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PubMed2001

Crystal structure of the hexameric replicative helicase RepA of plasmid RSF1010.

Unwinding of double-stranded DNA into single-stranded intermediates required for various fundamental life processes is catalyzed by helicases, a family of mono-, di- or hexameric motor proteins fueled by nucleoside triphosphate hydrolysis. The three-dimensional crystal structure of the hexameric helicase RepA encoded by plasmid RSF1010 has been determined by X-ray diffraction at 2.4 A resolution. The hexamer shows an annular structure with 6-fold rotational symmetry and a approximately 17 A wide central hole, suggesting that single-stranded DNA may be threaded during unwinding. Homologs of all five conserved sequence motifs of the DnaB-like helicase family are found in RepA, and the topography of the monomer resembles RecA and the helicase domain of the bacteriophage T7 gp4 protein. In a modeled complex, ATP molecules are located at the subunit interfaces and clearly define adenine-binding and ATPase catalytic sites formed by amino acid residues located on adjacent monomers; most remarkable is the "arginine finger" Arg207 contributing to the active site in the adjacent monomer. This arrangement of active-site residues suggests cooperativity between monomers in ATP hydrolysis and helicase activity of RepA. The mechanism of DNA unwinding remains elusive, as RepA is 6-fold symmetric, contrasting the recently published asymmetric structure of the bacteriophage T7 gp4 helicase domain.

Adenosine Triphosphatases↗
PubMed1996

[Molecular genetic study of the strain Streptomyces virginiae-- the producer of virginiamycin].

The level and the character of the Streptomyces virginiae virginiamycin-producing strain's resistance to self-produced antibiotic and a number of antibiotics from different groups were determined. S. virginiae was shown to display constitutive and inducible resistance to self-produced antibiotic. The phenomenon of cross-inducible resistance of the strain to virginiamycin and the antibiotics erythromycin, oleandomycin, and thiostrepton was demonstrated. The pTO1 and pVGTB24 plasmids were introduced into the strain by the method of intergeneric conjugation with Escherichia coli. Site-specific integration of the pTO1 vector into the S. virginiae chromosomal attB site without disturbance of growth, differentiation, and productivity of the strain was shown. The multicopy autonomously replicating plasmids pIJ699, pIJ702, pWOR109, and the integrative pZAT22 plasmid were introduced into the strain via electrotransformation of germinating spores. The efficiency of transformation was 1-5 x 10(3) transformants per 1 microgram DNA. The stable inheritance of the plasmids in S. virginiae without structural rearrangements was shown. These results allow the use of these plasmids to clone genes into S. virginiae.

Anti-Bacterial Agents↗
PubMed2005

Binding of six nucleotide cofactors to the hexameric helicase RepA protein of plasmid RSF1010. 2. Base specificity, nucleotide structure, magnesium, and salt effect on the cooperative binding of the cofactors.

Interactions of the RepA hexameric helicase with nucleotide cofactors have been examined using nucleotide analogues, TNP-ADP and TNP-ATP, and unmodified nucleotides. Thermodynamic parameters for the interactions of modified and unmodified nucleotides have been obtained using quantitative fluorescence titration and competition titration methods. The intrinsic binding constant of ATP is by a factor of approximately 10 and approximately 1000 higher than the value observed for ADP and PO(4)(-). The data suggest that helicase acquires free-energy transducing capabilities when associated with the ssDNA, thus, forming a "holoenzyme". ATP binding is characterized by significantly stronger negative cooperativity than ADP. The cooperative interactions are predominantly induced through the specific interactions of the gamma phosphate and the ribose with the protein. The salt effect on cofactor binding indicates a very different nature of the intrinsic and cooperative interactions. Surprisingly, binding of Mg(2+), to both the cofactor and helicase, predominantly controls the ADP-RepA interactions. Mg(2+) cations seem to play a role in affecting the distribution of high and low ssDNA-affinity states, through the strong effect on the diphosphate versus triphosphate binding. The data indicate that Mg(2+) has a dual function in nucleotide-helicase interactions. At low [Mg(2+)], NTP binds stronger than NDP and the enzyme is predominantly in the high ssDNA-affinity state. At higher [Mg(2+)], NTP binds weaker than NDP and the helicase subunits can exist in alternating low- and high-affinity states that facilitate the efficient dsDNA unwinding. The RepA helicase shows a preference toward purine nucleotides. The cooperative interactions are independent of the type of the base.

Adenosine Diphosphate↗
PubMed1992

Structural and functional analysis of two cryptic plasmids from Lactobacillus pentosus MD353 and Lactobacillus plantarum ATCC 8014.

The DNA sequences of a 2.4 kb plasmid (p353-2) from Lactobacillus pentosus MD353 and a 1.9 kb plasmid (p8014-2) from Lactobacillus plantarum ATCC 8014 show 81.5% overall similarity. Both plasmids carry elements (replication protein gene, plus-origin and minus-origin of replication), which are typical of plasmids that replicate via a rolling-circle mechanism of replication (RCR). Direct evidence for an RCR mechanism was obtained by showing the accumulation of single-stranded plasmid intermediates in the presence of rifampicin. A minus-origin of replication was defined for plasmids p353-2 and p8014-2 based on DNA sequence analysis and on its ability to convert single-stranded into double-stranded plasmid DNA. Plasmids pLPE323, pLPE350 and pLPC37 that are derived from the p353-2 or p8014-2 replicon are structurally and segregationally stable in L. pentosus MD353, L. plantarum ATCC 8014 and in Lactobacillus casei ATCC 393. The presence of Escherichia coli or lambda DNA fragments in vectors derived from p353-2 or p8014-2 does not affect the structural stability but results in segregational instability of the vectors. The instability increases with increasing size of the inserted DNA fragment. Since vectors based on these replicons can be efficiently propagated in a wide variety of Lactobacillus species, they are highly suitable for cloning and expression of foreign DNA in Lactobacillus, provided that selective pressure is applied.

Amino Acid Sequence↗
PubMed1983

Cointegrational transduction and mobilization of gentamicin resistance plasmid pWP14a is mediated by IS140.

The structures of two R-plasmids pWP14a and pWP12a (Tra-, Ap, Gm; 21 kb) and of several cointegrates they form with bacteriophages P1Cm and P1-15 were analyzed. In each case, replicon fusion was mediated by the element IS140 (about 0.8 kb), one copy of which resides on both plasmids adjacent to the gentamicin resistance determinant (AAC(3)-III). pWP14a cointegrated preferentially into or near the invertible C-loop structure of the P1 genome. Cointegrational mobilization of pWP14a was observed also with several conjugative R-factors. The process of replicon fusion is independent of the host's rec+ functions. Sequences homologous to IS140 are constituents of many R-factors, including RA1, R40a, R124, R144, Rts1, N3, and pJR255. IS140 also shows homology to two other sequences, IS15 delta and Tn2680, but not to other, well studied transposable elements. The ampicillin resistance determinant of pWP14a is within a Tn3-like transposon, Tn3651.

Chromosome Mapping↗
PubMed1994

A novel structural form of the 2 micron plasmid of the yeast Saccharomyces cerevisiae.

DNA was isolated from cells of Saccharomyces cerevisiae incubated under conditions that enriched for DNA replication intermediates. A novel form of the 2 microns plasmid was detected, in which two monomeric or dimeric circles were joined by a linear double-stranded segment of variable length. We suggest that this molecule is a consequence of site-specific recombination within a dimeric DNA molecule during DNA replication. The existence of this molecule provides supporting physical evidence for a variant of the model of 2 mu plasmid amplification first proposed by Futcher (1986).

DNA Replication↗
PubMed2003

Analysis of the structure and inheritance of a linear plasmid from the obligate biotrophic fungus Blumeria graminis f. sp. hordei.

A linear plasmid is widespread among isolates of the obligate biotrophic fungus Blumeria graminis f.sp. hordei (synonym Erysiphe graminis) (Bgh), the organism that causes the disease powdery mildew on barley. We cloned and sequenced the entire plasmid of 7965 bp. The plasmid contains two identical terminal inverted repeats (TIR) of 610 bp. Two ORFs are present on opposite strands, one encoding a phage-type DNA polymerase and the other a phage-type RNA polymerase. Two large transcripts of approximately 4.2 and 5.6 kb were identified in conidia, germinating conidia and Bgh -infected barley leaves, indicating that the polymerases are transcribed at most stages of the lifecycle. The transcription start sites were localised within the TIR regions, where a putative 11-bp ARS consensus sequence was also identified. To follow the sexual transmission of the plasmid we screened 27 Bgh isolates for mitochondrial polymorphisms. One polymorphism allowed us to carry out a cross between two isolates that differed in both mitochondrial genotype and presence/absence of the Bgh plasmid. The plasmid was transmitted independently of the origin of the mitochondria. No transfer of the plasmid was observed between two Bgh isolates that were co-cultivated for 1.5 years on a common susceptible barley variety. The plasmid appears to be an autonomous replicon with no phenotypic effect on Bgh.

Ascomycota↗
PubMed1986

Structure and function of the F plasmid genes essential for partitioning.

The F plasmid in Escherichia coli has its own partition mechanism controlled by the sopA and sopB genes, and by the cis-acting sopC region. The DNA sequence of the entire partition region and its flanking regions is described here. Two large open reading frames coding for 43,700 Mr and 35,400 Mr proteins correspond to sopA and sopB, respectively. The sopB reading frame is located immediately downstream from the sopA reading frame. Twelve 43 base-pair direct repeats exist in the sopC region without any spacer regions, and one pair of seven base-pair inverted repeats exists in each of the direct repeats. Analysis of deletions in the sopC region showed that the direct repeats play an important role in plasmid partition and IncD incompatibility. IncG incompatibility is exhibited by pBR322 derivatives carrying the sopB gene alone. When compared with the partition genes parA and parB of plasmid P1, homology in amino acid sequence was found between the SopA protein of F and the ParA protein of P1, and also between SopB protein of F and ParB protein of P1. In addition, homology was found between Rep proteins of F and P1.

Amino Acid Sequence↗
PubMed1983

The structure of cloned hemolysin DNA from plasmid pHly185.

The complete DNA segment coding for hemolysis on Escherichia coli plasmid, pHly185, on to the vector pBR322 has been cloned. The recombinant plasmid, pJS204, coded the production of externalized hemolysin and contained four contiguous EcoRI fragments from the original plasmid. A restriction map of pJS204 was generated with six endonucleases. Deletion mutations were constructed from the recombinant plasmid by excision with either BamHI and PstI and religation. The BamHI deletion removed a 3680-bp portion from the recombinant plasmid and prevented the synthesis of active hemolytic activity. The PstI-generated deletion removed 7800 bp from the opposite end of the Hly sequence and resulted in a plasmid coding for the production of active intracellular hemolytic activity but contained no information for the export of hemolysin. We examined the ability of the deletion mutants of pJS204 to complement each other, and Tn5 insertion mutations in the hly region of the parent plasmid, pHly185. The complementation studies indicated the presence of three separate cistrons able to complement in trans in a recA E. coli host. Two cistrons were found to be required for synthesis and one cistron for export of the hemolysin. The presence of two promoters and the directions of transcription for the synthesis of the hly gene products was also inferred from the complementation studies.

Bacterial Proteins↗
PubMed1988

Functional and structural map of pLST1000: a multiresistance plasmid widely distributed in Enterobacteriaceae.

pLST1000, an 80-kb plasmid found in Enterobacteriaceae in North and South America, harbors the aadB and several other resistance genes. We suggested earlier that, because of its widespread distribution, pLST1000 could act as a carrier plasmid, bringing the aadB gene to new locations. This paper presents the restriction enzyme recognition site and functional map of the plasmid. The resistance genes lie in a discreet region. The aadB and aadA genes form an operon with the aadB gene promoter proximal. This operon is flanked by bla-TEM and bla-OXA2 genes, the former located in a functional Tn3-like transposon. This arrangement is similar to that of relatives of the transposon TN21, where additional resistance genes are precisely inserted in recombinational "hot spot" sequences that flank the aadA gene. We were not able to demonstrate transposition of the aadB gene in Escherichia coli. A sul gene and mer operon lie beyond the bla-OXA2 gene. The transfer genes form a single region, defined by insertions of Tn5-132 that give the Tra- phenotype.

Drug Resistance, Microbial↗
PubMed1989

Structural organization of pLP1, a cryptic plasmid from Lactobacillus plantarum CCM 1904.

To construct shuttle vectors based on an endogenous replicon, we isolated a small cryptic plasmid (pLP1) from Lactobacillus plantarum CCM 1904. The nucleotide sequence (2093 bp, 38.25 GC mol%) revealed one major open reading frame encoding for a 317 amino acid protein (Rep). Comparisons with proteins encoded by other Gram-positive bacteria plasmids strongly suggest that the protein encoded by pLP1 has a replicative role. The presence of a consensus sequence including a tyrosine residue known to be the replication protein binding site to the DNA (in phage phi X174) strengthens this hypothesis. The DNA sequence contains also a sequence similar to the pC194 origin nick sequence, which initiates the plasmid replication at the plus origin, characteristic of plasmids which replicate following a rolling circle mechanism via single-stranded DNA intermediates. A set of 13 direct repeats of 17 bp could be involved in the expression of the incompatibility or in the copy number control as in the other plasmids. A promoter sequence located at the rep 5' region has been identified and is functional in Bacillus subtilis.

Amino Acid Sequence↗
PubMed1992

Construction and characterization of shuttle plasmids for lactic acid bacteria and Escherichia coli.

The chimeric plasmid pBN183 was first constructed in Escherichia coli by ligating the BamHI-digested E. coli plasmid pBR322 and a Bg/II-linearized streptococcal plasmid, pNZ18. The pBN183 transformed E. coli to ApR at a frequency of (8.2 +/- 1.2) x 10(5) colony forming units (CFU)/microgram DNA. Electrotransformation of Streptococcus thermophilus with pBN183 yielded CmR, ApS clones at a frequency of (2.6 +/- 0.3) x 10(1) CFU/microgram DNA. Plasmid screening with pBN183-transformed S. thermophilus clones revealed that ca. 70% of these transformants contained deleted plasmids. Plasmid pBN183A, a pBN183 deletion mutant lacking one copy of a tandemly arranged, highly homologous DNA sequence, was isolated for further study. It transformed E. coli to ApR and S. thermophilus to CmR with frequencies of (4.8 +/- 0.1) x 10(5) and (8.1 +/- 0.2) x 10(2) CFU/microgram DNA, respectively. Screening of S. thermophilus transformants did not show the presence of deleted plasmids. Based on the structure of pBN183A, a new shuttle plasmid, pDBN183, was constructed from pBN183 by removal of the small (1.2 kb) Sa/I fragment. Transformation frequencies of pDBN183 were (5.0 +/- 1.3) x 10(5) and (4.6 +/- 0.2) x 10(2) CFU/microgram DNA with E. coli and S. thermophilus, respectively. In contrast to the parent pBN183, only 17% of the pDBN183-transformed S. thermophilus contained deleted plasmids. Plasmid copy numbers of the three vectors in E. coli were estimated at 17-18 per chromosome. The three plasmids conferred ApR and CmR to E. coli, but only CmR to S. thermophilus. The insertion of a Streptomyces cholesterol oxidase gene (choA) into pDBN183 did not affect the plasmid's stability in Lactobacillus casei, but resulted in deletion of the recombinant DNA in S. thermophilus.

Cholesterol Oxidase↗
PubMed1993

Comparison of three typing methods in hospital outbreaks of Acinetobacter calcoaceticus infection.

During a period of 11 months Acinetobacter baumanii was isolated from 27 patients and 21 environmental samples in the Intensive Care Unit (ICU) and in one surgical unit of a University Hospital. The isolates were characterized by biotyping, antibiograms and plasmid profiles and compared with co-isolates. Plasmid fingerprinting distinguished three outbreaks, whereas other typing methods were less sensitive and discriminatory. Although plasmid profiles seem to be a simple and reproducible marker for epidemiological studies with acinetobacter strains, it might be useful to combine at least two typing methods since plasmids are unstable genetic structures, and not all strains possess plasmids.

Acinetobacter Infections↗
PubMed1991

DNA-binding properties and antitumour activity of monofunctional alkylating groups attached to the DNA-intercalating chromophore phenanthridine: n-bromoalkylphenanthridinium bromides.

We have synthesised an homologous series of n-bromoalkylphenanthridinium bromides and studied their DNA-binding and antitumour properties. Each of these compounds has the capacity both to intercalate and alkylate DNA. Dialysis measurements reveal a relatively high affinity for calf thymus DNA, being about 10(5) M-1 at ionic strength 0.01. Incubating calf thymus DNA-ligand complexes having a ligand-to-basepair ratio of 0.4 at 37 degrees C for 18 h leads to maximum alkylation levels of about one ligand molecule bound irreversibly per 40 basepairs. The reactivity of these compounds towards DNA is chain-length dependent, the n-decyl compound, for example, requiring about 10-times the ligand-to-basepair input ratio of the n-hexyl derivative to reach the same level of alkylation. The limited degree of alkylation is a consequence of conversion of the alkylbromides to the less reactive alkylchlorides in the buffer medium. The results of DNA sequencing experiments indicate that the n-hexyl derivative alkylates at guanines occurring in 5'-GT-3' sequences and in runs of guanines [(Gp)n]. The corresponding n-decyl compound, on the other hand, is highly selective for guanines in 5'-GT-3' sequences only and also reacts weakly with some adenines. None of the phenanthridinium compounds showed significant antitumour activity in the P388 murine leukaemia test system.

Alkylating Agents↗
PubMed2003

Synthesis, structure and nuclease properties of several ternary copper(II) peptide complexes with 1,10-phenanthroline.

Three new ternary peptide-Cu(II)-1,10-phenanthroline (phen) complexes, [Cu(L-ala-gly)(phen)].3.5H(2)O 1, [Cu(L-val-gly)(phen)] 2 and [Cu(gly-L-trp)(phen)].2H(2)O 3, have been prepared and structurally characterised. These compounds exist as distorted square pyramidal complexes with the five co-ordination sites occupied by the tridentate peptide dianion and the two heterocyclic nitrogens of the phenanthroline ligand. The bulk of the lateral chain in the peptide moiety determines the relative disposition of the phen ligand. Thus, in [Cu(L-val-gly)(phen)] 2, the phenanthroline plane is deviated towards the opposite side of the isopropyl group of the L-valine moiety. On the other hand, in [Cu(gly-L-trp)(phen)].2H(2)O 3 the absence of stacking interactions between phen and indole rings and the presence of an intramolecular CH...pi interaction should be pointed out. These complexes exhibit significant differences in their nuclease activity which depends on the nature of the peptidic moiety, the complex [Cu(gly-L-trp) (phen)].2H(2)O 3 being the most active.

Copper↗