Selection for mutants altered in the expression or export of outer membrane porin OmpF.

Strains in which the lacZ gene (which specifies beta-galactosidase) is fused to a gene encoding an envelope protein often exhibit a phenotype termed overproduction lethality. In such strains, high-level synthesis of the cognate hybrid protein interferes with the process of protein export, and this leads ultimately to cell death. A variation of this phenomenon has been discovered with lacZ fusions to the gene specifying the major outer membrane porin protein OmpF. In this case, we find that lambda transducing phage carrying an ompF-lacZ fusion will not grow on a host strain that constitutively overexpresses ompF. We have exploited this observation to develop a selection for ompF mutants. Using this protocol, we have isolated mutants altered in ompF expression and have identified mutations that block OmpF export. Our results suggest that it should be possible to adapt this selection for use with other genes specifying exported proteins.     

Analysis of protein localization by use of gene fusions with complementary properties.

This report describes a new transposon designed to facilitate the combined use of beta-galactosidase and alkaline phosphatase gene fusions in the analysis of protein localization. The transposon, called TnlacZ, is a Tn5 derivative that permits the generation of gene fusions encoding hybrid proteins carrying beta-galactosidase at their C termini. In tests with plasmids, TnlacZ insertions that led to high cellular beta-galactosidase activity were restricted to sequences encoding either cytoplasmic proteins or cytoplasmic segments of a membrane protein. The fusion characteristics of TnlacZ are thus complementary to those of TnphoA, a transposon able to generate alkaline phosphatase fusions whose high-activity insertion sites generally correspond to periplasmic sequences. The structure of TnlacZ allows the conversion of a TnlacZ fusion into the corresponding TnphoA fusion (and vice versa) through recombination or in vitro manipulation in a process called fusion switching. Fusion switching was used to generate the following two types of fusions with unusual properties: a low-specific-activity beta-galactosidase-alkaline phosphatase gene fusion and two toxic periplasmic-domain serine chemoreceptor-beta-galactosidase gene fusions. The generation of both beta-galactosidase and alkaline phosphatase fusions at exactly the same site in a protein permits a comparison of the two enzyme activities in evaluating the subcellular location of the site, such as in studies of membrane protein topology. In addition, fusion switching makes it possible to generate gene fusions whose properties should facilitate the isolation of mutants defective in the export or membrane anchoring of different cell envelope proteins.     

The membrane domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase confers endoplasmic reticulum localization and sterol-regulated degradation onto beta-galactosidase

A hybrid gene has been constructed consisting of coding sequence for the membrane domain of the endoplasmic reticulum protein 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase linked to the coding sequence for the soluble enzyme Escherichia coli beta-galactosidase. Expression of the hybrid gene in transfected Chinese hamster ovary cells results in the production of a fusion protein (HMGal) which is localized in the endoplasmic reticulum. The fusion protein contains the high-mannose oligosaccharides characteristic of HMG-CoA reductase. Importantly the beta-galactosidase activity of HMGal decreases when low density lipoprotein is added to the culture media. Therefore, the membrane domain of HMG-CoA reductase is sufficient to determine both correct intracellular localization and sterol-regulation of degradation. Mutant fusion proteins which lack 64, 85, or 98 amino acid residues from within the membrane domain of HMG-CoA reductase are found to be localized in the endoplasmic reticulum and to retain beta-galactosidase activity. However, sterol-regulation of degradation is abolished.     

Molecular components of the signal sequence that function in the initiation of protein export

We are studying the mechanism by which the LamB protein is exported to the outer membrane of Escherichia coli. Using two selection procedures based on gene fusions, we have identified a number of mutations that cause alterations in the LamB signal sequence. Characterization of the mutant strains revealed that although many such mutations block LamB export to greater than 95%, others have essentially no effect. These results allow an analysis of the functions performed by the various molecular components of the signal sequence. Our results suggest that a critical subset of four amino acids is contained within the central hydrophobic core of the LamB signal sequence. If this core can assume an alpha-helical conformation, these four amino acids comprise a recognition site that interacts with a component of the cellular export machinery. Since mechanisms of protein localization appear to have been conserved during evolution, the principles established by these results should be applicable to similar studies in eukaryotic cells.     

Genetic analysis of the membrane insertion and topology of MalF, a cytoplasmic membrane protein of Escherichia coli

MalF is an essential cytoplasmic membrane protein of the maltose transport system of Escherichia coli. We have developed a general approach for analysis of the mechanism of integration of membrane proteins and their membrane topology by characterizing a series of fusions of beta-galactosidase to MalF. The properties of the fusion proteins indicate the following. (1) The first two presumed transmembrane segments of MalF are sufficient to anchor beta-galactosidase firmly to the inner membrane. (2) Hybrid proteins with beta-galactosidase fused to a presumed cytoplasmic domain of MalF have high beta-galactosidase specific activity; fusions to periplasmic domains have low activity. We propose therefore, that periplasmic and cytoplasmic domains of integral membrane proteins can be distinguished by the enzymatic properties of such hybrid proteins. In general, it appears that cleaved or non-cleaved signal sequences when attached to beta-galactosidase cause it to become embedded in the membrane, and this results in the inability of the hybrid proteins to assemble into active enzyme. Additional properties of these fusion proteins contribute to our understanding of the regulation of MalF synthesis. The MalF protein, synthesized as part of the malEFG operon of E. coli, is approximately 30-fold less abundant in the cell than MalE protein (the maltose-binding protein). Differential amounts of the fusion proteins indicate that a regulatory signal occurs within the malF gene that is responsible for the step-down in expression from the malE gene to the malF gene.     

A lacZ-pbpB gene fusion coding for an inducible hybrid protein that recognizes localized sites in the inner membrane of Escherichia coli.

An in-phase gene fusion consisting of the 5'-terminal 1,314 base pairs (bp) of the structural gene for beta-galactosidase (lacZ) and the 3'-terminal 1,644 bp of the structural gene coding for penicillin-binding protein 3 (pbpB) of Escherichia coli was constructed and cloned in the plasmid pDIAM64. The product of the fusion gene was a remarkably stable protein with an apparent molecular weight of 110,000 (p110) that retained the ability to covalently interact with beta-lactam antibiotics. The fusion protein was found associated with the membrane at low levels of induction, but it accumulated in the cytoplasm of cells induced for a long time as inclusion bodies of high density. Inclusion bodies were localized at defined positions corresponding to septal sites in all of the pDIAM64-containing strains tested except PAT84 and GD113 (which carry the ftsZ84 mutant allele). These findings indicate a possible role of the FtsZ protein in the integration of Pbp3 into the membrane and in septum localization during the cell division cycle.     

A novel triple fusion reporter system for use in gene trap mutagenesis

Gene trapping is an insertional mutagenesis strategy that allows for simultaneous gene identification and mutation in embryonic stem (ES) cells. Gene trap vectors both disrupt coding sequence and report on the genes' endogenous expression. The most popular gene trap reporter to date combines beta-galactosidase expression with neomycin resistance in a fusion protein known as beta-geo. Here we describe a refinement to this reporter that also incorporates real time fluorescent readouts. We have constructed a series of gene trap vectors incorporating a novel tripartite fusion protein consisting of EGFP, beta-galactosidase, and the neomycin or hygromycin resistance activities. Our results indicate that these triple fusions can function efficiently as reporters of endogenous trapped gene expression and subcellular localization. We show that these fusion proteins constitute versatile gene trap reporters whose activity can be detected in real time by fluorescence and in fixed tissue with a sensitive enzymatic activity.     

Five SWI genes are required for expression of the HO gene in yeast

High-frequency mating type interconversion in yeast requires the HO gene, which encodes a site-specific endonuclease that initiates the switching process. We have isolated and analyzed switching-defective mutants. These mutants define five complementation and linkage groups, SWI 1 to SWI 5. We have shown by two assays, Northern hybridization and beta-galactosidase activity in strains containing an HO-lacZ fusion, that mutants defective any SWI gene fail to express the HO gene. In addition, all of the swi mutants exhibit other phenotypes, the most notable being the inviability of double mutants defective in SWI 4 and in either SWI 1, SWI 2 or SWI 3. These results indicate that the SWI genes function in some way as positive regulators of HO expression and have additional cellular roles.     

Altered translation initiation factor 2 in the cold-sensitive ssyG mutant affects protein export in Escherichia coli.

The Escherichia coli gene secY (pr1A) codes for an integral membrane protein that plays an essential role in protein export. We previously isolated cold-sensitive mutations (ssy) as extragenic suppressors of temperature-sensitive secY24 mutation. Now we show that the ssyG class of mutations are within infB coding for the translation initiation factor IF2. The mutants produce altered forms of IF2 with a cold-sensitive in vitro activity to form a translation initiation complex. The mutation suppresses not only secY24 but also other secretion-defective mutations such as secA51 and rp10215. The beta-galactosidase enzyme activity of the MalE-LacZ 72-47 hybrid protein is strikingly reduced in the ssyG mutant at the permissive high temperature, while the hybrid protein itself is normally synthesized. This effect, which was observed only for the hybrid protein with a functional signal sequence, may result from some alteration in the cellular localization of the protein. These results suggest that IF2 or the translation initiation step can modulate protein export reactions. The isolation of cold-sensitive ssyG mutations in infB provides genetic evidence that IF2 is indeed essential for normal growth of E. coli cells.     

Mutation prlF1 relieves the lethality associated with export of beta-galactosidase hybrid proteins in Escherichia coli.

The 42-1 lamB-lacZ gene fusion confers a conditionally lethal, export-dependent phenotype known as maltose sensitivity. A maltose-resistant mutant showing decreased beta-galactosidase activity of the hybrid protein, designated prlF1 (protein localization), was unlinked to the lamB-lacZ fusion. This mutation mapped at 70 min on the Escherichia coli linkage map and conferred maltose resistance, a 30-fold reduction in beta-galactosidase activity, and a 30% decrease in cellular growth rate at 30 degrees C that was independent of the presence of a gene fusion. prlF1 also decreased the beta-galactosidase activity and relieved the maltose sensitivity conferred by fusions of lacZ to the gene specifying the periplasmic maltose-binding protein, malE. The decrease in beta-galactosidase activity, however, was specific for exported hybrid proteins. When export of the hybrid protein was blocked by a signal sequence mutation, prlF1 decreased the beta-galactosidase activity only 2.5-fold. Similarly, prlF1 did not affect the beta-galactosidase activity of fusions of lacZ to a gene specifying a nonexported protein, malK.     

Isolation and Characterization of Dictyostelium Mutants Defective in Sexual Cell Fusion

Sexual cell fusion in the cellular slime mold Dictyostelium discoideum occurs between cells of opposite (heterothallic system) or same (homothallic system) mating types. It also requires certain environmental conditions such as darkness and abundance of water, and thus offers an interesting model system for analyzing mechanisms of cell recognition and of cellular response to environmental factors. We have been studying the mechanism of sexual cell fusion, using two heterothallic strains, NC4 and HM1 of D. discoideum. Two cell-surface glycoproteins, gp70 and gp138, have been identified as relevant molecules in the cell fusion of these strains. The former is specific to mat a cells (HM1) and the latter, common to both mat a and mat A (NC4). Involvement of cell-surface carbohydrates has also been suggested. However, the fuctions of the above fusion-related molecules are still elusive. In the present study, we isolated fusion-deficient mutants from a mutagenized mat A strain of D. discoideum to set up combined genetic and biochemical analyses. Among the three nonconditional mutants obtained, two were normal in the fruiting-body formation, asexual development, but one was aggregateless ( agg ⁻). Further analysis of these mutants would provide detailed information on the mechanism of sexual cell fusion.     

The Tsr chemosensory transducer of Escherichia coli assembles into the cytoplasmic membrane via a SecA-dependent process

The Tsr protein of Escherichia coli is a chemosensory transducer that mediates taxis toward serine and away from certain repellents. Like other bacterial transducers, Tsr spans the cytoplasmic membrane twice, forming a periplasmic domain of about 150 amino acids and a cytoplasmic domain of about 300 amino acids. The 32 N-terminal amino acids of Tsr resemble the consensus signal sequence of secreted proteins, but they are not removed from the mature protein. To investigate the function of this N-terminal sequence in the assembly process, we isolated translational fusions between tsr and the phoA and lacZ genes, which code for the periplasmic enzyme alkaline phosphatase and the cytoplasmic enzyme beta-galactosidase, respectively. All tsr-phoA fusions isolated code for proteins whose fusion joints are within the periplasmic loop of Tsr, and all of these hybrid proteins have high alkaline phosphatase activity. The most N-terminal fusion joint is at amino acid 19 of Tsr. Tsr-lacZ fusions were found throughout the tsr gene. The beta-galactosidase activity of the LacZ-fusion proteins varies greatly, depending on the location of the fusion joint. Fusions with low activity have fusion joints within the periplasmic loop of Tsr. The expression of these fusions is most likely reduced at the level of translation. In addition, one of these fusions markedly reduces the export and processing of the periplasmic maltose-binding protein and the outer membrane protein OmpA, but not of intact PhoA or of the outer membrane protein LamB. A temperature-sensitive secA mutation, causing defective protein secretion, stops expression of new alkaline phosphatase activity coded by a tsr-phoA fusion upon shifting to the nonpermissive temperature. The same secA mutation, even at the permissive temperature, increases the activity and the level of expression of LacZ fused to the periplasmic loop of Tsr relative to a secA+ strain. We conclude that the assembly of Tsr into the cytoplasmic membrane is mediated by the machinery responsible for the secretion of a subset of periplasmic and outer membrane proteins. Moreover, assembly of the Tsr protein seems to be closely coupled to its synthesis.     

Transition from reversible to irreversible attachment during biofilm formation by Pseudomonas fluorescens WCS365 requires an ABC transporter and a large secreted protein

We report the identification of an ATP-binding cassette (ABC) transporter and an associated large cell-surface protein that are required for biofilm formation by Pseudomonas fluorescens WCS365. The genes coding for these proteins are designated lap for large adhesion protein. The LapA protein, with a predicted molecular weight of approximately 900 kDa, is found to be loosely associated with the cell surface and present in the culture supernatant. The LapB, LapC and LapE proteins are predicted to be the cytoplasmic membrane-localized ATPase, membrane fusion protein and outer membrane protein component, respectively, of an ABC transporter. Consistent with this prediction, LapE, like other members of this family, is localized to the outer membrane. We propose that the lapEBC-encoded ABC transporter participates in the secretion of LapA, as strains with mutations in the lapEBC genes do not have detectable LapA associated with the cell surface or in the supernatant. The lap genes are conserved among environmental pseudomonads such as P. putida KT2440, P. fluorescens PfO1 and P. fluorescens WCS365, but are absent from pathogenic pseudomonads such as P. aeruginosa and P. syringae. The wild-type strain of P. fluorescens WCS365 and its lap mutant derivatives were assessed for their biofilm forming ability in static and flow systems. The lap mutant strains are impaired in an early step in biofilm formation and are unable to develop the mature biofilm structure seen for the wild-type bacterium. Time-lapse microscopy studies determined that the lap mutants are unable to progress from reversible (or transient) attachment to the irreversible attachment stage of biofilm development. The lap mutants were also found to be defective in attachment to quartz sand, an abiotic surface these organisms likely encounter in the environment.     

Enhanced export of beta-galactosidase fusion proteins in prlF mutants is Lon dependent.

We have used fusions of the outer membrane protein LamB to beta-galactosidase (encoded by lacZ) to study the protein export process. This LamB-LacZ hybrid protein blocks export when synthesized at high levels, as evidenced by inducer (maltose) sensitivity, a phenomenon termed LacZ hybrid jamming. The prlF1 mutation relieves LacZ hybrid jamming and allows localization of the fusion protein to a noncytoplasmic compartment. prlF1 and similar alleles are gain-of-function mutations. Null mutations in this gene confer no obvious phenotypes. Extragenic suppressors of a gain-of-function prlF allele have been isolated in order to understand how this gene product affects the export process. The suppressors are all lon null mutations, and they are epistatic to all prlF phenotypes tested. Lon protease activity has been measured in prlF1 cells and shown to be increased. However, the synthesis of Lon is not increased in a prlF1 background, suggesting a previously unidentified mechanism of Lon activation. Further analysis reveals that prlF1 activates degradation of cytoplasmically localized precursors in a Lon protease-dependent manner. It is proposed that accumulation of precursors during conditions of hybrid protein jamming titrates an essential export component(s), possibly a chaperone. Increased Lon-dependent precursor degradation would free this component, thus allowing increased protein export under jamming conditions.     

Studies with FtsA-LacZ protein fusions reveal FtsA located inner-outer membrane junctions

Four FtsA-LacZ translational gene fusions were constructed using a mini-Mu transposon (MudII 1734). FtsA-LacZ fusions and FtsA protein that were radioactively labelled using maxicell technique fractionated identically into membranes and cytoplasm. The FtsA-LacZ fusion proteins were also localized in wild type dividing cells using beta-galactosidase activity. Fractions from a modified sucrose equilibrium gradient exhibited beta-galactosidase activity in fractions corresponding to outer membrane-heavy (OMH) and outer membrane light (OML). The data are consistent with a model in which FtsA protein is incorporated into septal adhesion sites associated with cell division.     

Isolation of fla-lacZ fusions in Escherichia coli K-12: most fusions result in soluble beta-galactosidase

A series of fusions of flagellar genes to the lacZ gene was generated by insertion of Mu dII301 (Apr lac) bacteriophage into the genome of Escherichia coli. The beta-galactosidase activity in each resulting mutant was measured, and the location of the activity in the membrane, periplasmic, or cytoplasmic fraction of the cell was determined. There were three classes of mutants: those which had beta-galactosidase activity mainly in the membrane fraction, those which had it distributed in the soluble and membrane fractions, and those which had it in the cytoplasmic fraction only. The last, soluble-fraction-only, class was predominant in fla-lac gene fusions. In particular, the following mutants were shown to have beta-galactosidase activity in the membrane fractions: on the inner membrane, mutants with flaB fusions, and on the inner and outer membranes, mutants with flaA4850, flaM, and flaU4849 fusions. These results suggest that fla-lacZ gene fusions produce proteins which are able to detect the signals of the leader sequence and the membrane-anchoring region of the flagellar system.     

The hydrophobic domain of cytochrome b5 is capable of anchoring beta-galactosidase in Escherichia coli membranes.

Cytochrome b5 is inserted posttranslationally into membranes in vivo and spontaneously into liposomes in vitro by a short carboxyl-terminal hydrophobic membrane-anchoring sequence. DNA corresponding to this hydrophobic sequence has been synthesized, and two gene fusions with the Escherichia coli enzyme beta-galactosidase have been constructed by locating the hydrophobic domain in one case at the EcoRI site near the C terminus and in the other at the normal C terminus of the enzyme. The latter fusion protein was enzymatically active, having approximately 50% of the specific activity of beta-galactosidase, and cells expressing this protein grew normally with lactose as the sole carbon source. Both fusion proteins were localized to the E. coli inner membrane, converting beta-galactosidase from a cytoplasmic enzyme to a membrane-associated enzyme. The hydrophobic domain of cytochrome b5 therefore contains the information required to target polypeptides containing this domain to the membrane. Use of the cytochrome b5 hydrophobic peptide, either alone or in conjunction with other localizing sequences such as signal sequences, provides a general procedure for associating proteins with membranes. Polypeptides bearing this hydrophobic peptide may have considerable use as pharmaceuticals when associated with liposomes or cellular membranes.     

Stabilization of a HemA-LacZ hybrid protein against proteolysis during carbon starvation in atp mutants of Salmonella typhimurium.

Transposon insertions that stabilize the beta-galactosidase activity of a HemA-LacZ hybrid protein following carbon starvation were mapped to the atp operon of Salmonella typhimurium. This effect is similar to that seen with nuo mutants defective in the energy-conserving type I NADH dehydrogenase. Insertions in several other genes, including such highly pleiotropic mutants as rpoS, polA, and hfq, were isolated with the same phenotypic screen, but they do not affect the beta-galactosidase activity of HemA-LacZ. All of these mutants act indirectly to alter the colony color of many different fusion strains on indicator plates.