Table 1.
L. lactis strains and plasmids used in this study.
Fig 1.
Biogenesis and sequence analysis of ArgX.
(A) Schematic overview of the genomic locus in L. lactis of argR and ArgX. Black bent arrows indicate promoters. (B) Secondary structure of ArgX as predicted by Mfold [40]. (C) Northern analysis on an 8% polyacrylamide gel with a probe for ArgX (left) or for ArgR (right). Total RNA was used from exponential (OD600 of 1.0) and stationary phase (2h after an OD600 of 2.0 was reached) cultures, that were pooled in a 1:1 ratio after RNA isolation. The Northern analysis was repeated twice with identical results. (D) Northern hybridization analysis on an 12% polyacrylamide gel of ArgX using various growth phases/conditions and mutants of ArgX, showing that ArgX is derived solely from its own promoter and not from processing. A specific probe for ArgX was used. As a control for RNA quantity and quality, the 5S RNA was used as a control. (E) Nucleotide sequence of ArgX (black box) and its promoter region compared to ten L. lactis species. The black arrow indicates the transcription start of ArgX as determined in L. lactis MG1363. Asterisks: conserved nucleotides (in red), alternative nucleotides in blue or black, the promoter -10 box is indicated.
Fig 2.
(A) Phase contrast (top) and fluorescence microscopy images (bottom) of cells of L. lactis SVDM2006, carrying a chromosomally integrated PArgX-sfgfp fusion, grown under a low (2 mM) or high (25 mM) arginine concentration. The images depicts a representable situation of at least ten random fields of view. (B) Macroscopic pictures of colonies of PArgX-sfgfp expressing L. lactis SVDM2006 cells grown on a GM17 agar plate (M17 contains ~1.5 mM arginine). Bright fluorescent patches of cells with a high PArgX activity are indicated by red arrows. (C) Analysis of PArgX-sfgfp activity in L. lactis SVDM2006 cultures growing in CDMPC with the indicated concentration of arginine. The measurements were performed by a plate reader on cells growing in the stationary phase and were executed in quintuples. Standard deviations are indicated in the error bars. (D) GFP fluorescence in L. lactis SVDM2006 (control), SVDM2009 (ΔccpA), SVDM2010 (ΔargR) or SVDM2011 (ΔcodY), all carrying a chromosomal insertion of PArgX-sfgfp. The cells were grown to stationary phase in CDMPC with low (1 mM, red bars) or high (25 mM, blue bars) concentrations of arginine. Measurements were performed in triplicates in a plate reader and standard deviations are indicated in the error bars.
Fig 3.
Transcriptome and proteome analysis of the L. lactis ArgX deletion mutant SVDM2004.
(A) Volcano plots generated by T-REx [38], showing the RNA-seq results of the effect of ArgX deletion. Genes present outside the grey areas indicate a p-value of ≥ 0.05 and a fold change of ≥ 2. Genes outside dashed lines: p-value of ≥ 0.01 with fold change ≥ 5. Left: exponential phase, right: stationary phase. Yellow dots represent genes from the arginine catabolism (arc), blue dots those involved in arginine anabolism (arg). The shaded circles surrounding the genes provides a measure for the expression level. Two biological replicates were used for each strain. (B) Analysis by 2D gel electrophoresis of the proteomes of L. lactis SVDM2004 (left) compared and that of the wildtype strain, NZ9000 (right), grown in GM17 media in four biological replicates. Blue circles represent spots of arginine deaminase (ArcA), red circles represent the ornithine carbamolyltransferase (ArcB) enzyme, as determined by MALDI-TOF analysis.
Fig 4.
Influence of ArgX/ArgR overexpression on arc-sfgfp expression and the effect of ArgX on the growth of L. lactis.
(A) RNA duplex between ArgX and the arcC1 region containing the gene’s RBS, as predicted by TargetRNA2 [49]. The red box in the structure of ArgX shows the region involved in the postulated base pairing between ArgX and arcC1. Numbering in arcC1 counts from the start codon, numbering of ArgX from its TSS. (B) Schematic overview of a cell of the L. lactis strain designed to measure the effect of ArgX/ArgR overexpression on arc-sfgfp expression, measured by the development of GFP fluorescence. Lollipops: terminator structure; scissors: RNases; green cages: GFP. (C) Results of the experiment described. Blue bars: GFP fluorescence in un-induced cells; Red bars: GFP fluorescence in a culture of cells that were induced with 5 ng/ml of nisin to overexpress ArgX (SVDM2013), ArgR (SVDM2014) and ArgRΔstart (SVDM2015). L. lactis SVDM2012 is the empty vector control strain. Data derived from cells cultured in CDMPC containing 25 mM arginine, grown in the stationary phase, measured in a plate reader. The experiments were executed in quintuples and standard deviations are indicated in the error bars. (D) Growth effect of ArgX deletion mutant in CDMPC medium supplemented by 0.5% glucose and 0, 10 and 50 mM arginine. The red lines represent the deletion mutants of ArgX, blue lines represent the wildtype. Growth curves are the average of five cultures and were executed in a plate reader. The experiment is performed three times with consistent results and the standard error is indicated in the error bars.
Fig 5.
Model of arginine metabolism and its regulation in L. lactis.
Amino acids and (oligo)peptides can be taken up by L. lactis upon degradation of (milk) protein. CodY senses the intracellular pool of branched chain amino acids (BCAA) and represses arc and possibly ArgX expression. CcpA, in combination with Hpr-Ser46P, and ArgR/AhrC repress arc and ArgX expression by sensing fructose-1,6-diphosphate and arginine, respectively. ArgX represses arc by transcript stability (indicated by a scissor) and/or blocks the translation of arcC1 (indicated by a black schematic ribosome complex).