scholarly journals oriC Region and Replication Termination Site, dif, of the Xanthomonas campestris pv. campestris 17 Chromosome

2002 ◽  
Vol 68 (6) ◽  
pp. 2924-2933 ◽  
Author(s):  
Ming-Ren Yen ◽  
Nien-Tsung Lin ◽  
Chih-Hsin Hung ◽  
Ka-Tim Choy ◽  
Shu-Fen Weng ◽  
...  
Keyword(s):  
Xanthomonas Campestris ◽  
Xylella Fastidiosa ◽  
Filamentous Phage ◽  
Site Specific ◽  
E Coli ◽  
Site Specific Integration ◽  
Oric Region ◽  
Dna Fragment ◽  
And Function ◽  
Xanthomonas Campestris Pv Campestris

ABSTRACT A 13-kb DNA fragment containing oriC and the flanking genes thdF, orf900, yidC, rnpA, rpmH, oriC, dnaA, dnaN, recF, and gyrB was cloned from the gram-negative plant pathogen Xanthomonas campestris pv. campestris 17. These genes are conserved in order with other eubacterial oriC genes and code for proteins that share high degrees of identity with their homologues, except for orf900, which has a homologue only in Xylella fastidiosa. The dnaA/dnaN intergenic region (273 bp) identified to be the minimal oriC region responsible for autonomous replication has 10 pure AT clusters of four to seven bases and only three consensus DnaA boxes. These findings are in disagreement with the notion that typical oriCs contain four or more DnaA boxes located upstream of the dnaA gene. The X. campestris pv. campestris 17 attB site required for site-specific integration of cloned fragments from filamentous phage φLf replicative form DNA was identified to be a dif site on the basis of similarities in nucleotide sequence and function with the Escherichia coli dif site required for chromosome dimer resolution and whose deletion causes filamentation of the cells. The oriC and dif sites were located at 12:00 and 6:00, respectively, on the circular X. campestris pv. campestris 17 chromosome map, similar to the locations found for E. coli sites. Computer searches revealed the presence of both the dif site and XerC/XerD recombinase homologues in 16 of the 42 fully sequenced eubacterial genomes, but eight of the dif sites are located far away from the 6:00 point instead of being placed opposite the cognate oriC. The differences in the relative position suggest that mechanisms different from that of E. coli may participate in the control of chromosome replication.

scholarly journals Biosynthesis of Coenzyme Q in the Phytopathogen Xanthomonas campestris via a Yeast-Like Pathway

2019 ◽  
Vol 32 (2) ◽  
pp. 217-226 ◽  
Author(s):  
Lian Zhou ◽  
Ming Li ◽  
Xing-Yu Wang ◽  
Hao Liu ◽  
Shuang Sun ◽  
...  
Keyword(s):  
Xanthomonas Campestris ◽  
Coenzyme Q ◽  
Regulatory Mechanisms ◽  
Membrane Component ◽  
E Coli ◽  
Regulatory Complex ◽  
Iron Carboxylate ◽  
Xanthomonas Campestris Pv Campestris ◽  
Specific Inhibitors ◽  
Novel Protein

Coenzyme Q (CoQ) is a lipid-soluble membrane component found in organisms ranging from bacteria to mammals. The biosynthesis of CoQ has been intensively studied in Escherichia coli, where 12 genes (ubiA, -B, -C, -D, -E, -F, -G, -H, -I, -J, -K, and -X) are involved. In this study, we first investigated the putative genes for CoQ8 biosynthesis in the phytopathogen Xanthomonas campestris pv. campestris using a combination of bioinformatic, genetic, and biochemical methods. We showed that Xc_0489 (coq7Xc) encodes a di-iron carboxylate monooxygenase filling the E. coli UbiF role for hydroxylation at C-6 of the aromatic ring. Xc_0233 (ubiJXc) encodes a novel protein with an E. coli UbiJ-like domain organization and is required for CoQ8 biosynthesis. The X. campestris pv. campestris decarboxylase gene remains unidentified. Further functional analysis showed that ubiB and ubiK homologs ubiBXc and ubiKXc are required for CoQ8 biosynthesis in X. campestris pv. campestris. Deletion of ubiJXc, ubiBXc, and ubiKXc led to the accumulation of an intermediate 3-octaprenyl-4-hydroxybenzoic acid. UbiKXc interacts with UbiJXc and UbiBXc to form a regulatory complex. Deletion analyses of these CoQ8 biosynthetic genes indicated that they are important for virulence in Chinese radish. These results suggest that the X. campestris pv. campestris CoQ8 biosynthetic reactions and regulatory mechanisms are divergent from those of E. coli. The variations provide an opportunity for the design of highly specific inhibitors for the prevention of infection by the phytopathogen X. campestris pv. campestris.

scholarly journals Functional Characterization of Replication and Stability Factors of an Incompatibility Group P-1 Plasmid from Xylella fastidiosa

2010 ◽  
Vol 76 (23) ◽  
pp. 7734-7740 ◽  
Author(s):  
Min Woo Lee ◽  
Elizabeth E. Rogers ◽  
Drake C. Stenger
Keyword(s):  
Pseudomonas Syringae ◽  
Xanthomonas Campestris ◽  
Plasmid Stability ◽  
Xylella Fastidiosa ◽  
Shuttle Vector ◽  
Functional Characterization ◽  
Replication Initiation ◽  
Incompatibility Group ◽  
Antibiotic Selection ◽  
E Coli

ABSTRACT Xylella fastidiosa strain riv11 harbors a 25-kbp plasmid (pXF-RIV11) belonging to the IncP-1 incompatibility group. Replication and stability factors of pXF-RIV11 were identified and used to construct plasmids able to replicate in X. fastidiosa and Escherichia coli. Replication in X. fastidiosa required a 1.4-kbp region from pXF-RIV11 containing a replication initiation gene (trfA) and the adjacent origin of DNA replication (oriV). Constructs containing trfA and oriV from pVEIS01, a related IncP-1 plasmid of the earthworm symbiont Verminephrobacter eiseniae, also were competent for replication in X. fastidiosa. Constructs derived from pXF-RIV11 but not pVEIS01 replicated in Agrobacterium tumefaciens, Xanthomonas campestris, and Pseudomonas syringae. Although plasmids bearing replication elements from pXF-RIV11 or pVEIS01 could be maintained in X. fastidiosa under antibiotic selection, removal of selection resulted in plasmid extinction after 3 weekly passages. Addition of a toxin-antitoxin addiction system (pemI/pemK) from pXF-RIV11 improved plasmid stability such that >80 to 90% of X. fastidiosa cells retained plasmid after 5 weekly passages in the absence of antibiotic selection. Expression of PemK in E. coli was toxic for cell growth, but toxicity was nullified by coexpression of PemI antitoxin. Deletion of N-terminal sequences of PemK containing the conserved motif RGD abolished toxicity. In vitro assays revealed a direct interaction of PemI with PemK, suggesting that antitoxin activity of PemI is mediated by toxin sequestration. IncP-1 plasmid replication and stability factors were added to an E. coli cloning vector to constitute a stable 6.0-kbp shuttle vector (pXF20-PEMIK) suitable for use in X. fastidiosa.

scholarly journals Lessons in genome engineering: opportunities, tools and pitfalls

2019 ◽  
Author(s):  
Ingrid Poernbacher ◽  
Sam Crossman ◽  
Joachim Kurth ◽  
Hisashi Nojima ◽  
Alberto Baena-Lopez ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Gene Activity ◽  
Limited Information ◽  
Loss Of Function ◽  
Site Specific ◽  
Site Specific Integration ◽  
Regulatory Mutations ◽  
The Creation ◽  
And Function ◽  
Cautionary Tales

ABSTRACTCRISPR/Cas technology allows the creation of double strand breaks and hence loss of function mutations at any location in the genome. This technology is now routine for many organisms and cell lines. Here we describe how CRISPR/Cas can be combined with other DNA manipulation techniques (e.g. homology-based repair, site-specific integration and Cre or FLP-mediated recombination) to create sophisticated tools to measure and manipulate gene activity. In one class of applications, a single site-specific insertion generates a transcriptional reporter, a loss-of function allele, and a tagged allele. In a second class of modifications, essential sequences are deleted and replaced with an integrase site, which serves as a platform for the creation of custom reporters, transcriptional drivers, conditional alleles and regulatory mutations. We describe how these tools and protocols can be implemented easily and efficiently. Importantly, we also highlight unanticipated failures, which serve as cautionary tales, and suggest mitigating measures. Our tools are designed for use in Drosophila but the lessons we draw are likely to be widely relevant.AUTHOR SUMMARYThe genome contains all the information that an organism needs to develop and function throughout its life. One of the goal of genetics is to decipher the role of all the genes (typically several thousands for an animal) present in the genome. One approach is to delete each gene and assay the consequences. Deletion of individual genes is now readily achieved with a technique called CRISPR/Cas9. However, simple genetic deletion provides limited information. Here we describe strains and DNA vectors that streamline the generation of more sophisticated genetic tools. We describe general means of creating alleles (genetic variants) that enable gene activity to be measured and experimentally modulated in space and time. Although the tools we describe are universally applicable, each gene requires special consideration. Based on our experience of successes and failures, we suggest measures to maximise the chances that engineered alleles serve their intended purpose. Although our methods are designed for use in Drosophila, they could be adapted to any organism that is amenable to CRISPR/Cas9 genome modification.

The Blight-Associated Epitope and DNA Fragment from Xanthomonas campestris pv. campestris are not Required for Blight

Plant Biology ◽  
2001 ◽  
Vol 3 (1) ◽  
pp. 106-112
Author(s):  
T. Shigaki ◽  
D. W. Gabriel ◽  
S. S. Patil ◽  
D. Borthakur ◽  
J. H. Choi ◽  
...  
Keyword(s):  
Xanthomonas Campestris ◽  
Dna Fragment ◽  
Xanthomonas Campestris Pv Campestris

scholarly journals Site-Specific Recombination in the Cyanobacterium Anabaena sp. Strain PCC 7120 Catalyzed by the Integrase of Coliphage HK022

2009 ◽  
Vol 191 (13) ◽  
pp. 4458-4464 ◽  
Author(s):  
Olga Melnikov ◽  
Arieh Zaritsky ◽  
Aliza Zarka ◽  
Sammy Boussiba ◽  
Natalia Malchin ◽  
...  
Keyword(s):  
Integration Host Factor ◽  
Specific Gene ◽  
Recombination Reaction ◽  
Site Specific ◽  
Site Specific Recombination ◽  
E Coli ◽  
Site Specific Integration ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Pcc 7120

ABSTRACT The integrase (Int) of the λ-like coliphage HK022 catalyzes the site-specific integration and excision of the phage DNA into and from the chromosome of its host, Escherichia coli. Int recognizes two different pairs of recombining sites attP × attB and attL × attR for integration and excision, respectively. This system was adapted to the cyanobacterium Anabaena sp. strain PCC 7120 as a potential tool for site-specific gene manipulations in the cyanobacterium. Two plasmids were consecutively cointroduced by conjugation into Anabaena cells, one plasmid that expresses HK022 Int recombinase and the other plasmid that carries the excision substrate PglnA -attL-T1/T2-attR-lacZ, where T1/T2 are the strong transcription terminators of rrnB, to prevent expression of the lacZ reporter under the constitutive promoter PglnA . The Int-catalyzed site-specific recombination reaction was monitored by the expression of lacZ emanating as a result of T1/T2 excision. Int catalyzed the site-specific excision reaction in Anabaena cells when its substrate was located either on the plasmid or on the chromosome with no need to supply an accessory protein, such as integration host factor and excisionase (Xis), which are indispensable for this reaction in its host, E. coli.

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