RESUMO
The genetic manipulation of marine double-stranded DNA (dsDNA) bacteriophage PM2 (Corticoviridae) has been limited so far. The isolation of an autonomously replicating DNA element of Pseudoalteromonas haloplanktis TAC125 and construction of a shuttle vector replicating in both Escherichia coli and Pseudoalteromonas enabled us to design a set of conjugative shuttle plasmids encoding tRNA suppressors for amber mutations. Using a host strain carrying a suppressor plasmid allows the introduction and analysis of nonsense mutations in PM2. Here, we describe the isolation and characterization of a suppressor-sensitive PM2 sus2 mutant deficient in the structural protein P10. To infect and replicate, PM2 delivers its 10-kbp genome across the cell envelopes of two gram-negative Pseudoalteromonas species. The events leading to the internalization of the circular supercoiled dsDNA are puzzling. In a poorly understood process that follows receptor recognition, the virion capsid disassembles and the internal membrane fuses with the host outer membrane. While beginning to unravel the mechanism of this process, we found that protein P10 plays an essential role in the host cell penetration.
Assuntos
Corticoviridae/genética , Pseudoalteromonas/virologia , Proteínas do Capsídeo/genética , Corticoviridae/crescimento & desenvolvimento , Corticoviridae/isolamento & purificação , DNA Circular/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/virologia , Vetores Genéticos/genética , Genoma Viral/genética , Modelos Genéticos , Mutagênese Sítio-Dirigida , Mutação , Fenótipo , Plasmídeos/genética , Pseudoalteromonas/genética , RNA de Transferência/genética , Água do Mar/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Micro-organisms that thrive at low temperatures produce cold-adapted enzymes which display high catalytic efficiency, generally associated with low thermal stability. In the recent past, researchers and industries have focused the attention on cold-adapted enzymes, whose peculiar properties make them particularly interesting either for investigating stability/flexibility relationships, or for their potential application in industrial processes. Among these enzymes, lipases and esterases, have potential utilisations in a broad range of biotechnological applications. In fact, these biocatalysts represent the most widely used enzymes in biotechnology and organic chemistry. Modern methods of genetic engineering combined with growing knowledge of structure and function allow further adaptation to industrial needs and exploration of novel applications. Hence, in this review we attempt to offer an overview on some psychrophilic esterases and lipases; major details will be presented for ORF PSHAa0051 from Pseudoalteromonas haloplanktis TAC125, recently investigated by our team. In addition, potential biotechnological applications will be discussed.
Assuntos
Proteínas de Bactérias/metabolismo , Temperatura Baixa , Esterases/metabolismo , Lipase/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Esterases/química , Esterases/genética , Lipase/química , Lipase/genética , Modelos Químicos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Pseudoalteromonas/enzimologia , Pseudoalteromonas/genética , Pseudoalteromonas/crescimento & desenvolvimento , Especificidade por SubstratoRESUMO
The role of lipopolysaccharides (LPSs) in the biogenesis of outer membrane proteins have been investigated in several studies. Some of these analyses showed that LPS is required for correct and efficient folding of outer membrane proteins; other studies support the idea of independence of outer membrane proteins biogenesis from LPS structure. In this article, we investigated the involvement of LPS structure in the anomalous aggregation of outer membrane proteins in a E. coli mutant strain (S17-1(lambdapir)). To achieve this aim, the LPS structure of the mutant strain was carefully determined and compared with the E. coli K-12 one. It turned out that LPS of these two strains differs in the inner core for the absence of a heptose residue (HepIII). We demonstrated that this difference is due to a mutation in waaQ, a gene encoding the transferase for the branch heptose HepIII residue. The mutation was complemented to find out if the restoration of LPS structure influenced the observed outer membrane proteins aggregation. Data reported in this work demonstrated that, in E. coli S17-1(lambdapir) there is no influence of LPS structure on the outer membrane proteins inclusion bodies formation.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Escherichia coli K12/química , Corpos de Inclusão/química , Lipopolissacarídeos/química , Mutação/genética , Sequência de Bases , Clonagem Molecular , Proteínas de Escherichia coli/genética , Cromatografia Gasosa-Espectrometria de Massas , Glicosiltransferases/genética , Dados de Sequência Molecular , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The genome of the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 was searched for the presence of genes encoding ester-hydrolysing enzymes. Amongst the others, the gene PSHAa0051 coding for a putative secreted esterase/lipase was selected. The psychrophilic gene was cloned, functionally over-expressed in P. haloplanktis TAC125, and the recombinant product (after named PhTAC125 Lip1) was purified. PhTAC125 Lip1 was found to be associated to the outer membrane and exhibited higher enzymatic activity towards synthetic substrates with long acyl chains. A structural model was constructed using the structure of carboxylesterase Est30 from Geobacillus stearothermophilus as template. The model covered the central part of the protein with the exceptions of PhTAC125 Lip1 N- and C-terminal regions, where the psychrophilic protein displays extra-domains. The constructed model showed a typical alpha/beta-hydrolase fold, and confirmed the presence of a canonical catalytic triad consisting of Ser, Asp and His. The sequence analysis showed that PhTAC125 Lip1 is distantly related to other lipolytic enzymes, but closely related to other putative psychrophilic esterases/lipases. The aligned proteins share common features, such as: (1) a conserved new active-site pentapeptide motif (LGG(F/L/Y)STG); (2) the likely extra-cytoplasmic localization, (3) the absence of a typical calcium-binding pocket, and (4) the absence of a canonical lid. These observations strongly suggest that aligned proteins constitute a novel lipase family, typical of psychrophilic marine gamma-proteobacteria, and PhTAC125 Lip1 could be considered the first characterised member of this family.
Assuntos
Proteínas de Bactérias/metabolismo , Carboxilesterase/metabolismo , Temperatura Baixa , Lipase/metabolismo , Lipólise , Pseudoalteromonas/enzimologia , Adaptação Fisiológica , Sequência de Aminoácidos , Regiões Antárticas , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Carboxilesterase/química , Carboxilesterase/genética , Carboxilesterase/isolamento & purificação , Clonagem Molecular , Ativação Enzimática , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Vetores Genéticos , Concentração de Íons de Hidrogênio , Lipase/química , Lipase/genética , Lipase/isolamento & purificação , Fluidez de Membrana , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Pseudoalteromonas/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína , Especificidade por Substrato , TemperaturaRESUMO
The chemical structural variations induced by different growth temperatures in the lipooligosaccharide and exopolysaccharide components extracted from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC 125 are described. The increase in phosphorylation with the increase in growth temperature seems to be general, because it happens not only for the lipooligosaccharide but also for the exopolysaccharide. Structural variations in the lipid components of lipid A also occur. In addition, free lipid A is found at both 25 and 4 degrees C but not at 15 degrees C, which is the optimal growth temperature, suggesting a incomplete biosynthesis of the lipooligosaccharide component under the first two temperature conditions.