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PubMed2004

Dicistronic expression of the green fluorescent protein and antibiotic resistance genes in the plastid for selection and tracking of plastid-transformed cells in tobacco.

A plastid transformation vector was constructed for dicistronic expression of the aminoglycoside 3'-adenyltransferase (aadA) and green fluorescent protein (gfp) genes under the control of the plastid rrn promoter. Gold particles coated with the vector DNA were bombarded onto tobacco leaf explants using a particle delivery system. Leaf explants produced adventitious shoots when cultured on shoot-inducing medium containing 500 mg l(-1) spectinomycin. Shoots that exhibited green fluorescence under UV light were selected. Southern blot analysis detected the presence of the aadA and gfp genes between trnA and trnI in the plastid genome. Northern blot analysis revealed that the aadA and gfp genes were both properly transcribed into a dicistronic transcriptional unit. The expression of the gfp gene in the plastid enabled separation of transformed chloroplasts from wild-type chloroplasts in the protoplast under a fluorescent microscope. The overall results indicate that dicistronic expression of the aadA and gfp genes in the plastid simplifies gene manipulation, facilitating selection and tracking of plastid-transformed cells.

Chloroplasts↗
PubMed1998

Transcript analysis of the tobacco plastid operon rps2/atpI/H/F/A reveals the existence of a non-consensus type II (NCII) promoter upstream of the atpI coding sequence.

The plastid ATP synthase complex is composed of nine subunits, of which six are encoded in the plastome. The plastid-encoded genes are arranged in two transcriptional units: atpB/E and atpI/H/F/A. We have recently reported that besides containing four -10 and -35 consensus-type (CT) promoters, the atpB/E operon also contains a non-consensus type (NCII) promoter that alone is responsible for its expression in non-photosynthetic plastids. As the functionality of ATP synthase requires expression of all nine subunits, NCII promoter-driven transcription of the atpI/H/F/A operon is to be expected in non-photosynthetic plastids. Therefore, a detailed transcriptional analysis of this operon was carried out using RNA samples from tobacco leaf, cultured cells (BY-2) and seedlings grown on streptomycin and spectinomycin; which contain chloroplasts, translationally active non-photosynthetic plastids and translationally inactive plastids, respectively. We identified a total of three transcription initiation sites (TIS) and four transcript processing sites in the non-coding regions of this operon. Our results also demonstrate that rps2 is co-transcribed with the atpI/H/F/A genes. One of the TIS (-208 atpI) is characterized by an NCII type promoter, while other two primary transcripts (-131 atpI and -384 atpH) initiate from CT promoters. In non-photosynthetic plastids the atpI/H/F/A-specific transcript pool seems to be solely contributed by initiation at the -208 atpI (NCII type) promoter, because transcripts from CT promoters do not accumulate in these plastid types.

Base Sequence↗
PubMed1998

Mutagenesis of the genes encoding subunits A, C, H, I, J and K of the plastid NAD(P)H-plastoquinone-oxidoreductase in tobacco by polyethylene glycol-mediated plastome transformation.

Plastids contain a NAD(P)H-plastoquinone-oxidoreductase (NDH complex) which is homologous to the eubacterial and mitochondrial NADH-ubiquinone-oxidoreductase (complex I), but the metabolic function of the enzyme is unknown. The enzyme consists of at least eleven subunits (A-K), which are all encoded on the plastid chromosome. We have mutagenized ndhC and ndhJ by insertion, and ndhK and ndhA-I by deletion and insertion, of a cassette which carried a spectinomycin resistance gene as a marker. The transformation was carried out by the polyethylene glycol-mediated plastid transformation method. Southern analysis revealed that even after repeated regeneration cycles each of the four different types of transformants had retained 1-5% of wild-type gene copies. This suggests that complete deletion of ndh genes is not compatible with viability. The transformants displayed two characteristic phenotypes: (i) they lack the rapid rise in chlorophyll fluorescence in the dark after illumination with actinic light for 5 min; in the wild-type this dark-rise reflects a transient reduction of the plastoquinone pool by reduction equivalents generated in the stroma; and (ii) transformants with defects in the ndhC-K-J operon accumulate starch, indicating inefficient oxidation of glucose via glycolysis and the oxidative pentose phosphate pathway. Both observations support the theory of chlororespiration, which postulates that the NDH complex acts as a valve to remove excess reduction equivalents in the chloroplast.

Chloroplasts↗
PubMed1999

Transcripts containing the 5' untranslated regions of the plastid genes psbA and psbB from higher plants are unstable in Chlamydomonas reinhardtii chloroplasts.

The 5' regions of chloroplast genes contain cis-acting regulatory elements including promoters, and determinants of RNA stability and translation. In this work I examined whether the 5' regions of the spinach psbB and the wheat psbA genes can drive the expression of an aadA reporter gene in chloroplasts of the unicellar green alga Chlamydomonas reinhardtii. Both plant 5' sequences confer aadA-dependent, spectinomycin resistance on Escherichia coli, but not on the alga following integration into its chloroplast genome. Northern and run-on transcription analyses reveal that the plant promotors are active in C. reinhardtii but that the resulting chimeric transcripts are unstable. Therefore, the data suggest differences between higher plants and green algae with respect to the molecular mechanisms underlying plastid RNA metabolism.

5' Untranslated Regions↗
PubMed2000

Phenotypic and molecular characterization of conjugative antibiotic resistance plasmids isolated from bacterial communities of activated sludge.

In order to isolate antibiotic resistance plasmids from bacterial communities found in activated sludge, derivatives of the 3-chlorobenzoate-degrading strain Pseudomonas sp. B13, tagged with the green fluorescent protein as an identification marker, were used as recipients in filter crosses. Transconjugants were selected on agar plates containing 3-chlorobenzoate as the sole carbon source and the antibiotic tetracycline, streptomycin or spectinomycin, and were recovered at frequencies in the range of 10(-5) to 10(-8) per recipient. A total of 12 distinct plasmids, designated pB1-pB12, was identified. Their sizes ranged between 41 to 69 kb and they conferred various patterns of antibiotic resistance on their hosts. Two of the plasmids, pB10 and pB11, also mediated resistance to inorganic mercury. Seven of the 12 plasmids were identified as broad-host-range plasmids, displaying extremely high transfer frequencies in filter crosses, ranging from 10(-1) to 10(-2) per recipient cell. Ten of the 12 plasmids belonged to the IncP incompatibility group, based on replicon typing using IncP group-specific PCR primers. DNA sequencing of PCR amplification products further revealed that eight of the 12 plasmids belonged to the IncPbeta subgroup, whereas two plasmids were identified as IncPalpha plasmids. Analysis of the IncP-specific PCR products revealed considerable differences among the IncPbeta plasmids at the DNA sequence level. In order to characterize the gene "load" of the IncP plasmids, restriction fragments were cloned and their DNA sequences established. A remarkable diversity of putative proteins encoded by these fragments was identified. Besides transposases and proteins involved in antibiotic resistance, two putative DNA invertases belonging to the Din family, a methyltransferase of a type I restriction/modification system, a superoxide dismutase, parts of a putative efflux system belonging to the RND family, and proteins of unknown function were identified.

DNA Restriction Enzymes↗
PubMed2000

New colorimetric microdilution method for in vitro susceptibility testing of Borrelia burgdorferi against antimicrobial substances.

A newly developed colorimetric microdilution method was used to analyze the activity of 12 antimicrobial agents against nine Borrelia burgdorferi isolates, including all three genospecies pathogenic for humans. In addition, in vitro antimicrobial resistance patterns of Borrelia valaisiana and Borrelia bissettii tick isolates were investigated. The applied test system is based upon color changes that occur in the presence of phenol red and result from the accumulation of nonvolatile acid produced by actively metabolizing spirochetes. After 72 h of incubation, minimal inhibitory concentrations (MICs) were determined from the decrease of absorbance by software-assisted calculation of growth curves. MIC values were lowest for azlocillin (MIC, < or = 0.125 microg/ml), ceftriaxone (MIC range, < or = 0.015-0.06 microg/ ml), and azithromycin (MIC range, < or = 0.015-0.06 microg/ml). Whereas tobramycin (MIC range, 8-64 microg/ml) exhibited little activity, spectinomycin (MIC range, 0.25-2 microg/ml) showed in vitro antimicrobial activity against Borrelia burgdorferi. The MICs of penicillin G for Borrelia afzelii isolates were ten times higher than those for Borrelia burgdorferi, Borrelia valaisiana, and Borrelia bissettii isolates (P<0.05) and 100 times higher than those for isolates belonging to the genospecies Borrelia garinii (P < 0.05). Further significant differences with respect to the MIC values of the other antimicrobial agents tested were not noted. The colorimetric microdilution method offered the advantages of reliability, reproducibility, and convenience and could handle large numbers of isolates and antibiotics.

Anti-Bacterial Agents↗
PubMed2003

Tendency toward increase in the frequency of isolation of beta-lactamase-nonproducing Neisseria gonorrhoeae exhibiting penicillin resistance, and recent emergence of multidrug-resistant isolates in Japan.

Six hundred and ninety-nine strains of Neisseria gonorrhoeae were examined by the agar dilution method according to the M7-A5 guidelines established by the National Committee for Clinical Laboratory Standards (NCCLS) determine to their beta-lactamase production and susceptibility to penicillin G, cefixime, ceftriaxone, tetracycline, spectinomycin, ciprofloxacin, and azithromycin. The frequency of isolation of penicillinase-producing N. gonorrhoeae decreased gradually, from 7.3% of the test isolates (55 isolates) in 1995 to about 1% in 1998 and 1999. In contrast, while beta-lactamase-nonproducing N. gonorrhoeae exhibiting chromosomally mediated penicillin G resistance were not isolated from clinical specimens in 1995, the incidence of isolation of such resistant strains increased markedly, to 8.2% of 159 isolates, in 1997 and 14.9% of 242 isolates in 1999. The incidence of the isolation of tetracycline-resistant strains also increased between 1996 (none detected) and 1998-1999 (each 7%-8%), and a tendency towards an increase in the frequency of isolates of ciprofloxacin-resistant strains was also observed between 1995 (9.8%) and from 1997 to 1999 (more than 20%). There were no isolates resistant to any two antibiotics among penicillin G, tetracycline, and ciprofloxacin until 1997, but, in subsequent surveys in recent years, multidrug-resistant isolates (resistant to penicillin G, tetracycline, and ciprofloxacin) were detected in 1999.

Chromosomes, Bacterial↗
PubMed2006

Multiple doses of cefodizime are necessary for the treatment of Neisseria gonorrhoeae pharyngeal infection.

A single dose of cefodizime (CDZM), ceftriaxone (CTRX), or spectinomycin (SPCM) is recommended for the treatment of gonococcal urethritis or uterine cervicitis in the era of multidrug-resistant Neisseria gonorrhoeae; namely, cefozopran-resistant N. gonorrhoeae (CZRNG). N. gonorrhoeae pharyngeal infection is not so rare in Japan; however, the proper treatment regimen for this infection is not clear. We previously found that a single dose of CDZM completely eradicated multidrug-resistant N. gonorrhoeae in patients with urethritis and uterine cervicitis, so we tried a single 1.0-g dose of CDZM for the treatment of N. gonorrhoeae pharyngeal infection, including infections with CZRNG. The eradication rate of N. gonorrhoeae from the pharynx was 63.0% with a single 1.0-g dose of CDZM, while the rate for CZRNG with the same dose of CDZM was 38.5%. N. gonorrhoeae was completely eradicated from the pharynx when patients received one or two additional doses of CDZM. Therefore, we concluded that two to three doses of CDZM were necessary for the treatment of N. gonorrhoeae pharyngeal infection including infection with CZRNG.

Anti-Bacterial Agents↗
PubMed2005

Generation and analysis of soybean plastid transformants expressing Bacillus thuringiensis Cry1Ab protoxin.

We describe the generation of fertile and homoplasmic soybean plastid transformants, expressing the Bacillus thuringiensis insecticidal protoxin Cry1Ab. Transgenes were targeted in the intergenic region of Glycine max plastome, between the rps12/7 and trnV genes and selection was carried out using the aadA gene encoding spectinomycin resistance. Molecular analysis confirmed the integration of the cry1Ab and aadA expression cassettes at the expected location in the soybean plastome, and the transmission of the transgenes to the next generation. Western blot analyses showed that the Cry1Ab protoxin is highly expressed in leaves, stems and seeds, but not in roots. Its expression confers strong insecticidal activity to the generated transgenic soybean, as exemplified with velvetbean caterpillar (Anticarsia gemmatalis).

Animals↗
PubMed2006

Efficient and stable transformation of Lactuca sativa L. cv. Cisco (lettuce) plastids.

Transgenic plastids offer unique advantages in plant biotechnology, including high-level foreign protein expression. However, broad application of plastid genome engineering in biotechnology has been largely hampered by the lack of plastid transformation systems for major crops. Here we describe the development of a plastid transformation system for lettuce, Lactuca sativa L. cv. Cisco. The transforming DNA carries a spectinomycin-resistance gene (aadA) under the control of lettuce chloroplast regulatory expression elements, flanked by two adjacent lettuce plastid genome sequences allowing its targeted insertion between the rbcL and accD genes. On average, we obtained 1 transplastomic lettuce plant per bombardment. We show that lettuce leaf chloroplasts can express transgene-encoded GFP to approximately 36% of the total soluble protein. All transplastomic T0 plants were fertile and the T1 progeny uniformly showed stability of the transgene in the chloroplast genome. This system will open up new possibilities for the efficient production of edible vaccines, pharmaceuticals, and antibodies in plants.

Chloroplasts↗
PubMed2006

Expression of the cry9Aa2 B.t. gene in tobacco chloroplasts confers resistance to potato tuber moth.

We report here the control of potato tuber moth (Phthorimaea operculella) by incorporating a truncated Bacillus thuringiensis cry9Aa2 gene in the plastid genome. Plasmids pSKC84 and pSKC85 are derivatives of a new polycistronic plastid transformation vector, pPRV312L, that carries spectinomycin resistance (aadA) as a selective marker and targets insertions in the trnI-trnA intergenic region. The Cry9Aa2 N-terminal region (82.1 kDa; 734 amino acids) was expressed in a cassette, which consists of 49 nucleotides of the cry9Aa2 leader and the 3'-untranslated region of the plastid rbcL gene (TrbcL), and relies on readthrough transcription from the plastid rRNA operon. In a tobacco leaf bioassay, expression of Cry9Aa2 conferred resistance to potato tuber moth. In accordance, the Cry9Aa2 insecticidal protein accumulated to high levels, approximately 10% of the total soluble cellular protein and approximately 20% in the membrane fraction. However, high-level Cry9Aa2 expression significantly delayed plant development. Thus, a practical system to control potato tuber moth by Cry9Aa2 expression calls for down-regulation of its expression.

Animals↗
PubMed2006

Transformation of poplar (Populus alba) plastids and expression of foreign proteins in tree chloroplasts.

Plastid transformation offers several unique advantages compared with nuclear genome transformation, such as high level of transgene expression within plastids, expressing multiple transgenes as operons, lack of position effect due to site-specific transgene integration, and reducing risks of gene flow via pollen due to maternal inheritance of the plastid genome. Plastid transformation has been applied to several herbal species, but as yet there are no applications to tree species. We report here the first successful plastid transformation in a tree species, Populus alba. A vector for plastid transformation of poplar (Populus alba) was constructed, which carried the spectinomycin resistance gene and the green fluorescence protein gene as marker genes. In the regenerated shoots, the site-specific integration of foreign genes and the establishment of a high homoplastomic state were confirmed. Immunoblot analysis and histological observations corroborated the accumulation of green fluorescence protein in chloroplasts. The establishment of a plastid transformation system in poplar provides a novel tool for tree biotechnology.

Chloroplasts↗
PubMed2002

Update on Quinolone Resistance in Neisseria gonorrhoeae.

Quinolones are widely used for treating gonococcal infections, typically in single-dose, oral regimens. However, in the 1990s, quinolone-resistant Neisseria gonorrhoeae emerged, potentially compromising the utility of this drug class. In the past year, these strains have widely disseminated, accounting for over half of isolates in parts of Southeast Asia. The molecular mechanism of resistance has been localized to multiple mutations in genes coding for the bacterial DNA gyrase and topoisomerase enzymes. These mutations accumulate until the minimum inhibitory concentration is 4.0 g/mL or more, which in clinical studies appears to be the threshold for clinical treatment failure. Quinolone-resistant N. gonorrhoeae is independent from other plasmid- and chromosomally-mediated resistance determinants; nearly all isolates to date have been sensitive to cephalosporins and spectinomycin. Nevertheless, designing public health strategies to contain quinolone-resistant N. gonorrhoeae will be difficult.

Journal Article↗
PubMed1987

Norfloxacin in the treatment of uncomplicated gonococcal infections.

Norfloxacin, an oral fluoroquinolone antibacterial, is active in vitro against a variety of gram-positive and gram-negative pathogens, including both penicillinase-producing and non-penicillinase-producing strains of Neisseria gonorrhoeae. An earlier study demonstrated that a two-dose regimen of norfloxacin was as effective as standard therapy with spectinomycin for treating gonococcal urethritis, including infections caused by penicillinase-producing organisms. In this randomized study of treatment for uncomplicated gonococcal infection in men and women, three oral treatment regimens were compared: patients received either two doses of norfloxacin (600 mg twice daily), a single dose of norfloxacin (800 mg), or a single-dose ampicillin (3.5 g)/probenecid (1.0 g) regimen (as recommended by the Centers for Disease Control). All three treatment regimens achieved similar cure rates. Although the number of patients treated was too small to yield statistically significant conclusions, it appears that norfloxacin may be slightly better treatment for rectal and pharyngeal gonococcal infections than ampicillin and probenecid. Additionally, norfloxacin was well tolerated in this study. Thus, based on a review of these data, norfloxacin appears to be an alternative, single-dose, oral treatment regimen for uncomplicated gonococcal infection.

Adult↗
PubMed1991

The clinical experience with ofloxacin in the treatment of sexually transmitted diseases.

Sexually transmitted diseases are an important cause of morbidity in women. Some of the common causes of sexually transmitted disease include Neisseria gonorrhoeae, Chlamydia trachomatis, and in some patients Ureaplasma urealyticum. N. gonorrhoeae has become more resistant to traditional therapies including penicillin. Resistance to penicillin is mediated by the elaboration of beta-lactamase for most organisms and by chromosomal resistance in others. Resistance to tetracycline and spectinomycin has been clearly identified, as has an increase in the minimum inhibitory concentrations to some cephalosporins. Because of the potential for concomitant infection with N. gonorrhoeae and C. trachomatis, many patients are currently treated with a combination of a single dose of ceftriaxone plus a tetracycline for 7 days. Therefore it is significant that a single agent, ofloxacin, has been introduced recently for the treatment of such mixed infections. It also has the potential for treating N. gonorrhoeae resistant to other drugs.

Chlamydia Infections↗
PubMed1987

Aminoglycoside antibiotics preferentially increase permeability in phosphoinositide-containing membranes: a study with carboxyfluorescein in liposomes.

The rate of release from multilamellar liposomes of the fluorescent probe carboxyfluorescein was determined as a measure of membrane permeability. Liposomes of phosphatidylcholine and different anionic phospholipids were incubated with low (1 microM) and high (3 mM) concentrations of calcium in the absence or presence of aminoglycoside antibiotics. The leakage of carboxyfluorescein into the medium was not caused by liposomal fusion as no vesicle fusion was observed in experiments with terbium and dipicolinic acid-loaded liposomes. The basal rate of carboxyfluorescein release (in the absence or presence of 1 microM calcium) from all types of liposomes ranged from 0.1 to 0.3% of trapped carboxyfluorescein per hour. The presence of 3 mM calcium caused the greatest increase in the rate of carboxyfluorescein release (about 9-fold) in liposomes containing phosphatidylinositol 4,5-bisphosphate (PIP2) whereas liposomes containing the other anionic phospholipids (phosphatidylserine, phosphatidylinositol and phosphatidylinositol 4-phosphate) showed an approximate 5-fold increase. In the presence of 1 microM calcium, the aminoglycosides neomycin and gentamicin also increased the rate of carboxyfluorescein release, with PIP2-containing liposomes showing a 3-5-times greater response than the other liposomes, releasing up to 4.6% of trapped carboxyfluorescein per hour. This drug-induced release was dose-dependent and antagonized by calcium. In the presence of 3 mM calcium, 0.1 mM gentamicin or neomycin were ineffective while the drug at 1 mM affected carboxyfluorescein release from PIP2-liposomes only. The aminoglycoside antibiotics, neomycin, gentamicin, tobramycin, kanamycin, amikacin, netilmicin, as well as neamine and spectinomycin (all at 0.1 mM) showed a graded effect on the rate of carboxyfluorescein release from PIP2-containing vesicles in the presence of 0.1 mM calcium. The magnitude of the effect correlated well with the ototoxicity of the drugs previously determined directly in cochlear perfusions in the guinea pig. The study demonstrates that aminoglycoside antibiotics are capable of altering membrane permeabilities and that this effect is most pronounced if PIP2 is present in the bilayers. The excellent correlation between this membrane action and the in-situ toxicity of the drugs further establishes the specific role of PIP2 in the molecular mechanism of aminoglycoside-induced hearing loss. Moreover, it confirms the usefulness of such physicochemical models for the screening and prediction of aminoglycoside toxicity.

Aminoglycosides↗
PubMed1976

Effect of viomycin on dihydrostreptomycin binding to bacterial ribosomes.

Viomycin, a peptide antibiotic, reduced the amounts of dihydrostreptomycin bound to ribosomes of Myobacterium smegmatis and Escherichia coli, although they have different modes of action. The [3H]dihydrostreptomycin binding to ribosomes could not exchanged with streptomycin or dihydrostreptomycin, but not with unrelated antibiotics, namely, kanamycin, neomycin, spectinomycin, capreomycin, tuberactinomycin-N, chloramphenicol and erythromycin. We suggest that there is a significant interaction between the binding sites of viomycin and streptomycin on ribosomes.

Anti-Bacterial Agents↗
PubMed1983

Effects of aminoglycoside antibiotics on the coupling of protein and RNA syntheses in Escherichia coli.

The interdependency of protein and RNA syntheses was studied comparatively in bacteria confronted with amino acid starvation or treated separately with various aminoglycoside antibiotics. By contrast with the concomitant inhibition of macromolecular syntheses in cells deprived of an essential amino acid, RNA production was found to continue in drug-treated cells while protein synthesis was arrested. Such uncoupling process was also observed in bacteria subjected simultaneously to amino acid starvation and treatment with certain antibiotics (neomycin, gentamicin, spectinomycin and kasugamycin) but not with others (streptomycin and kanamycin). These results were related to the intracellular concentration of guanosine polyphosphates, ppGpp and pppGpp. They were discussed in terms of interaction of aminoglycosides with ribosomes.

Amino Acids↗