Biology subjects

Commichau, F. M.

Publications and source records attributed to Commichau, F. M..

6 recordsLinked to original sources

bioRxiv2026

The evolution of a Na+-sensitive Vibrio cholerae mutant unmasks the moonlighting aminopeptidase PepA as a regulator of nhaB Na+/H+ antiporter gene expression

The pathogenic bacterium Vibrio cholerae is native to seawater and can therefore be cultivated in nutrient media with increased salt concentration. To maintain osmotic balance, V. cholerae uses Na+/H+ antiporters and an Na+-translocating NADH:quinone oxidoreductase (Na+-NQR). The exact contribution of the various Na+/H+ antiporters to maintain Na+ and H+ homeostasis in V. cholerae is unclear. However, genetic studies indicate the major Na+/H+ antiporter NhaA and the Na+-NQR are required by the bacteria for Na+ resistance at alkaline conditions. Here we show that the growth defect of a V. cholerae nhaA nqr mutant at increased Na+ concentrations and alkaline pH is relieved by the rapid acquisition of suppressor mutations. The suppressor mutants could be assigned to two classes. We identified (i) mutations in the promoter of the nhaB gene encoding a Na+/H+ antiporter and (ii) mutations that either affect the expression level of the pepA gene or DNA binding activity of the encoded multifunctional aminopeptidase PepA. The characterization of the PnhaB and PpepA promoters of the suppressor mutants, membrane potential measurements and comparative proteome analyses identified PepA as a novel factor controlling Na+ homeostasis in V. cholerae. IMPORTANCEThe emergence and spread of multi-resistant bacteria are a major problem for humans and animals. Therefore, the identification of novel targets for the development of novel antibiotics to combat pathogenic bacteria is extremely important. Since sodium ion homeostasis is an essential process in many pathogenic bacteria, especially those living in marine environments, it represents an interesting target for antibiotics. The perturbation of sodium ion homeostasis does indeed impair the fitness of the human pathogenic bacterium Vibrio cholerae but can be restored by the rapid evolution of the bacteria. This suggests that inhibiting multiple targets with different antibiotics might be more effective in preventing the development of resistant bacteria.

microbiology↗

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↗

bioRxiv2023

Control of asparagine homeostasis in Bacillus subtilis: Identification of promiscuous amino acid importers and exporters

Amino acids are the main building block for proteins. The Gram-positive model bacterium B. subtilis is able to import all proteinogenic amino acids from the environment as well as to synthesize them. However, the players involved in the acquisition of asparagine have not yet been identified for this bacterium. In this work, we used D-asparagine as a toxic analog of L-asparagine to identify asparagine transporters. This revealed that D-but not L-asparagine is taken up by the malate/lactate antiporter MleN. Specific strains that are sensitive to the presence of L-asparagine due to the lack of the second messenger cyclic di-AMP or due to the intracellular accumulation of this amino acid were used to isolate and characterize suppressor mutants that were resistant to the presence of otherwise growth-inhibiting concentrations of L-asparagine. These screens identified the broad-spectrum amino acid importers AimA and BcaP as responsible for the acquisition of L-asparagine. The amino acid exporter AzlCD allows detoxification of L-asparagine in addition to 4-azaleucine and histidine. This work supports the idea that amino acids are often transported by promiscuous importers and exporters. However, our work also shows that even stereo-enantiomeric amino acids do not necessarily use the same transport systems. IMPORTANCETransport of amino acid is a poorly studied function in many bacteria, including the model organism Bacillus subtilis. The identification of transporters is hampered by the redundancy of transport systems for most amino acids as well as by the poor specificity of the transporters. Here, we apply several strategies to use the growth-inhibitive effect of many amino acids under defined conditions to isolate suppressor mutants that exhibit either reduced uptake or enhanced export of asparagine, resulting in the identification of uptake and export systems for L-asparagine. The approaches used here may be useful for the identification of transporters for other amino acids both in B. subtilis and other bacteria as well.

microbiology↗

bioRxiv2023

Metabolic rewiring compensates for the loss of glutamate and aspartate biosynthesis in Bacillus subtilis

Glutamate serves as the major cellular amino group donor. In Bacillus subtilis, glutamate is synthesized by the combined action of the glutamine synthetase and the glutamate synthase (GOGAT). The glutamate dehydrogenases are devoted to glutamate degradation in vivo. To keep the cellular glutamate concentration high, the genes and the encoded enzymes involved in glutamate biosynthesis and degradation need to be tightly regulated depending on the available carbon and nitrogen sources. Serendipitously, we found that the inactivation of the ansR and citG genes encoding the repressor of the ansAB genes and the fumarase, respectively, enables the GOGAT-deficient B. subtilis mutant to synthesize glutamate via a non-canonical fumarate-based ammonium assimilation pathway. We also show that the de-repression of the ansAB genes is sufficient to restore aspartate prototrophy of an aspB aspartate transaminase mutant. Moreover, with excess nitrogen, B. subtilis mutants lacking fumarase activity show a growth defect that can be relieved by aspB overexpression, by reducing arginine uptake and by decreasing the metabolic flux through the TCA cycle. It will be interesting to investigate whether the B. subtilis strain using the alternative glutamate biosynthesis route can evolve in such a way that it robustly grows during nitrogen limitation and excess.

microbiology↗

bioRxiv2022

Structural and functional analysis of YopR and identification of an additional key component of the SPβ phage lysis-lysogeny management system

Prophages need to tightly control their lifestyle to either be maintained within the host genome or enter the lytic cycle. The SP{beta} prophage present in the genome of Bacillus subtilis 168 was recently shown to possess an arbitrium system defining its replication stage. Using an historic B. subtilis strain harboring the heat-sensitive SP{beta} c2 mutant, we analyzed a key component of the lysis-lysogeny decision system called YopR, which is critical for maintenance of lysogeny. Here, we demonstrate that the heat-sensitive SP{beta} c2 phenotype is due to a single nucleotide exchange in the yopR gene, rendering the encoded YopRG136E protein temperature sensitive. Structural characterization of YopR revealed that the protein is a DNA-binding protein with an overall fold like tyrosine recombinases. Biochemical and functional analyses indicate that YopR has lost the recombinase function and the G136E exchange impairs its higher order structure and DNA binding activity. We further show that the heat-inducible SP{beta} excision of the c2 mutant still depends on the serine recombinase SprA. Finally, an evolution experiment identified the YosL protein of unknown function as a novel component of the lysis-lysogeny management system, as the presence of yosL is crucial for the induction of the lytic cycle of SP{beta}.

microbiology↗

bioRxiv2021

The Bacillus phage SPβ and its relatives: A temperate phage model system reveals new strains, species, prophage integration loci, conserved proteins and lysogeny management components

The Bacillus phage SP{beta} has been known for about 50 years, but only a few strains are avalible. We isolated four new wild type strains of the SPbeta species. Phage vB_BsuS-Goe14 introduces its prophage into the spoVK locus, previously not observed to be used by SP{beta}-like phages. We could also reveal the SP{beta}-like phage genome replication strategy, the genome packaging mode, and the phage genome opening point. We extracted 55 SP{beta}-like prophages from public Bacillus genomes, thereby discovering three more integration loci and one additional type of integrase. The identified prophages resembled four new species clusters and three species orphans in the genus Spbetavirus. The determined core proteome of all SP{beta}-like prophages consists of 38 proteins. The integration cassette proved to be not conserved even though present in all strains. It consists of distinct integrases. Analysis of SP{beta} transcriptomes revealed three conserved genes, yopQ, yopR, and yokI, to be transcribed from a dormant prophage. While yopQ and yokI could be deleted from the prophage without activating the prophage, damaging of yopR led to a clear-plaque phenotype. Under the applied laboratory conditions, the yokI mutant showed an elevated virion release implying the YokI protein being a component of the arbitrium system.

microbiology↗