Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium,Halomonas elongata

1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with l-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had K_ms of 9.1 mM for l-glutamate and 4.5 mM for dl-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a K_m of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.Halotolerance is of considerable interest scientifically and from the perspective of wide application in fermentation industries and in agriculture. When eubacteria are exposed to hyperosmotic stress, they accumulate various low-molecular-weight organic compounds, the so-called “compatible solutes” such as polyols, amino acids, sugars, and betaines (7–9, 13, 19, 48), because maintenance of turgor pressure is a prerequisite for growth under the conditions of elevated external osmotic pressure. Since Galinski et al. (14) discovered 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) as a compatible solute in Ectothiorhodospira halochloris, an extremely halophilic phototrophic eubacterium, ectoine has been found to be distributed widely in nature, largely in moderately halophilic eubacteria (3, 11, 12, 26, 38, 50). In addition, ectoine has been investigated as a new excellent universal osmoprotectant in this decade, since incorporation of external ectoine under hyperosmotic stress has been observed to confer protection on various nonhalotolerant eubacteria (16, 21, 44).We previously isolated a moderately halophilic eubacterium, Halomonas elongata (31), from dry salty land in Thailand. We identified ectoine and γ-N-acetyl-α,γ-diaminobutyric acid (ADABA), which is one of the cleavage structures of ectoine, as osmotically responding compounds in the cells grown in a glucose-mineral medium containing NaCl in a concentration range of 3 to 15% (31). To understand the accumulation mechanism of the intracellular ectoine, characterization of enzymes involved in the biosynthesis of ectoine is indispensable. Therefore, we have focused on the biosynthetic enzyme of ectoine in this organism. We observed that radioactivity from [1-¹⁴C]aspartate was most efficiently incorporated into ectoine and that the signal intensity was enriched preferentially from [1-¹³C]acetate into the methyl carbon at position 2′ and from [2-¹³C]acetate into the methine carbon at position 2 of the ectoine skeleton, respectively, in ¹³C nuclear magnetic resonance (NMR) spectroscopy (22). From these findings, we also hypothesized the following pathway essentially similar to that described by Peters et al. (34): aspartic β-semialdehyde (ASA) is converted to 2,4-diaminobutyric acid (DABA) by transamination, and DABA is converted to ADABA by acetylation with acetyl coenzyme A (CoA), which in turn yields ectoine by circularization (Fig. ​(Fig.1).1). The three enzymes involved in this pathway are DABA aminotransferase, DABA acetyltransferase, and ectoine synthase in order of the reactions to ectoine. Peters et al. (34) detected the activity of the first and the second of the three steps by using crude extracts of E. halochloris and H. elongata. However, the characterization of these enzymes was limited; in particular, their responses to various salt concentrations remained unknown. Open in a separate windowFIG. 1Proposed biosynthetic pathway of ectoine in H. elongata {"type":"entrez-protein","attrs":{"text":"OUT30018","term_id":"1200192464","term_text":"OUT30018"}}OUT30018.In this study, we confirmed the biosynthetic pathway of ectoine by using purified enzymes in H. elongata {"type":"entrez-protein","attrs":{"text":"OUT30018","term_id":"1200192464","term_text":"OUT30018"}}OUT30018 and characterized the three enzymes involved in the conversion of ASA to ectoine for the first time.     

Genetic and transcriptional analysis of a novel plasmid-encoded copper resistance operon from Lactococcus lactis

A plasmid-borne copper resistance operon (lco) was identified from Lactococcus lactis subsp. lactis LL58-1. The lco operon consists of three structural genes lcoABC. The predicted products of lcoA and lcoB were homologous to chromosomally encoded prolipoprotein diacylglyceral transferases and two uncharacterized proteins respectively, and the product of lcoC is similar to several multicopper oxidases, which are generally plasmid-encoded. This genetic organization represents a new combination of genes for copper resistance in bacteria. The three genes are co-transcribed from a copper-inducible promoter, which is controlled by lcoRS encoding a response regulator and a kinase sensor. The five genes are flanked by two insertion sequences, almost identical to IS-LL6 from L. lactis. Transposon mutagenesis and subcloning analysis indicated that the three structural genes were all required for copper resistance. Copper assay results showed that the extracellular concentration of copper of L. lactis LM0230 containing the lco operon was significantly higher than that of the host strain when copper was added at concentrations from 2 to 3 mM. The results suggest that the lco operon conferred copper resistance by reducing the intracellular accumulation of copper ions in L. lactis.     

Development of food-grade cloning and expression vectors for Lactococcus lactis

AIMS: To develop food-grade cloning and expression vectors for use in genetic modification of Lactococcus lactis. METHODS AND RESULTS: Two plasmid replicons and three dominant selection markers were isolated from L. lactis and used to construct five food-grade cloning vectors. These vectors were composed of DNA only from L. lactis and contained no antibiotic resistance markers. Three of the vectors (pND632, pND648 and pND969) were based on the same plasmid replicon and carried, either alone or in combination, the three different selectable markers encoding resistance to nisin, cadmium and/or copper. The other two (pND965DJ and pND965RS) were derived from a cadmium resistance plasmid, and carried a constitutive promoter and a copper-inducible promoter, respectively, immediately upstream of a multicloning site. All vectors were stable in L. lactis LM0230 for at least 40 generations without selection pressure. The two groups of vectors were compatible in L. lactis LM0230. The vectors pND648 and pND965RS, as representatives of the two groups, were transferred successfully by electroporation into and maintained in an industrial strain of L. lactis. The usefulness of the vectors was further demonstrated by expressing a phage resistance gene (abiI) in another industrial strain of L. lactis. CONCLUSIONS: The five food-grade vectors constructed are potentially useful for industrial strains of L. lactis. SIGNIFICANCE AND IMPACT OF THE STUDY: These vectors represent a new set of molecular tools useful for food-grade modifications of L. lactis.