pSW2, a Novel Low-Temperature-Inducible Gene Expression Vector Based on a Filamentous Phage of the Deep-Sea Bacterium Shewanella piezotolerans WP3

Abstract

A low-temperature-inducible protein expression vector (pSW2) based on a filamentous phage (SW1) of the deep-sea bacterium Shewanella piezotolerans WP3 was constructed. This vector replicated stably in Escherichia coli and Shewanella species, and its copy number increased at low temperatures. The pSW2 vector can be utilized as a complementation plasmid in WP3, and it can also be used for the production of complex cytochromes with multiple heme groups, which has the potential for application for metal ion recovery or bioremediation. Promoters of low-temperature-inducible genes in WP3 were fused into the vector to construct a series of vectors for enhancing protein expression at low temperature. The maximum green fluorescent protein intensity was obtained when the promoter for the hfq gene was used. The WP3/pSW2 system can efficiently produce a patatin-like protein (PLP) from a metagenomic library that tends to form inclusion bodies in E. coli. The yields of PLP in the soluble fraction were 8.3 mg/liter and 4.7 mg/liter of culture at 4°C and 20°C, respectively. Moreover, the pSW2 vector can be broadly utilized in other Shewanella species, such as S. oneidensis and S. psychrophila. The genus Shewanella can metabolize a broad range of metal and organic substances, such as U(VI), Tc(VII), and Cr(VI), trimethylamine-N-oxide (TMAO), dimethyl sulfoxide (DMSO), and fumarate (1). Therefore, Shewanella species are considered to be candidates for the bioremediation of metal and organic contaminants (2). Most of the earth's environments, such as the deep ocean and regions of permafrost, are at low temperatures; therefore, the application of Shewanella and/or other organisms for bioremediation in such environments would require strains that are adapted to low temperatures and an understanding of the effects of low temperatures on the metabolism of these organisms. Low-temperature-adapted (psychrophilic/psychrotrophic) Shewanella strains have been isolated from the deep sea, the Antarctic, and other cold environments (3-5). However, genetic manipulation tools or expression systems that can be utilized at low temperatures are limited (6), which has greatly hindered the research on low-temperature Shewanella organisms and their potential applications.Shewanella piezotolerans WP3 (here referred to as WP3) was isolated from deep-sea sediments. Although its optimal growth temperature is 20°C, it can grow within a temperature range of 0 to 28°C (7). WP3 contains a filamentous phage, named SW1 (7,718 bp) (8), that can exist as an extrachromosomal plasmid (doublestranded, replicative-form [RF] DNA) or integrate into the chromosome (7). The copy number of the SW1 RF DNA is temperature dependent and increases from approximately 10 copies at 20°C to approximately 30 copies at 4°C in the mid-stationary phase; the copy numbers peak in the late stationary phase at both temperatures (9). This low-temperature-induced SW1 phage could be used for the development of a genetic manipulation tool and a protein expression system.To our knowledge, mos...

Characterization of the relationship between polar and lateral flagellar structural genes in the deep-sea bacterium Shewanella piezotolerans WP3

“…The relative mRNA level of WP3 flagellar genes was quantified by the ΔΔC T method31. The amount of target was normalized to that of the reference gene swp2079 32, which has stable expression under various conditions relative to the calibrator (the transcription levels of the genes in WP3 wild-type strain were set as 1). The primer pairs (Table S1) used to amplify the selected genes via qPCR were designed using Primer Express software (Applied Biosystems, CA, USA).…”

Filamentous phages: masters of a microbial sharing economy

“…In the case of phage SW1 isolated from the deep‐sea bacterium Shewanella piezotolerans , the phage genes and virion secretion are induced at low temperatures and presence of the phage results in differential transcription of many genes in response to low temperature and high pressure including flagella genes which results in a reduced motility . A modified form of SW1 has been engineered into a low‐temperature inducible expression vector for biotechnology applications . Given the recent identification of filamentous phages from the hyperthermophile Thermus thermophilus , equivalent engineering could also generate high‐temperature expression vectors for biotechnology applications.…”

Enhancing the Adaptability of the Deep-Sea Bacterium Shewanella piezotolerans WP3 to High Pressure and Low Temperature by Experimental Evolution under H 2 O 2 Stress

“…The upregulated genes katE, ahpF, ahpC4, and ccpA2 in the ΔoxyRc strain were also found to be induced in OE100 at 20 MPa and 4°C, suggesting that these genes were under the control of OxyR and contribute to adaptation to HHP and LT. Complementation with OxyR did not restore the wild-type phenotype at 20 MPa and 4°C. Previously, the copy numbers of pSW2 in WP3 were determined (12 and 30 copies at 20°C and 4°C, respectively) (20). The transcription level of oxyR was upregulated by 18-fold in the complemented strain compared with the wild-type strain (Fig.…”