Identification and characterization of an aadB gene cassette at a secondary site in a plasmid from Acinetobacter

Transformation studies showed that an aminoglycoside resistance gene, aadB, is carried on a 6.0-kb plasmid (pRAY) in a clinical isolate of Acinetobacter (strain SUN). The gene was cloned and sequenced. An analysis of the DNA sequencing data showed that although the aadB gene is part of cassette, it is not associated with an integron. Rather, the aadB cassette has recombined at a secondary site downstream of putative promoters on pRAY.     

Inhibition of VirB-mediated transfer of diverse substrates from Agrobacterium tumefaciens by the IncQ plasmid RSF1010.

The transfer of DNA from Agrobacterium tumefaciens into a plant cell requires the activities of several virulence (vir) genes that reside on the tumor-inducing (Ti) plasmid. The putative transferred intermediate is a single-stranded DNA (T strand), covalently attached to the VirD2 protein and coated with the single-stranded DNA-binding protein, VirE2. The movement of this intermediate out of Agrobacterium cells and into plant cells requires the expression of the virB operon, which encodes 11 proteins that localize to the membrane system. Our earlier studies showed that the IncQ broad-host-range plasmid RSF1010, which can be transferred from Agrobacterium cells to plant cells, inhibits the transfer of T-DNA from pTiA6 in a fashion that is reversed by overexpression of virB9, virB10, and virB11. Here, we examined the specificity of this inhibition by following the transfer of other T-DNA molecules. By using extracellular complementation assays, the effects of RSF1010 on movement of either VirE2 or an uncoated T strand from A. tumefaciens were also monitored. The RSF1010 derivative plasmid pJW323 drastically inhibited the capacity of strains to serve as VirE2 donors but only partially inhibited T-strand transfer from virE2 mutants. Further, we show that all the virB genes tested are required for the movement of VirE2 and the uncoated T strand as assayed by extracellular complementation. Our results are consistent with a model in which the RSF1010 plasmid, or intermediates from it, compete with the T strand and VirE2 for a common transport site.     

Tn3 labeling of a cryptic plasmid found in the plant pathogenic bacterium Pseudomonas tabaci and mobilization of RSF1010 by donation.

pBPW1, a conjugative cryptic plasmid isolated from the plant pathogenic bacterium Pseudomonas tabaci BR2, was labeled with Tn3. pBPW1::Tn3 and RSF1010 mobilization into Pseudomonas mellea recipients were separate events, not involving recombination of the two plasmids during conjugation.     

Integron-associated gene cassettes in Halifax Harbour: assessment of a mobile gene pool in marine sediments

The integron/gene cassette systems identified in bacteria comprise a class of genetic elements that allow adaptation by acquisition of gene cassettes. Integron gene cassettes have been shown to facilitate the spread of drug resistance in human pathogens but their role outside a clinical setting has not been explored extensively. We sequenced 2145 integron gene cassettes from four marine sediment samples taken from the vicinity of Halifax Nova Scotia, Canada, increasing the number of gene cassettes obtained from environmental microbial communities by 10-fold. Sequence analyses reveals that the majority of these cassettes encode novel proteins and that this study is consistent with previous claims of high cassette diversity as we estimate a Chao1 diversity index of ∼3000 cassettes from these samples. The functional distribution of environmental cassettes recovered in this study, when compared with that of cassettes from the only other source with significant sampling ( Vibrio genomes) suggests that alternate selection regimes might be acting on these two gene pools. The majority of cassettes recovered in this study encode novel, unknown proteins. In instances where we obtained multiple alleles of a novel protein we demonstrate that non-synonymous versus synonymous substitution rates ratios suggest relaxed selection. Cassette-encoded proteins with known homologues represent a variety of functions and prevalent among these are isochorismatases; proteins involved in iron scavenging. Phylogenetic analysis of these isochorismatases as well as of cassette-encoded acetyltransferases reveals a patchy distribution, suggesting multiple sources for the origin of these cassettes. Finally, the two most environmentally similar sample sites considered in this study display the greatest overlap of cassette types, consistent with the hypothesis that cassette genes encode adaptive proteins.     

The Agrocybe aegerita mitochondrial genome contains two inverted repeats of the nad4 gene arisen by duplication on both sides of a linear plasmid integration site

The Agrocybe aegerita mitochondrial genome possesses two polB genes with linear plasmid origin. The cloning and sequencing of the regions flanking Aa-polB P1 revealed two large inverted repeats (higher than 2421 nt) separated by a single copy region of 5834 nt. Both repeats contain identical copies of the nad4 gene. The single copy region contains two disrupted genes with plasmid origin Aa-polB P1 and a small ORF homologous to a small gene described in two basidiomycete linear plasmids. The phylogenetic analyses argue in favor of a same plasmid origin for both genes but, surprisingly, these genes were separated by a mitochondrial tRNA-Met. Both strands of the complete region containing the two nad4 inverted copies and the tRNA-Met appear to be transcribed on large polycistronic mRNAs. A model summarizing the events that would have occurred is proposed: (1) capture of the tRNA by the plasmid before its integration in the mtDNA or acquisition of the tRNA gene by recombination after the plasmid integration, (2) integration of the plasmid in the mtDNA, accompanied by a large duplication containing the nad4 gene and (3) erosion of the plasmid sequences by large deletions and mutations.     

The Agrocybe aegerita mitochondrial genome contains two inverted repeats of the nad4 gene arisen by duplication on both sides of a linear plasmid integration site.

The Agrocybe aegerita mitochondrial genome possesses two polB genes with linear plasmid origin. The cloning and sequencing of the regions flanking Aa-polB P1 revealed two large inverted repeats (higher than 2421 nt) separated by a single copy region of 5834 nt. Both repeats contain identical copies of the nad4 gene. The single copy region contains two disrupted genes with plasmid origin Aa-polB P1 and a small ORF homologous to a small gene described in two basidiomycete linear plasmids. The phylogenetic analyses argue in favor of a same plasmid origin for both genes but, surprisingly, these genes were separated by a mitochondrial tRNA-Met. Both strands of the complete region containing the two nad4 inverted copies and the tRNA-Met appear to be transcribed on large polycistronic mRNAs. A model summarizing the events that would have occurred is proposed: (1) capture of the tRNA by the plasmid before its integration in the mtDNA or acquisition of the tRNA gene by recombination after the plasmid integration, (2) integration of the plasmid in the mtDNA, accompanied by a large duplication containing the nad4 gene and (3) erosion of the plasmid sequences by large deletions and mutations.     

Bacterial conjugation mediated by plasmid RP4: RSF1010 mobilization, donor-specific phage propagation, and pilus production require the same Tra2 core components of a proposed DNA transport complex.

DNA transfer by bacterial conjugation requires a mating pair formation (Mpf) system that specifies functions for establishing the physical contact between the donor and the recipient cell and for DNA transport across membranes. Plasmid RP4 (IncP alpha) contains two transfer regions designated Tra1 and Tra2, both of which contribute to Mpf. Twelve components are essential for Mpf, TraF of Tra1 and 11 Tra2 proteins, TrbB, -C, -D, -E, -F, -G, -H, -I, -J, -K, and -L. The phenotype of defined mutants in each of the Tra2 genes was determined. Each of the genes, except trbK, was found to be essential for RP4-specific plasmid transfer and for mobilization of the IncQ plasmid RSF1010. The latter process did not absolutely require trbF, but a severe reduction of the mobilization frequency occurred in its absence. Transfer proficiency of the mutants was restored by complementation with defined Tra2 segments containing single trb genes. Donor-specific phage propagation showed that traF and each of the genes encoded by Tra2 are involved. Phage PRD1, however, still adsorbed to the trbK mutant strain but not to any of the other mutant strains, suggesting the existence of a plasmid-encoded receptor complex. Strains containing the Tra2 plasmid in concert with traF were found to overexpress trb products as well as extracellular filaments visualized by electron microscopy. Each trb gene and traF are needed for the formation of the pilus-like structures. The trbK gene, which is required for PRD1 propagation and for pilus production but not for DNA transfer on solid media, encodes the RP4 entry-exclusion function. The components of the RP4 Mpf system are discussed in the context of related macromolecule export systems.     

Plasmid formation in Streptomyces: excision and integration of the SLP1 replicon at a specific chromosomal site

Summary We present data showing that the SLP1 plasmids found in Streptomyces lividans after mating with S. coelicolor strain A3(2) orginate as deletion mutants of a 17 kb segment of the S. coelicolor chromosome. Excision of the entire 17 kb segment yields a transiently existing plasmid containing a site for integration into the chromosome of recipient SLP1^- S. lividans strains at a unique locus that corresponds to the original chromosomal location of SLP1 in S. coelicolor. The deletion mutants of SLP1 lack the attachment site and/or other regions required for its integration, and thus persist in the recipient as autonomously replicating plasmids. Plasmids that contain the complete 17 kb sequence of the chromosomally integrated SLP1 segment were constructed in vitro by circularization of restriction endonuclease-generated fragements of chromosomal DNA carrying a tandemly-duplicated integrant of SLP1. Transformation of an SLP1^- S. lividans strain with such plasmids results in chromosomal integration of the SLP1 sequence at the same site at which it is integrated in S. lividans cells that acquire the sequence by mating with S. coelicolor. A model for the site-specific excision and integration of SLP1 is presented.     

Investigation of the polymorphic AvaII site by a PCR-based assay at the human CD18 gene locus

The AvaII polymorphic site within the human CD18 gene was investigated in the Japanese population. A distinct distribution pattern is observed in this population. This polymorphism provides a new genetic marker for the long arm of chromosome 21 and should be a useful marker of leukocyte adhesion deficiency caused by mutations of the CD18 gene.     

Production of acidocin B, a bacteriocin of Lactobacillus acidophilus M46 is a plasmid-encoded trait: Plasmid curing, genetic marking by in vivo plasmid integration, and gene transfer

Abstract Plasmid-curing studies suggest that acidocin B production is encoded by the 14-kb plasmid pCV461 in Lactobacillus acidophilus M46. Loss of pCV461 from the original producer strain M46 did not coincide with loss of immunity to acidocin B. Bacteriocin activity determination after SDS-PAGE showed that a substance of 2.4 kDa, absent in the culture supernatant of the mutant strain M46A2, lacking pCV461, represented acidocin B activity. In order to introduce a positive selection criterion, pCV461 was marked in vivo by the erythromycin resistance marker of pE194, present on pUC19 containing a 1.4-kb Hin dIII fragment of pCV461, after plasmid integration. Introduction of this recombinant plasmid into the mutant strain M46A2 or Lactobacillus plantarum resulted in erythromycin-resistant, acidocin B-producing transformants, showing unambiguously that acidocin B is encoded by pCV461.     

Identification of cis-diols as intermediates in the oxidation of aromatic acids by a strain of Pseudomonas putida that contains a TOL plasmid.

Pseudomonas putida BG1 was isolated from soil by enrichment with p-toluate and selection for growth with p-xylene. Other hydrocarbons that served as growth substrates were toluene, m-xylene, 3-ethyltoluene, and 1,2,4-trimethylbenzene. The enzymes responsible for growth on these substrates are encoded by a large plasmid with properties similar to those of TOL plasmids isolated from other strains of Pseudomonas. Treatment of P. putida BG1 with nitrosoguanidine led to the isolation of a mutant strain which, when grown with fructose, oxidized both p-xylene and p-toluate to (-)-cis-1,2-dihydroxy-4-methylcyclohexa-3,5-diene-1-carboxylic acid (cis-p-toluate diol). The structure of the diol was determined by conventional chemical techniques including identification of the products formed by acid-catalyzed dehydration and characterization of a methyl ester derivative. The cis-relative stereochemistry of the hydroxyl groups was determined by the isolation and characterization of an isopropylidene derivative. p-Xylene-grown cells contained an inducible NAD+-dependent dehydrogenase which formed catechols from cis-p-toluate diol and the analogous acid diols formed from the other hydrocarbon substrates listed above. The catechols were converted to meta ring fission products by an inducible catechol-2,3-dioxygenase which was partially purified from p-xylene-grown cells of P. putida BG1.     

Crystal structure of a conger eel galectin (congerin II) at 1.45A resolution: implication for the accelerated evolution of a new ligand-binding site following gene duplication

The crystal structure of congerin II, a galectin family lectin from conger eel, was determined at 1.45A resolution. The previously determined structure of its isoform, congerin I, had revealed a fold evolution via strand swap; however, the structure of congerin II described here resembles other prototype galectins. A comparison of the two congerin genes with that of several other galectins suggests acceralated evolution of both congerin genes following gene duplication. The presence of a Mes (2-[N-morpholino]ethanesulfonic acid) molecule near the carbohydrate-binding site in the crystal structure points to the possibility of an additional binding site in congerin II. The binding site consists of a group of residues that had been replaced following gene duplication suggesting that the binding site was built under selective pressure. Congerin II may be a protein specialized for biological defense with an affinity for target carbohydrates on parasites' cell surface.     

Characterization of the ATP-binding cassette transporter gene expression profile in Y79: a retinoblastoma cell line

The human TESTIN (TES) is a putative tumor suppressor and localizes to the cytoplasm as a component of focal adhesions and cell contacts. TES contains a PET domain in the NH(2)-terminus and three tandem LIM domains in the COOH-terminus. It has been hypothesized that interactions between two termini of TES might lead to a "closed" conformational state of the protein. Here, we provide evidence for different conformational states of TES. We confirmed that the NH(2)-terminus of TES can interact with its third LIM domain in the COOH-terminus by GST pull-down assays. In addition, antisera against the full-length or two truncations of TES were prepared to examine the relationship between the conformation and cellular distribution of the protein. We found that these antisera recognize different regions of TES and showed that TES is co-localised with the marker protein B23 in nucleolus, in addition to its localization in endoplasmic reticulum (ER). Furthermore, our co-immunoprecipitation (co-IP) analysis of TES and B23 demonstrated their co-existence in the same complex. Taken together, our results suggest that TES has different conformational states in different cellular compartments, and a "closed" conformational state of TES may be involved in nucleolar localization.     

The SOS response is induced by replication fork blockage at a Ter site located on a pUC-derived plasmid: dependence on the distance between ori and Ter sites

A new model system for the study of the SOS response has been developed. In this system the response is induced by blocking the replication fork at a Ter site located in pUC-derived plasmids. Blockage of the fork is dependent on the expression of the Ter binding protein, Tus, encoded on another plasmid, in which the tus gene is under the control of the ara promoter. SOS induction can, therefore, be controlled by arabinose. The extent of the SOS response was monitored by measuring the activity of β-galactosidase, expressed from a lacZ gene fused to the 5′ region of the sfiA gene, a typical SOS-responsive gene. Expression of the fusion gene is completely dependent on recA ⁺ and lexA ⁺ genes. Using this system, we found that the distance between the ori and Ter sites is directly correlated with the strength of SOS induction. The properties of this system are discussed.     

Nest‐site selection by crocodiles at a rocky site in the Australian tropics: Making the best of a bad lot

Most animals select nest sites non‐randomly, reflecting benefits of specific locations or incubation conditions for offspring viability as well as risks or costs to the reproducing adult. If few or no available nest sites offer suitable conditions, we expect animals to make the best of a bad lot, by selecting nest sites that provide the best conditions available. In tropical north‐western Australia, freshwater crocodiles (Crocodylus johnstoni: Crocodylidae) in a large artificial lake (Lake Argyle) experience this challenge: the types of nest sites used by this species in other parts of its range (moist, shaded sandy soils, far from the water's edge) are scarce. Measurements of 89 crocodile nests and 89 test holes (abandoned attempts at nesting) at Lake Argyle, and 28 nests on the nearby Ord River, show that most areas along the lakeshore are too steep and rocky for nesting. Crocodiles at the lake therefore are forced to nest at sites that are sun‐exposed, in dry gravelly substrates, and close to the water's edge. Comparisons of test holes and actual nests within such areas show that nesting crocodiles actively select sites that are less rocky, are suitable hydrically, and that provide stable thermal regimes. Those hydric and thermal attributes allow successful development of the offspring. The ability of freshwater crocodiles in Lake Argyle to flexibly modify their nest‐site selection criteria, under severe constraints enforced by this open rocky landscape, are critical to the species' success in exploiting the opportunity created by the dam's construction.     

Rearrangements at a hobo element inserted into the first intron of the singed gene in the unstable sn 49 system of Drosophila melanogaster

The cytological structure of the X chromosome and the DNA organisation of the singed locus were examined in five singed bristle mutants of Drosophila melanogaster. These mutants are all derived from the unstable mutant singed-49, isolated from a wild population in the Russian Far East in 1975. Rearrangements were found at a site within the first intron of the singed gene, where a hobo element is inserted in these mutants. One rearrangement, which is associated with a strong bristle phenotype, has an inversion between 2D and the location of singed at 7D, which separates the singed promoter from the singed coding region. Two phenotypically wild-type derivatives have smaller rearrangements within the first intron which do not appear to interfere with singed expression. Two derivatives with bristle phenotypes have more complex rearrangements, and one of them shows a dominant or antimorphic phenotype. DNA blotting and in situ hybridisation experiments show that, in addition to these rearrangements at a hobo element inserted at singed, other hobo elements in these strains have been mobilised. This system is therefore similar to others in which functional hobo elements continue to transpose, resulting in elevated rates of mutation and chromosome rearrangement.     

Contemporary evolution of resistance at the major insecticide target site gene Ace‐1 by mutation and copy number variation in the malaria mosquito Anopheles gambiae

Functionally constrained genes are ideal insecticide targets because disruption is often fatal, and resistance mutations are typically costly. Synaptic acetylcholinesterase (AChE) is an essential neurotransmission enzyme targeted by insecticides used increasingly in malaria control. In Anopheles and Culex mosquitoes, a glycine–serine substitution at codon 119 of the Ace‐1 gene confers both resistance and fitness costs, especially for 119S/S homozygotes. G119S in Anopheles gambiae from Accra (Ghana) is strongly associated with resistance, and, despite expectations of cost, resistant 119S alleles are increasing significantly in frequency. Sequencing of Accra females detected only a single Ace‐1 119S haplotype, whereas 119G diversity was high overall but very low at non‐synonymous sites, evidence of strong purifying selection driven by functional constraint. Flanking microsatellites showed reduced diversity, elevated linkage disequilibrium and high differentiation of 119S, relative to 119G homozygotes across up to two megabases of the genome. Yet these signals of selection were inconsistent and sometimes weak tens of kilobases from Ace‐1. This unexpected finding is attributable to apparently ubiquitous amplification of 119S alleles as part of a large copy number variant (CNV) far exceeding the size of the Ace‐1 gene, whereas 119G alleles were unduplicated. Ace‐1 CNV was detectable in archived samples collected when the 119S allele was rare in Ghana. Multicopy amplification of resistant alleles has not been observed previously and is likely to underpin the recent increase in 119S frequency. The large CNV compromised localization of the strong selective sweep around Ace‐1, emphasizing the need to integrate CNV analysis into genome scans for selection.