Plasmid mediated alterations in composition and structure of envelopes of Escherichia coli B/r.
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Bacterial Proteins↗
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Linear plasmids of Borrelia burgdorferi have a telomeric structure and sequence similar to those of a eukaryotic virus.
Spirochetes of the genus Borrelia have double-stranded linear plasmids with covalently closed ends. The physical nature of the terminal connections was determined for the 16-kb linear plasmid of the B31 strain of the Lyme disease agent Borrelia burgdorferi. Native telomeric fragments representing the left and right ends of this plasmid were isolated and subjected to Maxam-Gilbert sequence analysis. At the plasmid ends the two DNA strands formed an uninterrupted, perfectly palindromic, AT-rich sequence. This Borrelia linear plasmid consisted of a continuous polynucleotide chain that is fully base paired except for short single-stranded hairpin loops at each end. The left and right telomeres of the 16-kb plasmid were identical for 16 of the first 19 nucleotide positions and constituted an inverted terminal repeat with respect to each other. The left telomere of the 49-kb plasmid of strain B31 was identical to the corresponding telomere of the 16-kb plasmid. Different-sized plasmids of other strains of B. burgdorferi also contained sequences homologous to the left end of the 16-kb plasmid. When the borrelia telomeres were compared with telomeric sequences of other linear double-stranded DNA replicons, sequence similarities were noted with poxviruses and particularly with the iridovirus agent of African swine fever. The latter virus and a Borrelia sp. share the same tick vector. These findings suggest that the novel linear plasmids of Borrelia originated through a horizontal genetic transfer across kingdoms.
Animals↗
Antisense-RNA mediated transcriptional attenuation: importance of a U-turn loop structure in the target RNA of plasmid pIP501 for efficient inhibition by the antisense RNA.
Antisense-RNA mediated gene regulation has been found and studied in detail mainly in prokaryotic accessory DNA elements. In spite of different regulatory mechanisms, in all cases a rapid interaction between antisense and target RNA has been shown to be crucial for efficient regulation. Recently, a sequence comparison revealed in 45 antisense RNA control systems a 5' YUNR motif indicative for the formation of a U-turn structure in either an antisense or a target RNA loop and confirmed in the case of the hok/sok system of plasmid R1 its importance for regulation.Here, we demonstrate the importance of the 5' YUNR motif in the target RNA (RNAII) loop L1 of the replication control system of plasmid pIP501. The effect of four individual mutations in L1 was studied in vivo and in vitro. Mutations that maintained the putative U-turn or swapped it from sense to antisense RNA were silent, whereas mutations that eliminated the 5'-YUNR motif showed two- to threefold elevated copy numbers in vivo in correlation with three- to fourfold reduced inhibition rate constants of the complementary RNAIII species in vitro, whereas the half-lives of all RNAIII species were not affected. ENU probing experiments confirmed the U-turn structure for the silent mutation (N-C) and disruption of this structure upon alteration of the invariant U or inversion of the YUNR motif-containing loop. RNA secondary structure probing excluded loop size alterations as a reason for altered inhibition rates. Implications for the pathway and efficiency of RNAII/RNAIII interaction, and hence, pIP501 copy-number control, are discussed.
Bacillus subtilis↗
Enterococcus faecalis conjugative plasmid pAM373: complete nucleotide sequence and genetic analyses of sex pheromone response.
pAM373 is a 36.7 kb conjugative plasmid in Enterococcus faecalis that encodes a response to a peptide sex pheromone, cAM373, secreted by plasmid-free (recipient) strains of enterococci. It was identified over 15 years ago as one of five plasmids in E. faecalis strain RC73 and was of interest because a related pheromone activity could be detected in culture supernatants of Staphylococcus aureus and Streptococcus gordonii. Because of increased clinical concern relating to the possibility of mobilizing vancomycin resistance determinants from enterococci, where they are becoming common, into pathogens such as S. aureus, efforts were initiated to characterize pAM373 further. The results of a complete nucleotide sequence determination of pAM373, as well as a genetic analysis of key genes related to regulation of the pheromone response, are reported here. With regard to determinants related to conjugation, the plasmid has a structural organization similar to other known pheromone-responsive plasmids such as pAD1, pCF10 and pPD1; however, there are several unique features. Although there are significant homologues relating to a pheromone-binding surface protein (TraC) and a negatively regulating protein (TraA), there is an absence of a determinant equivalent to traB of pAD1 (reduces endogenous pheromone) and a determinant for surface-exclusion protein. The precursor structure of the inhibitor peptide iAM373 was identified, and its determinant (iam373) was found to be about 500 nt upstream of an apparent transcription terminator t1. Tn917-lac insertion analyses provided interesting insights into aspects of control of the pheromone response and showed that, although the traA product is sensitive to pheromone, it appears to act differently from the traA homologue of pAD1.
Adhesins, Bacterial↗
Nuclease recognition of an alternating structure in a d(AT)14 plasmid insert.
The nuclease reactivity and specificity of a cloned tract of poly X (dA-dT) X poly(dA-dT) has been explored. Digestion with DNAse I, Mung Bean nuclease, S1 nuclease, DNAse II, and copper (1,10-phenanthroline)2 on a 256 base pair restriction fragment containing d(AT)14A revealed a dinucleotide repeat structure for the alternating sequence. Furthermore, conditions which wind or unwind the linear DNA had little effect on the reactivity of the AT insert. These preferred cleavages offer insights to structural alterations within the DNA helix which differ from A, B, or Z-DNA. Nucleation into flanking sequences by this structural alteration was not observed.
Deoxyribonuclease I↗
Analysis of the first two genes of the CS1 fimbrial operon in human enterotoxigenic Escherichia coli of serotype 0139:H28.
An oligonucleotide, derived from the N-terminal amino acid sequence of the CS1 fimbrial subunit protein was used to identify the subunit gene on recombinant plasmid pDEP23 containing the structural genes of the CS1 fimbrial operon. The nucleotide sequence of the subunit gene (csoA), encoding a protein of 171 amino acids, was determined. Flanking it upstream, a gene (csoB) encoding a protein of 238 amino acids was found. The CsoB and CsoA proteins are homologous to the CfaA and CfaB proteins in the CFA/I fimbrial operon. For all the CS1 producing strains investigated the structural genes are located on plasmids. Like CFA/I fimbriae, CS1 fimbriae are only expressed in the presence of a positive regulator, CfaD for CFA/I and Rns for CS1, respectively. The promoter region upstream of the csoB gene was cloned in front of the promoterless alkaline phosphatase (phoA) gene of the promoter-probe vector pCB267. PhoA activity was enhanced approximately two-fold by the introduction of compatible plasmids containing either rns or cfaD.
Amino Acid Sequence↗
DNA phase transition promoted by replication initiator.
DNA is flexible and easily subjected to bending and wrapping via DNA/protein interaction. DNA supercoiling is known to play an important role in a variety of cellular events, such as transcription, replication, and recombination. It is, however, not well understood how the superhelical strain is efficiently redistributed during these reactions. Here we demonstrate a novel property of an initiator protein in DNA relaxation by utilizing a one-molecule-imaging technique, atomic force microscopy, combined with biochemical procedures. A replication initiator protein, RepE54 of bacterial mini-F plasmid (2.5 kb), binds to the specific sequences (iterons) within the replication region (ori2). When RepE54 binds to the iterons of the negatively supercoiled mini-F plasmid, it induces a dynamic structural transition of the plasmid to a relaxed state. This initiator-induced relaxation is mediated neither by the introduction of a DNA strand break nor by a local melting of the DNA double strand. Furthermore, RepE54 is not wrapped by DNA repeatedly. These data indicate that a local strain imposed by initiator binding can induce a drastic shift of the DNA conformation from a supercoiled to a relaxed state.
Adenosine Triphosphate↗
Characterization of hybrid plasmids carrying individual ribosomal ribonucleic acid transcription units of Escherichia coli.
We have screened the strains with ColE1 hybrid plasmids constructed by Clarke and Carbon (Cell 9:91-99, 1976) for the presence of ribosomal ribonucleic acid (rRNA) genes on the plasmids and identified 16 strains whose plasmids carry rRNA genes. The structures of these 16 plasmids were compared by heteroduplex analysis, and the plasmids were classified into six groups on the basis of their chromosomal origins. Homology with known transducing-phage deoxyribonucleic acids and genetic mapping have assigned locations on the Escherichia coli chromosome to three of the six groups. These are rrnB near rif at 88 min, rrnC near ilvE at 83 min, and rrnD near aroE at 71 min. A fourth group is probably rrnA at 85 min (T. Ikemura and M. Nomura, Cell, 11:779-793, 1977). We conclude that the minimum number of rRNA transcription units per haploid chromosomes is seven, that is, the six groups identified in this work plus a known operon (rrnE near metA at 89 min) that we failed to find among the hybrid plasmids. This heteroduplex analysis also suggests that there are only two kinds of rRNA operons with respect to their spacer region; three of the six rRNA operon groups studied here have one kind, whereas the remaining three have the other kind.
Bacterial Proteins↗
Micrococcal nuclease digestion of nuclei reveals extended nucleosome ladders having anomalous DNA lengths for chromatin assembled on non-replicating plasmids in transfected cells.
The chromatin structures of a variety of plasmids and plasmid constructions, transiently transfected into mouse Ltk- cells using the DEAE-dextran procedure, were studied by micrococcal nuclease digestion of nuclei and Southern hybridization. Although regularly arranged nucleosome-like particles clearly were formed on the transfected DNA, the nucleosome ladders, in some cases with 13-14 bands, were anomalous. Most often, a ladder of DNA fragments with lengths of approximately 300, 500, 700, 900 bp, etc. was generated. In contrast, typical 180-190 bp multiples were generated from bulk cellular or endogenous beta-actin gene chromatin. Very similar results were obtained with all DNA's transfected, and in a variety of cell lines, provided that plasmid replication did not occur. Additionally, after digestion of nuclei, about 90% of the chromatin fragments that contained transfected DNA sequences could not be solubilized at low ionic strength, in contrast with bulk cellular chromatin, suggesting association with nuclear structures or nuclear matrix. The remaining 10% of transfected DNA sequences, arising from soluble chromatin fragments, generated a typical nucleosome ladder. These results are consistent with the idea that assembly of atypical chromatin structures might be induced by proximity to elements of the nuclear pore complex or by nuclear compartmentalization.
Animals↗
Hairpin plasmid--a novel linear DNA of perfect hairpin structure.
The terminal structures of deletion derivatives of linear DNA killer plasmid from yeast were analyzed. The yeast Kluyveromyces lactis harbors two unique double-stranded linear DNA killer plasmids, pGKL1 of 8.9 kb and pGKL2 of 13.4 kb. The killer toxin and the resistance to the killer are coded by pGKL1, while pGKL2 is required for the maintenance of pGKL1 in the cell. When the pGKL plasmids from K. lactis were transferred into Saccharomyces cerevisiae by transformation, non-killer transformants harboring pGKL2 and new plasmids, F1 of 7.8 kb and F2 of 3.9 kb, were obtained. F2 was shown to be a linear DNA arising from a 5-kb deletion of the right part of pGKL1. F1 was an inverted dimer of F2. Here we show that F2 has two different terminal structures: one end has a protein attached at the 5' terminus whereas the two strands of duplex are linked together at the other end, thus forming a hairpin structure. This is a novel type of autonomously replicating DNA molecule.
Base Sequence↗
Modelling of genetically engineered microorganisms introduction in closed artificial microcosms.
The possibility of introducing genetically engineered microorganisms (GEM) into simple biotic cycles of laboratory water microcosms was investigated. The survival of the recombinant strain Escherichia coli Z905 (Apr, Lux+) in microcosms depends on the type of model ecosystems. During the absence of algae blooming in the model ecosystem, the part of plasmid-containing cells E. coli decreased fast, and the structure of the plasmid was also modified. In conditions of algae blooming (Ankistrodesmus sp.) an almost total maintenance of plasmid-containing cells was observed in E. coli population. A mathematics model of GEM's behavior in water ecosystems with different level of complexity has been formulated. Mechanisms causing the difference in luminescent exhibition of different species are discussed, and attempts are made to forecast the GEM's behavior in water ecosystems.
Animals↗
Cloning vectors based on cryptic plasmids isolated from lactic acid bacteria: their characteristics and potential applications in biotechnology.
Lactic acid bacteria (LAB) are Gram positive bacteria, widely distributed in nature, and industrially important as they are used in a variety of industrial food fermentations. The use of genetic engineering techniques is an effective means of enhancing the industrial applicability of LAB. However, when using genetic engineering technology, safety becomes an essential factor for the application of improved LAB to the food industry. Cloning and expression systems should be derived preferably from LAB cryptic plasmids that generally encode genes for which functions can be proposed, but no phenotypes can be observed. However, some plasmid-encoded functions have been discovered in cryptic plasmids originating from Lactobacillus, Streptococcus thermophilus, and Pediococcus spp. and can be used as selective marker systems in vector construction. This article presents information concerning LAB cryptic plasmids, and their structures, functions, and applications. A total of 134 cryptic plasmids collated are discussed.
Biotechnology↗
Structural and functional features of cis-acting sequences in the basic replicon of plasmid ColIb-P9.
We have structurally and functionally analyzed the cis-elements essential for ColIb-P9 plasmid DNA replication. The putative oriV region encompassed a region of 172 base pairs (bp) located 152 bp downstream of the repZ gene. A typical dnaA box found in this region proved nonessential for the DNA replication of ColIb-P9. The ssi signal of ColIb-P9 is a homologue of the G-sites of R1 and R100 plasmids. Deletion of the G-site led to 1.5-fold reduction of the copy number, suggesting that although this G-site is not essential, it is important for efficient ColIb-P9 DNA replication. In addition, the ColIb-P9 replicon is highly and extensively homologous with the P307 (RepFIC) replicon, and highly homologous with the R100 (RepFIIA) replicon around the G-site region. These facts imply a common ancestry from which the plasmids have evolved.
Base Sequence↗
Assembling the bacterial segrosome.
Genome segregation in prokaryotes is a highly ordered process that integrates with DNA replication, cytokinesis and other fundamental facets of the bacterial cell cycle. The segrosome is the nucleoprotein complex that mediates DNA segregation in bacteria, its assembly and organization is best understood for plasmid partition. The recent elucidation of structures of the ParB plasmid segregation protein bound to centromeric DNA, and of the tertiary structures of other segregation proteins, are key milestones in the path to deciphering the molecular basis of bacterial DNA segregation.
Centromere↗
The structural gene for tetanus neurotoxin is on a plasmid.
A pool of synthetic oligonucleotides was prepared based on the amino terminal amino acid sequence of tetanus toxin. This probe hybridized to plasmid DNA isolated from three toxigenic strains of Clostridium tetani but not to plasmid DNA from a nontoxigenic strain. These results show that the structural gene for the toxin is on the plasmid. The pCL1 plasmid from one of the toxigenic strains spontaneously deleted 22 kilobase pairs of DNA to form pCL2. Strains harboring this deleted plasmid are nontoxigenic. However, the probe mixture hybridized to pCL2, indicating that the DNA encoding the amino terminus of the toxin had not been deleted. Restriction endonuclease cleavage maps of pCL1 and pCL2 were constructed and indicate the approximate location and orientation of the structural gene for tetanus toxin.
Amino Acid Sequence↗
Plasmid P1 RepA is homologous to the F plasmid RepE class of initiators.
DNA replication of plasmid P1 requires a plasmid-encoded origin DNA-binding protein, RepA. RepA is an inactive dimer and is converted by molecular chaperones into an active monomer that binds RepA binding sites. Although the sequence of RepA is not homologous to that of F plasmid RepE, we found by using fold-recognition programs that RepA shares structural homology with RepE and built a model based on the RepE crystal structure. We constructed mutants in the two predicted DNA binding domains to test the model. As expected, the mutants were defective in P1 DNA binding. The model predicted that RepA binds the first half of the binding site through interactions with the C-terminal DNA binding domain and the second half through interactions with the N-terminal domain. The experiments supported the prediction. The model was further supported by the observation that mutants defective in dimerization map to the predicted subunit interface region, based on the crystal structure of pPS10 RepA, a RepE family member. These results suggest P1 RepA is structurally homologous to plasmid initiators, including those of F, R6K, pSC101, pCU1, pPS10, pFA3, pGSH500, Rts1, RepHI1B, RepFIB, and RSF1010.
Amino Acid Sequence↗
The nucleotide sequence of the structural gene for Escherichia coli tryptophanyl-tRNA synthetase.
The complete nucleotide sequence of trpS, the structural gene for Escherichia coli tryptophanyl-tRNA synthetase, was determined using a plasmid carrying the structural gene. From the single open reading frame of correct length and orientation we deduced an amino acid sequence consistent with the amino acid composition of the purified protein. In addition, previously sequenced peptides representing 52% of the protein were readily aligned with regions of the deduced sequence. The deduced amino acid sequence of the E. coli enzyme is 60% homologous with the sequence of the enzyme from Bacillus stearothermophilus. Using currently available procedures we predicted the secondary structure for the enzyme from each organism and compared these structures to those of the two aminoacyl-tRNA synthetases whose three-dimensional structures have been determined. We used a convenient plasmid recombination procedure to map the regional locations of missense mutations within trpS that have characteristic effects on the properties of the enzyme.
Amino Acid Sequence↗
Replication control of plasmid R1: disruption of an inhibitory RNA structure that sequesters the repA ribosome-binding site permits tap-independent RepA synthesis.
The replication frequency of plasmid R1 is controlled by an antisense RNA, CopA, that inhibits the synthesis of the replication initiator protein, RepA, at the post-transcriptional level. This inhibition is indirect and affects translation of a leader peptide reading frame (tap). Translation of tap is required for repA translation (Blomberg et al., 1992). Here we asked whether an RNA stem-loop sequestering the repA ribosome-binding site blocks tap translation-independent repA expression. Destabilization of this structure resulted in tap-independent RepA synthesis, concomitant with a loss of CopA-mediated inhibition; thus, CopA acts at the level of tap translation. Structure probing of RepA mRNAs confirmed that the introduced mutations induced a local destabilization in the repA ribosome-binding site stem-loop. An increased spacing between the repA Shine-Dalgarno region and the start codon permitted even higher repA expression. In Incl alpha/IncB plasmids, an RNA pseudoknot acts as an activator for rep translation. We suggest that the regulatory pathway in plasmid R1 does not involve an activator RNA pseudoknot.
Bacterial Proteins↗