GEN III BIOLOG Microplate tests (a) Utilization of sugars:
4.4.5 Classification of putatively novel Streptomyces strains
Phylogenetic analyses. The 10 Streptomyces isolates fell into two 16S rRNA subclades that were supported by all of the tree-making algorithms and very high bootstrap values (Figure 4.12). The 6 isolates assigned to the novel subclade had 16S rRNA gene sequences that were identical or almost identical (Table 4.16). These organisms were most closely related to the type strains of Streptomyces carpinensis (99.35-99.51%
similarity, 7-9 nt differences), Streptomyces cellulosae (99.35-99.50% similarity, 7-8 nt differences), Streptomyces gancidicus (99.49-99.64% similarity, 5-7 nt differences), Streptomyces levis (99.49-99.57% similarity, 6-8 nt differences), Streptomyces pseudogriseolus (99.30-99.57% similarity, 6-9 nt differences) and Streptomyces warraensis (99.20-99.36% similarity, 9-11 nt differences).
The S. fimbriatus 16S rRNA subclade encompasses considerable variation, as shown in Figure 4.8. Isolates C59 and KNN26.b formed a branch in the subclade together with the S. fimbriatus strain. Isolate C59 and the S. fimbriatus strain had identical 16S rRNA gene sequences, the corresponding value between isolate KNN 26.b and S.
fimbriatus NRBC 15411T is 99.64%, a value that corresponds to 16 nt differences at 1383 locations (Table 4.16). These strains are loosely associated with the isolates assigned to the novel subclade sharing 16S rRNA gene similarities with the latter within the range 98.62-99.11%, values that correspond to 18 and 12 nt differences at 1376 and 1353 sites.
Isolates KNN38.1b and KNN64.5b were also most closely related to the S. fimbriatus strain sharing 16S rRNA gene similarities with this organism of 97.38% and 98.84%, respectively, values that equate to 51 and 36 nt differences at 1382 and 1376 sites.
It is also clear from the analyses of the partial sequences of the house-keeping genes atpD, gyrB, recA, rpoB and trpB that isolates KNN 11.a, KNN 35.1b, KNN 35.2b, KNN42.f, KNN 48.3 and KNN 83.e belong to a distinct and homogeneous lineage in the Streptomyces MLSA gene tree that is supported by a 100% bootstrap value (Figure 4.13, Table 4.17). Members of this taxon are most closely related to the type strains of Streptomyces ghanaensis and Streptomyces viridosporus sharing MLSA distances with these organisms that fall within the range 0.037-0.038 and 0.038 and 0.040, respectively.
It is also clear from these analyses that the type strains of S. ghanaensis and S.
viridosporus are very closely related as they share an MLSA distance of 0.004.
167
Figure 4.13 Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing relationships between isolates C59, KNN6.11a, KNN26.b, KNN 35.1b, KNN 35.2b, KNN 38.1b, KNN 42.f, KNN 48.3, KNN64.5b and KNN83.e and between them and the type strains of closely related Streptomyces species. Asterisks indicate branches of the tree that were also recovered using the maximum-likelihood (ML) and maximum-parsimony (MP) tree-making methods. Numbers at the nodes indicate levels of bootstrap support based on a neighbour-joining analysies of 1000 resampled datasets, only values above 50% are shown. The scale bar indicates 0.005 substitutions per nucleotide position.
168
Table 4.16 Nucleotide similarities (%) and differences based on almost complete 16S rRNA gene sequences between isolates C59, KNN6.11a, KNN26.b, KNN 35.1b, KNN 35.2b, KNN 38.1b, KNN 42.f, KNN64.5b and KNN83.e and between them and the type strains of closely related Streptomyces species.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
1. Isolate KNN6.11a --- 2/1381 1/1382 1/1373 0/1407 0/1418 12/1353 17/1375 40/1373 32/1377 16/1405 9/1414 20/1402 20/1414 16/1409 13/1418 5/1403 9/1414 7/1414 7/1414 11/1413 58/1418 6/1409 8/1400 2. Isolate KNN35.1b 99.86 --- 1/1382 2/1373 2/1370 2/1382 13/1354 19/1376 41/1373 33/1377 18/1382 11/1382 22/1382 22/1382 18/1382 15/1382 7/1381 7/1382 9/1382 9/1382 13/1382 60/1382 8/1382 10/1382 3. Isolate KNN35.2b 99.93 99.93 --- 2/1373 1/1371 1/1383 13/1362 18/1376 41/1373 33/1377 17/1391 10/1391 21/1391 21/1391 17/1391 14/1391 6/1390 6/1391 8/1391 8/1391 12/1391 59/1391 7/1391 9/1391 4. Isolate KNN42.f 99.93 99.85 99.85 --- 1/1363 1/1373 13/1352 18/1373 41/1372 33/1371 17/1373 10/1373 21/1373 21/1373 17/1373 14/1373 6/1372 6/1373 8/1373 8/1373 12/1373 59/1373 7/1373 9/1373 5. Isolate KNN48.3 100 99.85 99.93 99.93 --- 0/1407 12/1342 17/1364 40/1363 32/1367 16/1394 9/1403 20/1391 20/1403 16/1398 13/1410 5/1392 9/1403 7/1403 7/1403 11/1402 57/1410 6/1398 8/1389 6. Isolate KNN83.e 100 99.86 99.93 99.93 100 --- 12/1354 17/1376 40/1373 32/1377 16/1406 9/1415 20/1403 20/1415 16/1410 13/1419 5/1404 9/1415 7/1415 7/1415 11/1414 58/1419 6/1410 8/1401 7. Isolate C59 99.11 99.04 99.05 99.04 99.11 99.11 --- 0/1358 34/1353 15/1355 0/1369 15/1365 16/1366 16/1366 18/1365 15/1365 15/1364 15/1365 15/1365 15/1365 15/1366 59/1367 16/1365 18/1365 8. Isolate KNN26.b 98.76 98.62 98.69 98.69 98.75 98.76 100 --- 41/1375 21/1377 5/1382 21/1378 22/1379 22/1379 24/1378 20/1376 20/1375 20/1376 20/1376 20/1376 20/1377 64/1378 21/1376 23/1376 9. Isolate KNN38.1b 97.09 97.01 97.01 97.01 97.07 97.09 97.49 97.02 --- 26/1375 36/1376 44/1375 45/1376 45/1376 47/1375 51/1373 45/1372 45/1373 45/1373 45/1373 46/1374 91/1374 46/1373 48/1373 10. Isolate KNN64.5b 97.68 97.6 97.6 97.59 97.66 97.68 98.89 98.47 98.11 --- 16/1383 33/1379 34/1380 34/1380 36/1379 35/1377 35/1376 35/1377 35/1377 35/1377 35/1378 79/1379 36/1377 38/1377 11. S. fimbriatus NBRC 15411T 98.86 98.7 98.78 98.76 98.85 98.86 100 99.64 97.38 98.84 --- 17/1455 18/1453 18/1456 20/1455 19/1453 19/1451 20/1453 19/1453 19/1453 19/1454 64/1455 20/1453 22/1448 12. S. werraensis NBRC 13404T 99.36 99.2 99.28 99.27 99.36 99.36 98.9 98.48 96.8 97.61 98.83 --- 19/1452 19/1464 11/1459 15/1463 4/1452 8/1463 14/1462 6/1463 16/1461 68/1462 5/1458 13/1448 13. S. griseostramineus NBRC 12781T 98.57 98.41 98.49 98.47 98.56 98.57 98.83 98.4 96.73 97.54 98.76 98.69 --- 0/1453 14/1452 25/1450 22/1449 24/1450 22/1450 22/1450 23/1451 69/1451 24/1450 22/1448 14. S griseomycini NBRC 12778T 98.59 98.41 98.49 98.47 98.57 98.59 98.83 98.4 96.73 97.54 98.76 98.7 100 --- 14/1459 25/1462 22/1451 27/1462 22/1462 22/1462 23/1462 69/1463 24/1457 22/1448 15. S. viridiviolaceus NBRC 13359T 98.86 98.7 98.78 98.76 98.86 98.87 98.68 98.26 96.58 97.39 98.63 99.25 99.04 99.04 --- 20/1457 15/1451 16/1457 17/1457 15/1457 17/1457 73/1457 16/1457 14/1448 16. S. caelestis NRRL 2418T 99.08 98.91 98.99 98.98 99.08 99.08 98.9 98.55 96.29 97.46 98.69 98.97 98.28 98.29 98.63 --- 11/1452 15/1463 18/1462 13/1463 16/1461 68/1474 12/1458 13/1448 17. S. gancidicus NBRC 15412T 99.64 99.49 99.57 99.56 99.64 99.64 98.9 98.55 96.72 97.46 98.69 99.72 98.48 98.48 98.97 99.24 --- 1/1452 10/1451 2/1452 12/1451 63/1451 1/1452 9/1447 18. S. pseudogriseolus NBRC12902T 99.36 99.49 99.57 99.56 99.36 99.36 98.9 98.55 96.72 97.46 98.62 99.45 98.34 98.15 98.9 98.97 99.93 --- 14/1462 6/1463 15/1461 68/1462 2/1458 10/1448 19. S. carpinensis NBRC 14214T 99.5 99.35 99.42 99.42 99.5 99.51 98.9 98.55 96.72 97.46 98.69 99.04 98.48 98.5 98.83 98.77 99.31 99.04 --- 10/1462 16/1461 66/1462 11/1457 7/1448 20. S. cellulosae NBRC 13027T 99.5 99.35 99.42 99.42 99.5 99.51 98.9 98.55 96.72 97.46 98.69 99.59 98.48 98.5 98.97 99.11 99.86 99.59 99.32 --- 12/1463 64/1463 3/1458 11/1448 21. S. afghaniensis NBRC12831T 99.22 99.06 99.14 99.13 99.22 99.22 98.9 98.55 96.65 97.46 98.69 98.9 98.41 98.43 98.83 98.9 99.17 98.97 98.9 99.18 --- 59/1463 11/1457 9/1448 22. S. capillispiralis NBRC 14222T 95.91 95.66 95.76 95.7 95.96 95.91 95.68 95.36 93.38 94.27 95.6 95.35 95.24 95.28 94.99 95.39 95.66 95.35 95.49 95.63 95.97 --- 64/1457 66/1448 23. S. levis NBRC 15423T 99.57 99.42 99.5 99.49 99.57 99.57 98.83 98.47 96.65 97.39 98.62 99.66 98.34 98.35 98.9 99.18 99.93 99.86 99.25 99.79 99.25 95.61 --- 8/1448 24. S. stelliscabiei CFBP 4521T 99.43 99.28 99.35 99.34 99.42 99.43 98.68 98.33 96.5 97.24 98.48 99.1 98.48 98.48 99.03 99.1 99.38 99.31 99.52 99.24 99.38 95.44 99.45
---169
Figure 4.14 Subtree from the Streptomyces phylogenetic tree inferred from concatenated partial sequences of the house-keeping genes atpD, gyrB, recA, rpoB and trpB in MEGA 6 (Tamura et al. 2013) using the maximum-likelihood method based on the General Time Reversible model (Nei and Kumar 2000). There were 2622 positions and 706 strains in the final dataset. Percentages at the nodes represent levels of bootstrap support from 1000 resampled datasets with values less than 60% not shown. Bar marker equals number of substitutions per site.
170
Table 4.17 MLSA distances for strains phylogenetically near to the novel Streptomyces subclade and related isolates. The distances between the isolates and the type strains greater than 0.007 which was defined by Rong and Huang (2012) as equivalent to 70% genomic DNA similarity.
S. prasinus NRRL B-12521TY25A1:Z26
S. prasinus NRRL B-2712T 0.003
S. emeiensis NRRL B-24621T 0.024 0.026
S. prasinopilosus NRRL B-2711T 0.022 0.024 0.003
S. herbaceus NRRL B-59128T 0.019 0.021 0.029 0.027
S. hirsutus NRRL B-3040T 0.029 0.031 0.043 0.042 0.030
S. hirsutus NRRL ISP-5095T 0.028 0.030 0.043 0.041 0.030 0.002
S. hirsutus NRRL B-2713T 0.028 0.030 0.043 0.041 0.030 0.002 0.000
S. chlorus KACC 20902T 0.035 0.037 0.041 0.040 0.033 0.026 0.025 0.025
S. chlorus NRRL B-24997T 0.035 0.037 0.041 0.040 0.033 0.026 0.025 0.025 0.000 S. viridis KACC 21003T 0.033 0.036 0.046 0.044 0.032 0.016 0.015 0.015 0.021 0.021 S. viridis NRRL B-59133T 0.034 0.036 0.047 0.044 0.032 0.017 0.016 0.016 0.021 0.021 0.000 S. incanus NRRL B-59129T 0.033 0.035 0.042 0.041 0.027 0.022 0.021 0.021 0.019 0.019 0.016 0.016 S. pratens NRRL B-59131T 0.036 0.037 0.043 0.043 0.032 0.024 0.023 0.023 0.022 0.022 0.021 0.021 0.017 S. daghestanicus NRRL_B-5418T 0.059 0.062 0.065 0.062 0.063 0.065 0.064 0.064 0.068 0.068 0.066 0.067 0.067 0.067 S. griseoviridis NRRL ISP-5229T 0.059 0.061 0.064 0.061 0.063 0.064 0.063 0.063 0.067 0.067 0.066 0.066 0.066 0.066 0.001 S. fumanus NRRL B-3898T 0.059 0.059 0.064 0.062 0.062 0.066 0.065 0.065 0.068 0.068 0.065 0.066 0.064 0.069 0.035 0.035 Streptomyces_species KNN 64.5b NRRL_B-65049 0.053 0.054 0.060 0.058 0.057 0.054 0.053 0.053 0.059 0.059 0.055 0.054 0.058 0.062 0.046 0.045 0.045 Streptomyces_species KNN 6.11 NRRL_B-65057 0.053 0.055 0.060 0.059 0.058 0.055 0.054 0.054 0.059 0.059 0.055 0.055 0.058 0.062 0.046 0.046 0.045 0.000 Streptomyces_species KNN 35.2b NRRL_B-65055 0.054 0.055 0.061 0.059 0.058 0.055 0.054 0.054 0.060 0.060 0.056 0.055 0.059 0.063 0.047 0.046 0.046 0.001 0.001 Streptomyces_species KNN 48.3e NRRL_B-65056 0.053 0.055 0.060 0.059 0.058 0.055 0.054 0.054 0.059 0.059 0.055 0.055 0.058 0.062 0.046 0.046 0.045 0.000 0.001 0.000 Streptomyces_species KNN 83.e NRRL_B-65058 0.053 0.055 0.060 0.059 0.058 0.055 0.054 0.054 0.059 0.059 0.055 0.055 0.058 0.062 0.046 0.046 0.045 0.000 0.001 0.000 0.000 Streptomyces_species KNN 38.1 NRRL_B-65050 0.053 0.054 0.060 0.058 0.057 0.054 0.053 0.053 0.059 0.059 0.054 0.054 0.057 0.062 0.045 0.045 0.044 0.000 0.001 0.001 0.001 0.001 S. ghanaensis NRRL B-12104T 0.055 0.056 0.064 0.062 0.059 0.056 0.055 0.055 0.059 0.059 0.057 0.057 0.061 0.063 0.054 0.054 0.055 0.037 0.038 0.038 0.038 0.038 0.037 S. ghanaensis ATCC 14672T 0.055 0.056 0.064 0.062 0.059 0.056 0.055 0.055 0.059 0.059 0.057 0.057 0.061 0.063 0.054 0.054 0.055 0.037 0.038 0.038 0.038 0.038 0.037 0.000 S. viridosporus NRRL ISP-5243T 0.053 0.054 0.062 0.060 0.057 0.053 0.053 0.053 0.058 0.058 0.056 0.056 0.059 0.062 0.054 0.053 0.055 0.039 0.039 0.040 0.039 0.039 0.038 0.004 0.004
171 Chemotaxonomy
All of the isolates and associated type strains contained LL-A2pm in whole-organism hydrolysates. It can be seen from Table 4.18 that 3 representatives of the novel Streptomyces taxon and the type strains of S. fimbriatus and S. ghanaensis contained complex mixture of saturated and branched chain fatty acids with predominant amounts of iso-C16:0 (21.3-33.3%). Quantitative differences were found in some components, as exemplified, by iso-C14:0 (3.3-8.1%), iso-C15:0 (5.2-11.3%) and anteiso-C17:0 (6.4-12.3%).
A small number of qualitative differences were also found, notably the presence of iso-C16:0 in isolates KNN 35.1b and KNN 35.2b and C16:0 in isolate KNN 35.2b and the type strain of S. ghanaensis.
Two representatives of the novel Streptomyces subclade, strains KNN 35.1b and KNN 35.2b, and the type strain of S. ghanaensis contained glucose, mannose, ribose and xylose in whole-organism hydrolysates; galactose was also detected in the latter. The polar lipid patterns of these strains consisted of diphosphotidylglycerol, phosphotidylethanolamine, phosphatidylinositol mannosides and a number of unidentified components, a trace of phosphatidylglycerol was detected in isolate KNN 35.1 (Figure 4.14). The predominant menaquinones of strain KNN35.1b consisted of MK9(H6) (34%), MK9(H8) (29%) and MK9(H4) (10%).
Isolates C59, KNN 38.1b, KNN 64.5b and the type strain of S. fimbriatus produced whole-organism hydrolysates containing glucose, mannose and ribose, those of isolate C59 and S. fimbriatus NRRL B-3175T also included galactose and a trace of xylose, respectively. These strains shared similar polar lipid patterns, they all contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides (Figure 4.15). Some polar lipids were discontinuously distributed, as exemplified by the presence of hydroxyl-phosphatidylethanolamine in the S. fimbriatus strain and several glycolipids in isolate C59. The major menaquinone detected in the type strain of S. fimbriatus and in isolate KNN 64.5b were MK9(H6) (61%) and MK9(H8) (15%), the predominant menaquinone in isolate C59 was MK9(H2) (66%).
172
Table 4.18 Fatty acid profiles (%) of Streptomyces isolates KNN35.1b, KNN 35.2b and KNN 42.f and the type strains of S. fimbriatus and S. ghanaensis.
Fatty acids Isolate KNN 35.1b Isolate KNN 35.2b Isolate KNN 42.f S. ghanaensis T NRRL B-12104 S. fimbriatus T NRRL B-3175
Trace proportions (< 0.9 %) are only cited for strains where other fatty acids were found beyond this cut-off point.
Summed feature 3, C16:1 w7c/C16:1 w6c; Summed feature 9, iso-C17:1 w9c.
KNN 35.2b: Summed feature 5, iso-C17:1 w9c.
KNN 42.f: Summed feature 5, C18:2 w6,9c/anteiso B.
S. fimbriatus: Summed feature 4, Iso- C17:1 I/anteiso B.
173
Figure 4.15 Two-dimensional thin-layer chromatography of polar lipids of Streptomyces isolates KNN 35.1b, KNN 35.2b and S. ghanaensis NRRL B-3175T stained with molybdenum blue spray (Sigma) using the solvent systems cited in the legend to Figure 4.3. Key: DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; methyl-PE, methyl- phosphatidylethanolamine;
OH-PE, hydroxyphosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PIM, phosphatidylinositol mannosides; AL, aminolipid;
GL, glycolipids, PL, phospholipid and L, unknown lipids.
174
Figure 4.16 Two-dimensional thin-layer chromatography of polar lipids of Streptomyces fimbriatus and closely related isolates stained with molybdenum blue spray (Sigma) using the solvent systems cited in the legend to Figure 4.3. Key: DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; methyl-PE, methyl- phosphatidylethanolamine; OH-PE, hydroxyphosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PIM, phosphatidylinositol mannosides; AGL, aminoglycolipid; AL, aminolipid; GL, glycolipids; PL, phospholipid and L, unknown lipids.
175
Phenotypic properties. The isolates assigned to the novel 16S rRNA subclade had many phenotypic features in common, some of which separate them from the type strain of S.
ghanaensis (Table 4.19). The isolates, unlike the Streptomyces ghanaensis strain, their nearest phylogenetic neighbour, produced N-acetyl-β-glucosaminidase, utilized D-fucose, butyric acid and α-keto-butyric acid and grew at 10oC. In contrast, only the S.
ghanaensis strain formed naphthol-AS-BI-phosphahydrolase and utilized-fucose, D-glucuronic acid and N-acetyl-β-D-mannosamine.
Identical results were obtained for all of the duplicated strains, apart from some of the GEN III BIOLOG microplate tests (Table 4.19 and Table 4.20). All of the isolates and marker strains produced acid phosphatase, cystine arylamidase , β-galactosidase, leucine arylamidase; hydrolysed aesculin and arbutin; degraded adenine, hypoxanthine, Tweens 20 and 40; grew between 20 and 40oC and utilized D-arabitol, ƴ-amino-butyric acid, β-bydroxy-butyric acid, N-acetyl-D-glucosamine, D-glucose, D-mannose, myo-inositol and L-rhamnose, sucrose and D-trehalose and were resistant to aztreonam, . In turn, all of the strains gave negative results for α-fucosidase, α-galactosidase, β-glucosidase and lipase (C14), for allantoin and urea hydrolysis, nitrate reduction, H2S production, for the degradation of cellulose, chitin, guanine and tributyrin, the utilization of glucuronamide, melibiose, 3-O-methyl-glucose, mucic acid, quinic acid, D-saccharic acid, D-serine #2, showed resistance to fusidic acid and minocycline and did not grow at 4 and 50oC.
176
Table 4.19 Phenotypic tests that distinguish isolates KNN 6.11a, KNN 35.1b, KNN 35.2b ,KNN 42.f,KNN 43.e KNN48.3e and KNN 83.e from one another and from S. ghanaensis NRRL B-12104T.
Characteristic
Isolate KNN 6.11a Isolate KNN 35.1b Isolate KNN 35.2b Isolate KNN 42.f Isolate KNN 43.e Isolate KNN 83.e S. ghanaensis NRRL B-12104T
API ZYM tests:
N-Acetyl-β-glucosaminidase + + + + + + -
α-Chymotrpsin - + - - - - -
Esterase (C4) + - - - - + -
α-Glucuronidase + - + - - + +
α-Mannosidase - + - + - - +
Naphthol-AS-BI-phosphohydrolase - - + - - - +
Trypsin - + - - - - -
GEN III BIOLOG Microplate tests (a) Utilization of sugars:
N-Acetyl-β-D-mannosamine - + - - - - +
N-Acetyl-D-galactosamine + - - - - + +
N-acetyl-neuraminic acid - - - + - - +
D-Fucose + + + + + + -
L-Fucose + + + - + + -
β-Gentiobiose + + + - + + +
177
D-Glucose-6-phosphate - + - + - - +
α-D-Lactose + + + - + - +
D-Maltose + + + - + + +
D-Mannitol + + + - + - +
β-Methyl-D-glucoside + + - - - - -
D-Salicin + - - - - + +
D-Sorbitol + + + - + + -
D-Turanose + + + - + + +
(b) Utilization of amino acids:
L-Alanine + + + + + - +
L-Arginine + + + + + - -
L-Histidine + + + + + - +/-
L-Serine + + - + - - +
(c) Utilization of organic acids:
Acetoacetic acid + + + + + - +
Bromo-succinic acid + + - - - - -
Butyric acid + + + + + + -
α-keto-Butyric acid + + + + + + -
Citric acid + + + + + +
-D-Galacturonic acid + - - - -
L-Galactonic acid-γ-lactone + - - - -
α-keto-Glutaric acid + - - + + + +
D-Glucuronic acid - - - +
α-Hydroxy-butyric acid + - - + - -
-p-Hydroxy-phenylacetic acid - - - + - - -
L-Lactic acid + - - + - - -
178
D-Malic acid + - - - -
L-Malic acid + - + + + + -
Methyl pyruvate - - - + - - -
L-Pyroglutamic acid + + + + + -
-(d) Utilization of other compounds:
Inosine + - + + + + +
Pectin + - - - -
(e) Growth in the presence of:
Aztreonam + + + + + + +
Guanidine hydrochloride + - - - -
Lincomycin + - - - - + -
Niaproof - - - + -
Potassium tellurite + + + - + + -
Rifamycin SV + + + - + + -
Sodium chloride (4%, w/v) + + + - + + -
Sodium formate - - - + - - -
Sodium lactate (1%) + + - + + + -
Tetrazolium blue + - - - - + -
Tetrazolium violet + - - - - + -
Troleandomycin + - - - - + -
Vancomycin + - - - -
(f) Growth at:
pH 5 + + - - + + -
Other phenotypic tests (a) Degradation tests:
179
Casein + + - - - - -
(b) Growth at:
10oC + + + + + + -
45 oC - - - + - + -
+, positive result; -, negative result.
Positive results recorded for all of the isolates and the Streptomyces ghanaensis type strain:
API ZYM tests: acid phosphatase, alkaline phosphatase, cystine arylamidase, esterase lipase (C8), β-galactosidase, leucine arylamidase and valine arylamidase.
GEN III BIOLOG microplate tests: utilization of acetic acid, N-acetyl-glucosamine, γ-amino-n-butyric acid, arabitol, L-aspartic acid, D-cellobiose, Dextrin, D-fructose, D-galactose, gelatin, D-gluconic acid, D-glucose, 3-O-methyl-D-glucose, L-glutamic acid, glycerol, glycl-L-proline, β-hydroxy-butyric acid, myo-inositol, D-mannose, D-melibiose, propionic acid, sucrose and D-trehalose, growth at pH6 and in the presence of aztreonam, lincomycin, nalidixic acid, sodium bromate and sodium chloride (1%, w/v).
Other phenotypic tests: aesculin hydrolysis, arbutin hydrolysis, degradation of adenine, elastin, hypoxanthine, starch, L-tyrosine, Tweens 40, 60 and 80 and growth at 20, 30 and 40oC.
Negative results recorded for all of the isolates and for the Streptomyces type strain:
API ZYM tests: α-fucosidase , α-galactosidase, β-glucosidase, β-glucuronidase and lipase (C14).
GEN III BIOLOG microplate tests: utilization of D-aspartic acid, D-fructose-6-phosphate, glucuronamide, D-lactic acid methyl ester, mucic acid, quinic acid, D-saccharic acid, D-serine #1, D-serine #2, stachyose and resistance to fusidic acid and minocycline
Other phenotypic tests: allantoin hydrolysis, nitrate reduction, H2S and urea hydrolysis, degradation of cellulose, chitin, guanine, tributyrin, uric acid, xanthine, xylan and growth in the presence of sodium chloride (8%, w/v) and at 4 and 50oC.
180
Table 4.20 Phenotypic profiles of Streptomyces fimbriatus NRRL-B3175T and related isolates classified in the Streptomyces fimbriatus 16S rRNA gene subclade.
Characteristics
Isolate C59 Isolate KNN 26.b Isolate KNN 38.1b Isolate KNN 64.5b S.fimbriatus NRRL B-3175T
API ZYM tests:
N-Acetyl-β-glucosaminidase - + + + +
Esterase (C4), esterase lipase (C8) + + - + +
α -Galactosidase - - + - -
α –Glucosidase, valine arylamidase - - + + -
β-Glucosidase - + - - +
Lipase (C14) + + + - -
α-Mannosidase + + - + +
Naphthol-AS-BI-phosphohydrolase - + - - +
GEN III BIOLOG Microplate tests (a) Utilization of sugars:
Acetic acid, D-arabitol, D-cellobiose + + + + -
N-Acetyl-β-D-mannosamine, L-fucose, D-glucose-6-phosphate, D-raffinose
- - - - +
D-Fructose, glycerol, D-trehalose + + + - +
D-Fructose-6-phosphate - - - - +
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D-Fucose + + - - +
D-Galactose - - + + -
D-Maltose + + - + -
D-Mannitol + + + + -
β-Methyl-D-glucoside, D-turanose - - - + -
Sucrose - - + - -
(b) Utilization of amino acids
L-Alanine - - + - -
L-Glutamic acid, L-serine + + + - +
(c) Utilization of organic acids:
Butyric acid + + - - +
α-keto-Butyric acid + + + - -
Citric acid + + - + +
D-Glucuronic acid - - + -
-L-Glutamic acid + - + + +
α-Hydroxy-butyric acid - + + + +
(d) Utilization of:
Gelatin + - + + +
Growth in the presence of:
Lithium chloride + + + - +
Nalidixic acid + + + - +
Sodium chloride (8%, w/v) + + - + -
Sodium lactate (1%) + + - + -
Growth at:
pH 5 + - + - -
182 Other phenotypic tests
(a) Degradation tests:
Arbutin - - + - -
Casein + - + - -
Elastin + + + + -
Starch + + + + -
L-Tyrosine - + + - -
Uric acid + + - - +
(b) Growth at:
10oC - - + + -
45 oC - - + + -
+, positive result; -, negative result.
Positive results recorded for all of the isolates and for the type strainof S. fimbriatus:
API ZYM tests: acid phosphatase, alkaline phosphatase, cystine arylamidase, β-galactosidase, α-glucuronidase and leucine arylamidase.
GEN III BIOLOG microplate tests: utilization of acetoacetic acid, N-acetyl-D-glucosamine, γ-amino-n-butyric acid, L-aspartic acid, dextrin, β-gentiobiose, gluconic acid, glucose, gly-pro, L-histidine, β-hydroxy-butyric acid, lactic acid methyl ester, α-lactose, mannose, D-mannitol, myo-inositol, propionic acid, L-pyroglutamic acid, L-rhamnose and resistance to aztreonam, growth at pH6 and in the presence of potassium tellurite and sodium chloride (4%, w/v).
Other phenotypic tests: aesculin and arbutin hydrolysis, degradation of adenine, hypoxanthine, Tweens 40, 60 and 80 and growth at 20, 30 and 40oC.
Negative results recorded for all of the isolates and for the type strain of S. fimbriatus:
API ZYM tests: α-chymotripsin, α-fucosidase, β-glucuronidase, lipase and trypsin.
GEN III BIOLOG microplate tests: utilization of N-acetyl-D-galactosamine, N-acetyl-neuraminic acid, L-arginine, D-aspartic acid, bromo-succinic acid, D-galacturonic acid, L-galactonic acid-γ-lactone, glucuronamide, α-keto-glutaric acid , p-hydroxy-phenylacetic acid, inosine,
3-O-methyl-D-183
glucose, L-lactic acid, malic acid, L-malic acid, melibiose, methyl pyruvate, mucic acid, pectin, quinic acid, salicin, saccharic acid, D-serine #1, D-D-serine #2, stachyose and resistance to fusidic acid, guanidine hydrochloride, lincomycin, minocycline, niaproof, rifamycin SV, tetrazolium blue, tetrazolium violet, troleandomycin and vancomycin and growth in the presence of sodium bromate and sodium formate.
Other phenotypic tests: Allantoin hydrolysis, nitrate reduction, H2S and urea hydrolysis, degradation of cellulose, chitin, guanine, xanthine, xylan and tributyrin and growth at 4 and 50oC.
Contrasting BIOLOG results were obtained for:
(a) Isolate C59: utilization of L-glutamic acid and L-serine.
(b) Isolate KNN 26.b: utilization of alanine, arginine, α-keto-butyric acid, citric acid, D-fucose, fucose, D-glucose-6-phosphate, D-raffinose, L-serine and growth in the presence of lithium chloride and 1% sodium lactate.
(c) Isolate KNN 38.1b: utilization of L-arginine, D-fucose, L-fucose, α-hydroxy-butyric acid and D-sorbitol.
(d) Isolate KNN 64.5b: utilization of L-alanine, butyric acid, citric acid, L-fucose, gelatin, L-glutamic acid and D-salicin.
(e) Streptomyces fimbriatus NRRL-B3175T: utilization of L-arginine, D-fucose, gelatin, D-glucuronic acid, D-salicin, L-serine and D-sorbitol.
184 4.5 Discussion
Twelve out of 16 Atacama Desert isolates assigned to genera other than Streptomyces were found to have 16S rRNA gene sequence similarities with the type strains of their closest phylogenetic neighbours that fell at or below 99.0% threshold which Meier-Kolthoff et al. (2013) recommended as the threshold value below which DNA:DNA relatedness assays were not required. Consequently, these islates were considered to be putatively novel species belonging to the genera Actinomadura, Amycolatopsis, Cryptosporangium and Pseudonocardia. Most of these isolates were compared with their nearest phylogenetic neighbours using a combination of chemotaxonomic, morphological and phenotypic properties designed to establish whether they merited recognition as novel species.
Two out of the three isolates classified in the genus Actinomadura, isolate H59 from the ALMA 2 environmental sample and isolate LB25 from Lomas Bayas soil had 16S rRNA gene sequence similarities against their nearest phylogenetic neighbours that were below 99.0% threshold mentioned above; the exception isolate LB54 from Lomas Bayas soil was very closely related to the type strains of A. coerulea and A.
verrucosispora. Isolate H59 was shown to have chemotaxonomic and morphological properties typical of the genus Actinomadura (Trujillo and Goodfellow 2012; Zhao et al.
2015; Abagana et al. 2016). This organism was distinguished readily from A. napierensis DSM 44846T and A. yumaensis DSM43931T, its closest phylogenetic neighbours, by a broad range of phenotypic properties. In light of these genotypic and phenotypic data isolate H59 is considered to be a new Actinomadura species. It seems likely that Actinomadura strains are common in Atacama Desert soils as isolates H59, LB25 and LB54 were representatives of multi-membered colour-groups; members of the genus have been isolated previously from hyper-arid Atacama Desert soil (Busarakam 2014). It is also interesting that an actinobacterium from Saharan soil has been validly published as Actinomadura algeriensis (Lahoum et al. 2016).
The 4 strains recovered within the evolutionary radiation encompassed by the genus Amycolatopsis were isolated from the ALMA 4 environmental sample on glucose-yeast extract agar. Isolate H6 was found to be most closely related to the type strain of A.
pretoriensis but the remaining strains, isolates H5, H97 and H101, were sharply separated from closely associated marker strains given the 99.0% 16S rRNA threshold recommended by Meier-Kolthoff et al. (2013). Isolate H5 was studied further and found to have chemotaxonomic and morphological properties characteristic of the genus Amycolatopsis (Tan and Goodfellow 2012; Klykleung et al. 2015) and was separated
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readily from A. mediterranei DSM 43304T and A. pretoriensis DSM 44654T, its closest phylogenetic relatives, by a combination of phenotypic features. These polyphasic taxonomic data indicate that isolate H5 belongs to a novel Amycolatopsis species. It seems likely given the present and previous data that Amycolatopsis strains are common in Atacama Desert habitats (Okoro et al. 2009; Busarakam 2014). Indeed, members of the rare thermophilic species A. ruani and A. thermalba have been shown to be major components of extreme hyper-arid soil from the Yungay core region (Busarakam et al.
2016a; Zucchi et al. 2012).
Strain H7 was isolated from an ALMA 4 environmental sample and assigned to colour-group 7 together with two other isolates. The assignment of these isolates to the genus Cryptosporangium is interesting as members of this poorly studied taxon have not been isolated previously from Atacama Desert habitats. Isolate H7 was most closely related to two of the six validly published Cryptosporangium species; that is, to C.
cibodasense and C. minutisporangium. The organism was shown to have chemotaxonomic and morphological properties consistent with its classification in the genus Cryptosporangium (Tamura et al. 1998) and was separated readily from C.
minutisporangium NBRC 15962T by a broad range of phenotypic properties. It seems likely that isolate H7 will be found to belong to a new Cryptosporangium species, but comparative phenotypic tests are needed to ensure that it can be seperated from C.
cibodasense NBRC 110976T, this organism was validly published as a new species after the experimental phase of this project had been completed.
All but one of the isolates recovered in the Pseudonocardia 16S rRNA gene tree were representatives of multi-membered taxa. It is particularly interesting that so many pseudonocardiae were isolated from environmental samples collected from Cerro Chajnantor and the Yungay core region as members of this taxon have only been isolated from the Atacama Desert on one previous occasion (Busarakam 2014). Nevertheless, it seems likely that Pseudonocardia strains are an integral part of actinobacterial communities in Atacama Desert landscapes, a point undelined in a culture-independent study which revealed that Pseudonocardia lineages were common in a high altitude debris field on Llallialaco Volcano in th viccinity of the Chilean-Argentine border (Lynch et al. 2012).
The 9 representative isolates assigned to the four Pseudonocardia 16S rRNA subclades along with type strains of associated marker strains were found to have chemotaxonomic and morphological properties in line with their classification in the genus Pseudonocardia (Huang and Goodfellow 2015). It seems likely that all but one of
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these isolates will be shown belong to novel Pseudonocardia species as they show relatively low 16S rRNA gene sequence similarities to their closest phylogenetic neighbours and can be distinguished from them by markedly different phenotypic properties; the exception, isolate H58, is most closely related to the type strain of P.
cypriaca sharing a 16S rRNA gene sequence with the latter of 99.58%.
A particularly convincing case can be made for cclassifying isolates ATK01, ATK03 and ATK17 as a novel spcecies of Pseudonocardia. These strains have key genotypic and phenotypic features in common which separate them from the type strains of P. bannaensis and P. xinjiangensis their nearest phylogenetic neighbours. It is particularly interesting that unlike the latter they produce spores in vesicles as well as in chains. It is, therefore, proposed that isolates ATK01, ATK03 and ATK17 can be recognized as a novel of Pseudonocardia species, Pseudonocardia yungayensis sp. nov..
A similar case can be made for recognising isolates H57, H69, H96, H99 and H215 as
A similar case can be made for recognising isolates H57, H69, H96, H99 and H215 as