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2.Method Marinobacter litoralis isolated from sea water
of the East Sea in Korea (SW-45=KCCM41591T=JCM11547T) Marinobacter litoralis (SW-45T=KCCM41591T=JCM11547T) isolated from sea water
of the East Sea in Korea M. hydrocarbonoclasticus
(DSM 8798T) M. aquaeolei
(DSM 11845T) isolated from an oilproducing well
on an offshore oil/gas platform
near a coastal town
in southern Vietnam Introduction 10학번 송영준(조장)
12학번 정혜윤
12학번 정혜진
12학번 조주현
12학번 조연정 Growth did not occur in the absence of NaCl in more than 19% NaCl in the presence of nitrate with acetate, citrate, succinate or glucose
as well as
in the absence of nitrate with acetate, citrate, succinate or glucose as sole carbon and energy sources under anaerobic conditions on MA
or on MA
or synthetic medium with nitrate Q-9 Strain SW-45T M. hydrocarbonoclasticus
DSM 8798T M. aquaeolei
DSM 11845T saturated unsaturated The cellular fatty acid profile of strain SW-45T M. hydrocarbonoclasticus
DSM 8798T M. aquaeolei
DSM 11845T Strain SW-45T showed oxidase and urease activities and weak catalase activity. Nitrate was reduced to nitrite. No acid was produced from the sugars used in this study. L-Glutamate and succinate were utilized as sole carbon and energy sources. Tween 80 was hydrolysed but aesculin, casein, gelatin, hypoxanthine, starch, tyrosine and xanthine were not. Marinobacter litoralis sp. nov., a moderately
halophilic bacterium isolated from sea water from
the East Sea in Korea 유비퀴논 코엔자임 큐텐(Coenzyme Q10)으로도 알려져 있으며 비단백질로서 무극성의 분자임. 세포호흡사슬에 관여하는 중요한 분자이며 주로 미토콘드리아에 존재함 The almost-complete 16S rDNA sequence of 1496 nt of strain SW-45T, representing approximately 96% of the Escherichia coli 16S rRNA sequence, was determined directly following PCR amplification. Strain SW-45T was found to have the highest 16S rDNA similarity to members of the c-subclass of the Proteobacteria. Strain SW-45T exhibited levels of 16S rDNA similarity of 94.9% and 95.3%, respectively, to the type strains of M. hydrocarbonoclasticus and M. aquaeolei. Levels of 16S rDNA similarity between strain SW-45T and other strains used in the phylogenetic analysis were less than 90.2%. Growth occurs Growth doesn't occur Optimal temperature : 30-37℃
(Grows at 4-46℃)

Optimal pH : 7.0-8.5

Optimal NaCl : 2–7% (w/v) NaCl
(Grows at 0.5–18% (w/v)NaCl) Methods temperature : above 47℃
pH : 4.5
NaCl : more than 19% (w/v) NaCl Isolation &
Culture conditions Morphological
characterization Marinobacter proposal strain SW-45T should be placed
in the genus Marinobacter
as a member of a novel species As part of this study.. the major respiratory lipoquinone of
M. hydrocarbonoclasticus and M. aquaeolei
was also found to be Q-9 isolated from sea water
near a petroleum refinery outlet Genus Marinobacter proposed with one species, M.hydrocarbonoclasticus. accommodates Gram-negative, aerobic,
motile and rod-shaped bacteria. Marinobacter species halophilic
degrade or utilize various hydrocarbons ☆ The results PART 2. 10학번 송영준 ☆ 16S rDNA ☆ SW-45T was found to be similar to those of Marinobacter species reported by Huu et al. (1999)


☆ Strain SW-45T has hydroxy fatty acid C12:0 3-OH (Special fatty acid in marinobacter)


☆ Respiratory lipoquinone


☆ The type strains of the two Marinobacter species have it.


☆ contain Q-9 as the major respiratory lipoquinone. Morphological Phenotypic Chemotaxonomic Phylogenetic genomic evidence We propose the name * Marinobacter litoralis sp. nov. * ☆ On the basis of the phenotypic, phylogenetic


☆ genomic evidence


☆strain SW-45T should be placed in the genus Marinobacter as a novel species The name Marinobacter litoralis sp. nov. *Result* 16 S rDNA Similarity ☆ low enough to except possibility, assigning strain SW-45T to one of the two Marinobacter species


☆ DNA–DNA similarity values were far below the threshold value suggested for species delineation (Wayne et al., 1987). ☆ Suggests two possibilities for the classification of strain SW-45T; as a member of the genus Marinobacter or as a member of a new genus. ☆ Based on 16S rDNA sequence data showing the phylogenetic position of strain SW-45T of the Y-subclass of the Proteobacteria.


☆ Bootstrap values greater than 50% are shown at branch points.


☆ 0.01 substitutions per nucleotide position.


☆ SW-45T joins the evolutionary clade comprising two species of the genus Marinobacter with a bootstrap resampling value of 100% ☆ Cell Gram-negative

0.5-0.8×1.5-3.0㎛

non-spore-forming rods

Motile by means of a single polar
flagellum Colony cream-coloured

smooth

circular to slightly irregular

low-convex

1.0-2.0㎜ in diameter after 3 days cultivation at 30℃ on MA Growth occur Growth doesn't occur Optimal temperature : 30-37℃
(Grows at 4-46℃)

Optimal pH : 7.0-8.5

Optimal NaCl : 2–7% (w/v) NaCl
(Grows at 0.5–18% (w/v) NaCl) temperature : above 47℃

pH : 4.5

NaCl : more than 19% (w/v) NaCl ↔ Anaerobic growth doesn't occur absence of nitrate with acetate

citrate

succinate

glucose presence of nitrate with acetate

citrate

succinate

glucose hydrolysed doesn't hydrolysed Tween 80 aesculin

casein

gelatin

hypoxanthine

starch

tyrosine

xanthine ↔ Acid is not produced adonitol

L-arabinose

D-cellobiose

D-fructose

D-galactose

D-glucose

lactose

maltose

D-mannitol

D-mannose,

D-melezitose melibiose

myo-inositol

D-raffinose

L-rhamnose

D-ribose

D-sorbitol

stachyose

sucrose

D-trehalose

D-xylose utilized as sole carbon and energy sources doesn't utilized as sole carbon and energy sources L-Glutamate

succinate Cellobiose

D-fructose

D-glucose

glycerol

D-mannitol

D-mannose
sucrose

D-gluconate

aspartate

DL-alanine

L-arginine

L-serine ↔ API ZYM test system present absent alkaline phosphatase

esterase (C4)

lipase (C8)

leucine arylamidase

acid phosphatase

naphthol-AS-BI-phosphohydrolase

N-acetyl-β-glucosaminidase
lipase (C14)

valine arylamidase

cystine arylamidase

trypsin

α-chymotrypsin

α-galactosidase

β-galactosidase

β-glucuronidase

α-glucosidase

β-glucosidase

α-mannosidase

α-fucosidase major respiratory lipoquinone ⇒ Q-9 predominant fatty acids ⇒ C18 : 1ω9c, C16 : 0, C16 : 1ω9c
and C12 : 0 3-OH DNA G+C content ⇒ 55 mol% (determined by HPLC) ☞SW-45T (=KCCM 41591T JCM 11547T) was isolated from sea water at Jungdongjin beach on the East Sea in Korea. Acknowledgements This work was supported by grant HSS0310134 and the NRL research program (grant M10104000294-01J0 00012800 and NLW0070111) from the Ministry of Science and Technology (MOST) of the Republic of Korea and by the research fund of the Probionic Corporation of Korea. References Al-Mallah, M., Goutx, M., Mille, G. & Bertrand, J. C. (1990).
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Alteromonas in seawater with eicosane as carbon source, a solid
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Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric
deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution
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Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a
maximum likelihood approach. J Mol Evol 17, 368–376.
Felsenstein, J. (1993). PHYLIP: phylogenetic inference package,
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University of Washington, Seattle, USA.
Gauthier, M. J., Lafay, B., Christen, R., Fernandez, L., Acquaviva, M.,
Bonin, P. & Bertrand, J.-C. (1992). Marinobacter hydrocarbonoclasticus
gen. nov., sp. nov., a new, extremely halotolerant, hydrocarbondegrading
marine bacterium. Int J Syst Bacteriol 42, 568–576.
Huu, N. B., Denner, E. B. M., Ha, D. T. C., Wanner, G. & Stan-Lotter,
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Leifson, E. (1963). Determination of carbohydrate metabolism of
marine bacteria. J Bacteriol 85, 1183–1184.
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406–425.
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based on 16S rDNA sequences. Int J Syst Bacteriol 48, 187–194. examined by light microscopy
transmission electron microscopy
(TEM) Physiological, Chemotaxonomic characterization -Catalase activity: bubble production in 3% (v/v) hydrogen peroxide solution.


-Oxidase activity: oxidation of 1% p-aminodimethylaniline oxalate. -Hydrolysis aesculin and nitrate reduction
casein, gelatin, starch
Tween 80 and urea
Hypoxanthine
tyrosine and xanthine
on MA plates Physiological, Chemotaxonomic characterization -Additional enzyme activities were determined by using the API ZYM system (bioMe´rieux).

-Acid production from carbohydrates: described by Leifson

-Tests for utilization of various substrates: described Gauthier et al

-Growth on MA with or without nitrate: determined after incubation in an anaerobic chamber -Anaerobic growth : determined in a synthetic medium

-Growth at various NaCl concentrations : investigated on MA or in MB.

-Growth at various temperatures : measured on MA at 4–55 °C. Physiological, Chemotaxonomic characterization DNA base composition -Chromosomal DNA : Yoon et al., 1996.

-ribonuclease T1 was used together with ribonuclease A.

-Respiratory lipoquinones: using reverse-phase HPLC.

-FAME: Microbial Identification
System (MIDI). reverse-phase HPLC G+C content -method of Tamaoka & Komagata.

-DNA was hydrolysed and the resultant nucleotides: reverse-phase HPLC. Microbial Identification System (MIDI) 16S rDNA seqence DNA–DNA hybridization -method of Ezaki et al.

-Using photobiotin-labelled DNA probes and microdilution wells. 3.Result www.strainInfo.net ☆ Introduction 12학번 조주현 ☆ Discription 12학번 정혜윤 "hydrocarbon dismantling"
they can degrade the major components of oil 시료를 투과시킨 전자선을
전자렌즈로 확대하여 관찰 1.Introduction ☆ Result 12학번 정혜진 Part 1. 화합물의 소수성 차이를 이용한 방법

고정상 비극성

이동상 극성

극성물질이 가장 빨리, 비극성 물질이 가장 늦게 용출 미생물을 동정하는 장치

미생물의 세포막에서 지방산을 추출

G+C함량으로 지방산 조성을 분석

분석데이터와 표준 미생물들의
지방산 조성과 비교 ☆ Method 12학번 조연정 Part 2. Method Part 1. Isolation &Culture conditions Morphological
characterization Physiological &
Chemotaxonomic
characterization DNA base composition Physiological&
Chemotaxonomic
characterization 16S rDNA sequencing
& phylogenetic analysis on a horizontal shaker at 150 r.p.m. cultivated
obtained
growing from agar plates for 3 days at 30 °C on MA urea hydrolysis -PCR에 의한 증폭 -QIAquick PCR purification kit
에 의한 정제 -CLUSTAL W software을
이용하여 배열 Phylogenetic trees -the neighbour-joining

-maximum-likelihood

-maximum-parsimony Q&A
QUESTION
AND
ANSWER thank you No growth under anaerobic conditions on MA
or on MA or synthetic medium with nitrate. Oxidase- and urease Catalase ⇒positive ⇒weakly positive ※Nitrate is reduced to nitrite starch hydrolysis 계통수를 작성할 때 현재 사용되고 있는 방법이며 대부분 무근 계통수를 그려준다. 이들 계통수는 서열간의 유사성을 정량적으로 알기에는 충분하다고 할 수 있으나, 시간 축에 대응한 진화의 방향성을 보기에는 적합하지 않다. Phylogenetic trees PHYLIP package -세계에서 가장 널리 사용되고 있는 계통분석용 프로그램 패키지 중의 하나이다. 이 페키지안에 위의 3개의 서로 다른 알고리즘을 갖는 계통수 작성 프로그램이 들어있다.


-CLUSTAL W software 보다 정밀도가 높은 최우도 추정법을 적용하고 싶을떄 사용을 권장한다. 4.Discription
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