Shuang-Jiang Liu

Functional Identification of Novel Genes Involved in the Glutathione-Independent Gentisate Pathway in Corynebacterium glutamicum

ABSTRACT Corynebacterium glutamicum used gentisate and 3-hydroxybenzoate as its sole carbon and energy source for growth. By genome-wide data mining, a gene cluster designated ncg12918-ncg12923 was proposed to encode putative proteins involved in gentisate/3-hydroxybenzoate pathway. Genes encoding gentisate 1,2-dioxygenase (ncg12920) and fumarylpyruvate hydrolase (ncg12919) were identified by cloning and expression of each gene in Escherichia coli. The gene of ncg12918 encoding a hypothetical protein (Ncg12918) was proved to be essential for gentisate-3-hydroxybenzoate assimilation. Mutant strain RES167Δncg12918 lost the ability to grow on gentisate or 3-hydroxybenzoate, but this ability could be restored in C. glutamicum upon the complementation with pXMJ19-ncg12918. Cloning and expression of this ncg12918 gene in E. coli showed that Ncg12918 is a glutathione-independent maleylpyruvate isomerase. Upstream of ncg12920, the genes ncg12921-ncg12923 were located, which were essential for gentisate and/or 3-hydroxybenzoate catabolism. The Ncg12921 was able to up-regulate gentisate 1,2-dioxygenase, maleylpyruvate isomerase, and fumarylpyruvate hydrolase activities. The genes ncg12922 and ncg12923 were deduced to encode a gentisate transporter protein and a 3-hydroxybenzoate hydroxylase, respectively, and were essential for gentisate or 3-hydroxybenzoate assimilation. Based on the results obtained in this study, a GSH-independent gentisate pathway was proposed, and genes involved in this pathway were identified.

Key Role of Cysteine Residues in Catalysis and Subcellular Localization of Sulfur Oxygenase-Reductase of Acidianus tengchongensis

ABSTRACT Analysis of known sulfur oxygenase-reductases (SORs) and the SOR-like sequences identified from public databases indicated that they all possess three cysteine residues within two conserved motifs (V-G-P-K-V-C31 and C101-X-X-C104; numbering according to the Acidianus tengchongensis numbering system). The thio-modifying reagent N-ethylmaleimide and Zn2+ strongly inhibited the activities of the SORs of A. tengchongensis, suggesting that cysteine residues are important. Site-directed mutagenesis was used to construct four mutant SORs with cysteines replaced by serine or alanine. The purified mutant proteins were investigated in parallel with the wild-type SOR. Replacement of any cysteine reduced SOR activity by 98.4 to 100%, indicating that all the cysteine residues are crucial to SOR activities. Circular-dichroism and fluorescence spectrum analyses revealed that the wild-type and mutant SORs have similar structures and that none of them form any disulfide bond. Thus, it is proposed that three cysteine residues, C31 and C101-X-X-C104, in the conserved domains constitute the putative binding and catalytic sites of SOR. Furthermore, enzymatic activity assays of the subcellular fractions and immune electron microscopy indicated that SOR is not only present in the cytoplasm but also associated with the cytoplasmic membrane of A. tengchongensis. The membrane-associated SOR activity was colocalized with the activities of sulfite:acceptor oxidoreductase and thiosulfate:acceptor oxidoreductase. We tentatively propose that these enzymes are located in close proximity on the membrane to catalyze sulfur oxidation in A. tengchongensis.

Production of poly(malic acid) from different carbon sources and its regulation in Aureobasidium pullulans

Aureobasidium pullulans CBS 591.75 produced poly(malic acid) (PMA) from glucose, sucrose and succinate. A suitable glucose concentration for PMA production was about 50 g/L. Trifluoroacetic acid inhibited while malonate, maleate, succinate and malate increased the production of PMA; for example supplementation of succinate up to 6.5 g/L to the medium for example increased PMA production by 44%. Co-feeding of trifluoroacetic acid plus succinate or malate relieved the inhibition by trifluoroacetic acid. These results showed that PMA synthesis is probably related to the citric acid cycle and also indicated that isocitrate lyase and malate synthase participate in PMA biosynthesis.

Rhodopseudomonas faecalis sp. nov., a phototrophic bacterium isolated from an anaerobic reactor that digests chicken faeces.

Five isolates of a previously undescribed species of purple non-sulfur phototrophic bacteria were characterized. They were Gram-negative, had mobile, budding vibrioid cells and contained lamellar intracytoplasmic membranes. Cultures produced red pigments in the light. Live cells of photosynthetic cultures exhibited absorption maxima at 382, 460-464, 494-496, 534-538, 596, 804-806 and 870-874 nm, indicating the presence of bacteriochlorophyll a and carotenoids belonging to the spirilloxanthin series in cells. The new isolates grew anaerobically or microaerobically in the light, but not aerobically in the dark. Optimal growth occurred at 35-40 degrees C and at pH 6.5-8.5. Various organic compounds were used as photosynthetic electron donors and carbon sources. Sulfate was used as sulfur source for growth. Ubiquinone 10 was synthesized as the major quinone. A phylogenetic analysis based on 16S rRNA gene sequences revealed that strain gc(T), a representative of the new isolates, was closest to Rhodopseudomonas palustris, with a similarity of 97.5%. DNA-DNA hybridization further distinguished strain gc(T) from Rhodopseudomonas palustris at the species level. Therefore, the name Rhodopseudomonas faecalis sp. nov. was proposed for the new isolates. The type strain is gc(T) ( = AS1.2176(T) = JCM 11668(T)).

Novel post-translational modification system of NifF protein exists in Klebsiella pneumoniae which is different from that in Escherichia coli

In Klebsiella pneumonia, regulation of the NifF protein (involved in N2-fixation) was not disscussed at the post-translational level until the recent discovery in Esherichia coli. Using a new K. pneumoniae strain MF, expressing high levels of NifF protein, we report a post-translational modification of NifF, which is different from that of E.coli. K. pneumoniae MF cultivated in L-broth and induced by IPTG had two forms of NifF protein (NifFα and NifFβ) that behaved similarly to the two forms of E. coli 71–18 NifF proteins (NifFI and NifFII) in non-SDS-PAGE. However, they behaved differently in response to the reduction by β-mercaptoethanol. NifFβ in the strain MF could not be reduced to NifFα, while NifFII from E. coli could be reduced to NifFI. Detection of NifF protein in different N2-fixing cells of K. pneumonia showed that only NifFβ existed, suggesting that this is the active form during N2-fixation. We conclude that post-translational control system of NifF during N2-fixation in K. pneumoniae is possibly different from that of E. coli.

Mechanistic studies of O2-resistant N2-fixation in N2-fixing Escherichia coli

Escherichia coli carrying the entire nif gene cluster from Klebsiella pneumoniae on a multicopy plasmid becomes more O2-resistant in a N-free medium as a result of the integration of the nif gene cluster into the chromosome and the loss of the plasmid (H.Iwahashi and J.Someya, Biochem. Biophys. Res. Comm. 1990, 168: 288–294). Our purpose is to characterize the physiological reason why the strain became O2-resistant by measuring the levels of nif proteins in cells under microaerobic conditions. The O2-resistant strain had a higher amount of NifH and a lower amount of NifL under microaerobic conditions (compared to that under anaerobic conditions), while the parent strain showed the opposite characteristics. Thus, the biochemical mechanism of the O2-resistant strain is attributed to the strains ability to synthesize and maintain a high amount of NifH and a low amount of NifL under microaerobic conditions.