Construction and characterization of Methylomicrobium alcaliphilum 20Z knockout mutants defective in sucrose and ectoine biosynthesis genes

253 Sucrose, the key plant carbohydrate, is synthesized by many phototrophic microorganisms and is supp posed to act as an osmoprotectant in cyanobacteria [1]. Sucrose was also found in halotolerant methyy lotrophic bacteria, where it accumulated in response to increasing salinity of the growth medium, which is in agreement with a potential osmoprotective function of this disaccharide [2]. It was also hypothesized that in some methanotrophs sucrose biosynthesis could act as a sink for excessively toxic formaldehyde [3]. Higher plants and cyanobacteria synthesize sucrose from UDPPglucose and fructosee66phosphate with intermediate formation of sucrosee66phosphate by sucrose phosphate synthase (SPS) and sucrose phosphate phosphatase (SPP) and/or from UDPPgluu cose and fructose with the involvement of sucrose synn thase [4]. Recently we have revealed a cluster of the spssspppfruKKams genes in the genome of the halotoll erant methanotroph Methylomicrobium alcaliphilum 20Z. The products of these genes were homologous to the characterized SPS and SPP of plants and cyanoo bacteria, as well as to fructokinases (FruK) and amyy losucrases (Ams) of heterotrophic bacteria utilizing sucrose as a growth substrate [5]. Although the arrangement of the sucrose metabolism genes within a single locus on the chromosome of Mm. alcaliphilum 20Z suggested the biochemical pathway of its biosynn thesis and subsequent decomposition, the role of sucrose was not quite clear, especially since ectoine is the predominant osmoprotectant in this organism [2]. In order to answer this intriguing question, we have obtained and characterized the Mm. alcaliphilum 20Z mutants defective in the sps and ams genes. We have also obtained the mutants with insertion of the kanaa mycin cassette in the ectBC genes encoding the enzymes of ectoine biosynthesis; their properties demm onstrated the osmoprotective function of sucrose. Mm. alcaliphilum 20Z (VKM BB2133 = NCIMB 14124) was grown in 7500mL shaker flasks containing 200 mL of the 2P medium [2] with different salt con centrations (1–7% NaCl). The sterile solution of 1 M NaHCO 3 (5 mL per 100 mL of the medium) was added as well. Methanol at a final concentration of 0.2% (vol/vol) was added as a carbon source. DNA was isolated from the cells by the standard methods [6]. The knockout mutants were obtained by double homologous recombination [7]. DNA fragg ments with the nucleotide sequences flanking the sps, ams, ectB, and ectC genes were cloned in the suicidal vector pCM184. The obtained plasmids pCMsps, pCMams, or pCMectBC were used to transform the cells …