Cheng-Ying Jiang

Novel Partial Reductive Pathway for 4-Chloronitrobenzene and Nitrobenzene Degradation in Comamonas sp. Strain CNB-1

ABSTRACT 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-tRNAAsn/Glu-tRNAGln 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.

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.

The Complete Genome of Comamonas testosteroni Reveals Its Genetic Adaptations to Changing Environments

ABSTRACT Members of the gram-negative, strictly aerobic genus Comamonas occur in various environments. Here we report the complete genome of Comamonas testosteroni strain CNB-2. Strain CNB-2 has a circular chromosome that is 5,373,643 bp long and has a G+C content of 61.4%. A total of 4,803 open reading frames (ORFs) were identified; 3,514 of these ORFs are functionally assigned to energy production, cell growth, signal transduction, or transportation, while 866 ORFs encode hypothetical proteins and 423 ORFs encode purely hypothetical proteins. The CNB-2 genome has many genes for transportation (22%) and signal transduction (6%), which allows the cells to respond and adapt to changing environments. Strain CNB-2 does not assimilate carbohydrates due to the lack of genes encoding proteins involved in glycolysis and pentose phosphate pathways, and it contains many genes encoding proteins involved in degradation of aromatic compounds. We identified 66 Tct and nine TRAP-T systems and a complete tricarboxylic acid cycle, which may allow CNB-2 to take up and metabolize a range of carboxylic acids. This nutritional bias for carboxylic acids and aromatic compounds enables strain CNB-2 to occupy unique niches in environments. Four different sets of terminal oxidases for the respiratory system were identified, and they putatively functioned at different oxygen concentrations. This study conclusively revealed at the genomic level that the genetic versatility of C. testosteroni is vital for competition with other bacteria in its special niches.

Genetic Characterization of the Resorcinol Catabolic Pathway in Corynebacterium glutamicum

ABSTRACT Corynebacterium glutamicum grew on resorcinol as a sole source of carbon and energy. By genome-wide data mining, two gene clusters, designated NCgl1110-NCgl1113 and NCgl2950-NCgl2953, were proposed to encode putative proteins involved in resorcinol catabolism. Deletion of the NCgl2950-NCgl2953 gene cluster did not result in any observable phenotype changes. Disruption and complementation of each gene at NCgl1110-NCgl1113, NCgl2951, and NCgl2952 indicated that these genes were involved in resorcinol degradation. Expression of NCgl1112, NCgl1113, and NCgl2951 in Escherichia coli revealed that NCgl1113 and NCgl2951 both coded for hydroxyquinol 1,2-dioxygenases and NCgl1112 coded for maleylacetate reductases. NCgl1111 encoded a putative monooxygenase, but this putative hydroxylase was very different from previously functionally identified hydroxylases. Cloning and expression of NCgl1111 in E. coli revealed that NCgl1111 encoded a resorcinol hydroxylase that needs NADPH as a cofactor. E. coli cells containing Ncgl1111 and Ncgl1113 sequentially converted resorcinol into maleylacetate. NCgl1110 and NCgl2950 both encoded putative TetR family repressors, but only NCgl1110 was transcribed and functional. NCgl2953 encoded a putative transporter, but disruption of this gene did not affect resorcinol degradation by C. glutamicum. The function of NCgl2953 remains unclear.

Nucleotide Sequence of Plasmid pCNB1 from Comamonas Strain CNB-1 Reveals Novel Genetic Organization and Evolution for 4-Chloronitrobenzene Degradation

ABSTRACT The nucleotide sequence of a new plasmid pCNB1 from Comamonas sp. strain CNB-1 that degrades 4-chloronitrobenzene (4CNB) was determined. pCNB1 belongs to the IncP-1β group and is 91,181 bp in length. A total of 95 open reading frames appear to be involved in (i) the replication, maintenance, and transfer of pCNB1; (ii) resistance to arsenate and chromate; and (iii) the degradation of 4CNB. The 4CNB degradative genes and arsenate resistance genes were located on an extraordinarily large transposon (44.5 kb), proposed as TnCNB1. TnCNB1 was flanked by two IS1071 elements and represents a new member of the composite I transposon family. The 4CNB degradative genes within TnCNB1 were separated by various truncated genes and genetic homologs from other DNA molecules. Genes for chromate resistance were located on another transposon that was similar to the Tn21 transposon of the class II replicative family that is frequently responsible for the mobilization of mercury resistance genes. Resistance to arsenate and chromate were experimentally confirmed, and transcriptions of arsenate and chromate resistance genes were demonstrated by reverse transcription-PCR. These results described a new member of the IncP-1β plasmid family, and the findings suggest that gene deletion and acquisition as well as genetic rearrangement of DNA molecules happened during the evolution of the 4CNB degradation pathway on pCNB1.

Alicyclobacillus ferrooxydans sp. nov., a ferrous-oxidizing bacterium from solfataric soil

A novel mesophilic and acidophilic Gram-positive bacterium, designated strain TC-34(T), was isolated from solfataric soil. Strain TC-34(T) grew aerobically at 17-40 degrees C and pH 2.0-6.0, and optimally at 28 degrees C and pH 3.0. Analysis based on 16S rRNA gene sequences showed that strain TC-34(T) was phylogenetically related to members of the genus Alicyclobacillus, with the highest similarity (94.8 %) to Alicyclobacillus pomorum. Strain TC-34(T) showed a range of phenotypic characteristics that differentiated it from recognized Alicyclobacillus species, including growth temperature, assimilation of carbon sources and production of acids from a range of compounds. Strain TC-34(T) was able to oxidize ferrous iron and its growth was correlated with the oxidation of Fe(2+) in culture medium. omega-Alicyclic fatty acids were not detected. On the basis of these results, it was concluded that strain TC-34(T) represents a novel species of the genus Alicyclobacillus, for which the name Alicyclobacillus ferrooxydans (type strain TC-34(T)=JCM 15090(T)=CGMCC 1.6357(T)) is proposed.

Novel bacterial sulfur oxygenase reductases from bioreactors treating gold-bearing concentrates

The microbial community and sulfur oxygenase reductases of metagenomic DNA from bioreactors treating gold-bearing concentrates were studied by 16S rRNA library, real-time polymerase chain reaction (RT-PCR), conventional cultivation, and molecular cloning. Results indicated that major bacterial species were belonging to the genera Acidithiobacillus, Leptospirillum, Sulfobacillus, and Sphingomonas, accounting for 6.3, 66.7, 18.8, and 8.3%, respectively; the sole archaeal species was Ferroplasma sp. (100%). Quantitative RT-PCR revealed that the 16S rRNA gene copy numbers (per gram of concentrates) of bacteria and archaea were 4.59 × 109 and 6.68 × 105, respectively. Bacterial strains representing Acidithiobacillus, Leptospirillum, and Sulfobacillus were isolated from the bioreactors. To study sulfur oxidation in the reactors, pairs of new PCR primers were designed for the detection of sulfur oxygenase reductase (SOR) genes. Three sor-like genes, namely, sorFx, sorSA, and sorSB were identified from metagenomic DNAs of the bioreactors. The sorFx is an inactivated SOR gene and is identical to the pseudo-SOR gene of Ferroplasma acidarmanus. The sorSA and sorSB showed no significant identity to any genes in GenBank databases. The sorSB was cloned and expressed in Escherichiacoli, and SOR activity was determined. Quantitative RT-PCR determination of the gene densities of sorSA and sorSB were 1,000 times higher than archaeal 16S rRNA gene copy numbers, indicating that these genes were mostly impossible from archaea. Furthermore, with primers specific to the sorSB gene, this gene was PCR-amplified from the newly isolated Acidithiobacillus sp. strain SM-1. So far as we know, this is the first time to determine SOR activity originating from bacteria and to document SOR gene in bioleaching reactors and Acidithiobacillus species.

Alicyclobacillus aeris sp nov., a novel ferrous- and sulfur-oxidizing bacterium isolated from a copper mine

A novel mesophilic, acidophilic, endospore-forming bacterium, designated strain ZJ-6(T), was isolated from Zi-Jin copper mine in Inner Mongolia, China. Cells of strain ZJ-6(T) were rod-shaped, stained Gram-positive or were Gram-variable, and grew aerobically at 25-35 degrees C (optimum, 30 degrees C) and pH 2.0-6.0 (optimum, pH 3.5). 16S rRNA gene sequence analysis showed that strain ZJ-6(T) was related phylogenetically to members of the genus Alicyclobacillus, with 16S rRNA gene sequence similarities of 89.5-94.2 %. Cells contained MK-7 as the major quinone and the DNA G+C content was 51.2 mol%. Strain ZJ-6(T) possessed a number of phenotypic characteristics that differentiated it from recognized Alicyclobacillus species, including its growth temperature, assimilation of various carbon sources, production of acids from a range of compounds, and the ability to grow chemoautotrophically using ferrous iron, elemental sulfur and tetrathionate as electron donors. The predominant cellular fatty acids of strain ZJ-6(T) were anteiso-C(15 : 0) (67.1 %), iso-C(16 : 0) (7.7 %) and anteiso-C(17 : 0) (7.4 %); omega-alicyclic fatty acids were not found. On the basis of these results, it is concluded that strain ZJ-6(T) represents a novel species within the genus Alicyclobacillus, for which the name Alicyclobacillus aeris sp. nov. is proposed; the type strain is ZJ-6(T) (=CGMCC 1.7072(T)=NBRC 104953(T)).

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.

Comamonas testosteroni uses a chemoreceptor for tricarboxylic acid cycle intermediates to trigger chemotactic responses towards aromatic compounds.

Bacterial chemotaxis towards aromatic compounds has been frequently observed; however, knowledge of how bacteria sense aromatic compounds is limited. Comamonas testosteroni CNB‐1 is able to grow on a range of aromatic compounds. This study investigated the chemotactic responses of CNB‐1 to 10 aromatic compounds. We constructed a chemoreceptor‐free, non‐chemotactic mutant, CNB‐1Δ20, by disruption of all 19 putative methyl‐accepting chemotaxis proteins (MCPs) and the atypical chemoreceptor in strain CNB‐1. Individual complementation revealed that a putative MCP (tagged MCP2201) was involved in triggering chemotaxis towards all 10 aromatic compounds. The recombinant sensory domain of MCP2201 did not bind to 3‐ or 4‐hydroxybenzoate, protocatechuate, catechol, benzoate, vanillate and gentisate, but bound oxaloacetate, citrate, cis‐aconitate, isocitrate, α‐ketoglutarate, succinate, fumarate and malate. The mutant CNB‐1ΔpmdF that lost the ability to metabolize 4‐hydroxybenzoate and protocatechuate also lost its chemotactic response to these compounds, suggesting that taxis towards aromatic compounds is metabolism‐dependent. Based on the ligand profile, we proposed that MCP2201 triggers taxis towards aromatic compounds by sensing TCA cycle intermediates. Our hypothesis was further supported by the finding that introduction of the previously characterized pseudomonad chemoreceptor (McpS) for TCA cycle intermediates into CNB‐1Δ20 likewise triggered chemotaxis towards aromatic compounds.