Inducible-Expression Plasmid for Rhodobacter sphaeroides and Paracoccus denitrificans

Abstract

We have developed a stable isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible-expression plasmid, pIND4, which allows graduated levels of protein expression in the alphaproteobacteria Rhodobacter sphaeroides and Paracoccus denitrificans. pIND4 confers kanamycin resistance and combines the stable replicon of pMG160 with the lacI q gene from pYanni3 and the lac promoter, P A1/04/03 , from pJBA24.Rhodobacter sphaeroides and Paracoccus denitrificans are often used for the study of bacterial metabolism, bioenergetics (8), and signal transduction (11). Although inducible-expression plasmids are available for these organisms, e.g., pRKSK1 (5) and pRECTX (9), these plasmids suffer from one or more of the following problems. First, continuous antibiotic selection is essential for maintaining the plasmid in the population, e.g., plasmid pRK415 (7), which is the vector backbone for most of the available expression vectors for these species, is retained by only approximately 10% of the population after 40 generations without antibiotic selection (6) (segregational instability). Second, the inducer affects the expression of many endogenous genes; for example, several R. sphaeroides vectors use either light-or oxygeninducible promoters to deliver high levels of protein expression (5). However, light and oxygen affect the expression of over 35% of the endogenous genes in this organism (2), which limifts the use of these vectors in functional studies.The plasmid developed in this study, pIND4 ( Fig. 1), uses the pMG170 vector backbone (6), the lacI q gene from pYanni3 (4), and the isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible-expression cassette from pJBA24, which includes the P A1/04/03 promoter, a ribosome binding site, a polylinker, and two transcriptional terminators (1). pMG170 confers kanamycin resistance and is an Escherichia coli shuttle cloning vector derived from naturally occurring plasmid pMG160 of Rhodobacter blasticus, which replicates and is segregationally stable in several other members of the Rhodobacteraceae (6). In E. coli, pMG170 has a high copy number, replicating using the ColE1 origin, while in R. sphaeroides, the pMG160 origin delivers a copy number of 18 to 23 (6).Vector construction. The construction of pIND4 is described in the supplemental material.Testing of the expression plasmid. The coding sequence for the R. sphaeroides cheY6 gene was cloned into pIND4, generating pIND4-Y6. The plasmid was introduced into R. sphaeroides strain JPA1336 (⌬cheY6 derivative of WS8N) and P. denitrificans strain PD1222 (wild type) via conjugation with the E. coli donor strain S17-1 pir (10). Cells containing the plasmid were grown from single colonies under aerobic conditions with shaking (225 rpm) in succinate medium containing 25 g/ml kanamycin. The effects of different concentrations of IPTG (Fig. 2) and different induction times (Fig. 3) on CheY6 protein accumulation were investigated.At IPTG concentrations of less than 1 M, no expression of CheY6 was detectable in R. sphaeroides (the minimum detection l...

Optogenetic Manipulation of Cyclic Di-GMP (c-di-GMP) Levels Reveals the Role of c-di-GMP in Regulating Aerotaxis Receptor Activity in Azospirillum brasilense

“…EB1 senses light via the flavin chromophore and is activated by violet-to-blue light (360 to 450 nm) (20). The engineered bphS-bphO operon expressing the diguanylate cyclase (plasmid pRed-DGC) or the bphS-bphO-eb1 operon expressing both the cyclase and phosphodiesterase (plasmid pBlue-PDE) was cloned downstream of the isopropyl-␤-Dthiogalactopyranoside (IPTG)-inducible P lac promoter into a broad-host-range vector, pIND4 (22). The plasmid containing the bphS-bphO-eb1 operon was initially designed to provide the ability to control both c-di-GMP synthesis and degradation, with light, from a single plasmid (Fig.…”

A Cardiolipin-Deficient Mutant of Rhodobacter sphaeroides Has an Altered Cell Shape and Is Impaired in Biofilm Formation

“…The primers used were NdeI-cls (5=-GGA GAA ATT AAC ATA TGA TCG ACG ACT GGC TGG GC-3=) and cls-BglII (5=-GAT GGT GAT GAG ATC TGA GGT AGC TCT GGA TCG G-3=). pIND5sp is a derivative of pIND4sp (a variant of pIND4 [17] with a spectinomycin resistance cassette) in which the NcoI site was replaced with an NdeI site by using the Stratagene QuikChange XL site-directed mutagenesis kit in accordance with the manufacturer's protocol. The primers used were pIND5sp (5=-GTG ATG GTG ATG AGA TCT GGA TCC TCC ATA TGT TAA TTT CTC CTC TTT AAT TCT AGA TG-3=) and pIND5sp-anti (5=-CAT CTA GAA TTA AAG AGG AGA AAT TAA CAT ATG GAG GAT CCA GAT CTC ATC ACC ATC AC-3=).…”

Signal-dependent turnover of the bacterial flagellar switch protein FliM

“…A construct containing the last 500 bp of fliM, followed by yPet, a repeat of the last 9 codons of FliM and 500 bp downstream of fliM, was inserted into the chromosome of E. coli (wild-type and ΔcheY) by allelic exchange as described in SI Text. CheY D13K∕Y106W and CheY D57A were overexpressed from pIND4 (27) in the ΔcheY strain.…”