Immunochemical comparison of phosphoribosylanthranilate isomerase-indoleglycerol phosphate synthetase among the Enterobacteriaceae.

The bifunctional enzyme of the tryptophan operon, phosphoribosylanthranilate isomerase-indoleglycerol phosphate synthetase (PRAI-InGPS;EC 4.1.1.48), was characterized by an immunochemical study of six representative members of the Enterobacteriaceae: Escherichia coli, Salmonella typhimurium, Enterobacter aerogenes, Serratia marcescens, Erwinia carotovora, and Proteus vulgaris. PRAI-InGPS was purified from E. coli, and antisera were prepared in rabbits. These antisera were utilized in quantitative microcomplement fixation allowing for a comparison of the overall antigenic surface structure of the various homologous enzymes. These data showed E. coli PRAI-InGPS and S. marcescens and E. carotovora PRAI-InGPS (taken as a group) to have an index of dissimilarity of approximately 10, whereas the other organisms had values intermediate. In addition, antiserum to E. coli tryptophan synthetase beta2 subunit was used in microcomplement fixation to extend the previous comparison of this subunit (Rocha, Crawford, and Mills, 1972) to E. carotovora and P. vulgaris. Indexes of dissimilarity for E. coli compared to P. vulgaris of E. carotovora were 1.0 and 1.7, respectively. Agar immunodiffusion using PRAI-Ingps antisera showed significant cross-reaction among E. coli, E. aerogenes, S. typhimurium, and P. vulgaris whereas the enzymes from S. marcescens and E. carotovora cross-reacted to a lesser extent, with the latter reaction being quite weak. Comparative enzyme neutralization using E. coli PRAI-InGPS antisera showed significant cross-reactions among the enzymes in that all were neutralized at least 25%. The data taken together indicate that the trpC gene products in the Enterobacteriaceae are a homologous group of proteins, that the genetic divergene of the trpC gene is basically the same as the trpA gene, and that both are less conserved than the trpB gene. Furthermore, the PRAI-InGPS, enzyme active site appears to represent a more evolutionarily conserved region of the protein. These findings indicate that, with respect to PRAI-InGPS, similarity to E. coli among the organisms examined is in the following order: (E. aerogenes, S. typhimurium, P. vulgaris) greater than (S. marcescens, E. carotovora).     

Monoclonal antibodies for detection of the H7 antigen of Escherichia coli.

Two murine monoclonal antibodies (MAbs) (2B7 and 46E9-9) reactive with the H7 flagellar antigen of Escherichia coli were produced and characterized. A total of 217 E. coli strains (48 O157:H7, 4 O157:NM, 23 O157:non-H7, 22 H7:non-O157, and 120 non-O157:nonH7), 17 Salmonella serovars, and 29 other gram-negative bacteria were used to evaluate the reactivities of the two MAbs by indirect enzyme-linked immunosorbent assay (ELISA). Both MAbs reacted strongly with all E. coli strains possessing the H7 antigen and with H23- and H24-positive E. coli strains. Indirect ELISA MAb specificity was confirmed by inhibition ELISA and by Western blotting (immunoblotting), using partially purified flagellins from E. coli O157:H7 and other E. coli strains. On a Western blot, MAb 46E9-9 was more reactive against H7 flagellin of E. coli O157:H7 than against H7 flagellin of E. coli O1:K1:H7. Competition ELISA suggested that MAbs 2B7 and 46E9-9 reacted with closely related H7 epitopes. When the ELISA reactivities of the MAbs and two commercially available polyclonal anti-H7 antisera were compared, both polyclonal antisera and MAbs reacted strongly with E. coli H7 bacteria. However, the polyclonal antisera cross-reacted strongly both with non-H7 E. coli and with many non-E. coli bacteria. The polyclonal antisera also reacted strongly with H23 and H24 E. coli isolates. The data suggest the need to define serotype-specific epitopes among H7, H23, and H24 E. coli flagella. The anti-H7 MAbs described in this report have the potential to serve as high-quality diagnostic reagents, used either alone or in combination with O157-specific MAbs, to identify or detect E. coli O157:H7 in food products or in human and veterinary clinical specimens.     

A method for efficient gene isolation from phage lambda gt11 libraries: use of antisera to denatured, acetone-precipitated proteins

Experience with cloning pseudorabies virus (PRV) DNA in the lambda gt11 phage vector has shown that there are special requirements for the antisera used in screening the libraries, in addition to the requirement that the antisera recognize proteins on a Western blot. Initial screening of a lambda gt11 library of sheared PRV DNA fragments in Escherichia coli for expression of PRV antigens using PRV hyperimmune antisera was unsuccessful. It was only after screening the library with antisera raised against PRV proteins eluted from sodium dodecyl sulfate (SDS)-polyacrylamide (PA) gels that positive results were obtained. These "gel-slice" antisera (GSA) were equivalent in potency to hyperimmune antisera in standard immunoassays (including ELISA, immunoprecipitation, Western blots, and neutralization of virus), but only the GSA could recognize PRV fusion proteins expressed by recombinant lambda gt11 phage. This difference was seen despite the fact that hyperimmune antisera performed satisfactorily on Western blots of denatured PRV-infected cell extracts. These results show that the efficiency of screening expression libraries in E. coli can be improved if antibodies are raised against denatured proteins.     

analysis of genomic rearrangements associated with two variable antigen genes of Trypanosoma brucei.

Some variable surface glycoprotein (VSG) genes of Trypanosoma brucei undergo duplication and transposition when they are expressed. We report here the cloning of cDNAs coding for two VSGs from the ILtar 1 repertoire. Analysis of the genomes of trypanosomes expressing these and other antigens shows that there is no additional copy of the sequences coding for eight VSG in expressing clones of trypanosomes, and reveals rearrangements analogous to those previously described for the gene for another VSG from this antigen repertoire. The data indicate that duplication does not accompany the expression of these VSG genes. Transposition to a specific expression site cannot be excluded, but would have to involve either a much larger segment of DNA, or movement to a region of much greater homology with the previous flanking sequences, than is observed for VSG genes that are duplicated when expressed. It is reasoned that the control of expression by coupled duplication and transposition is not sufficient to account for the selection of a single VSG gene for expression.     

Cloning and characterization of a variant surface glycoprotein expression site from Trypanosoma equiperdum.

Variant surface glycoprotein (VSG) genes of African trypanosomes are expressed when they are inserted into one of several telomere-linked expression sites. We cloned and characterized an 11-kilobase (kb) DNA fragment located upstream of an expressed VSG gene. A DNA sequence of 1.8 kb that is located immediately upstream of the inserted VSG gene contains sequences homologous to the 76-base-pair repeats described as being upstream of VSG genes in Trypanosoma brucei (D. A. Campbell, M. P. Van Bree, and J. C. Boothroyd, Nucleic Acids Res. 12:2759-2774). There are no such sequences elsewhere in the 11-kb cloned region. Southern blot analysis using probes from the cloned region revealed multiple unlinked copies of the same or very similar regions. At least three of these are located near telomeres, and two have been shown to be used for the expression of known Trypanosoma equiperdum VSG genes. Like VSG genes, the upstream sequences themselves can be duplicated and deleted. The choice of expression site to be used by a duplicated VSG gene is nonrandom; the site used for expression of the parental VSG gene is strongly favored for use in the daughter variant. Furthermore, even when the parental expression site is not used, the VSG gene occupying it is replaced. Thus, an active expression site is a preferential target for gene conversion in the next variation event.     

Telomere conversion in trypanosomes.

Activation of the gene coding for variant surface glycoprotein (VSG) 118 in Trypanosoma brucei proceeds via a duplicative transposition to a telomeric expression site. The resulting active expression-linked extra copy (ELC) is usually flanked by DNA that lacks sites for most restriction enzymes and that is thought to interfere with the cloning of the ELC as recombinant DNA in Escherichia coli. We have circumvented this problem by cloning an aberrant 118 ELC gene, flanked at the 3'-side by at least 1 kb DNA, that contains restriction enzyme sites. Our analysis shows that this DNA and the 3'-end of the 118 ELC gene are derived from another VSG gene (1.1006) that is permanently located at a telomeric position. We propose that the 3'-end of the 1.1006 gene and (all of) its 3' flanking sequence moved to the expression site by a telomere conversion. Such a telomere conversion can also account for the appearance of an extra copy of the 1.1006 gene detected in a sub-population of our trypanosome strain.     

Differential expression of GABAA receptor alpha-subunits in rat brain during development.

Unique cytoplasmic loop regions of the alpha 1, alpha 2, alpha 3, and alpha 5 subunits of the GABAA receptor have been expressed in E. coli and used to generate polyclonal antisera specific for these subunits. The antibodies identify proteins by SDS-polyacrylamide gel electrophoresis and western blotting of molecular size 51 kDa, 53 kDa, 59 kDa and 55 kDa, respectively, which show differential patterns of expression during development. Whereas the alpha 2 and alpha 3 subunits are present at early stages, the expression of alpha 1 and alpha 3 subunits is low at birth and increases with age. This differential expression could be correlated with previous studies examining the developmental expression of BZ1 and BZ2 benzodiazepine binding sites.     

Cloning, expression, and purification of the uropathogenic Escherichia coli invasin DraD.

In this study we presented a very efficient expression system, based on pET30LIC/Ek vector, for producing DraD invasin of the uropathogenic Escherichia coli and a one-step chromatography purification procedure for obtaining pure recombinant protein (DraD-C-His(6)). This protein has a molecular weight of 14,818 and calculated pI of 6.6. It contains a polyhistidine tag at the C-terminus (13 additional amino acids) that allowed single-step isolation by Ni affinity chromatography. Also, we obtained specific antibodies against DraD invasin to develop tools for characterizing the expression and biological function of this protein. The amount and quality of DraD-C-His(6) fusion protein purified from E. coli overexpression system seems to be fully appropriate for crystallographic studies (soluble form), and for establishing role of the protein in bacterium (cultured cell line interaction and in the internalization process) and for obtaining rabbit polyclonal antisera (insoluble form).     

Cross-neutralization reactions of Escherichia coli and Vibrio cholerae enterotoxins as studied by rabbit skin inoculation test.

Neutralization of enterotoxins of Vibrio cholerae 569 B and Escherichia coli 10407 by antitoxins to V. cholerae 569 B, E. coli 334, 408-3 and 10407 was studies by intradermal inoculation test in the rabbit. Neutralization of V. cholerae enterotoxin by homologous as well as heterologous antisera of E. coli was observed, except that there was no neutralization of the enterotoxin by antiserum to E. coli 408-3 enterotoxin. Neutralization of E. coli enterotoxin to a varied extent by homologous as well as all heterologous antisera, including that of V. cholerae 569 B antitoxin, was also observed.     

Novel structure of the recA locus of Mycobacterium tuberculosis implies processing of the gene product.

A fragment of Mycobacterium tuberculosis DNA containing recA-like sequences was identified by hybridization with the Escherichia coli recA gene and cloned. Although no expression was detected from its own promoter in E. coli, expression from a vector promoter partially complemented E. coli recA mutants for recombination, DNA repair, and mutagenesis, but not for induction of phage lambda. This clone produced a protein which cross-reacts with antisera raised against the E. coli RecA protein and was approximately the same size. However, the nucleotide sequence of the cloned fragment revealed the presence of an open reading frame for a protein about twice the size of other RecA proteins and the cloned product detected by Western blotting (immunoblotting). The predicted M. tuberculosis RecA protein sequence was homologous with RecA sequences from other bacteria, but this homology was not dispersed; rather it was localized to the first 254 and the last 96 amino acids, with the intervening 440 amino acids being unrelated. Furthermore, the junctions of homology were in register with the uninterrupted sequence of the E. coli RecA protein. Identical restriction fragments were found in the genomic DNAs of M. tuberculosis H37Rv and H37Ra and of M. bovis BCG. It is concluded that the ancestral recA gene of these species diversified via an insertional mutation of at least 1,320 bp of DNA. Possible processing mechanisms for synthesizing a normal-size RecA protein from this elongated sequence are discussed.     

Duplicative activation mechanisms of two trypanosome telomeric VSG genes with structurally simple 5'' flanks.

In the mammalian bloodstream, African trypanosomes express variant surface glycoprotein (VSG) genes from a family of long and complex telomeric expression sites. VSG switching generally occurs by the duplication of different VSG genes into these sites by gene conversion involving a series of 70 base pair (70bp) repeats in the 5' flank. In contrast, when VSG is first synthesised by trypanosomes in the tsetse fly at the metacyclic stage, a separate set of telomeric expression sites is activated. These latter telomeres appear not to act as recipients in gene conversion. We have found that the structure of two such expression sites is simple, with very short 70bp repeat regions and very little other sequence in common with bloodstream expression sites. However, the two telomeres readily act as donors in VSG gene conversion in the bloodstream and we show for one a consistent association of the conversion 5' end point with the short 70bp repeat region. These findings help explain why a very predictable set of VSGs is expressed in the tsetse fly and have implications for VSG gene conversion mechanisms.     

Molecular cloning and genetic analysis of the rfb region from Shigella flexneri type 6 in Escherichia coli K-12

The rfb gene cluster which determines the biosynthesis of the Shigella flexneri serotype 6 O-antigen specificity has been cloned in pHC79, generating plasmids pPM3115 and pPM3116. These plasmids mediate expression, in Escherichia coli K-12, of lipopolysaccharides (LPS) immunologically similar to the S. flexneri type 6 LPS as judged by SDS-PAGE and Western-immunoblot analysis using S. flexneri type 6 specific antisera. Thus, unlike other S. flexneri serotypes, no additional loci are required for serotype specificity. This expression is independent of E. coli K-12 rfb genes. Southern-hybridization analysis using the 16.2-kb BglII probe from S. flexneri type 6 rfb region detected very little sequence homology in S. flexneri serotypes 1-5, however, some homology was detected with E. coli O2 and O18, but not in E. coli 0101 strains, Salmonella and Vibrio cholerae.     

The nuclear:organelle distribution of chloroplast ribosomal proteins genes. Features of a cDNA clone encoding the cytoplasmic precursor of L11.

The majority of chloroplast ribosomal proteins are encoded in the nuclear genome. In order to characterize these proteins through their mRNA, we have previously constructed a spinach cDNA expression library and raised antisera to several spinach chloroplast ribosomal proteins. Here we describe the immuno isolation of cDNA clones encoding protein L11 and its chloroplast-targeting presequence. The cytoplasmic precursor form of L11 is 224 amino acid residues long (Mr 23,662); the mature L11 and the transit sequence are predicted to be of approximately 159 and approximately 65 residues, respectively. The predicted chloroplast L11 is significantly longer than the E coli L11, but similar (in size) to archaebacterial and yeast cytoplasmic L11. In sequence it is closer to E coli L11 (54% identity) than to the archaebacterial (32%) or yeast (23%) proteins. These results and the conservation of the contexts of the 3 methyl modified residues found in E coli L11 are discussed in the light of the endosymbiont theory and nuclear relocation of the rp/KAJL gene cluster.     

Identification of subunits of gonococcal RNA polymerase by immunoblot analysis: evidence for multiple sigma factors.

Heparin-agarose and single-stranded DNA-cellulose chromatography were used to purify RNA polymerase 25-fold from Neisseria gonorrhoeae, and the activity of the polymerase was characterized in altered assay systems. The core subunits (beta, beta', and alpha) were tentatively identified as major proteins copurifying with polymerase activity. The identification of the core subunits was confirmed by Western (immunoblot) analysis with polyclonal antisera to Escherichia coli core RNA polymerase. Gonococcal sigma factor heterogeneity was examined by Western blot analysis with polyclonal antiserum to the major E. coli sigma factor, sigma 70, to the E. coli heat shock sigma factor, sigma 32, and with a monoclonal antiserum to Salmonella typhimurium NtrA (sigma 54). Purified RNA polymerase and whole-cell extracts from type 1, type 4, heat-shocked, and anaerobically grown gonococci were examined. Four putative gonococcal sigma factors were detected in purified RNA polymerase preparations and in whole-cell extracts from all cell types. Two of these bands appeared as a doublet, which had an estimated Mr of 80,000. A single lower-Mr band, estimated to be 40,000, was also present. All three of these bands reacted with antisera to E. coli sigma 70 and to E. coli sigma 32. A fourth gonococcal protein reacted solely with a highly specific monoclonal antibody to sigma 54 and had an Mr of 90,000. We conclude that N. gonorrhoeae may contain multiple sigma factors, which it may use to regulate gene expression.     

Expression of whole and hybrid genes in Trypanosoma equiperdum antigenic variation.

A rapid technique involving the S1 nuclease resistance of RNA:DNA duplexes has been used to screen four Trypanosoma equiperdum variant surface glycoprotein (VSG) genes for evidence of hybrid gene structure in their transcribed regions. The results suggest that VSGs appearing early in a chronic infection each have a complete co-linear basic copy (BC) of their expressed gene while VSGs appearing later in infection are particularly associated with BC genes which are recombined before being expressed. The intensities of the S1-protected bands from hybrid VSGs indicate that the basic and expression linked copies are present in equivalent gene dosages. In addition, studies are presented on the expression of two additional VSG genes in T. equiperdum, VSG 4 and VSG 78, which (i) show that the basic copies are not located on telomeres even though one (VSG 4) is expressed early in infection and (ii) emphasize the role of a predominant expression site in T. equiperdum while nevertheless confirming the presence of multiple expression sites.     

Gene conversions mediating antigenic variation in Trypanosoma brucei can occur in variant surface glycoprotein expression sites lacking 70-base-pair repeat sequences.

African trypanosomes undergo antigenic variation of their variant surface glycoprotein (VSG) coat to avoid immune system-mediated killing by their mammalian host. An important mechanism for switching the expressed VSG gene is the duplicative transposition of a silent VSG gene into one of the telomeric VSG expression sites of the trypanosome, resulting in the replacement of the previously expressed VSG gene. This process appears to be a gene conversion reaction, and it has been postulated that sequences within the expression site may act to initiate and direct the reaction. All bloodstream form expression sites contain huge arrays (many kilobase pairs) of 70-bp repeat sequences that act as the 5' boundary of gene conversion reactions involving most silent VSG genes. For this reason, the 70-bp repeats seemed a likely candidate to be involved in the initiation of switching. Here, we show that deletion of the 70-bp repeats from the active expression site does not affect duplicative transposition of VSG genes from silent expression sites. We conclude that the 70-bp repeats do not appear to function as indispensable initiation sites for duplicative transposition and are unlikely to be the recognition sequence for a sequence-specific enzyme which initiates recombination-based VSG switching.     

The use of incomplete genes for the construction of a Trypanosoma equiperdum variant surface glycoprotein gene.

The expression of Trypanosoma equiperdum variant surface protein (VSG) 78 is accomplished by the duplicative transposition of silent basic copy (BC) genes into a telomer-linked expression site to form an expression-linked copy (ELC). In two independent isolates expressing VSG 78, the ELC is a composite gene. The analysis of VSG 78 cDNA clones from these two Bo Tat 78 isolates and the respective BC genes revealed that both ELCs were constructed from the same three BC genes, a 3' BC which donated the last 255 bp of each ELC and two closely related 5' BCs. Although sequences of both 5' BC genes were found in each ELC, the junction with the 3' BC was provided by the same 5' BC in both cases. This 5' BC is an incomplete gene with insufficient open reading frame to code for a complete VSG and thus can only be used when joined to a competent 3' end. Furthermore, both 5' BC genes lack a conserved 14 nucleotide sequence found on all VSG mRNAs. These results support a model in which composite gene formation plays a role in the determination of the order of VSG expression. They also illustrate similarities between immunoglobulin gene and VSG gene construction.     

Serological cross-reaction between the lipopolysaccharide O-polysaccharaide antigens of Escherichia coli O157:H7 and strains of Citrobcter freundii and Citrobacter sedlakii

A strain of Citrobacter sedlakii showing serological cross-reaction with Escherichia coli O157 antisera was demonstrated to produce a lipopolysaccharide O-antigen having an identical structure with that of the E. coli O157 O-antigen. A strain of Citrobacter freunndii showing similar cross-reaction with E. coli O157 specific monoclonal antibody was shown to produce a lipopolysaccharide O-antigen composed of a trisaccharide repeating unit having the structure [ 2)-alpha-D Rhap-(1-3)-beta-D-Rhap-(1-4)-beta-D-Glcp-(1-]. This O-antigen differs from that of the E. coli O157 O-antigen and also lacks a component 2-substituted 4-amino-4,6-dideoxy-alpha-D-mannopyranosyl residue implicated as the common epitope in the lipopolysaccharide O-antigens of previously investigated bacterial species showing serological cross-reactivity with E. coli O157 antisera. The C freundii O-antigen presents an interesting example of structural mimicry within a bacterial polysaccharide antigen.