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Deoxyribonucleic acid hybridization and plasmid studies in the genus Lactobacillus

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  • Using thermal denaturation methodology, the nucleic acid base composition of deoxyribonucleic acid (DNA) isolated from type, neotype and stock strains of Lactobacillus species was determined. The genus was found to be heterogeneous in base composition, ranging from 33.1 to 50.0 moles % guanine plus cytosine (GC). Genus members fell into three groups based on DNA GC content. Group I included species with a GC content between 33.1 and 39. 7 moles %: L. salivarius, L. jensenii, L. acidophillus, L. piscium sp. nov. (fish isolate), L. helveticus, and L. sanfrancisco (sourdough bread isolate). Group II included species with a GC content between 44. 0 and 48. 0 moles %: L. plantarum, L. rhamnosus (human isolate) and L. casei var. casei. Group III included organisms with a GC content between 48.4 and 50.0 moles %: L. leichmanii and L. fermenti. Several well known Lactobacillus species were given varietal status. These three groups were further divided into homofermentative and heterofermentative subgroups leading to the formation of six groups. Speciation of group members was determined from DNA, DNA hybridization results as follows: Homofermentative group I - L. salivarius, L. jensenii, L. acidophillus, L. helveticus, and L. piscium sp. nov. Heterofermentative group I - L. sanfrancisco Homofermentative group II - L. plantarum, L. casei and L. rhamnosus (formerly L. casei var. rhamnosus) Heterofermentative group II - L. brevis and L. viridescens. Homofermentative group III - L. leichrnanii, including the varieties lactis, bulgaricus and delbrueckii Heteromentative group III - L. fermenti, including the cellobiosus variety This classification, while differing significantly from the traditional taxonomic system for these organisms based on biochemical and serological properties, nevertheless constitutes a firm basis for species identification. This is substantiated from these homology values: 1. With L. salivarius 11 742 as reference organism, the following homology values for the indicated organisms were obtained: L. helveticus 15009, 10%; L. jensenii 25258, 0%, L. acidophilus 4356, 9%. 2. With L. piscium as the reference organism, the following homology values for the indicated organisms were obtained: L. piscium LRPKI-70, 92 %, L. salivarius 11742, 2, 45%; L. jensenii 25258, 22%; L. helveticus 8018, 16%; L. jugurt 521, 32%; Erysipelotrix insidiosa 19414, 14%. 3. With L. helveticus 15009 as reference organism, the following homology values for the indicated organisms were obtained: L. jugurt 521, 79%; L. jensenii 25258, 0%; L. acidophilus 4356, 0%; L. salivarius 11742, 9%; L. sanfrancisco WRRL-L, 0%; L. piscium 0%; 4. With L. sanfrancisco WRRL B as reference organism, the following homology values for the indicated organisms were obtained: L. sanfrancisco WRRL C, 89%; L. sanfracisco WRRL L, 95%; L. . sanfrancisco WRRL T95 %; L. acidophilus 4536, 23%; L. salivarius 11742, 12 %; L. jugurt 521, 6%; L. helveticus 8014, 32%; L. brevis NCDO 473, 39%. 5. With L. plantarurn 1491 7 as the reference organism, the following homology values for the indicated organisms were obtained: L. casei 393, 1 7%; L. casei 7469, 0%; L. brevis NCDO 473, 0%; L. lactis 1 231 5, 0%; L. fermenti NCDO 215, 2%; S. inulinus, 0%; L. helveticus 1 5009, 6%; Lactobacillus MSH, 0%; Lactobacillus Sardo 6, 84%; Lactobacillus DS46F, 88%. 6. With. L. casei 393 as the reference organism, the following homology values for the indicated organisms were obtained: L. casei 7469, 14%; L. plantarum 14917, 43%; L. brevis NCDO 473, 16%; L. fermenti NCDO 215, 4%; L. helveticus 15009, 17%; L. salivarius 11 742, 10%; S. inulinus, 21%; L. lactis 1 231 5, 4%; Lactobacillus Sardo 6,36%; Lactobacillus MSH, 41%; Lactobacillus DS46F, 24%. 7. With L. lactis 1 231 5 as the reference organism, the following homology values for the indicated organisms were obtained: L. bulgaricus 11842, 78%; L. delbrueckii 9649, 85%; L. leich.- manii 4797, 78%; L, fermenti, NCDO 215, 11%; L. cellobiosus NCDO 9 27, 8%; L. helveticus 1 5009, 30%. 8. With L. fermenti NCDO 215 as the reference organism the following homology values for the indicated organisms were obtained: L. cellobiosus NCDO 927, 83%; L. delbrueckii 9649, 0%; L. leichmanii 4797, 0%; L. lactis 1 2315, 6%; L. bulgaricus 11842, 11%; L. helveticus 15009, 21%. These homology values were obtained from experiments done at the stringent hybridization conditions of Tm 15 C. Genome size determinations also were carried out and two findings emerged: one, lactobacilli have approximately half the genetic capability of Escherichia coli and two, Sporolactobacillus inulinus has a genome which is as large as E. coli and similar to members of the Bacillus genus. Cesium chloride density gradient centrifugation and agarose gel electrophoresis revealed the presence of supercoiled (plasmid) DNA in L. rhamnosus MSH. The plasmid was approximately one million daltons in molecular weight. DNA hybridization studies of a Lactobacillus associated with fish kidney disease revealed that it is a new member of the genus. It was assigned the name L. piscium.
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