A class of Escherichia coli proteins controlled by the hflA locus

The HflA protein of Escherichia coli is critical for the choice of the lytic or lysogenic pathway by bacteriophage lambda. To investigate whether HflA plays a regulatory role in E. coli, we used two-dimensional gel electrophoresis to compare the distribution of E. coli proteins in hflA+ and hflA- cells. We found at least 13 proteins that are present in hflA- strains, but absent or very low in hflA+ strains. This observation indicates that HflA might be involved in regulation of a large class of E. coli proteins. Because of prior work implicating the Crp/cAMP system in Hfl-mediated regulation of lambda, we also studied the distribution of E. coli proteins in cya- strains unable to synthesize cAMP. Some of the same proteins found in hflA- are elicited by the addition of cAMP to cya- cells.     

hflB, a new Escherichia coli locus regulating lysogeny and the level of bacteriophage lambda cII protein

The level of the viral cII protein has been proposed to be the crucial determinant in the lysis-lysogeny decision of bacteriophage lambda. A new Escherichia coli locus (hflB) has been identified in which a mutation (hflB29) leads to high frequency of lysogeny by lambda. A double mutant defective in both hflB and the previously identified hflA gene displays a more severe Hfl- phenotype than either single mutant. The hflB locus is at 69 minutes on the E. coli map, 85% co-transducible with argG. The hflB29 mutation results in increased stability of the phage cII protein (increasing its half-life twofold) and is recessive to hflB+. We conclude that the hflB+ locus is a negative regulator of cII, perhaps coding for or regulating a protease that acts on cII. In addition, we observe that the can1 mutation, an alteration of the cII gene that results in enhanced lysogenization, leads to increased stability of cII protein. These observations reinforce the view that the level of cII is a key factor in the lysis-lysogeny decision of lambda.     

The HflB protease of Escherichia coli degrades its inhibitor lambda cIII.

The cIII protein of bacteriophage lambda is known to protect two regulatory proteins from degradation by the essential Escherichia coli protease HflB (also known as FtsH), viz., the lambda cII protein and the host heat shock sigma factor sigma32. lambda cIII, itself an unstable protein, is partially stabilized when the HflB concentration is decreased, and its half-life is decreased when HflB is overproduced, strongly suggesting that it is degraded by HflB in vivo. The in vivo degradation of lambda cIII (unlike that of sigma32) does not require the molecular chaperone DnaK. Furthermore, the half-life of lambda cIII is not affected by depletion of the endogenous ATP pool, suggesting that lambda cIII degradation is ATP independent (unlike that of lambda cII and sigma32). The lambda cIII protein, which is predicted to contain a 22-amino-acid amphipathic helix, is associated with the membrane, and nonlethal overproduction of lambda cIII makes cells hypersensitive to the detergent sodium dodecyl sulfate. This could reflect a direct lambda cIII-membrane interaction or an indirect association via the membrane-bound HflB protein, which is known to be involved in the assembly of certain periplasmic and outer membrane proteins.     

ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification.

Bacteriophage T7 RNA polymerase is stable in Escherichia coli but very susceptible to cleavage by at least one endoprotease after cell lysis. The major source of this endoprotease activity was found to be localized to the outer membrane of the cell. A rapid whole-cell assay was developed to screen different strains for the presence of this proteolytic activity. Using this assay, we identified some common laboratory strains that totally lack the protease. Genetic and Southern analyses of these null strains allowed us to conclude that the protease that cleaves T7 RNA polymerase is OmpT (formerly termed protein a), a known outer membrane endoprotease, and that the null phenotype results from deletion of the OmpT structural gene. A recombinant plasmid carrying the ompT gene enables these deletion strains to synthesize OmpT and converts them to a protease-positive phenotype. The plasmid led to overproduction of OmpT protein and protease activity in the E. coli K-12 and B strains we used, but only weak expression in the E. coli C strain, C1757. This strain-dependent difference in ompT expression was investigated with respect to the known influence of envZ on OmpT synthesis. A small deletion in the ompT region of the plasmid greatly diminishes the amount of OmpT protein and plasmid-encoded protease present in outer membranes. Use of ompT deletion strains for production of T7 RNA polymerase from the cloned gene has made purification of intact T7 RNA polymerase routine. Such strains may be useful for purification of other proteins expressed in E. coli.     

Detection of large COOH-terminal domains processed from the precursor of Serratia marcescens serine protease in the outer membrane of Escherichia coli.

The Serratia marcescens serine protease gene encoding a 1,045-amino-acid precursor protein of 112 kDa directs excretion of the mature protease of ca. 58 kDa through the outer membrane of Escherichia coli. A typical signal peptide of 27 amino acids and a large COOH-terminal domain of the precursor are both functionally essential for the excretion of the mature protease into the medium. Sequence analysis of the fragment peptides of the mature protease as well as site-directed mutagenesis indicated that the COOH-terminus of the mature enzyme was Asp645. By using the polyclonal antibody against the 112-kDa precursor protein, not only the intact precursor but also two proteins, C-1 (40 kDa) and C-2 (38 kDa), corresponding to the processed COOH-terminal domains were detected in the insoluble fraction of E. coli cells. Further fractionation by sucrose density gradient centrifugation showed that C-1 and C-2 were localized in the outer membrane. The NH2-terminal residues of C-1 and C-2 were determined to be Ala702 and Phe717, respectively. All these data suggest that the precursor is cleaved at three positions, between Asp645-Ser646, Glu701-Ala702, and Gly716-Phe717, probably by the self-processing activity in the normal excretion pathway through the outer membrane.     

Control of bacteriophage lambda CII activity by bacteriophage and host functions

We have studied the regulation of the lambda cII gene in vivo using cloned lambda fragments. Lambda N protein stimulated cII expression. Surprisingly, although very high cII protein levels were detected by gel electrophoresis, little cII protein activity, measured as stimulation of the lambda pI and pE promoters, was observed. The half-life of cII protein depended critically on its initial level. At low concentrations its half-life was as short as 1.5 min, whereas at high cII protein levels, it could be as long as 22 min. The Escherichia coli mutant ER437 directs lambda towards lysogeny; cII protein was more stable in this strain than in the wild type. On the other hand, although cyclic AMP is required for efficient lysogeny, it did not appear to influence the synthesis, stability, or activity of cII protein.     

In vivo analysis reveals substrate-gating mutants of a rhomboid intramembrane protease display increased activity in living cells

Intramembrane proteases hydrolyze peptide bonds within cell membranes. Recent crystal structures revealed that rhomboid intramembrane proteases contain a hydrated active site that opens to the outside of the cell, but is protected laterally from membrane lipids by protein segments. Using Escherichia coli rhomboid (GlpG) structures as a guide, we previously took a mutational approach to identify the GlpG gating mechanism that allows substrates to enter the active site laterally from the membrane. Mutations that weaken contacts keeping the gate closed increase enzyme activity and implicate transmembrane segment 5 as the substrate gate. Since these analyses were performed in vitro with pure proteins in detergent micelles, we have now examined GlpG in its natural environment, within the membrane of live E. coli cells. In striking congruity with in vitro analysis, gate-opening mutants in transmembrane segment 5 display up to a 10-fold increase in protease activity in living cells. Conversely, mutations in other parts of the protease, including the membrane-inserted L1 loop previously thought to be the gate, decrease enzyme activity. These observations provide evidence for the existence of both closed and open forms of GlpG in cells, and show that inter-conversion between them via substrate gating is rate limiting physiologically.     

Molecular construction of a multidrug exporter system, AcrAB: molecular interaction between AcrA and AcrB, and cleavage of the N-terminal signal sequence of AcrA

The AcrAB system of Escherichia coli is an intrinsic efflux protein with a broad substrate specificity. AcrA was thought to be localized in the periplasmic space, and to be linked to AcrB and TolC. The AcrAB-TolC system directly exports diverse substrates from the cell interior to the medium. In this study, we have determined the cellular localization of AcrA. By using the osmotic shock method, sucrose density gradient centrifugation, urea washing and Western blotting analysis, we reveal that AcrA is a peripheral inner membrane protein. A mutant plasmid encoding both the AcrA-TetBCt fusion protein and the AcrB-His fusion protein was constructed. Membrane vesicles prepared from cells expressing these fusion proteins were solubilized and AcrB-His was immunoprecipitated with an anti-polyhistidine antibody. After SDS-PAGE, Western blotting was performed with anti-TetBCt antiserum, resulting in the appearance of a 40 kDa band, indicating that AcrA co-precipitated with AcrB. Next we performed site-directed chemical labeling of Cys-introduced mutants of AcrA with [(14)C]N-ethylmaleimide. As judged from the labeling pattern and the molecular mass shift, the N-terminus of AcrA was removed and the mature protein is on the periplasmic surface. On the other hand, C25A mutants retained the N-terminal signal sequence on the cytoplasmic side of the membrane. We conclude that AcrA exists as a complex with AcrB on the periplasmic surface of the inner membrane after removal of the signal sequence.     

Immunocytochemical localization of P170 at the plasma membrane of multidrug-resistant human cells

P170 (P-glycoprotein) is a membrane protein found in high levels in multidrug-resistant cultured cell lines. We have localized this protein using monoclonal antibody MRK16 by immunofluorescence and electron microscopy in the multidrug-resistant human carcinoma cell line KB-C4. The P170 determinant recognized by antibody MRK16 was found on drug-resistant KB-C4 cells, but not on parental drug-sensitive KB-3-1 cells. The determinant was present on the external surface of the plasma membrane and on the luminal side of Golgi stack membranes. P170 was excluded from coated pits at the plasma membrane and absent from endocytic vesicles and lysosomes. This determinant was detected only in small amounts in the endoplasmic reticulum. The high protein concentration of P170 in the plasma membrane is consistent with a role of this protein as a drug efflux pump at the cell surface.     

Organic solvent-tolerant elastase efficiently hydrolyzes insoluble, cross-linked, protein fiber of eggshell membranes

Eggshell membrane is a mechanically stable and insoluble cross-linked fibrous protein. Pseudomonas aeruginosa strain ME-4 synthesizes a metalloprotease that degrades the eggshell membrane. We cloned the encoding gene in Escherichia coli. The recombinant protease, over-expressed in E. coli, was inactive but addition of acetone to crude cell extracts restored the activity and removed many E. coli proteins. We purified the active, acetone-treated protease to homogeneity in a single chromatography step with 57% recovery. The recombinant protease partially hydrolyzed eggshell membrane and produced more soluble peptides and proteins than commercial elastase, α-chymotrypsin, and collagenase. The soluble peptides produced from hydrolyzed eggshell membrane inhibited angiotensin-I-converting enzyme activity. The degradation of eggshell membrane by the recombinant elastase could be applied to the production of soluble bioactive peptides.     

Inner membrane protease I, an enzyme mediating intramitochondrial protein sorting in yeast.

Several precursors transported from the cytoplasm to the intermembrane space of yeast mitochondria are first cleaved by the MAS-encoded protease in the matrix space and then by additional proteases that have not been characterized. We have now developed a specific assay for one of these other proteases. The enzyme is an integral protein of the inner membrane; it requires divalent cations and acidic phospholipid for activity, and is defective in yeast mutant pet ts2858 which accumulates an incompletely processed cytochrome b2 precursor. The protease contains a 21.4 kd subunit whose C-terminal part is exposed on the outer face of the inner membrane. An antibody against this polypeptide inhibits the activity of the protease. As overproduction of the polypeptide does not increase the activity of the protease in mitochondria, the enzyme may be a hetero-oligomer. This 'inner membrane protease I' shares several key features with the leader peptidase of Escherichia coli and the signal peptidase of the endoplasmic reticulum.     

Identification of sat, an autotransporter toxin produced by uropathogenic Escherichia coli

Urinary tract infection (UTI) is a very common extraintestinal infection, and Escherichia coli is by far the most common causative organism. Uropathogenic E. coli possess traits that distinguish them from commensal strains of E. coli, such as secretion systems that allow virulence factors to be targeted to extracytoplasmic compartments. One of at least five characterized secretion mechanisms is the autotransporter system, which involves translocation of a protein across the inner membrane, presumably via the sec system, and across the outer membrane through a beta-barrel porin structure formed by the carboxy-terminus autotransporter domain. We identified a 107 kDa protein that was expressed significantly more often by E. coli strains associated with the clinical syndrome of acute pyelonephritis than by faecal strains (P = 0.029). We isolated the protein from E. coli CFT073, a strain cultured from the blood and urine of a patient with acute pyelonephritis. The N-terminal amino acid sequence showed highest similarity to two known SPATE (serine protease autotransporters of Enterobacteriaceae) proteins, Pet and EspC. Using a 509 bp probe from the 5' region of pet, 10 cosmid clones of an E. coli CFT073 gene library were positive for hybridization. From one cosmid clone, a 7.5 kb EcoRI restriction fragment, which reacted strongly with the probe, was shown to include the entire 3885 bp gene. The predicted 142 kDa protein product possesses the three domains that are typical of SPATE autotransporters: an unusually long signal sequence of 49 amino acids; a 107 kDa passenger domain containing a consensus serine protease active site (GDSGSG); and a C-terminal autotransporter domain of 30 kDa. The protein exhibited serine protease activity and displayed cytopathic activity on VERO primary kidney, HK-2 bladder and HEp-2 cell lines; the name Sat (secreted autotransporter toxin) was derived from these properties. In addition, Sat antibodies were present in the serum of mice infected with E. coli CFT073. Based upon its association with pathogenic isolates, its cytopathic phenotype and its ability to elicit a strong antibody response after infection, we postulate that Sat represents a novel virulence determinant of uropathogenic E. coli.     

Anchorage of cyclodextrin glucanotransferase on the outer membrane of Escherichia coli

The gene encoding cyclodextrin glucanotransferase (CGTase) was successfully cloned from B. macerans by PCR. A recombinant plasmid pCS005 with a gene encoding the Lpp-OmpA-CGTase trifusion protein was constructed and transformed into E. coli for the surface display of CGTase. Results of immunoblotting analysis and protease accessibility on the fractionated cell membranes confirmed that the Lpp-OmpA-CGTase trifusion protein was successfully anchored on the outer membrane of E. coli. However, only 50% of the membrane-anchored trifusion proteins were displayed on the outer surface of E. coli with the remaining 50% un-translocated. The low efficiency of surface display is attributed to the large size of CGTase. Only a trace amount of CGTase activity was detected for both the whole cells and the cell debris fractions. Because the results of the protease accessibility study suggested that the trypsin-resistant conformation of CGTase was preserved in the membrane-anchored CGTase, we believe that the lack of enzyme activity is mainly due to the inaccessibility of the CGTase active site, near the N-terminus, for substrate molecules. It can be estimated that the critical size for surface display of protein in E. coli is approximately 70 kDa.     

Surface display of antibodies

To screen antibody libraries that contain many millions of different clones, a selection system is required with an efficiency comparable to that of the immune system. This can be achieved by displaying antibodies on the surface of microorganisms containing the antibody's gene, analogous to the expression of the IgM antigen receptor on the surface of unactivated B-lymphocytes. Specific clones can then be selected using immobilized antigens. The minor coat protein of filamentous phages, pIII, which initiates the infection of E.coli by binding to their F-pili, and the major coat protein, pVIII, have been used as carriers for displaying antibodies on the phage surface. Recombinant antibodies have also been targeted to the cell surface of bacteria by fusing them with outer membrane components derived from lipoproteins, OmpA and an IgA protease. However, only the pIII system has been routinely used for screening antibody libraries. Here we describe the various antibody surface display systems and the screening of antibody libraries generated from the gene repertoire of lymphocytes and by gene synthesis. Finally, we have made a short comparison of the bacterial production of Fabs versus single chain antibodies (scFv).     

Molecular origin of plasma membrane citrate transporter in human prostate epithelial cells

The prostate is a highly specialized mammalian organ that produces and releases large amounts of citrate. However, the citrate release mechanism is not known. Here, we present the results of molecular cloning of a citrate transporter from human normal prostate epithelial PNT2‐C2 cells shown previously to express such a mechanism. By using rapid amplification of cDNA ends PCR, we determined that the prostatic carrier is an isoform of the mitochondrial transporter SLC25A1 with a different first exon. We confirmed the functionality of the clone by expressing it in human embryonic kidney cells and performing radiotracer experiments and whole‐cell patch‐clamp recordings. By using short interfering RNAs targeting different parts of the sequence, we confirmed that the cloned protein is the main prostatic transporter responsible for citrate release. We also produced a specific antibody and localized the cloned transporter protein to the plasma membrane of the cells. By using the same antibody, we have shown that the cloned transporter is expressed in non‐malignant human tissues.     

VIP36, a novel component of glycolipid rafts and exocytic carrier vesicles in epithelial cells.

In simple epithelial cells, apical and basolateral proteins and lipids in transit to the cell surface are sorted in the trans-Golgi network. We have recently isolated detergent-insoluble complexes from Madin-Darby canine kidney cells that are enriched in glycosphingolipids, apical cargo and a subset of the proteins of the exocytic carrier vesicles. The vesicular proteins are thought to be involved in protein sorting and include VIP21-caveolin. The vesicular protein VIP36 (36 kDa vesicular integral membrane protein) has been purified from a CHAPS-insoluble residue and a cDNA encoding VIP36 has been isolated. The N-terminal 31 kDa luminal/exoplasmic domain of the encoded protein shows homology to leguminous plant lectins. The transiently expressed protein is localized to the Golgi apparatus, endosomal and vesicular structures and the plasma membrane, as predicted for a protein involved in transport between the Golgi and the cell surface. It is diffusely localized on the plasma membrane but can be redistributed by antibody modulation into caveolae and clathrin-coated pits. We speculate that VIP36 binds to sugar residues of glycosphingolipids and/or glycosylphosphatidyl-inositol anchors and might provide a link between the extracellular/luminal face of glycolipid rafts and the cytoplasmic protein segregation machinery.     

Defective plasmid partition in ftsH mutants of Escherichia coli

FtsH is an ATP-dependent protease that is essential for cell viability in Escherichia coli. The essential function of FtsH is to maintain the proper balance of biosynthesis of major membrane components, lipopolysaccharide and phospholipids. F plasmid uses a partitioning system and is localized at specific cell positions, which may be related to the cell envelope, to ensure accurate partitioning. We have examined the effects of ftsH mutations on the maintenance of a mini-F plasmid, and have found that temperature-sensitive ftsH mutants are defective in mini-F plasmid partition, but not replication, at permissive temperature for cell growth. A significant fraction of replicated plasmid molecules tend to localize close together on one side of the cell, which may result in failure to pass the plasmid to one of the two daughter cells upon cell division. By contrast, an ftsH null mutant carrying the suppressor mutation sfhC did not affect partitioning of the plasmid. The sfhC mutation also suppressed defective maintenance in temperature-sensitive ftsH mutants. Using this new phenotype caused by ftsH mutations, we also isolated a new temperature-sensitive ftsH mutant. Mutations in ftsH cause an increase in the lipopolysaccharide/ phospholipid ratio due to stabilization of the lpxC gene product, which is involved in lipopolysaccharide synthesis and is a substrate for proteolysis by the FtsH protease. It is likely that altered membrane structure affects the localization or activity of a putative plasmid partitioning apparatus located at positions equivalent to 1/4 and 3/4 of the cell length.