Stulke, J. | bioRxiv Science

bioRxiv2024

RfaA (YqhY), a novel adaptor protein, controls metabolite-sensitive protein degradation in Bacillus subtilis

The carboxylation of acetyl-CoA is the committed step of fatty acid synthesis catalyzed by the multisubunit acetyl-CoA carboxylase (ACCase). However, the mechanisms that control the activity of this enzyme are poorly understood. Here, we identify the so far unknown protein RfaA (YqhY) of the model bacterium Bacillus subtilis as a regulator of fatty acid acquisition that targets the AccC subunit of ACCase for degradation. RfaA interacts with both AccC and the ClpE unfoldase subunit of the ClpEP protease complex. While the former interaction is not sensitive to the physiological conditions, RfaA interacts with ClpE only in the absence of the global amino group donor glutamate. This results in the degradation of AccC in the absence of glutamate. The inactivation of the rfaA gene results in the accumulation of fatty acids in the cell and in the formation of lipid droplets which are toxic for the bacteria. The reduced fatty acid synthesis in the absence of glutamate as a result of RfaA-and ClpEP-dependent degradation of AccC thus prevents intoxication of the cells by fatty acids. Our findings suggest that the degradation of enzymes that catalyze the committed step of biosynthetic pathways might be an important mechanism to control cellular homeostasis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/580884v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1f09555org.highwire.dtl.DTLVardef@1dc12e8org.highwire.dtl.DTLVardef@15d6961org.highwire.dtl.DTLVardef@1b1923e_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

How to deal with toxic amino acids: the bipartite AzlCD complex exports histidine in Bacillus subtilis

In the Gram-positive model bacterium Bacillus subtilis, the presence of the amino acid glutamate triggers potassium uptake due to the glutamate-mediated activation of the potassium channel KtrCD. As a result, the intracellular accumulation of glutamate is toxic in strains lacking the second messenger cyclic di-AMP since these cells are unable to limit potassium uptake. We observed that the presence of histidine, which is degraded to glutamate, is also toxic for a B. subtilis strain that lacks all three c-di-AMP synthesizing enzymes. However, suppressor mutants emerged, and whole genome sequencing revealed mutations in the azlB gene encoding the repressor of the azl operon. This operon encodes an exporter and an importer for branched-chain amino acids. The suppressor mutations result in overexpression of the azl operon. Deletion of the azlCD genes encoding the branched-chain amino acid exporter restored the toxicity of histidine indicating that this exporter is required for histidine export and resistance to otherwise toxic levels of the amino acid. The higher abundance of the amino acid exporter AzlCD increased the extracellular concentration of histidine, thus confirming the new function of AzlCD as a histidine exporter. Unexpectedly, AzlB-mediated repression of the operon remains active even in the presence of amino acids suggesting that expression of the azl operon requires mutational inactivation of AzlB. IMPORTANCEAmino acids are building blocks for protein biosynthesis in each living cell. However, due to their reactivity as well as the similarity between several amino amino acids, they may also be involved in harmful reactions or in non-cognate interactions and thus be toxic. Bacillus subtilis can deal with otherwise toxic histidine by overexpressing a bipartite amino acid exporter AzlCD. Although encoded in an operon that also contains a gene for an amino acid importer, the corresponding genes are not expressed, irrespective of the availability or not of amino acids in the medium. This suggests that the azl operon is a last resort to deal with histidine stress that can be expressed due to mutational inactivation of the cognate repressor, AzlB.

microbiology↗