scholarly journals Identification and Characterization of the Nickel Uptake System for Urease Biogenesis in Streptococcus salivarius 57.I

2003 ◽  
Vol 185 (23) ◽  
pp. 6773-6779 ◽  
Author(s):  
Yi-Ywan M. Chen ◽  
Robert A. Burne
Keyword(s):  
Urease Activity ◽  
Streptococcus Thermophilus ◽  
Uptake System ◽  
Streptococcus Salivarius ◽  
Reading Frame ◽  
Related Organism ◽  
Nickel Uptake ◽  
Insertional Inactivation ◽  
Cobalamin Biosynthesis

ABSTRACT Ureases are multisubunit enzymes requiring Ni2+ for activity. The low pH-inducible urease gene cluster in Streptococcus salivarius 57.I is organized as an operon, beginning with ureI, followed by ureABC (structural genes), and ureEFGD (accessory genes). Urease biogenesis also requires a high-affinity Ni2+ uptake system. By searching the partial genome sequence of a closely related organism, Streptococcus thermophilus LMG18311, three open reading frame (ORFs) homologous to those encoding proteins involved in cobalamin biosynthesis and cobalt transport (cbiMQO) were identified immediately 3′ to the ure operon. To determine whether these genes were involved in urease biogenesis by catalyzing Ni2+ uptake in S. salivarius, regions 3′ to ureD were amplified by PCRs from S. salivarius by using primers identical to the S. thermophilus sequences. Sequence analysis of the products revealed three ORFs. Reverse transcriptase PCR was used to demonstrate that the ORFs are transcribed as part of the ure operon. Insertional inactivation of ORF1 with a polar kanamycin marker completely abolished urease activity and the ability to accumulate 63Ni2+ during growth. Supplementation of the growth medium with NiCl2 at concentrations as low as 2.5 μM partially restored urease activity in the mutant. Both wild-type and mutant strains showed enhanced urease activity when exogenous Ni2+ was provided at neutral pH. Enhancement of urease activity by adding nickel was regulated at the posttranslational level. Thus, ORF1, ORF2, and ORF3 are part of the ure operon, and these genes, designated ureM, ureQ, and ureO, respectively, likely encode a Ni2+-specific ATP-binding cassette transporter.

scholarly journals Characterization of Recombinant, Ureolytic Streptococcus mutans Demonstrates an Inverse Relationship between Dental Plaque Ureolytic Capacity and Cariogenicity

2000 ◽  
Vol 68 (5) ◽  
pp. 2621-2629 ◽  
Author(s):  
K. Anne Clancy ◽  
Sylvia Pearson ◽  
William H. Bowen ◽  
Robert A. Burne
Keyword(s):  
Dental Caries ◽  
Streptococcus Mutans ◽  
Urease Activity ◽  
Dental Health ◽  
Oral Bacteria ◽  
Streptococcus Salivarius ◽  
Specific Pathogen ◽  
The Relationship

ABSTRACT Dental caries results from prolonged plaque acidification that leads to the establishment of a cariogenic microflora and demineralization of the tooth. Urease enzymes of oral bacteria hydrolyze urea to ammonia, which can neutralize plaque acids. To begin to examine the relationship between plaque ureolytic activity and the incidence of dental caries, recombinant, ureolytic strains ofStreptococcus mutans were constructed. Specifically, theureABCEFGD operon from Streptococcus salivarius57.I was integrated into the S. mutans chromosome in such a way that the operon was transcribed from a weak, cognate promoter inS. mutans ACUS4 or a stronger promoter in S. mutans ACUS6. Both strains expressed NiCl2-dependent urease activity, but the maximal urease levels in ACUS6 were threefold higher than those in ACUS4. In vitro pH drop experiments demonstrated that the ability of the recombinant S. mutans strains to moderate a decrease in pH during the simultaneous metabolism of glucose and urea increased proportionately with the level of urease activity expressed. Specific-pathogen-free rats that were infected with ACUS6 and fed a cariogenic diet with drinking water containing 25 mM urea and 50 μM NiCl2 had relatively high levels of oral urease activity, as well as dramatic decreases in the prevalence of smooth-surface caries and the severity of sulcal caries, relative to controls. Urease activity appears to influence plaque biochemistry and metabolism in a manner that reduces cariogenicity, suggesting that recombinant, ureolytic bacteria may be useful to promote dental health.

scholarly journals Galactose and Lactose Genes from the Galactose-Positive Bacterium Streptococcus salivarius and the Phylogenetically Related Galactose-Negative Bacterium Streptococcus thermophilus: Organization, Sequence, Transcription, and Activity of the gal Gene Products

2002 ◽  
Vol 184 (3) ◽  
pp. 785-793 ◽  
Author(s):  
Katy Vaillancourt ◽  
Sylvain Moineau ◽  
Michel Frenette ◽  
Christian Lessard ◽  
Christian Vadeboncoeur
Keyword(s):  
Gene Cluster ◽  
Streptococcus Thermophilus ◽  
Gene Clusters ◽  
Northern Blotting ◽  
Gene Products ◽  
Streptococcus Salivarius ◽  
Negative Bacterium ◽  
Positive Bacterium ◽  
Identical Nucleotide

ABSTRACT Streptococcus salivarius is a lactose- and galactose-positive bacterium that is phylogenetically closely related to Streptococcus thermophilus, a bacterium that metabolizes lactose but not galactose. In this paper, we report a comparative characterization of the S. salivarius and S. thermophilus gal-lac gene clusters. The clusters have the same organization with the order galR (codes for a transcriptional regulator and is transcribed in the opposite direction), galK (galactokinase), galT (galactose-1-P uridylyltransferase), galE (UDP-glucose 4-epimerase), galM (galactose mutarotase), lacS (lactose transporter), and lacZ (β-galactosidase). An analysis of the nucleotide sequence as well as Northern blotting and primer extension experiments revealed the presence of four promoters located upstream from galR, the gal operon, galM, and the lac operon of S. salivarius. Putative promoters with virtually identical nucleotide sequences were found at the same positions in the S. thermophilus gal-lac gene cluster. An additional putative internal promoter at the 3′ end of galT was found in S. thermophilus but not in S. salivarius. The results clearly indicated that the gal-lac gene cluster was efficiently transcribed in both species. The Shine-Dalgarno sequences of galT and galE were identical in both species, whereas the ribosome binding site of S. thermophilus galK differed from that of S. salivarius by two nucleotides, suggesting that the S. thermophilus galK gene might be poorly translated. This was confirmed by measurements of enzyme activities.

Cloning and characterization of the genes required for inorganic carbon transport of Synechocystis PCC6803

1991 ◽  
Vol 69 (5) ◽  
pp. 951-956 ◽  
Author(s):  
Teruo Ogawa
Keyword(s):  
Nadh Dehydrogenase ◽  
Inorganic Carbon ◽  
Wild Type ◽  
Reading Frame ◽  
Synechocystis Pcc6803 ◽  
Dna Libraries ◽  
Insertional Inactivation ◽  
Inorganic Carbon Transport ◽  
Carbon Transport

Transformation of the high CO2-requiring mutants of Synechocystis PCC6803 defective in inorganic carbon (Ci) transport (RKa and RKb) by wild type (WT) DNA libraries restored their ability to grow under air levels of CO2. Two clones (PK-1 and HP-1), which complement RKa and RKb, respectively, were isolated from the libraries. PK-1 contained an 11.8-kilobase pair (kbp) DNA insert. The sequence of amino acid coded in the DNA in the region of the mutation showed an extensive homology to that of the ndh2 gene product of liverwort chloroplasts, which is suspected to be the subunit 2 of NADH dehydrogenase. Based on the result, we designated the gene mutated in RKa as ndh2. Inactivation of the ndh2 gene in the WT cells by inserting an aminoglycoside-3′-phosphotransferase gene generated a mutant (M57) that was unable to grow under low CO2 conditions. HP-1 contained a 5.4-kbp DNA insert. Sequencing of nucleotides in the region of the mutation revealed an open reading frame that codes a hydrophobic protein that consists of 80 amino acids. Insertional inactivation of this putative Ci transport gene, designated ictA, generated a high CO2-requiring mutant (M9). All these mutants (RKa, RKb, M9, and M57) showed very low activity of CO2 uptake into the intracellular Ci pools. The activity of HCO3− uptake was negligibly low in RKb, M9, M57 and high CO2-grown cells of RKa, and was about 10% the activity of wild type cells in low CO2-adapted cells of RKa. Key words: CO2-concentrating mechanism, inorganic carbon transport, Synechocystis PCC6803, mutant, NADH dehydrogenase, insertional inactivation.

scholarly journals Phosphorylation of Streptococcus salivarius Lactose Permease (LacS) by HPr(His∼P) and HPr(Ser-P)(His∼P) and Effects on Growth

2003 ◽  
Vol 185 (23) ◽  
pp. 6764-6772 ◽  
Author(s):  
Christian Lessard ◽  
Armelle Cochu ◽  
Jean-Dominique Lemay ◽  
Denis Roy ◽  
Katy Vaillancourt ◽  
...  
Keyword(s):  
Streptococcus Thermophilus ◽  
Lactose Permease ◽  
Streptococcus Salivarius ◽  
Phosphorylated Form ◽  
Generation Times ◽  
Related Organism ◽  
Oral Bacterium ◽  
Mutant Strains ◽  
Enzyme I ◽  
Phosphotransferase Transport System

ABSTRACT The oral bacterium Streptococcus salivarius takes up lactose via a transporter called LacS that shares 95% identity with the LacS from Streptococcus thermophilus, a phylogenetically closely related organism. S. thermophilus releases galactose into the medium during growth on lactose. Expulsion of galactose is mediated via LacS and stimulated by phosphorylation of the transporter by HPr(His∼P), a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). Unlike S. thermophilus, S. salivarius grew on lactose without expelling galactose and took up galactose and lactose concomitantly when it is grown in a medium containing both sugars. Analysis of the C-terminal end of S. salivarius LacS revealed a IIA-like domain (IIALacS) almost identical to the IIA domain of S. thermophilus LacS. Experiments performed with purified proteins showed that S. salivarius IIALacS was reversibly phosphorylated on a histidine residue at position 552 not only by HPr(His∼P) but also by HPr(Ser-P)(His∼P), a doubly phosphorylated form of HPr present in large amounts in rapidly growing S. salivarius cells. Two other major S. salivarius PTS proteins, IIABL Man and IIABH Man, were unable to phosphorylate IIALacS. The effect of LacS phosphorylation on growth was studied with strain G71, an S. salivarius enzyme I-negative mutant that cannot synthesize HPr(His∼P) or HPr(Ser-P)(His∼P). These results indicated that (i) the wild-type and mutant strains had identical generation times on lactose, (ii) neither strain expelled galactose during growth on lactose, (iii) both strains metabolized lactose and galactose concomitantly when grown in a medium containing both sugars, and (iv) the growth of the mutant was slightly reduced on galactose.

scholarly journals Characterization of the Binding Protein-Dependent Cellobiose and Cellotriose Transport System of the Cellulose Degrader Streptomyces reticuli

1999 ◽  
Vol 65 (6) ◽  
pp. 2636-2643 ◽  
Author(s):  
Andreas Schlösser ◽  
Jens Jantos ◽  
Karl Hackmann ◽  
Hildgund Schrempf
Keyword(s):  
Abc Transporter ◽  
Binding Protein ◽  
Regulatory Protein ◽  
Transport Systems ◽  
Uptake System ◽  
Gene Products ◽  
Abc Transport ◽  
Insertional Inactivation ◽  
Atp Binding Protein

ABSTRACT Streptomyces reticuli has an inducible ATP-dependent uptake system specific for cellobiose and cellotriose. By reversed genetics a gene cluster encoding components of a binding protein-dependent cellobiose and cellotriose ABC transporter was cloned and sequenced. The deduced gene products comprise a regulatory protein (CebR), a cellobiose binding lipoprotein (CebE), two integral membrane proteins (CebF and CebG), and the NH2-terminal part of an intracellular β-glucosidase (BglC). The gene for the ATP binding protein MsiK is not linked to the ceb operon. We have shown earlier that MsiK is part of two different ABC transport systems, one for maltose and one for cellobiose and cellotriose, in S. reticuli and Streptomyces lividans. Transcription of polycistronic cebEFG and bglC mRNAs is induced by cellobiose, whereas the cebR gene is transcribed independently. Immunological experiments showed that CebE is synthesized during growth with cellobiose and that MsiK is produced in the presence of several sugars at high or moderate levels. The described ABC transporter is the first one of its kind and is the only specific cellobiose/cellotriose uptake system of S. reticuli, since insertional inactivation of the cebEgene prevents high-affinity uptake of cellobiose.

scholarly journals Characterization of a Novel Methyl-Accepting Chemotaxis Gene,dmcB, from the Oral Spirochete Treponema denticola

1999 ◽  
Vol 67 (2) ◽  
pp. 694-699 ◽  
Author(s):  
Hong Li ◽  
Shinichi Arakawa ◽  
Qing-Dong Deng ◽  
Howard Kuramitsu
Keyword(s):  
Northern Blot Analysis ◽  
Nucleotide Sequencing ◽  
Treponema Denticola ◽  
Reading Frame ◽  
Nutrient Supplements ◽  
Gene Coding ◽  
Insertional Inactivation ◽  
Significant Homology ◽  
Chemotaxis Proteins

ABSTRACT Immediately downstream from the previously isolated Treponema denticola ATCC 35405 prtB gene coding for a chymotrypsinlike protease activity, an open reading frame, ORF3, was identified which shared significant homology with the highly conserved domains (HCDs) of bacterial methyl-accepting chemotaxis proteins (MCPs). Nucleotide sequencing of this ORF revealed that the gene would code for a protein with a size of approximately 41 kDa. In addition, this sequence contained a domain which was virtually identical to the HCD of a recently characterized MCP, DmcA, of strain 35405. Therefore, this ORF was named dmcB. Northern blot analysis suggested that dmcB was part of an operon structure containing prtB. Insertional inactivation ofdmcB utilizing an ermF-ermAM cassette resulted in a mutant with decreased chemoattraction toward nutrient supplements. In addition, the mutant displayed an altered pattern of methylated proteins under conditions of chemotaxis. Inactivation of thedmcB gene also attenuated the methylation of the DmcA protein. These results suggest that the dmcB gene codes for an MCP in T. denticola which may interact with other MCPs in these organisms.

scholarly journals Antigenic Characterization of ORF2 and ORF3 Proteins of Hepatitis E Virus (HEV)

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1385
Author(s):  
Giulia Pezzoni ◽  
Lidia Stercoli ◽  
Eleonora Pegoiani ◽  
Emiliana Brocchi
Keyword(s):  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Recombinant Antigen ◽  
Open Reading Frame ◽  
Reading Frame ◽  
E Coli ◽  
Antigenic Characterization ◽  
Antigenic Properties ◽  
Open Reading Frame 2

To evaluate the antigenic properties of Hepatitis E Virus (HEV) Open Reading Frame 2 and 3 (ORF2 and ORF3) codified proteins, we expressed different portions of ORF2 and the entire ORF3 in E. coli, a truncated ORF2, was also expressed in baculovirus. A panel of 37 monoclonal antibodies (MAbs) was raised against ORF2 (1–660 amino acids) and MAbs were mapped and characterized using the ORF2 expressed portions. Selected HEV positive and negative swine sera were used to evaluate ORF2 and ORF3 antigens’ immunogenicity. The MAbs were clustered in six groups identifying six antigenic regions along the ORF2. Only MAbs binding to the sixth ORF2 antigenic region (394–608 aa) were found to compete with HEV positive sera and efficiently catch the recombinant antigen expressed in baculovirus. The ORF2 portion from 394–608 aa demonstrated to include most immunogenic epitopes with 85% of HEV positive swine sera reacting against the region from 461–544 aa. Only 5% of the selected HEV sera reacted against the ORF3 antigen.

scholarly journals Molecular cloning and analysis of l(1)ogre, a locus of Drosophila melanogaster with prominent effects on the postembryonic development of the central nervous system.

Genetics ◽  
1990 ◽  
Vol 126 (4) ◽  
pp. 1033-1044 ◽  
Author(s):  
T Watanabe ◽  
D R Kankel
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Postembryonic Development ◽  
P Element ◽  
Reading Frame ◽  
Isolation And Characterization ◽  
The Central Nervous System ◽  
Highly Hydrophobic ◽  
Conceptual Translation

Abstract Previous genetic studies have shown that wild-type function of the l(1)ogre (lethal (1) optic ganglion reduced) locus is essential for the generation and/or maintenance of the postembryonic neuroblasts including those from which the optic lobe is descended. In the present study molecular isolation and characterization of the l(1)ogre locus was carried out to study the structure and expression of this gene in order to gain information about the nature of l(1)ogre function and its relevance to the development of the central nervous system. About 70 kilobases (kb) of genomic DNA were isolated that spanned the region where l(1)ogre was known to reside. Southern analysis of a l(1)ogre mutation and subsequent P element-mediated DNA transformation mapped the l(1)ogre+ function within a genomic fragment of 12.5 kb. Northern analyses showed that a 2.9-kb message transcribed from this 12.5-kb region represented l(1)ogre. A 2.15-kb portion of a corresponding cDNA clone was sequenced. An open reading frame (ORF) of 1,086 base paris was found, and a protein sequence of 362 amino acids with one highly hydrophobic segment was deduced from conceptual translation of this ORF.

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