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.

No abstract

We determined the sequence of the 152,372-bp genome of ϕYS40, a lytic tailed bacteriophage of Thermus thermophilus. The genome contains 170 putative open reading frames and three tRNA genes. Functions for 25% of ϕYS40 gene products were predicted on the basis of similarity to proteins of known function from diverse phages and bacteria. ϕYS40 encodes a cluster of proteins involved in nucleotide salvage, such as flavin-dependent thymidylate synthase, thymidylate kinase, ribonucleotide reductase, and deoxycytidylate deaminase, and in DNA replication, such as DNA primase, helicase, type A DNA polymerase, and predicted terminal protein involved in initiation of DNA synthesis. The structural genes of ϕYS40, most of which have no similarity to sequences in public databases, were identified by mass-spectrometric analysis of purified virions. Various ϕYS40 proteins have different phylogenetic neighbors, including Myovirus, Podovirus, and Siphovirus gene products, bacterial genes, and in one case, a dUTPase from a eukaryotic virus. ϕYS40 has apparently arisen through multiple acts of recombination between different phage genomes as well as through acquisition of bacterial genes.

No abstract

We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases (Pavlov et. al., (2002) Proc. Natl. Acad. Sci. USA 99, 13510–13515). The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various non-specific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting Helix-hairpin-Helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species, but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of TopoV HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105°C by maintaining processivity of DNA synthesis at high temperatures. We also found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding templates to DNA polymerases.

Although ␣-and ␤-linked 3-deoxy-D-manno-octulosonic acid (KDO) is found in lipopolysaccharides (LPSs) of Gram-negative bacteria, capsular polysaccharides of microorganisms, and plants, very little is known about its degradation. Using both thin-layer chromatography and the periodate-thiobarbituric acid reaction, we found that the hepatopancreas of oyster (Crassostrea virginica) contained an enzyme (␣-KDOase) capable of releasing ␣-linked KDO from LPSs. To facilitate the studies of ␣-KDOase, we have carried out the synthesis of 4-methylumbelliferyl-␣-KDO (␣-KDO-MU) by conjugating the glycosyl chloride of the per-O-acetylated methylester of KDO with methylumbelliferone by the S N 2 type reaction and the catalyzed phase-transfer. In both cases, the ␤-anomer was obtained as the major product with a yield of about 80%, whereas the yield of ␣-anomer was only about 7%. Attempts to increase the yield of ␣-anomer were not successful. ␣-KDO-MU was used as substrate to follow the purification of ␣-KDOase from oyster hepatopancreas. The pH optimum for oyster ␣-KDOase was determined to be 4.5 using Re-LPS as substrate and 3.0 using ␣-KDO-MU as substrate. The enzyme was found to be stable in the pH range of 3-8. This enzyme released KDO from different LPSs, including Re-LPS from Escherichia coli and Salmonella minnesota, Rd-LPS from S. minnesota, and de-O-acyl-Re-LPS (Kiang, J., Szu, S. C., Wang, L.X., Tang, M., and Lee, Y. C. (1997) Anal. Biochem. 245, 97-101). 3-Deoxy-D-manno-octulosonic acid (KDO)1 is a major component of LPSs in Gram-negative bacteria (1-3). In addition, KDO was also found in bacterial capsular polysaccharides (4, 5) and plant cell walls (6). Despite the wide occurrence of KDO, nothing is known about its degradation. Using both TLC and the periodate-thiobarbituric acid reaction (7), we found that the hepatopancreas of oyster, Crassostrea virginica, contained an enzyme (␣-KDOase) capable of releasing ␣-linked KDO from LPSs. To facilitate the studies of ␣-KDOase, we have carried out the synthesis of ␣-KDO-MU by conjugating the glycosyl chloride of per-O-acetylated methylester of KDO with methylumbelliferone using the S N 2 coupling reaction and the catalyzed phase-transfer reaction. In both cases, the ␤-anomer was obtained as the major product. This paper describes the preparation of ␣-and ␤-KDO-MU (Fig. 1) and the use of ␣-KDO-MU as a substrate to follow the isolation of ␣-KDOase from oyster hepatopancreas. EXPERIMENTAL PROCEDURES MaterialsKDO (ammonium salt) was prepared by alkaline conjugation of oxalacetic acid and D-arabinose (7). The following were purchased from commercial sources: oysters (C. virginica) were from P&J Oyster Company (New Orleans, LA); KDO, octyl-Sepharose, and phenylmethylsulfonyl fluoride were from Sigma; precoated silica gel-60 and 60 F 254 TLC plates, silica gel, and Fractogel EMD SP-650(S) were from E. Merck (Darmstadt, Germany); and Sephacryl S 200-SF and Con A-Sepharose were from Pharmacia LKB Biotechnology. Re-LPS from Escherichia coli K12, D31 m4, lot 5 (KDO content 11.7%); R...

No abstract