ABSTRACT
We studied a sample from the GISP 2 (Greenland Ice Sheet Project) ice core to determine the diversity and survival of microorganisms trapped in the ice at least 120,000 years ago. Previously, we examined the phylogenetic relationships among 16S ribosomal DNA (rDNA) sequences in a clone library obtained by PCR amplification from genomic DNA extracted from anaerobic enrichments. Here we report the isolation of nearly 800 aerobic organisms that were grouped by morphology and amplified rDNA restriction analysis patterns to select isolates for further study. The phylogenetic analyses of 56 representative rDNA sequences showed that the isolates belonged to four major phylogenetic groups: the high-G+C gram-positives, low-G+C gram-positives, Proteobacteria, and the Cytophaga-Flavobacterium-Bacteroides group. The most abundant and diverse isolates were within the high-G+C gram-positive cluster that had not been represented in the clone library. The Jukes-Cantor evolutionary distance matrix results suggested that at least 7 isolates represent new species within characterized genera and that 49 are different strains of known species. The isolates were further categorized based on the isolation conditions, temperature range for growth, enzyme activity, antibiotic resistance, presence of plasmids, and strain-specific genomic variations. A significant observation with implications for the development of novel and more effective cultivation methods was that preliminary incubation in anaerobic and aerobic liquid prior to plating on agar media greatly increased the recovery of CFU from the ice core sample.
Subject(s)
Bacteria/classification , Fossils , Geologic Sediments/microbiology , Ice , Phylogeny , Bacteria/genetics , Bacteria/isolation & purification , Culture Media , DNA, Bacterial/analysis , DNA, Ribosomal/analysis , Greenland , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Ribosomal, 16S/analysis , RNA, Ribosomal, 16S/genetics , Restriction MappingABSTRACT
A small cryptic Lactobacillus helveticus plasmid, pLBL4, was able to reveal restriction fragment length polymorphism in different bacterial species including Lactobacillus species, Bacillus species, and Escherichia coli when used as a DNA probe. The observed polymorphism was a result of the combined hybridization of several microsatellite sequences. The 6-bp sequence (TTGTTT) was repeated 12 times, seven of which were concentrated within the region between 1791 and 1997 bp of the plasmid sequence. The polymorphic patterns generated with pLBL4 differed from those obtained with M13 DNA in the larger number of bands observed. The results presented here open the possibility of using pLBL4 as a new broad-spectrum polymorphic DNA probe for fingerprint analysis.
Subject(s)
Bacteria/genetics , DNA Probes , Lactobacillus/genetics , Plasmids/genetics , Polymorphism, Restriction Fragment Length , Bacteria/classification , Base Sequence , Molecular Sequence Data , Nucleic Acid Hybridization , Restriction MappingABSTRACT
The influence of nigericin (nandavir) on the organism producing it (Streptomyces hygroscopicus 155) was studied with respect to the synthesis of protein, RNA and DNA. The resistance of the mycelium of different ages to the exogenic antibiotic was investigated. Exogenic nigericin had a more marked inhibitory effect on the macromolecular synthesis in a nonproductive variant of the strain. The antibiotic sensitivity of the mycelium in both the variants (productive and nonproductive) depended on the culture age. It was suggested that there were not less than two mechanisms of the protection from the own antibiotic in the organism.
Subject(s)
Genetic Variation , Nigericin/pharmacology , Streptomyces/drug effects , Drug Resistance, Microbial/genetics , Microbial Sensitivity Tests , Nigericin/biosynthesis , Streptomyces/genetics , Streptomyces/metabolismABSTRACT
The optimal conditions for the application of M13 DNA fingerprinting to the genus Lactobacillus were determined. Comparative fingerprint analysis of representative strains of Lactobacillus delbrueckii subsp. delbrueckii, Lact. delbrueckii subsp. lactis, Lact. delbrueckii subsp. bulgaricus, Lact. helveticus and Lact. casei permitted the differentiation of species, subspecies and individual strains and the quantitative determination of their genetic relatedness. The results confirm the high specificity of M13 DNA fingerprinting and indicate that it might be used in the classification of Lactobacillus spp.
Subject(s)
DNA Fingerprinting , Lactobacillus/classification , Lactobacillus/isolation & purification , Blotting, Southern , DNA Fingerprinting/methods , DNA Probes , DNA, Bacterial/isolation & purification , Electrophoresis, Polyacrylamide Gel , Lactobacillus/genetics , Nucleic Acid Hybridization , Species SpecificityABSTRACT
Hypervariable nucleotide sequences were detected in Bacillus sphaericus by hybridization with radioactively labelled M13 DNA. Different serotypes could be distinguished by their hybridization profiles. The appearance of bands common for mosquito-pathogenic strains and their absence in an apathogenic strain opens the probability that M13 could hybridize to specific alleles, related to insect toxicity.
Subject(s)
Bacillus/genetics , DNA, Bacterial/analysis , Alleles , Animals , Bacillus/classification , DNA Probes , Nucleic Acid Hybridization , Nucleotide Mapping , Polymorphism, Restriction Fragment Length , Restriction Mapping , SerotypingABSTRACT
A small cryptic plasmid of Bacillus thuringiensis subsp. israelensis was labelled in vitro with two genetic markers. One of the recombinant plasmids was mapped and transformed in Escherichia coli, Bacillus subtilis and Bacillus thuringiensis. This and similar shuttle plasmids could be very useful as vectors for the investigation of the toxin genes in their own host.