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 Hypersensitivity 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 Escherichia coli significantly protected the recombinant host against cold and freeze stress.
BackgroundPenicillin 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.ResultsFusions 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.ConclusionsThis 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.
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.
Screening of an Antarctic soil functional fosmid metagenomic library identified a novel bacterial gene, homologous to known Water Hypersensitivity (WHy) domains. The WHy domain is a typical component of Late Embryogenesis Abundant (LEA) proteins which occurs widely in both prokaryotes and in plant eukaryotes and are expressed under various stress conditions [1]. A phylogenetic analysis of multiple WHy homologues from different species suggested that the ancestral origin of this protein gene lies within the ancient archaea [1]. Our previous studies have shown that this bacterial protein elicits significant protection against freeze and cold stress in recombinant E. coli [2]. Expression of the WHy gene in Arabidopsis resulted in a wide range of statistically significant stress-tolerant phenotypic properties. These included an increase of up to 6-fold higher germination efficiency of transgenic recombinant seeds compared to the WT, and a 100 % survival rate of WHy gene-expressing plants compared to 0 % survival of adult WT plants after freeze shock. Similar improvements in survival rates were observed for recombinant plants in drought stress experiments.
References
Mertens J, Aliyu H, Cowan DA (2018). Applied and environmental microbiology, AEM-00539.
Anderson D, Ferreras E, Trindade M, Cowan D (2015). FEMS Microbiology Letters, 362(15):fnv110.
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