Identification of Thai isolates assigned to the genus Gluconobacter based on 16S-23S rDNA ITS restriction analysis

Forty-four Thai isolates phenotypically assigned to the genus Gluconobacter were examined for 16S-23S rDNA ITS restriction analysis by MboII and SduI (=Bsp1286I) digestions. The Thai isolates tested were divided into seven groups: Group I for fourteen isolates, Group IX for one isolate, Group X for two isolates, Group V-2 for four isolates, Group XI for three isolates, Group IV for one isolate, and Group III for nineteen isolates. There were no isolates of either Group II or Group V-1 that were identified as G. cerinus. The isolates of Group III, Group IV, and Group XI were subjected to an additional 16S-23S rDNA ITS restriction analysis by AvaII, TaqI, BsoBI, and BstNI digestions. The isolates of Group III were divided into three groups and two subgroups: Group III-2 for five isolates, Group III-6 for two isolates, and Group III-4, which was divided into two subgroups, Subgroup III-4a for four isolates and Subgroup III-4b for eight isolates. The fourteen isolates of Group I were identified as G. oxydans, and the two isolates of Group X were temporarily identified as G. oxydans. The five isolates of Group III-2 and the one isolate of Group IV were identified as G. frateurii. The remaining twenty-two isolates of Group V-2, Group III-4, Group III-6, Group IX, and Group XI were not identified but are candidates for several new species.     

Identification of Thiobacillus ferrooxidans strains based on restriction fragment length polymorphism analysis of 16S rDNA.

The 16S rDNA sequences from ten strains of Thiobacillus ferrooxidans were amplified by PCR. The products were compared by performing restriction fragment length polymorphism (RFLP) analysis with restriction endonucleases Alu I, Hap II, Hha I, and Hae III. The RFLP patterns revealed that T. ferrooxidans could be distinguished from other iron- or sulphur-oxidizing bacteria such as T. thiooxidans NB1-3, T. caldus GO-1, Leptospirillum ferrooxidans and the marine iron-oxidizing bacterium strain KU2-11. The RFLP patterns obtained with Alu I, Hap II, and Hae III were the same for nine strains of T. ferrooxidans except for strain ATCC 13661. The RFLP patterns for strains NASF-1 and ATCC 13661 with Hha I were distinct from those for other T. ferrooxidans strains. The 16S rDNA sequence of T. ferrooxidans NASF-1 possessed an additional restriction site for Hha I. These results show that iron-oxidizing bacteria isolated from natural environments were rapidly identified as T. ferrooxidans by the method combining RFLP analysis with physiological analysis.     

Identification of strains assigned to the genus Gluconobacter Asai 1935 based on the sequence and the restriction analyses of the 16S-23S rDNA internal transcribed spacer regions

Thirteen reference strains, including the type strains of the type species of the genus Gluconobacter, Gluconobacter oxydans (NBRC 14819T), Gluconobacter cerinus (NBRC 3267T), and Gluconobacter frateurii (IFO 3264T) were examined for their species identification based on the sequence and the restriction analyses of the 16S-23S rDNA internal transcribed spacer (ITS) regions. A phylogenetic tree constructed by the neighbor-joining method represented three clusters corresponding respectively to the three species, G. oxydans, G. cerinus, and G. frateurii. The type strain of Gluconobacter asaii (NBRC 3276T), which is a junior subjective synonym of G. cerinus, was included completely in the G. cerinus cluster. Several restriction endonucleases discriminating the three species from one another were selected by computer analyses: Bsp1286I, MboII, SapI, Bpu10I, EarI, BsiHKAI, and FatI. On digestion of the PCR products with restriction endonucleases Bsp1286I and MboII, all the restriction patterns coincided with those of the type strains of the three species except for strain NBRC 3251. This strain gave a different pattern from the type strain of G. frateurii, when digested with MboII. However, strain 3251 was included phylogenetically in the G. frateurii cluster. All the reference strains were thus identified at the species level by the sequence and the restriction analyses of the 16S-23S rDNA ITS regions.     

Heterogeneity of strains assigned to Gluconobacter frateurii Mason and Claus 1989 based on restriction analysis of 16S-23S rDNA internal transcribed spacer regions

Twenty-three strains, which were assigned to Gluconobacter frateurii and maintained at Culture Collection NBRC, were re-identified at the species level on the basis of restriction analysis of 16S-23S rDNA ITS regions by digestion with six restriction endonucleases: Bsp1286I, MboII, AvaII, TaqI, BsoBI, and BstNI. The strains examined were divided into six groups, Group III-1, Group III-2, Group III-3, Group III-4, Group III-5, and Group IV. Group III-1 and Group III-4 respectively were divided into two subgroups, Subgroup III-1a, Subgroup III-1b and Subgroup III-4a, Subgroup III-4b. Gluconobacter frateurii NBRC 3264(T) was included in Group III-2, along with strains NBRC 3265 and NBRC 3270, and G. thailandicus BCC 14116(T) was included in Group III-3, along with strains NBRC 3254, NBRC 3256, NBRC 3258, NBRC 3255, and NBRC 3257. These groupings were supported by a phylogenetic tree based on 16S-23S rDNA ITS sequences. Strains of group III-2 and Group IV were unequivocally re-identified as G. frateurii, but strains of Group III-3, Group III-4, and Group III-5 were not necessarily re-identified as G. frateurii. The results obtained indicate that the 23 strains have a taxonomically heterogeneous nature, and they are referred to as the G. frateurii complex.     

Re-identification of Gluconobacter strains based on restriction analysis of 16S-23S rDNA internal transcribed spacer regions

Thirty Gluconobacter strains maintained at Culture Collection NBRC were re-identified at the species level on the basis of restriction analysis of 16S-23S rDNA internal transcribed spacer (ITS) regions by digestion with two restriction endonucleases MboII and Bsp1286I. The strains examined were divided into seven groups, designated as Group I and Group III-VIII, by the combination of the restriction patterns obtained with the two restriction endonucleases. Group I included seven strains, which gave "G. oxydans patterns" with the two restriction endonucleases and were re-identified as G. oxydans. Group III included 12 strains, which gave "G. frateurii patterns" and were re-identified as G. frateurii. Group IV included six strains, which gave "G. cerinus pattern" with MboII and "G. frateurii pattern" with Bsp1286I and were re-identified as G. frateurii. Group V included one strain (NBRC 3274), which gave respectively "G. frateurii pattern" and "G. cerinus pattern" and was re-identified as G. cerinus. Group VI included one strain (NBRC 3990), which gave respectively "G. oxydans pattern" and an unidentified restriction pattern and was re-identified temporarily as G. oxydans. Group VII included two strains (NBRC 3250 and NBRC 3273), which gave respectively an unidentified restriction pattern and "G. oxydans pattern." Group VIII included one strain (NBRC 3266), which gave unidentified restriction patterns. The three strains of Group VII and Group VIII were suggested to constitute new taxa by sequencing of 16S-23S rDNA ITS regions.     

Phylogeny of the genus Azospirillum based on 16S rDNA sequence

Abstract The 16S rDNA of 17 strains of Azospirillum , 14 assigned to one of the known species A. amazonense A. brasilense A. halopraeferens A. irakense and A. lipoferum , and the other three of uncertain taxonomic position, was sequenced after polymerase chain reaction amplification and analysed in order to investigate the phylogenetic relationships at the intra-generic and super-generic level. The phylogenetic analysis confirms that the genus Azospirillum constitutes a phylogenetically separate entity within the a subclass of Proteobacteria and that the five species are well defined. A. brasilense and A. lipoferum are closely related species and form one cluster together with A. halopraeferens ; the pair of species A. amazonense and A. irakense forms a second cluster in which Rhodospirillum centenum is also placed.     

The phylogeny of the genus Yersinia based on 16S rDNA sequences

Abstract The unique antibacterial properties of Fe-protoporphyrin (haemin) on Staphylococcus aureus , compared to Co-protoprophyrin (Co-PP), Mg-protoporphyrin (Mg-PP) and Zn-protoporphyrin (Zn-PP) are described. Only haemin (20 μM) exhibits a strong light-independent antibacterial effect on S. aureus ; the other metalloporphyrins, Co-PP, Mg-PP or Zn-PP, have no antibacterial effect in the dark. Only light photosensitization of Mg-PP-treated cells resulted in the inhibition of the bacterial growth, while Co-PP or Zn-PP were photodynamically inactive. A notable effect of haemin on inactivation of S. aureus was the induction of immediate ion fluxes as determined by X-ray microanalysis (XRMA) of fast-frozen cells. A marked efflux of K (96%) and Cl (94%) was expressed immediately as determined by X-ray microanalysis of S. aureus cells treated with haemin for 5 min. Only 48% loss of Na was detected in the cells under these treatment conditions, while P content was increased by 150%. Electron microscopy analysis revealed the appearance of a mesosome-like structure connected to the new septa, filmentous chromosome and arrays of aggregated ribosomes in the cytoplasm. We propose that haemin has multiple cellular targets for its oxidative effect in S. aureus .     

16S rDNA sequence analysis of Xylella fastidiosa strains

The 16S rDNA encoding the small subunit ribosomal RNA were amplified by PCR, cloned, and sequenced from 16 strains of Xylella fastidiosa originating from nine different hosts. In pair-wise comparisons, X. fastidiosa strains showed a maximum variation of 1.0% or 14 nucleotide positions. When all 16 sequences were considered as a set, 54 variable positions were found. Analysis of the sequence data indicated that the X. fastdiosa strains formed three rDNA groups. Group one includes Pierce's disease and mulberry leaf scorch strains; Group two, periwinkle wilt, plum leaf scald, phony peach, oak leaf scorch, and elm leaf scorch strains; and Group three, citrus variegated chlorosis and coffee leaf scorch strains. All X. fastidiosa strains exhibited significantly higher levels of sequence heterogeneity (63 to 83 nucleotide positions) when compared to species from Xanthomonas and Stenotrophomonas. Our data demonstrate that 16S rDNA sequence data could provide valuable information for future classification of X. fastidiosa at the sub-species level.     

Genotypic diversity of oscillatoriacean strains belonging to the genera Geitlerinema and Spirulina determined by 16S rDNA restriction analysis

Genotypic diversity of several cyanobacterial strains mostly isolated from marine or brackish waters, belonging to the genera Geitlerinema and Spirulina, was investigated by amplified 16S ribosomal DNA restriction analysis and compared with morphological features and response to salinity. Cluster analysis was performed on amplified 16S rDNA restriction profiles of these strains along with profiles obtained from sequence data of five Spirulina-like strains, including three representatives of the new genus Halospirulina. Our strains with tightly coiled trichomes from hypersaline waters could be assigned to the Halospirulina genus. Among the uncoiled strains, the two strains of hypersaline origin clustered together and were found to be distant from their counterparts of marine and freshwater habitat. Moreover, another cluster, formed by alkali-tolerant strains with tightly coiled trichomes, was well delineated.     

Identification of Azospirillum strains by restriction fragment length polymorphism of the 16S rDNA and of the histidine operon

Abstract DNA fingerprints of several Azospirillum strains, belonging to the five known species A. amazonense, A. brasilense, A. halopraeferens, A. irakense and A. lipoferum , were obtained by restriction analysis of the amplified 16S rDNA and by restriction fragment length polymorphism of the histidine biosynthetic genes. Data obtained showed that amplified rDNA restriction analysis is an easy, fast, reproducible and reliable tool for identification of Azospirillum strains, mainly at the species level, whereas restriction fragment length polymorphism could, in some cases, differentiate strains belonging to the same species. Moreover, both analyses gave congruent results in grouping strains and in the assignment of new strains to a given species.     

Identification ofEnterococcus sp. from midgut of silkworm based on biochemical and 16S rDNA sequencing analysis

Enterococcus sp. was isolated from the midgut of silkworm against the germination ofNosema bombycis spores. Identification was based on the biochemical characteristics, 16S rDNA sequences analysis and species-specific probes ofEnterococcus spp. The isolated strains fermented sorbitol and arabinose but did not ferment raffinose.Enterococcus sp. was clustered together withEnterococcus mundtii ATCC 43188 and 100% sequence homology was found by 16S rDNA sequences BLAST analysis and constructing the phylogenetic tree. Comparison of the sequences of the 16S rDNA species-specific probes ofEnterococcus spp. with the 16S rDNA sequence of isolate revealed similar segment to the species-specific probe ofE. mundtii. So, we can make conclusion the 16S rDNA segment ofEnterococcus sp. can hybridise with species-specific probe ofE. mundtii. Enterococcus mundtii was detected for the first time in the intestine of silkworm.     

Identification of Enterococcus spp. based on specific hybridisation with 16S rDNA probes

The conventional methods for routine enterococci species identification are usually based on phenotypic characteristics. However, in recent years, some studies have defined specific probes based on both 16S and 23S rRNA genes for the identification of some Enterococcus spp. A set of probes based on the 16S rRNA gene has been developed in order to evaluate the usefulness of a six-step biochemical key for species level identification of enterococci. Probe specificity has been evaluated with type collection and environmental strains by dot blot hybridisation. A high correlation was obtained between biochemical key and hybridisation identifications. This set of probes provides a confirmative method for phenotypic species identification.     

16SrDNA同源性所揭示的双歧杆菌与有关细菌的亲缘关系

本研究测定了低ＧＣ含量的双歧杆菌（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ ｉｎｏｐｉｎａｔｕｍ）和新种Ｂ。ｔｈｅｒｍｏｃｉ．ｄｏｐｈｉｌｕｍ的１６ＳｒＤＮＡ全序列,在同另外１９个双歧杆菌及８个相关细菌的１６ＳｒＮＡ同源笥分析的基础上构建了系统发育树。结果表明：除低ＧＣ含蜈的Ｂ．ｉｎｏｐｉｎａｔｕｍ外,所有双杆菌的种在１６ＳｒＤＮＡ序列相似性≥８２％的水平上聚类为一个簇群。尽管Ｂ．ｉｎｏｐｉｎａｉｕｍ与其它     

Structure analysis of 16S rDNA sequences from strains of Acidithiobacillus ferrooxidans

Four strains of Acidithiobacillus ferrooxidans with different iron oxidation capacity were isolated from different mine drainage stations. The 16S rRNA gene of these strains were cloned and sequenced. Based on our sequences analysis on the four strain and the data on the other strains deposited in Genbank, all A. ferrooxidans may be classified into three phylogenetic groups. The analysis data showed that nucleotide variables (signature sites) were detected in 21 positions, and most of them were found in the first 800 bp from 5' terminal except position 970 and 1375. Interestingly, the first 13 signature sites were located in two main regions: the first region (position 175-234) located in V2 while the second region (position 390-439) were detected in constant region between V2 and V3. Furthermore, the secondary structure and minimal free energy were determined in two regions among strains of three groups. These results may be useful in characterizing the microevolutionary mechanisms of species formation and monitoring in biohydrometallurgical application.     

Evolutionary relationships among Magnetospirillum strains inferred from phylogenetic analysis of 16S rDNA sequences.

We have investigated the evolutionary relationships between two facultatively anaerobic Magnetospirillum strains (AMB-1 and MGT-1) and fastidious, obligately microaerophilic species, such as Magnetospirillum magnetotacticum, using a molecular phylogenetic approach. Genomic DNA from strains MGT-1 and AMB-1 was used as a template for amplification of the genes coding for 16S rRNA (16S rDNA) by the polymerase chain reaction. Amplified DNA fragments were sequenced (1,424 bp) and compared with sequences for M. magnetotacticum MS-1 and Magnetospirillum gryphiswaldense MSR-1. Phylogenetic analysis of the aligned 16S rDNA sequences indicated that the two new magnetic spirilla, AMB-1 and MGT-1, lie within the alpha subdivision (alpha-1) of the eubacterial group Proteobacteria and are closely related to Rhodospirillum fulvum and to several endosymbiotic bacteria. Strains AMB-1, MGT-1, and MS-1 formed a cluster, termed group I, in which they were more closely related to each other than to group II, which contained M. gryphiswaldense MSR-1. Group I strains were also physiologically distinct from strain MSR-1. Sequence alignment studies allowed elucidation of genus-specific regions of the 16S rDNA, and oligonucleotide primers complementary to two of these regions were used to develop a specific polymerase chain reaction assay for detection of magnetic spirilla in natural samples.