RESUMO
Maltodextrin is a mixture of maltooligosaccharides, which are produced by the degradation of starch or glycogen. They are mostly composed of α-1,4- and some α-1,6-linked glucose residues. Genes presumed to code for the Enterococcus faecalis maltodextrin transporter were induced during enterococcal infection. We therefore carried out a detailed study of maltodextrin transport in this organism. Depending on their length (3 to 7 glucose residues), E. faecalis takes up maltodextrins either via MalT, a maltose-specific permease of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), or the ATP binding cassette (ABC) transporter MdxEFG-MsmX. Maltotriose, the smallest maltodextrin, is primarily transported by the PTS permease. A malT mutant therefore exhibits significantly reduced growth on maltose and maltotriose. The residual uptake of the trisaccharide is catalyzed by the ABC transporter, because a malT mdxF double mutant no longer grows on maltotriose. The trisaccharide arrives as maltotriose-6â³-P in the cell. MapP, which dephosphorylates maltose-6'-P, also releases Pi from maltotriose-6â³-P. Maltotetraose and longer maltodextrins are mainly (or exclusively) taken up via the ABC transporter, because inactivation of the membrane protein MdxF prevents growth on maltotetraose and longer maltodextrins up to at least maltoheptaose. E. faecalis also utilizes panose and isopanose, and we show for the first time, to our knowledge, that in contrast to maltotriose, its two isomers are primarily transported via the ABC transporter. We confirm that maltodextrin utilization via MdxEFG-MsmX affects the colonization capacity of E. faecalis, because inactivation of mdxF significantly reduced enterococcal colonization and/or survival in kidneys and liver of mice after intraperitoneal infection.IMPORTANCE Infections by enterococci, which are major health care-associated pathogens, are difficult to treat due to their increasing resistance to clinically relevant antibiotics, and new strategies are urgently needed. A largely unexplored aspect is how these pathogens proliferate and which substrates they use in order to grow inside infected hosts. The use of maltodextrins as a source of carbon and energy was studied in Enterococcus faecalis and linked to its virulence. Our results demonstrate that E. faecalis can efficiently use glycogen degradation products. We show here that depending on the length of the maltodextrins, one of two different transporters is used: the maltose-PTS transporter MalT, or the MdxEFG-MsmX ABC transporter. MdxEFG-MsmX takes up longer maltodextrins as well as complex molecules, such as panose and isopanose.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Enterococcus faecalis/enzimologia , Enterococcus faecalis/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Polissacarídeos/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Enterococcus faecalis/genética , Enterococcus faecalis/crescimento & desenvolvimento , Rim/microbiologia , Fígado/microbiologia , Maltose/farmacologia , Proteínas de Membrana Transportadoras/genética , Camundongos , Mutação , Oligossacarídeos/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Trissacarídeos/farmacologiaRESUMO
The bacterial phosphotransferase system (PTS) transports and phosphorylates sugars, but also carries out numerous regulatory functions. The ß-proteobacterium Neisseria meningitidis possesses an incomplete PTS unable to transport carbon sources because it lacks a membrane component. Nevertheless, the residual phosphorylation cascade is functional and the meningococcal PTS was therefore expected to carry out regulatory roles. Interestingly, a ΔptsH mutant (lacks the PTS protein HPr) exhibited reduced virulence in mice and after intraperitoneal challenge it was rapidly cleared from the bloodstream of BALB/c mice. The rapid clearance correlates with lower capsular polysaccharide production by the ΔptsH mutant, which is probably also responsible for its increased adhesion to Hec-1-B epithelial cells. In addition, compared to the wild-type strain more apoptotic cells were detected when Hec-1-B cells were infected with the ΔptsH strain. Coimmunoprecipitation revealed an interaction of HPr and P-Ser-HPr with the LysR type transcription regulator CrgA, which among others controls its own expression. Moreover, ptsH deletion caused increased expression of a ΦcrgA-lacZ fusion. Finally, the presence of HPr or phospho-HPr's during electrophoretic mobility shift assays enhanced the affinity of CrgA for its target sites preceding crgA and pilE, but HPr did not promote CrgA binding to the sia and pilC1 promoter regions.
Assuntos
Aderência Bacteriana , Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Neisseria meningitidis/metabolismo , Neisseria meningitidis/patogenicidade , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/química , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Fatores de Transcrição/metabolismo , Animais , Apoptose , Proteínas de Bactérias/genética , Células Epiteliais , Regulação Bacteriana da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Neisseria meningitidis/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Fosfotransferases/química , Fosfotransferases/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , VirulênciaRESUMO
Maltose and maltodextrins are formed during the degradation of starch or glycogen. Maltodextrins are composed of a mixture of maltooligosaccharides formed by α-1,4- but also some α-1,6-linked glucosyl residues. The α-1,6-linked glucosyl residues are derived from branching points in the polysaccharides. In Enterococcus faecalis, maltotriose is mainly transported and phosphorylated by a phosphoenolpyruvate:carbohydrate phosphotransferase system. The formed maltotriose-6â³-phosphate is intracellularly dephosphorylated by a specific phosphatase, MapP. In contrast, maltotetraose and longer maltooligosaccharides up to maltoheptaose are taken up without phosphorylation via the ATP binding cassette transporter MdxEFG-MsmX. We show that the maltose-producing maltodextrin hydrolase MmdH (GenBank accession no. EFT41964) in strain JH2-2 catalyzes the first catabolic step of α-1,4-linked maltooligosaccharides. The purified enzyme converts even-numbered α-1,4-linked maltooligosaccharides (maltotetraose, etc.) into maltose and odd-numbered (maltotriose, etc.) into maltose and glucose. Inactivation of mmdH therefore prevents the growth of E. faecalis on maltooligosaccharides ranging from maltotriose to maltoheptaose. Surprisingly, MmdH also functions as a maltogenic α-1,6-glucosidase, because it converts the maltotriose isomer isopanose into maltose and glucose. In addition, E. faecalis contains a glucose-producing α-1,6-specific maltodextrin hydrolase (GenBank accession no. EFT41963, renamed GmdH). This enzyme converts panose, another maltotriose isomer, into glucose and maltose. A gmdH mutant had therefore lost the capacity to grow on panose. The genes mmdH and gmdH are organized in an operon together with GenBank accession no. EFT41962 (renamed mmgT). Purified MmgT transfers glucosyl residues from one α-1,4-linked maltooligosaccharide molecule to another. For example, it catalyzes the disproportionation of maltotriose by transferring a glucosyl residue to another maltotriose molecule, thereby forming maltotetraose and maltose together with a small amount of maltopentaose.IMPORTANCE The utilization of maltodextrins by Enterococcus faecalis has been shown to increase the virulence of this nosocomial pathogen. However, little is known about how this organism catabolizes maltodextrins. We identified two enzymes involved in the metabolism of various α-1,4- and α-1,6-linked maltooligosaccharides. We found that one of them functions as a maltose-producing α-glucosidase with relaxed linkage specificity (α-1,4 and α-1,6) and exo- and endoglucosidase activities. A third enzyme, which resembles amylomaltase, exclusively transfers glucosyl residues from one maltooligosaccharide molecule to another. Similar enzymes are present in numerous other Firmicutes, such as streptococci and lactobacilli, suggesting that these organisms follow the same maltose degradation pathway as E. faecalis.
Assuntos
Proteínas de Bactérias/metabolismo , Enterococcus faecalis/enzimologia , Hidrolases/metabolismo , Polissacarídeos/biossíntese , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/genética , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Hidrolases/genética , Maltose/metabolismo , Oligossacarídeos/metabolismo , Óperon , Trissacarídeos/metabolismoRESUMO
Numerous bacteria possess transcription activators and antiterminators composed of regulatory domains phosphorylated by components of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). These domains, called PTS regulation domains (PRDs), usually contain two conserved histidines as potential phosphorylation sites. While antiterminators possess two PRDs with four phosphorylation sites, transcription activators contain two PRDs plus two regulatory domains resembling PTS components (EIIA and EIIB). The activity of these transcription regulators is controlled by up to five phosphorylations catalyzed by PTS proteins. Phosphorylation by the general PTS components EI and HPr is usually essential for the activity of PRD-containing transcription regulators, whereas phosphorylation by the sugar-specific components EIIA or EIIB lowers their activity. For a specific regulator, for example the Bacillus subtilis mtl operon activator MtlR, the functional phosphorylation sites can be different in other bacteria and consequently the detailed mode of regulation varies. Some of these transcription regulators are also controlled by an interaction with a sugar-specific EIIB PTS component. The EIIBs are frequently fused to the membrane-spanning EIIC and EIIB-mediated membrane sequestration is sometimes crucial for the control of a transcription regulator. This is also true for the Escherichia coli repressor Mlc, which does not contain a PRD but nevertheless interacts with the EIIB domain of the glucose-specific PTS. In addition, some PRD-containing transcription activators interact with a distinct EIIB protein located in the cytoplasm. The phosphorylation state of the EIIB components, which changes in response to the presence or absence of the corresponding carbon source, affects their interaction with transcription regulators. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).
Assuntos
Proteínas de Bactérias/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Transativadores/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Modelos Genéticos , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Fosforilação , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transativadores/genéticaRESUMO
In most firmicutes expression of the mannitol operon is regulated by MtlR. This transcription activator is controlled via phosphorylation of its regulatory domains by components of the phosphoenolpyruvate : carbohydrate phosphotransferase system (PTS). We found that activation of Bacillus subtilis MtlR also requires an interaction with the EIIB(Mtl) domain of the mannitol permease MtlA (EIICB(Mtl) ). The constitutive expression of the mtlAFD operon in an mtlF mutant was prevented when entire mtlA or only its 3' part (EIIB(Mtl) ) were deleted. Yeast two-hybrid experiments revealed a direct interaction of the EIIB(Mtl) domain with the two C-terminal domains of MtlR. Complementation of the Δ3'-mtlA ΔmtlF or ΔmtlAFD mutants with mtlA restored constitutive MtlR activity, whereas complementation with only 3'-mtlA had no effect. Moreover, synthesis of EIIB(Mtl) in strains producing constitutively active MtlR caused MtlR inactivation. Interestingly, EIIB(Mtl) fused to the trans-membrane protein YwqC restored constitutive MtlR activity in the above mutants. Replacing the phosphorylatable Cys with Asp in MtlA or soluble EIIB(Mtl) lowered MtlR activation, indicating that MtlR does not interact with phosphorylatyed EIIB(Mtl) . Induction of the B. subtilis mtl operon therefore follows a novel regulation mechanism where the transcription activator needs to be sequestered to the membrane by unphosphorylated EIICB(Mtl) in order to be functional.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/química , Óperon , Proteínas Repressoras/metabolismo , Bacillus subtilis/química , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Membrana Celular/química , Membrana Celular/enzimologia , Membrana Celular/genética , Manitol/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Repressoras/genéticaRESUMO
Listeria monocytogenes transports glucose/mannose via non-PTS permeases and phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTS). Two mannose class PTS are encoded by the constitutively expressed mpoABCD and the inducible manLMN operons. The man operon encodes the main glucose transporter because manL or manM deletion significantly slows glucose utilization, whereas mpoA deletion has no effect. The PTS(Mpo) mainly functions as a constitutively synthesized glucose sensor controlling man operon expression by phosphorylating and interacting with ManR, a LevR-like transcription activator. EIIB(Mpo) plays a dual role in ManR regulation: P~EIIB(Mpo) prevailing in the absence of glucose phosphorylates and thereby inhibits ManR activity, whereas unphosphorylated EIIB(Mpo) prevailing during glucose uptake is needed to render ManR active. In contrast to mpoA, deletion of mpoB therefore strongly inhibits man operon expression and glucose consumption. A ΔptsI (EI) mutant consumes glucose at an even slower rate probably via GlcU-like non-PTS transporters. Interestingly, deletion of ptsI, manL, manM or mpoB causes elevated PrfA-mediated virulence gene expression. The PTS(Man) is the major player in glucose-mediated PrfA inhibition because the ΔmpoA mutant showed normal PrfA activity. The four mutants showing PrfA derepression contain no or only little unphosphorylated EIIAB(Man) (ManL), which probably plays a central role in glucose-mediated PrfA regulation.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Glucose/metabolismo , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Fatores de Terminação de Peptídeos/metabolismo , Fatores de Virulência/metabolismo , Proteínas de Bactérias/genética , Deleção de Genes , Listeria monocytogenes/crescimento & desenvolvimento , Manose/metabolismo , Redes e Vias Metabólicas , Modelos Biológicos , Fatores de Terminação de Peptídeos/genética , Virulência , Fatores de Virulência/genéticaRESUMO
Many bacteria transport mannitol via the mtlAF-encoded phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). In most firmicutes the transcriptional activator MtlR controls expression of the mtl operon. MtlR possesses an N-terminal DNA binding domain, two PTS regulation domains (PRDs), an EIIB(Gat)- and EIIA(Mtl)-like domain. These four regulatory domains contain one or two potential PTS phosphorylation sites. Replacement of His-342 or His-399 in PRD2 with Ala prevented the phosphorylation of Bacillus subtilis MtlR by PEP, EI and HPr. These mutations as well as EI inactivation caused a loss of MtlR function in vivo. In contrast, phosphomimetic replacement of His-342 with Asp rendered MtlR constitutively active. The absence of phosphorylation in PRD2 serves as catabolite repression mechanism. When EIIA(Mtl) and the soluble EIIB(Mtl) domain of the EIICB(Mtl) permease were included in the phosphorylation mixture, His-599 in the EIIA-like domain of MtlR also became phosphorylated. Replacement of His-599 with Asp rendered MtlR inactive, while His599Ala replacement caused slightly constitutive, glucose-repressible MtlR activity. Doubly mutated His342Ala/His599Ala MtlR was still phosphorylated by EI, HPr and EIIA(Mtl) at Cys-419 in the EIIB(Gat)-like domain. Cys419Ala replacement and deletion of EIIA(Mtl) caused strong constitutive glucose-repressible MtlR activity. This is the first report that Cys phosphorylation controls PRD-containing transcriptional activators.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Óperon , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transcrição Gênica , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Cisteína/metabolismo , Genes Reporter , Glucose/metabolismo , Manitol/metabolismo , Fosforilação , Proteínas Repressoras/químicaRESUMO
The entire genome of Lactobacillus casei BL23, a strain with probiotic properties, has been sequenced. The genomes of BL23 and the industrially used probiotic strain Shirota YIT 9029 (Yakult) seem to be very similar.
Assuntos
Genoma Bacteriano/genética , Lacticaseibacillus casei/genética , Dados de Sequência MolecularRESUMO
Identifying the mechanisms of eukaryotic genome evolution by comparative genomics is often complicated by the multiplicity of events that have taken place throughout the history of individual lineages, leaving only distorted and superimposed traces in the genome of each living organism. The hemiascomycete yeasts, with their compact genomes, similar lifestyle and distinct sexual and physiological properties, provide a unique opportunity to explore such mechanisms. We present here the complete, assembled genome sequences of four yeast species, selected to represent a broad evolutionary range within a single eukaryotic phylum, that after analysis proved to be molecularly as diverse as the entire phylum of chordates. A total of approximately 24,200 novel genes were identified, the translation products of which were classified together with Saccharomyces cerevisiae proteins into about 4,700 families, forming the basis for interspecific comparisons. Analysis of chromosome maps and genome redundancies reveal that the different yeast lineages have evolved through a marked interplay between several distinct molecular mechanisms, including tandem gene repeat formation, segmental duplication, a massive genome duplication and extensive gene loss.
Assuntos
Evolução Molecular , Genes Fúngicos/genética , Genoma Fúngico , Leveduras/classificação , Leveduras/genética , Cromossomos Fúngicos/genética , Sequência Conservada/genética , Duplicação Gênica , Dados de Sequência Molecular , RNA Ribossômico/genética , RNA de Transferência/genética , Proteínas de Saccharomyces cerevisiae/genética , Sintenia/genética , Sequências de Repetição em Tandem/genéticaRESUMO
BACKGROUND: Many bacteria transport cellobiose via a phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). In Listeria monocytogenes, two pairs of soluble PTS components (EIIACel1/EIIBCel1 and EIIACel2/EIIBCel2) and the permease EIICCel1 were suggested to contribute to cellobiose uptake. Interestingly, utilization of several carbohydrates, including cellobiose, strongly represses virulence gene expression by inhibiting PrfA, the virulence gene activator. RESULTS: The LevR-like transcription regulator CelR activates expression of the cellobiose-induced PTS operons celB1-celC1-celA1, celB2-celA2, and the EIIC-encoding monocistronic celC2. Phosphorylation by Pâ¼His-HPr at His550 activates CelR, whereas phosphorylation by Pâ¼EIIBCel1 or Pâ¼EIIBCel2 at His823 inhibits it. Replacement of His823 with Ala or deletion of both celA or celB genes caused constitutive CelR regulon expression. Mutants lacking EIICCel1, CelR or both EIIACel exhibitedslow cellobiose consumption. Deletion of celC1 or celR prevented virulence gene repression by the disaccharide, but not by glucose and fructose. Surprisingly, deletion of both celA genes caused virulence gene repression even during growth on non-repressing carbohydrates. No cellobiose-related phenotype was found for the celC2 mutant. CONCLUSION: The two EIIA/BCel pairs are similarly efficient as phosphoryl donors in EIICCel1-catalyzed cellobiose transport and CelR regulation. The permanent virulence gene repression in the celA double mutant further supports a role of PTSCel components in PrfA regulation.
Assuntos
Proteínas de Bactérias/metabolismo , Celobiose/metabolismo , Listeria monocytogenes/enzimologia , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Proteínas de Bactérias/genética , Transporte Biológico , Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Fosforilação , VirulênciaRESUMO
We identified the genes encoding the proteins for the transport of glucose and maltose in Neisseria meningitidis strain 2C4-3. A mutant deleted for NMV_1892(glcP) no longer grew on glucose and deletion of NMV_0424(malY) prevented the utilization of maltose. We also purified and characterized glucokinase and α-phosphoglucomutase, which catalyze early catabolic steps of the two carbohydrates. N. meningitidis catabolizes the two carbohydrates either via the Entner-Doudoroff (ED) pathway or the pentose phosphate pathway, thereby forming glyceraldehyde-3-P and either pyruvate or fructose-6-P, respectively. We purified and characterized several key enzymes of the two pathways. The genes required for the transformation of glucose into gluconate-6-P and its further catabolism via the ED pathway are organized in two adjacent operons. N. meningitidis also contains genes encoding proteins which exhibit similarity to the gluconate transporter (NMV_2230) and gluconate kinase (NMV_2231) of Enterobacteriaceae and Firmicutes. However, gluconate might not be the real substrate of NMV_2230 because N. meningitidis was not able to grow on gluconate as the sole carbon source. Surprisingly, deletion of NMV_2230 stimulated growth in minimal medium in the presence and absence of glucose and drastically slowed the clearance of N. meningitidis cells from transgenic mice after intraperitoneal challenge.
Assuntos
Glucose/metabolismo , Maltose/metabolismo , Redes e Vias Metabólicas/genética , Neisseria meningitidis/metabolismo , Transporte Biológico , Deleção de Genes , Neisseria meningitidis/genética , ÓperonRESUMO
Transposon insertion into Listeria monocytogenes lmo2665, which encodes an EIIC of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), was found to prevent D-arabitol utilization. We confirm this result with a deletion mutant and show that Lmo2665 is also required for D-xylitol utilization. We therefore called this protein EIICAxl. Both pentitols are probably catabolized via the pentose phosphate pathway (PPP) because lmo2665 belongs to an operon, which encodes the three PTSAxl components, two sugar-P dehydrogenases, and most PPP enzymes. The two dehydrogenases oxidize the pentitol-phosphates produced during PTS-catalyzed transport to the PPP intermediate xylulose-5-P. L. monocytogenes contains another PTS, which exhibits significant sequence identity to PTSAxl. Its genes are also part of an operon encoding PPP enzymes. Deletion of the EIIC-encoding gene (lmo0508) affected neither D-arabitol nor D-xylitol utilization, although D-arabitol induces the expression of this operon. Both operons are controlled by MtlR/LicR-type transcription activators (Lmo2668 and Lmo0501, respectively). Phosphorylation of Lmo0501 by the soluble PTSAxl components probably explains why D-arabitol also induces the second pentitol operon. Listerial virulence genes are submitted to strong repression by PTS sugars, such as glucose. However, D-arabitol inhibited virulence gene expression only at high concentrations, probably owing to its less efficient utilization compared to glucose.
Assuntos
Metabolismo dos Carboidratos , Listeria monocytogenes/metabolismo , Álcoois Açúcares/metabolismo , Xilitol/metabolismo , Transporte Biológico , Biotransformação , Deleção de Genes , Listeria monocytogenes/enzimologia , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genéticaRESUMO
The hexitol D-mannitol is transported by many bacteria via a phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). In most Firmicutes, the transcription activator MtlR controls the expression of the genes encoding the D-mannitol-specific PTS components and D-mannitol-1-P dehydrogenase. MtlR contains an N-terminal helix-turn-helix motif followed by an Mga-like domain, two PTS regulation domains (PRDs), an EIIB(Gat)- and an EIIA(Mtl)-like domain. The four regulatory domains are the target of phosphorylation by PTS components. Despite strong sequence conservation, the mechanisms controlling the activity of MtlR from Lactobacillus casei, Bacillus subtilis and Geobacillus stearothermophilus are quite different. Owing to the presence of a tyrosine in place of the second conserved histidine (His) in PRD2, L. casei MtlR is not phosphorylated by Enzyme I (EI) and HPr. When the corresponding His in PRD2 of MtlR from B. subtilis and G. stearothermophilus was replaced with alanine, the transcription regulator was no longer phosphorylated and remained inactive. Surprisingly, L. casei MtlR functions without phosphorylation in PRD2 because in a ptsI (EI) mutant MtlR is constitutively active. EI inactivation prevents not only phosphorylation of HPr, but also of the PTS(Mtl) components, which inactivate MtlR by phosphorylating its EIIB(Gat)- or EIIA(Mtl)-like domain. This explains the constitutive phenotype of the ptsI mutant. The absence of EIIB(Mtl)-mediated phosphorylation leads to induction of the L. caseimtl operon. This mechanism resembles mtlARFD induction in G. stearothermophilus, but differs from EIIA(Mtl)-mediated induction in B. subtilis. In contrast to B. subtilis MtlR, L. casei MtlR activation does not require sequestration to the membrane via the unphosphorylated EIIB(Mtl) domain.
Assuntos
Proteínas de Bactérias/metabolismo , Sequência Conservada , Regulação Bacteriana da Expressão Gênica , Lacticaseibacillus casei/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Proteínas Repressoras/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/metabolismo , Lacticaseibacillus casei/genética , Manitol/metabolismo , Óperon , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Fosforilação , Ligação Proteica , Proteínas Repressoras/genéticaRESUMO
The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components.
Assuntos
Bactérias/metabolismo , Metabolismo dos Carboidratos , Fosfoenolpiruvato/metabolismo , Fosfotransferases/metabolismo , Proteínas de Bactérias/metabolismo , Transporte Biológico , Fosforilação , Ligação ProteicaRESUMO
Bacillus licheniformis alpha-amylase (BLA) is a highly thermostable starch-degrading enzyme that has been extensively studied in both academic and industrial laboratories. For over a decade, we have investigated BLA thermal properties and identified amino acid substitutions that significantly increase or decrease the thermostability. This paper describes the cumulative effect of some of the most beneficial point mutations identified in BLA. Remarkably, the Q264S-N265Y double mutation led to a rather limited gain in stability but significantly improved the amylolytic function. The most hyperthermostable variants combined seven amino acid substitutions and inactivated over 100 times more slowly and at temperatures up to 23 degrees C higher than the wild-type enzyme. In addition, two highly destabilizing mutations were introduced in the metal binding site and resulted in a decrease of 25 degrees C in the half-inactivation temperature of the double mutant enzyme compared with wild-type. These mutational effects were analysed by protein modelling based on the recently determined crystal structure of a hyperthermostable BLA variant. Our engineering work on BLA shows that the thermostability of an already naturally highly thermostable enzyme can be substantially improved and modulated over a temperature range of 50 degrees C through a few point mutations.