Expression of urease does not affect the ability of Bordetella bronchiseptica to colonise and persist in the murine respiratory tract

To investigate the role played by urease during the Bordetella bronchiseptica infection process, the ability to colonise and persist in the mouse respiratory tract of a urease-negative B. bronchiseptica BB7865 and a BB7865 derivative constitutively expressing urease was compared with that of the wild-type strain. The results obtained showed that neither constitutive expression nor abolishment of urease activity had any significant effect on the course of B. bronchiseptica infection. Therefore, under our experimental conditions, urease is not essential for B. bronchiseptica to colonise and persist within the murine host.     

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.     

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.     

Survival of diverse Bacillus thuringiensis strains in gypsy moth (Lepidoptera: Lymantriidae) is correlated with urease production

Bacillus thuringiensis is an entomopathogenic bacterium that can kill a variety of pests, but seldom causes epizootics because it replicates poorly in insects. We have tested lepidopteran-toxic B. thuringiensis strains with diverse substrate utilization profiles for the ability to survive repeated passages through larvae of the gypsy moth, Lymantria dispar, without intervening growth on artificial media. These experiments have revealed a remarkable correlation between the production of urease by the bacteria and its ability to survive repeated passages through larvae. Of 26 urease-positive strains tested, 23 were capable of surviving five passages through gypsy moth larvae. In contrast, none of the 24 urease-negative strains tested survived to the 4th passage, with only three strains surviving to the 3rd passage. Selection of B. thuringiensis strains with phenotypic traits favoring replication in the environment, such as urease production, may improve their efficacy as biological control agents.     

Representational difference analysis identifies a strain-specific LPS biosynthesis locus in Bordetella spp.

Bordetella pertussis and B. bronchiseptica are genetically very closely related but differ significantly in their virulence properties. Using Representational Difference Analysis (RDA), 11 DNA fragments specific for B. pertussis Tohama I or B. bronchiseptica BB7865 were identified. All B. bronchiseptica BB7865-derived fragments also hybridized with chromosomal DNA from B. parapertussis but not from the B. pertussis strains Tohama I and W28, underlining the close phylogenetic relationship between B. bronchiseptica and B. parapertussis. The B. pertussis type strain BP18323 is a special case, as it contains DNA sequences characteristic for both B. pertussis and B. bronchiseptica. As demonstrated by pulsed-field gel electrophoresis, several of the BB7865-derived fragments are present on a single 30-kb XbaI fragment. Based on the sequences of putative coding regions, four of these fragments may code for proteins involved in carbohydrate metabolism or transport. In agreement with this notion, a mutant for one of these loci synthesizes a significantly altered lipopolysaccharide that lacks the O-specific side chains. The analysis of the corresponding genomic region in various Bordetella species showed that this locus is present in B. bronchiseptica and B. parapertussis but not in B. pertussis. This confirms that the RDA approach has identified a novel strain-specific LPS biosynthesis locus which accounts for the differences between the LPS structures elaborated by different Bordetella species. Received: 24 February 1999 / Accepted: 17 May 1999     

Isolation and preliminary characterization of plasmids in Bacillus badius

The presence of urease and NADPH-specific glutamate dehydrogenase (GDH) coding plasmid in Bacillus badius was suggested by the loss of enzyme activites with a mitomycin C treatment and by the presence of 3 plasmids in urease-active strains compared to 2 plasmids in urease-negative strains. Furthermore two-dimensional protein gel electrophoresis showed that the urease-positive strains had some additional proteins compared to urease-negative strains. The two common plasmids had a size of 14.7 kb and 5.9 kb. The plasmid that was missing in the urease-negative strains had a size of 44.0 kb. The plasmids were purified and restriction map for AvaI, Bam HI and EcoRI was constructed for each plasmid. Hybridization tests showed that all three plasmids in Bacillus badius were independent entities.     

Phosphorylation chemistry of the Bordetella PlrSR TCS and its contribution to bacterial persistence in the lower respiratory tract

Bordetella species cause lower respiratory tract infections in mammals. B. pertussis and B. bronchiseptica are the causative agents of whooping cough and kennel cough, respectively. The current acellular vaccine for B. pertussis protects against disease but does not prevent transmission or colonization. Cases of pertussis are on the rise even in areas of high vaccination. The PlrSR two-component system, is required for persistence in the mouse lung. A partial plrS deletion strain and a plrS H521Q strain cannot survive past 3 days in the lung, suggesting PlrSR works in a phosphorylation-dependent mechanism. We characterized the biochemistry of B. bronchiseptica PlrSR and found that both proteins function as a canonical two-component system. His521 was essential and Glu522 was critical for PlrS autophosphorylation. Asn525 was essential for phosphatase activity. The PAS domain was critical for both PlrS autophosphorylation and phosphatase activities. PlrS could both phosphotransfer to and exert phosphatase activity toward PlrR. Unexpectedly, PlrR formed a tetramer when unphosphorylated and a dimer upon phosphorylation. Finally, we demonstrated the importance of PlrS phosphatase activity for persistence within the murine lung. By characterizing PlrSR we hope to guide future in vivo investigation for development of new vaccines and therapeutics.     

Cloning, expression and sequencing of Helicobacter felis urease genes

Urease genes from Helicobacter felis were cloned and expressed in Escherichia coli cells. A genomic bank of Sau3A-digested H. felis chromosomal DNA was created using a cosmid vector. Cosmid clones were screened for urease activity following subculture on a nitrogen-limiting medium. Subcloning of DNA from an urease-positive cosmid cione led to the construction of plLL205 (9.5 kb) which conferred a urease activity of 1.2±0.5 μmole urea min^-1 mg^-1 bacterial protein to E. coli HB101 bacteria grown on a nitrogen-limiting medium. Random mutagenesis using a MiniTn3-Km transposable element permitted the identification of three DNA regions on plLL205 which were necessary for the expression of an urease-positive phenotype in E. coii clones. To localize the putative structural genes of H. felis on plLL205, extracts of clones harbouring the mutated copies of the plasmid were analysed by Western blotting with anti-H. felis rabbit serum. One mutant cione did not synthesize the putative UreB subunit of H. felis urease and it was postulated that the transposable element had disrupted the corresponding structural gene. By sequencing the DNA region adjacent to the transposon insertion site two open reading frames, designated ureA and ureB, were identified. The polypeptides encoded by these genes had caicuiated moiecuiar masses of 26 074 and 61 663 Da, respectively, and shared 73.5% and 88.2% identity with the corresponding gene products of Helicobacter pylori urease.     

一株支气管炎鲍特杆菌国家标准菌种的再鉴定

通过生化试验、质谱鉴定、16S rRNA基因序列分析、全基因组序列测定及生物信息学分析等方法对国家标准菌种CMCC(B)40001进行再鉴定。对菌株的毒力、耐药和MLST进行分析，并对鲍特杆菌（Bordetella spp.）的群体进化进行分析。结合不同方法，CMCC(B)40001再鉴定为支气管炎鲍特杆菌（Bordetella bronchiseptica），且其作为标准菌种能够发挥国家标准描述的质控作用。同时获取该菌株的新ST型，在进化关系上，其与百日咳鲍特杆菌(Bordetella pertussis)和副百日咳鲍特杆菌(Bordetella parapertussis)遗传距离近。本研究再鉴定了国家标准菌种CMCC(B)40001为支气管炎鲍特杆菌，并分析了其遗传特征，为国家标准菌种的应用提供资源描述支持。     

Multiple proteins encoded within the urease gene complex of Proteus mirabilis.

Chromosomal DNA fragments from a uropathogenic isolate of Proteus mirabilis were inserted into the cosmid vector pHC79 to construct a genomic library in Escherichia coli HB101. A urease-positive recombinant cosmid, designated pSKW1, was recovered. Sequential recombinant manipulation of pSKW1 yielded a 10.2-kilobase plasmid, designated pSKW4, which encoded three urease isozymes with electrophoretic mobilities identical to those of the donor P. mirabilis strain. Plasmid pSKW4 gene sequences encode seven proteins designated 68K (apparent molecular weight, of 68,000), 28K, 25K, 22.5K, 18.5K, 7.5K, and 5.2K within the limits of the urease gene complex. Insertion mutations in genes encoding the 68K, 28K, 25K, 22.5K, 7.5K, and 5.2K proteins resulted in complete or partial (22.5K) loss of urease activity. There was no reduction in urease activity when the gene encoding the 18.5K protein was inactivated.     

A newly constructed primer pair for the PCR amplification,cloningand sequencing of the flagellin (<Emphasis Type="Italic">flaA</Emphasis>) gene from isolatesof urease-negative <Emphasis Type="Italic">Campylobacter lari</Emphasis>

A newly constructed primer pair (lari-Af/lari-Ar) designed to generate a product of the flagellin (flaA) gene for urease-negative Campylobacter lari produced a PCR amplicon of about 1700 bp for 16 isolates from 7 seagulls, 5 humans, 3 food animals and one mussel in Japan and Northern Ireland. Nucleotide sequencing and alignments of the flaA amplicons from these isolates demonstrated that the deduced amino acid sequences of the possible open reading frame were 564–572 amino acid residues in length with calculated molecular weights of 58,804 to 59,463. The deduced amino acid sequence similarity analysis strongly suggested that the ORF of the flaA from the 16 isolates showed 70–75% sequence similarities to those of Campylobacter jejuni isolates. The approximate Mr of the flagellin purified from some of the isolates of urease-negative C. lari was estimated to range from 59.6 to 61.8 kDa. Thus, flagellin from the isolates of urease-negative C. lari was shown for the first time to have a molecular size similar to those of C. jejuni and Campylobacter coli isolates, but to be different from the shorter flaA and smaller flagellin of urease-positive thermophilic Campylobacter (UPTC) isolates. Flagellins from C. lari spp., consisting of the two representative taxa of urease-negative C. lari and UPTC, thus show genotypic and phenotypic diversity.     

Representational difference analysis identifies a strain-specific LPS biosynthesis locus in Bordetella spp.

Bordetella pertussis and B. bronchiseptica are genetically very closely related but differ significantly in their virulence properties. Using Representational Difference Analysis (RDA), 11 DNA fragments specific for B. pertussis Tohama I or B. bronchiseptica BB7865 were identified. All B. bronchiseptica BB7865-derived fragments also hybridized with chromosomal DNA from B. parapertussis but not from the B. pertussis strains Tohama I and W28, underlining the close phylogenetic relationship between B. bronchiseptica and B. parapertussis. The B. pertussis type strain BP18323 is a special case, as it contains DNA sequences characteristic for both B. pertussis and B. bronchiseptica. As demonstrated by pulsed-field gel electrophoresis, several of the BB7865-derived fragments are present on a single 30-kb XbaI fragment. Based on the sequences of putative coding regions, four of these fragments may code for proteins involved in carbohydrate metabolism or transport. In agreement with this notion, a mutant for one of these loci synthesizes a significantly altered lipopolysaccharide that lacks the O-specific side chains. The analysis of the corresponding genomic region in various Bordetella species showed that this locus is present in B. bronchiseptica and B. parapertussis but not in B. pertussis. This confirms that the RDA approach has identified a novel strain-specific LPS biosynthesis locus which accounts for the differences between the LPS structures elaborated by different Bordetella species.     

Cell-wall structure,mitosis and urease activity in Torulopsis species of high GC content

Five Torulopsis species, in which, characteristically, the mean molar percentage of guanine plus cytosine of the DNA bases (GC content) is above 50%, were examined to determine their cell-wall structure; the mode of bud formation; course of mitosis and their urease activity. The two urease-negative species, T. gropengiesseri (% GC 57.1) and T. silvatica (% GC 56.3), were found to possess affinitive properties which characterize perfect ascomycetous yeast species. In the urease-positive species, T. fujisanensis (% GC 65.0–66.1), T. ingeniosa (% GC 55.6) and T. philyla (% GC 63.9), there appeared to be a correlation between pronounced urease activity and the affinitive properties which characterize perfect basidiomycetous yeast species. Pronounced urease activity has, however, also been found in the ascomycetous species Schizosaccharomyces pombe. Consequently neither high GC ratios nor the presence of urease activity when considered individually, can be taken as reliable criteria when attempting to establish the affinity of yeasts the perfect states of which are not known, with the Basidiomycetes. A strain of the ascomycetous species Sclerotinia trifoliorum was examined by electron microscopy for comparative purposes.     

Characteristics of a clinical isolate of urease-negative Helicobacter pylori and its ability to induce gastric ulcers in Mongolian gerbils

BACKGROUND: We clinically obtained urease-negative mutant strains of Helicobacter pylori. The goal of this study was to investigate the ability of the urease-negative strain to colonize and subsequently damage the gastric mucosa in Mongolian gerbils. In addition, the genes encoding the urease production in the test strain were analyzed, and other genes encoding the virulence factors, cytotoxin-associated protein and vacuolating-cytotoxin were evaluated. MATERIALS AND METHODS: The character of urease-negative isolates of H. pylori was defined. The identification of H. pylori was confirmed by polymerase chain reaction (PCR). The H. pylori isolate was transfected into Mongolian gerbils as previously described, which were followed up to 42 weeks, and the changes in their gastric mucosa were examined histologically. RESULTS AND CONCLUSION: Fifteen Mongolian gerbils orally infected with 10(7) colony forming units of urease-negative H. pylori were killed at 4, 12, 24, 36 and 42 weeks (n = 3) after infection. Culture medium without urease-negative H. pylori was given to the Mongolian gerbils as control. H. pylori continued to exist in the subject's stomach and gastric ulceration was observed and compared with the control. Clinically obtained urease-negative H. pylori continued to exist for at least 42 weeks in the subject's stomach and it induced gastric ulcers. These data demonstrated that the urease in H. pylori was not a necessary factor in the formation of gastric ulcers in the Mongolian gerbil model.     

Evolutionary relationships in the genus Bordetella

The nucleotide sequence of the pertussis toxin operon of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica, has shown that the last two species contain many common mutations and are likely to derive from a common ancestor (Arico and Rappuoli, 1987). To elucidate further the evolutionary relationships between the Bordetella species, we have cloned and sequenced the promoter region and the gene coding for the S1 subunit of pertussis toxin from additional B. pertussis strains, such as the type strain BP 18323 and two recent clinical isolates, namely strain BP 13456 from Sweden and strain BP SA1 from Italy. While the strains BP SA1 and BP 13456 are shown to differ from the published B. pertussis sequences by only one base pair, the type strain BP 18323 contains a total of 11 base-pair substitutions. Remarkably, 9 of the 11 substitutions found in BP 18323 are also common to B. parapertussis and B. bronchiseptica, strongly suggesting that this strain derives from the same ancestor as B. parapertussis and B. bronchiseptica. Computer analysis of the sequence data allows the construction of an evolutionary ‘tree’ showing that the B. pertussis strains are very homogeneous and significantly distant from B. parapertussis and B. bronchiseptica. Therefore the proposed conversion from B. parapertussis to B. pertussis appears highly improbable.     

Identification,cloning, and characterization of a multicomponent biphenyl dioxygenase from <Emphasis Type="Italic">Sphingobium yanoikuyae</Emphasis> B1

Sphingobium yanoikuyae B1 utilizes both polycyclic aromatic hydrocarbons (biphenyl, naphthalene, and phenanthrene) and monocyclic aromatic hydrocarbons (toluene, m- and p-xylene) as its sole source of carbon and energy for growth. The majority of the genes for these intertwined monocyclic and polycyclic aromatic pathways are grouped together on a 39 kb fragment of chromosomal DNA. However, this gene cluster is missing several genes encoding essential enzymatic steps in the aromatic degradation pathway, most notably the genes encoding the oxygenase component of the initial polycyclic aromatic hydrocarbon (PAH) dioxygenase. Transposon mutagenesis of strain B1 yielded a mutant blocked in the initial oxidation of PAHs. The transposon insertion point was sequenced and a partial gene sequence encoding an oxygenase component of a putative PAH dioxygenase identified. A cosmid clone from a genomic library of S. yanoikuyae B1 was identified which contains the complete putative PAH oxygenase gene sequence. Separate clones expressing the genes encoding the electron transport components (ferredoxin and reductase) and the PAH dioxygenase were constructed. Incubation of cells expressing the dioxygenase enzyme system with biphenyl or naphthalene resulted in production of the corresponding cis-dihydrodiol confirming PAH dioxygenase activity. This demonstrates that a single multicomponent dioxygenase enzyme is involved in the initial oxidation of both biphenyl and naphthalene in S. yanoikuyae B1.