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An Acidic Exopolysaccharide from Haloarcula hispanica ATCC33960 and Two Genes Responsible for Its Syn 时间:2017-07-04 09:45 来源:未知作者:admin 点击: 次 Abstract:A 1.1 × 106 Da acidic exopolysaccharide (EPS) was purified from an extremely halophilic archaeon Haloarcula hispanica ATCC33960 with a production of 30 mg L−1 when grown in AS-168 medium, which mainly composed of mannose and galactose with a small amount of glucose in a molar ratio of 55.9 : 43.2 : 0.9. Two glycosyltransferase genes (HAH_1662 and HAH_1667) were identified to be responsible for synthesis of the acidic EPS. Deletion of either HAH_1662 or HAH_1667 led to loss of the acidic EPS. The mutants displayed a different cell surface morphology, retarded growth in low salty environment, an increased adhesion, and swimming ability. Our results suggest that biosynthesis of the acidic EPS might act as an adaptable mechanism to protect the cells against harsh environments.
1. Introduction
Microorganisms produce EPSs as a strategy for growing, adhering to solid surfaces, protective barrier, a reserve nutrient, and formation of a biofilm as an adaptive lifestyle to encourage the survival in harsh environments and under changing environmental conditions [1–4]. EPS produced by bacteria has a wide range of potential applications in many industrial fields in which emulsifying, viscosifying, antioxidant, and chelating agents are required [5–7].
In order to find EPSs with novel and valuable properties, several EPSs from haloarchaea have been isolated and investigated, such as Haloferax, Halococcus, Haloarcula, Natronococcus, Haloterrigena, and Halobacterium [8–12]. The structures of several haloarchaeal EPSs have been solved but little is known about their biosynthesis [13]. The repeat unit of EPS from Haloferax gibbonsii ATCC33959 contains one main chain and two branches. The main chain is composed of two mannosyl and two galactosyl moieties; one branch contains one glucosyl moiety and the other branch is composed of one galactosyl and one rhamnosyl moiety [10]. The EPS from Haloferax mediterranei ATCC 33500 was identified to be a heteropolysaccharide containing mannose as the major component [14]. The repeat unit of EPS in H. mediterranei contains one mannosyl and two N-acetyl-glucosaminuronyl moieties, and one N-acetyl-glucosaminuronyl group is modified by a sulfonic group [15]. Based on the complete genome sequence of H. mediterranei, a gene cluster involved in EPS biosynthesis in H. mediterranei was identified [16]. Deletion of the gene cluster eliminated EPS synthesis. The mutant strain deficient of EPS biosynthesis showed a remarkable decrease in viscosity and foaming propensity of culture broth, increase in content of dissolved oxygen, and enhanced production of PHBV [17].
Haloarcula hispanica is an extremely halophilic archaeon, originally isolated from a solar saltern in Spain, and a producer of an extracellular polymer that gave a typical mucous character of the colonies [18]. Har. hispanica displays particularly low restriction activity and is therefore one of the most tractable haloarchaea for archaeal genetic research [19]. In this study, we isolated and purified an acidic EPS from Har. hispanica ATCC33960. By the gene deletion method, HAH_1662 and HAH_1667 were identified to be responsible for biosynthesis of this acidic EPS. Also, the impact of the acidic EPS on growth of Har. hispanica was evaluated.
2. Materials and Methods
2.1. Strains and Culture Conditions
Haloarcula hispanica ATCC 33960 and its mutant strains were cultured in AS-168 medium (per 1 L, 5 g Bacto Casamino acids, 5 g Bacto yeast extract, 1 g sodium glutamate, 3 g trisodium citrate, 20 g MgSO4·7H2O, 2 g KCl, 200 g NaCl, 50 mg FeSO4·7H2O, 0.36 mg MnCl2·4H2O, pH 7.0). Plates contained 1% agar unless mentioned otherwise. Mevinolin (Sigma) was added to a final concentration of 5 μg/mL in AS-168 medium for the screening of pUBP pop-in strains and pWL102 complementary strains. For transformation of pWL-CBD plasmid, novobiocin (Calbiochem) was added to a final concentration of 0.16 μg/mL in AS-168 medium. Escherichia coli JM110 was grown in LB medium. When needed, ampicillin was added to a final concentration of 100 μg/mL for E. coli.