Identification and Characterization of the Conjugal Transfer Region of the pCg1 plasmid from Naphthalene-Degrading Pseudomonas putida Cg1

ABSTRACT Hybridization and restriction fragment length polymorphism data (K. G. Stuart-Keil, A. M. Hohnstock, K. P. Drees, J. B. Herrick, and E. L. Madsen, Appl. Environ. Microbiol. 64:3633-3640, 1998) have shown that pCg1, a naphthalene catabolic plasmid carried by Pseudomonas putida Cg1, is homologous to the archetypal naphthalene catabolic plasmid, pDTG1, in P. putida NCIB 9816-4. Sequencing of the latter plasmid allowed PCR primers to be designed for amplifying and sequencing the conjugal transfer region in pCg1. The mating pair formation (mpf) gene, mpfA encoding the putative precursor of the conjugative pilin subunit from pCg1, was identified along with other trb-like mpf genes. Sequence comparison revealed that the 10 mpf genes in pCg1 and pDTG1 are closely related (61 to 84% identity) in sequence and operon structure to the putative mpf genes of catabolic plasmid pWW0 (TOL plasmid of P. putida) and pM3 (antibiotic resistance plasmid of Pseudomonas. spp). A polar mutation caused by insertional inactivation in mpfA of pCg1 and reverse transcriptase PCR analysis of mRNA showed that this mpf region was involved in conjugation and was transcribed from a promoter located upstream of an open reading frame adjacent to mpfA. lacZ transcriptional fusions revealed that mpf genes of pCg1 were expressed constitutively both in liquid and on solid media. This expression did not respond to host exposure to naphthalene. Conjugation frequency on semisolid media was consistently 10- to 100-fold higher than that in liquid media. Thus, conjugation of pCg1 in P. putida Cg1 was enhanced by expression of genes in the mpf region and by surfaces where conditions fostering stable, high-density cell-to-cell contact are manifest.

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Identification of Functional mob Regions in Rhizobium etli: Evidence for Self-Transmissibility of the Symbiotic Plasmid pRetCFN42d

Journal of Bacteriology ◽

10.1128/jb.186.17.5753-5761.2004 ◽

2004 ◽

Vol 186 (17) ◽

pp. 5753-5761 ◽

Cited By ~ 34

Author(s):

Daniel Pérez-Mendoza ◽

Ana Domínguez-Ferreras ◽

Socorro Muñoz ◽

María José Soto ◽

José Olivares ◽

...

Keyword(s):

Bacterial Genome ◽

Donor Cell ◽

Conjugal Transfer ◽

Rhizobium Etli ◽

Wild Type ◽

Reading Frame ◽

Laboratory Conditions ◽

Symbiotic Plasmid ◽

Transfer Region ◽

Multiple Copies

ABSTRACT An approach originally designed to identify functional origins of conjugative transfer (oriT or mob) in a bacterial genome (J. A. Herrera-Cervera, J. M. Sanjuán-Pinilla, J. Olivares, and J. Sanjuán, J. Bacteriol. 180:4583-4590, 1998) was modified to improve its reliability and prevent selection of undesired false mob clones. By following this modified approach, we were able to identify two functional mob regions in the genome of Rhizobium etli CFN42. One corresponds to the recently characterized transfer region of the nonsymbiotic, self-transmissible plasmid pRetCFN42a (C. Tun-Garrido, P. Bustos, V. González, and S. Brom, J. Bacteriol. 185:1681-1692, 2003), whereas the second mob region belongs to the symbiotic plasmid pRetCFN42d. The new transfer region identified contains a putative oriT and a typical conjugative (tra) gene cluster organization. Although pRetCFN42d had not previously been shown to be self-transmissible, mobilization of cosmids containing this tra region required the presence of a wild-type pRetCFN42d in the donor cell; the presence of multiple copies of this mob region in CFN42 also promoted conjugal transfer of the Sym plasmid pRetCFN42d. The overexpression of a small open reading frame, named yp028, located downstream of the putative relaxase gene traA, appeared to be responsible for promoting the conjugal transfer of the R. etli pSym under laboratory conditions. This yp028-dependent conjugal transfer required a wild-type pRetCFN42d traA gene. Our results suggest for the first time that the R. etli symbiotic plasmid is self-transmissible and that its transfer is subject to regulation. In wild-type CFN42, pRetCFN42d tra gene expression appears to be insufficient to promote plasmid transfer under standard laboratory conditions; gene yp028 may play some role in the activation of conjugal transfer in response to as-yet-unknown environmental conditions.

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Transactivation of Latent Marek's Disease Herpesvirus Genes in QT35, a Quail Fibroblast Cell Line, by Herpesvirus of Turkeys

Journal of Virology ◽

10.1128/jvi.74.21.10176-10186.2000 ◽

2000 ◽

Vol 74 (21) ◽

pp. 10176-10186 ◽

Cited By ~ 31

Author(s):

T. Yamaguchi ◽

S. L. Kaplan ◽

P. Wakenell ◽

K. A. Schat

Keyword(s):

Cell Line ◽

Fibroblast Cell ◽

Northern Blotting ◽

Marek's Disease ◽

Pcr Analysis ◽

Marek’S Disease ◽

Reading Frame ◽

Reverse Transcription Pcr ◽

Herpesvirus Of Turkeys ◽

Serotype 1

ABSTRACT The QT35 cell line was established from a methylcholanthrene-induced tumor in Japanese quail (Coturnix coturnix japonica) (C. Moscovici, M. G. Moscovici, H. Jimenez, M. M. Lai, M. J. Hayman, and P. K. Vogt, Cell 11:95–103, 1977). Two independently maintained sublines of QT35 were found to be positive for Marek's disease virus (MDV)-like genes by Southern blotting and PCR assays. Sequence analysis of fragments of the ICP4, ICP22, ICP27, VP16, meq, pp14, pp38, open reading frame (ORF) L1, and glycoprotein B (gB) genes showed a strong homology with the corresponding fragments of MDV genes. Subsequently, a serotype 1 MDV-like herpesvirus, tentatively name QMDV, was rescued from QT35 cells in chicken kidney cell (CKC) cultures established from 6- to 9-day-old chicks inoculated at 8 days of embryonation with QT35 cells. Transmission electron microscopy failed to show herpesvirus particles in QT35 cells, but typical intranuclear herpesvirus particles were detected in CKCs. Reverse transcription-PCR analysis showed that the following QMDV transcripts were present in QT35 cells: sense and antisense meq, ORF L1, ICP4, and latency-associated transcripts, which are antisense to ICP4. A transcript of approximately 4.5 kb was detected by Northern blotting using total RNA from QT35 cells. Inoculation of QT35 cells with herpesvirus of turkeys (HVT)-infected chicken embryo fibroblasts (CEF) but not with uninfected CEF resulted in the activation of ICP22, ICP27, VP16, pp38, and gB. In addition, the level of ICP4 mRNA was increased compared to that in QT35 cells. The activation by HVT resulted in the production of pp38 protein. It was not possible to detect if the other activated genes were translated due to the lack of serotype 1-specific monoclonal antibodies.

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A K+ Uptake Protein, TrkA, Is Required for Serum, Protamine, and Polymyxin B Resistance in Vibrio vulnificus

Infection and Immunity ◽

10.1128/iai.72.2.629-636.2004 ◽

2004 ◽

Vol 72 (2) ◽

pp. 629-636 ◽

Cited By ~ 40

Author(s):

Yu-Chung Chen ◽

Yin-Ching Chuang ◽

Chun-Chin Chang ◽

Chii-Ling Jeang ◽

Ming-Chung Chang

Keyword(s):

Human Serum ◽

Vibrio Vulnificus ◽

Polymyxin B ◽

Wild Type ◽

Gene Encoding ◽

Reading Frame ◽

Isogenic Mutant ◽

Virulence Attenuation ◽

Insertional Inactivation ◽

Transposon Mutants

ABSTRACT Vibrio vulnificus, a highly virulent marine bacterium, is the causative agent of both serious wound infections and fatal septicemia in many areas of the world. To identify the genes required for resistance to human serum, we constructed a library of transposon mutants of V. vulnificus and screened them for hypersensitivity to human serum. Here we report that one of the isolated serum-susceptible mutants had a mutation in an open reading frame identified as trkA, a gene encoding an amino acid sequence showing high identity to that of TrkA of Vibrio alginolyticus, a protein required for the uptake of potassium. A trkA isogenic mutant was constructed via insertional inactivation, and it was significantly more easily killed by human serum, protamine, or polymyxin B than was the wild type. At K+ concentrations of 1 to 20 mM, this isogenic mutant showed attenuated growth compared to the wild-type strain. In addition, infection experiments demonstrated virulence attenuation when this mutant was administered intraperitoneally or subcutaneously to both normal and iron-treated mice, indicating that TrkA may modulate the transport of potassium and resistance to host innate defenses and that it is important for virulence in mice.

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A novel miniature transposon-like element discovered in the coding sequence of a gene that encodes for 5-formyltetrahydrofolate in wheat

BMC Plant Biology ◽

10.1186/s12870-019-2034-1 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 2

Author(s):

Katherine Domb ◽

Danielle Keidar-Friedman ◽

Khalil Kashkush

Keyword(s):

Stop Codon ◽

Structural Similarity ◽

Wheat Genome ◽

Wild Emmer ◽

Pcr Analysis ◽

Wheat Species ◽

Wheat Evolution ◽

Reading Frame ◽

Specific Pcr ◽

Pol Iii

Abstract Background Transposable elements (TEs) comprise over 80% of the wheat genome and usually possess unique features for specific super-families and families. However, the role of TEs in wheat evolution and reshaping the wheat genome remains largely unclear. Results In this study, we discovered a miniature (307 bp in length) TE-like sequence in exon 6 of a gene that encodes for 5-formyltetrahydrofolate, in two accessions of wild emmer wheat (T. turgidum ssp. dicoccoides) and has interfered with the gene translation by creating a shorter reading frame as a result of a stop codon. The sequence that was termed Mariam, does not show any structural similarity to known TEs. It does not possess terminal inverted repeats (TIRs) that would allow us to assign this element to one of the TIR DNA super-families, and it does not possess characteristic features of SINE, such as a Pol-III promotor or a poly-A tail. In-silico analysis of five publicly available genome drafts of Triticum and Aegilops species revealed that Mariam element appears in a very low copy number (1–3 insertions) in diploid wheat species and ~ 12 insertions in tetraploid and hexaploidy wheat species. In addition, Mariam element was found to be unique to wheat, as it was not found in other plant genomes. The dynamic nature of Mariam in the wheat genome was assessed by site-specific PCR analysis and revealed that it retained activity in wild emmer populations in a population-specific manner. Conclusions This study provides additional insight into the evolutionary impact of TEs in wheat.

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Identification and characterization of a circadian clock-associated pseudo-response regulator 7 gene from trifoliate orange

Notulae Botanicae Horti Agrobotanici Cluj-Napoca ◽

10.15835/nbha48111785 ◽

2020 ◽

Vol 48 (1) ◽

pp. 128-139

Author(s):

Yu-E DING ◽

Wenkai HUANG ◽

Bo SHU ◽

Ying-Ning ZOU ◽

Qiang-Sheng WU ◽

...

Keyword(s):

Circadian Clock ◽

Response Regulator ◽

Poncirus Trifoliata ◽

Pcr Analysis ◽

Trifoliate Orange ◽

Drought Treatment ◽

Reading Frame ◽

Rhythmic Expression ◽

Pseudo Response Regulator ◽

Main Component

Circadian clock is usually involved in many physiological processes of plants, including responses to abiotic stress, whilst pseudo-response regulator 7 (PRR7) gene is the main component of the circadian clock. In this study, the cDNA of the PRR7 gene was obtained from trifoliate orange (Poncirus trifoliata). Based on the sequence analysis, the PtPRR7 gene had an open reading frame of 2343 bp, encoded 780 amino acids, and contained proteins of the REC and CCT domains. Subcellular localization indicated that PtPRR7 was mainly localized in the nucleus and a small amount of cytoplasm. qRT-PCR analysis revealed the highest expression level of PtPRR7 in roots than in both shoots and leaves. The PtPRR7 gene during 24 hours of soil water deficit exhibited a circadian rhythmic expression pattern: the expression peak at 9:00 am in leaves and at 21:00 pm in roots. Drought treatment affected PtPRR7 gene expression. Such data provide important references for understanding the characteristics of PtPRR7 gene in citrus plants.

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Chromosomal gene capture mediated by the Pseudomonas putida TOL catabolic plasmid.

Journal of Bacteriology ◽

10.1128/jb.176.15.4635-4641.1994 ◽

1994 ◽

Vol 176 (15) ◽

pp. 4635-4641 ◽

Cited By ~ 26

Author(s):

M I Ramos-González ◽

M A Ramos-Díaz ◽

J L Ramos

Keyword(s):

Pseudomonas Putida ◽

Chromosomal Gene ◽

Gene Capture ◽

Catabolic Plasmid

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Molecular and Insecticidal Characterization of a Cry1I Protein Toxic to Insects of the Families Noctuidae, Tortricidae, Plutellidae, and Chrysomelidae

Applied and Environmental Microbiology ◽

10.1128/aem.02861-05 ◽

2006 ◽

Vol 72 (7) ◽

pp. 4796-4804 ◽

Cited By ~ 32

Author(s):

Iñigo Ruiz de Escudero ◽

Anna Estela ◽

Manuel Porcar ◽

Clara Martínez ◽

José A. Oguiza ◽

...

Keyword(s):

Specific Activity ◽

Membrane Vesicles ◽

Lobesia Botrana ◽

Water Soluble ◽

Insect Species ◽

Pcr Analysis ◽

Cry Protein ◽

Toxic Activity ◽

Reading Frame

ABSTRACT The most notable characteristic of Bacillus thuringiensis is its ability to produce insecticidal proteins. More than 300 different proteins have been described with specific activity against insect species. We report the molecular and insecticidal characterization of a novel cry gene encoding a protein of the Cry1I group with toxic activity towards insects of the families Noctuidae, Tortricidae, Plutellidae, and Chrysomelidae. PCR analysis detected a DNA sequence with an open reading frame of 2.2 kb which encodes a protein with a molecular mass of 80.9 kDa. Trypsin digestion of this protein resulted in a fragment of ca. 60 kDa, typical of activated Cry1 proteins. The deduced sequence of the protein has homologies of 96.1% with Cry1Ia1, 92.8% with Cry1Ib1, and 89.6% with Cry1Ic1. According to the Cry protein classification criteria, this protein was named Cry1Ia7. The expression of the gene in Escherichia coli resulted in a protein that was water soluble and toxic to several insect species. The 50% lethal concentrations for larvae of Earias insulana, Lobesia botrana, Plutella xylostella, and Leptinotarsa decemlineata were 21.1, 8.6, 12.3, and 10.0 μg/ml, respectively. Binding assays with biotinylated toxins to E. insulana and L. botrana midgut membrane vesicles revealed that Cry1Ia7 does not share binding sites with Cry1Ab or Cry1Ac proteins, which are commonly present in B. thuringiensis-treated crops and commercial B. thuringiensis-based bioinsecticides. We discuss the potential of Cry1Ia7 as an active ingredient which can be used in combination with Cry1Ab or Cry1Ac in pest control and the management of resistance to B. thuringiensis toxins.

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Cloning of Acyl-ACP Thioesterase FatA fromArachis hypogaeaL. and Its Expression inEscherichia coli

Journal of Biomedicine and Biotechnology ◽

10.1155/2012/652579 ◽

2012 ◽

Vol 2012 ◽

pp. 1-5 ◽

Cited By ~ 3

Author(s):

Gao Chen ◽

Zhen-ying Peng ◽

Lei Shan ◽

Ning Xuan ◽

Gui-ying Tang ◽

...

Keyword(s):

Oleic Acid ◽

Palmitoleic Acid ◽

Membrane Lipid ◽

Pcr Analysis ◽

Fatty Acid Profiles ◽

Reading Frame ◽

E Coli ◽

Real Time Quantitative Pcr ◽

Immature Seeds ◽

Expression Inescherichia Coli

In this study, a full-length cDNA of the acyl-ACP thioesterase,AhFatA, was cloned from developing seeds ofArachis hypogaeaL. by 3′-RACE. Sequence analysis showed that the open reading frame encodes a peptide of 372 amino acids and has 50–70% identity with FatA from other plants. Real-time quantitative PCR analysis revealed thatAhFatA was expressed in all tissues ofA. hypogaeaL., but most strongly in the immature seeds harvested at 60 days after pegging. Heterologous expression ofAhFatA inEscherichia coliaffected bacterial growth and changed the fatty acid profiles of the membrane lipid, resulting in directed accumulation towards palmitoleic acid and oleic acid. These results indicate that AhFatA is at least partially responsible for determining the high palmitoleic acid and oleic acid composition ofE. coli.

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Complementation of a Nonmotile flaB Mutant ofBorrelia burgdorferi by Chromosomal Integration of a Plasmid Containing a Wild-Type flaB Allele

Journal of Bacteriology ◽

10.1128/jb.183.22.6558-6564.2001 ◽

2001 ◽

Vol 183 (22) ◽

pp. 6558-6564 ◽

Cited By ~ 32

Author(s):

Marina L. Sartakova ◽

Elena Y. Dobrikova ◽

M. Abdul Motaleb ◽

Henry P. Godfrey ◽

Nyles W. Charon ◽

...

Keyword(s):

Dna Hybridization ◽

Kanamycin Resistance ◽

Functional Restoration ◽

Pcr Analysis ◽

Null Mutant ◽

Wild Type ◽

Erythromycin Resistance ◽

Cloning Vectors ◽

Insertional Inactivation ◽

Wave Morphology

ABSTRACT With the recent identification of antibiotic resistance phenotypes, the use of reporter genes, the isolation of null mutants by insertional inactivation, and the development of extrachromosomal cloning vectors, genetic analysis of Borrelia burgdorferi is becoming a reality. A previously described nonmotile, rod-shaped, kanamycin-resistant B. burgdorferi flaB::Km null mutant was complemented by electroporation with the erythromycin resistance plasmid pED3 (a pGK12 derivative) containing the wild-typeflaB sequence and 366 bp upstream from its initiation codon. The resulting MS17 clone possessed erythromycin and kanamycin resistance, flat-wave morphology, and microscopic and macroscopic motility. Several other electroporations with plasmids containing wild-type flaB and various lengths (198, 366, or 762 bp) of sequence upstream from the flaB gene starting codon did not lead to functional restoration of the nonmotileflaB null mutant. DNA hybridization, PCR analysis, and sequencing indicated that the wild-type flaB gene in nonmotile clones was present in the introduced extrachromosomal plasmids, while the motile MS17 clone was a merodiploid containing single tandem chromosomal copies of mutatedflaB::Km and wild-type flaBwith a 366-bp sequence upstream from its starting codon. Complementation was thus achieved only when wild-typeflaB was inserted into the borrelial chromosome. Several possible mechanisms for the failure of complementation for extrachromosomally located flaB are discussed.

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The Largest Open Reading Frame (pks12) in the Mycobacterium tuberculosis Genome Is Involved in Pathogenesis and Dimycocerosyl Phthiocerol Synthesis

Infection and Immunity ◽

10.1128/iai.71.7.3794-3801.2003 ◽

2003 ◽

Vol 71 (7) ◽

pp. 3794-3801 ◽

Cited By ~ 49

Author(s):

Tatiana D. Sirakova ◽

Vinod S. Dubey ◽

Hwa-Jung Kim ◽

Michael H. Cynamon ◽

Pappachan E. Kolattukudy

Keyword(s):

Fatty Acids ◽

Mycobacterium Tuberculosis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Immunoblot Analysis ◽

Open Reading Frame ◽

Pcr Analysis ◽

Reading Frame ◽

Sds Page

ABSTRACT The cell wall lipids in Mycobacterium tuberculosis are probably involved in pathogenesis. The largest open reading frame in the genome of M. tuberculosis H37Rv, pks12, is unique in that it encodes two sets of domains needed to produce fatty acids. A pks12-disrupted mutant was produced, and disruption was confirmed by both PCR analysis and Southern blotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that a 430-kDa protein band present in the wild type was missing in the mutant. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS) and liquid chromatography (LC)-MS analysis of tryptic peptides showed that 54 peptides distributed throughout this protein matched the pks12-encoded sequence. Biochemical analysis using [1-14C]propionate as the radiotracer showed that the pks12 mutant was deficient in the synthesis of dimycocerosyl phthiocerol (DIM). SDS-PAGE, immunoblot analysis of proteins, and analysis of fatty acids showed that the mutant can produce mycocerosic acids. Thus, the pks12 gene is probably involved in the synthesis of phthiocerol, the diol required for DIM synthesis. Growth of the pks12 mutant was attenuated in mouse alveolar macrophage cell line MH-S, and the virulence of the mutant in vivo was highly attenuated in a murine model. Thus, pks12 probably participates in DIM production and its expression is involved in pathogenesis.

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