Mutations in the major gas vesicle protein GvpA and impacts on gas vesicle formation in Haloferax volcanii.

Gas vesicles are proteinaceous, gas-filled nanostructures produced by some bacteria and archaea. The hydrophobic major structural protein GvpA forms the ribbed gas vesicle wall. An in-silico 3D-model of GvpA of the predicted coil-α1-β1-β2-α2-coil structure is available and implies that the two β-chains constitute the hydrophobic interior surface of the gas vesicle wall. To test the importance of individual amino acids in GvpA we performed 85 single substitutions and analysed these variants in Haloferax volcanii ΔA+Amut transformants for their ability to form gas vesicles (Vac(+) phenotype). In most cases, an alanine substitution of a nonpolar residue did not abolish gas vesicle formation, but the replacement of single non-polar by charged residues in β1 or β2 resulted in Vac(-) transformants. A replacement of residues near the β-turn altered the spindle-shape to a cylindrical morphology of the gas vesicles. Vac(-) transformants were also obtained with alanine substitutions of charged residues of helix α1 suggesting that these amino acids form salt-bridges with another GvpA monomer. In helix α2, only the alanine substitution of His53, or Tyr54, led to Vac(-) transformants, whereas most other substitutions had no effect. We discuss our results in respect to the GvpA structure and data available from solid-state NMR. This article is protected by copyright. All rights reserved

The transcriptional regulator LysG (Rv1985c) of Mycobacterium tuberculosis activates lysE (Rv1986) in a lysine-dependent manner.

The Mycobacterium tuberculosis protein encoded by the Rv1986 gene is a target for memory T cells in patients with tuberculosis, and shows strong similarities to a lysine exporter LysE of Corynebacterium glutamicum. During infection, the pathogen Mycobacterium tuberculosis adapts its metabolism to environmental changes. In this study, we found that the expression of Rv1986 is controlled by Rv1985c. Rv1985c is located directly upstream of Rv1986 with an overlapping promoter region between both genes. Semiquantitative reverse transcription PCR using an isogenic mutant of Mycobacterium tuberculosis lacking Rv1985c showed that in the presence of lysine, Rv1985c protein positively upregulated the expression of Rv1986. RNA sequencing revealed the transcription start points for both transcripts and overlapping promoters. An inverted repeat in the center of the intergenic region was identified, and binding of Rv1985c protein to the intergenic region was confirmed by electrophoretic mobility shift assays. Whole transcriptome expression analysis and RNAsequencing showed downregulated transcription of ppsBCD in the Rv1985c-mutant compared to the wild type strain. Taken together, our findings characterize the regulatory network of Rv1985c in Mycobacterium tuberculosis. Due to their similarity of an orthologous gene pair in Corynebacterium glutamicum, we suggest to rename Rv1985c to lysG(Mt), and Rv1986 to lysE(Mt)

The oligonucleotides used in this study.

<p>The oligonucleotides used in this study.</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

Sequence accession number received from the manufacturer.

<p>Sequence accession number received from the manufacturer.</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

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