DNA restriction endonuclease cleavage patterns,DNA sequence similarity and phenotypical characteristics in some strains of Lactobacillus helveticus and Lactobacillus jugurti

Physiological characteristics, deoxyribonucleic acid (DNA) base composition (% guanine + cytosine; % GC), DNA sequence similarity (% DNA-DNA hybridization) and DNA restriction endonuclease cleavage patterns of two strains of Lactobacillus helveticus and four strains of Lactobacillus jugurti were examined.All the strains investigated were closely related genetically, having DNA-DNA hybridization values ranging from 89–100%. Nevertheless, these strains can be differentiated from one another on the basis of the digestion of their DNA by specific restriction endonucleases, such as Bam HI, Eco RI and Hind III. The DNA of these strains shows clear, reproducible and distinct cleavage patterns. Cleavage patterns of DNA from strains L. jugurti S.35.19 and S.36.2 were found to be similar. These findings suggest that fingerprinting of DNA by restriction endonuclease cleavage might provide, in addition to the conventional methods, a useful tool for the characterization of closely related microorganisms at the strain level.     

Application of a colorimetric DNA-DNA hybridization method for identification ofAeromonas genomic species,isolated from diarrheal stools

The colorimetric DNA-DNA hybridization method for the identification of 18 strains ofAeromonas spp. isolated from human stools was used. Bacterial isolates were also examined by phenotypic characteristics. On the basis of biochemical tests 13 strains were included in phenogroupA. caviœ and 5 strains inA. sobria. Identification to the species level was obtained by colorimetric hybridization method. DNA-DNA similarity values showed that isolates ofA. caviœ group belong to hybridization group (HG) 4 whereas isolates ofA. sobria belong to HG 8/10. DNA relatedness results obtained by the colorimetric method showed good agreement with values detected by the spectrophotometric method. The background in the colorimetric method is lower than in the spectrophotometric one. Results of this study indicate the usefulness of the colorimetric DNA-DNA hybridization in microplates method for the identification ofAeromonas genomic species, isolated from human diarrheal stools.     

Three new species ofBullera isolated from leaves in the Ogasawara Islands

Thirteen strains of ballistoconidium-forming yeasts were isolated from leaves collected in the Ogasawara Islands, Japan. They represent three different species in the genusBullera on the basis of morphological, physiological, and biochemical characteristics, analyses of the sequences of internal transcribed spacer regions and small subunit ribosomal DNA, and a nuclear DNA-DNA hybridization study. Three new species,Bullera boninensis (five strains),B. waltii (seven strains), andB. schimicola (one strain), are proposed for these 13 strains.     

Novel Endophytes of Rice form a Taxonomically Distinct Subgroup of Serratia marcescens

Six endophytic strains isolated from surface-sterilized rice roots and stems of different rice varieties grown in the Philippines were characterized. They were analyzed by physiological and biochemical tests, SDS-PAGE of whole-cell protein patterns, DNA-DNA hybridization and 16S rDNA sequencing. SDS-PAGE of whole-cell patterns showed that the six isolates fell into two subgroups which were similar but not identical in protein patterns to S. marcescens. The phylogenetic analysis of 16S rDNA sequences of two representative strains IRBG 500 and IRBG 501 indicated that they were closely related to S. marcescens(more than 99% identity). Physiological and biochemical tests corroborated that the isolates were highly related to each other and to S. marcescens. In cluster analysis, all six isolates were clustered together at 93% similarity level and grouped closely with Serratia marcescens at 86% similarity level. DNA-DNA hybridization studies revealed that the isolates shared high similarity levels with S. marcescens(≥86% DNA-DNA binding), indicating they belong to the same species. However, the isolates differed in several biochemical characteristics from the type strain. They produce urease and utilize urea and L(+) sorbose as a substrate, which is different from all known Serratia reference strains. These results suggest that the six endophytic isolates represent a novel, non-pigmented subgroup of S. marcescens.     

Molecular characterization of Acidithiobacillus ferrooxidans and A. thiooxidans strains isolated from mine wastes in Brazil

Nineteen strains of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans, including 12 strains isolated from coal, copper, gold and uranium mines in Brazil, strains isolated from similar sources in other countries and the type strains of the two species were characterized together with the type strain of A. caldus by using a combination of molecular systematic methods, namely ribotyping, BOX- and ERIC-PCR and DNA-DNA hybridization assays. Data derived from the molecular fingerprinting analyses showed that the tested strains encompassed a high degree of genetic variability. Two of the Brazilian A. ferrooxidans organisms (strains SSP and PCE) isolated from acid coal mine waste and uranium mine effluent, respectively, and A. thiooxidans strain DAMS, isolated from uranium mine effluent, were the most genetically divergent organisms. The DNA-DNA hybridization data did not support the allocation of Acidithiobacillus strain SSP to the A. ferrooxidans genomic species, as it shared only just over 40% DNA relatedness with the type strain of the species. Acidithiobacillus strain SSP was not clearly related to A. ferrooxidans in the 16S rDNA tree.     

DNA base composition of some sheathed bacteria

The DNA base composition of five recently isolated Haliscomenobacter hydrossis strains were compared with those of Sphaerotilus natans, Leptothrix cholodnii and Leptothrix discophora. The DNA base composition of H. hydrossis strains ranged from 48.3 to 49.7% GC, whereas the % GC values of S. natans, L. cholodnii and L. discophora were found to be 69.7, 69.6 and 71.2, respectively. These results indicate that Haliscomenobacter species and the microorganisms of the Sphaerotilus-Leptothrix group should not be classified into the same genus, as they are genetically at best remotely related.The reallocation of some Leptothrix species into the genus Sphaerotilus is not in contradiction with their DNA base composition.     

Classification of the genus Humicola Traaen: II. The DNA base composition of some strains within the genus

The base composition of DNA (GC content) of 25 strains, morphologically referred to the genusHumicola Traaen, is studied. The range of GC% variation is about 23 % (from 28,5 % to 51,6). Two prominent groups of strains with similar GC content may be distinguished: one ranging from 28 % to 37 % and the other ranging from 40 % to 49 %. The aleuriospore size is not related to the DNA base composition, but a group of strains with prevalently coloured hyphae, with aleuriospores of similar size and with high GC content is recognized. Several previous literature reports on the taxonomy ofHumicola are discussed.     

A comparison of strains of King's group IIb of Flavobacterium with Flavobacterium meningosepticum

Twenty-two strains of Flavobacterium recently isolated from patients and other sources were compared with 6 strains of Flavobacterium meningosepticum and 3 strains of King's Flavobacterium group IIb. The field strains were found to resemble group IIb in their characteristics.All 31 strains of Flavobacterium gave similar results in 28 phenotypic tests; the DNA base compositions of 18 phenotypically representative strains ranged from 35 to 39% GC. Within this group, the 6 strains of F. meningosepticum were phenotypically homogeneous, had a % GC of 36.9, and differed consistently from the 25 strains of group IIb only in the pale colour of their pigment, slowness of pigment production, and inability to hydrolyse starch. All 25 strains of group IIb differed in at least 6 tests from the 6 strains of F. meningosepticum, although not the same 6 tests in each case. Antisera to F. meningosepticum agglutinated 10 strains of group IIb.     

Polyphasic characterization of water bloom forming Raphidiopsis species (cyanobacteria) from central China

Two strains of Raphidiopsis Fritsch et Rich were isolated from a fishpond in Wuhan city, China and rendered axenic, and characterized by a combination of morphological, physiological, biochemical and genetic methods. Morphologically the strains were identified as Raphidiopsis mediterranea Skuja (straight trichomes) and R. curvata Fritsch et Rich (coiled trichomes). These two strains demonstrated slight differences in optimal temperature range and GC content, while sharing some common characteristics including inability to grow hetertrophically, similar salinity tolerance (up to 0.78%) and an identical fatty acid composition. Cyanotoxins were not found in the strain of R. mediterranea, however, the strain of R. curvata contained both deoxy-cylindrospermopsin and cylindrospermopsin. Phylogenetic affiliations inferred from 16S rRNA gene sequences demonstrated that both Raphidiopsis strains clustered with Cylindrospermopsis, demonstrating their phylogenetic ties to Nostocaceae.     

Taxonomic study of extreme halophilic archaea isolated from the "Salar de Atacama", Chile

A large number of halophilic bacteria were isolated in 1984-1992 from the Atacama Saltern (North of Chile). For this study 82 strains of extreme halophilic archaea were selected. The characterization was performed by using the phenotypic characters including morphological, physiological, biochemical, nutritional and antimicrobial susceptibility test. The results, together with those from reference strains, were subjected to numerical analysis, using the Simple Matching (S(SM)) coefficient and clustered by the unweighted pair group method of association (UPGMA). Fifteen phena were obtained at an 70% similarity level. The results obtained reveal a high diversity among the halophilic archaea isolated. Representative strains from the phena were chosen to determine their DNA base composition and the percentage of DNA-DNA similarity compared to reference strains. The 16S rRNA studies showed that some of these strains constitutes a new taxa of extreme halophilic archaea.     

Nucleotide composition and homologies in the DNA of new extreme halophilic soil archaebacteria

DNA nucleotide composition was studied in extreme halophilic bacteria belonging to the genera Halobacterium, Halococcus, Natronobacterium and Natronococcus. The cultures were shown to be a monolithic group of microorganisms with the content of GC pairs typical of extreme halophilic archebacteria. The difference between the content of DNA major and minor components was twice as high in Halobacterium distributus strains isolated from sulfate saline soils as compared to cultures of this species isolated from natural waters with a high salinity. DNA minor components were not found in haloalkalophilic microorganisms from soda saline soils in contrast to those from soda lakes. The results of DNA-DNA hybridization indicate that the Halobacterium genus is highly heterogeneous. The newly isolated strains of extremely halophilic H. distributus are characterized by the low homology of their DNAs both among themselves and with other species of the genus. However, the hybridization data for the collection strains H. vallismortis 1398 and H. halobium 996 from the National Collection of Microorganisms are indicative of a high homology (80-100%) which is not characteristic of cultures belonging to different species. These results as well as some phenotypical properties of H. vallismortis 1398 different from those of this species type strain support the data reported in the literature about the genetic instability of extreme halophilic archebacteria. The analysis of homologies in DNA nucleotide sequences may be used to study the taxonomy of extreme halophilic archebacteria.     

DNA base composition and taxonomy of someAcinetobacter strains

The DNA base composition of a group of numerically analyzed acinetobacters was determined. Seven strains ofAcinetobacter anitratus appear to form a homogeneous group with S>90% and ca. 42.1 % GC.A. Iwoffii is less homogeneous. Most of the strains have around 46.6 % GC and a broad compositional distribution with 2=3.8 C. Two other small groups have a % GC of 44.8±0.3 and 42.1±1 % GC. The DNA characteristics of the latter group are indistinguishable fromA. anitratus-DNA. ThreeAlcaligenes faecalis strains have 58.8 % GC. Numerical analysis suggests that they might be related toBordetella bronchiseptica, but the % GC of the latter (69.5) shows that the relationship is only remote.     

A comparison of interfertility and in vitro DNA-DNA reassociation as criteria for speciation in the genus Kluyveromyces

Hybridization studies based on the use of the prototrophic selection technique were undertaken to compare interfertility and DNA-DNA reassociation as criteria for speciation purposes in the yeast genus Kluyveromyces. Degrees of DNA-DNA reassociation > 70% between strains as reported in the literature, were found to correlate with high recombination frequencies. Degrees of DNA-DNA reassociation < 20% between strains did however, not invariably coincide with the absence of interfertility between strains. If interfertility is accepted as criterion for conspecificity, the hitherto reported low degrees ( < 20%) of DNA-DNA reassociation in Kluyveromyces cannot confidently be employed for speciation purposes.     

Chemical Composition and Antimicrobial Activity of Essential Oils from Scabiosa arenaria Forssk. Growing Wild in Tunisia

The essential oils isolated from three organs, i.e., fruits, stems and leaves, and flowers, of the endemic North African plant Scabiosa arenaria Forssk . were screened for their chemical composition, as well as their possible antibacterial, anticandidal, and antifungal properties. According to the GC‐FID and GC/MS analyses, 61 (99.26% of the total oil composition), 79 (98.43%), and 51 compounds (99.9%) were identified in the three oils, respectively. While α‐thujone (34.39%), camphor (17.48%), and β‐thujone (15.29%) constituted the major compounds of the fruit oil, chrysanthenone (23.43%), together with camphor (12.98%) and α‐thujone (10.7%), were the main constituents of the stem and leaf oil. In the case of the flower oil, also chrysanthenone (38.52%), camphor (11.75%), and α‐thujone (9.5%) were identified as the major compounds. Furthermore, the isolated oils were tested against 16 Gram‐positive and Gram‐negative bacteria, four Candida species, and nine phytopathogenic fungal strains. It was found that the oils exhibited interesting antibacterial and anticandidal activities, comparable to those of thymol, which was used as positive control, but no activity against the phytopathogenic fungal strains was observed.     

Relationships among isolates of oral haemophili as determined by DNA-DNA hybridization

In order to assess the relationships among strains of the genera Actinobacillus and Haemophilus, DNAs from 50 strains of these genera were isolated and purified. The guanine plus cytosine (G+C) content of DNAs from strains of Haemophilus segnis and Haemophilus para-influenzae were determined by thermal denaturation. DNA-DNA homologies were measured using labelled probes from one strain representing Haemophilus segnis (strain ATCC 10977), and two strains representing Haemophilus parainfluenzae (strains ATCC 9796 and ATCC 7901). Strains isolated as H. segnis had a G+C content of 39.0 to 42.9% and were 49–92% homologous with the ATCC 10977 DNA probe. All of the strains freshly isolated as H. parainfluenzae were 70–81% homologous with the ATCC 9796 DNA probe and had a G+C content of 34.9 to 38.3%. Strain ATCC 7901 was 11% homologous with the ATCC 9796 DNA probe, had a G+C content of 42.4%, and was 65–78% homologous to DNA from strains identified as Haemophilus aphrophilus and Haemophilus paraphrophilus. From these results we conclude that strain ATCC 7901 is a mislabelled strain of H. paraphrophilus. The results of multiple DNA-DNA hybridizations indicated that separate species designations were appropriate for H. segnis, H. parainfluenzae, Actinobacillus actinomycetemcomitans (Haemophilus actinomycetemcomitans), and H. aphrophilus. H. aphrophilus and H. paraphrophilus were closely related organisms and did not fulfill the generally accepted criteria for designation as separate species.     

A New Enzyme,d-Fructose Dehydrogenase

Genetic relatedness of 14 yeast strains and 2 mold strains was studied by the DNA-DNA hybridization method. The hybridization was performed between mitochondrial-DNA-free, ³²p-labeled DNA of Saccharomyces cerevisiae IAM 4009 and cold DNA of other strains. The DNA homology indices deviated considerably even among S. cerevisiae strains having similar GC contents, but, in general, yeast strains known to be able to mate with S. cerevisiae, showed high homology indices (35∽70%). Other species of Saccharomycetaceae and 6 asporogenous yeast strains exhibited values of 10∽20%. The relatedness suggested from these results was confirmed by the competition experiments and also by the hybridization with ³²P-DNA of Candida pulcherrima IFO 0561. DNA’s of Aspergillus oryzae I and Neurospora crassa IFO 6067 also exhibited low but appreciable homology indices (5∽7%). These results were discussed from the aspects of phylogenetics and also of gene conservation in microorganisms.     

Deoxyribonucleic acid relatedness among strains of the moderately halophilic speciesDeleya halophila

The genomic relatedness among 16 strains assigned to the moderately halophilic speciesDeleya halophila and other 20 representative strains of halophilic and nonhalophilic species was estimated by determination of deoxyribonucleic acid (DNA) base composition and by DNA-DNA hybridization studies. The guanine-plus-cytosine (G + C) base contents, determined from the melting temperature of DNAs ofD. halophila strains, were 66.0–68.8 mol %. DNA-DNA homology studies, determined by membrane filter technique, indicate that the 16 strains ofD. halophila comprise a genetically homogeneous group. High homology (70–100%) was obtained between the type strainD. halophila CCM 3662 and the otherD. halophila strains studied; however, very low DNA relatedness was found between the representative strains ofD. halophila and otherDeleya species (13-0%), as well as other moderately halophilic, marine, or nonhalophilic bacteria investigated.     

Genetic relationships among clinical and environmental isolates ofVibrio cholerae from Australia

DNA-DNA homology among twenty-nine isolates having the phenotypic properties ofVibrio cholerae was studied using the S1 endonuclease method. Ten strains ofV. cholerae O1 isolated from patients and from the environment in Australia showed greater than 88% homology with the neotype strain ofV. cholerae NCTC 8021. Strains of the non-O1 serotype isolated from a variety of clinical and environmental sources also showed a high level of relatedness, including four luminescent strains and a reference strain of the biotypealbensis. A group of sucrose-negative strains showed a low level of homology (40 to 43%) withV. cholerae, but 75% and 82% homology within the group.     

Isolation of bacteria of the genus <Emphasis Type="Italic">Variovorax</Emphasis> from the <Emphasis Type="Italic">Thioploca</Emphasis> mats of Lake Baikal

Three strains of gram-negative bacteria were isolated from the mats of colorless sulfur bacteria Thioploca (Lake Baikal). The cells of new strains are motile with peritrichous flagella. Bacteria are aerobic, obligate chemoorganoheterotrophs growing within the pH range of 3.0–8.8 with the optimum at 8.3 and within the temperature range of 5–42°C with the optimum at 28°C. The cells contained menaquinones MK-8 H2 as the major component, as well as MK-7 H2 (less than 15%), while the content of ubiquinone Q8 was at least an order of magnitude lower. The G+C content of DNA in the new strains varied from 67.4 to 69.9 mol %. The level of DNA-DNA hybridization between the strains ranged from 80 to 94%, indicating that all the isolates belonged to one species. Analysis of the 16S rRNA gene nucleotide sequences of the type strain (Gen-Bank HQ400611) revealed close homologues among the known species of the genus Variovorax: 98% resemblance with the type strains of the species V. paradoxus, V. soli, V. ginsengisoli, and V. boronicumulans and 96% similarity with the type strain of V. dokdonensis. However, since the isolates differed significantly in the composition of fatty acids and isoprenoid quinones from the nearest neighbors in the phylogenetic tree, they cannot be related implicitly to the known species.     

Enterococcus seriolicida Is a Junior Synonym of Lactococcus garvieae,a Causative Agent of Septicemia and Meningoencephalitis in Fish

The reference strains of Enterococcus seriolicida (ATCC 49156^T) (T = type strain) and of Lactococcus garvieae (ATCC 43921^T) and 30 field strains of Gram-positive cocci isolated from diseased rainbow trout in Italy were found to be phenotypically (API 20 STREPT and API 50 CH) and genetically (DNA-DNA hybridization) similar. The high DNA-DNA homologies (70–100%) and the low ΔT_m(e) (less than 1.1°C) among these strains showed that Enterococcus seriolicida and Lactococcus garvieae are synonyms, describing a single bacterial species. E. seriolicida strains should be classified as L. garvieae, which must be considered as a major pathogen of freshwater and salt water fish with a world-wide distribution.