Clinical isolates of E. faecalis and E. faecium harboring virulence genes show the concomitant presence of CRISPR loci and antibiotic resistance determinants.

In this study, we evaluated the antimicrobial susceptibility, the presence of gene-encoding virulence factors and CRISPR systems, as well as the ability to produce lytic enzymes among clinical E. faecalis and E. faecium isolates (n = 44). All enterococci isolates showed phenotypes of multidrug resistance. E. faecalis and E. faecium isolates exhibited high-level aminoglycoside resistance phenotype, several of them harboring the aac(6')Ie-aph(2″)Ia and aph(3')-IIIa genes. The gene vanA was the most frequent among vancomycin-resistant E. faecium. High prevalence of the virulence genes esp and efaA were observed; hyl gene was more associated with E. faecium, while ace and efaA genes were more frequently detected in E. faecalis. Caseinase activity was frequently detected among the isolates. Gelatinase and DNAse activities predominated among E. faecalis, while hemolytic capability was frequent among E. faecium isolates. Twenty-nine isolates showed at least one CRISPR system investigated. Several enterococci isolates harbored the aac(6')-Ie-aph(2″)-Ia or aph(3')-IIIa genes and a CRISPR loci. CRISPR loci were positively correlated to efaA and gelE genes, and gelatinase and DNAse activities, while CRISPR loci absence was related to hyl gene presence. These results show that clinical isolates of E. faecalis and E. faecium harboring virulence genes show the concomitant presence of CRISPR loci and antibiotic resistance determinants.

Antibiotic resistance and inhibition mechanism of novel aminoglycoside phosphotransferase APH(5) from B. subtilis subsp. subtilis strain RK.

Bacterial resistance towards aminoglycoside antibiotics mainly occurs because of aminoglycoside phosphotransferases (APHs). It is thus necessary to provide a rationale for focusing inhibitor development against APHs. The nucleotide triphosphate (NTP) binding site of eukaryotic protein kinases (ePKs) is structurally conserved with APHs. However, ePK inhibitors cannot be used against APHs due to cross reactivity. Thus, understanding bacterial resistance at the atomic level could be useful to design new inhibitors against such resistant pathogens. Hence, we carried out in vitro studies of APH from newly deposited multidrug-resistant organism Bacillus subtilis subsp. subtilis strain RK. Enzymatic modification studies of different aminoglycoside antibiotics along with purification and characterization revealed a novel class of APH, i.e., APH(5), with molecular weight 27 kDa approximately. Biochemical analysis of virtually screened inhibitor ZINC71575479 by coupled spectrophotometric assay showed complete enzymatic inhibition of purified APH(5). In silico toxicity study comparison of ZINC71575479 with known inhibitor of APH, i.e., tyrphostin AG1478, predicted its acceptable values for 96 h fathead minnow LC50, 48 h Tetrahymena pyriformis IGC50, oral rat LD50, and developmental toxicity using different QSAR methodologies. Thus, the present study gives novel insight into the aminoglycoside resistance and inhibition mechanism of APH(5) by applying experimental and computational techniques synergistically.

Intercistronic expression elements (IEE) from the chloroplast of Chlamydomonas reinhardtii can be used for the expression of foreign genes in synthetic operons.

KEY MESSAGE: Two intercistronic regions were identified as functional intercistronic expression elements (IEE) for the simultaneous expression of aphA-6 and gfp in a synthetic operon in the chloroplast of C. reinhardtii. Chlamydomonas reinhardtii, a biflagellate photosynthetic microalga, has been widely used in basic and applied science. Already three decades ago, Chlamydomonas had its chloroplast genome transformed and to this day constitutes the only alga routinely used in transplastomic technology. Despite the fact that over a 100 foreign genes have been expressed from the chloroplast genome, little has been done to address the challenge of expressing multiple genes in the form of operons, a development that is needed and crucial to push forward metabolic engineering and synthetic biology in this organism. Here, we studied five intercistronic regions and investigated if they can be used as intercistronic expression elements (IEE) in synthetic operons to drive the expression of foreign genes in the chloroplast of C. reinhardtii. The intercistronic regions were those from the psbB-psbT, psbN-psbH, psaC-petL, petL-trnN and tscA-chlN chloroplast operons, and the foreign genes were the aminoglycoside 3'-phosphotransferase (aphA-6), which confers resistance to kanamycin, and the green fluorescent protein gene (gfp). While all the intercistronic regions yielded lines that were resistant to kanamycin, only two (obtained with intercistronic regions from psbN-psbH and tscA-chlN) were identified as functional IEEs, yielding lines in which the second cistron (gfp) was translated and generated GFP. The IEEs we have identified could be useful for the stacking of genes for metabolic engineering or synthetic biology circuits in the chloroplast of C. reinhardtii.

mRNA imaging in the chloroplast of Chlamydomonas reinhardtii using the light-up aptamer Spinach.

Light-up aptamers are practical tools to image RNA localization in vivo. A now classical light-up aptamer system is the combination of the 3,5-difluoro-4-hydroxybenzylidene (DFHBI) fluorogen and the RNA aptamer Spinach, which has been successfully used in bacterial and mammalian cells. However, light-up aptamers have not been used in algae. Here, we show that a simple vector, carrying Spinach, transcriptionally fused to the aphA-6 gene, can be effectively used to generate a functional light-up aptamer in the chloroplast of Chlamydomonas reinhardtii. After incubation with DFHBI, lines expressing the aphA-6/Spinach mRNA were observed with laser confocal microscopy to evaluate the functionality of the light-up aptamer in the chloroplast of C. reinhardtii. Clear and strong fluorescence was localized to the chloroplast, in the form of discrete spots. There was no background fluorescence in the strain lacking Spinach. Light-up aptamers could be further engineered to image RNA or to develop genetically encoded biosensors in algae.

A promoter trap vector for knocking out bovine myostatin gene with high targeting efficiency.

With the development of gene targeting approaches, genomic mutation technologies in livestock animals such as gene trapping, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats and their associated systems have been improved. Although ZFNs have been used for gene targeting in many species, the off-target sites are still present. Using gene trapping, the workload of screening of targeted clones was decreased by generating a smaller number of drug-resistant clones. Determining whether the efficiency of gene trapping is lower than that of ZFNs for a specific gene has been challenging. In this study, to knock out the bovine myostatin gene, we constructed a promoter trap vector and compared its efficiency with that of ZFNs. The promoter trap vector contained a green fluorescent protein sequence without the promoter and a neomycin phosphotransferase (neo(R)) cassette driven by the phosphoglycerate kinase promoter. When the trapping vector was inserted downstream of the endogenous promoter, the fluorescent protein gene was expressed. The targeted-positive cell clones were identified based on green fluorescence and G418 double selection, followed by polymerase chain reaction analysis and sequencing. The targeting efficiency reached 5%. Compared with the efficiency of ZFN pairs (5.17 and 2.86%), the promoter trap vector PIII-myostatin could knock out the bovine myostatin gene. Therefore, gene trapping may be an effective tool for genomic modification.

Expression and genomic integration of transgenes after Agrobacterium-mediated transformation of mature barley embryos.

Mature embryos in tissue cultures are advantageous because of their abundance and rapid germination, which reduces genomic instability problems. In this study, 2-day-old isolated mature barley embryos were infected with 2 Agrobacterium hypervirulent strains (AGL1 and EHA105), followed by a 3-day period of co-cultivation in the presence of L-cystein amino acid. Chimeric expression of the b-glucuronidase gene (gusA) directed by a viral promoter of strawberry vein banding virus was observed in coleoptile epidermal cells and seminal roots in 5-day-old germinated seedlings. In addition to varying infectivity patterns in different strains, there was a higher ratio of transient b-glucuronidase expression in developing coleoptiles than in embryonic roots, indicating the high competency of shoot apical meristem cells in the mature embryo. A total of 548 explants were transformed and 156 plants developed to maturity on G418 media after 18-25 days. We detected transgenes in 74% of the screened plant leaves by polymerase chain reaction, and 49% of these expressed neomycin phosphotransferase II gene following AGL1 transformation. Ten randomly selected T0 transformants were analyzed using thermal asymmetric interlaced polymerase chain reaction and 24 fragments ranged between 200-600 base pairs were sequenced. Three of the sequences flanked with transferred-DNA showed high similarity to coding regions of the barley genome, including alpha tubulin5, homeobox 1, and mitochondrial 16S genes. We observed 70-200-base pair filler sequences only in the coding regions of barley in this study.

Physicochemical and biochemical characterization of transgenic papaya modified for protection against Papaya ringspot virus.

BACKGROUND: Papaya, a nutritious tropical fruit, is consumed both in its fresh form and as a processed product worldwide. Major quality indices which include firmness, acidity, pH, colour and size, are cultivar dependent. Transgenic papayas engineered for resistance to Papaya ringspot virus were evaluated over the ripening period to address physicochemical quality attributes and food safety concerns. RESULTS: With the exception of one transgenic line, no significant differences (P > 0.05) were observed in firmness, acidity and pH. Lightness (L*) and redness (a*) of the pulps of non-transgenic and transgenic papaya were similar but varied over the ripening period (P < 0.05). Fruit mass, though non-uniform (P < 0.05) for some lines, was within the range reported for similar papaya cultivars, as were shape indices of female fruits. Transgene proteins, CP and NPTII, were not detected in fruit pulp at the table-ready stage. CONCLUSION: The findings suggest that transformation did not produce any major unintended alterations in the physicochemical attributes of the transgenic papayas. Transgene proteins in the edible fruit pulp were low or undetectable.

The prevalence of aminoglycoside-modifying enzyme genes (aac (6')-I, aac (6')-II, ant (2")-I, aph (3')-VI) in Pseudomonas aeruginosa.

INTRODUCTION: Pseudomonas aeruginosa (P. aeruginosa) is one of the primary opportunistic pathogens responsible for nosocomial infections. Aminoglycosides are an import ant component of antipseudomonal chemotherapy. The inactivation of drugs by modifying enzymes is the most common mechanism of aminoglycoside resistance. OBJECTIVES: The inactivation of aminoglycosides by modifying enzymes is the primary resistance mechanism employed by P. aeruginosa. The aim of the present study was to investigate the occurrence of aminoglycoside resistance and the prevalence of four import ant modifying enzyme genes (aac (6')-I, aac (6')-II, ant (2")-I, aph (3')-VI) in P. aeruginosa in Iran. METHODS: A total of 250 clinical isolates of P. aeruginosa were collected from several hospitals in seven cities in Iran. Antimicrobial susceptibility tests (using the disk diffusion method and E-tests) were performed for all 250 isolates. In addition, all isolates were screened for the presence of modifying enzyme genes by polymerase chain reaction. RESULTS: The resistance rates, as determined by the disk diffusion method, were as follows: gentamicin 43%, tobramycin 38%, and amikacin 24%. Of the genes examined, aac (6')-II (36%) was the most frequently identified gene in phenotypic resist ant isolates, followed by ant (2")-I, aph (3')-VI, and aac (6')-I. CONCLUSIONS: Aminoglycoside resistance in P. aeruginosa remains a significant problem in Iran. Therefore, there is considerable local surveillance of aminoglycoside resistance.

Cloning of the nptII gene of Escherichia coli and construction of a recombinant strain harboring functional recA and nptII antibiotic resistance.

In an attempt to clone the ORF of the nptII gene of Escherichia coli K12 (ATCC 10798), two degenerate primers were designed based on the nptII sequence of its Tn5 transposon. The nptII ORF was placed under the control of the E. coli hybrid trc promoter, in the pKK388-1 vector, transformed into E. coli DH5α ΔrecA (recombinant, deficient strain). Transferred cells were tested for ampicillin, tetracycline, kanamycin, neomycin, geneticin, paromomycin, penicillin, and UV resistance. The neomycin phosphotransferase gene of E. coli was cloned successfully and conferred kanamycin, neomycin, geneticin, and paromomycin resistance to recombinant DH5α; this did not inhibit insertion of additional antibiotic resistance against ampicillin and tetracycline, meaning the trc promoter can express two different genes carried by two different plasmids harbored in the same cell. This resistance conferral process could be considered as an emulation of horizontal gene transfer occurring in nature and would be a useful tool for understanding mechanisms of evolution of multidrug-resistant strains.

The prevalence of aminoglycoside-modifying enzyme genes (aac (6')-I, aac (6')-II, ant (2")-I, aph (3')-VI) in Pseudomonas aeruginosa

INTRODUCTION: Pseudomonas aeruginosa (P. aeruginosa) is one of the primary opportunistic pathogens responsible for nosocomial infections. Aminoglycosides are an import ant component of antipseudomonal chemotherapy. The inactivation of drugs by modifying enzymes is the most common mechanism of aminoglycoside resistance. OBJECTIVES: The inactivation of aminoglycosides by modifying enzymes is the primary resistance mechanism employed by P. aeruginosa. The aim of the present study was to investigate the occurrence of aminoglycoside resistance and the prevalence of four import ant modifying enzyme genes (aac (6')-I, aac (6')-II, ant (2")-I, aph (3')-VI) in P. aeruginosa in Iran. METHODS: A total of 250 clinical isolates of P. aeruginosa were collected from several hospitals in seven cities in Iran. Antimicrobial susceptibility tests (using the disk diffusion method and E-tests) were performed for all 250 isolates. In addition, all isolates were screened for the presence of modifying enzyme genes by polymerase chain reaction. RESULTS: The resistance rates, as determined by the disk diffusion method, were as follows: gentamicin 43 percent, tobramycin 38 percent, and amikacin 24 percent. Of the genes examined, aac (6')-II (36 percent) was the most frequently identified gene in phenotypic resist ant isolates, followed by ant (2")-I, aph (3')-VI, and aac (6')-I. CONCLUSIONS: Aminoglycoside resistance in P. aeruginosa remains a signific ant problem in Iran. Therefore, there is considerable local surveillance of aminoglycoside resistance.

A reproducible genetic transformation system for cultivated Phaseolus acutifolius (tepary bean) and its use to assess the role of arcelins in resistance to the Mexican bean weevil.

A reproducible Agrobacterium tumefaciens-mediated genetic transformation method that delivers fertile and morphologically normal transgenic plants was developed for cultivated tepary bean (Phaseolus acutifolius L. Gray). Factors contributing to higher transformation efficiencies include (1) a low initial concentration of bacteria coupled with a longer cocultivation period with callus, (2) an initial selection of callus on a medium containing low levels of the selectable agent, (3) omission of the selectable agent from the medium during callus differentiation to shoots and (4) the efficient conversion of transgenic shoots into fertile plants. All plants regenerated with this procedure (T0) were stably transformed, and the introduced foreign genes were inherited in a Mendelian fashion in most of the 33 independent transformants. Integration, stable transmission and high expression levels of the transgenes were observed in the T1 and/or T3 progenies of the transgenic lines. The binary transformation vectors contained the beta-glucuronidase reporter gene, the neomycin phosphotransferase II selectable marker gene and either an arcelin 1 or an arcelin 5 gene. Arcelins are seed proteins that are very abundant in some wild P. vulgaris L. genotypes showing resistance to the storage insect Zabrotes subfasciatus (Boheman) (Coleoptera, Bruchidae). Transgenic beans from two different cultivated P. acutifolius genotypes with high arcelin levels were infested with Z. subfasciatus, but they were only marginally less susceptible to infestation than the non-transgenic P. acutifolius. Hence, the arcelin genes tested here are not major determinants of resistance against Z. subfasciatus.

Specificity of modified Drosophila mariner transposons in the identification of Leishmania genes.

Genetic manipulation of the protozoan Leishmania has led to a better understanding of the survival and development of these pathogens within their hosts. The association of the Leishmania genome sequencing information with the ability of transposons to introduce or destroy phenotypes allows a global perspective on the role and importance of genes in cellular pathways. Herein we report the construction and testing of mariner transposable elements carrying the neomycin phosphotransferase, green fluorescent protein, or beta-glucuronidase genes as reporters for translational fusion events. We demonstrate that the expression of the reporter genes will occur only when the genes are inserted in-frame within predicted genes. Our results not only add to the mariner toolkit for gene manipulation but also strengthen the evidence that the mariner system is a reliable means for the study of gene expression in Leishmania.

Inhibition of gene expression in Entamoeba histolytica with antisense peptide nucleic acid oligomers.

Peptide nucleic acids (PNAs) may be a potent tool for gene function studies in medically important parasitic organisms, especially those that have not before been accessible to molecular genetic knockout approaches. One such organism is Entamoeba histolytica, the causative agent of amebiasis, which infects about 500 million people and is the cause of clinical disease in over 40 million each year, mainly in the tropical and subtropical world. We used PNA antisense oligomers to inhibit expression of an episomally expressed gene (neomycin phosphorotransferase, NPT) and a chromosomal gene (EhErd2, a homolog of Erd2, a marker of the Golgi system in eukaryotic cells) in axenically cultured trophozoites of E. histolytica. Measurement of NPT enzyme activity and EhErd2 protein levels, as well as measurement of cellular proliferation, revealed specific decreases in expression of the target genes, and concomitant inhibition of cell growth, in trophozoites treated with micromolar concentrations of unmodified antisense PNA oligomers.

A family of removable cassettes designed to obtain antibiotic-resistance-free genomic modifications of Escherichia coli and other bacteria.

Modifications of microbial genomes often require the use of the antibiotic-resistance (Anb(R))-encoding genes and other easily selectable markers. We have developed a set of such selectable markers (Cm(R), Km(R) and Gm(R)), which could easily be inserted into the genome and subsequently removed by using the Cre/loxP site-specific recombination system of bacteriophage P1. In this manner the same marker could be used more than once in the same background, while the resulting strain could or would remain Anb(R) marker-free. Three plasmids were constructed, each containing a cassette consisting of the Cm(R), Km(R), or Gm(R) gene flanked by two parallel loxP sites and two polylinkers (MCS). To test insertion and excision, cassettes were inserted into the lacZ or galE genes carried on an origamma/pir-dependent suicide plasmid, which contained a dominant Sm(R) gene. The cassettes were crossed into the E. coli genome by homologous recombination (allelic exchange), in a manner analogous to that described by Pósfai et al. [Nucl. Acids Res. 22 (1994) 2392-2398], selecting for the Cm(R), Km(R), or Gm(R), for the LacZ(-) or GalE(-) and for the Sm(S) phenotypes (the latter to assure allelic exchange rather than insertion of the entire plasmid). When required, after selecting the strain with the desired modification, the Cm(R), Km(R), or Gm(R) marker was excised by supplying the Cre function. Cre was provided by the thermosensitive plasmid pJW168, which was transformed into the Anb(R) host at 30 degrees C, and was subsequently eliminated at 42 degrees C. Thus the Anb(R) marker was removed, whereas the lacZ or galE gene remained interrupted by the retained loxP site.

Colony polymerase chain reaction of stably transfected trypanosoma cruzi grown on solid medium.

Tools for the genetic manipulation of Trypanosoma cruzi are largely unavailable, although several vectors for transfection of epimastigotes and expression of foreign or recombinant genes have been developed. We have previously constructed several plasmid vectors in which recombinant genes are expressed in T. cruzi using the rRNA promoter. In this report, we demonstrate that one of these vectors can simultaneously mediate expression of neomycin phosphotransferase and green fluorescent protein when used to stably transfect cultured epimastigotes. These stably transfected epimastigotes can be selected and cloned as unique colonies on solid medium. We describe a simple colony PCR approach to the screening of these T. cruzi colonies for relevant genes. Thus, the methodologies outlined herein provide important new tools for the genetic dissection of this important parasite.