Engineering of insecticidal hybrid gene into potato chloroplast genome exhibits promising control of Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).

The potato chloroplast was transformed with codon optimized synthetic hybrid cry gene (SN19) to mitigate crop losses by Colorado potato beetle (CPB). The bombarded explants (leaves and internode) were cultured on MS medium supplemented with BAP (2.0 mg/l), NAA (0.2 mg/l), TDZ (2.0 mg/l) and GA3 (0.1 mg/l); spectinomycin 50 mg/l was used as a selection agent in the medium. Leaf explants of cultivar Kuroda induced highest percentage (92%) of callus where cultivar Santae produced the highest percentage (85.7%) of transplastomic shoots. Sante and Challenger showed 9.6% shoot regeneration efficiency followed by cultivar Simply Red (8.8%). PCR amplification yielded 16 postive transplastomic plantlets out of 21 spectinomycin resistant ones. Target gene integration was confirmed by PCR and Southern blot, whereas RT-qPCR was used to assess the expression level of transgene. The localization of visual marker gene gfp was tracked by laser scanning confocal microscopy which confirmed its expression in chloroplasts of leaf cells. The transplastomic plants ensured high mortality to both larvae and adult CPB. Foliage consumption and weight gain of CPB fed on transplastomic leaves were lower compared to the control plants. Sucessful implementation of current research findings can lead to a viable solution to CPB mediated potato losses globally.

CpPosNeg: A positive-negative selection strategy allowing multiple cycles of marker-free engineering of the Chlamydomonas plastome.

The chloroplast represents an attractive compartment for light-driven biosynthesis of recombinant products, and advanced synthetic biology tools are available for engineering the chloroplast genome ( = plastome) of several algal and plant species. However, producing commercial lines will likely require several plastome manipulations. This presents issues with respect to selectable markers, since there are a limited number available, they can be used only once in a serial engineering strategy, and it is undesirable to retain marker genes for antibiotic resistance in the final transplastome. To address these problems, we have designed a rapid iterative selection system, known as CpPosNeg, for the green microalga Chlamydomonas reinhardtii that allows creation of marker-free transformants starting from wild-type strains. The system employs a dual marker encoding a fusion protein of E. coli aminoglycoside adenyltransferase (AadA: conferring spectinomycin resistance) and a variant of E. coli cytosine deaminase (CodA: conferring sensitivity to 5-fluorocytosine). Initial selection on spectinomycin allows stable transformants to be established and driven to homoplasmy. Subsequent selection on 5-fluorocytosine results in rapid loss of the dual marker through intramolecular recombination between the 3'UTR of the marker and the 3'UTR of the introduced transgene. We demonstrate the versatility of the CpPosNeg system by serial introduction of reporter genes into the plastome.

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43.

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, studies in this serotype have been hindered by the lack of genetic tools to modify it. In this study, we describe a method to genetically modify a serotype 1 clinical isolate (strain 519/43). Interestingly, this was achieved by exploiting the Pneumococcus' ability to naturally acquire DNA. However, unlike most pneumococci, the use of linear DNA was not successful; to mutate this important strain, a suicide plasmid had to be used. This methodology has provided the means for a deeper understanding of this elusive serotype, both in terms of its biology and pathogenicity. To validate the method, the major known pneumococcal toxin, pneumolysin, was mutated because it has a well-known and easy to follow phenotype. We showed that the mutant, as expected, lost its ability to lyse red blood cells. By being able to mutate an important gene in the serotype of interest, we were able to observe different phenotypes for loss of function mutants upon intraperitoneal and intranasal infections from the ones observed for other serotypes. In summary, this study proves that strain 519/43 (serotype 1) can be genetically modified.

Taxonomic assignment of uncultured prokaryotes with long range PCR targeting the spectinomycin operon.

The taxonomic assignment of uncultured prokaryotes to known taxa is a major challenge in microbial systematics. This relies usually on the phylogenetic analysis of the ribosomal small subunit RNA or a few housekeeping genes. Recent works have disclosed ribosomal proteins as valuable markers for systematics and, due to the boom in complete genome sequencing, their use has become widespread. Yet, in the case of uncultured strains, for which complete genome sequences cannot be easily obtained, sequencing many markers is complicated and time consuming. Taking the advantage of the organization of ribosomal protein coding genes in large gene clusters, we amplified a 32 kb conserved region encompassing the spectinomycin (spc) operon using long range PCR from isolated and from uncultured nodular endophytic Frankia strains. The phylogenetic analysis of the 27 ribosomal protein genes contained in this region provided a robust phylogenetic tree consistent with phylogenies based on larger set of markers, indicating that this subset of ribosomal proteins contains enough phylogenetic signal to address systematic issues. This work shows that using long range PCR could break down the barrier preventing the use of ribosomal proteins as phylogenetic markers when complete genome sequences cannot be easily obtained.

A new subclass of intrinsic aminoglycoside nucleotidyltransferases, ANT(3")-II, is horizontally transferred among Acinetobacter spp. by homologous recombination.

The emergence and spread of antibiotic resistance among Acinetobacter spp. have been investigated extensively. Most studies focused on the multiple antibiotic resistance genes located on plasmids or genomic resistance islands. On the other hand, the mechanisms controlling intrinsic resistance are still not well understood. In this study, we identified the novel subclass of aminoglycoside nucleotidyltransferase ANT(3")-II in Acinetobacter spp., which comprised numerous variants distributed among three main clades. All members of this subclass can inactivate streptomycin and spectinomycin. The three ant(3")-II genes, encoding for the three ANT(3")-II clades, are widely distributed in the genus Acinetobacter and always located in the same conserved genomic region. According to their prevalence, these genes are intrinsic in Acinetobacter baumannii, Acinetobacter pittii, and Acinetobacter gyllenbergii. We also demonstrated that the ant(3")-II genes are located in a homologous recombination hotspot and were recurrently transferred among Acinetobacter species. In conclusion, our findings demonstrated a novel mechanism of natural resistance in Acinetobacter spp., identified a novel subclass of aminoglycoside nucleotidyltransferase and provided new insight into the evolutionary history of intrinsic resistance genes.

Tissue Penetration of a Novel Spectinamide Antibiotic for the Treatment of Tuberculosis.

The in vivo biodistribution and pharmacokinetics of 1329, a novel spectinamide antibiotic with anti-tubercular activity, were studied during intravenous administration of an tritium-labeled compound for nine consecutive, 12-hourly doses to rats. Serial blood samples were collected after the first and the eighth dose, and major organs and tissues were collected 1 h after the ninth dose. Urinary and fecal excretion was monitored throughout the dosing period. Radioactivity in the collected samples was assessed by scintillation counting. During the course of treatment, 86.6% of the administered radioactivity was recovered in urine, feces, organs, and muscle tissue. Urinary excretion was the major route of elimination, with 70% of radioactivity recovered from urine and 12.6% from feces. The time profiles of radioactivity in serum after the first and the eighth dose were identical for the first 2 h post-dose, with similar Cmax (3.39 vs. 3.55 mCi/L) and AUC0-τ (5.08 vs. 5.17 mCi • h/L), indicating no substantial accumulation of 1329 during multiple dosing. Radioactivity in major target organs for pulmonary tuberculosis infection, the lungs and spleen, was 2.79- and 3.06-fold higher than in the blood. Similarly, the intracellular uptake of 1329 into macrophages was sixfold higher than for streptomycin. Overall, these observations suggest biodistribution properties favorable for targeting pulmonary tuberculosis infections.

Molecular mechanics of 30S subunit head rotation.

During ribosomal translocation, a process central to the elongation phase of protein synthesis, movement of mRNA and tRNAs requires large-scale rotation of the head domain of the small (30S) subunit of the ribosome. It has generally been accepted that the head rotates by pivoting around the neck helix (h28) of 16S rRNA, its sole covalent connection to the body domain. Surprisingly, we observe that the calculated axis of rotation does not coincide with the neck. Instead, comparative structure analysis across 55 ribosome structures shows that 30S head movement results from flexing at two hinge points lying within conserved elements of 16S rRNA. Hinge 1, although located within the neck, moves by straightening of the kinked helix h28 at the point of contact with the mRNA. Hinge 2 lies within a three-way helix junction that extends to the body through a second, noncovalent connection; its movement results from flexing between helices h34 and h35 in a plane orthogonal to the movement of hinge 1. Concerted movement at these two hinges accounts for the observed magnitudes of head rotation. Our findings also explain the mode of action of spectinomycin, an antibiotic that blocks translocation by binding to hinge 2.

Probing RNA-antibiotic interactions: a FTIR study.

The crystal structure determination of antibiotic binding sites on the 30S ribosomal subunit and the increasing demand for developing RNA-based drugs has prompted us to study the direct binding of spectinomycin, vancomycin and bleomycin with yeast total RNA using Fourier transform infrared (FTIR) spectroscopy. We report that the OH of spectinomycin and the peptide group of vancomycin can bind to the bases of RNA, which might depend on Mg2+ concentration. Bleomycin on the other hand does not show such a drastic effect on yeast total RNA. This study might help in developing innovative strategies utilizing RNA molecules to perform a variety of essential biological functions.

A steric block in translation caused by the antibiotic spectinomycin.

The widely used antibiotic spectinomycin inhibits bacterial protein synthesis by blocking translocation of messenger RNA and transfer RNAs on the ribosome. Here, we show that in crystals of the Escherichia coli 70S ribosome spectinomycin binding traps a distinct swiveling state of the head domain of the small ribosomal subunit. Spectinomycin also alters the rate and completeness of reverse translocation in vitro. These structural and biochemical data indicate that in solution spectinomycin sterically blocks swiveling of the head domain of the small ribosomal subunit and thereby disrupts the translocation cycle.

The relationship between aminoglycosides' RNA binding proclivity and their antiplasmid effect on an IncB plasmid.

Bacteria routinely become resistant to antibiotics through the uptake of plasmids that encode resistance-mediating proteins. Such plasmid-based resistance is seen extensively in clinical settings and has been documented for a wide variety of bacterial infections from both Gram-positive and Gram-negative organisms. We recently reported that a small molecule could be used to mimic a natural process of plasmid elimination, called plasmid incompatibility, and that the addition of this compound causes elimination of an IncB plasmid from E. coli and a subsequent resensitization to antibiotics [DeNap, Thomas, Musk, and Hergenrother (2004) J. Am. Chem. Soc. 126, 15402-15404]. Described herein is a further substantiation and validation of the notion that plasmid incompatibility can be mimicked with small molecules that bind to important RNA targets controlling plasmid replication. In this study, the dissociation constant and stoichiometry of RNA binding are determined for 12 aminoglycosides with stem-loop I (SLI) of the IncB replication machinery. Importantly, it is found that compounds that do not bind to this RNA replication control element fail to induce plasmid loss in vivo, whereas those that do bind to the RNA typically cause measurable plasmid loss. These results highlight the potential for targeting key RNA regions for induction of plasmid loss and the subsequent resensitization of bacteria to antibiotics.

In vivo selection of spectinomycin-binding RNAs.

The folding of even short RNA molecules in a random library can produce a huge number of possible macromolecular structures. Using this principle, we have designed selections to seek non-coding RNA transcripts capable of interfering with specific macromolecules such as transcription factors in living bacterial cells. Here we show that such selections can uncover an unexpected class of RNAs. In the present case, we report short RNA transcripts whose expression confers bacterial resistance to the antibiotic spectinomycin. We provide evidence that such RNAs cause drug resistance by direct antibiotic binding, demonstrating a class of spectinomycin-specific functional molecular decoys built from RNA.

Construction of a stable non-mucoid deletion mutant of the Streptococcus equi Pinnacle vaccine strain.

Streptococcus equi causes equine strangles, a purulent lymphadenopathy of the head and neck. An avirulent, non-encapsulated strain (Pinnacle) has been used widely in North America as an intranasal vaccine. The aim of the study was to create a specific mutation of the hyaluronate synthase (hasA) gene in Pinnacle to permanently abolish the production of capsule and provide an easily recognisable genetic marker. An internal fragment of hasA was generated by PCR and cloned into pTW100 (Microscience, UK). An encapsulated revertant of Pinnacle was then transformed with the recombinant plasmid by electroporation and cultured under conditions to promote homologous recombination. Among 90 spectinomycin resistant transformants observed, one non-mucoid (non-encapsulated) spectinomycin resistant colony was detected. The presence of plasmid sequence within the hasA gene was confirmed by the PCR. After six passages in antibiotic-free medium, four non-mucoid spectinomycin sensitive colonies were found. Sequence analysis of one of these clones, designated Pinnacle HasNeg, revealed loss of the 3' end of the hasA and the 5' end of the hasB genes. This deletion mutant should serve as a useful candidate to replace Pinnacle since it cannot revert to a mucoid phenotype and can be distinguished genetically from wild type strains.

Enzymatic synthesis of aminoglycoside antibiotics: novel adenosylmethionine:2-deoxystreptamine N-methyltransferase activities in hygromycin B- and spectinomycin-producing Streptomyces spp. and uses of the methylated products.

Aminocyclitols structurally related to streptamine, a 1,3-diaminocyclitol, are common components of the RNA-binding aminoglycoside antibiotics. The respective aminocyclitol cores of hygromycin B and spectinomycin are N(3)-methyl-2-deoxy-D-streptamine and N(1),N(3)-dimethyl-2-epi-streptamine. Adenosyl[methyl-(14)C]methionine:2-deoxystreptamine N-methyltransferase activities were detected in extracts of early-stationary-phase mycelia of the hygromycin B producer Streptomyces hygroscopicus subsp. hygroscopicus ATCC 27438 and the spectinomycin producer Streptomyces flavopersicus ATCC 19756. Extracts of both strains methylated the N(1)- and N(3)-amino groups of 2-deoxystreptamine, streptamine, and 2-epi-streptamine; the N(1)-amino group of N(3)-methyl-2-deoxy-D-streptamine, and the N(3)-amino group of N(1)-ethyl-2-deoxy-D-streptamine, the semisynthetic aminocyclitol of netilmicin. The mono[(14)C]methyl derivatives of 2-deoxystreptamine, streptamine, and 2-epi-streptamine were excellent substrates for L-glutamine:aminocyclitol aminotransferase and thereby provided a sensitive assay for derepression of this key enzyme, a generic biosynthetic marker that we have shown to be the only enzyme common to the biosyntheses of all major aminoglycoside antibiotics. Other prospective uses for these methyl-labeled 2-deoxystreptamine analogs are also described.

Bifidobacterium longum as a delivery system for cancer gene therapy: selective localization and growth in hypoxic tumors.

A fundamental obstacle in gene therapy for cancer is the specific delivery of an anticancer gene product to a solid tumor, and yet no systemic delivery system that specifically targets solid tumors currently exists. A strain of domestic bacteria, Bifidobacterium longum, which is nonpathogenic and anaerobic, selectively localized and proliferated in several types of mouse solid tumors after systemic application. In this report, we further describe a novel approach to cancer gene therapy in which genetically engineered Bifidobacterium is used as a tumor-specific vector. Similarly to wild-type B. longum, genetically engineered B. longum could be detected in tumor tissue only and was not found in a large survey of normal mouse tissues after intravenous injection. This finding strongly suggests that obligate anaerobic bacteria such as Bifidobacterium can be used as highly specific gene delivery vectors for cancer gene therapy.

The Escherichia coli cmlA gene encodes the multidrug efflux pump Cmr/MdfA and is responsible for isopropyl-beta-D-thiogalactopyranoside exclusion and spectinomycin sensitivity.

Expression of cloned genes from isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoters is lowered when the Escherichia coli CmlA/Cmr/MdfA efflux pump is overexpressed, probably due to IPTG exclusion from the cytoplasm. The previously reported cmlA1 mutation confers a similar phenotype. cmlA1 contains an IS30 insertion upstream of cmr/mdfA, which creates a putative promoter. CmlA overproduction also causes spectinomycin hypersensitivity.

Activity-dependent release of brain-derived neurotrophic factor underlies the neuroprotective effect of N-methyl-D-aspartate.

The molecular mechanism(s) of N-methyl-D-aspartate (NMDA) neuroprotective properties were investigated in primary cultures of cerebellar granule cell neurons. Granule cells express the neurotrophin receptor TrkB but not TrkA or TrkC. In these cells, the TrkB ligand brain-derived neurotrophic factor (BDNF) prevents glutamate toxicity. Therefore, we have tested the hypothesis that NMDA activates synthesis and release of BDNF, which may prevent glutamate toxicity by an autocrine loop. Exposure of granule cells for 2 and 5 min to a subtoxic concentration of NMDA (100 microM) evoked an accumulation of BDNF in the medium without concomitant changes in the intracellular levels of BDNF protein or mRNA. The increase in BDNF in the medium is followed by enhanced TrkB tyrosine phosphorylation, suggesting that NMDA increases the release of BDNF and therefore the activity of TrkB receptors. To examine whether BDNF and TrkB signaling play a role in the NMDA-mediated neuroprotective properties, neurons were exposed to soluble trkB receptor-IgG fusion protein, which is known to inhibit the activity of extracellular BDNF, and to K252a, a tyrosine kinase inhibitor. Both compounds blocked the NMDA-mediated TrkB tyrosine phosphorylation and subsequently its neuroprotective properties. We suggest that NMDA activates the TrkB receptor via a BDNF autocrine loop, resulting in neuronal survival.

Shine-Dalgarno-like sequences are not required for translation of chloroplast mRNAs in Chlamydomonas reinhardtii chloroplasts or in Escherichia coli.

Initiation of translation in Escherichia coli and related eubacteria involves well-defined interactions between a conserved Shine-Dalgarno (SD) sequence immediately upstream of the initiation codon in the mRNA leader and an equally conserved anti-SD sequence at the 3' end of the 16S rRNA. SD-like sequences found in the leaders of many, but not all, mRNAs from cyanobacteria and chloroplasts are hypervariable in location, size, and base composition compared to those in E. coli, while anti-SD sequences in the respective 16S rRNAs remain highly conserved. We have examined the function of the SD-like sequences found in the leaders of four chloroplast genes of the green alga Chlamydomonas reinhardtii using replacement mutagenesis to eliminate complementarity with the anti-SD sequences and insertion of canonical SD sequences (GGAGG) at positions -9 to -5 relative to the initiation codon. Promoter-leader regions of the atpB, atpE, rps4, and rps7 genes representing the diversity of chloroplast SD-like sequences were fused to aadA and uidA reporter genes encoding spectinomycin resistance and GUS activity respectively. Analysis of chloroplast transformants of C. reinhardtii and transformants of E. coli carrying the wild-type and mutant reporter constructs revealed that mutagenic replacement of the putative SD sequences had no effect on the expression of either the aadA or uidA reporter genes. Chloroplast transformants with the canonical SD sequence also showed no differences in reporter gene expression, whereas expression of the reporter genes was increased by 10 to 30% in the E. coli transformants. Collectively our results suggest that even though SD-dependent initiation predominates in E. coli, this bacterium also has the capacity to initiate translation by an SD-independent mechanism. In contrast, plant chloroplasts, and very probably their cyanobacterial ancestors, appear to have adopted the SD-independent mechanism for translational initiation of most mRNAs.

Isolation of a gene encoding a novel spectinomycin phosphotransferase from Legionella pneumophila.

A gene capable of conferring spectinomycin resistance was isolated from Legionella pneumophila, the agent of Legionnaires' disease. The gene (aph) encoded a 36-kDa protein which has similarity to aminoglycoside phosphotransferases. Biochemical analysis confirmed that aph encodes a phosphotransferase which modifies spectinomycin but not hygromycin, kanamycin, or streptomycin. The strain that was the source of aph demonstrated resistance to spectinomycin, and Southern hybridizations determined that aph also exists in other legionellae.

The selection in vivo and characterization of an RNA recognition motif for spectinomycin.

Ribonucleoprotein (RNP) complexes participate in almost all macromolecular processes, including RNA processing, protein synthesis, and the signal recognition of proteins targeted for export. An understanding of these processes requires detailed knowledge of interactions at the molecular level, which has evidently been difficult due to the size and complexity of the particles. Fragmentation of large RNP complexes into functional subdomains is proven to be a successful in vitro strategy to probe ligand interactions at the molecular level. We reasoned that RNA molecules expressed in vivo may fold in such a manner as to mimic a drug binding site present on the intact ribosome. If expressed at sufficient levels, the RNA would sequester the antibiotic thereby permitting the continued function of the ribosome and consequently allow the cell to survive in the presence of the drug. Evidence is presented here in support of this RNA fragment-rescue concept following the selection and characterization of RNA fragments that confer resistance to the antibiotic spectinomycin.

Site-specific deletion and rearrangement of integron insert genes catalyzed by the integron DNA integrase.

Deletion of individual antibiotic resistance genes found within the variable region of integrons is demonstrated. Evidence for gene duplications and rearrangements resulting from the insertion of gene units at new locations is also presented. Deletion, duplication, and rearrangement occur only in the presence of the integron-encoded DNA integrase. These events are precise and involve loss or gain of one or more complete insert units or gene cassettes. This confirms the recent definition of gene cassettes as consisting of the gene coding sequences, all except the last 7 bases of the 59-base element found at the 3' end of the gene, and the core site located 5' to the gene (Hall et al., Mol. Microbiol. 5:1941-1959, 1991) and demonstrates that individual gene cassettes are functional units which can be independently mobilized. Both deletions and duplications can be generated by integrase-mediated cointegrate formation followed by integrase-mediated resolution involving a different pair of sites. However, deletion occurs 10 times more frequently than duplication, and we propose that the majority of deletion events are likely to involve integrase-dependent excision of the gene unit to generate a circular gene cassette. The implications of these findings in understanding the evolution of integrons and the spread of antibiotic resistance genes in bacterial populations is discussed.