Development of integrative vectors for Actinomycetes--producers of antibiotics]

The integrative vectors pSU 475 and pSU 476 with variable numbers of copies per genome were developed for antibiotic producing actinomycetes. For this, the amplifying sequence AUD-Sr 1 of Streptomyces rimosus and the BamHIB fragment of the eSA 1 genetic element from Streptomyces antibioticus were used. The eSA 1 fragment was an element required for integration of a vector to the actinomycete chromosomes since it was homologous with the chromosomal DNAs of S. lividans, S. erythraeus and S. antibioticus. At the first stage the AUD-Sr 1 sequence within the actinomycete plastid pSU 23 was cloned by the vector pUC 19 to E coli. In that experiment the 12.4-kb plasmid pSU 449 was isolated. At the second stage the BamHIB-fragment of the eSA 1 element was incorporated into the resultant hybrid plasmid pSU 449. The 16.5-kb hybrid plasmids pSU 475 and pSU 476 were isolated. In these plasmids the BamHIB fragment of eSA 1 was present in two orientations. The developed vectors were useful in cloning DNA to S. lividans and S. erythraeus.     

Location of determinants of stability to neomycin and kanamycin as well as DNA segments, responsible for amplification in Streptomyces plasmids pSU3 and pSU10

The S. rimosus amplifying sequence AUD-Sr1 encodes kanamycin and neomycin resistance, defined in the case of neomycin by aminoglycoside phosphotransferase. Its cloning on plasmid SLP1.2 makes possible the co-amplification of the obtained hybrid plasmids in S. lividans. In our study the regions responsible for resistance to aminoglycoside antibiotics and the capacity for amplification the two hybrid plasmids pSU10 and pSU3 were determined. Experiments on subcloning of the AUD-Sr1 sequence fragments on vector pIJ702 revealed localization of kanamycin and neomycin resistance determinants between PvuII(6) and BglII(7) on the AUD-Sr1 sequence fragments of 2.0 kb length. Two regions responsible for amplification of the hybrid plasmids were detected with deletion and insertion mapping. The first region is localized in the region of the plasmid SLP1.2 BamHI site and the second region is localized on the PstI(4)-PvuII(6) of the AUD-Sr1 sequence fragment of 1.1 kb length.     

Study of the structure of amplifying sequence of Streptomyces antibioticus

A low productive laboratory strain of S. antibioticus and a strain with an increased productivity of oleandomycin derived from it were studied comparatively with using restriction analysis and blotting hybridization. Amplification, site specific integration and segregation of the DNA sequence 32.0 kb in size were detected in the strains. The chromosomes of the laboratory strain contained one copy of the amplifying sequence AUD. After uniting of the end sequences AUD appeared to be capable of segregating from the chromosomes and its one copy per five genomes was present in the form of an extrachromosomal genetic element eSA1. The genome of the strain with increased productivity of oleandomycin contained in its chromosomes sequence ADS-Sa1 amplified to 150 copies and the eSA1 extrachromosomal genetic element in the form of mono-, di- and trimeric structures in the quantity of approximately one copy per genome. The BamHIB fragment of the eSA1 DNA 4 kb in size was identified. The fragment was able to participate in segregation or integration of eSA1 from or into the chromosomes since its subfragments were flanking AUD and ADS-SA1 in the chromosomes. The BamHIB fragment was hybridizing with a number of fragments of the chromosomal DNA of S. antibioticus, S. erythraeus. S. lividans and other strains of streptomycetes. It probably contained an IS-like element or a dispersed genetic element of another class. The DNA sequence of the eSA1 genetic element contained regions homologous to the sequence of the Erm E gene in S. erythraeus NRRL 2338.     

Characterization of plasmids and plasmid-borne macrolide resistance from Lactobacillus sp. strain 100-33

Lactobacillus sp. strain 100-33 is resistant to macrolides, lincosamides, and streptogramin B-type antibiotics (MLSR) and appears to contain several major and minor plasmids. One of these plasmids, pLAR33, is approximately 18 kbp in size. When cells of strain 100-33 were protoplasted and regenerated, an MLSS isolate was derived. The derivative, designated strain ES1, contained a unique plasmid complement in which it had apparently lost the major plasmids of the parental strain, including pLAR33, and retained only a minor plasmid seen in low concentrations in strain 100-33. The MLSR determinant was cloned from plasmid DNA of strain 100-33 on a 3-kbp EcoRV fragment into pBR322 and localized to pLAR33. The determinant expressed macrolide and lincosamide resistance in Escherichia coli HB101, was localized to approximately 1 kbp on the cloned sequence, and is apparently under the control of its own promoter. MLSR electroporants were derived from strain ES1 electroporated with plasmid DNA from strain 100-33; these MLSR isolates had acquired a plasmid complement similar to that of strain 100-33, including pLAR33. Endonuclease digestion and Southern analysis of plasmid DNA from both strains indicated that the major plasmids are multimeric and deleted forms of one archetypal extrachromosomal element.     

Amplification on the Amycolatopsis (Nocardia) mediterranei plasmid pMEA100: sequence similarities to actinomycete att sites

An amplification of a 2.0-kb fragment was found on the plasmid pMEA100 isolated from a subculture of the wild-type strain LBG A3136 of Amycolatopsis (Nocardia) mediterranei. Plasmid preparations contained a mixture of molecules with copy numbers of the amplified unit in the range of 2 to 10. The amplification on pMEA100 was stable; propagation of cells for many generations did not change the pattern of the amplified DNA. Fragments of the plasmids containing the amplifiable unit of DNA (AUD) and the amplified DNA sequence (ADS) were subcloned and characterized. Sequencing of the AUD terminal regions and the junction between ADS units showed that the amplifiable unit of DNA was flanked by 12-bp direct repeats. The DNA segments adjacent to the 12-bp sequence common to the left and right AUD terminal regions also showed significant similarities. In addition, the left AUD terminal region flanking the 12-bp repeat exhibited considerable sequence similarity to actinomycete plasmid attachment sites, particularly to the pMEA 100 att site.     

Cloning of genes for bacterial glycosyltransferases. I. Selection of hybrid plasmids carrying genes for two glucosyltransferases.

A method of identifying plasmids containing genes responsible for synthesis of nucleotide sugar:lipopolysaccharide glycosyltransferases is described. Hybrid ColE1 plasmids containing random fragments of the chromosome of Escherichia coli K12 were introduced into an indicator strain of Salmonella typhimurium which lacks UDP-glucose:lipopolysaccharide glucosyltransferase I due to an rfaG mutation. Plasmids capable of correcting the transferase defect were identified by their ability to convert the bacteriophage sensitivity pattern of the recipient strain from Ffm-sensitive to Ffm-resistant. Analysis of the lipopolysaccharide of the S. typhimurium/ColE1 hybrid strains and assay of cell extracts defined the new enzyme activities. Two plasmids were identified which carried the rfaG+ gene; one of these plasmids also contained genetic information for a second glucosyltransferase, the E. coli glucosyltransferase II, which normally is not present in S. typhimurium.     

The influenza virus haemagglutinin gene: cloning and characterisation of a double-stranded DNA copy.

A protocol has been developed for the synthesis of a double-stranded DNA (dsDNA) copy of the influenza virus RNA genome segment which codes for the major surface antigen, haemagglutinin (HA). This dsDNA copy was inserted, after digestion with S1 nuclease and poly (dC) tailing with terminal transferase, into poly(dG)-tailed, PstI-cut, pBR322 DNA, and used to transform E. coli RR1. Tetracycline-resistant bacterial colonies were screened for the presence of plasmid containing the copied HA gene by testing their ability to hybridise to a specific, 32P-labelled, single-stranded DNA probe. Four cloned hybrid plasmids, containing DNA complementary to the HA gene of the influenza strain 29C (a laboratory derivative of influenza A/NT/60/68 (1)) were analysed by restriction enzyme mapping. Each contained a dsDNA insert equivalent to a full length copy of the HA gene. The nucleotide sequence of a selected restriction fragment from the DNA inserted in one of these cloned plasmids (C89) was determined. The amino acid sequence deduced from these data agreed with the amino acid sequence determined for the corresponding region of HA from the influenza strain A/Mem/102/72, another member of the Hong Kong subtype, identifying the inserted dsDNA of C89 as an authentic copy of the influenza HA gene.     

Cloning of a yeast gene coding for the glutamate synthase small subunit (GUS2) by complementation of Saccharomyces cerevisiae and Escherichia coli glutamate auxotrophs

A Saccharomyces cerevisiae glutamate auxotroph, lacking NADP-glutamate dehydrogenase (NADP-GDH) and glutamate synthase (GOGAT) activities, was complemented with a yeast genomic library. Clones were obtained which still lacked NADP-GDH but showed GOGAT activity. Northern analysis revealed that the DNA fragment present in the complementing plasmids coded for a 1.5kb mRNA. Since the only GOGAT enzyme so far purified from S. cerevisiae is made up of a small and a large subunit, the size of the mRNA suggested that the cloned DNA fragment could code for the GOGAT small subunit. Plasmids were purified and used to transform Escherichia coli glutamate auxotrophs. Transformants were only recovered when the recipient strain was an E. coli GDH-less mutant lacking the small GOGAT subunit. These data show that we have cloned the structural gene coding for the yeast small subunit (GUS2). Evidence is also presented indicating that the GOGAT enzyme which is synthesized in the E. coli transformants is a hybrid comprising the large E. coli subunit and the small S. cerevisiae subunit.     

Fidelity of DNA recognition by the EcoRV restriction/modification system in vivo

The EcoRV restriction/modification system consists of two enzymes that recognize the DNA sequence GATATC. The EcoRV restriction endonuclease cleaves DNA at this site, but the DNA of Escherichia coli carrying the EcoRV system is protected from this reaction by the EcoRV methyltransferase. However, in vitro, the EcoRV nuclease also cleaves DNA at most sites that differ from the recognition sequence by one base pair. Though the reaction of the nuclease at these sites is much slower than that at the cognate site, it still appears to be fast enough to cleave the chromosome of the cell into many fragments. The possibility that the EcoRV methyltransferase also protects the noncognate sites on the chromosome was examined. The modification enzyme methylated alternate sites in vivo, but these were not the same as the alternate sites for the nuclease. The excess methylation was found at GATC sequences, which are also the targets for the dam methyltransferase of E. coli, a protein that is homologous to the EcoRV methyltransferase. Methylation at these sites gave virtually no protection against the EcoRV nuclease: even when the EcoRV methyltransferase had been overproduced, the cellular DNA remained sensitive to the EcoRV nuclease at its noncognate sites. The viability of E. coli carrying the EcoRV restriction/modification system was found instead to depend on the activity of DNA ligase. Ligase appears to proofread the EcoRV R/M system in vivo: DNA, cut initially in one strand at a noncognate site for the nuclease, is presumably repaired by ligase before the scission of the second strand.     

Heterogeneity of Escherichia coli phages encoding Vero cytotoxins: comparison of cloned sequences determining VT1 and VT2 and development of specific gene probes

Phages coding for production of Vero cytotoxins VT1 or VT2 in strains of Escherichia coli serotype O157.H7 or O157.H- were morphologically indistinguishable. Their genome size and restriction enzyme digests of the phage DNA were similar. These phages were clearly different in these respects from a VT1-encoding phage isolated from a strain of E. coli O26.H11 (H19). However the VT1 region cloned from the phage originating in the E. coli O157.H7 strain was identical to the VT1 region previously cloned from the phage carried by H19. Sequences encoding VT2 that were cloned from the phage in E. coli O157.H- have been mapped and the VT2 region identified by transposon insertion. The cloned regions coding for VT1 or VT2 production had no similarities in the presence of restriction enzyme sites over a distance of about 2 kb, and two VT1-specific probes spanning a region of about 1.4 kb did not hybridize under stringent conditions with cloned VT2 DNA. A 2 kb HincII fragment contained the VT2 genes but hybridized to VT1-encoding phages and recombinant plasmids via flanking phage DNA. A 0.85 kb AvaI-PstI fragment was a specific probe for VT2 sequences and did not hybridize under stringent conditions to phages or plasmid recombinants encoding VT1.     

Identification of restriction fragments from two cryptic Clostridium butyricum plasmids that promote the establishment of a replication-defective plasmid in Bacillus subtilis

Clostridium butyricum NCIB 7423 carries two cryptic plasmids, pCB101 (6.05 kbp) and pCB102 (7.8 kbp). Sites for the restriction enzymes EcoRI, EcoRV, HindIII, ClaI and PstI have been found in one or both of these plasmids and their relative positions determined. Restriction fragments from both plasmids have been inserted into a vector plasmid (pJAB1) that is able to replicate in Escherichia coli but not in Bacillus subtilis and the recombinant plasmids have been established in E. coli. A 3.3 kbp Sau3A fragment of pCB101 conferred upon the vector the ability to transform both Rec+ and Rec- strains of B. subtilis. Plasmid pRB1, a representative chimaera carrying only the 3.3 kbp Sau3A fragment of pCB101, was successfully transferred from B. subtilis back to E. coli. Plasmid pRB1 was readily lost from B. subtilis in the absence of selection. This evidence, together with the results of hybridization experiments, suggests that pRB1 is present as a weakly replicating autonomous element in B. subtilis. A recombinant plasmid carrying a 2.0 kbp Sau3A fragment of pCB102 underwent integration into the B. subtilis chromosome.     

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.     

Metronidazole activation and isolation of Clostridium acetobutylicum electron transport genes

An Escherichia coli F19 recA, nitrate reductase-deficient mutant was constructed by transposon mutagenesis and shown to be resistant to metronidazole. This mutant was a most suitable host for the isolation of Clostridium acetobutylicum genes on recombinant plasmids, which activated metronidazole and rendered the E. coli F19 strain sensitive to metronidazole. Twenty-five E. coli F19 clones containing different recombinant plasmids were isolated and classified into five groups on the basis of their sensitivity to metronidazole. The clones were tested for nitrate reductase, pyruvate-ferredoxin oxidoreductase, and hydrogenase activities. DNA hybridization and restriction endonuclease mapping revealed that four of the C. acetobutylicum insert DNA fragments on recombinant plasmids were linked in an 11.1-kb chromosomal fragment. DNA sequencing and amino acid homology studies indicated that this DNA fragment contained a flavodoxin gene which encoded a protein of 160 amino acids that activated metronidazole and made the E. coli F19 mutant very sensitive to metronidazole. The flavodoxin and hydrogenase genes which are involved in electron transfer systems were linked on the 11.1-kb DNA fragment from C. acetobutylicum.     

Cloning and expression of a nitroaryl reductase gene from Streptomyces aminophilus strain MCMB411 in E. coli JM109 and Streptomyces lividans TK64

A partial genomic library was prepared in E. coli JM109 using pBR322 as vector and 2.4 kb Sau 3A I chromosomal fragment, encoding a nitroaryl reductase (nbr A) gene, from Streptomyces aminophilus strain MCMB 411. From the library, 2.4 kb fragment was recloned in E. coli JM109 and S. lividans TK64 using pUC18 and pIJ702 as vectors respectively. The recombinant plasmids pSD103 and pSD105 expressed the reductase gene and exported the enzyme in periplasmic space of E. coli and in cytoplasm of S. lividans TK64. The proteins expressed by E. coli and S. lividans had the same molecular mass (70 kD) as that expressed by parent strain, which suggested that the enzyme was processed similarly by all strains. Activities of the enzymes cloned in E. coli JM109 and S. lividans TK64 containing recombinant plasmids pSD103 and pSD105 respectively were optimum at 30 degrees C and pH 9 and requirement of cofactors was same as that of the parent strain.     

An Mr 29000 protein is essential for mini-F maintenance in E. coli

Plasmids consisting of mini-F inserted into multicopy vectors were constructed. Derivatives of these hybrid replicons were isolated which contained the transposon Tn5. The polypeptides encoded by these plasmids were identified by Escherichia coli minicell analysis. We show that a previously unidentified polypeptide of 29000 Mr is encoded by the mini-F gene E between 45.1 and 46.2 F kb on the mini-F plasmid map, and that this coding sequence (E gene) is transcribed rightward. Hybrid plasmids carrying Tn5 inserted into the E gene are unable to replicate in a polA- strain. Hence the E protein is essential for mini-F replication. Mutations in the A and B genes of mini-F affect E gene expression, and the results suggest that E protein synthesis is stimulated by A protein.     

The replication origin of Azotobacter vinelandii

The putative replication origin of Azotobacter vinelandii was cloned as an autonomously replicating fragment after ligation to an antibiotic resistance cartridge. The resulting plasmids could be isolated and labelled by Southern hybridisation with the antibiotic resistance cartridge as probe and also visualised by electron microscopy. These plasmids integrated into the chromosome after a few generations, even in the recA mutant of A. vinelandii. The integrated copy of the plasmid was re-isolated from the chromosome and the DNA and its subfragments were cloned in the plasmid vector pBR322. A 200-bp DNA fragment was sufficient to allow the replication of pBR322 in an Escherichia coli polA strain. Electron microscopic analysis of this plasmid showed that replication initiated mostly within the A. vinelandii DNA fragment. The nucleotide sequence of the putative replication origin and its flanking regions was determined. In the sequence of the 200-bp fragment many of the distinctive features found in other replication origins are lacking. A greater variation from the consensus DnaA binding sequence was observed in A. vinelandii. Direct sequencing of the relevant genomic fragment was also carried after amplifying it from A. vinelandii chromosomal DNA by PCR. This confirmed that no rearrangements had taken place while the cloned fragment was resident in E. coli. It was shown by hybridisation that the 200-bp chromosomal origin fragment of A. vinelandii was present in three other field strains of Azotobacter spp.