RESUMEN
The enzyme telomerase ensures the integrity of linear chromosomes by maintaining telomere length. As a hallmark of cancer, cell immortalization and unlimited proliferation is gained by reactivation of telomerase. However, a significant fraction of cancer cells instead uses alternative telomere lengthening mechanisms to ensure telomere function, collectively known as Alternative Lengthening of Telomeres (ALT). Although the budding yeast Naumovozyma castellii (Saccharomyces castellii) has a proficient telomerase activity, we demonstrate here that telomeres in N. castellii are efficiently maintained by a novel ALT mechanism after telomerase knockout. Remarkably, telomerase-negative cells proliferate indefinitely without any major growth crisis and display wild-type colony morphology. Moreover, ALT cells maintain linear chromosomes and preserve a wild-type DNA organization at the chromosome termini, including a short stretch of terminal telomeric sequence. Notably, ALT telomeres are elongated by the addition of â¼275 bp repeats containing a short telomeric sequence and the subtelomeric DNA located just internally (TelKO element). Although telomeres may be elongated by several TelKO repeats, no dramatic genome-wide amplification occurs, thus indicating that the repeat addition may be regulated. Intriguingly, a short interstitial telomeric sequence (ITS) functions as the initiation point for the addition of the TelKO element. This implies that N. castellii telomeres are structurally predisposed to efficiently switch to the ALT mechanism as a response to telomerase dysfunction.
Asunto(s)
Saccharomycetales/genética , Homeostasis del Telómero , Telómero/genética , Cromosomas Fúngicos , Genoma Fúngico , Genómica/métodos , Humanos , Telomerasa/metabolismoRESUMEN
The bacterial endospore is a dormant and heat-resistant form of life. StoA (SpoIVH) in Bacillus subtilis is a membrane-bound thioredoxin-like protein involved in endospore cortex synthesis. It is proposed to reduce disulphide bonds in hitherto unknown proteins in the intermembrane compartment of developing forespores. Starting with a bioinformatic analysis combined with mutant studies we identified the sporulation-specific, high-molecular-weight, class B penicillin-binding protein SpoVD as a putative target for StoA. We then demonstrate that SpoVD is a membrane-bound protein with two exposed redox-active cysteine residues. Structural modelling of SpoVD, based on the well characterized orthologue PBP2x of Streptococcus pneumoniae, confirmed that a disulphide bond can form close to the active site of the penicillin-binding domain restricting access of enzyme substrate or functional association with other cortex biogenic proteins. Finally, by exploiting combinations of mutations in the spoVD, stoA and ccdA genes in B. subtilis cells, we present strong in vivo evidence that supports the conclusion that StoA functions to specifically break the disulphide bond in the SpoVD protein in the forespore envelope. The findings contribute to our understanding of endospore biogenesis and open a new angle to regulation of cell wall synthesis and penicillin-binding protein activity.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , Disulfuros/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Unión a las Penicilinas/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Esporas Bacterianas/enzimología , Secuencia de Aminoácidos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Dominio Catalítico , Proteínas de Transporte de Membrana/genética , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Missense , Proteínas de Unión a las Penicilinas/química , Proteínas de Unión a las Penicilinas/genética , Proteína Disulfuro Isomerasas/genética , Estructura Terciaria de Proteína , Alineación de Secuencia , Esporas Bacterianas/química , Esporas Bacterianas/metabolismo , Streptococcus pneumoniae/química , Streptococcus pneumoniae/genéticaRESUMEN
We present a mass spectrometry-based method for the identification and quantification of membrane proteins using the low-specificity protease Proteinase K, at very high pH, to digest proteins isolated by a modified SDS-PAGE protocol. The resulting peptides are modified with a fragmentation-directing isotope labeled tag. We apply the method to quantify differences in membrane protein expression of Bacillus subtilis grown in the presence or absence of glucose.
Asunto(s)
Proteínas de la Membrana/análisis , Fragmentos de Péptidos/análisis , Péptido Hidrolasas/metabolismo , Proteómica/métodos , Bacillus subtilis/química , Proteínas Bacterianas/análisis , Proteínas Bacterianas/efectos de los fármacos , Electroforesis en Gel de Poliacrilamida , Endopeptidasa K/metabolismo , Glucosa/farmacología , Humanos , Espectrometría de MasasRESUMEN
BdbD is a thiol:disulfide oxidoreductase (TDOR) from Bacillus subtilis that functions to introduce disulfide bonds in substrate proteins/peptides on the outside of the cytoplasmic membrane and, as such, plays a key role in disulfide bond management. Here we demonstrate that the protein is membrane-associated in B. subtilis and present the crystal structure of the soluble part of the protein lacking its membrane anchor. This reveals that BdbD is similar in structure to Escherichia coli DsbA, with a thioredoxin-like domain with an inserted helical domain. A major difference, however, is the presence in BdbD of a metal site, fully occupied by Ca(2+), at an inter-domain position some 14 A away from the CXXC active site. The midpoint reduction potential of soluble BdbD was determined as -75 mV versus normal hydrogen electrode, and the active site N-terminal cysteine thiol was shown to have a low pK(a), consistent with BdbD being an oxidizing TDOR. Equilibrium unfolding studies revealed that the oxidizing power of the protein is based on the instability introduced by the disulfide bond in the oxidized form. The crystal structure of Ca(2+)-depleted BdbD showed that the protein remained folded, with only minor conformational changes. However, the reduced form of Ca(2+)-depleted BdbD was significantly less stable than reduced Ca(2+)-containing protein, and the midpoint reduction potential was shifted by approximately -20 mV, suggesting that Ca(2+) functions to boost the oxidizing power of the protein. Finally, we demonstrate that electron exchange does not occur between BdbD and B. subtilis ResA, a low potential extra-cytoplasmic TDOR.
Asunto(s)
Proteínas Bacterianas/química , Calcio/química , Proteína Disulfuro Reductasa (Glutatión)/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Calcio/metabolismo , Cristalización , Conformación Molecular , Datos de Secuencia Molecular , Oxidación-Reducción , Unión Proteica , Proteína Disulfuro Reductasa (Glutatión)/genética , Proteína Disulfuro Reductasa (Glutatión)/metabolismo , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Bacillus subtilis StoA is an extracytoplasmic thiol-disulfide oxidoreductase (TDOR) important for the synthesis of the endospore peptidoglycan cortex protective layer. Here we demonstrate that StoA is membrane-associated in B. subtilis and report the crystal structure of the soluble protein lacking its membrane anchor. This showed that StoA adopts a thioredoxin-like fold with N-terminal and internal additions that are characteristic of extracytoplasmic TDORs. The CXXC active site of the crystallized protein was found to be in a mixture of oxidized and reduced states, illustrating that there is little conformational variation between redox states. The midpoint reduction potential was determined as -248 mV versus normal hydrogen electrode at pH 7 consistent with StoA fulfilling a reductive role in endospore biogenesis. pK(a) values of the active site cysteines, Cys-65 and Cys-68, were determined to be 5.5 and 7.8. Although Cys-68 is buried within the structure, both cysteines were found to be accessible to cysteine-specific alkylating reagents. In vivo studies of site-directed variants of StoA revealed that the active site cysteines are functionally important, as is Glu-71, which lies close to the active site and is conserved in many reducing extracytoplasmic TDORs. The structure and biophysical properties of StoA are very similar to those of ResA, a B. subtilis extracytoplasmic TDOR involved in cytochrome c maturation, raising important general questions about how these similar but non-redundant proteins achieve specificity. A detailed comparison of the two proteins demonstrates that relatively subtle differences, largely located around the active sites of the proteins, are sufficient to confer specificity.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas de la Membrana/química , Esporas Bacterianas/química , Secuencia de Aminoácidos , Biofisica/métodos , Dominio Catalítico , Citocromos c/química , Ácido Glutámico/química , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/fisiología , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Plásmidos/metabolismo , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The trxA gene is regarded as essential in Bacillus subtilis, but the roles of the TrxA protein in this gram-positive bacterium are largely unknown. Inactivation of trxA results in deoxyribonucleoside and cysteine or methionine auxotrophy. This phenotype is expected if the TrxA protein is important for the activity of the class Ib ribonucleotide reductase and adenosine-5'-phosphosulfate/3'-phosphoadenosine-5'-phosphosulfate reductase. We demonstrate here that a TrxA deficiency in addition causes defects in endospore and cytochrome c synthesis. These effects were suppressed by BdbD deficiency, indicating that TrxA in the cytoplasm is the primary electron donor to several different thiol-disulfide oxidoreductases active on the outer side of the B. subtilis cytoplasmic membrane.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/fisiología , Tiorredoxinas/fisiología , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cisteína/metabolismo , Citocromos c/metabolismo , Electroforesis en Gel de Poliacrilamida , Modelos Biológicos , Mutación , Temperatura , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMEN
Thiol-disulfide oxidoreductases catalyze formation, disruption, or isomerization of disulfide bonds between cysteine residues in proteins. Much is known about the functional roles and properties of this class of redox enzymes in vegetative bacterial cells but their involvement in sporulation has remained unknown until recently. Two membrane-embedded thiol-disulfide oxidoreductases, CcdA and StoA/SpoIVH, conditionally required for efficient production of Bacillus subtilis heat-resistant endospores, have now been identified. Properties of mutant cells lacking the two enzymes indicate new aspects in the molecular details of endospore envelope development. This mini-review presents an overview of membrane-bound thiol-disulfide oxidoreductases in the Gram-positive bacterium B. subtilis and endospore synthesis. Accumulated experimental findings on CcdA and StoA/SpoIVH are reviewed. A model for the role of these proteins in endospore cortex biogenesis in presented.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bacterias Formadoras de Endosporas/enzimología , Proteínas de la Membrana/metabolismo , Proteína Disulfuro Reductasa (Glutatión)/metabolismo , Esporas Bacterianas/metabolismo , Bacillus subtilis/enzimología , Bacillus subtilis/ultraestructura , Disulfuros/metabolismo , Bacterias Formadoras de Endosporas/ultraestructura , Estructura Molecular , Oxidación-Reducción , Peptidoglicano/química , Peptidoglicano/metabolismo , Esporas Bacterianas/ultraestructuraRESUMEN
Bacillus subtilis is an endospore-forming bacterium. There are indications that protein disulfide linkages occur in spores, but the role of thiol-disulfide chemistry in spore synthesis is not understood. Thiol-disulfide oxidoreductases catalyze formation or breakage of disulfide bonds in proteins. CcdA is the only B. subtilis thiol-disulfide oxidoreductase that has previously been shown to play some role in endospore biogenesis. In this work we show that lack of the StoA (YkvV) protein results in spores sensitive to heat, lysozyme, and chloroform. Compared to CcdA deficiency, StoA deficiency results in a 100-fold-stronger negative effect on sporulation efficiency. StoA is a membrane-bound protein with a predicted thioredoxin-like domain probably localized in the intermembrane space of the forespore. Electron microscopy of spores of CcdA- and StoA-deficient strains showed that the spore cortex is absent in both cases. The BdbD protein catalyzes formation of disulfide bonds in proteins on the outer side of the cytoplasmic membrane but is not required for sporulation. Inactivation of bdbD was found to suppress the sporulation defect of a strain deficient in StoA. Our results indicate that StoA is a thiol-disulfide oxidoreductase that is involved in breaking disulfide bonds in cortex components or in proteins important for cortex synthesis.