Evaluation of a structural model of Pseudomonas aeruginosa outer membrane protein OprM, an efflux component involved in intrinsic antibiotic resistance

The outer membrane protein OprM of Pseudomonas aeruginosa is involved in intrinsic and mutational multiple-antibiotic resistance as part of two resistance-nodulation-division efflux systems. The crystal structure of TolC, a homologous protein in Escherichia coli, was recently published (V. Koronakis, A. Sharff, E. Koronakis, B. Luisl, and C. Hughes, Nature 405:914-919, 2000), demonstrating a distinctive architecture comprising outer membrane beta-barrel and periplasmic helical-barrel structures, which assemble differently from the common beta-barrel-only conformation of porins. Based on their sequence similarity, a similar content of alpha-helical and beta-sheet structure determined by circular dichroism spectroscopy, and our observation that OprM, like TolC, reconstitutes channels in planar bilayer membranes, OprM and TolC were considered to be structurally homologous, and a model of OprM was constructed by threading its sequence to the TolC crystal structure. Residues thought to be important for the TolC structure were conserved in space in this OprM model. Analyses of deletion mutants and previously isolated insertion mutants of OprM in the context of this model allowed us to propose roles for different protein domains. Our data indicate that the helical barrel of the protein is critical for both the function and the integrity of the protein, while a C-terminal domain localized around the equatorial plane of this helical barrel is dispensable. Extracellular loops appear to play a lesser role in substrate specificity for this efflux protein compared to classical porins, and there appears to be a correlation between the change in antimicrobial activity for OprM mutants and the pore size. Our model and channel formation studies support the "iris" mechanism of action for TolC and permit us now to form more focused hypotheses about the functional domains of OprM and its related family of efflux proteins.     

Amino acids at positions 3 and 4 determine the membrane specificity of Pseudomonas aeruginosa lipoproteins

Escherichia coli lipoproteins with Asp at position 2 remain in the inner membrane, whereas those having other amino acids are targeted to the outer membrane by the Lol system. However, inner membrane lipoproteins without Asp at position 2 are found in other Gram-negative bacteria. MexA of Pseudomonas aeruginosa, an inner membrane-specific lipoprotein involved in multidrug efflux, has Gly at position 2. To identify the residue or region of MexA that functions as an inner membrane retention signal, we constructed chimeric lipoproteins comprising various regions of MexA and an outer membrane lipoprotein, OprM, and analyzed their membrane localization. Lys and Ser at positions 3 and 4, respectively, were found to be critical for the inner membrane localization of MexA in P. aeruginosa. Substitution of these residues with Leu and Ile, which are present in OprM, was sufficient to target the chimeric lipoprotein to the outer membrane and to abolish the ability of MexA to confer drug resistance. The membrane specificity of a model lipoprotein, lipoMalE, a lipidated variant of the periplasmic maltose-binding protein of E. coli, was also determined by the residues at positions 3 and 4 in P. aeruginosa. In contrast to the widely accepted "+2 rule" for E. coli lipoproteins, these results suggest a new "+3, +4 rule" for lipoprotein sorting in P. aeruginosa, namely, the final destination of lipoproteins is determined by the residues at positions 3 and 4.     

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.     

Analysis of the interaction of 16S rRNA and cytoplasmic membrane with the C-terminal part of the Streptococcus pneumoniae Era GTPase.

Era, an essential GTPase, plays a regulatory role in several cellular processes. The Era protein of Streptococcus pneumoniae has recently been shown to bind to 16S rRNA and the cytoplasmic membrane. However, exact locations of Era responsible for RNA- and membrane-binding were unknown. To identify the regions in Era that interact with the RNA and membrane, the C-terminal part of S. pneumoniae Era was systematically deleted while the N-terminal part, responsible for the GTPase activity of the protein, was kept intact. The resulting truncated Era proteins were purified and characterized. The C-terminal deletion of 9 or 19 amino-acid residues did not affect 16S rRNA-binding activity while further deletions of the C-terminus (29-114 amino-acid residues) abolished the activity. These results indicate that the integrity of the putative KH domain of Era, spanning the amino-acid residues between approximately 22-83 from the C-terminus, is required for 16S rRNA-binding. Furthermore, the Era proteins with a deletion up to 45 residues from the C-terminus retained membrane-binding activity, but longer deletions significantly reduced the activity. These results indicate that part of the putative KH domain is also required for membrane-binding. Thus, these results indicate for the first time that the regions critical for the membrane- and 16S rRNA-binding activities of Era overlap. The era gene with a deletion of 9 or 19 codons from its 3' terminus complemented an Escherichia coli mutant strain deficient in Era production whereas the genes with longer deletions failed to do so, thereby indicating that the KH domain is essential for Era function. Taken together, the results of this study indicate that the putative KH domain is required for 16S rRNA-binding activity and that part of the KH domain is also required for membrane-binding activity. The results also suggest that the interaction between Era and 16S rRNA is essential for bacterial growth.     

Overexpression, refolding, and purification of the histidine-tagged outer membrane efflux protein OprM of Pseudomonas aeruginosa

This paper describes the overproduction and purification of the C-terminus polyhistidine-tagged outer membrane protein OprM, which is a part of the MexA-MexB-OprM active efflux system of Pseudomonas aeruginosa. Renaturation of the protein from inclusion bodies of Escherichia coli was achieved using guanidine-HCl as denaturing agent and n-octylpolyoxyethylene (C8POE) and n-octyltetraoxyethylene (C8E4) as nonionic detergents. The refolded protein was purified by ion-exchange and nickel-affinity chromatography. The final yield was 6 mg of pure histidine-tagged OprM per liter of E. coli culture. Renaturation was monitored by the effects of heating prior to SDS-PAGE, using a typical and exclusive property of outer membrane proteins. Immunoblotting revealed that the recombinant protein is addressed to the outer membrane of E. coli, after maturation by excision of its N-terminal signal sequence. Complementation of an oprM deletion mutant with the plasmid encoded histidine-tagged OprM protein restored antibiotic susceptibilities to wild-type levels, demonstrating functionality of recombinant OprM.     

The C-terminal Domain of the Escherichia coli WaaJ glycosyltransferase is important for catalytic activity and membrane association

The waaJ gene encodes an alpha-1,2-glucosyltransferase involved in the synthesis of the outer core region of the lipopolysaccha-ride of some Escherichia coli and Salmonella isolates. WaaJ belongs to glycosyltransferase CAZy family 8, characterized by the GT-A fold, a DXD motif, and by retention of configuration at the anomeric carbon of the donor sugar. Detailed kinetic and structural information for bacterial family 8 glycosyltransferases has resulted from studies of Neisseria meningitidis LgtC. As many as 28 amino acids could be deleted from the C terminus of LgtC without affecting its in vitro catalytic behavior. This C-terminal domain has a high ratio of positively charged and hydrophobic residues, a feature conserved in WaaJ and some other family 8 representatives. Unexpectedly, deletion of as few as five residues from the C terminus of WaaJ resulted in substantially reduced in vivo activity. With deletions of 15 residues or less, activity was only detected when levels of expression were elevated. No in vivo activity was detected after the removal of 20 amino acids, regardless of expression levels. Longer deletions (20 residues and greater) compromised the ability of WaaJ to associate with the membrane. However, the reduced in vivo activity in enzymes lacking 5-12 C-terminal residues also reflected a dramatic drop in catalytic activity in vitro (a 294-fold decrease in the apparent kcat/Km,LPS). Deletions removing 20 or more residues resulted in a protein showing no detectable in vitro activity. Therefore, the C-terminal domain of WaaJ plays a critical role in enzyme function.     

Analysis of E1A-mediated growth regulation functions: binding of the 300-kilodalton cellular product correlates with E1A enhancer repression function and DNA synthesis-inducing activity.

Adenovirus E1A transforming function requires two distinct regions of the protein. Transforming activity is closely linked with the presence of a region designated conserved domain 2 and the ability of this region to bind the product of the cellular retinoblastoma tumor suppressor gene. We have investigated the biological properties of the second transforming region of E1A, which is located near the N terminus. Transformation-defective mutants containing deletions in the N terminus (deletion of residues between amino acids 2 and 36) were deficient in the ability to induce DNA synthesis and repress insulin enhancer-stimulated activity. The function of the N-terminal region correlated closely with binding of the 300-kilodalton E1A-associated protein and not with binding of the retinoblastoma protein. These results indicate that transformation by E1A is mediated by two functionally independent regions of the protein which interact with different specific cellular proteins and suggest that the 300-kilodalton E1A-associated protein plays a major role in E1A-mediated cell growth control mechanisms.     

Role of the membrane fusion protein in the assembly of resistance-nodulation-cell division multidrug efflux pump in Pseudomonas aeruginosa

The tripartite xenobiotic-antibiotic transporter of Pseudomonas aeruginosa consists of the inner membrane transporter (e.g., MexB, MexY), the periplasmic membrane-fusion-protein (e.g., MexA, MexX), and the outer membrane channel protein (e.g., OprM). These subunits were assumed to assemble into a transporter unit during export of the substrates. However, subunit interaction and their specificity in native form remained to be elucidated. To address these important questions, we analyzed the role of the individual subunits for the assembly of MexAB-OprM by pull-down assay tagging only one of the subunits. We found stable MexA-MexB-OprM complex without chemical cross-linking that withstand all purification procedures. Results of bi-partite interactions analysis showed tight association between MexA and OprM in the absence of MexB, whereas the expression systems lacking MexA failed to co-purify MexB or OprM. None of the heterologous subunit combinations such as MexA+MexY(his)+OprM and MexX+MexB(his)+OprM showed interaction. These results implied that the membrane fusion protein is central to the tripartite xenobiotic transporter assembly.     

Regions of toxin A involved in toxin A excretion in Pseudomonas aeruginosa.

Toxin A is excreted by Pseudomonas aeruginosa as a mature 66,583-dalton protein. In this study, we used molecular cloning and deletion analysis to define specific regions of the toxin molecule involved in its excretion. Subclones that express either the amino terminus, the carboxy terminus, or toxin A molecules with internal deletions were constructed. The hypotoxigenic mutant PAO-T1 was used as a host for the expression of the toxin constructs. When overexpressed (by the presence of extra copies of the toxin A-positive regulatory gene, regA, in trans), toxin A-cross-reactive materials produced by most of these constructs were detected in the supernatant of PAO-T1. The supernatant of P. aeruginosa PAO-T1 contained proteolytic activity that degraded toxin A-derived products but not the intact toxin molecule. A single SalI intragenic deletion (coding for the leader peptide, the first 30 amino acids, and the last 305 amino acids of the toxin) resulted in a relatively stable product in the supernatant of PAO-T1. The product of the carboxy terminus construct (which codes for the last 305 amino acids of the toxin) was detected in the lysate of PAO-T1 only. The data suggest that the amino terminus region of toxin A (the leader peptide plus the first 30 amino acid of the mature protein) is sufficient for its excretion, and that a second region, amino acids 309 through 413, protects an internally truncated toxin A molecule from the proteolytic activity in the supernatant of P. aeruginosa PAO-T1.     

The outer membrane component of the multidrug efflux pump from Pseudomonas aeruginosa may be a gated channel.

OprM, the outer membrane component of the MexAB-OprM multidrug efflux pump of Pseudomonas aeruginosa, has been assumed to facilitate the export of antibiotics across the outer membrane of this organism. Here we purified to homogeneity the OprM protein, reconstituted it into liposome membranes, and tested its channel activity by using the liposome swelling assay. It was demonstrated that OprM is a channel-forming protein and exhibits the channel property that amino acids diffuse more efficiently than saccharides. However, antibiotics showed no significant diffusion through the OprM channel in the liposome membrane, suggesting that OprM functions as a gated channel. We reasoned that the protease treatment may cause the disturbance of the gate structure of OprM. Hence, we treated OprM reconstituted in the membranes with alpha-chymotrypsin and examined its solute permeability. The results demonstrated that the protease treatment caused the opening of an OprM channel through which antibiotics were able to diffuse. To elucidate which cleavage is intimately related to the opening, we constructed mutant OprM proteins where the amino acid at the cleavage site was replaced with another amino acid. By examining the channel activity of these mutant proteins, it was shown that the proteolysis at tyrosine 185 and tyrosine 196 of OprM caused the channel opening. Furthermore, these residues were shown to face into the periplasmic space and interact with other component(s). We considered the possible opening mechanism of the OprM channel based on the structure of TolC, a homologue of OprM.     

Interchangeable enzyme modules. Functional replacement of the essential linker of the biotinylated subunit of acetyl-CoA carboxylase with a linker from the lipoylated subunit of pyruvate dehydrogenase

Biotin carboxyl carrier protein (BCCP) is the small biotinylated subunit of Escherichia coli acetyl-CoA carboxylase, the enzyme that catalyzes the first committed step of fatty acid synthesis. E. coli BCCP is a member of a large family of protein domains modified by covalent attachment of biotin. In most biotinylated proteins, the biotin moiety is attached to a lysine residue located about 35 residues from the carboxyl terminus of the protein, which lies in the center of a strongly conserved sequence that forms a tightly folded anti-parallel beta-barrel structure. Located upstream of the conserved biotinoyl domain sequence are proline/alanine-rich sequences of varying lengths, which have been proposed to act as flexible linkers. In E. coli BCCP, this putative linker extends for about 42 residues with over half of the residues being proline or alanine. I report that deletion of the 30 linker residues located adjacent to the biotinoyl domain resulted in a BCCP species that was defective in function in vivo, although it was efficiently biotinylated. Expression of this BCCP species failed to restore normal growth and fatty acid synthesis to a temperature-sensitive E. coli strain that lacks BCCP when grown at nonpermissive temperatures. In contrast, replacement of the deleted BCCP linker with a linker derived from E. coli pyruvate dehydrogenase gave a chimeric BCCP species that had normal in vivo function. Expression of BCCPs having deletions of various segments of the linker region of the chimeric protein showed that some deletions of up to 24 residues had significant or full biological activity, whereas others had very weak or no activity. The inactive deletion proteins all lacked an APAAAAA sequence located adjacent to the tightly folded biotinyl domain, whereas deletions that removed only upstream linker sequences remained active. Deletions within the linker of the wild type BCCP protein also showed that the residues adjacent to the tightly folded domain play an essential role in protein function, although in this case some proteins with deletions within this region retained activity. Retention of activity was due to fusion of the domain to upstream sequences. These data provide new evidence for the functional and structural similarities of biotinylated and lipoylated proteins and strongly support a common evolutionary origin of these enzyme subunits.     

Characterization of the structure of a low Km, rolipram-sensitive cAMP phosphodiesterase. Mapping of the catalytic domain.

Considerable structural similarities are present in a region of approximately 270 amino acids in most known cyclic nucleotide phosphodiesterase (PDE) sequences, opening the possibility that this region encodes the catalytic domain of the enzyme. To test this hypothesis, the structure of a high affinity cAMP PDE (cAMP-PDE) was analyzed by deletion mutations and site-directed mutagenesis. A ratPDE3 cDNA was mutated using a strategy based on fragment amplification by polymerase chain reaction. The effect of the introduced mutations was determined by expressing wild type and mutated proteins in prokaryotic and eukaryotic cells. The level of expression of the PDE protein was monitored by immunoblot analysis using two specific cAMP-PDE polyclonal antibodies and by measuring the PDE activity. After removal of a 99-amino acid region at the carboxyl terminus flanking the conserved domain, the protein retains its catalytic activity even though its Km and velocity were changed. Internal deletions at the amino terminus of this PDE showed that the enzyme activity was increased when a 97-amino acid fragment (from Tyr49 to Lys145) was removed. Further deletions within the amino terminus produced inactive proteins. Within the domain that appears essential for catalysis, 1 threonine and 2 serine residues are conserved in all PDEs. Substitutions of the invariant threonine (Thr349) present in the most conserved region with alanine, proline, or serine yielded proteins of the correct size and a level of expression comparable to the wild type PDE. However, in both expression systems used, proteins were completely devoid of the ability to hydrolyze cyclic nucleotides, except when the threonine was substituted with a serine. Conversely, mutations of 2 other conserved serine residues (Ser305 and Ser398) present in the catalytic domain either had no effect or produced changes only in Km and Vmax, but did not abolish catalytic activity. In addition, 2 histidine residues (His278 and His311) present in proximity to Thr349 appeared to be essential for the structure of the catalytic domain, since any substitution performed in these residues yielded an inactive enzyme. Mutations of a serine residue (Ser295) in the region homologous to the cAMP binding site of the regulatory subunit of the cAMP-dependent protein kinase demonstrated that this region does not have the same function in the two proteins. These data provide direct evidence that a 37-kDa domain, which in part corresponds to the region of conservation in all PDEs, contains the catalytic domain, and that threonine and histidine residues are probably involved in catalysis and/or are essential for the conformation of an active enzyme.     

Analysis of the conserved cysteine periodicity of Paramecium variable surface antigens

The major surface antigens expressed by free-living and parasitic protozoa commonly contain repeating cysteine motifs. Despite the common occurrence of these repeats their functional significance remains largely unexplored. In this paper we investigate the conserved cysteine repeats within the variable surface antigens of Paramecium tetraurelia. We show that deletion of 2 entire repeating units or portions of repeats near the N-terminus does not prevent expression of the A51 variable surface antigen. Alteration of a single cysteine to serine residue also has no effect on A51 expression. In contrast, deletions near the C-terminus of the protein have identified a small segment within the repeats that is required for expression on the surface. The required region contains a number of conserved amino acid residues, yet site-directed mutagenesis of two residues (serine and threonine to alanine) did not prevent expression. These studies demonstrate the feasibility of using deletion analysis to identify regions critical for the expression of cysteine-rich surface antigens. The relationship of these results to the structure and expression of cysteine-rich surface proteins in other protozoa is discussed.     

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.     

Localization of the active site of the alpha subunit of the Escherichia coli DNA polymerase III holoenzyme.

Using a deletion approach on the alpha subunit of DNA polymerase III from Escherichia coli, we show that there is an N-proximal polymerase domain which is distinct from a more C-proximal tau and beta binding domain. Although deletion of 60 residues from the alpha N terminus abolishes polymerase activity, deletions of 48, 169, and 342 amino acids from the C terminus progressively impair its catalytic efficiency but preserve an active site. Deletion of 342 C-terminal residues reduces k(cat) 46-fold, increases the Km for gapped DNA 5.5-fold, and increases the Km for deoxynucleoside triphosphates (dNTPs) twofold. The 818-residue protein with polymerase activity displays typical Michaelis-Menten behavior, catalyzing a polymerase reaction that is saturable with substrate and linear with time. With the aid of newly acquired sequences of the polymerase III alpha subunit from a variety of organisms, candidates for two key aspartate residues in the active site are identified at amino acids 401 and 403 of the E. coli sequence by inspection of conserved acidic amino acids. The motif Pro-Asp-X-Asp, where X is a hydrophobic amino acid, is shown to be conserved among all known DnaE proteins, including those from Bacillaceae, cyanobacteria, Mycoplasma, and mycobacteria. The E. coli DnaE deletion protein with only the N-terminal 366 amino acids does not have polymerase activity, consistent with the proposed position of the active-site residues.     

Mechanism of signal peptide cleavage in the biosynthesis of the major lipoprotein of the Escherichia coli outer membrane

On treatment of Escherichia coli cells with globomycin, a glyceride-containing precursor of the major outer membrane lipoprotein accumulates in the cytoplasmic membrane (Hussain, M., Ichihara, S., and Mizushima, S. (1980) J. Biol. Chem. 255, 3707-3712). When the envelope fraction from such cells was incubated in a suitable buffer, this precursor could be processed to the mature lipoprotein. The processing involved removal of the signal peptide and subsequent acylation of the NH2 terminus thus bared. Two types of peptidase and an acylation enzyme(s) were found to be involved in these processes. The enzyme that cleaves the signal peptide, called signal peptidase in this paper, had many unique properties: being highly resistant to high temperature, having a wide optimum pH range, and being highly sensitive to detergents. The other peptidase(s), called signal peptide peptidase in this paper, was assumed to be responsible for the digestion of the signal peptide that had been cleaved from the precursor lipoprotein. This enzyme was rather heat-sensitive. Thus the processing from the precursor to the mature lipoprotein at a high temperature resulted in accumulation of a peptide that was most probably the intact signal peptide. The third enzyme(s) involved in the processing was the one that is responsible for acylation of the newly bared NH2 terminus of the lipoprotein. The enzyme activity was also lost at 80 degrees C. In the light of these findings, the biosynthetic pathway of the lipoprotein is discussed.     

Molecular analysis of the Escherichia coli ferric enterobactin receptor FepA

In Escherichia coli, the outer membrane protein FepA is a receptor for the siderophore complex ferric enterobactin and for colicins B and D. To identify protein domains important for FepA activity, the effects of deletion and linker insertion mutations on receptor structure and function were examined. In-frame internal deletion mutations removing sequences encoding up to 304 amino acid residues resulted in functionally defective FepA polypeptides, although most were translocated efficiently to the outer membrane. One exception, a derivative lacking 87 internal amino acid residues near the N terminus, showed an inability to transport ferric enterobactin but retained limited colicin receptor function. Analysis of cells carrying 3'-terminal fepA deletion mutations suggested that residues within the C terminus of FepA may be involved in secretion and proper translocation of the protein to the outer membrane. Introduction of the peptide Leu-Glu after FepA residues 55, 142, or 324 severely impaired receptor function for all three ligands, while the same insertion after residues 339 or 359 had virtually no detrimental effect on FepA function. Foreign peptides inserted after residues 204 or 635 restricted colicin B and D function only, leaving ferric enterobactin transport ability at near wild-type levels. The results presented in this study have identified key regions of FepA potentially involved in receptor function and demonstrate the presence of both shared and unique ligand-responsive domains.     

Information within the mature LamB protein necessary for localization to the outer membrane of E coli K12

It has been proposed that the efficient localization of the outer membrane protein LamB requires a functional signal sequence and at least two additional regions contained within the mature protein. We define these regions more precisely by deletion analysis, and we describe methods for cloning deleterious lacZ fusions onto high-copy-number plasmids and generating in-frame deletions. Analysis of the effects of a series of internal lamB deletions on the export of a LamB-LacZ hybrid protein and of the LamB protein itself indicates that necessary informational signal(s) required for localization lie at the amino-terminal end of the protein. In addition, our analysis indicates that there is a region of information close to or within the fusion joint of the largest lamB-lacZ fusion that increases the efficiency of the export process. A unique deletion that removes a protein segment from amino acid 70 to 200 appears to prevent proteolytic removal of the signal sequence. Nevertheless, the mutant protein is exported to the outer membrane.