Urease Characteristics and Phylogenetic Status of Bacillus paralicheniformis.

In 2015, Bacillus paralicheniformis was separated from B. licheniformis on the basis of phylogenomic and phylogenetic studies, and urease activity was reported as a phenotypic property able to differentiate between the two species. Subsequently, we have found that the urease activity of B. paralicheniformis is strain-specific, and does not reliably discriminate between species, as strains having the same urease gene cluster were identified in B. licheniformis and B. sonorensis, the closest relatives of B. paralicheniformis. We developed a multilocus sequence typing scheme using eight housekeeping genes, adk, ccpA, glpF, gmk, ilvD, pur, spo0A, and tpi to clearly identify B. paralicheniformis from closely related Bacillus species and to find a molecular marker for the rapid identification of B. paralicheniformis. The scheme differentiated 33 B. paralicheniformis strains from 90 strains formerly identified as B. licheniformis. Among the eight housekeeping genes, spo0A possesses appropriate polymorphic sites for the design of a B. paralichenofomis-specific PCR primer set. The primer set designed in this study perfectly separated B. paralicheniformis from B. licheniformis and B. sonorensis.

The genome of Rhizobiales bacteria in predatory ants reveals urease gene functions but no genes for nitrogen fixation.

Gut-associated microbiota of ants include Rhizobiales bacteria with affiliation to the genus Bartonella. These bacteria may enable the ants to fix atmospheric nitrogen, but no genomes have been sequenced yet to test the hypothesis. Sequence reads from a member of the Rhizobiales were identified in the data collected in a genome project of the ant Harpegnathos saltator. We present an analysis of the closed 1.86 Mb genome of the ant-associated bacterium, for which we suggest the species name Candidatus Tokpelaia hoelldoblerii. A phylogenetic analysis reveals a relationship to Bartonella and Brucella, which infect mammals. Novel gene acquisitions include a gene for a putative extracellular protein of more than 6,000 amino acids secreted by the type I secretion system, which may be involved in attachment to the gut epithelium. No genes for nitrogen fixation could be identified, but genes for a multi-subunit urease protein complex are present in the genome. The urease genes are also present in Brucella, which has a fecal-oral transmission pathway, but not in Bartonella, which use blood-borne transmission pathways. We hypothesize that the gain and loss of the urease function is related to transmission strategies and lifestyle changes in the host-associated members of the Rhizobiales.

Archaeal amoA and ureC genes and their transcriptional activity in the Arctic Ocean.

Thaumarchaeota and the gene encoding for a subunit of ammonia monooxygenase (amoA) are ubiquitous in Polar Seas, and some Thaumarchaeota also have a gene coding for ureC, diagnostic for urease. Using quantitative PCR we investigated the occurrence of genes and transcripts of ureC and amoA in Arctic samples from winter, spring and summer. AmoA genes, ureC genes and amoA transcripts were always present, but ureC transcripts were rarely detected. Over a 48 h light manipulation experiment amoA transcripts persisted under light and dark conditions, but not ureC transcripts. In addition, maxima for amoA transcript were nearer the surface compared to amoA genes. Clone libraries using DNA template recovered shallow and deep amoA clades but only the shallow clade was recovered from cDNA (from RNA). These results imply environmental control of amoA expression with direct or indirect light effects, and rare ureC expression despite its widespread occurrence in the Arctic Ocean.

Enzymatic, immunological and phylogenetic characterization of Brucella suis urease.

BACKGROUND: The sequenced genomes of the Brucella spp. have two urease operons, ure-1 and ure-2, but there is evidence that only one is responsible for encoding an active urease. The present work describes the purification and the enzymatic and phylogenomic characterization of urease from Brucella suis strain 1330. Additionally, the urease reactivity of sera from patients diagnosed with brucellosis was examined. RESULTS: Urease encoded by the ure-1 operon of Brucella suis strain 1330 was purified to homogeneity using ion exchange and hydrophobic interaction chromatographies. The urease was purified 51-fold with a recovery of 12% of the enzyme activity and 0.24% of the total protein. The enzyme had an isoelectric point of 5, and showed optimal activity at pH 7.0 and 28-35 degrees C. The purified enzyme exhibited a Michaelis-Menten saturation kinetics with a Km of 5.60 +/- 0.69 mM. Hydroxyurea and thiourea are competitive inhibitors of the enzyme with Ki of 1.04 +/- 0.31 mM and 26.12 +/- 2.30 mM, respectively. Acetohydroxamic acid also inhibits the enzyme in a competitive way. The molecular weight estimated for the native enzyme was between 130-135 kDa by gel filtration chromatography and 157 +/- 7 kDa using 5-10% polyacrylamide gradient non-denaturing gel. Only three subunits in SDS-PAGE were identified: two small subunits of 14,000 Da and 15,500 Da, and a major subunit of 66,000 Da. The amino terminal sequence of the purified large subunit corresponded to the predicted amino acid sequence encoded by ureC1. The UreC1 subunit was recognized by sera from patients with acute and chronic brucellosis. By phylogenetic and cluster structure analyses, ureC1 was related to the ureC typically present in the Rhizobiales; in contrast, the ureC2 encoded in the ure-2 operon is more related to distant species. CONCLUSION: We have for the first time purified and characterized an active urease from B. suis. The enzyme was characterized at the kinetic, immunological and phylogenetic levels. Our results confirm that the active urease of B. suis is a product of ure-1 operon.

Sensitive pattern discovery with 'fuzzy' alignments of distantly related proteins.

MOTIVATION: Evolutionary comparison leads to efficient functional characterisation of hypothetical proteins. Here, our goal is to map specific sequence patterns to putative functional classes. The evolutionary signal stands out most clearly in a maximally diverse set of homologues. This diversity, however, leads to a number of technical difficulties. The targeted patterns-as gleaned from structure comparisons-are too sparse for statistically significant signals of sequence similarity and accurate multiple sequence alignment. RESULTS: We address this problem by a fuzzy alignment model, which probabilistically assigns residues to structurally equivalent positions (attributes) of the proteins. We then apply multivariate analysis to the 'attributes x proteins' matrix. The dimensionality of the space is reduced using non-negative matrix factorization. The method is general, fully automatic and works without assumptions about pattern density, minimum support, explicit multiple alignments, phylogenetic trees, etc. We demonstrate the discovery of biologically meaningful patterns in an extremely diverse superfamily related to urease.

Molecular analysis of Vibrio parahaemolyticus isolated from human patients and shellfish during US Pacific north-west outbreaks.

AIMS: The objective of this study was to investigate the occurrence and distribution of haemolysin genes, plasmid profile, serogroup analysis and cellular urease activity for Vibrio parahaemolyticus isolates from infected human patients and oysters from the Pacific north-western United States between 1988 and 1997. METHODS AND RESULTS: All of the clinical and environmental isolates tested in this study exhibited the presence of the thermolabile haemolysin gene, tl, confirming that all of the isolates were V. parahaemolyticus. Furthermore, the V. parahaemolyticus isolates that contained either the thermostable direct haemolysin gene, tdh, or the thermostable direct haemolysin-related gene, trh, or both, were also positive for urease. Isolates from infected human patients belong to serogroups O1 and O4, whereas, the isolates from oysters belong to serogroups O1, O4 and O5. These results suggest that the presence of a V. parahaemolyticus serogroup O1 and O4 could indicate the presence of a virulent strain of this pathogen. In this study, the presence of the haemolysin genes, serogroup profiles and urease production in V. parahaemolyticus isolated from human patients correlated with the oysters collected during the outbreaks. However, no significant correlation of the plasmid profiles was detected, based on their distribution and molecular weights, between V. parahaemolyticus isolated from infected human patients and from oysters collected during this outbreak. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF STUDY: It is apparent from this study that the identification of the haemolysin genes by multiplex PCR amplification, in conjunction with serogroup analysis and urease production, can be used to monitor shellfish for the presence of potentially pathogenic strains of V. parahaemolyticus.

Immunization of BALB/c mice against Helicobacter felis infection with Helicobacter pylori urease.

BACKGROUND/AIMS: Because Helicobacter pylori is a potentially dangerous human pathogen, the protective potential of oral immunization with H. pylori urease and its subunits was evaluated in an animal model. METHODS: Mice were orally immunized with H. pylori sonicate, urease, or recombinant enzymatically inactive urease subunits and then challenged with Helicobacter felis. Control mice were sham-immunized. RESULTS: H. felis colonization was present 5 days after challenge in 9 of 10 sham-immunized, 6 of 9 sonicate-immunized, and 3 of 10 urease-immunized animals (P = 0.031 vs. sham-immunized). Twelve days after challenge, urease B-immunized mice had a weaker colonization than sham-immunized controls, whereas urease A had no effect. After 70 days, most urease A- and urease B-immunized mice had cleared the colonization (10/17: P = 0.0019; 16/20: P = 0.00002 vs. sham-immunized). In urease B-immunized animals, protection was often associated with corpus gastritis. CONCLUSIONS: Oral immunization with H. pylori urease protects mice against H. felis infection. Enzymatically inactive urease A and B subunits contain protective epitopes. It is unclear whether protection depends on the development of a mononuclear inflammatory response in the gastric corpus. Our observations should encourage the development of a human vaccine.

Cloning, sequencing, and expression of thermophilic Bacillus sp. strain TB-90 urease gene complex in Escherichia coli.

The urease of thermophilic Bacillus sp. strain TB-90 is composed of three subunits with molecular masses of 61, 12, and 11 kDa. By using synthetic oligonucleotide probes based on N-terminal amino acid sequences of each subunit, we cloned a 3.2-kb EcoRI fragment of TB-90 genomic DNA. Moreover, we cloned two other DNA fragments by gene walking starting from this fragment. Finally, we reconstructed in vitro a 6.2-kb DNA fragment which expressed catalytically active urease in Escherichia coli by combining these three DNA fragments. Nucleotide sequencing analysis revealed that the urease gene complex consists of nine genes, which were designed ureA, ureB, ureC, ureE, ureF, ureG, ureD, ureH, and ureI in order of arrangement. The structural genes ureA, ureB, and ureC encode the 11-, 12-, and 61-kDa subunits, respectively. The deduced amino acid sequences of UreD, UreE, UreF, and UreG, the gene products of four accessory genes, are homologous to those of the corresponding Ure proteins of Klebsiella aerogenes. UreD, UreF, and UreG were essential for expression of urease activity in E. coli and are suggested to play important roles in the maturation step of the urease in a co- and/or posttranslational manner. On the other hand, UreH and UreI exhibited no significant similarity to the known accessory proteins of other bacteria. However, UreH showed 23% amino acid identity to the Alcaligenes eutrophus HoxN protein, a high-affinity nickel transporter.