Single-Cell Genomics and the Oral Microbiome.

The human oral cavity is one of the first environments where microbes have been discovered and studied since the dawn of microbiology. Nevertheless, approximately 200 types of bacteria from the oral microbiota have remained uncultured in the laboratory. Some are associated with a healthy oral microbial community, while others are linked to oral diseases, from dental caries to gum disease. Single-cell genomics has enabled inferences on the physiology, virulence, and evolution of such uncultured microorganisms and has further enabled isolation and cultivation of several novel oral bacteria, including the discovery of novel interspecies interactions. This review summarizes some of the more recent advances in this field, which is rapidly moving toward physiologic characterization of single cells and ultimately cultivation of the yet uncultured. A combination of traditional microbiological approaches with genomic-based physiologic predictions and isolation strategies may lead to the oral microbiome being the first complex microbial community to have all its members cultivable in the laboratory. Studying the biology of the individual microbes when in association with other members of the community, in controlled laboratory conditions and in vivo, should lead to a better understanding of oral dysbiosis and its prevention and reversion.

Inter-field variability in the microbial communities of hydrothermal vent deposits from a back-arc basin.

Diverse microbial communities thrive on and in deep-sea hydrothermal vent mineral deposits. However, our understanding of the inter-field variability in these communities is poor, as limited sampling and sequencing efforts have hampered most previous studies. To explore the inter-field variability in these communities, we used barcoded pyrosequencing of the variable region 4 (V4) of the 16S rRNA gene to characterize the archaeal and bacterial communities of over 30 hydrothermal deposit samples from six vent fields located along the Eastern Lau Spreading Center. Overall, the bacterial and archaeal communities of the Eastern Lau Spreading Center are similar to other active vent deposits, with a high diversity of Epsilonproteobacteria and thermophilic Archaea. However, the archaeal and bacterial communities from the southernmost vent field, Mariner, were significantly different from the other vent fields. At Mariner, the epsilonproteobacterial genus Nautilia and the archaeal family Thermococcaceae were prevalent in most samples, while Lebetimonas and Thermofilaceae were more abundant at the other vent fields. These differences appear to be influenced in part by the unique geochemistry of the Mariner fluids resulting from active degassing of a subsurface magma chamber. These results show that microbial communities associated with hydrothermal vent deposits in back-arc basins are taxonomically similar to those from mid-ocean ridge systems, but differences in geologic processes between vent fields in a back-arc basin can influence microbial community structure.

Complete and draft genome sequences of six members of the Aquificales.

The Aquificales are widespread in marine and terrestrial hydrothermal environments. Here, we report the complete and draft genome sequences of six new members of the Aquificales: two marine species, Persephonella marina strain EX-H1 and Hydrogenivirga strain 128-5-R1 (from the East Pacific Rise, 9 degrees 50.3'N, 104 degrees 17.5'W, and the Eastern Lau Spreading Center, 176 degrees 11.5'W, 20 degrees 45.8'S, respectively), and four terrestrial isolates, Sulfurihydrogenibium azorense strain Az-Fu1, Sulfurihydrogenibium yellowstonense strain SS-5, and Sulfurihydrogenibium strain Y03AOP1 (from Furnas, Azores, Portugal, and Calcite Springs and Obsidian Pool in Yellowstone National Park, United States, respectively), and the only thermoacidophilic isolate, Hydrogenobaculum strain Y04AAS1 (from a stream adjacent to Obsidian Pool). Significant differences among the different species exist that include nitrogen metabolism, hydrogen utilization, chemotaxis, and signal transduction, providing insights into their ecological niche adaptations.

A molecular phylogenetic framework for the phylum Ctenophora using 18S rRNA genes.

This paper presents the first molecular phylogenetic analysis of the phylum Ctenophora, by use of 18S ribosomal RNA sequences from most of the major taxa. The ctenophores form a distinct monophyletic group that, based on this gene phylogeny, is most closely related to the cnidarians. Our results suggest that the ancestral ctenophore was tentaculate and cydippid-like and that the presently recognized order Cydippida forms a polyphyletic group. The other ctenophore orders that we studied (Lobata, Beroida, and Platyctenida) are secondarily derived from cydippid-like ancestors, a conclusion that is also supported by developmental and morphological data. The very short evolutionary distances between characterized ctenophore 18S rRNA gene sequences suggests that extant ctenophores are derived from a recent common ancestor. This has important consequences for future studies and for an understanding of the evolution of the metazoans.

Photocrosslinking of 4-thio uracil-containing RNAs supports a side-by-side arrangement of domains 5 and 6 of a group II intron.

Previous studies suggested that domains 5 and 6 (D5 and D6) of group II introns act together in splicing and that the two helical structures probably do not interact by helix stacking. Here, we characterized the major Mg2+ ion- and salt-dependent, long-wave UV light-induced, intramolecular crosslinks formed in 4-thiouridine-containing D56 RNA from intron 5gamma (aI5gamma) of the COXI gene of yeast mtDNA. Four major crosslinks were mapped and found to result from covalent bonds between nucleotides separating D5 from D6 [called J(56)] and residues of D6 near and including the branch nucleotide. These findings are extended by results of similar experiments using 4-thioU containing D56 RNAs from a mutant allele of aI5gamma and from the group IIA intron, aI1. Trans-splicing experiments show that the crosslinked wild-type aI5gamma D56 RNAs are active for both splicing reactions, including some first-step branching. An RNA containing the 3-nt J(56) sequence and D6 of aI5gamma yields one main crosslink that is identical to the most minor of the crosslinks obtained with D56 RNA, but in this case in a cation-independent fashion. We conclude that the interaction between J(56) and D6 is influenced by charge repulsion between the D5 and D6 helix backbones and that high concentrations of cations allow the helices to approach closely under self-splicing conditions. The interaction between J(56) and D6 appears to be a significant factor establishing a side-by-side (i.e., not stacked) orientation of the helices of the two domains.

More than one way to splice an RNA: branching without a bulge and splicing without branching in group II introns.

Domain 6 (D6) of group II introns contains a bulged adenosine that serves as the branch-site during self-splicing. In addition to this adenosine, other structural features in D6 are likely to contribute to the efficiency of branching. To understand their role in promoting self-splicing, the branch-site and surrounding nucleotides were mutagenized. Detailed kinetic analysis on the self-splicing efficiency of the mutants revealed several interesting features. First, elimination of the branch-site does not preclude efficient splicing, which takes place instead through a hydrolytic first step. Second, pairing of the branch-site does not eliminate branching, particularly if the adenosine is involved in a mispair. Third, the G-U pairs that often surround group II intron branch-points contribute to the efficiency of branching. These results suggest that there is a strong driving force for promoting self-splicing by group II introns, which employ a versatile set of different mechanisms for ensuring that splicing is successful. In addition, the behavior of these mutants indicates that a bulged adenosine per se is not the important determinant for branch-site recognition in group II introns. Rather, the data suggest that the branch-site adenosine is recognized as a flipped base, a conformation that can be promoted by a variety of different substructures in RNA and DNA.

The two steps of group II intron self-splicing are mechanistically distinguishable.

The two transesterification reactions catalyzed by self-splicing group II introns take place in either two active sites or two conformations of a single active site involving rearrangements of the positions of the reacting groups. We have investigated the effects on the rates of the chemical steps of the two reactions due to sulfur substitution of nonbridging oxygens at both the 5' and 3' splice sites as well as the deoxyribose substitution of the ribose 2' hydroxyl group at the 5' splice site. The data suggest that the two active sites differ in their interactions with several of these groups. Specifically, sulfur substitution of the pro-Sp nonbridging oxygen at the 5' splice site reduces the chemical rate of the step one branching reaction by at least 250-fold, whereas substitution of the pro-Sp oxygen at the 3' splice site has only a 4.5-fold effect on the chemical rate of step two. Previous work demonstrated that the Rp phosphorothioate substitutions at both the 5' and 3' splice sites reduced the rate of both steps of splicing to an undetectable level. These results suggest that either two distinct active sites catalyze the two steps or that more significant alterations must be made in a single bifunctional active site to accommodate the two different reactions.

Domain 5 binds near a highly conserved dinucleotide in the joiner linking domains 2 and 3 of a group II intron.

Photocrosslinking has identified the joiner between domains 2 and 3 [J(23)] as folding near domain 5 (D5), a highly conserved helical substructure of group II introns required for both splicing reactions. D5 RNAs labeled with the photocrosslinker 4-thiouridine (4sU) reacted with highly conserved nucleotides G588 and A589 in J(23) of various intron acceptor transcripts. These conjugates retained some ribozyme function with the lower helix of D5 crosslinked to J(23), so they represent active complexes. One partner of the gamma x gamma' tertiary interaction (A587 x U887) is also in J(23); even though gamma x gamma' is involved in step 2 of the splicing reaction, D5 has not previously been found to approach gamma x gamma'. Similar crosslinking patterns between D5 and J(23) were detected both before and after step 1 of the reaction, indicating that the lower helix of D5 is positioned similarly in both conformations of the active center. Our results suggest that the purine-rich J(23) strand is antiparallel to the D5 strand containing U32 and U33. Possibly, the interaction with J(23) helps position D5 correctly in the ribozyme active site; alternatively, J(23) itself might participate in the catalytic center.

Group II intron splicing in vivo by first-step hydrolysis.

Group I, group II and spliceosomal introns splice by two sequential transesterification reactions. For both spliceosomal and group II introns, the first-step reaction occurs by nucleophilic attack on the 5' splice junction by the 2' hydroxyl of an internal adenosine, forming a 2'-5' phosphodiester branch in the intron. The second reaction joins the two exons with a 3'-5' phosphodiester bond and releases intron lariat. In vitro, group II introns can self-splice by an efficient alternative pathway in which the first-step reaction occurs by hydrolysis. The resulting linear splicing intermediate participates in normal second-step reactions, forming spliced exon and linear intron RNAs. Here we show that the group II intron first-step hydrolysis reaction occurs in vivo in place of transesterification in the mitochondria of yeast strains containing branch-site mutations. As expected, the mutations block branching, but surprisingly still allow accurate splicing. This hydrolysis pathway may have been a step in the evolution of splicing mechanisms.

Mutations of the two-nucleotide bulge of D5 of a group II intron block splicing in vitro and in vivo: phenotypes and suppressor mutations.

Domain 5 (D5) is a highly conserved substructure of group II introns that is essential for catalysis of both steps of the splicing pathway. Here we studied the effects of mutations of the conserved 2-nt bulge in the binding and catalytic functions of D5 of intron aI5gamma of yeast mitochondrial DNA. Sequence variants of the 2-nt bulge reduced the rate of self-splicing somewhat. Deletion of one or both bulge nucleotides inhibited splicing more than 10(4)-fold, whereas mutants with a 3-nt bulge were much less debilitated. A novel allele with a symmetrical internal loop in place of the bulge self-splices nearly as actively as the control. Trans-splicing assays of D5 function showed some mutations primarily affect the catalytic function of D5, whereas others chiefly affect its binding function. Representative alleles were transformed into mtDNA and each inhibited splicing and respiratory growth. Pseudo-revertants included suppressor mutations nearby in D5 and one mutant yielded a dominant nuclear suppressor. These experiments provide new evidence that the D5 bulge is crucial for D5 binding and catalysis. It is possible that the bulge bends the D5 helix and that the extent of bending is especially important for D5 binding. The presence and exact nature of the bulge is also likely to have local structural consequences (besides bending) that influence the participation of specific backbone groups in binding and catalysis.

A UV-induced, Mg(2+)-dependent crosslink traps an active form of domain 3 of a self-splicing group II intron.

In vitro irradiation of a15 gamma group II intron RNA with low doses of 254 nm UV light induces a single major crosslink. This crosslink was mapped within the domain 3 substructure of this RNA and one of the participating nucleotides was identified. When an RNA containing only the domain 3 substructure is irradiated under the same conditions, an intramolecular crosslink forms between two specific pyrimidines, one of them identical to the nucleotide crosslinked in the full-length intron RNA. In both RNAs, the crosslink is magnesium ion-dependent and photoreversible. A trans assay for domain 3 function was developed and used to find that the crosslinked domain 3 RNA remains highly reactive. This suggests that crosslinking has trapped a functional, Mg(2+)-induced folded state of this group II intron substructure and that this folding is probably independent of the other domains of the intron.

Stereochemical selectivity of group II intron splicing, reverse splicing, and hydrolysis reactions.

We have previously shown, using phosphorothioate substitutions at splice site, that both transesterification steps of group II intron self-splicing proceed, by stereochemical inversion, with an Sp but not an Rp phosphorothioate. Under alternative reaction conditions or with various intron fragments, group II introns can splice following hydrolysis at the 5' splice site and can also hydrolyze the bond between spliced exons (the spliced-exon reopening reaction). In this study, we have determined the stereochemical specificities of all of the major model hydrolytic reactions carried out by the aI5 gamma intron from Saccharomyces cerevisiae mitochondria. For all substrates containing exon 1 and most of the intron, the stereospecificity of hydrolysis is the same as for the step 1 transesterification reaction. In contrast, the spliced-exon reopening reaction proceeds with an Rp but not an Sp phosphorothioate at the scissile bond, as does true reverse splicing. Thus, by stereochemistry, this reaction appears to be related to the reverse of step 2 of self-splicing. Finally, a substrate RNA that contains the first exon and nine nucleotides of the intron, when reacted with the intron ribozyme, releases the first exon regardless of the configuration of the phosphorothioate at the 5' splice site, suggesting that this substrate can be cleaved by either the step 1 or the step 2 reaction site. Our findings clarify the relationships of these model reactions to the transesterification reactions of the intact self-splicing system and permit new studies to be interpreted more rigorously.

The stereochemical course of group II intron self-splicing.

The stereochemical specificities and reaction courses for both self-splicing steps of a group II intron have been determined by phosphorothioate substitution at the 5' and 3' splice site phosphodiester bonds. Both steps of the splicing reaction proceeded with a phosphorothioate in the Sp configuration but were blocked by the Rp diastereomer. Both steps also proceeded with inversion of stereochemical configuration around phosphorus, consistent with a concerted transesterification reaction. These results are identical to those found for nuclear precursor mRNA (pre-mRNA) splicing and provide support for the hypothesis that group II introns and nuclear pre-mRNA introns share a common evolutionary history.

Regulatory elements involved in tax-mediated transactivation of the HTLV-I LTR.

HTLV-I is the etiologic agent of adult T-cell leukemia. In this study, we investigated the regulatory elements and cellular transcription factors which function in modulating HTLV-I gene expression in response to the viral transactivator protein, tax. Transfection experiments into Jurkat cells of a variety of site-directed mutants in the HTLV-1 LTR indicated that each of the three motifs A, B, and C within the 21-bp repeats, the binding sites for the Ets family of proteins, and the TATA box all influenced the degree of tax-mediated activation. Tax is also able to activate gene expression of other viral and cellular promoters. Tax activation of the IL-2 receptor and the HIV-1 LTR is mediated through NF-kappa B motifs. Interestingly, sequences in the 21-bp repeat B and C motifs contain significant homology with NF-kappa B regulatory elements. We demonstrated that an NF-kappa B binding protein, PRDII-BF1, but not the rel protein, bound to the B and C motifs in the 21-bp repeat. PRDII-BF1 was also able to stimulate activation of HTLV-I gene expression by tax. The role of the Ets proteins on modulating tax activation was also studied. Ets 1 but not Ets 2 was capable of increasing the degree of tax activation of the HTLV-I LTR. These results suggest that tax activates gene expression by either direct or indirect interaction with several cellular transcription factors that bind to the HTLV-I LTR.