High-affinity iron uptake systems present in Erwinia carotovora subsp. carotovora include the hydroxamate siderophore aerobactin.

The phytopathogenic bacterium Erwinia carotovora subsp. carotovora W3C105 produced the hydroxamate siderophore aerobactin under iron-limiting conditions. A survey of 22 diverse strains of E. carotovora revealed that strain W3C105 alone produced aerobactin. The ferric-aerobactin receptor of strain W3C105 was an 80-kDa protein, identified by immunoblots of Sarkosyl-soluble proteins obtained from E. carotovora cells grown in iron-depleted medium and probed with antiserum raised against the 74-kDa ferric-aerobactin receptor encoded by the pColV-K30 plasmid of Escherichia coli. Genes determining aerobactin biosynthesis and uptake were localized to an 11.3-kb EcoRI-HindIII chromosomal fragment of strain W3C105. A 10-kb subclone of the fragment conferred on E. coli DH5 alpha both aerobactin biosynthesis and uptake, determined by cloacin DF13 sensitivity, the presence of the 80-kDa receptor protein, and iron-independent growth of E. coli clones. The aerobactin biosynthesis genes of E. carotovora W3C105 hybridized to those of the pColV-K30 plasmid of E. coli, but the restriction patterns of the aerobactin regions of E. coli and E. carotovora differed. Although the aerobactin region of enteric bacteria is commonly flanked by IS1-like sequences, IS1 sequences were not detected in the genomic DNA or the cloned aerobactin region of E. carotovora. E. coli DH5 alpha cells harboring cloned aerobactin biosynthesis genes from E. carotovora W3C105 produced greater quantities of aerobactin and the 80-kDa ferric-aerobactin receptor when grown in iron-limited than in iron-replete medium. Strain W3C105 grew on an iron-limited medium, whereas derivatives that lacked a functional aerobactin iron acquisition system did not grow on the medium. These results provide evidence for the occurrence and heterogeneity of aerobactin as a high-affinity iron uptake system of both clinical and phytopathogenic species of the Enterobacteriaceae. Although future studies may reveal a role for aerobactin in the virulence or ecology of strain W3C105, a functional aerobactin iron acquisition system is not necessary for the pathogenicity of E. carotovora.     

Molecular cloning, expression, and regulation in Escherichia coli K-12 of a chromosome-mediated aerobactin iron transport system from a human invasive isolate of E. coli K1.

We have cloned chromosomal genes determining the aerobactin iron transport system from the Escherichia coli K1 strain VW187. Mapping and hybridization experiments showed that the VW187 aerobactin region was identical to that of the plasmid ColV-K30. However, in the E. coli K-12 background, the biosynthesis of both siderophore and ferric aerobactin receptor encoded by the VW187-derived recombinant plasmids was not repressed by iron to the same extent found when a recombinant plasmid derived from pColV-K30 was used. RNA-DNA dot-blot hybridization experiments demonstrated that the aerobactin-specific mRNA synthesized by the VW187-derived clones was not iron regulated in E. coli K-12. In contrast, the synthesis of aerobactin and its receptor in strain VW187 was completely repressed by iron regardless of whether the recombinant plasmids originated from VW187 or pColV-K30. Similar results were obtained with gene fusions in which a promoterless lac operon was placed under the control of aerobactin promoter regions of either chromosome- or plasmid-mediated aerobactin systems. DNA sequencing of the chromosomal aerobactin promoter region showed changes in bases located immediately upstream to the -35 region compared with the corresponding region in pColV-K30, which is known to be part of the binding site for the Fur repressor protein.     

New aerobactin-mediated iron uptake system in a septicemia-causing strain of Enterobacter cloacae.

Unlike the great majority of the aerobactin-producing enteric bacteria documented in the literature, Enterobacter cloacae EK33, isolated from a case of human neonatal meningitis, did not show any homology at the DNA level with the prototype aerobactin system encoded by the ColV-K30 plasmid. However, both the nuclear magnetic resonance spectrum and fast-atom bombardment mass spectrometry of the siderophore purified from EK33 confirmed its identity with aerobactin. Bioassay screening of a gene library of total DNA of EK33 led to the isolation of several aerobactin-positive clones. Under conditions of iron limitation, these clones expressed in Escherichia coli a protein of 72 kilodaltons that reacted with antiserum raised against the pColV-K30 74-kilodalton aerobactin receptor, while the original E. cloacae strain synthesized an 85-kilodalton protein which also cross-reacted with the antiserum. Restriction endonuclease analysis of the cloned DNA confirmed the structural differences between the two aerobactin genetic systems.     

Production of the siderophore aerobactin by a halophilic pseudomonad.

A bacterial strain, isolated from a cyanobacterial culture, was identified as Pseudomonas sp. strain X40. Under iron-limiting conditions, the Pseudomonas sp. produced aerobactin, a dihydroxamate siderophore previously found only in the family Enterobacteriaceae. Aerobactin was identified by electrophoretic mobility, spectrophotometric titration, proton nuclear magnetic resonance spectroscopy, mass spectrometry, acid hydrolysis, and biological activity. Aerobactin was used as a siderophore in the Pseudomonas sp. and Escherichia coli. Two iron-repressed outer membrane proteins were observed in the Pseudomonas sp., neither of which had electrophoretic mobility identical to that of the aerobactin outer membrane receptor protein from E. coli. DNA hybridization assays showed no hybridization to the aerobactin genes from the E. coli plasmid pColV, indicating that the genetic determinants for aerobactin production by Pseudomonas strain X40 differ substantially from those found in the archetypic enteric plasmid pColV-K30.     

Production of the siderophore aerobactin by a halophilic pseudomonad.

A bacterial strain, isolated from a cyanobacterial culture, was identified as Pseudomonas sp. strain X40. Under iron-limiting conditions, the Pseudomonas sp. produced aerobactin, a dihydroxamate siderophore previously found only in the family Enterobacteriaceae. Aerobactin was identified by electrophoretic mobility, spectrophotometric titration, proton nuclear magnetic resonance spectroscopy, mass spectrometry, acid hydrolysis, and biological activity. Aerobactin was used as a siderophore in the Pseudomonas sp. and Escherichia coli. Two iron-repressed outer membrane proteins were observed in the Pseudomonas sp., neither of which had electrophoretic mobility identical to that of the aerobactin outer membrane receptor protein from E. coli. DNA hybridization assays showed no hybridization to the aerobactin genes from the E. coli plasmid pColV, indicating that the genetic determinants for aerobactin production by Pseudomonas strain X40 differ substantially from those found in the archetypic enteric plasmid pColV-K30.     

Transport of ferric-aerobactin into the periplasm and cytoplasm of Escherichia coli K12: role of envelope-associated proteins and effect of endogenous siderophores.

Purified [14C]aerobactin, supplied exogenously to non-growing bacteria, was translocated via the periplasm into the cytoplasm of Escherichia coli K12 strains expressing the aerobactin receptor protein IutA. No significant uptake was observed into either compartment of strains lacking the iutA gene or specifically defective in tonB. Uptake into both compartments was markedly reduced, but not abolished, in an exb mutant. Accumulation of [14C]aerobactin in the periplasm of fhuD, fhuB or fhuC mutant strains was not significantly lower than in the wild-type strain, but entry into the cytoplasm was greatly reduced in all cases. Uptake of aerobactin by strains wild-type for all transport functions occurred most efficiently in strains either lacking or specifically defective in the genetic determinants for aerobactin biosynthesis; significantly lower levels of exogenous 14C-labelled siderophore were observed in both compartments of strains producing aerobactin. Aerobactin-mediated 59Fe uptake, however, was not inhibited by the presence of endogenous aerobactin. Endogenous enterochelin did not affect aerobactin uptake.     

Binding of basement-membrane laminin by Escherichia coli

An invasive Escherichia coli (EIEC) isolate was found to bind basement-membrane laminin in a saturable and time-dependent manner. Excess of unlabelled laminin inhibited the binding of the radioactively labelled protein. Non-invasive E. coli K-12 exhibited only low-level laminin binding but introduction of the virulence-associated plasmid from the EIEC isolate led to high-level binding. Expression of a receptor for laminin on the bacteria was therefore associated with the presence of the virulence plasmid. Scatchard plot analysis indicated approximately 1000 receptors per bacterial cell, and a Kd of high-affinity binding of 0.5 pM. A laminin-binding protein which correlated with the presence of the plasmid was isolated and characterized. Its sequence of the eight amino-terminal amino acids was identical to that of the LamB protein of E. coli, although the molecular mass of the two in sodium dodecyl sulphate/polyacrylamide gel (SDS-PAGE) appeared to be slightly different. Both proteins reacted in immunoblot assays with polyclonal antisera raised against either protein, and both proteins bound laminin. Southern-blot hybridization analysis established that both the EIEC strain and the K-12 strains with or without the virulence plasmid contained one lamB gene only, and no laminin-binding protein appeared when the virulence plasmid was introduced into bacteria deleted for the lamB gene. On the basis of these results we suggest that native LamB protein of E. coli or a modified variant of it serves as a major receptor for laminin binding and is present at an increased level in invasive E. coli containing the virulence plasmid.     

Novel aerobactin receptor in Klebsiella pneumoniae

Several Klebsiella pneumoniae strains which produced enterochelin but not aerobactin were nevertheless sensitive to cloacin DF13. In contrast, a strain of serotype K1:O1 which produced both siderophores was cloacin-resistant. Loss by mutation of the O1 but not K1 antigen rendered this strain cloacin-sensitive, indicating that the O1 antigen prevented access of cloacin to the cloacin/aerobactin receptor. Unlike the K1:O1 strain, the aerobactin-negative strains failed to hybridize in a colony blot assay with an aerobactin receptor gene probe prepared from pColV-K30. However, antisera raised against the 74 kDa pColV-K30 aerobactin receptor cross-reacted with a 76 kDa outer-membrane protein in each K. pneumoniae strain. In addition to the 76 kDa protein, the K1:O1 strain also produced a strongly cross-reacting 74 kDa protein. To determine whether these aerobactin-negative strains could use aerobactin, mutants unable to synthesize siderophores were isolated. Aerobactin promoted the growth of these mutants in iron-deficient media. The evidence presented suggests that some K. pneumoniae strains produce an aerobactin iron-uptake system without apparent production of aerobactin and which is probably based on a 76 kDa receptor, the gene for which does not hybridize with aerobactin receptor gene encoded on pColV-K30.     

Flanking and internal regions of chromosomal genes mediating aerobactin iron uptake systems in enteroinvasive Escherichia coli and Shigella flexneri

We have investigated the presence of the aerobactin system and the location of the aerobactin genes in enteroinvasive strains of Escherichia coli. Also, we cloned the aerobactin region and its flanking sequences from the chromosome of a strain of Shigella flexneri and compared the molecular organization of the aerobactin genes in the two genera. Of the 11 enteroinvasive E. coli strains studied, 5 possessed the aerobactin genes, which were located on the chromosome in each case. These strains produced and utilized aerobactin and also were susceptible to the bacteriocin cloacin-DF13. Restriction endonuclease mapping and hybridization experiments showed that the regions corresponding to the aerobactin-specific sequences were very similar in both enteroinvasive E. coli and S. flexneri. However, differences were found in the region corresponding to the aerobactin receptor gene. The regions flanking the aerobactin system in enteroinvasive E. coli and S. flexneri exhibited some similarities but were different from those in pColV-K30. Under iron-limiting conditions, aerobactin-producing enteroinvasive E. coli and S. flexneri synthesized outer-membrane proteins of 76 and 77 kDa, respectively, which cross-reacted immunologically with rabbit antiserum raised against the 74 kDa pColV-K30-encoded ferric aerobactin receptor.     

Utilization of enterobactin and other exogenous iron sources by Haemophilus influenzae, H. parainfluenzae and H. paraphrophilus

The ability of Haemophilus influenzae, H. parainfluenzae and H. paraphrophilus to utilize iron complexes, iron-proteins and exogenous microbial siderophores was evaluated. In a plate bioassay, all three species used not only ferric nitrate but also the iron chelates ferric citrate, ferric nitrilotriacetate and ferric 2,3-dihydroxybenzoate. Each Haemophilus species examined also used haemin, haemoglobin and haem-albumin as iron sources although only H. influenzae could acquire iron from transferrin or from haemoglobin complexed with haptoglobin. None of the haemophili obtained iron from ferritin or lactoferrin or from the microbial siderophores aerobactin or desferrioxamine B. However, the phenolate siderophore enterobactin supplied iron to both H. parainfluenzae and H. paraphrophilus, and DNA isolated from both organisms hybridized with a DNA probe prepared from the Escherichia coli ferric enterobactin receptor gene fepA. In addition, a monospecific polyclonal antiserum raised against the E. coli 81 kDa ferric enterobactin receptor (FepA) recognized an iron-repressible outer membrane protein (OMP) in H. parainfluenzae of between 80 and 82 kDa (depending on the strain). This anti-FepA serum did not cross-react with any of the OMPs of H. paraphrophilus or H. influenzae. The OMPs of each Haemophilus species were also probed with antisera raised against the 74 kDa Cir or 74 kDa IutA (aerobactin receptor) proteins of E. coli. Apart from one H. parainfluenzae strain (NCTC 10665), in which an OMP of about 80 kDa cross-reacted with the anti-IutA sera, no cross-reactivity was observed between Cir, IutA and the OMPs of H. influenzae, H. parainfluenzae or H. paraphrophilus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasmid-determined cloacin DF13-susceptibility inEnterobacter cloacae andKlebsiella edwardsii; identification of the cloacin DF13/aerobactin outer membrane receptor proteins

BothEnterobacter cloacae H478 andKlebsiella edwardsii S15 were shown to harbour a relatively large conjugative plasmid that coded for cloacin DF13-susceptibility and the production and uptake of a hydroxamate iron chelator, most probably aerobactin. Protein-blotting experiments with antiserum raised against the purified cloacin DF13/aerobactin receptor protein fromEscherichia coli (Co1V-K30) revealed that the corresponding outer membrane receptor proteins ofEnt. cloacae H478 andK. edwardsii S15 had apparent mol wts of 85 000 and 76000, respectively.E. coli transconjugants harbouring either the plasmid fromEnt. cloacae H478 orK. edwardsii S15 expressed a cloacin DF13/aerobactin outer membrane receptor protein with a mol wt of 74000. The receptor protein encoded by theEnt. cloacae andK. edwardsii plasmids were immunologically more related to each other than to the pCo1V-K30-encoded receptor protein.     

Cloning and expression in Escherichia coli of a recA-like gene from Bacteroides fragilis

The recombinant plasmid pHG100, containing a 5.2-kb DNA fragment from Bacteroides fragilis, complemented defects in homologous recombination, DNA repair and prophage induction to various levels in an Escherichia coli recA mutant strain. There was no DNA homology between the cloned B. fragilis recA-like gene and E. coli chromosomal DNA. pHG100 produced two proteins with Mr of approx. 39,000 and 37,000 which cross-reacted with antibodies raised against E. coli RecA protein. The production of these proteins was not increased after UV induction. The cloned B. fragilis recA-like gene product did not enhance the production of native but defective E. coli RecA protein after UV irradiation.     

Cloning and expression in Escherichia coli of a recA-like gene from the acidophilic autotroph Thiobacillus ferrooxidans

A recombinant plasmid, pRSR100, containing the functional analogue of the Escherichia coli recA gene was isolated from a genomic library of Thiobacillus ferrooxidans ATCC 33020. The plasmid complemented defects in DNA repair and homologous recombination in E. coli recA mutant strains. Antiserum raised against E. coli RecA protein reacted with the native but defective E. coli HB101 RecA protein; it did not react with protein extracts from the recA deletion mutant E. coli JK696, but it reacted with two protein bands in extracts of E. coli JK696(pRSR100). A single band with an apparent Mr equal to the higher-Mr band in E. coli JK696(pRSR100) was detected in T. ferrooxidans cell extracts with the E. coli RecA antiserum.     

Cloning and characterization of the iutA gene which encodes ferric aerobactin receptor from marine Vibrio species

The iutA gene from marine Vibrio species SD004, which encoded a ferric aerobactin receptor for the uptake of iron(III), was cloned onto a multicopy plasmid, pUC 18, in Escherichia coli. Identification of the positive clone was achieved on the basis of its deferrization activity and was detected as a halo formation on the chrome azurol S (CAS)-containing selective plate. Nucleotide sequence analysis of the cloned DNA fragment revealed an open reading frame (ORF) which encoded a polypeptide of 706 amino acid residues, and the deduced molecular mass of this polypeptide was 77.906 kD. The amino acid sequence showed a 41% homology with that of the lutA protein from E. coli. The cloned gene was iutA, which encoded the ferric aerobactin receptor. Another incomplete ORF was found 100 bp upstream of the iutA gene, which was homologous (31 out of 49 amino acids) with the C-terminal region of the luc D protein of E. coli. It is suggested that aerobactin biosynthesis and the transport genes are located tandemly on the Vibrio chromosome and may form an aerobactin operon.     

Characterization of antibodies to chicken riboflavin carrier protein. Immunoneutralizing ability of antibodies to a sequence-specific region of the protein.

The properties of antibodies generated in rabbits against native riboflavin carrier protein (cRCP), riboflavin carrier protein that had been denatured/renatured by SDS treatment (SDS-RCP) or disulphide-bond-reduced then S-carboxymethylated (Carb-RCP) were studied. SDS-RCP could displace native RCP in radioimmunoassay (r.i.a.), whereas Carb-RCP could not. By using antibodies raised in five different rabbits against native cRCP, 125I-labelled Carb-RCP could bind between 0 and 30% of the native antibodies. Antibodies raised against native RCP appear to be largely directed towards specific conformational determinants of RCP. Carb-RCP displaced native RCP in an r.i.a. using antibodies raised against SDS-RCP. SDS denaturation presumably unmasks cryptic epitopes in native RCP. Carb-RCP was a weak immunogen and elicited, presumably, antibodies to sequential epitope/epitopes. When injected into pregnant mice the antibodies caused neutralization of RCP, leading to termination of pregnancy, indicating highly conserved sequential epitopes in chicken and rodent RCP. Antibodies raised against Carb-RCP or native RCP reacted with CNBr fragments of native RCP, further confirming the presence of sequence-specific antibodies elicited by Carb-RCP.     

Role of TraT protein, an anticomplementary protein produced in Escherichia coli by R100 factor, in serum resistance.

Escherichia coli K12 strain W3110/SM bearing a plasmid containing the traT gene (traT+ strain) was more resistant to the bactericidal activity of guinea pig serum than the same strain bearing this plasmid without the traT gene (traT- strain). A murine mAb was generated against synthetic TraT peptide (86-99). This antibody reacted only with denatured TraT protein, but it was used for monitoring TraT protein by immunoblotting during purification of the protein. Six mAb were then generated against partially purified traT protein from the solubilized membrane fraction of the traT+ strain. These mAb reacted with the native protein even on living cells, and their F(ab) fragments were found to suppress the inhibitory effect of the TraT protein on the bactericidal activity of serum. TraT protein was purified from solubilized membranes of the traT+ strain by ion exchange and gel filtration chromatographies. The purified TraT protein inhibited the lysis of sensitized erythrocytes by serum complement. Its inhibitory action was mainly on the C6 step. It strongly inhibited the reaction of C6 with EAC14b2a3b and excess C5, C7, C8, and C9. TraT protein also inhibited the reaction of C7-deficient human serum with guinea pig erythrocytes when it was activated by cobra venom factor. It did not inhibit the reaction of preformed C5b6 complexes. However, TraT did not have any effect on the cleavage of 125I[C5] to 125I[C5b] in similar conditions. It also partially inhibited the reaction steps of C4, C5, and factor B and limited guinea pig complement serum in 0.1% gelatin veronal buffered saline, pH 7.4, containing 10 mM EDTA with their respective preceding intermediate cells. It had no effect on either the binding of C3 to EAC14b2a or the cleavage of C3b by factors H and I. TraT protein probably inhibits the formation of C5b6 complex or causes structural alteration of the complex to a nonfunctional form.     

大肠杆菌K88ac菌毛操纵子fae全基因的克隆、表达及生物学活性初步研究

目的：在体外克隆和表达猪肠产毒性大肠杆菌（ETEC）K88ae菌毛操纵子,触结构基因,并检测重组菌毛的相关生物学活性。方法：利用长PCR技术以猪ETECK88ae株C83902基因组DNA为模板扩增编码K88菌毛操纵子触基因,克隆入表达质粒载体pBR322,构建和筛选重组质粒pBR322-fae,转化至不含任何菌毛的大肠杆菌EP株;电镜观察重组菌表面菌毛表达情况;用热抽提法提纯表达的重组菌毛;用纯化菌毛免疫小鼠制备高效价抗血清;用SDS-PAGE和Western blot检测重组菌毛的抗原性,用细胞黏附和黏附抑制试验检测其生物学活性。结果和结论：在电镜下观察到重组菌表面大量表达K88ae菌毛,该重组菌与兔抗K88ae菌毛单因子阳性血清、鼠抗K88ac菌毛单克隆抗体均产生凝集反应;纯化菌毛经SDS-PAGE,结构单位菌毛呈单一的相对分子质量约26×10^3的蛋白条带;纯化菌毛免疫小鼠后可制备出高效价的鼠抗血清,玻板凝集试验和Western blot结果表明体外表达的K88ae菌毛具有与K88ae野生菌毛相同的抗原性;猪小肠上皮细胞系黏附和黏附抑制实验结果表明重组EP菌和野生菌株一样具有较强的黏附猪小肠上皮细胞系的能力,而且提纯重组菌毛制备出的鼠抗血清能有效抑制上述重组菌或野生菌株对猪小肠上皮细胞系的黏附结合。     

Novel hemoglobins to enhance microaerobic growth and substrate utilization in Escherichia coli

Limited oxygen availability is a prevalent problem in microbial biotechnology. Recombinant Escherichia coli expressing the hemoglobin from Vitreoscilla (VHb) or the flavohemoglobin from Ralstonia eutropha (formerly Alcaligenes eutrophus) (FHP) demonstrate significantly increased cell growth and productivity under microaerobic conditions. We identify novel bacterial hemoglobin-like proteins and examine if these novel bacterial hemoglobins can elicit positive effects similar to VHb and FHP and if these hemoglobins alleviate oxygen limitation under microaerobic conditions when expressed in E. coli. Several finished and unfinished bacterial genomes were screened using R. eutropha FHP as a query sequence for genes (hmp) encoding hemoglobin-like proteins. Novel hmp genes were identified in Pseudomonas aeruginosa, Salmonella typhi, Klebsiella pneumoniae, Deinococcus radiodurans, and Campylobacter jejuni. Previously characterized hmp genes from E. coli and Bacillus subtilis and the novel hmp genes from P. aeruginosa, S. typhi, C. jejuni, K. pneumoniae, and D. radiodurans were PCR amplified and introduced into a plasmid for expression in E. coli. Biochemically active hemoproteins were expressed in all constructs, as judged by the ability to abduct carbon monoxide. Growth behavior and byproduct formation of E. coli K-12 MG1655 cells expressing various hemoglobins were analyzed in microaerobic fed-batch cultivations and compared to plasmid-bearing control and to E. coli cells expressing VHb. The clones expressing hemoglobins from E. coli, D. radiodurans, P.aeruginosa, and S. typhi reached approximately 10%, 27%, 23%, and 36% higher final optical density values, respectively, relative to the plasmid bearing E. coli control (A(600) 5.5). E. coli cells expressing hemoproteins from P. aeruginosa, S. typhi, and D. radiodurans grew to similar final cell densities as did the strain expressing VHb (A(600) 7.5), although none of the novel constructs was able to outgrow the VHb-expressing E. coli strain. Additionally, increased yield of biomass on glucose was measured for all recombinant strains, and an approximately 2-fold yield enhancement was obtained with D. radiodurans hemoprotein-expressing E. coli relative to the E. coli control carrying the parental plasmid without any hemoglobin gene.     

Reconstitution of binding protein dependent ribose transport in spheroplasts derived from a binding protein negative escherichia coli K12 mutant and from salmonella typhimurium

Highly purified ribose-binding protein from Escherichia coli has been used to reconstitute a binding-protein-dependent ribose transport in spheroplasts derived from a binding-protein-deficient mutant of E coli K 12, and in spheroplasts derived from Salmonella typhimurium. The cross-species reconstitution was nearly as efficient as the reconstitution of the E coli strain from which the binding protein was derived. Antibody raised against the ribose binding protein completely prevented reconstitution, whereas it had no effect on whole cells. The reconstitution procedure has been improved by generating spheroplasts from cells grown in a rich medium and by reducing the background uptake in spheroplasts through a special washing procedure. Rapid purification of ribose binding protein by high pressure liquid chromatography is also described.     

Expression of adenovirus type 12 E1b 58-kDa protein in Escherichia coli and production of antibodies raised against a 58-kDa::beta-galactosidase fusion protein

DNA fragments coding for the N-terminal 185 amino acids (aa) and for the entire coding region of the adenovirus (Ad)12 E1b 58-kDa protein have been cloned in a prokaryotic expression vector. The N-terminal region of the 58-kDa viral protein (aa 21-205) is expressed as a beta-galactosidase (beta Gal) fusion protein encoded by plasmid pB58Ngal. Escherichia coli strains transformed with this plasmid synthesize a full-length fusion protein of 150-kDa and two truncated proteins: a 140-kDa protein containing aa 64-205 and a 120-kDa polypeptide containing aa 158-205 of the E1b 58-kDa protein. Antibodies raised against purified fusion proteins specifically immunoprecipitate the E1b 58-kDa protein from Ad12-infected and transformed cells. Bacteria transformed with plasmid pB58 carrying the entire E1b 58-kDa coding region (minus the first N-terminal 20 aa which are replaced by 4 aa of beta Gal) showed dramatically reduced growth properties after induction of 58K gene expression. We have not been able to detect substantial amounts of the 58-kDa protein in these cells. However, the viral 58-kDa polypeptide could be synthesized in vitro from plasmid pB58 in a DNA-dependent translation system from E. coli.