Genetic characterization and mutational analysis of the role of Streptococcus gordonii adc and cop operons in biofilm formation and detachment

PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact open-help@bu.edu.Thesis (D.Sc.D.)--Boston University, Goldman School of Dental Medicine, 2006 (Pediatric Dentistry).Includes bibliographical references: leaves 149-165.Pioneer oral bacteria, including Streptococcus gordonii, initiate the formation of oral biofilms on tooth surfaces, which requires differential expression of genes that recognize unique environmental cues. Three S. gordonii::TN917-lac biofilm-defective mutants were isolated using an in vitro biofilm formation assay. Identified the transposon insertion site for each biofilm-defective mutant. The first one encodes a protein homologous to a Streptococcus pneumoniae repressor, AdcR. The S. gordonii adc operon, consists of four genes adcR, adcC, adcB and adcA. Expression of adcR, measured by the [Beta]-galactosidase activity of the adcR::Tn917-lac mutant, was growth phase dependent and increased when the mutant was grown in media with high levels of manganese ([greater than] 1 mM) and to a lesser extent zinc, indicating that AdcR may be a regulator at high levels of extracellular manganese. A non-polar inactivation of adeR resulted in a biofilm- and competence-defective phenotype. The biofilm-defective phenotype observed suggests AdcR is an active repressor when synthesized and acts at distant site/s on the chromosome. Thus the adc person is involved in manganese acquisition in S. gordonii and manganese homeostasis and appears to modulate sessile growth in this bacterium. The second biofilm-defective mutant contains an insertion in a gene that encodes a protein homologous to a Streptococcus mutans FruK. Three genes, fruR, fruK and frul, encoding polypeptides that are part of the fluctose-phosphotransferase system (PTS) in S. gordonii and are homoIogous to the fruRKI operon of S. mutans, which plays a role in fructose transport. Expression of fruK, measured by the [Beta]-galactosidase activity of the fruK::Tn917-lac mutant, was observed to be growth phase dependent and was enhanced when the mutant was grown in media with high levels of fructose, sucrose, xylitol and human serum, indicating that the fructose PTS operon was fructose- and xylitol-inducible, similar to the S. mutans fructose PTS system. Non-polar inactivation of the fruR gene in the fruK:: Tn917-lac mutant resulted in a greater increase in [square]-galactosidase activity when grown in media supplemented with fructose, confirming that fruR is a transcriptional repressor of the fructose PTS operon, and this repressor activity may be an important regulator of biofilm formation in S. gordonii. This results suggest that regulation of fructose transport and metabolism in S. gordonii is intricately tied to carbon catabolite control and its ability to form bilfilms. The last biogfilm-defective mutant contains an insertion in a gene encodes a homolog of NosX of Ralstonia eutropha, a putative maturation factor of nitrous oxide reductase. Located downstream are two genes, qor1 and qor2, predicted to encode two putative NADPH quinone oxidoreductases. These three are cotranscribed, forming a putative oxidative stress response (osr)operon in S. gordonii. Inactivation of nosX, qor1, or qor2 resulted in biofilm- defective phenotypes. NosX expression was increased in biofilm cells compared to planktonic cells. These genes may be part of an island that deals with oxidoreductive response, some of which may be important in S. gordonii biofilm formation. Located downstream of adc operon in S. gordonii, three genes are homologous to the copYAZ genes in S. mutans. The copY gene shared homology with a negative transcriptional regulator of a copper-transport operon, copA is a copper-transporting P-type ATPase and copZ is a putative metallochapalone. Each of the copYAZ gene was inactivated using a spectinomycin antibiotic cassette by PCR ligation mutagenesis. All S. gordonii copYAZ deletion mutants are non-polar and were biofilm-positive as determined by biofilm assay and by phase-contrast microscopy of biofilm formation on glass coverslips. Similar to S. mutans, mutations in copY and copA and copZ of S. gordonii resulted in increased resistance to higher levels of extracellular copper when compared to the parent strain, indicating that the cop operon in S. gordonii is involved in copper homeostasis. However unlike other transition elements, copper does not play a role in biofilm formation in S. gordonii