The protein regulator ArgR and the sRNA derived from the 3'-UTR region of its gene, ArgX, both regulate the arginine deiminase pathway in Lactococcus lactis

Small regulatory RNAs (sRNAs) and their enormous potential and versatility have provided us with an astounding insight in the complexity of bacterial transcriptomes. sRNAs have been shown to be involved in a variety of cellular processes that range from stress to general metabolism. Here we report that the gene encoding the transcriptional regulator ArgR is immediately followed by the gene of the small regulatory RNA ArgX. The latter is transcribed from its own promoter. The production of ArgX is induced by increasing arginine concentrations and repressed by CcpA. Previously, ArgR was shown to act as a transcriptional repressor of the catabolic arginine deiminase pathway (arc operon) by binding in the promoter region of arcA. Here we demonstrate that ArgX downregulates arc mRNA levels. Furthermore, ArgX putatively blocks the translation of one of the genes in the operon, arcC1, a process that would redirect an intermediate in arginine degradation, carbamoyl phosphate, towards pyrimidine synthesis. Our findings exemplify, for the first time, the combinatorial power of a transcription factor and a small regulatory RNA derived from the 3'-UTR region. The regulators ArgR and ArgX share a common target, but act on transcription and on RNA level, respectively

Binding of MSA2-LysM_AcmA and MSA2-LysM_AcmD to <i>L. lactis</i> cells.

<p><b>a</b>. Anti-MSA2 antibody-treated Western blot showing binding of MSA2-LysM_AcmA, MSA2-LysM_AcmD and MSA2 to non-treated (lanes 1, 2 and 3) and TCA-treated (lanes 4, 5 and 6) <i>L. lactis</i> cells, respectively. <b>b</b>. Western blot treated with anti-MSA2 antibody. Lanes 1 and 3 indicate MSA2-LysM_AcmD bound to TCA-treated <i>L. lactis</i> cells at pH 6.2 and 3.2, respectively. The unbound protein at pH 6.2 and 3.2 is shown in lanes 2 and 4, respectively. Lane 5, positive control: MSA2-LysM_AcmA bound to <i>L. lactis</i> at pH 6.2. Arrow points out Pro-MSA2-LysM_AcmD and * indicates mature MSA2-LysM_AcmD. The percentage of both MSA2-LysM variants bound to <i>L. lactis</i> cells at pH 6.2 and 3.2 were semi-quantified (as the number of pixels present per mm² of both bands in lanes 1 or 3) divided by the total signal of the cell and supernatant fractions (number of pixels per mm² of all bands in lanes 1+2 or 3+4, respectively) is shown at the bottom of lanes 1 and 3. <b>c</b>. Transmission electron microscopic images of <i>L. lactis</i> incubated with MSA2 or MSA2 fusion proteins and subsequently incubated with rabbit anti-MSA2 antibodies and finally decorated with goat anti-rabbit IgG gold marker. Picture 1: non-treated <i>L. lactis</i> cells incubated with MSA2-LysM_AcmA. Pictures 2, 3 and 4 show TCA-treated <i>L. lactis</i> cells incubated with MSA2-LysM_AcmA, MSA2-LysM_AcmD and MSA2 proteins, respectively. Arrows indicate immunogold particles detected on the cells.</p

AcmD of <i>L. lactis</i> is involved in cell separation.

<p>(<b>a</b>) Turbidity and cell sedimentation of <i>L. lactis</i> IL1403 (1), IL1403<i>acmA</i>::IS<i>S1</i> (2) and IL1403<i>acmA</i>::IS<i>S1acm</i>D::<i>myc</i> (3) after overnight growth at 30° C in GM17 broth. (<b>b</b>) Light microscopic views of <i>L. lactis</i> IL1403, IL1403<i>acmA</i>::IS<i>S1</i> and IL1403<i>acmA</i>::IS<i>S1acmD</i>::<i>myc</i> after overnight growth at 30° C as standing cultures in GM17 medium. Magnification: 1250x in all three frames; representative views. (<b>c</b>) Phase contrast microscopic views of uninduced (left) and nisin (10 ng/ml)-induced (right) IL1403<i>acmA</i>::IS<i>S1acmD</i>::<i>myc</i> harboring plasmids pNGAcmD (AcmD) and pNZ9530 (nisRK). (<b>d</b>) Quantification of chain length in <i>L. lactis</i> IL1403, IL1403<i>acmA</i>::IS<i>S1</i>, IL1403<i>acmA</i>::IS<i>S1acm</i>D::<i>myc</i>, and nisin (10 ng/ml)-induced IL1403<i>acmA</i>::IS<i>S1acmD</i>::<i>myc</i> (pNGacmD) (the complemented double mutant). Number of diplococci per chain was counted and the mean of 30 chains were depicted. Standard deviation (***) indicates the significance as analyzed by Bonferroni’s multiple comparison test (<i>p</i><0.05) using one-way ANOVA.</p

Additional file 1: of Adaption to glucose limitation is modulated by the pleotropic regulator CcpA, independent of selection pressure strength

Additional materials and methods and supplementary figures 1â 9. (ZIP 12575 kb

AcmD, a Homolog of the Major Autolysin AcmA of Lactococcus lactis, Binds to the Cell Wall and Contributes to Cell Separation and Autolysis

<p>Lactococcus lactis expresses the homologous glucosaminidases AcmB, AcmC, AcmA and AcmD. The latter two have three C-terminal LysM repeats for peptidoglycan binding. AcmD has much shorter intervening sequences separating the LysM repeats and a lower iso-electric point (4.3) than AcmA (10.3). Under standard laboratory conditions AcmD was mainly secreted into the culture supernatant. An L. lactis acmAacmD double mutant formed longer chains than the acmA single mutant, indicating that AcmD contributes to cell separation. This phenotype could be complemented by plasmid-encoded expression of AcmD in the double mutant. No clear difference in cellular lysis and protein secretion was observed between both mutants. Nevertheless, overexpression of AcmD resulted in increased autolysis when AcmA was present (as in the wild type strain) or when AcmA was added to the culture medium of an AcmA-minus strain. Possibly, AcmD is mainly active within the cell wall, at places where proper conditions are present for its binding and catalytic activity. Various fusion proteins carrying either the three LysM repeats of AcmA or AcmD were used to study and compare their cell wall binding characteristics. Whereas binding of the LysM domain of AcmA took place at pHs ranging from 4 to 8, LysM domain of AcmD seems to bind strongest at pH 4.</p>