A vector for the construction of translational fusions to TEM beta-lactamase and the analysis of protein export signals and membrane protein topology

A plasmid vector, pJBS633, that facilitates the construction of translational fusions of genes of interest to the coding region of the mature form of TEM beta-lactamase has been developed. Transformants containing in-frame fusions can be identified by their ability to grow when plated at high inocula on agar containing ampicillin (Ap). The cellular location of the beta-lactamase moiety of the fusion proteins can then be determined since only those that direct the translocation of the beta-lactamase across the cytoplasmic membrane to the periplasm result in the ability of individual cells of Escherichia coli to form isolated colonies in the presence of Ap. Conversely, those fusion proteins in which the beta-lactamase moiety remains cytoplasmic do not protect individual cells against Ap. Transformants expressing the latter class of fusion proteins can, however, be identified when plated at high inocula since, as cells start to lyse, the cytoplasmic beta-lactamase activity is released and provides Ap resistance to the surrounding cells. The vector contains the origin of replication of f1 phage so that single-stranded plasmid DNA can be obtained in the appropriate orientation to allow sequencing across the fusion junction using a universal primer complementary to the start of the coding region of mature TEM beta-lactamase. pJBS633 should be useful as a general vector for the construction of beta-lactamase fusions and, in particular, for the analysis of protein export signals and the determination of the organisation of proteins in the E. coli cytoplasmic membrane.     

Induction of beta-lactamase influences the course of development in Myxococcus xanthus.

Myxococcus xanthus is a gram-negative bacterium that develops in response to starvation on a solid surface. The cells assemble into multicellular aggregates in which they differentiate from rod-shaped cells into spherical, environmentally resistant spores. Previously, we have shown that the induction of beta-lactamase is associated with starvation-independent sporulation in liquid culture (K. A. O'Connor and D. R. Zusman, Mol. Microbiol. 24:839-850, 1997). In this paper, we show that the chromosomally encoded beta-lactamase of M. xanthus is autogenously induced during development. The specific activity of the enzyme begins to increase during aggregation, before spores are detectable. The addition of inducers of beta-lactamase in M. xanthus, such as ampicillin, D-cycloserine, and phosphomycin, accelerates the onset of aggregation and sporulation in developing populations of cells. In addition, the exogenous induction of beta-lactamase allows M. xanthus to fruit on media containing concentrations of nutrients that are normally too high to support development. We propose that the induction of beta-lactamase is an integral step in the development of M. xanthus and that this induction is likely to play a role in aggregation and in the restructuring of peptidoglycan which occurs during the differentiation of spores. In support of this hypothesis, we show that exogenous induction of beta-lactamase can rescue aggregation and sporulation of certain mutants. Fruiting body spores from a rescued mutant are indistinguishable from wild-type fruiting body spores when examined by transmission electron microscopy. These results show that the signal transduction pathway leading to the induction of beta-lactamase plays an important role in aggregation and sporulation in M. xanthus.     

Folding and aggregation of beta-lactamase in the periplasmic space of Escherichia coli

High level expression of TEM beta-lactamase results in the accumulation of precursor and mature protein in the insoluble fraction of Escherichia coli. The mature polypeptide is sequestered in protein aggregates (inclusion bodies) located within the periplasmic space whereas the insoluble precursor is present in the cytoplasm. With the native beta-lactamase, aggregation is observed when the rate of expression exceeds 2.5% of the total protein synthesis rate. Substitution of the native signal sequence with the outer membrane protein A (OmpA) leader peptide results in extensive aggregation of only the mature protein. Furthermore, for OmpA-beta-lactamase, the accumulation of mature insoluble protein is independent of the rate of protein synthesis. These observations cannot be accounted by the kinetics of export of the OmpA-beta-lactamase and the native precursor, therefore suggesting that the signal sequence affects the conformation of the newly secreted mature polypeptide and in turn, the folding pathway. Previously, we have shown that the aggregation of the mature protein secreted using its own signal sequence can be inhibited by growing the cells in the presence of non-metabolizable sugars such as sucrose (Bowden, G., and Georgiou, G. (1988) Biotechnol. Prog. 4, 97-101). We show here that this phenomenon is not related to osmotic effects, changes in beta-lactamase translation or precursor processing. It follows that the addition of sugars exerts a direct effect on the in vivo pathway of aggregation and folding, in analogy with the well characterized effect of sugars in vitro.     

Membrane topology of penicillin-binding protein 3 of Escherichia coli

The beta-lactamase fusion vector, pJBS633, has been used to analyse the organization of penicillin-binding protein 3 (PBP3) in the cytoplasmic membrane of Escherichia coli. The fusion junctions in 84 in-frame fusions of the coding region of mature TEM beta-lactamase to random positions within the PBP3 gene were determined. Fusions of beta-lactamase to 61 different positions in PBP3 were obtained. Fusions to positions within the first 31 residues of PBP3 resulted in enzymatically active fusion proteins which could not protect single cells of E. coli from killing by ampicillin, indicating that the beta-lactamase moieties of these fusion proteins were not translocated to the periplasm. However, all fusions that contained greater than or equal to 36 residues of PBP3 provided single cells of E. coli with substantial levels of resistance to ampicillin, indicating that the beta-lactamase moieties of these fusion proteins were translocated to the periplasm. PBP3 therefore appeared to have a simple membrane topology with residues 36 to the carboxy-terminus exposed on the periplasmic side of the cytoplasmic membrane. This topology was confirmed by showing that PBP3 was protected from proteolytic digestion at the cytoplasmic side of the inner membrane but was completely digested by proteolytic attack from the periplasmic side. PBP3 was only inserted in the cytoplasmic membrane at its amino terminus since replacement of its putative lipoprotein signal peptide with a normal signal peptide resulted in a water-soluble, periplasmic form of the enzyme. The periplasmic form of PBP3 retained its penicillin-binding activity and appeared to be truly water-soluble since it fractionated, in the absence of detergents, with the expected molecular weight on Sephadex G-100 and was not retarded by hydrophobic interaction chromatography on Phenyl-Superose.     

Secretion cloning vectors in Escherichia coli

The DNA fragment coding for the signal peptide of the OmpA protein, a major outer membrane protein of Escherichia coli, has been inserted into the high-level expression vectors, pIN-III. A foreign DNA fragment can be cloned in any one of the three reading frames at the unique EcoRI, HindIII or BamHI sites immediately after the ompA signal peptide coding sequence. The cloned foreign gene is under the control of both the lpp promoter and the lac promoter-operator. The expression of the gene is regulated by the lac repressor produced by the same vectors. Using the pIN-III-ompA vector, the DNA fragment coding for only the mature portion of beta-lactamase was inserted into the EcoRI site. Upon induction of gene expression, beta-lactamase was secreted into the periplasmic space. The ompA signal peptide was correctly removed resulting in the production of beta-lactamase with four extra amino acid residues (Gly-Ile-Pro-Gly) at its amino terminus due to the linker sequence in the vector. After a 3-h induction, beta-lactamase was accumulated to 20% of total cellular protein without any detectable accumulation of pro-beta-lactamase. Using oligonucleotide-directed site-specific mutagenesis, we have also removed the linker sequence and upon induction of gene expression, beta-lactamase with the authentic NH2-terminal sequence was produced, in even larger amounts than the beta-lactamase with the linker sequence.     

Identification of amino acid sequences that can function as translocators of β-lactamase in Escherichia coli

A plasmid vector, pYZ1, was constructed which lacks most of the beta-lactamase signal-peptide coding region, but has a unique EcoRI site spanning codons 2 and 3 of the resultant cytoplasmic beta-lactamase derivative. Short quasi-random DNA sequences were cloned into the EcoRI site and Escherichia coli transformants in which some translocation of beta-lactamase across the cytoplasmic membrane was restored were selected by their ability to survive and form colonies on plates containing a low level of ampicillin. About 15-20% of all in-frame inserts restored some beta-lactamase translocation and the salient feature of these sequences was their marked hydrophobicity. These results are discussed in the light of a similar study in which sequences able to function as translocators of invertase in yeast were cloned and analysed (Kaiser et al., 1987).     

Expression in Escherichia coli of the carboxy terminal domain of the BLAR sensory-transducer protein of Bacillus licheniformis as a water-soluble Mr 26 000 penicillin-binding protein

A cloning vector has been constructed which allows production and export by Escherichia coli of the Met346-Arg601 carboxy terminal domain of the 601 amino acid BLAR sensory-transducer involved in beta-lactamase inducibility in Bacillus licheniformis. The polypeptide, referred to as BLAR-CTD, accumulates in the periplasm of E. coli in the form of a water-soluble, Mr 26,000 penicillin-binding protein. These data and homology searches suggest that BLAR has a membrane topology similar to that of other sensory-transducers involved in chemotaxis.     

Folding and aggregation of TEM beta-lactamase: analogies with the formation of inclusion bodies in Escherichia coli.

The enzyme TEM beta-lactamase has been used as a model for understanding the pathway leading to formation of inclusion bodies in Escherichia coli. The equilibrium denaturation of TEM beta-lactamase revealed that an intermediate that has lost enzymatic activity, native protein fluorescence, and UV absorption, but retains 60% of the native circular dichroism signal, becomes populated at intermediate (1.0-1.4 M) concentrations of guanidium chloride (GdmCl). This species exhibits a large increase in bis-1-anilino-8-naphthalene sulfonic acid fluorescence, indicating the presence of exposed hydrophobic surfaces. When TEM beta-lactamase was unfolded in different initial concentrations of GdmCl and refolded to the same final conditions by dialysis a distinct minimum in the yield of active protein was observed for initial concentrations of GdmCl in the 1.0-1.5 M range. It was shown that the lower reactivation yield was solely due to the formation of noncovalently linked aggregates. We propose that the aggregation of TEM beta-lactamase involves the association of a compact state having partially exposed hydrophobic surfaces. This hypothesis is consistent with our recent findings that TEM beta-lactamase inclusion bodies contains extensive secondary structure (Przybycien TM, Dunn JP, Valax P, Georgiou G, 1994, Protein Eng 7:131-136). Finally, we have also shown that protein aggregation was enhanced at higher temperatures and in the presence of 5 mM dithiothreitol and was inhibited by the addition of sucrose. These conditions exert a similar effect on the formation of inclusion bodies in vivo.     

Export of beta-lactamase is independent of the signal recognition particle

In Escherichia coli, three different types of proteins engage the SecY translocon of the inner bacterial membrane for translocation or insertion: 1) polytopic membrane proteins that prior to their insertion into the membrane are targeted to the translocon using the bacterial signal recognition particle (SRP) and its receptor; 2) secretory proteins that are targeted to and translocated across the SecY translocon in a SecA- and SecB-dependent reaction; and 3) membrane proteins with large periplasmic domains, requiring SRP for targeting and SecA for the translocation of the periplasmic moiety. In addition to its role as a targeting device for membrane proteins, a function of the bacterial SRP in the export of SecB-independent secretory proteins has also been postulated. In particular, beta-lactamase, a hydrolytic enzyme responsible for cleavage of the beta-lactam ring containing antibiotics, is considered to be recognized and targeted by SRP. To examine the role of the SRP pathway in beta-lactamase targeting and export, we performed a detailed in vitro analysis. Chemical cross-linking and membrane binding assays did not reveal any significant interaction between SRP and beta-lactamase nascent chains. More importantly, membrane vesicles prepared from mutants lacking a functional SRP pathway did block the integration of SRP-dependent membrane proteins but supported the export of beta-lactamase in the same way as that of the SRP-independent protein OmpA. These data demonstrate that in contrast to previous results, the bacterial SRP is not involved in the export of beta-lactamase and further suggest that secretory proteins of Gram-negative bacteria in general are not substrates of SRP.     

Use of a beta-lactamase fusion vector to investigate the organization of penicillin-binding protein 1B in the cytoplasmic membrane of Escherichia coli

The coding region for the mature form of TEM beta-lactamase was fused to random positions within the coding region of the penicillin-binding protein 1B (PBP 1B) gene and the nucleotide sequences across the fusion junctions of 100 in-frame fusions were determined. All fusion proteins that contained at least the NH2-terminal 94 residues of PBP 1B provided individual cells of E. coli with substantial levels of ampicillin resistance, suggesting that the beta-lactamase moiety had been translocated to the periplasm. Fusion proteins that contained less than or equal to 63 residues of PBP 1B possessed beta-lactamase activity, but could not protect single cells of E. coli from ampicillin, indicating that the beta-lactamase moiety of these fusion proteins remained in the cytoplasm. The beta-lactamase fusion approach suggested a model for the organization of PBP 1B in which the protein is embedded in the cytoplasmic membrane by a single hydrophobic transmembrane segment (residues 64-87), with a short NH2-terminal domain (residues 1-63), and the remainder of the polypeptide (residues 88-844) exposed on the periplasmic side of the cytoplasmic membrane. The proposed model for the organization of PBP 1B was supported by experiments which showed that the protein was completely digested by proteinase K added from the periplasmic side of the cytoplasmic membrane but was only slightly reduced in size by protease attack from the cytoplasmic side of the membrane.     

Membrane topology of the ArsB protein, the membrane subunit of an anion-translocating ATPase.

The ars operon of the conjugative R-factor R773 encodes an oxyanion pump that catalyzes extrusion of arsenicals from cells of Escherichia coli. The oxyanion translocation ATPase is composed of two polypeptides, the catalytic ArsA protein and the intrinsic membrane protein, ArsB. The topology of regions of the ArsB protein in the inner membrane was determined using a variety of gene fusions. Random gene fusions with lacZ and phoA were generated using transposon mutagenesis. A series of gene fusions with blaM were constructed in vitro using a beta-lactamase fusion vector. To localize individual segments of the ArsB protein, a ternary fusion method was developed, where portions of the arsB gene were inserted in-frame between the coding regions for two heterologous proteins, in this case a portion of a newly identified arsD gene and the blaM sequence encoding the mature beta-lactamase. The location of a periplasmic loop was determined from V8 protease digestion of an ArsA-ArsB chimera. From analysis of data from 26 fusions, a topological model of the ArsB protein with 12 membrane-spanning regions is proposed.     

Correct insertion of a simple eukaryotic plasma-membrane protein into the cytoplasmic membrane of Escherichia coli

A genetic system for directly synthesizing eukaryotic membrane proteins in Escherichia coli and assessing their ability to insert into the bacterial cytoplasmic membrane is described. The components of this system are the direct expression vector, pYZ4, and the mature beta-lactamase (BlaM) cassette plasmid, pYZ5, that can be used to generate translational fusions of BlaM to any synthesized membrane protein. The beta-subunit of sheep-kidney Na,K-ATPase (beta NKA), a class-II plasma membrane protein, was synthesized in E. coli using pYZ4, and BlaM was fused to a normally extracellular portion of it. The fusion protein conferred ampicillin resistance on individual host cells, indicating that the BlaM portion had been translocated to the bacterial periplasm, and that, by inference, the eukaryotic plasma-membrane protein can insert into the bacterial cytoplasmic membrane. A series of 31 beta NKA::BlaM fusion proteins was isolated and characterised to map the topology of the eukaryotic plasma membrane protein with respect to the bacterial cytoplasmic membrane. This analysis revealed that the organisation of the beta NKA in the E. coli cytoplasmic membrane was indistinguishable from that in its native plasma membrane.     

Evidence for posttranslational translocation of beta-lactamase across the bacterial inner membrane

Secretion of beta-lactamase was studied in Salmonella typhimurium infected with P22 phage carrying wild-type and mutant alleles of the structural gene. Cellular location of precursor and mature products of wild-type and temperature-sensitive and chain-terminating mutants was analyzed by cell fractionation and by trypsin accessibility in intact and lysed spheroplasts. The precursors of wild-type and all these mutants (none of which alter the signal peptide) are found sequestered within the cell, while all the mature forms have at least partially been translocated across the inner membrane. Thus most beta-lactamase molecules traverse the membrane after completion of their translation. It seems that the carboxyl terminus of beta-lactamase is not required for translocation across the inner membrane but is required for the protein to appear in the periplasm as a soluble species.     

β-lactamase as a probe of membrane protein assembly and protein export

The enzyme TEM beta-lactamase constitutes a versatile gene-fusion marker for studies on membrane proteins and protein export in bacteria. The mature form of this normally periplasmic enzyme displays readily detectable and distinctly different phenotypes when localized to the bacterial cytoplasm versus the periplasm, and thus provides a useful alternative to alkaline phosphatase for probing the topology of cytoplasmic membrane proteins. Cells producing translocated forms of beta-lactamase can be directly selected as ampicillin-resistant colonies, and consequently a beta-lactamase fusion approach can be used for positive selection for export signals, and for rapid assessment of whether any protein expressed in Escherichia coli inserts into the bacterial cytoplasmic membrane. The level of ampicillin resistance conferred on a cell by an extracytoplasmic beta-lactamase derivative depends on its level of expression, and therefore a beta-lactamase fusion approach can be used to directly select for increased yields of any periplasmic or membrane-bound gene products expressed in E. coli.     

Penetratin-induced aggregation and subsequent dissociation of negatively charged phospholipid vesicles

The interaction of the cellular delivery vector penetratin with a model system consisting of negatively charged phospholipid vesicles has been studied. Above a certain peptide to lipid molar ratio, the cationic oligopeptide induces vesicle aggregation. Interestingly, the aggregation is followed by spontaneous disaggregation, which may be related to membrane translocation of the peptide. Circular dichroism (CD) measurements indicate a conformational transition, from alpha-helix to antiparallel beta-pleated sheet, which is simultaneous with the aggregation process. The potential influence of spectroscopic artifacts on CD data due to the drastically increased turbidity during aggregation is discussed.     

Beta-lactamase III of Bacillus cereus 569: membrane lipoprotein and secreted protein

A third beta-lactamase in Bacillus cereus 569 has been identified and characterized. It corresponds to gamma-penicillinase reported by Pollock [Pollock, M. R. (1956) J. Gen. Microbiol. 15, 154-169] but whose existence has been questioned since then. It will be called beta-lactamase III. It resembles the class A beta-lactamases but is immunologically distinct from the major class A secreted beta-lactamase I of B. cereus. As with several other Gram-positive beta-lactamases it occurs in two forms, membrane bound as a glyceride-cysteine lipoprotein and as a hydrophilic secreted protein formed by cleavage on the carboxyl side of the modified cysteine that is the membrane attachment site. It is produced in all B. cereus 569 strains tested but is absent in B. cereus 5/b. Antibody to beta-lactamase III interacts to varying degrees with all the known class A beta-lactamases, most strongly with that of B. licheniformis 749/C.     

Development of an assay for beta-lactam hydrolysis using the pH-dependence of enhanced green fluorescent protein

An assay has been developed utilizing the pH-dependent fluorescence of enhanced green fluorescent protein (EGFP). This photoprotein allows for the study of kinetic properties of hydrolytic enzymes based on the production of protons. As a model system, beta-lactamase, a well-characterized enzyme responsible for antibiotic resistance in many bacteria, was used. More specifically, EGFP and beta-lactamase were genetically fused using overlap extension PCR and incorporated into a bacterial expression vector. The vector was subsequently transformed into Escherichia coli, and the fusion protein was expressed and purified. beta-Lactamase catalyzes the hydrolysis of the beta-lactam ring of ampicillin. This causes a decrease in the local pH, which in turn changes the spectral properties of EGFP. This property was utilized to perform enzyme kinetic studies on the new fusion protein as well as on the beta-lactamase inhibitor, sulbactam. The assay can be used to evaluate substrates and inhibitors of beta-lactamase in a format that should be amenable to high-throughput screening.     

Gene fusion analysis of membrane protein topology: a direct comparison of alkaline phosphatase and beta-lactamase fusions.

To compare two approaches to analyzing membrane protein topology, a number of alkaline phosphatase fusions to membrane proteins were converted to beta-lactamase fusions. While some alkaline phosphatase fusions near the N terminus of cytoplasmic loops of membrane proteins have anomalously high levels of activity, the equivalent beta-lactamase fusions do not. This disparity may reflect differences in the folding of beta-lactamase and alkaline phosphatase in the cytoplasm.     

Stability of the lactose permease in detergent solutions

Protein stability, as measured by irreversible protein aggregation, is one of the central difficulties in the handling of detergent-solubilized membrane proteins. We present a quantitative analysis of the stability of the Escherichia coli lactose (lac) permease and a series of lac permease fusion proteins containing an insertion of cytochrome(b562), T4 lysozyme or beta-lactamase in the central hydrophilic loop of the permease. The stability of the proteins was evaluated under a variety of storage conditions by both a qualitative SDS-PAGE assay and by a quantitative hplc assay. Long-chain maltoside detergents were more effective at maintaining purified protein in solution than detergents with smaller head groups and/or shorter alkyl tails. A full factorial experiment established that the proteins were insensitive to sodium chloride concentrations, but greatly stabilized by glycerol, low temperature and the combination of glycerol and low temperature. The accurate quantitation of the protein by absorbance spectroscopy required exclusion of all contact with clarified polypropylene or polyvinyl chloride (PVC) materials. Although some of the fusion proteins were more prone to aggregation than the wild-type permease, the stability of a fusion protein containing a cytochrome(b562) insertion was indistinguishable from that of native lac permease.