YjbH-enhanced proteolysis of Spx by ClpXP in Bacillus subtilis is inhibited by the small protein YirB (YuzO)

The Spx protein of Bacillus subtilis is a global regulator of the oxidative stress response. Spx concentration is controlled at the level of proteolysis by the ATP-dependent protease ClpXP and a substrate-binding protein, YjbH, which interacts with Spx. A yeast two-hybrid screen was carried out using yjbH as bait to uncover additional substrates or regulators of YjbH activity. Of the several genes identified in the screen, one encoded a small protein, YirB (YuzO), which elevated Spx concentration and activity in vivo when overproduced from an isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible yirB construct. Pulldown experiments using extracts of B. subtilis cells producing a His-tagged YirB showed that native YjbH interacts with YirB in B. subtilis. Pulldown experiments using affinity-tagged Spx showed that YirB inhibited YjbH interaction with Spx. In vitro, YjbH-mediated proteolysis of Spx by ClpXP was inhibited by YirB. The activity of YirB is similar to that of the antiadaptor proteins that were previously shown to reduce proteolysis of a specific ClpXP substrate by interacting with a substrate-binding protein.     

Clp-dependent proteolysis down-regulates central metabolic pathways in glucose-starved Bacillus subtilis

Entry into stationary phase in Bacillus subtilis is linked not only to a redirection of the gene expression program but also to posttranslational events such as protein degradation. Using ³⁵S-labeled methionine pulse-chase labeling and two-dimensional polyacrylamide gel electrophoresis we monitored the intracellular proteolysis pattern during glucose starvation. Approximately 200 protein spots diminished in the wild-type cells during an 8-h time course. The degradation rate of at least 80 proteins was significantly reduced in clpP, clpC, and clpX mutant strains. Enzymes of amino acid and nucleotide metabolism were overrepresented among these Clp substrate candidates. Notably, several first-committed-step enzymes for biosynthesis of aromatic and branched-chain amino acids, cell wall precursors, purines, and pyrimidines appeared as putative Clp substrates. Radioimmunoprecipitation demonstrated GlmS, IlvB, PurF, and PyrB to be novel ClpCP targets. Our data imply that Clp proteases down-regulate central metabolic pathways upon entry into a nongrowing state and thus contribute to the adaptation to nutrient starvation. Proteins that are obviously nonfunctional, unprotected, or even “unemployed” seem to be recognized and proteolyzed by Clp proteases when the resources for growth become limited.     

Characterization of Bacillus subtilis recombinational pathways

Recombination in Bacillus subtilis requires the products of numerous rec loci. To dissect the various mechanisms which may be involved in genetic recombination, we constructed a series of isogenic strains containing more than one mutant rec allele. On the basis of their impairment in genetic exchange, the various loci (represented by specific rec alleles) were classified into different epistatic groups. Group alpha consists of rec genes represented by recB, recD, recF, recG, recL, and recR mutations, while group beta comprises the addA and addB mutations. Group gamma consists of the recH and recP mutations. These results suggest that B. subtilis has multiple pathways for genetic recombination and that the products of the genes within the alpha, beta, and gamma epistatic groups are involved in these alternative recombination pathways. The RecA protein is required in all three pathways of intermolecular recombination.     

Multiple catalases in Bacillus subtilis.

Vegetative cells of Bacillus subtilis in logarithmic growth phase produced one catalase, labeled catalase 1, with a nondenatured molecular weight of 205,000. As growth progressed, other activity bands with slower electrophoretic mobilities on polyacrylamide gels appeared, including a series of bands with a common nondenatured molecular weight of 261,000, collectively labeled catalase 2, and a minor band, with a molecular weight of 387,000, labeled catalase 3. Purified spores contained only catalase 2, and it was not produced in spo0A- or spo0F-containing mutants. Strains deficient in catalase 1 or catalase 2 or both were selected after mutagenesis. Sensitivities of the two main catalases to NaCN, NaN3, hydroxylamine, and temperature were similar, but the apparent Kms for H2O2 differed, being 36.6 and 64.4 mM, respectively, for catalase 1 and catalase 2. The levels of catalase 1 increased 15-fold during growth into stationary phase and could be increased 30-fold by the addition of H2O2 to the medium. Catalase 2, which was not affected by H2O2, appeared only after the cells had reached stationary phase, and the maximum levels were only half of the basal level of catalase 1.     

Characteristics of intracellular proteolysis in the cells of Bacillus subtilis

The rate of total protein degradation down to acid-soluble products in the B. subtilis cells growing on a minimal medium is about 4--5% per hour. Under amino acid deficiency the rate of proteolysis depends on the allelic state of the relA gene, so that in the rel+ cells it increases two-fold, while in the rel- cells it remains low. Elimination of NH4+, PO43- and Mg2+ from the culture medium or an addition of NaN3 (8 mM) or 2,4-dinitrophenol (2 mM) results in 1.5--2.0-fold stimulation of proteolysis independently of the relA gene. In all cases studied the rate of proteolysis decreases after addition of chloramphenicol (100 micrograms/ml). It is proposed that chloramphenicol decreases the intracellular concentration of ppGpp, which is believed to exert pleiotropic alterations of cellular metabolism under condition of growth limitation. Quite different is the case of degradation of anomalous proteins synthesized in the presence of the lysine analog--S-2-aminoethylcystein. Degradation of anomalous proteins proceeds very rapidly (about 70% per hour); chloramphenicol (100 micrograms/ml) decreases the rate of proteolysis only two-fold. It was found that tetracycline (100 micrograms/ml) effectively inhibits the degradation of anomalous proteins. This activity of tetracycline was not observed in the presence of 50 mM of Mg2+ and seems to be dependent on the capacity of the antibiotic to form complexes with bivalent cations. These results reveal common features of control of proteolysis in the cells of B. subtilis and E. coli.     

Localization of general and regulatory proteolysis in Bacillus subtilis cells

Protein degradation mediated by ATP-dependent proteases, such as Hsp100/Clp and related AAA+ proteins, plays an important role in cellular protein homeostasis, protein quality control and the regulation of, e.g. heat shock adaptation and other cellular differentiation processes. ClpCP with its adaptor proteins and other related proteases, such as ClpXP or ClpEP of Bacillus subtilis, are involved in general and regulatory proteolysis. To determine if proteolysis occurs at specific locations in B. subtilis cells, we analysed the subcellular distribution of the Clp system together with adaptor and general and regulatory substrate proteins, under different environmental conditions. We can demonstrate that the ATPase and the proteolytic subunit of the Clp proteases, as well as the adaptor or substrate proteins, form visible foci, representing active protease clusters localized to the polar and to the mid-cell region. These clusters could represent a compartmentalized place for protein degradation positioned at the pole close to where most of the cellular protein biosynthesis and also protein quality control are taking place, thereby spatially separating protein synthesis and degradation.     

Substrate requirements for regulated intramembrane proteolysis of Bacillus subtilis pro-sigmaK

During sporulation of Bacillus subtilis, pro-sigmaK is activated by regulated intramembrane proteolysis (RIP) in response to a signal from the forespore. RIP of pro-sigmaK removes its prosequence (amino acids 1 to 20), releasing sigmaK from the outer forespore membrane into the mother cell cytoplasm, in a reaction catalyzed by SpoIVFB, a metalloprotease in the S2P family of intramembrane-cleaving proteases. The requirements for pro-sigmaK to serve as a substrate for RIP were investigated by producing C-terminally truncated pro-sigmaK fused at different points to the green fluorescent protein (GFP) or hexahistidine in sporulating B. subtilis or in Escherichia coli engineered to coexpress SpoIVFB. Nearly half of pro-sigmaK (amino acids 1 to 117), including part of sigma factor region 2.4, was required for RIP of pro-sigmaK-GFP chimeras in sporulating B. subtilis. Likewise, pro-sigmaK-hexahistidine chimeras demonstrated that the N-terminal 117 amino acids of pro-sigma(K) are sufficient for RIP, although the N-terminal 126 amino acids, which includes all of region 2.4, allowed much better accumulation of the chimeric protein in sporulating B. subtilis and more efficient processing by SpoIVFB in E. coli. In contrast to the requirements for RIP, a much smaller N-terminal segment (amino acids 1 to 27) was sufficient for membrane localization of a pro-sigmaK-GFP chimera. Addition or deletion of five amino acids near the N terminus allowed accurate processing of pro-sigmaK, ruling out a mechanism in which SpoIVFB measures the distance from the N terminus to the cleavage site. A charge reversal at position 13 (substituting glutamate for lysine) reduced accumulation of pro-sigmaK and prevented detectable RIP by SpoIVFB. These results elucidate substrate requirements for RIP of pro-sigmaK by SpoIVFB and may have implications for substrate recognition by other S2P family members.     

Characterization of Bacillus subtilis glutamine synthetase by limited proteolysis.

The inactivation of native glutamine synthetase (GS) from Bacillus subtilis by trypsin, chymotrypsin, or subtilisin followed pseudo-fast order kinetics. Trypsin cleaved the polypeptide chain of GS into two principal fragments, one of about 43,000 (Mr) and the other of smaller than 10,000. Chymotrypsin and subtilisin caused similar cleavage of GS. A large fragment (Mr 35,000) and one smaller than 10,000 were detected on SDS-PAGE. The nicked protein remained dodecameric, as observed on gel filtration, electrophoresis, and electron micrography. In the presence of glutamate, ATP, and Mn2+, the digestion of GS by each of the three proteases was retarded completely; however, the presence of one substrate, L-glutamate, ATP+Mn2+, or ATP+Mg2+ led to partial protection. The product, L-glutamine, did not retard but altered the susceptibility of the protease sensitive sites. Amino acid sequence analysis of the two smaller polypeptide fragments showed that the nicked region was around serine 375 and serine 311, respectively, and that both large fragments (43,000 and 35,000) were N-terminal polypeptides of GS. The serine 311 region was involved in the formation of the enzyme-substrate complex. Tyrosine 372 near serine 375 corresponded to tyrosine 397 which was adenylylated by adenyltransferase in Escherichia coli GS.     

Insertion mutagenesis in Bacillus subtilis (Marmur)

A method for obtaining mutations in Bacillus subtilis using integration into the chromosome of the plasmid pHV60 ligated to chromosomal DNA fragments was developed. Auxotrophic mutants acquire, in addition, chloramphenicol-resistance, due to insertion of appropriate plasmid determinants. Chromosomal localization was established and the properties of several mutants were studied.     

Limited proteolysis of the pyruvate dehydrogenase multienzyme complex of Bacillus subtilis.

Limited digestion of the pyruvate dehydrogenase complex of Bacillus subtilis with either trypsin or chymotrypsin at 0 degrees C inhibited its ability to decarboxylate pyruvate and 2-oxoisovalerate oxidatively, without causing disassembly of the complex. The proteinases selectively cleaved the E1 alpha subunits to form two fragments of Mr 31500 and approx. 9500, as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both fragments remaining bound to the complex. Trypsin also caused a much slower cleavage of the E2 subunits, to form a fragment of apparent Mr 34000. The inhibition of overall dehydrogenase-complex activity was accompanied by the apparent loss of the pyruvate-driven and 2-oxoisovalerate-driven E1 activities, which was found to be due to a large increase in the Km for the 2-oxo acids: this change was correlated with the cleavage of the E1 alpha subunit.