RepID represses megakaryocytic differentiation by recruiting CRL4A-JARID1A at DAB2 promoter.

BACKGROUND: Megakaryocytes (MKs) are platelet precursors, which arise from hematopoietic stem cells (HSCs). While MK lineage commitment and differentiation are accompanied by changes in gene expression, many factors that modulate megakaryopoiesis remain to be uncovered. Replication initiation determinant protein (RepID) which has multiple histone-code reader including bromodomain, cryptic Tudor domain and WD40 domains and Cullin 4-RING E3 ubiquitin ligase complex (CRL4) recruited to chromatin mediated by RepID have potential roles in gene expression changes via epigenetic regulations. We aimed to investigate whether RepID-CRL4 participates in transcriptional changes required for MK differentiation. METHODS: The PCR array was performed using cDNAs derived from RepID-proficient or RepID-deficient K562 erythroleukemia cell lines. Correlation between RepID and DAB2 expression was examined in the Cancer Cell Line Encyclopedia (CCLE) through the CellMinerCDB portal. The acceleration of MK differentiation in RepID-deficient K562 cells was determined by estimating cell sizes as well as counting multinucleated cells known as MK phenotypes, and by qRT-PCR analysis to validate transcripts of MK markers using phorbol 12-myristate 13-acetate (PMA)-mediated MK differentiation condition. Interaction between CRL4 and histone methylation modifying enzymes were investigated using BioGRID database, immunoprecipitation and proximity ligation assay. Alterations of expression and chromatin binding affinities of RepID, CRL4 and histone methylation modifying enzymes were investigated using subcellular fractionation followed by immunoblotting. RepID-CRL4-JARID1A-based epigenetic changes on DAB2 promoter were analyzed by chromatin-immunoprecipitation and qPCR analysis. RESULTS: RepID-deficient K562 cells highly expressing MK markers showed accelerated MKs differentiation exhibiting increases in cell size, lobulated nuclei together with reaching maximum levels of MK marker expression earlier than RepID-proficient K562 cells. Recovery of WD40 domain-containing RepID constructs in RepID-deficient background repressed DAB2 expression. CRL4A formed complex with histone H3K4 demethylase JARID1A in soluble nucleus and loaded to the DAB2 promoter in a RepID-dependent manner during proliferation condition. RepID, CRL4A, and JARID1A were dissociated from the chromatin during MK differentiation, leading to euchromatinization of the DAB2 promoter. CONCLUSION: This study uncovered a role for the RepID-CRL4A-JARID1A pathway in the regulation of gene expression for MK differentiation, which can form the basis for the new therapeutic approaches to induce platelet production. Video Abstract.

RepID represses megakaryocytic differentiation by recruiting CRL4A-JARID1A at DAB2 promoter.

Background Megakaryocytes (MKs) are platelet precursors, which arise from hematopoietic stem cells (HSCs). While MK lineage commitment and differentiation are accompanied by changes in gene expression, many factors that modulate megakaryopoiesis remain to be uncovered. Replication origin binding protein (RepID) which has multiple histone-code reader including bromodomain, cryptic Tudor domain and WD40 domains and Cullin 4-RING ubiquitin ligase complex (CRL4) recruited to chromatin mediated by RepID have potential roles in gene expression changes via epigenetic regulations. We aimed to investigate whether RepID-CRL4 participates in transcriptional changes required for MK differentiation. Methods The PCR array was performed using cDNAs derived from RepID-proficient or RepID-deficient K562 erythroleukemia cell lines. Correlation between RepID and DAB2 expression was examined in the Cancer Cell Line Encyclopedia (CCLE) through the CellMinerCDB portal. The acceleration of MK differentiation in RepID-deficient K562 cells was determined by estimating cell sizes as well as counting multinucleated cells known as MK phenotypes, and by qRT-PCR analysis to validate transcripts of MK markers using phorbol 12-myristate 13-acetate (PMA)-mediated MK differentiation condition. Interaction between CRL4 and histone methylation modifying enzymes were investigated using BioGRID database, immunoprecipitation and proximity ligation assay. Alterations of expression and chromatin binding affinities of RepID, CRL4 and histone methylation modifying enzymes were investigated using subcellular fractionation followed by immunoblotting. RepID-CRL4-JARID1A-based epigenetic changes on DAB2 promoter were analyzed by chromatin-immunoprecipitation and qPCR analysis. Results RepID-deficient K562 cells highly expressing MK markers showed accelerated MKs differentiation exhibiting increases in cell size, lobulated nuclei together with reaching maximum levels of MK marker expression earlier than RepID-proficient K562 cells. Recovery of WD40 domain-containing RepID constructs in RepID-deficient background repressed DAB2 expression. CRL4A formed complex with histone H3K4 demethylase JARID1A in soluble nucleus and loaded to the DAB2 promoter in a RepID-dependent manner during proliferation condition. RepID, CRL4A, and JARID1A were dissociated from the chromatin during MK differentiation, leading to euchromatinization of the DAB2 promoter. Conclusion This study uncovered a role for the RepID-CRL4A-JARID1A pathway in the regulation of gene expression for MK differentiation, which can form the basis for the new therapeutic approaches to induce platelet production.

Complete genome sequence of a novel species of Brevundimonas (strain NIBR10).

The complete genome sequence of strain NIBR10 was sequenced using PacBio RS II (Pacific Biosciences) sequencing platform. The 4,006,378-bp genome has a G + C content of 66.89% and around 3,832 coding sequences. Genomic data will provide valuable research for natural taxonomy and comparative genomics of the genus Brevundimonas.

Resistant starch utilization by Bifidobacterium, the beneficial human gut bacteria.

Resistant starch (RS) reaches the large intestine largely intact, where it is fermented by the gut microbiota, resulting in the production of short-chain fatty acids (SCFAs) that have beneficial effects on the human body. Bifidobacteria are a major species widely used in the probiotic field, and are increased in the gut by RS, indicating their importance in RS metabolism in the intestine. Bifidobacteria have a genetic advantage in starch metabolism as they possess a significant number of starch-degrading enzymes and extraordinary three RS-degrading enzymes, allowing them to utilize RS. However, to date, only three species of RS-degrading bifidobacteria have been reported as single isolates B. adolescentis, B. choerinum, and B. pseudolongum. In this review, we describe recent studies on RS utilization by Bifidobacterium, based on their biochemical characteristics and genetic findings. This review provides a crucial understanding of how bifidobacteria survive in specific niches with abundant RS such as the human gut.

4,4'-Diaponeurosporene Production as C30 Carotenoid with Antioxidant Activity in Recombinant Escherichia coli.

Carotenoids, a group of isoprenoid pigments, are naturally synthesized by various microorganisms and plants, and are industrially used as ingredients in food, cosmetic, and pharmaceutical product formulations. Although several types of carotenoids and diverse microbial carotenoid producers have been reported, studies on lactic acid bacteria (LAB)-derived carotenoids are relatively insufficient. There is a notable lack of research focusing on C30 carotenoids, the functional characterizations of their biosynthetic genes and their mass production by genetically engineered microorganisms. In this study, the biosynthesis of 4,4'-diaponeurosporene in Escherichia coli harboring the core biosynthetic genes, dehydrosqualene synthase (crtM) and dehydrosqualene desaturase (crtN), from Lactiplantibacillus plantarum subsp. plantarum KCCP11226 was constructed to evaluate and enhance 4,4'-diaponeurosporene production and antioxidant activity. The production of 4,4'-diapophytoene, a substrate of 4,4'-diaponeurosporene, was confirmed in E. coli expressing only the crtM gene. In addition, recombinant E. coli carrying both C30 carotenoid biosynthesis genes (crtM and crtN) was confirmed to biosynthesize 4,4'-diaponeurosporene and exhibited a 6.1-fold increase in carotenoid production compared to the wild type and had a significantly higher antioxidant activity compared to synthetic antioxidant, butylated hydroxytoluene. This study presents the discovery of an important novel E. coli platform in consideration of the industrial applicability of carotenoids.

Differential dynamics of cullin deneddylation via COP9 signalosome subunit 5 interaction.

Cullin-RING E3 ubiquitin ligases (CRLs) spatiotemporally regulate the proteasomal degradation of numerous cellular proteins involved in cell cycle control, DNA replication, and maintenance of genome stability. Activation of CRLs is controlled via neddylation by NEDD8-activating, -conjugating, and -attaching enzymes to the C-terminus of scaffold cullins (CULs), whereas the COP9 signalosome (CSN) inactivates CRLs via deneddylation. Here, we show that the deneddylation rate of each CUL is differentially modulated. Dose- or time-dependent treatment with pevonedistat, a small molecule inhibitor of NEDD8-activating enzyme (NAE), rapidly inhibits neddylation in most CULs, including CUL1, CUL3, CUL4A/B, and CUL5, whereas the deneddylation of CUL2 is slowly increased. We revealed that the different deneddylation speeds of each CUL depend on its binding strength with CSN5, the catalytic core of the CSN complex. Immunoprecipitation analysis revealed that CUL2 has a lower binding affinity for CSN5 than other CULs. Consistently, released cells treated with CSN5 inhibitor showed that CUL2 was slowly converted to the deneddylated form compared to the rapid deneddylation of other CULs. These findings provide mechanistic insights into the different dynamics of CULs in neddylation-deneddylation conversion.

The differentially expressed gene signatures of the Cullin 3-RING ubiquitin ligases in neuroendocrine cancer.

Cullin-RING ubiquitin E3 ligase (CRLs) composed of four components including cullin scaffolds, adaptors, substrate receptors, and RING proteins mediates the ubiquitination of approximately 20% of cellular proteins that are involved in numerous biological processes. While CRLs deregulation contributes to the pathogenesis of many diseases, including cancer, how CRLs deregulation occurs is yet to be fully investigated. Here, we demonstrate that components of CRL3 and its transcriptional regulators are possible prognosis marker of neuroendocrine (NE) cancer. Analysis of Cancer Cell Line Encyclopedia (CCLE) through the CellMinerCDB portal revealed that expression of CRL3 scaffold Cullin 3 (CUL3) highly correlates with NE signature, and CUL3 silencing inhibited NE cancer proliferation. Moreover, subset of 151 BTB (Bric-a-brac, Tramtrack, Broad complex) domain-containing proteins that have dual roles as substrate receptors and adaptor subunits in CRL3, as well as the expression of transcription factors (TFs) that control the transcription of BTB genes were upregulated in NE cancer. Analysis using published ChIP-sequencing data in small cell lung cancer (SCLC), including NE or non-NE SCLC verified that gene promoter of candidates which show high correlation with NE signature enriched H3K27Ac. These observations suggest that CRL3 is a master regulator of NE cancer and knowledge of specifically regulated CRL3 genes in NE cancer may accelerate new therapeutic approaches.

Fictibacillus marinisediminis sp. nov., a nitrate-reducing bacterium isolated from marine sediment in Hupo Basin, Republic of Korea.

An isolate, designated strain KIGAM418T was isolated from marine mud below 192 m depth in the Hupo Basin, Republic of Korea. Strain KIGAM418T was Gram-stain positive, spore-forming, rod-shaped, facultatively anaerobic, and grew at 10‒45 °C, in 0‒2% (w/v) NaCl at pH 4.0‒12.0. The strain tested positive for catalase, oxidase, and motility. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain KIGAM418T was related to the genus Fictibacillus. The strain showed the highest similarity to Fictibacillus rigui WPCB074T (98.0-98.1%) and Fictibacillus solisalsi YC1T (97.2-97.8%). The diagnostic diamino acid of the cell wall was meso-diaminopimelic acid. The major fatty acids were characterized as anteiso-C15:0 and iso-C15:0. Strain KIGAM418T possessed diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine as the major polar lipids and menaquinone-7 as the predominant menaquinone. The genome size and G + C content were 4.56 Mb and 43.2 mol%, respectively. According to predicted functional genes of the genome, the category of amino acid transport and metabolism was mainly distributed. Based on the polyphasic taxonomic data, strain KIGAM418T represents a novel species of the genus Fictibacillus, for which the name Fictibacillus marinisediminis sp. nov. is proposed. The type strain is KIGAM418T (= KCTC 43291 T = JCM 34437 T).

Genomic and Physiological Characterization of Metabacillus flavus sp. nov., a Novel Carotenoid-Producing Bacilli Isolated from Korean Marine Mud.

The newly isolated strain KIGAM252T was found to be facultatively anaerobic, Gram-stain-positive, spore-forming, and rod-shaped. They grew at 10-45 °C, pH 6.0-10.0, and were able to tolerate up to 6% NaCl in the growth medium. Phylogenetic analysis indicated that the KIGAM252T strain was related to the genus Metabacillus. The cell membrane fatty acid composition of strain KIGAM252T included C15:0 anteiso and C15:0 iso (25.6%) as the major fatty acids, and menaquinone 7 was the predominant isoprenoid quinone. The major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. The size of the whole genome was 4.30 Mbp, and the G + C content of the DNA was 43.8%. Average nucleotide and amino acid identity and in silico DNA-DNA hybridization values were below the species delineation threshold. Pan-genomic analysis revealed that 15.8% of all genes present in strain KIGAM252T was unique to the strain. The analysis of the secondary biosynthetic pathway predicted the carotenoid synthetic gene cluster in the strain KIGAM252T. Based on these current polyphasic taxonomic data, strain KIGAM252T represents a novel species of the genus Metabacillus that produces carotenoids, for which we propose the name Metabacillus flavus sp. nov. The type of strain was KIGAM252T (=KCTC 43261T = JCM 34406T).

Fibrivirga algicola gen. nov., sp. nov., an algicidal bacterium isolated from a freshwater river.

An aerobic, gram-stain-negative, pink-colored, non-motile and rod-shaped algicidal bacterium, designated as JA-25T was isolated from freshwater in Geumgang River, Republic of Korea. Strain JA-25T grew at 15-30 °C and pH 6-9, and did not require NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain JA-25T belongs to the family 'Spirosomaceae' and is most closely related to Fibrella aestuarina BUZ 2T (93.6%). Strain JA-25T showed < 90% sequence similarity to other members of the family 'Spirosomaceae'. The average nucleotide identity(ANI), in silico DNA-DNA hybridization and average amino acid identity(AAI) values based on the genomic sequences of JA-25T and F. aestuarina BUZ 2T were 74.4, 20.5, and 73.6%, respectively. Strain JA-25T showed an algicidal effect on the marine flagellate alga Heterocapsa triquetra, but no effect on fresh water cyanobacterium (Nostoc). In genome analysis, RIPP-like peptides were detected and predicted to resemble the indolmycin biosynthetic gene cluster, which possibly influence its algicidal effect. Furthermore, a bacteriorhodopsin gene with photoheterotrophic characteristics was detected. The genomic DNA G + C content was 52.5 mol%. The major cellular fatty acids were summed feature 3 (C16:1 ω6c/C16:1 ω7c), C16:1 ω5c, C16:0 (> 10%). The major respiratory quinone was menaquinone 7 and major polar lipids were phosphatidylethanolamine, two unidentified aminolipids, two phospholipids, and five unidentified lipids. Considering the phylogenetic inference, phenotypic, and chemotaxonomic data, strain JA-25T should be classified as a novel species in the novel genus Fibrivirga, with the proposed name Fibrivirga algicola sp. nov. The type strain is JA-25T (= KCCM 43334T = NBRC 114259T).

Expression, purification, and characterization of glutamate decarboxylase from human gut-originated Lactococcus garvieae MJF010.

Human gut-originated lactic acid bacteria were cultivated, and high γ-aminobutyric acid (GABA)-producing Lactococcus garvieae MJF010 was identified. To date, despite the importance of GABA, no studies have investigated GABA-producing Lactococcus species, except for Lc. lactis. A recombinant glutamate decarboxylase of the strain MJF010 (rLgGad) was successfully expressed in Escherichia coli BL21(DE3) with a size of 53.9 kDa. rLgGad could produce GABA, which was verified using the silylation-derivative fragment ions of GABA. The purified rLgGad showed the highest GABA-producing activity at 35 °C and pH 5. rLgGad showed a melting temperature of 43.84 °C. At 30 °C, more than 80% of the activity was maintained even after 7 h; however, it rapidly decreased at 50 °C. The kinetic parameters, Km, Vmax, and kcat, of rLgGad were 2.94 mM, 0.023 mM/min, and 12.3 min- 1, respectively. The metal reagents of CaCl2, MgCl2, and ZnCl2 significantly had positive effects on rLgGad activity. However, most coenzymes including pyridoxal 5'-phosphate showed no significant effects on enzyme activity. In conclusion, this is the first report of Gad from Lc. garvieae species and provides important enzymatic information related to GABA biosynthesis in the Lactococcus genus.

Human gut commensal bacterium Ruminococcus species FMB-CY1 completely degrades the granules of resistant starch.

Resistant starch (RS) in the diet reaches the large intestine and is fermented by the gut microbiota, providing beneficial effects on human health. The human gut bacterium FMB-CY1 was isolated and identified as a new species closest to Ruminococcus bromii. Ruminococcus sp. FMB-CY1 completely degraded RS including commercial RS types 2, 3, and 4, and generated glucose and maltose; however, it did not assimilate glucose. Genome analysis revealed 15 amylolytic enzymes (Amy) present in FMB-CY1. The evolutionary trees revealed that the Amys were well divided each other. All Amys (4, 9, 10, 12, and 16) containing cohesin and/or dockerin and scaffolding proteins known to be involved in constituting the amylosome, were identified. A new species of Ruminococcus, strain FMB-CY1, was considered to have the ability to form amylosomes for the degradation of RS. This new RS-degrading Ruminococcus species provides insights into the mechanism(s) underlying RS degradation in the human gut. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-021-01027-2.

Enzymatic analysis of truncation mutants of a type II pullulanase from Bifidobacterium adolescentis P2P3, a resistant starch-degrading gut bacterium.

A putative type II pullulanase gene, pulP, was identified in Bifidobacterium adolescentis P2P3. PulP possesses an α-amylase domain at the N-terminus and a pullulanase type I domain at the C-terminus, as well as three carbohydrate-binding modules (one CBM25 and two CBM41s) between them. The native PulP and four truncated mutant recombinant proteins (PulPΔCΔP, PulPΔP, PulPΔAΔC, and PulPΔA), in which each of the two catalytic domains and/or the CBMs were deleted, were produced in Escherichia coli and their specific properties were characterized. The removal of either catalytic domain abolished the corresponding catalytic activity of the wild-type enzyme. Deletion of the C-terminal domain resulted in a drastic decrease in the optimal temperature and thermostability, indicating that the pullulanase domain might be related to the temperature dependency of the enzyme. In addition, the elimination of the CBMs in the mutant proteins led to a loss of binding affinity toward raw substrates as well as the loss of their hydrolysis activities compared to the wild-type enzyme. HPAEC and TLC analyses proved that PulP and its mutants could hydrolyze α-glucans into maltotriose as their main product. These results suggest that PulP may play an important role in α-glucan metabolism in B. adolescentis P2P3.

Limnovirga soli gen. nov., sp. nov., isolated from river sediment.

A cream-coloured, Gram-stain-negative, rod-shaped bacterium, designated strain KSC-6T, was isolated from soil sampled at the Gapcheon River watershed in Daejeon, Republic of Korea. The organism does not require NaCl for growth and grows at pH 6.0-8.0 (optimum, pH 7.0) and 10-37 °C (optimum, 25 °C). Phylogenetic trees based on the 16S rRNA gene sequences reveal that strain KSC-6T belongs to the family Chitinophagaceae within the order Chitinophagales and is most closely related to Panacibacter ginsenosidivorans Gsoil 1550T (95.9% similarity). The genomic DNA G+C content was 38.9 mol%. The major cellular fatty acids (>8 %) of strain KCS-6T were iso-C15:0, iso-C15 : 1 G and iso-C17 : 0 3-OH. The predominant respiratory quinone was menaquinone 7 and the predominant polar lipids were phosphatidylethanolamine, five unidentified aminolipids and two unidentified lipids. Based on genome analyses, low digital DNA-DNA hybridization, average nucleotide identity and average amino acid identity values with closely related genera, and differential chemotaxonomic and physiological properties, we suggest that strain KCS-6T represents a novel species in a new genus in the family Chitinophagaceae, for which the name Limnovirga soli gen. nov., sp. nov. (type strain KCS-6T=KCCM 43337T=NBRC 114336T) is proposed.

Genome analysis of 1-deoxynojirimycin (1-DNJ)-producing Bacillus velezensis K26 and distribution of Bacillus sp. harboring a 1-DNJ biosynthetic gene cluster.

1-Deoxynojirumycin (1-DNJ) is a representative iminosugar with α-glucosidase inhibition (AGI) activity. In this study, the full genome sequencing of 1-DNJ-producing Bacillus velezensis K26 was performed. The genome consists of a circular chromosome (4,047,350 bps) with two types of putative virulence factors, five antibiotic resistance genes, and seven secondary metabolite biosynthetic gene clusters. Genomic analysis of a wide range of Bacillus species revealed that a 1-DNJ biosynthetic gene cluster was commonly present in four Bacillus species (B. velezensis, B. pseudomycoides, B. amyloliquefaciens, and B. atrophaeus). In vitro experiments revealed that the increased mRNA expression levels of the three 1-DNJ biosynthetic genes were closely related to increased AGI activity. Genomic comparison and alignment of multiple gene sequences indicated the conservation of the 1-DNJ biosynthetic gene cluster in each Bacillus species. This genomic analysis of Bacillus species having a 1-DNJ biosynthetic gene cluster could provide a basis for further research on 1-DNJ-producing bacteria.

4,4'-Diaponeurosporene from Lactobacillus plantarum subsp. plantarum KCCP11226: Low Temperature Stress-Induced Production Enhancement and In Vitro Antioxidant Activity.

Carotenoids, which have biologically beneficial effects and occur naturally in microorganisms and plants, are pigments widely applied in the food, cosmetics and pharmaceutical industries. The compound 4,4'-diaponeurosporene is a C30 carotenoid produced by some Lactobacillus species, and Lactobacillus plantarum is the main species producing it. In this study, the antioxidant activity of 4,4'-diaponeurosporene extracted from L. plantarum subsp. plantarum KCCP11226 was examined. Maximum carotenoid content (0.74 ± 0.2 at A470) was obtained at a relatively low temperature (20°C). The DPPH radical scavenging ability of 4,4'-diaponeurosporene (1 mM) was approximately 1.7-fold higher than that of butylated hydroxytoluene (BHT), a well-known antioxidant food additive. In addition, the ABTS radical scavenging ability was shown to be 2.3- to 7.5-fold higher than that of BHT at the range of concentration from 0.25 mM to 1 mM. The FRAP analysis confirmed that 4,4'- diaponeurosporene (0.25 mM) was able to reduce Fe3+ by 8.0-fold higher than that of BHT. Meanwhile, 4,4'-diaponeurosporene has been confirmed to be highly resistant to various external stresses (acid/bile, high temperature, and lysozyme conditions). In conclusion, L. plantarum subsp. plantarum KCCP11226, which produces 4,4'-diaponeurosporene as a functional antioxidant, may be a potentially useful strain for the development of functional probiotic industries.

The presence of resistant starch-degrading amylases in Bifidobacterium adolescentis of the human gut.

Resistant starch (RS) is a complex prebiotic carbohydrate beneficial to the human gut. In the present study, four genes encoding for putative amylolytic enzymes, likely to be responsible for RS-degradation, were identified in the genome of Bifidobacterium adolescentis P2P3 by comparative genomic analysis. Our results showed that only three enzymes (RSD1, RSD2, and RSD3) exhibited non-gelatinized high amylose corn starch (HACS)-degrading activity in addition to typical α-amylase activity. These three RS-degrading enzymes (RSD) were composed of multiple domains, including signal peptide, catalytic domain, carbohydrate binding domains, and putative cell wall-anchoring domains. Typical catalytic domains were conserved by exhibiting seven typical conserved regions (I-VII) found mostly in α-amylases. Analysis of enzymatic activity revealed that RSD2 displayed stronger activity toward HACS-granules than RSD1 and RSD3. Comparative genomics in combination with enzymatic experiments confirmed that RSDs might be the key enzymes used by RS-degrading bifidobacteria to degrade RS in a particular ecological niche, such as the human gut.

Genome analysis of Lactobacillus plantarum subsp. plantarum KCCP11226 reveals a well-conserved C30 carotenoid biosynthetic pathway.

Carotenoids are group of colored terpenoids with antioxidant properties and widespread in nature including in microorganisms. Lactobacillus plantarum subsp. plantarum KCCP11226 was previously isolated from kimchi, while exhibiting the production of 4,4'-diaponeurosporene as a C30 carotenoid. In this study, full genome sequencing of the strain KCCP11226 was performed. Genome analysis revealed that the dehydrosqualene synthase (crtM) and dehydrosqualene desaturase (crtN) genes, which are major genes for biosynthesis of 4,4'-diaponeurosporene, were shown to act as an operon in most L. plantarum strains, but they were uncommon in other Lactobacillus species. In vitro experiments revealed that the production of 4,4'-diaponeurosporene was greatly increased by oxidative stress. In this situation, mRNA expressions of crtN and crtM were also significantly increased. In conclusion, genome analysis of L. plantarum subsp. plantarum KCCP11226 suggested the presence of a well-conserved C30 carotenoid biosynthetic pathway that includes the crtM-crtN operon. The genomic information on L. plantarum subsp. plantarum KCCP11226 could further elucidate the functions of genes involved in isoprenoid biosynthetic pathway, especially in C30 carotenoid biosynthesis.

Comparative study on four amylosucrases from Bifidobacterium species.

Amylosucrase (ASase) is α-glucan-producing enzyme. Four putative ASase genes (bdas, blas, bpas, and btas) were cloned from Bifidobacterium sp. and expressed in Escherichia coli. All ASases from Bifidobacterium sp. (BAS) displayed typical ASase properties with slightly different characteristics. Among the BASs studied, BdAS and BpAS showed maximal enzyme activities at 35 and 30 °C, respectively, whereas BlAS and BtAS were maximally active at higher temperatures, i.e., 45 and 50 °C, respectively. BpAS exhibited optimum pH under slightly basic conditions (pH 8.0), while BdAS, BlAS, and BtAS preferred weakly acidic conditions (pH 5.0-6.0). All BASs showed higher isomerization activities. Particularly, BlAS produced more trehalulose than turanose. Although polymerization was the highest for BtAS, BtAS synthesized α-1, 4-glucans with a lower degree of polymerization than that of the other BASs. The versatile properties of the BASs described could contribute to the efficient production of highly valuable biomaterials for the agriculture, food, and pharmaceutical industries.

Hymenobacter lutimineralis sp. nov., belonging to the family Hymenobacteraceae, isolated from zeolite.

A red-pigmented bacterial strain, designated KIGAM108T, within the family Hymenobacteraceae was isolated from zeolite in the Gampo-41 mine of the Gyeongju, Republic of Korea. This strain was a Gram-negative, strictly aerobic, non-spore forming, rod-shaped bacterium. Phylogenetic analysis of the 16S rRNA gene sequence of strain KIGAM108T found that it was related to the genus Hymenobacter, with similarities of 96.6, 96.4, 95.5, and 95.0% to H. fastidiosus VUG-A124T, H. algoricola VUG-A23aT, H. crusticola MIMBbqt21T, and H. daecheongensis DSM 21074T, respectively. Strain KIGAM108T grew in the presence of 0-0.5% (w/v) NaCl at 4-37 °C and pH 6.0-10.0. This isolate contained MK-7 as a respiratory quinone. The polar lipids of strain KIGAM108T were identified as phosphatidylethanolamine, two unidentified aminophospholipids, one unidentified phospholipid and five unidentified lipids. The major fatty acids profile showed summed feature 3 (C16:1ω6c and/or C16:1ω7c) (22.3%), anteiso-C15:0 (17.1%), C16:1ω5c (13.3%), and iso-C15:0 (11.0%). The genomic DNA G + C content was 60.0 mol%. Based on the polyphasic taxonomic data, strain KIGAM108T is considered to represent a novel species of the genus Hymenobacter, for which the name Hymenobacter lutimineralis sp. nov. is proposed. The type strain is KIGAM108T (=KCTC 72263T =JCM 33444T).