An avian pathogenic Escherichia coli strain produces a hemolysin, the expression of which is dependent on cyclic AMP receptor protein gene function

An avian pathogenic Escherichia coli strain M1000 showed a clear zone of erythrocyte lysis on sheep blood agar plates. The hemolytic activity was not detected in the culture supernatant nor was any DNA sequence homologous to the E. coli alpha-hemolysin gene detected in the chromosome or plasmid DNA of the strain, indicating that the observed hemolysis was different from alpha-type. To identify the genetic determinant responsible for the hemolysis, we performed random Tn5 insertional mutagenesis and obtained one mutant, named M5005, that totally lacked the hemolytic activity. Cloning and nucleotide sequencing of the region flanking the transposon insertion site in the M5005 chromosome revealed that the transposon was inserted within an open reading frame of the cyclic AMP receptor protein (CRP) gene, which is involved in one of the global regulatory networks of gene expression in E. coli. Nucleotide sequence analysis of the intact crp gene of the strain M1000 showed that the CRP protein of M1000 is 99% identical to that of K-12. Introduction of the intact crp gene on a low copy plasmid into the mutant M5005 restored the hemolytic phenotype, confirming that the mutation site in M5005 was in the crp gene. CRP plays a central role in catabolite repression, the phenomenon by which the synthesis of many enzymes required to metabolize various sugars is repressed in the presence of glucose. When the hemolytic activity of E. coli M1000 grown in the presence of glucose was examined, the hemolysis was totally impaired. These results indicate that the avian pathogenic E. coli strain M1000 produces a hemolysin the expression of which is dependent on crp gene function.     

Hemolytic properties and riboflavin synthesis of Helicobacter pylori: cloning and functional characterization of the ribA gene encoding GTP-cyclohydrolase II that confers hemolytic activity to Escherichia coli

Various strains of Helicobacter pylori were able to lyse erythrocytes from sheep, horse, and human when grown on blood agar. The hemolysis did not depend on the production of the vacuolating cytotoxin VacA as demonstrated by the hemolytic behavior of an isogenic vacA-negative mutant strain. The hemolytic activity could be detected in cell-free supernatants and was not regulated by iron. To isolate genes coding for proteins involved in the destruction of erythrocytes, a plasmid-based DNA library was screened for expression of lytic activity on blood agar. This approach revealed that the H. pylori ribA gene confers hemolytic properties to Escherichia coli. The ribA gene encodes the enzyme GTP-cyclohydrolase II [EC 3.5.4.25] that catalyzes the initial step in the synthesis of riboflavin. The predicted amino acid sequence of the H. pylori RibA protein showed a high degree of similarity to equivalent enzymes from microorganisms and from plants. The single gene on a plasmid restored riboflavin synthesis in a ribA mutant of E. coli and induced hemolytic activity. Furthermore, ribA overexpression was associated with the production of a fluorescent yellow molecule that was not identical with riboflavin. Hemolysis was also seen for the ribA gene from E. coli, indicating that this feature was not specific for the H. pylori gene. The presence of ribA in various H. pylori strains was confirmed by Southern blot hybridization and by polymerase chain reaction with specific primers. This analysis revealed that microdiversity exists within the DNA region upstream from ribA, which was further confirmed by nucleotide sequence analysis.     

Identification and characterization of an immunogenic outer membrane protein of Campylobacter jejuni

We cloned and expressed in Escherichia coli a gene encoding an 18-kDa outer membrane protein (Omp18) from Campylobacter jejuni ATCC 29428. The nucleotide sequence of the gene encoding Omp18 was determined, and an open reading frame of 165 amino acids was revealed. The amino acid sequence had the typical features of a leader sequence and a signal peptidase II cleavage site at the N-terminal part of Omp18. Moreover, the sequence had a high degree of similarity to the peptidoglycan-associated outer membrane lipoprotein P6 of Haemophilus influenzae and the peptidoglycan-associated lipoprotein PAL of E. coli. Southern blot analysis in which the cloned gene was used as a probe revealed genes similar to that encoding Omp18 in all species of the thermophilic group of campylobacters as well as Campylobacter sputorum. All campylobacters tested expressed a protein with a molecular mass identical to that of Omp18. The protein reacted immunologically with polyclonal antibodies directed against Omp18 from C. jejuni. PCR amplification of the gene encoding Omp18 with specific primers and subsequent restriction enzyme analysis of the amplified DNA fragments showed that the gene for Omp18 is highly conserved in C. jejuni strains isolated from humans, dogs, cats, calves, and chickens but is different in other Campylobacter species. In order to obtain pure recombinant Omp18 protein for serological assays, the cloned gene for Omp18 was genetically modified by replacing the signal sequence with a DNA segment encoding six adjacent histidine residues. Expression of this construct in E. coli allowed purification of the modified protein (Omp18-6xHis) by metal chelation chromatography. Sera from patients with past C. jejuni infection reacted positively with Omp18-6xHis, while sera from healthy blood donors showed no reaction with this antigen. Omp18, which is an outer membrane protein belonging to the family of PALs is well conserved in C. jejuni and is highly immunogenic. It is therefore a good candidate as an antigen for the serological diagnosis of past C. jejuni infections.     

Characterization of the thermal stress response of Campylobacter jejuni

Campylobacter jejuni, a microaerophilic, gram-negative bacterium, is a common cause of gastrointestinal disease in humans. Heat shock proteins are a group of highly conserved, coregulated proteins that play important roles in enabling organisms to cope with physiological stresses. The primary aim of this study was to characterize the heat shock response of C. jejuni. Twenty-four proteins were preferentially synthesized by C. jejuni immediately following heat shock. Upon immunoscreening of Escherichia coli transformants harboring a Campylobacter genomic DNA library, one recombinant plasmid that encoded a heat shock protein was isolated. The recombinant plasmid, designated pMEK20, contained an open reading frame of 1,119 bp that was capable of encoding a protein of 372 amino acids with a calculated molecular mass of 41,436 Da. The deduced amino acid sequence of the open reading frame shared similarity with that of DnaJ, which belongs to the Hsp-40 family of molecular chaperones, from a number of bacteria. An E. coli dnaJ mutant was successfully complemented with the pMEK20 recombinant plasmid, as judged by the ability of bacteriophage lambda to form plaques, indicating that the C. jejuni gene encoding the 41-kDa protein is a functional homolog of the dnaJ gene from E. coli. The ability of each of two C. jejuni dnaJ mutants to form colonies at 46 degreesC was severely retarded, indicating that DnaJ plays an important role in C. jejuni thermotolerance. Experiments revealed that a C. jejuni DnaJ mutant was unable to colonize newly hatched Leghorn chickens, suggesting that heat shock proteins play a role in vivo.     

Prevalence of cytolethal distending toxin production in Campylobacter jejuni and relatedness of Campylobacter sp. cdtB gene

Campylobacter jejuni produces a toxin called cytolethal distending toxin (CDT). The genes encoding this toxin in C. jejuni 81-176 were cloned and sequenced. The nucleotide sequence of the genes revealed that there are three genes, cdtA, cdtB, and cdtC, encoding proteins with predicted sizes of 30,11-6, 28,989, and 21,157 Da, respectively. All three proteins were found to be related to the Escherichia coli CDT proteins, yet the amino acid sequences have diverged significantly. All three genes were required for toxic activity in a HeLa cell assay. HeLa cell assays of a variety of C. jejuni and C. coli strains suggested that most C. jejuni strains produce significantly higher CDT titers than do C. coli strains. Southern hybridization experiments demonstrated that the cdtB gene is present on a 6.0-kb ClaI fragment in all but one of the C. jejuni strains tested; the cdtB gene was on a 6.9-kb ClaI fragment in one strain. The C. jejuni 81-176 cdtB probe hybridized weakly to DNAs from C. coli strains. The C. jejuni 81-176 cdtB probe did not hybridize to DNAs from representative C. fetus, C. lari, C. "upsaliensis," and C. hyointestinalis strains, although the HeLa cell assay indicated that these strains make CDT. PCR experiments indicated the probable presence of cdtB sequences in all of these Campylobacter species.     

Cloning and sequence analysis of the gbpC gene encoding a novel glucan-binding protein of Streptococcus mutans

We have isolated dextran-aggregation-negative mutants of Streptococcus mutans following random mutagenesis with plasmid pVA891 clone banks. A chromosomal region responsible for this phenotype was characterized in one of the mutants. A 2.2-kb fragment from the region was cloned in Escherichia coli and sequenced. A gene specifying a putative protein of 583 amino acid residues with a calculated molecular weight of 63,478 was identified. The amino acid sequence deduced from the gene exhibited no similarity to the previously identified S. mutans 74-kDa glucan-binding protein or to glucan-binding domains of glucosyltransferases but exhibited similarity to surface protein antigen (Spa)-family proteins from streptococci. Extract from an E. coli clone of the gene exhibited glucan-binding activity. Therefore, the gene encoded a novel glucan-binding protein.     

PCR detection, identification to species level, and fingerprinting of Campylobacter jejuni and Campylobacter coli direct from diarrheic samples

Three sets of primers were designed for PCR detection and differentiation of Campylobacter jejuni and Campylobacter coli. The first PCR assay was designed to coidentify C. jejuni and C. coli based on their 16S rRNA gene sequences. The second PCR assay, based on the hippuricase gene sequence, identified all tested reference strains of C. jejuni and also strains of that species which lack detectable hippuricase activity. The third PCR assay, based on the sequence of a cloned (putative) aspartokinase gene and the downstream open reading frame, identified all tested reference strains of C. coli. The assays will find immediate application in the rapid identification to species level of isolates. The assays combine with a protocol for purification of total DNA from fecal samples to allow reproducible PCR identification of campylobacters directly from stools. Of 20 clinical samples from which campylobacters had been cultured, we detected C. jejuni in 17, C. coli in 2, and coinfection of C. jejuni and Campylobacter hyointestinalis in 1. These results were concordant with culture and phenotypic identification to species level. Strain typing by PCR-restriction fragment length polymorphism of the flagellin (flaA) gene detected identical flaA types in fecal DNA and the corresponding campylobacter isolate. Twenty-five Campylobacter-negative stool samples gave no reaction with the PCR assays. These PCR assays can rapidly define the occurrence, species incidence, and flaA genotypes of enteropathogenic campylobacters.     

Identification of the enteropathogens Campylobacter jejuni and Campylobacter coli based on the cadF virulence gene and its product

Campylobacter jejuni and Campylobacter coli are common causes of gastroenteritis in humans. Infection with C. jejuni or C. coli is commonly acquired by eating undercooked chicken. The goal of this study was to develop specific detection assays for C. jejuni and C. coli isolates based on the cadF virulence gene and its product. The cadF gene from C. jejuni and C. coli encodes a 37-kDa outer membrane protein that promotes the binding of these pathogens to intestinal epithelial cells. A fragment of approximately 400 bp was amplified from 38 of 40 (95%) C. jejuni isolates and 5 of 6 (83.3%) C. coli isolates with primers designed to amplify an internal fragment of the cadF gene. PCR was then used to amplify Campylobacter DNA from store-bought chickens. A 400-bp band was amplified from 26 of the 27 chicken carcasses tested by the PCR-based assay. The CadF protein was detected in every C. jejuni and C. coli isolate tested, as judged by immunoblot analysis with a rabbit anti-C. jejuni 37-kDa serum. In addition, methanol-fixed samples of whole-cell C. jejuni and C. coli were detected with the rabbit anti-37-kDa serum by using an indirect-immunofluorescence microscopy assay. These findings indicate that the cadF gene and its product are conserved among C. jejuni and C. coli isolates and that a PCR assay based on the cadF gene may be useful for the detection of Campylobacter organisms in food products.     

Specific identification of the enteropathogens Campylobacter jejuni and Campylobacter coli by using a PCR test based on the ceuE gene encoding a putative virulence determinant

A PCR method for the rapid identification and discrimination of thermophilic Campylobacter jejuni and Campylobacter coli was developed by using a gene encoding a protein involved in siderophore transport (ceuE). A nucleotide sequence divergence of approximately 13% in the ceuE genes of C. jejuni and C. coli facilitated the design of two species-specific PCR primer sets. The specificity of the PCR amplification reactions was confirmed by using two nonradioactively labelled species-specific internal oligonucleotide hybridization probes for each of these species.     

A cloned Erwinia chrysanthemi Hrp (type III protein secretion) system functions in Escherichia coli to deliver Pseudomonas syringae Avr signals to plant cells and to secrete Avr proteins in culture

The Hrp (type III protein secretion) system is essential for the plant parasitic ability of Pseudomonas syringae and most Gram-negative bacterial plant pathogens. AvrB and AvrPto are two P. syringae proteins that have biological activity when produced via heterologous gene expression inside plant cells or when produced by Hrp+ bacteria. Avr-like proteins, presumably injected by the Hrp system on bacterial contact with plant cells, appear to underlie pathogenic interactions, but none has been observed outside of the bacterial cytoplasm, and identifying novel genes encoding them is tedious and uncertain without a phenotype in culture. Here we describe a cloned Hrp secretion system that functions heterologously in Escherichia coli to secrete AvrB and AvrPto in culture and to promote AvrB and AvrPto biological activity in inoculated plants. The hrp gene cluster, carried on cosmid pCPP2156, was cloned from Erwinia chrysanthemi, a pathogen that differs from P. syringae in being host promiscuous. E. coli DH5alpha carrying pCPP2156, but not related Hrp-deficient cosmids, elicited a hypersensitive response in Nicotiana clevelandii only when also expressing avrB in trans. The use of pAVRB-FLAG2 and pAVRPTO-FLAG, which produce Avr proteins with a C-terminal FLAG-epitope fusion, enabled immunoblot detection of the secretion of these proteins to E. coli(pCPP2156) culture media. Secretion was Hrp dependent, occurred without leakage of a cytoplasmic marker, and did not occur with E. coli(pHIR11), which encodes a functional P. syringae Hrp system. E. coli(pCPP2156) will promote investigation of Avr protein secretion and systematic prospecting for the effector proteins underlying bacterial plant pathogenicity.     

Identification and analysis of a gene encoding L-2,4-diaminobutyrate:2-ketoglutarate 4-aminotransferase involved in the 1,3-diaminopropane production pathway in Acinetobacter baumannii

The ca. 2.2-kbp region upstream of the ddc gene encoding L-2,4-diaminobutyrate decarboxylase in Acinetobacter baumannii was sequenced and found to contain another open reading frame of 1,338 nucleotides encoding a protein with a deduced molecular mass of 47,423 Da. Analysis of the homologies observed from the deduced amino acid sequence indicated that the gene product is an enzyme belonging to subgroup II of the aminotransferases. This was first verified when examination of the crude extracts from Escherichia coli transformants led to detection of a novel aminotransferase activity catalyzing the following reversible reactions: L-2,4-diaminobutyric acid + 2-ketoglutaric acid<-->L-glutamic acid + L-aspartic beta-semialdehyde. Further confirmation was obtained when the gene was overexpressed in E. coli and the corresponding protein was purified to homogeneity. It catalyzed the same reactions and its N-terminal amino acid sequence was consistent with that deduced from the nucleotide sequence. Therefore, the gene and its product were named dat and L-2,4-diaminobutyrate:2-ketoglutarate 4-aminotransferase (DABA AT), respectively. Feeding experiments of A. baumannii with L-[U-14C]aspartic acid resulted in the incorporation of the label into 1,3-diaminopropane. Apparent homologs of dat and DABA AT were detected in other Acinetobacter species by PCR amplification and Western blotting. These results indicate that the dat gene (as well as the ddc gene) participates in the synthesis of 1,3-diaminopropane, the only diamine found in this genus. However, the biological role, if one exists, of 1,3-diaminopropane synthesis is unknown.     

Purification and characterization of an isoaspartyl dipeptidase from Escherichia coli

We have identified a gene (iadA) in Escherichia coli encoding a 41-kDa polypeptide that catalyzes the hydrolytic cleavage of L-isoaspartyl, or L-beta-aspartyl, dipeptides. We demonstrate at least a 3000-fold purification of the enzyme to homogeneity from crude cytosol. From the amino-terminal amino acid sequence obtained from this preparation, we designed an oligonucleotide that allowed us to map the gene to the 98-min region of the chromosome and to clone and obtain the DNA sequence of the gene. Examination of the deduced amino acid sequence revealed no similarities to other peptidases or proteases, while a marked similarity was found with several dihydroorotases and imidases, reflecting the similarity in the structures of the substrates for these enzymes. Using an E. coli strain containing a plasmid overexpressing this gene, we were able to purify sufficient amounts of the dipeptidase to characterize its substrate specificity. We also examined the phenotype of two E. coli strains where this isoaspartyl dipeptidase gene was deleted. We inserted a chloramphenicol cassette into the disrupted coding region of iadA in both a parent strain (MC1000) and a derivative strain (CL1010) lacking pcm, the gene encoding the L-isoaspartyl methyltransferase involved in the repair of isomerized proteins. We found that the iadA deletion does not result in reduced stationary phase or heat shock survival. Analysis of isoaspartyl dipeptidase activity in the deletion strain revealed a second activity of lower native molecular weight that accounts for approximately 31% of the total activity in the parent strain MC1000. The presence of this second activity may account for the absence of an observable phenotype in the iadA mutant cells.     

Cloning and sequencing of the gene encoding a 31-kilodalton antigen of Haemophilus somnus

Immunoblots using bovine antibody against Haemophilus somnus as the primary antibody consistently identified 31-, 40- and 78-kDa proteins in Sarkosyl-insoluble extracts of H. somnus. A genomic library of H. somnus 8025 DNA was constructed in plasmid pUC19, and 45 recombinants expressed proteins which were recognized by bovine antiserum in Western blots (immunoblots). Ten of the recombinants expressing a 31-kDa protein caused the lysis of bovine erythrocytes. Restriction endonuclease mapping indicated that the hemolytic recombinants shared an approximately 1.7-kb BglII fragment. Southern blot analysis using the BglII fragment as a probe revealed homology among the recombinants and the presence of an identically sized BglII fragment in the chromosome of all H. somnus isolates tested. Sequence analysis indicated the presence of an 822-bp open reading frame within the 1.7-kb BglII fragment. Deletion of this open reading frame resulted in the loss of hemolytic activity and protein expression in recombinant Escherichia coli, suggesting the possible role of the 31-kDa protein as a hemolysin. An amino acid sequence deduced from the DNA sequence shared homology with outer membrane protein A of E. coli, Salmonella typhimurium, and Shigella dysenteriae, with P6 of Haemophilus influenzae, and with PIII of Neisseria gonorrhoeae. An amino acid analysis of the recombinant 31-kDa protein agreed with the amino acid composition deduced from the DNA sequence.     

Characterization of degQ and degS, Escherichia coli genes encoding homologs of the DegP protease

The degQ and degS genes of Escherichia coli encode proteins of 455 and 355 residues, respectively, which are homologs of the DegP protease. The purified DegQ protein has the properties of a serine endoprotease and is processed by the removal of a 27-residue amino-terminal signal sequence. A plasmid expressing degQ rescues the temperature-sensitive phenotype of a strain bearing the degP41 deletion, implying that DegQ, like DegP, functions as a periplasmic protease in vivo. Deletions in the degQ gene cause no obvious growth defect, while those in the degS gene result in a small-colony phenotype. The latter phenotype is rescued by a plasmid expressing the degS gene but not by plasmids expressing the degQ or degP genes. This result and the inability of a plasmid expressing degS to rescue the temperature-sensitive degP41 phenotype indicate that the DegS protein is functionally different from the DegQ and DegP proteins.     

Activity and cellular location in Saccharomyces cerevisiae of chimeric mouse/yeast and Bacillus subtilis/yeast ferrochelatases

We have constructed a series of chimeric yeast/mouse and yeast/Bacillus subtilis ferrochelatase genes in order to investigate domains of the ferrochelatase that are important for activity and/or association with the membrane. These genes were expressed in a Saccharomyces cerevisiae mutant in which the endogenous ferrochelatase gene (HEM15) had been deleted, and the phenotypes of the transformants were characterized. Exchanging the approximately 40-amino-acid C-terminus between the yeast and mouse ferrochelatases caused a total loss of activity and the hybrid proteins were unstable when overproduced in Escherichia coli. The water-soluble ferrochelatase of B. subtilis did not complement the yeast mutant, although a large amount of active protein accumulated in the cytosol. Addition of the N-terminal leader sequence of yeast ferrochelatase to the B. subtilis enzyme targeted the fusion protein to mitochondria, but both the precursor and the mature forms of the enzyme were inactive in vivo and had residual activity when measured in vitro. An internal approximately 45-amino-acid segment located at the N-terminus of yeast ferrochelatase was identified, which, when replaced with the corresponding 30-amino-acid segment of the B. subtilis enzyme, caused the yeast enzyme to be located in the mitochondrial matrix as a soluble protein. The fusion protein was inactive in vivo and had residual activity in vitro. We speculate that this segment, which shows the greatest variability between species, is responsible for the association of the enzyme with the membrane.     

Characterization of the avian pathogenic Escherichia coli hemagglutinin Tsh, a member of the immunoglobulin A protease-type family of autotransporters

We reported earlier that a single gene, tsh, isolated from a strain of avian pathogenic Escherichia coli (APEC) was sufficient to confer on E. coli K-12 a hemagglutinin-positive phenotype and that the deduced sequence of the Tsh protein shared homology to the serine-type immunoglobulin A (IgA) proteases of Neisseria gonorrhoeae and Haemophilus influenzae. In this report we show that E. coli K-12 containing the recombinant tsh gene produced two proteins, a 106-kDa extracellular protein and a 33-kDa outer membrane protein, and was also able to agglutinate chicken erythrocytes. N-terminal sequence data indicated that the 106-kDa protein, designated Tshs, was derived from the N-terminal end of Tsh after the removal of a 52-amino-acid N-terminal signal peptide, while the 33-kDa protein, designated Tshbeta, was derived from the C-terminal end of Tsh starting at residue N1101. The Tshs domain contains the 7-amino-acid serine protease motif that includes the active-site serine (S259), found also in the secreted domains of the IgA proteases. However, site-directed mutagenesis of S259 did not abolish the hemagglutinin activity or the extracellular secretion of Tshs indicating that host-directed proteolysis was mediating the release of Tshs. Studies with an E. coli K-12 ompT mutant strain showed that the surface protease OmpT was not needed for the secretion of Tshs. Tsh belongs to a subclass of the IgA protease family, which also includes EspC of enteropathogenic E. coli, EspP of enterohemorragic E. coli, and SepA and VirG of Shigella flexneri, which seem to involve a host endopeptidase to achieve extracellular release of their N-terminal domains. In proteolytic studies conducted in vitro, Tshs did not cleave the substrate of the IgA proteases, human IgA1 or chicken IgA, and did not show proteolytic activity in a casein-based assay. Correlation of Tsh expression and hemagglutination activity appears to be a very complex phenomenon, influenced by strain and environmental conditions. Nevertheless, for both APEC and recombinant E. coli K-12 strains containing the tsh gene, it was only the whole bacterial cells and not the cell-free supernatants that could confer hemagglutinin activity. Our results provide insights into the expression, secretion, and proteolytic features of the Tsh protein, which belongs to the growing family of gram-negative bacterial extracellular virulence factors, named autotransporters, which utilize a self-mediated mechanism to achieve export across the bacterial cell envelope.     

The Bacillus subtilis nucleotidyltransferase is a tRNA CCA-adding enzyme

There has been increased interest in bacterial polyadenylation with the recent demonstration that 3' poly(A) tails are involved in RNA degradation. Poly(A) polymerase I (PAP I) of Escherichia coli is a member of the nucleotidyltransferase (Ntr) family that includes the functionally related tRNA CCA-adding enzymes. Thirty members of the Ntr family were detected in a search of the current database of eubacterial genomic sequences. Gram-negative organisms from the beta and gamma subdivisions of the purple bacteria have two genes encoding putative Ntr proteins, and it was possible to predict their activities as either PAP or CCA adding by sequence comparisons with the E. coli homologues. Prediction of the functions of proteins encoded by the genes from more distantly related bacteria was not reliable. The Bacillus subtilis papS gene encodes a protein that was predicted to have PAP activity. We have overexpressed and characterized this protein, demonstrating that it is a tRNA nucleotidyltransferase. We suggest that the papS gene should be renamed cca, following the notation for its E. coli counterpart. The available evidence indicates that cca is the only gene encoding an Ntr protein, despite previous suggestions that B. subtilis has a PAP similar to E. coli PAP I. Thus, the activity involved in RNA 3' polyadenylation in the gram-positive bacteria apparently resides in an enzyme distinct from its counterpart in gram-negative bacteria.