RESUMO
A new superior bacteria complementation model was achieved for testing antifolate compounds and investigating antifolate resistance in the dihydrofolate reductase (DHFR) enzyme of the malaria parasite. Earlier models depended on the addition of trimethoprim (TMP) to chemically suppress the host Escherichia coli (Ec) DHFR function. However, incomplete suppression of EcDHFR and potential interference of antibiotics needed to maintain plasmids for complementary gene expression can complicate the interpretations. To overcome such limitations, the folA (F) and thyA (T) genes were genetically knocked out (Δ) in E. coli BL21(DE3). The resulting EcΔFΔT cells were thymidine auxotroph where thymidine supplementation or functional complementation with heterologous DHFR-thymidylate synthase (TS) is needed to restore the loss of gene functions. When tested against pyrimethamine (PYR) and its analogs designed to target Plasmodium falciparum (Pf) DHFR-TS, the 50 % inhibitory concentration values obtained from EcΔFΔT surrogates expressing wildtype (PfTM4) or double mutant (PfK1) DHFR-TS showed strong correlations to the results obtained from the standard in vitro P. falciparum growth inhibition assay. Interestingly, while TMP had little effect on the susceptibility to PYR and analogs in EcΔFΔT expressing PfDHFR-TS, it hypersensitized the chemically knockdown E. coli BL21(DE3) expressing PfTM4 DHFR-TS but desensitized the one carrying PfK1 DHFR-TS. The low intrinsic expression level of PfTM4 in E. coli BL21(DE3) by western blot analysis may explain the hypersensitive to antifolates of chemical knockdown bacteria surrogate. These results demonstrated the usefulness of EcΔFΔT surrogate as a new tool for antifolate antimalarial screening with potential application for investigation of antifolate resistance mechanism.
Assuntos
Escherichia coli , Antagonistas do Ácido Fólico , Técnicas de Inativação de Genes , Plasmodium falciparum , Pirimetamina , Tetra-Hidrofolato Desidrogenase , Timidilato Sintase , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Antagonistas do Ácido Fólico/farmacologia , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/genética , Timidilato Sintase/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Pirimetamina/farmacologia , Antimaláricos/farmacologia , Concentração Inibidora 50 , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Resistência a Medicamentos/genética , Teste de Complementação Genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Complexos MultienzimáticosRESUMO
The efficient natural transformation of Neisseria meningitidis allows the rapid construction of bacterial mutants in which the genes of interest are interrupted or replaced by antibiotic-resistance cassettes. However, this proved to be a double-edged sword, i.e., although facilitating the genetic characterization of this important human pathogen, it has limited the development of strategies for constructing markerless mutants without antibiotic-resistance markers. In addition, efficient tools for complementation or labeling are also lacking in N. meningitidis. In this study, we significantly expand the meningococcal genetic toolbox by developing new and efficient tools for the construction of markerless mutants (using a dual counterselection strategy), genetic complementation (using integrative vectors), and cell labeling (using a self-labeling protein tag). This expanded toolbox paves the way for more in-depth genetic characterization of N. meningitidis and might also be useful in other Neisseria species.IMPORTANCENeisseria meningitidis and Neisseria gonorrhoeae are two important human pathogens. Research focusing on these bacteria requires genetic engineering, which is facilitated by their natural ability to undergo transformation. However, the ease of mutant engineering has led the Neisseria community to neglect the development of more sophisticated tools for gene editing, particularly for N. meningitidis. In this study, we have significantly expanded the meningococcal genetic toolbox by developing novel and efficient tools for markerless mutant construction, genetic complementation, and cell tagging. This expanded toolbox paves the way for more in-depth genetic characterization of N. meningitidis and might also be useful in other Neisseria species.
Assuntos
Edição de Genes , Neisseria meningitidis , Neisseria meningitidis/genética , Edição de Genes/métodos , Teste de Complementação GenéticaRESUMO
Relapsing fever (RF), a vector-borne disease caused by Borrelia spp., is characterized by recurring febrile episodes due to repeated bouts of bacteremia. RF spirochetes can be geographically and phylogenetically divided into two distinct groups; Old World RF Borrelia (found in Africa, Asia, and Europe) and New World RF Borrelia (found in the Americas). While RF is a rarely reported disease in the Americas, RF is prevalent in endemic parts of Africa. Despite phylogenetic differences between Old World and New World RF Borrelia and higher incidence of disease associated with Old World RF spirochete infection, genetic manipulation has only been described in New World RF bacteria. Herein, we report the generation of genetic tools for use in the Old World RF spirochete, Borrelia duttonii. We describe methods for transformation and establish shuttle vector- and integration-based approaches for genetic complementation, creating green fluorescent protein (gfp)-expressing B. duttonii strains as a proof of principle. Allelic exchange mutagenesis was also used to inactivate a homolog of the Borrelia burgdorferi p66 gene, which encodes an important virulence factor, in B. duttonii and demonstrate that this mutant was attenuated in a murine model of RF. Finally, the B. duttonii p66 mutant was complemented using shuttle vector- and cis integration-based approaches. As expected, complemented p66 mutant strains were fully infectious, confirming that P66 is required for optimal mammalian infection. The genetic tools and techniques reported herein represent an important advancement in the study of RF Borrelia that allows for future characterization of virulence determinants and colonization factors important for the enzootic cycle of Old World RF spirochetes.
Assuntos
Borrelia , Febre Recorrente , Animais , Febre Recorrente/microbiologia , Borrelia/genética , Borrelia/classificação , Camundongos , Feminino , Teste de Complementação Genética , Proteínas de Fluorescência Verde/genética , HumanosRESUMO
The PhoPR system is a master regulator in Mycobacterium tuberculosis. A key difference between M. tuberculosis and Mycobacterium bovis is a G71I substitution in the M. bovis PhoR orthologue. Functional studies of the M. bovis PhoPR system have generated conflicting findings, with some research suggesting that the M. bovis PhoPR is defective while others indicate it is functional. We sought to revisit the functionality of the M. bovis PhoPR system. To address this, we constructed a phoPR mutant in the reference strain M. bovis AF2122/97. We employed a combination of growth assays and transcriptomics analyses to assess the phenotype of the mutant vs wild type and complemented strains. We found that the M. bovis AF2122/97 ΔphoPR mutant showed a growth defect on solid and liquid media compared to the wild type and complemented strains. The transcriptome of the M. bovis AF2122/97 ΔphoPR mutant was also altered as compared to wild type, including differential expression of genes involved in lipid metabolism and secretion. Our work provides further insight into the activity of PhoPR in M. bovis and underlines the importance of the PhoPR system as a master regulator of gene expression in the Mycobacterium tuberculosis complex.
Assuntos
Proteínas de Bactérias , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Mutação , Mycobacterium bovis , Mycobacterium bovis/genética , Mycobacterium bovis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Perfilação da Expressão Gênica/métodos , Fenótipo , Transcriptoma , Metabolismo dos Lipídeos/genética , Teste de Complementação GenéticaRESUMO
OBJECTIVES: The mechanisms underlying chromosomally encoded colistin resistance in Escherichia coli remain insufficiently investigated. In this study, we investigated the contribution of various pmrB mutations from E. coli clinical isolates to colistin resistance. METHODS: The resistance mechanisms in eight mcr-negative colistin-resistant E. coli isolates obtained from a nationwide surveillance program in Taiwan using recombinant DNA techniques and complementary experiments were investigated. The minimal inhibitory concentrations (MICs) of colistin in the recombinant strains were compared with those in the parental strains. The expression levels of pmrA and pmrK (which are part of the pmrCAB and pmrHFIJKLM operons associated with colistin resistance) were measured using reverse transcription-quantitative real-time polymerase chain reaction. RESULTS: In the complementation experiments, various mutated pmrB alleles from the eight mcr-negative colistin-resistant E. coli strains were introduced into an ATCC25922 mutant with a PmrB deletion, which resulted in colistin resistance. The MIC levels of colistin in the most complemented strains were comparable to those of the parental colistin-resistant strains. Increased expression levels of pmrA and pmrK were consistently detected in most complemented strains. The impact for colistin resistance was confirmed for various novel amino acid substitutions, P94L, G19E, L194P, L98R and R27L in PmrB from the parental clinical strains. The detected amino acid substitutions are distributed in the different functional domains of PmrB. CONCLUSIONS: Colistin resistance mediated by amino acid substitutions in PmrB is a major chromosomally encoded mechanism in E. coli of clinical origin.
Assuntos
Antibacterianos , Colistina , Farmacorresistência Bacteriana , Escherichia coli , Testes de Sensibilidade Microbiana , Mutação , Colistina/farmacologia , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Antibacterianos/farmacologia , Humanos , Proteínas de Bactérias/genética , Infecções por Escherichia coli/microbiologia , Taiwan , Cromossomos Bacterianos/genética , Proteínas de Escherichia coli/genética , Teste de Complementação Genética , Regulação Bacteriana da Expressão Gênica , Fatores de TranscriçãoRESUMO
The picornavirus genome encodes a large, single polyprotein that is processed by viral proteases to form an active replication complex. The replication complex is formed with the viral genome, host proteins, and viral proteins that are produced/translated directly from each of the viral genomes (viral proteins provided in cis). Efficient complementation in vivo of replication complex formation by viral proteins provided in trans, thus exogenous or ectopically expressed viral proteins, remains to be demonstrated. Here, we report an efficient trans complementation system for the replication of defective poliovirus (PV) mutants by a viral polyprotein precursor in HEK293 cells. Viral 3AB in the polyprotein, but not 2BC, was processed exclusively in cis. Replication of a defective PV replicon mutant, with a disrupted cleavage site for viral 3Cpro protease between 3Cpro and 3Dpol (3C/D[A/G] mutant) could be rescued by a viral polyprotein provided in trans. Only a defect of 3Dpol activity of the replicon could be rescued in trans; inactivating mutations in 2CATPase/hel, 3B, and 3Cpro of the replicon completely abrogated the trans-rescued replication. An intact N-terminus of the 3Cpro domain of the 3CDpro provided in trans was essential for the trans-active function. By using this trans complementation system, a high-titer defective PV pseudovirus (PVpv) (>107 infectious units per mL) could be produced with the defective mutants, whose replication was completely dependent on trans complementation. This work reveals potential roles of exogenous viral proteins in PV replication and offers insights into protein/protein interaction during picornavirus infection. IMPORTANCE: Viral polyprotein processing is an elaborately controlled step by viral proteases encoded in the polyprotein; fully processed proteins and processing intermediates need to be correctly produced for replication, which can be detrimentally affected even by a small modification of the polyprotein. Purified/isolated viral proteins can retain their enzymatic activities required for viral replication, such as protease, helicase, polymerase, etc. However, when these proteins of picornavirus are exogenously provided (provided in trans) to the viral replication complex with a defective viral genome, replication is generally not rescued/complemented, suggesting the importance of viral proteins endogenously provided (provided in cis) to the replication complex. In this study, I discovered that only the viral polymerase activity of poliovirus (PV) (the typical member of picornavirus family) could be efficiently rescued by exogenously expressed viral proteins. The current study reveals potential roles for exogenous viral proteins in viral replication and offers insights into interactions during picornavirus infection.
Assuntos
Poliovirus , Proteínas Virais , Replicação Viral , Poliovirus/genética , Poliovirus/fisiologia , Replicação Viral/genética , Humanos , Proteínas Virais/genética , Proteínas Virais/metabolismo , Células HEK293 , Mutação , Teste de Complementação Genética , Poliproteínas/metabolismo , Poliproteínas/genética , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/genética , Proteases Virais 3CRESUMO
In this study, we examined the role of the lipopolysaccharide (LPS) core of Rhizobium etli in facilitating the adsorption and infection of phages with broad host range. When the plasmid-encoded LPS biosynthesis genes, wreU and wreV, were disrupted, distinct and contrasting effects on phage infection were observed. The wreU mutant strains exhibited wild-type adsorption and infection properties, whereas the wreV mutant demonstrated resistance to phage infection, but retained the capacity to adsorb phages. Complementation of the wreV mutant strains with a recombinant plasmid containing the wreU and wreV, restored the susceptibility to the phages. However, the presence of this recombinant plasmid in a strain devoid of the native lps-encoding plasmid was insufficient to restore phage susceptibility. These results suggest that the absence of wreV impedes the proper assembly of the complete LPS core, potentially affecting the formation of UDP-KdgNAg or KDO precursors for the O-antigen. In addition, a protein not yet identified, but residing in the native lps-encoding plasmid, may be necessary for complete phage infection.
Assuntos
Bacteriófagos , Especificidade de Hospedeiro , Lipopolissacarídeos , Plasmídeos , Rhizobium etli , Lipopolissacarídeos/biossíntese , Bacteriófagos/genética , Rhizobium etli/genética , Rhizobium etli/virologia , Rhizobium etli/metabolismo , Plasmídeos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ligação Viral , Teste de Complementação GenéticaRESUMO
Knowing the genes involved in quantitative traits provides an entry point to understanding the biological bases of behavior, but there are very few examples where the pathway from genetic locus to behavioral change is known. To explore the role of specific genes in fear behavior, we mapped three fear-related traits, tested fourteen genes at six quantitative trait loci (QTLs) by quantitative complementation, and identified six genes. Four genes, Lamp, Ptprd, Nptx2, and Sh3gl, have known roles in synapse function; the fifth, Psip1, was not previously implicated in behavior; and the sixth is a long non-coding RNA, 4933413L06Rik, of unknown function. Variation in transcriptome and epigenetic modalities occurred preferentially in excitatory neurons, suggesting that genetic variation is more permissible in excitatory than inhibitory neuronal circuits. Our results relieve a bottleneck in using genetic mapping of QTLs to uncover biology underlying behavior and prompt a reconsideration of expected relationships between genetic and functional variation.
Assuntos
Medo , Locos de Características Quantitativas , Animais , Feminino , Masculino , Camundongos , Comportamento Animal/fisiologia , Mapeamento Cromossômico , Medo/fisiologia , Camundongos Endogâmicos C57BL , Teste de Complementação GenéticaRESUMO
The extremely halophilic archaeon Haloarcula japonica accumulates the C50 carotenoid, bacterioruberin (BR). To reveal the BR biosynthetic pathway, unidentified phytoene desaturase candidates were functionally characterized in the present study. Two genes encoding the potential phytoene desaturases, c0507 and d1086, were found from the Ha. japonica genome sequence by a homology search using the Basic Local Align Search Tool. Disruption mutants of c0507 and d1086 and their complemented strains transformed with expression plasmids for c0507 and d1086 were subsequently constructed. High-performance liquid chromatography (HPLC) ana-lyses of carotenoids produced by these strains revealed that C0507 and D1086 were both bifunctional enzymes with the same activities as both phytoene desaturase (CrtI) and 3,4-desaturase (CrtD). C0507 and D1086 complemented each other during BR biosynthesis in Ha. japonica. This is the first study to identify two distinct enzymes with both CrtI and CrtD activities in an extremely halophilic archaeon.
Assuntos
Carotenoides , Haloarcula , Oxirredutases , Carotenoides/metabolismo , Haloarcula/genética , Haloarcula/enzimologia , Haloarcula/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Vias Biossintéticas/genética , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Teste de Complementação Genética , FilogeniaRESUMO
bb0616 of Borrelia burgdorferi, the Lyme disease pathogen, encodes a hypothetical protein of unknown function. In this study, we showed that BB0616 was not surface-exposed or associated with the membrane through localization analyses using proteinase K digestion and cell partitioning assays. The expression of bb0616 was influenced by a reduced pH but not by growth phases, elevated temperatures, or carbon sources during in vitro cultivation. A transcriptional start site for bb0616 was identified by using 5' rapid amplification of cDNA ends, which led to the identification of a functional promoter in the 5' regulatory region upstream of bb0616. By analyzing a bb0616-deficient mutant and its isogenic complemented counterparts, we found that the infectivity potential of the mutant was significantly attenuated. The inactivation of bb0616 displayed no effect on borrelial growth in the medium or resistance to oxidative stress, but the mutant was significantly more susceptible to osmotic stress. In addition, the production of global virulence regulators such as BosR and RpoS as well as virulence-associated outer surface lipoproteins OspC and DbpA was reduced in the mutant. These phenotypes were fully restored when gene mutation was complemented with a wild-type copy of bb0616. Based on these findings, we concluded that the hypothetical protein BB0616 is required for the optimal infectivity of B. burgdorferi, potentially by impacting B. burgdorferi virulence gene expression as well as survival of the spirochete under stressful conditions.
Assuntos
Proteínas de Bactérias , Borrelia burgdorferi , Regulação Bacteriana da Expressão Gênica , Doença de Lyme , Borrelia burgdorferi/genética , Borrelia burgdorferi/patogenicidade , Borrelia burgdorferi/metabolismo , Animais , Camundongos , Doença de Lyme/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regiões Promotoras Genéticas , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Virulência , Camundongos Endogâmicos C3H , Fator sigma/genética , Fator sigma/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Sítio de Iniciação de Transcrição , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Teste de Complementação Genética , Concentração de Íons de HidrogênioRESUMO
Soluble di-iron monooxygenases (SDIMOs) are multi-component enzymes catalysing the oxidation of various substrates. These enzymes are characterized by high sequence and functional diversity that is still not well understood despite their key role in biotechnological processes including contaminant biodegradation. In this study, we analysed a mutant of Rhodoccocus aetherivorans BCP1 (BCP1-2.10) characterized by a transposon insertion in the gene smoA encoding the alpha subunit of the plasmid-located SDIMO SmoABCD. The mutant BCP1-2.10 showed a reduced capacity to grow on propane, lost the ability to grow on butane, pentane and n-hexane and was heavily impaired in the capacity to degrade chloroform and trichloroethane. The expression of the additional SDIMO prmABCD in BCP1-2.10 probably allowed the mutant to partially grow on propane and to degrade it, to some extent, together with the other short-chain n-alkanes. The complementation of the mutant, conducted by introducing smoABCD in the genome as a single copy under a constitutive promoter or within a plasmid under a thiostreptone-inducible promoter, allowed the recovery of the alkanotrophic phenotype as well as the capacity to degrade chlorinated n-alkanes. The heterologous expression of smoABCD allowed a non-alkanotrophic Rhodococcus strain to grow on pentane and n-hexane when the gene cluster was introduced together with the downstream genes encoding alcohol and aldehyde dehydrogenases and a GroEL chaperon. BCP1 smoA gene was shown to belong to the group 6 SDIMOs, which is a rare group of monooxygenases mostly present in Mycobacterium genus and in a few Rhodococcus strains. SmoABCD originally evolved in Mycobacterium and was then acquired by Rhodococcus through horizontal gene transfer events. This work extends the knowledge of the biotechnologically relevant SDIMOs by providing functional and evolutionary insights into a group 6 SDIMO in Rhodococcus and demonstrating its key role in the metabolism of short-chain alkanes and degradation of chlorinated n-alkanes.
Assuntos
Alcanos , Oxigenases de Função Mista , Alcanos/metabolismo , Oxigenases de Função Mista/metabolismo , Oxigenases de Função Mista/genética , Teste de Complementação Genética , Mutagênese Insercional , Biotransformação , Elementos de DNA Transponíveis , Hidrocarbonetos Clorados/metabolismoRESUMO
Endosperm is the main storage organ in cereal grain and determines grain yield and quality. The molecular mechanisms of heat shock proteins in regulating starch biosynthesis and endosperm development remain obscure. Here, we report a rice floury endosperm mutant flo24 that develops abnormal starch grains in the central starchy endosperm cells. Map-based cloning and complementation test showed that FLO24 encodes a heat shock protein HSP101, which is localized in plastids. The mutated protein FLO24T296I dramatically lost its ability to hydrolyze ATP and to rescue the thermotolerance defects of the yeast hsp104 mutant. The flo24 mutant develops more severe floury endosperm when grown under high-temperature conditions than normal conditions. And the FLO24 protein was dramatically induced at high temperature. FLO24 physically interacts with several key enzymes required for starch biosynthesis, including AGPL1, AGPL3 and PHO1. Combined biochemical and genetic evidence suggests that FLO24 acts cooperatively with HSP70cp-2 to regulate starch biosynthesis and endosperm development in rice. Our results reveal that FLO24 acts as an important regulator of endosperm development, which might function in maintaining the activities of enzymes involved in starch biosynthesis in rice.
Assuntos
Endosperma , Oryza , Proteínas de Plantas , Amido , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/biossíntese , Endosperma/genética , Endosperma/crescimento & desenvolvimento , Endosperma/metabolismo , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Mutação/genética , Oryza/genética , Oryza/metabolismo , Oryza/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Plastídeos/metabolismo , Ligação Proteica , Amido/biossíntese , Amido/genética , Termotolerância , Fatores de TranscriçãoRESUMO
BACKGROUND: Practical resistance of Helicoverpa zea to Cry proteins has become widespread in the US, making Vip3Aa the only effective Bacillus thuringiensis (Bt) protein for controlling this pest. Understanding the genetic basis of Vip3Aa resistance in H. zea is essential in sustaining the long-term efficacy of Vip3Aa. The objectives of this study were to characterize the inheritance of Vip3Aa resistance in four distinct field-derived H. zea strains (M1-RR, AC4-RR, R2-RR and R15-RR), and to test for shared genetic basis among these strains and a previously characterized Texas resistant strain (LT#70-RR). RESULTS: Maternal effects and sex linkage were absent, and the effective dominance level (DML) was 0.0 across Vip3Aa39 concentrations ranging from 1.0 to 31.6 µg cm-2, in all H. zea resistant strains. Mendelian monogenic model tests indicated that Vip3Aa resistance in each of the four strains was controlled by a single gene. However, interstrain complementation tests indicated that three distinct genetic loci are involved in Vip3Aa resistance in the five resistant H. zea strains: one shared by M1-RR and LT#70-RR; another shared by R2-RR and R15-RR; and a distinct one for AC4-RR. CONCLUSION: Results of this study indicate that Vip3Aa resistance in all H. zea strains was controlled by a single, recessive and autosomal gene. However, there were three distinct genetic loci associated with Vip3Aa resistance in the five resistant H. zea strains. The information generated from this study is valuable for exploring mechanisms of Vip3Aa resistance, monitoring the evolution of Vip3Aa resistance, and devising effective strategies for managing Vip3Aa resistance in H. zea. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
Assuntos
Proteínas de Bactérias , Resistência a Medicamentos , Mariposas , Mariposas/efeitos dos fármacos , Mariposas/genética , Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Resistência a Medicamentos/genética , Controle de Pragas/métodos , Dose Letal Mediana , Teste de Complementação Genética , Genes Recessivos/genética , AnimaisRESUMO
Peptidoglycan is an essential component of the bacterial cell envelope that contains glycan chains substituted by short peptide stems. Peptide stems are polymerized by D,D-transpeptidases, which make bonds between the amino acid in position four of a donor stem and the third residue of an acceptor stem (4-3 cross-links). Some bacterial peptidoglycans also contain 3-3 cross-links that are formed by another class of enzymes called L,D-transpeptidases which contain a YkuD catalytic domain. In this work, we investigate the formation of unusual bacterial 1-3 peptidoglycan cross-links. We describe a version of the PGFinder software that can identify 1-3 cross-links and report the high-resolution peptidoglycan structure of Gluconobacter oxydans (a model organism within the Acetobacteraceae family). We reveal that G. oxydans peptidoglycan contains peptide stems made of a single alanine as well as several dipeptide stems with unusual amino acids at their C-terminus. Using a bioinformatics approach, we identified a G. oxydans mutant from a transposon library with a drastic reduction in 1-3 cross-links. Through complementation experiments in G. oxydans and recombinant protein production in a heterologous host, we identify an L,D-transpeptidase enzyme with a domain distantly related to the YkuD domain responsible for these non-canonical reactions. This work revisits the enzymatic capabilities of L,D-transpeptidases, a versatile family of enzymes that play a key role in bacterial peptidoglycan remodelling.
Assuntos
Proteínas de Bactérias , Gluconobacter oxydans , Modelos Moleculares , Peptidoglicano , Peptidil Transferases , Aminoácidos/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico/genética , Peptidoglicano/química , Peptidoglicano/genética , Peptidoglicano/metabolismo , Peptidil Transferases/química , Peptidil Transferases/genética , Peptidil Transferases/metabolismo , Software , Gluconobacter oxydans/enzimologia , Gluconobacter oxydans/genética , Biologia Computacional , Teste de Complementação Genética , Estrutura Terciária de ProteínaRESUMO
BRCA2 is a tumor-suppressor gene that is normally expressed in the breast and ovarian tissue of mammals. The BRCA2 protein mediates the repair of double-strand breaks (DSBs) using homologous recombination, which is a conserved pathway in eukaryotes. Women who express missense mutations in the BRCA2 gene are predisposed to an elevated lifetime risk for both breast cancer and ovarian cancer. In the present study, the efficiency of human BRCA2 (hBRCA2) in DSB repair was investigated in the budding yeast Saccharomyces cerevisiae. While budding yeast does not possess a true BRCA2 homolog, they have a potential functional homolog known as Rad52, which is an essential repair protein involved in mediating homologous recombination using the same mechanism as BRCA2 in humans. Therefore, to examine the functional overlap between Rad52 in yeast and hBRCA2, we expressed the wild-type hBRCA2 gene in budding yeast with or without Rad52 and monitored ionizing radiation resistance and DSB repair efficiency. We found that the expression of hBRCA2 in rad52 mutants increases both radiation resistance and DSB repair frequency compared to cells not expressing BRCA2. Specifically, BRCA2 improved the protection against ionizing radiation by at least 1.93-fold and the repair frequency by 6.1-fold. In addition, our results show that homology length influences repair efficiency in rad52 mutant cells, which impacts BRCA2 mediated repair of DSBs. This study provides evidence that S. cerevisiae could be used to monitor BRCA2 function, which can help in understanding the genetic consequences of BRCA2 variants and how they may contribute to cancer progression.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Animais , Feminino , Humanos , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Reparo do DNA/genética , Genes BRCA2 , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Teste de Complementação GenéticaRESUMO
N-terminal acetylation is a conserved protein modification among eukaryotes. The yeast Saccharomyces cerevisiae is a valuable model system for studying this modification. The bulk of protein N-terminal acetylation in S. cerevisiae is catalyzed by the N-terminal acetyltransferases NatA, NatB, and NatC. Thus far, proteome-wide identification of the in vivo protein substrates of yeast NatA and NatB has been performed by N-terminomics. Here, we used S. cerevisiae deleted for the NatC catalytic subunit Naa30 and identified 57 yeast NatC substrates by N-terminal combined fractional diagonal chromatography analysis. Interestingly, in addition to the canonical N-termini starting with ML, MI, MF, and MW, yeast NatC substrates also included MY, MK, MM, MA, MV, and MS. However, for some of these substrate types, such as MY, MK, MV, and MS, we also uncovered (residual) non-NatC NAT activity, most likely due to the previously established redundancy between yeast NatC and NatE/Naa50. Thus, we have revealed a complex interplay between different NATs in targeting methionine-starting N-termini in yeast. Furthermore, our results showed that ectopic expression of human NAA30 rescued known NatC phenotypes in naa30Δ yeast, as well as partially restored the yeast NatC Nt-acetylome. Thus, we demonstrate an evolutionary conservation of NatC from yeast to human thereby underpinning future disease models to study pathogenic NAA30 variants. Overall, this work offers increased biochemical and functional insights into NatC-mediated N-terminal acetylation and provides a basis for future work to pinpoint the specific molecular mechanisms that link the lack of NatC-mediated N-terminal acetylation to phenotypes of NatC deletion yeast.
Assuntos
Acetiltransferases N-Terminal , Saccharomyces cerevisiae , Humanos , Acetilação , Cromatografia Líquida , Sequência Conservada , Teste de Complementação Genética , Metionina/metabolismo , Acetiltransferase N-Terminal C/genética , Acetiltransferase N-Terminal C/metabolismo , Acetiltransferase N-Terminal E , Acetiltransferases N-Terminal/deficiência , Acetiltransferases N-Terminal/genética , Acetiltransferases N-Terminal/metabolismo , Fenótipo , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Especificidade por SubstratoRESUMO
Oxidative genome damage is an unavoidable consequence of cellular metabolism. It arises at gene regulatory elements by epigenetic demethylation during transcriptional activation1,2. Here we show that promoters are protected from oxidative damage via a process mediated by the nuclear mitotic apparatus protein NuMA (also known as NUMA1). NuMA exhibits genomic occupancy approximately 100 bp around transcription start sites. It binds the initiating form of RNA polymerase II, pause-release factors and single-strand break repair (SSBR) components such as TDP1. The binding is increased on chromatin following oxidative damage, and TDP1 enrichment at damaged chromatin is facilitated by NuMA. Depletion of NuMA increases oxidative damage at promoters. NuMA promotes transcription by limiting the polyADP-ribosylation of RNA polymerase II, increasing its availability and release from pausing at promoters. Metabolic labelling of nascent RNA identifies genes that depend on NuMA for transcription including immediate-early response genes. Complementation of NuMA-deficient cells with a mutant that mediates binding to SSBR, or a mitotic separation-of-function mutant, restores SSBR defects. These findings underscore the importance of oxidative DNA damage repair at gene regulatory elements and describe a process that fulfils this function.
Assuntos
Proteínas de Ciclo Celular , Dano ao DNA , Reparo do DNA , Estresse Oxidativo , Regiões Promotoras Genéticas , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Genes , Teste de Complementação Genética , Mitose , Mutação , Estresse Oxidativo/genética , Diester Fosfórico Hidrolases/metabolismo , Poli ADP Ribosilação , Regiões Promotoras Genéticas/genética , RNA/biossíntese , RNA/genética , RNA Polimerase II/metabolismo , Fuso Acromático/metabolismo , Sítio de Iniciação de TranscriçãoRESUMO
Rice, as one of the most aluminium (Al)-resistant cereal crops, has developed more complicated Al resistance mechanisms than others. By using forward genetic screening from a rice ethyl methanesulfonate mutant library, we obtained a mutant showing specifically high sensitivity to Al. Through MutMap analysis followed by a complementation test, we identified the causal gene, Al-related Protein Kinase (ArPK) for Al-sensitivity. ArPK expression was induced by a relatively longer exposure to high Al concentration in the roots. The result of RNA-sequencing indicated the functional disorder in arginine metabolism pathway with downregulation of N-acetylornithine deacetylase (NAOD) expression and upregulation of Ornithine decarboxylase1 (ODC1) expression in arpk mutant. Al specifically and rapidly upregulated ODC1 expression and causes overaccumulation of putrescine (Put), whereas the ODC inhibitor difluoromethylornithine reverted Al-sensitive phenotype of arpk, suggesting that overaccumulation of endogenous Put might be harmful for root growth, and that ArPK seems to act as an endogenous inhibitor of ODC1 action to maintain suitable endogenous Put level under Al treatment. Overall, we identified ArPK and its putative repressive role in controlling a novel ODC-dependent Put biosynthesis pathway specifically affecting rice Al resistance, thus enriching the fundamental understanding of plant Al resistance.