Evaluation of Streptomyces Species-Groups by Pyrolysis Mass Spectrometry

Zbl. Bakt. 285,169-181 (1997) © Gustav Fischer Verlag, Jena

Evaluation of Streptomyces Species-Groups by Pyrolysis Mass Spectrometry ELAYNE V. FERGUSOW, ALAN C. WARD!, JEAN-JACQUES SANGLIER2, and MICHAEL GOODFELLOW 1 1

2

Department of Microbiology, The Medical School, Newcastle-upon-Tyne, UK Preclinical Research, Sandoz Pharma Ltd., Basel, Switzerland

Summary Curie-point pyrolysis mass spectrometry was used to evaluate the taxonomic integrity of three subclusters, provisionally labelled S. albidoflavus, S. anulatus and S. halstedii, which formed a species-group in an extensive numerical phenetic survey of the genus Streptomyces. Excellent agreement was found between the results of the triplicate analyses of each strain while the duplicated strains clustered adjacent to one another. Sequential principal component-canonical variates analysis of the experimental data collected on the 32 representative organisms showed that the S. albidoflavus strains formed a distinct group. This result taken together with earlier chemical, molecular and numerical taxonomic data indicates that the S. albidoflavus subcluster corresponds to a distinct species. In contrast, the subclusters equated with S. anulatus and S. halstedii were found to be heterogeneous and hence in need of further study. However, it is evident from the present investigation that Curiepoint pyrolysis mass spectrometry provides a rapid and reproducible way of evaluating the taxonomic integrity of Streptomyces species-groups. Introduction The application of modern taxonomic methods has led to significant improvements in streptomycete systematics (10, 22). Nevertheless, the taxonomic integrity of most validly described species of Streptomyces recognised in the current edition of Bergey's Manual of Systematic Bacteriology (37) is based primarily on the numerical taxonomic survey of Williams et al. (36). In this study, the type strains of over 300 species of Streptomyces were assigned to 41 minor (2 to 5 strains) and 22 single membered clusters, which were equated with species and 20 major (6 to 71 strains) clusters that were provisionally considered to be species-groups. The largest cluster, the S. albidoflavus species-group, encompassed three subgroups which were later considered to correspond to the species S. albidoflavus (Rossi-Doria 1891 [27]), Waksman and Henrici 1948 (35), S. anulatus (Beijerinck 1912 [1]), Waksman 1957 (33) and S. halstedii (Waksman and Curtis 1916 [34]), Waksman and Henrici 1948 (35). Members of these taxa were assigned to well-defined clusters in subsequent numerical phenetic studies

(10,15).

Relatively few attempts have been made to evaluate the taxonomic status of clusters defined in numerical taxonomic investigations (9, 10, 15,22,36). However, good con-

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E. V. Ferguson, A. C. Ward, J.-J. Sanglier, and M. Goodfellow

gruence has been found between numerical phenetic and DNA:DNA relatedness data for taxa equated with S. albidoflavus (25) and S. ipomoea (16). It is also encouraging that RFLP profiles from pulse field gel electrophoresis of DNA digests of S. ipomoea strains correlate with DNA:DNA relatedness data (2). Streptomyces acidoscabies strains from the same genomic species (13) have also been shown to be closely related on the basis of phenetic data (4). In contrast, some numerically circumscribed speciesgroups have been found to contain several genomic species (17, 18, 19,38). The rationale of using DNA:DNA relatedness data as the gold standard for the delineation of bacterial species is based on the results of numerous investigations where good congruence has been found between DNA relatedness values and results based on other taxonomic methods, including numerical taxonomy (12). The advantages of DNA: DNA pairing analyses outweigh their limitations though such studies are technically demanding and hence cannot readily be applied to resolving relationships within complex taxa such as Streptomyces. Analytical chemical methods, such as Curiepoint pyrolysis mass spectrometry (PyMS), may provide a way of circumventing this problem as DNA:DNA and PyMS studies give similar patterns of relatedness (7, 28). The speed and reproducibility of Curie-point PyMS make it an attractive method for clarifying the taxonomy of Streptomyces species and species-groups (11, 22, 28). It is necessary to further unravel the intrageneric taxonomic structure of the genus Streptomyces given the need to establish the clinical, ecological and industrial importance of individual species. The popular view that streptomycetes from clincal material can be dismissed as harmless saprophytes is being questioned (23,24). Strains labelled to S. albus, S. anulatus (griseus), S.lavendulae, S. rimosus, S. somaliensis and S. violaceoruber have all been isolated from clinical specimens. The aim of the present investigation was to determine the value of the Curie-point PyMS technique in resolving relationships within the S. albidoflavus species-group, a taxon which includes organisms of potential medical importance. Materials and Methods Strains and cultivation. The test strains (Table 1) were grown, in duplicate from glycerol cultures, for 7 days at 25·C on a non-sporulating agar (NSA) medium designed to inhibit sporulation of streptomycetes (28). Growth from the plates was spread over sterile polycarbonate filters (0.45 Ilm, 47 mm. diam., Millipore) placed over NSA plates; the latter were incubated as before. This exercise was repeated twice and growth from the final preparations used for PyMS. Pyrolysis mass spectrometry. For each strain, biomass (ca. 50 Ilg) from polycarbonate filters was smeared uniformly over the upper surfaces of ferro-nickel alloy pyrolysis foils (Horizon Instruments Ltd., Heathfield, East Sussex, UK) which had been partially inserted into 100 ilL pyrolysis tubes (Horizon Instruments Ltd.). The assembled inoculated foils were dried in a hot air oven at 80·C for 15 minutes then tamped down into the pyrolysis tubes, using a stainless steel depth gauge, so that the tip of each foil lay about 10 mm from the mouth of the tube. Viton O-ring collars (Horizon Instruments Ltd.) were then positioned approximately 2 mm from the edge of the pyrolysis tubes and the latter loaded onto the carousel as a sequential series of replicates (A1, B1, C1, Al, B2, C2, A3, B3, C3). Triplicated samples of each of the duplicated strains were investigated and all test strains were analysed under a code which was broken after the data had been analysed. The samples were examined as a single batch using a Horizon Instruments 200X pyrolysis mass spectrometer. The inlet heater had been set at 160·C and the tube loader heated to 120·C. Curie-point pyrolysis occurred at 530·C for 3 seconds, under vacuum, with a

PyMS of Streptomyces Species-Groups

171

Table 1. Designation, source and sample codes of representatives of the Streptomyces albidoflavus species-group" ISP number

Sample codes

Name

Source

Subcluster 1A: Streptomyces albidoflavus (Rossi-Doria 1891 [27]) Waksman and Henrici 1948 AL (35). 5445T 3,4 S. albidoflavus A. Seino, KCC S-0446 (ATCC 25422) 5001 T Z,Z S. canescens H.J. Kutzner, DSM 40001 (ATCC 19736) "S. citreus" 5364 9,16 A. Seino, KCC S-0464 (ATCC 25441) 5233 T 10,11 A. Seino, KCC S-0065 (ATCC 23899) S. coelicolor 5485 X,x "S. coriofaciens" A. Seino, KCC S-0741 (ATCC 14155) "S. craterifer" 5296 1,2 T. Cross, CUB 302 (ATCC 25445) S. felleus A. Seino, KCC S-0368 (ATCC 19752) W,w 5130T S.limosus 5131 T 5,6 H.J. Kutzner, DSM 40131 (ATCC 19778) 12,13 A. Seino, KCC S-0803 (ATCC 6246) 5347T S.odorifer S. rutgersensis Y,y H.J. Kutzner, DSM 40077 (ATCC 3350) 5077T 5394T 14,15 S. sampsonii A. Seino, KCC S-0515 (ATCC 25495) "S. tetanusemus" A. Seino, KCC S-0839 (ATCC 27471) 5585 7,8 Subcluster 1B: Streptomyces anulatus (Beijerinck 1912 [1]) Waksman 1957AL (33). 5320 I,i "S. albidus" S. T. Williams, (LIV) A45 (ATCC 25423) 5043T B,b S. alboniger A. Seino, KCC S-0563 (ATCC 12461) 5326T H,h S. alboviridis A. Seino, KCC S-0449 (ATCC 25425) F,f S. anulatus A. Seino, KCC S-0721 (ATCC 27416) 5361 T 5598 T 0,0 S. bacillaris S. T. Williams, (LIV) A179 (ATCC 15855) 5128 D,d S. chrysomallus H.J. Kutzner, DSM 40128 (ATCC 23209) 5203T E,e:N,n S. fluorescens I.J. Bousfield, NCIB 9851 (ATCC 15860) 5199T J,j S. globisporus I.J. Bousfield, NCIB 9796 (ATCC 15864) K,k S. griseobrunneus H.J. Kutzner, DSM 40066 (ATCC 19762) 5066T 5088 T M,m S. niveus I.J. Bousfield, NCIB 9219 (ATCC 19793) "S. oligocarbophilus" A. Seino, KCC S-0804 (ATCC 27453) 5589 G,g 5019T L,l S. pluricolorescens I.J. Bousfield, NCIB 9813 (ATCC 19798) 5255T C,c S. sindenensis A. Seino, KCC S-0669 (ATCC 23963) 5292T A,a S. spheroides S. T. Williams, (LIV) A116 (ATCC 23965) Subcluster 1C: Streptomyces halstedii Waksman & Curtis 1916 [34]) Waksman and Henrici 1948 AL (35). A. Seino, KCC S-0748 (ATCC 11635) S. erythraeus 5517T V,V S. griseolus S. T Williams, DSM 40062 (ATCC 3320) 5067T T,t S. halstedii 5068 T R,r I.J. Bousfield, NCIB 9839 (ATCC 10897) "S. humifer" U,U A. Seino, KCC S-0770 (ATCC 13748) 5602 5023 T P,p S. nitrosporeus H.J. Kutzner, DSM 40023 (ATCC 12769) 5072T Q,q S. olivaceus H.J. Kutzner, DSM 40072 (ATCC 3335) .. Species-group defined in the numerical taxonomic study of Williams et al. (36). Key: T type strain; Binomials in inverted commas were not on Approved Lists of Bacterial Names (29) and have not been validated subsequently. Abbreviations: ATCC, American Type Culture Collection, Rockville, Md., USA; CUB, School of Biological Sciences, University of Bradford, Bradford, UK; DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany; KCC, Collection of the Kaken Chemical Co. Ltd., Tokyo, Japan; LIV, Department of Genetics and Microbiology, University of Liverpool, Liverpool, UK; NCIB, National Collection of Industrial Bacteria, Aberdeen, UK.

172

E. V. Ferguson, A. C. Ward, ].-J. Sanglier, and M. Goodfellow

temperature rise time of 0.6 seconds. The resultant pyrolysate was ionised by collision with a low energy (25eV) electron beam and the ions separated in a quadrupole mass spectrometer which scanned the pyrolysate at 0.35 second intervals. Total ion counts, pyrolysis sequence numbers and integrated ion counts at unit mass intervals from 51 to 200 were recorded for each sample on floppy disk. The time required for each individual sample to be pyrolysed was approximately 2 minutes. Data analysis. Mass ion counts of less than 5 x 105 and greater than 6 x 106 were removed from the dataset to reduce distorting statistical effects due to poor ion reproducibility. Variations within the remaining mass spectra due to inconsistencies in inoculum size were normalised by iterative normalisation (14). The individual masses were then ranked according to their characteristicity values (5), a procedure which allows the selection of masses showing good reproducibility within a group of sample replicates (in this case triplicates) and good discrimination between triplicate groups. Duplicate triplicates were used to validate the reproducibility between separately grown and pyrolysed samples of the same organism. The PyMS data were then subjected to principal component analysis. Principal components (PC) accounting for over 0.1 % of the total variance were used as input data for canonical variate analysis (CVA) and the results from the combined study presented as two dimensional ordination diagrams. Confidence limits (95%) represented by circles were plotted around the replicate samples to distinguish between the resultant groups. Clusters of similar organisms were identified by sequential PC-CVA analysis, in which, disseminated groups are sequentially removed from the dataset which is then re-analysed to achieve finer discrimination (6). Membership of each of the two resultant clusters was confirmed by testing the addition of each duplicate triplicate to the cluster and the omission of each cluster member, individually, from the cluster.

Results Excellent agreement was found between the results of the triplicate analyses of each strain, that is, the members of each triplicate set were either superimposed or occupied adjacent positions on the ordination plot (Fig. 1). Similarly, as anticipated, the duplicated cultures clustered adjacent to one another. It is also evident from the figure that S. nitrosporeus ISP 5023 T is sharply separated from all of the test strains. In subsequent analyses, the same proved to be true for S. pluricolorescens ISP 5019T and S. sindenensis ISP 5255T • Most of the remaining strains fall into two phena, groups lA and IB, which can be equated with subclusters lA and IB (Table 1). The representatives of the remaining taxon, subcluster lC, either show a close relationship with group IB ("S. humifer" ISP 5602 and S.olivaceus ISP 50nT) or lie between groups lA and IB (S. erythraeus ISP 5517T , S. griseolus ISP 5067T and S. halstedii ISP 5068T ; since the former two strains occupy adjacent positions only the data for the latter organism are shown in Figure 1). Sequential PC-CVA analysis shows that all of the subcluster lA strains (Table 1) form a distinct taxon, namely group lA (Fig. 2). Streptomyces albidoflavus ISP 5445T is an outlying member of this group though the 95% confidence limit for the data on this strain overlap in all orientations of the three dimensional ordination diagram. A view of the data with the sharpest separation of the S. albidoflavus strain is shown in Figure 2. All but one of the representatives of subcluster IB were found to be clearly distinct from the group lA strains, a typical result is shown for S. globisporus ISP 5199T (Fig. 3). The exception, S. chrysomallus ISP 5128, was recovered with the group lA strains (Fig.4). Once again, the S.albidoflavus strain was found on the periphery of group lA. However, on the basis of 95% confidence limits this organism is a bona

1

PyMS of Streptomyces Species-Groups

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173

nitrosporeus

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Fig. 1. PC-CVA ordination diagram of pyrolysis mass spectrometry data for the Streptomyces albidoflavus species-group of Williams et al. (36). Sample codes for the strains are given in Table 1.

fide member of this group. Streptomyces anulatus ISP 5361, a member of subcluster 1B, showed a relatively close relationship with the group 1A strains. The representatives of subcluster 1B (Table 1) formed a relatively heterogeneous assemblage (Fig. 5). Streptomyces griseobrunneus ISP 5066T, S. pluricolorescens ISP 50l9T and S. sindenensis ISP 5255 T fall outside group 1B based on 95% confidence circles. In contrast, S. alboviridis ISP 5326\ S. chrysomallus ISP 5128, S. niveus ISP 5088 T and S. spheroides ISP 5292T lie on the periphery of the main body of group lB. Streptomyces albidoflavus ISP 5445T was the member of group lA which showed the closest affinity to the group lB strains (data not shown). The subgroup Ie strains also formed an heterogeneous assemblage (Fig. 1). Streptomyces erythraeus ISP 5517\ S. griseolus ISP 5067T and S. halstedii ISP 5068 T were found to be closely related to one another and to S. chrysomallus ISP 5128. However, none of these strains joined group lA when tested separately from the S. chrysomallus strain though S. olivaceus ISP 50nT was associated with group lA through a link with S. alboniger ISP 5043 T • "Streptomyces humifer" ISP 5062 fell just outside group lB. The final subcluster lC strain, S. nitrosporeus ISP 5072T, formed a distinct single membered group.

E. V. Ferguson, A. C. Ward, ].-J. Sanglier, and M. Goodfellow

174

S.

anulatus

GROUP 1A

S.

albidoflavus

Fig. 2. PC-CVA ordination diagram of pyrolysis mass spectrometry data for subcluster lA. On the basis of the 95% confidence limits S. albidoflavus ISP 5445 T is an outlying member group lA while S. anulatus ISP 5361 is not a member. Discussion Streptomycete systematics began as largely an empirical discipline but is becoming increasingly objective due to the application of modern chemical, molecular and numerical taxonomic procedures (10, 17, 18, 19,22,37,38). However, it is becoming increasingly clear that the various methods currently used to define bacterial species have individual flaws as well as recognised strengths (12,30). Consequently, the most reliable and comprehensive approach to the delineation of streptomycete species should be based on the integrated use of genotypic and phenotypic data (22). This approach, which is known as polyphasic taxonomy, was introduced by Colwell (3) to signify successive or simultaneous taxonomic studies on bacterial groups using methods designed to yield genetic and phenetic data (32). Polyphasic taxonomic studies can be expected to yield well circumscribed species and an improved nomenclature (12,26). The results of successive comparative taxonomic analyses carried out on representatives of subcluster 1A (S. albidoflavus; 36) are shown in Table 2. It is clear from the PyMS, chemical (21), DNA:DNA relatedness (25) and numerical taxonomic data (10, 15) that members of this subcluster belong to a well defined species, namely

PyMS of Streptomyces Species-Groups

175

S.globisporus

Fig. 3. PC-CVA ordination diagram of sub-cluster lA with a typical representative of subcluster IB (S. globisporus ISP 5199T ).

S. albido{lavus (Rossi-Doria 1891 [27]), Waksman and Henrici 1948 AL (35). The remaining validly described species which can be assigned to this taxon need to be formally reduced to synonyms of S. albido{lavus. Further comparative studies are required to clarify the taxonomic status of "S. craterifer" ISP 5296 and S. tetanus emus ISP 5585. The results of the present study when taken with these earlier investigations (Table 2) show that subclusters 1B (S. anulatus; 36) and lC (S. halstedii; 36) contain several centres of taxonomic variation. It is clear that additional comparative studies need to be carried out on well chosen strains to unravel the taxonomy of strains assigned to these taxa in successive numerical taxonomic surveys (10, 15, 36). It is evident from the present study that PyMS should feature in further polyphasic taxonomic studies designed to clarify relationships between strains assigned to Streptomyces species-groups. The congruence found between the PyMS and other types of taxonomic data used to define S. albido{lavus is particularly encouraging and is in line with the results of corresponding analyses performed on several bacterial taxa (11, 20, 31). However, the speed and reproducibility of PyMS give it the edge over other modern taxonomic methods used to gain a first approximation of the integrity of clusters defined in numerical taxonomic surveys. The results of the present investigation confirm the preliminary observation of Sanglier et al. (28) that it is possible to sep~rate subcluster 1A (S. albido{lavus; 36) and 1B strains (S. anulatus; 36) by PyMS. However, it is also evident from the present analysis that the resolution of the technique can be influenced by minor changes in the composition of the test strains studied. The position of S. chrysomallus ISP 5128 was found to especially sensitive to small changes in test strain composition. It is also possible that the PyMS data were distorted by the production of different types of secondary me-

176

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Fig. 4. PC-CVA ordination diagram of sub-cluster 1A with S. chrysomallus ISP 5128. This strain clusters with group 1B in the overall analysis but is indistinguishable from group 1A when analysed with that group. tabolites as the strains were grown for 7 days prior to analysis. The ability to detect secondary metabolites produced by streptomycetes may be advantageous to the microbial technologist but not necessarily to the taxonomist. In subsequent studies on streptomycetes, experiments should be carried out after incubation for both 3 and 7 days in order to detect differences which may be due to secondary metabolite production (8).

Acknowledgements. The authors are indebted to colleagues who provided strains (Table 1). E.V.F. and M.G. are grateful to Sandoz AG, Basle, Switzerland for financial support.

griseobrunneus

S.

chrysamllus

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Fig. 5. PC-CVA ordination diagram and 95% confidence circles for sub-cluster lB. Individual strains form single member clusters and many strains are outliers from the centre of group lB.

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lA lA IB 2 2 2

IB: Streptomyces anulatus (Beijerinck 1912 [1]) Waksman 1957AL (33).

1 1 1 1 1 ND 1 1 1 1 1 ND

* Subcluster

lA lA lA lA lA IB lA lA lA+ lA lA IB+

S. albidoflavus S. canescens "S. citreus" S. coelicolor "S. coriofaciens" "S. craterifer" S. felleus S.limosus S.odorifer S. rutgersensis S. sampsonii "S. tetanusemus"

5445 5001 5364 5233 5485 5296 5130 5131 5347 5077 5394 5585

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s. anulatus and S. halstedii using different taxonomic methods

* Subcluster lA: Streptomyces albjdoflavus (Rossi-Doria [27]) Waksman and Henrici 1948 AL (35).

Strain ISP number

Table 2. Classification of representative strains of s. albidoflavus,

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lA lA lA lA lA lA lA lA lA lA lA lA

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ND 1B+ LR ND 1B+ 1B+ LR 1B LR LR LR 2 2 ND ND 2 ND ND 2 2 2 2

SMC 1-3 1-3 1-3 1-3 1-3 SMC 1-3 1-3 1-3 SMC

S. erythraeus S. griseolus S. halstedii "S. humifer" S. nitrosporeus S. olivaceus

LR LR LR LR LR LR 3 3 3 3 ND 3

ND 1-2 1-2 35 ND SMC

ND ND ND ND ND ND

VIII V ND V V ND ND ND V ND IV

1C 1C 1C SMG SMG 1A

1B SMG 1A and 1B 1B 1B SMG 1B 1B SMG SMG 1B

* Cluster composition according.to Williams et ai., (36); + Relatively high DNA relatedness values shown to appropriate reference strains. Abbreviations: LR, low DNA relatedness shown to all of the reference strains; ND, not determined; SMC single member cluster; SMG, single member group.

5517 5067 5068 5602 5023 5072

* Subcluster 1C: Streptomyces halstedii Waksman & Curtis 1916 [34]) Waksman and Henrici 1948 AL (35).

5361 5598 5128 5203 5199 5066 5088 5589 5019 5255 5292

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References

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Prof. M. Goodfellow, Department of Microbiology, The Medical School, Framlington Place, Newcastle-upon-Tyne, NE2 4HH, UK, Tel.: +191-2227706, Fax: +191-2227736