An nptI-sacB-sacR cartridge for constructing directed, unmarked mutations in Gram-negative bacteria by marker exchange-eviction mutagenesis

Ried, Jeffrey L.; Collmer, Alan

doi:10.1016/0378-1119(87)90127-2

Cited by 343 publications

(257 citation statements)

References 23 publications

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“…Gene deletion of cydAB was performed by amplifying a 1,029-bp fragment upstream of cydA using primers Cydleftfor and Cydleftrev and a 1,036-bp fragment downstream of cydB using primers Cydrightfor and Cydrightrev, followed by insertion of the respective fragments into the XbaI/BamHI and SacI/ EcoRI sites of pUC19 to generate pCyd1. The BamHI-digested npt1/sacRB cassette from pUM24Cm (Ried and Collmer, 1987) was inserted into the BamHI site between the upstream and downstream fragments in pCOX1, pCta1, and pCyd1 to generate pCOX2, pCta2, and pCyd2, respectively.…”

Section: Plasmid Constructionmentioning

confidence: 99%

Thylakoid Terminal Oxidases Are Essential for the CyanobacteriumSynechocystissp. PCC 6803 to Survive Rapidly Changing Light Intensities

et al. 2013

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Cyanobacteria perform photosynthesis and respiration in the thylakoid membrane, suggesting that the two processes are interlinked. However, the role of the respiratory electron transfer chain under natural environmental conditions has not been established. Through targeted gene disruption, mutants of Synechocystis sp. PCC 6803 were generated that lacked combinations of the three terminal oxidases: the thylakoid membrane-localized cytochrome c oxidase (COX) and quinol oxidase (Cyd) and the cytoplasmic membrane-localized alternative respiratory terminal oxidase. All strains demonstrated similar growth under continuous moderate or high light or 12-h moderate-light/dark square-wave cycles. However, under 12-h high-light/dark square-wave cycles, the COX/Cyd mutant displayed impaired growth and was completely photobleached after approximately 2 d. In contrast, use of sinusoidal light/dark cycles to simulate natural diurnal conditions resulted in little photobleaching, although growth was slower. Under high-light/dark square-wave cycles, the COX/Cyd mutant suffered a significant loss of photosynthetic efficiency during dark periods, a greater level of oxidative stress, and reduced glycogen degradation compared with the wild type. The mutant was susceptible to photoinhibition under pulsing but not constant light. These findings confirm a role for thylakoid-localized terminal oxidases in efficient dark respiration, reduction of oxidative stress, and accommodation of sudden light changes, demonstrating the strong selective pressure to maintain linked photosynthetic and respiratory electron chains within the thylakoid membrane. To our knowledge, this study is the first to report a phenotypic difference in growth between terminal oxidase mutants and wild-type cells and highlights the need to examine mutant phenotypes under a range of conditions.

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Section: Plasmid Constructionmentioning

confidence: 99%

Thylakoid Terminal Oxidases Are Essential for the CyanobacteriumSynechocystissp. PCC 6803 to Survive Rapidly Changing Light Intensities

et al. 2013

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“…To analyze the effect of virE1 (coding for the VirE2 chaperone protein) on translocation of VirE2, we constructed a precise in frame deletion of virE1 by using the marker exchange-eviction mutagenesis method as described by Ried and Collmer (1987). This resulted in a double crossover event at the virE operon of the A. tumefaciens Ti-plasmid of both wild-type A. tumefaciens (LBA1010; Table I) and its disarmed derivative LBA1100.…”

Section: Construction Of An a Tumefaciens Vire1 Deletion Strainmentioning

confidence: 99%

“…tumefaciens strain LBA1100 (C58C1 with a disarmed octopine-type pTiB6 plasmid [Beijersbergen et al, 1992]) was used for protein transport experiments to plant and yeast cells. A precise virE1 deletion mutant was constructed from LBA1100 using the marker exchange-eviction mutagenesis method as described by Ried and Collmer (1987). To this end, two primers were designed to amplify a 5Ј fragment of virE2 introducing an NdeI site (underlined) at the ATG start codon.…”

Section: Bacterial Strainsmentioning

confidence: 99%

“…A BglII/ SacI fragment, containing about 400 bp of flanking DNA sequences of virE1 on either side was cloned in pSDM3005 (pSDM3600). Plasmid pSDM3005 was the result of cloning a PstI fragment from pUM24 (Ried and Collmer, 1987) into the PstI site of pBGS19 (Spratt et al, 1986). pSDM3005 contains the Bacillus subtilis sacR/B gene and a kanamycin resistance marker and does not replicate in A. tumefaciens to allow easy selection for double crossover events in A. tumefaciens.…”

Section: Bacterial Strainsmentioning

confidence: 99%

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Recognition of the Agrobacterium tumefaciens VirE2 Translocation Signal by the VirB/D4 Transport System Does Not Require VirE1

et al. 2003

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Agrobacterium tumefaciens uses a type IV secretion system to deliver a nucleoprotein complex and effector proteins directly into plant cells. The single-stranded DNA-binding protein VirE2, the F-box protein VirF and VirE3 are delivered into host cells via this VirB/D4 encoded translocation system. VirE1 functions as a chaperone of VirE2 by regulating its efficient translation and preventing VirE2-VirE2 aggregation in the bacterial cell. We analyzed whether the VirE1 chaperone is also essential for transport recognition of VirE2 by the VirB/D4 encoded type IV secretion system. In addition, we assayed whether translocation of VirF and VirE3, which also forms part of the virE operon, is affected by the absence of VirE1. We employed the earlier developed CRAFT (Cre recombinase Reporter Assay For Translocation) assay to detect transfer of Cre::Vir fusion proteins from A. tumefaciens into plants, monitored by stable reconstitution of a kanamycin resistance marker, and into yeast, screened by loss of the URA3 gene. We show that the C-terminal 50 amino acids of VirE2 and VirE3 are sufficient to mediate Cre translocation into host cells, confirming earlier indications of a C-terminal transport signal. This transfer was independent of the presence or absence of VirE1. Besides, the translocation efficiency of VirF is not altered in a virE1 mutant. The results unambiguously show that the VirE1 chaperone is not essential for the recognition of the VirE2 transport signal by the transport system and the subsequent translocation across the bacterial envelope into host cells.Agrobacterium tumefaciens causes crown gall disease on a wide range of plants by genetic transformation of host cells with a piece of its oncogenic plasmidborne transfer (T)-DNA (for most recent review, see Gelvin, 2003). The expression of the genes located on the T-DNA, which contain plant transcription and translation signals, results in overproduction of the plant hormones auxin and cytokinin and hence increased cell division and the tumor phenotype. A set of accessory virulence or effector proteins (encoded by the vir region on the tumor inducing [Ti] plasmid) is also transported into the transformed cells to confer full virulence. The host range is not confined to the plant kingdom, because A. tumefaciens can also transform yeast (Bundock et al., 1995), fungi (de Groot et al., 1998), and mammalian cells (Kunik et al., 2000).The translocated proteins of A. tumefaciens described to date include VirD2, VirE2, VirE3, and VirF. In the bacterium, VirD2 introduces a nick at the border sequences surrounding the T-DNA and by replacement synthesis a single-stranded (ss) DNA copy of the bottom strand is released. VirD2 acts as a pilot protein as it remains covalently attached to the 5Ј end of the T-strand during transport of the T-DNA into the host cell nucleus (Ward and Barnes, 1988). The ssDNA-binding protein VirE2 binds cooperatively to the T-strand and thereby protects it from degradation in the host cell (Rossi et al., 1996). Both VirD2 and VirE2 contain nucl...

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“…Selection with sacB, which encodes the secretory levansucrase from Bacillus subtilis (Ried and Collmer, 1987), has been successfully used in cyanobacteria (Andersson et al, 2000;Cai and Wolk, 1990). First, a copy of sacB was inserted onto cosmid 2F10, in which the toxin gene in the sepA1T1 operon is partially deleted and the antitoxin gene is intact (Fig.…”

Section: Both Toxin-antitoxin Cassettes Are Necessary For Exclusion-tmentioning

confidence: 99%

The complete sequence and functional analysis of pANL, the large plasmid of the unicellular freshwater cyanobacterium Synechococcus elongatus PCC 7942

Chen

Holtman

Magnuson

et al. 2008

Plasmid

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Two endogenous plasmids are present in Synechococcus elongatus PCC 7942, a model organism for studying photosynthesis and circadian rhythms in cyanobacteria. The large plasmid, pANL, was shown previously to be involved in adaptation of S. elongatus cells to sulfur starvation, which provided the first evidence of cellular function of a cyanobacterial plasmid. Here, we report the complete sequence of pANL, which is 46,366 bp in length with 53% GC content and encodes 58 putative ORFs. The pANL plasmid can be divided into four structural and functional regions: the replication origin region, a signal transduction region, a plasmid maintenance region, and a sulfurregulated region. Cosmid-based deletion analysis suggested that the plasmid maintenance and replication origin regions are required for persistence of pANL in the cells. Transposon-mediated mutagenesis and complementation-based pANL segregation assays confirmed that two predicted toxin-antitoxin cassettes encoded in the plasmid maintenance region, belonging to PemK and VapC families, respectively, are necessary for plasmid exclusion. The compact and efficient organization of sulfur-related genes on pANL may provide selective advantages in environments with limited sulfur.

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An nptI-sacB-sacR cartridge for constructing directed, unmarked mutations in Gram-negative bacteria by marker exchange-eviction mutagenesis

Cited by 343 publications

References 23 publications

Thylakoid Terminal Oxidases Are Essential for the CyanobacteriumSynechocystissp. PCC 6803 to Survive Rapidly Changing Light Intensities

Thylakoid Terminal Oxidases Are Essential for the CyanobacteriumSynechocystissp. PCC 6803 to Survive Rapidly Changing Light Intensities

Recognition of the Agrobacterium tumefaciens VirE2 Translocation Signal by the VirB/D4 Transport System Does Not Require VirE1

The complete sequence and functional analysis of pANL, the large plasmid of the unicellular freshwater cyanobacterium Synechococcus elongatus PCC 7942

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