Design–Build–Test of Synthetic Promoters for Inducible Gene Regulation in Alphaproteobacteria

Abstract

Inducible gene expression is useful for biotechnological applications and for studying gene regulation and function in bacteria. Many inducible systems that perform in model organisms such as the Gammaproteobacterium Escherichia coli do not perform well in other bacteria that are of biotechnological interest. Typical problems include weak or leaky expression. Here, we describe an invention named ACIT (Alphaproteobacteria chromosomally integrating transcription-control cassette) that is carried on a suicide plasmid to enable insertion into the chromosome of the host. ACIT consists of multiple DNA fragments specifically arranged in a cassette that allows tight transcription control over any gene or gene cluster of interest following homologous recombination. At the heart of the invention is the ability to modify or exchange parts, e.g., promoters, to suit particular bacteria and growth conditions, allowing for customized gene expression control. Furthermore, ACIT provides a basis for a design–build–test approach for controlling gene expression in less studied bacteria. We describe examples of its control over pigment and exopolysaccharide production, growth, cell form, and social behavior in various Alphaproteobacteria.

Function of the RNA-targeting class 2 type VI CRISPR Cas system of Rhodobacter capsulatus

“…The inducible promoter is a synthetic construct consisting of the 16S gene promoter from R. sphaeroides flanked by Lac operators. This promoter, together with the lacI gene from E. coli , was previously tested in R. capsulatus ( Kretz et al, 2023 ) and found to have at least a 100-fold induction capacity in gene expression. Furthermore, to observe effects of the absence of Cas13a activity in vivo , we generated an additional R. capsulatus strain by inserting a transcription terminator between the cas13a gene and its promoter on the chromosome.…”

Tools for Genetic Engineering and Gene Expression Control inNovosphingobium aromaticivoransandRhodobacter sphaeroides

“…Most inducible promoters are based on the consensus sequence from E. coli σ 70 ; however, it is unknown how well these elements function in high GC-Alphaproteobacteria, such as N. aromaticivorans (63% GC) and R. sphaeroides (68% GC). For instance, the wild-type lac promoter from E. coli is reported to function poorly in R. sphaeroides (26). While no inducible promoters have been examined in N. aromaticivorans to our knowledge, promoters inducible by light, oxygen, crystal violet, and IPTG have been tested in R. sphaeroides (26)(27)(28)(29).…”

“…For instance, the wild-type lac promoter from E. coli is reported to function poorly in R. sphaeroides (26). While no inducible promoters have been examined in N. aromaticivorans to our knowledge, promoters inducible by light, oxygen, crystal violet, and IPTG have been tested in R. sphaeroides (26)(27)(28)(29). Although these foreign promoters can respond to an array of possible inducers, some inducers inhibit R. sphaeroides growth (crystal violet) (26) or substantially alter physiological processes of interest (light, oxygen).…”

“…While no inducible promoters have been examined in N. aromaticivorans to our knowledge, promoters inducible by light, oxygen, crystal violet, and IPTG have been tested in R. sphaeroides (26)(27)(28)(29). Although these foreign promoters can respond to an array of possible inducers, some inducers inhibit R. sphaeroides growth (crystal violet) (26) or substantially alter physiological processes of interest (light, oxygen). Existing IPTG inducible promoters are derivatives of the 16S rRNA promoter (11,26) which is regulated according to nutrient availability by several cellular factors including CarD, ppGpp, and DksA in Alphaproteobacteria and some other species (30)(31)(32)(33).…”

“…Although these foreign promoters can respond to an array of possible inducers, some inducers inhibit R. sphaeroides growth (crystal violet) (26) or substantially alter physiological processes of interest (light, oxygen). Existing IPTG inducible promoters are derivatives of the 16S rRNA promoter (11,26) which is regulated according to nutrient availability by several cellular factors including CarD, ppGpp, and DksA in Alphaproteobacteria and some other species (30)(31)(32)(33). Synthetic, IPTGinducible promoters have a potential advantage of not being inherently regulated by nutrient conditions and of utilizing an inducer that is likely less physiologically disruptive to the host.…”