Construction and evaluation of a plasmid vector for the expression of recombinant lipoproteins in Escherichia coli

Outer membrane lipoproteins are emerging as key targets for protective immunity to many bacterial pathogens. Heterologous expression of lipoproteins in Escherichia coli does not always result in high level expression of acylated recombinant protein. Thus, these proteins do not take up their correct membrane topology and are lacking the immunostimulatory properties endowed by the lipid. To this end, we have designed a lipoprotein expression vector (pDUMP) that results in the production of fusion proteins containing the E. coli major outer membrane lipoprotein (Lpp) signal sequence, lipoprotein signal peptidase recognition site, and the +2 outer membrane sorting signal at their N termini. To test the ability of pDUMP to express lipoproteins from heterologous hosts, the surface lipoprotein PsaA from the Gram-positive organism Streptococcus pneumoniae and the outer membrane lipoproteins MlpA from the Gram-negative Pasteurella multocida and BlpA from the spirochete Brachyspira hyodysenteriae were cloned into both hexahistidine fusion vectors and pDUMP. High level expression of antigenically active protein from both the hexahistidine fusion vectors and pDUMP resulted in abundant bands of the predicted molecular masses when analyzed by SDS-PAGE. When grown in the presence of 3[H]palmitic acid, proteins encoded by pDUMP were observed to incorporate palmitic acid whilst the hexahistidine fusion proteins did not. Using mass spectrometry and image analysis we determined the efficiency of lipidation between the three clones to vary from 31.7 to 100%. In addition, lipidated, but not hexahistidine, forms of the proteins were presented on the E. coli surface.     

Production and characterisation of a monoclonal antibody to Serpulina hyodysenteriae

Abstract A monoclonal antibody (mAb) directed against Serpulina hyodysenteriae , the causative agent of swine dysentery, was produced and characterised. The mAb (BJL/SH1) reacted in Western blots with a protein with a molecular mass of about 30 kDa in outer membrane preparations from a range of S. hyodysenteriae isolates of different serotypes. It did not react with preparations made from a variety of non- S. hyodysenteriae intestinal spirochaetes. Immunogold labelling was used to confirm the location of the reactive epitope on the cell outer membrane. The mAb agglutinated and produced fluorescence only with strains of S. hyodysenteriae , and should prove to be a useful reagent for identification of S. hyodysenteriae .     

Presence of 22-kDa protein reacting with sera in piglets experimentally infected with Brachyspira hyodysenteriae

In order to examine the effect of spectinomycin on outbreaks of swine dysentery, experimental infection of piglets with Brachyspira hyodysenteriae was carried out. Feed with and without spectinomycin (SP) was given to each piglet ad libitum and the susceptibility of the piglets to infection with B. hyodysenteriae was compared between SP-treated and untreated piglets. The results showed that the SP-treated piglets did not display clinical signs of swine dysentery unlike the untreated piglets. The sera obtained from these piglets were examined by the microscopic agglutination test and antibodies to B. hyodysenteriae in both groups of experimentally infected piglets were detected and the reaction was serogroup-specific. The agglutination titers were very high in the untreated piglets with dysentery while the titers in the SP-treated piglets were lower than those in the untreated piglets. In addition, the immunoblotting technique was applied and the results demonstrated that 22- and 17-kDa proteins in strain ATCC 31212 (serogroup B) reacted strongly with the sera from the untreated piglets but not with the sera from the SP-treated piglets. The 22- and 17-kDa proteins also reacted with strain ATCC 27164 (serogroup A) which belongs to a different serogroup. The 22- and 17-kDa proteins were also confirmed in six other strains of B. hyodysenteriae which belong to six different serogroups. These proteins were sensitive to proteinase K. These results indicate that the 22- and 17-kDa proteins are common to eight strains of B. hyodysenteriae which differ serologically from each other.     

Cloning, expression and immunogenicty analysis of five outer membrane proteins of Vibrio parahaemolyticus zj2003

Genes of five outer membrane proteins of Vibrio parahaemolyticus zj2003, including OmpW, OmpV, OmpK, OmpU and TolC, were cloned and expressed as N-terminal His(6)-tagged proteins in Escherichia coli. The recombinant fusion proteins were purified with nickel chelate affinity chromatography. To analyze the immunogenicity of these proteins, large yellow croaker (Pseudosciaena crocea) were immunized by intraperitoneal injection. Antibody response was assessed by method of enzyme-linked immunosorbent assay. Titres to all five recombinant proteins increased during 4 to 8 weeks post immunization, within the range of log 2 values of 5.75 to 10.8. Recorded relative survival percent (RPS) of the vaccinated groups varied from 80% to 90%, while 10 fish in control group all died. Western blot tests were undertaken with the serum of survival fish after experimental infection. Except for recombinant TolC, the other four recombinant proteins were recognized by the serum. It is indicated that four outer membrane proteins of V. parahaemolyticus zj2003, including OmpW, OmpV, OmpU and OmpK, are immunogenic during in vivo infection, which would be of some significance in developing efficient vaccine in aquaculture. This is the first report of successful vaccination against V. parahaemolyticus with purified recombinant outer membrane proteins.     

The Salmonella typhimurium virulence plasmid complement resistance gene rck is homologous to a family of virulence-related outer membrane protein genes, including pagC and ail.

A fragment of the Salmonella typhimurium virulence plasmid containing the rck locus, when cloned in the recombinant cosmid pADE016, was shown previously to confer high-level complement resistance on both rough and smooth Escherichia coli, Salmonella minnesota, and S. typhimurium and was associated with the production of an outer membrane protein. We determined the nucleotide sequence of the fragment containing the rck locus. Mutations in the two major open reading frames confirmed that the complement resistance mediated by pADE016 was due to a single 555-bp rck gene encoding a 17-kDa outer membrane protein. Analysis of the rck gene revealed that the Rck outer membrane protein consisted of 185 amino acid residues, with a calculated postcleavage molecular mass of 17.4 kDa. Rck is homologous to a family of outer membrane proteins expressed in gram-negative bacteria, two of which have been associated with virulence-related phenotypes: PagC, required by S. typhimurium for survival in macrophages and for virulence in mice; and Ail, a product of the Yersinia enterocolitica chromosome capable of mediating bacterial adherence to and invasion of epithelial cell lines. Rck, most closely related to PagC, represents the third outer membrane protein in this five-member family with a distinct virulence-associated phenotype.     

Expression of the Chlamydia trachomatis major outer membrane protein-encoding gene in Escherichia coli: role of the 3' end in mRNA stability

The major outer membrane protein (MOMP)-encoding gene (omp1) of Chlamydia trachomatis has been cloned into Escherichia coli and partially sequenced. This recombinant gene expresses a full-length 40-kDa product, which is recognized by a monoclonal antibody directed against the species-specific epitope of MOMP. The recombinant omp1 is expressed in either insertion orientation, indicating that it utilizes its own promoter system. The endogenous omp1 promoter possesses a relatively low activity despite the high level of MOMP expression. Deletion of a 520-bp fragment at the 3' end encoding 39 amino acids (aa) at the C terminus and the remainder of the noncoding region leads to a significant decrease in mRNA stability and loss of protein synthesis. When the MOMP-encoding plasmid was introduced into E. coli minicells, it expressed 40- and 43-kDa proteins; however, inhibition of post-translational processing by ethanol revealed only a 43-kDa protein. These data indicate that the unprocessed omp1 gene product contains a 22-aa leader sequence which is cleaved during translocation to the outer membrane, to yield a processed 40-kDa protein. The recombinant MOMP was localized to the outer membrane E. coli fraction, comparable to the location of the native C. trachomatis protein.     

Antigenic variability of the outer membrane antigens of Legionella pneumophila serogroups 1 to 8

The outer membrane proteins of Legionella pneumophila serogroups 1 to 8 were prepared from broken cells by selective solubilization using sodium lauryl sarcosinate. The isolated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose sheets. Rabbit antisera against each of the eight serogroups of L. pneumophila were obtained by immunizing each animal with live bacteria. The transferred proteins were revealed using these antisera and peroxidase-labeled swine anti-rabbit immunoglobulins. Antigenic determinants common to all eight serogroups were found in at least three outer membrane antigens (19, 29, and 45 kilodaltons (kDa)). However, cross-absorption experiments revealed that these three antigens were immunologically related, but not identical among serogroups. The antigenic relationships observed with two of these three antigens correlated well with cross-reactions observed in immunofluorescence. When a monoclonal antibody directed against L. pneumophila serogroup 1 lipopolysaccharide was used to reveal a blot of serogroup 1 outer membrane antigens, the 29- and 45-kDa bands appeared. This demonstrates a strong association between lipopolysaccharide and outer membrane proteins.     

Cloning and sequencing of a gene encoding a 21-kilodalton outer membrane protein from Bordetella avium and expression of the gene in Salmonella typhimurium.

Three gene libraries of Bordetella avium 197 DNA were prepared in Escherichia coli LE392 by using the cosmid vectors pCP13 and pYA2329, a derivative of pCP13 specifying spectinomycin resistance. The cosmid libraries were screened with convalescent-phase anti-B. avium turkey sera and polyclonal rabbit antisera against B. avium 197 outer membrane proteins. One E. coli recombinant clone produced a 56-kDa protein which reacted with convalescent-phase serum from a turkey infected with B. avium 197. In addition, five E. coli recombinant clones were identified which produced B. avium outer membrane proteins with molecular masses of 21, 38, 40, 43, and 48 kDa. At least one of these E. coli clones, which encoded the 21-kDa protein, reacted with both convalescent-phase turkey sera and antibody against B. avium 197 outer membrane proteins. The gene for the 21-kDa outer membrane protein was localized by Tn5seq1 mutagenesis, and the nucleotide sequence was determined by dideoxy sequencing. DNA sequence analysis of the 21-kDa protein revealed an open reading frame of 582 bases that resulted in a predicted protein of 194 amino acids. Comparison of the predicted amino acid sequence of the gene encoding the 21-kDa outer membrane protein with protein sequences in the National Biomedical Research Foundation protein sequence data base indicated significant homology to the OmpA proteins of Shigella dysenteriae, Enterobacter aerogenes, E. coli, and Salmonella typhimurium and to Neisseria gonorrhoeae outer membrane protein III, Haemophilus influenzae protein P6, and Pseudomonas aeruginosa porin protein F. The gene (ompA) encoding the B. avium 21-kDa protein hybridized with 4.1-kb DNA fragments from EcoRI-digested, chromosomal DNA of Bordetella pertussis and Bordetella bronchiseptica and with 6.0- and 3.2-kb DNA fragments from EcoRI-digested, chromosomal DNA of B. avium and B. avium-like DNA, respectively. A 6.75-kb DNA fragment encoding the B. avium 21-kDa protein was subcloned into the Asd+ vector pYA292, and the construct was introduced into the avirulent delta cya delta crp delta asd S. typhimurium chi 3987 for oral immunization of birds. The gene encoding the 21-kDa protein was expressed equivalently in B. avium 197, delta asd E. coli chi 6097, and S. typhimurium chi 3987 and was localized primarily in the cytoplasmic membrane and outer membrane. In preliminary studies on oral inoculation of turkey poults with S. typhimurium chi 3987 expressing the gene encoding the B. avium 21-kDa protein, it was determined that a single dose of the recombinant Salmonella vaccine failed to elicit serum antibodies against the 21-kDa protein and challenge with wild-type B. avium 197 resulted in colonization of the trachea and thymus with B. avium 197.     

大肠杆菌脑微血管内皮细胞侵袭基因ibeB的编码产物为外膜蛋白前体

为进一步探讨大肠杆菌脑微血管内皮细胞侵袭基因ibeB的生物学特性 ,将ibeB基因克隆到pET2 8a(+)载体 ,以E .coliBL2 1 (DE3)为宿主菌 ,经IPTG诱导后 ,通过Ni2 + NTA树脂提纯IbeB蛋白 .SDS PAGE确定纯化蛋白的分子量 ;应用无蛋白酶的体外转录和翻译系统进一步鉴定ibeB基因表达蛋白的分子量 ;通过 [3 5S]Met标记的体内T7表达体系并结合膜蛋白分离技术定位IbeB蛋白在细菌中的亚细胞分布 ;利用细菌侵袭实验分析IbeB蛋白抗体对E .coliK1侵袭人脑微血管内皮细胞的封闭作用 .结果发现 ,ibeB基因的重组蛋白表达纯化产物呈现出 5 0kD和 34kD两种分子量大小 ,5 0kD存在于表达细菌的可溶性部分 ,而 34kD则存在于包涵体中 ;体外翻译实验也显示出较弱的 5 0kD和较浓的 34kD两个蛋白带 ;体内T7表达体系实验显示 34kD的IbeB成熟蛋白定位于E .coli的外膜 ;抗 34kDIbeB蛋白抗体能封闭E .coli对人脑微血管内皮细胞的侵袭 .这些结果提示 ,大肠杆菌脑微血管内皮细胞侵袭基因ibeB的编码产物为 5 0kD的外膜蛋白前体 ,该前体可通过分子内剪接形成成熟的 34kDIbeB蛋白     

Outer Membrane Proteins of Escherichia coli VII. Evidence That Bacteriophage-Directed Protein 2 Functions as a Pore

Protein 1, a major protein of the outer membrane of Escherichia coli, has been shown to be the pore allowing the passage of small hydrophilic solutes across the outer membrane. In E. coli K-12 protein 1 consists of two subspecies, 1a and 1b, whereas in E. coli B it consists of a single species which has an electrophoretic mobility similar to that of 1a. K-12 strains mutant at the ompB locus lack both proteins 1a and 1b and exhibit multiple transport defects, resistance to toxic metal ions, and tolerance to a number of colicins. Mutation at the tolF locus results in the loss of 1a, in less severe transport defects, and more limited colicin tolerance. Mutation at the par locus causes the loss of protein 1b, but no transport defects or colicin tolerance. Lysogeny of E. coli by phage PA-2 results in the production of a new major protein, protein 2. Lysogeny of K-12 ompB mutants resulted in dramatic reversal of the transport defects and restoration of the sensitivity to colicins E2 and E3 but not to other colicins. This was shown to be due to the production of protein 2, since lysogeny by phage mutants lacking the ability to elicit protein 2 production did not show this effect. Thus, protein 2 can function as an effective pore. ompB mutations in E. coli B also resulted in loss of protein 1 and similar multiple transport defects, but these were only partially reversed by phage lysogeny and the resulting production of protein 2. When the ompB region from E. coli B was moved by transduction into an E. coli K-12 background, only small amounts of proteins 1a and 1b were found in the outer membrane. These results indicate that genes governing the synthesis of outer membrane proteins may not function interchangeably between K-12 and B strains, indicating differences in regulation or biosynthesis of these proteins between these strains.     

Extracellular accumulation of recombinant protein by Escherichia coli in a defined medium

Extracellular accumulation of recombinant proteins in the culture medium of Escherichia coli is desirable but difficult to obtain. The inner or cytoplasmic membrane and the outer membrane of E. coli are two barriers for releasing recombinant proteins expressed in the cytoplasm into the culture medium. Even if recombinant proteins have been exported into the periplasm, a space between the outer membrane and the inner membrane, the outer membrane remains the last barrier for their extracellular release. However, when E. coli was cultured in a particular defined medium, recombinant proteins exported into the periplasm could diffuse into the culture medium automatically. If a nonionic detergent, Triton X-100, was added in the medium, recombinant proteins expressed in the cytoplasm could also be released into the culture medium. It was then that extracellular accumulation of recombinant proteins could be obtained by exporting them into the periplasm or releasing them from the cytoplasm with Triton X-100 addition. The tactics described herein provided simple and valuable methods for achieving extracellular production of recombinant proteins in E. coli.     

Identification of the vibriobactin receptor of Vibrio cholerae.

Vibrio cholerae produces the novel phenolate siderophore vibriobactin and several outer membrane proteins in response to iron starvation. To determine whether any of these iron-regulated outer membrane proteins serves as the receptor for vibriobactin, the classical V. cholerae strain 0395 was mutagenized by using TnphoA, and iron-regulated fusions were analyzed for vibriobactin transport. One mutant, MBG14, was unable to bind or utilize exogenous vibriobactin and did not grow in low-iron medium. However, synthesis of the siderophore and transport of other iron complexes, including ferrichrome, hemin, and ferric citrate, were unaffected in MBG14. Analysis of membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the loss from the mutant of a 74-kDa iron-regulated outer membrane protein present in the parental strain when grown in iron-limiting conditions. This protein partitioned into the detergent phase during Triton X-114 extraction, suggesting that it is a hydrophobic membrane protein. DNA sequences encoding the gene into which TnphoA had inserted, designated viuA (vibriobactin uptake), restored the wild-type phenotype to the mutant; the complemented mutant expressed the 74-kDa outer membrane protein under iron-limiting conditions and possessed normal vibriobactin binding and uptake. These data indicate that the 74-kDa outer membrane protein of V. cholerae serves as the vibriobactin receptor.     

Expression and purification of polyhistidine-tagged rotavirus NSP4 proteins in insect cells

The rotavirus nonstructural NSP4 protein, a transmembrane endoplasmic reticulum-specific glycoprotein, has been described as the first viral enterotoxin. Purified NSP4 or a peptide corresponding to NSP4 residues 114-135 induces diarrhea in young mice. NSP4 has a membrane-destabilizing activity and causes an increase in intracellular calcium levels and chloride secretion by a calcium-dependent signalling pathway in eucaryotic cells. In this study, four recombinant baculoviruses were generated expressing the rotavirus NSP4 glycoprotein from the human strains Wa and Ito, the porcine strain OSU, and the simian strain SA11, which belong to two different NSP4 genotypes, A and B. The recombinant glycoproteins, expressed as polyhistidine-tagged molecules, were analyzed by Western blotting and immunoprecipitation. Newborn mice responded with diarrhea after inoculation with each of the recombinant NSP4 proteins.     

Cloning and functional analysis of the P97 swine cilium adhesin gene of Mycoplasma hyopneumoniae.

Colonization of the swine respiratory tract by Mycoplasma hyopneumoniae is accomplished by specific binding to the cilia of the mucosal epithelial cells. Previous studies have implicated a 97-kDa outer membrane-associated protein, P97, that appeared to mediate this interaction. In order to further define the role of P97 in adherence to porcine cilia, the structural gene was cloned and sequenced, and the recombinant products were analyzed. Monoclonal antibodies were used to identify recombinant clones in a genomic library expressed in an opal suppressor host because of alternate codon usage by mycoplasmas. The gene coding for P97 was then identified by Tn1000 mutagenesis of recombinant clones. DNA sequence analysis revealed an open reading frame coding for a 124.9-kDa protein with a hydrophobic transmembrane spanning domain. The N-terminal sequence of purified P97 mapped at amino acid position 195 of the translated sequence, indicating that a processing event had occurred in M. hyopneumoniae. Both recombinant P97 protein expressed in an Escherichia coli opal suppressor host and M. hyopneumoniae bound specifically to swine cilia, and the binding was inhibited by heparin and fucoidan, thus supporting the hypothesis that P97 was actively involved in binding to swine cilia in vivo.     

Purified outer membranes of Serpulina hyodysenteriae contain cholesterol.

We have isolated outer and inner membranes of Serpulina hyodysenteriae by using discontinuous sucrose density gradients. The outer and inner membrane fractions contained less than 1 and 2%, respectively, of the total NADH oxidase activity (soluble marker) in the cell lysate. Various membrane markers including lipooligosaccharide (LOS), the 16-kDa outer membrane lipoprotein (SmpA), and the C subunit of the F1F0 ATPase indicated that the lowest-density membrane fraction contained outer membranes while the high-density membrane fraction contained inner membranes and that both are essentially free of contamination by the periplasmic flagella, a major contaminant of membranes isolated by other techniques. The outer membrane fractions (rho = 1.10 g/cm3) contained 0.25 mg of protein/mg (dry weight), while the inner membrane samples (rho = 1.16 g/cm3) contained significantly more protein (0.55 mg of protein/mg [dry weight]). Lipid analysis revealed that the purified outer membranes contained cholesterol as a major component of the membrane lipids. Treatment of intact S. hyodysenteriae with different concentrations of digitonin, a steroid glycoside that interacts with cholesterol, indicated that the outer membrane could be selectively removed at concentrations as low as 0.125%.     

Characterization of an OmpA-like outer membrane protein of the acidophilic iron-oxidizing bacterium, <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

An OmpA family protein (FopA) previously reported as one of the major outer membrane proteins of an acidophilic iron-oxidizing bacterium Acidithiobacillus ferrooxidans was characterized with emphasis on the modification by heat and the interaction with peptidoglycan. A 30-kDa band corresponding to the FopA protein was detected in outer membrane proteins extracted at 75°C or heated to 100°C for 10 min prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, the band was not detected in outer membrane proteins extracted at ≤40°C and without boiling prior to electrophoresis. By Western blot analysis using the polyclonal antibody against the recombinant FopA, FopA was detected as bands with apparent molecular masses of 30 and 90 kDa, suggesting that FopA existed as an oligomeric form in the outer membrane of A. ferrooxidans. Although the fopA gene with a sequence encoding the signal peptide was successfully expressed in the outer membrane of Escherichia coli, the recombinant FopA existed as a monomer in the outer membrane of E. coli. FopA was detected in peptidoglycan-associated proteins from A. ferrooxidans. The recombinant FopA also showed the peptidoglycan-binding activity.     

Variation in the expression of pili and outer membrane protein by Neisseria meningitidis during the course of meningococcal infection

The occurrence of antigenic shift during meningococcal infection has been investigated by comparison of paired isolates obtained from the blood, cerebrospinal fluid or nasopharynx of patients. Isolates from any individual produced identical DNA 'fingerprints' and showed stability in expression of both class 2 outer membrane protein and an antigen common to pathogenic Neisseria, confirming their origin as a single strain. One of the four strains examined produced variants which differed in the molecular mass of their class 5 outer membrane proteins. Three of the strains produced pili containing the epitope recognized by monoclonal antibody SM1 and two of these gave rise to variants which expressed pili of differing subunit molecular masses. The two variants of the remaining strain produced pilins lacking the common epitope detected by antibody SM1 but radioimmune precipitation with polyclonal anti-pilus antiserum revealed that variation in the molecular mass of the pilin expressed also occurred with this second class of pili. Antigenic variation in expression of both class 5 outer membrane proteins and pili therefore appears to be a common occurrence during meningococcal infection.     

The cellular location of a foreign B cell epitope expressed by recombinant bacteria determines its T cell-independent or T cell-dependent characteristics.

We have targeted two foreign B cell antigenic determinants to different locations in the Escherichia coli cell to examine what effect this had on antibody responses elicited by the recombinant bacteria. The two epitopes were the 132-145 peptide from the PreS2 region of hepatitis B virus and the C3 neutralization epitope of poliovirus type 1. They were each expressed in two forms either on the surface, as part of the outer-membrane protein LamB, or soluble in the periplasm, as part of the periplasmic protein MalE. When live bacteria expressing the foreign epitope at the cell surface were used for immunization of mice, they induced T cell-independent antibody responses characterized by a rapid induction of IgM and IgG antibodies. In contrast, when the same foreign epitope was inserted into the MalE protein, the antibody response was only detectable after 3 wk, belonged only to the IgG class and was strictly T cell dependent. This study has therefore identified two major pathways by which epitopes expressed by bacterial cells can stimulate specific antibody responses. The first pathway is mediated by direct activation of B cells by bacterial cell-surface Ag and does not require T cell help. The second pathway is T cell dependent and concerns Ag that can be released from the bacteria in a soluble form. We have also studied the effect of the exact position of the B cell antigenic determinant within the LamB protein and with respect to the outer membrane by comparing the immunogenicity of the PreS epitope inserted at three different permissive sites of LamB. The data indicated that to obtain an antibody response with intact bacteria, the epitope must be protruding sufficiently from the outside of the outer membrane. In contrast, when semipurified hybrid proteins were used as immunogen, the exact position of the B cell antigenic determinant within solubilized LamB protein does not influence its immunogenicity.     

Involvement of ExbB and TonB in transport across the outer membrane of Escherichia coli: phenotypic complementation of exb mutants by overexpressed tonB and physical stabilization of TonB by ExbB.

The exb locus in Escherichia coli consists of two genes, termed exbB and exbD. Exb functions are related to TonB function in that most TonB-dependent processes are enhanced by Exb. Like tonB mutants, exb mutants were resistant to colicin M and albomycin but, in contrast to tonB mutants, showed only reduced sensitivity to colicins B and D. Overexpressed tonB on the multicopy vector pACYC177 largely restored the sensitivity of exb mutants to colicins B, D, and M but only marginally increased sensitivity to albomycin. Suppression of the btuB451 mutation in the structural gene for the vitamin B12 outer membrane receptor protein by a mutation in tonB occurred only in an exb+ strain. Degradation of the unstable overproduced TonB protein was prevented by overproduced ExbB protein. The ExbB protein also stabilized the ExbD protein. Pulse-chase experiments with radiolabeled ferrichrome revealed release of ferrichrome from exbB, tonB, and fhuC mutants, showing that ferrichrome had not crossed the cytoplasmic membrane. It is concluded that the ExbB and ExbD proteins contribute to the activity of TonB and, like TonB, are involved in receptor-dependent transport processes across the outer membrane.