(Received for publication, November 7, 1995; and in revised form, January 19, 1996)
From the
A 0.972-kilobase pair DNA fragment from Streptomyces lividans that induces the production of the blue-pigmented antibiotic actinorhodine in S. lividans when cloned on a multicopy plasmid has led to the isolation of a 4-kilobase pair DNA fragment from Streptomyces coelicolor containing homologous sequence. Computer-assisted analysis of the DNA sequence revealed three putative open reading frames (ORFs), ORF1, ORF2, and ORF3. ORF2 extends beyond the sequenced DNA fragment, and its deduced product shares no similarities with any other known proteins in the data bases. ORF3 is also truncated, and its 41-amino acid C-terminal product is identical to the S. coelicolor adenine phosphoribosyltransferase. The 847-amino acid ORF1 protein, with a predicted molecular mass of 94.2 kDa, strongly resembled the relA and spoT gene products from Escherichia coli and the homologs from Vibrio sp. strain S14, Haemophilus influenzae, Streptococcus equisimilis H46A, and Mycoplasma genitalium. Unlike these proteins, the ORF1 amino acid sequence analysis revealed the presence of a putative ATP/GTP-binding domain. A mutant was generated by deleting most of the ORF1 gene that showed an actinorhodine-nonproducing phenotype, while undecylprodigiosin and the calcium-dependent antibiotic were unaffected. The mutant strain grew at a much lower rate than the wild-type strain, and spore formation was delayed. When the gene was propagated on a low copy number vector, not only was actinorhodine production restored, but actinorhodine and undecylprodigiosin production was enhanced in both the mutant and wild-type strains and morphological differentiation returned to wild-type characteristics. (p)ppGpp synthetase activity was not detected in purified ribosomes from the ORF1-deleted mutant, while it was restored by complementation of this strain.
Streptomyces species have a complex cell life cycle that involves morphological and biochemical differentiation. Antibiotic production is usually initiated at the transition between vegetative growth and the development of the spore-bearing aerial mycelium(1, 2) , suggesting that there may be a close relationship between both processes. During this developmental regulation, antibiotic biosynthesis is controlled by a series of metabolites and regulatory gene products operating at different levels, with their final target being the structural genes of the antibiotic pathway.
Streptomyces coelicolor, the genetically most
studied Streptomyces species, produces at least four
structurally different antibiotics: actinorhodine(3) ,
undecylprodigiosin(4) , methylenomycin(5) , and the
calcium-dependent antibiotic (CDA)()(6) . For the
first three compounds, it has been reported that their biosynthesis is
controlled by the action of specific regulatory genes located within
the particular biosynthetic
clusters(4, 7, 8, 9) . Such
regulatory genes have also been identified in other antibiotic
clusters, such as bialaphos(10) ,
streptomycin(11, 12) , and daunorubicin(13) .
In S. coelicolor, several so-called pleiotropic genes outside the biosynthetic clusters have been implicated in the regulation of the multiple antibiotic pathways; mutations in absA(14) and absB(15) completely abolish the biosynthesis of all four antibiotics, from which the production of both pigmented antibiotics is restored by the afsQ1-afsQ2 gene pair in absA but not absB mutants(16) . Neither actinorhodine nor undecylprodigiosin (as well as reduced amounts of methylenomycin and CDA) could be detected in afsB mutants(17) , which were suppressed by the afsR gene(18, 19) . In abaA-ORFB (20) mutants, actinorhodine and undecylprodigiosin production is blocked, while the level of CDA is reduced and methylenomycin remains unaffected. The bld (bldA-D and bldF-G) (21, 22) genes have been described as being required for both antibiotic production and aerial mycelium formation. The bldA gene encodes a leucyl-tRNA (23) that recognizes the UUA codon (extremely rare in Streptomyces mRNA because of the high G + C content of their DNA), and the suggestion has been made that this gene might constitute a translational regulatory mechanism controlling sporulation genes and some antibiotic pathways(9, 24) .
The stringent response and (p)ppGpp formation have been extensively studied in Escherichia coli(25, 26) . These polyphosphorylated nucleotides are synthesized by at least two possible routes. The main one is attributed to the (p)ppGpp synthetase I activity, which is encoded by the relA gene and operates on ribosomes under amino acid deprivation when codon-specified uncharged tRNAs are bound to the ribosomal acceptor site(27) . The reaction involves a pyrophosphoryl transfer from ATP to GTP or GDP. The transient (p)ppGpp accumulation leads to complex regulatory adjustments such as a reduction in stable RNA transcription rate (28, 29, 30, 31, 32) and an increase in expression of certain amino acid operons(33, 34) . Defective ribosomal (p)ppGpp synthesis was observed in relC mutants, which have an altered L11 protein in the 50 S ribosomal unit, the same subunit implicated in the binding of the RelA protein(35, 36) . A putative relC mutant of S. coelicolor has been isolated(37) . The strain is deficient in the production of actinorhodine and undecylprodigiosin as well as in its ability to form aerial mycelium. In this relaxed mutant, there is a 10-fold reduction of (p)ppGpp upon amino acid starvation when compared with the parental strain. Based on this observation and the isolation and characterization of relaxed mutants from other Streptomyces species (38, 39, 40, 41) and Bacillus subtilis(42) , a correlation was suggested between the stringent response at either the onset of secondary metabolism because of (p)ppGpp formation or morphological differentiation due to the reduction of the intracellular GTP level (40) . The second route for (p)ppGpp synthesis in E. coli, mediated by a ribosome-independent enzyme, (p)ppGpp synthetase II activity (spoT gene product)(43) , is deduced to occur because during carbon and energy source deprivation, (p)ppGpp does accumulate in relA-deleted strains(44) , while it is no longer detectable in strains carrying deletions of both the relA and spoT genes(45) .
Recently, a (p)ppGpp synthetase from Streptomyces antibioticus has been purified and characterized (46, 47) and shown to possess differential catalytic properties, which raises the possibility that the reported enzyme would not represent the analog of the RelA (or SpoT) protein from E. coli; nevertheless, the presence in S. antibioticus of a pathway similar to that of relA in E. coli for (p)ppGpp synthesis could not be excluded. The isolation of relC mutants in S. antibioticus(41, 48) supports this hypothesis.
We report here the isolation and characterization of a new gene that strongly resembles relA and spoT and that is implicated in the regulation of antibiotic production in S. coelicolor.
For high resolution S1 mapping, the method of Murray (69) was used. For actI-ORF1(70) , a 798-base pair SphI-SacI fragment (from positions 13.4 to 14.1) containing the actI-ORF1 promoter region uniquely labeled at the 5`-end of the SacI site within the actI-ORF1 coding region was used as probe. For actVI-ORF1(71) , a 847-base pair KpnI-BssHII fragment (nucleotides 1406-2252) that contained the actVI-ORF1 promoter region labeled at the 5`-end of the BssHII site within the internal actVI-ORF1 coding region was used. For actII-ORF4 (9) , the actII-ORF4 promoter region included in a 635-base pair fragment (nucleotides 4824-5458) was uniquely labeled at the 5`-end of the XhoI site (nucleotide 5458) within the actII-ORF4 coding region and was used as probe. RNA was extracted as described (61) from 3-day-old mycelium grown on the surface of cellophane discs on R5 agar plates as described(72) .
Southern blotting of BamHI-digested chromosomal DNA from S. lividans TK21 and S. coelicolor J1501, probed with pSCNB079, showed a single hybridizing band of the same intensity in both strains of 1 kb in S. lividans and of 3.3 kb in S. coelicolor. This confirmed that the sequenced DNA was not the result of rearrangement during the cloning experiments and indicated the presence of a similar gene in S. coelicolor.
Figure 1: Restriction map of the recombinant phage and organization of deduced ORF genes as revealed by DNA sequencing. Only relevant restriction sites are shown. Chromosomal DNAs are represented by thick bars; plasmid and phage sequences are represented by thin lines; and vectors are given in brackets. pSCNB080 consisted of the Sau3AI-BamHI fragment (nucleotides 1-4064; previously cloned in pSU21 and rescued with EcoRI) blunt-ended and cloned into the EcoRV site of pIJ941. The limits of the internal ORF1 region deleted in the 18J strain are indicated by arrowheads. A frameshift in the resulting ORF1-deleted mutant is introduced in the chromosome at the BamHI site, generating a possible fusion protein of 241 amino acid residues that contained the first 166 residues of the N terminus of the ORF1 protein.
Computer-assisted analysis of the resulting 4-kb DNA sequence revealed three possible ORFs (Fig. 1), which were named ORF1, ORF2, and ORF3. No other putative ORF could be deduced by consideration of Streptomyces codon usage.
The most likely translation initiation codon for ORF1 is at position 311 (a TTG codon), as deduced by its overall distribution of GC content in the third position, the codon usage within ORF, and the presence of a good putative ribosome-binding site (GAGGAG, nucleotides 300-305) at an appropriate distance(82) . The similarities observed between the putative ORF1 product and other known proteins (see below) were used as additional criteria. The stop codon (TAG) is located at position 2852.
ORF1 encodes a protein of 847 amino acids
with a predicted molecular mass of 94.2 kDa. Comparison of the ORF1 product showed a strong resemblance to the following proteins:
RelA from E. coli (38% identity, 61% similarity)(27) ,
RelA from Vibrio sp. strain S14 (38% identity, 60%
similarity)(78) , SpoT from E. coli (43% identity, 62%
similarity)(43) , RelA from H. influenzae (37%
identity, 60% similarity) (79) , SpoT from H. influenzae (38% identity, 60% similarity)(79) , the Rel-like proteins
from S. equisimilis H46A (40% identity, 63% similarity) (80) and from M. genitalium (25% identity, 50%
similarity)(81) , and a putative SpoT from M. leprae cosmid B1177 (62% identity, 77% similarity). Additionally, the 166-amino acid N-terminal ORF1 product
was shown to be almost identical to the translated DNA extreme region
near the secD and secF genes from S. coelicolor A3(2). (
)There is a conserved mismatch in amino acid
sequence at position 24, alanine instead of proline, due to a change of
a guanine to a cytosine nucleotide in the DNA sequence. This difference
could be attributed to the different strains used. The N-terminal
region of the ORF1 protein is
90 amino acid residues longer than
the homologous ones. Six nucleotides were different between S.
lividans and S. coelicolor within the original fragment,
while the corresponding products were almost identical with only a
conserved change (leucine instead of valine at position 197).
The amino acid sequence of the ORF1 protein reveals a particularly well conserved ATP/GTP-binding domain (amino acids 458-465). This sequence motif, (A/G)XXXXGK(S/T), generally referred to as the ``A'' consensus sequence (83) or the ``P-loop''(84) , is not present in RelA or SpoT proteins(27, 43, 78, 79, 80, 81) and represents a gap in these proteins when aligned with the ORF1 product (Fig. 2).
Figure 2: Polypeptide sequence alignments of the ORF1 gene product with other proteins. 01, ORF1; 02, M. leprae cosmid B1177 (p)ppGpp 3`-pyrophosphohydrolase (see Footnote 3); 03, E. coli ATP:GTP 3`-pyrophosphotransferase(27) ; 04, E. coli (p)ppGpp 3`-pyrophosphohydrolase (43) ; 05, Vibrio sp. strain S14 ATP:GTP 3`-pyrophosphotransferase(78) ; 06, H. influenzae ATP:GTP 3`-pyrophosphotransferase(79) ; 07, H. influenzae (p)ppGpp 3`-pyrophosphohydrolase(79) ; 08, S. equisimilis H46A Rel-like protein(80) ; 09, M. genitalium Rel-like protein(81) ; 10, consensus. Plurality is 6. Amino acids are numbered according to their original positions in the proteins.
The second ORF (ORF2, nucleotides 2933-4066) extends beyond the sequenced DNA. Comparison of its 378-amino acid C-terminal product with protein sequences contained in data bases gave no similarities to other known proteins and therefore no clue as to its possible function.
The third ORF (ORF3,
nucleotides 2-127) is incomplete, and translation of this short
DNA sequence was shown to be identical to that of the 41-amino acid C
terminus of the adenine phosphoribosyltransferase from S.
coelicolor reported in the data base. Based on the
observed similarities, we infer that the DNA sequence reported here is
adjacent to the secD and secF region.
To delete ORF1, a clone was constructed by sequentially ligating the 0.805-kb Sau3AI-BamHI fragment (nucleotides 1-805) and the 1.429-kb XhoI fragment (nucleotides 2567-3995) in the same relative orientation as in the chromosome into the BamHI and SalI sites of E. coli vector pIJ2925, respectively; the resulting fragment, carrying the intended deletion, was rescued by digestion with BglII and ligated to the PM1 vector previously digested with BglII and BamHI, which replaces the thiostrepton resistance marker with the recombinant fragment. Insertion of the phage through one of the flanking fragments was confirmed by Southern blotting. One of the lysogens was spread on agar plates without selection, and spores from this first unselected round were analyzed on plates for hygromycin sensitivity. The hygromycin-sensitive colonies are expected either to carry the internal deletion, after double crossover with the prophage fragment, or to have simply lost the prophage from the chromosome. Six different hygromycin-sensitive colonies were analyzed by Southern blotting in order to determine their chromosomal structure. Three of them were shown to carry the expected physical deletion; two still contained the prophage (their sensitivity may be the result of the generation of a mutation on the hygromycin resistance gene); and the last gave the same pattern as the wild type. These deleted mutants (named 18J strain) were shown to grow slower than and not to sporulate as well as the wild type. Actinorhodine production was almost abolished, while undecylprodigiosin and CDA production was little affected (Table 1). Normal sporulation rate and actinorhodine biosynthesis were restored by introducing plasmid pSCNB080, which contained the complete region (Fig. 1). However, the amount of both pigmented antibiotics seemed to be higher in both S. coelicolor strains 18J and J1501 when transformed with pSCNB080 (Table 1). Interestingly, transformation of the 18J strain with actII-ORF4 in plasmid pPAS4 led to actinorhodine production (see below), without affecting the low growth rate and the deficiency in sporulation (data not shown).
Figure 3: High resolution S1 mapping of act cluster genes. Shown are the results from the transcriptional analysis of actI-ORF1 (A), actVI-ORF1 (B), and actII-ORF4 (C). RNAs were extracted from the following S. coelicolor strains: strain J1501 (lane 1), strain 18J containing the pPAS3 control plasmid (lane 2), and strain 18J containing the actII-ORF4 gene in plasmid pPAS4 (lane 3). E. coli tRNA was used a control (lane 4). Protected fragments of the expected size are indicated with arrows. End-labeled HinfI-digested pBR329 was used as size marker.
Figure 4: Synthesis of phosphorylated guanine nucleotides by purified ribosomes from S. coelicolor and E. coli. Reaction conditions were as specified under ``Experimental Procedures'': without ribosomes (lane 1) and with ribosomes from E. coli JM101 (lane 2), E. coli JM101 carrying the pGG21 plasmid (lane 3), S. coelicolor strain 18J harboring either the pPAS3 (lane 4) or pSCNB080 (lane 5) plasmid, S. coelicolor strain J1501 carrying either the pPAS3 (lane 6) or pSCNB080 (lane 7) plasmid, and S. coelicolor strain J1501 carrying the pSCNB080 plasmid either with 4% dimethyl sulfoxide (lane 8) or 2 µM thiostrepton (lane 9). Final protein concentrations were 1.36, 1.35, 1.8, 1.18, and 1.53 mg/ml (lanes 2-6, respectively) and 1.14 mg/ml (lanes 7-9). Preincubation of isolated ribosomes with either thiostrepton dissolved in dimethyl sulfoxide at a final concentration of 2 µM or with dimethyl sulfoxide at an equivalent final concentration (4%) was allowed to proceed for 30 min at 4 °C. The migration positions of pppGpp, ppGpp, GTP, and GDP are indicated.
The
(p)ppGpp formation in S. coelicolor was 25-fold lower
than that in E. coli JM101 (Table 2), with a correlation
between the copy number of the ORF1 gene and (p)ppGpp
synthetic activity in the former strain (Table 2). Interestingly,
a ppGpp/pppGpp ratio of 3.22 was found in the reaction with ribosomes
from the J1501 strain with the pPAS3 control plasmid, while values of
0.75 and 1.18 were observed with the 18J and J1501 strains harboring
extra copies of the ORF1 gene, respectively. This difference
was also observed with ribosomes from E. coli when compared
with the ribosomes from the same strain carrying extra copies of the relA gene (pGG21) (ppGpp/pppGpp ratios of 3.90 and 1.35,
respectively).
By selecting a clone from S. lividans that stimulated actinorhodine production in S. lividans, we have isolated a gene in S. coelicolor that seems to be involved in the control of antibiotic biosynthesis in this bacterium. The ORF1-encoded 847-amino acid protein strongly resembles a group of enzymes involved in the biosynthesis of (p)ppGpp compounds, being produced under stringent conditions and generally referred to as (p)ppGpp synthetases.
The RelA and SpoT proteins of E. coli have been studied in some detail(26) , and RelA from Vibrio sp. strain S14 (78) and the (p)ppGpp
synthetases from Bacillus sp. (85, 86) and
from S. antibioticus(46, 47) have also been
characterized. The predicted ORF1 product has a molecular mass
of 94.2 kDa, which is close to that described for E. coli RelA
(84,000 Da) and SpoT (79,000 Da)(27, 43) , for Vibrio RelA (84,500 Da)(78) , for the
ribosome-dependent (p)ppGpp synthetase from Bacillus
stearothermophilus (86,000 Da)(86) , and for the
ribosome-independent (p)ppGpp synthetase I from S. antibioticus (88,000 Da)(46) . Nevertheless, the ORF1 protein is
90 amino acids longer at its N terminus than the homologs so far
sequenced.
That the cloned ORF1 gene codes for a (p)ppGpp
synthetase is also supported by the measurements of this activity in
purified ribosomes from S. coelicolor. In this context, it
should be emphasized that no (p)ppGpp formation was detected with
ribosomes from the S. coelicolor ORF1-deleted mutant, while
the activity was restored by complementation of the 18J strain. Like
the ribosome-dependent (p)ppGpp synthetase I (RelA) from E.
coli(26) , the enzyme from S. coelicolor was
inhibited by thiostrepton (Table 2), its activity was detected in
the presence of 18% methanol, and Mg ions were
absolutely required, not being replaced by Mn
or
Zn
(data not shown).
The increase in the relative proportion of ppGpp with respect to pppGpp in ribosomes from the J1501 strain harboring the control plasmid (pPAS3) (Table 2) when compared with either the 18J or J1501 strain carrying extra copies of the ORF1 gene (pSCNB080) might be consistent with the observation that ppGpp is synthesized from pppGpp in E. coli(87) . Nevertheless, the direct pyrophosphoryl transfer from ATP to the GDP formed from GTP by ribosomal nucleotidases cannot yet be excluded and might also account for the observed differences.
An interesting feature of the ORF1-deduced
product is the presence of a putative ATP/GTP-binding
motif(83) , (A/G)XXXXGK(S/T), which has not been
described in any other known protein related to (p)ppGpp metabolism.
The presence of this conserved motif in the ORF1 protein is in
agreement with its biochemical function because ATP and GTP are both
substrates of the reaction catalyzed by the (p)ppGpp synthetase.
Additionally, a consensus GTP-binding domain has been proposed by Dever et al.(88) to be composed of three conserved
elements: (A/G)XXXXGK, DXX(A/G), and NKXD,
with a spacing of either 40-80 or 130-170 amino acid
residues between the first and second elements and of 40-80
residues between the second and third elements. The first two elements
are involved in interactions with the phosphate portion of the GTP
molecule, and the last element is involved in nucleotide
specificity(89) . Although with a mismatch, this conserved
GTP-binding fingerprint is observed in the ORF1 protein (amino acids
458-464, APKSSGK; amino acids 513-516, DVIA; and amino
acids 587-590, NKIR, with spacings of 48 and 70 amino acids,
respectively), suggesting that it could be specifically involved in GTP
binding. The deviation in the consensus sequence of the last element in
the ORF1 protein (the aspartic acid of NKXD is replaced by
arginine, NKIR) might be related to a lower affinity for GTP, as has
been demonstrated by Feig et al.(90) for the
p21 protein, in which the mutation of this residue to
asparagine resulted in a reduction in affinity for GTP by a factor of
100 (K
= 10
to
10
M). Thus, it is still reasonable to
suggest that the ORF1 protein could have the capacity to bind GTP.
Further biochemical studies of this (p)ppGpp synthetase as well as the
characterization of mutations within the putative nucleotide-binding
domain will be of particular interest for understanding its biochemical
function and are currently in progress.
One out of three antibiotics produced by S. coelicolor, actinorhodine, was severally affected in the 18J strain. This dramatic reduction is due to a decrease in the specific mRNA level of the transcriptional activator gene of the act cluster (actII-ORF4). Surprisingly, undecylprodigiosin production is only slightly reduced in the 18J mutant, although both pathways are controlled by their respective positive regulators(8, 9) , which are very similar to each other. Nevertheless, a possible role for the ORF1 gene in the regulation of both pathways is suggested since extra copies of this gene resulted in an increase in both actinorhodine and undecylprodigiosin in both the J1501 and 18J strains. It is well known that several metabolites and regulatory genes operate at different points and in a particular mode on antibiotic biosynthesis, giving rise to a signaling within an intricate regulatory network. An alternatively acting signal or any other factor independent of the ORF1 mechanism could be sufficient to trigger undecylprodigiosin biosynthesis, in contrast to the ORF1 requirement for actinorhodine production. The fact that actinorhodine and undecylprodigiosin are not equally reduced in the ORF1-deleted mutant, while both of them are enhanced by extra copies of this gene, is an interesting observation, and the mechanism of these differences needs to be studied in more detail.
The production of actinorhodine in strain 18J could be restored by extra copies of either ORF1 or actII-ORF4. An effect similar to that observed with the actII-ORF4 gene has been described previously in absA and absB mutants(91, 92) . These data also support the suggestion that the ActII-ORF4 protein is by itself sufficient to activate transcription of the biosynthetic genes, and if any other additional factor is required, either it does not constitute a limitation or its action might be overtaken by the overproduced ActII-ORF4 protein(93) . As expected, deficiencies in morphological differentiation of strain 18J carrying extra copies of actII-ORF4 cannot be complemented, and they are only completely restored in trans by the ORF1 gene.
We
do not yet know why the disruptants in the ORF1 gene showed an
apparently normal phenotype. A residual (p)ppGpp synthetase activity
cannot be excluded in these lysogens. This question is of interest not
only for defining putative functional peptides, but also for
understanding the activation of actinorhodine production in S.
lividans by the original BamHI fragment (internal region
of the ORF1 gene), which allowed us to isolate the gene. In E. coli, the relA1 gene products (- and
-fragments) have been shown to complement each other in trans to yield some (p)ppGpp synthetic activity, while overexpressed
RelA1
-fragment abolishes (p)ppGpp formation of a relA
strain, probably due to its competition
with the wild-type gene product for ribosomal binding(44) .
Furthermore, a C-terminally truncated RelA protein is still
active(94, 95) , although it then becomes relC-independent, unlike the wild-type protein(94) .
The differences in phenotype observed between the lysogens and the ORF1-deleted mutant might be interpreted in this context.
The involvement of (p)ppGpp in the stringent response has been studied in E. coli in some detail(25, 26) . Depletion of amino acids leads to the synthesis of these polyphosphorylated nucleotides by (p)ppGpp synthetase I activity, which apparently mediates several complex changes in gene expression, due to the inability of the cell to maintain sufficient aminoacylated tRNAs for the demands of protein synthesis. There are several reports that (p)ppGpp formation takes place during stringent response in several Streptomyces species(37, 38, 39, 40, 41, 96, 97, 98, 99) . A relaxed (presumptively relC) mutant in S. coelicolor has been isolated (37) in which the onset of aerial mycelium formation was delayed and the production of actinorhodine and undecylprodigiosin was abnormal. The 18J strain reported here was shown to grow slower than the wild type and to have a reduced formation of spores (in agreement with the observations of Ochi (37) ), but only actinorhodine was severally affected in our mutant. In addition, the relaxed mutant isolated by Ochi (37) did produce actinorhodine after 10 days on agar plates, while no such effect could be detected in our ORF1-deleted mutant under the same conditions. Thus, we cannot yet exclude that the observed differences could reflect either the existence of alternative effects on the (p)ppGpp biosynthetic pathway or the presence of more than one pathway for (p)ppGpp formation in S. coelicolor.
Differentiation and production of secondary metabolites start concomitantly in response to nutrient limitation, and although a possible role of (p)ppGpp in initiating antibiotic biosynthesis has been suggested(37, 38, 39, 40, 41, 96, 98) , no direct link was established by others(99-101). Furthermore, (p)ppGpp accumulation due to nutritional shiftdown or serine hydroxamate treatment does not seem to be sufficient to trigger antibiotic production(99-101), suggesting that sensing of growth rate or growth cessation may be of critical importance(101) . Further biochemical and genetic characterization of the ORF1 protein and the deleted mutant will provide some insight into the role played by (p)ppGpp levels in the onset of antibiotic biosynthesis as well as in other regulatory events in the cellular physiology of Streptomyces strains.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X92519 [GenBank]and X92520[GenBank].