A tunable anthranilate-inducible gene expression system for Pseudomonas species

Abstract

Pseudomonads are among the most common bacteria in soils, limnic ecosystems, and human, animal, or plant host environments, including intensively studied species such as Pseudomonas aeruginosa, P. putida, or P. fluorescens. Various gene expression systems are established for some species, but there is still a need for a simple system that is suitable for a wide range of pseudomonads and that can be used for physiological applications, i.e., with a tuning capacity at lower expression levels. Here, we report the establishment of the anthranilate-dependent PantA promoter for tunable gene expression in pseudomonads. During studies on P. fluorescens, we constructed an anthranilate-inducible AntR/PantA-based expression system, named pUCP20-ANT, and used GFP as reporter to analyze gene expression. This system was compared with the rhamnose-inducible RhaSR/PrhaB-based expression system in an otherwise identical vector background. While the rhamnose-inducible system did not respond to lower inducer concentrations and always reached high levels over time when induced, expression levels of the pUCP20-ANT system could be adjusted to a range of distinct lower or higher levels by variation of anthranilate concentrations in the medium. Importantly, the anthranilate-inducible expression system worked also in strains of P. aeruginosa and P. putida and therefore will be most likely useful for physiological and biotechnological purposes in a wide range of pseudomonads. Key points • We established an anthranilate-inducible gene expression system for pseudomonads. • This system permits tuning of gene expression in a wide range of pseudomonads. • It will be very useful for physiological and biotechnological applications.

HutT functions as the major L‐histidine transporter in Pseudomonas putida KT2440

“…However, in M9 minimal medium supplemented with histidine and NH 4 Cl as sources of carbon and nitrogen, respectively, growth of the ∆ hutT strain was strongly impaired compared to wild‐type. Expression of hutT from plasmid pUCP20‐ANT2‐ hutT6H stimulated growth of the ∆ hutT strain compared to the ∆ hutT strain transformed with the empty plasmid [28]. However, wild‐type growth behavior was only partially restored (Fig.…”

“…For overproduction and purification of HutT, hutT was cloned into pET21a yielding plasmid pET21a-hutT6H. For transport assays and growth measurements with P. putida, hutT together with a sequence encoding a 6His tag was cloned into pUCP20-ANT2-MCS [28] yielding plasmid pUCP20-ANT2-hutT6H. Cloning was achieved by performing PCRs with the primers listed in Table S3, and restriction of amplicons and plasmid vectors with enzymes BamHI, XhoI, or NdeI followed by ligation with T4 ligase and transformation of E. coli DH5a.…”

The ABC transporter family efflux pump PvdRT‐OpmQ of Pseudomonas putida KT2440: purification and initial characterization

“…For overproduction and purification of PvdT, the gene was cloned into pUCP20‐ANT2‐MCS [20] using the multiple cloning site and restriction enzymes. Additionally, the sequence encoding a SUMO tag was amplified from a pET‐ SUMO system from Thermo Fisher.…”

“…During amplification by PCR, a histidine tag (7His) and a glycine‐serine (G‐S) linker were added to the SUMO tag by primers. All fragments were cloned into pUCP20‐ANT2‐MCS [20] yielding plasmid pUCP20‐ANT2‐ 7His‐SUMO‐pvdT using the restrictions enzymes Bam HI and Nde I for the 7His‐ SUMO tag. For the pvdT amplicon, restriction enzymes Nde I and Xba I were used, respectively.…”

PvdM of fluorescent pseudomonads is required for the oxidation of ferribactin by PvdP in periplasmic pyoverdine maturation

“…The coding region of pvdM , including a C-terminal Strep -tag used for later Western blot detections, was amplified from the plasmid pEXPT7- pvdM -strep ( 13 ) by PCR with the primer pair SpeI -RBS- PvdM -F-MS and pEXPT7-Strep-term-R-MS (see Table 1 for primers) and subcloned into pUCP20-ANT2-MCS ( 35 ), using SpeI/HindIII restriction sites, resulting in the plasmid pUCP20-ANT2- pvdM -strep-term for expression in P. fluorescens , and into pEXH5- tac ( 36 ) using NdeI/HindIII restriction sites, resulting in the plasmid pEX- pvdM -strep-term-tac for expression in E. coli . Single amino acid exchanges in the globular domain of PvdM were introduced by QuikChange mutagenesis (Stratagene) of pUCP20-ANT2- pvdM -strep-term, using the primers pvdM -M246A-F-MS or pvdM -H266A-F-MS in conjunction with the reverse primers that cover the identical sequence region, resulting in pUCP20-ANT2- pvdM M246A-strep-term and pUCP20-ANT2 -pvdM H266A-strep-term.…”