Kae Kyoung Kwon

Formate-driven growth coupled with H2 production

Although a common reaction in anaerobic environments, the conversion of formate and water to bicarbonate and H2 (with a change in Gibbs free energy of ΔG° = +1.3 kJ mol−1) has not been considered energetic enough to support growth of microorganisms. Recently, experimental evidence for growth on formate was reported for syntrophic communities of Moorella sp. strain AMP and a hydrogen-consuming Methanothermobacter species and of Desulfovibrio sp. strain G11 and Methanobrevibacter arboriphilus strain AZ. The basis of the sustainable growth of the formate-users is explained by H2 consumption by the methanogens, which lowers the H2 partial pressure, thus making the pathway exergonic. However, it has not been shown that a single strain can grow on formate by catalysing its conversion to bicarbonate and H2. Here we report that several hyperthermophilic archaea belonging to the Thermococcus genus are capable of formate-oxidizing, H2-producing growth. The actual ΔG values for the formate metabolism are calculated to range between −8 and −20 kJ mol−1 under the physiological conditions where Thermococcus onnurineus strain NA1 are grown. Furthermore, we detected ATP synthesis in the presence of formate as a sole energy source. Gene expression profiling and disruption identified the gene cluster encoding formate hydrogen lyase, cation/proton antiporter and formate transporter, which were responsible for the growth of T. onnurineus NA1 on formate. This work shows formate-driven growth by a single microorganism with protons as the electron acceptor, and reports the biochemical basis of this ability.

The complete genome sequence of Thermococcus onnurineus NA1 reveals a mixed heterotrophic and carboxydotrophic metabolism.

Members of the genus Thermococcus, sulfur-reducing hyperthermophilic archaea, are ubiquitously present in various deep-sea hydrothermal vent systems and are considered to play a significant role in the microbial consortia. We present the complete genome sequence and feature analysis of Thermococcus onnurineus NA1 isolated from a deep-sea hydrothermal vent area, which reveal clues to its physiology. Based on results of genomic analysis, T. onnurineus NA1 possesses the metabolic pathways for organotrophic growth on peptides, amino acids, or sugars. More interesting was the discovery that the genome encoded unique proteins that are involved in carboxydotrophy to generate energy by oxidation of CO to CO(2), thereby providing a mechanistic basis for growth with CO as a substrate. This lithotrophic feature in combination with carbon fixation via RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) introduces a new strategy with a complementing energy supply for T. onnurineus NA1 potentially allowing it to cope with nutrient stress in the surrounding of hydrothermal vents, providing the first genomic evidence for the carboxydotrophy in Thermococcus.

Crystal structure of Lon protease: molecular architecture of gated entry to a sequestered degradation chamber

Lon proteases are distributed in all kingdoms of life and are required for survival of cells under stress. Lon is a tandem fusion of an AAA+ molecular chaperone and a protease with a serine‐lysine catalytic dyad. We report the 2.0‐Å resolution crystal structure of Thermococcus onnurineus NA1 Lon (TonLon). The structure is a three‐tiered hexagonal cylinder with a large sequestered chamber accessible through an axial channel. Conserved loops extending from the AAA+ domain combine with an insertion domain containing the membrane anchor to form an apical domain that serves as a gate governing substrate access to an internal unfolding and degradation chamber. Alternating AAA+ domains are in tight‐ and weak‐binding nucleotide states with different domain orientations and intersubunit contacts, reflecting intramolecular dynamics during ATP‐driven protein unfolding and translocation. The bowl‐shaped proteolytic chamber is contiguous with the chaperone chamber allowing internalized proteins direct access to the proteolytic sites without further gating restrictions.

Approaches for novel enzyme discovery from marine environments

The enormous pool of biodiversity in marine ecosystems is an excellent natural reservoir for acquiring an inventory of enzymes with potential for biotechnological applications. Moreover, the opportunity for sustainable resource management has been greatly enhanced by recent advances in culturing methods for recalcitrant microbes. In this review, we will focus primarily on successful examples in culturing marine microbes and provide an overview of work examining the biotechnological potential of the marine reservoir, mainly through genomic strategies, such as activity-based functional screening of genomic and metagenomic libraries and homology-driven screening of enormous amounts of sequence data.

Genome Sequence of Benzo(a)pyrene-Degrading Bacterium Novosphingobium pentaromativorans US6-1

Novosphingobium pentaromativorans US6-1 showed a good ability to degrade high-molecular-weight polycyclic aromatic hydrocarbons. We report the draft genome sequence of strain US6-1, which contains a main chromosome (5,096,413 bp, G+C content of 63.1%) and two plasmids (188,476 and 60,085 bp). The majority of the aromatic-hydrocarbon-degrading genes are encoded in the larger plasmid.

Lacinutrix algicola sp. nov. and Lacinutrix mariniflava sp. nov., two novel marine alga- associated bacteria and emended description of the genus Lacinutrix

Two heterotrophic, aerobic, yellow-pigmented, Gram-negative, non-gliding bacteria, designated AKS293(T) and AKS432(T), isolated from a red alga, were analysed using a polyphasic taxonomic approach. 16S rRNA gene sequence analysis revealed that the novel strains were affiliated to the genus Lacinutrix, a member of the family Flavobacteriaceae, showing sequence similarities of 96.1-96.4 % with respect to the type strain of Lacinutrix copepodicola. The two novel isolates shared 99.5 % 16S rRNA gene sequence similarity and 55.0 % DNA-DNA relatedness. They grew optimally at 17.5 degrees C and pH 6.5. The main cellular fatty acids of strain AKS293(T) were iso-C(15 : 0), iso-C(15 : 0) 3-OH and iso-C(16 : 0) 3-OH, while those of strain AKS432(T) were anteiso-C(15 : 0), iso-C(15 : 0), iso-C(15 : 1) and iso-C(15 : 0) 3-OH. In both cases, the major isoprenoid quinone was MK-6. The DNA G+C contents were 34.7 and 37.0 mol% for strains AKS293(T) and AKS432(T), respectively. The phylogenetic evidence, phenotypic data and DNA-DNA hybridization results support the differentiation of strains AKS293(T) and AKS432(T) from each other and from their closest relative, L. copepodicola DJ3(T). Therefore, strains AKS293(T) and AKS432(T) represent two novel species, for which the names Lacinutrix algicola sp. nov. and Lacinutrix mariniflava sp. nov. are proposed, respectively. The type strain of L. algicola sp. nov. is AKS293(T) (=KCCM 42313(T)=JCM 13825(T)) and the type strain of L. mariniflava sp. nov. is AKS432(T) (=KCCM 42306(T)=JCM 13824(T)). An emended description of the genus Lacinutrix is also proposed.

Description of Croceitalea gen. nov. in the family Flavobacteriaceae with two species, Croceitalea eckloniae sp. nov. and Croceitalea dokdonensis sp. nov., isolated from the rhizosphere of the marine alga Ecklonia kurome

Two novel bacterial strains, designated DOKDO 025(T) and DOKDO 023(T), were isolated on Dokdo Island, Korea, from the rhizosphere of the brown alga Ecklonia kurome. The strains were subjected to a polyphasic taxonomy study and were found to be Gram-negative, aerobic, rod-shaped, non-motile and orange-coloured. The isolates shared 96.3 % 16S rRNA gene sequence similarity. They showed 93.8-95.6 % 16S rRNA gene sequence similarity with respect to members of the genus Muricauda in the family Flavobacteriaceae, but formed a distinct phyletic line. Moreover, the cellular appendages reported for all Muricauda species were absent from strains DOKDO 025(T) and DOKDO 023(T). The predominant cellular fatty acids of strain DOKDO 025(T) were iso-C(15 : 0), iso-C(15 : 1) and one with an equivalent chain-length of 13.565 and those of strain DOKDO 023(T) were iso-C(15 : 0), iso-C(15 : 1) and iso-C(17 : 0) 3-OH. The DNA G+C content of strains DOKDO 025(T) and DOKDO 023(T) were 59.5 and 66.5 mol%, respectively, higher than any values found in recognized members of the family Flavobacteriaceae. The major respiratory quinone was MK-6. On the basis of evidence from the polyphasic study, strains DOKDO 025(T) and DOKDO 023(T) represent two novel species in a new genus, Croceitalea gen. nov., for which the names Croceitalea eckloniae sp. nov. (the type species) and Croceitalea dokdonensis sp. nov. are proposed. The type strain of Croceitalea eckloniae sp. nov. is DOKDO 025(T) (=KCCM 42309(T) =JCM 13827(T)) and that of Croceitalea dokdonensis sp. nov. is DOKDO 023(T) (=KCCM 42308(T) =JCM 13826(T)).

Brumimicrobium mesophilum sp. nov., isolated from a tidal flat sediment, and emended descriptions of the genus Brumimicrobium and Brumimicrobium glaciale

A Gram-stain-negative, aerobic, rod-shaped, non-motile and orange-coloured marine bacterium, YH207(T), was isolated from a tidal flat at Yeongheung-do on the coast of the Yellow Sea, Korea. 16S rRNA gene sequence analysis revealed that strain YH207(T) was affiliated with the family Cryomorphaceae and showed highest similarity to Brumimicrobium glaciale IC156(T) (95.4 %). Growth was observed at 11-36 °C, at pH 6.5-10.0 and with 0.4-7.0 % NaCl. The predominant cellular fatty acids when grown at 20 °C were iso-C15 : 0 (44.2 %), iso-C15 : 1 G (34.3 %), iso-C17 : 0 3-OH (8.7 %) and summed feature 3 (comprising iso-C15 : 0 2-OH and/or C16 : 1ω7c; 2.3 %). The major respiratory quinone was MK-6. Phosphatidylethanolamine, phosphatidylglycerol, three unidentified lipids, three unidentified aminophospholipids, one unidentified phospholipid, four unidentified aminolipids and three unidentified glycolipids were identified as major polar lipids. The DNA G+C content was 34.3 mol%. On the basis of the data from our polyphasic taxonomic study, strain YH207(T) should be classified in a novel species in the genus Brumimicrobium, for which the name Brumimicrobium mesophilum sp. nov. is proposed. The type strain is YH207(T) ( = KCCM 42331(T)  = JCM 14063(T)). Emended descriptions of the genus Brumimicrobium and Brumimicrobium glaciale Bowman et al. 2003 are also given.

Proteomic Characterization of Plasmid pLA1 for Biodegradation of Polycyclic Aromatic Hydrocarbons in the Marine Bacterium, Novosphingobium pentaromativorans US6-1

Novosphingobium pentaromativorans US6-1 is a halophilic marine bacterium able to degrade polycyclic aromatic hydrocarbons (PAHs). Genome sequence analysis revealed that the large plasmid pLA1 present in N. pentaromativorans US6-1 consists of 199 ORFs and possess putative biodegradation genes that may be involved in PAH degradation. 1-DE/LC-MS/MS analysis of N. pentaromativorans US6-1 cultured in the presence of different PAHs and monocyclic aromatic hydrocarbons (MAHs) identified approximately 1,000 and 1,400 proteins, respectively. Up-regulated biodegradation enzymes, including those belonging to pLA1, were quantitatively compared. Among the PAHs, phenanthrene induced the strongest up-regulation of extradiol cleavage pathway enzymes such as ring-hydroxylating dioxygenase, putative biphenyl-2,3-diol 1,2-dioxygenase, and catechol 2,3-dioxygenase in pLA1. These enzymes lead the initial step of the lower catabolic pathway of aromatic hydrocarbons through the extradiol cleavage pathway and participate in the attack of PAH ring cleavage, respectively. However, N. pentaromativorans US6-1 cultured with p-hydroxybenzoate induced activation of another extradiol cleavage pathway, the protocatechuate 4,5-dioxygenase pathway, that originated from chromosomal genes. These results suggest that N. pentaromativorans US6-1 utilizes two different extradiol pathways and plasmid pLA1 might play a key role in the biodegradation of PAH in N. pentaromativorans US6-1.

Carboxylicivirga gen. nov. in the family Marinilabiliaceae with two novel species, Carboxylicivirga mesophila sp. nov. and Carboxylicivirga taeanensis sp. nov., and reclassification of Cytophaga fermentans as Saccharicrinis fermentans gen. nov., comb. nov.

Two facultatively anaerobic mesophilic bacteria, strains MEBiC 07026(T) and MEBiC 08903(T), were isolated from two different tidal flat sediments and both strains showed approximately 92.2 % 16S rRNA gene sequence similarity with [Cytophaga] fermentans DSM 9555(T). 16S rRNA gene sequence similarity between the two new isolates was 97.5 % but levels of DNA-DNA relatedness between the two were 31.3-31.8 %. Phylogenetic analysis revealed that the two isolates and [Cytophaga] fermentans DSM 9555(T) were affiliated with the family Marinilabiliaceae in the class Bacteroidia. The dominant fatty acids of strains MEBiC 07026(T), MEBiC 08903(T) and [Cytophaga] fermentans DSM 9555(T) were branched-type or hydroxylated C15 : 0, but [Cytophaga] fermentans DSM 9555(T) contained a higher proportion of anteiso-branched fatty acids. The two new isolates contained a markedly higher proportion of monounsaturated fatty acids than other members of the family Marinilabiliaceae. The major respiratory quinone of the strains was MK-7. Strains MEBiC07026(T) and MEBiC08903(T) utilized a wide range of carboxylic acids whereas [Cytophaga] fermentans DSM 9555(T) utilized carbohydrates rather than carboxylic acids. The DNA G+C content of the novel strains was about 44 mol% but that of [Cytophaga] fermentans DSM 9555(T) revealed from the genome sequence was 37.6 mol%. Based on evidence from this polyphasic taxonomic study, a novel genus, Carboxylicivirga gen. nov., is proposed in the family Marinilabiliaceae with two novel species, Carboxylicivirga mesophila sp. nov. with type strain MEBiC 07026(T) ( = KCCM 42978(T) = JCM 18290(T)) and Carboxylicivirga taeanensis sp. nov. with type strain MEBiC 08903(T) ( = KCCM 43024(T) = JCM 19490(T)). Additionally, [Cytophaga] fermentans DSM 9555(T) ( = ATCC 19072(T)) is reclassified as Saccharicrinis fermentans gen. nov., comb. nov.