The role of pyridoxal phosphate in the function of EspB, a protein secreted by enteropathogenic Escherichia coli

The sequence of EspB, a secreted protein required for virulence of enteropathogenic Escherichia coli (EPEC), reveals a motif common to enzymes that bind pyridoxal phosphate. Pyridoxal phosphate was not found by fluorometry in concentrated supernatants of EPEC cultures that contain EspB. Plasmids containing cloned espB, in which the lysine residue conserved in the motif was substituted with either an arginine or methionine residue, remained capable of complementing an espB deletion mutant to restore EspB function. The results of these studies do not support a role for pyridoxal phosphate in EspB function.     

Characterization of fimbriae produced by enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) express rope-like bundles of filaments, termed bundle-forming pili (BFP) (J. A. Girón, A. S. Y. Ho, and G. K. Schoolnik, Science 254:710-713, 1991). Expression of BFP is associated with localized adherence to HEp-2 cells and the presence of the EPEC adherence factor plasmid. In this study, we describe the identification of rod-like fimbriae and fibrillae expressed simultaneously on the bacterial surface of three prototype EPEC strains. Upon fimbrial extraction from EPEC B171 (O111:NM), three fimbrial subunits with masses of 16.5, 15.5, and 14.7 kDa were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their N-terminal amino acid sequence showed homology with F9 and F7(2) fimbriae of uropathogenic E. coli and F1845 of diffuse-adhering E. coli, respectively. The mixture of fimbrial subunits (called FB171) exhibited mannose-resistant agglutination of human erythrocytes only, and this activity was not inhibited by alpha-D-Gal(1-4)-beta-Gal disaccharide or any other described receptor analogs for P, S, F, M, G, and Dr hemagglutinins of uropathogenic E. coli, which suggests a different receptor specificity. Hemagglutination was inhibited by extracellular matrix glycoproteins, i.e., collagen type IV, laminin, and fibronectin, and to a lesser extent by gangliosides, fetuin, and asialofetuin. Scanning electron microscopic studies performed on clusters of bacteria adhering to HEp-2 cells revealed the presence of structures resembling BFP and rod-like fimbriae linking bacteria to bacteria and bacteria to the eukaryotic cell membrane. We suggest a role of these surface appendages in the interaction of EPEC with eukaryotic cells as well as in the overall pathogenesis of intestinal disease caused by EPEC.     

A novel sensor-regulator protein that belongs to the homologous family of signal-transduction proteins involved in adaptive responses in Escherichia coli

Expression of the Escherichia coli outer membrane porins, OmpC and OmpF, is regulated in response to changes in the medium osmolarity through the functions of the regulatory factors, EnvZ and OmpR. A 3.0 kilobase pair DNA fragment cloned from E. coli is able phenotypically to suppress the defect in ompC and ompF expression caused by an envZ deletion mutation, provided that a certain gene located in this fragment is expressed on a high copy-number plasmid. Nucleotide sequencing revealed that the putative gene encodes a protein of 102,452 Da. The deduced amino acid sequence of the protein shows a high degree of homology to those of both EnvZ and OmpR, i.e. it contains both a 'sensory kinase domain' and a 'response regulator domain' in its primary amino acid sequence. The protein identified in this study is probably a novel member of the homologous family of proteins involved in bacterial adaptive responses. Hence, the gene encoding this novel sensor-regulator protein was designated as barA (bacterial adaptive responses) and mapped at 60 min on the E. coli genetic map. The BarA protein in isolated membranes was demonstrated in vitro to undergo phosphorylation in the presence of ATP.     

DNA-binding by Haemophilus influenzae and Escherichia coli YbaB, members of a widely-distributed bacterial protein family

Background  

Genes orthologous to the ybaB loci of Escherichia coli and Haemophilus influenzae are widely distributed among eubacteria. Several years ago, the three-dimensional structures of the YbaB orthologs of both E. coli and H. influenzae were determined, revealing a novel "tweezer"-like structure. However, a function for YbaB had remained elusive, with an early study of the H. influenzae ortholog failing to detect DNA-binding activity. Our group recently determined that the Borrelia burgdorferi YbaB ortholog, EbfC, is a DNA-binding protein. To reconcile those results, we assessed the abilities of both the H. influenzae and E. coli YbaB proteins to bind DNA to which B. burgdorferi EbfC can bind.     

NhaR, a protein homologous to a family of bacterial regulatory proteins (LysR), regulates nhaA, the sodium proton antiporter gene in Escherichia coli.

On the basis of protein homology, nhaR has previously been shown to belong to a large family of regulatory proteins, the LysR family (Henikoff, S., Haughn, G.W., Calvo, J.M., and Wallace, J.C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6602-6606). In this work we show that nhaR is a regulator of nhaA, a gene encoding a Na+/H+ antiporter in Escherichia coli. Multicopy plasmid bearing nhaR enhances the Na(+)-dependent induction of a chromosomal nhaA'-'lacZ fusion. Extracts derived from cells overexpressing nhaR exhibit specific DNA binding capacity to the upstream sequences of nhaA. Construction of an nhaR deletion mutant (OR100) shows that nhaR is required in addition to nhaA to tolerate the extreme conditions under which nhaA is indispensable. Whereas OR100 grows like the wild type at neutral pH even at high Na+ concentrations (700 mM), it becomes much more sensitive to Na+ (greater than 300 mM) at pH 8.5; furthermore, OR100 is more sensitive to Li+ (100 mM) than the wild type. Nevertheless, the phenotype of OR100, which is more resistant to Na+, Li+, and alkaline pH than a delta nhaA strain (NM81), implies that the regulation exerted by nhaR is not complete and that some expression of nhaA exists in OR100. Accordingly, the effect of nhaR in cells is dependent on the level of nhaA. OR200, a nhaA and nhaR deletion mutant, has the same phenotype as NM81. Multicopy plasmid bearing nhaR does not change the phenotype of either OR200 or NM81. On the other hand, multicopy nhaA renders the cells Li(+)- and and Na(+)-resistant even without nhaR.     

Identification and characterization of an outer membrane protein, OmpX, in Escherichia coli that is homologous to a family of outer membrane proteins including Ail of Yersinia enterocolitica.

We previously reported that a region of the Escherichia coli chromosome at 18 min increased E sigma E activity when cloned in multicopy (J. Mecsas, P. E. Rouviere, J. W. Erickson, T. J. Donohue, and C. A. Gross, Genes Dev. 7:2618-2628, 1993). In the present report, we identify and characterize the gene responsible for the increase in E sigma E activity. This gene is in a monocistronic operon with two promoters and a rho-independent terminator. Sequence analysis of this gene indicated that it encodes an outer membrane protein which is 83% identical to OmpX in Enterobacter cloacae, leading us to name this gene ompX. There are four other proteins that are homologous to OmpX. Several of these proteins, Ail of Yersinia enterocolitica and Rck and PagC of Salmonella typhimurium, have properties that allow bacteria to adhere to mammalian cells, survive exposure to human serum, and/or survive within macrophages. We therefore characterized strains deleted for ompX for their growth phenotypes, E sigma E activity, serum resistance, and adherence to mammalian cells. No differences in growth rates, serum resistance, or adherence to mammalian cells were observed; however, E sigma E activity was dependent on expression of OmpX in certain strain backgrounds.     

A 20-kilodalton protein is required for efficient production of the Bacillus thuringiensis subsp. israelensis 27-kilodalton crystal protein in Escherichia coli.

The 27-kilodalton (kDa) mosquitocidal protein gene from Bacillus thuringiensis subsp. israelensis has been cloned as a 10-kilobase (kb) HindIII fragment from plasmid DNA; efficient expression in Escherichia coli KM1 depends on a region of DNA located approximately 4 kb upstream (K. McLean and H. R. Whiteley, J. Bacteriol. 169:1017-1023, 1987). We have cloned the upstream DNA region and show that it contains a complete open reading frame (ORF) encoding a protein with a molecular mass of 19,584 Da. Sequencing of adjacent stretches of DNA revealed two partial ORFs: one has 55.2% identity in an overlap of 319 amino acids to the putative transposase of IS231 of B. thuringiensis subsp. thuringiensis, and the other, a 78-codon partial ORF, may be the carboxyl terminus of the 67-kDa protein previously observed in maxicells of strain KM1. A 0.8-kb fragment containing only the 20-kDa protein gene greatly enhanced the expression of the 27-kDa protein in E. coli. The introduction of nonsense codons into the 20-kDa protein gene ORF abolished this effect, indicating that the gene product, not the mRNA or DNA, is required for the enhancement. The effect of the 20-kDa protein gene on various fusions of lacZ to the 27-kDa protein gene suggests that the 20-kDa protein acts after the initiation of translation of the 27-kDa protein gene.     

Characterization of the ves gene, which is expressed at a low temperature in Escherichia coli

A gene, designated ves, that is expressionally responsive to temperature was found in Escherichia coli. Experiments with a single-copy lacZ operon fusion and primer extension analysis revealed that ves was expressed at a low temperature with a peak around 25 degrees C but was hardly expressed at 42 degrees C. After a temperature downshift, the mRNA level increased until 6 to 12 h and then decreased. Consistently, an A + T-rich sequence similar to UP elements seen in cold-shock inducible cold-shock protein (Csp) genes was found up-stream of the ves promoter, and its 5'-untranslated region was found to share similarity with those of the cold-shock inducible and cold-adaptive cspA and cspB genes. Additionally, a putative down-stream box, which also exists in cold-inducible proteins, was found. The ves product was identified by overproduction and determination of its N-terminal sequence. Similarity of the C-terminal portion of Ves to the CspA family suggests that Ves belongs to this family. The results of gene-disruption experiments suggest that ves is not essential for E. coli.     

Purification of a diagnostic, secreted cysteine protease-like protein from the hookworm Ancylostoma caninum

The enteric infection of humans with the canine hookworm Ancylostoma caninum varies in its clinical presentation, ranging from asymptomatic to eosinophilic gastroenteritis requiring surgical intervention. Infections are not patent, but can be diagnosed immunologically by detecting antibodies to an immunodominant secreted hookworm protein termed Ac68. To characterise Ac68, we purified the native protein from A. caninum excretory/secretory products using size exclusion followed by anion exchange chromatography. The epitopes in the purified protein recognised by human infection sera were shown to be proteins and not carbohydrates. The N-terminal amino acid sequence of the purified Ac68 was determined and six of the 11 residues obtained were shared with a previously characterised cysteine protease of A. caninum, AcCP1.     

Structure-function analysis of BfpB, a secretin-like protein encoded by the bundle-forming-pilus operon of enteropathogenic Escherichia coli

Production of type IV bundle-forming pili by enteropathogenic Escherichia coli (EPEC) requires BfpB, an outer-membrane lipoprotein and member of the secretin protein superfamily. BfpB was found to compose a ring-shaped, high-molecular-weight outer-membrane complex that is stable in 4% sodium dodecyl sulfate at temperatures of < or = 65 degrees C. Chemical cross-linking and immunoprecipitation experiments disclosed that the BfpB multimeric complex interacts with BfpG, and mutational studies showed that BfpG is required for the formation and/or stability of the multimer but not for the outer-membrane localization of BfpB. Formation of the BfpB multimer also does not require BfpA, the repeating subunit of the pilus filament. Functional studies of the BfpB-BfpG complex revealed that its presence confers vancomycin sensitivity, indicating that it may form an incompletely gated channel through the outer membrane. BfpB expression is also associated with accumulation of EPEC proteins in growth medium, suggesting that it may support both pilus biogenesis and protein secretion.     

Identity of the 17-kilodalton protein, a DNA-binding protein from Escherichia coli, and the firA gene product.

The 17-kilodalton protein, a DNA-binding protein encoded by the skp gene of Escherichia coli, was found to be identical to histonelike protein I, the product of the firA gene. This conclusion was reached after chromosomal localization, using the recently constructed high- and low-resolution E. coli restriction maps, and by direct comparison of the N-terminal amino acid sequence of histonelike protein I and the 17-kilodalton protein.     

A dominant mutation in Escherichia coli OmpR lies within a domain which is highly conserved in a large family of bacterial regulatory proteins

Summary We have fortuitously created an in-frame insertion mutation in the cloned ompR gene of Escherichia coli in the course of an experiment involving linker insertion mutagenesis. According to the DNA sequence, the mutant protein has an insertion at the 53rd amino acid residue, which replaced the original valine, with the sequence Ala-Leu-Glu. The expression level of the mutant protein, OmpRX6, in a minicell system, is similar to that of the wild-type protein and the size of the mutant is slightly larger than the wild type by approxiately 300 daltons. This mutant was completely unable to activate porin expression as the wildtype does, and in addition, this phenotype was shown to be dominant over the wild type. Comparison of the amino acid sequence of OmpRX6 with those of a family of homologous bacterial regulatory proteins revealed that the mutation lies in a domain which is highly conserved among these proteins.     

A human DNA editing enzyme homologous to the Escherichia coli DnaQ/MutD protein.

Mammalian DNA polymerases alpha and beta lack 3' exonuclease activity and are unable to edit errors after DNA synthesis. However, editing exonucleases can be functions of separate polypeptides. We isolated a widely distributed DNA-specific 3' exonuclease from rabbit liver nuclei, sequenced tryptic peptides by mass spectrometry, and identified the corresponding human open reading frame. The protein expressed from the cloned human sequence exhibits 3' exonuclease activity. The human clone shares sequence homology with the editing function of the Escherichia coli DNA polymerase III holoenzyme, i.e., the DnaQ/MutD protein, and weakly with the editing 3' exonuclease domain of eukaryotic DNA polymerase epsilon. The gene maps to human chromosome 3p21.2-21.3. In a reconstituted human DNA repair system containing DNA polymerase beta and DNA ligase III-XRCC1, accurate rejoining of a 3' mismatched base residue at a single-strand break is dependent on addition of the exonuclease.