Instrumentos de aplicación practica de las tarifas

Phylogenetic analyses. Three isolates, strains H59, LB25 and LB54, were found to form distinct phyletic lines within the evolutionary radiation occupied by the genus Actinomadura (Figure 4.1). Strain H59 was isolated from the ALMA 2 environmental sample on glucose-yeast extract agar and is one of three representatives of multi-membered colour-group 52 (Appendix 1). Isolates LB25 and LB54 were isolated from a Lomas Bayas environmental sample and represent multi-membered colour-groups 14 and 10, which encompass 3 and 2 isolates, respectively (Appendix 1).

Isolate H59 is closely related to Actinomadura yumaensis JCM 3369^T, this relationship is supported by all the tree-making algorithms and by a 99% bootstrap value;

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these organisms share a 16S rRNA gene sequence similarity of 97.0%, a value which corresponds to 44 nt differences at 1441 locations (Table 4.2). However, isolate H59 is marginally more closely related to Actinomadura napierensis B60^T; these organisms share a 97.3% 16S rRNA gene sequence similarity which equates to 36 nt differences albeit at only 1339 locations. In turn, isolate LB25 forms a loose subclade together with Actinomadura geliboluensis A8036^T, an association that is supported by all the tree-making algorithms and by a 100% bootstrap value; these strains share a 16S rRNA gene sequence similarity of 97.8%, a value that corresponds to 30 nt differences at 1364 locations (Table 4.2). The remaining strain, isolate LB54, lies towards the periphery of a 16S rRNA gene subclade that includes Actinomadura citrea IFO 14678^T, Actinomadura coerulea IFO 14679^T, Actinomadura glauciflava AS 4.1202^T, Actinomadura luteofluorescens IFO 13057^T, Actinomadura mexicana A290^T and Actinomadura verrucisospora NBRC 14100^T; the status of this taxon is supported by all of the tree-making algorithms and by a 95% bootstrap value. Isolate LB54 was found to be most closely related to the type strains of Actinomadura coerulea and Actinomadura verrucosospora, it shares a 16S rRNA gene similarity of 99.2% with these strains, a value that equates to 11 nt differences at 1369 and 1368 sites, respectively.

Characterization of strain H59. This isolate was studied further as it clearly forms a distinct branch in the Actinomadura 16S rRNA gene tree and hence may be the nucleus of a novel Actinomadura species.

Cultural and morphological properties. The isolate grew moderately well on most of the ISP media but poorly on ISP 5 and 7 (Table 4.3). It is also evident from this Table that the aerial hyphae were predominantly white and the substrate mycelia mainly yellow white; colonies were leathery, irregular, umbonate with undulate margins. The strain formed an extensively branched substrate mycelium that bore aerial hyphae on oatmeal agar, some of the aerial hyphae differentiated into chains of smooth spores (Figure 4.2).

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Figure 4.1 Neighbour-joining phylogenetic tree based on almost complete 16S rRNA gene sequences showing relationships between isolates H59, LB25 and LB54 and between them and the type strains of closely related Actinomadura species. Asterisks indicate branches of the tree that were also recovered using the maximum-likelihood and maximum-parsimony tree-making methods.

Numbers at the nodes indicate levels of bootstrap support based on a neighbour-joining analysis of 1000 resampled datasets, only values above 50% are shown. The scale bar indicates 0.005 substitutions per nucleotide position.

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Table 4.2 Nucleotide similarities (%) and differences based on almost complete 16S rRNA gene sequences showing relationships between isolates H59, LB25 and LB54 and between them and their nearest phylogenetic neighbours.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

1. Isolate H59 --- 67/1358 43/1367 44/1441 53/1428 49/1403 75/1393 57/1428 51/1420 65/1436 36/1339 67/1443 64/1444 57/1429 67/1437 60/1445 49/1403 48/1413 49/1404 50/1413 50/1404 119/1435 2. Isolate LB25 95.1 --- 56/1360 58/1363 60/1362 56/1358 84/1360 52/1362 53/1363 50/1354 53/1342 46/1364 30/1364 42/1361 53/1361 44/1363 58/1358 54/1359 61/1361 57/1359 58/1359 107/1359 3. Isolate LB54 96.9 95.9 --- 33/1372 29/1371 11/1368 60/1358 32/1368 29/1369 30/1363 32/1342 21/1372 28/1369 23/1366 29/1368 23/1372 17/1368 15/1366 16/1368 12/1369 11/1369 85/1362 4. A. yumaensis JCM 3369^T 97.0 95.7 97.6 --- 30/1430 35/1408 61/1399 33/1434 32/1421 36/1438 29/1342 43/1444 33/1445 29/1437 35/1440 30/1447 35/1408 28/1420 38/1408 36/1420 36/1409 89/1437 5. A. vinacea JCM 3325^T 96.3 95.6 97.9 97.9 --- 30/1407 60/1396 33/1417 29/1429 29/1432 32/1342 30/1440 32/1438 29/1421 31/1431 22/1441 32/1407 29/1419 35/1408 34/1418 32/1408 90/1429 6. A. verrucosispora NBRC 14100^T 96.5 95.9 99.2 97.5 97.9 --- 65/1381 32/1404 29/1397 30/1399 35/1338 25/1408 28/1405 24/1403 29/1404 23/1408 9/1409 18/1402 13/1406 18/1403 6/1407 91/1397 7. A. scrupuli R-Ac121^T 94.6 93.8 95.6 95.6 95.7 95.3 --- 53/1394 62/1399 63/1389 53/1342 66/1399 60/1399 61/1396 65/1396 57/1398 71/1381 64/1394 73/1383 69/1395 68/1382 94/1394 8. A. nitritigenes DSM 44137^T 96.0 96.2 97.7 97.7 97.7 97.7 96.2 --- 25/1409 33/1425 30/1344 34/1432 26/1434 27/1428 32/1431 27/1434 36/1404 28/1417 37/1406 37/1413 33/1405 78/1426 9. A. xylanilytica BK147^T 96.4 96.1 97.9 97.8 98.0 97.9 95.6 98.2 --- 29/1440 25/1343 32/1431 26/1441 24/1414 29/1422 23/1431 30/1397 24/1410 29/1399 30/1411 30/1398 83/1443 10. A. chokoriensis 3-45-a/11^T 95.5 96.3 97.8 97.5 98.0 97.9 95.5 97.7 98.0 --- 24/1334 31/1444 26/1455 20/1427 27/1435 7/1449 32/1399 31/1412 35/1400 34/1411 32/1400 89/1455 11. A. napierensis B60^T 97.3 96.1 97.6 97.8 97.6 97.4 96.1 97.8 98.1 98.2 --- 30/1344 27/1344 25/1340 29/1343 20/1343 37/1338 30/1341 36/1341 34/1339 36/1339 84/1338 12. A. formosensis JCM 7474^T 95.4 96.6 98.5 97.0 97.9 98.2 95.3 97.6 97.8 97.9 97.8 --- 18/1452 26/1435 31/1445 25/1453 29/1408 22/1421 25/1410 27/1420 25/1409 93/1444 13. A. geliboluensis A8036^T 95.6 97.8 98.0 97.7 97.8 98.0 95.7 98.2 98.2 98.2 98.0 98.8 --- 11/1437 25/1443 16/1455 30/1405 26/1419 33/1408 32/1417 30/1406 89/1457 14. A. meyerae A288^T 96.0 96.9 98.3 98.0 98.0 98.3 95.6 98.1 98.3 98.6 98.1 98.2 99.2 --- 22/1432 13/1436 26/1403 23/1416 30/1405 29/1414 26/1404 86/1429 15. A. madurae DSM 43067^T 95.3 96.1 97.9 97.6 97.8 97.9 95.3 97.8 98.0 98.1 97.8 97.9 98.3 98.5 --- 20/1444 31/1404 21/1421 30/1405 33/1416 31/1405 85/1435 16. A. bangladeshensis 3-46-b3^T 95.9 96.8 98.3 97.9 98.5 98.4 95.9 98.1 98.4 99.5 98.5 98.3 98.9 99.1 98.6 --- 25/1408 24/1421 29/1409 27/1420 25/1409 83/1446 17. A. luteofluorescens IFO 13057^T 96.5 95.7 98.8 97.5 97.7 99.4 94.9 97.4 97.9 97.7 97.2 97.9 97.9 98.2 97.8 98.2 --- 18/1402 13/1406 16/1403 10/1407 93/1397

18. A. mexicana A290^T 96.6 96.0 98.9 98.0 98.0 98.7 95.4 98.0 98.3 97.8 97.8 98.5 98.2 98.4 98.5 98.3 98.7 --- 14/1403 16/1414 16/1403 88/1411

19. A. citrea IFO 14678^T 96.5 95.5 98.8 97.3 97.5 99.1 94.7 97.4 97.9 97.5 97.3 98.2 97.7 97.9 97.9 97.9 99.1 99.0 --- 19/1403 11/1407 93/1399

20. A. glauciflava AS 4.1202^T 96.5 95.8 99.1 97.5 97.6 98.7 95.1 97.4 97.9 97.6 97.5 98.1 97.7 98.0 97.7 98.1 98.9 98.9 98.7 --- 16/1404 94/1408

21. A. coerulea IFO 14679^T 96.4 95.7 99.2 97.4 97.7 99.6 95.1 97.7 97.9 97.7 97.3 98.2 97.9 98.2 97.8 98.2 99.3 98.9 99.2 98.9 --- 92/1397

22. Nonomuraea maritima FXJ7.203^T 91.7 92.1 93.8 93.8 93.7 93.5 93.3 94.5 94.3 93.9 93.7 93.6 93.9 94.0 94.1 94.3 93.3 93.8 93.4 93.3 93.4

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Table 4.3 Growth and cultural characteristics of isolate H59 on ISP media after incubation for 14 days at 28^oC.

Inorganic salts-starch agar (ISP medium 4)

++ Light olive gray Yellow white

Oatmeal agar (ISP medium 3) ++ White Yellow white

Peptone-yeast extract-iron agar (ISP medium 6)

++ White Light brown

Tryptone-yeast extract agar (ISP medium 1)

++ White Gray yellow

Tyrosine agar (ISP medium 7) + None Yellow white

Yeast extract-malt extract agar (ISP medium 2)

++ None Light yellow

brown ++ moderate growth; + good growth; + poor growth. Diffusible pigments were not produced on any of the media.

Figure 4.2 Scanning electron micrograph of Actinomadura isolate H59 showing chains of smooth spores following growth on oatmeal agar after incubation at 28^oC for 10 days. Bar 1µm.

Chemotaxonomy. The whole-organism hydrolysates of isolate H59 were found to contain meso-A2pm and galactose, glucose, mannose and ribose. The polar lipid pattern consisted of diphosphatidylglycerol, methyl- phosphatidylethanolamine, phosphatidylglycerol, aminoglycolipid, aminolipid, aminophospholipid and phospholipids (Figure 4.3) and the major menaquinones was MK9(H8) (52%), other

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components were MK10(H6) (15%), MK9(H6) (13%) together with a small proportion of MK9(H4) (4%).

Phenotypic properties. Identical results were obtained for the duplicated phenotypic tests carried out on isolate H59, A. napierensis DSM 44846^T and A.yumaensis DSM 43931^T, apart for some of the BIOLOG tests. It is evident from Table 4.4 that the isolate can be differentiated readily from its closest phylogenetic neighbours using a range of phenotypic properties though it is also apparent that all three strains have many properties in common. The isolate, unlike the type strain of A. napierensis, produces α-chymotrypsin and metabolizes D-fructose, D-fucose, β-gentiobiose, D-mannose, glycerol, L-rhamnose, myo-inositol and a number of other compounds. In contrast, the A. napierensis strain, unlike isolate H59, produces α-glucuronidase and naphthol-AS-BI-phosphohydrolase (API tests), degrades starch and uric acid and utilizes D-maltose, L-alanine, L-arginine, L-histidine and L-serine and a number of other compounds and was resistant to aztreonam, lincomycin, minocycline and troleandomycin. In turn, isolate H59 can be differentiated from the type strain of A. yumaensis by its ability to produce β-glucosidase, utilize D-fructose-6-phosphate, D-glucose-6-phosphate, glycl-proline, D- and L-fucose, N-acetyl-D-galactosamine, N-acetyl-β-D-mannosamine and a number of other compounds. In contrast, only the A.yumaensis strain metabolized, for example, D-maltose, D-mannitol, L-alanine, D-turanose, is resistant to aztreonam, fusidic acid, guanidine hydrochloride, minocycline and vancomycin and grows in the presence of up to 8% w/v, sodium chloride and at 45^oC.

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Figure 4.3 Two-dimensional thin-layer chromatography of polar lipids of isolate H7 stained with molybdenum blue spray (Sigma). Chloroform : methanol : water (32.5:12.5:2.0, v/v) was used in the first direction, followed by chloroform : acetic acid : methanol : water (40.0:7.5:6.0:2.0, v/v) in the second direction.

DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; PG, phosphatodylglycerol, PL, phospholipids; AL, aminolipid; AGL, aminoglycolipid and APL, aminophospholipid.

Table 4.4 Phenotypic characteristics that differentiate isolate H59 from Actinomadura napierensis DSM 44846^T and Actinomadura yumaensis DSM 43931^T

Characteristics Isolate