The roles of intermediates in biodegradation of benzene,toluene, and p-xylene by Pseudomonas putida F1

Several types of biodegradation experiments with benzene, toluene, or p-xylene show accumulation of intermediates by Pseudomonas putida F1. Under aerobic conditions, the major intermediates identified for benzene, toluene, and p-xylene are catechol, 3-methylcatechol, and 3,6-dimethylcatechol, respectively. Oxidations of catechol and 3-methylcatechol are linked to biomass synthesis. When oxygen is limited in the system, phenol (from benzene) and m-cresol and o-cresol (from toluene) accumulate.     

Role of extracellular polymeric substances in Cu(II) adsorption on Bacillus subtilis and Pseudomonas putida

The effect of extracellular polymeric substances (EPS) of Gram-positive Bacillus subtilis and Gram-negative Pseudomonas putida on Cu(II) adsorption was investigated using a combination of batch adsorption, potentiometric titrations, Fourier transform infrared spectroscopy. Both the potentiometric titrations and the Cu(II) adsorption experiments indicated that the presence of EPS in a biomass sample significantly enhance Cu(II) adsorption capacity. Surface complexation modeling showed that the pK_a values for the three functional groups (carboxyl, phosphate and hydroxyl) were very similar for untreated and EPS-free cells, indicating no qualitative difference in composition. However, site concentrations on the untreated cell surface were found to be significantly higher than those on the EPS-free cell surface. Infrared analysis provided supporting evidence and demonstrated that carboxyl and phosphate groups are responsible for Cu(II) adsorption on the native and EPS-free cells.     

The interplay of the EIIA component of the nitrogen-related phosphotransferase system (PTS) of Pseudomonas putida with pyruvate dehydrogenase

Background

Pseudomonas putida KT2440 is endowed with a variant of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS^Ntr), which is not related to sugar transport but believed to rule the metabolic balance of carbon vs. nitrogen. The metabolic targets of such a system are largely unknown.

Methods

Dielectric breakdown of P. putida cells grown in rich medium revealed the presence of forms of the EIIA^Ntr (PtsN) component of PTS^Ntr, which were strongly associated to other cytoplasmic proteins. To investigate such intracellular partners of EIIA^Ntr, a soluble protein extract of bacteria bearing an E epitope tagged version of PtsN was immunoprecipitated with a monoclonal anti-E antibody and the pulled-down proteins identified by mass spectrometry.

Results

The E1 subunit of the pyruvate dehydrogenase (PDH) complex, the product of the aceE gene, was identified as a major interaction partner of EIIA^Ntr. To examine the effect of EIIA^Ntr on PDH, the enzyme activity was measured in extracts of isogenic ptsN⁺/ptsN⁻P. putida strains and the role of phosphorylation was determined. Expression of PtsN and AceE proteins fused to different fluorescent moieties and confocal laser microscopy indicated a significant co-localization of the two proteins in the bacterial cytoplasm.

Conclusion

EIIA^Ntr down-regulates PDH activity. Both genetic and biochemical evidence revealed that the non-phosphorylated form of PtsN is the protein species that inhibits PDH.

General significance

EIIA^Ntr takes part in the node of C metabolism that checks the flux of carbon from carbohydrates into the Krebs cycle by means of direct protein–protein interactions with AceE. This type of control might connect metabolism to many other cellular functions. This article is part of a Special Issue entitled: Systems Biology of Microorganisms.     

Conversion of sodium cyanide to carbon dioxide and ammonia by immobilized cells ofPseudomonas putida

Summary Pseudomonas putida, isolated from contaminated industrial wastewaters and soil sites, was found to utilize sodium cyanide (NaCN) as a sole source of carbon and nitrogen. Cells, immobilized in calcium alginate beads (1–2 mm diameter) were aerated in air-uplift-type fluidized batch bioreactor containing 100–400 ppm of NaCN. Degradation of NaCN was monitored for 168 h by analyzing gaseous and dissolved ammonia (NH₃), CO₂, pH and optical density. The results indicated that the alginate-immobilized cells ofP. putida were able to degrade NaCN into NH₃ and CO₂ in a time-dependent manner.     

Removal of malachite green from aqueous solution by immobilized Pseudomonas sp. DY1 with Aspergillus oryzae

Triphenylmethane dyes are considered to be one of the most recalcitrant pollutants in the environment. Malachite Green (MG) was successfully removed from aqueous solution by Pseudomonas sp. DY1 immobilization with Aspergillus oryzae. Inhibition test in the presence of sodium azide and nystatin indicated that A. oryzae was a natural immobilization reagent, and removal of MG by the immobilized cell pellets was attributed to the biodegradation by Pseudomonas sp. DY1. Optimum conditions of immobilization for maximum biodegradation were obtained using Taguchi design at 37 °C, inoculation size of Pseudomonas sp. DY1 (dry cell mass) 0.01 g, of A. oryzae (spore number) 1.0 × 10⁹, initial pH 6.5. Decolorization and biodegradation of MG by immobilized pellets under optimum conditions were 99.5% and 93.3%, respectively. Immobilized pellets exhibited more than 96% decolorization after 16 days in batch condition, indicating it had stable and high biodegradation capabilities when immobilized for long-term operation.     

Further diterpenoids from Plectranthus ornatus and P. grandidentatus

A new halimane diterpenoid and the previously known labdane rhinocerotinoic acid were isolated from Plectranthus ornatus Codd. In addition, a new monoacetyl derivative of the abietane coleon U was found among the constituents of Plectranthus grandidentatus Gürke. These diterpenes could be significant from both chemotaxonomic and biogenetic points of view.     

A two-step model for the kinetics of BTX degradation and intermediate formation by Pseudomonas putida F1

A two-step model is developed for the aerobic biodegradation of benzene,toluene, and p-xylene (BTX) by Pseudomonas putida F1. The model contains three unique features. First, an initial dioxygenation step transforms BTX into their catechol intermediates, but does not support biomassgrowth. Second, the benzene or toluene intermediates are mineralized, which supports biomass synthesis. Third, BTX exhibit competitive inhibition on each other's transformation, while toluene and benzenenoncompetitively inhibit the mineralization of their catechol intermediate. A suite of batch and chemostat experiments is used to systematically measure the kinetic parameters for the two-step transformations and the substrate interactions.     

A novel Pseudomonas putida strain with high levels of hydantoin-converting activity, producing -amino acids

Optically pure chiral amino acids and their derivatives can be efficiently synthesised by the biocatalytic conversion of 5-substituted hydantoins in reactions catalysed by stereo-selective microbial enzymes: initially a hydantoinase catalyses the cleavage of the hydantoin producing an N-carbamyl amino acid. In certain bacteria where an N-carbamyl amino acid amidohydrolase (NCAAH) is present, the N-carbamyl amino acid intermediate is further converted to amino acid, ammonia and CO₂. In this study we report on a novel Pseudomonas putida strain which exhibits high levels of hydantoin-converting activity, yielding -amino acid products including alanine, valine, and norleucine, with bioconversion yields between 60% and 100%. The preferred substrates are generally aliphatic, but not necessarily short chain, 5-alkylhydantoins. In characterizing the enzymes from this microorganism, we have found that the NCAAH has -selectivity, while the hydantoinase is non-stereoselective. In addition, resting cell reactions under varying conditions showed that the hydantoinase is highly active, and is not subject to substrate inhibition, or product inhibition by ammonia. The rate-limiting reaction appears to be the NCAAH-catalysed conversion of the intermediate. Metal-dependence studies suggest that the hydantoinase is dependent on the presence of magnesium and cobalt ions, and is strongly inhibited by the presence of copper ions. The relative paucity of -selective hydantoin-hydrolysing enzyme systems, together with the high level of hydantoinase activity and the unusual substrate selectivity of this P. putida isolate, suggest that is has significant potential in industrial applications.     

Isolation and Characterization of a Pseudomonas putida Strain able to Grow with Trimethyl-1,2-dihydroxy-propyl-ammonium as Sole Source of Carbon, Energy and Nitrogen

Trimethyl-1,2-dihydroxypropyl-ammonium (TM) originates from the hydrolysis of the parent esterquat surfactant, which is widely used as softener in fabric care. Based on test procedures mimicking complex biological systems, TM is supposed to degrade completely when reaching the environment. However, no organisms able to degrade TM were isolated nor has the degradation pathway been elucidated so far. We isolated a Gram-negative rod able to grow with TM as sole source of carbon, energy and nitrogen. The strain reached a maximum specific growth rate of 0.4 h^–1 when growing with TM as the sole source of carbon, energy and nitrogen. TM was degraded to completion and surplus nitrogen was excreted as ammonium into the growth medium. A high percentage of the carbon in TM (68% in continuous culture and 60% in batch culture) was combusted to CO₂ resulting in a low yield of 0.54 mg cell dry weight per mg carbon during continuous cultivation and 0.73 mg cell dry weight per mg carbon in batch cultures. Choline, a natural structurally related compound, served as a growth substrate, whereas a couple of similar other quaternary aminoalcohols also used in softeners did not. The isolated bacterium was identified by 16S-rDNA sequencing as a strain of Pseudomonas putida with a difference of only one base pair to P. putida DSM 291^T. Despite their high identity, the reference strain P. putida DSM 291^T was not able to grow with TM and the two strains differed even in shape when growing on the same medium. This is the first microbial isolate able to degrade a quaternary ammonium softener head group to completion. Previously described strains growing on quaternary ammonium surfactants (decyltrimethylammonium, hexadecyltrimethylammonium and didecyldimethylammonium) either excreted metabolites or a consortium of bacteria was required for complete degradation.     

Catechol 1,2-dioxygenase from the new aromatic compounds - Degrading Pseudomonas putida strain N6

This study aimed to characterization of catechol 1,2-dioxygenase from a Gram-negative bacterium, being able to utilize a wide spectrum of aromatic substrates as a sole carbon and energy source. Strain designated as N6, was isolated from the activated sludge samples of a sewage treatment plant at Bentwood Furniture Factory Jasienica, Poland. Morphology, physio-biochemical characteristics and phylogenetic analysis based on 16S rDNA sequence indicate that strain belongs to Pseudomonas putida. When cells of strain N6 grown on protocatechuate or 4-hydroxybenzoic acid mainly protocatechuate 3,4-dioxygenase was induced. The activity of catechol 1,2-dioxygenase was rather small. The cells grown on benzoic acid, catechol or phenol showed high activity of only catechol 1,2-dioxygenase. This enzyme was optimally active at 35 °C and pH 7.4. Kinetic studies showed that the value of K_m and V_max was 85.19 ??M and 14.54 ??M min⁻¹ respectively. Nucleotide sequence of gene encoding catechol 1,2-dioxygenase in strain N6 has 100% identity with catA genes from two P. putida strains. The deduced 301-residue sequence of enzyme corresponds to a protein of molecular mass 33.1 kDa. The deduced molecular structure of the catechol 1,2-dioxygenase from P. putida N6 was very similar and characteristic for the other intradiol dioxygenases.     

Intracellular degradation of two structurally different polyhydroxyalkanoic acids accumulated in Pseudomonas putida and Pseudomonas citronellolis from mixtures of octanoic acid and 5-phenylvaleric acid

From a set of mixed carbon sources, 5-phenylvaleric acid (PV) and octanoic acid (OA), polyhydroxyalkanoic acid (PHA) was separately accumulated in the two pseudomonads Pseudomonas putida BM01 and Pseudomonas citronellolis (ATCC 13674) to investigate any structural difference between the two PHA accumulated under a similar culture condition using one-step culture technique. The resulting polymers were isolated by chloroform solvent extraction and characterized by fractional precipitation and differential scanning calorimetry. The solvent fractionation analysis showed that the PHA synthesized by P. putida was separated into two fractions, 3-hydroxy-5-phenylvalerate (3HPV))-rich PHA fraction in the precipitate phase and 3-hydroxyoctanoate (3HO)-rich PHA fraction in the solution phase whereas the PHA produced by P. citronellolis exhibited a rather little compositional separation into the two phases. According to the thermal analysis, the P. putida PHA exhibited two glass transitions indicative of the PHA not being homogeneous whereas the P. citronellolis PHA exhibited only one glass transition. It was found that the structural heterogeneity of the P. putida PHA was caused by a significant difference in the assimilation rate between PV and OA. The structural heterogeneity present in the P. putida PHA was also confirmed by a first order degradation kinetics analysis of the PHA in the cells. The two different first-order degradation rate constants (k₁), 0.087 and 0.015/h for 3HO- and 3HPV-unit, respectively, were observed in a polymer system over the first 20 h of degradation. In the later degradation period, the disappearance rate of 3HO-unit was calculated to be 0.020 h. The k₁ value of 0.083/h, almost the same as for the 3HO-unit in the P. putida PHA, was obtained for the P(3HO) accumulated in P. putida BM01 grown on OA as the only carbon source. In addition, the k₁ value of 0.015/h for the 3HPV-unit in the P. putida PHA, was also close to 0.019/h for the P(3HPV) homopolymer accumulated in P. putida BM01 grown on PV plus butyric acid. On the contrary, the k₁ values for the P. citronellolis PHA were determined to be 0.035 and 0.029/h for 3HO- and 3HPV-unit, respectively, thus these two relatively close values implying a random copolymer nature of the P. citronellolis PHA. In addition, the faster degradation of P(3HO) than P(3HPV) by the intracellular P. putida PHA depolymerase indicates that the enzyme is more specific against the aliphatic PHA than the aromatic PHA.     

Hydrocarbon degradation and bioemulsifier production by thermophilic Geobacillus pallidus strains

Geobacillus pallidus XS2 and XS3 were isolated from oil contaminated soil samples in Yumen oilfield, China, and were able to produce bioemulsifiers on different hydrocarbons. Biodegradation assays exhibited that approximately 70% of PAH (250 mg/L) or 85% of crude oil (500 mg/L) was removed by the thermophilic bacteria after 20 days. The bioemulsifiers of the two strains were isolated and obtained a productive yield of 4.24 ± 0.08 and 3.82 ± 0.11 g/L, respectively. GPC analysis revealed that the number-average molecular weights (M_n) of the two bioemulsifiers were 271,785 Da and 526,369 Da, with PDI values of 1.104 and 1.027, respectively. Chemical composition studies exhibited that the bioemulsifier XS2 consisted of carbohydrates (68.6%), lipids (22.7%) and proteins (8.7%) while the bioemulsifier XS3 was composed by carbohydrates (41.1%), lipids (47.6%) and proteins (11.3%). Emulsification assays approved the effectiveness of bioemulsifiers over a wide range of temperature, pH and salinity.     

Localization and organization of phenol degradation genes ofPseudomonas putida strain H

The genetic organization of the DNA region encoding the phenol degradation pathway ofPseudomonas putida H has been investigated. This strain can utilize phenol or some of its methylated derivatives as its sole source of carbon and energy. The first step in this process is the conversion of phenol into catechol. Catechol is then further metabolized via themeta-cleavage pathway into TCA cycle intermediates. Genes encoding these enzymes are clustered on the plasmid pPGH1. A region of contiguous DNA spanning about 16 kb contains all of the genetic information necessary for inducible phenol degradation. The analysis of mutants generated by insertion of transposons and cassettes indicates that all of the catabolic genes are contained in a single operon. This codes for a multicomponent phenol hydroxylase andmeta-cleavage pathway enzymes. Catabolic genes are subject to positive control by the gene product(s) of a second locus.     

AFLPs detect low genetic diversity for Phytophthora nemorosa and P. pseudosyringae in the US and Europe

In California and Oregon, two recently described oomycete forest pathogens, Phytophthora nemorosa and P. pseudosyringae, overlap in their host and geographic ranges with the virulent P. ramorum, causal agent of “sudden oak death.” Epidemiological observations, namely broader geographic distribution and lack of landscape-level mortality, led to the hypothesis they are native to this region, whereas multiple lines of evidence indicate P. ramorum is exotic to North America. We used AFLP analysis to measure genetic variability in the homothallic P. nemorosa and P. pseudosyringae and to evaluate the hypothesis of endemism. We analysed 39 P. nemorosa and 48 P. pseudosyringae isolates (29 American and 19 European) from throughout their geographic and host ranges. In the US, both P. nemorosa and P. pseudosyringae have a dominant AFLP clone with several closely related variants. There is no evidence that genetic diversity is partitioned by host or location in P. nemorosa, but the US P. pseudosyringae clonal lineage is largely nested within a more genetically variable European group. Though the absence of highly variable sampled source populations does not allow us to determine whether each species is native or introduced in the western US with certainty, the results are most consistent with the hypothesis that both are introduced — P. pseudosyringae perhaps from Europe. Invasive Phytophthora species are increasingly being implicated in emergent forest diseases, highlighting the need to identify and characterize both native and previously unknown introduced forest Phytophthoras.     

Nitrile degradation by free and immobilized cells of the thermophile Bacillus sp. UG-5B, isolated from polluted industrial waters

A moderately thermophilic Gram-positive, sporulating, rod-shaped strain of Bacillus with nitrile-degrading activity was isolated from polluted industrial waters. Whole cells and cell-free extracts from the end of exponential growth phase expressed 7.6 nkat mg⁻¹ and 2.0 nkat mg⁻¹ benzonitrile-degrading activity, respectively, after cultivation in a fermentor with complex medium containing benzonitrile as an inducer. The benzonitrile degradation took place via the nitrilase pathway directly to benzoic acid without intermediate formation of benzamide. Samples with benzonitrilase activity of 7.6 nkat mg⁻¹ converted 3 mg benzonitrile in 1 h at 45°C. The half-life of benzonitrilase activity for a whole cell suspension and for cells immobilized in 2% agar was 4.5 min and 6 min at 70°C without substrate and 3 min at 90°C with substrate, respectively. The nitrilase had a broad substrate spectrum. The active biocatalyst obtained by immobilization was used in a continuous process and total biodegradation of 14.1 mM benzonitrile and 37.2 mM 4-cyanopyridine in a column bioreactor at 50°C for 5 h was achieved.     

WLIP and tolaasin I, lipodepsipeptides from Pseudomonas reactans and Pseudomonas tolaasii, permeabilise model membranes

The activity of the White Line Inducing Principle (WLIP) and tolaasin I, produced by virulent strains of Pseudomonas reactans and Pseudomonas tolaasii, respectively, was comparatively evaluated on lipid membranes. Both lipodepsipeptides were able to induce the release of calcein from large unilamellar vesicles. Their activity was dependent on the toxin concentration and liposome composition and in particular it increased with the sphingomyelin content of the membrane. Studies of dynamic light scattering suggested a detergent-like activity for WLIP at high concentration (> 27 μM). This effect was not detected for tolaasin I at the concentrations tested (< 28 μM). Differences were also observed in lipodepsipeptides secondary structure. In particular, the conformation of the smaller WLIP changed slightly when it passed from the buffer solution to the lipid environment. On the contrary, we observed a valuable increment in the helical content of tolaasin I which was inserted in the membrane core and oriented parallel to the lipid acyl chains.     

Pseudomonas putida KT2440低特异性L-苏氨酸醛缩酶的表达、酶学性质及温度稳定性提高

【目的】从Pseudomonas putida KT2440基因组中,钓取低特异性L-苏氨酸醛缩酶基因(lta E),构建重组大肠杆菌。研究目标酶的酶学性质,和关键氨基酸位点突变对酶活和温度稳定性的影响。【方法】以P. putida KT2440基因组DNA为模板,PCR扩增出lta E基因,构建重组表达质粒p ET28a-KT2440并转化Escherichia coli BL21 (DE3),获得重组菌E. coli BL21 (DE3)/p ET-KT2440,利用Ni~(2+)柱亲和层析纯化低特异性L-苏氨酸醛缩酶(LTA),对关键氨基酸位点Thr206和Lys207实施定点突变。【结果】SDS-PAGE结果表明LTA在大肠杆菌中获得高效表达,分子量为40k Da左右,与理论值大小相符。Ni~(2+)柱亲和层析纯化LTA,获得单一条带。利用双酶耦联法测得LTA酶活为5577.3U/mg,最适反应温度为50°C,最适p H为8.0。在温度低于45°C,p H 5.0-9.0时,重组酶较稳定。LTA酶的Km和kcat值为23.95 mmol/L和19216.6 s–1。Mg~(2+)、Ca~(2+)金属离子对LTA有明显的促进作用,而Ni~(~(2+))、Cu~(2+)、Zn~(2+)、Fe~(2+)等对酶有明显的抑制作用。该酶在叔丁基甲基醚溶剂中具有良好的耐受性,在叔丁基甲基醚中保存1h后仍保留90%以上的酶活。Thr206Ser突变明显提高了酶对温度的稳定性。Lys207对酶催化功能是必需的,该位点突变对酶活都是致死的。【结论】克隆并表达P. putida KT2440的LTA酶,研究了酶学性质,通过定点改造提高了酶的温度稳定性,筛选获得一种酶耐受性好的有机溶剂,为LTA酶在有机溶剂中高效稳定催化β-羟基-α-氨基酸奠定了较坚实的研究基础。     

Phylogeny of Phaeomollisia piceae gen. sp. nov.: a dark, septate, conifer-needle endophyte and its relationships to Phialocephala and Acephala

Dark, septate endophytes (DSE) were isolated from roots and needles of dwarf Picea abies and from roots of Vaccinium spp. growing on a permafrost site in the Jura Mountains in Switzerland. Two of the isolates sporulated after incubation for more than one year at 4 °C. One of them was a hitherto undescribed helotialean ascomycete Phaeomollisia piceae gen. sp. nov., the other was a new species of Phialocephala, P. glacialis sp. nov. Both species are closely related to DSE of the Phialocephala fortinii s. lat.-Acephala applanata species complex (PAC) as revealed by phylogenetic analyses of the ITS and 18S rDNA regions. Morphologically dissimilar fungi, such as Vibrissea and Loramyces species, are phylogenetically also closely linked to the new species and the PAC. Cadophora lagerbergii and C. (Phialophora) botulispora are moved to Phialocephala because Phialocephala dimorphospora and P. repens are the closest relatives. Several Mollisia species were closely related to the new species and the PAC according to ITS sequence comparisons. One DSE from needles of Abies alba and one from shoots of Castanea sativa formed Cystodendron anamorphs in culture. Their identical 18S sequences and almost identical ITS sequences indicated Mollisia species as closest relatives, suggesting that Mollisia species are highly euryoecious.