Luteimonas granuli sp. nov., Isolated from Granules of the Wastewater Treatment Plant.

A Gram-reaction negative, aerobic, non-motile, light yellow colored, and rod-shaped bacterium (designated Gr-4T) isolated from granules of a wastewater treatment plant, was characterized by a polyphasic approach to clarify its taxonomic position. Strain Gr-4T was observed to grew optimally at 30 ºC and at pH 7.0 on R2A medium. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain Gr-4T belongs to the genus Luteimonas of the family Xanthomonadaceae and was most closely related to Luteimonas padinae CDR SL 15T (99.1%), Luteimonas terricola DSM 22344T (98.5%) and Luteimonas arsenica 26-35T (97.6). The genome comprises 2,917,404 bp with a G+C content of 70.5 mol%. The ANI value between strain Gr-4T and Luteimonas padinae CDR SL 15T was 87.3%. The DNA-DNA relatedness value between strain Gr-4T and Luteimonas padinae CDR SL 15T, Luteimonas terricola DSM 22344T was 36.4 ± 1.3% and 14.2 ± 1.7%, respectively. The predominant quinone was Q-8. The major fatty acids were iso-C15:0, iso-C16:0 and summed feature 9 (comprising iso-C17:1ω9c and/or C16:0 10-methyl) supported the affiliation of strain Gr-4T to the genus Luteimonas. Moreover, the physiological, biochemical results, and low level of ANI and DNA-DNA relatedness value allowed the phenotypic and genotypic differentiation of strains Gr-4T from other Luteimonas species with validly published names. The novel isolate therefore represents a novel species, for which the name Luteimonas granuli sp. nov. is proposed, with the type strain Gr-4T (=KACC 16614T = JCM 18203T).

Lysobacter lacus sp. nov., isolated from from lake sediment.

An aerobic and Gram-stain-negative bacterial strain, designated UKS-15T, was isolated from lake water in the Republic of Korea. Results of 16S rRNA gene sequence and phylogenetic analyses indicated that the novel isolate belongs to the genus Lysobacter and was most closely related to Lysobacter xinjiangensis RCML-52T (98.0 %), Lysobacter mobilis 9 NM-14T (97.4 %) and Lysobacter humi FJY8T (97.2 %). The DNA G+C content was 69.1 mol%. Strain UKS-15T possessed ubiquinone-8 (Q-8) as the sole respiratory quinone and the fatty acid profile comprised iso-C15 : 0, iso-C17 : 0 and summed feature 9 (iso-C17 : 1 ω9c and/or C16 : 0 10-methyl) as its major components. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and one unidentified aminophospholipid. Moreover, the physiological and biochemical results and low level of DNA-DNA relatedness (<22.0 %) allowed the phenotypic and genotypic differentiation of strain UKS-15T from other Lysobacter species. Therefore, on the basis of the data from this polyphasic taxonomic study, strain UKS-15T should represent a novel species of the genus Lysobacter, for which the name Lysobacter lacus sp. nov. is proposed. The type strain is UKS-15T (=JCM 30983T=KACC 18719T).

Actinomadura hankyongense sp. nov. Isolated From Soil of Ginseng Cultivating Field.

A Gram-positive, rod-shaped, non-spore-forming, and aerobic bacterium (Gsoil 556T) was isolated from soil of a ginseng field and subjected to its taxonomic position. Based on 16S rRNA gene sequence similarity, strain Gsoil 556T was shown to belong to the genus Actinomadura of the family Thermomonosporaceae and was closely related to A. montaniterrae CYP1-1BT (99.3%), A. nitritigenes DSM 44137T (98.7%), and A. rudentiformis HMC1T (98.5%), while it showed less than 98.4% sequence similarity to the other species of this genus. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that it is most closely related to A. rudentiformis HMC1T and A. nitritigenes DSM 44137T. The DNA G+C content was 73.1 mol%. The peptidoglycan was meso-diaminopimelic acid and the whole-cell sugar contained fucose, galactose, glucose, mannose, and ribose. The predominant menaquinone (KK) was MK-9(H8) [55%] and MK-9(H6) [45%]. The major cellular fatty acids were C14:0, C16:0, C18:1 ω9c and summed feature 3 (C16:1 ω6c/C16:1 ω7c). All these data supported the affiliation of strain Gsoil 556T to the genus Actinomadura. The DNA-DNA hybridization between strain Gsoil 556T and its phylogenetically closest relatives were less than 40%. Furthermore, the results of physiological and biochemical tests enabled strain Gsoil 556T to be differentiated genotypically and phenotypically from currently known Actinomadura species. Therefore, strain Gsoil 556T represents a novel species of the genus Actinomadura, for which the name Actinomadura hankyongense sp. nov. is proposed. The type strain Gsoil 556T (=KACC 19438T=LMG 30327T).

Effects of Increased NADPH Concentration by Metabolic Engineering of the Pentose Phosphate Pathway on Antibiotic Production and Sporulation in Streptomyces lividans TK24.

Most of the biosynthetic pathways for secondary metabolites are influenced by carbon metabolism and supply of cytosolic NADPH. We engineered carbon distribution to the pentose phosphate pathway (PPP) and redesigned the host to produce high levels of NADPH and primary intermediates from the PPP. The main enzymes producing NADPH in the PPP, glucose 6-phosphate dehydrogenase (encoded by zwf1 and zwf2) and 6-phosphogluconate dehydrogenase (encoded by zwf3), were overexpressed with opc encoding a positive allosteric effector essential for Zwf activity in various combinations in Streptomyces lividans TK24. Most S. lividans transformants showed better cell growth and higher concentration of cytosolic NADPH than those of the control, and S. lividans TK24/pWHM3-Z23O2 containing zwf2+zwf3+opc2 showed the highest NADPH concentration but poor sporulation in R2YE medium. S. lividans TK24/pWHM3-Z23O2 in minimal medium showed the maximum growth (6.2 mg/ml) at day 4. Thereafter, a gradual decrease of biomass and a sharp increase of cytosolic NADPH and sedoheptulose 7-phosphate between days 2 and 4 and between days 1 and 3, respectively, were observed. Moreover, S. lividans TK24/pWHM3-Z23O2 produced 0.9 times less actinorhodin but 1.8 times more undecylprodigiosin than the control. These results suggested that the increased NADPH concentration and various intermediates from the PPP specifically triggered undecylprodigiosin biosynthesis that required many precursors and NADPH-dependent reduction reaction. This study is the first report on bespoke metabolic engineering of PPP routes especially suitable for producing secondary metabolites that need diverse primary precursors and NADPH, which is useful information for metabolic engineering in Streptomyces.

[Production of Antibody against Helicobacter pylori HP0231.].

BACKGROUND: Stool antigen detection kits for diagnosis of infection of Helicobacter pylori have been widely used for their convenience, but are mostly imported. Since Helicobacter pylori strains show a distinctive genetic diversity, it is important to find a protein that is a common antigen among various strains and shows a strong immunogenicity for the development of a stool antigen detection kit. HP0231 protein strongly reacts with the sera of patients suffering from gastritis and peptic ulcer. Therefore, HP0231 is an excellent candidate as a target gene for this study. METHODS: Chromosomal DNA from H. pylori was isolated. HP0231 gene was amplified by PCR, cloned into pET28a(+) vector, and overexpressed using isopropyl-beta-D-thiogalactopyranoside in E. coli BL21 (DE3). HP0231 protein was purified by Ni-NTA affinity chromatography followed by electroelution after SDS-PAGE. Rabbits were immunized with the purified HP0231 protein for the production of antibodies. Rabbit anti-HP0231 antibody was partially purified and tested for the sensitivity and specificity using ELISA and Western Blot Analysis. RESULTS: The sequence of the cloned HP0231 gene was identical with the gene sequence from Genbank (AA216016). HP0231 gene was overexpressed and HP0231 protein was purified. Rabbit anti-HP0231 antibody produced after immunization with the purified HP0231 protein reacted with the purified HP0231 protein, cell extracts from cultured H. pylori, and stomach biopsy tissue from patients, but not with cell extracts from cultured E. coli used as a negative control. After 1 million fold dilution, rabbit anti-HP0231 antibody still reacted with 1 microg of HP0231 protein. CONCLUSIONS: Rabbit anti-HP0231 antibody was produced to detect HP0231 protein of H. pylori and will be tested for the development of a stool antigen detection kit for H. pylori.

Sensitization of vanilloid receptor involves an increase in the phosphorylated form of the channel.

A vanilloid receptor (VR1, now known as TRPV1) is an ion channel activated by vanilloids, including capsaicin (CAP) and resiniferatoxin (RTX), which are pungent ingredients of plants. Putative endogenous activators (anandamide and metabolites of arachidonic acid) are weak activators of VR1 compared to capsaicin and RTX, and the concentrations of the physiological condition of those activators are not sufficient to induce significant activation of VR1. One way to overcome the weak activation of endogenous activators would be the sensitization of VR1, with the phosphorylation of the channel being one possibility. The phosphorylation of VR1 by several kinases has been reported, mostly by indirect evidence. Here, using an in vivo phosphorylation method, the VR1 channel was shown to be sensitized by phosphorylation of the channel itself by multiple pathways involving PKA, PKC and acid. Also, in sensitizing VR1, BK appeared to show activation of PKC for the sensitization of VR1 by phosphorylation of the channel.