Dataset Information

Identification and biochemical evidence of a medium-chain-length polyhydroxyalkanoate depolymerase in the Bdellovibrio bacteriovorus predatory hydrolytic arsenal.

ABSTRACT: The obligate predator Bdellovibrio bacteriovorus HD100 shows a large set of proteases and other hydrolases as part of its hydrolytic arsenal needed for its predatory life cycle. We present genetic and biochemical evidence that open reading frame (ORF) Bd3709 of B. bacteriovorus HD100 encodes a novel medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase (PhaZ(Bd)). The primary structure of PhaZ(Bd) suggests that this enzyme belongs to the ?/?-hydrolase fold family and has a typical serine hydrolase catalytic triad (serine-histidine-aspartic acid) in agreement with other PHA depolymerases and lipases. PhaZ(Bd) has been extracellularly produced using different hypersecretor Tol-pal mutants of Escherichia coli and Pseudomonas putida as recombinant hosts. The recombinant PhaZ(Bd) has been characterized, and its biochemical properties have been compared to those of other PHA depolymerases. The enzyme behaves as a serine hydrolase that is inhibited by phenylmethylsulfonyl fluoride. It is also affected by the reducing agent dithiothreitol and nonionic detergents like Tween 80. PhaZ(Bd) is an endoexohydrolase that cleaves both large and small PHA molecules, producing mainly dimers but also monomers and trimers. The enzyme specifically degrades mcl-PHA and is inactive toward short-chain-length polyhydroxyalkanoates (scl-PHA) like polyhydroxybutyrate (PHB). These studies shed light on the potentiality of these predators as sources of new biocatalysts, such as an mcl-PHA depolymerase, for the production of enantiopure hydroxyalkanoic acids and oligomers as building blocks for the synthesis of biobased polymers.

SUBMITTER: Martinez V

PROVIDER: S-EPMC3416617 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Identification and biochemical evidence of a medium-chain-length polyhydroxyalkanoate depolymerase in the Bdellovibrio bacteriovorus predatory hydrolytic arsenal.

Martínez Virginia V Martínez Virginia V de la Peña Fernando F García-Hidalgo Javier J de la Mata Isabel I García José Luis JL Prieto María Auxiliadora MA

Applied and environmental microbiology 20120615 17

The obligate predator Bdellovibrio bacteriovorus HD100 shows a large set of proteases and other hydrolases as part of its hydrolytic arsenal needed for its predatory life cycle. We present genetic and biochemical evidence that open reading frame (ORF) Bd3709 of B. bacteriovorus HD100 encodes a novel medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase (PhaZ(Bd)). The primary structure of PhaZ(Bd) suggests that this enzyme belongs to the α/β-hydrolase fold family and has a typical seri ...[more]

PMID: 22706067

Similar Datasets

Project description:UnlabelledOver the course of the last 3 decades the role of the second messenger cyclic di-GMP (c-di-GMP) as a master regulator of bacterial physiology was determined. Although the control over c-di-GMP levels via synthesis and breakdown and the allosteric regulation of c-di-GMP over receptor proteins (effectors) and riboswitches have been extensively studied, relatively few effectors have been identified and most are of unknown functions. The obligate predatory bacterium Bdellovibrio bacteriovorus has a peculiar dimorphic life cycle, in which a phenotypic transition from a free-living attack phase (AP) to a sessile, intracellular predatory growth phase (GP) is tightly regulated by specific c-di-GMP diguanylate cyclases. B. bacteriovorus also bears one of the largest complement of defined effectors, almost none of known functions, suggesting that additional proteins may be involved in c-di-GMP signaling. In order to uncover novel c-di-GMP effectors, a c-di-GMP capture-compound mass-spectroscopy experiment was performed on wild-type AP and host-independent (HI) mutant cultures, the latter serving as a proxy for wild-type GP cells. Eighty-four proteins were identified as candidate c-di-GMP binders. Of these proteins, 65 did not include any recognized c-di-GMP binding site, and 3 carried known unorthodox binding sites. Putative functions could be assigned to 59 proteins. These proteins are included in metabolic pathways, regulatory circuits, cell transport, and motility, thereby creating a potentially large c-di-GMP network. False candidate effectors may include members of protein complexes, as well as proteins binding nucleotides or other cofactors that were, respectively, carried over or unspecifically interacted with the capture compound during the pulldown. Of the 84 candidates, 62 were found to specifically bind the c-di-GMP capture compound in AP or in HI cultures, suggesting c-di-GMP control over the whole-cell cycle of the bacterium. High affinity and specificity to c-di-GMP binding were confirmed using microscale thermophoresis with a hypothetical protein bearing a PilZ domain, an acyl coenzyme A dehydrogenase, and a two-component system response regulator, indicating that additional c-di-GMP binding candidates may be bona fide novel effectors.ImportanceIn this study, 84 putative c-di-GMP binding proteins were identified in B. bacteriovorus, an obligate predatory bacterium whose lifestyle and reproduction are dependent on c-di-GMP signaling, using a c-di-GMP capture compound precipitation approach. This predicted complement covers metabolic, energy, transport, motility and regulatory pathways, and most of it is phase specific, i.e., 62 candidates bind the capture compound at defined modes of B. bacteriovorus lifestyle. Three of the putative binders further demonstrated specificity and high affinity to c-di-GMP via microscale thermophoresis, lending support for the presence of additional bona fide c-di-GMP effectors among the pulled-down protein repertoire.

Project description:Bdellovibrio bacteriovorus is a small Gram-negative, obligate predatory bacterium that is largely found in wet, aerobic environments (e.g., soil). This bacterium attacks and invades other Gram-negative bacteria, including animal and plant pathogens. The intriguing life cycle of B. bacteriovorus consists of two phases: a free-living nonreplicative attack phase, in which the predatory bacterium searches for its prey, and a reproductive phase, in which B. bacteriovorus degrades a host's macromolecules and reuses them for its own growth and chromosome replication. Although the cell biology of this predatory bacterium has gained considerable interest in recent years, we know almost nothing about the dynamics of its chromosome replication. Here, we performed a real-time investigation into the subcellular localization of the replisome(s) in single cells of B. bacteriovorus Our results show that in B. bacteriovorus, chromosome replication takes place only during the reproductive phase and exhibits a novel spatiotemporal arrangement of replisomes. The replication process starts at the invasive pole of the predatory bacterium inside the prey cell and proceeds until several copies of the chromosome have been completely synthesized. Chromosome replication is not coincident with the predator cell division, and it terminates shortly before synchronous predator filament septation occurs. In addition, we demonstrate that if this B. bacteriovorus life cycle fails in some cells of Escherichia coli, they can instead use second prey cells to complete their life cycle.IMPORTANCE New strategies are needed to combat multidrug-resistant bacterial infections. Application of the predatory bacterium Bdellovibrio bacteriovorus, which kills other bacteria, including pathogens, is considered promising for combating bacterial infections. The B. bacteriovorus life cycle consists of two phases, a free-living, invasive attack phase and an intracellular reproductive phase, in which this predatory bacterium degrades the host's macromolecules and reuses them for its own growth. To understand the use of B. bacteriovorus as a "living antibiotic," it is first necessary to dissect its life cycle, including chromosome replication. Here, we present a real-time investigation into subcellular localization of chromosome replication in a single cell of B. bacteriovorus This process initiates at the invasion pole of B. bacteriovorus and proceeds until several copies of the chromosome have been completely synthesized. Interestingly, we demonstrate that some cells of B. bacteriovorus require two prey cells sequentially to complete their life cycle.

Project description:The development of therapeutic and diagnostic antibodies is a rapidly growing field of research, being the fastest expanding group of products on the pharmaceutical market, and appropriate quality controls are crucial for their application. We have identified and characterized the serine protease termed BspK (Bdellovibrio serine protease K) from Bdellovibrio bacteriovorus and here show its activity on antibodies. Mutation of the serine residue at position 230 rendered the protease inactive. Further investigations of BspK enzymatic characteristics revealed autoproteolytic activity, resulting in numerous cleavage products. Two of the autoproteolytic cleavage sites in the BspK fusion protein were investigated in more detail and corresponded to cleavage after K28 and K210 in the N- and C-terminal parts of BspK, respectively. Further, BspK displayed stable enzymatic activity on IgG within the pH range of 6.0 to 9.5 and was inhibited in the presence of ZnCl2 BspK demonstrated preferential hydrolysis of human IgG1 compared to other immunoglobulins and isotypes, with hydrolysis of the heavy chain at position K226 generating two separate Fab fragments and an intact IgG Fc domain. Finally, we show that BspK preferentially cleaves its substrates C-terminally to lysines similar to the protease LysC. However, BspK displays a unique cleavage profile compared to several currently used proteases on the market. The rapid development of novel therapeutic antibodies is partly hindered by difficulties in assessing their quality and safety. The lack of tools and methods facilitating such quality controls obstructs and delays the process of product approval, eventually affecting the patients in need of treatment. These difficulties in product evaluations indicate a need for new and comprehensive tools for such analysis. Additionally, recent concerns raised regarding the limitations of established products on the market (e.g., trypsin) further highlight a general need for a larger array of proteases with novel cleavage profiles to meet current and future needs, within both the life science industry and the academic research community.

Dataset Information

Identification and biochemical evidence of a medium-chain-length polyhydroxyalkanoate depolymerase in the Bdellovibrio bacteriovorus predatory hydrolytic arsenal.

Publications

Identification and biochemical evidence of a medium-chain-length polyhydroxyalkanoate depolymerase in the Bdellovibrio bacteriovorus predatory hydrolytic arsenal.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets