Download Polyacrylamide Gel Electrophoresis Analysis of Ribosomal Protein

Vol. 41, No. 2
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JOURNALOF SYSTEMATIC
BACTERIOLOGY,
Apr. 1991, p. 234-239
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Polyacrylamide Gel Electrophoresis Analysis of Ribosomal Protein
AT-L30 as a Novel Approach to Actinomycete Taxonomy:
Application to the Genera Actinomadura and Micvotetraspova
KOZO OCHI,l* SHINJI MIYADOH,2 AND TOSHIAKI TAMURA'
Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 5-2-3 Tokodai, Tsukuba, Ibaraki 300-26,' and Pharmaceutical
Research Center, Meiji Seika Kaisha, Ltd., Morooka, Kohoku-ku, Yokohama 222,2 Japan
Actinomycete ribosomal protein AT-L30 exhibits electrophoretic mobility that is specific for each genus. On
the basis of this fact, we analyzed ribosomal AT-L30 proteins from 26 type strains of species belonging to the
genera Actinomadura and Microtetraspora. The electrophoretic mobilities of AT-L30 preparations from these
strains, as determined by two-dimensional polyacrylamide gel electrophoresis, revealed that they could be
divided into two groups, one group with relative electrophoretic mobilities of 14.0 to 41.5 and another group
with relative electrophoretic mobilities of -6.5 to 0. The first group corresponded to the genus Actinomadura,
and the second group corresponded to the genus Microtetraspora. Partial amino acid sequencing of AT-L30
preparations from several strains proved that we were indeed dealing with the specified protein homologous to
ribosomal protein L30 of Escherichia coli. Our results strongly supported the conclusions of previous work and
thus proved the efficacy of ribosomal protein analysis as a novel approach for taxonomy of actinomycetes.
The ribosomal proteins of Escherichia coli have been
studied in great detail (26-28). However, only few reports
are available on the ribosomal proteins of actinomycetes (16,
23). The use of two-dimensional separation of ribosomal
proteins for identification and classification has been extended to the family Enterobacteriaceae, the family Bacillaceae, and several other bacteria (2, 4, 5). Despite the
similarity of ribosomal protein patterns within related genera
or species, Ochi (19,21) has found that several Streptomyces
species exhibit considerable electrophoretic variability in
ribosomal protein patterns and has proposed that the members of the genus Streptomyces have ribosomal protein
patterns that are specific to each taxon. The practical application of ribosomal protein patterns to Streptomyces taxonomy has also been demonstrated (19, 20). In the course of
such studies, it has been discovered that there is striking
variability in the electrophoretic mobilities of certain ribosomal proteins (the AT-L30 proteins) among genera of
actinomycetes; nevertheless, these ribosomal proteins appear to be highly conserved (with respect to electrophoretic
mobility) within each genus (21a). Therefore, it should be
possible to utilize this fact in the taxonomy of actinomycetes. To demonstrate the efficacy of this new method,
we performed an electrophoretic analysis of ribosomal ATL30 proteins to establish the taxonomic status of the genera
Actinomadura and Microtetraspora, whose taxonomy has
been much confused to date. Goodfellow (8) has recommended that the genus Actinomadura should be restricted to
Actinomadura madurae and related species (the A. madurae
group) and that Actinomadura pusilla and related taxa (the
A. pusilla group) merit separate generic status. Subsequently, Miyadoh et al. (18) suggested that the A. pusilla
group and the genus Microtetraspora should be combined at
the genus level on the basis of chemotaxonomic and DNADNA hybridization results. More recently, Kroppenstedt et
al. (15) proposed a revision of the genus Actinomadura; on
* Corresponding author.
the basis of chemical, molecular, and numerical taxonomic
evidence, they proposed that the A. pusilla group and the
genus Microtetraspora should be combined at the genus
level. The results of our ribosomal protein AT-L30 analysis
are consistent with these previous proposals. In this study
we demonstrated the efficacy of this novel method in the
taxonomy of actinomycetes.
MATERIALS AND METHODS
Bacterial strains. The strains used in this study are listed in
Table 1. All were type strains obtained from the Japan
Collection of Microorganisms (Saitama, Japan), the Institute
for Fermentation (Osaka, Japan), and the American Type
Culture Collection (Rockville, Md.).
Preparation of total ribosomal proteins. Since the members
of the genera Actinomadura and Microtetraspora generally
grew slowly in a soluble starch-peptone-yeast extract medium (19), a medium suitable for the growth of other actinomycetes, all of the strains used in this study were grown in a
glucose-yeast extract medium (18) on a rotary shaker (220
rpm) at 30°C. After cultivation for 20 to 30 h (mid-exponential phase), the cells were collected, washed, and then
disrupted by sonication for 3 to 5 min, as described previously (19). The cells of several Actinomadura species were
hardly disrupted by sonic treatment, but the cells were not
treated for more than 5 min to avoid destruction of ribosomes. The 70s ribosomes were pelleted by centrifugation at
110,000 X g for 4 h. Ribosomal proteins were prepared from
the 70s ribosomes by extraction with acetic acid, as previously described (19), using the method of Hardy et al. (10).
The total ribosomal protein samples obtained in this way
contained 5 to 12 mg of protein per ml. The relatively low
protein concentrations in the samples compared with samples from other actinomycete genera (20 to 30 mg of protein
per ml) (19) were apparently due to the difficulty encountered in disrupting cells of Actinomadura species,
Two-dimensional PAGE. For polyacrylamide gel electrophoresis (PAGE), the method of Kaltschmidt and Wittmann
234
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PAGE ANALYSIS OF PROTEIN AT-L30
VOL.41, 1991
(14) was used, as described in detail previously (19). The gels
were run twice for each ribosomal protein sample to confirm
the reproducibility of the results obtained.
Determination of amino acid sequences. After two-dimensional PAGE, the spots (usually containing 20 to 50 kg of
protein) assigned to AT-L30 were cut from the gels and
washed with 100 ml of a solution containing 10% (vol/vol)
acetic acid and 50% (vol/vol) methanol with gentle shaking
for 5 days. This procedure was effective for removing the
pigment (Coomassie brilliant blue) which bound to the
protein. The proteins were extracted from the gels by
electrophoresis. Sequence analyses of the extracted proteins
were performed with model 470A protein sequencer (Applied Biosystems). Complete details of the sequencing procedures will be given elsewhere.
RESULTS
Two-dimensional PAGE analysis of ribosomal proteins. All
of the organisms belonging to the genera Actinomadura and
Microtetraspora examined were type strains (Table 1);the
nomenclature for these bacteria was based on the proposal
by Kroppenstedt et al. (15). The ribosomal proteins of these
strains were extracted with acetic acid and separated by
two-dimensional PAGE. A few examples of the results of
this analysis are shown in Fig. 1A through I (the spots
assigned to ribosomal protein AT-L30 are indicated by
arrows). The results for Saccharomonospora viridis, whose
protein AT-L30 was taken as unity for determining relative
electrophoretic mobilities (REMs), are shown in Fig. 1J.
Figure 1 shows that the species which we tested could be
divided into two clusters, one group in which little or no
AT-L30 mobility occurred in the first dimension of gel
electrophoresis and another group in which AT-L30 did
migrate to the cathode side (the right side in Fig. 1). The
former corresponded to the genus Microtetraspora, and the
latter corresponded to the genus Actinomadura. The distances (in millimeters) that AT-L30 moved in the first
dimension in the original slab gels are shown in Table 1.
Table 1 also shows the electrophoretic mobility of AT-L30
from each species relative to the electrophoretic mobility of
S. viridis AT-L30, which displayed the greatest mobility
among all of the actinomycetes examined (21a). The relative
electrophoretic mobility (REM) defined in this way could
make it possible to compare the AT-L30 mobilities in different laboratories. For members of the genus Actinomadura, the experimental error for AT-L30 mobility, as determined from several gel runs of the same sample, was at most
1 to 2 mm (equivalent to an error of 10% in REM). In
contrast, the AT-L30 mobility for members of the genus
Microtetraspora differed in different gel runs, with a maximum error of 4 mm, but the moving distances were always
within the range of -4.5 to 0 mm (confirmed by several gel
runs for each sample). The greater fluctuation in AT-L30
mobility for Microtetraspora species in different gel runs
was apparently due to higher sensitivity to slight changes in
the pH of the separation gel and/or the electrode buffer
prepared for each experiment. Thus, the genus Microtetraspora (REMs, -6.5 to 0) and the genus Actinomadura
(REMs, 14.0 to 41.5) were sharply separated by the electrophoretic properties of AT-L30 (Table 1).
Several species of Microtetraspora (Microtetraspora pusilla, Microtetraspora roseoviolacea, Microtetraspora helvatu, Microtetraspora fastidiosa, Microtetraspora spiralis,
Microtetraspora roseola, Microtetraspora ferruginea , and
Microtetraspora salmonea) were classified in the genus
235
TABLE 1. Electrophoretic mobilities of ribosomal AT-L30
proteins from the type strains of members of the genera
Actinomadura and Microtetraspora
Strain"
Microtetraspora niveoalba JCM 3149Td
Microtetruspora pusilla (Actinomadura
pusilla) ATCC 27296T
Microtetraspora glauca JCM 3300'
Microtetraspora fusca ATCC 2305gT
Microtetrasporu roseoviolacea (Actinomadura roseoviolacea) JCM 3145=
Microtetraspora helvata (Actinomadura
helvata) JCM 3143T
Microtetraspora fastidiosa (Actinomadura
fastidiosa) JCM 3321T
Micro tetra sp ora s p iralis ( Ac tinoma du ra
spiralis) IF0 14097T
Microtetraspora roseola (Actinomadura
roseola) JCM 3323T
Microtetraspora africana (Nocardiopsis
africana) JCM 6240T
Microtetraspora ferruginea (Actinomadura
ferruginea) JCM 3283T
Microtetraspora salmoneu (Actinomadura
salmonea) JCM 3324'
Microtetraspora angiospora (Micropolyspora angiospora) JCM 3109=
Actinomadura spadix IF0 14099'
Actinomadura kijaniata JCM 3306T
"Parvopolyspora pallida" JCM 6053T
Actinomadura atramentaria JCM 6250T
Actinomadura madurae ATCC 19425'
Actinomadura malachitica JCM 3297T
Actinomadura cremea IF0 14182T
Actinomadura libanotica IF0 14095'
Actinomadura vinacea JCM 3325'
Actinomadura coerulea JCM 3320T
Actinomadura luteofluorescens JCM 4203'
Actinomadura pelletieri JCM 338gT
Actinomadura verrucosospora JCM 3147'
Nocardia asteroides JCM 3384'
Escherichia coli ATCC 11775T
Saccharomonospora viridis JCM 3036'
Mobility of
AT-L30 (mm)'
REM"
-4.5
-4.0
-6.5
-6.0
-3.5
-3.0
-3.0
-5.0
-4.5
-4.5
-2.0
-3.0
-1.0
-1.5
-1.0
-1.5
- 1.0
-1.5
-1.0
-1.5
0.0
0.0
0.0
0.0
0.0
0.0
9.5
11.0
11.0
11.5
13.0
14.5
19.5
25.0
25.5
28.5
28.5
28.5
28.5
40.5
50.0
68.5
14.0
16.0
16.0
16.5
19.0
21.0
28.5
36.5
37.0
41.5
41.5
41.5
41.5
59.0
73.0
100.0
The nomenclature of bacteria is based on the proposal of Kroppenstedt et
al. (15); the old names are given in parentheses.
Mobility of AT-L30 in the first dimension. Negative values indicate that
the protein moved toward the reverse side (the anode side).
" The mobility of protein AT-L30 from S. viridis JCM 3036= was defined as
unity (100).
T = type strain.
'
Actinomadura until Kroppenstedt et al. (15) revised the
taxonomic status of these species. It is striking that these
species all had REMs similar to those obtained for authentic
Microtetraspora species (e .g., Microtetraspora glauca) but
not for members of the genus Actinomadura sensu stricto.
These results strongly support the previous proposal of
Kroppenstedt et al. (15). Microtetraspora angiospora and
Microtetraspora africana have been previously called Micropolyspora angiospora and Nocardiopsis africana, respectively. Chemotaxonomic studies of these species have
shown that they are closely related to Microtetraspora
pusilla and related species (9, 22), and recently Kroppenstedt et al. (15) validly classified these two species in the
genus Microtetraspora. Our results supported this proposal,
since the REMs for these organisms were similar to the
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:236
OCHI ET AL.
INT. J. SYST. BACTERIOL.
REMs for Microtetraspora species (Table 1). “Parvopolyspora pallida,” which was presumed to belong to the genus
Actinomadura by Itoh et al. (13) and Miyadoh et al. (17), was
found to have an REM (16.0) in the range observed for the
genus Actinomadura (Table l),supporting the presumption
of Itoh et al. and Miyadoh et al. Thus, despite the entirely
different taxonomic approaches used in the different laboratories, we reached the same conclusion.
Amino acid sequence of the protein assigned to AT-L30. The
complete amino acid sequence of E . coli protein L30 is
known, as reported by Ritter and Wittmann-Liebold (24); its
N-terminal amino acid sequence is shown in Fig. 2. This
protein contains 58 amino acid residues, and the molecular
weight calculated from the sequence is 6,411. We determined the N-terminal amino acid sequences of AT-L30
proteins from several species of the genera Actinomadura
and Microtetraspora. As shown in Fig. 2, the same amino
acid sequence (Lys-Ile-Thr-Gln-Thr) was found in both E .
coli and Actinomadura rnalachitica. These results provide
strong evidence that the protein assigned to AT-L30 in A .
malachitica is indeed homologous to protein L30 of E . coli.
Almost no similarity was found between L30 from E . coli
and AT-L30 from Microtetraspora helvata or Microtetraspora glauca. Nevertheless, these AT-L30 proteins can be
considered homologous since they share a sequence signature in their N termini (Fig. 2, box). In addition, a comparison of the sequence of each AT-L30 protein with the known
sequences of E . coli ribosomal proteins other than L30
showed no homology at all. Thus, it is evident that we were
indeed dealing with the proteins (named AT-L30 proteins)
homologous to protein L30 of E . coli.
It should also be emphasized that Microtetraspora helvata
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VOL. 41, 1991
PAGE ANALYSIS OF PROTEIN AT-L30
237
FIG. 1. Two-dimensional PAGE of total ribosomal proteins from actinomycetes. The gel system was based on the system of Kaltschmidt
and Wittmann (14). 0 in panel A indicates the origin in the first dimension. The arrows indicate the positions of AT-L30 proteins. Strains were
arranged in order of protein AT-L30 mobility, with less mobility to the cathode side. (A) Microtetraspora pusilla. (B) Microtetraspora
niveoalba. (C) Microtetraspora glauca. (D) Microtetraspora fusca. (E) Microtetraspora helvata. (F) Microtetraspora ferruginea. (G)
Actinomadura madurae. (H) Actinomadura malachitica. (I) Actinomadura pelletieri. (J) Saccharomonospora viridis.
and Microtetraspora glauca, a single generic taxon, exhibited an extremely high level of homology in the amino acid
sequences of their AT-L30 proteins; in contrast, there was
little homology between A . malachitica and these two organisms (Fig. 2). This implies that there is an evolutionally
estranged relationship between the genus Microtetraspora
and the genus Actinomadura. Also, although the sequencing
experiment was conducted for only one-third of the whole
AT-L30 protein, the high frequency of acidic amino acids
and the low frequency of basic amino acids in Microtetraspora glauca compared with E. coli account for the observed
low electrophoretic mobility of the AT-L30 protein of Microtetraspora glauca (Fig. 1C).
5
DISCUSSION
Bacterial ribosomes consist of three species (5S, 16S, and
23s) of RNA and more than 50 species of ribosomal proteins
(for reviews, see references 26 through 28). Because of many
structural and functional constraints, the variability of ribosomal proteins (and also rRNA) is more limited than the
variability of other proteins; thus, ribosomal proteins have
much lower evolutionary rates than other proteins (11, 12).
Therefore, analysis of ribosomal proteins and rRNAs could
be an excellent approach for studying the evolutionary
relationships among organisms. Indeed, classification of
20
15
10
1
Ala-Glu-Ile Val-Asp-Ala-Leu-Gln-Phe-Val-
? -Gly-Glu-Arg-Gly-Phe-Tyr-Phe-Asn
Ala- ? -1le Val- ? -Ala- ? -Gln-Phe-Val- ? -Gly- ?
-?-?-
? -Tyr
Ala-Gln-Leu Lys-Ile-Thr-Gln-Thr-Lys-Ala-Val-Ile-Asn
4)
Ala-Lys-Thr-I1 e-Lys-Ile-Thr-Gln-Thr-Arg-Ser-Ala-Ile-Gly-Arg-Leu-Pro-Lys-His-Lys
1
5
10
15
20
FIG. 2. Primary structures of N termini of AT-L30 proteins from several species of the genera Actinomadura and Microtetraspora. The
data for E. coli are from reference 24. The common sequence found in E . coli and other strains is indicated by underlining. ?, Not determined;
1, Microtetraspora glauca; 2, Microtetraspora helvata; 3, Actinomadura malachitica; 4, Escherichia coli K-12.
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238
INT. J . SYST.BACTERIOL.
OCHI ET AL.
actinomycetes has been attempted by partial sequencing of
16s rRNAs (3, 7, 25).
In our analysis of ribosomal protein AT-L30 by twodimensional PAGE, we demonstrated that the members of
the genus Actinomadura and the members of the genus
Microtetraspora can be separated clearly by their AT-L30
REMs. Since the genera of actinomycetes have been shown
to have REMs that are specific to each genus (21a), it seems
reasonable to propose that the genera Actinomadura and
Microtetraspora should be viewed as independent taxa. To
our knowledge, this is the first study to deal with ribosomal
protein analysis for classification of actinomycetes ; the
efficacy of this method was predicted by Ochi previously
(19). It is especially noteworthy that several species of
Actinomadura (for example, Actinomadura kijaniata , Actinomadura libanotica, and Actinomadura spadix) whose
classification in the genus Actinomadura or the genus Microtetraspora has been ambiguous to date were all clearly
assigned to the former genus on the basis of the REMs of
their AT-L30 proteins (Table 1).It should also be pointed out
that there were great similarities in the ribosomal protein
patterns for the three Microtetraspora species (Microtetraspora glauca , Microtetraspora fusca , and Microtetraspora
niveoalba) (Fig. 1B through D). These results, together with
the results of a DNA-DNA pairing test (18), suggest that
these three species might be combined into a single species.
The extensive similarities were especially impressive for
Microtetraspora roseoviolacea, Microtetraspora salmonea,
and Microtetraspora angiospora and for Microtetraspora
roseola and Microtetraspora africana, implying that these
organisms are closely related species. In the genus Actinomadura, only one similar example was found (Actinomadura
luteojluorescens and Actinomadura verrucosospora) . Thus,
our results prove that ribosomal protein analysis (especially
analysis of AT-L30 proteins) is a very useful approach for
both classification and identification of actinomycetes. Several attempts have been made to utilize PAGE analysis of
ribosomal proteins for classification of bacteria, yeast, and
fungi (1, 2, 4 6 ) , but it has generally been difficult to obtain
unambiguous conclusions, perhaps because of the extensive
similarities in the ribosomal protein patterns of bacteria.
Apparently, our success in classification of actinomycetes
depended solely on the striking variability of the ribosomal
proteins of actinomycetes. In bacteria such as E . coli and
Bacillus subtilis, no such variability occurs, at least with
respect to electrophoretic mobility, even in the L30 protein
corresponding to the AT-L30 protein (21a). It is clear that
the variability of amino acid sequences (Fig. 2) is involved in
part (if not solely) in the diversity of AT-L30 electrophoretic
mobilities. However, we cannot explain why actinomycetes
evolved AT-L30 proteins that are so divergent with respect
to electrophoretic mobility. A sequence analysis of several
whole AT-L30 proteins will be required for further understanding.
In contrast to the narrow ranges of REMs in the genera
Streptomyces (14.5 to 25.5), Streptosporangium (21.0 to
30.5), Saccharomonospora (97.0 to loo), and Microtetraspora (-6.5 to 0), the range of REMs for members of the
genus Actinomadura (14.0 to 41.5) is wide (21a; this study).
Although the values are continuous within this range (Table
l), the data may imply that this taxon is heterologous.
Perhaps the greater variability in the ribosomal protein
patterns in the genus Actinomadura than in the genus
Microtetraspora (see above) reflects the wide range of
REMs.
As will be reported elsewhere, analysis of AT-L30 pro-
teins by two-dimensional PAGE has been used successfully
to establish the taxonomic status of the genera Microbispora
and Streptosporangium. Therefore, it is evident that ribosomal protein analysis will be very helpful in generating
polyphasic taxonomies of actinomycete taxa.
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