Department of Microbiology, Michigan State University, East Lansing, MI 48824-1101, USA1
Author for correspondence: Wendy Champness. Tel: +1 517 353 9770. Fax: +1 517 353 8957. e-mail: champnes{at}pilot.msu.edu
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ABSTRACT |
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Keywords: Streptomyces coelicolor, antibiotic biosynthesis and regulation, two-component system, genetic suppression
Abbreviations: Act, actinorhodin; CDA, calcium-dependent antibiotic; Mmy, methylenomycin; NF, normal fertility; Red, undecylprodigiosin; UF, ultra fertility
a Present address: TerraGen Diversity Inc., Vancouver, British Columbia, Canada.
b Present address:Department of Molecular Biology and Microbiology, Tufts University, Boston, MA 02111, USA.
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INTRODUCTION |
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In S. coelicolor, coupling of antibiotic production to sporulation is easily observed visually because two of the four antibiotics produced are pigments. Actinorhodin (Act) is blue or red, depending on pH, and undecylprodigiosin (Red) is red or yellow, its colour also varying with pH. Early genetic studies, using colour phenotypes to define the genes required for Act and Red synthesis (Rudd & Hopwood, 1979 , 1980
), showed that these genes comprised two clusters, act and red, respectively. The other two S. coelicolor antibiotics, methylenomycin (Mmy) and calcium-dependent antibiotic (CDA), have also been genetically characterized (Chater & Bruton, 1985
; Chong et al., 1998
).
Molecular characterization of the antibiotic gene clusters has included cloning, sequencing, definition of transcripts and identification of cluster-specific regulatory genes. The act cluster consists of at least six transcripts and 20 ORFs. The act- and red-specific regulators [ActII-ORF4 (Fernández-Moreno et al., 1991 ; Gramajo et al., 1993
) and RedD (Takano et al., 1992
; Narva & Feitelson, 1990
)] are the best understood. They are both activators with considerable amino acid sequence similarity and are members of the growing SARP family (streptomycete antibiotic regulatory protein) of transcriptional regulators (Wietzorrek & Bibb, 1997
).
Growth-phase-regulated expression of these antibiotic-specific regulators is an important component of antibiotic regulation (Bibb, 1996 ). Approaches to defining the genetic mechanisms responsible (reviewed by Champness, 1999a
) have included analysis of cloned genes that enhance antibiotic production, evaluation of the roles of metabolic regulators such as relA through gene disruption (e.g. Chakraburtty & Bibb, 1997
), and screening for mutations that perturb antibiotic regulation (reviewed by Champness, 1999b
; Chater & Bibb, 1997
).
In one genetic analysis of S. coelicolor antibiotic regulation, some mutations that blocked production of all four known antibiotics (Abs- phenotype, for antibiotic synthesis deficient) were found to define the absA locus (Adamidis et al., 1990 ). This locus was subsequently shown to encode a two-component signal transduction system (Brian et al., 1996
) composed of AbsA1, a homologue of orthodox histidine kinase sensor-transmitter proteins, and AbsA2, a putative DNA-binding response regulator with the amino acid sequences conserved in orthodox response-regulator proteins (Parkinson, 1995
; Stock et al., 1995
). The absA locus regulates transcription of the actII-ORF4 and redD pathway-specific activators (Aceti & Champness, 1998
).
The absA mutants isolated on the basis of their Abs- phenotype were shown, by marker rescue experiments (Brian et al., 1996 ), to carry mutations of the absA1 gene. In contrast, disruptions of the locus resulted in a phenotype of early onset enhanced antibiotic production. This phenotype was associated with disruption of both absA1 and absA2 or of absA2 alone. Hence, we proposed (Brian et al., 1996
) that the absA locus exerts a negative regulatory effect on antibiotic production and that the Abs- absA strains are mutationally locked into the negatively acting state.
Two Abs- absA mutant strains, C542 and C577, were phenotypically characterized in detail. One shared trait is spontaneous apparent reversion. We were interested in determining the genetic events underlying formation of the apparent revertants because if these strains contained suppressive mutations, the suppressors might provide insights into the mechanisms of AbsA1/AbsA2 function or identify other genetic elements functioning in the absA pathway.
Here, we present a genetic analysis of the absA pseudoreversion phenomenon. We first define the absA1 mutational alterations in Abs- strains and then demonstrate that a set of pseudorevertant strains carries second-site suppressive mutations, sab (for suppressor of absA). We use plasmid-mediated and protoplast fusion mapping techniques to locate sab alleles and then use a specialized transducing phage to show that some sab mutations map to the absA locus. By sequence analysis, we define the mutational alterations to the AbsA1 and AbsA2 proteins.
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METHODS |
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Isolation of sab strains.
Pigmented strains were cultured from spore to spore a minimum of three times and then acid-treated (0·01 M HCl, pH 2·0, for 10 min), followed by neutralization with an equal volume of 0·01 M NaOH to kill any contaminating mycelia that might carry multiple genomes, and therefore carry a sab+ allele.
sab strains were isolated from spontaneously arising pigmented sectors in plates of strains C542 and C577. To ensure that independent sab mutations were isolated, Abs- spores were plated and the resulting individual colonies were cultured through several rounds of streaking before pigmented sectors were selected. Each sab strain was isolated from a different Abs- progenitor colony.
Genetic mapping techniques.
Crosses and data analysis were carried out as described previously (Champness, 1988 ). For plasmid-mediated mating, chromosomal recombination was mediated primarily by plasmid SCP1 integrated at 9 oclock on the genetic map to give the NF type (Hopwood & Chater, 1974
). In an NFxSCP1- cross (also referred to as NFxUF), close to 100% of the progeny will be NF (Hopwood et al., 1969
). Several phenotypes are associated with the NF state: (1) NF strains are Aga- and fail to sink into the agar whereas Aga+ strains sink (Hodgson & Chater, 1981
); and (2) NF strains (including Abs- strains) are usually Mmy-resistant due to the presence of the mmy gene cluster on SCP1 (Kirby & Hopwood, 1977
).
The frequencies of markers donated by the NF parent decrease in both clockwise and anticlockwise directions from the SCP1 insertion region at 9 oclock (Hopwood et al., 1985 ). If the SCP1- parent is J1501 (Table 1
), the hisA1 and strA1 alleles will be present in >95% of spore progeny plated nonselectively.
Protoplast fusions.
PEG-mediated protoplast fusions were performed as described by Hopwood et al. (1985 ). Spores from regenerated protoplasts were plated on selective media.
Actinophage transductions.
Transductions were carried out essentially as described by Piret & Chater (1985 ). Briefly, phage suspensions were mixed with spores of the recipient strain on R5 agar. After colonies had sporulated, they were replicated to R5 agar containing thiostrepton (50 µg ml-1) to select lysogens. Phage released from lysogens were isolated as plaques formed in S. lividans soft agar overlays poured on to 1-d-old lysogenic cultures streaked on nutrient agar. Nutrient agar and phage propagation techniques were those described by Hopwood et al. (1985
).
DNA sequencing of absA1 and sab mutant alleles.
The absA1 alleles of Abs- strains C542 and C577 and the presumptive sab mutations were sequenced from nested PCR-generated fragments. The absA1 gene (Brian et al., 1996 ) was amplified from primers A15a (5'-CATCTGGCGGCGATCGGCAACGACCG-3'; nt 5471 upstream of the absA1 translation start site) and A13e (5'-CGCGAATCATCCGATCGTTCCCTGGTG-3'; nt 228 downstream of the absA1 stop codon). The PCR reaction mixtures (100 µl) contained 200 ng genomic DNA template, 0·3 µmol of each primer and 2·5 U Pfu polymerase (Stratagene). Template denaturation for 5 min at 95 °C was followed by 30 cycles of 95 °C for 1 min, 65 °C for 1·5 min and 72 °C for 1 min, and a final extension at 72 °C for 10 min. The 1·8 kb absA1 product was purified by agarose gel electrophoresis, digested with BamHI and XhoI, and cloned in pBluescript II SK(+) (Stratagene). Because previous genetic mapping had localized the absA1 mutations to the 1·45 kb XhoIBamHI segment of absA1 (Brian et al., 1996
), sequence analysis was restricted to this region. The end regions were sequenced from standard vector primers, while the internal region was sequenced from primer A15c (5'-CGCTACATCGCCGACCAG-3'; nt 546563 of absA1) (Iowa State University DNA Sequencing Facility, Ames, IA, USA). The absA1 XhoI/BamHI regions of sab mutants C542S3 and C542S11 were sequenced in the same manner. In addition, the N- and C-termini of these alleles were confirmed by direct sequencing of the PCR product using primers A15a and A13e.
The sab absA2 sequences were generated with primers A25a (5'-CAGGGAAGGATCCGATGATTCGCG-3'; nt -18 to +10 of absA2) and A23a (5'-GGGCGACCGGCGGATCCGCCTC-3'; nt 80101 downstream of the absA2 stop codon), both of which contain BamHI restriction sites. Each reaction mixture (100 µl) contained 200 ng genomic DNA template, 0·3 µmol of each primer and 2·5 U Taq polymerase (Gibco-BRL). Samples were denatured at 95 °C for 5 min followed by 30 cycles of 95 °C for 1 min, 65 °C for 1·5 min and 72 °C for 1 min, and a final extension at 72 °C for 10 min. The 0·8 kb product was purified on a Qiagen PCR purification column, digested with BamHI, purified again and cloned in pBluescript II SK(+). Convergent overlapping sequences were obtained from standard primers contained on the vector. All mutations were verified by sequencing at least two independently generated PCR products. Sequences were analysed using the Wisconsin GCG package.
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RESULTS AND DISCUSSION |
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While culturing Abs- strains, we observed that both C542 and C577 appeared to revert spontaneously, acquiring pigmented sectors that were stably culturable. These could be sorted into two phenotypic groups: Type I produced a colony very similar to that of the J1501 parent in both pigmentation and morphology; Type II produced a colony that was very deeply pigmented and also was altered in morphology, with a crenulated surface and few aerial hyphae. The crenulation was especially noticeable in colonies grown at low density. Both C542 and C577 produced the two types of pigmented strains.
The frequency of phenotypic reversion in C542 and C577, although not quantified, appeared to be much higher than for other S. coelicolor non-antibiotic-producing mutants, including absB, bldB, bldG, bldH (W. Champness, unpublished) and bldA (Guthrie et al., 1998 ; W. Champness, unpublished). By 2 weeks, many absA mutant colonies contained a pigmented sector, whereas cultures of the latter set of mutants typically contained only a few pigmented sectors per plate. Moreover, the pigmented sectors that arose spontaneously in absB and bld mutants were mostly of the Cms (chloramphenicol-sensitive), scarlet type (Sermonti et al., 1977
), a pigmentation phenotype associated with DNA rearrangements (Dyson & Schrempf, 1987
) but otherwise poorly understood. In contrast, the apparent revertant strains isolated from C542 and C577 were as Cmr as J1501 (data not shown).
Four Type I and seven Type II pigmented strains (Table 1), all independently isolated, were evaluated in plate-grown cultures for production of the S. coelicolor antibiotics Act and Red. Each strain produced both antibiotics. All strains except C577S20 and C577S25 produced the CDA. An SCP1NF strain, C5422S1, produced Mmy. A notable characteristic of the Type II strains was that they produced Act (especially
-actinorhodin; Bystrykh et al., 1996
) and Red about 1 d earlier than did J1501. Below we refer to this Type II phenotype as Pha (precocious hyperproduction of antibiotics).
Genetic evidence for second-site suppressor mutations
For initial genetic characterization, Pha Type II strain C542S11 was chosen; spores were purified by acid treatment (see Methods) designed to kill any mycelial (multi-genomic) fragments and ensure genetic homogeneity.
To distinguish whether restoration of antibiotic production was due to a reverting mutation or to a second-site suppressing mutation, strain C542S11 was crossed to a standard mapping strain, J650, and progeny were examined for the Abs- phenotype, occurrence of which would indicate that C542S11 carried both the absA1-542 mutant site and a suppressive mutant site, sab (for suppressor of abs). Abs- colonies were obtained at a frequency of about one in 5000 among Strr progeny from the cross. In contrast, Abs- colonies could not be found in a screen of approximately 200000 colonies plated from C542S11 spores. Together, these results suggested that the Abs- colonies arising from the C542S11xJ650 cross were likely absA1-542 sab+ recombinants. To verify that the recombinants Abs- phenotypes were due to absA mutant alleles, several Abs- strains from the C542S11xJ650 cross were characterized in detail: their phenotypes were identical to that of C542, and genetic crosses demonstrated that each carried a mutant absA allele (data not shown).
Genetic mapping of sab suppressor mutations
The recovery of Abs- recombinants from the C542S11xJ650 cross was strong evidence for the existence of a sab mutant allele in C542S11, and the very low frequency of Abs- recombinants from the cross suggested that the sab mutation site was very close to the absA1-542 mutant site. To map the putative sab locus, additional crosses were performed.
(i) Evidence consistent with a sab locus in the 10 oclock region, where absA is also located, came from crossing strain C5422S1 with C542 (Table 1). The results are shown in Fig. 1(a
). C5422S1 was a Type II sab mutant isolated in a genetic background (C5422 in Table 1
) useful for mapping in the vicinity of absA; this strain had the genotype sab absA1-542 cysA15 proA1 argA1 strA1 SCP1NF. Strain C5422S1 carried SCP1 integrated in the 9 oclock region, whereas C542 lacked SCP1. The fertility characteristics of this type of cross, referred to as NFxUF (Hopwood & Chater, 1974
), result in a curious bias in the alleles that are recovered in progeny spores, even in the absence of selection: all progeny inherit the integrated SCP1 plasmid but almost all carry genomic markers from the SCP1- parent for the region extending clockwise from approximately 11 oclock to about 7 oclock. In Fig. 1(a
), this effect can be observed for the hisA and argA loci: 66/68 spores were hisA1 arg+. Because 34/68 spores were Pgm+ (e.g. sab mutant) and the other 34 were Pgm- (e.g. sab+), the sab locus appeared to be close to SCP1NF, either between SCP1NF and cysA or between SCP1NF and uraA. Four additional sab mutants isolated from strain C5422 gave similar results.
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(iii) Crosses like that shown in Fig. 2(a) mapped additional sab mutations in some of the sab strains listed in Table 1
. Before being used in crosses, each strain was purified as described in Methods, and its phenotypic stability was evaluated: no Abs- colonies were found among approximately 200000 colonies screened by microscopic analysis of plates containing several thousand colonies each.
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A sab locus maps close to the absA locus
As tabulated in Fig. 2(a), crosses with the pseudorevertant strains all yielded a low percentage of Abs- progeny. Difficulty in detecting the infrequent absA1 sab+ recombinants precluded making significant distinctions between the locations of the various sab mutations. Because protoplast fusions are potentially capable of increasing the accuracy of recombination analysis due to the higher recombination frequency in such crosses (Hopwood et al., 1977
), we explored this procedure. As in NFxUF plasmid-mediated crosses, SCP1NFxUF protoplast fusions typically result in acquisition of SCP1NF in all progeny (Hopwood et al., 1977
). However, in contrast to NFxUF matings, the frequency of unselected parental markers at other loci is not biased toward either parental genotype.
Protoplasts of sab strains C542S3 or C542S9 were mixed with J650 protoplasts and recombinant progeny were analysed. Two classes of recombinants were selected: Strr Ura+ and Strr His+. Among these, Abs- colonies were found at higher frequencies than in plasmid-mediated crosses, as predicted (e.g. 0·6% vs 0·0025% for the strA1 his+ SCP1NF selection with strain C542S3). Mock protoplast fusions using only the individual sab strains, without J650, yielded no Abs- colonies.
The recombination data for C542S9 suggested the relative order for the sab and absA alleles shown in Fig. 2(b). This order would be consistent with the higher frequency of Abs- recombinants found in the Strr Ura+ selection compared to the Strr His+ selection: in the former, two recombination events, in intervals 1 and 4, could produce Abs- progeny whereas in the latter, three recombination events, in intervals 1 or 2, 4 and 5, would be necessary. The data for C542S3, however, showed no significant difference in recombinant frequencies for the two selections. This observation is discussed further below.
The observed frequencies of recombination between the sab and absA1 alleles did not provide sufficient information to accurately determine their physical distances, and therefore to suggest whether sab alleles were intragenic or extragenic. Very little data correlating recombination frequencies with physical distance are available for streptomycetes. To make one such comparison, we measured the recombination frequency between mutations in the act gene cluster, which has been completely cloned and sequenced. Using protoplast fusions of strains TK16 and TK18 (Table 1), we obtained 3·7% act+ recombinants between the act-141 and act-117 mutations, which affect the actIII and actIV loci, respectively, and so the mutations would be approximately 5 kb apart in the act cluster (Malpartida & Hopwood, 1986
). Considering these data with respect to the sab-absA1 recombination frequencies suggested that the sab mutant sites could lie within the same gene as the absA1-542 mutation or very close by.
Isolation of sab alleles on specialized transducing phage
The mapping results for the sab mutations suggested that they might be close enough to absA to lie within the region carried by an 11 kb absA1-542-complementing clone, pWC3151, that we had obtained in other experiments (Brian et al., 1996 ). Accordingly we subcloned DNA from pWC3151 in a temperate phage vector, KC516, for marker rescue experiments. To establish lysogens in absA1-542 sab strains, we used phage RS100, which carries a 3·2 kb XhoI fragment (Fig. 5a
) spanning the absA1 mutant sites (Brian et al., 1996
), and also including absA2. Fig. 3
illustrates the genotypes expected in such lysogens. In this example, a sab mutant site is hypothesized in absA2 (see below) and three intervals for recombination are delineated. Two considerations are important in interpreting the lysogen phenotypes. First, the RS100 cloned fragment lacks the absA1 N-terminus and transcription start site; second, absA1 and absA2 are predicted to be cotranscribed (T. Anderson, unpublished). Lysogens, therefore, would contain one expressed copy of the absA locus and one non-expressed copy; those formed by recombination in interval II would carry the sab mutant site in the non-expressed copy and the absA1-542 sab+ alleles in the expressed copy. Hence, recovery of Abs- lysogens following infection of a sab strain by RS100 would indicate that the sab mutant site was covered by the cloned XhoI fragment. For four strains shown in Fig. 3
, recovery of Abs- lysogens following this protocol indicated that the sab mutant sites were located within the 3·2 kb XhoI interval; for C542S9, the result was confirmed by marker exchange, recovering the sab allele on the cloned 3·2 kb XhoI fragment.
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In previous genetic studies, two kinds of disruption mutations in absA caused a Pha sab-Type II-like phenotype (Brian et al., 1996 ; Aceti & Champness, 1998
). These mutations (Fig. 5a
) were (1) an ermE insertion/deletion in strain C420, eliminating much of the absA1 gene and probably also expression of the downstream absA2 gene, and (2) an insertional disruption (in strain C430) created by single-crossover integration of a phage-cloned fragment internal to the absA transcript (phage RS500 in Fig. 5a
). The latter mutation primarily disrupted absA2 expression; it also truncated the C-terminus of absA1, but complementation results suggested that the truncated AbsA1 protein was active in vivo. Because both disruptions eliminated absA2 function, we proposed (Brian et al., 1996
) that AbsA2 functions as a negative regulator of antibiotic synthesis.
The absA alleles that cause an Abs- phenotype mapped to absA1. One hypothesis for their effects is that they lock the absA two-component system into a negatively regulating mode. Disruption of absA2 might thus be epistatic to the absA1-542 and absA1-577 mutations. The Pha phenotype of RS500 lysogens established in C542 and C577 (see Methods) confirmed this prediction.
Sequence analysis of Abs- mutant strains
In previous work (Brian et al., 1996 ), marker rescue experiments were used to localize absA mutations responsible for the Abs- phenotype to the XhoIBamHI interval within absA1 (Fig. 5a
). To define the mutant sites present in strains C542 and C577, the absA1 gene was sequenced.
The absA1 mutations found in C542 and C577 are shown in Fig. 5(b). C577 contained a single amino acid change, L253R. C542 contained two amino acid changes (I360L and R365Q). We do not know if both mutations in C542 are required to cause the Abs- phenotype. Sequencing absA2 in C542 and C577 confirmed that in these strains absA2 contained no mutations.
The C542 and C577 mutations lie in regions of AbsA1 that are conserved in the histidine kinase sensor-transmitter family: the C542 mutations alter the G box, which is involved in nucleotide binding (Stock et al., 1995 ), and the C577 mutation alters a region, provisionally named the X box, and recently proposed to be involved in the aspartylphosphatase activity common to many sensor-transmitters (Hsing et al., 1998
).
Sequence analysis of sab mutations
To locate sab mutations potentially within the absA locus, we amplified regions of the absA1 and absA2 genes by PCR, and then sequenced the products from two independent amplifications of each mutant strain. Fig. 5(b) shows the sab mutant sites found in this analysis.
absA2 was sequenced in 10 sab strains, including those genetically analysed in the experiments discussed above. In C542S9, sequencing revealed an S171W change at the N-terminal end of the predicted helixturnhelix region.
In one sab strain, C577S25, the absA locus had undergone a deletion that started within the absA1 gene and extended through absA2 into the neighbouring ORF, d9 (Fig. 5a).
Both C542S9 and C577S25 were Type II sab strains with a Pha phenotype. Interestingly, C542S9 was even more strongly pigmented than C577S25 and produced more -actinorhodin than any other sab strain. This was surprising because if the absA system functions as a negative regulator, the phenotype of a
absA1absA2 mutation might be expected to overproduce more strongly than an absA2 point mutation. Thus this result hints at a complex role for the AbsA2 response regulator in antibiotic production. Another possibility is that deletion of the neighbouring uncharacterized DNA modulates the Pha effect in C577S25.
One Type I strain, C577S20, contained an absA2 mutation, V29A, that appeared to restore at least some normal AbsA function. It did not alter any of the domains known to be involved in phosphotransfer reactions but affected a region that was expected from the NarL response regulator crystal structure (Baikalov et al., 1996 ) to be
-helical.
As mentioned above, the strains C577S20 and C577S25 were CDA-. For C577S25, this can be explained by the observation that the sab mutation is a deletion which extends into a cda biosynthetic region (Fig. 5a, b). For C577S20 we do not have an explanation for the CDA- phenotype at this time.
No absA2 mutations were found in the other strains sequenced. However, the marker rescue experiments discussed above had strongly predicted that the sab alleles in the strains tested lay in the 3·2 kb XhoI interval (Fig. 3), and it was possible that absA1 could be mutant in some sabs. Therefore, we sequenced absA1 in two strains: C542S3, a Type I strain that had been used in all of the genetic experiments; and C542S11, a Type II strain used in initial investigations of the pseudoreversion phenomenon. Both strains proved to contain sab mutations in absA1, as well as in the absA1-542 mutations discussed above.
Strain C542S3 contained the mutation G252V. This amino acid is in the same region of AbsA1 in which the Abs- strain C577 is mutant. In fact, the two mutations affect adjacent residues, posing the questions about whether the sab3 mutation could suppress the absA1-577 mutation and whether the absA1-577 mutation could suppress the absA1-542 mutation. However, we have not yet tested these possibilities or otherwise addressed whether the various sab mutations are allele-specific.
The Type II strain C542S11 contained an absA1 nonsense mutation in amino acid residue 360. The Pha phenotype may have been due to a polar effect on absA2 expression. Alternatively, if the nonsense mutation was not polar, the antibiotic might be overproduced because phosphorylation of AbsA2 is necessary for its negatively regulating function, and the mutant lacks AbsA1 kinase activity.
Do sab mutations occur outside the absA locus?
Two of 10 sab strains in which absA2 was sequenced contained absA2 mutations and two more strains in which absA1 was sequenced contained absA1 mutations. Of the remaining strains, two (C542S2 and C54210) were used in the marker rescue mapping experiments that predicted close linkage of the absA1-542 and sab alleles. Thus, these would be expected to contain mutations in absA1, but could contain mutations in the 2·7 kb uncharacterized region (rightwards of absA2 in Fig. 5a) cloned in phage RS100 [however, the relatively low recombination frequency of Abs- lysogens (Fig. 3
) compared to the sab9 mutation in absA2 argues against this possibility]. Altogether, 11 sab strains have been used in one of the types of crosses illustrated in Figs 13
and in all cases there was evidence for a sab mutation close to absA. Nevertheless, the analysis discussed here does not exclude the possibility that sab mutations could occur outside absA.
Correlation of mapping data with sequencing data and the physical map
The protoplast fusion results with strain C542S9 led us to predict in Fig. 2(b) that the relative order for absA1-542 and sab, with respect to other genetic markers, would be (in anticlockwise order), hisAabsA1saburaA. Considering that sab9 lies in absA2, this would give a relative gene order for absA1 and absA2 of hisAabsA1absA2uraA. We have located the absA locus on the ordered cosmid E8 (Redenbach et al., 1996
), and the order has been confirmed during the ongoing genomic sequencing (www.sanger.ac.uk/pub/s_coelicolor/sequence).
The protoplast fusion results (Fig. 2b) did not predict a relative order for the absA1 and sab3 mutations in strain C542S3, since there was no significant difference in the frequency of absA1-542 sab+ recombinants for the two selections. In light of the close linkage of absA1-542 and sab3 in the absA1 sequence (Fig. 5b
), gene conversion events could have contributed significantly to production of recombinant genotypes, and so distorted apparent recombination frequencies. The marker rescue experiment in Fig. 3
and the plasmid-mediated recombination experiment in Fig. 2
would also be subject to gene conversion effects that could affect ordering of mutations that are close together.
Conclusions
(i) Non-antibiotic-producing (Abs-) mutants of the absA locus, which seem to lock the AbsA regulatory system into a negatively regulating mode, contain point mutations in conserved domains of the AbsA1 histidine kinase sensor-transmitter protein.
(ii) The absA1 mutants spontaneously acquire suppressive mutations that restore antibiotic synthesis.
(iii) Plasmid-mediated and protoplast fusion mapping techniques were useful for genetic analysis of suppressive (sab) mutations, locating some close to absA.
(iv) Actinophage C31-derived vectors were useful for marker rescue and marker exchange experiments that verified the existence and location of sab mutations and allowed transduction of sab mutations from strain to strain.
(v) Sequence analysis defined sab mutant residues in the absA two-component system. Some sab alleles (Type I) restore apparently normal AbsA function since they restore a wild-type phenotype to Abs- mutants, whereas some (Type II) cause antibiotic overproduction.
(vi) Antibiotic overproduction in sab strains can result from deletion of absA, consistent with absAs proposed role as a negative regulator, but the most strongly pigmented sab strain contains a point mutation in the AbsA2 response regulator, suggesting a complex role for the absA locus in production of antibiotics.
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ACKNOWLEDGEMENTS |
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Received 8 March 1999;
revised 26 May 1999;
accepted 2 June 1999.