Homology between the genes of octopine catabolism of Rhizobium meliloti A3 and corresponding genes from the Ti plasmid

1993 ◽  
Vol 39 (11) ◽  
pp. 1041-1050 ◽  
Author(s):  
Janique Bergeron ◽  
Carole Beaulieu ◽  
Roger C. Levesque ◽  
Adam Kondorosi ◽  
Patrice Dion
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Type Strain ◽  
Wild Type Strain ◽  
Genomic Library ◽  
Rhizobium Meliloti ◽  
Ti Plasmid ◽  
Wild Type ◽  
Carbon And Nitrogen ◽  
Plasmid Genes ◽  
Ornithine Cyclodeaminase

The ability to catabolize crown-gall opines is found in Agrobacterium tumefaciens and various types of nonagrobacteria. Among the 75 Rhizobium meliloti strains tested in this work, 6 utilized the opines octopine and octopinic acid as the sole carbon and nitrogen source. From a genomic library of one of these six strains, R. meliloti A3, a clone conferring the octopine catabolism (Occ) phenotype was identified and named pJMA. A different Occ clone, which had been obtained from R. meliloti Rm41, did not hybridize with clone pJMA from strain A3. However, some fragments of clone pJMA hybridized to a 20-kilobase KpnI fragment containing the Ti plasmid genes of octopine catabolism (occ genes) from A. tumefaciens 15955. Shorter probes carrying the octopine permease genes or part of the octopine oxidase and ornithine cyclodeaminase genes from A. tumefaciens also hybridized with pJMA. The R. meliloti DNA carried by pJMA was localized to a megaplasmid of the wild-type strain A3. Thus, it appears possible that genes represented on the Occ clone from strain A3 share a common origin with the corresponding genes from the Ti plasmid.Key words: Rhizobium meliloti, Agrobacterium tumefaciens, octopine catabolism.

scholarly journals Cloning, Sequencing, and Characterization of the SdeAB Multidrug Efflux Pump of Serratia marcescens

2005 ◽  
Vol 49 (4) ◽  
pp. 1495-1501 ◽  
Author(s):  
Ayush Kumar ◽  
Elizabeth A. Worobec
Keyword(s):  
Serratia Marcescens ◽  
Type Strain ◽  
Wild Type Strain ◽  
Genomic Library ◽  
Efflux Pump ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Diverse Range ◽  
Mutant Strains ◽  
Encoding Genes

ABSTRACT Serratia marcescens is an important nosocomial agent known for causing various infections in immunocompromised individuals. Resistance of this organism to a broad spectrum of antibiotics makes the treatment of infections very difficult. This study was undertaken to identify multidrug resistance efflux pumps in S. marcescens. Three mutant strains of S. marcescens were isolated in vitro by the serial passaging of a wild-type strain in culture medium supplemented with ciprofloxacin, norfloxacin, or ofloxacin. Fluoroquinolone accumulation assays were performed to detect the presence of a proton gradient-dependent efflux mechanism. Two of the mutant strains were found to be effluxing norfloxacin, ciprofloxacin, and ofloxacin, while the third was found to efflux only ofloxacin. A genomic library of S. marcescens wild-type strain UOC-67 was constructed and screened for RND pump-encoding genes by using DNA probes for two putative RND pump-encoding genes. Two different loci were identified: sdeAB, encoding an MFP and an RND pump, and sdeCDE, encoding an MFP and two different RND pumps. Northern blot analysis revealed overexpression of sdeB in two mutant strains effluxing fluoroquinolones. Analysis of the sdeAB and sdeCDE loci in Escherichia coli strain AG102MB, deficient in the RND pump (AcrB), revealed that gene products of sdeAB are responsible for the efflux of a diverse range of substrates that includes ciprofloxacin, norfloxacin, ofloxacin, chloramphenicol, sodium dodecyl sulfate, ethidium bromide, and n-hexane, while those of sdeCDE did not result in any change in susceptibilities to any of these agents.

scholarly journals The Stringent Response Is Required for Amino Acid and Nitrate Utilization, Nod Factor Regulation, Nodulation, and Nitrogen Fixation in Rhizobium etli

2005 ◽  
Vol 187 (15) ◽  
pp. 5075-5083 ◽  
Author(s):  
Arturo Calderón-Flores ◽  
Gisela Du Pont ◽  
Alejandro Huerta-Saquero ◽  
Horacio Merchant-Larios ◽  
Luis Servín-González ◽  
...  
Keyword(s):  
Amino Acids ◽  
Type Strain ◽  
Wild Type Strain ◽  
Stringent Response ◽  
Insertion Mutant ◽  
Rhizobium Etli ◽  
Wild Type ◽  
Nod Factor ◽  
Truncated Protein ◽  
Carbon And Nitrogen

ABSTRACT A Rhizobium etli Tn5 insertion mutant, LM01, was selected for its inability to use glutamine as the sole carbon and nitrogen source. The Tn5 insertion in LM01 was localized to the rsh gene, which encodes a member of the RelA/SpoT family of proteins. The LM01 mutant was affected in the ability to use amino acids and nitrate as nitrogen sources and was unable to accumulate (p)ppGpp when grown under carbon and nitrogen starvation, as opposed to the wild-type strain, which accumulated (p)ppGpp under these conditions. The R. etli rsh gene was found to restore (p)ppGpp accumulation to a ΔrelA ΔspoT mutant of Escherichia coli. The R. etli Rsh protein consists of 744 amino acids, and the Tn5 insertion in LM01 results in the synthesis of a truncated protein of 329 amino acids; complementation experiments indicate that this truncated protein is still capable of (p)ppGpp hydrolysis. A second rsh mutant of R. etli, strain AC1, was constructed by inserting an Ω element at the beginning of the rsh gene, resulting in a null allele. Both AC1 and LM01 were affected in Nod factor production, which was constitutive in both strains, and in nodulation; nodules produced by the rsh mutants in Phaseolus vulgaris were smaller than those produced by the wild-type strain and did not fix nitrogen. In addition, electron microscopy revealed that the mutant bacteroids lacked poly-β-hydroxybutyrate granules. These results indicate a central role for the stringent response in symbiosis.

scholarly journals Pigment and Virulence Deficiencies Associated with Mutations in the aroE Gene of Xanthomonas oryzaepv. oryzae

2001 ◽  
Vol 67 (1) ◽  
pp. 245-250 ◽  
Author(s):  
Ajay Kumar Goel ◽  
Lakshmi Rajagopal ◽  
R. V. Sonti
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Genomic Library ◽  
Bacterial Species ◽  
Aromatic Amino Acids ◽  
Dna Helicase ◽  
Single Mutation ◽  
Wild Type ◽  
Shikimate Dehydrogenase ◽  
In The Wild

ABSTRACT Xanthomonadins are yellow, membrane-bound pigments produced by members of the genus Xanthomonas. We identified an ethyl methanesulfonate-induced Xanthomonas oryzae pv. oryzae mutant (BXO65) that is deficient for xanthomonadin production and virulence on rice, as well as auxotrophic for aromatic amino acids (Pig− Vir− Aro−). Reversion analysis indicated that these multiple phenotypes are due to a single mutation. A genomic library of the wild-type strain was used to isolate a 7.0-kb clone that complements BXO65. By transposon mutagenesis, marker exchange, sequence analysis, and subcloning, the complementing activity was localized to a 849-bp open reading frame (ORF). This ORF is homologous to the aroE gene, which encodes shikimate dehydrogenase in various bacterial species. Shikimate dehydrogenase activity was present in the wild-type strain and the mutant with the complementing clone, whereas no activity was found in BXO65. This clone also complemented an Escherichia coli aroE mutant for prototrophy, indicating that aroE is functionally conserved in X. oryzae pv. oryzae and E. coli. The nucleotide sequence of the 2.9-kb region containing aroErevealed that a putative DNA helicase gene is located adjacent toaroE. Our results indicate that aroE is required for normal levels of virulence and xanthomonadin production inX. oryzae pv. oryzae.

scholarly journals Functional Analysis of Fructosyl-Amino Acid Oxidases of Aspergillus oryzae

2004 ◽  
Vol 70 (10) ◽  
pp. 5882-5890 ◽  
Author(s):  
Shin-ichi Akazawa ◽  
Tetsuya Karino ◽  
Nobuyuki Yoshida ◽  
Tohoru Katsuragi ◽  
Yoshiki Tani
Keyword(s):  
Amino Acid ◽  
Aspergillus Oryzae ◽  
Type Strain ◽  
Wild Type Strain ◽  
Nitrogen Sources ◽  
Amino Acid Sequences ◽  
Wild Type ◽  
Amino Acid Oxidase ◽  
Carbon And Nitrogen Sources ◽  
Carbon And Nitrogen

ABSTRACT Three active fractions of fructosyl-amino acid oxidase (FAOD-Ao1, -Ao2a, and -Ao2b) were isolated from Aspergillus oryzae strain RIB40. N-terminal and internal amino acid sequences of FAOD-Ao2a corresponded to those of FAOD-Ao2b, suggesting that these two isozymes were derived from the same protein. FAOD-Ao1 and -Ao2 were different in substrate specificity and subunit assembly; FAOD-Ao2 was active toward N ε-fructosyl N α-Z-lysine and fructosyl valine (Fru-Val), whereas FAOD-Ao1 was not active toward Fru-Val. The genes encoding the FAOD isozymes (i.e., FAOAo1 and FAOAo2) were cloned by PCR with an FAOD-specific primer set. The deduced amino acid sequences revealed that FAOD-Ao1 was 50% identical to FAOD-Ao2, and each isozyme had a peroxisome-targeting signal-1, indicating their localization in peroxisomes. The genes was expressed in Escherichia coli and rFaoAo2 showed the same characteristics as FAOD-Ao2, whereas rFaoAo1 was not active. FAOAo2 disruptant was obtained by using ptrA as a selective marker. Wild-type strain grew on the medium containing Fru-Val as the sole carbon and nitrogen sources, but strain ΔfaoAo2 did not grow. Addition of glucose or (NH4)2SO4 to the Fru-Val medium did not affect the assimilation of Fru-Val by wild-type, indicating glucose and ammonium repressions did not occur in the expression of the FAOAo2 gene. Furthermore, conidia of the wild-type strain did not germinate on the medium containing Fru-Val and NaNO2 as the sole carbon and nitrogen sources, respectively, suggesting that Fru-Val may also repress gene expression of nitrite reductase. These results indicated that FAOD is needed for utilization of fructosyl-amino acids as nitrogen sources in A. oryzae.

scholarly journals Enhanced Nitrogen Fixation in a glgX-Deficient Strain of Cyanothece sp. Strain ATCC 51142, a Unicellular Nitrogen-Fixing Cyanobacterium

2019 ◽  
Vol 85 (7) ◽  
Author(s):  
Michelle Liberton ◽  
Anindita Bandyopadhyay ◽  
Himadri B. Pakrasi
Keyword(s):  
Nitrogen Fixation ◽  
Type Strain ◽  
Wild Type Strain ◽  
Light Period ◽  
Strain Atcc ◽  
Nitrogen Fixing ◽  
Wild Type ◽  
Modification System ◽  
Carbon And Nitrogen ◽  
Global Carbon

ABSTRACT Cyanobacteria are oxygenic photosynthetic prokaryotes with important roles in the global carbon and nitrogen cycles. Unicellular nitrogen-fixing cyanobacteria are known to be ubiquitous, contributing to the nitrogen budget in diverse ecosystems. In the unicellular cyanobacterium Cyanothece sp. strain ATCC 51142, carbon assimilation and carbohydrate storage are crucial processes that occur as part of a robust diurnal cycle of photosynthesis and nitrogen fixation. During the light period, cells accumulate fixed carbon in glycogen granules to use as stored energy to power nitrogen fixation in the dark. These processes have not been thoroughly investigated, due to the lack of a genetic modification system in this organism. In bacterial glycogen metabolism, the glgX gene encodes a debranching enzyme that functions in storage polysaccharide catabolism. To probe the consequences of modifying the cycle of glycogen accumulation and subsequent mobilization, we engineered a strain of Cyanothece 51142 in which the glgX gene was genetically disrupted. We found that the ΔglgX strain exhibited a higher growth rate than the wild-type strain and displayed a higher rate of nitrogen fixation. Glycogen accumulated to higher levels at the end of the light period in the ΔglgX strain, compared to the wild-type strain. These data suggest that the larger glycogen pool maintained by the ΔglgX mutant is able to fuel greater growth and nitrogen fixation ability. IMPORTANCE Cyanobacteria are oxygenic photosynthetic bacteria that are found in a wide variety of ecological environments, where they are important contributors to global carbon and nitrogen cycles. Genetic manipulation systems have been developed in a number of cyanobacterial strains, allowing both the interruption of endogenous genes and the introduction of new genes and entire pathways. However, unicellular diazotrophic cyanobacteria have been generally recalcitrant to genetic transformation. These cyanobacteria are becoming important model systems to study diurnally regulated processes. Strains of the Cyanothece genus have been characterized as displaying robust growth and high rates of nitrogen fixation. The significance of our study is in the establishment of a genetic modification system in a unicellular diazotrophic cyanobacterium, the demonstration of the interruption of the glgX gene in Cyanothece sp. strain ATCC 51142, and the characterization of the increased nitrogen-fixing ability of this strain.

Disruption of MRG19 results in altered nitrogen metabolic status and defective pseudohyphal development in Saccharomyces cerevisiae

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 91-98 ◽  
Author(s):  
Maitreyi Das ◽  
Paike Jayadeva Bhat
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nitrogen Metabolism ◽  
Type Strain ◽  
Wild Type Strain ◽  
Glucose Repression ◽  
Metabolic Status ◽  
Wild Type ◽  
Developmental Pathway ◽  
Carbon And Nitrogen ◽  
Carbon And Nitrogen Metabolism

It was previously shown that MRG19 downregulates carbon metabolism in Saccharomyces cerevisiae upon glucose exhaustion, and that the gene is glucose repressed. Here, it is shown that glucose repression of MRG19 is overcome upon nitrogen withdrawal, suggesting that MRG19 is a regulator of carbon and nitrogen metabolism. β-Galactosidase activity fostered by the promoter of GDH1/3, which encode anabolic enzymes of nitrogen metabolism, was altered in an MRG19 disruptant. As compared to the wild-type strain, the MRG19 disruptant showed a decrease in the ratio of 2-oxoglutarate to glutamate under nitrogen-limited conditions. MRG19 disruptants showed reduced pseudohyphal formation and enhanced sporulation, a phenomenon that occurs under conditions of both nitrogen and carbon withdrawal. These studies revealed that MRG19 regulates carbon and nitrogen metabolism, as well as morphogenetic changes, suggesting that MRG19 is a component of the link between the metabolic status of the cell and the corresponding developmental pathway.

scholarly journals Dual Control of Quorum Sensing by Two TraM-Type Antiactivators in Agrobacterium tumefaciens Octopine Strain A6

2006 ◽  
Vol 188 (7) ◽  
pp. 2435-2445 ◽  
Author(s):  
Chao Wang ◽  
Hai-Bao Zhang ◽  
Guozhou Chen ◽  
Lingling Chen ◽  
Lian-Hui Zhang
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Quorum Sensing ◽  
Type Strain ◽  
Specific Binding ◽  
Ti Plasmid ◽  
Single Amino Acid ◽  
Spontaneous Mutant ◽  
Gene Promoters ◽  
Dual Control ◽  
Wild Type

ABSTRACT Agrobacterium tumefaciens wild-type strains have a unique quorum-sensing (QS)-dependent Ti plasmid conjugative transfer phenotype in which QS signaling is activated by corresponding conjugative opine inducers. Strain K588, with a nopaline-type chromosomal background harboring an octopine-type Ti plasmid, however, is a spontaneous mutant displaying a constitutive phenotype in QS. In this study, we show that a single amino acid mutation (L54P) in the QS antiactivator TraM encoded by the traM gene of Ti plasmid is responsible for the constitutive phenotype of strain K588. Introduction of the L54P point mutation to the TraM of wild-type strain A6 by allelic replacement, however, failed to generate the expected constitutive phenotype in this octopine-type strain. Intriguingly, the QS-constitutive phenotype appeared when the pTiA6 carrying the mutated traM was placed in the chromosomal background of the nopaline-type strain C58C1RS, suggesting an unknown inhibitory factor(s) encoded by the chromosomal background of strain A6 but not by C58C1RS. Low-stringency Southern blotting analysis showed that strain A6, but not strain C58 and its derivatives, contains a second traM homologue. The homologue, designated traM2, has 64% and 65% identities with traM at the DNA and peptide levels, respectively. Similar to TraM, TraM2 is a potent antiactivator that functions by blocking TraR, the QS activator, from specific binding to the tra gene promoters. Deletion of traM2 in strain A6 harboring the mutated traM confers a constitutive QS phenotype. The results demonstrate that the QS system in strain A6 is subjected to the dual control of TraM and TraM2.

Identification and analysis of a siderophore biosynthetic gene cluster from Agrobacterium tumefaciens C58

Microbiology ◽  
2004 ◽  
Vol 150 (11) ◽  
pp. 3857-3866 ◽  
Author(s):  
Michelle R. Rondon ◽  
Katie S. Ballering ◽  
Michael G. Thomas
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Gene Cluster ◽  
Type Strain ◽  
Wild Type Strain ◽  
Biosynthetic Gene Cluster ◽  
Filamentous Cyanobacterium ◽  
Wild Type ◽  
Reactive Material ◽  
Iron Deficient ◽  
Mutant Strains

Using the complete genome sequence from Agrobacterium tumefaciens C58, the authors identified a secondary metabolite gene cluster that encodes the biosynthesis of a metabolite with siderophore activity. Support for this conclusion came from genetic and regulatory analysis of the gene cluster, along with the purification of a metabolite from A. tumefaciens C58 with iron-chelating activity. Genetic analysis of mutant strains disrupted in this gene cluster showed that these strains grew more slowly than the wild-type strain in medium lacking iron. Additionally, the mutant strains failed to produce a chrome-azurol-S-reactive material in liquid or solid medium, and failed to produce the metabolite with iron-chelating characteristics that was identified in the wild-type strain. Addition of this purified metabolite to the growth medium of a mutant strain restored its ability to grow in iron-deficient medium. Furthermore, expression of this gene cluster was induced by growth under iron-limiting conditions, suggesting that expression of this gene cluster occurs when iron is scarce. These data are all consistent with the proposal that the proteins encoded by this gene cluster are involved in the production of a siderophore. Interestingly, these proteins show the highest level of amino acid similarity to proteins from a gene cluster found in the filamentous cyanobacterium Nostoc sp. PCC7120, rather than to known siderophore biosynthetic enzymes. Given these properties, it is proposed that the siderophore produced by A. tumefaciens C58 will have a unique chemical structure. Production of the siderophore was not required for virulence of A. tumefaciens when tested with a standard stem inoculation assay.

scholarly journals An Oxidoreductase Is Involved in Cercosporin Degradation by the Bacterium Xanthomonas campestris pv. zinniae

2006 ◽  
Vol 72 (9) ◽  
pp. 6070-6078 ◽  
Author(s):  
Tanya V. Taylor ◽  
Thomas K. Mitchell ◽  
Margaret E. Daub
Keyword(s):  
Type Strain ◽  
Xanthomonas Campestris ◽  
Wild Type Strain ◽  
Genomic Library ◽  
Transcriptional Regulator ◽  
Pcr Analysis ◽  
Breakdown Product ◽  
Wild Type ◽  
Rt Pcr ◽  
Degrading Bacteria

ABSTRACT The polyketide toxin cercosporin plays a key role in pathogenesis by fungal species of the genus Cercospora. The bacterium Xanthomonas campestris pv. zinniae is able to rapidly degrade this toxin. Growth of X. campestris pv. zinniae strains in cercosporin-containing medium leads to the breakdown of cercosporin and to the formation of xanosporic acid, a nontoxic breakdown product. Five non-cercosporin-degrading mutants of a strain that rapidly degrades cercosporin (XCZ-3) were generated by ethyl methanesulfonate mutagenesis and were then transformed with a genomic library from the wild-type strain. All five mutants were complemented with the same genomic clone, which encoded a putative transcriptional regulator and an oxidoreductase. Simultaneous expression of these two genes was necessary to complement the mutant phenotype. Sequence analysis of the mutants showed that all five mutants had point mutations in the oxidoreductase gene and no mutations in the regulator. Quantitative reverse transcription-PCR (RT-PCR) showed that the expression of both of these genes in the wild-type strain is upregulated after exposure to cercosporin. Both the oxidoreductase and transcriptional regulator genes were transformed into three non-cercosporin-degrading bacteria to determine if they are sufficient for cercosporin degradation. Quantitative RT-PCR analysis confirmed that the oxidoreductase was expressed in all transconjugants. However, none of the transconjugants were able to degrade cercosporin, suggesting that additional factors are required for cercosporin degradation. Further study of cercosporin degradation in X. campestris pv. zinniae may allow for the engineering of Cercospora-resistant plants by using a suite of genes.

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