Psychrophilic Pseudomonas syringae requires trans-monounsaturated fatty acid for growth at higher temperature

Abstract

A psychrophilic bacterium, Pseudomonas syringae (Lz4W) from Antarctica, was used as a model system to establish a correlation, if any, between thermal adaptation, trans-fatty acid content and membrane fluidity. In addition, attempts were made to clone and sequence the cti gene of P. syringae (Lz4W) so as to establish its characteristics with respect to the cti of other Pseudomonas spp. and also to in vitro mutagenize the cti gene so as to generate a cti null mutant. The bacterium showed increased proportion of saturated and trans-monounsaturated fatty acids when grown at 28 degrees C compared to cells grown at 5 degrees C, and the membrane fluidity decreased with growth temperature. In the mutant, the trans-fatty acid was not synthesized, and the membrane fluidity also decreased with growth temperature, but the decrease was not to the extent that was observed in the wild-type cells. Thus, it would appear that synthesis of trans-fatty acid and modulation of membrane fluidity to levels comparable to the wild-type cells is essential for growth at higher temperatures since the mutant exhibits growth arrest at 28 degrees C. In fact, the cti null mutant-complemented strain of P. syringae (Lz4W-C30b) that was capable of synthesizing the trans-fatty acid was indeed capable of growth at 28 degrees C, thus confirming the above contention. The cti gene of P. syringae (Lz4W) that was cloned and sequenced exhibited high sequence identity with the cti of other Pseudomonas spp. and exhibited all the conserved features.

Importance of trmE for Growth of the Psychrophile Pseudomonas syringae at Low Temperatures

“…Southern analysis using genomic DNA of CSM1 (one of the cold-sensitive mutants) and ␣-32 P-labeled pOT182 (35,36) showed that CSM1 produced two bands, confirming that a single copy of the transposon was integrated (data not shown). The disrupted gene was identified by inverse PCR (46) using 35 cycles and transposon-specific outward primers (primers Ash 1 and Ash 3 for upstream regions and primers Ash 1 and Ash 2 for downstream regions) ( Table 2).…”

“…P. syringae was mutagenized with a Tn5 transposon-based suicide plasmid vector (pOT182) (35,42), and cold-sensitive mutants were identified based on their inability to grow or their delayed growth on plates incubated at 4°C for 1 week. Growth characteristics were also analyzed using a UV-visible spectrophotometer (Shimadzu, Kyoto, Japan) (36,37).…”

“…The disrupted gene was identified by inverse PCR (46) using 35 cycles and transposon-specific outward primers (primers Ash 1 and Ash 3 for upstream regions and primers Ash 1 and Ash 2 for downstream regions) ( Table 2). PCR-amplified products were purified and sequenced with a ABI Prism 3700 DNA sequencer (Applied Biosystems, California) (12,13,36,37) using Ash 1 and Ash 3.…”

“…For reverse transcription PCR RNA was isolated from P. syringae (1,13,36), and first-strand cDNA was synthesized by RT. The cDNA was then amplified using Ash 71 and Ash 72 for the ␤-galactosidase gene and Ash 5 and Ash 44 for trmE for 21 cycles (Table 2).…”

Marinomonas polaris sp. nov., a psychrohalotolerant strain isolated from coastal sea water off the subantarctic Kerguelen islands

“…For biochemical tests, cultures were grown at 22 uC on marine agar 2216 and tests were performed as described by Baumann et al (1984) and Smibert & Krieg (1994) potassium chloride and 0?028 g ferrous sulphate, supplemented with 0?2 % of the filter-sterilized carbon source (Romanenko et al, 2003). Fatty acid methyl esters were prepared from cells grown at 22 u C for 48 h according to the method of Sato & Murata (1988) and analysed as described by Kiran et al (2004). The modified method of Bligh & Dyer (1959) was employed to extract polar lipids and molybdenum blue reagent was used to detect lipids containing phosphate esters.…”