Identification ofStreptomyces violaceoruber Tü22 genes involved in the biosynthesis of granaticin

A 50 kb region of DNA fromStreptomyces violaceoruber Tü22, containing genes encoding proteins involved in the biosynthesis of granaticin, was isolated. The DNA sequence of a 7.3 kb fragment from this region, located approximately 10 kb from the genes that encode the polyketide synthetase responsible for formation of the benzoisochromane quinone skeleton, revealed five open reading frames (ORF1-ORF5). The deduced amino acid sequence of GraE, encoded by ORF2, shows 60.8% identity (75.2% similarity) to a dTDP-glucose dehydratase (StrE) fromStreptomyces griseus. Cultures ofEscherichia coli containing plasmids with ORF2, on a 2.1 kbBamHI fragment, were able to catalyze the formation of dTDP-4-keto-6-deoxy-d-glucose from dTDP-glucose at 5 times the rate of control cultures, confirming that ORF2 encodes a dTDP-glucose dehydratase. The amino acid sequence encoded by ORF3 (GraD) is 51.4% identical (69.9% similar) to that of StrD, a dTDP-glucose synthase fromStreptomyces griseus. The amino acid sequence encoded by ORF4 shares similarities with proteins that confer resistance to tetracycline and methylenomycin, and is suggested to be involved in transporting granaticin out of the cells by an active efflux mechanism.     

Saccharopolyspora hirsuta 367 encodes clustered genes similar to ketoacyl synthase,ketoacyl reductase,acyl carrier protein,and biotin carboxyl carrier protein

The actI gene, encoding a component of the actinorhodin polyketide synthase of Streptomyces coelicolor, was used to identify and clone a homologous 11.7 kb BamHI DNA fragment from Saccharopolyspora hirsuta 367. The cloned fragment complemented actinorhodin production in a strain of Streptomyces coelicolor bearing a mutant actI gene. The DNA sequence of a 5.1 kb fragment revealed 6 open reading frames (ORF). ORF1 does not resemble any known DNA or deduced protein sequence, while the deduced protein sequence of ORF2 resembles that of biotin carboxyl carrier proteins. Based on the similarity to deduced protein sequences from cloned genes of polyketide producers, ORF3 would code for a ketoreductase, ORF4 and ORF5 for the putative heterodimeric β-ketoacyl synthase, and ORF6 for an acyl carrier protein.     

Identification of Streptomyces violaceoruber Tü22 genes involved in the biosynthesis of granaticin

A 50 kb region of DNA fromStreptomyces violaceoruber Tü22, containing genes encoding proteins involved in the biosynthesis of granaticin, was isolated. The DNA sequence of a 7.3 kb fragment from this region, located approximately 10 kb from the genes that encode the polyketide synthetase responsible for formation of the benzoisochromane quinone skeleton, revealed five open reading frames (ORF1-ORF5). The deduced amino acid sequence of GraE, encoded by ORF2, shows 60.8% identity (75.2% similarity) to a dTDP-glucose dehydratase (StrE) fromStreptomyces griseus. Cultures ofEscherichia coli containing plasmids with ORF2, on a 2.1 kbBamHI fragment, were able to catalyze the formation of dTDP-4-keto-6-deoxy-d-glucose from dTDP-glucose at 5 times the rate of control cultures, confirming that ORF2 encodes a dTDP-glucose dehydratase. The amino acid sequence encoded by ORF3 (GraD) is 51.4% identical (69.9% similar) to that of StrD, a dTDP-glucose synthase fromStreptomyces griseus. The amino acid sequence encoded by ORF4 shares similarities with proteins that confer resistance to tetracycline and methylenomycin, and is suggested to be involved in transporting granaticin out of the cells by an active efflux mechanism. Dedicated to Professor Satoshi Ōmura, a pioneer in the field of antibiotics, on the occasion of his 60th birthday     

Cloning of the gene encoding avermectin B 5-O-methyltransferase in avermectin-producing Streptomyces avermitilis

Complementation of a mutant lacking avermectin B 5-O-methyltransferase (AveD) of Streptomyces avermitilis, which catalyses the methylation of the hydroxyl group at the C5 position of avermectin B compounds, revealed that the gene encoding AveD is in a 1.25-kb SalI–EcoNI fragment in the left region of the gene cluster for avermectin biosynthesis. The nucleotide sequence of this fragment predicted a 283-aa gene product homologous to several methyltransferases requiring S-adenosyl-l-methionine as a cofactor. After cloning of the aveD region from mutant not producing AveD, the complementation experiments were performed using a pair of hybrid fragments (AveD⁺/AveD⁻ and AveD⁻/AveD⁺). They suggest that the mutation(s) is in the N-terminus of AveD. SSCP analysis of amplified DNA of the aveD region derived from both wild type and mutant strains supports the results of the complementation experiments. Sequence analysis of the aveD region of the mutant strain revealed that a point mutation is within ORF, being Thr23→Ile substitution. This mutation causes the inactivation of O-methyltransferase activity of AveD.     

Saccharopolyspora hirsuta 367 encodes clustered genes similar to ketoacyl synthase, ketoacyl reductase, acyl carrier protein, and biotin carboxyl carrier protein

The actI gene, encoding a component of the actinorhodin polyketide synthase of Streptomyces coelicolor, was used to identify and clone a homologous 11.7 kb BamHI DNA fragment from Saccharopolyspora hirsuta 367. The cloned fragment complemented actinorhodin production in a strain of Streptomyces coelicolor bearing a mutant actI gene. The DNA sequence of a 5.1 kb fragment revealed 6 open reading frames (ORF). ORF1 does not resemble any known DNA or deduced protein sequence, while the deduced protein sequence of ORF2 resembles that of biotin carboxyl carrier proteins. Based on the similarity to deduced protein sequences from cloned genes of polyketide producers, ORF3 would code for a ketoreductase, ORF4 and ORF5 for the putative heterodimeric -ketoacyl synthase, and ORF6 for an acyl carrier protein.     

Cloning and Analysis of a DNA Fragment Stimulating Avermectin Production in Various Streptomyces avermitilis Strains

To isolate a gene for stimulating avermectin production, a genomic library of Streptomyces avermitilis ATCC 31267 was constructed in Streptomyces lividans TK21 as the host strain. An 8.0-kb DNA fragment that significantly stimulated actinorhodin and undecylprodigiosin production was isolated. When wild-type S. avermitilis was transformed with the cloned fragment, avermectin production increased approximately 3.5-fold. The introduction of this fragment into high-producer (ATCC 31780) and semi-industrial (L-9) strains also resulted in an increase of avermectin production by more than 2.0- and 1.4-fold, respectively. Subclones were studied to locate the minimal region involved in stimulation of pigmented-antibiotic and avermectin production. An analysis of the nucleotide sequence of the entire DNA fragment identified eight complete and one incomplete open reading frame. All but one of the deduced proteins exhibited strong homology (68 to 84% identity) to the hypothetical proteins of Streptomyces coelicolor A3(2). The orfX gene product showed no significant similarity to any other protein in the databases, and an analysis of its sequence suggested that it was a putative membrane protein. Although the nature of the stimulatory effect is still unclear, the disruption of orfX revealed that this gene was intrinsically involved in the stimulation of avermectin production in S. avermitilis.     

Molecular analysis of some genes from plasmid p19 of the Bacillus subtilis 19 soil strain involved in conjugation

Two fragments of conjugative plasmid p19 (95 kb) from the Bacillus subtilis 19 soil strain were cloned and sequenced; these fragments carry genes, products of which are indispensable for the conjugative transfer. One of the fragments 4518 bp in size carries five open reading frames and their fragments (ORF1–ORF5). The protein corresponding to ORF4 is homologous to proteins from the family VirD4. Inactivation of ORF4 and ORF1 by insertional mutagenesis caused a three-to-fivefold decrease in the frequency of plasmid p19 conjugative transfer. Another 2932-bp fragment of p19 was shown to possess a rep region homologous to the rep region of plasmid pBS72 from the B. subtilis 72 soil strain and a novel ORF (ORF6); the protein corresponding to this ORF contains the HTH motif typical for DNA-binding proteins.     

Cloning and expression of a gene from Streptomyces scabies encoding a putative pathogenicity factor.

We cloned a 9.4-kb DNA fragment from Streptomyces scabies ATCC 41973 that allows the nonpathogen Streptomyces lividans 66 TK24 to necrotize and colonize potato tuber slices and produce scab-like symptoms on potato minitubers. Deletion analysis demonstrated that activity was conferred by a 1.6-kb DNA region. Sequence analysis of a 2.4-kb DNA fragment spanning the DNA region necessary for activity revealed three open reading frames (ORFs). The deduced amino acid sequence of ORF1, designated ORFtnp, showed high levels of identity with the first 233 amino acids of the putative transposases of the IS1164 elements from Rhodococcus rhodochrous (71%) and Mycobacterium bovis (68%), members of the Staphylococcus aureus IS256 family of transposases. No significant homologies to ORF2 and ORF3 were found in the nucleic acid and protein databases. ORFtnp is located 5' of ORF3. ORF2 is incomplete and is located 3' of ORF3. Subcloning of the individual ORFs demonstrated that ORF3, designated nec1, is sufficient for necrotizing activity in S. lividans 66 TK24. S. lividans 66 TK24 expressing nec1 does not produce thaxtomin A but produces an unidentified extracellular water-soluble compound that causes necrosis on potato tuber discs. The G+C content of nec1 suggests that it has moved horizontally from another genus. Southern analysis of ORFtnp and nec1 demonstrate that these genes are physically linked in Streptomyces strains, including S. scabies and Streptomyces acidiscabies strains, that are pathogenic on potato and that produce the phytotoxin thaxtomin A. These data suggest that nec1 may have been mobilized into S. scabies through a transposition event mediated by ORFtnp.     

Sequence Analysis of the Cryptic Plasmid pMG101 from Rhodopseudomonas palustris and Construction of Stable Cloning Vectors

A 15-kb cryptic plasmid was obtained from a natural isolate of Rhodopseudomonas palustris. The plasmid, designated pMG101, was able to replicate in R. palustris and in closely related strains of Bradyrhizobium japonicum and phototrophic Bradyrhizobium species. However, it was unable to replicate in the purple nonsulfur bacterium Rhodobacter sphaeroides and in Rhizobium species. The replication region of pMG101 was localized to a 3.0-kb SalI-XhoI fragment, and this fragment was stably maintained in R. palustris for over 100 generations in the absence of selection. The complete nucleotide sequence of this fragment revealed two open reading frames (ORFs), ORF1 and ORF2. The deduced amino acid sequence of ORF1 is similar to sequences of Par proteins, which mediate plasmid stability from certain plasmids, while ORF2 was identified as a putative rep gene, coding for an initiator of plasmid replication, based on homology with the Rep proteins of several other plasmids. The function of these sequences was studied by deletion mapping and gene disruptions of ORF1 and ORF2. pMG101-based Escherichia coli-R. palustris shuttle cloning vectors pMG103 and pMG105 were constructed and were stably maintained in R. palustris growing under nonselective conditions. The ability of plasmid pMG101 to replicate in R. palustris and its close phylogenetic relatives should enable broad application of these vectors within this group of α-proteobacteria.     

Complete Sequence of the Enterocin Q-Encoding Plasmid pCIZ2 from the Multiple Bacteriocin Producer Enterococcus faecium L50 and Genetic Characterization of Enterocin Q Production and Immunity

The locations of the genetic determinants for enterocin L50 (EntL50A and EntL50B), enterocin Q (EntQ), and enterocin P (EntP) in the multiple bacteriocin producer Enterococcus faecium strain L50 were determined. These bacteriocin genes occur at different locations; entL50AB (encoding EntL50A and EntL50B) are on the 50-kb plasmid pCIZ1, entqA (encoding EntQ) is on the 7.4-kb plasmid pCIZ2, and entP (encoding EntP) is on the chromosome. The complete nucleotide sequence of pCIZ2 was determined to be 7,383 bp long and contains 10 putative open reading frames (ORFs) organized in three distinct regions. The first region contains three ORFs: entqA preceded by two divergently oriented genes, entqB and entqC. EntqB shows high levels of similarity to bacterial ATP-binding cassette (ABC) transporters, while EntqC displays no significant similarity to any known protein. The second region encompasses four ORFs (orf4 to orf7), and ORF4 and ORF5 display high levels of similarity to mobilization proteins from E. faecium and Enterococcus faecalis. In addition, features resembling a transfer origin region (oriT) were found in the promoter area of orf4. The third region contains three ORFs (orf8 to orf10), and ORF8 and ORF9 exhibit similarity to the replication initiator protein RepE from E. faecalis and to RepB proteins, respectively. To clarify the minimum requirement for EntQ synthesis, we subcloned and heterologously expressed a 2,371-bp fragment from pCIZ2 that encompasses only the entqA, entqB, and entqC genes in Lactobacillus sakei, and we demonstrated that this fragment is sufficient for EntQ production. Moreover, we also obtained experimental results indicating that EntqB is involved in ABC transporter-mediated EntQ secretion, while EntqC confers immunity to this bacteriocin.     

Gene controlled by promoter--PTH4 depending on whiG of Streptomyces coelicolor

The downstream gene controlled by promoter--P_TH4 which is related to Streptomyces differentiation was cloned, and its sequence was determined by the dideoxy chain termination method. The results indicated that the 1597 bp of DNA fragment conferred a complete open reading frame (ORF). In searches of databases, the deduced product of the ORF was not homologous with any known proteins; it may be a new protein. The function of the gene was studied using the strategy of gene disruption; the actinorhodin could not be produced when this gene was disrupted. Therefore, this gene may be related to actinorhodin biosynthesis in Streptomyces coelicolor, and the result also shows that this gene may play a role in multiple level regulation of differentiation genes in Streptomyces.     

Cloning of an avilamycin biosynthetic gene cluster from Streptomyces viridochromogenes Tü57.

A 65-kb region of DNA from Streptomyces viridochromogenes Tü57, containing genes encoding proteins involved in the biosynthesis of avilamycins, was isolated. The DNA sequence of a 6.4-kb fragment from this region revealed four open reading frames (ORF1 to ORF4), three of which are fully contained within the sequenced fragment. The deduced amino acid sequence of AviM, encoded by ORF2, shows 37% identity to a 6-methylsalicylic acid synthase from Penicillium patulum. Cultures of S. lividans TK24 and S. coelicolor CH999 containing plasmids with ORF2 on a 5.5-kb PstI fragment were able to produce orsellinic acid, an unreduced version of 6-methylsalicylic acid. The amino acid sequence encoded by ORF3 (AviD) is 62% identical to that of StrD, a dTDP-glucose synthase from S. griseus. The deduced amino acid sequence of AviE, encoded by ORF4, shows 55% identity to a dTDP-glucose dehydratase (StrE) from S. griseus. Gene insertional inactivation experiments of aviE abolished avilamycin production, indicating the involvement of aviE in the biosynthesis of avilamycins.     

The Linear Plasmid SCP1 of Streptomyces coelicolor A3(2) Possesses a Centrally Located Replication Origin and Shows Significant Homology to the Transposon Tn4811

The linear plasmid SCP1 of Streptomyces coelicolor A3(2) is one of the genetically more studied linear streptomycete replicons. Although the genetics of SCP1 and its interaction with the host chromosome have been analyzed for nearly three decades no information exists on its replication. With the help of an ordered cosmid contig for the complete 360-kb element, we have localized a 5439-bp fragment from the central region that confers autonomous replication in Streptomyces lividans. The minimal origin contains two overlapping ORFs which are separated from an AT-rich region which might correspond to the replication start point. ORF1 revealed intensive similarity to a class of DNA-primase/helicases of actinophages and archael plasmids. In addition, we have identified a region in both terminal inverted repeats of SCP1 that shows significant homology to the transposable element Tn4811 located near the ends of the S. lividans 66 chromosome.     

A novel gene—samfR involved in early stage ofStreptomyces ansochromogenes differentiation

A 4.6 kb DNA fragment was cloned from the DNA library ofStreptomyces ansochromogenes using a partial DNA fragment located in the downstream of promoter-P_TH4 as probe. The experiments revealed that this DNA fragment consists ofsaw D gene and a 1.4 kbPvu II fragment which can accelerate mycelium formation ofS. ansochromogenes. The nucleotide sequence of 1.4 kb DNA fragment was determined and analysed; the result indicated that the fragment contains one complete open reading frame (ORF) which encodes a protein with 213 amino acids, and this gene was designated assamfR. The deduced protein has 36% amino acid identities and 52% amino acid similarities in comparison with that encoded byhppR gene, which is involved in the regulation of catabolism for 3-(3-hydroxyphenyl) propionate (3HPP) inRhodococcus globerulus. The function ofsamfR gene was studied using strategy of gene disruption, and the resultingsamfR mutant failed to form aerial hyphae and spores, its development and differentiation stopped at the stage of substrate mycelium in contrast with wild type strain. The results showed that thesamfR gene is closely related toS. ansochromogenes differentiation.     

The heterocyst-specific fdxH gene product of the cyanobacterium Anabaena sp. PCC 7120 is important but not essential for nitrogen fixation

To clarify the role of the heterocyst-specific [2Fe-2S] ferredoxin in cyanobacterial nitrogen fixation, mutational analysis of the Anabaena 7120 fdxH gene region was carried out. First, the DNA sequence of the wild-type 3509-bp EcoRI fragment downstream of the fdxH gene was determined. Genes homologous to ORF3 from the fdxH gene regions of A. variabilis and Plectonemaboryanum, the mop genes of Clostridiumpasteurianum encoding molybdo-pterin binding proteins, and ORF3 from the A. variabilis hydrogenase gene cluster were identified within the sequenced region. For mutational analysis the Anabaena 7120 mutant strains LAK4, BMB92, and KSH10 were constructed. In LAK4 the fdxH coding region is disrupted by an interposon, whereas BMB92 is deleted for a 2799-bp NheI fragment encompassing fdxH, ORF3, mop, ORF4, and ORF5. Mutant strain KSH10 is a derivative of BMB92, complemented for fdxH but not for the other genes located further downstream. Analysis of the Nif phenotype of these mutant strains showed that FdxH is necessary for maximum nitrogenase activity and optimal growth under nitrogen-fixing conditions, but not absolutely essential for diazotrophic growth. The role of alternative electron donors for nitrogenase, which might substitute for FdxH, is discussed. Iron concentrations (1μM Fe) sufficient to induce synthesis of the vegetative cell flavodoxin did not stimulate diazotrophic growth of the fdxH mutant strains, suggesting that FdxH was not replaced by a NifJ-flavodoxin system. Comparison of LAK4 and BMB92 indicated that one of the genes located downstream of fdxH might also play a (minor) role in nitrogen fixation.     

A method for the identification of promoters recognized by RNA polymerase containing a particular sigma factor: cloning of a developmentally regulated promoter and corresponding gene directed by the Streptomyces aureofaciens sigma factor RpoZ

We have developed a method for the identification of promoters recognized by a particular sigma factor of RNA polymerase, based on a two-compatible plasmid system in Escherichia coli (Ec). Using the method, a DNA fragment containing the promoter, P_REN40, recognized by sporulation-specific Streptomyces aureofaciens (Sa) sigma factor RpoZ, was cloned. High-resolution S1 nuclease mapping using RNA prepared from Ec, and Sa from various developmental stages has shown a high degree of similarity of P_REN40 to consensus sequence of flagellar and chemotaxis promoters. The promoter was induced at the time of aerial mycelium formation, and was off in the Sa strain with the rpoZ-disrupted gene. A promoter-bearing DNA fragment was inserted into the promoter-probe plasmid pARC1 to give expression patterns consistent with the results of direct RNA analysis. The region downstream of the promoter was cloned in Sa. Sequence analysis revealed an open reading frame (ORF) of 283 amino acids (M_r 30 006), encoding a highly basic (pI 12.35) protein with high percentage of serine, threonine and alanine (41.8%).     

Molecular characterization of a gene from Saccharopolyspora erythraea (Streptomyces erythraeus) which is involved in erythromycin biosynthesis

A 7.3 kbp DNA fragment, encompassing the erythromycin (Em) resistance gene (ermE) and a portion of the gene cluster encoding the biosynthetic genes for erythromycin biosynthesis in Saccharopolyspora erythraea (formerly Streptomyces erythraeus) has been cloned in Streptomyces lividans using the plasmid vector pIJ702, and its nucleotide sequence has been determined using a modified dideoxy chain-termination procedure. In particular, we have examined the region immediately 5′ of the resistance determinant, where the tandem promoters for ermE overlap the promoters for a divergently transcribed coding sequence (ORF). Disruption of this ORF using an integrational pIJ702-based plasmid vector gave mutants which were specifically blocked in erythromycin biosynthesis, and which accumulated 3-O-α-L-mycarosylerythronolide B: this behaviour is identical to that of previously described eryC1 mutants. The eryC1-gene product, a protein of subunit M_r 39200, is therefore involved either as a structural or as a regulatory gene in the formation of the deoxyamino-sugar desosamine or in its attachment to the macro-lide ring.     

Menaquinone (vitamin K2) biosynthesis: localization and characterization of the menE gene from Escherichia coli

In Escherichia coli, the biosynthesis of the electron carrier menaquinone (vitamin K₂) involves at least seven identified enzymatic activities, five of which are encoded in the men cluster. One of these, the conversion of o-succinylbenzoic acid to 1,4-dihydroxy-2-naphthoic acid, requires the formation of o-succinylbenzoyl-CoA (OSB-CoA) as an intermediate. Formation of the intermediate is mediated by OSB-CoA synthetase encoded by the menE locus known to be located either 5′ of menB, or 3′ of menC. A DNA fragment overlapping the 3′ end of menC is shown by enzymatic complementation to elevate OSB-CoA synthetase activity. Nucleotide sequence analysis of the fragment identified a 1.355-kb open reading frame (ORF) which, when deleted at either the 5′ or 3′ end, failed to generate increased enzymatic activity. The ORF is preceded by a consensus ribosome-binding site, but no apparent σ⁷⁰ promoter. An oppositely transcribed unidentified gene cluster follows the menE ORF. The region 5′ of menB contains an additional ORF of unknown function (orf241) and establishes the order of genes in the men cluster as menD, orf241, menB, menC and menE. All loci are transcribed counter-clockwise.     

An ABC-transporter from Streptomyces longisporoflavus confers resistance to the polyether-ionophore antibiotic tetronasin

Streptomyces longisporoflavus produces the poly-ketide-polyether antibiotic, tetronasin, which acts as an ionophore and depolarizes the membrane of bacteria sensitive to the drug. A genomic library of S. longisporoflavus DN A was cloned in Streptomyces Uvldans and screened to identify tetronasin-resistance determinants. The inclusion of 0.2 M NaCl in the growth medium with tetronasin markedly improved the sensitivity of the screen. Two different resistance determinants, designated tnrB (ptetR51) and tnrA (ptetR11) respectively, were identified. The determinant tnrB (ptetR51) but not tnrA (ptetR11), also conferred resistance to tetronasin when cloned into Streptomyces albus. The tnrB determinant was further localized, by subcloning, to a 2.8 kb Kpnl fragment. DNA sequence analysis of this insert revealed one incomplete and two complete open reading frames (ORFs 1, 2 and 3). The deduced sequence of the gene product of ORF2 (TnrB2) revealed significant similarity to the ATP-binding domains of the ABC (A TP b inding c assette) superfamily of transport-related proteins. The adjacent gene, ORF3, is translationally coupled to ORF2 and would encode a hydrophobic protein (TnrB3) with six transmembrane helices which probably constitutes the integral membrane component of the transporter. The mechanism of tetronasin resistance mediated by tnrB is probably an ATP-dependent efflux system.