Genome of the opportunistic pathogen Streptococcus sanguinis

The genome of Streptococcus sanguinis is a circular DNA molecule consisting of 2,388,435 bp and is 177 to 590 kb larger than the other 21 streptococcal genomes that have been sequenced. The G+C content of the S. sanguinis genome is 43.4%, which is considerably higher than the G+C contents of other streptococci. The genome encodes 2,274 predicted proteins, 61 tRNAs, and four rRNA operons. A 70-kb region encoding pathways for vitamin B(12) biosynthesis and degradation of ethanolamine and propanediol was apparently acquired by horizontal gene transfer. The gene complement suggests new hypotheses for the pathogenesis and virulence of S. sanguinis and differs from the gene complements of other pathogenic and nonpathogenic streptococci. In particular, S. sanguinis possesses a remarkable abundance of putative surface proteins, which may permit it to be a primary colonizer of the oral cavity and agent of streptococcal endocarditis and infection in neutropenic patients.     

Characterization of Hydrogen Peroxide-Induced DNA Release by Streptococcus sanguinis and Streptococcus gordonii

Extracellular DNA (eDNA) is produced by several bacterial species and appears to contribute to biofilm development and cell-cell adhesion. We present data showing that the oral commensals Streptococcus sanguinis and Streptococcus gordonii release DNA in a process induced by pyruvate oxidase-dependent production of hydrogen peroxide (H₂O₂). Surprisingly, S. sanguinis and S. gordonii cell integrity appears unaffected by conditions that cause autolysis in other eDNA-producing bacteria. Exogenous H₂O₂ causes release of DNA from S. sanguinis and S. gordonii but does not result in obvious lysis of cells. Under DNA-releasing conditions, cell walls appear functionally intact and ribosomes are retained over time. During DNA release, intracellular RNA and ATP are not coreleased. Hence, the release mechanism appears to be highly specific for DNA. Release of DNA without detectable autolysis is suggested to be an adaptation to the competitive oral biofilm environment, where autolysis could create open spaces for competitors to invade. Since eDNA promotes cell-to-cell adhesion, release appears to support oral biofilm formation and facilitates exchange of genetic material among competent strains.The release of bacterial DNA into the environment is of recent interest since this polymer is now recognized to stabilize cell-to-cell adherence and biofilm architecture (1, 35, 37). Treatment of extracellular DNA (eDNA) with DNase results in reduced intercellular stickiness, consistent with an adhesive function for eDNA. Furthermore, eDNA from Neisseria meningitis appears to have sufficient structural integrity to transform competent strains (11), indicating chromosomal origin. Since the abundance of eDNA is influenced by growth conditions, DNA release can also be regulated (40).DNA release is typically a consequence of cell lysis. Linked to DNA release, genetic transformation is the natural ability of competent bacterial species to take up DNA from the environment (13, 34, 42). During competence development, Streptococcus pneumoniae DNA is released by lysis of a subpopulation of cells (30, 42). Cell lysis and DNA release are controlled in a cell density-dependent signal transduction process. The S. pneumoniae comX regulon, carrying late competence genes, also includes the murein hydrolase genes lytA and cbpD (19, 42). Murein hydrolases digest structural components of the peptidoglycan, contributing to remodeling, recycling, and daughter cell separation. Furthermore, murein hydrolases trigger autolytic cell wall digestion, leading to release of DNA and other cellular content into the environment (36). The autolysis of bacterial cells as part of a regulated death program seems to be an important source for eDNA in diverse species, including Staphylococcus aureus (4, 36, 37), Staphylococcus epidermidis (35), Enterococcus faecalis (44), and Pseudomonas aeruginosa (1). In these species, the eDNA contributes to biofilm formation as a component of the extracellular biofilm matrix (35, 37, 44).Unlike for cell lysis-dependent release, the oral streptococci appear to induce eDNA release by a novel mechanism. In dual-species cultures, the oral commensals Streptococcus sanguinis and Streptococcus gordonii release eDNA in a manner dependent on pyruvate oxidase (Pox) generation of hydrogen peroxide (H₂O₂) under the control of ambient oxygen (23). In this report, we now provide direct evidence of selective H₂O₂-induced eDNA release by these oral commensal streptococci.     

Streptococcal antagonism in oral biofilms: Streptococcus sanguinis and Streptococcus gordonii interference with Streptococcus mutans

Biofilms are polymicrobial, with diverse bacterial species competing for limited space and nutrients. Under healthy conditions, the different species in biofilms maintain an ecological balance. This balance can be disturbed by environmental factors and interspecies interactions. These perturbations can enable dominant growth of certain species, leading to disease. To model clinically relevant interspecies antagonism, we studied three well-characterized and closely related oral species, Streptococcus gordonii, Streptococcus sanguinis, and cariogenic Streptococcus mutans. S. sanguinis and S. gordonii used oxygen availability and the differential production of hydrogen peroxide (H(2)O(2)) to compete effectively against S. mutans. Interspecies antagonism was influenced by glucose with reduced production of H(2)O(2). Furthermore, aerobic conditions stimulated the competence system and the expression of the bacteriocin mutacin IV of S. mutans, as well as the H(2)O(2)-dependent release of heterologous DNA from mixed cultures of S. sanguinis and S. gordonii. These data provide new insights into ecological factors that determine the outcome of competition between pioneer colonizing oral streptococci and the survival mechanisms of S. mutans in the oral biofilm.     

Structure and evolution of a novel dimeric enzyme from a clinically important bacterial pathogen

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of the lysine biosynthetic pathway. The tetrameric structure of DHDPS is thought to be essential for enzymatic activity, as isolated dimeric mutants of Escherichia coli DHDPS possess less than 2.5% that of the activity of the wild-type tetramer. It has recently been proposed that the dimeric form lacks activity due to increased dynamics. Tetramerization, by buttressing two dimers together, reduces dynamics in the dimeric unit and explains why all active bacterial DHDPS enzymes to date have been shown to be homo-tetrameric. However, in this study we demonstrate for the first time that DHDPS from methicillin-resistant Staphylococcus aureus (MRSA) exists in a monomer-dimer equilibrium in solution. Fluorescence-detected analytical ultracentrifugation was employed to show that the dimerization dissociation constant of MRSA-DHDPS is 33 nm in the absence of substrates and 29 nm in the presence of (S)-aspartate semialdehyde (ASA), but is 20-fold tighter in the presence of the substrate pyruvate (1.6 nm). The MRSA-DHDPS dimer exhibits a ping-pong kinetic mechanism (k(cat)=70+/-2 s(-1), K(m)(Pyruvate)=0.11+/-0.01 mm, and K(m)(ASA)=0.22+/-0.02 mm) and shows ASA substrate inhibition with a K(si)(ASA) of 2.7+/-0.9 mm. We also demonstrate that unlike the E. coli tetramer, the MRSA-DHDPS dimer is insensitive to lysine inhibition. The near atomic resolution (1.45 A) crystal structure confirms the dimeric quaternary structure and reveals that the dimerization interface of the MRSA enzyme is more extensive in buried surface area and noncovalent contacts than the equivalent interface in tetrameric DHDPS enzymes from other bacterial species. These data provide a detailed mechanistic insight into DHDPS catalysis and the evolution of quaternary structure of this important bacterial enzyme.     

Oxanine DNA glycosylase activity from Mammalian alkyladenine glycosylase

Oxanine (Oxa) is a deaminated base lesion derived from guanine in which the N(1)-nitrogen is substituted by oxygen. This work reports the mutagenicity of oxanine as well as oxanine DNA glycosylase (ODG) activities in mammalian systems. Using human DNA polymerase beta, deoxyoxanosine triphosphate is only incorporated opposite cytosine (Cyt). When an oxanine base is in a DNA template, Cyt is efficiently incorporated opposite the template oxanine; however, adenine and thymine are also incorporated opposite Oxa with an efficiency approximately 80% of a Cyt/Oxa (C/O) base pair. Guanine is incorporated opposite Oxa with the least efficiency, 16% compared with cytosine. ODG activity was detected in several mammalian cell extracts. Among the known human DNA glycosylases tested, human alkyladenine glycosylase (AAG) shows ODG activity, whereas hOGG1, hNEIL1, or hNEIL2 did not. ODG activity was detected in spleen cell extracts of wild type age-matched mice, but little activity was observed in that of Aag knock-out mice, confirming that the ODG activity is intrinsic to AAG. Human AAG can excise Oxa from all four Oxa-containing double-stranded base pairs, Cyt/Oxa, Thy/Oxa, Ade/Oxa, and Gua/Oxa, with no preference to base pairing. Surprisingly, AAG can remove Oxa from single-stranded Oxa-containing DNA as well. Indeed, AAG can also remove 1,N(6)-ethenoadenine from single-stranded DNA. This study extends the deaminated base glycosylase activities of AAG to oxanine; thus, AAG is a mammalian enzyme that can act on all three purine deamination bases, hypoxanthine, xanthine, and oxanine.     

A novel uracil-DNA glycosylase family related to the helix-hairpin-helix DNA glycosylase superfamily

Cytosine bases can be deaminated spontaneously to uracil, causing DNA damage. Uracil-DNA glycosylase (UDG), a ubiquitous uracil-excising enzyme found in bacteria and eukaryotes, is one of the enzymes that repair this kind of DNA damage. To date, no UDG-coding gene has been identified in Methanococcus jannaschii, although its entire genome was deciphered. Here, we have identified and characterized a novel UDG from M.jannaschii designated as MjUDG. It efficiently removed uracil from both single- and double-stranded DNA. MjUDG also catalyzes the excision of 8-oxoguanine from DNA. MjUDG has a helix–hairpin–helix motif and a [4Fe–4S]-binding cluster that is considered to be important for the DNA binding and catalytic activity. Although MjUDG shares these features with other structural families such as endonuclease III and mismatch-specific DNA glycosylase (MIG), unique conserved amino acids and substrate specificity distinguish MjUDG from other families. Also, a homologous member of MjUDG was identified in Aquifex aeolicus. We report that MjUDG belongs to a novel UDG family that has not been described to date.     

Structure of formamidopyrimidine-DNA glycosylase covalently complexed to DNA

Formamidopyrimidine-DNA glycosylase (Fpg) is a DNA repair enzyme that excises oxidized purines from damaged DNA. The Schiff base intermediate formed during this reaction between Escherichia coli Fpg and DNA was trapped by reduction with sodium borohydride, and the structure of the resulting covalently cross-linked complex was determined at a 2.1-A resolution. Fpg is a bilobal protein with a wide, positively charged DNA-binding groove. It possesses a conserved zinc finger and a helix-two turn-helix motif that participate in DNA binding. The absolutely conserved residues Lys-56, His-70, Asn-168, and Arg-258 form hydrogen bonds to the phosphodiester backbone of DNA, which is sharply kinked at the lesion site. Residues Met-73, Arg-109, and Phe-110 are inserted into the DNA helix, filling the void created by nucleotide eversion. A deep hydrophobic pocket in the active site is positioned to accommodate an everted base. Structural analysis of the Fpg-DNA complex reveals essential features of damage recognition and the catalytic mechanism of Fpg.     

ATP-dependent protein kinase activities in the oral pathogen Streptococcus mutans

ATP-dependent protein kinase activities were detected in both membrane and cytoplasmic fractions from the oral pathogen Streptococcus mutans. Different polypeptides were phosphorylated by endogenous kinase(s) in the two fractions. In membranes, five phosphoproteins were detected with apparent masses of 82, 37, 22, 12, and 10 kilodaltons (KD). In cytoplasm, two major acid-stable phosphoproteins were found. One was identified as HPr of the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS), while the other had an apparent mass of 61 KD. Both of these proteins were phosphorylated on a seryl residue. Fructose 1,6-bisphosphate stimulated phosphorylation of HPr by the kinase and inhibited phosphorylation of the 61-KD protein. In contrast, fructose 1-phosphate, 2-phosphoglycerate, 3-phosphoglycerate, and dihydroxyacetone phosphate inhibited phosphorylation of HPr and stimulated phosphorylation of the 61-KD protein. Several other glycolytic intermediates as well as inorganic phosphate inhibited phosphorylation of either or both proteins. Preincubation of cytoplasm with PEP prior to incubation with ATP reduced the amount of phospho-(seryl)-HPr formed, but not that of the 61-KD phosphoprotein. The latter protein has not yet been identified but has properties that suggest that it may be the protein kinase itself. These results provide evidence for one or more soluble ATP-dependent protein kinases in S mutans that are regulated by glycolytic intermediates and that may play a role in the modulation of carbohydrate uptake and metabolism in this organism. A model for feedback regulation of sugar transport in S mutans, mediated by an allosterically regulated kinase, is presented.     

Synthesis and molecular modeling of novel HSV1 uracil-DNA glycosylase inhibitors.

In a recent paper the first selective inhibitors of HSV1 uracil-DNA glycosylase (UDG) acting in the micromolar range have been reported. A 28.5 kDa catalytic fragment of HSV1 UDG has been crystallized in the presence of uracil, and the structure was recently solved. Starting with the optimized model of binding between 6-(4'-n-octylanilino)uracil (octAU) and UDG some new derivatives have been predicted to be active. In vitro studies with the novel synthetized compounds confirm the plausibility of the model and define the structure features for UDG inhibitors.     

Role of Streptococcus sanguinis sortase A in bacterial colonization

Streptococcus sanguinis, a normal inhabitant of the human oral cavity, has low cariogenicity, though colonization on tooth surfaces by this bacterium initiates aggregation by other oral bacteria and maturation of dental plaque. Additionally, S. sanguinis is frequently isolated from infective endocarditis patients. We investigated the functions of sortase A (SrtA), which cleaves LPXTG-containing proteins and anchors them to the bacterial cell wall, as a possible virulence factor of S. sanguinis. We identified the srtA gene of S. sanguinis by searching a homologous gene of Streptococcus mutans in genome databases. Next, we constructed an srtA-deficient mutant strain of S. sanguinis by insertional inactivation and compared it to the wild type strain. In the case of the mutant strain, some surface proteins could not anchor to the cell wall and were partially released into the culture supernatant. Furthermore, adherence to saliva-coated hydroxyapatite beads and polystyrene plates, as well as adherence to and invasion of human epithelial cells were reduced significantly in the srtA-deficient strain when compared to the wild type. In addition, antiopsonization levels and bacterial survival of the srtA-deficient mutant were decreased in human whole blood. This is the first known study to report that SrtA contributes to antiopsonization in streptococci. Our results suggest that SrtA anchors surface adhesins as well as some proteins that function as antiopsonic molecules as a means of evading the human immune system. Furthermore, they demonstrate that SrtA of S. sanguinis plays important roles in bacterial colonization.     

Human DNA glycosylase enzyme TDG repairs thymine mispaired with exocyclic etheno-DNA adducts

Lipid peroxidation directly reacts with DNA and produces various exocyclic etheno-base DNA adducts, some of which are considered to contribute to carcinogenesis. However, the system for repairing them in humans is largely unknown. We hypothesized that etheno-DNA adducts are repaired by base excision repair initiated by DNA glycosylase. To test this hypothesis, we examined the activities of the DNA glycosylase proteins OGG1, SMUG1, TDG, NEIL1, MUTYH, NTH1, MPG, and UNG2 against double-stranded oligonucleotides containing 1,N⁶-ethenoadenine (εA), 3,N⁴-ethenocytosine (εC), butanone-ethenocytosine (BεC), butanone-ethenoguanine (BεG), heptanone-ethenocytosine (HεC), or heptanone-ethenoguanine (HεG) using a DNA cleavage assay. We found that TDG is capable of removing thymine that has mispaired with εC, BεC, BεG, HεC, or HεG in vitro. We next examined the effect of TDG against etheno-DNA adducts in human cells. TDG-knockdown cells exhibited the following characteristics: (a) higher resistance to cell death caused by the induction of etheno-DNA adducts; (b) lower repair activity for εC; and (c) a modest acceleration of mutations caused by εC, compared with the rate in control cells. All these characteristics suggest that TDG exerts a repair activity against etheno-DNA adducts in human cells. These results suggest that TDG has novel repair activities toward etheno-DNA adducts.     

Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase

The hydrolytic deamination of cytosine and 5-methylcytosine drives many of the transition mutations observed in human cancer. The deamination-induced mutagenic intermediates include either uracil or thymine adducts mispaired with guanine. While a substantial array of methods exist to measure other types of DNA adducts, the cytosine deamination adducts pose unusual analytical problems, and adequate methods to measure them have not yet been developed. We describe here a novel hybrid thymine DNA glycosylase (TDG) that is comprised of a 29-amino acid sequence from human TDG linked to the catalytic domain of a thymine glycosylase found in an archaeal thermophilic bacterium. Using defined-sequence oligonucleotides, we show that hybrid TDG has robust mispair-selective activity against deaminated U:G and T:G mispairs. We have further developed a method for separating glycosylase-released free bases from oligonucleotides and DNA followed by GC–MS/MS quantification. Using this approach, we have measured for the first time the levels of total uracil, U:G, and T:G pairs in calf thymus DNA. The method presented here will allow the measurement of the formation, persistence, and repair of a biologically important class of deaminated cytosine adducts.     

The novel DNA glycosylase,NEIL1, protects mammalian cells from radiation-mediated cell death

DNA damage mediated by reactive oxygen species generates miscoding and blocking lesions that may lead to mutations or cell death. Base excision repair (BER) constitutes a universal mechanism for removing oxidatively damaged bases and restoring the integrity of genomic DNA. In Escherichia coli, the DNA glycosylases Nei, Fpg, and Nth initiate BER of oxidative lesions; OGG1 and NTH1 proteins fulfill a similar function in mammalian cells. Three human genes, designated NEIL1, NEIL2 and NEIL3, encode proteins that contain sequence homologies to Nei and Fpg. We have cloned the corresponding mouse genes and have overexpressed and purified mNeil1, a DNA glycosylase that efficiently removes a wide spectrum of mutagenic and cytotoxic DNA lesions. These lesions include the two cis-thymineglycol(Tg) stereoisomers, guanine- and adenine-derived formamidopyrimidines, and 5,6-dihydrouracil. Two of these lesions, fapyA and 5S,6R thymine glycol, are not excised by mOgg1 or mNth1. We have also used RNA interference technology to establish embryonic stem cell lines deficient in Neil1 protein and showed them to be sensitive to low levels of gamma-irradiation. The results of these studies suggest that Neil1 is an essential component of base excision repair in mammalian cells; its presence may contribute to the redundant repair capacity observed in Ogg1 -/- and Nth1 -/- mice.     

Purification and characterization of a novel UV lesion-specific DNA glycosylase/AP lyase from Bacillus sphaericus

The purification and characterization of a pyrimidine dimer-specific glycosylase/AP lyase from Bacillus sphaericus (Bsp-pdg) are reported. Bsp-pdg is highly specific for DNA containing the cis-syn cyclobutane pyrimidine dimer, displaying no detectable activity on oligonucleotides with trans-syn I, trans-syn II, (6-4), or Dewar photoproducts. Like other glycosylase/AP lyases that sequentially cleave the N--glycosyl bond of the 5' pyrimidine of a cyclobutane pyrimidine dimer, and the phosphodiester backbone, this enzyme appears to utilize a primary amine as the attacking nucleophile. The formation of a covalent enzyme-DNA imino intermediate is evidenced by the ability to trap this protein-DNA complex by reduction with sodium borohydride. Also consistent with its AP lyase activity, Bsp-pdg was shown to incise an AP site-containing oligonucleotide, yielding beta- and delta-elimination products. N-terminal amino acid sequence analysis of this 26 kDa protein revealed little amino acid homology to any previously reported protein. This is the first report of a glycosylase/AP lyase enzyme from Bacillus sphaericus that is specific for cis-syn pyrimidine dimers.     

Involvement of NADH Oxidase in Biofilm Formation in Streptococcus sanguinis

Biofilms play important roles in microbial communities and are related to infectious diseases. Here, we report direct evidence that a bacterial nox gene encoding NADH oxidase is involved in biofilm formation. A dramatic reduction in biofilm formation was observed in a Streptococcus sanguinis nox mutant under anaerobic conditions without any decrease in growth. The membrane fluidity of the mutant bacterial cells was found to be decreased and the fatty acid composition altered, with increased palmitic acid and decreased stearic acid and vaccenic acid. Extracellular DNA of the mutant was reduced in abundance and bacterial competence was suppressed. Gene expression analysis in the mutant identified two genes with altered expression, gtfP and Idh, which were found to be related to biofilm formation through examination of their deletion mutants. NADH oxidase-related metabolic pathways were analyzed, further clarifying the function of this enzyme in biofilm formation.     

Structure modeling and inhibitor prediction ofNADP oxidoreductase enzyme from Methanobrevibacter smithii

The F420-dependent NADP oxidoreductase enzyme from Methanobrevibacter smithii catalyzes the important electron transfer step during methanogenesis. Therefore, it may act as potential target for blocking the process of methane formation. Its protein sequence is available in GenBank (accession number: ABQ86254.1) however no report has been found about its 3D protein structure. In this work, we first time claim 3D model structure of F420-dependent NADP oxidoreductase enzyme from Methanobrevibacter smithii by comparative homology modeling method. Swiss model and ESyPred3d (via Modeller 6v2) software's were generated the 3D model by detecting 1JAX (A) as template along with sequence identities of 34.272% and 35.40%. Furthermore, PROCHECK with Ramachandran plot and ProSA analysis revealed that swiss model produced better model than Modeller6v2 with 98.90% of residues in favored and additional allowed regions (RM plot) as well as with ProSA Z score of -7.26. In addition, we investigated that the substrate F420 bound at the cavity of the model. Subsequently, inhibitor prediction study revealed that Lovastatin (-22.07 Kcal/mol) and Compactin (Mevastatin) (-21.91 Kcal/mol) produced more affinity for model structure of NADP oxidoreducatse as compared to F420 (-14.40 Kcal/mol). It indicates that the Lovastatin and Compactin (Mevastatin) compounds (Negative regulator) may act as potential inhibitor of F420 dependent NADP oxidoreducatse protein.     

极端嗜热古菌尿嘧啶DNA糖苷酶的研究进展

高温会加快碱基脱氨基反应形成损伤碱基的速率,进一步对脱氨基的碱基进行复制会导致突变。因此,极端嗜热古菌基因组的稳定性面临着其生存高温环境的挑战。胞嘧啶脱氨基形成尿嘧啶,是常见的脱碱基类型,复制DNA中尿嘧啶会造成GC→AT的突变。尿嘧啶DNA糖苷酶(Uracil DNA glycosylase,UDG)是修复DNA中尿嘧啶的关键酶。基于识别底物的特异性,UDG分为6个家族,广泛分布在细菌、古菌、真核生物以及一些病毒中。基因组序列显示,极端嗜热古菌至少编码一种UDG。目前,对于细菌和真核生物的UDG已进行了大量的研究,但是关于极端嗜热古菌UDG的研究相对较少,尚处于初期阶段。本文综述了极端嗜热古菌UDG的研究进展,并对今后的研究提出了展望。     

Thermostable uracil-DNA glycosylase from Thermotoga maritima a member of a novel class of DNA repair enzymes.

Uracil-DNA glycosylase (UDG) is a ubiquitous enzyme found in eukaryotes and prokaryotes [1][2][3]. This enzyme removes uracil bases that are present in DNA as a result of either deamination of cytosine or misincorporation of dUMP instead of dTMP [4] [5], and it is the primary activity in the DNA base excision repair pathway. Although UDG activities have been shown to be present in several thermophiles [6][7][8], no sequences have been found that are complementary to the Escherichia coli ung gene, which encodes UDG [9]. Here, we describe a UDG from the thermophile Thermotoga maritima. The T. maritima UDG gene has a low level of homology to the E. coli G-T/U mismatch-specific DNA glycosylase gene (mug). The expressed protein is capable of removing uracil from DNA containing either a U-A or a U-G base pair and is heat-stable up to 75 degrees C. The enzyme is also active on single-stranded DNA containing uracil. Analogous genes appear to be present in several prokaryotic organisms, including thermophilic and mesophilic eubacteria as well as archaebacteria, the human-disease pathogens Treponema palladium and Rickettsia prowazekii, and the extremely radioresistant organism Deinococcus radiodurans. These findings suggest that the T. maritima UDG is a member of a new class of DNA repair enzymes.