7.3 E NRICHMENT EXPERIMENTS
7.3.5 Dietzia sp. strain 2
7.3.5.2 Dietzia sp. strain 2 with the intermediates of cholate degradation in strain Chol1
We analyzed the capability of Dietzia sp. strain 2 to degrade DHOCTO, DHOPDC, DHADD and THSATD as well as its ability to degrade cholate both in the presence of these compounds.
Dietzia sp. strain 2 could neither grow with nor degrade the above-mentioned compounds.
In the presence of DHOCTO, Dietzia sp. strain 2 could not use cholate, which might be due to either DHOCTO is being toxic or DHOCTO blocking the uptake of cholate into the cells.
Discussion
Pseudomonassp. strain Chol1 Dietziasp. strain 2
OH
OH
S-CoA O
Figure 7-4: Biochemical pathways of cholate degradation in Pseudomonas sp. strain Chol1 and Dietzia sp. strain 2, drawn in red and blue colors, respectively. The activities of 3-hydroxy steroid dehydrogenase (3-Hsd), and 3-keto steroid dehydrogenase (3-Ksdh) could not be detected in Dietzia sp. strain 2. Cholyl-CoA ligase could be detected in Dietzia sp. strain 2. DHOPDC and DHADD are potentially interfering with the biochemical pathway of cholate degradation in Dietzia sp. strain 2 and lead to the accumulation of compound X and related compounds in the culture supernatant of Dietzia sp. strain 2. Pathways are based on metabolites detected in culture supernatants during growth with cholate. CoA-esters of the respective compounds are not shown for simplicity except for cholyl-CoA in Dietzia sp. strain 2.
Discussion
131
Figure 7-5: Potential structure analogues of compounds X1 and X2 accumulated in the culture supernatant of Dietzia sp. strain 2. (a) 3,12-dioxo-23,24-dinorchola-4,6-dienoic acid (Mukherjee et al., 1993), (b) 4,6-androstadiene-3,17-dione (Zalewski and Dunn, 1970), (c) 1,4,6-androstatriene-3,17-dione, (d) Equilin (Deghengh et al., 1967), (e) Equilenin (Zderic et al., 1958), (f) 12β-hydroxy-1,4,6-androstatriene-3,17-dione, (g) 12α-hydroxy-3-oxo-4,6-choladien-24-oic-acid, (h) 3,12-dioxo-4,6-choladien-24-oic acid (Hayakawa, 1982). λmax of the respective are indicated. a and f were dissolved in methanol while b dissolved in water.
Discussion
132
In M. tuberculosis strain H37Rv, the mce4 locus, a part of mce (mammalian cell entry) operon was identified in involving the uptake of cholesterol (Senaratne et al., 2008; Van der Geize et al., 2007). The mce4 locus was also identified in Rhodococcus jostii strain RHA1, responsible for uptake of cholesterol. However, the growth of the mutant (∆mce4) of R. jostii was not affected with cholate, suggested that mce4 might not be involved in transporting cholate into the cell (Mohn et al., 2008). So far, no cholate specific transporters and the compound blocking the transport of cholate into the cells are known.
The growth of Dietzia sp. strain 2 ceased with cholate in the presence of DHOPDC. Further, we saw the accumulation of an array of compounds similar to compound X in the culture supernatant (Fig. 6-32b). All of these compounds were showing a similar UV-absorption spectrum to compound X and eluted at different time points (Fig. 6-32b and c), suggesting that their core part responsible for absorbing light might be similar, while they may differ in length of their side chain. The existing differences in length of their side chain caused them to elute at different time points. Such steroids having an oxidized A- and B-rings differing from each other by the length of the acyl side chain have earlier been reported in A. simplex (Mukherjee et al., 1993) and S. rubescens (Hayakawa, 1982). In Dietzia sp. strain 2, there is a possibility for the A/-and B-rings to be broken before the activation/or oxidation of the acyl side chain during cholate degradation. If this is the case in Dietzia sp. strain 2, steroid compounds without the acyl side chain (e.g. DHADD and THSATD) could neither be a substrate for growth nor transform into some other compounds.
Dietzia sp. strain 2 could not grow with cholate in the presence of DHOPDC which might be due to DHOPDC is blocking either the enzyme responsible for converting compound X into further products of degradation or blocking the transport of compound X into the cells.
In some instances, while in the presence of DHADD, Dietzia sp. strain 2 could not degrade cholate and accumulated compounds X1 and X2 in the culture supernatant. In this situation, the growth of Dietzia sp. strain 2 ceased. In other instances, Dietzia sp. strain 2 could degrade cholate in the presence of DHADD, and transiently accumulating compounds X1 and X2 in the culture supernatant, which were degraded later.
Discussion
133
In this situation, the growth of Dietzia sp. strain 2 did not cease. Altogether, these results suggest that DHADD could also potentially interfere with the biochemical pathway of Dietzia sp.
strain 2.
Concerning the growth of Dietzia sp. strain 2 with THSATD, there was a slight increase in OD600
in the growth experiment, which was due to the presence of ∆1/∆4-monoene of 3-ketocholate in the medium along with THSATD. We have shown that Dietzia sp. strain 2 is accumulating
∆1/∆4-monoene of 3-ketocholate and ∆1,4-3-ketocholate in the culture supernatant during growth with cholate.
By analyzing the culture supernatant of these experiments, we found that Dietzia sp. strain 2 did not grow with THSATD, because the concentration of THSATD remained at the end of the growth experiment while ∆1,4-3-ketocholate was completely utilized by Dietzia sp. strain 2 (Fig. 6-36a).
In the presence of THSATD, Dietzia sp. strain 2 is able to grow with cholate and degraded it completely. Compounds X1 and X2 were transiently accumulating in the culture supernatant.
Apparently, THSATD is not toxic to Dietzia sp. strain 2 and it has no influence on cholate degradation of Dietzia sp. strain 2.
It is confirmed that Dietzia sp. strain 2 is not releasing DHADD and THSATD in the culture supernatant and it could not degrade these compounds. Dietzia sp. strain 2 is transiently releasing two unknown compounds during cholate degradation, which are to be further analyzed structurally. It is possible that DHADD and DHOPDC are potentially interfering with the biochemical pathway of cholate degradation in Dietzia sp. strain 2.
The activity of cholyl-CoA ligase was determined in the cell extracts of Dietzia sp. strain 2, and further oxidation of cholyl-CoA is yielding a new compound, which did not co-elute with 3-ketocholyl-CoA despite the fact that its UV-spectrum was similar to 3-ketocholyl-CoA.
However, we could not detect the activity of cholate oxidizing enzymes in the cell extracts of Dietzia sp. strain 2, but we did monitor the formation of an unknown product (D:P9; Fig. 6-27a), produced by the action of an enzyme involved in oxidizing CoA-activated cholate.
Discussion
134
Although enzymes oxidizing cholate are prevalent in many actinobacteria, such as Streptomyces lividans (Choi et al., 1995), Nocardia opaca (Drobnic et al., 1993), Nocardia corallina (Hatta et al., 1991) and Rhodococcus rhodochrous (Morii et al., 1998). These enzyme(s) could not be detected in Dietzia sp. strain 2.
It might be that oxidation of cholate is only possible if it is first activated with CoA and this CoA activation might be a prerequisite essential reaction for cholate oxidation in Dietzia sp. strain 2.
All these results strongly suggest that Dietzia sp. strain 2 is degrading cholate completely through a novel degradation pathway, which has so far not been proved in any other steroid-degrading bacteria. Few other genera of actinobacteria, Arthrobacter sp. (Dutta et al., 1992), Mycobacterium sp. (Hu et al., 2010), Nocardia sp. (Strijewski, 1982) and Rhodococcus sp. (Yam et al., 2011) are using the 9,10-seco pathway for degrading steroids. Nevertheless, Dietzia sp.
strain 2 is not using the 9,10-seco pathway for cholate degradation. The pathway for degrading cholate in Dietzia sp. strain 2 might be diverged after the formation of ∆1,4-3-ketocholate in comparing to the pathway of cholate degradation strain Chol1.
Taken all together, we were successful in isolating different strains of bacteria capable of degrading cholate. We were able to show that the abundance of bacteria capable for degrading cholate in the littoral sediment of Lake Constance. Among the cultivable bacteria present in the littoral zone of Lake Constance, approximately more than 1 % of bacteria are being capable of degrading cholate. The capability of bacteria degrading cholate is prevalent in the bacterial communities living in the littoral zone of Lake Constance. These strains of bacteria, which were investigated, all belong to different phylogenetic groups and approximately, they did not show any unity in their biochemical pathways for degrading cholate. Dietzia sp. strain 2 degrades cholate via an unexplored pathway and investigating the biochemistry, physiology and genetics of Dietzia sp. strain 2 will provide more insights of steroid degradation.
---The end
---Appendix
135
8 A
PPEN DIXBlast analysis of 16S rDNA sequence of strain 1
Zoogloea caeni strain EMB 43 16S ribosomal RNA gene, partial sequence Length=1460 Score = 2364 bits (1280)
Query 1 ATCGGAACGTACCCAGTCGTGGGGGATAACGTAGCGAAAGTTACGCTAATACCGCATACG 60 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 91 ATCGGAACGTACCCAGTCGTGGGGGATAACGTAGCGAAAGTTACGCTAATACCGCATACG 150 Query 61 TCCTGAGGGAGAAAGCGGGGGACCGTAAGGCCTCGCGCGATTGGAGCGGCCGATGTCGGA 120 |||||||||||||||||||||||||||||||| |||||||||||||||||||||||||||
Sbjct 151 TCCTGAGGGAGAAAGCGGGGGACCGTAAGGCCCCGCGCGATTGGAGCGGCCGATGTCGGA 210 Query 121 TTAGCTAGTTGGTAGGGTAAAGGCCTACCAAGGCGACGATCCGTAGCGGGTCTGAGAGGA 180 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 211 TTAGCTAGTTGGTAGGGTAAAGGCCTACCAAGGCGACGATCCGTAGCGGGTCTGAGAGGA 270 Query 181 TGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGA 240 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 271 TGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGA 330 Query 241 ATTTTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGAGTGAAGAAGGCCTTCG 300 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 331 ATTTTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGAGTGAAGAAGGCCTTCG 390 Query 301 GGTTGTAAAGCTCTTTCAGCCGGAAAGAAATCGCGCAGGATAATACTCTGCGTGGATGAC 360 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 391 GGTTGTAAAGCTCTTTCAGCCGGAAAGAAATCGCGCAGGATAATACTCTGCGTGGATGAC 450 Query 361 GGTACCGGAAGAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGT 420 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 451 GGTACCGGAAGAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGT 510 Query 421 GCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTATGTAAGACAGAT 480 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 511 GCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTATGTAAGACAGAT 570 Query 481 GTGAAATCCCCGGGCTCAACCTGGGAACTGCGTTTGTGACTGCATAACTAGAGTACGGCA 540 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 571 GTGAAATCCCCGGGCTCAACCTGGGAACTGCGTTTGTGACTGCATAACTAGAGTACGGCA 630 Query 541 GAGGGAGGTGGAATTCCGCGTGTAGCAGTGAAATGCGTAGATATGCGGAGGAACACCGAT 600 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 631 GAGGGAGGTGGAATTCCGCGTGTAGCAGTGAAATGCGTAGATATGCGGAGGAACACCGAT 690 Query 601 GGCGAAGGCAGCCTCCTGGGCCAGTACTGACGCTCATGCACGAAAGCGTGGGGAGCAAAC 660 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 691 GGCGAAGGCAGCCTCCTGGGCCAGTACTGACGCTCATGCACGAAAGCGTGGGGAGCAAAC 750 Query 661 AGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAGTTGTTCGGTGAGGA 720 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 751 AGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAGTTGTTCGGTGAGGA 810
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Appendix
136 Query 721 GACTCATTGAGTAACGCAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGCCGCAAG 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 811 GACTCATTGAGTAACGCAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGCCGCAAG 870 Query 781 GTTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTC 840 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 871 GTTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTC 930 Query 841 GATGCAACGCGAAAAACCTTACCTACCCTTGACATGCCAGGAACTTGCCAGAGATGGCTT 900 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 931 GATGCAACGCGAAAAACCTTACCTACCCTTGACATGCCAGGAACTTGCCAGAGATGGCTT 990 Query 901 GGTGCTCGAAAGAGAGCCTGGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGA 960 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 991 GGTGCTCGAAAGAGAGCCTGGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGA 1050 Query 961 GATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCGTTAATTGCCATCATTAAGTT 1020 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1051 GATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCGTTAATTGCCATCATTAAGTT 1110 Query 1021 GGGCACTTTAGCGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCC 1080 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1111 GGGCACTTTAGCGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCC 1170 Query 1081 TCATGGCCCTTATGGGTAGGGCTTCACACGTCATACAATGGTCGGTACAGAGGGTTGCCA 1140 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1171 TCATGGCCCTTATGGGTAGGGCTTCACACGTCATACAATGGTCGGTACAGAGGGTTGCCA 1230 Query 1141 AGCCGCGAGGTGGAGCCAATCCCAGAAAGCCGATCGTAGTCCGGATTGGAGTCTGCAACT 1200 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1231 AGCCGCGAGGTGGAGCCAATCCCAGAAAGCCGATCGTAGTCCGGATTGGAGTCTGCAACT 1290 Query 1201 CGACTCCATGAAGTCGGAATCGCTAGTAATCGCAGATCAGCATGCTGCGGTGAATACGTT 1260 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1291 CGACTCCATGAAGTCGGAATCGCTAGTAATCGCAGATCAGCATGCTGCGGTGAATACGTT 1350 Query 1261 CCCGGGTCTTGTACACACCGCCC 1283
|||||||||||||||||||||||
Sbjct 1351 CCCGGGTCTTGTACACACCGCCC 1373
Table 8-1: BLAST analysis of partial 16S rDNA of strain 1 No of nucleotides used for analysis: 1283
Differences in the query nucleotides are highlighted red in color
Appendix
137 Blast names color map
bacteria b-proteobacteria unknown unclassified
Table 8-2: Distance tree map of strain 1
Appendix
138
BLAST analysis of 16S rDNA sequences of strain 2
Dietzia natronolimnaea strain W5044 16S ribosomal RNA gene, partial sequence Length=1439 Score = 2481 bits (1343)
Query 6 ATGC-AGTCG-ACGGT-AGGCCCTTTCGGGGGTACACGAGTGGCGAACGGGTGAGTAACA 62 |||| ||||| ||||| |||||||||||||||||||||||||||||||||||||||||||
Sbjct 28 ATGCAAGTCGAACGGTAAGGCCCTTTCGGGGGTACACGAGTGGCGAACGGGTGAGTAACA 87 Query 63 CCTGGGTAATCTGCCCTGCACTTCGGGATAAGCCTGGGAAACCGGGTCTAATACCGGATA 122 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 88 CGTGGGTAATCTGCCCTGCACTTCGGGATAAGCCTGGGAAACCGGGTCTAATACCGGATA 147
Query 123 TGAGCTCCTGCCGCATGGTGGGGGTTGGAAAGTTTTTCGGTGCAGGATGAGTCCGCGGCC 182 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 148 TGAGCTCCTGCCGCATGGTGGGGGTTGGAAAGTTTTTCGGTGCAGGATGAGTCCGCGGCC 207 Query 183 TATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAG 242 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 208 TATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAG 267 Query 243 GGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGG 302 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 268 GGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGG 327 Query 303 GAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGGGGGATGACGGTCTT 362 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 328 GAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGGGGGATGACGGTCTT 387 Query 363 CGGATTGTAAACTCCTTTCAGTAGGGACGAAGCGAAAGTGACGGTACCTGCAGAAGAAGC 422 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 388 CGGATTGTAAACTCCTTTCAGTAGGGACGAAGCGAAAGTGACGGTACCTGCAGAAGAAGC 447 Query 423 ACCGGCCAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTGTCCGGAAT 482 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 448 ACCGGCCAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTGTCCGGAAT 507 Query 483 TACTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCACGTCGTCTGTGAAATCCTCCAGCTCA 542 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 508 TACTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCACGTCGTCTGTGAAATCCTCCAGCTCA 567 Query 543 ACTGGGGGCGTGCAGGCGATACGGGCAGACTTGAGTACTACAGGGGAGACTGGAATTCCT 602 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 568 ACTGGGGGCGTGCAGGCGATACGGGCAGACTTGAGTACTACAGGGGAGACTGGAATTCCT 627 Query 603 GGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTG 662 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 628 GGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTG 687 Query 663 GGTAGTAACTGACGCTGAGGAGCGAAAGCATGGGGAGCAAACAGGATTAGATACCCTGGT 722 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 688 GGTAGTAACTGACGCTGAGGAGCGAAAGCATGGGGAGCAAACAGGATTAGATACCCTGGT 747 Query 723 AGTCCATGCCGTAAACGGTGGGCGCTAGGTGTGGGGTCCTTCCACGGATTCCGTGCCGTA 782 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 748 AGTCCATGCCGTAAACGGTGGGCGCTAGGTGTGGGGTCCTTCCACGGATTCCGTGCCGTA 807
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Appendix
139 Query 783 GCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAAT 842
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 808 GCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAAT 867 Query 843 TGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACC 902 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 868 TGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACC 927 Query 903 TTACCTAGGCTTGACATATACAGGACGACGGCAGAGATGTCGTTTCCCTTGTGGCTTGTA 962 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 928 TTACCTAGGCTTGACATATACAGGACGACGGCAGAGATGTCGTTTCCCTTGTGGCTTGTA 987 Query 963 TACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAA 1022 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 988 TACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAA 1047 Query 1023 CGAGCGCAACCCCTGTCTCATGTTGCCAGCACGTTATGGTGGGGACTCGTGAGAGACTGC 1082 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1048 CGAGCGCAACCCCTGTCTCATGTTGCCAGCACGTTATGGTGGGGACTCGTGAGAGACTGC 1107 Query 1083 CGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCTAGG 1142 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1108 CGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCTAGG 1167 Query 1143 GCTTCACACATGCTACAATGGCTAGTACAGAGGGCTGCGAGACCGCGAGGTGGAGCGAAT 1202 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1168 GCTTCACACATGCTACAATGGCTAGTACAGAGGGCTGCGAGACCGCGAGGTGGAGCGAAT 1227 Query 1203 CCCTTAAAGCTAGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGT 1262 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1228 CCCTTAAAGCTAGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGT 1287 Query 1263 CGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACC 1322 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1288 CGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACC 1347 Query 1323 GCCCGGCGCGTCATGAAAGTCGGTAACACCCGAAGCCGGTG-CCTA 1367
||||| | ||||||||||||||||||||||||||||||||| ||||
Sbjct 1348 GCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCCGGTGGCCTA 1393
Table 8-3: BLAST analysis of partial 16S rDNA of strain 2 No. of nucleotides used for analysis: 1367
Differences in the query nucleotides are highlighted red in color
Appendix
140
Table 8-4: Distance tree map of strain 2
Blast names color map bacteria
b-proteobacteria unknown unclassified
Appendix
141
BLAST analysis of 16S rDNA sequences of strain 9
Pseudomonas putida strain c84 16S ribosomal RNA gene, partial sequence Length=1443 Score = 2333 bits (1263), Identities = 1346/1383 (98%)
Query 11 AACATGC-AGTCGAGCGGATGACGGGAGCTTGCTCCTTGATTCAGCGGCGGACGGGTGAG 69 ||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 18 AACATGCGAGTCGAGCGGATGACGGGAGCTTGCTCCTTGATTCAGCGGCGGACGGGTGAG 77 Query 70 TAATACCTAGGAATCTGCCTGATAGTGGGGGACAACGTTTCGAAAGGAACGCTAATACCG 129 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 78 TAATACCTAGGAATCTGCCTGATAGTGGGGGACAACGTTTCGAAAGGAACGCTAATACCG 137 Query 130 CATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAG 189 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 138 CATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAG 197 Query 190 GTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTG 249 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 198 GTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTG 257 Query 250 AGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAG 309 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 258 AGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAG 317 Query 310 TGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGG 369 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 318 TGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGG 377 Query 370 TCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGCTAATACCTTGCTGT 429 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 378 TCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGCTAATACCTTGCTGT 437 Query 430 TTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACA 489 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 438 TTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACA 497 Query 490 GAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCGTTAAG 549 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 498 GAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCGTTAAG 557 Query 550 TTGAATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCGAGCTAGAGT 609 ||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 558 TTGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCGAGCTAGAGT 617 Query 610 AGGGCAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAAC 669 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 618 AGGGCAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAAC 677 Query 670 ACCAGTGGCGAAGGCGACCACCTGGGCTCATACTGACACTGAGGTGCGAAAGCGTGGGGA 729 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 678 ACCAGTGGCGAAGGCGACCACCTGGGCTCATACTGACACTGAGGTGCGAAAGCGTGGGGA 737
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Appendix
142 Query 730 GCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGA 789
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 738 GCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGA 797 Query 790 ATCCTTGAGATTTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCC 849 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 798 ATCCTTGAGATTTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCC 857 Query 850 GCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTT 909 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 858 GCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTT 917 Query 910 AATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATCCAATGAACTTTCCAGAGAT 969 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 918 AATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATCCAATGAACTTTCCAGAGAT 977 Query 970 GGATTGGTGCCTTCGGGAACATTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCG 1029 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 978 GGATTGGTGCCTTCGGGAACATTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCG 1037 Query 1030 TGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTT 1089 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1038 TGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTT 1097 Query 1090 ATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCA 1149 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1098 ATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCA 1157 Query 1150 AGTCATCATGGCCCTTACGGCCTGGGCTACCCACGTGCTACAATGGTCCGGTACAAAGGG 1209 |||||||||||||||||||||||||||||| ||||||||||||||||| |||||| ||||
Sbjct 1158 AGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTC-GGTACAGAGGG 1216 Query 1210 TTGCCAAGCCGCGAGGTGGAACTAATCTCAAAAAACCGATCGGATTCCGGAACCGCATTC 1269 |||||||||||||||||||| ||||||||| ||||||||||| | |||||| | ||| ||
Sbjct 1217 TTGCCAAGCCGCGAGGTGGAGCTAATCTCACAAAACCGATCGTAGTCCGGATC-GCAGTC 1275 Query 1270 TGCAACTCGAATGGCTTGATGTCGGAATCCCTAATAATCCCGAAATCCAAAATTGCTC-C 1328 |||||||||| || | ||| ||||||||| ||| ||||| |||| || | ||| | || | Sbjct 1276 TGCAACTCGACTG-CGTGAAGTCGGAATCGCTAGTAATCGCGAA-TC-AGAAT-G-TCGC 1330 Query 1329 GGTGAATAACTTTCCCGGAGCCTTTGTA-ACCCGCGCCCGTCACCACT-TGGGGAATGGG 1386 |||||||| | ||||||| ||||| ||| || | |||||||||| || |||| | ||||
Sbjct 1331 GGTGAATA-CGTTCCCGG-GCCTT-GTACACAC-CGCCCGTCAC-ACCATGGG-AGTGGG 1384 Query 1387 TTG 1389
|||
Sbjct 1385 TTG 1387
Table 8-5: BLAST analysis of partial 16S rDNA of strain 9 No. of nucleotides used for analysis: 1389
Differences in the query nucleotides are highlighted red in color
Appendix
143
Table 8-6: Distance tree map of strain 9
Blast names color map
bacteria b-proteobacteria unknown unclassified
Appendix
144
8.1 NAME O F CO M PO UN DS AND T H E IR RE S PE CT IVE PE AK S; IDE NT I FIE D AND DE RIVE D FRO M DIFFE RE NT B ACT E RIAL S T RAINS DE SCRIB E D IN T H IS T H ES IS
Pseudomonas sp. strain Chol1 Zoogloea sp. strain 1 Dietzia sp. strain 2 Pseudomonas sp. strain 9 Name of
peaks Name of compounds‡ Name of
peaks Name of compounds Name of
peaks Name of compounds Name of
peaks Name of compounds
C:P1 DHOPDC Z:P1 DHOPDC D:P1 New compound P:P1 New compound
C:P2 ∆1/∆4 monoene of
DHOPDC Z:P2 ∆1/∆4 monoene of
DHOPDC D:P2 New compound P:P2 New compound
C:P3 DHADD Z:P3a DHADD D:P3 New compound P:P a DHOPDC
C:P4 ∆1,4-3-ketocholate Z:P4b ∆1,4-3-ketocholate D:P4c ∆1,4-3-ketocholate P:P4b ∆1/∆4 monoene of DHOPDC C:P5 ∆1/∆4 monoene of
3-ketocholate Z:P5b ∆1/∆4 monoene of
3-ketocholate D:P5b ∆1/∆4 monoene of
3-ketocholate P:P5b ∆1/∆4 monoene of 3-ketocholate
C:P6 THSATD Z:P6 a THSATD D:P6 Compound X1 P:P6 New compound
C:P7 ∆1,4-3-ketocholyl-CoA D:P7 Compound X2
C:P8 ∆1/∆4 monoene of
3-ketocholyl-CoA D:P9 New compound
C:P9 3-ketocholyl-CoA D:P10a Cholyl-CoA
C:P10 Cholyl-CoA
‡ Standards compounds
a Compounds confirmed by co-elution and UV-spectral analysis
b Compounds confirmed by co-elution and UV-spectra analysis, but their UV-spectra are not shown c Compounds confirmed by co-elution, UV-spectra and LC/MS analysis
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