was first demonstratedby Adams and Bradley .... rib-2. Riboflavin thi-1 .. Thiamine ura-1 .. Uracil str-2 .. (Streptomycin-resistant; resistant to concentrations up to ...
JOURNAL OF BACTERIOLOGY, Jan. 1975. Copyright 0 1975 American Society for Microbiology
Vol. 121, No. 1 Printed in U.SA.
Genetic Recombination in Nocardia mediterranei T. SCHUPP,' R. HUTTER,* AND D. A. HOPWOOD Department of Microbiology, Swiss Federal Institute of Technology, Zurich, Switzerland,* and Department of Genetics, John Innes Institute, Norwich, England Received for publication 23 September 1974
A system of genetic recombination in Nocardia mediterranei ATCC 13685 is described. This strain produces a mixture of several rifamycin antibiotics. Using haploid recombinant selection and analysis procedures similar to those applied to Streptomyces coelicolor A3(2), 14 auxotrophic markers and 1 streptomycin resistance marker were located on a circular linkage map. The linkage map of N. mediterranei seems to be similar to that of S. coelicolor A3(2).
Genetic recombination in the genus Nocardia was first demonstrated by Adams and Bradley (4), who showed that mixed cultures of mutants of an N. erythropolis strain and mutants of a second strain classified as N. canicruria yielded prototrophic recombinants. Later, the two strains were both classified as N. erythropolis (3). Since then, the system of mating between these two nocardias (both strains are self-sterile) has been studied further. The results of these studies were summarized recently (18). Mating was also found to occur between a third isolate, N. restrictus (also self-sterile), and the N. canicruria strain (32). N. mediterranei was isolated in 1957 from a soil sample collected near St. Raphael, France (24). N. mediterranei ATCC 13685 produces a mixture of several rifamycin antibiotics (30). Originally, N. mediterranei was classified as Streptomyces mediterranei (24). However, because of its invariably poor sporulation (23) and the tendency of the mycelium to fragment into short rodlike units (25), Thiemann et al. (31) reexamined the taxonomic position of the organism. Chemical analysis showed clearly that S. mediterranei has a cell wall typical of the genus Nocardia, and Thiemann et al. (31) therefore proposed its classification as Nocardia mediterranei. The work described here was performed primarily in the hope that knowledge of genetic recombination and of the linkage map of N. mediterranei may later be used in a genetic approach to the problem of improving, both qualitatively and quantitatively, rifamycin production by industrial strains of the organism, and to study the biosynthesis of this antibiotic. ' Present address: Research Laboratories of the Pharmaceutical Division of Ciba-Geigy Ltd., Basel, Switzerland.
128
MATERIALS AND METHODS Strains. All strains were derived from a culture of N. mediterranei (Margalith and Beretta) Thiemann et al. (31) (ATCC 13685). Media. Complete medium (CM) was used for maintenance of stock cultures, isolation of mutant strains, and crosses. CM agar was that of Pridham et al. (28) and contained (per liter): yeast extract (Oxoid Co., Newcastle, England), 4 g; malt extract (Oxoid), 10 g; glucose, 4 g; and agar No. 3 (Oxoid), 20 g (omitted for liquid CM). The pH was adjusted to 7.3 with 1 N KOH. For the growth of auxotrophic mutants requiring aromatic amino acids or uracil, CM agar was supplemented with tryptophan, tyrosine, and phenylalanine (50 lAg of each per ml) or uracil (10 Mg/ml). Minimal medium (MM), modified from the salt starch agar of Pridham et al. (28), contained (per liter): K2HPO4, 1 g; (NH4)2 SO4, 2 g; Mg SO4 .7H30, 1 g; NaCl, 1 g; CaCOs, 1 g; trace salt solution (FeSO4.7H20, 0.1%; MnCl2.4H20, 0.1%; ZnSO4. 7H20, 0.1%), 1 ml; agar No. 3 (Oxoid), 20 g; glucose, 10 g (autoclaved separately as a 50% [wt/voll solution and added to the medium before use). Supplements to MM agar were stored as sterile solutions and added to the medium after sterilization to give the following concentrations (per milliliter): amino acids, 50 Ag; purines and pyrimidines, 10 Ag; vitamins, 1 Mg; and streptomycin sulfate, 25 ug. MM agar was used (appropriately supplemented) for the detection and characterization of auxotrophic mutants and recombinants. All incubations were at 27 C. Isolation of mutants. Auxotrophic mutants were induced by exposing mycelial suspensions to ultraviolet light or N-methyl-N'-nitro-N-nitrosoguanidine. Mycelial suspensions for mutagenic treatment were prepared from liquid cultures (20 ml of liquid CM in 100-ml Erlenmeyer flasks inoculated with a loopful of a slant culture and grown for 4 days on a reciprocating shaker at 120 strokes per min). Samples (10 ml) of the cultures were then shaken vigorously for several minutes in small bottles containing quartz pebbles (2 to 3 mm in diameter) to break up large mycelial fragments, and the suspensions were filtered through cotton wool, centrifuged, and suspended in distilled
GENETIC RECOMBINATION IN N. MEDITERRANEI
VOL. 121, 1975
129
water. The resulting suspensions contained a viable count of 109 to 4 x 109 mycelial fragments per ml, A% of which were less than 5 jsm long. Ultraviolet irradiation was done by exposing 20 ml of a suspension of mycelial fragment (2 x 108/ml) in a petri dish to a dose of 600 to 800 ergs/mm2, giving a survival of 1.0 to 0.1%. Treatment with nitrosoguanidine was performed under the conditions recommended by Delic et al. (12), using 1 mg of nitrosoguanidine per ml in 0.05 M tris(hydroxymethyl)aminomethane-hydrochloride-maleic acid buffer (pH 9.0) and a 60- or 120-min incubation period. After mutagenic treatment, dilutions were plated on CM agar, giving about 100 survivors per plate. After 7 days of incubation, auxotrophic colonies were detected by replica plating on MM and CM agar and comparing replicas after 3 to 4 days. After purification by streaking, auxotrophic mutants were characterized by replication to MM agar supplemented with groups of growth factors (14). Ultraviolet irradiation yielded about 0.6% auxotrophs among survivors and nitrosoguanidine yielded about 0.2%. Spontaneously occurring streptomycin-resistant mutants were obtained by plating a suspension of 108 mycelial fragments on CM agar containing 25 Ag of streptomycin sulfate per ml. The wild-type strain is inhibited by streptomycin concentrations above 3
optical density of the suspensions, and spread (0.2 ml) on a large slant of CM agar (15 by 2 cm tube containing 15 ml of medium). After 5 days of incubation, a mycelial suspension was prepared as follows. The surface of the mixed culture was scraped with a loop into 10 ml of water. The resulting suspension was shaken vigorously with quartz pebbles, filtered through cotton wool, and washed by centrifugation and resuspension in water. This suspension was then plated (0.1 ml) at 10-1, 10-2, and 10-' dilutions on supplemented MM plates, each medium selective for one allele from each parent, and at 10-5 and 10' dilutions on appropriately supplemented MM plates to determine the titers of the parents. Random samples of colonies (100 to 200 per selection) appearing on the selective plates after 7 to 9 days were picked with sterile toothpicks and streaked in a regular pattern on master plates (30 or 50 per plate) containing the same selective medium. After 4 days of incubation, the master plates were replicated to a series of diagnostic plates of suitably supplemented MM agar to determine the phenotypes of recombinants. The diagnostic plates were examined after 3 to 5 days of incubation. Recombinant colonies arose with frequencies of 10-9 to 10-4 of the total population of
;Ig/ml.
RESULTS evidence of linkage. Linkage Preliminary was first studied by means of crosses in which the two parents, differing in four markers, each contained two selectable alleles. These four-factor crosses were analyzed by the procedure of Hopwood (15, 19). Table 2 shows the results of a cross between strains T2 (aro-1 ura-l+ leu-l+ str-2+) and T41 (aro-1+ ura-1 leu-1 str-2). Equal samples of mycelial fragments from the mixed culture of the two strains were plated on the four possible media that selected for one allele from each parent. A random sample of the colonies arising on each selective medium was
Genetic markers. The auxotrophic and streptomycin resistance markers used for the mapping experiments are listed in Table 1. The auxotrophic markers were satisfactorily stable, showing reversion frequences of 10-7 to 10-9. Crossing procedure. The method of crossing and analysis of recombinants were adapted from those used for Streptomyces coelicolor (16). Parental cultures, each derived from a purified clone, were grown for 4 days on slants of CM agar (5 ml per tube). To prepare mycelial suspensions, the surfaces of parental cultures were scraped with a loop into 5 ml of water, the suspensions were centrifuged, and the pellets were resuspended in water. The parental mycelial suspensions were mixed in an approximate 1:1 ratio based on
mycelial fragments.
TABLE 1. List of markers used for mapping experiments Requirement
Marker pur-5
..
aro-1
..
his-2
..
Isoleucine + valine Leucine Leucine
..
Lysine Methionine
.. ..
Pyridoxine Pyridoxine Riboflavin
rib-2
thi-1 ura-1 str-2
phenylalanine
Histidine
..
met-12
pdx-1 pdx-2
+
Cysteine
cys-3
ilv-1 leu-1 leu-2 Iys-1
Purines Tryptophan + tyrosine
.. ..
Thiamine Uracil
..
(Streptomycin-resistant; resistant to concentrations up to 200 gg/ml)
130
J. BACTERIOL.
SCHUPP, HOTWER, AND HOPWOOD
analyzed with respect to the two nonselected markers (column A, Table 2). In this way, 9 of the possible 16 genotypes of progeny could be recovered. These included one or two members of each pair of complementary genotypes, except the parental pair. The frequency of each
per unit volume (column B, Table 2) then calculated from its frequency in the classified sample and the total recombinant count on each medium. In addition to colonies that behaved ias pure haploid recombinants, there was a variable minority of mixed colonies
genotype
was
TAaL 2. Analysis of a four-factor cross: T2 aro-1 ura-l+ leu-1+ str-2+ x T41 aro-1+ ura-1 leu-1 str-2 Resultsa on selective media supplemented with: Genotypes of
Aromatic amino acids, uracil, and streptomycin
Leucine
Uracil
progeny aro ura leu
str
A
B
B
A
B
A
Aromatic amino acids, leucine, and streptomycin
+
3
15
30
18
+
r
0
0
1
1
-a+
r
84
412
x+
I+
Parents
+
+ +
.
1
16
30
488
0
0
17
3
296 + r
4
Total
r
9
44
14
160
784
90
450 296
29612 ~~~~~0 ~ 0 450
4 50
564 53 40313
1
Mixed C
1
17
+7 9
C
B
A
-|r + |+ +
-
Average of frequency each pair of complementary genotypes
8
10
160 796
30
21 0 503
54
90
1,460
82
500
1
1
Recombination frequencies between the six pairs of loci" aro-1
aro-I
-str-2
ura-1
leu-1
leu-1
ura-)
str-2 aro-1
ura-1 leu-1
~~17 ~~~4 ~~450 ~~296
4 450 503 1
4 19 503 296
17 503 296 1
450 19 296 1
958
822
817
766
c
D:
E:
17 4 19 1
41
2.5%
767
str-2
50%
a(A) Numbers of colonies of each genotype found in the random sample tested; column totals are the sample sizes. (B) Frequencies of the genotypes per unit volume; column totals are the colony counts on each medium. (f) Paris of complementary genotypes. I(C) Average values for the frequencies in columns B. (D) Relative recombination frequencies. (E) Absolute recombination frequencies.
VOL. 121, 1975
131
GENETIC RECOMBINATION IN N. MEDITERRANEI
each containing more than one genotype. These mixed colonies were normally indistinguishable from the pure clones on the original plates but showed a mixture of two or more phenotypes on replicas from the master plates. The frequencies of the same genotypes on different media were in fairly good agreement and, in the two cases where complementary genotypes could be recovered, these had similar frequencies (Table 2). It can be assumed, therefore, that good reciprocity also occurs in the other cases, i.e., where only one genotype of a complementary pair is recovered (15). This allows us to calculate the frequencies of the seven pairs of complementary recombinant genotypes by taking the average of the values in column B of the genotypes (column C, Table 2). From the average frequencies of the seven pairs of complementary genotypes, the relative recombination frequencies between the six pairs of loci can be calculated (Table 2, bottom). The recombination frequencies between members of five pairs of loci are high and nearly identical. By setting the average of these values at 50% recombination, the recombination frequency of the remaining pair, aro-1 - ura-1, can be calculated as 2.5%. To confirm the result of the cross discussed above, two recombinants, T47 (ura-1 aro-l leu-1 + str-2+) and T48 (ura-1 + aro-1 leu-1 str-2), were isolated and used as parents in a cross with the same four markers in a different coupling arrangement. This cross confirmed the close linkage of aro-1 and ura-1 and the absence of detectable linkage between the other five pairs of loci (data not shown). We can thus conclude that aro-1 and ura-1 are rather closely linked and leu-1 and str-2 are not close to them. This first indication of consistent linkage confirmed the qualitative conclusion from the finding of recombinants inheriting auxotrophic markers, which are unlikely to be dominant, from both parents (for example aro ura+ leu str+ in Table 2) that we are dealing with a process of true recombination in which substitution of segments of genetic material occurs. Ordering of a set of four loci. In several crosses that were analyzed as described above, no close linkage between other loci was found. To obtain more information from these crosses, 2 x 2 tables were constructed to analyze the segregation of the two pairs of nonselected alleles on each of the four selective media (17, 19). The following result is expected if the four loci lie on a circular linkage map. When the two nonselected loci are separated by the selected loci, segregation of the nonselected loci should
be nearly independent; when the selected loci are adjacent, segregation of the nonselected loci should depart very markedly from independence, with one genotype much rarer than the others, representing the multiple crossover class. Table 3 shows the 2 x 2 data for a cross between strains T55 (leu-1 ura-1+ lys-l+ str-2+) and T73 (leu-1+ ura-1 lys-1 str-2). It is apparent TABLE 3. Segregation of nonselected alleles in a cross: T55 leu-1 ura-l+ lys-1+ str-2+ x T73 leu-1+ ura-i lys-1 str-2 on the four selective mediaa Selection
Iys-1+/
ura+
ura-
25 (1,2)
(2,4)c
46 (1,3)
(3,4)
Selection ura-l1+ Ieu-I+
ys+
lys
str+
33 (1,2)
(1,2,3,4)
Ieu-l+
str+
strf
str'
Selection lys-l+/
22b
4c+
90
1+ str-2
ura-I
2
46
83
(1,3)
(1,4)
ura+
urac
sr2
ure-1
str-2
leu+ leu-
Selection ura-1+/ str-2
leu+ leu
31
91
(1,3)
(3,4)
36 (2,3)
1 (1,2,3,4)
Iys+
lys-
35
63
(1,3)
(1,4)
65 (2,3)
32 (2,4)
4+
+
+
str -2
3Y+
+
+
str-2
(A) Selected alleles. b Numbers of each genotype in the random sample tested. cCrossing-over in intervals numbered as in the a
diagrams.
132
SCHUPP, H10TER, AND HOPWOOD
that selections Iys-1+/leu-1+ and ura-1 +Istr-2 result in a nearly independent segregation of the nonselected loci, whereas selections ura-1+/Ieu1 and lys-1+/str-2 result in a segregation departing markedly from independence. This result indicates circular linkage of the four loci in the order shown in Table 3. By performing two further crosses with -the same four markers in other coupling arrangements, the consistency of the gene sequence derived from the cross discussed above (Table 3) was tested (Table 4). All three crosses indicated the same sequence for the four loci, thus leaving no doubt about its validity. Multifactor crosses. After the analysis of four-factor crosses had revealed the sequence of four loci on a circular linkage map, the position of further loci was deduced by five- and six-factor crosses. With mutant and recombinant strains, the crosses were chosen in such a way that the position of only one marker in each cross was unknown. Table 5 shows the results of a six-factor cross in which the position of cys-3 was unknown. The rationale of the analysis,
J. BACTERIOL.
TABLE 4. Mode of segregation of the nonselected alleles in two crosses: T87 lys-l ura-1 + leu-1 + str-2+ x T41 lys-l+ ura-1 leu-1 str-2 and T5 lys-l + ura-l leu-l + str-2+ x T86 lys-l ura-l+ leu-1 str-2 Segregation of the nonselected alleles
Selected alleles
Cross: T87 x T41
lys-l+/ura-l+ lys-l+/leu-l+ ura-l+/Ieu-l leu-l+/str-2 lys-l+/str-2 ura-l+/str-2
Cross: T5 x T86
Dependent
Dependent
Independent
Dependent Dependent Dependent
Dependent
Independent
UM1+
Marker arrange-
,"
+
+ ;' + u-,
ments
deduced Or~~~-2
s1~~~~2
'2Marker arrangements have to be in agreement with the fact that selection for nonadjacent loci results in independent segregation, whereas selection for adjacent loci results in a segregation departing markedly from independence.
TABLE 5. Location of cys-3 determined by analysis of the cross T69 ura-1 cys-3 Iys-1+ aro-1+ str-2+ x T76 ura-1+ cys-3+ lys-l aro-1 leu-1 str-2a
(A) Selection aro-l+/str-2
(B) Selection lys-l+/ura-l+ Crossover in intervals
Crossover in intervals
No. GenotypeP
ura ura ura ura ura ura ura +
+ + cys cys cys cys + +
lys leu str lys + str + leu str + + str lys + str lys leu str + leu str lys leu str
62 20 14 55 3 1 1
3
Genotype
Position 1
Position 2
1,5 2,5 1,3 2,3 2,4 1,4 1,3,4,5 1,6
1,5 2,5 1,2,3,4
Poition 1
1,2,3,5 1,4 1,6
+ aro + +
159
e1 lea-i
2 +
str-2 159
3,5 2,5
29 2 5
3,4 2,4 1,4 5,6 1,2,3,5 1,2,3,4
3 2
+ aro + str
1
3,5 2,5 1,5 4,5 2,3,4,5 1,3,4,5 5,6 1,2,3,5 1,2,4,5
1,5
PosItion 4
3
~~~~~~~~~~~~~~~~~~~~~127 124 +~~~~~~ o 0 *j-i 6
78
127
+ 6
81
+ ly. 8970 ~~~~~~~~ 2 eOU-i 9730
0
47 16 22
+ +
Positon
Position 4
124
+ ~~~~~~~~~urUra-iur3 79159 1-3
are-i 78
Position 3
cys aro + str
cys +
Pwetbn 2
ar-
lyi
cys aro leu str cys aro leu + cys aro + + + aro leu str + aro leu +
3,4 3,5
3 3 0 + 0+
cys-
J No.
-i
lye-i
+
+
127
33
cys -3 4
0
~~~+3 str- 2
159
48
+
str-2 79
eou-i 94
lysi
127 +
+
033
IeU1 94
90ce24+ et r7
'The frequency of each allele, calculated from the genotype frequencies, is indicated in the diagrams. The possible positions of cys-3 are chosen so that the allele frequency at the cys-3 locus falls within the gradient of allele frequencies at the other loci. This gives two possible positions for cys-3 for each of the two selections. Position 1 is equivalent to position 3 and is chosen as correct (see text). Triangles indicate selected alleles. 'Wild-type alleles omitted.
133
GENETIC RECOMBINATION IN N. MEDITERRANEI
VOL. 121, 1975
which is similar to that used for S. coelicolor (16), is as follows. The suspension from the cross is plated Qn two selective media, each selecting for one allele from each parent. For each selection, the allele frequencies at all loci are calculated from the numbers of each genotype in the sample analyzed. Since the allele frequencies must form a continuous gradient in each arc, falling from the maximum at the selected allele to zero at the counter-selected allele (16), two possible positions of cys-3, one in each arc, are indicated for each selection (Table 5). Considering the two possible positions for each of the two selections, we see that the location of cys-3 between lys-1 and ura-1 is compatible with the results of both selections (position 1 = position 3), whereas the other two locations, positions 2 and 4, of cys-3 are mutually exclusive. Moreover, positions 1 and 3 require fewer multiple crossovers than positions 2 and 4 to explain the genotypes found (Table 5). The location of cys-3 between Iys-i and ura-1 is therefore chosen as correct.
Linkage map of N. mediterranei. By analyzing many five- and six-factor crosses, markers were progressively added to the linkage map, and the positions of loci already mapped were confirmed, e.g., the close linkage of ura-1 and aro-1. The results of these crosses indicated a unique sequence of 15 loci on a circular linkage map (Fig. 1). Since mapping of the kind performed in this study gives definite information only on marker sequence, but not on the relative lengths of map intervals (see below), all loci cys
-
3
pdx-2
except rib-2, ura-1 and aro-1 are arbitrarily spaced at equal intervals in Fig. 1. The loci rib-2/ura-1 and ura-1/aro-1 are closely linked to each other (for ura-llaro-1 see Table 2) and are therefore drawn closer together. The fact that those crossovers that lead to selected progeny are not randomly distributed is the reason the mapping procedure does not give exact information on internal lengths. The segregation of nonselected alleles in four-factor crosses gave the first indication of a mutual influence of crossovers in zygotes of N. mediterranei. In Table 3 we see that the two "independent" segregations (selections Iys-1+1 leu-l+ and ura-1+/str-2) are disturbed, since the values for crossovers in nonadjacent intervals (intervals 1,3 and 2,4) are too low. This kind of disturbance was found in all four-factor crosses and indicated a consistent excess of associated crossing-over in adjacent map intervals; in other words, negative interference was found. The same effect was found in multifactor crosses. For example, the data for selection aro-1+/str-2 of the six-factor cross shown in Table 5 are analyzed in Table 6 and demonstrate a correlation of crossing-over in nearby map intervals. Crossing-over in interval 1 is positively correlated with crossing-over in intervals 5 or 6, whereas crossing-over in interval 2 tends to be associated with exchanges in intervals 3 or 4 (see crossover ratios in the lower part of Table 6). As pointed out by Hopwood (16), the most likely explanation for negative interference over fairly long intervals, as found in these crosses, is the formation of incompletely diploid zygotes (merozygotes).
DISCUSSION This study has shown that genetic recombination occurs when two marked strains of N. mediterranei ATCC 13685 are grown in mixed culture. In contrast to N. erythropolis, N. canicruria, and N. restrictus (1, 32), which are
met-12
thi-1
TABLE 6. Mutual influence of two crossovers: selection aro-l+/str-2" pur-5
No. of crossovers in interval:
Crossovers in interval: str- 2
ilv-l
FIG. 1. Linkage map of Nocardia mediterranei ATCC 13685. The loci, except rib-2, ura-1, and aro-1, are arbitrarily spaced at equal intervals. rib-2, ura-1, aro-1, which are closely linked, are separated by shorter intervals. For explanation of locus symbols see Table 1.
1 2
Crossover ratio: interval 1/ interval 2
4
3
14 55
0.25
aData from Table 5.
1 3
0.33
5 62 20 3.1
6 3 0 >3
134
SCHUPP, HO1TTER, AND HOPWOOD
self-sterile, N. mediterranei is self-fertile, yielding 1 to 10 recombinants per 10' cells of parental genotype under our conditions. The majority of the recombinants were interpretable as stable haploid genotypes arising by substitution of a segment of genetic material from one parent by a homologous segment from the other. Because recombinants differing from both parents by more than one allele occurred fairly frequently, it can be concluded that rather long fragments of the chromosome are transferred between strains. This makes it most unlikely that a process of transformation or transduction is responsible for the transfer of genetic material; recombination in N. mediterranei, as in other mesophilic actinomycetes, presumably results from a process of "conjugation" (18). A method of linkage analysis based on haploid recombinant selection, similar to that used for S. coelicolor (16) and S. rimosus (13), proved effective in N. mediterranei and led to the construction of the beginnings of a linkage map for this strain, bearing all the markers so far investigated. Circularity of the map of N. mediterranei was apparent from the results of the first four-factor crosses and was later confirmed by the results of further crosses involving progressively more markers. A comparison of the linkage map of N. mediterranei with that of N. erythropolis is premature since there are not enough presumptively homologous loci mapped in both strains; in N. erythropolis many of the markers involved drug resistance (8), whereas in N. mediterranei nearly all were auxotrophic. On the other hand, a comparison of the linkage maps of S. coelicolor and N. mediterranei (Fig. 2) shows what appears to be a more than random correspondence in the sequence of markers that might be homologous. This very interesting finding, if confirmed by the location of further markers, would indicate that the similarity of the linkage maps found in the genus Streptomyces, involving S. coelicolor (20), S. rimosus (5, 13), S. bikiniensis (11), S. olivaceus (26), and S. glaucescens (6), extends over the generic boundary, as defined by cell wall composition, into the related genus Nocardia. The features of the crosses of N. mediterranei are very similar to those of the streptomycetes. Incompletely diploid zygotes are formed and each parent can function as the donor of the fragment of genetic material (as in crosses between strains of S. coelicolor of the same fertility type [20]). The main difference is the occurrence of a variable but significant proportion of recombinant colonies that show a mix-
J. BACTE:RIOL.
FIG. 2. Comparison of the linkage maps of Nocardia mediterranei (outer circle) and Streptomyces coelicolor A3(2) (inner circle). All 15 markers of N. mediterranei are included, but only possibly homologous loci of S. coelicolor (20). The map intervals between the loci of N. mediterranei were adjusted to correspond with those of the S. coelicolor map. The order of bracketed loci is not known.
ture of phenotypes; these may be due to the frequently multinucleate nature of the fragments of mycelium that serve as plating units. The fertility of crosses was fairly constant, although their polarity, in terms of the donation of chromosome fragments by one or other parent, was variable; however, this appeared to be a function of crossing conditions rather than being characteristic of particular strains or markers. The conclusion can therefore be drawn that no fertility types, such as have been found in S. coelicolor (33, 34) and N. erythropolis (8, 9), have so far segregated among our mutant and recombinant strains of N. mediterranei. It is not possible to judge to what extent the apparent differences in the genetic systems of N. mediterranei and N. erythropolis are significant. This is because of the different systems of selection and analysis used in the crosses and the added complication of self-sterility of the strains of N. erythropolis, which makes it necessary to use two strains with incompletely homologous genomes (10). This system was originally analyzed on the assumption of complete zygote formation and a linear linkage group (7);
VOL. 121, 1975
GENETIC RECOMBINATION IN N. MEDITERRANEI
however, later work (2) threw some doubt on the assumption of completely diploid zygotes, and it may be that the mating process in N. erythropolis will turn out to be basically similar to that of N. mediterranei and the streptomycetes.
The finding of a readily controlled and reproducible system of genetic recombination in N. mediterranei is significant in view of the industrial importance of this organism as the producer of the rifamycin class of antibiotics (27, 35). The goals of strain improvement in this organism will doubtless involve not only increases in the yield of rifamycins, but also changes in the relative proportions of the various members of the class and possibly the evolution of new rifamycins. In addition to the classical procedures of mutation and selection, recombination is a potentially useful tool in such programs, particularly if recombination between strains of diverse origin becomes a possibility. The development of such wide crossing procedures may depend on further insight into the control of gene transfer in N. mediterranei and the possible involvement in it of plasmids, as in the genus Streptomyces (21, 22, 29, 33). ACKNOWLEDGMENT We gratefully acknowledge financial support for this work from Ciba-Geigy AG, Basel, Switzerland. LITERATURE CITED 1. Adams, J. N. 1964. Recombination between Nocardia erythropolis and Nocardia canicruria. J. Bacteriol. 88:865-876. 2. Adams, J. N. 1968. Partial exclusion of the Nocardia erythropolis chromosome in nocardial recombinants. J. Bacteriol. 96:1750-1759. 3. Adams, M. M., J. N. Adams, and G. H. Brownell. 1970. The identification of Jensenia canicruria Bisset and Moore as a mating type of Nocardia erythropolis (Gray and Thornton) Waksman and Henrici. Int. J. Syst. Bacteriol. 20:133-147. 4. Adams, J. N., and S. G. Bradley. 1963. Recombination events in the bacterial genus Nocardia. Science 140:1392-1394. 5. Alacevic, M., M. Strasek-Vesligaj, and G. Sermonti. 1973. The circular linkage map of Streptomyces rimosus. J. Gen. Microbiol. 77:173-185. 6. Baumann, R., R. Hiitter, and D. A. Hopwood. 1974. Genetic analysis of a melanin-producing streptomycete, Streptomyces glaucescens. J. Gen. Microbiol. 81:463-474. 7. Brownell, G. H., and J. N. Adams. 1967. Linkage and segregation of unselected markers in matings of Nocardia erythropolis with Nocardia canicruria. J. Bacteriol. 94:650-659. 8. Brownell, G. H., and J. N. Adams. 1968. Linkage and segregation of a mating type specific phage and resistance characters in nocardial recombinants. Genetics 60:437-448. 9. Brownell, G. H., and K. L. Kelly. 1969. Inheritance of
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