Corynebacterium glutamicum promoters: a practical approach

Abstract

Transcription initiation is the key step in gene expression in bacteria, and it is therefore studied for both theoretical and practical reasons. Promoters, the traffic lights of transcription initiation, are used as construction elements in biotechnological efforts to coordinate 'green waves' in the metabolic pathways leading to the desired metabolites. Detailed analyses of Corynebacterium glutamicum promoters have already provided large amounts of data on their structures, regulatory mechanisms and practical capabilities in metabolic engineering. In this minireview the main aspects of promoter studies, the methods developed for their analysis and their practical use in C. glutamicum are discussed. These include definitions of the consensus sequences of the distinct promoter classes, promoter localization and characterization, activity measurements, the functions of transcriptional regulators and examples of practical uses of constitutive, inducible and modified promoters in biotechnology. The implications of the introduction of novel techniques, such as in vitro transcription and RNA sequencing, to C. glutamicum promoter studies are outlined.

Figure 1

Mapping of TSPs by primer extension (PEX) and deducing respective promoters. Analysis of C. glutamicum dnaK gene transcription inducible by heat shock is shown (Barreiro et al., 2004).A. Determination of two dnaK TSPs by non-radioactive PEX technique. The bottom peaks (PEX) represent cDNA synthesized by reverse transcription using RNA from C. glutamicum. The peaks generated by the automatic sequencer (T, G, C, A) represent the products of sequencing reactions run with the same fluorescein-labelled primer as that used for PEX. The TSP nucleotides were determined by comparing the positions of the primer extension products and the sequencing signal.B. Quantitative comparison of signals representing reverse dnaK transcripts synthesized on RNA templates isolated from cells cultivated under various conditions (30°C or 40°C).C. Defining two dnaK promoters. The nucleotides of TSPs and the core promoter sequences are underlined, the sequences representing the σ^H-dependent promoter (P2) are shown in bold and the sequences of the σ^A-dependent promoter (P1) are in plain font. The boxes represent the approximate regions of the P1 promoter (thin line) and the P2 promoter (thick line).

Figure 2

Alignment of dnaK promoter regions of five species of genus Corynebacterium. The conserved putative −10 and −35 sequences of σ^A-dependent promoter P1 and σ^H-dependent promoter P2 are shaded. The −10 core hexamer of P1 is underlined. The experimentally determined TSPs pertinent to the C. glutamicum P1 (T) and P2 (A) promoters (Barreiro et al., 2004) are in bold and underlined. The boxes represent the conserved HAIR sequences (binding sites for the HspR repressor). The positions with identical nucleotides in all sequences are indicated with an asterisk (*); positions with identical nucleotides in 4/5 and 3/5 sequences are indicated with a colon (:) and a dot (.) respectively. The consensus sequences of C. glutamicum σ^A-and σ^H-dependent promoters are shown in the IUPAC code (K = G or T; Y = C or T; R = A or G; W = A or T; N = A,G,T,C) above the alignment (Pátek and Nešvera, ; Busche et al., 2012).

Figure 3

General workflow for transcriptome sequencing (RNA-seq) in bacteria.

Figure 4

Determination of TSPs of the C. glutamicum dnaK gene by transcriptome sequencing. The reads (sequences) derived from RNA-seq experiments that map the 5′-ends of the transcripts driven from the σ^A-and the σ^H-specific promoters are shown. The y axis denotes relative transcription efficiency as the number of reads normalized to the total number of reads in the respective RNA-seq experiment. The C. glutamicum ΔsigH mutant and the C. glutamicum ΔrshA mutant (with inactivated anti-sigma factor RshA) exhibit a different activity of the σ^H-specific P2dnaK promoter, whereas the activity of the σ^A-driven P1dnaK promoter remained unaffected in the ΔsigH strain (T. Busche and J. Kalinowski, unpublished).

Figure 5

Redirection of metabolite flow in biosynthesis pathway of aspartate-derived amino acids using the propionate-induced PprpD2 promoter.A. Insertion of the PprpD2 promoter upstream of the hom-thrB operon in lysine-producing strain ST06, resulting in the strain JP20. hom^fbr, the hom mutant gene coding for feedback-resistant homoserine dehydrogenase; P + OprpD2, promoter and operator of the prpD2 gene.B. Metabolic pathway showing increased flux of metabolites from aspartate-4-semialdehyde to P-homoserine due to propionate-induced overexpression of hom and thrB genes coding for homoserine dehydrogenase and homoserine kinase, respectively, in the strain JP20.C. Concentrations of excreted amino acid after addition of propionate to both cultures of ST06 and JP20, indicating higher synthesis of homoserine, threonine and isoleucine in the strain JP20 (Plassmeier et al., 2012a).

Figure 6

Bionsensor system for visualizing intracellular amino acid concentration within a single C. glutamicum cell using eyfp reporter gene.A. A sensor cell with low amino acid level exhibiting only background level of reporter gene expression.B. Induction of reporter gene expression, estimated as fluorescence increase, due to increased concentration of amino acid enabling its interaction with the Lrp activator (biosensor) and consequently resulting in the activation of the PbrnF promoter. The thick arrows representing genes (empty or hatched) and promoters (short filled) indicate the direction of transcription. The thin bent arrows represent the mRNA transcripts (the dashed bent arrow indicates the low basal level of the transcript). Lrp, transcriptional regulator (sensor); YFP, yellow fluorescent protein (reporter); circled A; amino acid (methionine or a branched-chain amino acid) (adapted according to Mustafi et al., 2012).