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.     

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.     

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.     

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.     

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.     

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.     

Synthesis of Deoxyribonucleic Acid After Ultraviolet Irradiation of Sensitive and Resistant Haemophilus influenzae

Synthesis of deoxyribonucleic acid (DNA) has been measured as a function of ultraviolet (UV) radiation dose in wild-type and seven UV-sensitive strains of Haemophilus influenzae. At the UV doses used, all strains were able to resume DNA synthesis, even those which are unable to excise pyrimidine dimers from their DNA. These excisionless strains showed longer UV-induced delays in DNA synthesis than all but one of the other strains. The longest delay was shown by DB117, a strain which can excise dimers but which is recombination deficient and unable to rejoin X ray-induced single-strand breaks. All strains showed a progressive decrease in sensitivity as they approached the stationary phase.     

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).     

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.     

Identification of novel virulence-associated loci in uropathogenic Escherichia coli by suppression subtractive hybridization

To identify novel virulence-associated genes in uropathogenic Escherichia coli (UPEC) strains, a suppression subtractive hybridization strategy was applied to genomic DNA of four clinical UPEC isolates from patients suffering from cystitis or pyelonephritis. The genomic DNA of four isolates (tester strains) was subtracted from the DNA of two different driver strains, the well characterized UPEC strain CFT073 and the non-pathogenic E. coli K-12 strain MG1655. We determined the sequence of 172 tester strain-specific DNA fragments, 86 of which revealed only low or no homology to nucleotide sequences of public databases. We further determined the virulence association of the 86 novel DNA fragments using each DNA fragment as a probe in Southern hybridizations of a reference strain collection consisting of 60 extraintestinal pathogenic E. coli isolates, and 40 non-virulent E. coli strains from stool samples. From this, 19 novel DNA fragments were demonstrated to be significantly associated with virulent strains and thus may represent new virulence traits. Our results support the idea of a considerable genetic variability among UPEC strains and suggest that novel genomic determinants might contribute to virulence of UPEC.     

一种适用于PCR反应的酵母菌、无绿藻及丝状真菌DNA提取方法

目的 介绍一种从酵母、无绿藻及丝状真菌中提取DNA以用于PCR反应的方法。方法 所用菌种包括临床分离的未知菌株和保藏菌株共23株：未知酵母菌（5株）、真皮毛孢子菌（1株）、糠秕马拉色菌（1株）、季也蒙念珠菌（5株）、未知丝状真菌（6株）、无绿藻（1株）、烟曲霉（2株）、拟青霉菌（1株）、茎点霉（1株）。用溶细胞酶（lyticase）结合Biospin真菌基因组DNA提取试剂盒提取基因组DNA,A260／A280检测纯度并计算质量浓度,用真菌通用引物ITS1／ITS4扩增真菌核糖体基因（rDNA）内转录间区ITS基因,经PCR扩增检验所提取的DNA质量。结果 成功提取所有23株真菌基因组DNA,其纯度及质量浓度能满足PCR反应的要求。结论 用溶细胞酶结合Biospin真菌基因组DNA提取试剂盒从酵母菌、无绿藻及丝状真菌提取的DNA可用于PCR反应。     

Natural plasmid transformation in a high-frequency-of-transformation marine Vibrio strain

The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 X 10(-9) and 3.4 X 10(-7) transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42, 857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 X 10(-8) to 1.3 X 10(-4) transformants per recipient with plasmid DNA and at an average frequency of 8.3 X 10(-5) transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [3H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations.     

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).     

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.     

Natural plasmid transformation in a high-frequency-of-transformation marine Vibrio strain.

The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 X 10(-9) and 3.4 X 10(-7) transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42, 857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 X 10(-8) to 1.3 X 10(-4) transformants per recipient with plasmid DNA and at an average frequency of 8.3 X 10(-5) transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [3H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations.     

Differentiation of mosquito-pathogenic strains of Bacillus sphaericus from non-toxic varieties by ribosomal RNA gene restriction patterns.

DNA from 17 strains of Bacillus sphaericus, including representatives of all the established DNA homology groups, was cleaved with EcoRI or HindIII and fragments were separated by agarose gel electrophoresis. Southern blots of this DNA were hybridized to a radioactively labelled DNA probe prepared from the cloned 16S rrnB ribosomal RNA operon of Escherichia coli. Banding patterns of the chromosomal DNA digests and the autoradiograms were specific to DNA homology groups I (B. sphaericus sensu stricto), IIA (mosquito-pathogenic strains), IIB (B. fusiformis) and V, but groups III and IV were not clearly distinguished. This suggests that the mosquito-pathogenic strains represent a separate subspecies.     

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.     

Role of bacteriophages in genomic variability of related coagulase-negative staphylococci

Abstract DNA analysis using pulsed-field gel electrophoresis (PFGE) has emerged as one of the most sensitive epidemiological tools for the characterization of coagulase-negative staphylococci (CNST). The significance of some minor differences observed between the DNA restriction pulsed patterns of two CNST strains are difficult to interpret since they can theoretically be due to minor chromosomal rearrangements or to phage DNA integration. The latter possibility was investigated by comparing DNA restriction patterns of Staphylococcus epidermidis strains with those of their lysogenized derivatives. In vitro lysogenisation was obtained by exposing the strains to phage 118II. The pulsed patterns of the lysogenized strains were compared to those of their parental strains, revealing a shift in size of approximately 50 kb in a single band which was shown by Southern blotting to contain prophage. One strain was lysogenized ten times, revealing a potential preferref attachment site for phage 118II. These results confirm that chromosomal integration of a phage can be responsible for minor stanle variations in DNA restriction patterns.