A conserved function of YidC in the biogenesis of respiratory chain complexes.

The Escherichia coli inner membrane protein (IMP) YidC is involved in the membrane integration of IMPs both in concert with and independently from the Sec translocase. YidC seems to be dispensable for the assembly of Sec-dependent IMPs, and so far it has been shown to be essential only for the proper Sec-independent integration of some phage coat proteins. Here, we studied the physiological consequences of YidC depletion in an effort to understand the essential function of YidC. The loss of YidC rapidly and specifically induced the Psp stress response, which is accompanied by a reduction of the proton-motive force. This reduction is due to defects in the functional assembly of cytochrome o oxidase and the F(1)F(o) ATPase complex, which is reminiscent of the effects of mutations in the yidC homologue OXA1 in the yeast mitochondrial inner membrane. The integration of CyoA (subunit II of the cytochrome o oxidase) and F(o)c (membrane subunit of the F(1)F(o) ATPase) appeared exceptionally sensitive to depletion of YidC, suggesting that these IMPs are natural substrates of a membrane integration and assembly pathway in which YidC plays an exclusive or at least a pivotal role.

Purification of the autotransporter protein Hbp of Escherichia coli.

The enzyme Hbp (hemoglobin protease) of the pathogenic Escherichia coli strain EB1 has been purified to homogeneity by gel filtration chromatography. The purified protein is capable of binding heme and shows hemoglobin protease activity. Our method of purification is applicable not only to Hbp but also to other autotransporter proteins and will contribute to a better understanding of the function-structure relationship of this family of proteins.

Sec-dependent membrane protein insertion: sequential interaction of nascent FtsQ with SecY and YidC.

Recent studies identified YidC as a novel membrane factor that may play a key role in membrane insertion of inner membrane proteins (IMPs), both in conjunction with the Sec-translocase and as a separate entity. Here, we show that the type II IMP FtsQ requires both the translocase and, to a lesser extent, YidC in vivo. Using photo-crosslinking we demonstrate that the transmembrane (TM) domain of the nascent IMP FtsQ inserts into the membrane close to SecY and lipids, and moves to a combined YidC/lipid environment upon elongation. These data are consistent with a crucial role for YidC in the lateral transfer of TM domains from the Sec translocase into the lipid bilayer.

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.

Analysis of the type IV fimbrial-subunit gene fimA of Xanthomonas hyacinthi: application in PCR-mediated detection of yellow disease in Hyacinths.

A sensitive and specific detection method was developed for Xanthomonas hyacinthi; this method was based on amplification of a subsequence of the type IV fimbrial-subunit gene fimA from strain S148. The fimA gene was amplified by PCR with degenerate DNA primers designed by using the N-terminal and C-terminal amino acid sequences of trypsin fragments of FimA. The nucleotide sequence of fimA was determined and compared with the nucleotide sequences coding for the fimbrial subunits in other type IV fimbria-producing bacteria, such as Xanthomonas campestris pv. vesicatoria, Neisseria gonorrhoeae, and Moraxella bovis. In a PCR internal primers JAAN and JARA, designed by using the nucleotide sequences of the variable central and C-terminal region of fimA, amplified a 226-bp DNA fragment in all X. hyacinthi isolates. This PCR was shown to be pathovar specific, as assessed by testing 71 Xanthomonas pathovars and bacterial isolates belonging to other genera, such as Erwinia and Pseudomonas. Southern hybridization experiments performed with the labelled 226-bp DNA amplicon as a probe suggested that there is only one structural type IV fimbrial-gene cluster in X. hyacinthi. Only two Xanthomonas translucens pathovars cross-reacted weakly in PCR. Primers amplifying a subsequence of the fimA gene of X. campestris pv. vesicatoria (T. Ojanen-Reuhs, N. Kalkkinen, B. Westerlund-Wikström, J. van Doorn, K. Haahtela, E.-L. Nurmiaho-Lassila, K. Wengelink, U. Bonas, and T. K. Korhonen, J. Bacteriol. 179: 1280-1290, 1997) were shown to be pathovar specific, indicating that the fimbrial-subunit sequences are more generally applicable in xanthomonads for detection purposes. Under laboratory conditions, approximately 1,000 CFU of X. hyacinthi per ml could be detected. In inoculated leaves of hyacinths the threshold was 5,000 CFU/ml. The results indicated that infected hyacinths with early symptoms could be successfully screened for X. hyacinthi with PCR.

Biosynthesis of K88 fimbriae in Escherichia coli: interaction of tip-subunit FaeC with the periplasmic chaperone FaeE and the outer membrane usher FaeD.

K88 fimbriae are ordered polymeric protein structures at the surface of enterotoxigenic Escherichia coli cells. Their production and assembly requires a molecular chaperone located in the periplasm (FaeE) and a molecular usher located in the outer membrane (FaeD). FaeC is the tip component of the K88 fimbriae. We studied the expression of the subcloned faeC gene, the subcellular localization of FaeC and its interaction with the chaperone and the outer membrane usher. In the absence of the chaperone or the usher, FaeC could not be detected in E. coli cells harbouring the faeC gene and its ribosome binding site under contol of the IPTG inducible lpp/lac promoter/operator. The expression of FaeC was detectable in the presence of chaperone FaeE, but a direct interaction between the chaperone and FaeC was not found. The expression of FaeC was also detectable in cells co-expressing the outer membrane usher FaeD. Overexpression of FaeC after changing the faeC ribosome binding site appeared to induce lethality. Expression of subcloned FaeC in the absence of FaeE or FaeD could be detected when faeC was cloned under the tight control of the ara promoter/operator and when lethality induction was avoided. The direct interaction of FaeC with outer membranes containing the usher FaeD was studied by cell fractionation, isopycnic sucrose density gradient centrifugation, SDS-PAGE and immunoblotting. FaeC was found to bind to outer membranes containing FaeD or a FaeD-PhoA hybrid construct containing 215 amino-terminal residues of FaeD. This binding was not observed when control outer membranes without FaeD were used. No other K88 specific proteins were required for this interaction. The direct interaction between FaeC and FaeD in the outer membranes was shown by affinity blotting experiments. FaeE was not required for this interaction. Together these data indicate that the minor fimbrial subunit FaeC, unlike FaeG, H and F, does not have a strong interaction with the chaperone FaeE in the E. coli periplasm, but directly binds to the outer membrane molecular usher FaeD.

Nascent Lep inserts into the Escherichia coli inner membrane in the vicinity of YidC, SecY and SecA.

Targeting and assembly of the Escherichia coli inner membrane protein leader peptidase (Lep) was studied using a homologous in vitro targeting/translocation assay. Assembly of full-length Lep was efficient in the co-translational presence of membrane vesicles and hardly occurred when membranes were added post-translationally. This is consistent with the signal recognition particle-dependent targeting of Lep. Crosslinking experiments showed that the hydrophilic region P1 of nascent membrane-inserted Lep 100-mer was in the vicinity of SecA and SecY, whereas the first transmembrane domain H1 was in the vicinity of YidC. These results suggested that YidC, together with the Sec translocase, functions in the assembly of Lep. YidC might be a more generic component in the assembly of inner membrane proteins.

The F4 fimbrial antigen of Escherichia coli and its receptors.

F4 or K88 fimbriae are long filamentous polymeric surface proteins of enterotoxigenic Escherichia coli (ETEC), consisting of so-called major (FaeG) and minor (FaeF, FaeH, FaeC, and probably FaeI) subunits. Several serotypes of F4 have been described, namely F4ab, F4ac, and F4ad. The F4 fimbriae allow the microorganisms to adhere to F4-specific receptors present on brush borders of villous enterocytes and consequently to colonize the small intestine. Such ETEC infections are responsible for diarrhea and mortality in neonatal and recently weaned pigs. In this review emphasis is put on the morphology, genetic configuration, and biosynthesis of F4 fimbriae. Furthermore, the localization of the different a, b, c, and d epitopes, and the localization of the receptor binding site on the FaeG major subunit of F4 get ample attention. Subsequently, the F4-specific receptors are discussed. When the three variants of F4 (F4ab, F4ac, and F4ad) are considered, six porcine phenotypes can be distinguished with regard to the brush border adhesiveness: phenotype A binds all three variants, phenotype B binds F4ab and F4ac, phenotype C binds F4ab and F4ad, phenotype D binds F4ad, phenotype E binds none of the variants, and phenotype F binds F4ab. The following receptor model is described: receptor bcd is found in phenotype A pigs, receptor bc is found in phenotype A and B pigs, receptor d is found in phenotype C and D pigs, and receptor b is found in phenotype F pigs. Furthermore, the characterization of the different receptors is described in which the bcd receptor is proposed as collection of glycoproteins with molecular masses ranging from 45 to 70 kDa, the bc receptor as two glycoproteins with molecular masses of 210 an 240 kDa, respectively, the b receptor as a glycoprotein of 74 kDa, and the d receptor as a glycosphingolipid with unknown molecular mass. Finally, the importance of F4 fimbriae and their receptors in the study of mucosal immunity in pigs is discussed.

Anionic phospholipids are involved in membrane association of FtsY and stimulate its GTPase activity.

FtsY, the Escherichia coli homologue of the eukaryotic signal recognition particle (SRP) receptor alpha-subunit, is located in both the cytoplasm and inner membrane. It has been proposed that FtsY has a direct targeting function, but the mechanism of its association with the membrane is unclear. FtsY is composed of two hydrophilic domains: a highly charged N-terminal domain (the A-domain) and a C-terminal GTP-binding domain (the NG-domain). FtsY does not contain any hydrophobic sequence that might explain its affinity for the inner membrane, and a membrane-anchoring protein has not been detected. In this study, we provide evidence that FtsY interacts directly with E.coli phospholipids, with a preference for anionic phospholipids. The interaction involves at least two lipid-binding sites, one of which is present in the NG-domain. Lipid association induced a conformational change in FtsY and greatly enhanced its GTPase activity. We propose that lipid binding of FtsY is important for the regulation of SRP-mediated protein targeting.

YidC, the Escherichia coli homologue of mitochondrial Oxa1p, is a component of the Sec translocase.

In Escherichia coli, both secretory and inner membrane proteins initially are targeted to the core SecYEG inner membrane translocase. Previous work has also identified the peripherally associated SecA protein as well as the SecD, SecF and YajC inner membrane proteins as components of the translocase. Here, we use a cross-linking approach to show that hydrophilic portions of a co-translationally targeted inner membrane protein (FtsQ) are close to SecA and SecY, suggesting that insertion takes place at the SecA/Y interface. The hydrophobic FtsQ signal anchor sequence contacts both lipids and a novel 60 kDa translocase-associated component that we identify as YidC. YidC is homologous to Saccharomyces cerevisiae Oxa1p, which has been shown to function in a novel export pathway at the mitochondrial inner membrane. We propose that YidC is involved in the insertion of hydrophobic sequences into the lipid bilayer after initial recognition by the SecAYEG translocase.

SecA is not required for signal recognition particle-mediated targeting and initial membrane insertion of a nascent inner membrane protein.

In Escherichia coli, signal recognition particle (SRP)-dependent targeting of inner membrane proteins has been described. In vitro cross-linking studies have demonstrated that short nascent chains exposing a highly hydrophobic targeting signal interact with the SRP. This SRP, assisted by its receptor, FtsY, mediates the transfer to a common translocation site in the inner membrane that contains SecA, SecG, and SecY. Here we describe a further in vitro reconstitution of SRP-mediated membrane insertion in which purified ribosome-nascent chain-SRP complexes are targeted to the purified SecYEG complex contained in proteoliposomes in a process that requires the SRP-receptor FtsY and GTP. We found that in this system SecA and ATP are dispensable for both the transfer of the nascent inner membrane protein FtsQ to SecY and its stable membrane insertion. Release of the SRP from nascent FtsQ also occurred in the absence of SecYEG complex indicating a functional interaction of FtsY with lipids. These data suggest that SRP/FtsY and SecB/SecA constitute distinct targeting routes.

Development and application of pathovar-specific monoclonal antibodies that recognize the lipopolysaccharide O antigen and the type IV fimbriae of Xanthomonas hyacinthi.

The objective of this study was to develop a specific immunological diagnostic assay for yellow disease in hyacinths, using monoclonal antibodies (MAbs). Mice were immunized with a crude cell wall preparation (shear fraction) from Xanthomonas hyacinthi and with purified type IV fimbriae. Hybridomas were screened for a positive reaction with X. hyacinthi cells or fimbriae and for a negative reaction with X. translucens pv. graminis or Erwinia carotovora subsp. carotovora. Nine MAbs recognized fimbrial epitopes, as shown by immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy; however, three of these MAbs had weak cross-reactions with two X. translucens pathovars in immunoblotting experiments. Seven MAbs reacted with lipopolysaccharides and yielded a low-mobility ladder pattern on immunoblots. Subsequent analysis of MAb 2E5 showed that it specifically recognized an epitope on the O antigen, which was found to consist of rhamnose and fucose in a 2:1 molar ratio. The cross-reaction of MAb 2E5 with all X. hyacinthi strains tested showed that this O antigen is highly conserved within this species. MAb 1B10 also reacted with lipopolysaccharides. MAbs 2E5 and 1B10 were further tested in ELISA and immunoblotting experiments with cells and extracts from other pathogens. No cross-reaction was found with 27 other Xanthomonas pathovars tested or with 14 other bacterial species from other genera, such as Erwinia and Pseudomonas, indicating the high specificity of these antibodies. MAbs 2E5 and 1B10 were shown to be useful in ELISA for the detection of X. hyacinthi in infected hyacinths.

Identification of Tn10 insertions in the rfaG, rfaP, and galU genes involved in lipopolysaccharide core biosynthesis that affect Escherichia coli adhesion.

Escherichia coli was used as a model to study initial adhesion and early biofilm development to abiotic surface. Tn10 insertion mutants of Escherichia coli K-12 W3110 were selected for altered abilities to adhere to a polystyrene surface. Seven insertion mutants that showed a decrease in adhesion harbored insertions in genes involved in lipopolysaccharide (LPS) core biosynthesis. Two insertions were located in the rfaG gene, two in the rfaP gene, and three in the galU gene. These adhesion mutants were found to exhibit a deep-rough phenotype and to be reduced, at different levels, in type 1 fimbriae production and motility. The loss of adhesion exhibited by these mutants was associated with either the affected type 1 fimbriae production and/or the dysfunctional motility. Apart from the pleiotropic effect of the mutations affecting LPS on type 1 fimbriae and flagella biosynthesis, no evidence for an involvement of the LPS itself in adhesion to polystyrene surface could be observed.

Identification of Tn10 insertions in the dsbA gene affecting Escherichia coli biofilm formation.

Escherichia coli was used as model to study initial adhesion and early biofilm development to an abiotic surface. Tn10 insertion mutants with reduced attachment to a polystyrene surface were isolated. Three adhesion mutants harbored the transposon in the dsbA gene, whose product, DsbA, catalyses folding of numerous extracytoplasmic disulfide bond-containing proteins. All three mutants were weakly adherent and grew poorly. Cell surface structure analysis showed that motility. type 1 fimbriation and lipopolysaccharide structure were affected in these mutants. The pleiotropic effect of the dsbA mutations on biofilm formation is discussed.

mrp, a multigene, multifunctional locus in Bacillus subtilis with roles in resistance to cholate and to Na+ and in pH homeostasis.

A 5.9-kb region of the Bacillus subtilis chromosome is transcribed as a single transcript that is predicted to encode seven membrane-spanning proteins. Homologues of the first gene of this operon, for which the designation mrp (multiple resistance and pH adaptation) is proposed here, have been suggested to encode an Na+/H+ antiporter or a K+/H+ antiporter. In the present studies of the B. subtilis mrp operon, both polar and nonpolar mutations in mrpA were generated. Growth of these mutants was completely inhibited by concentrations of added Na+ as low as 0.3 M at pH 7.0 and 0.03 M at pH 8.3; there was no comparable inhibition by added K+. A null mutant that was constructed by full replacement of the mrp operon was even more Na+ sensitive. A double mutant with mutations in both mrpA and the multifunctional antiporter-encoding tetA(L) gene was no more sensitive than the mrpA mutants to Na+, consistent with a major role for mrpA in Na+ resistance. Expression of mrpA from an inducible promoter, upon insertion into the amyE locus, restored significant Na+ resistance in both the polar and nonpolar mrpA mutants but did not restore resistance in the null mutant. The mrpA disruption also resulted in an impairment of cytoplasmic pH regulation upon a sudden shift in external pH from 7.5 to 8.5 in the presence of Na+ and, to some extent, K+ in the range from 10 to 25 mM. By contrast, the mrpA tetA(L) double mutant, like the tetA(L) single mutant, completely lost its capacity for both Na+- and K+-dependent cytoplasmic pH regulation upon this kind of shift at cation concentrations ranging from 10 to 100 mM; thus, tetA(L) has a more pronounced involvement than mrpA in pH regulation. Measurements of Na+ efflux from the wild-type strain, the nonpolar mrpA mutant, and the complemented mutant indicated that inducible expression of mrpA increased the rate of protonophore- and cyanide-sensitive Na+ efflux over that in the wild-type in cells preloaded with 5 mM Na+. The mrpA and null mutants showed no such efflux in that concentration range. This is consistent with MrpA encoding a secondary, proton motive force-energized Na+/H+ antiporter. Studies of a polar mutant that leads to loss of mrpFG and its complementation in trans by mrpF or mrpFG support a role for MrpF as an efflux system for Na+ and cholate. Part of the Na+ efflux capacity of the whole mrp operon products is attributable to mrpF. Neither mrpF nor mrpFG expression in trans enhanced the cholate or Na+ resistance of the null mutant. Thus, one or more other mrp gene products must be present, but not at stoichiometric levels, for stability, assembly, or function of both MrpF and MrpA expressed in trans. Also, phenotypic differences among the mrp mutants suggest that functions in addition to Na+ and cholate resistance and pH homeostasis will be found among the remaining mrp genes.

Molecular characterization of Escherichia coli FtsE and FtsX.

The genes ftsE and ftsX are organized in one operon together with ftsY. FtsY codes for the receptor of the signal recognition particle (SRP) that functions in targeting a subset of inner membrane proteins. We have found no indications for a structural relationship between FtsE/X and FtsY. Evidence is presented that FtsE and FtsX form a complex in the inner membrane that bears the characteristics of an ATP-binding cassette (ABC)-type transporter. FtsE is a hydrophilic nucleotide-binding protein that has a tendency to dimerize and associates with the inner membrane through an interaction with the integral membrane protein FtsX. An FtsE null mutant showed filamentous growth and appeared viable on high salt medium only, indicating a role for FtsE in cell division and/or salt transport.

Epitope tagging analysis of the outer membrane folding of the molecular usher FaeD involved in K88 fimbriae biosynthesis in Escherichia coli.

To analyse the outer membrane folding of the molecular usher FaeD, tagged derivatives were prepared and their expression, tag-localisation and functioning in K88 fimbriae biosynthesis was studied. A semi-random insertion mutagenesis approach with factor Xa cleavage sites yielded six tagged FaeD derivatives. A site-directed mutagenesis approach in which c-myc epitopes were inserted yielded twenty-one different derivatives. Four tagged FaeD constructs were not expressed in the outer membrane as full-sized proteins to levels that could be detected by using immunoblotting analyses. Two of these had an insertion in the amino-terminal part of FaeD, whereas the other two had a tag inserted in the carboxyl-terminal part. The latter ones yielded stable carboxyl-terminally shortened truncates of about 70 kDa, as did other mutations in this region. Six tagged derivatives were expressed but the location of the tag with respect to the outer membrane could not be determined, possibly due to shielding. Functional analysis showed that insertion of a tag in two regions of FaeD, a central region of approximately 200 amino acid residues (a.a. 200-400) and the carboxyl-terminal region (a.a. 600-end), resulted in a defective K88 fimbriae biosynthesis. In-frame deletions in the amino-terminal region of FaeD abolished fimbriae production. The integrity of these regions is obviously essential for fimbriae biosynthesis. Based on the results and with the aid of a computer analysis programme for the prediction of outer membrane beta-strands, a folding model with 22 membrane spanning beta-strands and two periplasmioc domains has been developed.

Characterization of a hemoglobin protease secreted by the pathogenic Escherichia coli strain EB1.

Many pathogenic bacteria can use heme compounds as a source of iron. Pathogenic Escherichia coli strains are capable of using hemoglobin as an iron source. However, the mechanism of heme acquisition from hemoglobin is not understood for this microorganism. We present the first molecular characterization of a hemoglobin protease (Hbp) from a human pathogenic E. coli strain. The enzyme also appeared to be a heme-binding protein. Affinity purification of this bifunctional protein enabled us to identify the extracellular gene product, and to clone and analyze its gene. A purification procedure developed for Hbp allowed us to perform functional studies. The protein interacted with hemoglobin, degraded it and subsequently bound the released heme. These results suggest that the protein is involved in heme acquisition by this human pathogen. Hbp belongs to the so-called IgA1 protease-like proteins, as indicated by the kinetics of its membrane transfer and DNA sequence similarity. The gene of this protein appears to be located on the large pColV-K30 episome, that only has been isolated from human and animal pathogens. All these characteristics indicate that Hbp may be an important virulence factor that may play a significant role in the pathogenesis of E. coli infections.

The Escherichia coli SRP and SecB targeting pathways converge at the translocon.

Two distinct protein targeting pathways can direct proteins to the Escherichia coli inner membrane. The Sec pathway involves the cytosolic chaperone SecB that binds to the mature region of pre-proteins. SecB targets the pre-protein to SecA that mediates pre-protein translocation through the SecYEG translocon. The SRP pathway is probably used primarily for the targeting and assembly of inner membrane proteins. It involves the signal recognition particle (SRP) that interacts with the hydrophobic targeting signal of nascent proteins. By using a protein cross-linking approach, we demonstrate here that the SRP pathway delivers nascent inner membrane proteins at the membrane. The SRP receptor FtsY, GTP and inner membranes are required for release of the nascent proteins from the SRP. Upon release of the SRP at the membrane, the targeted nascent proteins insert into a translocon that contains at least SecA, SecY and SecG. Hence, as appears to be the case for several other translocation systems, multiple targeting mechanisms deliver a variety of precursor proteins to a common membrane translocation complex of the E.coli inner membrane.

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.