Bacterial diversity within the human subgingival crevice

Molecular, sequence-based environmental surveys of microorganisms have revealed a large degree of previously uncharacterized diversity. However, nearly all studies of the human endogenous bacterial flora have relied on cultivation and biochemical characterization of the resident organisms. We used molecular methods to characterize the breadth of bacterial diversity within the human subgingival crevice by comparing 264 small subunit rDNA sequences from 21 clone libraries created with products amplified directly from subgingival plaque, with sequences obtained from bacteria that were cultivated from the same specimen, as well as with sequences available in public databases. The majority (52.5%) of the directly amplified 16S rRNA sequences were <99% identical to sequences within public databases. In contrast, only 21.4% of the sequences recovered from cultivated bacteria showed this degree of variability. The 16S rDNA sequences recovered by direct amplification were also more deeply divergent; 13.5% of the amplified sequences were more than 5% nonidentical to any known sequence, a level of dissimilarity that is often found between members of different genera. None of the cultivated sequences exhibited this degree of sequence dissimilarity. Finally, direct amplification of 16S rDNA yielded a more diverse view of the subgingival bacterial flora than did cultivation. Our data suggest that a significant proportion of the resident human bacterial flora remain poorly characterized, even within this well studied and familiar microbial environment.

The extended environment of mononuclear metal centers in protein structures

The objectives of this and the following paper are to identify commonalities and disparities of the extended environment of mononuclear metal sites centering on Cu, Fe, Mn, and Zn. The extended environment of a metal site within a protein embodies at least three layers: the metal core, the ligand group, and the second shell, which is defined here to consist of all residues distant less than 3.5 Å from some ligand of the metal core. The ligands and second-shell residues can be characterized in terms of polarity, hydrophobicity, secondary structures, solvent accessibility, hydrogen-bonding interactions, and membership in statistically significant residue clusters of different kinds. Findings include the following: (i) Both histidine ligands of type I copper ions exclusively attach the Nδ1 nitrogen of the histidine imidazole ring to the metal, whereas histidine ligands for all mononuclear iron ions and nearly all type II copper ions are ligated via the Nɛ2 nitrogen. By contrast, multinuclear copper centers are coordinated predominantly by histidine Nɛ2, whereas diiron histidine contacts are predominantly Nδ1. Explanations in terms of steric differences between Nδ1 and Nɛ2 are considered. (ii) Except for blue copper (type I), the second-shell composition favors polar residues. (iii) For blue copper, the second shell generally contains multiple methionine residues, which are elements of a statistically significant histidine–cysteine–methionine cluster. Almost half of the second shell of blue copper consists of solvent-accessible residues, putatively facilitating electron transfer. (iv) Mononuclear copper atoms are never found with acidic carboxylate ligands, whereas single Mn2+ ion ligands are predominantly acidic and the second shell tends to be mostly buried. (v) The extended environment of mononuclear Fe sites often is associated with histidine–tyrosine or histidine–acidic clusters.

PEA3 sites within the progression elevated gene-3 (PEG-3) promoter and mitogen-activated protein kinase contribute to differential PEG-3 expression in Ha-ras and v-raf oncogene transformed rat embryo cells

Transformation of normal cloned rat embryo fibroblast (CREF) cells with cellular oncogenes results in acquisition of anchorage-independent growth and oncogenic potential in nude mice. These cellular changes correlate with an induction in the expression of a cancer progression-promoting gene, progression elevated gene-3 (PEG-3). To define the mechanism of activation of PEG-3 as a function of transformation by the Ha-ras and v-raf oncogenes, evaluations of the signaling and transcriptional regulation of the ~2.0 kb promoter region of the PEG-3 gene, PEG-Prom, was undertaken. The full-length and various mutated regions of the PEG-Prom were linked to a luciferase reporter construct and tested for promoter activity in CREF and oncogene-transformed CREF cells. An analysis was also performed using CREF cells doubly transformed with Ha-ras and the Ha-ras specific suppressor gene Krev-1, which inhibits the transformed phenotype in vitro. These assays document an association between expression of the transcription regulator PEA3 and PEG-3. The levels of PEA3 and PEG-3 RNA and proteins are elevated in the oncogenically transformed CREF cells, and reduced in transformation and tumorigenic suppressed Ha-ras/Krev-1 doubly transformed CREF cells. Enhanced tumorigenic behavior, PEG-3 promoter function and PEG-3 expression in Ha-ras transformed cells were all dependent upon increased activity within the mitogen-activated protein kinase (MAPK) pathway. Electrophoretic mobility shift assays and DNase I footprinting experiments indicate that PEA3 binds to sites within the PEG-Prom in transformed rodent cells in an area adjacent to the TATA box in a MAPK-dependent fashion. These findings demonstrate an association between Ha-ras and v-raf transformation of CREF cells with elevated PEA3 and PEG-3 expression, and they implicate MAPK signaling via PEA3 as a signaling cascade involved in activation of the PEG-Prom.

Oxygen as a key developmental regulator of Rhizobium meliloti N2-fixation gene expression within the alfalfa root nodule.

The symbiotic pattern of expression of Rhizobium meliloti N2-fixation genes is tightly coupled with the histological organization of the alfalfa root nodule and thus is under developmental control. N2-fixation gene expression is induced very sharply at a particular zone of the nodule called interzone II-III that precedes the zone where N2 fixation takes place. We show here that this coupling can be disrupted, hereby resulting in ectopic expression of N2-fixation genes in the prefixing zone II of the nodule. Uncoupling was obtained either by using a R. meliloti strain in which a mutation rendered N2-fixation gene expression constitutive with respect to oxygen in free-living bacterial cultures or by placing nodules induced by a wild-type R. meliloti strain in a microoxic environment. These results implicate oxygen as a key determinant of the symbiotic pattern of N2-fixation gene expression.