Lactic acid-producing bacteria are associated with various plant and animal niches and play a key role in the production of fermented foods and beverages. We report nine genome sequences representing the phylogenetic and functional diversity of these bacteria. The small genomes of lactic acid bacteria encode a broad repertoire of transporters for efficient carbon and nitrogen acquisition from the nutritionally rich environments they inhabit and reflect a limited range of biosynthetic capabilities that indicate both prototrophic and auxotrophic strains. Phylogenetic analyses, comparison of gene content across the group, and reconstruction of ancestral gene sets indicate a combination of extensive gene loss and key gene acquisitions via horizontal gene transfer during the coevolution of lactic acid bacteria with their habitats. evolutionary genomics ͉ fermentation L actic acid bacteria (LAB) are historically defined as a group of microaerophilic, Gram-positive organisms that ferment hexose sugars to produce primarily lactic acid. This functional classification includes a variety of industrially important genera, including Lactococcus, Enterococcus, Oenococcus, Pediococcus, Streptococcus, Leuconostoc, and Lactobacillus species. The seemingly simplistic metabolism of LAB has been exploited throughout history for the preservation of foods and beverages in nearly all societies dating back to the origins of agriculture (1). Domestication of LAB strains passed down through various culinary traditions and continuous passage on food stuffs has resulted in modern-day cultures able to carry out these fermentations. Today, LAB play a prominent role in the world food supply, performing the main bioconversions in fermented dairy products, meats, and vegetables. LAB also are critical for the production of wine, coffee, silage, cocoa, sourdough, and numerous indigenous food fermentations (2).LAB species are indigenous to food-related habitats, including plant (fruits, vegetables, and cereal grains) and milk environments. In addition, LAB are naturally associated with the mucosal surfaces of animals, e.g., small intestine, colon, and vagina. Isolates of the same species often are obtained from plant, dairy, and animal habitats, implying wide distribution and specialized adaptation to these diverse environments. LAB species employ two pathways to metabolize hexose: a homofermentative pathway in which lactic acid is the primary product and a heterofermentative pathway in which lactic acid, CO 2 , acetic acid, and͞or ethanol are produced (3).Complete genome sequences have been published for eight fermentative and commensal LAB species: Lactococcus lactis, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus acidophilus, Lactobacillus sakei, Lactobacillus bulgaricus, Lactobacillus salivarius, and Streptococcus thermophilus (4-11). This study examines nine other LAB genomes representing the phylogenetic and functional diversity of lactic acid-producing microorganisms. The LAB have small genomes encoding a range of biosynthe...

Background: Bifidobacteria are frequently proposed to be associated with good intestinal health primarily because of their overriding dominance in the feces of breast fed infants. However, clinical feeding studies with exogenous bifidobacteria show they don't remain in the intestine, suggesting they may lose competitive fitness when grown outside the gut.

The genomes of two closely related lytic Thermus thermophilus siphoviruses with exceptionally long (~800 nm) tails, bacteriophages P23-45 and P74-26, were completely sequenced. The P23-45 genome consists of 84,201 bp with 117 putative ORFs (Open Reading Frames), and the P74-26 genome has 83,319 bp and 116 putative ORFs. The two genomes are 92% identical with 113 ORFs shared. Only 25% of phage gene product functions can be predicted from similarities to proteins and protein domains with known functions. The structural genes of P23-45, most of which have no similarity to sequences from public databases, were identified by mass-spectrometric analysis of virions. An unusual feature of the P23-45 and P74-26 genomes is the presence, in their largest intergenic regions, of long polypurine-polypyrimidine (R-Y) sequences with mirror repeat symmetry. Such sequences, abundant in eukaryotic genomes but rare in prokaryotes, are known to form stable triple helices that block replication and transcription and induce genetic instability. Comparative analysis of the two phage genomes shows that the area around the triplex-forming elements is enriched in mutational Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access

Triplex-forming oligonucleotides (TFO) are designed to bind sequence specifically to their DNA targets without a significant disturbance of the double helix. They have been proposed to deliver DNA-reactive agents to specific DNA sequences for gene targeting applications. We suggested the use of 125I-labeled TFO for delivery of the energy of radioiodine decay to specific genes. This approach is called antigene radiotherapy. Here we demonstrate the ability of 125I-labeled TFO to produce sequence-specific breaks within a target in the human mdrl gene in cultured cells. TFO and TFO conjugated with a nuclear localization signal peptide (NLS) were delivered into cells using cationic liposomes. This was done either alone or in the presence of an excess of a "ballast" oligonucleotide with an unrelated sequence. In all cases, nuclear localization of TFO and survival of the cells after treatment has been confirmed. Breaks in the gene target were analyzed by restriction enzyme digestion of the DNA recovered from the TFO-treated cells followed by Southern hybridization with DNA probes flanking the target sequence. We have found that TFO/NLS conjugates cleave the target in a concentration-dependent manner regardless of the presence of the "ballast" oligonucleotide. In contrast, TFO without NLS cleaved the target only in the presence of an excess of the "ballast." We hypothesize that TFO and TFO/NLS are delivered into the nucleus by different pathways. These results provide a new insight into the mechanism of intracellular transport of oligonucleotides and open new avenues for improvement of the efficacy of antigene therapies.

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Folding of the DNA and RNA strands in an arrested T7 RNA polymerase (RNAP) transcription complex was studied by radioprobing, a novel method based on an analysis of the strand breaks produced by decay of the iodine-125 incorporated at the C5 position of cytosine. (125)I-labeled cytosines were incorporated into transcripts at different positions relative to the site of the arrest. The intensities of the DNA breaks inversely correlate with the distances from the (125)I decay site, so the radioprobing data provide information about the spatial RNA/DNA folding during transcription. We found that the yield of DNA strand breaks is significantly higher in the template than the non-template strand. This is consistent with local opening of the DNA duplex and formation of a hybrid between RNA and the template DNA strand. Our data demonstrate that the RNA-DNA hybrid has a nonuni form A-like structure. When the (125)I is incorporatedseven nucleotides from the active center of RNAP, the yield of strand breaks is substantially lower than if (125)I is positioned at the ends of the hybrid. Consequently, the DNA and RNA strands are located closer to each other at the ends of the hybrid and somehow separated in the middle. Surprisingly, the (125)I-induced breaks were detected in both DNA strands upstream from the transcription "bubble" indicating that DNA and RNA are closely associated outside the RNAP cleft. Thus, radioprobing data imply that the RNA/DNA fold in the complex with T7 RNAP is more complicated than had been anticipated by the existing models. Based on the present data, we suggest a sterically feasible model explaining how formation of the long RNA-DNA hybrid can result in the initiation-to-elongation switch in the T7 transcription complex. According to this model, the topological linkage between the DNA and RNA strands provides the necessary stability for the elongation complex, while permitting movement of the polymerase along the DNA duplex.

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A new Lactobacillus acidophilus strain, FSI4, isolated from yogurt, was isolated and sequenced in our laboratory. Our data, although supportive of previous conclusions regarding the remarkable stability of L. acidophilus species, indicate accumulating mutations in commercial L. acidophilus strains that warrant further study of the effect of damaged genes on the competitiveness of these bacteria in gut microbiota.