Creating a bifunctional protein by insertion of beta-lactamase into the maltodextrin-binding protein.

Hybrid proteins were generated by inserting the penicillin-hydrolyzing enzyme, TEM beta-lactamase (Bla), into the maltodextrin-binding protein (MalE). The inserted Bla was functionally accommodated by MalE when it was placed within permissive sites. The maltose binding and penicillinase activities of purified hybrids were indistinguishable from those of the wild-type MalE and Bla proteins. Moreover, these hybrids displayed an additional unexpected property: maltose stabilized the active site of inserted Bla.

N-tail translocation of mature beta-lactamase across the Escherichia coli cytoplasmic membrane.

Mature beta-lactamase was attached to the N-terminus of human glycophorin C, an N-out membrane protein lacking a cleavable signal peptide (an N-tail membrane protein). When synthesised in Escherichia coli more than 30% of the intact mature beta-lactamase-glycophorin C molecules assembled N-out, C-in into the cytoplasmic membrane. The N-tail translocated beta-lactamase folded into an enzymatically active form, but it was more susceptible to proteolysis than the equivalent portion of beta-lactamase-glycophorin C synthesised with an N-terminal signal peptide. Its translocation was virtually abolished when the N-out domain of glycophorin C was truncated or when the basic residues C-terminally flanking the glycophorin C membrane-spanning segment were replaced with neutral ones.

Secretion of active beta-lactamase to the medium mediated by the Escherichia coli haemolysin transport pathway.

An in frame gene fusion containing the coding region for mature beta-lactamase and the 3'-end of hylA encoding the haemolysin secretion signal, was constructed under the control of a lac promoter. The resulting 53 kDa hybrid protein was specifically secreted to the external medium in the presence of the haemolysin translocator proteins, HlyB and HlyD. The specific activity of the beta-lactamase portion of the secreted protein (measured by the hydrolysis of penicillin G), approximately 1 U/microgram protein, was close to that of authentic, purified TEM-beta-lactamase. This is an important example of a hybrid protein that is enzymatically active, and secreted via the haemolysin pathway. Previous studies have indicated that haemolysin is secreted directly into the medium, bypassing the periplasm, to which beta-lactamase is normally targeted. This study indicated, therefore, that normal folding of an active beta-lactamase, can occur, at least when fused to the HlyA C-terminus, without the necessity of entering the periplasm. Despite the secretion of approximately 5 micrograms/ml levels of the active beta-lactamase fusion into the medium, there was maximally only a 50% detectable increase in the LD50 for resistance to ampicillin at the individual cell level. This result suggests that, normally, resistance to ampicillin requires a high concentration of the enzyme close to killing targets, i.e. in the periplasm, in order to achieve significant levels of protection.

Cleavable signal peptides are rarely found in bacterial cytoplasmic membrane proteins (review).

Most proteins destined for secretion are synthesized with amino-terminal extensions, known as signal peptides, which play a vital role in their translocation across the membrane bordering the cytoplasm. Following translocation across the eukaryotic endoplasmic reticulum (ER) membrane or the bacterial cytoplasmic membrane, signal peptides are proteolytically removed from the preproteins. The process of membrane protein assembly can be likened to that of protein export in that it involves the translocation of portions of proteins across membranes. Moreover, the topological similarities between eukaryotic ER and plasma membrane proteins and bacterial cytoplasmic membrane proteins suggest that the mechanisms of membrane protein assembly may, like those of protein export, share fundamental similarities in eukaryotic and bacterial cells. However, whilst many of the ER and plasma membrane proteins of higher eukaryotes are synthesized with cleavable signal peptides, the same is true of only very few bacterial cytoplasmic membrane proteins. This fact is not widely appreciated, probably because certain exceptional (signal peptide-containing) bacterial membrane proteins, such as the major coat protein of bacteriophage M13, have been the subject of extensive investigations. In this review we highlight this anomaly and discuss it within the general context of membrane protein topology.

Assembly of eukaryotic class III (N-out, C-in) membrane proteins into the Escherichia coli cytoplasmic membrane.

Class III membrane proteins lack cleavable signal peptides but adopt an N-out, C-in topology with respect to their native membranes. We have analysed the fate of two eukaryotic class III plasma membrane proteins, human erythrocyte glycophorin C and influenza A virus M2 protein, in Escherichia coli. The N-terminal domains of both proteins were efficiently localised to the extracytoplasmic side of the bacterial cytoplasmic membrane. When beta-lactamase was fused to the C-terminus of glycophorin C it was localised to the cytoplasm, and protease treatment of spheroplasts caused a reduction in size of the fusion protein consistent with glycophorin C adopting its native topology in E. coli.

TnblaM: a transposon for directly tagging bacterial genes encoding cell envelope and secreted proteins.

A transposon, TnblaM, designed for the direct selection of bacterial mutants with insertions in genes encoding cell envelope and secreted proteins, was constructed and subcloned into plasmid and bacteriophage lambda delivery vectors. TnblaM is a spectinomycin-resistant derivative of Tn5 with an unexpressed open reading frame encoding mature beta-lactamase (BlaM) at its left end. Therefore, when it inserts into genes in the correct orientation and reading frame, gene fusions encoding hybrid proteins are generated. By introducing TnblaM into bacterial cells and selecting ampicillin-resistant (ApR) colonies, the subset of isolates producing extracytoplasmic BlaM, and hence containing TnblaM inserted in genes encoding secreted proteins and cell envelope proteins, can be directly selected. TnblaM, like TnphoA, can therefore be used to preferentially mutagenise genes encoding extracytoplasmic proteins, but it has the advantage over TnphoA that the desired mutants can be isolated by direct selection (as ApR colonies) rather than by phenotypic screening. Isolates in which TnblaM occupies sites in the chromosome from which it can transpose at high frequency are readily identifiable, and constitute TnblaM donors, with which to simply and efficiently generate rare types of insertion mutants. Moreover, the ApR selection that is used with TnblaM can be fine-tuned to obtain blaM fusions to poorly or well-expressed genes.

Analysis of the membrane organization of an Escherichia coli protein translocator, HlyB, a member of a large family of prokaryote and eukaryote surface transport proteins.

Haemolysin B (HlyB) is essential for secretion of the 107 x 10(3) Mr haemolysin A protein from Escherichia coli and is a member of a family of highly conserved, apparently ATP-dependent surface proteins in many organisms. We have shown in this study that both HlyB and HlyD fractionate primarily with the cytoplasmic membrane of E. coli and are accessible to proteases after removal of the outer membrane. We have measured experimentally the topological organization of HlyB within the membrane by construction of fusions to beta-lactamase as a reporter. The predicted folding of HlyB, with a minimum of six transmembrane segments, does not always coincide with regions of highest average hydrophobicity. This suggests that HlyB may have a novel organization within the bilayer. From our data and comparative sequence analysis, we have been able to predict very similar topological models for the other members of the HlyB family.

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.

Beta-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.

A simple method for maximizing the yields of membrane and exported proteins expressed in Escherichia coli.

The feasibility of using a beta-lactamase fusion approach for maximizing the levels of periplasmic or membrane-bound proteins expressed in Escherichia coli was investigated. The coding region for mature TEM beta-lactamase was fused after the signal peptide and aminoterminal portion of the coding region of a weakly expressed periplasmic protein, PBP3*. The resultant plasmid was mutagenized and transformants expressing increased levels of ampicillin resistance were selected. The PBP3* gene of the unmutagenized beta-lactamase fusion plasmid, and of two mutant derivatives encoding increased ampicillin resistance, were then reassembled and the latter constructs were found to express increased levels of PBP3*. The applications of a beta-lactamase fusion approach in monitoring and optimizing levels of extracytoplasmic gene products expressed in E. coli are considered.

Identification of amino acid sequences that can function as translocators of beta-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).

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.

Kanamycin-resistant vectors that are analogues of plasmids pUC8, pUC9, pEMBL8 and pEMBL9.

Analogues of the cloning vectors pUC8, pUC9, pEMBL8 +/- and pEMBL9 +/- that have kanamycin resistance (KmR) instead of ampicillin resistance (ApR) as the selectable marker have been developed. HindIII and SmaI sites within the KmR gene have been removed so that all of the cloning sites in the multi-linker region of these plasmids may be used except the AccI site.

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.

Lysis of Escherichia coli by beta-lactam antibiotics: deletion analysis of the role of penicillin-binding proteins 1A and 1B.

Deletions of the ponA and ponB genes of Escherichia coli have been constructed in vitro and recombined into the chromosome to produce strains that completely lack penicillin-binding protein 1A or penicillin-binding protein 1B. In each case a DNA fragment internal to the gene was replaced by a fragment encoding an antibiotic resistance. The ponA and ponB deletions can therefore be readily introduced into other E. coli strains by P1 transduction of the antibiotic resistance. Although the complete absence of penicillin-binding protein 1A or penicillin-binding protein 1B was tolerated, the absence of both of these proteins was shown to result in bacterial lysis.

Construction of a mutant of Escherichia coli that has deletions of both the penicillin-binding protein 5 and 6 genes.

A mutant of Escherichia coli has been constructed with deletions of the genes encoding penicillin-binding protein 5 (dacA) and penicillin-binding protein 6 (dacC). The construction of this mutant establishes that the complete loss of the two most abundant species of penicillin-binding proteins can be tolerated by E. coli. Moreover, the double deletion mutant had the same growth rate and morphology as an isogenic dacA+ dacC+ strain.