Five strains of thermotolerant methylotrophic yeasts isolated in Thailand were found to represent three new species in the genera Pichia and Candida, based on phylogenetic analysis of D1/D2 domain of 26S rDNA, in addition to the morphological, physiological, biochemical and chemotaxonomic characterization. Three strains, FS96 and FS101 from flowers and M02 from tree flux, were characterized by ubiquinone Q7, multilateral budding, and the formation of hat-shaped ascospores that are liberated at maturation. These strains showed identical nucleotide sequences in the D1/D2 domain and formed a cluster with Candida thermophila, “Pichia salicis” and Pichia angusta. They differed by 1.9% of nucleotide substitutions from Candida thermophila, the nearest species. They were considered to represent a single new species and are described as Pichia siamensis sp. nov. Two strains, N051 and S023, isolated from soil did not produce ascospores, proliferated by multilateral budding, did not demonstrate urease or DBB color reaction, and lacked sexual stages. These characteristics correspond to the genus Candida. Strains N051 and S023 differed by 2.8% and 1.9% of nucleotide substitutions in the D1/D2 domain from the nearest species, Candida nemodendra and Candida ovalis, respectively, and are considered to represent respective new species. N051 and S023 are described as Candida krabiensis sp. nov. and Candida sithepensis sp. nov., respectively.
Phylogenetic analyses of the 16S rRNA gene sequence indicate that the genus Derxia forms a distinct lineage in the β-Proteobacteria. On the NJ tree Derxia has a low bootstrap value (30.9%) with Alcaligeneceae, and on the ML tree it shows an independent cluster separated from other families. Moreover, there is below 93.4% 16S rDNA sequence similarity between genus Derxia and the genera of the β-Proteobacteria. These facts reveal that Derxia is not grouped with any known family of β-Proteobacteria and should be placed as a separate genus of β-Proteobacteria. The data on high G+C content (71 mol%), the cellular fatty acid composition, and the physiological characteristics of facultative hydrogen autotrophy and nitrogen fixation are unique for Derxia. The nifH gene sequence was found in this genus and phylogenetically compared among nitrogen-fixing bacteria to indicate that Derxia is clustered with the diazotrophs of β-Proteobacteria.
The Vibrio harveyi hemolysin gene (vhh), which encodes for a virulence factor involved in pathogenicity to fish and shellfish species, may be targeted for species detection or strain differentiation. Primers designed for this gene were used in detection studies of V. harveyi strains from various hosts. One primer set among four tested, could amplify the expected gene fragment in PCR using templates from all 11 V. harveyi strains studied. Detection of the presence of the hemolysin gene could therefore serve as a suitable detection marker of Vibrio harveyi isolates potentially pathogenic to fish and shrimps.
A taxonomic study was carried out on a bacterial strain designated as Jip2T isolated from a soil sample mixed with rotten rice straw. It was a Gram-negative, aerobic, motile, and rod-shaped bacterium. It grew well on nutrient agar medium and utilized a fairly narrow spectrum of carbon source. The G+C content of the genomic DNA was 65.3 mol%. The major ubiquinone was Q-8. The major fatty acids were branched fatty acids, especially large amounts of iso C15:0 and iso C17:1 ω9c were detected in the cells grown on TSA agar for 24 h. Comparative 16S rDNA study showed a clear affiliation of this bacterium to the genus Rhodanobacter. The 16S rDNA sequence of strain Jip2T showed 96.4% sequence similarity to that of Rhodanobacter lindaniclasticus RP5575T. On the basis of phenotypic characteristics and 16S rDNA sequence analysis, strain Jip2T is clearly distinct from Rhodanobacter lindaniclasticus. We propose the name Rhodanobacter fulvus sp. nov. for strain Jip2T (=IAM 15025T=KCTC 12098T).
The cell-free supernatant containing bacteriocin ST13BR, produced by Lactobacillus plantarum ST13BR, inhibits the growth of L. casei, Pseudomonas aeruginosa, Enterococcus faecalis, Klebsiella pneumoniae and Escherichia coli. Based on tricine-SDS-PAGE, bacteriocin ST13BR is 10 kDa in size. Complete inactivation or significant reduction in bacteriocin activity was observed after treatment with Proteinase K, trypsin and pronase, but not with catalase or α-amylase. Low bacteriocin activity (200 AU/ml) was recorded in BHI medium, M17 broth, 10% (w/v) soy milk, and 2% and 10% (w/v) molasses, despite good growth. Maximal bacteriocin activity (6,400 AU/ml) was recorded after 23 h in MRS broth, but only at 30°C. Tween 80 in MRS broth increased bacteriocin production by more than 50%. Meat extract or yeast extract as sole nitrogen source, or a combination of the two (1 : 1) in MRS broth, stimulated bacteriocin production (6,400 AU/ml). Only 50% activity (3,200 AU/ml) was recorded with tryptone as sole nitrogen source, whereas a combination of tryptone, meat extract and yeast extract yielded 6,400 AU/ml. Bacteriocin production was not stimulated by the addition of glucose at 2.0% w/v (3,200 AU/ml), nor 2% (w/v) fructose, sucrose, lactose or mannose, respectively (800 AU/ml). Activity levels less than 200 AU/ml were recorded in the presence of 0.05% to 0.5% (w/v) maltose. Maximal bacteriocin production (6,400 AU/ml) was recorded in the presence of 2% (w/v) maltose. Maltose at 4.0% (w/v) led to a 50% reduction of bacteriocin activity. The presence of 1.0% (w/v) and higher KH2PO4, or glycerol at 0.2% (w/v) suppressed bacteriocin production.
Four strains of acetic acid bacteria were isolated from flowers collected in Thailand. In phylogenetic trees based on 16S rRNA gene sequences and 16S–23S rDNA internal transcribed spacer (ITS) region sequences, the four isolates were located in the lineage of the genus Gluconobacter and constituted a separate cluster from the known Gluconobacter species, Gluconobacter oxydans, Gluconobacter cerinus, and Gluconobacter frateurii. In addition, the isolates were distinguished from the known species by restriction analysis of 16S–23S rDNA ITS region PCR products using three restriction endonucleases Bsp1286I, MboII, and AvaII. The DNA base composition of the isolates ranged from 55.3–56.3 mol% G+C. The four isolates constituted a taxon separate from G. oxydans, G. cerinus, and G. frateurii on the basis of DNA-DNA similarities. Morphologically, physiologically, and biochemically, the four isolates were very similar to the type strains of G. oxydans, G. cerinus, and G. frateurii; however, the isolates were discriminated in their growth at 37°C from the type strains of G. cerinus and G. frateurii, and in their growth on L-arabitol and meso-ribitol from the type strain of G. oxydans. The isolates showed no acid production from myo-inositol or melibiose, which differed from the type strains of the three known species. The major ubiquinone homologue was Q-10. On the basis of the results obtained, Gluconobacter thailandicus sp. nov. was proposed for the four isolates. The type strain is isolate F149-1T (=BCC 14116T=NBRC 100600T=JCM 12310T=TISTR 1533T=PCU 225T), which had 55.8 mol% G+C, isolated from a flower of the Indian cork tree (Millingtonia hortensis) collected in Bangkok, Thailand.
Strain improvement through random mutagenesis, screening and selection has provided us with spontaneous mutants which could produce more ML-236B than the original isolate, Penicillium citrinum SANK18767. The objective of the present study is to clarify how a high-producing mutant No. 41520 acquired the ability to produce 500 times more ML-236B than the original isolate on a molecular basis. Southern blot analysis and sequence comparison revealed that amplification of the ML-236B biosynthetic gene cluster and alteration of nucleotides within the loci had not occurred in the genome of No. 41520. On the other hand, a differential hybridization and Northern blot analysis showed that expression levels of the nine biosynthetic genes mlcA to mlcH and mlcR in No. 41520 increased greatly as compared to those in the original isolate. These data suggested that the increase in ML-236B production was partly due to increased expression of genes involved in ML-236B biosynthesis. Morphological differences and higher consumption of carbon source would also affect ML-236B production in No. 41520. Functional analysis revealed that a gene, orf1 next to mlcR, was not involved in the ML-236B biosynthesis, but it was involved in the transcriptional activation of genes along with the ML-236B gene cluster. Titer enhanced mutations might have occurred in the regulation system for transcription activation of the ML-236B biosynthetic genes in the mutants of P. citrinum.