Membrane topology of the Escherichia coli TolR protein required for cell envelope integrity.

TolR is a 142-amino-acid protein required for the import of colicins and bacteriophage and for maintenance of cell envelope integrity. The topology of TolR in the inner membrane was analyzed by two methods. First, bacteria expressing a series of TolR-beta-galactosidase, TolR-alkaline phosphatase, and TolR-beta-lactamase fusions were assayed for the appropriate enzymatic activity. Second, the accessibility of TolR to proteinase K was determined in permeabilized cells and everted vesicles with an antibody elicited against the carboxyl-terminal 70% of TolR. The results are consistent with TolR spanning the inner membrane once via residues 23 to 43 and with the carboxyl-terminal moiety being exposed to the periplasm. Quantitative studies with the anti-TolR antibody indicated the presence of 2 x 10(3) to 3 x 10(3) TolR molecules per cell.     

Phospholipase-A-independent damage caused by the colicin A lysis protein during its assembly into the inner and outer membranes of Escherichia coli

The requirement for the activation of phospholipase A by the colicin A lysis protein (Cal) in the efficient release of colicin A by Escherichia coli cells containing colicin A plasmids was studied. In particular, we wished to determine if this activation is the primary effect of Cal or whether it reflects more generalized damage to the envelope caused by the presence of large quantities of this small acylated protein. E. coli tolQ cells, which were shown to be leaky for periplasmic proteins, were transduced to pldA and then transformed with the recombinant colicin A plasmid pKA. Both the pldA and pldA+ strains released large quantities of colicin A following induction, indicating that in these cells phospholipase A activation is not required for colicin release. This release was, however, still dependent on a functioning Cal protein. The assembly and processing of Cal in situ in the cell envelope was studied by combining pulse-chase labelling with isopycnic sucrose density gradient centrifugation of the cell membranes. Precursor Cal and lipid-modified precursor Cal were found in the inner membrane at early times of chase, and gave rise to mature Cal which accumulated in both the inner and outer membrane after further chase. The signal peptide was also visible on these gradients, and its distribution too was restricted to the inner membrane. Gradient centrifugation of envelopes of cells which were overproducing Cal resulted in very poor separation of the membranes. The results of these studies provide evidence that the colicin A lysis protein causes phospholipase A-independent alterations in the integrity of the E. coli envelope.(ABSTRACT TRUNCATED AT 250 WORDS)     

Escherichia coli K-12 envelope proteins specifically required for ferrienterobactin uptake.

Escherichia coli genes specifically required for transport of iron by the siderophore enterobactin are designated fep. The studies reported here were initiated to identify and localize the fepB product. The plasmid pCP111, which consisted of an 11-kilobase E. coli DNA fragment containing fepB ligated to pACYC184, was constructed. The fepB gene was subcloned; in the process, complementation tests and Tn5 mutagenesis results provided evidence for the existence of a new fep gene, fepC. The order of the transport genes in the ent gene cluster is as follows: fepA fes entF fepC fepB entE. Minicell, maxicell, and in vitro DNA-directed protein synthesizing systems were used to identify the fepB and fepC products. The fepC polypeptide was 30,500 daltons in standard sodium dodecyl sulfate-polyacrylamide gels. The fepB gene was responsible for the appearance of three or four bands (their apparent molecular weights ranged from 31,500 to 36,500) in sodium dodecyl sulfate-polyacrylamide gels, depending on the gel system employed. The largest of these was tentatively designated proFepB, since it apparently had a leader sequence. Localization experiments showed that FepC was a membrane constituent and that mature FepB was present in the periplasm. An additional polypeptide (X) was also encoded by the bacterial DNA of pCP111, but its relationship to iron transport is unknown. The results indicated that ferrienterobactin uptake is mediated by a periplasmic transport system and that genes coding for outer membrane (fepA), periplasmic (fepB), and cytoplasmic membrane (fepC) components have now been identified.     

A domain of RecC required for assembly of the regulatory RecD subunit into the Escherichia coli RecBCD holoenzyme

The heterotrimeric RecBCD enzyme of Escherichia coli is required for the major pathway of double-strand DNA break repair and genetic exchange. Assembled as a heterotrimer, the enzyme has potent nuclease and helicase activity. Analysis of recC nonsense and deletion mutations revealed that the C terminus of RecC is required for assembly of the RecD subunit into RecBCD holoenzyme but not for recombination proficiency; the phenotype of these mutations mimics that of recD deletion mutations. Partial proteolysis of purified RecC polypeptide yielded a C-terminal fragment that corresponds to the RecD-interaction domain. RecD is essential for nuclease activity, regulation by the recombination hotspot Chi, and high affinity for DNA ends. The RecC-RecD interface thus appears critical for the regulation of RecBCD enzyme via the assembly and, we propose, disassembly or conformational change of the RecD subunit.     

Localization of the dicarboxylate binding protein in the cell envelope of Escherichia coli K12

Examination of the localization of the dicarboxylate binding protein (DBP) in the cell envelope of Escherichia coli K12 reveals that this protein is present on the cell surface, and also in the inner and outer regions of the periplasmic space. The cell surface DBP is release by treating the cells with EDTA. This protein can be surface labeled by lactoperoxidase radioiodination, and by diazo[125I]iodosulfanilic acid in whole cells. It also binds tightly, but not covalently, to lipopolysaccharide. The DBP located in the outer region of the periplasmic space is released when the outer membrane is dissociated by EDTA-osmotic shock treatment. The DBP located in the inner region of the periplasmic space is released only when the EDTA-osmotic shocked cells are subjected to lysozyme treatment. At the moment, it is not certain whether this protein is bound to or trapped by the peptidoglycan network. This protein cannot be surface labeled in whole cells or in EDTA-osmotic shock treated cells; and it is not associated with lipopolysaccharide. Analysis of transport mutants indicates that these DBP are coded by the same gene.     

Identification of a region of Escherichia coli ribosomal protein L2 required for the assembly of L16 into the 50 S ribosomal subunit

In vitro mutagenesis of rplB was used to generate changes in a conserved region of Escherichia coli ribosomal protein L2 between Gly221 and His231. Mutants were selected by temperature sensitivity using an inducible expression system. A mutant L2 protein with the deletion of Thr222 to Asp228 was readily distinguishable from wild-type L2 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and ribosomes from the strain overexpressing this mutant protein were characterized by sucrose density gradient centrifugation and protein composition. In addition to 30 S and 50 S ribosomal subunits, cell lysates contained a new component that sedimented at 40 S in 1 mM Mg2+ and at 48 S in 10 mM Mg2+. These particles contained mutant L2 protein exclusively, completely lacked L16, and had reduced amounts of L28, L33, and L34. They did not reassociate with 30 S ribosomal subunits and were inactive in polyphenylalanine synthesis. Other mutants in the same conserved region, including the substitution of His229 by Gln229, produced similar aberrant 50 S particles that sedimented at 40 S and failed to associate with 30 S subunits.     

Topology analysis of the colicin V export protein CvaA in Escherichia coli.

The antibacterial protein toxin colicin V is secreted from Escherichia coli cells by a dedicated export system that is a member of the multicomponent ATP-binding cassette (ABC) transporter family. At least three proteins, CvaA, CvaB, and TolC, are required for secretion via this signal sequence-independent pathway. In this study, the subcellular location and transmembrane organization of membrane fusion protein CvaA were investigated. First, a series of CvaA-alkaline phosphatase (AP) protein fusions was constructed. Inner and outer membrane fractionations of cells bearing these fusions indicated that CvaA is inner membrane associated. To localize the fusion junctions, the relative activities of the fusion proteins, i.e., the amounts of phosphatase activity normalized to the rate of synthesis of each protein, as well as the stability of each fusion, were determined. These results indicated that all of the fusion junctions occur on the same side of the inner membrane. In addition, the relative activities were compared with that of native AP, and the protease accessibility of the AP moieties in spheroplasts and whole cells was analyzed. The results of these experiments suggested that the fusion junctions occur within periplasmic regions of CvA. We conclude that CvaA is an inner membrane protein with a single transmembrane domain near its N terminus; the large C-terminal region extends into the periplasm. This study demonstrates the application of AP fusion analysis to elucidate the topology of a membrane-associated protein having only a single transmembrane domain.     

Membrane topology and mutational analysis of the TolQ protein of Escherichia coli required for the uptake of macromolecules and cell envelope integrity.

TolQ is a 230-amino-acid protein required to maintain the integrity of the bacterial envelope and to facilitate the import of both filamentous bacteriophage and group A colicins. Cellular fractionation experiments showed TolQ to be localized to the cytoplasmic membrane. Bacteria expressing a series of TolQ-beta-galactosidase and TolQ-alkaline phosphatase fusion proteins were analyzed for the appropriate enzyme activity, membrane location, and sensitivity to exogenously added protease. The results are consistent with TolQ being an integral cytoplasmic membrane protein with three membrane-spanning regions. The amino-terminal 19 residues as well as a small loop in the 155 to 170 residue region appear exposed in the periplasm, while the carboxy terminus and a large loop after the first transmembrane region are cytoplasmic. Amino-terminal sequence analysis of TolQ purified from the membrane revealed the presence of the initiating formyl methionine group, suggesting a rapid translocation of the amino-terminal region across the cytoplasmic membrane. Analysis of various tolQ mutant strains suggests that the third transmembrane region as well as parts of the large cytoplasmic loop are necessary for activity.     

Maturation and localization of the TolB protein required for colicin import.

The tolB gene has been shown previously to encode two proteins of 47.5 kDa (TolB) and 43 kDa (TolB*). To explain the presence of these two forms, two hypotheses have been proposed: TolB might be posttranslationally processed to TolB*, or an internal in-frame translation initiation resulting in TolB* may occur (S. K. Levengood and R. E. Webster, J. Bacteriol. 171:6600-6609, 1989). To address this question, TolB was tagged by inserting in its C-terminal region an epitope recognized by monoclonal antibody 1C11 without altering the function of TolB. It was then demonstrated that the functional protein corresponded to TolB*, the mature periplasmic protein, and that TolB was its precursor form, which was observed only when the protein was overexpressed. These two forms were purified by immunoprecipitation, and their N-terminal sequences were determined. An antibody directed against TolB was raised, which confirmed the results obtained with the tagged TolB.     

Localization of acyl carrier protein in Escherichia coli.

Acyl carrier protein was localized by immunoelectron microscopy in the cytoplasm of Escherichia coli. These data are inconsistent with the previous report of an association between acyl carrier protein and the inner membrane (H. Van den Bosch, J.R. Williamson, and P.R. Vagelos, Nature [London] 228:338-341, 1970). Moreover, bacterial membranes did not bind a significant amount of acyl carrier protein or its thioesters in vitro. A thioesterase activity specific for long-chain acyl-acyl carrier protein was associated with the inner membrane.     

Factors affecting the entry of antibiotics into Escherichia coli

Factors affecting the entry into Escherichia coli of diverse antibacterial agents, especially beta-lactams were investigated. Agents of greater than a critical molecular weight (approximately 600 Daltons) penetrated extremely poorly. However, there was little correlation between penetrative ability and molecular weight for substances below the critical size. Within classes of related antibiotics (e.g. cephalosporins) penetrative ability was highly dependent on hydrophobicity. The relationship was parabolic rather than linear in nature. The proposal that the envelope of E. coli preferentially excludes hydrophobic molecules is to some extent an artefact arising from pre-selection of the agents used. For unrelated antibiotics hydrophobic nature was a poor guide to penetrative ability. A rather empirical property, diffusion ability through agar, was found to show good inverse correlation with penetrative ability for many unrelated antibiotics.     

Imp/OstA is required for cell envelope biogenesis in Escherichia coli

In Gram-negative bacteria, all components of the outer membrane are synthesized in the cytoplasm or the cytoplasmic leaflet of the inner membrane and must thus traverse the inner membrane and the periplasm on the way to their final destination. In this study, we show Imp/OstA to have characteristics typical for proteins involved in envelope biogenesis. Imp is essential and forms a high-molecular-weight disulphide-bonded complex in the outer membrane. Upon depletion of Imp, lipids and outer membrane proteins appear in a novel membrane fraction with higher density than the outer membrane. We propose Imp to be part of a targeting/usher system for components of the outer membrane.     

NarJ is a specific chaperone required for molybdenum cofactor assembly in nitrate reductase A of Escherichia coli

The formation of active membrane-bound nitrate reductase A in Escherichia coli requires the presence of three subunits, NarG, NarH and NarI, as well as a fourth protein, NarJ, that is not part of the active nitrate reductase. In narJ strains, both NarG and NarH subunits are associated in an unstable and inactive NarGH complex. A significant activation of this complex was observed in vitro after adding purified NarJ-6His polypeptide to the cell supernatant of a narJ strain. Once the apo-enzyme NarGHI of a narJ mutant has become anchored to the membrane via the NarI subunit, it cannot be reactivated by NarJ in vitro . NarJ protein specifically recognizes the catalytic NarG subunit. Fluorescence, electron paramagnetic resonance (EPR) spectroscopy and molybdenum quantification based on inductively coupled plasma emission spectroscopy (ICPES) clearly indicate that, in the absence of NarJ, no molybdenum cofactor is present in the NarGH complex. We propose that NarJ is a specific chaperone that binds to NarG and may thus keep it in an appropriate competent-open conformation for the molybdenum cofactor insertion to occur, resulting in a catalytically active enzyme. Upon insertion of the molybdenum cofactor into the apo-nitrate reductase, NarJ is then dissociated from the activated enzyme.     

Release of colicin E2 from Escherichia coli.

Treatment of Escherichia coli K-12(ColE2.P9) with 500 ng of mitomycin C per ml resulted in rapid and almost synchronous colicin E2 production. Colicin accumulated outside the cytoplasmic membrane, most probably in the periplasmic space. Colicin release occurred during a period in which the turbidity of the culture declined markedly. Periplasmic alkaline phosphatase was released during the same period, but cytoplasmic beta-galactosidase release was delayed.     

Temperature-sensitive, colicin M-tolerant mutant of Escherichia coli.

A mutant sensitive to colicin M at 30 degrees C and tolerant at 42 degrees C to high concentrations of colicin M was isolated from Escherichia coli K-12. A temperature shift from 30 to 42 degrees C rescued all cells up to the time they started to lyse at 30 degrees C (25 min after addition of colicin M). The growth rate at 42 degrees C remained unaffected by colicin M. AT 42 degrees C the cell-bound colicin M was inactivated by trypsin, sodium dodecyl sulfate, and antiserum against colicin M. Ferrichrome competed with colicin M at 42 degrees C only during the initial adsorption to the common receptor protein in the outer membrane. Since cells lysed earlier at 30 degrees C when they had been preincubated with colicin M at 42 degrees C, we conclude that the process leading finally to cell lysis is initiated at 42 degrees C and stops at a later stage of colicin M trypsin, dodecyl sulfate, and antiserum when cells were transferred from 30 to 42 degrees C, we assume that colicin M is translocated from its target site towards the cell surface. The mutation conferring tolerance was mapped close to the rpsL gene.     

The dynamics of assembly of a cytoplasmic membrane protein in Escherichia coli.

The topology of integral cytoplasmic membrane proteins can be analyzed using alkaline phosphatase fusions by determining which constructs have low and which have high specific activity. We show that in all cases the enzymatic activity is due to the fraction of the alkaline phosphatase moiety of the fusion protein localized to the periplasm. We present evidence that these fusions can also be used to analyze the process of assembly of cytoplasmic proteins into the membrane. The rate of acquisition of protease resistance of the alkaline phosphatase moiety of such hybrid proteins is compared for fusions to periplasmic and cytoplasmic domains. We show that this process, which is assumed to be representative of export of alkaline phosphatase, is significantly slower for fusions to cytoplasmic and certain periplasmic domains than for most periplasmic domains. These results are discussed in the context of the normal assembly of integral membrane proteins.     

Regulation of a membrane component required for protein secretion in Escherichia coli

We have previously described a gene, secA, which may code for a component of the secretion machinery of E. coli. Temperature-sensitive mutations in this gene lead to the cytoplasmic accumulation of precursors to a number of secreted proteins. In this paper, we describe the use of antibody to the SecA protein to characterize the cellular location and regulation of the protein. The antibody was elicited in response to a SecA-LacZ hybrid protein, produced by a strain carrying a secA-lacZ gene fusion. The secA gene product is a 92 kd polypeptide that is present in small amounts in the cell and that fractionates as a peripheral cytoplasmic membrane protein. The synthesis of the SecA protein is greatly derepressed (at least tenfold) when secretion in E. coli is blocked either in a secAts mutant or in the presence of a MalE-LacZ hybrid protein. We suggest that components of the secretion machinery of E. coli, such as the SecA protein, may be regulated in response to the secretion needs of the cell. When suppression of a secAam mutant is eliminated, leading to the absence of SecA protein, the synthesis of maltose-binding protein is greatly reduced. These results support a mechanism in which secretion and translation are coupled.     

Structural evidence that colicin A protein binds to a novel binding site of TolA protein in Escherichia coli periplasm

The Tol assembly of proteins is an interacting network of proteins located in the Escherichia coli cell envelope that transduces energy and contributes to cell integrity. TolA is central to this network linking the inner and outer membranes by interactions with TolQ, TolR, TolB, and Pal. Group A colicins, such as ColA, parasitize the Tol network through interactions with TolA and/or TolB to facilitate translocation through the cell envelope to reach their cytotoxic site of action. We have determined the first structure of the C-terminal domain of TolA (TolAIII) bound to an N-terminal ColA polypeptide (TA(53-107)). The interface region of the TA(53-107)-TolAIII complex consists of polar contacts linking residues Arg-92 to Arg-96 of ColA with residues Leu-375-Pro-380 of TolA, which constitutes a β-strand addition commonly seen in more promiscuous protein-protein contacts. The interface region also includes three cation-π interactions (Tyr-58-Lys-368, Tyr-90-Lys-379, Phe-94-Lys-396), which have not been observed in any other colicin-Tol protein complex. Mutagenesis of the interface residues of ColA or TolA revealed that the effect on the interaction was cumulative; single mutations of either partner had no effect on ColA activity, whereas mutations of three or more residues significantly reduced ColA activity. Mutagenesis of the aromatic ring component of the cation-π interacting residues showed Tyr-58 of ColA to be essential for the stability of complex formation. TA(53-107) binds on the opposite side of TolAIII to that used by g3p, ColN, or TolB, illustrating the flexible nature of TolA as a periplasmic hub protein.