Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli

Background: Penicillin acylases (PACs) are enzymes of industrial relevance in the manufacture of beta-lactam antibiotics. Development of a PAC with a longer half-life under the reaction conditions used is essential for the improvement of the operational stability of the process. A gene encoding a homologue to Escherichia coli PAC was found in the genome of the thermophilic bacterium Thermus thermophilus (Tth) HB27. Because of the nature of this PAC and its complex maturation that is crucial to reach its functional heterodimeric final conformation, the overexpression of this enzyme in a heterologous mesophilic host was a challenge. Here we describe the purification and characterization of the PAC protein from Tth HB27 overexpressed in Escherichia coli. Results: Fusions to a superfolder green fluorescent protein and differential membrane solubilization assays indicated that the enzyme remains attached through its amino-terminal end to the outer side of the cytoplasmic membrane of Tth cells. In order to overexpress this PAC in E. coli cells, a variant of the protein devoid of its membrane anchoring segment was constructed. The effect of the co-expression of chaperones and calcium supplementation of the culture medium was investigated. The total production of PAC was enhanced by the presence of DnaK/J and GrpE and even more by trigger factor and GroEL/ES. In addition, 10 mM calcium markedly improved both PAC specific and volumetric activities. Recombinant PAC was affinity-purified and proper maturation of the protein was confirmed by SDS-PAGE and MALDI-TOF analysis of the subunits. The recombinant protein was tested for activity towards several penicillins, cephalosporins and homoserine lactones. Hydrophobic acyl-chain penicillins were preferred over the rest of the substrates. Penicillin K (octanoyl penicillin) was the best substrate, with the highest specificity constant value (16.12 mM-1.seg-1). The optimum pH was aprox. 4 and the optimum temperature was 75 [DEGREE SIGN]C. The half-life of the enzyme at this temperature was 9.2 h. Conclusions: This is the first report concerning the heterologous expression of a pac gene from a thermophilic microorganism in the mesophilic host E. coli. The recombinant protein was identified as a penicillin K-deacylating thermozymeL.T., A.H. and J.B. acknowledge funding from the Spanish Ministry of Science (grants CIT 010000-2009-29, RyC2006-02441) and an institutional grant from Fundación Ramón Areces to CBMS

A novel bacterial Water Hypersensitivity-like protein shows in vivo protection against cold and freeze damage

Metagenomic library screening, by functional or sequence analysis, has become an established method for the identification of novel genes and gene products, including genetic elements implicated in microbial stress response and adaptation. We have identified, using a sequence based approach, a fosmid clone from an Antarctic desert soil metagenome library containing a novel gene which codes for a protein homologous to a Water Hypersensitive domain (WHy). The WHy domain is typically found as a component of specific LEA (Late Embryogenesis Abundant) proteins, particularly the LEA-14 (LEA-8) variants, which occur widely in plants, nematodes, bacteria and archaea and which are typically induced by exposure to stress conditions. The novel Why-like protein, (165 amino acid, 18.6 kDa) exhibits a largely invariant NPN motif at the N-terminus and has high sequence identity to genes identified in Pseudomonas genomes. Expression of this protein in E. coli significantly protected the recombinant host against cold and freeze stress.South African National Antarctic Program of the National Research Foundation.http://femsle.oxfordjournals.org2016-08-31hb201

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Functional expression of a penicillin acylase from the extreme thermophile <it>Thermus thermophilus</it> HB27 in <it>Escherichia coli</it>

Abstract Background Penicillin acylases (PACs) are enzymes of industrial relevance in the manufacture of β-lactam antibiotics. Development of a PAC with a longer half-life under the reaction conditions used is essential for the improvement of the operational stability of the process. A gene encoding a homologue to Escherichia coli PAC was found in the genome of the thermophilic bacterium Thermus thermophilus (Tth) HB27. Because of the nature of this PAC and its complex maturation that is crucial to reach its functional heterodimeric final conformation, the overexpression of this enzyme in a heterologous mesophilic host was a challenge. Here we describe the purification and characterization of the PAC protein from Tth HB27 overexpressed in Escherichia coli. Results Fusions to a superfolder green fluorescent protein and differential membrane solubilization assays indicated that the native enzyme remains attached through its amino-terminal end to the outer side of the cytoplasmic membrane of Tth cells. In order to overexpress this PAC in E. coli cells, a variant of the protein devoid of its membrane anchoring segment was constructed. The effect of the co-expression of chaperones and calcium supplementation of the culture medium was investigated. The total production of PAC was enhanced by the presence of DnaK/J and GrpE and even more by trigger factor and GroEL/ES. In addition, 10 mM calcium markedly improved both PAC specific and volumetric activities. Recombinant PAC was affinity-purified and proper maturation of the protein was confirmed by SDS-PAGE and MALDI-TOF analysis of the subunits. The recombinant protein was tested for activity towards several penicillins, cephalosporins and homoserine lactones. Hydrophobic acyl-chain penicillins were preferred over the rest of the substrates. Penicillin K (octanoyl penicillin) was the best substrate, with the highest specificity constant value (16.12 mM-1.seg-1). The optimum pH was aprox. 4 and the optimum temperature was 75 °C. The half-life of the enzyme at this temperature was 9.2 h. Conclusions This is the first report concerning the heterologous expression of a pac gene from a thermophilic microorganism in the mesophilic host E. coli. The recombinant protein was identified as a penicillin K-deacylating thermozyme.</p

Draft genome sequence of Microbacterium sp. strain CH12i, isolated from shallow groundwater in Cape Hallett, Antarctica

The Antarctic continent is largely covered by an expansive ice sheet, but it harbors diverse terrestrial and aquatic habitats in the coastal ice-free continental margins. Here we present the draft genome of Microbacterium sp. CH12i, which was isolated from hypersaline, alkaline, and nutrient-rich groundwater from Cape Hallett, northern Victoria Land, Antarctica.Fil: Ferreras, Eloy R.. University of Pretoria; SudáfricaFil: De Maayer, Pieter. University of Pretoria; SudáfricaFil: Makhalanyane, Thulani P.. University of Pretoria; SudáfricaFil: Guerrero, Leandro Demián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of Pretoria; SudáfricaFil: Aislabie, Jackie M.. Crown Research Institutes. Landcare Research; Nueva ZelandaFil: Cowan, Don A.. University of Pretoria; Sudáfric

Spatial organization, group living and ecological correlates in low-density populations of Eurasian badgers, Meles meles

1. Territoriality and group living are described in a low-density population of Eurasian badgers, Meles meles L., by studying the patterns of spatial grouping and territory marking, as well as the differences between individuals in some of their characteristics (body condition and dispersal) and in their space use (seasonally, periods of activity and interaction between pairs of individuals) under strong seasonal fluctuations in the availability of the key resource ( young rabbits, Oryctolagus cuniculus L.). Finally, the role of the spatial distribution of the main prey ( young rabbits) in the development of sociality was also studied in order to test some of the assumptions and predictions of the resource dispersion hypothesis (RDH). 2. Badgers were territorial, showing a flexible system of territory marking, which includes the marking of the most used areas (sett-latrines at the centres of activity) and additionally, at the smaller territories, a system of border-latrines in the areas of contact between territories. The maximum use of border-latrines was associated with the reproductive season, and that of sett-latrines with the season of food scarcity. 3. In the study area where badgers had rabbits as main prey, territories were occupied by small groups of animals, formed by one adult female who reproduced, one adult male who also showed signs of reproductive activity, the cubs of the year (if there was reproduction) and some animals born during previous years, which remained in their natal territory until their dispersal (normally during the mating season of their third or fourth year of life). This system was not strictly fixed as males, given the opportunity, expanded their territories to encompass additional females. Territories in another study site were occupied by one adult female (marked), plus the cubs of the year and another adult individual (unmarked). 4. In winter and spring dominant females and subordinates used only a small fraction of their territories, moved short distances, at a low speed and covering small areas per night. These seasons corresponded with the reproduction of rabbits (highest food availability). Dominant females were the only individuals using all the territory available in the summer (lowest food availability), when badgers had the worst body condition. Food availability increased again in autumn, as did body condition, while range sizes were again reduced. Dominant males used the same proportion of their territories over all seasons. However, in winter (reproductive season) they moved faster, over longer distances, and covered larger areas per period of activity. These results indicate that use of space by dominant males was affected by different factors from that of dominant females and subordinates. 5. RDH does not seem to explain group living in our populations because: (a) territori- ality in each pair of primary animals was driven by different factors (trophic resources for females and females for males); (b) dominant males acted as expansionists; and (c) territory size was related to its richness and not to patch dispersion.6. We propose an integrative hypothesis to explain not only group formation but also interpopulation variability in the social organization of badgers within ecological, demographic and behavioural constraints and in the light of current theory on delayed dispersalPeer reviewe