Genetic analysis of an Escherichia coli urease locus: evidence of DNA rearrangement.

Ureolytic Escherichia coli strains are uncommon clinical isolates. The urease phenotype in a large percentage of these isolates is unstable and lost upon storage. We examined two urease-positive uropathogenic E. coli isolates that give off urease-negative segregants and determined that the urease phenotype was chromosomally encoded. The urease phenotype was cloned from E. coli 1021 and found to be encoded on a 9.4-kilobase HindIII restriction fragment. Transposon mutagenesis indicated that at least 3.2 kilobases of this fragment were necessary for production of urease. The urease recombinant plasmid pURE coded for at least four insert-specific polypeptides as determined by maxicell analysis. Disruption of the region encoding two of these polypeptides (67 and 27 kilodaltons) abolished urease activity. Analysis by Southern hybridization of urease-positive E. coli 1021 and seven independently isolated urease-negative segregants showed that a DNA rearrangement was associated with the urease-negative phenotype.     

A bifunctional urease enhances survival of pathogenic Yersinia enterocolitica and Morganella morganii at low pH.

To infect a susceptible host, the gastrointestinal pathogen Yersinia enterocolitica must survive passage through the acid environment of the stomach. In this study, we showed that Y. enterocolitica serotype O8 survives buffered acidic conditions as low as pH 1.5 for long periods of time provided urea is available. Acid tolerance required an unusual cytoplasmically located urease that was activated 780-fold by low-pH conditions. Acid tolerance of Helicobacter species has also been attributed to urease activity, but in that case urease was not specifically activated by low-pH conditions. A ure mutant strain of Y. enterocolitica was constructed which was hypersensitive to acidic conditions when urea was available and, unlike the parental strain, was unable to grow when urea was the sole nitrogen source. Examination of other urease-producing gram-negative bacteria indicated that Morganella morganii survives in acidic conditions but Escherichia coli 1021, Klebsiella pneumoniae, Proteus mirabilis, Providencia stuartii, and Pseudomonas aeruginosa do not. Consistent with these results, biochemical evidence demonstrated that Y. enterocolitica and M. morganii ureases were activated in vitro by low pH with an unusually low activity optimum of pH 5.5. In whole cells activation occurred as medium values decreased below pH 3.0 for Y. enterocolitica and pH 5.5 for M. morganii, suggesting that in vivo activation occurs as a result of cytoplasmic acidification. DNA sequence analysis of portions of the M. morganii ure locus showed that the predicted primary structure of the enzyme structural subunits is most similar to those of Y. enterocolitica urease. One region of similarity between these two ureases located near the active site is distinct from most other ureases but is present in the urease of Lactobacillus fermentum. This region of similarity may be responsible for the unique properties of the Y. enterocolitica and M. morganii ureases since the L. fermentum urease also has been shown to have a low pH optimum for activity.     

Expression of Helicobacter pylori urease genes in Escherichia coli grown under nitrogen-limiting conditions.

Helicobacter pylori produces a potent urease that is believed to play a role in the pathogenesis of gastroduodenal diseases. Four genes (ureA, ureB, ureC, and ureD) were previously shown to be able to achieve a urease-positive phenotype when introduced into Campylobacter jejuni, whereas Escherichia coli cells harboring these genes did not express urease activity (A. Labigne, V. Cussac, and P. Courcoux, J. Bacteriol. 173:1920-1931, 1991). Results that demonstrate that H. pylori urease genes could be expressed in E. coli are presented in this article. This expression was found to be dependent on the presence of accessory urease genes hitherto undescribed. Subcloning of the recombinant cosmid pILL585, followed by restriction analyses, resulted in the cloning of an 11.2-kb fragment (pILL753) which allowed the detection of urease activity (0.83 +/- 0.39 mumol of urea hydrolyzed per min/mg of protein) in E. coli cells grown under nitrogen-limiting conditions. Transposon mutagenesis of pILL753 with mini-Tn3-Km permitted the identification of a 3.3-kb DNA region that, in addition to the 4.2-kb region previously identified, was essential for urease activity in E. coli. Sequencing of the 3.3-kb DNA fragment revealed the presence of five open reading frames encoding polypeptides with predicted molecular weights of 20,701 (UreE), 28,530 (UreF), 21,744 (UreG), 29,650 (UreH), and 19,819 (UreI). Of the nine urease genes identified, ureA, ureB, ureF, ureG, and ureH were shown to be required for urease expression in E. coli, as mutations in each of these genes led to negative phenotypes. The ureC, ureD, and ureI genes are not essential for urease expression in E. coli, although they belong to the urease gene cluster. The predicted UreE and UreG polypeptides exhibit some degree of similarity with the respective polypeptides encoded by the accessory genes of the Klebsiella aerogenes urease operon (33 and 92% similarity, respectively, taking into account conservative amino acid changes), whereas this homology was restricted to a domain of the UreF polypeptide (44% similarity for the last 73 amino acids of the K. aerogenes UreF polypeptide). With the exception of the two UreA and UreB structural polypeptides of the enzyme, no role can as yet be assigned to the nine proteins encoded by the H. pylori urease gene cluster.     

Pseudomonas aeruginosa plasmids as suicide vectors in Escherichia coli: resolution of genomic cointegrates through short regions of homology.

Pseudomonas aeruginosa plasmids which cannot replicate in Escherichia coli have been used to introduce specific modifications into the E. coli chromosome by homologous recombination ('gene targeting'). The E. coli gene (gpt) encoding guanine-xanthine phosphoribosyltransferase (Gpt) was used for initial targeting studies owing to the availability of a powerful positive selection for loss of the Gpt+ phenotype (6-thioguanine resistance or 6TGR or Gpt-). P. aeruginosa plasmids containing selectable markers flanked by gpt sequences were introduced as supercoiled DNA into an E. coli strain which contained a normal gpt locus. Primary cointegration of such plasmids into the E. coli genome results in a gene duplication event which maintains Gpt function; a secondary recombinational event which resolves the cointegrate either reverses the primary event or results in replacement of the original gpt copy with the modified version. A 316-bp region of homology was sufficient for cointegrate formation, and resolution of the cointegrates through a shorter (92 bp) homologous flank was selectable through loss of Gpt function. The frequency of cointegrate resolution under these conditions was significantly above the spontaneous gpt mutational loss rate.     

The large plasmids found in enterohemorrhagic and enteropathogenic Escherichia coli constitute a related series of transfer-defective Inc F-IIA replicons.

Forty-six of 52 (88.5%) enterohemorrhagic Escherichia coli (EHEC) strains screened carried a "common" plasmid of about 90 kb which encoded sequences homologous to the Inc F-IIA replicon. A similarly high incidence of Inc F-IIA plasmid-containing strains was observed in other groups of diarrheagenic E. coli, but not in random environmental coliform isolates. Enteropathogenic E. coli (EPEC) contain plasmids of similar properties and share a 23-kb DNA fragment with plasmids from EHEC. The common region encodes the F-IIA replication region and sequences homologous to the transfer operon of the Inc F-II plasmid R1. Sequence homology varied between plasmids isolated from different EHEC/EPEC strains with > 80% showing homology to the regions encoding the rep and par genes. Only 5% of plasmids from EHEC strains had intact sequences homologous to the DNA between these two regions, including the oriT site. Some plasmids with an apparently intact tra operon still failed to plaque F-pilus-specific phages. This is consistent with observations that the large plasmids of EHEC and EPEC are phenotypically nonconjugative. These results suggest that the large plasmids of EHEC/EPEC constitute a family of transfer-deficient Inc F-IIA plasmids with varying degrees of deletion in tra function. The evolutionary ramifications of this finding are considered.     

Insertional inactivation of an Escherichia coli urease gene by IS3411.

Ureolytic Escherichia coli are unusual clinical isolates that are found at various extraintestinal sites of infection, predominantly the urinary tract. The urease-positive phenotype is unstable in approximately 25% of these isolates, and urease-negative segregants are produced at a high frequency. We have studied the nature of the urease-positive-to-negative transition in one of these isolates, designated E. coli 1021. Southern hybridization experiments with genomic DNA extracted from seven independent E. coli 1021 urease-negative segregants revealed the presence of a 1.3-kb DNA insertion in the urease gene cluster. A DNA fragment containing the DNA insertion was cloned from one of the urease-negative segregants. This cloned DNA fragment was capable of mediating cointegrate formation with the conjugative plasmid pOX38, suggesting that the DNA insertion was a transposable element. The insert was identified as an IS3411 element in ureG by DNA sequence analysis. A 3-bp target duplication (CTG) flanking the insertion element was found. DNA spanning the insertion site was amplified from the other six urease-negative segregants by using the polymerase chain reaction. The DNA sequence of the amplified fragments indicated that an IS3411 element was found in an identical site in all urease-negative segregants examined. These data suggest that in E. coli 1021, IS3411 transposes at a high frequency into ureG at a CTG site, disrupting this gene and eliminating urease activity.     

Metronidazole activation and isolation of Clostridium acetobutylicum electron transport genes

An Escherichia coli F19 recA, nitrate reductase-deficient mutant was constructed by transposon mutagenesis and shown to be resistant to metronidazole. This mutant was a most suitable host for the isolation of Clostridium acetobutylicum genes on recombinant plasmids, which activated metronidazole and rendered the E. coli F19 strain sensitive to metronidazole. Twenty-five E. coli F19 clones containing different recombinant plasmids were isolated and classified into five groups on the basis of their sensitivity to metronidazole. The clones were tested for nitrate reductase, pyruvate-ferredoxin oxidoreductase, and hydrogenase activities. DNA hybridization and restriction endonuclease mapping revealed that four of the C. acetobutylicum insert DNA fragments on recombinant plasmids were linked in an 11.1-kb chromosomal fragment. DNA sequencing and amino acid homology studies indicated that this DNA fragment contained a flavodoxin gene which encoded a protein of 160 amino acids that activated metronidazole and made the E. coli F19 mutant very sensitive to metronidazole. The flavodoxin and hydrogenase genes which are involved in electron transfer systems were linked on the 11.1-kb DNA fragment from C. acetobutylicum.     

Structuro-functional organization of segments of Co1A plasmid DNA, determining its stable inheritance

Two par regions were localized within the structure of a small colicinogenic plasmid ColA. One of them functions at the expense of plasmid multimere resolution. Analysis of the nucleotide sequence of the region revealed the existence of essential homology with the par locus of plasmid ColE1. As compared to E. coli C600, the function of multimere forms' resolution of plasmid DNA in E. coli C is reduced or absent due to par regions of the ColE1 type. Par regions of various degrees of homology with the par locus of ColE1 were localized by Southern hybridization within the structure of colicinogenic plasmids ColN and ColD. The stabilization of the colicinogenic plasmids is believed to be also determined by the functioning of genes connected with the synthesis and action of colicin.     

Characterization of a plasmid-encoded urease gene cluster found in members of the family Enterobacteriaceae.

Plasmid-encoded urease gene clusters found in uropathogenic isolates of Escherichia coli, Providencia stuartii, and Salmonella cubana demonstrated DNA homology, similar positions of restriction endonuclease cleavage sites, and manners of urease expression and therefore represent the same locus. DNA sequence analysis indicated that the plasmid-encoded urease genes are closely related to the Proteus mirabilis urease genes.     

Distribution and diversity of hsd genes in Escherichia coli and other enteric bacteria.

We screened Salmonella typhimurium, Citrobacter freundii, Klebsiella pneumoniae, Shigella boydii, and many isolates of Escherichia coli for DNA sequences homologous to those encoding each of two unrelated type I restriction and modification systems (EcoK and EcoA). Both K- and A-related hsd genes were identified, but never both in the same strain. S. typhimurium encodes three restriction and modification systems, but its DNA hybridized only to the K-specific probe which we know to identify the StySB system. No homology to either probe was detected in the majority of E. coli strains, but in C. freundii, we identified homology to the A-specific probe. We cloned this region of the C. freundii genome and showed that it encoded a functional, A-related restriction system whose specificity differs from those of known type I enzymes. Sequences immediately flanking the hsd K genes of E. coli K-12 and the hsd A genes of E. coli 15T- were shown to be homologous, indicating similar or even identical positions in their respective chromosomes. E. coli C has no known restriction system, and the organization of its chromosome is consistent with deletion of the three hsd genes and their neighbor, mcrB.     

Cloning and characterization of the hemolysin determinants from Vibrio cholerae RV79(Hly+), RV79(Hly-), and 569B

The Hly region from the chromosome of Vibrio cholerae El Tor strain RV79(Hly-) and the nonhemolytic classical strain 569B were cloned into plasmid vector pBR322. Escherichia coli K-12 transformants possessing these recombinant plasmids were nonhemolytic and were detected with a 32P-labeled hly-specific DNA probe. Restriction endonuclease Sau3AI digestions of the cloned hly loci of two independently obtained RV79(Hly+) convertants, when compared with the digests of cloned RV79(Hly-) loci, revealed that an apparent alteration (10 to 15 base pairs) had occurred. In contrast, an apparent 20-base-pair deletion was present in the cloned hly locus of the classical biotype V. cholerae strain 569B. Maxicell analysis and immunoprecipitation of labeled proteins of E. coli which are encoded by the cloned hly loci of RV79(Hly+) and from nuclease BAL 31-deleted plasmids, as well as immunoprecipitation of [35S]methionine-labeled V. cholerae proteins, suggest that the hemolysin is an 84,000-dalton polypeptide.     

Formation of genes coding for hybrid proteins by recombination between related, cloned genes in E. coli.

We describe a method for the formation of hybrid genes by in vivo recombination between two genes with partial sequence homology. DNA structures consisting of plasmid vector sequences, flanked by the alpha 2 interferon gene on the one side and a portion of the alpha 1 interferon gene (homology about 80%) on the other, were transfected into E. coli SK1592. Appropriate resistance markers allowed the isolation of colonies containing circular plasmids which arose by in vivo recombination between the partly homologous interferon gene sequences. Eleven different recombinant genes were identified, six of which encoded new hybrid interferons not easily accessible by recombinant DNA techniques.     

Characterization of the Azorhizobium sesbaniae ORS571 genomic locus encoding NADPH-glutamate synthase.

Sixteen independent Azorhizobium sesbaniae ORS571 vector insertion (Vi) mutants defective in ammonium assimilation (Asm-) were selected; genomic DNA sequences flanking the insertion endpoints were cloned directly. Resulting recombinant plasmids were used to identify, by hybridization, corresponding wild-type DNA sequences from an A. sesbaniae lambda EMBL3 genomic library (lambda Asm phages). All 16 Asm- Vi mutants physically mapped to a single genomic locus. Plasmid subclones of recombinant phage lambda Asm152 were able to complement both Escherichia coli gltB and A. sesbaniae Asm- Vi mutants; NADPH-glutamate synthase activity was detected in all such strains complemented to Asm+. Heterologous and homologous complementations required both A. sesbaniae gltA+ and (inferred) gltB+ genes. Eleven A. sesbaniae Asm- Vi mutants mapped to a 4-kilobase-pair (kbp) DNA region that exhibited homology with Bacillus subtilis gltA+. In E. coli maxicell labeling experiments, this 4-kbp DNA region encoded a 165-kilodalton polypeptide that was inferred to be the product of the A. sesbaniae gltA+ gene (glutaminase NADPH-dependent L-glutamate synthase subunit). Site-directed Tn5-lacZ mutagenesis of a glt plasmid subclone identified a region that bisected this locus into (at least) two cistrons. Because the remaining five A. sesbaniae Asm- mutants mapped to a 1.5-kbp region adjacent to gltA+, these mutants probably define a single gltB+ gene (glutamate dehydrogenase NADPH-dependent L-glutamate synthase subunit); this region did not exhibit homology with the B. subtilis gltB+ gene.     

Cloning of chromosomal DNA encoding the F41 adhesin of enterotoxigenic Escherichia coli and genetic homology between adhesins F41 and K88.

The genetic determinant for production of the adhesive antigen F41 was isolated from a porcine enterotoxigenic Escherichia coli strain by cosmid cloning. The cloned DNA included sequences homologous to those of hybridization probes prepared from the K88 adhesive antigen operon. Transposon insertions which inactivated F41 production mapped to the same region of DNA showing homology with the K88 genes, demonstrating the genetic relatedness of F41 and K88. Hybridization of a K88 gene probe to plasmid and total DNA from the porcine E. coli isolate from which the F41 gene was cloned indicated that F41 is chromosomally encoded by this strain. This observation was extended to other F41-producing animal isolates. A large number of animal E. coli isolates were examined with K88, F41, and K99 gene probes and for mannose-resistant hemagglutination of human group O erythrocytes and K88 and F41 antigen production. All K88 and F41 antigen producers possessed genetic homology with the K88 and F41 gene probes. Most, but not all, F41-producing strains possessed homology to the K99 gene probe, reflecting the previously observed association of F41 and K99 antigen production. In the strains examined, homology with the K99 gene probe was plasmid associated, whereas homology with the F41 gene probe was chromosomal. The K88 antigen-producing strains showed no homology with the K99 probe. A number of strains possessed homology with the K88 and F41 gene probes and were mannose-resistant hemagglutination positive, but did not produce K88 or F41 antigens. This suggests that there are adhesins among animal isolates of E. coli which are genetically related to but antigenically distinct from K88 and F41.     

Controlled and functional expression of the Pseudomonas oleovorans alkane utilizing system in Pseudomonas putida and Escherichia coli

The OCT plasmid encodes enzymes for alkane hydroxylation and alkanol dehydrogenation. Structural components are encoded on the 7.5-kilobase pair alkBAC operon, whereas positive regulatory components are encoded by alkR. We have constructed plasmids containing fusions of cloned alkBAC and alkR DNA and used these fusion plasmids to study the functional expression of the alkBAC operon and the regulatory locus alkR in Pseudomonas putida and in Escherichia coli. Growth on alkanes requires a functional chromosomally encoded fatty acid degradation system in addition to the plasmid-borne alk system. While such a system is active in P. putida, it is active in E. coli only in fadR mutants in which fatty acid degradation enzymes are expressed constitutively. Using such mutants, we found that E. coli as well as P. putida grew on octane as the sole source of carbon and energy when they were supplied with the cloned complete alk system. The alkR locus was strictly necessary in E. coli as well as in P. putida for expression of the alkBAC operon. The alkBAC operon could, however, be further reduced to a 5-kilobase pair operon without affecting the Alk phenotype in either species to a significant extent. Although with this reduction the plasmid-encoded alkanol dehydrogenase activity was lost, chromosomally encoded alkanol dehydrogenases in P. putida and E. coli compensated for this loss. The induction kinetics of the alk system was studied in detail in P. putida and E. coli. We used specific antibodies raised against alkane hydroxylase to follow the appearance of this protein following induction with octane. We found the induction kinetics of alkane hydroxylase to be similar in both species. A steady-state level was reached after about 2 h of induction in which time the alkane hydroxylase accounted for about 1.5% of total newly synthesized protein. Thus, alkBAC expression is very efficient and strictly regulated to both P. putida and E. coli.     

Construction and characterization of autonomously replicating plasmids in the green unicellular alga Chlamydomonas reinhardii

Plasmids that replicate autonomously in Chlamydomonas reinhardii were constructed by inserting random DNA fragments from this alga into a plasmid containing the yeast ARG4 locus. Arginine prototrophy was used as a selective marker. The presence of free plasmids in the DNA of the transformants was demonstrated by hybridization with a specific plasmid probe and by recovering these plasmids in E. coli after transformation. Four of them were characterized. Their inserts of 415, 257, 153, and 102 by all hybridize to chloroplast DNA and were localized on the physical map of the chloroplast genome. One of these plasmids also promotes autonomous replication in yeast. Sequence analysis of the inserts of the plasmids reveals several short direct and inverted repeats and two semiconserved AT-rich elements of 19 and 12 by that may play a role in promoting autonomous replication in C. reinhardii.     

Cloning and expression of Acinetobacter calcoaceticus catechol 1,2-dioxygenase structural gene catA in Escherichia coli.

Catechol 1,2-dioxygenase (EC 1.13.1.1), the product of the catA gene, catalyzes the first step in catechol utilization via the beta-ketoadipate pathway. Enzymes mediating subsequent steps in the pathway are encoded by the catBCDE genes which are carried on a 5-kilobase-pair (kbp) EcoRI restriction fragment isolated from Acinetobacter calcoaceticus. This DNA was used as a probe to identify Escherichia coli colonies carrying recombinant pUC19 plasmids with overlapping sequences. Repetition of the procedure yielded an A. calcoaceticus 6.7-kbp EcoRI restriction fragment which contained the catA gene and bordered the original 5-kbp EcoRI restriction fragment. When the catA-containing fragment was placed under the control of the lac promoter on pUC19 and induced with isopropylthiogalactopyranoside, catechol dioxygenase was formed in E. coli at twice the level found in fully induced cultures of A. calcoaceticus. A. calcoaceticus strains with mutations in the catA gene were transformed to wild type by DNA from lysates of E. coli strains carrying the catA gene on recombinant plasmids. Thus, A. calcoaceticus strains with a mutated gene can be used in a transformation assay to identify E. coli clones in which at least part of the wild-type gene is present but not necessarily expressed.     

Evidence for two phosphonate degradative pathways in Enterobacter aerogenes.

We screened mini-Mu plasmid libraries from Enterobacter aerogenes IFO 12010 for plasmids that complement Escherichia coli phn mutants that cannot use phosphonates (Pn) as the sole source of phosphorus (P). We isolated two kinds of plasmids that, unexpectedly, encode genes for different metabolic pathways. One kind complements E. coli mutants with both Pn transport and Pn catalysis genes deleted; these plasmids allow degradation of the 2-carbon-substituted Pn alpha-aminoethylphosphonate but not of unsubstituted alkyl Pn. This substrate specificity is characteristic of a phosphonatase pathway, which is absent in E. coli. The other kind complements E. coli mutants with Pn catalysis genes deleted but not those with both transport and catalysis genes deleted; these plasmids allow degradation of both substituted and unsubstituted Pn. Such a broad substrate specificity is characteristic of a carbon-phosphorus (C-P) lyase pathway, which is common in gram-negative bacteria, including E. coli. Further proof that the two kinds of plasmids encode genes for different pathways was demonstrated by the lack of DNA homology between the plasmids. In particular, the phosphonatase clone from E. aerogenes failed to hybridize to the E. coli phnCDEFGHIJKLMNOP gene cluster for Pn uptake and degradation, while the E. aerogenes C-P lyase clone hybridized strongly to the E. coli phnGHIJKLM genes encoding C-P lyase but not to the E. coli phnCDE genes encoding Pn transport. Specific hybridization by the E. aerogenes C-P lyase plasmid to the E. coli phnF, phnN, phnO, and phnP genes was not determined. Furthermore, we showed that one or more genes encoding the apparent E. aerogenes phosphonatase pathway, like the E. coli phnC-to-phnP gene cluster, is under phosphate regulon control in E. coli. This highlights the importance of Pn in bacterial P assimilation in nature.     

Functional and phylogenetic analysis of ureD in Shiga toxin-producing Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that can cause severe health complications and utilizes a much lower infectious dose than other E. coli pathotypes. Despite having an intact ure locus, ureDABCEFG, the majority of EHEC strains are phenotypically urease negative under tested conditions. Urease activity potentially assists with survival fitness by enhancing acid tolerance during passage through the stomach or by aiding with colonization in either human or animal reservoirs. Previously, in the EHEC O157:H7 Sakai strain, a point mutation in ureD, encoding a urease chaperone protein, was identified, resulting in a substitution of an amber stop codon for glutamine. This single nucleotide polymorphism (SNP) is observed in the majority of EHEC O157:H7 isolates and correlates with a negative urease phenotype in vitro. We demonstrate that the lack of urease activity in vitro is not solely due to the amber codon in ureD. Our analysis has identified two additional SNPs in ureD affecting amino acid positions 38 and 205, in both cases determining whether the encoded amino acid is leucine or proline. Phylogenetic analysis based on Ure protein sequences from a variety of urease-encoding bacteria demonstrates that the proline at position 38 is highly conserved among Gram-negative bacteria. Experiments reveal that the L38P substitution enhances urease enzyme activity; however, the L205P substitution does not. Multilocus sequence typing analysis for a variety of Shiga toxin-producing E. coli isolates combined with the ureD sequence reveals that except for a subset of the O157:H7 strains, neither the in vitro urease-positive phenotype nor the ureD sequence is phylogenetically restricted.