Bacterial cell division is facilitated by a molecular machine--the divisome--that assembles at mid-cell in dividing cells. The formation of the cytokinetic Z-ring by the tubulin homologue FtsZ is ...regulated by several factors, including the divisome component EzrA. Here we describe the structure of the 60-kDa cytoplasmic domain of EzrA, which comprises five linear repeats of an unusual triple helical bundle. The EzrA structure is bent into a semicircle, providing the protein with the potential to interact at both N- and C-termini with adjacent membrane-bound divisome components. We also identify at least two binding sites for FtsZ on EzrA and map regions of EzrA that are responsible for regulating FtsZ assembly. The individual repeats, and their linear organization, are homologous to the spectrin proteins that connect actin filaments to the membrane in eukaryotes, and we thus propose that EzrA is the founding member of the bacterial spectrin family.
Bacillus subtilis possesses different enzymes for the utilization of plant cell wall polysaccharides. This includes a gene cluster containing galactan degradation genes (ganA and ganB), two ...transporter component genes (ganQ and ganP), and the sugar-binding lipoprotein-encoding gene ganS (previously known as cycB). These genes form an operon that is regulated by GanR. The degradation of galactan by B. subtilis begins with the activity of extracellular GanB. GanB is an endo-β-1,4-galactanase and is a member of glycoside hydrolase (GH) family 53. This enzyme was active on high-molecular-weight arabinose-free galactan and mainly produced galactotetraose as well as galactotriose and galactobiose. These galacto-oligosaccharides may enter the cell via the GanQP transmembrane proteins of the galactan ABC transporter. The specificity of the galactan ABC transporter depends on the sugar-binding lipoprotein, GanS. Purified GanS was shown to bind galactotetraose and galactotriose using thermal shift assay. The energy for this transport is provided by MsmX, an ATP-binding protein. The transported galacto-oligosaccharides are further degraded by GanA. GanA is a β-galactosidase that belongs to GH family 42. The GanA enzyme was able to hydrolyze short-chain β-1,4-galacto-oligosaccharides as well as synthetic β-galactopyranosides into galactose. Thermal shift assay as well as electrophoretic mobility shift assay demonstrated that galactobiose is the inducer of the galactan operon regulated by GanR. DNase I footprinting revealed that the GanR protein binds to an operator overlapping the -35 box of the σ(A)-type promoter of Pgan, which is located upstream of ganS IMPORTANCE: Bacillus subtilis is a Gram-positive soil bacterium that utilizes different types of carbohydrates, such as pectin, as carbon sources. So far, most of the pectin degradation systems and enzymes have been thoroughly studied in B. subtilis Nevertheless, the B. subtilis utilization system of galactan, which is found as the side chain of the rhamnogalacturonan type I complex in pectin, has remained partially studied. Here, we investigated the galactan utilization system consisting of the ganSPQAB operon and its regulator ganR This study improves our knowledge of the carbohydrate degradation systems of B. subtilis, especially the pectin degradation systems. Moreover, the galactan-degrading enzymes may be exploited for the production of galacto-oligosaccharides, which are used as prebiotic substances in the food industry.
Bacterial spores can rapidly exit dormancy through the process of germination. This process begins with the activation of nutrient receptors embedded in the spore membrane. The prototypical germinant ...receptor in Bacillus subtilis responds to l-alanine and is thought to be a complex of proteins encoded by the genes in the
operon:
,
, and
. The GerAB subunit has recently been shown to function as the nutrient sensor, but beyond contributing to complex stability, no additional functions have been attributed to the other two subunits. Here, we investigate the role of GerAA. We resurrect a previously characterized allele of
(termed
) that carries a mutation in
and show that it constitutively activates germination even in the presence of a wild-type copy of
. Using an enrichment strategy to screen for suppressors of
, we identified mutations in all three
genes that restore a functional receptor. Characterization of two distinct
suppressors revealed that one (
reduces the GerA complex's ability to respond to l-alanine, while another (
) disrupts the germinant signal downstream of l-alanine recognition. These data argue against models in which GerAA is directly or indirectly involved in germinant sensing. Rather, our data suggest that GerAA is responsible for transducing the nutrient signal sensed by GerAB. While the steps downstream of
have yet to be uncovered, these results validate the use of a dominant-negative genetic approach in elucidating the
signal transduction pathway.
Endospore formers are a broad group of bacteria that can enter dormancy upon starvation and exit dormancy upon sensing the return of nutrients. How dormant spores sense and respond to these nutrients is poorly understood. Here, we identify a key step in the signal transduction pathway that is activated after spores detect the amino acid l-alanine. We present a model that provides a more complete picture of this process that is critical for allowing dormant spores to germinate and resume growth.
Spx is a global transcriptional regulator that orchestrates the Bacillus subtilis response to disulfide stress. The YjbH (SpxH) protein adapts Spx for ClpXP‐mediated degradation, playing a critical ...role in the regulation of the cellular Spx levels. Upon stress, YjbH forms aggregates by a yet unknown mechanism, resulting in increased Spx levels due to reduced proteolysis. Here, we studied how individual cells use the Spx‐YjbH system to respond to disulfide stress. We show, using fluorescent reporters, a correlation between the Spx levels and the amount of YjbH, as well as a transient growth inhibition upon disulfide stress. The in vivo dynamics and inheritance of YjbH aggregates are characterized by a bipolar distribution over time and appear to be entropy‐driven by nucleoid exclusion. Moreover, we reveal that the population following disulfide stress is highly heterogenous in terms of aggregate load and that the aggregate load has strong implications for cellular fitness. We propose that the observed heterogeneity could be a mechanism to ensure population survival during stress. Finally, we find that the two YjbH domains (DsbA‐like domain and winged‐helix domain) contribute to its aggregation function, and show that the aggregation of the DsbA‐like domain is conserved among other studied orthologs, whereas important differences are observed for the winged‐helix domain.
Upon stress, the adaptor protein YjbH forms aggregates. YjbH aggregation promotes increased levels of Spx, a key transcriptional regulator that orchestrates gene expression to mitigate stress. We investigated the localization and in vivo dynamics of YjbH aggregates, the impact of aggregates on cellular fitness, and the contributions of the two domains of YjbH to aggregation. Furthermore, we demonstrate that the aggregation properties of YjbH are conserved among members of the Bacillota (Firmicutes) phylum.
There is a great need for efficient acetonitrile removal technology in wastewater treatment to reduce the discharge of this pollutant in untreated wastewater. In this study, a nitrilase gene (nit) ...isolated from a nitrile-degrading bacterium (Rhodococcus rhodochrous BX2) was cloned and transformed into a biofilm-forming bacterium (Bacillus subtilis N4) that expressed the recombinant protein upon isopropylthio-β-galactoside (IPTG) induction. The recombinant bacterium (B. subtilis N4-pHT01-nit) formed strong biofilms and had nitrile-degrading capability. Further testing demonstrated that biofilms formed by B. subtilis N4-pHT01-nit were highly resistant to loading shock from acetonitrile and almost completely degraded the initial concentration of acetonitrile (800 mg L−1) within 24 h in a moving bed biofilm reactor (MBBR) after operation for 35 d. The bacterial composition of the biofilm, identified by high-throughput sequencing, in a reactor in which the B. subtilis N4-pHT01-nit bacterium was introduced indicated that the engineered bacterium was successfully immobilized in the reactor and became dominant genus. This work demonstrates that an engineered bacterium with nitrile-degrading and biofilm-forming capacity can improve the degradation of contaminants in wastewater. This approach offers a novel strategy for enhancing the biological oxidation of toxic pollutants in wastewater.
•A recombinant bacterium, Bacillus subtilis N4-pHT01-nit was established.•Strain N4-pHT01-nit has two functions: biofilm formation and nitrile degradation.•Strain N4-pHT01-nit had a strong resistance to loading shock from acetonitrile.•Strain N4-pHT01-nit improved the degradation of acetonitrile in wastewater treatment.•Strain N4-pHT01-nit was successfully immobilized in the reactor.
Fengycin is a cyclic lipopeptide used as an agricultural fungicide. It is synthesized by Bacillus subtilis as an immune response against fungal infection and functions by damaging the target’s cell ...membrane. Previous molecular dynamics simulations and experiments have led to the hypothesis that the aggregation of fengycins on the membrane surface plays a key role in cell disruption. Here, we used microsecond-scale all-atom molecular dynamics simulations to understand the specificity, selectivity, and structure of fengycin oligomers. Our simulations suggest that fengycin is more likely to form stable oligomers in model fungal membranes (phosphatidylcholine) compared to the model bacterial membranes (phosphatidylethanolamine:phosphatidylglycerol). Furthermore, we characterize the differences in the structure and kinetics of the membrane-bound aggregates and discuss their functional implications.
The DctSR two-component system of Bacillus subtilis controls the expression of the aerobic C4-dicarboxylate transporter DctA. Deletion of DctA leads to an increased dctA expression. The inactivation ...of DctB, an extracellular binding protein, is known to inhibit the expression of dctA. Here, interaction between the sensor kinase DctS and the transporter DctA as well as the binding protein DctB was demonstrated in vivo using streptavidin (Strep) or His protein interaction experiments (mSPINE or mHPINE), and the data suggest that DctA and DctB act as cosensors for DctS. The interaction between DctS and DctB was also confirmed by the bacterial two-hybrid system (BACTH). In contrast, no indication was obtained for a direct interaction between the transporter DctA and the binding protein DctB. Activity levels of uptake of 14Csuccinate by bacteria that expressed DctA from a plasmid were similar in the absence and the presence of DctB, demonstrating that the binding protein DctB is not required for transport. Thus, DctB is involved not in transport but in cosensing with DctS, highlighting DctB as the first example of a TRAP-type binding protein that acts as a cosensor. The simultaneous presence of DctS/DctB and DctS/DctA sensor pairs and the lack of direct interaction between the cosensors DctA and DctB indicate the formation of a tripartite complex via DctS. It is suggested that the DctS/DctA/DctB complex forms the functional unit for C4-dicarboxylate sensing in B. subtilis.
Bacillus subtilis bacteria play an important role in veterinary medicine, medicine, and biotechnology, and the permanently growing demand for biotechnological products fuels the improvement of the ...properties of biotechnological strains. B. subtilis strains with improved characteristics maybe obtained by rational design and the directed evolution technologies, or be found among newly described strains. In the course of the long-term microbiome composition studies in the Russian segment of the International Space Station, the B. subtilis 20 strain was isolated, this strain shows the capacity for rapid growth and considerable biomass accumulation, as well as increased resistance to acidification of the environment in comparison to the "terrestrial" B. subtilis 168 strain. What is more, B. subtilis 20 is hyperresistant to the DNA and protein damaging factors that are linked to the overexpression of the genes controlling DNA repair, hydrogen sulfide production, and reactive oxygen species neutralization. The described properties of B. subtilis 20 are indicative of its considerable potential as a promising producer of biologically active compounds.
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