Kanamycin Resistance Determinant Research Articles

Novel plasmid conferring kanamycin and tetracycline resistance in the turkey-derived Campylobacter jejuni strain 11601MD

In Campylobacter spp., resistance to the antimicrobials kanamycin and tetracycline is frequently associated with plasmid-borne genes. However, relatively few plasmids of Campylobacter jejuni have been fully characterized to date. A novel plasmid (p11601MD; 44,095nt) harboring tet(O) was identified in C. jejuni strain 11601MD, which was isolated from the jejunum of a turkey produced conventionally in North Carolina. Analysis of the p11601MD sequence revealed the presence of a high-GC content cassette with four genes that included tet(O) and a putative aminoglycoside transferase gene (aphA-3) highly similar to kanamycin resistance determinants. Several genes putatively involved in conjugative transfer were also identified on the plasmid. These findings will contribute to a better understanding of the distribution of potentially self-mobilizing plasmids harboring antibiotic resistance determinants in Campylobacter spp. from turkeys and other sources.

MiniUIB, a Novel Minitransposon-Based System for Stable Insertion of Foreign DNA into the Genomes of Gram-Negative and Gram-Positive Bacteria

Transposition of the insertion sequence (IS) ISPpu12 is actively induced after conjugative interaction. The transposase of this IS can act in trans on structures flanked by inverted repeats similar to those of the transposon. Based on that fact, an ISPpu12-based minitransposon, miniUIB, has been constructed in order to biotechnologically exploit the self-regulation of ISPpu12 and its increased activity after conjugative interaction. Mobilization of the miniUIB structure into the genome of Pseudomonas stutzeri AN10 after conjugative interaction was demonstrated. A single gene, i.e., the kanamycin resistance determinant, or large genetic structures of >12 kb, i.e., alkBFGHJKL and alkST operons of Pseudomonas putida TF4-1L (GPo1), have been easily integrated in P. stutzeri AN10 by an RP4-based delivery system. Therefore, the integration of the alk determinants by use of the miniUIB system has extended the biodegradation capabilities of this strain. Plasmid pJOC100, containing the transposase and regulator genes of ISPpu12 adjacent to the miniUIB structure, was constructed in order to extend the host range of this biotechnologically useful genetic tool to other model and real-world bacteria. The effectiveness of the system for random mutagenesis in a phylogenetic wide range of bacteria and for the insertion of novel functions has been demonstrated, even in successive steps.

Revisiting the Nucleotide and Aminoglycoside Substrate Specificity of the Bifunctional Aminoglycoside Acetyltransferase(6′)-Ie/Aminoglycoside Phosphotransferase(2″)-Ia Enzyme

The bifunctional aminoglycoside-modifying enzyme aminoglycoside acetyltransferase(6')-Ie/aminoglycoside phosphotransferase(2″)-Ia, or AAC(6')-Ie/APH(2″)-Ia, is the major source of aminoglycoside resistance in gram-positive bacterial pathogens. In previous studies, using ATP as the cosubstrate, it was reported that the APH(2″)-Ia domain of this enzyme is unique among aminoglycoside phosphotransferases, having the ability to inactivate an unusually broad spectrum of aminoglycosides, including 4,6- and 4,5-disubstituted and atypical. We recently demonstrated that GTP, and not ATP, is the preferred cosubstrate of this enzyme. We now show, using competition assays between ATP and GTP, that GTP is the exclusive phosphate donor at intracellular nucleotide levels. In light of these findings, we reevaluated the substrate profile of the phosphotransferase domain of this clinically important enzyme. Steady-state kinetic characterization using the phosphate donor GTP demonstrates that AAC(6')-Ie/APH(2″)-Ia phosphorylates 4,6-disubstituted aminoglycosides with high efficiency (k(cat)/K(m) = 10(5)-10(7) M(-1) s(-1)). Despite this proficiency, no resistance is conferred to some of these antibiotics by the enzyme in vivo. We now show that phosphorylation of 4,5-disubstituted and atypical aminoglycosides are negligible and thus these antibiotics are not substrates. Instead, these aminoglycosides tend to stimulate an intrinsic GTPase activity of the enzyme. Taken together, our data show that the bifunctional enzyme efficiently phosphorylates only 4,6-disubstituted antibiotics; however, phosphorylation does not necessarily result in bacterial resistance. Hence, the APH(2″)-Ia domain of the bifunctional AAC(6')-Ie/APH(2″)-Ia enzyme is a bona fide GTP-dependent kinase with a narrow substrate profile, including only 4,6-disubstituted aminoglycosides.

Target Site Recognition by a Diversity-Generating Retroelement

Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacterial, phage, and plasmid genomes. They function to introduce vast amounts of targeted diversity into protein-encoding DNA sequences via mutagenic homing. Adenine residues are converted to random nucleotides in a retrotransposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using the Bordetella bacteriophage BPP-1 element as a prototype, we have characterized requirements for DGR target site function. Although sequences upstream of VR are dispensable, a 24 bp sequence immediately downstream of VR, which contains short inverted repeats, is required for efficient retrohoming. The inverted repeats form a hairpin or cruciform structure and mutational analysis demonstrated that, while the structure of the stem is important, its sequence can vary. In contrast, the loop has a sequence-dependent function. Structure-specific nuclease digestion confirmed the existence of a DNA hairpin/cruciform, and marker coconversion assays demonstrated that it influences the efficiency, but not the site of cDNA integration. Comparisons with other phage DGRs suggested that similar structures are a conserved feature of target sequences. Using a kanamycin resistance determinant as a reporter, we found that transplantation of the IMH and hairpin/cruciform-forming region was sufficient to target the DGR diversification machinery to a heterologous gene. In addition to furthering our understanding of DGR retrohoming, our results suggest that DGRs may provide unique tools for directed protein evolution via in vivo DNA diversification.

Contribution of AhyR to virulence of Aeromonas hydrophila J-1

Aeromonas hydrophila is a broad-host-range pathogen and its pathogenesis is multifactorial. A regulatory mechanism known as quorum sensing has been found to be involved in the regulation of virulence in many bacteria. In A. hydrophila the ahyR gene encodes LuxR-type response regulator. Here we describe the inactivation of the ahyR gene of A. hydrophila J-1 by the insertion of a DNA fragment containing a kanamycin resistance determinant and reintroduced by allelic exchange into the chromosome of A. hydrophila J-1 by means of the suicide plasmid pJP5603. Cytotoxic effects on EPC cells assay and LD 50 determinations in fish demonstrated that the ahyR mutant was highly attenuated relative to the wild-type strain. Compared with the parent strain, some characteristics, such as biochemical characters and outer membrane protein profiles, had changed. Some main virulent determinants could not be detected, including proteases, amylase, Dnase, hemolysin and S layer. This article confirmed the important function of AhyR in the pathogenesis of A. hydrophila J-1.

Genetic tools for highly pathogenic Francisella tularensis subsp. tularensis

This paper is the first detailed description of the development and use of new genetic tools specifically for the safe manipulation of highly pathogenic Francisella tularensis subsp. tularensis. Most of these tools are also demonstrated to work with other F. tularensis subspecies. Kanamycin and hygromycin resistance determinants that function as genetic markers in F. tularensis subsp. tularensis strain Schu and sets of episomal shuttle vectors that are either unstable or stably maintained in the absence of selection were developed. In addition, the hyg gene, expressed from the F. tularensis groESL promoter, was successfully used as a marker for transposon mutagenesis. This work also includes the development of sacB-based suicide plasmids expressing kanamycin resistance that can be used for electroporation-mediated allelic exchange of unmarked mutations in Schu and the F. tularensis live vaccine strain (LVS). Using these plasmids, the two predicted beta-lactamase genes, blaA and blaB, in Schu and LVS were deleted. Only the Delta blaB1 mutants had increased susceptibility to ampicillin, and this phenotype was complemented by a plasmid expressing blaB+. The results suggest that the beta-lactam antibiotic resistance phenotype of Schu and LVS is likely due to only one of the two beta-lactamase genes present and that ampicillin resistance can be used as an additional selectable marker in beta-lactamase deletion mutants. The collection of tools presented in this report will be helpful for the genetic analyses of F. tularensis subsp. tularensis pathogenesis.

Analysis of the Salmonella typhimurium Proteome through Environmental Response toward Infectious Conditions

Salmonella enterica serovar Typhimurium (also known as Salmonella typhimurium) is a facultative intracellular pathogen that causes approximately 8,000 reported cases of acute gastroenteritis and diarrhea each year in the United States. Although many successful physiological, biochemical, and genetic approaches have been taken to determine the key virulence determinants encoded by this organism, the sheer number of uncharacterized reading frames observed within the S. enterica genome suggests that many more virulence factors remain to be discovered. We used a liquid chromatography-mass spectrometry-based "bottom-up" proteomic approach to generate a more complete picture of the gene products that S. typhimurium synthesizes under typical laboratory conditions as well as in culture media that are known to induce expression of virulence genes. When grown to logarithmic phase in rich medium, S. typhimurium is known to express many genes that are required for invasion of epithelial cells. Conversely stationary phase cultures of S. typhimurium express genes that are needed for both systemic infection and growth within infected macrophages. Lastly bacteria grown in an acidic, magnesium-depleted minimal medium (MgM) designed to mimic the phagocytic vacuole have been shown to up-regulate virulence gene expression. Initial comparisons of protein abundances from bacteria grown under each of these conditions indicated that the majority of proteins do not change significantly. However, we observed subsets of proteins whose expression was largely restricted to one of the three culture conditions. For example, cells grown in MgM had a higher abundance of Mg(2+) transport proteins than found in other growth conditions. A second more virulent S. typhimurium strain (14028) was also cultured under these same growth conditions, and the results were directly compared with those obtained for strain LT2. This comparison offered a unique opportunity to contrast protein populations in these closely related bacteria. Among a number of proteins displaying a higher abundance in strain 14028 were the products of the pdu operon, which encodes enzymes required for propanediol utilization. These pdu operon proteins were validated in culture and during macrophage infection. Our work provides further support for earlier observations that suggest pdu gene expression contributes to S. typhimurium pathogenesis.

Chloramphenicol and kanamycin resistance among porcine Escherichia coli in Ontario

The purpose of this study was to compare the distribution of chloramphenicol and kanamycin resistance genes across three populations of porcine Escherichia coli. PCR was used to assess the distribution of the major chloramphenicol and kanamycin resistance genes catA1, cmlA and floR, and aphA1, aphA2 and aadB in enterotoxigenic E. coli (ETEC), non-ETEC isolates from cases of diarrhoea and commensal E. coli from healthy pigs. Associations between these genes and resistance genes for other antimicrobials or virulence genes were assessed. The chloramphenicol and kanamycin resistance genes were distributed differently among the three E. coli populations. While aphA1, aphA2 and aadB were evenly distributed among resistant ETEC, non-ETEC and commensals, the catA1 gene was significantly more frequent in ETEC than in non-ETEC and commensals. Transformation experiments confirmed statistical associations by demonstrating that elt, estB, astA, aadA and sul1 were located with catA1 on a large ETEC plasmid. Plasmids carrying cmlA also carried sul3 and aadA. Other plasmids carrying floR and aadB also carried tet(A), sul2, strA/strB, bla(CMY-2) and occasionally aac(3)IV. The clustering of genes observed is a likely cause for chloramphenicol resistance persistence. Similar to tetracycline, chloramphenicol resistance genes are physically linked to virulence genes. This is not the case for kanamycin resistance determinants, which were linked to other resistance genes only.

ATP Hydrolysis Is Essential for the Function of the Uup ATP-binding Cassette ATPase in Precise Excision of Transposons

In Escherichia coli K-12, the RecA- and transposase-independent precise excision of transposons is thought to be mediated by the slippage of the DNA polymerase between the two short direct repeats that flank the transposon. Inactivation of the uup gene, encoding an ATP-binding cassette (ABC) ATPase, led to an important increase in the frequency of precise excision of transposons Tn10 and Tn5 and a defective growth of bacteriophage Mu. To provide insight into the mechanism of Uup in transposon excision, we purified this protein, and we demonstrated that it is a cytosolic ABC protein. Purified recombinant Uup binds and hydrolyzes ATP and undergoes a large conformational change in the presence of this nucleotide. This change affects a carboxyl-terminal domain of the protein that displays predicted structural homology with the socalled little finger domain of Y family DNA polymerases. In these enzymes, this domain is involved in DNA binding and in the processivity of replication. We show that Uup binds to DNA and that this binding is in part dependent on its carboxyl-terminal domain. Analysis of Walker motif B mutants suggests that ATP hydrolysis at the two ABC domains is strictly coordinated and is essential for the function of Uup in vivo.

Potential use of a Yersinia ruckeri O1 auxotrophic aroA mutant as a live attenuated vaccine

The aroA gene of Yersinia ruckeri, which encodes 5-enolpyruvylshikimate 3-phosphate synthase, was insertionally inactivated with a DNA fragment containing a kanamycin resistance determinant and reintroduced by allelic exchange into the chromosome of Y. ruckeri 21102 O1 by means of the suicide vector pIVET8. The Y. ruckeri aroA::Kan(r) mutant was highly attenuated when inoculated intraperitoneally into rainbow trout, with a 50% lethal dose of >5 x 10(7) CFU. The mutants were not recoverable from the internal organs 48 h post-inoculation or later. The vaccination of rainbow trout with the AroA mutant as a live vaccine conferred significant protection (relative percentage survival = 90%) against the pathogenic wild-type strain of Y. ruckeri.

Analysis of the Escherichia coli Alp Phenotype: Heat Shock Induction in ssrA Mutants

The major phenotypes of lon mutations, UV sensitivity and overproduction of capsule, are due to the stabilization of two substrates, SulA and RcsA. Inactivation of transfer mRNA (tmRNA) (encoded by ssrA), coupled with a multicopy kanamycin resistance determinant, suppressed both lon phenotypes and restored the rapid degradation of SulA. This novel protease activity was named Alp but was never identified further. We report here the identification, mapping, and characterization of a chromosomal mutation, faa (for function affecting Alp), that leads to full suppression of a Deltalon ssrA::cat host and thus bypasses the requirement for multicopy Kan(r); faa and ssrA mutants are additive in their ability to suppress lon mutants. The faa mutation was mapped to the C terminus of dnaJ(G232); dnaJ null mutants have similar effects. The identification of a lon suppressor in dnaJ suggested the possible involvement of heat shock. We find that ssrA mutants alone significantly induce the heat shock response. The suppression of UV sensitivity, both in the original Alp strain and in faa mutants, is reversed by mutations in clpY, encoding a subunit of the heat shock-induced ClpYQ protease that is known to degrade SulA. However, capsule synthesis is not restored by clpY mutants, probably because less RcsA accumulates in the Alp strain and because the RcsA that does accumulate is inactive. Both ssrA effects are partially relieved by ssrA derivatives encoding protease-resistant tags, implicating ribosome stalling as the primary defect. Thus, ssrA and faa each suppress two lon mutant phenotypes but by somewhat different mechanisms, with heat shock induction playing a major role.

Characterization of plasmid-mediated aphA-3 kanamycin resistance in Campylobacter jejuni.

A total of 254 isolates of Campylobacter jejuni and three isolates of Campylobacter coli, isolated from Sweden, Canada, and Egypt, were screened for kanamycin resistance. Eight strains of C. jejuni contained large plasmids that carried the aphA-3 kanamycin-resistance marker. In six plasmids, the aphA-3 gene was located downstream of an apparent insertion sequence, designated IS607*, which showed a considerable similarity to IS607, characterized on the chromosome of some Helicobacter pylori strains. In contrast, the other plasmids carried the aphA-3 gene as a part of a resistance cluster. This included three resistance markers encoding 6'-adenylyltransferase (aadE), streptothricin acetyltransferase (sat), and 3'-aminoglycoside phosphotransferase type III (aphA-3). The genetic organization of this resistance cluster suggests that it has been acquired by C. jejuni from a Gram-positive organism. The IS607* element was also observed in kanamycin-susceptible strains of C. jejuni on plasmids mediating tetracycline resistance. The kanamycin-resistance phenotype transferred along with tetracycline resistance by conjugation from four representative C. jejuni strains to a recipient strain of C. jejuni. The kanamycin-resistance determinant (aphA-3) was stably transferred from one of the four C. jejuni strains to a recipient strain of Escherichia coli. However, the C. jejuni plasmid, which also carries the tetO gene, was not maintained in E. coli. Pulsed-field gel electrophoresis revealed the integration of approximately 50 kb of the plasmid into the chromosome of the E. coli recipient.

Class 1 integron containing a new gene cassette, aadA10, associated with Tn1404 from R151.

The carbenicillin, gentamicin, kanamycin, streptomycin, spectinomycin, sulfonamide, and tobramycin resistance determinants found on Pseudomonas aeruginosa plasmid R151 have previously been shown to translocate to another plasmid, R388, and it was inferred that a transposon, Tn1404, carried the resistance determinants. Sequencing of the cassette array from the plasmid known as R388::Tn1404 revealed two known gene cassettes, oxa10 and aadB, and a previously unidentified cassette determining resistance to streptomycin and spectinomycin, here designated aadA10, in the order oxa10-aadB-aadA10. These cassettes replaced the dfrB2-orfA cassette array of R388, indicating that movement of the resistance determinants from R151 to R388 resulted from recombinational exchange between two class 1 integrons rather than transposition. The AadA10 protein is most closely related to AadA6 (85% identical) and AadA7 (80% identical). The aadA10 cassette found here has only a simple site containing a 7-bp spacer derived from attI1 in place of a 59-base element and is likely to represent a derivative of the complete cassette. IntI1-mediated deletion of the aadA10 cassette was not detected, indicating that this single simple site is either inactive or only weakly active.

Sudden Increase in Pseudomonas aeruginosa Nosocomial Strains with Broad Host Range Transfer of Antibiotic Resistance

We investigated transfer of antibiotic resistance from 51 multiply resistant strains of Pseudomonas aeruginosa isolated from seriously ill patients in the Frankfurt University Clinics. Nine isolates directly transferred resistance to three recipient strains used. Ticarcillin and cephalothin resistance determinants were accepted from eight isolates, and in one case a kanamycin resistance determinant was transferred. The total spectrum of resistance transferred demonstrated that several donor strains transferred a different set of resistance determinants to all three recipient strains. Two P. aeruginosa isolates transferred spectrum of seven resistance determinants including ceftazidime, cefepime and aztreonam, three isolates transferred five determinants and four isolates transferred four resistance determinants. The fact that identical spectra of multiple drug resistance were transferred to recipient strains belonging to three different species (Escherichia coli, Proteus mirabilis and P. aeruginosa), indicates a broad host range in all three transferable genetic elements not observed in previous transfers from P. aeruginosa strains.

PUB2380: Characterization of a ColD-like Resistance Plasmid

A detailed analysis of the mobilizable, ColE1-like resistance plasmid, pUB2380, is reported. The 8.5-kb genome encodes six (possibly seven) major functions: (1) a ColD-like origin of replication, oriV, with associated replication functions, RNAI and RNAII; (2) a set of active mobilization functions highly homologous to that of ColE1, including the origin of transfer, oriT; (3) a ColE1-like multimer resolution site (cer); (4) a kanamycin-resistance determinant, aph, encoding an aminoglycoside-3′-phosphotransferase type 1; (5) an insertion sequence, IS1294; and (6) two genes, probably cotranscribed, of unknown function(s). The GC content of the various parts of the genome indicates that the plasmid is a hybrid structure assembled from DNA from at least three different sources, of which the replication region, the mobilization functions, and the resistance gene are likely to have originated in the enterobacteriaceae.

Genetic characterization of antimicrobial resistance in Canadian isolates of Salmonella serovar Typhimurium DT104.

PCR was used to identify antibiotic resistance determinants in 31 Canadian Salmonella serovar Typhimurium DT104 isolates. Genes encoding resistance to ampicillin (pse1 or blaP1), chloramphenicol (pasppflo-like), streptomycin-spectinomycin (aadA2), sulfonamide (sulI), and tetracycline [tet(G)] were mapped to a 13-kb region of DNA of one isolate. Two copies of sulI were identified and mapped to the 3' end of either pse1 or aadA2 integrons. The two integrons were separated by the pasppflo-like gene and the tet(G) gene. The kanamycin resistance determinant (aphA-1) was present on a 2.0-MDa plasmid (five isolates) or on the chromosome (three isolates).

A single-step transconjugation system for the introduction of unmarked deletions into Actinobacillus pleuropneumoniae serotype 7 using a sucrose sensitivity marker

Research on the porcine respiratory tract pathogen Actinobacillus pleuropneumoniae requires the availability of improved genetic tools. Therefore, using the sacB gene of Bacillus subtilis, we developed a sucrose-based counterselection system that allows rapid curing of an Escherichia coli-A. pleuropneumoniae shuttle vector as well as the introduction of unmarked mutations into the A. pleuropneumoniae chromosome. A cassette containing the Tn 903 kanamycin resistance determinant (km r) and the sacB gene expressed from the A. pleuropneumoniae omlA promoter was introduced by homologous recombination into the ureC gene of A. pleuropneumoniae. The resultant stable plasmid cointegrates were kanamycin-resistant, sucrose-sensitive, and urease-positive. A simple counterselection on sucrose-containing agar plates without an additional transconjugation step allowed the efficient isolation of urease-negative A. pleuropneumoniae mutants that had lost the km r- sacB cassette.

A single-step transconjugation system for the introduction of unmarked deletions intoActinobacillus pleuropneumoniaeserotype 7 using a sucrose sensitivity marker

Research on the porcine respiratory tract pathogen Actinobacillus pleuropneumoniae requires the availability of improved genetic tools. Therefore, using the sacB gene of Bacillus subtilis, we developed a sucrose-based counterselection system that allows rapid curing of an Escherichia coli-A. pleuropneumoniae shuttle vector as well as the introduction of unmarked mutations into the A. pleuropneumoniae chromosome. A cassette containing the Tn903 kanamycin resistance determinant (km(r)) and the sacB gene expressed from the A. pleuropneumoniae omlA promoter was introduced by homologous recombination into the ureC gene of A. pleuropneumoniae. The resultant stable plasmid cointegrates were kanamycin-resistant, sucrose-sensitive, and urease-positive. A simple counterselection on sucrose-containing agar plates without an additional transconjugation step allowed the efficient isolation of urease-negative A. pleuropneumoniae mutants that had lost the km(r)-sacB cassette.

Growth properties of afolAnull mutant ofEscherichia coliK12

In Escherichia coli, dihydrofolate reductase is required for both the de novo synthesis of tetrahydrofolate and the recycling of dihydrofolate produced during the synthesis of thymidylate. The coding region of the dihydrofolate reductase gene, folA, was replaced with a kanamycin resistance determinant. Unlike earlier deletions, this mutation did not disrupt flanking genes. When the mutation was transferred into a wild-type strain and a thymidine- (thy) requiring strain, the resulting strains were viable but slow growing on rich medium. Both synthesized less folate than their parents, as judged by the incorporation of radioactive para-aminobenzoic acid. The derivative of the wild-type strain did not grow on any defined minimal media tested. In contrast, the derivative of the thy-requiring strain grew slowly on minimal medium with thy but exhibited auxotrophies on some combinations of supplements. These results suggest that when folates are limited, they can be distributed appropriately to folate-dependent biosynthetic reactions only under some conditions. Key words: dihydrofolate reductase, Escherichia coli, biosynthesis, folates, one-carbon metabolism.

Genetic and physiologic characterization of urease of Actinomyces naeslundii.

Ammonia production from urea by ureolytic oral bacteria is believed to have a significant impact on oral health and the ecological balance of oral microbial populations. In this study we cloned and characterized the urease gene cluster of Actinomyces naeslundii, which is one of the pioneer organisms in the oral cavity and a significant constituent of supragingival and subgingival dental plaque in children and adults. An internal fragment of the ureC gene of A. naeslundii WVU45 was initially amplified by PCR with degenerate primers derived from conserved amino acid sequences of the large catalytic subunit of urease in bacteria and plants. The PCR product was then used as a probe to identify recombinant bacteriophages carrying the A. naeslundii urease gene cluster and roughly 30 kbp of flanking DNA. Nucleotide sequence analysis demonstrated that the gene cluster was comprised of seven contiguously arranged open reading frames with significant homologies at the protein and nucleotide sequence levels to the ureABCEFGD genes from other organisms. By using primer extension, a putative transcription initiation site was mapped at 66 bases 5' to the start codon of ureA. A urease-deficient strain was constructed by insertion of a kanamycin resistance determinant within the ureC gene via allelic replacement. In contrast to the wild-type organism, the isogenic mutant was unable to grow in a semidefined medium supplemented with urea as the nitrogen source and was not protected by the addition of urea against killing in moderately acidic environments. These data indicated that urea can be effectively utilized as a nitrogen source by A. naeslundii via a urease-dependent pathway and that ureolysis can protect A. naeslundii against environmental acidification at physiologically relevant pH values. Therefore, urease could confer to A. naeslundii critical selective advantages over nonureolytic organisms in dental plaque, constituting an important determinant of plaque ecology.