfamily and consists of an ATPase (OpuAA), an integral membrane protein (OpuAB), and a hydrophilic polypeptide (OpuAC) that shows the signature sequence of lipoproteins (B. Kempf and E. Bremer, J. Biol. Chem. 270:16701-16713, 1995). The OpuAC protein might thus serve as an extracellular substrate binding protein anchored in the cytoplasmic membrane via a lipid modification at an amino-terminal cysteine residue. A malE-opuAC hybrid gene was constructed and used to purify a lipidless OpuAC protein. The purified protein bound radiolabeled glycine betaine avidly and exhibited a KD of 6 microM for this ligand, demonstrating that OpuAC indeed functions as the substrate binding protein for the B. subtilis OpuA system. We have selectively expressed the opuAC gene under T7 phi10 control in Escherichia coli and have demonstrated through its metabolic labeling with [3H]palmitic acid that OpuAC is a lipoprotein. A mutant expressing an OpuAC protein in which the amino-terminal cysteine residue was changed to an alanine (OpuAC-3) was constructed by oligonucleotide site-directed mutagenesis. The OpuAC-3 protein was not acylated by [3H]palmitic acid, and part of it was secreted into the periplasmic space of E. coli, where it could be released from the cells by cold osmotic shock. The opuAC-3 mutation was recombined into an otherwise wild-type opuA operon in the chromosome of B. subtilis. Unexpectedly, this mutant OpuAC system still functioned efficiently for glycine betaine acquisition in vivo under high-osmolarity growth conditions. In addition, the mutant OpuA transporter exhibited kinetic parameters similar to that of the wild-type system. Our data suggest that the lipidless OpuAC-3 protein is held in the cytoplasmic membrane of B. subtilis via its uncleaved hydrophobic signal peptide.     

A novel outer membrane lipoprotein, LolB (HemM), involved in the LolA (p20)-dependent localization of lipoproteins to the outer membrane of Escherichia coli.

The Escherichia coli major outer membrane lipoprotein (Lpp) is released from the inner membrane into the periplasm as a complex with a carrier protein, LolA (p20), and is then specifically incorporated into the outer membrane. An outer membrane protein playing a critical role in Lpp incorporation was identified, and partial amino acid sequences of the protein, named LolB, were identical to those of HemM, which has been suggested to play a role in 5-aminolevulinic acid synthesis in the cytosol. In contrast to this suggested role, the deduced amino acid sequence of HemM implied that the gene encodes a novel outer membrane lipoprotein. Indeed, an antibody raised against highly purified LolB revealed its outer membrane localization, and inhibited in vitro Lpp incorporation into the outer membrane. Furthermore, LolB was found to be synthesized as a precursor with a signal sequence and then processed to a lipid-modified mature form. An E.coli strain possessing chromosomal hemM under the control of the lac promoter-operator required IPTG for growth, indicating that hemM (lolB) is an essential gene. Outer membrane prepared from LolB-depleted cells did not incorporate Lpp. When the Lpp-LolA complex was incubated with a water-soluble LolB derivative, Lpp was transferred from LolA to LolB. Based on these results, the outer membrane localization pathway for E.coli lipoprotein is discussed with respect to the functions of LolA and LolB.     

The role of the lipoprotein sorting signal (aspartate +2) in pullulanase secretion

The analyses of hybrid proteins and of deletion and insertion mutations reveal that the only amino acid at the amino-proximal end of the cell surface lipoprotein pullulanase that is specifically required for its extracellular secretion is an aspartate at position +2, immediately after the fatty acylated amino-terminal cysteine. To see whether the requirement for this amino acid is related to its proposed role as a cyto-plasmic membrane lipoprotein sorting signal, we used sucrose gradient floatation analysis to determine the subcellular location of pullulanase variants (with or without the aspartate residue) that accumulated in cells lacking the pullulanase-specific secretion genes. A non-secretable pullulanase variant with a serine at position +2 cofractionated mainly with the major peak of outer membrane porin. In contrast, most (55%) of a pullulanase variant with an aspartate at position +2 cofractionated with slightty lighter fractions that contained small proportions of both outer membrane porin and the cytoplasmic membrane marker NADH oxidase. Only 5% of this pullulanase variant cofractionated with the major NADH oxidase peak, while the rest (c. 40%) remained at the bottom of the gradient in fractions totally devoid of porin and NADH oxidase. When analysed by sedimentation through sucrose gradients, however, a large proportion of this variant was recovered from fractions near the top of the gradient that also contained the major NADH oxidase peak. When this peak fraction was applied to a floatation gradient, the pullulanase activity remained at the bottom while the NADH oxidase floated to the top. Thus, there is no evidence that lipoproteins that cofractionate with the cytoplasmic membrane under certain conditions are actually associated with the membrane. Instead, the results support our previous proposal that lipoproteins with an aspartate +2 residue are specifically enriched in a distinct domain of the cell envelope that contains material from both the cytoplasmic and the outer membranes. Possible explanations for the requirement for the aspartate residue in pullulanase secretion are discussed.     

Prolipoprotein signal peptidase of Escherichia coli requires a cysteine residue at the cleavage site

A signal peptidase specifically required for the secretion of the lipoprotein of the Escherichia coli outer membrane cleaves off the signal peptide at the bond between a glycine and a cysteine residue. This cysteine residue was altered to a glycine residue by guided site-specific mutagenesis using a synthetic oligonucleotide and a plasmid carrying an inducible lipoprotein gene. The induction of mutant lipoprotein production was lethal to the cells. A large amount of the prolipoprotein was accumulated in the outer membrane fraction. No protein of the size of the mature lipoprotein was detected. These results indicate that the prolipoprotein signal peptidase requires a glyceride modified cysteine residue at the cleavage site.     

Neither lipid modification nor processing of prolipoprotein is essential for the formation of murein-bound lipoprotein in Escherichia coli.

The relationship between the modification and processing of prolipoprotein and the formation of murein-bound lipoprotein has been investigated using Escherichia coli mutants altered in the signal sequence of prolipoprotein and an E. coli strain producing OmpF-Lpp hybrid protein. The glyceride-modified prolipoprotein in mutant lppT20 and in globomycin-treated wild-type strain were covalently attached to the peptidoglycan. Likewise, the unmodified prolipoproteins in mutants lppL20, lppV20, and lppG21 were attached to the peptidoglycan. The OmpF-Lpp hybrid protein that is processed but not modified with lipid due to the absence of the cysteine-containing modification site in the hybrid protein was also covalently linked to the peptidoglycan. These results indicate that neither lipid modification nor the processing of prolipoprotein is essential for the formation of murein-bound lipoprotein in E. coli. In contrast, introduction of a charged amino acid residue such as Asp or Arg at the 14th position of prolipoprotein affected not only the lipid modification and processing of the mutant prolipoprotein but also the formation of murein-bound lipoprotein. Replacement of the Gly14 with Glu or Lys partially affected the lipid modification and processing of prolipoprotein; the peptidoglycan of the lppE14 and lppK14 mutants contained a reduced amount of mature lipoprotein but no mutant prolipoprotein. In addition, lpp mutants A20I23I24 and A20I23K24 were found to be defective in both lipid modification/processing of prolipoprotein and the formation of murein-bound lipoprotein. The defective formation of murein-bound lipoprotein in the latter mutants may be related to an alteration in the secondary structure at the modification/processing site of the mutant prolipoproteins.     

Lethality of the covalent linkage between mislocalized major outer membrane lipoprotein and the peptidoglycan of Escherichia coli.

The major outer membrane lipoprotein (Lpp) of Escherichia coli possesses serine at position 2, which is thought to function as the outer membrane sorting signal, and lysine at the C terminus, through which Lpp covalently associates with peptidoglycan. Arginine (R) is present before the C-terminal lysine in the wild-type Lpp (LppSK). By replacing serine (S) at position 2 with aspartate (D), the putative inner membrane sorting signal, and by deleting lysine (K) at the C terminus, Lpp mutants with a different residue at either position 2 (LppDK) or the C terminus (LppSR) or both (LppDR) were constructed. Expression of LppSR and LppDR little affected the growth of E. coli. In contrast, the number of viable cells immediately decreased when LppDK was expressed. Prolonged expression of LppDK inhibited separation of the inner and outer membranes by sucrose density gradient centrifugation, whereas short-term expression did not. Pulse-labeled LppDK and LppDR were localized in the inner membrane, indicating that the amino acid residue at position 2 functions as a sorting signal for the membrane localization of Lpp. LppDK accumulated in the inner membrane covalently associated with the peptidoglycan and thus prevented the separation of the two membranes. Globomycin, an inhibitor of lipoprotein-specific signal peptidase II, was lethal for E. coli only when Lpp possessed the C-terminal lysine. Taken together, these results indicate that the inner membrane accumulation of Lpp per se is not lethal for E. coli. Instead, a covalent linkage between the inner membrane Lpp having the C-terminal lysine and the peptidoglycan is lethal for E. coli, presumably due to the disruption of the cell surface integrity.     

A novel periplasmic carrier protein involved in the sorting and transport of Escherichia coli lipoproteins destined for the outer membrane.

Lipoproteins are localized in the outer or inner membrane of Escherichia coli, depending on the species of amino acid located next to the N-terminal fatty acylated Cys. The major outer membrane lipoprotein (Lpp) expressed in spheroplasts was, however, retained in the inner membrane as a mature form. A novel protein that is essential for the release of Lpp from the inner membrane was discovered in the periplasm and purified. The partial amino acid sequence of this 20 kDa protein (p20) was determined and used to clone a gene for p20. Sequencing of the gene revealed that p20 is synthesized as a precursor with a signal sequence. p20 formed a soluble complex only with outer membrane-directed lipoproteins such as Lpp, indicating that p20 plays a critical role in the sorting of lipoproteins. Lpp released from the inner membrane in the presence of p20 was specifically assembled into the outer membrane in vitro. These results indicate that p20 is a periplasmic carrier protein involved in the translocation of lipoproteins from the inner to the outer membrane.     

Comparison of the lipoprotein gene among the enterobacteriaceae. DNA sequence of Morganella morganii lipoprotein gene and its expression in Escherichia coli

A DNA sequence of 532 base pairs encompassing the entire Morganella morganii lipoprotein gene (lpp) was determined. Sequence comparisons of the M. morganii lpp gene with the lpp genes from Escherichia coli, Serratia marcescens, and Erwinia amylovora reveal that the M. morganii lpp gene is more distantly related to the E. coli lpp gene than any of the other lpp genes examined. Between the E. coli and M. morganii lpp genes, the following homologies were found: 44% in the promoter region (bases, -45 to -1), 88% in the 5'-end untranslated region of the mRNA, 58% in the signal sequence coding region, 75% in the coding region for the first 51 and 43% for the last 7 amino acid residues. Upstream of the promoter region and downstream of the termination codon, there are extensive insertions, deletions, and base substitutions. In spite of the differences in the DNA sequences, the lipoprotein structure was found to be highly conserved except for the carboxyl-terminal sequence of 7 amino residues. The coding region of the M. morganii lpp gene including the signal sequence was inserted into an expression cloning vector so that the production of the M. morganii lipoprotein could be induced in E. coli by a lac inducer, isopropyl-beta-D-thioglactoside. It was found that when induced, the M. morganii prolipoprotein was apparently secreted normally across the E. coli cytoplasmic membrane, modified with glycerol and palmitic acid, processed to the mature lipoprotein, and assembled in the E. coli outer membrane. The bound form covalently linked to the peptidoglycan was also found.     

Overproduction of NlpE, a new outer membrane lipoprotein, suppresses the toxicity of periplasmic LacZ by activation of the Cpx signal transduction pathway.

The LamB-LacZ-PhoA tripartite fusion protein is secreted to the periplasm, where it exerts a toxicity of unknown origin during high-level synthesis in the presence of the inducer maltose, a phenotype referred to as maltose sensitivity. We selected multicopy suppressors of this toxicity that allow growth of the tripartite fusion strains in the presence of maltose. Mapping and subclone analysis of the suppressor locus identified a previously uncharacterized chromosomal region at 4.7 min that is responsible for suppression. DNA sequence analysis revealed a new gene with the potential to code for a protein of 236 amino acids with a predicted molecular mass of 25,829 Da. The gene product contains an amino-terminal signal sequence to direct the protein for secretion and a consensus lipoprotein modification sequence. As predicted from the sequence, the suppressor protein is labeled with [3H]palmitate and is localized to the outer membrane. Accordingly, the gene has been named nlpE (for new lipoprotein E). Increased expression of NlpE suppresses the maltose sensitivity of tripartite fusion strains and also the extracytoplasmic toxicities conferred by a mutant outer membrane protein, LamBA23D. Suppression occurs by activation of the Cpx two-component signal transduction pathway. This pathway controls the expression of the periplasmic protease DegP and other factors that can combat certain types of extracytoplasmic stress.     

Generalized transposon shuttle mutagenesis in Neisseria gonorrhoeae: a method for isolating epithelial cell invasion-defective mutants

Hybrid genes were constructed to express bifunctional hybrid proteins in which staphyloccal nuclease A with or without an amino-terminai OmpA signal sequence was fused with TEM β-lactamase (at the carboxyl terminal side) using the signal peptide of the major outer membrane lipoprotein of Escherichia coli as an internal linker. The hybrid proteins were found to be inserted in the membrane. Orientation of the hybrid protein with the OmpA signal peptide showed that the nuclease was translocated into the periplasm and the β-lactamase remained in the cytoplasm. This indicates that the cleavable OmpA signal peptide served as a secretory signal for nuclease and the internal lipoprotein signal served as the transmembrane anchor, in the absence of the OmpA signal sequence the topology of the hybrid protein was reversed indicating that the internal lipoprotein signal peptide initially served as the signal peptide for the secretion of the carboxy terminal β-lactamase domain across the membrane and subsequently as a membrane anchoring signal. The role of charged amino acids in the translocation and transmembrane orientation of membrane proteins was also analysed by introducing charged amino acids to either or both sides of the internal lipoprotein signal sequence in the bifunctional hybrid proteins in the absence of the amino-terminal signal sequence. Introduction of two lysine residues at the carboxy-terminal side of the internal signal sequence reversed the topology of the transmembrane protein by translocating the aminoterminal nuclease domain across the membrane, leaving the carboxyl terminal β-actamase domain in the cytoplasm. When three more lysine residues were added to the amino-terminal side of the internal signal sequence of the same construct the membrane topology flipped back to the original orientation. A similar reversion of the topology could be obtained by introducing negatively charged residues at the amino-terminal side of the internal signal sequence. Present results demonstrate for the first time that a bifunctional transmembrane protein can be engineered to assume either of the two opposite orientations and that charge balance around the transmembrane domain is a major factor in controlling the topology of a transmembrane protein.     

Mechanism underlying the inner membrane retention of Escherichia coli lipoproteins caused by Lol avoidance signals

Escherichia coli lipoproteins are localized to either the inner or outer membrane depending on the residue at position 2. The inner membrane retention signal, Asp at position 2 in combination with certain residues at position 3, functions as a Lol avoidance signal, i.e. the signal inhibits the recognition of lipoproteins by LolCDE that releases lipoproteins from the inner membrane. To understand the role of the residue at position 2, outer membrane-specific lipoproteins with Cys at position 2 were subjected to chemical modification followed by the release reaction in reconstituted proteoliposomes. Sulfhydryl-specific introduction of nonprotein molecules or a negative charge to Cys did not inhibit the LolCDE-dependent release. In contrast, oxidation of Cys to cysteic acid resulted in generation of the Lol avoidance signal, indicating that the Lol avoidance signal requires a critical length of negative charge at the second residue. Furthermore, not only modification of the carboxylic acid of Asp at position 2 but also that of the amine of phosphatidylethanolamine abolished the Lol avoidance function. Based on these results, the Lol avoidance mechanism is discussed.     

Function of the membrane fusion protein, MexA, of the MexA, B-OprM efflux pump in Pseudomonas aeruginosa without an anchoring membrane

Resistance of Pseudomonas aeruginosa to multiple species of antibiotics is largely attributable to expression of the MexA, B-OprM efflux pump. The MexA protein is thought to be located at the inner membrane and has been assumed to link the xenobiotics-exporting subunit, MexB, and the outer membrane channel protein, OprM. To verify this assumption, we analyzed membrane anchoring and localization of the MexA protein. n-[9, 10-(3)H]Palmitic acid incorporation experiments revealed that MexA was radiolabeled with palmitic acid, suggesting that the MexA anchors the inner membrane via the fatty acid moiety. To evaluate the role of lipid modification and inner membrane anchoring, we substituted cysteine 24 with phenylalanine or tyrosine and tested whether or not these mutant MexAs function properly. When the mutant mexAs were expressed in the strain lacking chromosomal mexA in the presence of n-[9,10-(3)H]palmitic acid, we found undetectable radiolabeling at the MexA band. These transformants restored antibiotic resistance to the level of the wild-type strain, indicating that lipid modification is not essential for MexA function. These mutant strains contained both processed and unprocessed forms of the MexA proteins. Cellular fractionation experiments revealed that an unprocessed form of MexA anchored the inner membrane probably via an uncleaved signal sequence, whereas the processed form was undetectable in the membrane fraction. To assure that the lipid-free MexA polypeptide could be unbound to the membrane, we analyzed the two-dimensional membrane topology by the gene fusion technique. A total of 78 mexA-blaM fusions covering the entire MexA polypeptide were constructed, and all fusion sites were shown to be located at the periplasm. To answer the question of whether or not membrane anchoring is essential for the MexA function, we replaced the signal sequence of the MexA protein with that of the azurin protein, which contains a cleavable signal sequence but no lipid modification site. The signal sequence of the azurin-MexA hybrid protein was properly processed and bore the mature MexA, which was fully recovered in the soluble fraction. The transformant, which expressed azurin-MexA hybrid protein restored the antibiotic resistance to a level indistinguishable from that of the wild-type strain. We concluded from these results that the MexA protein is fully functional as expressed in the periplasmic space without anchoring the inner membrane. This finding questioned the assumption that the membrane fusion proteins connect the inner and outer membranes.     

Molecular cloning of the gene encoding an outer-membrane-associated beta-N-acetylglucosaminidase involved in chitin degradation system of Alteromonas sp. strain O-7

The gene encoding beta-N-acetylglucosaminidase (GlcNAcaseA) was cloned using PCR with degenerate oligonucleotide primers from the partial amino acid sequence of the enzyme. The gene encoded a polypeptide of 863 amino acids with a predicted molecular mass of 97kDa. A characteristic signal peptide, which was present at the amino-terminus of the precursor protein, contained four amino acids (Ala-Gly-Cys-Ser) identical in sequence and location to the processing and modification sites of the outer membrane lipoprotein of Escherichia coli, indicating that the mature GlcNAcaseA is a lipoprotein the N-terminal cysteine residue of which would be modified by the fatty acid that anchors the protein in the membrane. The predicted amino acid sequence of GlcNAcaseA showed similarity to bacterial beta-N-acetylglucosaminidases belonging to the family 20 glycosyl hydrolases.