Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria

Herrero, Marta; Lorenzo, Vı́ctor de; Timmis, Kenneth N.

doi:10.1128/jb.172.11.6557-6567.1990

Cited by 1,454 publications

(806 citation statements)

References 44 publications

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“…To screen for novel transcriptional regulators of the phlACBD operon, the 2P24 strain of P. fluorescens was subjected to random Tn5 insertional mutagenesis using plasmid pUT-Km (Herrero et al, 1990). Four out of about 4000 kanamycin-resistant colonies of this 2P24 strain showed increased red pigment production, which is a characteristic phenotype known to be linked with 2,4-DAPG production (Bangera & Thomashow, 1999;Raaijmakers et al, 1997).…”

Section: Methodsmentioning

confidence: 99%

The resistance-nodulation-division efflux pump EmhABC influences the production of 2,4-diacetylphloroglucinol in Pseudomonas fluorescens 2P24

Tian

Duan

et al. 2010

Microbiology

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The polyketide metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) plays a major role in the biological control of soil-borne plant diseases by Pseudomonas fluorescens 2P24. Two mutants (PM810 and PM820) with increased extracellular accumulation of 2,4-DAPG were isolated using transposon mutagenesis. The disrupted genes in these two mutants shared .80 % identity with the genes of the EmhR-EmhABC resistance-nodulation-division (RND) efflux system of P. fluorescens cLP6a. The deletion of emhA (PM802), emhB (PM803) or emhC (PM804) genes in strain 2P24 increased the extracellular accumulation of 2,4-DAPG, whereas the deletion of the emhR (PM801) gene decreased the biosynthesis of 2,4-DAPG. The promoter assay confirmed the elevated transcription of emhABC in the EmhR disrupted strain (PM801) and an indirect negative regulation of 2,4-DAPG biosynthetic locus transcription by the EmhABC efflux pump. Induction by exogenous 2,4-DAPG led to remarkable differences in transcription of chromosomeborne phlA : : lacZ fusion in PM901 and PM811 (emhA " ) strains. Additionally, the EmhABC system in strain 2P24 was involved in the resistance to a group of toxic compounds, including ampicillin, chloramphenicol, tetracycline, ethidium bromide and crystal violet. In conclusion, our results suggest that the EmhABC system is an important element that influences the production of antibiotic 2,4-DAPG and enhances resistance to toxic compounds in P. fluorescens 2P24.

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Section: Methodsmentioning

confidence: 99%

The resistance-nodulation-division efflux pump EmhABC influences the production of 2,4-diacetylphloroglucinol in Pseudomonas fluorescens 2P24

Tian

Duan

et al. 2010

Microbiology

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“…Bacteria were electrotransformed as described by Sharma & Schimke (1996). E. coli strains DH5a (Invitrogen) and CC118lpir (Herrero et al, 1990) were used for DNA cloning procedures; BL21(DE3) (Studier & Moffatt, 1986) was used for the overexpression of P. putida Fis by a previously published protocol (Teras et al, 2009).…”

Section: Methodsmentioning

confidence: 99%

Fis regulates the competitiveness of Pseudomonas putida on barley roots by inducing biofilm formation

et al. 2012

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An important link between the environment and the physiological state of bacteria is the regulation of the transcription of a large number of genes by global transcription factors. One of the global regulators, Fis (factor for inversion stimulation), is well studied in Escherichia coli, but the role of this protein in pseudomonads has only been examined briefly. According to studies in Enterobacteriaceae, Fis regulates positively the flagellar movement of bacteria. In pseudomonads, flagellar movement is an important trait for the colonization of plant roots. Therefore we were interested in the role of the Fis protein in Pseudomonas putida, especially the possible regulation of the colonization of plant roots. We observed that Fis reduced the migration of P. putida onto the apices of barley roots and thereby the competitiveness of bacteria on the roots. Moreover, we observed that overexpression of Fis drastically reduced swimming motility and facilitated P. putida biofilm formation, which could be the reason for the decreased migration of bacteria onto the root apices. It is possible that the elevated expression of Fis is important in the adaptation of P. putida during colonization of plant roots by promoting biofilm formation when the migration of bacteria is no longer favoured.

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“…The first pair amplifies a 930-bp fragment comprising the upstream region and first 11 codons of ddcA and introduces a BamHI site (underlined); the second pair amplifies a 660-bp fragment corresponding to the 3Ј end and downstream region of ddcA, also introducing a BamHI site. The resulting fragments were digested with EcoRV/BamHI and BamHI/SalI, respectively, and cloned into pUC18Not (22), giving rise to plasmids pME51 and pME52. The BamHI/SalI fragment of pME52 was introduced in pME51 to give pME512.…”

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confidence: 99%

Cell Density-Dependent Gene Contributes to Efficient Seed Colonization by Pseudomonas putida KT2440

Espinosa‐Urgel

Ramos

2004

Appl Environ Microbiol

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We have characterized the expression pattern of a gene, ddcA, involved in initial colonization of corn seeds by Pseudomonas putida KT2440. The ddcA gene codes for a putative membrane polypeptide belonging to a family of conserved proteins of unknown function. Members of this family are widespread among prokaryotes and include the products of a Salmonella enterica serovar Typhimurium gene expressed during invasion of macrophages and psiE, an Escherichia coli phosphate starvation-inducible gene. Although its specific role is undetermined, the presence of ddcA in multicopy restored the seed adhesion capacity of a KT2440 ddcA mutant. Expression of ddcA is growth phase regulated, being maximal at the beginning of stationary phase. It is independent of RpoS, nutrient depletion, or phosphate starvation, and it is not the result of changes in the medium pH during growth. Expression of ddcA is directly dependent on cell density, being also stimulated by the addition of conditioned medium and of seed exudates. This is the first evidence suggesting the existence of a quorum-sensing system in P. putida KT2440. The potential implication of such a signaling process in seed adhesion and colonization by the bacterium is discussed.

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Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria

Cited by 1,454 publications

References 44 publications

The resistance-nodulation-division efflux pump EmhABC influences the production of 2,4-diacetylphloroglucinol in Pseudomonas fluorescens 2P24

The resistance-nodulation-division efflux pump EmhABC influences the production of 2,4-diacetylphloroglucinol in Pseudomonas fluorescens 2P24

Fis regulates the competitiveness of Pseudomonas putida on barley roots by inducing biofilm formation

Cell Density-Dependent Gene Contributes to Efficient Seed Colonization by Pseudomonas putida KT2440

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