BACKGROUND
most of the commonly found structural changes in dna are due to methylation of selected bases.
in some viral dnas, certain bases may be hydroxymethylated or glucosylated  <cit> .
altered or unusual bases in dna molecules often have significant physiological implications, such as dna replication control, gene regulation, or protection of the respective organisms from invasion by foreign dna  <cit> .
in contrast to other types of dna modification, s, lividans has a site-specific and stereo-selective sulfur modification on the dna backbone termed phosphorothioation  <cit> .
this sulfur modification occurs specifically between two guanine nucleotides in s.lividans  <cit> .
the sulfur-modified dna suffers double-stranded cleavage at the modification sites during normal and pulsed-field gel electrophoresis  <cit> .
the dnd phenotype was proven to be a peracid-mediated, oxidative and amine-catalysed reaction to form a phosphorothioated dna backbone  <cit> .
the dnd phenotype can be overcome by replacing tris with hepes in the electrophoresis buffer or by adding a certain concentration of thiourea to tris-containing buffers  <cit> .
in s.lividans, this dna sulfur modification was found to be determined by a dnd gene cluster carrying five open reading frames   <cit> .
homologous dnd gene clusters and/or dnd phenotypes are found in many strains of streptomyces, e. coli, bacillus, salmonella, klebsiella, enterobacter, mycobacterium, vibrio, pseudomonas, pseudoalteromonas, hahella, oceanobacter, geobacter, pelagibacter, roseobacter, mesorhizobium, serratia, acinetobacter, and clostridium, as well as in certain archaea and unidentified marine microbes, indicating that dna sulfur modification is a widespread phenomenon in prokaryotes  <cit> .
here we attribute dna phosphorothioate modification to a dnd gene cluster consisting of a  <dig> -bp region of dna carrying just five genes.
we confirmed by transcriptional analysis that dndb-e constitute an operon, and made systematic in-frame deletion mutations within each gene or combinations of the five dnd genes before performing a series of complementation analyses to evaluate the roles of individual dnd genes in dna sulfur modification.
RESULTS
identification of a minimal dnd region
in an effort to precisely localize the region responsible for the dnd phenotype and obtain unambiguous evidence on the genes involved in dna phosphorothioation, we made a series of phz <dig> derivatives by removing end segments from a ca.
10-kb fragment of dna carrying some likely cis-acting elements using convenient restriction sites, thus identifying a core region carrying only five dnd genes.
a combination of restriction fragments  was incorporated into appropriate sites of integrative vector pset <dig>  <cit>  to produce four plasmids .
mediated by the attp site of streptomyces phage Øc <dig> present on pset <dig>  these vectors can site-specifically integrate into the attb site in the chromosome of s. lividans zx <dig>  <cit>  after transfer by conjugation from e. coli et12567/puz <dig> into zx <dig>  the dna of these zx1-derivative strains was either degraded  or stable  during electrophoresis .
the minimal dnd region conferring the dnd phenotype  was localised to a  <dig> -bp fragment on pjtu <dig>  the left and right borders of the minimal dnd cluster are only 4-bp and 472-bp from the stop codons of dnda and dnde , respectively, confirming that five genes are necessary and sufficient for dna phosphorothioation.
transcriptional analysis of the dnd genes
bioinformatic analysis of the  <dig> -bp region of pjtu <dig>  suggests that dnda and dndb-e are divergently transcribed.
the facts that the 3' end of dndb and the 5' end of dndc overlap by  <dig> bp , that the initiation codon  of dndd precedes the 3' end of dndc by  <dig> bp , and that the initiation codon of dnde  is  <dig> bp upstream of the stop codon of dndd  strongly imply that the dndb-e might constitute an operon.
to prove divergent transcription of dnda and a hypothetical dndb-e operon, we performed a transcriptional analysis on the minimal dnd cluster by rt-pcr.
rna was extracted from s. lividans  <dig> and amplified by rt-pcr using oligonucleotide primers depicted in fig.
2a.
the pcr products were fractionated by electrophoresis .
as an internal control, 16s rrna was amplified in all samples.
the appearance of dna bands , which were amplified using different sets of primers , unambiguously suggests that dndb-e are co-transcribed as a single operon in s. lividans  <dig>  the absence of dna bands using primers a <dig> and b <dig>  suggests a lack of co-transcription in the region between a <dig> and b <dig>  confirming independent transcription of dnda and dndb-e.
a mutation-integration system for functional analysis of individual dnd genes
as demonstrated by the transcriptional analysis, dndb-e constitute an operon.
we therefore inactivated each of the five dnd genes independently to examine their effect on the dnd phenotype in terms of dna phosphorothioation.
early experiments on disruption of dnda  and dndd  by a str/spc cassette clearly abolished the dnd phenotype  <cit>   but could not provide unambiguous evidence for the function of dndd as insertion of antibiotic resistant genes could block expression of downstream gene of an operon by a polar effect.
we attempted to make in-frame deletions internal to individual dnd genes at their corresponding chromosomal loci to avoid polar effects.
apart from the dndb in-frame deletion mutant hxy <dig>  <cit>  , extensive efforts to obtain mutants specific to other dnd genes directly on the wild-type s. lividans  <dig> chromosome failed for unknown reasons.
we therefore attempted to develop a mutation-integration system by first generating a complete set of in-frame deletions of individual dnd gene in vitro in e. coli.
these mutated dnd genes were then integrated back into the chromosome of s.lividans hxy <dig> .
a complete set of pset152-derived integration plasmids with targeted in-frame deletions of the five dnd genes was generated by pcr and cloned into e. coli .
these plasmids were introduced into hxy <dig> to obtain mutants xtg1-xtg <dig> with in-frame deletions in dnda-e in a uniform parental background.
isogenic mutant strains  were assayed for their dnd phenotype.
interestingly, while the dnd phenotype, as displayed by degradation of chromosomal  or plasmid phz <dig>  dna isolated from strains xtg <dig>  xtg <dig>  xtg <dig>  and xtg <dig>  was clearly abolished, dna isolated from xtg <dig> retained the dnd phenotype, clearly showing that dnda, c, d, and e are all essential for dna phosphorothioation.
single-stranded dna modification, which should be indicated by shifting of the covalently closed circular  to the open circular  form for plasmid phz <dig> dna if cleaved by the electrophoresis buffer, was not observed with these mutants , as also found for hxy <dig> .
a close comparison of the dnd phenotypes displayed by the wild-type  <dig> and the dndb mutant xtg <dig>  however, revealed a clear difference.
the degradation "smear" from the genomic dna of xtg <dig> migrated much faster than that from wild-type strain  <dig> .
smaller genomic dna fragments, or more frequently degraded genomic dnas, were observed in the mutant xtg <dig> than the wild-type strain  <dig>  in other words, the dndb mutation appeared to aggravate the dnd phenotype, in agreement with the enhanced dnd phenotype of another in-frame deletion mutant hxy <dig>  <cit> .
on the other hand, degradation of the circular plasmid phz <dig>  as shown by the relative intensities of the linearized phz <dig>  appeared to be more intense from xtg <dig> than from  <dig>  almost all the circular plasmid phz <dig> from xtg <dig> was degraded as linearized forms, but only about two-thirds of the circular plasmid phz <dig> from  <dig> was linearized .
rescue of the dnd phenotype of dnd mutants by complementation
the first direct evidence that the dnd phenotype, reflecting dna phosphorothioation, involves the combined action of five independent proteins  comes from complementation experiments using plasmids expressing individual dnd proteins.
this was achieved by the construction of individual dnd gene expression plasmids using phz <dig>  <cit> , an e. coli-streptomyces shuttle expression vector derived from pij <dig> with a strong thiostrepton-inducible ptipa promoter  <cit> .
firstly, dna fragments carrying individual dnda-e genes were cloned in-frame into phz <dig> to generate expression plasmids .
secondly, the expression plasmids were independently introduced by transformation into the corresponding mutant strains xtg <dig>   <dig>   <dig>   <dig>  and  <dig> .
even without induction of the ptipa promoter by addition of thiostrepton, strains xtg <dig>   <dig>   <dig>   <dig> carrying their counterpart expression plasmids recovered the dnd phenotype of the wild-type strain  <dig> , while xtg <dig> carrying pjtu <dig>  abolished enhanced dnd phenotype  with recovery of the original dnd phenotype  comparable with that of the wild-type strain  <dig> .
as additional evidence, we cloned dndd into pet15b to obtain an expression plasmid  for the production of an n-terminal his-tag fusion protein.
the purified dndd protein was then used for the production of rabbit anti-dndd polyclonal antibody.
when we used this antibody to detect native dndd protein expression, we observed identical bands with a size of  <dig>  kd in the expression strain xtg4/pjtu <dig>  and wild-type s. lividans  <dig> .
as a negative control, a  <dig> derivative with complete deletion of the dnd gene cluster  produced no signal in the corresponding position .
the protein size agrees well with our transcriptional analysis mentioned earlier and the dndd protein was correctly expressed in the complemented strain xtg4/pjtu <dig> .
these results suggest that all of the mutations in xtg1– <dig> are dnd-specific and the dnd proteins are correctly expressed in vivo.
in the meantime, these expression plasmids carrying individual dnd genes could also rescue the dnd phenotype of other corresponding mutants including hxy <dig> and hxy <dig> .
these complementation results from different mutants further support the conclusion that these mutations are not caused by secondary mutation elsewhere on the chromosome.
effect on cell viability by overdosage of dnd proteins in vivo
the above complementation assays for all of the dnd mutants were tested without induction by the addition of thiostrepton.
because each individual dnd gene is under the control of the thiostrepton-inducible promoter ptipa in the expression plasmids, a feature which could provide us a tool for testing the effect of the over-expressed dnd protein in cells, we induced dnd over-expression in the strains xtg1– <dig> carrying individual dnd gene expression plasmids by adding thiostrepton to a final concentration of  <dig> μg/ml in the normal culture medium.
surprisingly, xtg3/pjtu <dig>  and xtg4/pjtu <dig>  completely ceased growth, in sharp contrast to the normal growth of xtg1/pjtu <dig> , xtg2/pjtu <dig> , and xtg5/pjtu <dig> .
this result suggests that over-expression of dndc or dndd proteins in vivo has a detrimental effect on cell viability.
discussion
early predictions of genes involved in dna phosphorothioation and their organization as an operon within a region covering the cloned dnd gene cluster was mostly based on bioinformatic analysis, and no detailed experiments had been performed to provide direct evidence.
we refined the conclusions by first minimizing the responsible region to a ca.
<dig> -kb dna fragment carrying only five genes, which still retained the ability to confer the dnd phenotype on dnd- hosts.
we went on to confirm the expression of multiple and independent proteins encoded by an operon  using systematic mutagenesis either by targeted gene disruption and/or in-frame deletions internal to each protein.
we then introduced individual engineered constructs, each containing only one specific gene under the control of a common promoter, into the above mutants.
reversion of the dna shift from stable to degradation status or vice versa demonstrated unambiguously that dnda, c, d, and e are required in the biochemical pathway leading to the dnd phenotype.
the opposite effect of mutation in dndb to aggravate the dnd phenotype was at least partly attributed to the changes of the sequence recognition specificity surrounding the modification sites  <cit> .
the finding that excessive expression of dndc and dndd could affect cell growth and/or viability suggests that the in vivo level of the dnd system must be tightly regulated, consistent with our earlier observation that not all of the available sites could be modified  <cit> .
this agrees well with the fact that the dnd phenotype could only be detected when the entire dnd gene cluster was integrated into the chromosome or carried on a low copy-number plasmid in zx <dig>  but not on a plasmid with a copy-number of  <dig> or more  <cit> .
all of these phenomena suggested that either an unknown mechanism is present in the cell to tightly control dna phosphorothioation, or that over-expression of some of the proteins to override the regulation could be detrimental to the cells.
we propose that the dosage of the dnd proteins in the cells may not exceed the tolerable limit, and that the dnd proteins must be balanced so as to be expressed in a highly coordinated manner in the cells.
therefore, simultaneous and/or unbalanced over-expression of one or even all four of the dnd genes could seriously harm the cells, leading to inhibition of growth.
the present study, showing that strongly induced expression of dndd and dndc, but not the other dnd proteins, by the addition of thiostrepton, strongly suggests that these two proteins are the key determinants for the phenomenon.
being an iscs-like protein, dnda  <cit>  was suggested to provide sulfur via its l-cysteine desulfurase activity and to catalyze iron-sulfur cluster assembly of dndc  <cit> , probably by generating a persulfide  in dndc or dndd) in the modification process.
as such iscs-like proteins are also often required, as multi-functional proteins, for many other metabolic pathways  <cit> , the detrimental effect by over-expression of dndc and dndd could be attributed to deprivation of dnda which is vital for primary metabolism.
thus, the fact that dnda function could not be substituted by other iscs homologs, at least in s. lividans analyzed here, might be due to a failure of proper persulfide formation, which could subsequently be delivered to target the dna via dndc or dndd .
the exact mechanism of negative role of the over-expressed dndc and dndd proteins to cell viability remains, however, to be determined.
CONCLUSIONS
genetic determination of the dnd phenotype diagnostic for dna sulfur modification in s. lividans was unambiguously attributed to a  <dig> -bp dna region carrying five dnd genes, with dndb-e constituting an operon and dnda transcribed divergently.
mutations in each of four dnd genes  abolished the dnd phenotype while mutation of dndb aggravated the dnd phenotype.
the dnd phenotype of all mutants could be restored by complementation with the corresponding dnd gene, suggesting that they are essential for dna sulfur modification.
the fact that the cells ceased growth by overdosage of dndc or dndd in vivo suggests that the frequency of dna phosphorothioate modification is under strict control in the native host.

 most of the commonly found structural changes in dna are due to methylation of selected bases. in some viral dnas, certain bases may be hydroxymethylated or glucosylated. altered or unusual bases in dna molecules often have significant physiological implications, such as dna replication control, gene regulation, or protection of the respective organisms from invasion by foreign dna. in contrast to other types of dna modification, s, lividans has a site-specific and stereo-selective sulfur modification on the dna backbone termed phosphorothioation. this sulfur modification occurs specifically between two guanine nucleotides in s.lividans. the sulfur-modified dna suffers double-stranded cleavage at the modification sites during normal and pulsed-field gel electrophoresis. the dnd phenotype was proven to be a peracid-mediated, oxidative and amine-catalysed reaction to form a phosphorothioated dna backbone. the dnd phenotype can be overcome by replacing tris with hepes in the electrophoresis buffer or by adding a certain concentration of thiourea to tris-containing buffers. in s.lividans, this dna sulfur modification was found to be determined by a dnd gene cluster carrying five open reading frames. homologous dnd gene clusters and/or dnd phenotypes are found in many strains of streptomyces, e. coli, bacillus, salmonella, klebsiella, enterobacter, mycobacterium, vibrio, pseudomonas, pseudoalteromonas, hahella, oceanobacter, geobacter, pelagibacter, roseobacter, mesorhizobium, serratia, acinetobacter, and clostridium, as well as in certain archaea and unidentified marine microbes, indicating that dna sulfur modification is a widespread phenomenon in prokaryotes. here we attribute dna phosphorothioate modification to a dnd gene cluster consisting of a -bp region of dna carrying just five genes. we confirmed by transcriptional analysis that dndb-e constitute an operon, and made systematic in-frame deletion mutations within each gene or combinations of the five dnd genes before performing a series of complementation analyses to evaluate the roles of individual dnd genes in dna sulfur modification.  identification of a minimal dnd region in an effort to precisely localize the region responsible for the dnd phenotype and obtain unambiguous evidence on the genes involved in dna phosphorothioation, we made a series of phz derivatives by removing end segments from a ca. 10-kb fragment of dna carrying some likely cis-acting elements using convenient restriction sites, thus identifying a core region carrying only five dnd genes. a combination of restriction fragments was incorporated into appropriate sites of integrative vector pset to produce four plasmids. mediated by the attp site of streptomyces phage Øc present on pset these vectors can site-specifically integrate into the attb site in the chromosome of s. lividans zx after transfer by conjugation from e. coli et12567/puz into zx the dna of these zx1-derivative strains was either degraded or stable during electrophoresis. the minimal dnd region conferring the dnd phenotype was localised to a -bp fragment on pjtu the left and right borders of the minimal dnd cluster are only 4-bp and 472-bp from the stop codons of dnda and dnde, respectively, confirming that five genes are necessary and sufficient for dna phosphorothioation. transcriptional analysis of the dnd genes bioinformatic analysis of the -bp region of pjtu suggests that dnda and dndb-e are divergently transcribed. the facts that the 3' end of dndb and the 5' end of dndc overlap by bp, that the initiation codon of dndd precedes the 3' end of dndc by bp, and that the initiation codon of dnde is bp upstream of the stop codon of dndd strongly imply that the dndb-e might constitute an operon. to prove divergent transcription of dnda and a hypothetical dndb-e operon, we performed a transcriptional analysis on the minimal dnd cluster by rt-pcr. rna was extracted from s. lividans and amplified by rt-pcr using oligonucleotide primers depicted in fig. 2a. the pcr products were fractionated by electrophoresis. as an internal control, 16s rrna was amplified in all samples. the appearance of dna bands, which were amplified using different sets of primers, unambiguously suggests that dndb-e are co-transcribed as a single operon in s. lividans the absence of dna bands using primers a and b suggests a lack of co-transcription in the region between a and b confirming independent transcription of dnda and dndb-e. a mutation-integration system for functional analysis of individual dnd genes as demonstrated by the transcriptional analysis, dndb-e constitute an operon. we therefore inactivated each of the five dnd genes independently to examine their effect on the dnd phenotype in terms of dna phosphorothioation. early experiments on disruption of dnda and dndd by a str/spc cassette clearly abolished the dnd phenotype but could not provide unambiguous evidence for the function of dndd as insertion of antibiotic resistant genes could block expression of downstream gene of an operon by a polar effect. we attempted to make in-frame deletions internal to individual dnd genes at their corresponding chromosomal loci to avoid polar effects. apart from the dndb in-frame deletion mutant hxy, extensive efforts to obtain mutants specific to other dnd genes directly on the wild-type s. lividans chromosome failed for unknown reasons. we therefore attempted to develop a mutation-integration system by first generating a complete set of in-frame deletions of individual dnd gene in vitro in e. coli. these mutated dnd genes were then integrated back into the chromosome of s.lividans hxy. a complete set of pset152-derived integration plasmids with targeted in-frame deletions of the five dnd genes was generated by pcr and cloned into e. coli. these plasmids were introduced into hxy to obtain mutants xtg1-xtg with in-frame deletions in dnda-e in a uniform parental background. isogenic mutant strains were assayed for their dnd phenotype. interestingly, while the dnd phenotype, as displayed by degradation of chromosomal or plasmid phz dna isolated from strains xtg xtg xtg and xtg was clearly abolished, dna isolated from xtg retained the dnd phenotype, clearly showing that dnda, c, d, and e are all essential for dna phosphorothioation. single-stranded dna modification, which should be indicated by shifting of the covalently closed circular to the open circular form for plasmid phz dna if cleaved by the electrophoresis buffer, was not observed with these mutants, as also found for hxy. a close comparison of the dnd phenotypes displayed by the wild-type and the dndb mutant xtg however, revealed a clear difference. the degradation "smear" from the genomic dna of xtg migrated much faster than that from wild-type strain. smaller genomic dna fragments, or more frequently degraded genomic dnas, were observed in the mutant xtg than the wild-type strain in other words, the dndb mutation appeared to aggravate the dnd phenotype, in agreement with the enhanced dnd phenotype of another in-frame deletion mutant hxy. on the other hand, degradation of the circular plasmid phz as shown by the relative intensities of the linearized phz appeared to be more intense from xtg than from almost all the circular plasmid phz from xtg was degraded as linearized forms, but only about two-thirds of the circular plasmid phz from was linearized. rescue of the dnd phenotype of dnd mutants by complementation the first direct evidence that the dnd phenotype, reflecting dna phosphorothioation, involves the combined action of five independent proteins comes from complementation experiments using plasmids expressing individual dnd proteins. this was achieved by the construction of individual dnd gene expression plasmids using phz, an e. coli-streptomyces shuttle expression vector derived from pij with a strong thiostrepton-inducible ptipa promoter. firstly, dna fragments carrying individual dnda-e genes were cloned in-frame into phz to generate expression plasmids. secondly, the expression plasmids were independently introduced by transformation into the corresponding mutant strains xtg and. even without induction of the ptipa promoter by addition of thiostrepton, strains xtg carrying their counterpart expression plasmids recovered the dnd phenotype of the wild-type strain, while xtg carrying pjtu abolished enhanced dnd phenotype with recovery of the original dnd phenotype comparable with that of the wild-type strain. as additional evidence, we cloned dndd into pet15b to obtain an expression plasmid for the production of an n-terminal his-tag fusion protein. the purified dndd protein was then used for the production of rabbit anti-dndd polyclonal antibody. when we used this antibody to detect native dndd protein expression, we observed identical bands with a size of kd in the expression strain xtg4/pjtu and wild-type s. lividans. as a negative control, a derivative with complete deletion of the dnd gene cluster produced no signal in the corresponding position. the protein size agrees well with our transcriptional analysis mentioned earlier and the dndd protein was correctly expressed in the complemented strain xtg4/pjtu. these results suggest that all of the mutations in xtg1– are dnd-specific and the dnd proteins are correctly expressed in vivo. in the meantime, these expression plasmids carrying individual dnd genes could also rescue the dnd phenotype of other corresponding mutants including hxy and hxy. these complementation results from different mutants further support the conclusion that these mutations are not caused by secondary mutation elsewhere on the chromosome. effect on cell viability by overdosage of dnd proteins in vivo the above complementation assays for all of the dnd mutants were tested without induction by the addition of thiostrepton. because each individual dnd gene is under the control of the thiostrepton-inducible promoter ptipa in the expression plasmids, a feature which could provide us a tool for testing the effect of the over-expressed dnd protein in cells, we induced dnd over-expression in the strains xtg1– carrying individual dnd gene expression plasmids by adding thiostrepton to a final concentration of μg/ml in the normal culture medium. surprisingly, xtg3/pjtu and xtg4/pjtu completely ceased growth, in sharp contrast to the normal growth of xtg1/pjtu, xtg2/pjtu, and xtg5/pjtu. this result suggests that over-expression of dndc or dndd proteins in vivo has a detrimental effect on cell viability. discussion early predictions of genes involved in dna phosphorothioation and their organization as an operon within a region covering the cloned dnd gene cluster was mostly based on bioinformatic analysis, and no detailed experiments had been performed to provide direct evidence. we refined the conclusions by first minimizing the responsible region to a ca. -kb dna fragment carrying only five genes, which still retained the ability to confer the dnd phenotype on dnd- hosts. we went on to confirm the expression of multiple and independent proteins encoded by an operon using systematic mutagenesis either by targeted gene disruption and/or in-frame deletions internal to each protein. we then introduced individual engineered constructs, each containing only one specific gene under the control of a common promoter, into the above mutants. reversion of the dna shift from stable to degradation status or vice versa demonstrated unambiguously that dnda, c, d, and e are required in the biochemical pathway leading to the dnd phenotype. the opposite effect of mutation in dndb to aggravate the dnd phenotype was at least partly attributed to the changes of the sequence recognition specificity surrounding the modification sites. the finding that excessive expression of dndc and dndd could affect cell growth and/or viability suggests that the in vivo level of the dnd system must be tightly regulated, consistent with our earlier observation that not all of the available sites could be modified. this agrees well with the fact that the dnd phenotype could only be detected when the entire dnd gene cluster was integrated into the chromosome or carried on a low copy-number plasmid in zx but not on a plasmid with a copy-number of or more. all of these phenomena suggested that either an unknown mechanism is present in the cell to tightly control dna phosphorothioation, or that over-expression of some of the proteins to override the regulation could be detrimental to the cells. we propose that the dosage of the dnd proteins in the cells may not exceed the tolerable limit, and that the dnd proteins must be balanced so as to be expressed in a highly coordinated manner in the cells. therefore, simultaneous and/or unbalanced over-expression of one or even all four of the dnd genes could seriously harm the cells, leading to inhibition of growth. the present study, showing that strongly induced expression of dndd and dndc, but not the other dnd proteins, by the addition of thiostrepton, strongly suggests that these two proteins are the key determinants for the phenomenon. being an iscs-like protein, dnda was suggested to provide sulfur via its l-cysteine desulfurase activity and to catalyze iron-sulfur cluster assembly of dndc, probably by generating a persulfide in dndc or dndd) in the modification process. as such iscs-like proteins are also often required, as multi-functional proteins, for many other metabolic pathways, the detrimental effect by over-expression of dndc and dndd could be attributed to deprivation of dnda which is vital for primary metabolism. thus, the fact that dnda function could not be substituted by other iscs homologs, at least in s. lividans analyzed here, might be due to a failure of proper persulfide formation, which could subsequently be delivered to target the dna via dndc or dndd. the exact mechanism of negative role of the over-expressed dndc and dndd proteins to cell viability remains, however, to be determined.  genetic determination of the dnd phenotype diagnostic for dna sulfur modification in s. lividans was unambiguously attributed to a -bp dna region carrying five dnd genes, with dndb-e constituting an operon and dnda transcribed divergently. mutations in each of four dnd genes abolished the dnd phenotype while mutation of dndb aggravated the dnd phenotype. the dnd phenotype of all mutants could be restored by complementation with the corresponding dnd gene, suggesting that they are essential for dna sulfur modification. the fact that the cells ceased growth by overdosage of dndc or dndd in vivo suggests that the frequency of dna phosphorothioate modification is under strict control in the native host. 
a detailed mutational analysis of the individual genes within the dnd locus in streptomyces lividans responsible for dna phosphorothioation was performed and is described here. dna phosphorothioation is a multi-enzymatic and highly coordinated process controlled by five dnd genes. a -bp dna region carrying just five orfs was defined as the sole determinant for modification of the dna backbone in s. lividans to form phosphorothioate. interestingly, overdosage of dndc or dndd, but not other dnd proteins, in vivo was found to be detrimental to cell viability. overexpression of some proteins in vivo prevented growth of host strain, suggesting that expression of the gene cluster is strictly regulated in the native host. an efficient mutation-integration-complementation system was developed to allow for detailed functional analysis of these dnd genes. the dnd- phenotype caused by specific in-frame deletion of the dnda, c, d, and e genes or the enhanced dnd phenotype resulting from in-frame deletion of dndb could be restored by expression vectors carrying the corresponding dnd genes. while dnda is clearly transcribed independently, dndb-e constitute an operon, as revealed by rt-pcr analysis.
 a novel dna phosphorothioate modification, in which one of the non-bridging oxygen atoms in the phosphodiester bond linking dna nucleotides is exchanged by sulphur, was found to be genetically determined by dnd or dnd-counterpart loci in a wide spectrum of bacteria from diverse habitats. a detailed mutational analysis of the individual genes within the dnd locus in streptomyces lividans responsible for dna phosphorothioation was performed and is described here. it should be of great help for the mechanistic study of this intriguing system.  a -bp dna region carrying just five orfs was defined as the sole determinant for modification of the dna backbone in s. lividans to form phosphorothioate. this provides a diagnostically reliable and easily assayable dnd phenotype. while dnda is clearly transcribed independently, dndb-e constitute an operon, as revealed by rt-pcr analysis. an efficient mutation-integration-complementation system was developed to allow for detailed functional analysis of these dnd genes. the dnd- phenotype caused by specific in-frame deletion of the dnda, c, d, and e genes or the enhanced dnd phenotype resulting from in-frame deletion of dndb could be restored by expression vectors carrying the corresponding dnd genes. interestingly, overdosage of dndc or dndd, but not other dnd proteins, in vivo was found to be detrimental to cell viability.  dna phosphorothioation is a multi-enzymatic and highly coordinated process controlled by five dnd genes. overexpression of some proteins in vivo prevented growth of host strain, suggesting that expression of the gene cluster is strictly regulated in the native host. 