No 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 RES167⌬ncg12918 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.Many members of Corynebacterium occur in soil and sediments and take part in decomposition of aromatic compounds (3,7,8,11,28,47). Other Corynebacterium species have been isolated from human or animal specimen or agricultural products (6, 55). The universal occurrence and versatile metabolism of organisms belonging to the genus Corynebacterium make this group of bacteria ecologically, medically, and economically important. For example, Corynebacterium glutamicum produces several amino acids used commercially and is a model organism for studying the physiology and biochemistry of this group of bacteria. Recently, the entire genome of C. glutamicum ATCC 13032 was sequenced (26, 33), and several gene clusters encoding enzymes related to aromatic compounds of catabolism have been revealed, but the identities and functions of these genes, except for a catechol 1,2-dioxygenase gene (46), have not been characterized.Gentisate and substituted gentisates are key intermediates during aerobic degradation of many aromatic compounds, such as 3-hydroxybenzoate (18, 31, 43), 3,5-or 2,5-xylenol (29, 41), salicylate (27, 40, 42), 3,6-dichloro-2-methoxybenzoate (54), and naphthalene (17,19,37). In the gentisate pathway, maleylpyruvate is produced, following the aromatic ring cleavage catalyzed by gentisate 1,2-dioxygenase. Further conversion of maleylpyruvate to central metabolic pathways proceeds via (i) direct hydrolysis to pyruvate and maleate (4, 24, 41) or (ii) isomerization to fumarylpyruvate and subsequent hydrolysis to fumarat...

To date, there has been no way to examine induced human p53 gene mutations in cell cultures exposed to mutagenic factors, other than by restriction site analysis. Here, we used embryonic cells from our Hupki (human p53 knock-in) mouse strain to generate human p53 DNA-binding domain (DBD) mutations experimentally. Twenty cultures of untreated primary mouse Hupki fibroblasts and 20 short-wavelength UV light (UVC)-treated cultures (20J͞m 2 ) were passaged >20 times. Established Hupki embryonic fibroblast cell lines (HUFs) were genotyped by dideoxy DNA sequencing of p53 exons 4 -9. Seven of the HUFs harbored point mutations in the humanized p53 DBD. Of the 9 mutations (6 single-and 1 triple-site mutation), 2 were at the most frequently mutated codons in human cancers (c.248 and c.273). The Affymetrix p53 GeneChip assay also readily identified the 6 single-base substitutions. All mutations in HUFs from UV-treated cultures were at dipyrimidine sites, including 3 nontranscribed strand C 3 T transitions. The mutant HUFs were deficient in p53 transactivation function, and missense mutants had high levels of nuclear p53 protein. In a second experiment, primary Hupki cells were exposed to the carcinogen aristolochic acid I (AAI). Five of 10 cultures that became established within 2 months harbored p53 DBD mutations. All were transversions, including 4 A 3 T substitutions on the nontranscribed strand, a hallmark of DNA mutation by AAI. We conclude that establishment of Hupki mouse fibroblasts in culture readily selects for p53 DBD mutations found in human tumors, providing a basis for generating experimental mutation patterns in human p53.tumor suppressor ͉ mutation spectrum ͉ gene targeting ͉ carcinogen

Comamonas sp. strain CNB-1 grows on 4-chloronitrobenzene (4-CNB) and nitrobenzene as sole carbon and nitrogen sources. In this study, two genetic segments, cnbB-orf2-cnbA and cnbR-orf1-cnbCaCbDEFGHI, located on a newly isolated plasmid, pCNB1 (ca. 89 kb), and involved in 4-CNB/nitrobenzene degradation, were characterized. Seven genes (cnbA, cnbB, cnbCa, cnbCb, cnbD, cnbG, and cnbH) were cloned and functionally expressed in recombinant Escherichia coli, and they were identified as encoding 4-CNB nitroreductase (CnbA), 1-hydroxylaminobenzene mutase (CnbB), 2-aminophenol 1,6-dioxygenase (CnbCab), 2-amino-5-chloromuconic semialdehyde dehydrogenase (CnbD), 2-hydroxy-5-chloromuconic acid (2H5CM) tautomerase, and 2-amino-5-chloromuconic acid (2A5CM) deaminase (CnbH). In particular, the 2A5CM deaminase showed significant identities (31 to 38%) to subunit A of Asp-tRNA Asn /Glu-tRNA Gln amidotransferase and not to the previously identified deaminases for nitroaromatic compound degradation. Genetic cloning and expression of cnbH in Escherichia coli revealed that CnbH catalyzed the conversion of 2A5CM into 2H5CM and ammonium. Four other genes (cnbR, cnbE, cnbF, and cnbI) were tentatively identified according to their high sequence identities to other functionally identified genes. It was proposed that CnbH might represent a novel type of deaminase and be involved in a novel partial reductive pathway for chloronitrobenzene or nitrobenzene degradation.Chlorinated nitroaromatic compounds such as chloronitrobenzenes are massively produced and are widely used as intermediates for chemical syntheses of drugs, herbicides, dyes, etc. The natural formation of chlorinated nitroaromatic compounds is rare, and most of these compounds are from industrial productions and have been introduced into the environment for a relatively short period. Apparently, their occurrence in the environment has selected microorganisms that are able to utilize chlorinated nitroaromatic compounds as carbon and/or nitrogen sources for growth. Examples of such microorganisms are bacterial strain LW1 (15), a coculture of Pseudomonas putida and a Rhodococcus sp. (25), and recently Comamonas sp. strain CNB-1 (38).Nitroaromatic compounds and chlorinated nitroaromatic compounds are structurally analogs. The microbial degradation of nitroaromatic compounds has been extensively investigated and the removal of the nitro group(s) is carried out via oxidative pathways that initiate with monooxygenases (22, 31, 40) or dioxygenases (8,16,20,19,32) or a partial reductive pathway that initiates with nitroreductases (7-9, 17, 22, 29, 30). Although structurally related to the nitroaromatic compounds, the chlorinated nitroaromatic compounds are more resistant to microbial degradation due to the simultaneous existence of chlorine and nitro groups, and thus the knowledge of its microbial degradation is very limited.Previous studies revealed that reductive dehalogenization (35) and partial reduction of nitro groups (15, 39) might be involved in the initial steps during chlorina...

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Human p53 mutation spectra differ significantly from one cancer type to another. One possible reason is that carcinogenic risk factors differ, and these factors elicit distinct mutation patterns. There has been no mammalian assay, however, with which to generate mutation patterns in human p53 sequences experimentally, hampering interpretation of the human tumor spectra. We have designed a new mammalian cell assay using gene targeting technology that selects and scores human p53 gene sequence mutations in human-p53 knock-in (Hupki) murine embryonic fibroblasts (HUF) that have undergone immortalization. With the Hupki assay we examined here whether benzo(a)pyrene (BaP), a major tobacco smoke carcinogen could elicit p53 mutation patterns characterizing the human lung tumor p53 mutation spectrum. We found that, in contrast to unexposed HUFs or HUFs exposed to other carcinogenic agents, HUFs exposed to BaP acquire mutations that display major features of the human lung tumor p53 mutation spectrum: (a) predominance of G-to-T mutations, (b) unequivocal strand bias of the transversions, and (c) a mutation hotspot at codons 157 to 158. These data are consistent with the hypothesis that BaP has a direct role in causing smokers' lung tumor p53 mutations. The assay can be used to examine various hypotheses on the endogenous or exogenous factors responsible for the p53 mutations in human tumors arising in other tissues. (Cancer Res 2005; 65(7): 2583-7)

Novosphingobium taihuense sp. nov., a novel aromatic-compound-degrading bacterium isolated from Taihu Lake, China

dThe prokaryotic community composition and diversity and the distribution patterns at various taxonomic levels across gradients of salinity and physiochemical properties in the surface waters of seven plateau lakes in the Qaidam Basin, Tibetan Plateau, were evaluated using Illumina MiSeq sequencing. These lakes included Lakes Keluke (salinity, <1 g/liter), Qing (salinity, 5.5 to 6.6 g/liter), Tuosu (salinity, 24 to 35 g/liter), Dasugan (salinity, 30 to 33 g/liter), Gahai (salinity, 92 to 96 g/liter), Xiaochaidan (salinity, 94 to 99 g/liter), and Gasikule (salinity, 317 to 344 g/liter). The communities were dominated by Bacteria in lakes with salinities of <100 g/liter and by Archaea in Lake Gasikule. The clades At12OctB3 and Salinibacter, previously reported only in hypersaline environments, were found in a hyposaline lake (salinity, 5.5 to 6.6 g/liter) at an abundance of ϳ1.0%, indicating their ecological plasticity. Salinity and the concentrations of the chemical ions whose concentrations covary with salinity (Mg 2؉ , K ؉ , Cl ؊ , Na ؉ , SO 4 2؊ , and Ca 2؉ ) were found to be the primary environmental factors that directly or indirectly determined the composition and diversity at the level of individual clades as well as entire prokaryotic communities. The distribution patterns of two phyla, five classes, five orders, five families, and three genera were well predicted by salinity. The variation of the prokaryotic community structure also significantly correlated with the dissolved oxygen concentration, pH, the total nitrogen concentration, and the PO 4 3؊ concentration. Such correlations varied depending on the taxonomic level, demonstrating the importance of comprehensive correlation analyses at various taxonomic levels in evaluating the effects of environmental variable factors on prokaryotic community structures. Our findings clarify the distribution patterns of the prokaryotic community composition in plateau lakes at the levels of individual clades as well as whole communities along gradients of salinity and ionic concentrations.