| a new family of bacterial regulatory proteins. | a new family of bacterial regulatory proteins has been identified by sequence similarity. the family contains the repressor of the bacillus subtilis gluconate operon (gntr), the regulators for histidine utilization in pseudomonas putida (hutcpp) and klebsiella aerogenes (hutcka), the repressor (fadr) of fatty acid degradation in escherichia coli, a regulator involved in the conjugal transfer of the broad host range plasmid pij101 (kora), and three proteins of unidentified function in e. coli (ge ... | 1991 | 2060763 |
| mutational and functional analysis of the kora and korb gene products of streptomyces plasmid pij101. | the kora and korb loci of streptomyces lividans plasmid pij101 have previously been shown to control expression of the pij101 tra (formerly kila) and kilb genes at the transcriptional level. we show here that mutations in translational open reading frames (orfs) that map within the kor loci abolish repression of the s. lividans lac gene directed by the tra and kilb promoters. introduction of the kora and korb orfs into escherichia coli maxicells under control of an e. coli promoter gave rise to ... | 1990 | 2274034 |
| expression of a streptomyces plasmid promoter in escherichia coli. | a 166-bp dna fragment from the streptomyces multicopy plasmid pij101 with in vivo promoter activity both in streptomyces lividans and in escherichia coli was isolated. the start point of the rna transcribed from this fragment, determined by high resolution s1 nuclease mapping, was the same in s. lividans and in e. coli. this suggests that the e. coli rna polymerase recognizes the same sequence determinants and chooses the point of initiation of rna synthesis in the same way as the corresponding ... | 1986 | 2427388 |
| transfer of conjugative plasmids and mobilization of a nonconjugative plasmid between streptomyces strains on agar and in soil. | the conjugative plasmid pij101 and its conjugative nondeletion derivatives pij303 and pij211 were tested for their transferability between strains of streptomyces on laboratory media and in the soil environment. their roles in the mobilization of the cloning vector plasmid pij702, a nonconjugative deletion derivative of pij101, were also examined. biparental and triparental crosses were performed on agar slants and in sterile soil between the plasmid donor streptomyces lividans and several recip ... | 1988 | 2843092 |
| new multifunctional escherichia coli-streptomyces shuttle vectors allowing blue-white screening on xgal plates. | four new shuttle vectors for escherichia coli (ec) and streptomyces, puwl218, puwl219, puwl-sk and puwl-ks, which permit recognition of recombinant (re-) plasmids on xgal plates in ec, were constructed. these vectors contain the replication functions of the streptomyces wide-host-range multicopy plasmid pij101, the tsr gene conferring resistance to thiostrepton in streptomyces, the colei origin of replication from the puc plasmids for replication in ec and the bla gene conferring resistance to a ... | 1995 | 7489909 |
| sequences essential for replication of plasmid pij101 in streptomyces lividans. | a 2.1-kb sacii dna fragment of the high-copy-number streptomyces lividans plasmid pij101 previously has been shown to include the functions required for maintenance of pij101 as an extrachromosomal replicon. this fragment contains the open translational reading frames rep and orf 56 plus an intervening segment believed to be noncoding. using deletion mutations, we show that the pij101 replication origin and other cis-acting sites necessary and sufficient for replication map to a dna segment that ... | 1995 | 7568466 |
| mutational analysis of the tra locus of the broad-host-range streptomyces plasmid pij101. | the tra gene of streptomyces lividans plasmid pij101 encodes a 621-amino-acid protein that can mediate both plasmid transfer and the interbacterial transfer of chromosomal genes (i.e., chromosome-mobilizing ability [cma]) during mating. here we report the results of in-frame insertional mutagenesis studies aimed at defining regions of tra required for these functions. while hexameric linker insertions throughout the tra gene affected plasmid and chromosomal gene transfer, insertions in a 200-ami ... | 2000 | 10913083 |
| minimal and contributing sequence determinants of the cis-acting locus of transfer (clt) of streptomycete plasmid pij101 occur within an intrinsically curved plasmid region. | efficient interbacterial transfer of streptomycete plasmid pij101 requires the pij101 tra gene, as well as a cis-acting plasmid function known as clt. here we show that the minimal pij101 clt locus consists of a sequence no greater than 54 bp in size that includes essential inverted-repeat and direct-repeat sequences and is located in close proximity to the 3' end of the korb regulatory gene. evidence that sequences extending beyond the minimal locus and into the korb open reading frame influenc ... | 2000 | 11073933 |
| unraveling the essential role in conjugation of the tra protein of streptomyces lividans plasmid pij101. | plasmid pij101 from streptomyces lividans encodes a single gene, tra, that is essential for both plasmid transfer and mobilization of chromosomes during mating. the tra gene product (tra) is a membrane protein, a portion of which shows similarity to transfer proteins of other streptomycete plasmids as well as additional bacterial chromosome partitioning proteins. this paper reviews past and present work that has focused on elucidating the precise role of the tra protein of pij101 in conjugation ... | 2001 | 11816966 |
| common and distinguishing regulatory and expression characteristics of the highly related korb proteins of streptomycete plasmids pij101 and psb24.2. | the conjugative plasmid pij101 of the spore-forming bacterium streptomyces lividans contains a regulatory gene, korb, whose product is required to repress potentially lethal expression of the pij101 kilb gene. the korb protein also autoregulates korb gene expression and may be involved in control of pij101 copy number. korb (pij101) is expressed as a 10-kda protein in s. lividans that is immediately processed to a mature 6-kda repressor molecule. the conjugative streptomyces cyanogenus plasmid p ... | 2003 | 12813071 |
| [sequencing and analysis of thermophylic streptomyces plasmid ptsc2 and characterization of its rolling-circle replication]. | a streptomyces strain x3 -3, growing up to 50 degrees c, was isolated from the composts prepared from swine manure and straw. this strain harbored a -7-kb plasmid ptsc2. | 2010 | 20344939 |
| transposition of tn4560 of streptomyces fradiae in mycobacterium smegmatis. | tn4560 (8.6 kb) was derived from tn4556, a tn3-like element from streptomyces fradiae. it contains a viomycin resistance gene that has not been used previously for selection in mycobacteria. tn4560, cloned in a streptomyces plasmid, was introduced by electroporation into mycobacterium smegmatis mc(2)155. tn4560 transposed into the host genome: there was no obvious target sequence preference, and insertions were in or near several conserved open reading frames. the insertions were located far apa ... | 2002 | 11814670 |
| the tra locus of streptomycete plasmid pij101 mediates efficient transfer of a circular but not a linear version of the same replicon. | conjugal transfer of circular plasmids in streptomyces involves a unique mechanism employing few plasmid-encoded loci and the transfer of double-stranded dna by an as yet uncharacterized intercellular route. efficient transfer of the circular streptomycete plasmid pij101 requires only two plasmid loci: the pij101 tra gene, and as a cis-acting function known as clt. here, we compared the ability of the pij101 transfer apparatus to promote conjugal transfer of circular versus linear versions of th ... | 2010 | 20522498 |
| expression characteristics of the transfer-related kilb gene product of streptomyces plasmid pij101: implications for the plasmid spread function. | intermycelial transfer of streptomyces plasmid pij101 occurs prior to cellular differentiation and is mediated by plasmid functions that are also required for production of zones of growth-inhibited recipient cells (i.e., pocks) that develop around individual donors during mating on agar medium. several other pij101 functions, including that of the kilb gene, whose unregulated expression on pij101 is lethal, are required for normal pock size and so have been postulated to mediate intramycelial s ... | 2001 | 11157947 |
| replacement vectors for localized gene cloning in the specified region of streptomyces lividans 66 and model method for the screening of desired recombinants via counter-selection. | two dna fragments (3.5 kb and 3.8 kb in size) flanking both ends of the phi hau3r gene in the genome of streptomyces lividans 66 were determined and cloned in their natural relative orientations in pij653, a cosmid vector derived from the multi-copy streptomyces plasmid pij101, resulting in phz806. after insertion of spectinomycin/streptomycin (spc/str) resistance gene into the pij101 replication region in phz806 and insertion of a hygromycin (hyg) resistance gene between 3.5 kb and 3.8 kb dna f ... | 1999 | 10719626 |
| complementation of conjugation functions of streptomyces lividans plasmid pij101 by the related streptomyces plasmid psb24.2. | a database search revealed extensive sequence similarity between streptomyces lividans plasmid pij101 and streptomyces plasmid psb24. 2, which is a deletion derivative of streptomyces cyanogenus plasmid psb24.1. the high degree of relatedness between the two plasmids allowed the construction of a genetic map of psb24.2, consisting of putative transfer and replication loci. two psb24.2 loci, namely, the cis-acting locus for transfer (clt) and the transfer-associated korb gene, were shown to be ca ... | 1999 | 10419972 |
| novel site-specific dna modification in streptomyces: analysis of preferred intragenic modification sites present in a 5.7 kb amplified dna sequence. | both streptomyces lividans and streptomyces avermitilis encode similar systems of post-replicative dna modification which act site-specifically on closely opposed guanines on either strand. the modifications can be detected since they react in vitro with an oxidative derivative of tris, resulting in strand cleavage. previous analysis of the preferred modification site of plasmid pij101 indicated that extensive amounts of flanking sequence, including direct and inverted repeat structures, are req ... | 1998 | 9649620 |
| novel post-replicative dna modification in streptomyces: analysis of the preferred modification site of plasmid pij101. | both streptomyces lividans and streptomyces avermitilis have the ability to site specifically modify their dna, rendering it susceptible to in vitro tris-dependent double-strand cleavage. we have cloned a 160 bp fragment containing the preferred modification site of plasmid pij101 and, employing an in vitro primer extension assay, determined that the modifications occur at guanine residues on either strand separated by 3 bp. these guanines are located within a 6 bp palindromic 'core' sequence. a ... | 1998 | 9469833 |
| plasmid transfer and expression of the transfer (tra) gene product of plasmid pij101 are temporally regulated during the streptomyces lividans life cycle. | we previously have shown that a chromosomally integrated copy of the tra gene of plasmid pij101 under the control of the kora repressor protein, which regulates transcription of tra as well as its own synthesis, can promote the conjugal transfer of both chromosomal and plasmid genes in streptomyces lividans. using an antibody generated against a fusion protein containing the c-terminal portion of tra, we show here that this essential conjugation protein is present in membrane fractions of both s ... | 1996 | 8830269 |
| analysis of the site for second-strand initiation during replication of the streptomyces plasmid pij101. | the indigenous plasmid pij101 is the parent of many cloning vectors used in streptomyces. one early pij101 derivative, pij702, has been particularly widely used. pij702 lacks sti:cop/korb and accumulates single-stranded dna (ssdna). the 1.2 kb bcli-bcli sti:cop/korb and 0.7 kb spei-bcli sti regions were isolated from pij101 and cloned into pij702 at the psti site in both orientations. no ssdna was detected in constructs containing sti present in its correct orientation with respect to the basic ... | 1993 | 8515227 |
| identification of the minimal replicon of the streptomycete plasmid pij101. | the minimal replicon of pij101 was defined to a 2.0-kb bali-sacii fragment containing the rep orf and a non-coding region of dna, but not orf56 (pqr435). this non-coding region of dna was found to be crucial for plasmid replication in streptomyces lividans and may contain either the origin of replication or the rep promoter (or both). a 1.4-kb noti-sacii fragment containing only the rep orf produced a 54-kda protein in vitro when expressed from the lacz promoter. pqr435 was structurally unstable ... | 1993 | 8441769 |
| excisive recombination of the slp1 element in streptomyces lividans is mediated by int and enhanced by xis. | the functions mediating site-specific recombination of the slp1 element have been mapped to a 2.2-kb region that includes the site of integration (attp), a gene (int) that specifies a function both necessary and sufficient for integration of slp1, and an open reading frame, orf61, suspected of encoding a protein, xis, that shows limited similarity to the excisionases of other site-specific recombination systems. here we describe experiments that investigate the respective roles of orf61 and int ... | 1993 | 8387994 |
| relationship between the replication functions of streptomyces plasmids pjv1 and pij101. | the essential replication region of the streptomyces phaeochromogenes plasmid pjv1 was sequenced and compared to the equivalent region of the well-characterized streptomyces plasmid pij101. the sequence revealed a similar organization in both plasmids, including a conserved region within both plasmid origins, which is just upstream of a gene encoding the replication protein (rep). the rep proteins of pjv1 and pij101 are very similar, and both have a conserved motif, also present in rep proteins ... | 1993 | 8234485 |
| the active form of the korb protein encoded by the streptomyces plasmid pij101 is a processed product that binds differentially to the two promoters it regulates. | the korb gene of streptomyces lividans plasmid pij101 is known to encode an autoregulated protein that also represses transcription of a gene, kilb, implicated in pij101 transfer and in spreading of the plasmid along mycelia of the recipient. earlier work has indicated that the primary gene product of korb is a 10-kda protein predicted from the gene sequence (d.s. stein and s.n. cohen, mol. gen. genet. 222:337-344, 1990; s. zamen h. richards, and j. ward, nuleic acids res. 20:3693-3700, 1992). w ... | 1993 | 8226643 |
| rna polymerase-dna interactions in streptomyces. in vitro studies of a s. lividans plasmid promoter with s. coelicolor rna polymerase. | dna fragments of the streptomyces lividans plasmid pij101 have been tested for their ability to bind streptomyces coelicolor rna polymerase in vitro or to promote transcription in streptomyces in vivo. one dna fragment which does both was shown to encode a transcript which was expressed at low cell-density in cultures of pij101-containing cells. the transcript start was located on the dna sequence of the fragment by nucleotide-primed rna polymerase binding experiments and by s1 nuclease mapping. ... | 1985 | 4046037 |
| two promoters from the streptomyces plasmid pij101 and their expression in escherichia coli. | an rna polymerase-binding restriction fragment from the small, high-copy-number streptomyces plasmid pij101 has been shown to have promoter activity in vivo using a promoter-probe vector. the nucleotide sequence of the promoter (the pij101b promoter) and the approximate position of the transcription start point as identified by in vitro run-off transcription are presented. both the pij101b promoter and the previously characterised pij101a promoter were found to promote transcription in escherich ... | 1987 | 3297922 |
| complete nucleotide sequence of the streptomyces lividans plasmid pij101 and correlation of the sequence with genetic properties. | the complete nucleotide sequence of the multicopy streptomyces plasmid pij101 has been determined and correlated with previously published genetic data. the circular dna molecule is 8,830 nucleotides in length and has a g+c composition of 72.98%. the use of a computer program, frame, enabled identification in the sequence of seven open reading frames, four of which, tra (621 amino acids [aa]), spda (146 aa), spdb (274 aa), and kilb (177 aa), appear to be genes involved in plasmid transfer. at le ... | 1988 | 3170481 |
| "strong incompatibility" between derivatives of the streptomyces multi-copy plasmid pij101. | some derivatives of pij101, a 8.9 kb streptomyces multi-copy plasmid, can co-exist with each other at similar copy numbers but others are strongly incompatible. the dna sequence, sti, which causes this "strong incompatibility" was localised on a dna segment of about 200 bp which is not part of the essential replication region of pij101. the sti function is active only when the dna fragment carrying it is present in the natural orientation with respect to the basic replicon region of pij101. pair ... | 1988 | 3070352 |
| plasmid transfer in streptomyces lividans: identification of a kil-kor system associated with the transfer region of pij101. | the 8.9-kilobase streptomyces plasmid pij101 is self-transmissible at high frequency into recipient strains. by genetic analysis of the transfer region of the plasmid, we identified six plasmid-encoded loci involved in gene transfer and the associated pocking phenomenon. two loci, kila and kilb, could not be cloned into streptomyces lividans on a minimal pij101-based replicon unless suitable kil-override (kor) genes were present, either in cis or in trans. kora could control the lethal effects o ... | 1987 | 3040681 |
| activity of a streptomyces transcriptional terminator in escherichia coli. | a 205bp dna fragment from the streptomyces multi-copy plasmid pij101 has in vivo terminator activity both in streptomyces lividans and in escherichia coli. termination of rna synthesis, detected by high-resolution s1 nuclease mapping, occurs at precisely the same nucleotides in both organisms. this suggests that the e. coli rna polymerase recognizes the same sequence elements and chooses the point(s) of termination in the same way as the corresponding s. lividans enzyme. | 1987 | 3031607 |
| plasmid pij699, a multi-copy positive-selection vector for streptomyces. | a plasmid vector, pij699, which provides positive selection for cloned dna, was constructed using the replication functions of the streptomyces wide-host-range multi-copy plasmid pij101. the selection for inserts is based on the principle that plasmids with long uninterrupted perfect palindromes (inverted repeats) are 'not viable' in bacteria. for cloning, pij699 is digested with bglii. this produces two fragments, one of which is the linearized vector, with two arms of the palindrome at its end ... | 1988 | 2840358 |
| identification and analysis of transcriptional regulatory signals for the kil and kor loci of streptomyces plasmid pij101. | four regulated promoters that direct the transcription of genes (i.e., kora, tra, kilb, and korb) involved in the transfer of the streptomyces plasmid pij101 were isolated following the in vitro fusion of plasmid dna fragments to a promoterless gene encoding the s. lividans extracellular enzyme beta-galactosidase. introduction of pij101 into cells carrying each of these promoter-lac fusions resulted in decreased lac expression. the sites of initiation of transcription by the promoter sequences w ... | 1989 | 2681140 |
| expression and characterisation of the korb gene product from the streptomyces lividans plasmid pij101 in escherichia coli and determination of its binding site on the korb and kilb promoters. | a 0.5kb spel-bcli fragment containing the pij101 korb orf was cloned into puc8 under the control of the lacz promoter, creating pqr206. in vitro coupled transcription-translation of pqr206 identified a protein product of approximately 10kda, which corresponds to the predicted molecular weight deduced from the korb sequence. pqr206 was used to express the 10kda korb protein in vivo in e. coli. crude e. coli protein extracts containing korb were shown to bind to a 0.8kb kilb fragment and a 0.5kb k ... | 1992 | 1641335 |
| two phz1358-derivative vectors for efficient gene knockout in streptomyces. | deletion of sti on streptomyces plasmid pij101, made its derivative phz1358 an efficient vector for gene disruption and replacement. here, we further optimized phz1358 by the construction of a derivative plasmid pjtu1278, in which a cassette carrying multiple cloning sites and a lacz selection marker was introduced for convenient plasmid construction in e. coli. furthermore, we also deleted orit region of the pjtu1278, generated a vector (pjtu1289) that can be used specifically for pcr-targeting ... | 2010 | 20467238 |
| analysis of functions in plasmid phz1358 influencing its genetic and structural stability in streptomyces lividans 1326. | the complete dna sequence of plasmid phz1358, a widely used vector for targeted gene disruption and replacement experiments in many streptomyces hosts, has been determined. this has allowed a detailed analysis of the basis of its structural and segregational instability, compared to the high copy number plasmid pij101 of streptomyces lividans 1326 from which it was derived. a 574-bp dna region containing sti (strong incompatibility locus) was found to be a determinant for segregational instabili ... | 2009 | 19066884 |
| an in vivo assay for conjugation-mediated recombination yields novel results for streptomyces plasmid pij101. | efficient transmission of circular plasmids in streptomyces spp. proceeds by an uncharacterized mechanism that requires a cis-acting locus of transfer (clt) and often only a single plasmid-encoded protein. for circular plasmids from other bacteria, site- and strand-specific nicking takes place at the cis-acting orit locus via the plasmid-encoded relaxase protein prior to single-strand transfer. using an assay originally designed to demonstrate that conjugative transfer of plasmids containing tan ... | 2006 | 16388851 |
| separate and coordinate transcriptional control mechanisms link expression of the potentially lethal kilb spread locus to the upstream transmission operon on streptomyces plasmid pij101. | efficient conjugation of the high copy plasmid pij101 among members of the bacterial genus streptomyces depends on a single plasmid gene (tra) for initial inter-mycelial transfer, and involves three additional pij101 functions (spda, spdb, and kilb), which may promote intra-mycelial spread of the plasmid upon its entrance into the recipient. the genes tra, spda, and spdb are co-transcribed as part of an operon, whose expression is negatively controlled by the pij101 repressor kora. downstream of ... | 2003 | 14643654 |