Taxonomy of alkaliphilic Bacillus strains

The DNA base compositions of 78 alkaliphilic Bacillus strains were determined. These strains were grouped as follows: DNA group A, guanine-plus-cytosine (G+C) content of 34.0 to 37.5 mol% (17 strains); DNA group B, G+C content of 38.2 to 40.8 mol% (33 strains); and DNA group C, G+C content of 42.1 to 43.9 mol% (28 strains). DNA group A includes the type strain of Bacillus alcalophilus Vedder 1934. DNA-DNA hybridization studies with DNA group A strains revealed that only one strain, strain DSM 2526, exhibited a high level of DNA homology with B. alcalophilus DSM 485T (T = type strain). Neither strain DSM 485T nor any other DNA group A strain is homologous to any of the Bacillus type strains with comparable base compositions. Six strains formed a distinct group containing three highly homologous strains and three strains exhibiting greater than 50% DNA homology.     

DNA from diazotrophic Desulfovibrio strains is homologous to Klebsiella pneumoniae structural nif DNA and can be chromosomal or plasmid-borne

Abstract Genomic DNA from 13 diazotrophic strains of Desulfovibrio showed homology to structural nif DNA from Klebsiella pneumoniae ; DNA from 3 non-diazotrophic strains did not. The nif DNA is chromosomal in 10 strains, but is carried on 130-MDa plasmids in 3 strains of Desulfovibrio vulgaris .     

Lactic acid bacteria isolated from soy sauce mash in Thailand

Fourteen sphere-shaped and 30 rod-shaped lactic acid bacteria were isolated from soy sauce mash of two factories in Thailand. These strains were separated into two groups, Group A and Group B, by cell shape and DNA-DNA similarity. Group A contained 14 tetrad-forming strains, and these strains were identified as Tetragenococcus halophilus by DNA similarity. Group B contained 30 rod-shaped bacteria, and they were further divided into four Subgroups, B1, B2, B3, and B4, and three ungrouped strains by phenotypic characteristics and DNA similarity. Subgroup B1 contained 16 strains, and these strains were identified as Lactobacillus acidipiscis by DNA similarity. Subgroup B2 included two strains, and the strains were identified as Lactobacillus farciminis by DNA similarity. Subgroup B3 contained five strains. The strains had meso-diaminopimelic acid in the cell wall, and were identified as Lactobacillus pentosus by DNA similarity. The strains tested produced DL-lactic acid from D-glucose. Subgroup B4 contained four strains. The strains had meso-diaminopimelic acid in the cell wall, and they were identified as Lactobacillus plantarum by DNA similarity. Two ungrouped strains were homofermentative, and one was heterofermentative. They showed a low degree of DNA similarity with the type strains tested, and were left unnamed. The distribution of lactic acid bacteria in soy sauce mash in Thailand is discussed.     

Thermostabilities of DNA ligases and DNA polymerases from four genera of thermophilic eubacteria

Thermophilic eubacteria were screened for thermostability of DNA ligases and DNA polymerases. A total of 103 and 248 strains were screened respectively. The strains belonged to four distantly related genera of Thermus, Bacillus, Rhodothermus and Hydrogenobacter. Thermostable DNA ligases were found in 22% of the strains and thermostable DNA polymerase were found in 15% of the strains. Thermus strains gave the highest frequency of both heat tolerant enzymes.     

Clostridium sporogenes isolates and their relationship to C. botulinum based on deoxyribonucleic acid reassociation.

Sixty-two isolates of Clostridium sporogenes from canned foods were examined for cultural properties, heat resistance and DNA-DNA homology to Clostridium botulinum type A190. Sporulation was observed in most of 21 umbonate and rhizoidal colony-forming strains (colony-type I strains), but not in most of the 41 strains with convex and circular or crenate colonies with a mat to semi-glossy surface (colony-type II strains). More than half of the latter strains showed much higher heat resistance than the rhizoidal colony-forming strains. The DNA isolated from colony-type II strains was 81% or more homologous to C. botulinum A190 DNA, forming duplexes which had thermostabilities similar to homologous duplexes of strain A190 DNA. Colony-type I strains differed from C. botulinum by 30 to 40% DNA homology and the DNA duplexes formed between these strains and strain A190 showed deltaT m(e) values of 7-0 degrees C when compared with the T m(e) of homologous DNA duplexes of strain A190.     

Deoxyribonucleic acid homologies among strains of Bacteroides melaninogenicus and related species

Saccharolytic, black-pigmented Bacteroides strains, which at present belong to the species Bacteroides melaninogenicus were classified on the basis of deoxyribonucleic acid (DNA) base ratios and DNA hybridization studies. These strains were divided into several DNA homology groups, which showed no or low mutual DNA homology. A DNA homology group with a percentage guanine plus cytosine (G + C) of 42–43% was formed by three strains of Bact. melaninogenicus subsp. melaninogenicus ; the type strain of this subspecies, strain ATCC 25845, had about 60% DNA homology with this group. Strain ATCC 15930, which has been assigned to this subspecies, had a percentage G + C of 47% and showed no DNA homology with the former group. All strains of Bact. melaninogenicus subsp. intermedius had a percentage G + C of 39–45%. A DNA homology group was formed by eight strains of this subspecies. The type strain of Bact. melaninogenicus subsp. intermedius , ATCC 25611, showed relatively low DNA homology with this main DNA homology group. A strain of Bact. melaninogenicus subsp. intermedius serotype C1 showed no DNA homology with the other strains tested. Furthermore two strains labelled 'Bact. melaninogenicus subsp. levii' were found to form a distinct DNA homology group. On the basis of the DNA homology results, the strains, which at present are classified in the species Bact. melaninogenicus , were clearly distinguished from strains of Bact. asaccharolyticus and Bact. gingivalis , and also from strains of related non-pigmented Bacteroides species.     

Biogenesis of mitochondria

Summary The proportion of total cell DNA which is mitochondrial DNA was measured in haploid, diploid and tetraploid strains of S. cerevisiae grown under a standard set of conditions. For all strains tested the mitochondrial DNA level was in the range 16%–25% of total cell DNA. Repeated measurements of the cellular level of mitochondrial DNA in two haploid strains showed that these strains have measurably different cellular mitochondrial DNA levels (17% and 24% of total DNA, respectively) under our conditions. These two grande strains were used to investigate the role of the mitochondrial and nuclear genomes in the regulation of the mitochondrial DNA level. We have shown by genetic analysis that the difference between these two strains is determined by at least two nuclear genes. The mitochondrial genome is not involved in the regulation of cellular mitochondrial DNA levels.A number of purified petite clones derived from independent spontaneous petite isolates of the grande strain which contained 24% mitochondrial DNA were also studied. The mitochondrial DNA levels in all but one of these petites fell in the range 20–25% of total cell DNA. From these results we conclude that, in general, the mitochondrial DNA level in petite strains is controlled by the same mechanism as operates in grande strains.We propose a general model for the control of the cellular mitochondrial DNA level, in which the amount of mitochondrial DNA per cell is determined by regulation of the number of mitochondrial DNA molecules per cell. This regulation is mediated through the availability of a set of nuclear coded components, possibly a mitochondrial membrane site, which are required for the replication of mitochondrial DNA.     

Relatedness Among Rhizobium and Agrobacterium Species Determined by Three Methods of Nucleic Acid Hybridization

Deoxyribonucleic acid (DNA) was isolated from 20 strains of Rhizobium and Agrobacterium and from one strain of Serratia marcescens; the guanine plus cytosine content of each DNA sample was determined by thermal denaturation. Radioactive DNA was isolated from three reference strains following the uptake of [2-(14)C]thymidine in the presence of deoxyadenosine. Ribonucleic acid (RNA) polymerase was used to synthesize radioactive RNA on DNA templates from the three reference strains. Radioactive DNA and RNA from the three reference strains were each hybridized with filter-bound DNA from all of the 21 test strains in 6 x SSC (standard saline citrate) and 50% formamide at 43 C for 40 hr. DNA/DNA relatedness was also determined by spectrophotometric measurement of the rates of association of single-stranded DNA. The order of relatedness between strains was similar by each method. Overall standard deviations for the DNA/DNA and DNA/RNA membrane filter techniques were +/-0.87 and +/-1.03%, respectively; that for the spectrophotometric technique was +/-4.11%. The DNA/DNA membrane technique gave higher absolute values of hybridization than did the DNA/RNA technique. R. leguminosarum and R. trifolii could not be distinguished from each other by these techniques. These results also indicated close relationships between R. lupini and R. japonicum, and (with less certainty) between R. meliloti and R. phaseoli. Of all the rhizobia tested against the A. tumefaciens 371 reference strain, the R. japonicum strains were the most unrelated. The three Agrobacterium strains used were as related to the R. lupini and R. leguminosarum references as were several rhizobium strains.     

DNA relatedness among strains of the sweet potato pathogen Streptomyces ipomoea (Person and Martin 1940) Waksman and Henrici 1948.

DNA relatedness among 28 putative strains of Streptomyces ipomoea from geographically diverse locations and the type strain, NRRL B-12321, was determined spectrophotometrically. The data confirm that these 28 strains are not closely related genetically to the plant-pathogenic species Streptomyces scabies (39% DNA relatedness) or Streptomyces acidiscabies (17% DNA relatedness) or any other major blue-spored Streptomyces species (less than 30% DNA relatedness). Of the 29 strains examined, 4 could be clearly distinguished from S. ipomoea on the basis of morphological criteria, i.e., they had gray rather than blue spores and produced melanin pigment, and their low DNA relatedness to authentic S. ipomoea strains confirmed their original misidentification. The remaining 25 S. ipomoea strains exhibited high DNA relatedness among themselves (76 to 100% homology), even though they had been isolated in different locations throughout the United States and Japan. The avirulent type strain, NRRL B-12321, exhibited slightly lower DNA relatedness with the virulent strains of S. ipomoea (85% average DNA relatedness) than was observed among the virulent strains (average of 96% DNA relatedness).     

DNA relatedness among strains of the sweet potato pathogen Streptomyces ipomoea (Person and Martin 1940) Waksman and Henrici 1948.

DNA relatedness among 28 putative strains of Streptomyces ipomoea from geographically diverse locations and the type strain, NRRL B-12321, was determined spectrophotometrically. The data confirm that these 28 strains are not closely related genetically to the plant-pathogenic species Streptomyces scabies (39% DNA relatedness) or Streptomyces acidiscabies (17% DNA relatedness) or any other major blue-spored Streptomyces species (less than 30% DNA relatedness). Of the 29 strains examined, 4 could be clearly distinguished from S. ipomoea on the basis of morphological criteria, i.e., they had gray rather than blue spores and produced melanin pigment, and their low DNA relatedness to authentic S. ipomoea strains confirmed their original misidentification. The remaining 25 S. ipomoea strains exhibited high DNA relatedness among themselves (76 to 100% homology), even though they had been isolated in different locations throughout the United States and Japan. The avirulent type strain, NRRL B-12321, exhibited slightly lower DNA relatedness with the virulent strains of S. ipomoea (85% average DNA relatedness) than was observed among the virulent strains (average of 96% DNA relatedness).     

Construction and use of a computerized DNA fingerprint database for lactic acid bacteria from silage

Efficient selection of new silage inoculant strains from a collection of over 10,000 isolates of lactic acid bacteria (LAB) requires excellent strain discrimination. Toward that end, we constructed a GelCompar II database of DNA fingerprint patterns of ethidium bromide-stained EcoRI fragments of total LAB DNA separated by conventional agarose gel electrophoresis. We found that the total DNA patterns were strain-specific; 56/60 American Type Culture Collection strains of 33 species of LAB could be distinguished. Enterococcus faecium strains ATCC19434 and ATCC35667 had identical total DNA patterns and RiboPrints. Lactobacillus rhamnosus strains ATCC7469 and ATCC27773 also had identical total DNA patterns, but different RiboPrints. EcoRI RiboPrint patterns could distinguish only about 9/23 Lactobacillus plantarum strains and about 6/10 Lactobacillus buchneri strains, whereas all 33 strains could be distinguished by EcoRI total DNA patterns. Despite gel-to-gel variation, new DNA patterns can be readily grouped with existing patterns using GelCompar II. The database contains large homogenous clusters of L. plantarum, E. faecium, L. buchneri, Lactobacillus brevis and Pediococcus species that can be used for tentative taxonomic assignment. We routinely use the DNA fingerprint database to identify and characterize new strains, eliminate duplicate isolates and for quality control of inoculant product strains. The GelCompar II database has been in continuous use for 7 years and contains more than 3600 patterns representing approximately 700 unique patterns from over 300 gels and is the largest computerized DNA fingerprint database for LAB yet reported.     

Taxonomic relationships among strains of the anaerobic bacterium Bacteroides ruminicola determined by DNA and extracellular polysaccharide analysis.

DNA and extracellular polysaccharide (EPS) analyses were performed on 14 strains of Bacteroides ruminicola. The guanine-plus-cytosine (G+C) base contents, determined from the buoyant densities of chromosomal DNAs, showed a broad range of values, from 37.6 to 50.9 mol%. DNA hybridization showed generally low DNA relatedness among the strains. Seven strains formed two groups of closely related bacteria consisting of five (group 1) and two (group 2) strains, and another strain, E42g, showed moderate relatedness to group 1 strains. However, the remaining six strains were not related to any of the other strains. DNA reassociation indicates that the strains constitute a genetically diverse group representing as many as nine separate species. EPS analysis showed that the strains produced EPS with rather uniform sugar compositions, which did not correlate with strain relationships determined by DNA analysis. Four strains had EPS with acidic sugars or unknown compounds. The EPS of strain 20-63 contained the unusual acidic sugar 4-O-(1-carboxyethyl)-rhamnose. This monosaccharide has been shown to occur in nature in only one other bacterial species.     

Streptococcus uberis: an Approach to its Classification

Thirty-one strains of streptococci, selected because they were considered to be or were similar to Streptococcus uberis , were examined by physiological tests and for properties of their lactate dehydrogenases (LDHs) and deoxyribonucleic acid (DNA). Twenty-nine of the 31 strains fell into two main genotypes as judged by DNA/DNA hybridization. One genotype (Group I) included the type strain of Strep. uberis and most other strains in this group were physiologically similar to it and had the same percent guanine + cytosine (%GC) in their DNA. The other genotype (Group II) contained strains which were more variable physiologically and had DNA of a lower % GC than the type strain. The European strains examined contained two distinct LDH types, one being associated with the Group I and the other with Group II strains. Three American strains examined however had LDH's which were unlike those of the European strains.     

Characterization of Dutch porcine Serpulina (Treponema) isolates by restriction endonuclease analysis and DNA hybridization.

Genomes of 55 Dutch porcine Serpulina (Treponema) hyodysenteriae and non-pathogenic Serpulina isolates were characterized by restriction endonuclease analysis (REA) and DNA hybridization. The Dutch porcine isolates were compared with American Type Culture Collection (ATCC) strains of S. hyodysenteriae and S. innocens and isolates of S. hyodysenteriae with known serotypes (reference strains). REA of the Dutch S. hyodysenteriae isolates resulted in two main patterns, while the non-pathogenic isolates had many distinct REA patterns, all different from the S. hyodysenteriae strains. The S. hyodysenteriae reference strains all had distinct REA patterns, different from the Dutch strains. Upon Southern hybridization with a S. hyodysenteriae DNA fragment encoding a flagellar protein, all S. hyodysenteriae strains could be divided in two groups. The non-pathogenic Serpulina strains had many distinct hybridization patterns and hybridized less intensely. Upon hybridization with a S. hyodysenteriae DNA fragment encoding a haemolysin, DNA of all S. hyodysenteriae strains reacted in the same band. DNA of non-pathogenic Dutch Serpulina strains and S. innocens did not hybridize. It was concluded that there are two main genotypes of S. hyodysenteriae in the Netherlands. This could be of importance for recombinant DNA vaccine development.     

Plasmid-determined 2-hydroxypyridine utilization by Arthrobacter crystallopoietes.

Arthrobacter crystallopoietes has the ability to utilize 2-hydroxypyridine (2-HP) as a source of carbon and nitrogen and forms a blue extracellular pigment when grown in the presence of 2-HP. Ultracentrifugal analyses of pigment producing (Pig+) and pigment nonproducing (Pig-) strains of A. crystallopoietes revealed the presence of plasmid material in both strains. Recovery of plasmid DNA from Pig+ strains is two or three times greater than from Pig- strains. The molecular weight of plasmid DNA recovered from Pig+ strains (62 Mdaltons) is slightly higher than the molecular weight of plasmid DNA from Pig- strains. Consistent with the characterization of plasmid DNA from the two strains is that Pig+ strains contain a 63-Mdalton plasmid encoding 2-HP utilization as well as a cryptic plasmid of very nearly equal molecular weight. Pig- strains contain only the cryptic plasmid.     

Comparative study of the homology of DNA in thermophilic and mesophilic lactococcal strains of various origin

A comparative study of DNA homology among 12 strains of thermophilic streptococci currently used as ferments in the dairy industry in different regions of the CIS and 16 strains of mesophilic lactococci obtained from different sources was performed by the method of optical reassociation in solution. These strains were found to form three groups with DNA homology generally not exceeding 30-35% between each other. These data indicate that strains commonly classified as Streptococcus thermophilus may belong to different taxa. At the same time, the DNA base composition was similar in all these strains (the G + C content was 38-40 mol %). Among 16 strains of mesophilic lactococci DNA homology was generally higher than 50%, i.e., all these strains indeed belong to the same species, Lactococcus lactis.     

Conserved Plasmid Hydrogen-Uptake (hup)-Specific Sequences within HupRhizobium leguminosarum Strains

Thirteen Rhizobium leguminosarum strains previously reported as H(2)-uptake hydrogenase positive (Hup) or negative (Hup) were analyzed for the presence and conservation of DNA sequences homologous to cloned Bradyrhizobium japonicum hup-specific DNA from cosmid pHU1 (M. A. Cantrell, R. A. Haugland, and H. J. Evans, Proc. Natl. Acad. Sci. USA 80:181-185, 1983). The Hup phenotype of these strains was reexamined by determining hydrogenase activity induced in bacteroids from pea nodules. Five strains, including H(2) oxidation-ATP synthesis-coupled and -uncoupled strains, induced significant rates of H(2)-uptake hydrogenase activity and contained DNA sequences homologous to three probe DNA fragments (5.9-kilobase [kb] HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) from pHU1. The pattern of genomic DNA HindIII and EcoRI fragments with significant homology to each of the three probes was identical in all five strains regardless of the H(2)-dependent ATP generation trait. The restriction fragments containing the homology totalled about 22 kb of DNA common to the five strains. In all instances the putative hup sequences were located on a plasmid that also contained nif genes. The molecular sizes of the identified hup-sym plasmids ranged between 184 and 212 megadaltons. No common DNA sequences homologous to B. japonicum hup DNA were found in genomic DNA from any of the eight remaining strains showing no significant hydrogenase activity in pea bacteroids. These results suggest that the identified DNA region contains genes essential for hydrogenase activity in R. leguminosarum and that its organization is highly conserved within Hup strains in this symbiotic species.     

Taxonomic relationships among strains of the anaerobic bacterium Bacteroides ruminicola determined by DNA and extracellular polysaccharide analysis.

DNA and extracellular polysaccharide (EPS) analyses were performed on 14 strains of Bacteroides ruminicola. The guanine-plus-cytosine (G+C) base contents, determined from the buoyant densities of chromosomal DNAs, showed a broad range of values, from 37.6 to 50.9 mol%. DNA hybridization showed generally low DNA relatedness among the strains. Seven strains formed two groups of closely related bacteria consisting of five (group 1) and two (group 2) strains, and another strain, E42g, showed moderate relatedness to group 1 strains. However, the remaining six strains were not related to any of the other strains. DNA reassociation indicates that the strains constitute a genetically diverse group representing as many as nine separate species. EPS analysis showed that the strains produced EPS with rather uniform sugar compositions, which did not correlate with strain relationships determined by DNA analysis. Four strains had EPS with acidic sugars or unknown compounds. The EPS of strain 20-63 contained the unusual acidic sugar 4-O-(1-carboxyethyl)-rhamnose. This monosaccharide has been shown to occur in nature in only one other bacterial species.     

Sequence analysis of the hypervariable regions of the 56 kDa immunodominant protein genes of Orientia tsutsugamushi strains in Malaysia

The DNA sequences encompassing two hypervariable regions, VD II and III of the 56 kDa immunodominant protein gene of 21 Malaysian strains of Orientia tsutsugamushi were determined. Two strains demonstrated a 100% DNA homology with the Gilliam prototype strain, and one with TH1817 strain and TA678 strain respectively. High percentages of DNA similarity (95-99%) were observed with Karp (4 strains), Gilliam (2 strains), TH1817 (4 strains), TC586 (3 strains) and TA763 (1 strain). The remaining strains demonstrated the highest DNA similarity with TA763 (1 strain, 89%), TA678 (1 strain, 86%) and TA686 (1 strain, 87%). Our study provides additional evidence on the existence and the genetic heterogeneity of TA strains of the Southeast Asia and their closely related strains in Malaysia.