Journal of General Microbiol0g.v (X971), 68,187-197
Printed in Great Britain
The Linkage Map of Streptomyces rimosus
By E. J. F R I E N D
PJzer Ltd., Sandwich, Kent
D. A. HOPWOOD
John Innes Institute, Norwich NOR 70F
AND
(Acceptedfor publication I 3 July 197I)
SUMMARY
Haploid recombinant selection has been used to map the loci of 24 auxotrophic
mutations on a circular linkage group in an industrial strain of Streptomyces
rimosus. The features of crosses are similar to those in S . coelicolor A 3 (2), except for
the recovery of variable numbers of heterokaryons on selective media in S. rimosus
crosses. By a suitable choice of selected marker combinations, heterokaryons can
be largely eliminated. The linkage map of S. rimosus is extremely similar to that of
S. coelicolor ~ 3 ( 2 ) this
;
may indicate a conservation of gene arrangement in prokaryotes as already suggested by the similarity of linkage relationships in Escherichia
coli and Salmonella typhimurium. The S. rimosus map provides further support
for the idea of circular symmetry of the Streptomyces linkage map first proposed
for S. coelicolor A 3 (2).
INTRODUCTION
Until very recently, mapping studies in Streptomyces have been confined to a single strain,
Streptomyces coeZicolor ~ 3 ( 2 )(Hopwood, 1967b). It is of obvious interest to extend such
studies to antibiotic-producing strains so that knowledge of the linkage map could serve as
a starting-point for strain improvement by genetic manipulation. An additional interest
would be a comparison of the linkage maps of different streptomycetes, which would
indicate the extent to which gene arrangements have evolved or been conserved during the
evolution of the genus. This paper describes the results of linkage studies, started in April
I 967, with an industrial strain of S. rimosus producing oxytetracycline. The resulting linkage
map is compared with that of S. coelicolor A 3 (2), and with those of two other strains which
have been the subject of investigations by others (AlaEeviC, 1969, 1970; Coats & Roeser,
1971).
METHODS
Media. ‘Complete’ medium (CM) was employed for maintenance of stock cultures and
for crosses. CM was the medium of Emerson, Whiffen, Bohonos & DeBoer (1946) supplemented with additional growth factors. Each litre of medium contained: Difco Emerson
agar, 41.5 g.; Difco Casamino acids, 2 g.; vitamin solution, I ml.; yeast nucleic acid hydrolysate solution, 5 ml. Vitamin solution and yeast nucleic acid hydrolysate were as described
by Hopwood (19673).
Minimal medium (MM) was used for detection of auxotrophic mutants and, with appropriate additions, as diagnostic medium for the characterization of such mutants and for the
isolation of recombinants. MM contained the following ingredients per litre : sodium
chloride, 0.5 g. ; magnesium sulphate (7H,O), 0.5 g. ; ammonium chloride, I g. ; potassium
dihydrogen orthophosphate, 1.3 g. ;diammonium hydrogen orthophosphate, 2-4g. ;glucose,
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E. J. F R I E N D AND D. A. H O P W O O D
10g.; agar, 20 g. Glucose was autoclaved separately as a 50 yo (w/v) solution and added to
the medium before cooling.
Growth factors used to supplement MM were stored as sterile solutions and added to the
medium after sterilization to yield the following concentrations : individual amino acids,
50 pglml; adenine, guanine or uracil, 10pglml; individual vitamins, 5 ,ug/ml.
Strains. All strains of Streptomyces rimosus were derived from a prototrophic culture
used commercially for production of oxytetracycline.
Mutagenic treatments. Mutant strains were isolated after treatment of spore suspensions
either with ultraviolet light at 254 nm. or by exposure to N-methyl-N'-nitro-Jnitrosoguanidine using conditions described previously (Delib, Hopwood & Friend, I 970).
Genetic markers. Only auxotrophic markers were used ; their characteristics are shown in
Table I. The letters designating loci were assigned by analogy with comparably placed loci
in Streptomyces coelicolor A 3 (2) (see Discussion). A preliminary attempt to isolate mutant
strains resistant to a range of antibiotics was unsuccessful since the original culture was
resistant to high concentrations of streptomycin, kanamycin, lincomycin and amphotericin.
Table
I.
List of auxotrophic markers
Marker
Requirement
Marker
Requirement
adeAz
argA I
ath A 2
CYSD 3
guaA 2
guaBI
gluA I
hisA I
hisD 2
Purines
Arginine, citrulline or ornithine
Purines plus thiamine
Cysteine or S203,or Sz04or S,O,
Guanine
Guanine
Glutamic acid
Histidine
Histidine or histidinol (accumulation
of histidinol phosphate)
Isoleucine plus valine
Leucine
Leucine
/ysB2
metA I
metBz
nice2
panA I
P O A3
ribBI
serA I
sevCa
tJiiBa
thrA 6
uvuB5
Lysine
Methionine
Methionine or homocysteine
Nicotinamide
Pantothenic acid
Proline
Riboflavin
Serine or glycine
Serine or glycine
Thiamine
Threonine
Uracil
iZvB 4
ZeuA I
leuBz
Preparations for crossing. In the operations described below, all growing cultures were
incubated at 30". Two parent strains were chosen, each of which required at least two different
growth factors, and separate cultures of these strains were streaked on to plates of CM.
After four to six days' incubation, the sporulating colonies were replicated to appropriately
supplemented MM by means of velvet pads to confirm parental phenotypes. One colony of
each strain was then selected and placed in a 5 cm. x I cm. glass tube, where it was finely
ground using a glass rod. The fragments were suspended in 0.3 ml. of distilled water and
inoculated on to the surface of a slant of CM (17 ml. of medium in a 2 - 5 cm. x 15 cm. tube).
These pure parent cultures were incubated for four to six days until good sporulation was
evident.
Recombination conditions. To carry out a cross a 5 ml. hypodermic syringe fitted with an
18 cm. 13-gauge needle was filled with 2-5 ml. of distilled water and the needle, introduced
into one of the parent slants, was used to scrape the surface. Water expelled from the syringe
was used to wash the surface of the slant. This resulted in a suspension of spores and mycelial
fragments which was withdrawn into the syringe. The procedure was repeated for the second
slant using the same syringe and thus adding spores and mycelial fragments from the first
culture to the suspension of the second. The mixed suspension was inoculated on to slants
of CM at the rate of 0.3 ml. per slant. In general, the number of mixed slants prepared was
the same as the number of selective media.
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The linkage map of Streptomyces rimosus
189
Recovery and analysis of recombinants. A suspension of spores in 10ml. of water was
prepared from each mixed slant as described above. This suspension was agitated, filtered
through cotton wool to remove large mycelial fragments and centrifuged at 20008 for
15 min. The pellet was resuspended in 0.5 ml. of distilled water, the suspensions from
replicate slants were pooled, and two serial tenfold dilutions were made. Samples (0.1 ml.)
of the original suspension and of the two diluted suspensions were spread on to plates of
selective medium.
The plates were examined after two and three days’ incubation. If heavy background
growth from the parent strains had not developed, colonies varying in diameter between
0.5 and 2 mm. were easily distinguished and their positions were marked. The majority of
colonies recovered by this method proved, on subsequent analysis, to be recombinants;
occasionally a small proportion were shown to be heterokaryons. These were characterized
by their failure to grow when transferred to the medium on which they appeared originally
and by their giving rise to spores of the two parental phenotypes only.
A successful cross usually yielded up to IOO colonies on each plate inoculated with
undiluted suspension; this represented a recombination frequency of about I O - ~ to I O - ~ .
Less frequently the fertility was ten times higher. These frequencies are within the range
yielded by strains of IF (Initial Fertility) type in Streptomyces coelicolor (Vivian & Hopwood,
1970).
Incubation of plates was resumed for a further two or three days until recombinant
colonies were sporulating. Spores from a random sample of 50 colonies were inoculated on
to a master plate containing the same selective medium in a pattern to allow easy scoring of
the crosses. After incubation for four days, the master plate was replicated to a series of
diagnostic media; each medium in the series lacked one of the nutritional factors present in
the master plate. The diagnostic plates were examined after two days’ incubation and the
recombinants were classified according to their phenotypes.
RESULTS
Preliminary evidence of linkage. Linkage was first studied in four-factor crosses analysed
according to the procedure of Hopwood (1959). Crosses between pairs of double auxotrophs
were analysed by the isolation of recombinants on four different selective media, each containing a different non-parental combination of two growth factors. Table 2 summarizes
the results of a cross between strains ~ 2 6 ade-2
0
arg-if his-I Zys-a+ and ~ 2 6 ade-2+
1
arg-r
his-i+ lys-2. Spores from the mixed culture of the two strains were plated at equal concentrations on four selective media containing, respectively, adenine + arginine (selecting his+ and
Zys+) adenine +lysine (selecting arg+ and his+), arginine +histidine (selecting ade+ and l’,s+),
and histidine+lysine (selecting ade+ and arg+). The numbers of colonies arising on each
medium were counted. Samples of the colonies were then classified in respect of the two
non-selected markers on each medium, giving four possible genotypes on each, and nine
genotypes (out of a possible 16) taking the four media together. These included at least one
member of every pair of complementary genotypes, except the parental pair, with both
members of two pairs. The frequency of each genotype on each medium was then calculated
from the frequency in the classified sample and the total recombinant count on each medium.
As well as fully vigorous and well-replicating recombinant colonies, there was a variable
proportion of heterokaryons, recognized by their failure to replicate to the same medium
as the master plate.
The results (Table 2) show that the frequencies of the same genotype on different selective
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E. J. FRIEND A N D D. A. HOPWOOD
190
media were in general in fairly good agreement (within a factor of 2 to 3), indicating that
differential recovery of particular genotypes on different media was not an important source
of bias in the data. Moreover, in the two cases in which complementary genotypes could
be recovered, their frequencies were similar. As discussed by Hopwood (1g5g), the latter
finding allows a single member of a pair of complementary genotypes to be used as a measure
of the frequency of the pair. in those cases where one member alone is selectable. The seven
values, each representing the frequency of a different pair of complementary recombinant
genotypes, in the right-hand column of Table 2, were then used to calculate relative frequencies of recombination between each of the six pairs of markers in the cross. When this
was done (lower section of Table 2), it was found that the recombination frequency (87)
between arg-I and his-r was some fivefold less than between certain other pairs of loci. Two
Table 2 . Analysis of a four-factor cross: ~ 2 6 ade-2
0
arg-I+ his-r Zys-2+
x R 26 I ade-zf arg-r his-I+ Zys-2
a : Numbers of colonies of each progeny class in a random sample of 150 colonies from each
selectivemedium; b : number of colonies of each progeny class per unit volume of spore suspension,
derived from the figures in column a by scaling, using the total progeny on that medium; c : average
values for the figures in appropriate columns 6.
-
Selective media supplemented with
r
Genotypes of selectable
progeny
A
Adenine
and
arginine
Adenine
and
lysine
a
b
a
b
14
17
42
24
29
72
45
30
-
36
24
76
130
-
-
51
-
41
-
**
I
+
+
+
arg
ade
+
+
ade
-I-
ade
+
+
+
+
arg
+
+
+
+
+
-
his
+
+
his
+
-
arg
his
Heterokaryons
Sample size
Total progeny per unit
volume of suspension
Total
-
_
.
-
-
-
-
0
0
-
-
I
2
24
19
-
150
-
257
-
I20
-
Histidine
and
lysine
r--7
a
b
33
I4
-
150
-
48
86
-
112
202
-
-
-
-
2
o
150
-
a
6
92
3
16
-
33
b
26
393
I3
-I70
5
o
-
150
-
352
-
644
142
3
c
30
27
92
298
27
I00
3
-
Relative recombination frequencies in each interval
A
r
Components
-
Arginine
and
histidine
Average
frequency of
each pair of
complementary
genotypes
\
ade-a-arg-I
ade-2-his-I
ude-2-lys-2
urg-I-his-I
avg-I-iys-2
his-I-Eys-z
30
298
27
27
298
30
27
27
3
92
27
30
27
92
87
I00
3
30
92
298
3
455
428
423
I00
I00
3
I00
222
249
other values, for arg-r-Zys-a and his-I-Zys-2, were about half the maximum value of 423 to
455 shown by the remaining three pairs of loci; however, such a difference is best ignored
at this stage in view of the two- to threefold discrepancies between the various estimates of
the frequencies of particular pairs of recombinant genotypes (Table 2 ) . We thus concluded
from this analysis only that his-I and arg-r are linked, and that the other two loci are not
close to them, and certainly not between them.
It is significant that the frequency of the class of colonies diagnosed as heterokaryons
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The linkage map of Streptomyces rimosus
191
varies enormously on different selective media. This finding is entirely consistent with the
interpretation of these colonies as heterokaryons since it is to be expected that the vigour
of heterokaryotic growth, through complementation, would vary with the combination of
growth factors omitted from the selective medium. Thus heterokaryons would compete successfullywith parental background growth to give rise to visible colonies only on certain media.
Further information can be obtained from the data of this cross by constructing 2 x 2
tables to test the independence of segregation of each pair of non-selected alleles on each
of the four selective media. Clearly, in the absence of linkage between any of the four
loci, this segregation should always be independent ; different predictions follow from
various patterns of linkage. For the particular case of linkage of all four loci on a circular
map, Hopwood (1969) pointed out the unique expectation of a lack of independence when
the two non-selected loci are adjacent, and independence when they are separated by the
selected loci. Table 3 shows the 2 x 2 tabulations for each of the four selective media. Two
Table 3. Segregation of non-selected alleles in the data of Table 2
Each of the four sections of the table relates to the segregation of non-selected markers on one of
the four selective media. The frequency of each class of progeny is the actual number of colonies
in the classified sample; that is from columns a in Table 2 . The probability values for independent
segregation of each pair of non-selected alleles indicate independence for nde with arg and of his
with lys, and lack of independence of ade with lys and of arg with his.
adef
Crossovers*
adeCrossovers*
Probability of independence
Selected alleles
arg+
arg-
14
42
(293)
76
(44)
(I,3 )
17
(I,4)
(I,2)
30
(194)
0
(I,2, 39
his+/lys+
arg+
arg-
I4
86
(3>4)
*
6
92
(394)
(I,439 4)
< 0'001
adef llysf
See the map intervals in Fig.
lys-
3
(I,3)
(273)
2
4)
< 0'001
arg+/his+
lys'
48
(I,3)
51
45
(I,3)
0.30
his+
Crossovers*
hisCrossovers*
Probability of independence
Selected alleles
lys-
lys'
(I,2)
0-15
ade+largf
16
(44)
I.
of the four show an independent segregation of the alleles at the non-selected loci, while the
other two show a very marked lack of independence. This result indicates circular linkage
of the four markers in the order shown in Fig. I, such that non-selected loci separated from
each other by selected loci show independent segregation (ade-alarg-I and his-l/lys-2),
while adjacent non-selected loci show a lack of independence, with a deficiency of that combination requiring multiple crossing-over for its production ; the crossovers required to
generate each class of recombinants are indicated in Table 3.
The location of further markers. The success of four-factor crosses of the type described in
the preceding section in providing evidence for the linkage of markers on a circular map led
to many further crosses between recombinant and mutant strains, each cross normally
involving from five to eight markers. In this way, more markers were added to the map,
while the correctness of the first conclusions on linkage was confirmed. These multi-factor
crosses were not usually analysed by plating on all possible selective media; certain selections
were precluded by the leakiness of some markers, which could therefore be used only nonI3
MIC
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E. J. F R I E N D AND D. A. H O P W O O D
192
his-1
Fig. I. Arrangement of markers on a circular linkage map compatible with the data of Table 2,
analysed in Table 3. The outer circle represents parent strain ~ 2 6 0and
, the inner circle strain ~ 2 6 1 .
Table 4. Location of ser-r in a cross of ~ 2 6 ade-2
0
arg-r+ his-I lys-2+ ser-r+
and ~ 8 1 ade-2f
9
arg-r his-ri- Zys-2 ser-r
Two map locations for ser-I are consistent with the observed allele ratio Ser-IIser-if.The patterns
of crossing-over required to generate the observed recombinants on the two alternative locations
are indicated in the table. Position I is chosen (see text). Triangles in the diagrams indicate selected
alleles.
0
his-I
Genotype
ade-2 arg-1 ser-r
ade-a arg-r +
ade-2 + ser-I
ade-2 +
+
arg-I ser-r
arg-I +
+ ser-I
+
+
+
+
+
Total
+
Number
r-
I0
0
0
2
67
I
9
8
97
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The linkage map of Streptomyces rimosus
I93
selectively. In addition, certain combinations of markers, each selectable in other combinations, led to troublesome background growth or the presence of numerous heterokaryons ;
such difficultiesprevented the unbiased recognition of recombinant colonies. In any case,
one or two selections sufficed in later crosses to determine the map position of a new marker.
An example of this approach, using the rationale described by Hopwood (1967b), is in
Table 4. The cross involved the four markers previously discussed, and a fifth, s e w . Selection
was made for his-I+ and Zys-2+, with ade-a, arg-I and ser-I non-selected. The allele ratio
ser-1:ser-I' of 86: 11 serves to locate the new locus either between the loci of his-I and
arg-I (Position I) or between ade-2 and his-I (Position 11). These two possibilities are
considered in Table 4, and we see that the former (Position I) results in only one multiple
crossover recombinant, whereas the alternative (Position 11) requires 9 of the 11 ser-1'
recombinants to be multiples. Position I is therefore chosen.
serC
\
IiisD
thrA
Fig. 2 . Linkage map of Streptomyces rimosus. For explanation of locus symbols see Table I . The
order of the bracketed loci is unknown. Certain loci have not been ordered relative to loci covered
by broken lines. Loci are arbitrarily spaced at equal intervals.
The linkage map of Streptomyces rimosus. The procedure described in the preceding
paragraph was applied to several hundred crosses, most of which served to extend and
confirm the sequence of markers on the circular linkage map of the organism. The results
of such studies are summarized in Fig. 2. A unique sequence of most of the markers was
deduced from the crosses; only a few ambiguities remain. These are mainly due to lack of
strains bearing the particular combinations of markers that would have been required to
resolve particular ambiguities, or to a paucity of critical recombinant classes in certain
crosses.
13-2
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E. J. F R I E N D AND D. A. HOPWOOD
It should be emphasized that crosses of the kind performed in this study, while yielding
definitive information on marker sequence, are not adapted to the precise estimation of the
relative lengths of map intervals. This is because, when zygotes are incomplete diploids,
as in all bacterial systems so far investigated, including Streptomyces, crossovers leading
to selected progeny tend to be non-randomly arranged, being concentrated in the vicinity
of the selected markers (Hopwood, 19673). In Fig. 2 the loci are arbitrarily spaced equidistant from one another. No attempt has been made to study the partially diploid colonies
known as heteroclones which provided the basis for the estimation of map intervals in
Streptomyces coelicolor (Hopwood, I 966 b).
DISCUSSION
In this study it has been possible to obtain extensive information on the genetic map of
an industrial antibiotic-producing strain of Streptomyces rimosus by adapting techniques of
mutagenesis and genetic analysis already worked out for the non-antiobiotic-producing
S. coelicolor ~3 (2). Apart from relatively minor modifications in crossing technique, the
main operational difference from S. coelicolor is the frequent recovery of heterokaryons in
S. rimosus crosses. However, a judicious choice of marker selections, including at least
one non-leaky marker, allows most interference by heterokaryons to be avoided. In certain
cases the colonies picked from particular selective media include a proportion of heterokaryons (Table 2), but these can be recognized by their failure to replicate from the master
plates to the same selective medium; thus, although some effort may occasionally be wasted
in picking such colonies, they do not render genetic analysis ambiguous.
We have not attempted to select heteroclones in Streptomyces rimosus. However, AlaEeviC
(1969) has described recombinant colonies in another strain (ATCC 10970) of S. rimosus
which closely resemble the heteroclones of S. coelicolor (Hopwood, Sermonti & SpadaSermonti, 1963), so that, as pointed out by AlaEeviC (1969), the sexual cycle of S. rimosus
may be essentially similar to that of S. coelicolor (Hopwood, 19673, 1969).
Perhaps the most interesting feature of the present results is the striking resemblance of
the linkage map of our strain of Streptomyces rimosus to that of S. coelicolor A3(2). The
two maps are drawn concentricallyin Fig. 3. In this diagram the spacing of loci in S. rimosus
has been chosen to emphasize the similarity in gene sequence in the two species. No fewer
than 16 out of 24 loci in S. rimosus occur in a sequence corresponding to that of loci of the
same phenotypic class in S. coelicolor, with three further loci (gluA, guaA and thiB) which
are not precisely mapped in S. rimosus but which may correspond in position to the gluA,
guaA, thiB loci of S. coelicolor. We lack precise knowledge of the biosynthetic steps controlled by most of the loci, in either organism. This means that there could be ambiguity
in matching the two maps, since two alternative orientations are possible because of the
circular symmetry of the S. coelicolor linkage group (Hopwood, 1967a;see below). However,
the phenotypes of the mutations cys-3, his-I, his-a, met-r and met-2 in S. rimosus correspond
with those of mutations in cysl), hisA, hisl), metA and metB respectively in S. coelicolor
(Table 5). These findings lead to the relative orientation of the two maps indicated in Fig. 3.
There is no compelling evidence to indicate that the relative spacing of loci, in terms of
the probability of crossing-over, is the same in the two species. In particular the very long
'empty ' regions of the Streptomyces coelicoZor map, which may represent regions of unusually
high crossover incidence rather than long chromosomal regions devoid of known markers
(Hopwood, 1966a) may not occur in S. rimosus.
The correspondence of the genetic maps of the two organisms tends to emphasize the
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The linkage map of Streptomyces rimosus
I95
conservation of linkage relationships in prokaryotes. Hitherto, the only comparison of the
linkage maps of two bacteria that might be expected to be phylogenetically closely related
concerns Escherichia coli and Salmonella typhimurium. The linkage maps of these two enteric
bacteria are extremely similar (Sanderson, 1970;Taylor, 1970). The finding of a second
example of the same phenomenon, in Streptomyces coelicolor and S. rimosus, suggests a
remarkable evolutionary stability of gene arrangements on prokaryote genomes.
The data of others do not conflict with the conclusion of homology of the linkage maps
(serC)
Fig. 3. Comparison of the linkage maps of Streptomyces rimosus (outer circle) and S. coelicolor
A 3 ( 2 ) (inner circle). The S. rimosus map includes all known loci and is the map of Fig. z redrawn
with intervals adjusted to correspond with those of the S. coelicolor map. The S. coelicolor map,
from Hopwood (1967b),includesonly those loci for which a possible counterpart has been identified
in S. rimosus, plus two loci (lysA and nicB) which may be diametrically opposite lysB and nice of
S. rimosus (see text). For explanation of brackets and broken arcs, see caption to Fig. 2.
Table 5. Phenotypes of mutations in ‘homologous’ loci of Streptomyces coelicolor
A 3 ( 2 ) and S. rimosus
Mutation in
f
A
Streptomyces
coelicolor
cysD 18
hisA I
hisDj
metA5
metBq
-l
Streptomyces
rimosus
Phenotype
Grows
on
cysteine
or S,O, or S,O, or S 2 0 ,
cys-3
his-I
Grows on histidine but not histidinol
his-2
Grows on histidine or histidinol; accumulates
histidinol phosphate
Grows on methionine but not honiocysteine
met-I
Grows on methionine or homocysteine
met-z
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E. J. F R I E N D A N D D. A. HOPWOOD
196
of different streptomycetes. AlaEeviC (I 969), working with Streptomyces rimosus ATCC 19070,
used exclusively heteroclones in mapping studies and therefore concentrated on the study
of linkages over comparatively short regions of the map, since segregations in individual
heteroclones are too biased for confident mapping over long distances (Hopwood, 1969).
She presented data defining a linkage group consisting of four markers :pro-I-his-I-arg-Ityu-I. Although AlaEeviC did not make the comparison, this sequence could possibly have
corresponded to a sequence of loci in S. coelicolor A 3 ( 2 ) : proA-hisA-argA-aroA. In a later
summary of her mapping results, AlaEeviC (1970) stated that ‘the order of some markers
recalls the order of similar markers in S. coelicolor ~ 3 ( 2 ) ’but details have not so far been
given. In a recent study of S. bikiniensis var. zorbonensis (NRRL 3684, an antibiotic-producing
wild-type, Coats & Roeser (1971) mapped I I auxotrophic markers and streptomycinresistance by a procedure similar to the one described here. They made no comparisons
between their linkage map and that of S. coelicolor A 3 (2). However, as shown in Fig. 4, the
maps are by no means incompatible.
Fig. 4. Comparison of the linkage maps of Streptomyces bikiniensis var. zorbonensis (outer circle)
and S. coeZicoIor ~3(2)(inner circle). The S.bikiniensis map (from Coats & Roeser, 1971)includes
all known loci except those for antibiotic production. Map intervals have been drawn to show a
possible correspondenceof the two linkage maps; cysA, which lies between arg-3,4 and adeB, might
correspond with either cysE or cysD of S. coelicolor.
The present data on Streptomyces rimosus serve to strengthen the notion of circular
symmetry of the Streptomyces linkage map first proposed for S. coelicolor A3 (2) (Hopwood,
1967a).At that time, there were 10 examples of pairs of loci of the same phenotypic class
located diametrically. As we have just seen, 19 loci of S. rimosus may correspond in position
to comparable loci of S. coelicolor. The remaining five loci in S. rimosus (guaB, ZysB, nice,
panA and serC) at present have no counterpart in S. coelicolor. However, three of these
(guaB, lysB and nicC) occupy positions that may be diametrically opposite to loci of the
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The linkage map of Streptomyces rimosus
I97
same class in S. coelicolor (guaA, ZysA and nicB respectively). If we postulate that corre-
sponding loci to guaB, ZysB and nicC in S. coelicolor exist but remain to be identified, then
the S. rimosus loci add three further examples to the set of loci demonstrating circular
symmetry.
The letters assigned to the loci so far identified in Streptomyces rimosus have been chosen
not to conflict with the known loci of S. coelicolor A 3 (2). Thus, the first thi locus identified
in S. rimosus has been designated thiB, rather than thiA, since it is located in a position that
may correspond to that of thiB in S. coeticofor,while the second ser locus of S. rimosus has
been called serC since it is located in a region that does not (yet) contain a known ser locus
in S. coelicolor. This does not mean, of course, that loci with the same letter in the two
organisms are necessarily truly homologous ;nor is the converse necessarily true. However,
it seems preferable to adopt this convention rather than deliberately to designate by different
letters loci in the two organisms that might, from their map positions, be homologous.
We wish to thank the Directors of Pfizer Ltd for permission to publish these results. We
are also grateful to Mr D. Baldock and Dr A. D. Portno for their interest and encouragement and Mr H. G. Brett for skilled technical assistance.
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