Purification and characterization of a novel type of protocatechuate 3,4-dioxygenase with the ability to oxidize 4-sulfocatechol

4-Aminobenzenesulfonate is degraded via 4-sulfocatechol by a mixed bacterial culture that consists of Hydrogenophaga palleronii strain S1 and Agrobacterium radiobacter strain S2. From the 4-sulfocatechol-degrading organism A. radiobacter strain S2, a dioxygenase that converted 4-sulfocatechol to 3-sulfomuconate was purified to homogeneity. The purified enzyme also converted protocatechuate with a similar catalytic activity to 3-carboxy-cis,cis-muconate. Furthermore, the purified enzyme oxidized 3,4-dihydroxyphenylacetate, 3,4-dihydroxycinnamate, catechol, and 3- and 4-methylcatechol. The enzyme had a mol. wt. of about 97,400 as determined by gel filtration and consisted of two different types of subunits with mol. wt. of about 23,000 and 28,500. The NH₂-terminal amino acid sequences of the two subunits were determined. An isofunctional dioxygenase was partially purified from H. palleronii strain S1. A. radiobacter strain S2 also induced, after growth with 4-sulfocatechol, an „ordinary“ protocatechuate 3,4-dioxygenase that did not oxidize 4-sulfocatechol. This enzyme was also purified to homogeneity, and its catalytic and structural characteristics were compared to the „4-sulfocatechol-dioxygenase“ from the same strain. Received: 5 February 1996 / Accepted: 18 April 1996     

Degradation of 4-aminobenzenesulfonate by a two-species bacterial coculture

The mutualistic interactions in a 4-aminobenzenesulfonate (sulfanilate) degrading mixed bacterial culture were studied. This coculture consisted of Hydrogenophaga palleronii strain S1 and Agrobacterium radiobacter strain S2. In this coculture only strain S1 desaminated sulfanilate to catechol-4-sulfonate, which did not accumulate in the medium but served as growth substrate for strain S2. During growth in batch culture with sulfanilate as sole source of carbon, energy, nitrogen and sulfur, the relative cell numbers (colony forming units) of both strains were almost constant. None of the strains reached a cell number which was more than threefold higher than the cell number of the second strain. A mineral medium with sulfanilate was inoculated with different relative cell numbers of both strains (relative number of colony forming units S1:S2 2200:1 to 1:500). In all cases, growth was found and the proportion of both strains moved towards an about equal value of about 3:1 (strain S1:strain S2). In contrast to the coculture, strain S1 did not grow in a mineral medium in axenic culture with 4-aminobenzenesulfonate or any other simple organic compound tested. A sterile culture supernatant from strain S2 enabled strain S1 to grow with 4-aminobenzenesulfonate. The same growth promoting effect was found after the addition of a combination of 4-aminobenzoate, biotin and vitamin B₁₂. Strain S1 grew with 4-aminobenzenesulfonate plus the three vitamins with about the same growth rate as the mixed culture in a mineral medium. When (resting) cells of strain S1 were incubated in a pure mineral medium with sulfanilate, up to 30% of the oxidized sulfanilate accumulated as catechol-4-sulfonate in the culture medium. In contrast, only minor amounts of catechol-4-sulfonate accumulated when strain S1 was grown with 4ABS in the presence of the vitamins.Abbreviations 4ABS 4-aminobenzenesulfonate - CFU colony forming units - 4CS catechol-4-sulfonate - 4HB 4-hydroxybenzoate     

Isolation and characterization of polycyclic aromatic hydrocarbons-degrading Sphingomonas sp. strain ZL5

A bacterial strain ZL5, capable of growing on phenanthrene as a sole carbon and energy source but not naphthalene, was isolated by selective enrichment from crude-oil-contaminated soil of Liaohe Oil Field in China. The isolate was identified as a Sphingomonas sp. strain on the basis of 16S ribosomal DNA analysis. Strain ZL5 grown on phenanthrene exhibited catechol 2,3-dioxygenase (C23O) activity but no catechol 1,2-dioxygenase, gentisate 1,2-dioxygenase, protocatechuate 3,4-dioxygenase and protocatechuate 4,5-dioxygenase activities. This suggests that the mode of cleavage of phenanthrene by strain ZL5 could be meta via the intermediate catechol, which is different from the protocatechuate way of other two bacteria, Alcaligenes faecelis AFK2 and Nocardioides sp. strain KP7, also capable of growing on phenanthrene but not naphthalene. A resident plasmid (approximately 60 kb in size), designated as pZL, was detected from strain ZL5. Curing the plasmid with mitomycin C and transferring the plasmid to E. coli revealed that pZL was responsible for polycyclic aromatic hydrocarbons degradation. The C23O gene located on plasmid pZL was cloned and overexpressed in E. coli JM109(DE3). The ring-fission activity of the purified C23O from the recombinant E. coli on dihydroxylated aromatics was in order of catechol > 4-methylcatechol > 3-methylcatechol > 4-chlorocatechol > 3,4-dihydroxyphenanthrene > 3-chlorocatechol.     

Genetic and biochemical characterization of a 4-hydroxybenzoate hydroxylase from <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum uses 4-hydroxybenzoic acid (4HBA) as sole carbon source for growth. Previous studies showed that 4HBA was taken up into cells via PcaK, and the aromatic ring was cleaved via protocatechuate 3,4-dioxygenase. In this study, the gene pobA _Cg (ncgl1032) involved in the conversion of 4HBA into 3,4-dihydroxybenzoate (protocatechuate) was identified, and the gene product PobA _Cg was characterized as a 4HBA 3-hydroxylase, which is a homodimer of PobA_Cg. The pobA _Cg is physically associated with pcaK and formed a putative operon, but the two genes were located distantly to the pca cluster, which encode other enzymes for 4HBA/protocatechuate degradation. This new 4HBA 3-hydroxylase is unique in that it prefers NADPH to NADH as a cosubstrate, although its sequence is similar to other 4HBA 3-hydroxylases that prefer NADH as a cosubstrate. Sited-directed mutagenesis on putative NADPH-binding sites, D38 and T42, further improved its affinity to NADPH as well as its catalytic efficiency.     

Key enzymes of the protocatechuate branch of the β-ketoadipate pathway for aromatic degradation inCorynebacterium glutamicum

Although the protocatechuate branch of the β-ketoadipate pathway in Gram⁻ bacteria has been well studied, this branch is less understood in Gram⁺ bacteria. In this study,Corynebacterium glutamicum was cultivated with protocatechuate,p-cresol, vanillate and 4-hydroxybenzoate as sole carbon and energy sources for growth. Enzymatic assays indicated that growing cells on these aromatic compounds exhibited protocatechuate 3,4-dioxygenase activities. Data-mining of the genome of this bacterium revealed that the genetic locusncg12314-ncg12315 encoded a putative protocatechuate 3,4-dioxygenase. The genes,ncg12314 andncg12315, were amplified by PCR technique and were cloned into plasmid (pET21aP34D). RecombinantEscherichia coli strain harboring this plasmid actively expressed protocatechuate 3,4-dioxygenase activity. Further, when this locus was disrupted inC. glutamicum, the ability to degrade and assimilate protocatechuate,p-cresol, vanillate or 4-hydroxybenzoate was lost and protocatechuate 3,4-dioxygenase activity was disappeared. The ability to grow with these aromatic compounds and protocatechuate 3,4-dioxygenase activity ofC. glutamicum mutant could be restored by gene complementation. Thus, it is clear that the key enzyme for ring-cleavage, protocatechuate 3,4-dioxygenase, was encoded byncg12314 andncg12315. The additional genes involved in the protocatechuate branch of the β-ketoadipate pathway were identified by mining the genome data publically available in the Gen Bank. The functional identification of genes and their unique organization inC. glutamicum provided new insight into the genetic diversity of aromatic compound degradation.     

Mineralization of 4-sulfophthalate by aPseudomonas strain isolated from the River Elbe

The bacteriumPseudomonas sp. strain RW31 isolated from the river Elbe utilized the ammonium salt of 4-sulfophthalate (4SPA) as sole source of carbon, sulfur, nitrogen, and energy and grew also with phthalate (PA) and several other aromatic compounds as sole carbon and energy source. The xenobiotic sulfo group of 4SPA was eliminated as sulfite, which transiently accumulated in the culture supernatant up to about 10 µM and was slowly oxidized to the stoichiometrical amount of sulfate. Biodegradation routes of 4SPA as well as of PA converged into the protocatechuate pathway and from found activities for the decarboxylation of 4,5-dihydroxyphthalate we deduce this compound the first rearomaticized intermediate after initial dioxygenation. Protocatechuate then underwentmeta-cleavage mediated by a protocatechuate 4,5-dioxygenase activity which was competitively inhibited by the structurally related compound 3,4,5-trihydroxybenzoate; protocatechuate accumulated in the medium up to an about 2 mM concentration. Indications for the presence of selective transport systems are presented.     

Peptide mass fingerprinting- and 2-DE/MS-based analysis of the biodegradation potential for monocyclic aromatic hydrocarbons in <Emphasis Type="Italic">Pseudomonas</Emphasis> sp.

The combined analysis of peptide mass fingerprinting and 2-DE/MS using the induced and selected protein spots following growth of Pseudomonas sp. DU102 on benzoate or p-hydroxybenzoate revealed not only alpha- and beta-subunits of protocatechuate 3,4-dioxygenase but also catechol 1,2-dioxygenase responsible for ortho-pathway through ring-cleavage of aromatic compounds. Toluate 1,2-dioxygenase and p-hydroxybenzoate hydroxylase were also identified. Purification of intradiol dioxygenases such as catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase from the benzoate or p-hydroxybenzoate culture makes it possible to trace the biodegradation pathway of strain DU102 for monocyclic aromatic hydrocarbons. Interestingly, vanillin-induced protocatechuate 3,4-dioxygenase was identical in amino acid sequences with protocatechuate 3,4-dioxygenase from p-hydroxybenzoate.     

A proposed biochemical mechanism for citric acid accumulation by Aspergillus niger Yang No. 2 growing in solid state fermentation

Aspergillus niger Yang No. 2 and its mutant strain SL1 were grown in solid state fermentation. Samples were taken after 2, 4 and 6 days of incubation and the mycelia were analysed for their intracellular concentrations of some organic acids and adenylates and the activities of selected enzymes. Strain Yang No. 2 contained high concentrations of citrate with very little oxalate, while strain SL1 contained lower concentrations of citrate but considerably higher concentrations of oxalate. As the fermentation proceeded, strain Yang No. 2 showed a much higher ratio of ATP:AMP than did strain SL1. In addition, the enzyme ATP:citrate lyase became undetectable during citrate accumulation in strain Yang No. 2, while its activity remained high during oxalate accumulation in strain SL1. It is proposed that citrate accumulation by strain Yang No. 2 during solid state fermentation is due to blockage of its metabolism in the mitochondrion via inhibition of isocitrate dehydrogenase by the high ATP:AMP ratio, and in the cytosol by repression of ATP:citrate lyase activity.     

Biodegradation of mixture containing monohydroxybenzoate isomers by <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis>

A bacterial strain capable of utilizing a mixture containing 2-hydroxybenzoic acid (2-HBA), 3-hydroxybenzoic acid (3-HBA) and 4-hydroxybenzoic (4-HBA) acid was isolated through enrichment from a soil sample. Based on 16SrDNA sequencing, the microorganism was identified as Acinetobacter calcoaceticus. The sequence of biodegradation of the three isomers when provided as a mixture (0.025%, w/v each) was elucidated. The dihydroxylated metabolites formed from the degradation of 2-HBA, 3-HBA and 4-HBA were identified as catechol, gentisate and protocatechuate, respectively, using the cell-free supernatant and cell-free crude extracts. Monooxygenases and dioxygenases that were induced in the cells of Acinetobacter calcoaceticus in response to growth on mixture containing 2-HBA, 3-HBA and 4-HBA could be detected in cell-free extracts. These data revealed the pathways operating in Acinetobacter calcoaceticus for the sequential metabolism of monohydroxybenzoate isomers when presented as a mixture.     

Metabolism of dibutylphthalate and phthalate by Micrococcus sp. strain 12B.

Micrococcus sp. strain 12B was isolated by enriching for growth with dibutylphthalate as the sole carbon and energy source. A pathway for the metabolism of dibutylphthalate and phthalate by micrococcus sp. strain 12B is proposed: dibutylphthalate leads to monobutylphthalate leads to phthalate leads to 3,4-dihydro-3,4-dihydroxyphthalate leads to 3,4-dihydroxyphthalate leads to protocatechuate (3,4-dihdroxybenzoate). Protocatechuate is metabolized both by the meta-cleavage pathway through 4-carboxy-2-hydroxymuconic semialdehyde and 4-carboxy-2-hydroxymuconate to pyruvate and oxaloacetate and by the ortho-cleavage pathway to beta-ketoadipate. Dibutylphthalate- and phthalate-grown cells readily oxidized dibutylphthalate, phthalate, 3,4-dihydroxyphthalate, and protocatechuate. Extracts of cells grown with dibutylphthalate or phthalate contained the 3,4-dihydroxyphthalate decarboxylase and the enzymes of the protocatechuater 4,5-meta-cleavage pathway. Extracts of dibutylphthalate-grown cells also contained the protocatechuate ortho-cleavage pathway enzymes. The dibutylphthalate-hydrolyzing esterase and 3,4-dihydroxyphthalate decarboxylase were constitutively synthesized; phthalate-3,4-dioxygenase (and possibly the "dihydrodiol" dehydrogenase) was inducible by phthalate or a metabolite occurring before protocatechuate in the pathway; two protocatechuate oxygenases and subsequent enzymes were inducible by protocatechuate or a subsequent metabolic product. During growth at 37 degrees C, strain 12B gave clones at high frequency that had lost the ability to grow with phthalate esters. One of these nonrevertible mutants, strain 12B-Cl, lacked all of the enzymes required for the metabolism of dibutylphthalate through the protocatechuate meta-cleavage pathway. Enzymes for the metabolism of protocatechuate by the ortho-cleavage pathway were present in this strain grown with p-hydroxybenzoate or protocatechuate.     

Metabolism of dimethylterephthalate byAspergillus niger

Aspergillus niger (AG-1) metabolized dimethylterephthalate through monomethylterephthalate, terephthalate and protocatechuate. Degradation of dimethylterephthalate was followed by extraction of residual dimethylterephthalate from the spent medium. The quantitative UV analysis showed that 58% of the dimethylterephthalate supplement was taken up in 144 h. The metabolites were isolated from resting cell cultures. Thin layer chromatography analysis of the extract revealed the presence of two intermediates, monomethylterephthalate and terephthalate. Use of an inhibitor in resting cell culture experiment demonstrated the accumulation of protocatechuate. The time course of protocatechuate accumulation was also studied. Metabolites were identified by employing various physicochemical methods. Enzyme studies using cell-free extracts exhibited dimethylterephthalate esterase and protocatechuate dioxygenase activities. Protocatechuate was oxidized by themeta cleavage pathway. A tentative pathway for the degradation of DMTP has been proposed inA. niger.Abbreviations A. niger Aspergillus niger (AG1) - DMSO dimethyl sulfoxide - DMTP dimethylterephthalate - MMTP monomethylterephthalate - MS mass spectra - NMR nuclear magnetic resonance spectra - PCA protocatechuate - TLC thin layer chromatography - TP terephthalate - UV ultra violet spectra     

Pathways for 3-chloro- and 4-chlorobenzoate degradationin Pseudomonas aeruginosa 3mT

A bacterial isolate, Pseudomonas aeruginosa 3mT, exhibited the ability to degrade high concentrations of 3-chlorobenzoate (3-CBA, 8 g l^-1) and 4-chlorobenzoate (4-CBA 12 g l^-1) (Ajithkumar 1998). In this study, by delineating the initial biochemical steps involved in the degradation of these compounds, we investigated how this strain can do so well. Resting cells, permeabilised cells as well as cell-free extracts failed to dechlorinate both 3-CBA and 4-CBA under anaerobic conditions, whereas the former two readily degraded both compounds under aerobic conditions. Accumulation of any intermediary metabolite was not observed during growth as well as reaction with resting cells under highly aerated conditions. However, on modification of reaction conditions, 3-chlorocatechol (3-CC) and 4-chlorocatechol (4-CC) accumulated in 3-CBA and 4-CBA flasks, respectively. Fairly high titres of pyrocatechase II (chlorocatechol 1,2-dioxygenase) activity were obtained in extracts of cells grown on 3-CBA and 4-CBA. Meta-pyrocatechase (catechol 2,3-dioxygenase) activity against4-CC and catechol, but not against 3-CC, was also detected in low titres. Accumulation of small amounts of 2-chloro-5-hydroxy muconic semialdehyde, the meta-cleavage product of 4-CC, was detected in the medium, when 4-CBA concentration was 4 mM or greater, indicating the presence of a minor meta-pathway in strain 3mT. However, 3-CBA exclusively, and more than 99% of 4-CBA were degraded through the formation of the respective chlorocatechol, via a modified ortho-pathway. This defies the traditional view that the microbes that follow chlorocatechol pathways are not very good degraders of chlorobenzoates. 4-Hydroxybenzoatewas readily (and 3-hydroxybenzoate to a lesser extent) degraded by the strain, through the formation of protocatechuate and gentisate, respectively, as intermediary dihydroxy metabolites.     

Aromatic metabolism in Rhizobium trifolii —protocatechuate 3,4-dioxygenase

Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) has been purified 42-fold from 4-hydroxybenzoate-grown cells of Rhizobium trifolii TA1, where it constitutes about 2% of the cell protein. The dioxygenase has a molecular weight of 220,000, with two dissimilar sub-units of molecular weights 29,000 and 26,500, corresponding to an 44 composition. The enzyme is specific for protocatechuate, with a Km of 1.75×10^-5 M and maximum activity at pH 9.2. Metal removal and replacement studies indicate that the enzyme contains complexed Fe³⁺ which is required for activity. Direct atomic absorption analysis gave 1.3–1.5 g atoms Fe³⁺ per mole of isolated enzyme, but correction for metal-deficient proteins suggests that the value is close to 2.     

一株木质纤维素降解菌的筛选及其全基因组分析北大核心CSCD

【目的】筛选和鉴定有木质纤维素降解能力的1株细菌,测定其相关酶活力并进行全基因组分析,为构建木质纤维素降解工程菌提供依据。【方法】采用3种木质素类似物(天青-B;酚红;愈创木酚)的脱色/染色法,从腐木和被枝叶覆盖的土壤中分离和筛选出1株具有较强木质纤维素降解能力的细菌。通过16S r RNA基因和全基因组序列分析对该菌进行种属鉴定。使用紫外分光光度法测定其锰过氧化物酶(Mn P)、漆酶(Lac)、羧甲基纤维素酶(CMCase)以及滤纸酶(FPA)活力,了解该菌相关酶活力大小在一定时间内的变化趋势。使用Illumina Miseq和454 GS Junior测序平台获取该菌的全基因组序列,将其全基因组序列经过注释的基因蛋白质序列提交COG和KEGG数据库进行BLASTp比对分析,确定该菌潜在的重要酶类和代谢途径,并对部分注释基因进行定量RT-PCR验证。【结果】筛选得到1株优势菌株S12,该菌经鉴定后命名为解鸟氨酸拉乌尔菌(Raoultella ornithinolytica)。在液体CMC-Na培养基中发酵28 h,菌体生长达到稳定期,纤维素降解相关酶活力也在此时达到峰值。生物信息学分析结果表明,菌株S12具有木质素降解通路中重要酶类的编码基因,如过氧化物酶、Fe-Mn型超氧化物歧化酶、邻苯二酚1,2-双加氧酶和原儿茶酸-3,4-双加氧酶等,这些基因在以碱性木质素为碳源的培养条件下表达量不同程度地高于以葡萄糖为碳源的培养条件。另外,菌株S12具备完整的纤维素降解和乙醇生成通路。【结论】本研究首次揭示了Raoultella ornithinolytica S12具备有效的木质纤维素降解性能,这对于推动木质纤维素应用产业的发展具有重要意义。     

Physiological comparisons of root and stem nodules of Aeschynomene scabra and Sesbania rostrata

A few legume species possess the ability to form N₂-fixing nodules on stems as well as on roots. Little is known of the functional characteristics of stem nodules, or to what extent they differ from root nodules. Stem and root nodules of greenhouse-grown plants of Aeschynomene scabra (inoculated with the photosynthetic rhizobial strain BTAi 1) and Sesbania rostrata (inoculated with Azorhizobium caulinodans strain BTSr 3) were examined for assimilation of ¹⁴CO₂ in the light and dark, soluble carbohydrate and starch contents, acetylene reduction activity, relative efficiency of nitrogenase in terms of uptake-hydrogenase activity, glutamine synthetase and glutamate synthase, and reduced N and ureide contents. In general, stem nodules possessed higher enzyme activities and metabolite contents than did root nodules, suggesting that they fix N₂ with greater energy efficiency. This greater efficiency correlated with photosynthesis in the cortex of stem nodules. Differences in enzyme activities and metabolite contents between the stem nodules on A. scabra and those on S. rostrata probably result either from legume-species characteristics or from the photosynthetic capability of strain BTAi 1.     

Key enzymes of the protocatechuate branch of the β-ketoadipate pathway for aromatic degradation in Corynebacterium glutamicum

Although the protocatechuate branch of the β-ketoadipate pathway in Gram- bacteria has been well studied, this branch is less understood in Gram⁺ bacteria. In this study, Corynebacterium glutamicum was cultivated with protocatechuate, p-cresol, vanillate and 4-hydroxybenzoate as sole carbon and energy sources for growth. Enzymatic assays indicated that growing cells on these aromatic compounds exhibited protocatechuate 3,4-dioxygenase activities. Data-mining of the genome of this bacterium revealed that the genetic locus ncg12314-ncg12315 encoded a putative protocatechuate 3,4-dioxygenase. The genes, ncg12314 and ncg12315, were amplified by PCR technique and were cloned into plasmid (pET21aP34D). Recombinant Escherichia coli strain harboring this plasmid actively expressed protocatechuate 3,4-dioxygenase activity. Further, when this locus was disrupted in C. glutamicum, the ability to degrade and assimilate protocatechuate, p-cresol, vanillate or 4-hydroxybenzoate was lost and protocatechuate 3,4-dioxygenase activity was disappeared. The ability to grow with these aromatic compounds and protocatechuate 3,4-dioxygenase activity of C. glutamicum mutant could be restored by gene complementation. Thus, it is clear that the key enzyme for ring-cleavage, protocatechuate 3,4-dioxygenase, was encoded by ncg12314 and ncg12315. The additional genes involved in the protocatechuate branch of the β-ketoadipate pathway were identified by mining the genome data publically available in the GenBank. The functional identification of genes and their unique organization in C. glutamicum provided new insight into the genetic diversity of aromatic compound degradation.     

Genotoxic activation of 2-phenylbenzotriazole-type compounds by human cytochrome P4501A1 and N-acetyltransferase expressed in Salmonella typhimurium umu strains

Four 2-phenylbenzotriazole (PBTA)-type compounds (PBTA-4, PBTA-6, PBTA-7, and PBTA-8) were identified as major mutagens in blue cotton/rayon-adsorbed substances collected at sites below textile dyeing factories or municipal water treatment plants treating domestic waste and effluents from textile dyeing factories in several rivers in Japan. The main purpose of this study is to understand the basis of the roles of human cytochrome P450 (CYP) and N-acetyltransferases (NATs) in genotoxic activation of PBTA derivatives. We compared the induction of umuC gene expression as a measure of genotoxicity using Salmonella typhimurium TA1535/pSK1002 (parental strain), NM2009 (bacterial O-acetyltransferase-overexpressing strain) established in our laboratories. PBTA-4, PBTA-6, PBTA-7, and PBTA-8 induced the umuC gene expression more strongly in the bacterial O-acetyltransferase-overproducing strain than in the parental strain in the presence of rat S9 mix. We determined the activation of PBTA derivatives by cDNA-based recombinant (Trichoplusia ni) systems expressing human or rat cytochrome P450 enzymes (P450 or CYP) and NADPH-P450 reductase using S. typhimurium NM2009. The results showed that human recombinant CYP1A1 enzyme was much more active than CYP1A2 and CYP3A4 in the genotoxic activation of PBTA-4, PBTA-6, PBTA-7, and PBTA-8. Similarly, rat recombinant CYP1A1 enzyme catalyzed the activation of these chemicals at high rates. α-Naphthoflavone, a known inhibitor of CYP1A1, was found to inhibit genotoxic activation caused by PBTA derivatives. We further determined the activation of PBTA derivatives using S. typhimurium NM6001 (human NAT1-expressing strain), S. typhimurium NM6002 (human NAT2-expressing strain), and S. typhimurium NM6000 (O-AT-deficient parent strain) in the presence of S9 mix. PBTA-4 showed almost similar sensitivity in the NAT1-expressing strain and the NAT2-expressing strain, although NAT2-expressing strain exhibited relatively higher sensitivity to PBTA-6, PBTA-7, and PBTA-8 than NAT1-expressing strain. The results support the view that O-acetylation by human NAT1 and NAT2 enzymes is involved in the genotoxic activation of PBTA compounds. These results demonstrate for the first time that human P4501A1 and NATs (NAT1 and NAT2) contribute significantly to the activation of PBTA-type compounds to genotoxic metabolites that induce umuC gene expression in S. typhimurium tester strains.     

Involvement of human mucous saliva and salivary mucins in the aggregation of the oral bacteria Streptococcus sanguis,Streptococcus oralis,and Streptococcus rattus

The contribution of human parotid (Par) and submandibular/sublingual (SM/SL) saliva and of the human whole salivary mucin fraction (HWSM) to saliva-induced bacterial aggregation was studied for S. sanguis C476, S. oralis I581, and S. rattus HG 59. The mucous SM/SL saliva showed a much higher aggregation potency towards the S. sanguis and S. oralis strain than did the serous Par saliva. The SM/SL saliva-induced aggregation was observed after 30 min, at 60 min followed by the Par saliva-induced aggregation, and showed a 4-fold higher aggregation titer of 128 for S. sanguis, and an 8-fold higher titer of 516 for S. oralis. In contrast, the Par saliva showed a slightly higher aggregation activity than the SM/SL saliva towards S. rattus as judged by a twofold higher titer of 64. Morphologically, however, the SM/SL saliva-induced aggregation of S. rattus was far more pronounced as was also found for S. sanguis. Finally, the HWSM-induced aggregation showed a 4 to 8-fold higher titer than the originating salivary source, measuring 2048 for S. oralis and 128 for S. rattus. Moreover, no difference was observed in aggregation activity between the HWSM from whole saliva of a blood group O donor and the HWSM from SM/SL saliva of a blood group A donor. All the data point to an important, though not exclusive role of the human salivary mucin fraction in the saliva-induced aggregation of these strains.     

Induction and selection of formaldehyde-based resistance inPseudomonas aeruginosa

Summary A formaldehyde resistant (R) phenotype ofPseudomonas aeruginosa was isolated from a formaldehydesensitive (S) parent by sequential treatment with 1,3,5-tris-(ethyl)hexahydro-s-triazine (ET). The resistance of the (R) strain to treatment with ET was approximately 3-fold higher than the parental (S) strain. Two modes of resistance to ET, and simultaneous resistance to formaldehyde, are demonstrated: (1) transient or induced resistance is expressed during shor-term exposure to ET, and this resistance is gradually lost during subsequent growth in the absence of ET, and (2) resistance that results from a stable phenotypic change in the (S) strain following sequential treatment with ET ((R) strain phenotype). The observed activities of three forms of the formaldehyde oxidizing enzyme, formaldehyde dehydrogenase, are strongly correlated with the relative response of the (S) and (R) strains to treatment with ET. The observed resistance of the (R) strain appears to be due to high levels of an NAD⁺-linked, glutathione-dependent form of formaldehyde dehydrogenase as well as a dye-linked formaldehyde dehydrogenase. The transient or induced response of the (R) strain involves an increase in activity of the dye-linked formaldehyde dehydrogenase. The induced response of the (S) strain and an ATCC strain ofP. aeruginosa, however, is correlated with the two forms of the NAD⁺-linked enzyme (glutathione-dependent (EC 1.2.1.1) and independent (EC 1.2.1.46)) with no contribution from the dye-linked enzyme.     

Transient Accumulation of Metabolic Intermediates of p-Cresol in the Culture Medium by a Pseudomonas sp. Strain A Isolated from a Sewage Treatment Plant

Summary Activated sludge from a sewage treatment plant in Kanpur, India, was screened for bacterial strains metabolizing p-cresol exclusively under aerobic conditions. One such isolate was identified to be belonging to the genus Pseudomonas based on morphological and physiological criteria as well as 16S ribosomal RNA gene sequence analysis. Two intermediates were identified from the culture medium during the growth phase of Pseudomonas sp. strain A that indicated that the strain degraded p-cresol via the protocatechuate (PCA) pathway. p-Cresol was rapidly converted into p-hydroxybenzaldehyde (PHB) during early growth phase, which was later utilized after p-cresol depletion. p-Hydroxybenzoate (PHBA) accumulation was observed during the later stages of exponential growth phase. Kinetic constants for the degradation of p-cresol were determined using Haldane’s model. High μ_max and inhibitory constant (K_I) values along with the observed accumulation of significant amounts of PHB in culture filtrates seem to indicate that the isolated Pseudomonas sp. strain A may be of potential use in biotransformations.