Construction of two stable bifunctional plasmids for Streptomyces spp. and Escherichia coli

Two bifunctional plasmid vectors pZG5 (7.45 kb) and pZG6 (6.95 kb), for gene transfer between Streptomyces spp. and Escherichia coli have been constructed by fusion of the multicopy broad-host-range Streptomyces plasmid pIJ350 with E. coli plasmids Bluescribe M13- (pZG5) or pUC18 (pZG6). Both plasmids possess several unique restriction sites suitable for DNA cloning. Stable transformants of Streptomyces rimosus R6 and S. lividans 66 were obtained, harboring intact plasmids regardless of colony age or multiple subculturing. Moreover, pZG5 and pZG6 were successfully used to introduce several homologous transfer RNA genes into S. rimosus.     

Absence in Bacillus subtilis and Staphylococcus aureus of the sequence-specific deoxyribonucleic acid methylation that is conferred in Escherichia coli K-12 by the dam and dcm enzymes

Restriction analysis of plasmid pHV14 deoxyribonucleic acid isolated from Escherichia coli K-12, Bacillus subtilis, and staphylococcus aureus with restriction endonucleases MboI, Sau3AI, and EcoRII was used to study the methylation of those nucleotide sequences which in E. coli contain the major portions of N6-methyladenine and 5-methylcytosine. The results showed that neither B. subtilis nor S. aureus methylates deoxyribonucleic acid at the same sites and nucleotides which are recognized and methylated by dam and dcm enzymes in E. coli K-12.     

Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector.

An integration vector for gene analysis in Streptomyces has been constructed. This vector replicates in Escherichia coli, and integrates into Streptomyces by homologous recombination between a cloned fragment and the genome. To overcome methylation-specific restriction barriers, an E. coli mutant triply defective in DNA methylation was constructed as a source for the integration plasmids. The frequency of integration of pVE616 derivatives into the Streptomyces avermitilis genome was proportional to the size of the cloned DNA. Derivatives of pVE616, containing fragments from pVE650, a plasmid with a 24-kb insert of S. avermitilis DNA, were used in complementation analyses of seven S. avermitilis mutants defective in glycosylation of avermectin (Av). Three complementation groups, located in a 7-kb region, were identified. Derivatives of pVE616, containing fragments from the 18-kb of DNA adjacent to the glycosylation region, were integrated into an Av producer. Av produced from the integrants was substantially reduced, indicating that the 18 kb also encodes gene products which are involved in Av biosynthesis.     

Cloning and expression of a Streptomyces fradiae neomycin resistance gene in Escherichia coli

A Streptomyces fradiae DNA sequence, which codes for a neomycin phosphotransferase, has been subcloned from the Streptomyces recombinant plasmid pIJ2 [a chimera between the Streptomyces plasmid SLP1.2 and chromosomal DNA containing a neomycin (Nm) resistance gene] into the BamHI restriction enzyme site of pHV14. Three different recombinant plasmids (pWHR1, pWHR2, pWHR3) have been isolated which transform Escherichia coli to Nm resistance. Southern transfer hybridization experiments show that the recombinant plasmids contain the cloned Streptomyces Nm resistance gene, and lysates of E. coli containing the recombinant plasmids were shown to have Nm phosphotransferase activity, demonstrating that a gene from Streptomyces can be expressed in E. coli.     

The construction of Streptomyces cyaneus genomic libraries in Escherichia coli is dependent upon the use of Mcr-deficient strains.

Streptomyces cyaneus genomic DNA ligated into either lambda phage or plasmid vectors was very inefficiently cloned into standard Escherichia coli host strains. However, the same material could be efficiently cloned using Mcr-deficient E. coli strains. These results suggest that the S. cyaneus genome contains 5-methylcytosine residues, some of which occur within the recognition sequences of the E. coli Mcr restriction system.     

Salmonella typhimurium SA host specificity system is based on deoxyribonucleic acid-adenine methylation.

We have determined the nature of the deoxyribonucleic acid (DNA) modification governed by the SA host specificity system of Salmonella typhimurium. Two lines of evidence indicate that SA modification is based on methylation of DNA-adenine residues. (i) The SA+ locus of Salmonella was transferred into Escherichia coli B, a strain that does not contain 5-methylcytosine in its DNA; although the hybrid strain was able to confer SA modification, its DNA still did not contain 5-methylcytosine. (ii) the N6-methyladenine content of phage L DNA was measured after growth in various host strains; phage lacking SA modification contained fewer N6-methyladenine residues per DNA. We also investigated the possibility, suggested by others (32), that SA modification protects phage DNA against restriction by the RII host specificity system. Phages lambda, P3, and L were grown in various SA+ and SA- hosts and tested for their relative plating ability on strains containing or lacking RII restriction; the presence or absence of SA modification had no effect on RII restriation. In vitro studies revealed, however, that Salmonella DNA is protected against cleavage by purified RII restriction endonuclease (R-EcoRII). This protection is not dependent on SA modification; rather, it appears to be due to methylation by a DNA-cytosine methylase which has overlapping specificity with the RII modification enzyme, but which is not involved in any other known host specificity system.     

Restriction analysis and quantitative estimation of methylated bases of filamentous and unicellular cyanobacterial DNAs.

The DNAs of strains of three cyanobacterial genera (Anabaena, Plectonema, and Synechococcus) were found to be partially or fully resistant to many restriction endonucleases. This could be due to the absence of specific sequences or to modifications, rendering given sequences resistant to cleavage. The latter explanation is substantiated by the content of N6-methyladenine and 5-methylcytosine in these genomes, which is high in comparison with that in other bacterial genomes. dcm- and dam-like methylases are present in the three strains (based on the restriction patterns obtained with the appropriate isoschizomeric enzymes). Their contribution to the overall content of methyladenine and methylcytosine in the genomes was calculated. Partial methylation of GATC sequences was observed in Anabaena DNA. In addition, the GATC methylation patterns might not have been random in the three cyanobacterial DNA preparations, as revealed by the appearance of discrete fragments (possibly of plasmid origin) withstanding cleavage by DpnI (which requires the presence of methyladenine in the GATC sequence).     

On the nature of the cytosine-methylated sequence in DNA of Bacillus brevis var. G.-B.

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of [Me-3H]methionine, practically all the radioactivity incorporated into DNA is found to exist in 5-methylcytosine and N6-methyladenine. The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of 5-methylcytosine in R-m5 C-R and R-m5 C-T-R oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring 5-methylcytosine to be identified and shows that 5-methylcytosine localizes in G-m5 C-A and G-m5 C-Tr fragments. B. brevis S DNA methylase modifying cytosine residues recognizes the GCA/TGC degenerate nucleotide sequence which is a part of the following complementary structure with a two-fold rotational axis of symmetry: (5')...N'-G-C-T-G-C-N... (3') (3')...N-C-G-A-C-G-N'... (5') (Methylated cytosine residues are askerisked). Cytosine-modifying DNA methylase activity is isolated from B. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence DNA in bacterial cells can be undermethylated. This enzyme methylates cytosine residues in native and denatured DNA in the same nucleotide sequences. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA. DNA methylases of different variants of B. brevis (R, S, P+, P-)) methylate cytosine residues in the same nucleotide sequences. It means that specificity or methylation of DNA cytosine residues in the cells of different variants of B. brevis is the same.     

Development of Escherichia coli virus T1: escape from host restriction.

The bacterial virus T1 grows interchangeably on different Escherichia coli strains (C, B, and K). This implies that T1 has an efficient mechanism to overcome the host restriction barrier. The DNA of T1 was found to be methylated independently of the hosts. The percentage of N6-methyladenine varied from 1.6 to 1.8, and the 5-methylcytosine content varied from 0.1 to 0.4%. In contrast, the range in percentage of N6-methyladenine and 5-methylcytosine found in the hosts was 0.7 to 2.4 and 0.0 to 1.1, respectively.     

Site-specific methylases induce the SOS DNA repair response in Escherichia coli.

Expression of the site-specific adenine methylase HhaII (GmeANTC, where me is methyl) or PstI (CTGCmeAG) induced the SOS DNA repair response in Escherichia coli. In contrast, expression of methylases indigenous to E. coli either did not induce SOS (EcoRI [GAmeATTC] or induced SOS to a lesser extent (dam [GmeATC]). Recognition of adenine-methylated DNA required the product of a previously undescribed gene, which we named mrr (methylated adenine recognition and restriction). We suggest that mrr encodes an endonuclease that cleaves DNA containing N6-methyladenine and that DNA double-strand breaks induce the SOS response. Cytosine methylases foreign to E. coli (MspI [meCCGG], HaeIII [GGmeCC], BamHI [GGATmeCC], HhaI [GmeCGC], BsuRI [GGmeCC], and M.Spr) also induced SOS, whereas one indigenous to E. coli (EcoRII [CmeCA/TGG]) did not. SOS induction by cytosine methylation required the rglB locus, which encodes an endonuclease that cleaves DNA containing 5-hydroxymethyl- or 5-methylcytosine (E. A. Raleigh and G. Wilson, Proc. Natl. Acad. Sci. USA 83:9070-9074, 1986).     

Development of a system of plasmid transfer in the strains Streptomyces avermitilis ATCC 31272 and Saccharopolyspora erythraea ATCC 11635 by the method of intergeneric conjugation

A new method of plasmid DNA transfer from the donor strain Escherichia coli S17-1 to the erythomycin-producing strain Saccharopolyspora erythraea and avermectin-producing strain Streptomyces avermitilis via intergeneric conjugation was proposed. The optimal parameters of the method were chosen for increasing the efficiency of crosses and ensuring easily reproducible results. The behavior of the multicopy plasmid pPM803 and the integration vector pTO1 along with a number of new plasmids specially created by us, was examined in these strains. A new plasmid vector (pSI60) capable of integrating into the chromosome of actinomycetes at the integration site of the temperate actinophage phi C31 was constructed. This vector possesses unique sites convenient for cloning and may be stably maintained in exconjugants of S. avermitilis and in the model strain Streptomyces lividans. The gene encoding resistance to spectinomycin and streptomycin was cloned into the vector pSI60 in this strain. For cloning in strain Sac. erythraea, vectors pSI261-280, which integrate into the chromosome via homology with the cloned DNA and can be stably maintained in exconjugants, were constructed.     

Specificity of new restrictases and methylases. Unusual modification of cytosine at position 4

Fourteen restriction endonucleases and 4 methylases were isolated and purified from 14 strains of Citrobacter freundii and Escherichia coli, which were isolated from natural sources. To determine the nucleotide sequence recognized by the endonucleases a comparison of DNA cleavage patterns, the evaluation of the cleavage frequency of some DNA with known recognition sequences and mapping was used. It was determined that Cfr101 is a new enzyme recognizing 5'PuCCGGPy. Other restriction enzymes isolated were isoschizomers of: Cfr5I, Cfr11I, Eco60I, Eco61I--EcoRII; Cfr4I, Cfr8I, Cfr13I--Sau96I; Cfr6I--PvuII, Cfr9I--SmaI, Eco26I--HgiJII; Eco32I--EcoRV; Eco52I--XmaIII; Eco56I--NaeI. Some of the enzymes in C. freundii and E. coli were found for the first time. The methylases MCfrI; MCfr6I, MCfr9I and MCfr10I recognize the same nucleotide sequence as specific endonucleases isolated from the same strain. DNA modification in vitro by MCfrI and MCfr10I yields 5-methylcytosine and 4-methylcytosine by MCfr6I and MCfr9I.     

Cytokinins do not noticeably change the methylation of adenine residues of DNA in cultured Escherichia coli B

6-Benzylaminopurine (6-BAP) (1 mg/ml) does not influence the growth of E. coli B cell cultures or the number of [8-14C] labeled N6-methyladenine (m6A) residues in the total DNA [(100.m6A/(A x m6A) = 1.7]. The growth of bacterial cells in the presence of adenine or cytokinins (6-BAP, kinetin, zeatin) (1 mg/ml) was unaccompanied by significant changes in the intracellular content of plasmid pBR 322. The mode of restriction by endonuclease Cfu I hydrolyzing the Gm6ATC site of plasmids pBR 322 from E. coli B cells grown in the presence of adenine or one of the above-mentioned cytokinins is identical. These plasmids also have identical restriction products Mbo I or Sau 3AI. Thus, the cytokinins under study do not markedly affect the methylation of adenine residues in total DNA of E. coli B cell cultures and the GATC sequence in plasmids pBR 322 isolated from these cells.     

The Neisseria gonorrhoeae S.NgoVIII restriction/modification system: a type IIs system homologous to the Haemophilus parahaemolyticus HphI restriction/modification system.

Strains of Neisseria gonorrhoeae possess numerous restriction-modification (R-M) systems. One of these systems, which has been found in all strains tested, encodes the S. NgoVIII specificity (5'TCACC 3') R-M system. We cloned two adjacent methyltransferase genes (dcmH and damH), each encoding proteins whose actions protect DNA from digestion by R.HphI or R.Ngo BI (5'TCACC 3'). The damH gene product is a N 6-methyladenine methyltransferase that recognizes this sequence. We constructed a plasmid containing multiple copies of the S.NgoVIII sequence, grew it in the presence of damH and used the HPLC to demonstrate the presence of N 6-methyladenine in the DNA. A second plasmid, containing overlapping damH and Escherichia coli dam recognition sequences in combination with various restriction digests, was used to identify which adenine in the recognition sequence was modified by damH. The predicted dcmH gene product is homologous to 5-methylcytosine methyltransferases. The products of both the dcmH and damH genes, as well as an open reading frame downstream of the damH gene are highly similar to the Haemophilus parahaemolyticus hphIMC , hphIMA and hphIR gene products, encoding the Hph I Type IIs R-M system. The S.NgoVIII R-M genes are flanked by a 97 bp direct repeat that may be involved in the mobility of this R-M system.     

Use of cloned DNA methylase genes to increase the frequency of transfer of foreign genes into Xanthomonas campestris pv. malvacearum.

In vitro-packaged cosmid libraries of DNA from the bacterium Xanthomonas campestris pv. malvacearum were restricted 200- to 1,000-fold when introduced into Mcr+ strains of Escherichia coli compared with restriction in the Mcr- strain HB101. Restriction was predominantly associated with the mcrBC+ gene in E. coli. A plasmid (pUFR052) encoding the XmaI and XmaIII DNA methylases was isolated from an X. campestris pv. malvacearum library by a screening procedure utilizing Mcr+ and Mcr- E. coli strains. Transfer of plasmids from E. coli strains to X. campestris pv. malvacearum by conjugation was enhanced by up to five orders of magnitude when the donor cells contained pUFR052 as well as the plasmid to be transferred. Subcloning of pUFR052 revealed that at least two regions of the plasmid were required for full modification activity. Use of such modifier plasmids is a simple, novel method that may allow the efficient introduction of genes into any organism in which restriction systems provide a potent barrier to such gene transfer.     

Location of the Bases Modified by M.<Emphasis Type="Italic">Bco</Emphasis>KIA and M.<Emphasis Type="Italic">Bco</Emphasis>KIB Methylases in the Sequence 5′-CTCTTC-3′/5′-GAAGAG-3′

The strain Bacillus coagulans K contains two DNA-methyltransferases, M.BcoKIA and M.BcoKIB, which recognize the sequence 5 -CTCTTC-3 /5 -GAAGAG-3 and possess N4-methylcytosine and N6-methyladenine specificities, respectively. A special construct containing the recognition site of BcoKI and sites of four IIS restriction endonucleases (IIS restriction endonuclease cassette) was designed to locate the nucleotides modified by the methylases. The modified bases were determined as: 5 -m(4)CTCTTC-3 /5 -GAAGAm(6)G-3 .     

Study of the methylation process and DNA methylase specificity in Shigella]

The nature and content of minor bases in DNA of 3 Shigella strains are investigated. DNAs from Shigella stutzeri 2, Sh. sonnei 1188 and Sh. sonnei 311 are found to contain 0.43, 0.56 and 0.45 mol.% of N6-methyladenine respectively. 5-methylcytosine (0.16 mol.%) is discovered in Sh. sonnei 311. Substrate specificity of adenine methylase from Sh. sonnei 1188 with respect to phage DNAs of different host modification is investigated. Recognition sites for guanine methylase of DDVI phage and for adenine methylase of Sh. sonnei 1188 turned to be different. DNA of DDII phage grown in Sh. stutzeri 2 cells does not accept methyl groups under the treatment with Sh. sonnei 1188 extracts, but it is methylated by Escherichia coli extract. Adenine methylases of Sh. sonnei 1188 and Sh. stutzeri 2 are suggested to be either the same enzyme, or enzymes, which recognition sites are partially overlapped.     

Restriction of two-replicon shuttle Escherichia coli-Streptomyces plasmids in Streptomyces lividans strain 66

The shuttle Escherichia coli - Streptomyces plasmids were used to transform S. lividans 66. Plasmid DNAs isolated from this strain transform it 10-1000-fold more efficiently than DNAs from E. coli. Rare transformant cured from most restricted plasmid is more efficient recipient of plasmid DNA from E. coli and has the property of R +/- M+ mutant. Restriction in S. lividans 66 correlates with the appearance in DNA from E. coli of the sites susceptible to Scg2I restriction endonuclease. The latter was isolated earlier from recombinant strain Rcg2, a hybrid between S. griseus Kr. 15 and S. coelicolor A3(2). Scg2I possesses the recognition sequence CCTAGG, like EcoRII, MvaI and Eco dcm methylase. The DNA resistant to Scg2I cleavage retained this ability after in vitro modification by EcoRII methylase. So, the resistance of DNA to Scg2I cleavage is not connected with methylation at 4th and 5th position of second cytosine in the recognition sequence. Neither restriction of plasmid DNA in S. lividans 66 is dependent on dcm modification in E. coli, though its dependence on dam modification is not excluded. It is assumed that the restriction in S. lividans 66 is specified by endonuclease analogous to Scg2I.     

High-frequency mobilization of broad-host-range plasmids into Neisseria gonorrhoeae requires methylation in the donor.

Antibiotic resistance in Neisseria gonorrhoeae has been associated with the acquisition of R plasmids from heterologous organisms. The broad-host-range plasmids of incompatibility groups P (IncP) and Q (IncQ) have played a role in this genetic exchange in nature. We have utilized derivatives of RSF1010 (IncQ) and RP1 (IncP) to demonstrate that the plethora of restriction barriers associated with the gonococci markedly reduces mobilization of plasmids from Escherichia coli into strains F62 and PGH 3-2. Partially purified restriction endonucleases from these gonococcal strains can digest RSF1010 in vitro. Protection of RSF1010-km from digestion by gonococcal enzymes purified from strain F62 is observed when the plasmid is isolated from E. coli containing a coresident plasmid, pCAL7. Plasmid pCAL7 produces a 5'-MECG-3' cytosine methylase (M.SssI). The M.SssI methylase only partially protects RSF1010-km from digestion by restriction enzymes from strain PGH 3-2. Total protection of RSF1010-km from PGH 3-2 restriction requires both pCAL7 and a second coresident plasmid, pFnuDI, which produces a 5'-GGMECC-3' cytosine methylase. When both F62 and PGH 3-2 are utilized as recipients in heterospecific matings with E. coli, mobilization of RSF1010 from strains containing the appropriate methylases into the gonococci occurs at frequencies 4 orders of magnitude higher than from strains without the methylases. Thus, protection of RSF1010 from gonococcal restriction enzymes in vitro correlates with an increase in the conjugal frequency. These data indicate that restriction is a major barrier against efficient conjugal transfer between N. gonorrhoeae and heterologous hosts.     

Characterization and expression of the Escherichia coli Mrr restriction system.

The mrr gene of Escherichia coli K-12 is involved in the acceptance of foreign DNA which is modified. The introduction of plasmids carrying the HincII, HpaI, and TaqI R and M genes is severely restricted in E. coli strains that are Mrr+. A 2-kb EcoRI fragment from the plasmid pBg3 (B. Sain and N. E. Murray, Mol. Gen. Genet. 180:35-46, 1980) was cloned. The resulting plasmid restores Mrr function to mrr strains of E. coli. The boundaries of the mrr gene were determined from an analysis of subclones, and plasmids with a functional mrr gene produce a polypeptide of 33.5 kDa. The nucleotide sequence of the entire fragment was determined; in addition to mrr, it includes two open reading frames, one of which encodes part of the hsdR. By using Southern blot analysis, E. coli RR1 and HB101 were found to lack the region containing mrr. The acceptance of various cloned methylases in E. coli containing the cloned mrr gene was tested. Plasmid constructs containing the AccI, CviRI, HincII, Hinfl (HhaII), HpaI, NlaIII, PstI, and TaqI N6-adenine methylases and SssI and HhaI C5-cytosine methylases were found to be restricted. Plasmid constructs containing 16 other adenine methylases and 12 cytosine methylases were not restricted. No simple consensus sequence causing restriction has been determined. The Mrr protein has been overproduced, an antibody has been prepared, and the expression of mrr under various conditions has been examined. The use of mrr strains of E. coli is suggested for the cloning of N6-adenine and C5-cytosine methyl-containing DNA.