Additional file 1: of Sequence-based identification of inositol monophosphatase-like histidinol-phosphate phosphatases (HisN) in Corynebacterium glutamicum, Actinobacteria, and beyond

Supplementary Figures and Tables. The supplementary figures comprise the SDS-PAGE of purified HisN Cg and Cg0911, several alignments of IMPase-like proteins in C. glutamicum and other species, and the complementation assay for various hisN Cg gene mutants. Furthermore, supplementary tables carry the used strains, plasmids and primers. (DOCX 200 kb

Le Courrier

17 juillet 18331833/07/17 (N198)

Additional file 4: Table S4. of Detailed transcriptome analysis of the plant growth promoting Paenibacillus riograndensis SBR5 by using RNA-seq technology

Putative RNA motifs present in the genome of P. riograndensis SBR5. Highlighted cells are referent to riboswitches detected in the transcriptome analysis. (XLSX 22 kb

Additional file 2: Table S2. of Detailed transcriptome analysis of the plant growth promoting Paenibacillus riograndensis SBR5 by using RNA-seq technology

Delta OD, growth rate and OD of harvested cells of P. riograndensis SBR5 when cultivated in Paenibacillus minimal medium (PbMM) or lysogeny broth (LB) with a variation of growth parameters. Cells cultivated at 30 °C and transferred to LB medium at 4 °C or 50 °C for 5 min for application of treatment of *cold shock or +heat shock, respectively. (XLSX 14 kb

Data_Sheet_1_Functional Characterization of a Small Alarmone Hydrolase in Corynebacterium glutamicum.DOCX

<p>The (pp)pGpp metabolism is an important component of bacterial physiology as it is involved in various stress responses and mechanisms of cell homeostasis, e.g., the regulation of growth. However, in order to better understand the (pp)pGpp associated regulation, it is crucial to study the molecular mechanisms of (pp)pGpp metabolism. In recent years, bioinformatic analyses of the RelA/SpoT homolog (RSH) superfamily have led to the discovery of small monofunctional RSH derivatives in addition to the well-known bifunctional Rel proteins. These are also referred to as small alarmone synthetases (SASs) or small alarmone hydrolases (SAHs). In this study, the ORF cg1485 from C. glutamicum was identified as a putative SAH encoding gene, based on a high similarity of the corresponding amino acid sequence with the (pp)pGpp hydrolysis domain. The characterization of its gene product, designated as RelH_Cg, represents the first functional investigation of a bacterial representative of the SAH subfamily. The predicted pyrophosphohydrolase activity was demonstrated in vivo by expression in two E. coli strains, characterized by different alarmone basal levels, as well as by in vitro analysis of the purified protein. During the assay-based analysis of hydrolysis activity in relation to the three known alarmone species, both RelH_Cg and the bifunctional RSH enzyme Rel_Cg were found to exhibit a pronounced substrate inhibition for alarmone concentrations of more than 0.75 mM. This characteristic of (pp)pGpp hydrolases could be an important mechanism for realizing the bistable character of the (pp)pGpp metabolism between a (pp)pGpp basal level and stress-associated alarmone production. The deletion of relH_Cg caused only a minor effect on growth behavior in both wild-type background and deletion mutants with deletion of (pp)pGpp synthetases. Based on this observation, the protein is probably only present or active under specific environmental conditions. The independent loss of the corresponding gene in numerous representatives of the genus Corynebacterium, which was found by bioinformatic analyses, also supports this hypothesis. Furthermore, growth analysis of all possible deletion combinations of the three active C. glutamicum RSH genes revealed interesting functional relationships which will have to be investigated in more detail in the future.</p

Log-log plot of the whole transcriptome expression analysis.

<p>The log-log plot shows the signal intensities of the mutant strain Δ<i>lysG</i> plotted against those of the Wt <i>Mt</i> strain. Spots represent the average of log2 data for each gene from three independent experiments. High through low expression levels are presented as a three-color spectrum. The red color indicates high expression and the green color low expression.</p

Genes regulated by LysG(^Mt).

<p>Genes regulated by LysG(^Mt).</p

LysG(^Mt) upregulates <i>lysE</i>(^Mt) in the presence of lysine and histidine.

<p>Relative gene expression level of <i>lysE</i>(^Mt) in Wt <i>Mt</i> H37Rv, the knockout mutant Δ<i>lysG</i> and the complemented strain Δ<i>lysG</i>::<i>lysG</i> during incubation in a minimal medium with lysine, histidine, arginine, asparagine, aspartate and leucine as additional nitrogen source. For normalization, the expression level of the validated reference gene <i>sigA</i> and <i>sigE</i> were used. The cutoff for significant regulation was set at 2-fold change. Means were calculated from three independent experiments. Error bars represent standard errors of the mean.</p

Genomic organization suggest a LTTR-dependent regulation of <i>lysE</i>(^Mt).

<p>A 108 bp region separates <i>lysG</i>(^Mt) from <i>lysE</i>(^Mt). The <i>lysG</i>(^Mt) and <i>lysE</i>(^Mt) transcriptional start sites are indicated with +1 (in red and bold type). The coloured arrows denote the beginning of the ORFs. The promoter binding motifs (-10 and -35 motifs) are marked as boxes. The underlined base pairs represent a palindromic DNA sequence.</p

Transcriptome profiles of the DNA region from <i>fadD26</i> to <i>ppsE</i>.

<p>(A) Genomic organization of the genes <i>fadD26</i> to <i>ppsE</i> of <i>Mt</i> is shown. (B) and (C) Profiles of mapped reads from RNAseq using RNA samples from Wt <i>Mt</i> with ammonium (B) or lysine (C) as nitrogen source. (D) and (E) Profiles of mapped reads from RNAseq using RNA samples from the Δ<i>lysG</i> mutant strain with ammonium (D) or lysine (E) as nitrogen source.</p