Disruption of a Transcriptional Repressor by an Insertion Sequence Element Integration Leads to Activation of a Novel Silent Cellobiose Transporter in Lactococcus lactis MG1363

Abstract

subsp. strains typically carry many dairy niche-specific adaptations. During adaptation to the milk environment these former plant strains have acquired various pseudogenes and insertion sequence elements indicative of ongoing genome decay and frequent transposition events in their genomes. Here we describe the reactivation of a silenced plant sugar utilization cluster in an MG1363 derivative lacking the two main cellobiose transporters, PtcBA-CelB and PtcBAC, upon applying selection pressure to utilize cellobiose. A disruption of the transcriptional repressor gene by an insertion sequence (IS) element allows expression of the otherwise silent novel cellobiose transporter Llmg_1244 and leads to growth of mutant strains on cellobiose. Llmg_1239 was labeled CclR, forellobiose usterepressor. Insertion sequences (ISs) play an important role in the evolution of lactococci and other bacteria. They facilitate DNA rearrangements and are responsible for creation of new genetic variants with selective advantages under certain environmental conditions. MG1363 possesses 71 copies in a total of 11 different types of IS elements. This study describes yet another example of an IS-mediated adaptive evolution. An integration of IS or IS into a gene coding for a transcriptional repressor led to activation of the repressed gene cluster coding for a plant sugar utilization pathway. The expression of the gene cluster allowed assembly of a novel cellobiose-specific transporter and led to cell growth on cellobiose.

Natural diversity of lactococci in γ-aminobutyric acid (GABA) production and genetic and phenotypic determinants

“…This IS981 element creates a new − 35 box, leading to a "hybrid" P gadCB promoter with the − 10 box of native P gadCB,, which probably affects the transcription of the gadCB operon and hence GABA production by this strain. The effect of an IS981 insertion on transcription has been described previously for other genes [50][51][52][53].…”

Resistance to the Bacteriocin Lcn972 Deciphered by Genome Sequencing

“…The accumulation of mutations in related lactococcal strains has already been shown to occur, resulting in, for example, different carbohydrate fermentation patterns [ 36 , 37 ]. For L. cremoris MG1614, a preliminary insight into its fermentation profile did not reveal major changes compared to its ancestor ( Table S2 ).…”

“…celB was one of the repressed genes in L. cremoris D1 [ 23 ] and the IS9 905:: celB mutation could have been selected as a countermeasure. Activation of cellobiose metabolism appears to occur frequently in laboratory strains derived from L. cremoris MG1363 in multiple ways from single-nucleotide mutations in promoter regions to disruption of a transcriptional repressor [ 36 , 37 , 43 ].…”

Further Elucidation of Galactose Utilization in Lactococcus lactis MG1363

“…The gene is located next to genes coding for cellulase and a transcriptional regulator of the Lac-I family. This cluster was recently shown to be involved in cellobiose uptake ( Solopova et al, 2017 ). The second candidate, llmg_0963 is present in a similar metabolic gene cluster.…”

“…Additionally, compensating mutations elsewhere on the chromosome might occur and only the resequencing of the whole genome of the strain can pinpoint such mutation. Various cases demonstrated how an unidentified gene or even a pseudogene exposes its activity when a strong selection pressure is applied ( Vaughan et al, 2001 ; Bongers et al, 2003 ; Gaspar et al, 2007 ; Aleksandrzak-Piekarczyk et al, 2011 ; Solopova et al, 2012 , 2017 ), indicating that caution should be taken in the interpretation of knock-out results.…”