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

2004 ◽  
Vol 186 (17) ◽  
pp. 5753-5761 ◽  
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.

scholarly journals Conjugative Transfer of p42a from Rhizobium etli CFN42, Which Is Required for Mobilization of the Symbiotic Plasmid, Is Regulated by Quorum Sensing

2003 ◽  
Vol 185 (5) ◽  
pp. 1681-1692 ◽  
Author(s):  
Cristina Tun-Garrido ◽  
Patricia Bustos ◽  
Víctor González ◽  
Susana Brom
Keyword(s):  
Quorum Sensing ◽  
Regulatory Gene ◽  
Homoserine Lactone ◽  
Regulatory Genes ◽  
Conjugal Transfer ◽  
Conjugative Transfer ◽  
Rhizobium Etli ◽  
Regulation Scheme ◽  
Symbiotic Plasmid ◽  
Transfer Region

ABSTRACT Rhizobium etli CFN42 contains six plasmids. Only one of them, p42a, is self-conjugative at high frequency. This plasmid is strictly required for mobilization of the symbiotic plasmid (pSym). To study the transfer mechanism of p42a, a self-transmissible cosmid clone containing its transfer region was isolated. Its sequence showed that most of the tra genes are highly similar to genes of Agrobacterium tumefaciens pTiC58 and other related plasmids. Four putative regulatory genes were identified; three of these (traI, traR, and cinR) belong to the LuxR-LuxI family. Mutagenesis of these genes confirmed their requirement for p42a transfer. We found that the conjugative transfer of p42a is dependent on quorum sensing, and consequently pSym transfer also was found to be similarly regulated, establishing a complex link between environmental conditions and pSym transfer. Although R. etli has been shown to produce different N-acyl-homoserine lactones, only one of them, a 3-oxo-C8-homoserine lactone encoded by the traI gene described here, was involved in transfer. Mutagenesis of the fourth regulatory gene, traM, had no effect on transfer. Analysis of transcriptional fusions of the regulatory genes to a reporter gene suggests a complex regulation scheme for p42a conjugative transfer. Conjugal transfer gene expression was found to be directly upregulated by TraR and the 3-oxo-C8-homoserine lactone synthesized by TraI. The traI gene was autoregulated by these elements and positively regulated by CinR, while cinR expression required traI. Finally, we did not detect expression of traM, indicating that in p42a TraM may be expressed so weakly that it cannot inhibit conjugal transfer, leading to the unrepressed transfer of p42a.

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

2003 ◽  
Vol 69 (6) ◽  
pp. 3263-3271 ◽  
Author(s):  
Woojun Park ◽  
Che Ok Jeon ◽  
Amy M. Hohnstock-Ashe ◽  
Stephen C. Winans ◽  
Gerben J. Zylstra ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Pcr Primers ◽  
Conjugal Transfer ◽  
Pcr Analysis ◽  
Cell Contact ◽  
Reading Frame ◽  
Transfer Region ◽  
Catabolic Plasmid ◽  
Insertional Inactivation ◽  
High Density Cell

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.

scholarly journals Isolation and characterization of SGE1: a yeast gene that partially suppresses the gal11 mutation in multiple copies.

Genetics ◽  
1993 ◽  
Vol 134 (3) ◽  
pp. 675-683
Author(s):  
H Amakasu ◽  
Y Suzuki ◽  
M Nishizawa ◽  
T Fukasawa
Keyword(s):  
Carbon Sources ◽  
Northern Analysis ◽  
Wild Type ◽  
Reading Frame ◽  
Recessive Mutations ◽  
Isolation And Characterization ◽  
Multiple Copies ◽  
The Right ◽  
Inducible Genes

Abstract Recessive mutations of GAL11 in Saccharomyces cerevisiae cause pleiotropic defects that include weak fermentation of galactose, alpha-specific sterility and slow growth on nonfermentable carbon sources. Recent experiments suggest that Gal11P functions as a "co-activator" that links transcriptional activators, such as Gal4p and Grf1p/Rap1p/Tuf1p, with the basic transcription machinery. In the present experiments we isolated a gene, SGE1, that suppresses gal11 for growth on ethidium bromide/galactose agar when the gene was present in two or more copies. The other gal11 phenotypes were not suppressed by SGE1 in the multiple-copy state. Multiple copies of SGE1 increased expression of galactose-inducible genes in gal11 yeast, presumably at the level of transcription. When SGE1 was disrupted in wild-type yeast, the expression of galactose-inducible genes decreased to 50-60% of the wild-type level, presumably due to effect on transcription. Complete DNA sequence analysis revealed that SGE1 encodes a predicted protein of 543 amino acids. SGE1-specific mRNA of 1.8 kilonucleotides was detected by Northern analysis along the direction of the open reading frame. The gene mapped near RAD56, at the right end of chromosome 16.

scholarly journals Transfer of the Symbiotic Plasmid of Rhizobium etli CFN42 Requires Cointegration with p42a, Which May Be Mediated by Site-Specific Recombination

2004 ◽  
Vol 186 (22) ◽  
pp. 7538-7548 ◽  
Author(s):  
Susana Brom ◽  
Lourdes Girard ◽  
Cristina Tun-Garrido ◽  
Alejandro García-de los Santos ◽  
Patricia Bustos ◽  
...  
Keyword(s):  
Attachment Site ◽  
Conjugative Transfer ◽  
Rhizobium Etli ◽  
Wild Type ◽  
Specific System ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Symbiotic Plasmid ◽  
Recombination System ◽  
Attachment Sites

ABSTRACT Plasmid p42a from Rhizobium etli CFN42 is self-transmissible and indispensable for conjugative transfer of the symbiotic plasmid (pSym). Most pSym transconjugants also inherit p42a. pSym transconjugants that lack p42a always contain recombinant pSyms, which we designated RpSyms*. RpSyms* do not contain some pSym segments and instead have p42a sequences, including the replication and transfer regions. These novel recombinant plasmids are compatible with wild-type pSym, incompatible with p42a, and self-transmissible. The symbiotic features of derivatives simultaneously containing a wild-type pSym and an RpSym* were analyzed. Structural analysis of 10 RpSyms* showed that 7 shared one of the two pSym-p42a junctions. Sequencing of this common junction revealed a 53-bp region that was 90% identical in pSym and p42a, including a 5-bp central region flanked by 9- to 11-bp inverted repeats reminiscent of bacterial and phage attachment sites. A gene encoding an integrase-like protein (intA) was localized downstream of the attachment site on p42a. Mutation or the absence of intA abolished pSym transfer from a recA mutant donor. Complementation with the wild-type intA gene restored transfer of pSym. We propose that pSym-p42a cointegration is required for pSym transfer; cointegration may be achieved either through homologous recombination among large reiterated sequences or through IntA-mediated site-specific recombination between the attachment sites. Cointegrates formed through the site-specific system but resolved through RecA-dependent recombination or vice versa generate RpSyms*. A site-specific recombination system for plasmid cointegration is a novel feature of these large plasmids and implies that there is unique regulation which affects the distribution of pSym in nature due to the role of the cointegrate in conjugative transfer.

scholarly journals Chemical Elicitors Induce Rare Bioactive Secondary Metabolites in Deep-Sea Bacteria under Laboratory Conditions

Metabolites ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 107
Author(s):  
Rafael de Felício ◽  
Patricia Ballone ◽  
Cristina Freitas Bazzano ◽  
Luiz F. G. Alves ◽  
Renata Sigrist ◽  
...  
Keyword(s):  
Natural Products ◽  
Secondary Metabolites ◽  
Natural Product ◽  
Deep Sea ◽  
Chemical Space ◽  
Bacterial Genome ◽  
Vast Number ◽  
Bacterial Metabolites ◽  
Laboratory Conditions ◽  
Deep Sea Bacteria

Bacterial genome sequencing has revealed a vast number of novel biosynthetic gene clusters (BGC) with potential to produce bioactive natural products. However, the biosynthesis of secondary metabolites by bacteria is often silenced under laboratory conditions, limiting the controlled expression of natural products. Here we describe an integrated methodology for the construction and screening of an elicited and pre-fractionated library of marine bacteria. In this pilot study, chemical elicitors were evaluated to mimic the natural environment and to induce the expression of cryptic BGCs in deep-sea bacteria. By integrating high-resolution untargeted metabolomics with cheminformatics analyses, it was possible to visualize, mine, identify and map the chemical and biological space of the elicited bacterial metabolites. The results show that elicited bacterial metabolites correspond to ~45% of the compounds produced under laboratory conditions. In addition, the elicited chemical space is novel (~70% of the elicited compounds) or concentrated in the chemical space of drugs. Fractionation of the crude extracts further evidenced minor compounds (~90% of the collection) and the detection of biological activity. This pilot work pinpoints strategies for constructing and evaluating chemically diverse bacterial natural product libraries towards the identification of novel bacterial metabolites in natural product-based drug discovery pipelines.

scholarly journals The Cloning and Molecular Analysis of pawn-B in Paramecium tetraurelia

Genetics ◽  
2000 ◽  
Vol 155 (3) ◽  
pp. 1105-1117 ◽  
Author(s):  
W John Haynes ◽  
Kit-Yin Ling ◽  
Robin R Preston ◽  
Yoshiro Saimi ◽  
Ching Kung
Keyword(s):  
Genomic Library ◽  
Open Reading Frame ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Rt Pcr ◽  
Reading Frame ◽  
Close Proximity ◽  
In The Wild ◽  
Dna Fragment ◽  
Alternative Sites

Abstract Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca2+ current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca2+ current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5′ neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.

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

2004 ◽  
Vol 72 (2) ◽  
pp. 629-636 ◽  
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.

scholarly journals CZK3, a MAP Kinase Kinase Kinase Homolog in Cercospora zeae-maydis, Regulates Cercosporin Biosynthesis, Fungal Development, and Pathogenesis

2003 ◽  
Vol 16 (9) ◽  
pp. 760-768 ◽  
Author(s):  
Won-Bo Shim ◽  
Larry D. Dunkle
Keyword(s):  
Map Kinase ◽  
Map Kinases ◽  
Gray Leaf Spot ◽  
Open Reading Frame ◽  
Wild Type ◽  
Toxic Activity ◽  
Reading Frame ◽  
Cercospora Zeae Maydis ◽  
Map Kinase Kinase Kinase ◽  
Map Kinase Kinase

The fungus Cercospora zeae-maydis causes gray leaf spot of maize and produces cercosporin, a photosensitizing perylenequinone with toxic activity against a broad spectrum of organisms. However, little is known about the biosynthetic pathway or factors that regulate cercosporin production. Analysis of a cDNA subtraction library comprised of genes that are up-regulated during cercosporin synthesis revealed a sequence highly similar to mitogen-activated protein (MAP) kinases in other fungi. Sequencing and conceptual translation of the full-length genomic sequence indicated that the gene, which we designated CZK3, contains a 4,119-bp open reading frame devoid of introns and encodes a 1,373-amino acid sequence that is highly similar to Wis4, a MAP kinase kinase kinase in Schizosaccharomyces pombe. Targeted disruption of CZK3 suppressed expression of genes predicted to participate in cercosporin biosynthesis and abolished cercosporin production. The disrupted mutants grew faster on agar media than the wild type but were deficient in conidiation and elicited only small chlorotic spots on inoculated maize leaves compared with rectangular necrotic lesions incited by the wild type. Complementation of disruptants with the CZK3 open reading frame and flanking sequences restored wild-type levels of conidiation, growth rate, and virulence as well as the ability to produce cercosporin. The results suggest that cercosporin is a virulence factor in C. zeae-maydis during maize pathogenesis, but the pleiotropic effects of CZK3 disruption precluded definitive conclusions.

scholarly journals DAF1, a mutant gene affecting size control, pheromone arrest, and cell cycle kinetics of Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (11) ◽  
pp. 4675-4684 ◽  
Author(s):  
F R Cross
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Critical Size ◽  
Size Control ◽  
Mutant Gene ◽  
G1 Phase ◽  
Wild Type ◽  
Alpha Factor ◽  
Multiple Copies ◽  
Kinetics Of

The mating pheromone alpha-factor arrests Saccharomyces cerevisiae MATa cells in the G1 phase of the cell cycle. Size control is also exerted in G1, since cells do not exit G1 until they have attained a critical size. A dominant mutation (DAF1-1) which causes both alpha-factor resistance and small cell size (volume about 0.6-fold that of the wild type) has been isolated and characterized genetically and by molecular cloning. Several alpha-factor-induced mRNAs were induced equivalently in daf1+ and DAF1-1 cells. The DAF1-1 mutation consisted of a termination codon two-thirds of the way through the daf1+ coding sequence. A chromosomal deletion of DAF1 produced by gene transplacement increased cell volume about 1.5-fold; thus, DAF1-1 may be a hyperactive or deregulated allele of a nonessential gene involved in G1 size control. Multiple copies of DAF1-1 also greatly reduced the duration of the G1 phase of the cell cycle.

The Symbiotic Plasmid of Rhizobium Etli

1999 ◽  
pp. 163-165 ◽  
Author(s):  
J. G. Dávila ◽  
P. Bustos ◽  
M. A. Cevallos ◽  
M. L. Girard ◽  
R. Palacios ◽  
...  
Keyword(s):  
Rhizobium Etli ◽  
Symbiotic Plasmid
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