Search

On the basis of the Thermoanaerobacter tengcongensis genome, a novel type of gal operon was deduced. The gene expression and biochemical properties of this operon were further characterized. RT-PCR analysis of the intergenic regions suggested that the transcription of the gal operon was continuous. With gene cloning and enzyme activity assays, TTE1929, TTE1928 and TTE1927 were identified to be GAIT, GaIK and GaIE, respectively. Results elicited from polarimetry assays revealed that TTE1925, a hypothetical protein, was a novel mutarotase, termed MR-Tt. TTE1926 was identified as a regulator that could bind to two operators in the operon promoter. The transcriptional start sites were mapped, and this suggested that there are two promoters in this operon. Expression of the gal genes was significantly induced by galactose, whereas only MR-Tt expression was detected in glucose-cultured T. tengcongensis at both the mRNA and the protein level. In addition, the abundance of gal proteins was examined at different temperatures. At temperatures ranging from 60 to 80[degrees]C, the level of MR-Tt protein was relatively stable, but that of the other gal proteins was dramatically decreased. The operator-binding complexes were isolated and identified by electrophoretic mobility shift assay-liquid chromatography (EMSA-LC) MS-MS, which suggested that several regulatory proteins, such as GalR and a sensory histidine kinase, participate in the regulation of the gal operon.

Summary: Nα‐acetyl‐α‐lysine was found as a new type of compatible solutes that acted as an organic cytoprotectant in the strain of Salinicoccus halodurans H3B36. A novel lysine Nα‐acetyltransferase gene (shkat), encoding an enzyme that catalysed the acetylation of lysine exclusively at α position, was identified from this moderate halophilic strain and expressed in Escherichia coli. Sequence analysis indicated ShKAT contained a highly conserved pyrophosphate‐binding loop (Arg‐Gly‐Asn‐Gly‐Asn‐Gly), which was a signature of the GNAT superfamily. ShKAT exclusively recognized free amino acids as substrate, including lysine and other basic amino acids. The enzyme showed a wide range of optimal pH value and was tolerant to high‐alkali and high‐salinity conditions. As a new member of the GNAT superfamily, the ShKAT was the first enzyme recognized free lysine as substrate. We believe this work gives an expanded perspective of the GNAT superfamily, and reveals great potential of the shkat gene to be applied in genetic engineering for resisting extreme conditions. [ABSTRACT FROM AUTHOR]

Attempts to identify members of the antiporter complement of the alkali- and saline-adapted soda lake alkaliphile Alkalimonas amylolytica N10 have used screens of DNA libraries in antiporter-deficient Escherichia coli KNabc. Earlier screens used [Na.sup.+] or [Li.sup.+] for selection but only identified one NhaD-type antiporter whose properties were inconsistent with a robust role in pH homeostasis. Here, new screens using elevated pH for selection identified three other putative antiporter genes that conferred resistance to pH [greater than or equal to] 8.5 as well as [Na.sup.+] resistance. The three predicted gene products were in the calcium/cation antiporter (CaCA), cation/proton antiporter-2 (CPA2) and cation/proton antiporter-1 (CPA1) families of membrane transporters, and were designated Aa-CaxA, Aa-KefB and Aa-NhaP respectively, reflecting homology within those families. Aa-CaxA conferred the poorest [Na.sup.+] resistance and also conferred modest [Ca.sup.2+] resistance. Aa-KefB and Aa-NhaP inhibited growth of a [K.sup.+] uptake-deficient E. coli mutant (TK2420), suggesting that they catalysed [K.sup.+] efflux. For Aa-NhaP, the reversibility of the growth inhibition by high [K.sup.+] concentrations depended upon an organic nitrogen source, e.g. glutamine, rather than ammonium. This suggests that [NH.sup.+.sub.4] as well as [K.sup.+] efflux is catalysed by Aa-NhaP. Vesicles of E. coli KNabc expressing Aa-NhaP, which conferred the strongest alkali resistance, exhibited [K.sup.+]/[H.sup.+] antiport activity in a pH range from 7.5 to 9.5, and with an apparent [K.sub, m] for [K.sup.+] of 0.5 mM at pH 8.0. The properties of this antiporter are consistent with the possibility that this soda lake alkaliphile uses [K.sup.+] (NH +)/[H.sup.+] antiport as part of its alkaline pH homeostasis mechanism and part of its capacity to reduce potentially toxic accumulation of cytoplasmic [K.sup.+] or N[H.sup.+.sub.4] respectively, under conditions of high osmolarity or active amino acid catabolism.

In extreme alkaliphiles, [Na.sup.+]/[H.sup.+] antiporters play a central role in the [Na.sup.+] cycle that supports pH homeostasis, [Na.sup.+] resistance, solute uptake, and motility. Properties of individual antiporters have only been examined in extremely alkaliphilic soil Bacillus spp., whereas the most alkaline natural habitats usually couple high pH with high salinity. Here, studies were conducted on a [Na.sup.+]([Li.sup.+])/[H.sup.+] antiporter, NhaD, from the soda lake haloalkaliphile Alkalimonas amylolytica. The activity profile of A. amylolytica NhaD at different pH values and [Na.sup.+] concentrations reflects its unique natural habitat. In membrane vesicles from antiporter-deficient Escherichia coli EP432 ([DELTA]nhaA [DeLTA]nhaB), the pH optimum for NhaD-dependent [Na.sup.+]([Li.sup.+])/[H.sup.+] antiport was at least 9.5, the highest pH that could be tested; no activity was observed at pH [less than or equal to]8.5. NhaD supported low [Na.sup.+]/[H.sup.+] antiport activity at pH 9.5 that was detectable over a range of [Na.sup.+] concentrations from 10 mM to at least 800 mM, with a 600 mM optimum. Although A. amylolytica nhaD was isolated by complementing the [Li.sup.+] sensitivity of the triple mutant E. coli strain KNabc ([DELTA]nhaA [DELTA]nhaB [DELTA]chaA), sustained propagation of nhaD-bearing plasmids in this strain resulted in a glycine ([GlyL.sup.327])[right arrow]serine mutation in a putative cytoplasmic loop of the mutant transporter. The altered activity profile of NhaD-G327S appears to be adaptive to the E. coli setting: a much higher activity than wild-type NhaD at [Na.sup.+] concentrations up to 200 mM but lower activity at 400 to 600 mM [Na.sup.+], with a pH optimum and minimal pH for activity lower than those of wild-type NhaD.

3-Ketosteroid-Δ1-dehydrogenases (KstDs [EC 1.3.99.4]) catalyze the Δ1-dehydrogenation of steroids and are a class of important enzymes for steroid biotransformations. In this study, nine putative kstD genes from different origins were selected and overexpressed in Escherichia coli BL21(DE3). These recombinant enzymes catalyzed the Δ1-desaturation of a variety of steroidal compounds. Among them, the KstD from Propionibacterium sp. (PrKstD) displayed the highest specific activity and broad substrate spectrum. The detailed catalytic characterization of PrKstD showed that it can convert a wide range of 3-ketosteroid compounds with diverse substituents, ranging from substituents at the C9, C10, C11 and C17 position through substrates without C4-C5 double bond, to previously inactive C6-substituted ones such as 11β,17-dihydroxy-6α-methyl-pregn-4-ene-3,20-dione. Reaction conditions were optimized for the biotransformation of hydrocortisone in terms of pH, temperature, co-solvent and electron acceptor. By using 50 g/L wet resting E. coli cells harboring PrKstD enzyme, the conversion of hydrocortisone was about 92.5% within 6 h at the substrate concentration of 80 g/L, much higher than the previously reported results, demonstrating the application potential of this new KstD. [ABSTRACT FROM AUTHOR]