The early interaction of the outer membrane protein phoe with the periplasmic chaperone Skp occurs at the cytoplasmic membrane.

Spheroplasts were used to study the early interactions of newly synthesized outer membrane protein PhoE with periplasmic proteins employing a protein cross-linking approach. Newly translocated PhoE protein could be cross-linked to the periplasmic chaperone Skp at the periplasmic side of the inner membrane. To study the timing of this interaction, a PhoE-dihydrofolate reductase hybrid protein was constructed that formed translocation intermediates, which had the PhoE moiety present in the periplasm and the dihydrofolate reductase moiety tightly folded in the cytoplasm. The hybrid protein was found to cross-link to Skp, indicating that PhoE closely interacts with the chaperone when the protein is still in a transmembrane orientation in the translocase. Removal of N-terminal parts of PhoE protein affected Skp binding in a cumulative manner, consistent with the presence of two Skp-binding sites in that region. In contrast, deletion of C-terminal parts resulted in variable interactions with Skp, suggesting that interaction of Skp with the N-terminal region is influenced by parts of the C terminus of PhoE protein. Both the soluble as well as the membrane-associated Skp protein were found to interact with PhoE. The latter form is proposed to be involved in the initial interaction with the N-terminal regions of the outer membrane protein.

Optimization of bacteriocin release protein (BRP)-mediated protein release by Escherichia coli: random mutagenesis of the pCloDF13-derived BRP gene to uncouple lethality and quasi-lysis from protein release.

Bacteriocin release proteins (BRPs) can be used for the release of heterologous proteins from the Escherichia coli periplasm into the culture medium. However, high-level expression of BRP causes apparent lysis of the host cells in liquid cultures (quasi-lysis) and inhibition of growth on broth agar plates (lethality). To optimize BRP-mediated protein release, the pCloDF13 BRP gene was subjected to random mutagenesis by using PCR techniques. Mutated BRPs with a strongly reduced capacity to cause growth inhibition on broth agar plates were selected, analyzed by nucleotide sequencing, and further characterized by performing growth and release experiments in liquid cultures. A subset of these BRP derivatives did not cause quasi-lysis and had only a small effect on growth but still functioned in the release of the periplasmic protein beta-lactamase and the periplasmic K88 molecular chaperone FaeE and in the release of the bacteriocin cloacin DF13 into the culture medium. These BRP derivatives can be more efficiently used for extracellular production of proteins by E. coli than can the original BRP.

Cell surface expression of receptor protein tyrosine phosphatase RPTP mu is regulated by cell-cell contact.

RPTP mu is a transmembrane protein tyrosine phosphatase with an adhesion molecule-like ectodomain. It has recently been shown that RPTP mu mediates homophilic interactions when expressed in insect cells. In this study, we have examined how RPTP mu may function as a cell contact receptor in mink lung epithelial cells, which express RPTPmu endogenously, as well as in transfected 3T3 cells. We find that RPTP mu has a relatively short half-life (3-4 hours) and undergoes posttranslational cleavage into two noncovalently associated subunits, with both cleaved and uncleaved molecules being present on the cell surface (roughly at a 1:1 ratio); shedding of the ectodomain subunit is observed in exponentially growing cells. Immunofluorescence analysis reveals that surface expression of RPTPmu is restricted to regions of tight cell-cell contact. RPTPmu surface expression increases significantly with increasing cell density. This density-induced upregulation of RPTP mu is independent of its catalytic activity and is also observed when transcription is driven by a constitutive promoter, indicating that modulation of RPTPmu surface expression occurs posttranscriptionally. Based on our results, we propose the following model of RPTP mu function: In the absence of cell-cell contact, newly synthesized RPTP mu molecules are rapidly cleared from the cell surface. Cell-cell contact causes RPTPmu to be trapped at the surface through homophilic binding, resulting in accumulation of RPTP mu at intercellular contact regions. This contact-induced clustering of RPTPmu may then lead to tyrosine dephosphorylation of intracellular substrates at cell-cell contacts.

Homophilic interactions mediated by receptor tyrosine phosphatases mu and kappa. A critical role for the novel extracellular MAM domain.

The receptor-like protein tyrosine phosphatases (RPTP) mu and RPTP kappa have a modular ectodomain consisting of four fibronectin type III-like repeats, a single Ig-like domain, and a newly identified N-terminal MAM domain. The function of the latter module, which comprises about 160 amino acids and is found in diverse transmembrane proteins, is not known. We previously reported that both RPTP mu and RPTP kappa can mediate homophilic cell interactions when expressed in insect cells. Here we show that despite their striking structural similarity, RPTP mu and RPTP kappa fail to interact in a heterophilic manner. To examine the role of the MAM domain in homophilic binding, we expressed a mutant RPTP mu lacking the MAM domain in insect Sf9 cells. Truncated RPTP mu is properly expressed at the cell surface but fails to promote cell-cell adhesion. Homophilic cell adhesion is fully restored in a chimeric RPTP mu molecule containing the MAM domain of RPTP kappa. However, this chimeric RPTP mu does not interact with either RPTP mu or RPTP kappa. These results indicate that the MAM domain of RPTP mu and RPTP kappa is essential for homophilic cell-cell interaction and helps determine the specificity of these interactions.

The use of a hybrid genetic system to study the functional relationship between prokaryotic and plant multi-enzyme fatty acid synthetase complexes.

Fatty acid synthesis in bacteria and plants is catalysed by a multi-enzyme fatty acid synthetase complex (FAS II) which consists of separate monofunctional polypeptides. Here we present a comparative molecular genetic and biochemical study of the enoyl-ACP reductase FAS components of plant and bacterial origin. The putative bacterial enoyl-ACP reductase gene (envM) was identified on the basis of amino acid sequence similarities with the recently cloned plant enoyl-ACP reductase. Subsequently, it was unambiguously demonstrated by overexpression studies that the envM gene encodes the bacterial enoyl-ACP reductase. An anti-bacterial agent called diazaborine was shown to be a specific inhibitor of the bacterial enoyl-ACP reductase, whereas the plant enzyme was insensitive to this synthetic antibiotic. The close functional relationship between the plant and bacterial enoyl-ACP reductases was inferred from genetic complementation of an envM mutant of Escherichia coli. Ultimately, envM gene-replacement studies, facilitated by the use of diazaborine, demonstrated for the first time that a single component of the plant FAS system can functionally replace its counterpart within the bacterial multienzyme complex. Finally, lipid analysis of recombinant E. coli strains with the hybrid FAS system unexpectedly revealed that enoyl-ACP reductase catalyses a rate-limiting step in the elongation of unsaturated fatty acids.

The relation of proton motive force, adenylate energy charge and phosphorylation potential to the specific growth rate and efficiency of energy transduction in Bacillus licheniformis under aerobic growth conditions.

The magnitude of the proton motive force (delta p) and its constituents, the electrical (delta psi) and chemical potential (-Z delta pH), were established for chemostat cultures of a protease-producing, relaxed (rel-) variant and a not protease-producing, stringent (rel+) variant of an industrial strain of Bacillus licheniformis (respectively referred to as the A- and the B-type). For both types, an inverse relation of delta p with the specific growth rate mu was found. The calculated intracellular pH (pHin) was not constant but inversely related to mu. This change in pHin might be related to regulatory functions of metabolism but a regulatory role for pHin itself could not be envisaged. Measurement of the adenylate energy charge (EC) showed a direct relation with mu for glucose-limited chemostat cultures; in nitrogen-limited chemostat cultures, the EC showed an approximately constant value at low mu and an increased value at higher mu. For both limitations, the ATP/ADP ratio was directly related to mu. The phosphorylation potential (delta G'p) was invariant with mu. From the values for delta G'p and delta p, a variable -->H+/ATP-stoichiometry was inferred: -->H+/ATP = 1.83 +/- 0.52 mu, so that at a given -->H+/O-ratio of four (4), the apparent P/O-ratio (inferred from regression analysis) showed a decline of 2.16 to 1.87 for mu = 0 to mu max (we discuss how more than half of this decline will be independent of any change in internal cell-volume). We propose that the constancy of delta G'p and the decrease in the efficiency of energy-conservation (P/O-value) with increasing mu are a way in which the cells try to cope with an apparent less than perfect coordination between anabolism and catabolism to keep up the highest possible mu with a minimum loss of growth-efficiency. Protease production in nitrogen-limited cultures as compared to glucose-limited cultures, and the difference between the A- and B-type, could not be explained by a different energy-status of the cells.

cDNA cloning and expression of Brassica napus enoyl-acyl carrier protein reductase in Escherichia coli.

The onset of storage lipid biosynthesis during seed development in the oilseed crop Brassica napus (rape seed) coincides with a drastic qualitative and quantitative change in fatty acid composition. During this phase of storage lipid biosynthesis, the enzyme activities of the individual components of the fatty acid synthase system increase rapidly. We describe a rapid and simple purification procedure for the plastid-localized NADH-dependent enoyl-acyl carrier protein reductase from developing B. napus seed, based on its affinity towards the acyl carrier protein (ACP). The purified protein was N-terminally sequenced and used to raise a potent antibody preparation. Immuno-screening of a seed-specific lambda gt11 cDNA expression library resulted in the isolation of enoyl-ACP reductase cDNA clones. DNA sequence analysis of an apparently full-length cDNA clone revealed that the enoyl-ACP reductase mRNA is translated into a precursor protein with a putative 73 amino acid leader sequence which is removed during the translocation of the protein through the plastid membrane. Expression studies in Escherichia coli demonstrated that the full-length cDNA clone encodes the authentic B. napus NADH-dependent enoyl-ACP reductase. Characterization of the enoyl-ACP reductase genes by Southern blotting shows that the allo-tetraploid B. napus contains two pairs of related enoyl-ACP reductase genes derived from the two distinct genes found in both its ancestors, Brassica oleracea and B. campestris. Northern blot analysis of enoyl-ACP reductase mRNA steady-state levels during seed development suggests that the increase in enzyme activity during the phase of storage lipid accumulation is regulated at the level of gene expression.

Formation of fermentation products and extracellular protease during anaerobic growth of Bacillus licheniformis in chemostat and batch-culture.

For a relaxed (rel-), protease producing (A-type) and a stringent (rel+), not-protease producing (B-type) variant of Bacillus licheniformis we determined fermentation patterns and products, growth parameters and alkaline protease-production (if any) in anaerobic, glucose-grown chemostats and batch-cultures. Glucose is dissimilated via glycolysis and oxidative pentose phosphate pathway simultaneously; the relative share of these two routes depends on growth phase (in batch) and specific growth rate (in chemostat). Predominant products are lactate, glycerol and acetaldehyde for A-type batches and acetaldehyde, ethanol, acetate and lactate for B-type batches. Both types show a considerable acetaldehyde production. In chemostat cultures, the fermentation products resemble those in batch-culture. From the anaerobic batches and chemostats, we conclude that the A-type (with low ATP-yield) will have a YATPmax of probably 12.9 g/mol and the B-type (with high ATP-yield) a YATPmax of about 10.1 g/mol. For batch-cultures, both types have about the same, high Yglucose (12 g/mol). So, the slow-growing A-type has a relatively high efficiency of anaerobic growth (i.e. an efficient use of ATP) and the fast-growing B-type a relatively low efficiency of anaerobic growth. In aerobic batch-cultures, we found 48, respectively 41% glucose-carbon conversion into mainly glycerol and pyruvate, respectively acetate as overflow metabolites in the A- and B-type. In both aerobic and anaerobic batch-cultures of the A-type, protease is produced predominantly in the logarithmic and early stationary phase, while a low but steady production is maintained in the stationary phase. Protease production occurs via de novo synthesis; up to 10% of the total protease in a culture is present in a cell-associated form. Although anaerobic protease production (expressed as protease per amount of biomass) is much higher than for aerobic conditions, specific rates of production are in the same range as for aerobic conditions while, most important, the substrate costs of anaerobic production are very much higher than for aerobic conditions.

Instability of protease production in a rel+/rel- -pair of Bacillus licheniformis and associated morphological and physiological characteristics.

A naturally occurring relaxed/protease-producing (A-type) versus stringent/not protease-producing (B-type) pair of an industrial Bacillus licheniformis has been characterized; either of the two types can convert into the other. Both types can sporulate, grow anaerobically, grow at 56 degrees C and reduce nitrate; morphologically, they can easily be distinguished by cell- and colony-shape. They differ in the ability to use 12 substrates, as determined in API-tests. The two types are remarkably different in their content of extrachromosomal elements (A-type: 2; B-type: 4); furthermore, they differ in their rel-status (A-type: relaxed; B-type: stringent). We propose that the differences in the ability of the two types to use different substrates probably are due to integration/extrusion of the extrachromosomal elements in and out of the chromosome, distorting or restoring a number of genes, together with induction of certain catabolic genes that are under control of the rel-system.

Uptake of cloacin DF13 by susceptible cells: removal of immunity protein and fragmentation of cloacin molecules.

Monoclonal antibodies (MAb) directed against different epitopes on the equimolar complex of cloacin and immunity protein (cloacin DF13) were isolated, characterized, and used to study the uptake of cloacin DF13 by susceptible cells. Four MAbs recognized the amino-terminal part, one MAb recognized the central part, and three MAbs recognized the carboxyl-terminal part of the cloacin molecule. Three MAbs reacted with the immunity protein. Five MAbs inhibited the lethal action of cloacin DF13, but none of the MAbs inhibited the binding of cloacin DF13 to its purified outer membrane receptor protein or the in vitro inactivation of ribosomes. Binding of cloacin DF13 to susceptible cells cultured in broth resulted in a specific, time-dependent dissociation of the complex and a fragmentation of the cloacin molecules. Increasing amounts of immunity protein were detected in the culture medium from about 20 min after the addition of cloacin DF13. Cloacin was fragmented into two carboxyl-terminal fragments with relative molecular masses of 50,000 and 10,000. The larger fragment was detected 5 min after the binding of the bacteriocin complex to the cells. The smaller fragment was detected after 10 min. Both fragments were associated with the cells and could not be detected in the culture supernatant fraction. Cells grown in brain heart infusion were much less susceptible to cloacin DF13 than cells grown in broth, although they possessed a similar number of outer membrane receptor molecules. This decreased susceptibility correlated with a decreased translocation, dissociation, and fragmentation of cloacin DF13.

Plasmid-determined cloacin DF13-susceptibility in Enterobacter cloacae and Klebsiella edwardsii; identification of the cloacin DF13/aerobactin outer membrane receptor proteins.

Both Enterobacter cloacae H478 and Klebsiella edwardsii S15 were shown to harbour a relatively large conjugative plasmid that coded for cloacin DF13-susceptibility and the production and uptake of a hydroxamate iron chelator, most probably aerobactin. Protein-blotting experiments with antiserum raised against the purified cloacin DF13/aerobactin receptor protein from Escherichia coli (Co1V-K30) revealed that the corresponding outer membrane receptor proteins of Ent. cloacae H478 and K. edwardsii S15 had apparent mol wts of 85 000 and 76 000, respectively. E. coli transconjugants harbouring either the plasmid from Ent. cloacae H478 or K. edwardsii S15 expressed a cloacin DF13/aerobactin outer membrane receptor protein with a mol wt of 74 000. The receptor protein encoded by the Ent. cloacae and K. edwardsii plasmids were immunologically more related to each other than to the pCo1V-K30-encoded receptor protein.

Subcloning of the cloacin DF13/aerobactin receptor protein and identification of a pColV-K30-determined polypeptide involved in ferric-aerobactin uptake.

A plasmid containing a pColV-K30 fragment that encoded only for the cloacin DF13/aerobactin receptor protein was constructed. Escherichia coli cells harboring this plasmid were sensitive to cloacin DF13 but were unable to take up ferric-aerobactin. Another pColV-K30-determined polypeptide (molecular weight, 50,000), localized in the membrane fraction, was essential for the uptake of ferric-aerobactin.