Physiology and biochemical diversity of bacterial cholate degradation
Dissertation
Zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.)
vorgelegt von
Vemparthan Suvekbala
an der
Mathematisch-Naturwissenschafliche Sektion Fachbereich Biologie
Konstanz, Mai 2011
Tag der mündlichen Prüfung: 24.06.2011 Prüfungkommission: Prof. Dr. Bernhard Schink
PD. Dr. Bodo Philipp Prof. Dr. Iwona Adamska
Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-139818
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1 T
ABLE OFC
O NTEN TS2 ZUSMMENFASSUNG ...7
3 SUMMARY IN ENGLISH ... 10
4 INTRODUCTION 4.1 STEROIDS ... 12
4.2 STEROIDS IN THE ENVIRONMENT ... 12
4.2.1 Impact of steroids in the environment... 14
4.3 BACTERIAL DEGRADATION OF STEROIDS ... 16
4.4 BILE SALTS ... 20
4.4.1 Bacterial degradation of bile salts ... 20
4.4.2 Cholate degradation by Pseudomonas sp. strain Chol1 ... 20
4.5 MICROCOSM AND ENRICHMENT FOR CHOLATE DEGRADING BACTERIA PRESENT IN THE LITTORAL ZONE OF LAKE CONSTANCE ... 26
4.6 AIMS OF THIS DISSERTATION ... 27
5 MATERIALS AND METHODS 5.1 MICROBIOLOGICAL METHODS 5.1.1 Bacterial strains ... 28
5.1.2 Media ... 28
5.1.3 Growth experiments ... 29
5.1.4 Preparation of DHADD from Pseudomonas sp. strain Chol1 ... 31
5.1.5 Cell-suspension experiments ... 31
5.1.6 Microcosm experiments ... 33
5.1.7 Quantitative enrichment experiments for the isolation of cholate-degrading bacteria from the littoral zone of Lake Constance ... 35
5.1.8 Isolation and maintenance of pure cultures ... 35
5.1.9 Characterization of strains 1, 2 and 9 ... 36
5.2 BIOCHEMICAL METHODS 5.2.1 Preparation of cell extracts ... 36
5.2.2 Enzyme assays ... 37
5.2.3 Measuring the activities of enzymes oxidizing cholate in strains 1, 2 and 9 ... 40
5.2.4 Measuring the activities of enzyme activating cholate in strain 2 ... 40
5.2.5 Protein quantification ... 40
5.3 ANALYTICAL METHODS 5.3.1 Wavelength scan analysis ... 41
5.3.2 HPLC ... 41
5.3.3 LC/MS ... 42
5.3.4 Chemicals ... 42
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6 RESULTS PART I
6.1 GROWTH OF PSEUDOMONAS SP. STRAIN CHOL1 WITH PROPIONATE AND ACETATE ... 43
6.2 CELL SUSPENSION EXPERIMENTS FOR CO-INDUCTION OF CHOLATE AND PROPIONATE METABOLISM IN STRAIN CHOL1 ... 43
6.3 STUDIES ON PROPIONATE METABOLISM IN CELL EXTRACTS OF STRAIN CHOL1 ... 46
6.4 STUDIES OF CENTRAL METABOLISM IN PSEUDOMONAS SP. STRAIN CHOL1 ... 49
6.5 STUDIES ON ACETYL-COA METABOLISM IN PSEUDOMONAS SP. STRAIN CHOL1 ... 51
6.6 STUDIES ON CARBON ANAPLEROSIS AND INDUCTION OF PROPIONATE OXIDATION IN PSEUDOMONAS SP. STRAIN CHOL1... 51
PART II SECTION - I 6.7 MICROCOSM EXPERIMENTS 6.7.1 Microcosm experiment 1 ... 55
6.7.2 Microcosm experiment 2 ... 57
6.7.3 Microcosm experiment 3 ... 60
6.7.4 Microcosm experiment 4 ... 63
SECTION - II 6.8 ENRICHMENT EXPERIMENTS ... 66
6.8.1 Physiological characterization of strains 1, 2 and 9... 67
6.8.2 Molecular characterization of strains 1, 2 and 9 ... 67
6.9 ZOOGLOEA SP. STRAIN 1 ... 69
6.9.1 Degradation of cholate by Zoogloea sp. strain 1 ... 69
6.9.2 Identification of the intermediates during cholate degradation in Zoogloea sp. strain 1 ... 71
6.9.3 Measurement of enzyme activities in Zoogloea sp. strain 1... 75
6.10 PSEUDOMONAS SP. STRAIN 9 ... 76
6.10.1 Cholate degradation by Pseudomonas sp. strain 9 ... 76
6.10.2 Identification of the intermediates during cholate degradation in Pseudomonas sp. strain 9 ... 77
6.11.1 Enzyme oxidizing the A-ring of cholate ... 79
6.12 DIETZIA SP. STRAIN 2 ... 80
6.12.1 Degradation of cholate by Dietzia sp. strain 2 ... 80
6.12.2 Identification of the intermediates during cholate degradation in Dietzia sp. strain 2 ... 82
6.12.3 LC/MS analysis of culture supernatant of Dietzia sp. strain 2 ... 83
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6.13 MEASUREMENTS OF ENZYME ACTIVITIES IN DIETZIA SP. STRAIN 2 ... 88
6.13.1 Enzymes oxidizing the A-ring of cholate in Dietzia sp. strain 2 ... 88
6.13.2 Enzymes activating cholate in Dietzia sp. strain 2 ... 88
6.13.3 In-vitro detection of cholyl-CoA in Dietzia sp. strain 2 ... 88
6.13.4 In-vitro detection of cholyl-CoA oxidation in Dietzia sp. strain 2 ... 90
6.14 GROWTH EXPERIMENTS WITH DIETZIA SP. STRAIN 2 IN PRESENCE OF THE KEY INTERMEDIATES OF CHOLATE DEGRADATION PRODUCED BY STRAIN CHOL1 ... 94
7 DISCUSSION 7.1 STUDIES ON PHYSIOLOGY AND BIOCHEMISTRY OF PRODUCTS FROM THE DEGRADATION OF SIDE CHAIN IN CHOLATE DEGRADING PSEUDOMONAS SP. STRAIN CHOL1 ... 109
7.1.1 Studies on propionyl-CoA metabolism in Pseudomonas sp. strain Chol1 ... 109
7.1.2 2-Methylcitrate cycle in Pseudomonas sp. strain Chol1 ... 110
7.1.3 Studies on other biochemical pathways for oxidizing propionyl-CoA in Pseudomonas sp. strain Chol1 ... 111
7.1.4 Studies on central metabolism in Pseudomonas sp. strain Chol1 ... 112
7.1.5 Induction of propionate oxidation in cholate-grown Pseudomonas sp. strain Chol1 ... 114
7.2 MICROCOSM EXPERIMENTS ... 117
7.3 ENRICHMENT EXPERIMENTS ... 119
7.3.1 Enrichments for cholate-degrading bacteria from the littoral zone of Lake Constance ... 120
7.3.2 New bacterial strains capable of degrading cholate from the littoral zone of Lake Constance ... 122
7.3.3 Zoogloea sp. strain 1 ... 122
7.3.4 Pseudomonas sp. strain 9 ... 123
7.3.5 Dietzia sp. strain 2 ... 127
7.3.5.1 Intermediates and biochemical pathway of cholate degradation in Dietzia sp. strain 2 ... 127
7.3.5.2 Dietzia sp. strain 2 with the intermediates of cholate degradation in strain Chol1... 129
8 APPENDIX 8.1 NAME OF COMPOUNDS AND THEIR RESPECTIVE PEAKS; IDENTIFIED AND DERIVED FROM DIFFERENT BACTERIAL STRAINS DESCRIBED IN THIS THESIS ... 144
9 REFERENCES ... 145
Contributions...153
ACKNOWLEDGEMENT ... 154
CURRICULUM VITAE ... 155
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Abbreviations
ADD Androstadienediones
ADP Adenosine diphosphate
AMP Adenosine monophosphate
ATP Adenosine-5'-triphosphate BLAST Basic local alignment search tool
CoA Coenzyme A
DAD Diode array detector
DAPI 4',6-diamidino-2-phenylindole
DCM Dichloromethane
DCPIP Dichlorophenolindophenol
DHADD 7,12-dihydroxy-1,4-androstadiene-3,17-dione
DHOCTO 7α,12α-dihydroxy-3-oxochola-1,4,22E-triene-24-oate DHOPDC 7,12-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate HEPES 4-(2-hydroxyethyl)-piperazineethanesulfonic acid HPLC High performance liquid chromatography
LC-MS Liquid chromatography - mass spectrophotometry
MPN Most probable number
MOPS 3-(N-morpholino) propanesulfonic acid NAD+ Nicotinamide adenine dinucleotide
NADP+ Nicotinamide adenine dinucleotide phosphate NADH Nicotinamide adenine dinucleotide reduced
NADPH Nicotinamide adenine dinucleotide phosphate reduced
TCA Tri-carboxylic acid
THOCDO 7α,12α,22-trihydroxy-3-oxochola-1,4-diene-24-oate
THSATD 3,7,12-trihydroxy-9,10-seco-1,3,5(10)-androstatriene-9,17-dione TNB2- Thio-nitrobenzoate
Tris-HCl Tris (hydroxymethyl) aminomethane
Summary
7
2 Z
USM MEN FASS UN GSteroide sind weit verbreitete Naturstoffe, welche vor allem in eukaryotischen Organismen vorkommen und dort sehr verschiedene Funktionen erfüllen. In die Umwelt gelangen diese Verbindungen v.a. durch Abbau von pflanzlichem und tierischem Material und durch Exkretion.
Bakterien können Steroide nur selten selbst bilden, jedoch sind viele Bakterien in der Lage, diese Verbindungen abzubauen oder zu transformieren. Über die Abundanz von Bakterien, welche Steroide in natürlichen Habitaten abbauen können, und über die Diversität der biochemischen Abbauwege ist bisher wenig bekannt. In dieser Arbeit wurde der bakterielle Steroidabbau anhand des Gallensalzes Cholat als Modellsubstanz untersucht. Cholat ist ein oberflächenaktives Steroid, welches eine C5 Acylseitenkette am Kohlenstoffatom C17 des Steroidgerüstes aufweist.
Pseudomonas sp. Stamm Chol1 kann mit Cholat als einziger Kohlenstoff- und Energiequelle wachsen und dieses unter aeroben Bedingungen komplett zu CO2 abbauen. Hierbei geschieht der aerobe Abbau über den sogenannten 9,10-Seco Weg, welcher bisher der einzige gut untersuchte Abbauweg für Steroide unter aeroben Bedingungen ist. Die Seitenkette von Cholat wird vermutlich über β-Oxidation abgebaut, wobei ein Acetyl- (C2) und ein Propionyl-CoA (C3) Rest abgespalten werden.
Im ersten Teil der Arbeit wurde der weitere Abbau von Acetyl- und Propionyl-CoA in Cholat gewachsenen Zellen von Stamm Chol1 untersucht. In Zellextrakten von Acetat-gewachsenen Zellen war die spezifische Aktivität der Isocitratlyase, des Schlüsselenzyms des Glyoxylatzyklus, 50-fach erhöht gegenüber Extrakten von Cholat gewachsenen Zellen. Dies zeigt, dass der Glyoxylatzyklus in Cholat-gewachsenen Zellen von Stamm Chol1 nicht induziert ist und Acetyl-CoA über den Citratzyklus abgebaut wird. Der Abbau von Propionyl-CoA war in Cholat- gewachsenen Zellen induziert, während dies in Succinat-gewachsenen Zellen, die auch nicht für den Abbau von Cholat induziert sind, nicht der Fall war. Weiterhin war die spezifische Aktivität der 2-Methylcitratsynthase, des Schlüsselenzyms des 2-Methylcitratzyklus, in Cholat- und Propionat-gewachsenen Zellen annähernd identisch, während die Aktivität in Succinat- gewachsenen Zellen 10-fach niedriger war. Dies bedeutet, dass Propionat, welches während des Cholatabbaus ensteht, durch den 2-Methylcitratzyklus abgebaut wird. In Transposonmutanten, welche die Seitenkette von Cholat nicht abbauen können und daher kein Propionyl-CoA bilden war die spezifische Aktivität der 2-Methylcitratsynthase sehr viel niedriger. Folglich induziert die Bildung von Propionyl-CoA während des Cholatabbaus durch Stamm Chol1 den 2-Methylcitratzyklus.
Summary
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Im zweiten Teil der Arbeit wurden die Abundanz und die Biochemie von cholatabbauenden Bakterien in der Littoralzone des Bodensees untersucht. Mithilfe von Mikrokosmos- experimenten konnten wir zeigen, dass die endogene Mikrobengemeinschaft im Littoralssediment des Bodensees unmittelbar in der Lage war, Cholat zu transformieren.
Weiterhin wurden quantitative Anreicherungsexperimente mit verdünnten Sedimentproben des Littorals des Bodensees als Inokulum durchgeführt, welche zeigten, dass mehr als 1 % der kultivierbaren Bakterien in der Lage sind, Cholat abzubauen. Von diesen wurden fünfzehn Stämme als Reinkulturen isoliert, wobei drei weiter charakterisiert wurden. Der erste Stamm, Zoogloea sp. Stamm 1, ein β-Proteobakterium, baut Cholat über den 9,10-Seco Weg ab, was sich an der vorrübergehenden Akkumulation der charakteristischen Intermediate dieses Weges, DHADD (7,12-Dihydroxy-1,4-androstadien-3,17-dion) und THSATD (3,7,12-Trihydroxy-9,10- secoandrosta-1,3,5(10)-trien-9,17-dion), im Kulturüberstand zeigte. Der zweite Stamm, Pseudomonas sp. Stamm 9, ein γ-Proteobakterium, akkumuliert während des Wachstums mit Cholat vorübergehend das ∆1/∆4-Monoen von 3-Ketocholat, 7,12-Dihydroxy-3-oxopregna-1,4- dien-20-carboxylat (DHOPDC) und das ∆1/∆4 Monoen von DHOPDC im Kulturüberstand. Dies deutet darauf hin, dass dieser Stamm ebenso den 9,10-Seco Weg für den Abbau von Cholat nutzt. Aktivitäten von Cholat-oxidierenden Enzymen (3-Hydroxysteroid Dehydrogenase [3-Hsd]
und 3-Ketosteroid Dehydrogenase [3Ksdh]) wurden in beiden Stämmen nachgewiesen. Während des Cholatabbaus durch den dritten Stamm, Dietzia sp. Stamm 2, ein amyceliales Actinobakterium, akkumulierten zwei bisher unbekannte Produkte (X1 und X2) im Kulturüberstand, während die typischen Intermediate des 9,10-Seco Weges nicht auftraten. Die UV Spektren dieser beiden Produkte unterschieden sich stark von den Spektren der Intermediate des 9,10-Seco Weges. Obwohl die Bildung von ∆1,4 3-Ketocholat in Dietzia sp. Stamm 2 durch HPLC und LC-MS Analyse nachgewiesen wurde, konnten keine Aktivitäten von 3-Hsd und 3-Ksdh in zellfreiem Extrakt gemessen werden. Jedoch wurde Aktivität einer Choly-CoA-Ligase in in vitro Experimenten gemessen. Dietzia sp. Stamm 2 konnte nicht mit DHOPDC, 7,12-Dihydroxy-3-oxochola-1,4,22-trien-24-oat (DHOCTO), DHADD und THSATD wachsen, welche charakteristische Intermediate des 9,10-Seco Weges in Stamm Chol 1 während des Abbaus von Cholat darstellen. Darüber hinaus inhibierten diese Verbindungen den Cholatabau durch Stamm 2 und förderten die Bildung der Produkte X1 und X2.
Summary
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Diese Ergebnisse zeigen, dass Dietzia sp. Stamm 2 einen Abbauweg für Cholat nutzt, der sich von dem bekannten 9,10-Seco Weg unterscheidet. Zusammengefasst konnten wir zeigen, dass die Fähigkeit, Steroidverbindungen abzubauen innerhalb der kultivierbaren Bakterien in der Littoralzone des Bodensees sehr weit verbreitet ist. Die Entdeckung einen neuen Cholat- Abbauweges in Dietzia sp. Stamm 2 zeigt außerdem, dass die Diversität metabolischer Abbauwege für Steroidverbindungen bisher unterschätzt wurde.
Summary
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3 S
UMMA RY INE
N GL ISHSteroids are ubiquitous natural compounds, present in eukaryotic organisms to fulfill various biological functions. Steroids enter into the environments via excretion by and decay of animals and plants. Steroids seldom occur in bacteria; however, bacteria are capable of transforming and degrading steroids. Knowledge regarding the abundance of bacteria present in a natural habitat capable of degrading steroids and the diversity of biochemical pathways for steroid degradation is scarce. In this thesis, bacterial steroid degradation was studied with the bile salt cholate as a model substance. Cholate is a surface-active steroid compound, in which a C5 acyl side chain is attached to the C17 position of the steroid skeleton. Pseudomonas sp. strain Chol1 is able to grow with cholate as the sole source of carbon and energy and degrades it completely to CO2
under oxic conditions via the 9,10-seco pathway, which is the only well-described pathway for aerobic degradation of steroid compounds. The C5 acyl side chain is presumably degraded by β-oxidation during which acetyl- (C2) and propionyl-CoA (C3) moieties are released.
In the first part of this thesis, we investigated the degradation of acetyl- and propionyl-CoA moieties in cholate-grown cells of strain Chol1. In cell extracts of acetate-grown cells of strain Chol1, the specific activity of isocitrate lyase, the key enzyme of glyoxylate cycle, was 50 times higher than in cell extracts of cholate-grown cells of strain Chol1. These results showed that the glyoxylate cycle was not induced in cholate-grown cells of strain Chol1 indicating that acetyl-CoA is degraded via the tricarboxylic acid cycle. The degradation of propionate was co-induced along with cholate degradation in cholate-grown cells of strain Chol1, while in succinate-grown cells of strain Chol1, which were not induced for cholate degradation, propionate degradation was not co-induced. The specific activity of 2-methylcitrate synthase, the key enzyme of 2-methylcitrate cycle was almost equal in cholate- and propionate-grown cells of strain Chol1 while it was 10 times lower in succinate-grown cells of strain Chol1. These results indicate that propionate is degraded through the 2-methylcitrate cycle during cholate degradation in strain Chol1. The specific activity of 2-methylcitrate synthase was much lower in transposon mutants, that are blocked in the degradation of acyl side chain and therefore do not release propionyl-CoA. Thus, propionyl-CoA is responsible for inducing 2-methylcitrate synthase in strain Chol1 during cholate degradation.
Summary
11
In the second part of this thesis, we investigated the abundance and biochemistry of bacteria present in the littoral zone of Lake Constance capable of degrading cholate. Microcosm experiments showed that the endogenous microflora present in the littoral zone of Lake Constance was able to readily transform cholate. Quantitative enrichment experiments performed with diluted littoral sediments sample of Lake Constance as inoculum showed the percentage of cholate-degrading bacteria among the cultivable bacteria was above 1 %. Fifteen different strains capable of degrading cholate were isolated. Three strains were characterized further. The first strain, Zoogloea sp. strain 1 (β-proteobacterium), degraded cholate via the 9,10-seco pathway as indicated by the transient accumulation of the characteristic intermediates DHADD (7,12-dihydroxy-1,4-androstadiene-3,17-dione) and THSATD (3,7,12-trihydroxy- 9,10-secoandrosta-1,3,5(10)-triene-9,17-dione) in the culture supernatant. The second strain, Pseudomonas sp. strain 9 (γ-proteobacterium), degraded cholate and transiently accumulated
∆1/∆4-monoene of 3-ketocholate, 7,12-dihydroxy-3-oxopregna-1,4-diene-20-carboylate
(DHOPDC) and ∆1/∆4 monoene of DHOPDC indicating that Pseudomonas sp. strain 9 also followed the 9,10-seco pathway. The activities of cholate oxidizing enzymes, 3-hydroxy steroid dehydrogenase (3-Hsd) and 3-keto steroid dehydrogenase (3-Ksdh), were detected in both strains. During cholate degradation in the third strain, Dietzia sp. strain 2 (amycelial actinobacteria), two so far unknown products (compounds X1 and X2) accumulated transiently while the characteristic intermediates of the 9,10-seco pathway were not detected. The UV-spectra of these two products were entirely different from the UV-spectra of steroid compounds occurring in the 9,10-seco pathway. Although ∆1,4-3-ketocholate was detected in Dietzia sp. strain 2 by HPLC and LC/MS analysis, the activities of 3-Hsd and 3-Ksdh could not be detected in cell extracts of Dietzia sp. strain 2. In the in-vitro assays, the activity of cholyl-CoA ligase was detected. Dietzia sp. strain 2 could not grow with DHOPDC, 7,12-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO), DHADD and THSATD, which are characteristic intermediates of the 9,10-seco pathway of cholate degradation in strain Chol1.
Besides, these compounds inhibited cholate degradation by Dietzia sp. strain 2 and promoted the accumulation of compound X1 and X2. These results clearly showed that Dietzia sp. strain 2 harbors a pathway for cholate degradation, which is different from the 9,10-seco pathway.
Altogether, we could show that the capability for cholate-degradation is widespread among the cultivable bacteria present in the littoral zone of Lake Constance. The discovery that Dietzia sp.
strain 2 degraded cholate via a new pathway suggests that the diversity of metabolic pathways for steroid degradation might be under-estimated.
Introduction
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4 I
NTRO DUCT ION 4.1 ST E RO IDSAn alicyclic organic compound with four condensed rings, referred as A, B, C and D and showing a cyclopentanoperhydrophenanthrene arrangement (a in Fig. 4-1) is named as steroid.
Numerous distinct steroids, such as cholesterol, bile salts (e.g. cholate and deoxycholate), sexual hormones (e.g. testosterone and estradiol) and adrenal corticosteroids (e.g. corticosterone) are reported to be present in animals to fulfill various biological functions. Steroids are also found in plants (e.g. β-sitosterol) and fungi (e.g. ergosterol), a principal component of membrane lipids.
Steroids seldom occur in bacteria, e.g. 7-cholesten-3β-ol in Stigmatella aurantiaca (Bode et al., 2003), in which the purpose and function of steroids remain unclear. The molecular structures of cholate, 7-cholesten-3β-ol, cholesterol, deoxycholate, estradiol, β-sitosterol, and testosterone are shown in Fig. 4-1, and they differ from each other by the presence or absence of the alkyl/acyl side chain attached to the D-ring, the length of the alkyl/acyl side chain and the oxidation status of their A- and B rings. Steroids are very stable and persist in the environment due to the presence of methyl groups located at C10 and C13 positions, with which these carbon atoms become quaternary, and the oxidation of these carbon atoms is difficult.
4.2 ST E RO IDS IN T H E E NVIRO NME NT
Steroids enter into the environment from various sources, such as decaying dead animals and plants and excretions from animals and humans (Shore and Shemesh, 2003). Natural steroids like, cholesterol, ergosterol, 17-α and -β estradiol, β-sitosterol and testosterone have been quantitatively determined in various environments e.g. soils of agricultural fields in Italy (Puglisi et al., 2003), an aquatic habitat situated in San Joaquin, California (Kolodziej et al., 2004), a fresh water River in Jordan (Barel-Cohen et al., 2006), the river streams of Pennsylvania (Velicu and Suri, 2009) and different locations of Pearl River, China (Yu et al., 2011). Sewage treatment plants situated at various places have been identified as major steroidal sinks for natural and synthetic steroids e.g. ethinylestradiol, a major compound in contraceptive pills (Gomes et al., 2009; Kolodziej et al., 2004; Streck, 2009) which might seep into the adjacent environment.
Introduction
13
The concentration of 17-α and -β estradiol, estriol, estrone, ethinylestradiol, progesterone and testosterone may vary in accordance with sampling time and the source, which is releasing these compounds into the environment. Wastewater treatment plants have also been identified as the potential steroid reserves (Streck, 2009; Ying et al., 2002). The concentration of estradiol is often found higher than other steroids in the environment.
Figure 4-1: Examples of different steroid compounds. (a) cyclopentanoperhydrophenanthrene, (b) cholate, (c) 7-cholesten-3β-ol, (d) cholesterol (e) deoxycholate, (f) estradiol, (g) β-sitosterol and (h) testosterone.
Introduction
14
There are many anthropogenic factors responsible for the accumulation of steroids in the environment, such as sewage water treatment plants (Ying et al., 2002), using manure as organic fertilizer (Hanselman et al., 2003), intensive poultry and cow farming practices (Lange et al., 2002) and a river sediments which receive the effluent of paper mills (Jenkins et al., 2003).
Recently, rangeland animal grazing has been recognized as one of the major human activities which is responsible for the accumulation of natural steroids in the environment (Kolodziej and Sedlak, 2007). Thus, it is presumed that natural steroids are ubiquitous in the environment.
4.2.1 IM P A C T OF S TE R O ID S I N TH E EN V IR O N MEN T
Hormonally active steroid compounds could cause serious effects to the endocrine systems of aquatic animals and birds and leading to the misbalance in their hormone system even with concentrations at the ng/l level (Hanselman et al., 2003; Sumpter and Johnson, 2005).
Feminization of male fish by estrogen present in aquatic habitats has been reported (Jobling et al., 2006). Androstenedione acts as a pheromone for goldfishes (Sorensen et al., 2005) and in the environment, it is likely subjected to microbial degradation. This may cause certain disturbance in their steroid- mediated communications. Recently, cholate has been found act as plant elicitor in the rice seedlings by promoting the secretion of phytoalexins (Koga et al., 2006; Shimizu et al., 2008). Hence, it has been proposed that the availability of cholate in garden soil enhances the survival fitness of plants against their pathogens.
4.2.2 FA T E O F S TE R O ID S I N TH E EN V IR O N ME N T
It is apparent that steroids are highly influential and elicits many ecological effects on animals and plants in the environment. However, in the environment, steroids are either transformed or degraded completely by the cooperation existing between non-biological (e.g. light, adsorption) and biological factors.
Introduction
15
4.2.2.1 NO N-B I OL O G I CA L RE M O VA L OF S T ER O I D S
Among the non-biological factors, photolysis of steroids and sorption of steroids to a solid material have been reported by several authors (Casey et al., 2005; Lee et al., 2003; Liu et al., 2003). It has been suggested that photolysis is the optimal way to remove steroids, especially for 17-β estradiol, from sewage treatment plants. The estrogenic activity of 17-β estradiol has been found to be reduced by ~ 50 % with UV-light in the presence of a catalyst (Coleman et al., 2004). Concerning the adsorption, the time taken for adsorbing to solid material for testosterone and estradiol has been reported (Casey et al., 2004; Casey et al., 2003). However, the mechanism of adsorption and its effectiveness in removing steroids from the environment remains unclear and is not completely understood.
4.2.2.2 BI O L OG I C AL RE M O VA L OF S T ER O I D S
In the environment, e.g. aquatic eco-system or soil, microbiological removal of steroids is likely the predominant way to remove these compounds completely from the environment as a part of the global carbon cycle. Microbial degradation of steroids has been the focus of study for several authors. Many authors have found different bacterial strains capable of degrading steroids, which are described below. Studying bacterial degradation of steroid is attractive because the hydroxylated or dehydroxylated and functional-group-modified steroid compounds have been emphasized for a long time because they show potential therapeutic properties, e.g. hydroxycorticoson, ethinylestradiol. Thus, studying the properties of the enzymes responsible for regio-stereo specific hydroxylation or dehydroxylation present in these steroid degrading bacteria would be important in order to produce clinically active compounds. These strains could be used to produce numerous active steroid compounds on the industrial scale, serving as a way to produce green chemicals. Thus, the bacteria capable of employing such regio and stereo specific enzymes acting on steroids at specific positions would be of particular interest to the biotechnology industries.
Introduction
16
4.3 BACT E RIAL DE G RADAT IO N O F S T E RO IDS
Extensive varieties of natural steroids have been identified in plants and animals and synthetic steroids are also being explored to fulfill the therapeutic demands of humans. All these steroids somehow enter into the environment. In the environment, hormonally active steroids are toxic to bacteria (Plotkin et al., 2003). However, many intestinal and environmental bacteria have been identified as they could degrade steroids and use them as the sole source of carbon and energy both under oxic and anoxic conditions, such as Bacteriodes sp. (Aries and Hill, 1970; Groh et al., 1993), Clostridium sp. (Goddard and Hill, 1972), Microbacterium sp. (Yu et al., 2007), Pseudomonas sp. (Leppik, 1989; Tenneson et al., 1979), Rhodococcus sp. (Watanabe et al., 1987) and Steroidobacter denitrificans (Fahrbach et al., 2010). A β-proteobacterium, Comamonas testosteroni has long been recognized as a model organism for studying bacterial steroid degradation and its regulation (Horinouchi et al., 2010).
Steroid degradation is generally initiated by oxidizing the A-ring of the steroid skeleton, and/or by CoA-activation of the acyl side chain. This is followed by the presumable β-oxidation of the acyl side chain leading to the formation of androstadienediones (ADDs), a central intermediate often found in the culture supernatants of steroid degrading bacteria (Hayakawa, 1982; Kieslich, 1985) in which the acyl side chain is completely removed and the A-ring of the steroid skeleton is oxidized. ADDs are further degraded through the so-called 9,10-seco pathway of steroid degradation. The 9,10-seco pathway for the degradation of dihydroxy-ADD is exemplified in Fig. 4-3. Many genes and enzymes involved in the 9,10-seco pathway have been documented in C. testosteroni, degrading-testosterone and Mycobacterium tuberculosis, which is utilizing- cholesterol.
After the formation of ADDs, the A-ring is further oxidized, and leading to a compound with an aromatized A-ring and concomitantly breaking the C-C bond between C9 and C10 position, by which the B-ring is opened (Park et al., 1986). Breaking the B-ring prior requires the hydroxylation at the C9 position by an oxygen-dependent 3-ketosteroid-9α-hydroxylase, aromatization of A-ring, and followed by retro-aldol rearrangement. A compound with a steroid skeleton, including a broken ring is called seco-steroid, hence this bacterial steroid degradation pathway is known as the 9,10-seco pathway.
Introduction
17
Figure 4-2: Aerobic bacterial steroid degradation is channeled into the formation of (ADDs), central intermediates and degraded further through 9,10-seco pathway. Adapted and modified from (Philipp, 2011)
The enzyme, 3-ketosteroid-9α-hydroxylase was purified from R. rhodochrous (Petrusuma et al., 2009), and identified in Arthrobacter oxydans (Dutta et al., 1992), R. erthropolis (Van der Geize et al., 2002), and M. tuberculosis (Hu et al., 2010).
Introduction
18
Once the B-ring is opened, the C4 position of the A-ring is hydroxylated, and leads to the steroid compound known as 3,4-dihydroxy-9,10-secoandrota-1,3,5(10)-triene-9,17-dione (3,4-DHSA, compound III; Fig. 4-3). A structurally similar compound to 3,4-DHSA has been identified in cholesterol-utilizing M. tuberculosis.
The subsequent degradation proceeds in a fashion similar to the oxidation of aromatic compounds by employing meta-cleaving extra diol oxygenases (Horinouchi et al., 2003a;
Horinouchi et al., 2003b; Horinouchi et al., 2001) and lead to break the A-ring, yielding 7,12-dihydroxy derivate of 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10)2-diene-4-oate (compound IV; Fig. 4-3). This is followed by a hydrolytic cleavage between former A- and B-rings. A carbon-carbon hydrolase from M. tuberculosis does this cleavage reaction (Lack et al., 2010). A similar hydrolase was also identified in mutant strain of C. testosteroni, degrading testosterone (Horinouchi et al., 2004). The products of this hydrolytic cleavage are an aliphatic 2-hydroxy-hexa-2,4-dienoate (HHD, compound V; Fig. 4-3) and 7,12-dihydroxy-9,17-dioxo- 1,2,3,4,10,19-hexanorandrostan-5-oate (7,12-dihydroxy DOHNAA, compound VII; Fig. 4-3).
The HHD is further degraded by hydradation and form 4-hydroxy-2-oxohexanoate (compound VI; Fig. 4-3), which is presumably converted into propionate and pyruvate and degraded completely.
The subsequent degradation of the other intermediate of this pathway (7-12-dihydroxy DOHNAA, compound VIII; Fig. 4-3) is not yet clear. A further mutant strain of C. testosteroni, interrupted a gene designated as ORF18 encoding a putative CoA-transferase accumulates 7,12-dihydroxy DOHNAA (Horinouchi et al., 2006), suggesting that CoA activation is required for degrading this 7-12-dihydroxy DOHNAA to CO2 and the subsequent biochemical reactions of the 9,10-seco pathway of steroid degradation are not still known.
Recently, Rhodococcus sp. and Mycobacterium sp. are also being utilized for studying the biochemical pathway and genetic regulation of cholesterol degradation (Van der Geize et al., 2007; Yam et al., 2011). In these organisms, authors have found the formation of common intermediates (ADD and seco steroid) and the release of propionyl- and acetyl-CoA moieties during cholesterol degradation.
Introduction
19
Figure 4-3: The 9,10-seco pathway for degradation of dihydroxy androstadienedione based on compounds identified in different steroid-degrading bacteria. (I) 7,12-dihydroxy-1,4-androstadiene-3,17-dione, (II) 3,7,12-trihydroxy-9,10-seco-1,3,5(10)-androstatriene-9,17-dione, (IV) 7,12-dihydroxy derivate of 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10)2-diene-4-oate, (V) 2-hydroxy-hexa-2,4-dienoate, (VI) 4-hydroxy-2-oxohexanoate, (VII) 7,12-dihydroxy-9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oate.
Compounds I and II have been identified in Pseudomonas sp. strain Chol1 during the degradation of cholate (Refer chapter 4.4.2 described below) and others have been identified in Comamonas testosteroni degrading testosterone.
Compounds indicated in red are hypothetical. A structural analogue of compound III has been detected in Mycobacterium tuberculosis utilizing cholesterol.
Introduction
20
4.4 BIL E S AL TS
Cholate and chenodeoxycholate are primary bile salts of mammals, derived from cholesterol.
Bile salts are surface-active steroids and act as detergents due to the presence of α-hydroxyl groups at C3, C7 and C12 positions, e.g. hydroxyl groups attached to C3, C7 and C12 positions in cholate are protruding in one direction, enable it to behave as detergent. Bile salts are highly water-soluble compounds. Bile salts are produced in the liver, stored and concentrated in the gall bladder from which they are discharged into the bile duct. Bile salts are not released as such into the intestine but appear as conjugates with either taurine or glycine. In the intestinal environment, primary bile salts are subjected to microbial transformation, which lead to the formation of secondary bile salts, such as deoxycholate and lithocholate. Bile salts emulsify dietary lipids and promote their digestion. Later, the unused bile salts are reabsorbed by enterohepatic circulation, nevertheless significant portion of bile salts are released through their fecal (0.3 - 0.6 g day-1) and urinary excretion (~ 4 mg day-1) (Hylemon and Harder, 1998; Ridlon et al., 2006).
4.4.1 BA C TE R IA L DE G R AD A TI O N OF B IL E S AL T S
A unifying scheme of degradation of bile salts by different types of bacteria has been proposed (Hayakawa, 1982). All the biochemically and physiologically characterized bacteria capable of degrading bile salts follow the 9,10-seco steroid pathway through the formation of the central intermediate ADD (Bortolini et al., 1997; Park et al., 1986; Philipp, 2011).
4.4.2 CH O L A T E D E G RA D A TI O N B Y PS EU D OM O NA S S P. S T RA I N CH OL1
The facultative anaerobic bacterium Pseudomonas sp. strain Chol1 was found to be able to grow with cholate (compound I; Fig. 4-4) and use it as the sole source of carbon and energy.
Pseudomonas sp. strain Chol1 could transform cholate into 7,12-dihydroxy-1,4-androstadiene- 3,17-dione (DHADD, compound XVII; Fig. 4-4) as the end product using nitrate as an electron acceptor under anoxic conditions. DHADD is further converted into 3,7,12-trihydroxy-9,10- seco-1,3,5(10)-androstatriene-9,17-dione (THSATD, compound XVIII; Fig. 4-4) under oxic conditions by an oxygenase-dependent reaction, and THSATD is subsequently degraded completely. THSATD is a seco-steroid.
Introduction
21
The strain Chol1 transiently accumulates DHADD and THSATD in the culture supernatant during cholate degradation under oxic conditions (Philipp et al., 2006) and the proven biochemical pathway of cholate degradation by strain Chol1 is shown in Fig. 4-4 (Birkenmaier et al., 2011). In strain Chol1, cholate oxidation is initiated by oxidizing the A-ring, and/or activating the acyl side chain of cholate with CoA either simultaneously or subsequently. The intermediates with an oxidized A-ring, 3-ketocholate (compound II; Fig. 4-4), ∆1/∆4 monoene of 3-ketocholate, ∆1,4-3-ketocholate (compound IV; Fig. 4-4) were identified earlier. The activated acyl side chain is further oxidized in a fashion as presumably similar to the β-oxidation of fatty acids, and is cleaved from the steroid skeleton of cholate skeleton thus, leading to the formation of DHADD.
A transposon mutant, Pseudomonas sp. strain G12 could grow with succinate in the presence of cholate and transformed cholate into three major products, accumulating in the culture supernatant. One major dead end product was identified as 7α,12α-dihydroxy-3-oxochola- 1,4,22E-triene-24-oate (DHOCTO, compound XI; Fig. 4-4), free salt of DHOCTO-CoA (compound IX; Fig. 4-4). DHOCTO-CoA was formed from ∆1,4-3-ketocholyl-CoA (compound VIII; Fig. 4-4) by the activity of dehydrogenase (Fig. 4-4).
The second minor compound was 7α,12α,22-trihydroxy-3-oxochola-1,4-diene-24-oate (THOCDO, compound XII; Fig. 4-4) and third was ∆1,4-3-ketocholate found the culture supernatant of strain G12. It was presumed that DHOCTO-CoA is hydrated and forms THOCDO-CoA, CoA ester of THOCDO (compound X; Fig. 4-4). A second dehydrogenase is catalyzing THOCDO-CoA into compound XIII.
During the oxidation of acyl side chain in cholate degradation, an acetyl-CoA (C2) residue is released at the point where a presumable β-keto thiolase is involved in converting compound XIII into CoA ester of 7,12-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC-CoA, compound XIV; Fig. 4-4, description about DHOPDC is followed). The gene responsible for this reaction is skt (steroid keto thiolase). This gene was recently identified in this transposon mutant Pseudomonas sp. strain G12, encoded a β-keto thiolase involving in the degradation of the acyl side chain of cholate (Birkenmaier et al., 2011).
Introduction
22
Figure 4-4: The proposed degradation pathway for cholate (I) by Pseudomonas sp. strain Chol1. (II) 3-ketocholate, (III) ∆4-3-ketocholate, (IV) ∆1,4-3-ketocholate, (V) cholyl-CoA, (VI) 3-ketocholyl-CoA (VII) ∆4-3-ketocholyl-CoA (VIII) ∆1,4-3-ketocholyl-CoA, (IX) DHOCTO-CoA, (XI) DHOCTO, (XII) THOCDO, (XIV) DHOPDC-CoA, (XV) DHOPDC, (XVII) DHADD and (XVIII) THSATD. Compounds indicated in red color (IX, X, XIII and XVI) have not been detected yet. Reactions indicated red in color are hypothetical. DHOPDC, DHOCTO and THOCDO are the products accumulating in the culture supernatant of strains R1 and G12, respectively. skt and acad are genes encoding steroid keto thiolase and acyl-CoA dehydrogenase, respectively involved in side chain degradation.
Acetyl- and propionyl-CoA are released during side chain degradation.
Introduction
23
Another transposon mutant, Pseudomonas sp. strain R1 could also grow with succinate in the presence of cholate. Cholate was transformed into a dead-end product accumulating in the culture supernatant, identified as 7,12-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC, compound XV; Fig. 4-4), free salt of DHOPDC-CoA (compound XIV; Fig. 4-4).
This is the product with the shorted acyl side chain during cholate degradation in strain Chol1.
A further gene, acad (acyl-CoA dehydrogenase) responsible for the side chain degradation was identified in this mutant, encoded a presumable acyl-CoA dehydrogenase involving in the conversion of DHOPDC-CoA into compound XVI (Birkenmaier et al., 2007).
Further, the C17 position of compound XVI is presumably dehydrogenated, subsequently hydratized, and followed by an aldol cleavage, and thereby releasing a propionyl-CoA (C3) residue. The remaining the part of the side chain is completely removed from the steroid skeleton of cholate, and leads to DHADD (Fig. 4-4).
Shortly, two genes involved in side chain degradation of cholate were identified in Pseudomonas sp. strain Chol1. 3-ketocholate, ∆1/∆4-monoene of 3-ketocholate, ∆1,4-3-ketocholate, DHOCTO, DHOPDC, DHADD and THSATD are the intermediates identified in strain Chol1 during cholate degradation. Acetyl- and propionyl- CoA residues are released from the steroid skeleton of cholate during the presumable β-oxidation of acyl-side chain. The strain G12 releases neither propionyl-CoA nor acetyl-CoA and the strain R1 only releases acetyl-CoA, not propionyl-CoA from the oxidation of acyl-side chain. There is no information available regarding the fate of these C3 and C2 residues released from the steroid skeleton of cholate in strain Chol1. Thus, the metabolism of acetyl- and propionyl-CoA residues are to be determined in strain Chol1 during cholate degradation. Following this elucidation, we want to determine the impact of these side chain residues in the central metabolism of strain Chol1.
There are many putative pathways for the oxidation of propionate in bacteria, starting with propionyl-CoA (Fig. 4-5). e.g. 2-methylcitrate cycle in Escherichia coli (Textor et al., 1997), a reductive carbonylation in a methanogenic culture (Tholozan et al., 1988) , α- and β-oxidation in Clostridium kluyveri (Schweiger and Buckel, 1985) and α-carbonylation in Propionibacterium sp.
Introduction
24 Propionate
CH2 COOH H3C
CH2 COSCoA H3C
Propionyl-CoA
CH2 COO- H2C O COO-
Oxalo acetate
2-methylcitrate
2-methylisocitrate
C COOH H3C
Succinate Fumarate
Malate
Pyruvate O
C COOH H2C
O H3C
H2C H3C C
H OH
C 2-Oxobutyrate
2-Hydroxybutyryl-CoA
Crotonyl-CoA SCoA O
CH
H3C CH COSCoA
CH2 CH COSCoA Acrylolyl-CoA
CHOH
H3C COSCoA Lactolyl-CoA
C COOH H3C
Pyruvate O
CH2OH CH2 COSCoA 3-Hydroxypropionyl-CoA
OHC CH2 Malonic semialdehyde
COOH
C CH2 COOH Malonyl-CoA HC
H3C
Methylmalonyl-CoA COOH
COSCoA Succinyl-CoA
a
b
c, d
c
d e
CoAS O MCS
MCD AC
MCL SDH
FUM
MDH HO C CH
COO-
COO- CH3 H2C
COO-
C
H C
-OOC
COO- CH3 H2C
COO- OH
Figure 4-5: Possible biochemical pathways for the oxidation of propionate in bacteria.
(a) 2-methylcitrate cycle (b) reductive carbonylation (c) α-oxidation (d) β-oxidation (e) carboxylation. Adapted and modified from (Textor et al., 1997). Alphabetical order of enzymes involved in these cycles: ACN (aconitase), FUM (fumarase), MDH (malate dehydrogenase), MCD (methylcitrate dehydratase), MCS (2-methylcitrate synthase), MCL (2-methylisocitrate lyase), and SDH (succinate dehydrogenase).
Introduction
25
Moreover, the release of propionyl-CoA was earlier observed in cholesterol-utilizing Mycobacterium tuberculosis, and the released propionyl-CoA is oxidized via the 2-methylcitrate cycle (Munoz-Elias et al., 2006) suggesting that cholate oxidizing strain Chol1 might also use a similar pathway for degrading propionate.
Later, 2-methylcitrate cycle was identified as a responsible pathway for the oxidation of propionyl-CoA in cholesterol-utilizing M. smegmatis and the role of this pathway was found to reduce the intracellular concentration of propionyl-CoA (Upton and McKinney, 2007). If strain Chol1 is also using the 2-methylcitrate cycle then the role of the 2-methylcitrate cycle in cholate- grown strain Chol1 could also be explained.
Besides, 2-methylcitrate was identified as a potent inhibitor of cell growth in Salmonella enterica (Horswill et al., 2001). Moreover, cholate itself is toxic to strain Chol1 (Philipp et al., 2006).
Thus, propionyl-CoA and the presumptive intermediates of the propionyl-CoA oxidation pathway(s) may also exert an effect in cholate-grown cells of strain Chol1. This makes it interesting to investigate the physiology of cholate degradation.
Besides, the bacteria capable of degrading steroids which have been identified and characterized are studied on the phenomenological level, e.g. for optimizing conditions to produce and accumulate more steroid compounds in their culture supernatants. The knowledge about the physiology and biochemistry of steroid degradation during growth with steroids is still limited.
Introduction
26
4.5 MICRO CO S M AND E NRICH ME NT FO R CH O L AT E DE G RADING B ACT E RIA PRE S E NT IN T H E L IT T O RAL ZO NE O F LAK E CO NS T ANCE It has been proven that fishes and aquatic animals release steroids into their eco-system (Kolodziej et al., 2004; Sorensen et al., 2005). However, it is a difficult task to collect data regarding the concentration of available steroids in a particular eco-system at a given point in time. This data might provide an idea about the probable number of bacteria that are present in a particular eco-system and which capable for degrading steroids. To our knowledge, there is no such data available for the littoral zone of Lake Constance. Nevertheless, we wanted to investigate the cholate degrading capacity of those bacteria, which are present in the littoral zone of Lake Constance.
Furthermore, our knowledge about microbial steroid degradation by environmental bacteria is limited. In particular, not much is known about the diversity, abundance and eco-physiology of steroid-degrading bacteria present in a particular environment, e.g. littoral zone of Lake Constance. Since steroids are ubiquitous in the environment, bacteria capable of degrading these steroids might also be ubiquitous. Two approaches were used to investigate this hypothesis. The following aspects of microbial steroid biodegradation are the focus of this study.
First, we wanted to determine the capability of bacteria present in the littoral zone of Lake Constance for cholate degradation. Second, we wanted to isolate cholate-degrading bacteria belonging to different phylogenetic groups, and study the biochemical-diversity in them.
Introduction
27
4.6 AIMS O F T H IS DIS S E RT AT IO N
Firstly, as described above studies on propionate degradation in cholate-grown cells of Pseudomonas sp. strain Chol1 were missing.
Secondly, not much is known about the abundance and biochemical-diversity of steroid- degrading bacteria in a given environment. Thus, the objectives of this PhD thesis are as follows
1) In the first part, we wanted to investigate and determine the fate of propionyl and acetyl-CoA moieties released from the steroid skeleton of cholate during the presumable β-oxidation of the acyl side chain in Pseudomonas strain sp. Chol1 and study the impact of propionyl-CoA in the central metabolism of strain Chol1.
2) In the second part, we wanted to investigate our hypothesis regarding the presence of steroid degrading bacteria in the littoral zone of Lake Constance. Two approaches were used to investigate our hypothesis. First, we wanted to determine the capability of the environmental bacteria present in the littoral zone of Lake Constance for cholate degradation.
3) Second, we wanted to scrutinize the diversity and abundance of steroid degrading bacteria present in the littoral zone of Lake Constance. For this, we set up quantitative enrichments for cholate-degrading bacteria from the littoral zone of Lake Constance and isolate pure cultures of cholate-degrading bacteria belonging to different phylogenetic groups. Subsequently, we wanted to study and analyze the biochemical pathways of cholate degradation in all these new strains and check whether all of them are following the similar 9,10-seco pathway for degrading cholate.
Materials and methods
28
5 M
ATERI ALS A NDM
ETH ODS5.1 MICRO B IO L OG ICAL ME T H O DS 5.1.1 BA C TE R IA L S TR A IN S
Pseudomonas sp. strain Chol1 was maintained on MMChol agar plate containing cholate (2 mM) as described earlier (Philipp et al., 2006). The transposon mutants Pseudomonas sp. strain R1 (Birkenmaier et al., 2007), and Pseudomonas sp. strain G12 (Birkenmaier et al., 2011) were maintained on MMChol agar plates containing cholate (2 mM) + succinate (12 mM) with the appropriate concentration of antibiotics. The plates are incubated at 28°C for 1 - 2 days and all those plates were preserved at 4°C and used as the parental inoculum for preparing the respective pre-cultures for growth experiments. Before preserving the plates, those were left at lab bench for ~ 24 hr.
Further bacterial strains, namely strains 3 - 8, and strains 10 - 15 were isolated from the littoral zone of Lake Constance and maintained on medium B (see below) agar plates containing cholate (1 mM), preserved at 4°C and used as the parental inoculum for preparing the respective pre-cultures for growth experiments. Sub-cultures of all these strains were prepared constantly for every two weeks. Zoogloea sp. strain 1, Dietzia sp. strain 2 and Pseudomonas sp. strain 9 were also preserved in the similar manner and before preserving the plates those were incubated at 28°C for 1 - 2, 2 - 3 and 1 - 2 days, respectively and were left at lab bench for ~ 24 hr.
Sub-cultures of Zoogloea sp. strain 1, Dietzia sp. strain 2 and Pseudomonas sp. strain 9 were prepared for every week.
5.1.2 ME DI A
For growth experiments with Pseudomonas sp. strain Chol1, R1, and G12, MMChol medium was used as described earlier (Birkenmaier et al., 2007; Philipp et al., 2006).
For enrichment and isolation of cholate degrading bacteria from the littoral zone of Lake Constance, medium B (Jagmann et al., 2010) was used with vitamins, trace elements solution SL10 (Widdel and Pfennig, 1981) and cholate (1 mM). Medium B is a HEPES-buffered minimal medium and contained the following ingredients: HEPES (50 mM), NH4Cl (5 mM),
Materials and methods
29
MgSO4 (0.5 mM), KCl (14 mM), NaCl (7.2 mM). The pH was adjusted to 7.0 with NaOH (5 M).
After autoclaving and cooling this medium, CaCl2 (0.01 mM) and phosphate solution [(0.15 mM pH 7.1, prepared with K2HPO4 x 3H2O (350 mM), NaH2PO4 x H2O (150 mM)] and trace element solution SL 10 were added. Cholate was added into this medium B from a sterile stock solution to serve as the sole source of carbon and energy. Medium B agar (1.5 %) plates with cholate (1 mM) were prepared to maintain strains 1 - 15.
5.1.3 GR O W T H EX PE R IM EN T S
All growth experiments were performed at 28°C. Growth was determined by measuring optical density at 600 nm (OD600).
5.1.3.1 PS E U D OM O NA S S P. ST R AI N S CH O L1, R1 A ND G12
To perform growth experiments with Pseudomonas sp. strain Chol1 in MMChol medium with cholate (2 mM) as the sole source of carbon and energy, the protocol described earlier was followed (Philipp et al., 2006). Either one of the following substrates, propionate (12 mM) succinate (12 mM), or acetate (24 mM) replaced cholate wherever necessary. Pre-cultures were prepared on MMChol medium with either propionate or succinate as the sole source of carbon and energy for 45 - 48 hr and 12 - 14 hr, respectively.
The transposon mutants Pseudomonas sp. strain R1 and strain G12 were grown in MMChol medium with cholate (2 mM) + succinate (12 mM) and kanamycin (10 µg/ml) as described earlier (Birkenmaier et al., 2007).
5.1.3.2 ZO O G LO EA S P. S T R AI N 1, DI E TZI A SP. S TR A I N 2 A N D PS E U DO MO N A S S P. S T RA I N 9 WI T H C H OL AT E
Strains 1 and 9 were grown in medium B with cholate (1 mM), and other experimental conditions and set-up were similar to Pseudomonas sp. strain Chol1 as described earlier (Philipp et al., 2006). In contrast to strain Chol1, the main cultures of strain 2 were incubated at 100 rpm on a shaker (Certomat, Braun).
Materials and methods
30
Immediately after inoculation, samples were withdrawn (time 0) from the main culture and at regular intervals thereafter for measuring OD600 and analyzing metabolites. The samples were centrifuged (10 min at 12,800 rpm in an Eppendorf), and the supernatant was stored at -20°C until HPLC analysis.
5.1.3.3 DI E TZI A S P. S T R AI N 2 WI T H T H E I N TER M E DI AT E S OF C H O LA T E D E G R A DA TI O N R E LE A S E D FR O M PS E UD O MO N A S S P. S T R AI N
CH O L1
Growth experiments of Dietzia sp. strain 2 were performed in medium B with either one of the following substrates as the sole source of carbon and energy namely, DHOCTO, DHOCTO + cholate (1 mM), DHOPDC (~ 1 mM), DHOPDC (~ 1 mM) + cholate (1 mM), DHADD (~ 1 mM), DHADD (~ 1 mM) + cholate (~ 1 mM), THSATD (~ 1 mM), THSATD (~ 1 mM) + cholate (~ 1 mM).
Growth experiments were performed in sterile test tubes (10 ml) containing 4 ml of medium B with the appropriate single and or combination of carbon source(s) and seeded with the inoculum of strain 2. Inoculum of strain 2 was prepared from one-week-old medium B parental agar plate containing cholate (1 mM) preserved at 4°C. Few loop-full of cells were taken and resuspended homogenously in a small volume (1.5 ml) of medium B without cholate. The appropriate volume of cell-suspension was transferred to test tubes containing 4 ml of medium B with cholate (1 mM) to get a final OD600 of ~ 0.05. Tubes were incubated at 180 rpm on a rotary mixer (CVM, Fröbel) for 7 - 8 days. Samples were withdrawn immediately after inoculation (time 0), at regular intervals thereafter and at the end of the growth experiments (time end).
Supernatants were prepared by centrifugation (10 min at 12,800 rpm in an Eppendorf) and stored at -20°C until HPLC analysis.
To check the physiological active state of inoculum of strain 2, the same volume of inoculum was transferred to medium B with cholate (1 mM). To check the quality of substrates used in this experiment that have been purified from strains Chol1, R1 and G12 (see below), growth of strain Chol1 was additionally monitored with medium B in the presence of the respective compounds.
Materials and methods
31
5.1.4 PR E P A R AT I ON O F DHADD F R O M PS E U DOM O NA S S P. S TR A IN CH O L1
Pseudomonas sp. strain Chol1 was grown in MMChol1 medium with 2 mM cholate under anoxic condition with nitrate (10 mM) as an electron acceptor as described earlier (Philipp et al., 2006). After a week of incubation, filter sterilized nitrate solution was added into the culture to transform all other intermediates in to DHADD in the culture supernatant. Cells were removed by centrifugation (5,900 x g for 15 min at room temperature), and extracted at least four times with dichloromethane (DCM) as described earlier (Philipp et al., 2006). After the evaporation of DCM, the residual solid was dissolved in medium B (35 ml) and sterilized by filtration. The purity of DHADD was checked by HPLC. The concentration of DHADD was determined by using extinction coefficient 14,860 M-1 cm-1 and λmax at 244 nm. λmax had been determined by wavelength scan analysis (200 - 450 nm) performed with three different dilutions (1:100, 1:200 and 1:400) of DHADD dissolved in methanol and medium B. The concentration of DHADD was ~ 1 mM.
5.1.5 CE L L-S U SP E N SI O N EX PE R IM EN T S
5.1.5.1 CE L L-S U SP E N SI O N EX PE R IM E NT W IT H PS E U D OM O NA S S P. S T RA I N
CH O L1 F O R DE G R A DA TI O N E XPE R IM E NT S
Cell-suspension experiments were performed to determine the fate of compounds released from side-chain degradation of cholate in Pseudomonas sp. strain Chol1.
Cholate-, propionate- and succinate-grown cells of Pseudomonas sp. strain Chol1 were harvested from the mid-exponential growth phase by centrifugation (5900 x g for 10 min at 4°C) under sterile conditions, and washed twice with sterile ice cold potassium-phosphate buffer (50 mM, pH 7.1, prepared by mixing the equimolar concentration of K2HPO4 x 3H2O and KH2PO4).
Cells were concentrated to the OD600 of 10 with the same buffer and transferred to a appropriate volume to freshly prepared MMChol medium (100 ml of medium in a 500 ml Erlenmeyer flask with baffled)containing either one of the following carbon sources, propionate (12 mM), lactate (12 mM), malonate (12 mM) and pyruvate (12 mM) to get a final OD600 of 1. Cell-suspensions was incubated for 6 - 8 hr on a shaker (Certomat, Braun) at 180 rpm.
Materials and methods
32
Samples were withdrawn immediately after the cells had been added into the medium (time 0) and thereafter at regular intervals. Cells were removed by centrifugation (5,900 x g for 15 min at room temperature) and the supernatant were stored at -20°C until HPLC analysis.
5.1.5.2 CE L L-S U SP E N SI O N EX PE R IM E NT W IT H PS E U D OM O NA S S P. S T RA I N
CH O L1 F O R T HE PR EP A RA T I ON O F THSATD
To prepare THSATD, further cell-suspension experiments were performed. Cholate-grown Pseudomonas sp. strain Chol1 were harvested, concentrated as described above from the mid- exponential phase and transferred to an appropriate volume of fresh MMChol medium containing cholate (2 mM) to get a final OD600 of 1. Cells-suspension of Pseudomonas sp. strain Chol1 was incubated on a shaker (Certomat, Braun) at 180 rpm for 2 - 2.5 hr. THSATD was extracted from the culture supernatant, concentrated and residual solid was dissolved in medium B (35 ml) and sterilized by filtration as described above. The purity of THSATD was checked by HPLC. The concentration of THSATD was determined by using extinction coefficient 10,964 M-1 cm-1 and λmax at 279 nm. λmax had been determined by wavelength scan analysis (200 - 350 nm) performed with three different dilutions (1:50, 1:100 and 1:200) of THSATD dissolved in methanol and medium B. The concentration of THSATD was ~ 1 mM.
5.1.5.3 CE L L-S U SP E N SI O N EX PE R IM E NT W IT H PS E U D OM O NA S S P. S T RA I N
G12 F O R T H E P RE PA R AT I O N OF DHOCTO
Pre-cultures (3 ml) were grown in MMChol medium with filter sterilized DHADD in the presence of kanamycin (10 µg/ml) on a rotary mixer (CVM, Fröbel) at 100 rpm for 2 - 3 days.
When the OD600 of the pre-culture reached ~ 0.25, cells were removed by centrifugation (5900 x g for 5 min at room temperature), resuspended in a small volume of medium B and transferred to fresh MMChol medium (100 ml) containing cholate (2 mM), succinate (12 mM) and kanamycin (10 µg/ml). This main culture was incubated on a shaker (Certomat, Braun) at 180 rpm (for 15 - 16 hr) until mid-exponential phase. Cell-suspension experiments were carried out further to produce DHOCTO as described above for THSATD in MMChol medium with cholate (2 mM) and succinate (12 mM) and this cells-suspension of Pseudomonas sp. strain G12 was incubated on a shaker (Certomat, Braun) at 180 rpm for 20 - 22 hr.
