A Broad-spectrum mer Operon in a Multi-drug Resistant Strain of the Fish Pathogen, Aeromonas salmonicida.

Aeromonas salmonicida AS03, a potential fish pathogen, was isolated from Atlantic salmon, Salmo salar, in 2003. This strain was found to be resistant to ≥1000 mM HgCl2 and ≥32 mM phenylmercuric acetate as well as multiple antimicrobials. Mercury (Hg) and antibiotic resistance genes are often located on the same mobile genetic elements, so the genetic determinants of both resistances and the possibility of horizontal gene transfer were examined. Specific PCR primers were used to amplify and sequence distinctive regions of the mer operon. A. salmonicida AS03 was found to have a pDU1358-like broad-spectrum mer operon, containing merB as well as merA, merD, merP, merR and merT, most similar to Klebsiella pneumonaie plasmid pRMH760. To our knowledge, the mer operon has never before been documented in Aeromonas spp. PCR and gene sequencing were used to identify class 1 integron associated antibiotic resistance determinants and the Tet A tetracycline resistance gene. The transposase and resolvase genes of Tn1696 were identified through PCR and sequencing with Tn21 specific PCR primers. We provide phenotypic and genotypic evidence that the mer operon, the aforementioned antibiotic resistances, and the Tn1696 transposition module are located on a single plasmid or conjugative transposon that can be transferred to E. coli DH5α through conjugation in the presence of low level Hg and absence of any antibiotic selective pressure. Additionally, the presence of low-level Hg or chloramphenicol in the mating media was found to stimulate conjugation, significantly increasing the transfer frequency of conjugation above the transfer frequency measured with mating media lacking both antibiotics and Hg. This research demonstrates that mercury indirectly selects for the dissemination of the antibiotic resistance genes of A. salmonicida AS03.

A broad-spectrum mer operon in a multi-drug resistant strain of the fish pathogen, Aeromonas salmonicida

Aeromonas salmonicida AS03, a potential fish pathogen, was isolated from Atlantic salmon, Salmo salar, in 2003. This strain was found to be resistant to ≥1000 mM HgCl2 and ≥32 mM phenylmercuric acetate as well as multiple antimicrobials. Mercury (Hg) and antibiotic resistance genes are often located on the same mobile genetic elements, so the genetic determinants of both resistances and the possibility of horizontal gene transfer were examined. Specific PCR primers were used to amplify and sequence distinctive regions of the mer operon. A. salmonicida AS03 was found to have a pDU1358-like broad-spectrum mer operon, containing merB as well as merA, merD, merP, merR and merT, most similar to Klebsiella pneumonaie plasmid pRMH760. To our knowledge, the mer operon has never before been documented in Aeromonas spp. PCR and gene sequencing were used to identify class 1 integron associated antibiotic resistance determinants and the Tet A tetracycline resistance gene. The transposase and resolvase genes of Tn1696 were identified through PCR and sequencing with Tn21 specific PCR primers. We provide phenotypic and genotypic evidence that the mer operon, the aforementioned antibiotic resistances, and the Tn1696 transposition module are located on a single plasmid or conjugative transposon that can be transferred to E. coli DH5α through conjugation in the presence of low level Hg and absence of any antibiotic selective pressure. Additionally, the presence of low-level Hg or chloramphenicol in the mating media was found to stimulate conjugation, significantly increasing the transfer frequency of conjugation above the transfer frequency measured with mating media lacking both antibiotics and Hg. This research demonstrates that mercury indirectly selects for the dissemination of the antibiotic resistance genes of A. salmonicida AS03.

The role of prophage in plant-pathogenic bacteria

A diverse set of phage lineages is associated with the bacterial plant-pathogen genomes sequenced to date. Analysis of 37 genomes revealed 5,169 potential genes (approximately 4.3 Mbp) of phage origin, and at least 50 had no function assigned or are nonessential to phage biology. Some phytopathogens have transcriptionally active prophage genes under conditions that mimic plant infection, suggesting an association between plant disease and prophage transcriptional modulation. The role of prophages within genomes for cell biology varies. For pathogens such as Pectobacterium, Pseudomonas, Ralstonia, and Streptomyces, involvement of prophage in disease symptoms has been demonstrated. In Xylella and Xanthomonas, prophage activity is associated with genome rearrangements and strain differentiation. For other pathogens, prophage roles are yet to be established. This review integrates available information in a unique interface (http://propnav.esalq.usp.br) that may be assessed to improve research in prophage biology and its association with genome evolution and pathogenicity. © Copyright ©2013 by Annual Reviews. All rights reserved.

Tropical Africa as a cradle for horizontal transfers of transposable elements between species of the genus Drosophila and Zaprionus

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Transposable elements as a potential source for understanding the fish genome

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Xylella fastidiosa comparative genomic database is an information resource to explore the annotation, genomic features, and biology of different strains

The Xylella fastidiosa comparative genomic database is a scientific resource with the aim to provide a user-friendly interface for accessing high-quality manually curated genomic annotation and comparative sequence analysis, as well as for identifying and mapping prophage-like elements, a marked feature of Xylella genomes. Here we describe a database and tools for exploring the biology of this important plant pathogen. The hallmarks of this database are the high quality genomic annotation, the functional and comparative genomic analysis and the identification and mapping of prophage-like elements. It is available from web site http://www.xylella.lncc.br.

Large scale variation in Enterococcus faecalis illustrated by the genome analysis of strain OG1RF.

BACKGROUND: Enterococcus faecalis has emerged as a major hospital pathogen. To explore its diversity, we sequenced E. faecalis strain OG1RF, which is commonly used for molecular manipulation and virulence studies. RESULTS: The 2,739,625 base pair chromosome of OG1RF was found to contain approximately 232 kilobases unique to this strain compared to V583, the only publicly available sequenced strain. Almost no mobile genetic elements were found in OG1RF. The 64 areas of divergence were classified into three categories. First, OG1RF carries 39 unique regions, including 2 CRISPR loci and a new WxL locus. Second, we found nine replacements where a sequence specific to V583 was substituted by a sequence specific to OG1RF. For example, the iol operon of OG1RF replaces a possible prophage and the vanB transposon in V583. Finally, we found 16 regions that were present in V583 but missing from OG1RF, including the proposed pathogenicity island, several probable prophages, and the cpsCDEFGHIJK capsular polysaccharide operon. OG1RF was more rapidly but less frequently lethal than V583 in the mouse peritonitis model and considerably outcompeted V583 in a murine model of urinary tract infections. CONCLUSION: E. faecalis OG1RF carries a number of unique loci compared to V583, but the almost complete lack of mobile genetic elements demonstrates that this is not a defining feature of the species. Additionally, OG1RF's effects in experimental models suggest that mediators of virulence may be diverse between different E. faecalis strains and that virulence is not dependent on the presence of mobile genetic elements.

PEG-3, a nontransforming cancer progression gene, is a positive regulator of cancer aggressiveness and angiogenesis

Cancer is a progressive disease culminating in acquisition of metastatic potential by a subset of evolving tumor cells. Generation of an adequate blood supply in tumors by production of new blood vessels, angiogenesis, is a defining element in this process. Although extensively investigated, the precise molecular events underlying tumor development, cancer progression, and angiogenesis remain unclear. Subtraction hybridization identified a genetic element, progression elevated gene-3 (PEG-3), whose expression directly correlates with cancer progression and acquisition of oncogenic potential by transformed rodent cells. We presently demonstrate that forced expression of PEG-3 in tumorigenic rodent cells, and in human cancer cells, increases their oncogenic potential in nude mice as reflected by a shorter tumor latency time and the production of larger tumors with increased vascularization. Moreover, inhibiting endogenous PEG-3 expression in progressed rodent cancer cells by stable expression of an antisense expression vector extinguishes the progressed cancer phenotype. Cancer aggressiveness of PEG-3 expressing rodent cells correlates directly with increased RNA transcription, elevated mRNA levels, and augmented secretion of vascular endothelial growth factor (VEGF). Furthermore, transient ectopic expression of PEG-3 transcriptionally activates VEGF in transformed rodent and human cancer cells. Taken together these data demonstrate that PEG-3 is a positive regulator of cancer aggressiveness, a process regulated by augmented VEGF production. These studies also support an association between expression of a single nontransforming cancer progression-inducing gene, PEG-3, and the processes of cancer aggressiveness and angiogenesis. In these contexts, PEG-3 may represent an important target molecule for developing cancer therapeutics and inhibitors of angiogenesis.

The affected gene underlying the class K glycosylphosphatidylinositol (GPI) surface protein defect codes for the GPI transamidase

The final step in glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins consists of a transamidation reaction in which preassembled GPI donors are substituted for C-terminal signal sequences in nascent polypeptides. In previous studies we described a human K562 cell mutant, termed class K, that accumulates fully assembled GPI units but is unable to transfer them to N-terminally processed proproteins. In further work we showed that, unlike wild-type microsomes, microsomes from these cells are unable to support C-terminal interaction of proproteins with the small nucleophiles hydrazine or hydroxylamine, and that the cells thus are defective in transamidation. In this study, using a modified recombinant vaccinia transient transfection system in conjunction with a composite cDNA prepared by 5′ extension of an existing GenBank sequence, we found that the genetic element affected in these cells corresponds to the human homolog of yGPI8, a gene affected in a yeast mutant strain exhibiting similar accumulation of GPI donors without transfer. hGPI8 gives rise to mRNAs of 1.6 and 1.9 kb, both encoding a protein of 395 amino acids that varies in cells with their ability to couple GPIs to proteins. The gene spans ≈25 kb of DNA on chromosome 1. Reconstitution of class K cells with hGPI8 abolishes their accumulation of GPI precursors and restores C-terminal processing of GPI-anchored proteins. Also, hGPI8 restores the ability of microsomes from the mutant cells to yield an active carbonyl in the presence of a proprotein which is considered to be an intermediate in catalysis by a transamidase.

CTXφ contains a hybrid genome derived from tandemly integrated elements

CTXφ is a filamentous, temperate bacteriophage whose genome includes ctxAB, the genes that encode cholera toxin. In toxigenic isolates of Vibrio cholerae, tandem arrays of prophage DNA, usually interspersed with the related genetic element RS1, are integrated site-specifically within the chromosome. We have discovered that these arrays routinely yield hybrid virions, composed of DNA from two adjacent prophages or from a prophage and a downstream RS1. Coding sequences are always derived from the 5′ prophage whereas most of an intergenic sequence, intergenic region 1, is always derived from the 3′ element. The presence of tandem elements is required for production of virions: V. cholerae strains that contain a solitary prophage rarely yield CTX virions, and the few virions detected result from imprecise excision of prophage DNA. Thus, generation of the replicative form of CTXφ, pCTX, a step that precedes production of virions, does not depend on reversal of the process for site-specific integration of CTXφ DNA into the V. cholerae chromosome. Production of pCTX also does not depend on RecA-mediated homologous recombination between adjacent prophages. We hypothesize that the CTXφ-specific proteins required for replication of pCTX can also function on a chromosomal substrate, and that, unlike the processes used by other integrating phages, production of pCTX and CTXφ does not require excision of the prophage from the chromosome. Use of this replication strategy maximizes vertical transmission of prophage DNA while still enabling dissemination of CTXφ to new hosts.

Interactions of the chaperone Hsp104 with yeast Sup35 and mammalian PrP

[PSI+] is a genetic element in yeast for which a heritable change in phenotype appears to be caused by a heritable change in the conformational state of the Sup35 protein. The inheritance of [PSI+] and the physical state of Sup35 in vivo depend on the protein chaperone Hsp104 (heat shock protein 104). Although these observations provide a strong genetic argument in support of the “protein-only” or “prion” hypothesis for [PSI+], there is, as yet, no direct evidence of an interaction between the two proteins. We report that when purified Sup35 and Hsp104 are mixed, the circular dichroism (CD) spectrum differs from that predicted by the addition of the proteins’ individual spectra, and the ATPase activity of Hsp104 is inhibited. Similar results are obtained with two other amyloidogenic substrates, mammalian PrP and β-amyloid 1-42 peptide, but not with several control proteins. With a group of peptides that span the PrP protein sequence, those that produced the largest changes in CD spectra also caused the strongest inhibition of ATPase activity in Hsp104. Our observations suggest that (i) previously described genetic interactions between Hsp104 and [PSI+] are caused by direct interaction between Hsp104 and Sup35; (ii) Sup35 and PrP, the determinants of the yeast and mammalian prions, respectively, share structural features that lead to a specific interaction with Hsp104; and (iii) these interactions couple a change in structure to the ATPase activity of Hsp104.

A candidate live inactivatable attenuated vaccine for AIDS.

The recent discovery of long term AIDS nonprogressors who harbor nef-attenuated HIV suggests that a naturally occurring live vaccine for AIDS may already exist. Animal models have shown that a live vaccine for AIDS, attenuated in nef, is the best candidate vaccine. There are considerable risks, real and perceived, with the use of live HIV vaccines. We have introduced a conditional lethal genetic element into HIV-1 and simian immunodeficiency virus (SIV) molecular clones deleted in nef. The antiviral strategy we employed targets both virus replication and the survival of the infected cell. The suicide gene, herpes simplex virus thymidine kinase (tk), was expressed and maintained in HIV over long periods of time. Herpes simplex virus tk confers sensitivity to the antiviral activity of acyclic nucleosides such as ganciclovir (GCV). HIV-tk and SIV-tk replication were sensitive to GCV at subtoxic concentrations, and virus-infected cells were eliminated from tumor cell lines as well as primary cell cultures. We found the HIV-tk virus to be remarkably stable even after being cultured in media containing a low concentration of GCV and then challenged with the higher dose and that while GCV resistant escape mutants did arise, a significant fraction of the virus remained sensitive to GCV.

Identification of interdomain sequences promoting the intronless evolution of a bacterial protein family.

In the evolution of eukaryotic genes, introns are believed to have played a major role in increasing the probability of favorable duplication events, chance recombinations, and exon shuffling resulting in functional hybrid proteins. As a rule, prokaryotic genes lack introns, and the examples of prokaryotic introns described do not seem to have contributed to gene evolution by exon shuffling. Still, certain protein families in modern bacteria evolve rapidly by recombination of genes, duplication of functional domains, and as shown for protein PAB of the anaerobic bacterial species Peptostreptococcus magnus, by the shuffling of an albumin-binding protein module from group C and G streptococci. Characterization of a protein PAB-related gene in a P. magnus strain with less albumin-binding activity revealed that the shuffled module was missing. Based on this fact and observations made when comparing gene sequences of this family of bacterial surface proteins interacting with albumin and/or immunoglobulin, a model is presented that can explain how this rapid intronless evolution takes place. A new kind of genetic element is introduced: the recer sequence promoting interdomain, in frame recombination and acting as a structure-less flexibility-promoting spacer in the corresponding protein. The data presented also suggest that antibiotics could represent the selective pressure behind the shuffling of protein modules in P. magnus, a member of the indigenous bacterial flora.

Malignant conversion of chemically transformed normal human cells.

Two structurally unrelated chemicals, aflatoxin B1 and propane sultone, transformed human foreskin cells to a stage of anchorage-independent growth. Isolation from agar and repopulation in monolayer culture of these transformed cells was followed by transfection with a cDNA library, which resulted in cells that exhibited an altered epithelioid morphology. Chemically transformed/nontransfected cells and transfected normal cells did not undergo a significant morphological change. These epithelioid-appearing, transfected cells, when inoculated into nude mice, form progressively growing tumors. The tumors are histopathologically interpreted as carcinomas. All of the first generation tumors in the surrogate hosts exhibited characteristic rates of growth similar to those of transplants of spontaneous human tumors. In the second generation of tumor xenografts, the progressively growing tumors derived from the transfected cells exhibited a more rapid rate of growth. Southern analysis and reverse transcription PCR confirmed that a 1.3-kb genetic element was integrated into the genome and was actively being transcribed. Examination of the metaphase chromosomes in normal human cells revealed that the genetic element responsible for this conversion was located at site 31-32 of the q arm of chromosome 7. The DNA sequence of this 1.3-kb genetic element contains a coding region for 79 amino acids and a long 3'-untranslated region and appears to be identical to CATR1.3 isolated from tumors produced by methyl methanesulfonate-converted, nontransplantable human tumor cells.

Heterogeneity of primer extension products in asymmetric PCR is due both to cleavage by a structure-specific exo/endonuclease activity of DNA polymerases and to premature stops.

In PCR, DNA polymerases from thermophilic bacteria catalyze the extension of primers annealed to templates as well as the structure-specific cleavage of the products of primer extension. Here we show that cleavage by Thermus aquaticus and Thermus thermophilus DNA polymerases can be precise and substantial: it occurs at the base of the stem-loop structure assumed by the single strand products of primer extension using as template a common genetic element, the promoter-operator of the Escherichia coli lactose operon, and may involve up to 30% of the products. The cleavage is independent of primer, template, and triphosphates, is dependent on substrate length and temperature, requires free ends and Mg2+, and is absent in DNA polymerases lacking the 5'-->3' exonuclease, such as the Stoffel fragment and the T7 DNA polymerase. Heterogeneity of the extension products results also from premature detachment of the enzyme approaching the 5' end of the template.