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Heterotrophic nitrifying and aerobic denitrifying bacteria that have been widely isolated from complicated activated sludge microflorae demonstrate dominant advantages in simultaneous removal of ammonium and nitrogen oxides under aerobic conditions. However, owing to the need of organic carbon to support bacterial growth, nitrogen removal of actual industrial wastewater with low carbon-to-nitrogen (C/N) ratio remains a challenge. Here, Pseudomonas mendocina Y7 was identified and presented to effectively remove nitrogen of actual refinery wastewater with low C/N ratio. The isolated bacterium showed high removal efficiency of NH 4 + –N, NO 2 − –N, and NO 3 − –N up to about 90% in single (100 mg/L) or mixed (200 mg/L) nitrogen source media at low C/N ratio of 6 when it was cultivated for 12 or 21 h. According to PCR amplification, the heterotrophic nitrification and aerobic denitrification capability of strain Y7 was attributed to the functional genes of amoA, hao, napA, and nirS. In activated sludge process for treating actual refinery wastewater with low C/N ratio, compared to abundant accumulation of NO 2 − –N and NO 3 − –N only using the activated sludge, strain Y7 significantly improved the removal efficiency of NH 4 + ‒N and total nitrogen (with influent concentrations of about 40 and 55 mg/L) from about 47% and 22% to about 85% and 73%, respectively, without the accumulation of nitrogen oxides. Microbial community structure analysis revealed that strain Y7 could coexist well with other microorganisms in the activated sludge and maintain highly efficient and steady nitrogen removal in continuous treatment system. This discovery provides a promising treatment approach toward actual nitrogen-rich industrial wastewater.

Even though organisms with squalene hopene cyclase activity involved in hopanoid synthesis has been reported earlier, their existence along with carotenoid synthesis is rarely reported. Here, we report the existence of hopanoid and C30 carotenoid biosynthetic pathway in Pseudomonas mendocina, the squalene hopene cyclase producing endophyte of the medicinal plant Murraya koenigii. The enzyme squalene hopene cyclase from Pseudomonas mendocina is involved in the synthesis of dehydrosqualene-mediated alternate pathway for carotenoid biosynthesis. The hopanoids are involved in membrane stability and integrity, and the carotene chromophores are involved in the photo protection of the cell. The orange-colored C30 carotenoid pigment 4-4' diaponeurosporenic acid in the extracellular extract of Pseudomonas mendocina with squalene cyclase activity was detected by the combination of UV/Vis spectrometry, FTIR, and Mass Spectrometry. 4-4' diaponeurosporenic acid could be traced as the end product of the carotenoid pathway and belonged to the xanthophyll group of carotenoids.

Tropical oleaginous seeds are an unexplored source for the discovery of novel lipolytic microorganisms, which could be applied to the bioremediation of agro-industrial oily wastes and solve numerous environmental issues. Such wastes hold potential to be revalorized towards a variety of products through microbial bioremediation. In this study, we investigate the microbial diversity and lipase activity from bacterial and fungal isolates obtained from the oil seeds of Elaeis guineensis, Ricinus communis, and Jatropha curcas L. from Costa Rica. A total of 27 strains were confirmed as lipase-producing strains via fluorogenic and colorimetric agar plate assays. The diversity of the isolates comprises 12 fungal ascomycetes from the genera Aspergillus and Fusarium and 15 bacterial isolates classified into four genera: Serratia, Proteus, Pseudomonas, and Bacillus. Microbial isolates from E. guineensis showed the highest diversity of lipolytic microorganisms (6 genera) followed by J. curcas (4 genera) and R. communis (2 genera). Isolates showing the highest activity in agar plates were tested further by submerged fermentation and the specific lipase activity was measured with 4-nitrophenyl laurate as substrate. Accordingly, the highest specific lipase activity was demonstrated by Bacillus pumilus B5 (24.98 U mg−1), Serratia marcescens B10 (17.65 U mg−1), Pseudomonas mendocina B16 (8.62 U mg−1), and Bacillus pumilus B1 (5.72 U mg−1) in submerged fermentation. These findings indicate the presence of a specialized microbial diversity in tropical oil seeds and highlight their potential to be applied in the bioremediation of agro-industrial oily wastes.

Metformin is a pharmaceutical most prescribed for type 2 diabetes and is now being examined for potential benefits to COVID-19 patients. People taking the drug pass it largely unchanged, and it subsequently enters wastewater treatment plants., The widely prescribed pharmaceutical metformin and its main metabolite, guanylurea, are currently two of the most common contaminants in surface and wastewater. Guanylurea often accumulates and is poorly, if at all, biodegraded in wastewater treatment plants. This study describes Pseudomonas mendocina strain GU, isolated from a municipal wastewater treatment plant, using guanylurea as its sole nitrogen source. The genome was sequenced with 36-fold coverage and mined to identify guanylurea degradation genes. The gene encoding the enzyme initiating guanylurea metabolism was expressed, and the enzyme was purified and characterized. Guanylurea hydrolase, a newly described enzyme, was shown to transform guanylurea to one equivalent (each) of ammonia and guanidine. Guanidine also supports growth as a sole nitrogen source. Cell yields from growth on limiting concentrations of guanylurea revealed that metabolism releases all four nitrogen atoms. Genes encoding complete metabolic transformation were identified bioinformatically, defining the pathway as follows: guanylurea to guanidine to carboxyguanidine to allophanate to ammonia and carbon dioxide. The first enzyme, guanylurea hydrolase, is a member of the isochorismatase-like hydrolase protein family, which includes biuret hydrolase and triuret hydrolase. Although homologs, the three enzymes show distinct substrate specificities. Pairwise sequence comparisons and the use of sequence similarity networks allowed fine structure discrimination between the three homologous enzymes and provided insights into the evolutionary origins of guanylurea hydrolase. IMPORTANCE Metformin is a pharmaceutical most prescribed for type 2 diabetes and is now being examined for potential benefits to COVID-19 patients. People taking the drug pass it largely unchanged, and it subsequently enters wastewater treatment plants. Metformin has been known to be metabolized to guanylurea. The levels of guanylurea often exceed that of metformin, leading to the former being considered a “dead-end” metabolite. Metformin and guanylurea are water pollutants of emerging concern, as they persist to reach nontarget aquatic life and humans, the latter if it remains in treated water. The present study has identified a Pseudomonas mendocina strain that completely degrades guanylurea. The genome was sequenced, and the genes involved in guanylurea metabolism were identified in three widely separated genomic regions. This knowledge advances the idea that guanylurea is not a dead-end product and will allow for bioinformatic identification of the relevant genes in wastewater treatment plant microbiomes and other environments subjected to metagenomic sequencing.

An arsenic resistant bacteria SMSKVR-3 has been isolated from the rhizospheric soil of the metal-contaminated site of khetri copper mines situated in the Jhunjhunu district of Rajasthan, India. The strain showed homology with Pseudomonas mendocina strain ATCC 25411. This gram-negative isolate exhibited optimal growth in M9 minimal media with temperature and salt concentration as 30 °C and 0.25% (w/v), respectively, at pH 7.0. The similar growth pattern and SEM analysis of this strain exposed to M9 minimal media alone, M9 media supplemented with 300 mM arsenate [As(V)] or M9 media supplemented with 1.34 mM arsenite [As(III)] indicate the existence of the strong arsenic resistance mechanism. The isolate was able to produce siderophores and was able to reduce As(V) to As(III). A decrease in polyP concentration from 354.8 µg/10

Maltooligosaccharides (MOS), oligosaccharides composed of 2 to 10 glucopyranose subunits linked by α-1,4 bonds, are promising ingredients possessing low sweetness, low osmolality, high water-holding capacity, and crystallization-prevention ability. For improved the cost effectiveness, a novel process for MOS preparation was developed by utilizing sucrose as a raw material instead of starch. In this work, a novel α-amylase from Pseudomonas mendocina (PM-Amy) was characterized and applied to produce the MOS from α-1,4-glucans obtained by the amylosucrase treatment of sucrose. PM-Amy showed a maximum activity at a pH of 6.0 and 55 °C. In the one-pot enzymatic reaction, a maximum MOS yield of 22.8% (w/w) was achieved, while the highest MOS yield (34.1%, w/w) was achieved from the two-step dual enzyme reaction.

Summary This study demonstrated the utilization of unprocessed biodiesel waste as a carbon feedstock for Pseudomonas mendocina CH50, for the production of PHAs. A PHA yield of 39.5% CDM was obtained using 5% (v/v) biodiesel waste substrate. Chemical analysis confirmed that the polymer produced was poly(3‐hydroxyhexanoate‐co‐3‐hydroxyoctanoate‐co‐3‐hydroxydecanoate‐co‐3‐hydroxydodecanoate) or P(3HHx‐3HO‐3HD‐3HDD). P(3HHx‐3HO‐3HD‐3HDD) was further characterized and evaluated for its use as a tissue engineering scaffold (TES). This study demonstrated that P(3HHx‐3HO‐3HD‐3HDD) was biocompatible with the C2C12 (myoblast) cell line. In fact, the % cell proliferation of C2C12 on the P(3HHx‐3HO‐3HD‐3HDD) scaffold was 72% higher than the standard tissue culture plastic confirming that this novel PHA was indeed a promising new material for soft tissue engineering.

Bioremediation usually exhibits low removal efficiency toward hexane because of poor water solubility, which limits the mass transfer rate between the substrate and microorganism. This work aimed to enhance the hexane degradation rate by increasing cell surface hydrophobicity (CSH) of the degrader, Pseudomonas mendocina NX-1. The CSH of P. mendocina NX-1 was manipulated by treatment with starch and chitosan solution of varied concentrations, reaching a maximum hydrophobicity of 52%. The biodegradation of hexane conformed to the Haldane inhibition model, and the maximum degradation rate (ν max) of the cells with 52% CSH was 0.72 mg (mg cell)−1·h−1 in comparison with 0.47 mg (mg cell)−1·h−1 for cells with 15% CSH. The production of CO2 by high CSH cells was threefold higher than that by cells at 15% CSH within 30 h, and the cumulative rates of O2 consumption were 0.16 and 0.05 mL/h, respectively. High CSH was related to low negative charge carried by the cell surface and probably reduced the repulsive electrostatic interactions between hexane and microorganisms. The FT-IR spectra of cell envelopes demonstrated that the methyl chain was inversely proportional to increasing CSH values, but proteins exhibited a positive effect to CSH enhancement. The ratio of extracellular proteins and polysaccharides increased from 0.87 to 3.78 when the cells were treated with starch and chitosan, indicating their possible roles in increased CSH.

Conditions for the optimal production of polyhydroxyalkanoate (PHA) by Pseudomonas mendocina PSU using a biodiesel liquid waste (BLW) were determined by response surface methodology. These were an initial carbon to nitrogen ratio (C/N) of 40 (mole/mole), an initial pH of 7.0, and a temperature of 35 °C. A biomass and PHA concentration of 3.65 g/L and about 2.6 g/L (77% DCW), respectively, were achieved in a growth associated process using 20 g/L glycerol in the BLW after 36 h of exponential growth. The PHA monomer compositions were 3HB (3-hydroxybutyrate), a short-chain-length-PHA, and the medium-chain-length-PHA e.g. 3-hydroxyoctanoate and 3-hydroxydecanoate. Both the phbC and phaC genes were characterized. The phbC enzyme had not been previously detected in a Pseudomonas mendocina species. A 2.15 g/L of an exopolysaccharide, alginate, was also produced with a similar composition to that of other Pseudomonas species.

Polyhydroxyalkanoates (PHAs) has been paid great attention because of its useful thermoplastic properties and complete degradation in various natural environments. But, at industrial level, the successful commercialization of PHAs is limited by the high production cost due to the expensive carbon source and recovery processes. Pseudomonas mendocina PSU cultured for 72 h in mineral salts medium (MSM) containing 2% (v/v) biodiesel liquid waste (BLW) produced 79.7 wt% poly(3-hydroxybutyrate) (PHB) at 72 h. In addition, this strain produced 43.6 wt% poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with 8.6 HV mol% at 60 h when added with 0.3% sodium propionate. The synthesized intracellular PHA granules were recovered and purified by the recently reported biological method using mealworms. The weight average molecular weight (Mw ) and number average molecular weight (Mn ) of the biologically extracted PHA were higher than that from the chloroform extraction with comparable melting temperature (Tm ) and high purity. This study has successfully established a low-cost process to synthesize PHAs from BLW and subsequently confirmed the ability of mealworms to extract PHAs from various kinds of bacterial cells.

To enhance the biosynthesis of medium-chain-length polyhydroxyalkanoates (PHAMCL) from glucose in Pseudomonas mendocina NK-01, metabolic engineering strategies were used to block or enhance related pathways. Pseudomonas mendocina NK-01 produces PHAMCL from glucose. Besides the alginate oligosaccharide biosynthetic pathway proved by our previous study, UDP-d-glucose and dTDP-l-rhamnose biosynthetic pathways were identified. These might compete for glucose with the PHAMCL biosynthesis. First, the alg operon, galU and rmlC gene were deleted one by one, resulting in NK-U-1(∆alg), NK-U-2 (∆alg∆galU), NK-U-3(alg∆galU∆rmlC). After fermentation for 36 h, the cell dry weight (CDW) and PHAMCL production of these strains were determined. Compared with NK-U: 1) NK-U-1 produced elevated CDW (from 3.19 ± 0.16 to 3.5 ± 0.11 g/l) and equal PHAMCL (from 0.78 ± 0.06 to 0.79 ± 0.07 g/l); 2) NK-U-2 produced more CDW (from 3.19 ± 0.16 to 3.55 ± 0.23 g/l) and PHAMCL (from 0.78 ± 0.06 to 1.05 ± 0.07 g/l); 3) CDW and PHAMCL dramatically decreased in NK-U-3 (1.53 ± 0.21 and 0.41 ± 0.09 g/l, respectively). Additionally, the phaG gene was overexpressed in strain NK-U-2. Although CDW of NK-U-2/phaG decreased to 1.29 ± 0.2 g/l, PHA titer (%CDW) significantly increased from 24.5 % up to 51.2 %. The PHAMCL biosynthetic pathway was enhanced by blocking branched metabolic pathways in combination with overexpressing phaG gene.

Enzyme catalysts will be vital in the development of synthetic biology approaches for converting pectinic monosaccharides from citrus and beet processing waste streams to value-added materials. We describe here the biophysical and mechanistic characterization of uronate dehydrogenases from a wide variety of bacterial sources that convert galacturonic acid, the predominate building block of pectin from these plant sources, and glucuronic acid to their corresponding dicarboxylic acids galactarate and glucarate, the latter being a DOE top value biochemical from biomass. The enzymes from Pseudomonas syringae and Polaromonas naphthalenivorans were found to have the highest reported kcat(glucuronic acid) values, on the order of 220-270 s(-1). The thermal stability of this enzyme type is described for the first time here, where it was found that the Kt((0.5)) value range was >20 °C, and the enzyme from Chromohalobacter was moderately thermostable with Kt((0.5))=62.2 °C. The binding mechanism for these bi-substrate enzymes was also investigated in initial rate experiments, where a predominately steady-state ordered binding pattern was indicated.

Successful colonization is the initial step for plant-bacteria interactions; therefore, the development of strategies to improve adherence to plant surfaces is critically important for environmental bacteria. Biofilm formation is thought to be one such strategy for bacteria to establish stable colonization on inert and living surfaces. Although biofilms play potential roles in enabling persistent bacterial colonization, little attention has been paid to biofilms formed by plant-associated bacteria. In this study, we characterized the biofilm-forming ability of 6 species of bacteria from the family Pseudomonadaceae: Pseudomonas protegens, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas mendocina, and Pseudomonas syringae. These strains exhibit different degrees of biofilm formation depending on incubation time and nutrient availability. Distinct preferences for growth media were observed, as biofilms were formed by P. protegens with rich nutrients and by P. fluorescens and P. putida with poor nutrients. Likewise, P. stutzeri did not form biofilms with rich nutrients but did form biofilms under nutrient-poor conditions. These observations indicate that particular components in media may influence biofilm formation. P. putida, one of the strains with high biofilm-forming ability, showed the highest ability for initial attachment, which may be mediated by the hydrophobicity of its cell surface. P. mendocina also has high ability for initial attachment, and this strain produces cell surface-attached extracellular polysaccharides that promote cell aggregation. Thus, each strain possesses different properties that facilitate biofilm formation. Shedding light on bacterial strategies for colonization via biofilm formation would enable a better understanding of plant-bacteria interactions.

Root exudates are chemical compounds that are released from living plant roots and provide significant energy, carbon, nitrogen and phosphorus sources for microbes inhabiting the rhizosphere. The exudates shape the microflora associated with the plant, as well as influences the plant health and productivity. Therefore, a better understanding of the trophic link that is established between the plant and the associated bacteria is necessary. In this study, a comprehensive survey on the utilization of grapevine and rootstock related organic acids were conducted on a vineyard soil isolate which is Pseudomonas mendocina strain S5.2. Phenotype microarray analysis has demonstrated that this strain can utilize several organic acids including lactic acid, succinic acid, malic acid, citric acid and fumaric acid as sole growth substrates. Complete genome analysis using single molecule real-time technology revealed that the genome consists of a 5,120,146 bp circular chromosome and a 252,328 bp megaplasmid. A series of genetic determinants associated with the carbon utilization signature of the strain were subsequently identified in the chromosome. Of note, the coexistence of genes encoding several iron–sulfur cluster independent isoenzymes in the genome indicated the importance of these enzymes in the events of iron deficiency. Synteny and comparative analysis have also unraveled the unique features of d-lactate dehydrogenase of strain S5.2 in the study. Collective information of this work has provided insights on the metabolic role of this strain in vineyard soil rhizosphere. Electronic supplementary material The online version of this article (doi:10.1186/s13568-017-0437-7) contains supplementary material, which is available to authorized users.

We optimized the culture medium for 3-hydroxycarboxylic acid production by Pseudomonas mendocina DS-04-T-biodegraded polyhydroxybutyrate (PHB) using the Plackett-Burman design, steepest ascent method, and Box-Behnken design. The optimized concentrations of the constituents of the culture medium were as follows: PHB (7.57 g/L), NH4 Cl (5.0 g/L), KH2 PO4 (2.64 g/L), Na2 HPO4 ·12H2 O (12 g/L), MgSO4 ·7H2 O (0.5 g/L), and CaCl2 ·2H2 O (5 mg/L). The yield of 3-hydroxycarboxylic acid obtained using the optimized culture medium was 56.8 ± 1.64%, which was 2.5-fold higher than that obtained when the unoptimized culture medium was used.

Four methyl tert-butyl ether (MTBE)-degrading bacteria were isolated from a processing plant in Malaysia. Based on 16S rDNA sequences, the four isolates were identified as Exiguobacterium profundum P1M-2, Bacillus megaterium P1M-11, Alishewanella sp. P2A-12 and Pseudomonas mendocina P2M-8. Each of the isolates obtained optimum growth using a different source of nitrogen (0.1- 0.03 % yeast or peptone) and all four isolates were able to biodegrade 92.05-99.98 % of MTBE within seven days. Amongst the four isolates, the highest percentage of MTBE degradation was achieved using B. megaterium P1M-11. The highest growth on tert-butyl alcohol (TBA), tert-amyl alcohol (TAA) and 2-hydroxyisobutyric acid (2-HIBA) was also observed in B. megaterium P1M-11. This study suggests MTBE degradation by each of the bacteria can be enhanced by choosing the right nitrogen source. Furthermore, the ability of B. megaterium P1M-11 to grow on primary metabolites of MTBE and other structurally related ethers suggests the secretion of diverse degradative enzymes, making this isolate a good candidate to be applied in MTBE bioremediation strategies.

A novel toluene monooxygenase (TMO) six-gene cluster from Bradyrhizobium sp. BTAi1 having an overall 35, 36, and 38 % protein similarity with toluene o-xylene monooxygenase (ToMO) of Pseudomonas sp. OX1, toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1, and toluene-para-monooxygenase (TpMO) of Ralstonia pickettii PKO1, respectively, was cloned and expressed in Escherichia coli TG1, and its potential activity was investigated for aromatic hydroxylation and trichloroethylene (TCE) degradation. The natural substrate toluene was hydroxylated to p-cresol, indicating that the new toluene monooxygenase (T4MO·BTAi1) acts as a para hydroxylating enzyme, similar to T4MO and TpMO. Some shifts in regiospecific hydroxylations were observed compared to the other wild-type TMOs. For example, wild-type T4MO·BTAi1 formed catechol (88 %) and hydroquinone (12 %) from phenol, whereas all the other wild-type TMOs were reported to form only catechol. Furthermore, it was discovered that TG1 cells expressing wild-type T4MO·BTAi1 mineralized TCE at a rate of 0.67 ± 0.10 nmol Cl(-)/h/mg protein. Saturation and site directed mutagenesis were used to generate eight variants of T4MO·BTAi1 at alpha-subunit positions P101, P103, and H214: P101T/P103A, P101S, P101N/P103T, P101V, P103T, P101V/P103T, H214G, and H214G/D278N; by testing the substrates phenol, nitrobenzene, and naphthalene, positions P101 and P103 were found to influence the regiospecific oxidation of aromatics. For example, compared to wild type, variant P103T produced four fold more m-nitrophenol from nitrobenzene as well as produced mainly resorcinol (60 %) from phenol whereas wild-type T4MO·BTAi1 did not. Similarly, variants P101T/P103A and P101S synthesized more 2-naphthol and 2.3-fold and 1.6-fold less 1-naphthol from naphthalene, respectively.

Highlights • Selective enrichment was used to isolate active biodegradative bacteria. • The role of chemotaxis in xenobiotic metabolism was elucidated. • Wastewater mesocosms were identified as a repository for biodegradative bacteria., Wastewater environment is a rich source of microorganisms with the capability for the degradation of malicious aromatic pollutants. Although wastewater could be regarded as both a resource and a problem, we intended to elucidate its beneficial aspect in this study sourcing for laccase-producing proteobacteria. Different wastewater samples, from selected wastewater treatment plants (WWTPs), were selectively enriched with some model compounds (vanillin, lignin and potassium hydrogen phthalate) to screen out bacterial isolates that possess excellent degradation potentials. Thereafter, positive isolates were screened for the production of laccase and degradation on phenolic (guaiacol, α-naphthol and syringaldazine) and non-phenolic (ABTS; 2,2 azino-bis -(3-ethylbenzothiazoline 6 sulphonic acid) and PFC; potassium ferrocyanoferrate) substrates characteristic of laccase oxidation. Remarkable laccase producers were identified based on their 16 S rRNA sequences and their secreted enzymes were subjected to substrate specificity test, employing laccase substrates; ABTS, PFC, guaiacol, α-naphthol, 2,6-dimethoxyphenol and pyrogallol. Results showed that wastewater and selective enrichment, in tandem, produced the gammaproteobacteria Pseudomonas aeruginosa DEJ16, Pseudomonas mendocina AEN16 and Stenotrophomonas maltophila BIJ16, which preferably oxidized the non-phenolic substrates. Units of extracellular laccase activity ranging between cca. 490 and cca. 600 U/mL were recorded for ABTS whereas outputs recorded from PFC catalysis ranged from cca. 320 to cca. 430 U/mL. Stenotrophomonas maltophila BIJ16 presented an unparalleled high laccase activity and had a responsive substrate specificity to aromatic and inorganic substrates, thereby suggesting its employment for in situ biodegradation studies. In conclusion, wastewater serves as an ideal milieu for the isolation of laccase producing bacteria.

Two polyhydroxyalkanoate depolymerases, PHAase I and PHAase II, were purified to homogeneity from the culture supernatant of an effective PHA-degrading bacterium, Pseudomonas mendocina DS04-T. The molecular masses of PHAase I and PHAase II were determined by SDS-PAGE as 59.4 and 33.8 kDa, respectively. Their optimum pH values were 8.5 and 8, respectively. Enzymatic activity was optimal at 50 °C. Both purified enzymes could degrade PHB, PHBV, and P(3HB-co-4HB). Addition of Na(+) and K(+) slightly increased the rate of PHAase II. EDTA significantly inhibited PHAase II but not PHAase I. Mercaptoethanol and H2O2 also inhibited the activities of both enzymes.

Glucosylglycerols (GGs) are known as compatible solutes accumulated by some bacteria including cyanobacteria as well as higher plants for their adaptations to salt or desiccation stresses. Since being identified in Japanese sake, their physiological effects and potential applications on human health cares have been explored in the following 15 years. Several different synthesis methods have been successively developed for the production of GGs. However, the efficiency of GG synthesis, especially biological synthesis, is still low. With the recent advances in genome sequencing and synthetic biology tools, systematical screening of enzyme candidates and metabolic engineering approaches is necessary for improving GG synthesis efficiency. In this review, we will summarize GG structure information, protective effects on human skin and digestive system as well as industrial enzymes, together with their synthesis by chemical, enzymatic, and biological in vivo approaches in detail, and provide some prospects on improving GG production.

Summary The two-component system TmoS/TmoT controls the expression of the toluene-4-monooxygenase pathway in Pseudomonas mendocina RK1 via modulation of PtmoX activity. The TmoS/TmoT system belongs to the family of TodS/TodT like proteins. The sensor kinase TmoS is a 108 kDa protein composed of seven different domains. Using isothermal titration calorimetry we show that purified TmoS binds a wide range of aromatic compounds with high affinities. Tightest ligand binding was observed for toluene (KD = 150 nM), which corresponds to the highest affinity measured between an effector and a sensor kinase. Other compounds with affinities in the nanomolar range include benzene, the 3 xylene isomers, styrene, nitrobenzene or p-chlorotoluene. We demonstrate that only part of the ligands that bind to TmoS increase protein autophosphorylation in vitro and consequently pathway expression in vivo. These compounds are referred to as agonists. Other TmoS ligands, termed antagonists, failed to increase TmoS autophosphorylation, which resulted in their incapacity to stimulate gene expression in vivo. We also show that TmoS saturated with different agonists differs in their autokinase activities. The effector screening of gene expression showed that promoter activity of PtmoX and PtodX (controlled by the TodS/TodT system) is mediated by the same set of 22 compounds. The common structural feature of these compounds is the presence of a single aromatic ring. Among these ligands, toluene was the most potent inducer of both promoter activities. Information on the TmoS/TmoT and TodS/TodT system combined with a sequence analysis of family members permits to identify distinct features that define this protein family.

Choline is abundant in association with eukaryotes and plays roles in osmoprotection, thermoprotection, and membrane biosynthesis in many bacteria. Aerobic catabolism of choline is widespread among soil proteobacteria, particularly those associated with eukaryotes. Catabolism of choline as a carbon, nitrogen, and/or energy source may play important roles in association with eukaryotes, including pathogenesis, symbioses, and nutrient cycling. We sought to generate choline analogues to study bacterial choline catabolism in vitro and in situ . Here we report the characterization of a choline analogue, propargylcholine, which inhibits choline catabolism at the level of Dgc enzyme-catalyzed dimethylglycine demethylation in Pseudomonas aeruginosa . We used genetic analyses and 13 C nuclear magnetic resonance to demonstrate that propargylcholine is catabolized to its inhibitory form, propargylmethylglycine. Chemically synthesized propargylmethylglycine was also an inhibitor of growth on choline. Bioinformatic analysis suggests that there are genes encoding DgcA homologues in a variety of proteobacteria. We examined the broader utility of propargylcholine and propargylmethylglycine by assessing growth of other members of the proteobacteria that are known to grow on choline and possess putative DgcA homologues. Propargylcholine showed utility as a growth inhibitor in P. aeruginosa but did not inhibit growth in other proteobacteria tested. In contrast, propargylmethylglycine was able to inhibit choline-dependent growth in all tested proteobacteria, including Pseudomonas mendocina , Pseudomonas fluorescens , Pseudomonas putida , Burkholderia cepacia , Burkholderia ambifaria , and Sinorhizobium meliloti . We predict that chemical inhibitors of choline catabolism will be useful for studying this pathway in clinical and environmental isolates and could be a useful tool to study proteobacterial choline catabolism in situ .

When Pseudomonas mendocina NK-01 was cultivated in a 200-L fermentor using glucose as carbon source, 0.316 g L(-1) medium-chain-length polyhydroxyalkanoate (PHA(MCL)) and 0.57 g L(-1) alginate oligosaccharides (AO) were obtained at the end of the process. GC/MS was used to characterize the PHA(MCL), which was found to be a polymer mainly consisting of 3HO (3-hydroxyoctanoate) and 3HD (3-hydroxydecanoate). T (m) and T (g) values for the PHA(MCL) were 51.03 °C and -41.21 °C, respectively, by DSC. Its decomposition temperature was about 300 °C. The elongation at break was 700% under 12 MPa stress. MS and GPC were also carried out to characterize the AO which had weight-average molecular weights of 1,546 and 1,029 Da, respectively, for the two main components at the end of the fermentation process. MS analysis revealed that the AO were consisted of β-D-mannuronic acid and/or α-L-guluronic acid, and the β-D-mannuronic acid and/or α-L-guluronic acid residues were partially acetylated at position C2 or C3.

A strain of Pseudomonas mendocina producing extracellular lipase was isolated from soil. The bacterium accumulates lipase in culture fluid when grown aerobically at 30 °C for 24 h in a medium composed of olive oil (1%) as substrate. Pseudomonas mendocina lipase was optimally active at pH 9.0, temperature of 50 °C and was found to be stable between pH 7.0-11.0. The lipase was inhibited by detergents such as SDS and Tween-80. The enzyme was stable in various organic solvents tested with maximum stability in chloroform followed by toluene and exhibited 1-3 regiospecificity for hydrolytic reaction. This lipase was capable of hydrolyzing a variety of lipidic substrates and is mainly active towards synthetic triglycerides and fatty acid esters that possess a butyryl group. Metal ions like Mg(2+), Ca(2+) and Na(+) stimulated lipase activity, whereas, Cu(2+), Mn(2+) and Hg(2+) ions caused inhibition.

Evident effect of an algicidal bacterium Pseudomonas mendocina on the growth and antioxidant system of Aphanizomenon flos-aquae was detected in this experiment. Seven parameters including the chlorophyll a contents, Fv/Fm values, reactive oxygen species (ROS), malonaldehyde (MDA), catalase (CAT), peroxide dismutase (POD), and superoxide dismutase (SOD) were tested in the cyanobacterium A. flos-aquae cells after inoculation with the algicidal bacterium Pseudomonas mendocina DC10. It was shown from the experiment that the growth of the treated cyanobacterium A. flos-aquae was significantly restrained, which was expressed as great reductions in the chlorophyll a contents and Fv/Fm values. At the same time, the treated cyanobacterial cells exhibited an obvious increase in the production of ROS and MDA compared with the control. CAT and POD activities in the treated group kept at high level, however, they both reduced significantly on day 6. SOD activities in the treated A. flos-aquae showed obvious declines after inoculation, and great augmentations on day 3 and 4, thereafter, they kept in a declining tendency. The results showed the oxidative stresses induced by the bacterium could be a killing agent of the cyanobacterium A. flos-aquae cells.

A Gram-negative, deep brown-pigmented Gammaproteobacteria, strain IPL-1(T), capable of oxidizing indole was isolated from a lindane-contaminated site and subjected to a polyphasic taxonomic study. Most of the physiological and biochemical properties, major fatty acids (C(18:1)omega7c, C(16:1)omega7c/iso C(15:0) 2OH and C(16:0)), estimated DNA G+C content (67.2mol%) and 16S rRNA gene sequence analysis showed that strain IPL-1(T) belonged to the genus Pseudomonas. Strain IPL-1(T) exhibited highest 16S rRNA gene sequence similarity with Pseudomonas pseudoalcaligenes (99.0%), followed by Pseudomonas alcaliphila (98.7%), Pseudomonas oleovorans (98.3%), Pseudomonas nitroreducens (98.0%), Pseudomonas mendocina (97.6%) and Pseudomonas stutzeri (97.4%). However, the DNA-DNA relatedness values between strain IPL-1(T) and the closely related taxa were between 22% and 61%. On the basis of differential phenotypic characteristics and genotypic distinctiveness, strain IPL-1(T) should be classified within the genus Pseudomonas as a novel species, for which the name Pseudomonas indoloxydans is proposed. The type strain is IPL-1(T) (=MTCC 8062(T)=JCM 14246(T)).

The growth and botulinic toxin production of Clostridium argentinense G 89 HT in co-culture with Pseudomonas mendocina were studied using two culture systems with carbon sources of rapid and slow utilization. Growth with glucose in homogeneous co-culture presented a dual-phase progression, with the toxin produced in the slow-growing phase. The extended 50 h growth period of the second phase at low specific growth rate was attributed to the combined metabolization of glucose and a complex carbon source of the alginate type produced by P. mendocina under strongly reducing conditions. With dextrin, the co-culture grew at the lower specific growth rate (μ = 0.03 h−1) for a period lasting 80 h. This fully enhanced the production of toxin with a specific toxicity 2.5 times higher than with glucose in a homogeneous system and 10.7 higher than that of C. argentinense G 89 HT single culture. The heterogeneous co-culture obtained with a dialysis membrane physically separating both bacteria (thereby eliminating the metabolic interaction) produced the lowest levels of growth and toxin of all the cases analyzed.

Five bacterial strains were isolated from wastewater treatment facilities which were able to use furfural as the sole carbon source. Based on 16S rRNA phylogenetic analysis, these strains were identified as Cupriavidus pinatubonensis (designated ALS1280), Pigmentiphaga sp. (ALS1172), Pseudomonas sp. BWDY (ALS1279), Pseudomonas mendocina (ALS1131), and Pseudomonas putida (ALS1267). In all cases, growth under oxygenated conditions on furfural was accompanied by the transient accumulation of 2-furoic acid (furoate) with no furfuryl alcohol observed. ALS1267 and ALS1279 were also able to metabolize 5-(hydroxymethyl)furfural. The five isolates and their phylogenetic near neighbors were compared for furfural dehydrogenase activity and tolerance to furfural and furoate in defined and complex media. P. putida ALS1267 was the most tolerant to furans and tolerated 17 mM furfural or 195 mM furoate before its growth rate was reduced by 50 % in a defined medium. This strain also had the greatest specific growth rate on furfural (0.6/h at 27-30 °C) and showed the highest specific activity of furfural dehydrogenase (170 mIU/mg) of any furfural-utilizing strain that has been characterized to date.

In this study alginate production by Pseudomonas mendocina in a laboratory-scale fermenter was investigated. In the experiments the effect of temperature (25–31°C) and agitation (500–620 rev min−1) at a constant air flow of 10 v/v/h were evaluated in relation to the rate of glucose bioconversion to alginate using response surface methodology (RSM). The fermenter configuration was also adapted to a system with a screw mixer and draft tube, due to the change in rheological characteristics of the fermentation broth. The adjusted model indicates a temperature of 29.1°C and agitation of 553 rev min−1 for optimum alginate synthesis. In this fermentation system a Yp/s of 44.8% was achieved. The alginate synthesized by P. mendocina showed a partially acetylated pattern as previously reported for alginates obtained from other Pseudomonas spp and Azotobacter vinelandii.

A two-phase system is developed here for converting: (1) benzene to phenol and (2) naphthalene to 2-naphthol, using whole cells expressing wild-type toluene 4-monooxygenase (T4MO) and the alpha subunit variant TmoA I100A from Pseudomonas mendocina KR1. Using the T4MO TmoA I100A variant, the solubility of naphthalene was enhanced and the toxicity of the naphthols was prevented by the use of a water/dioctyl phthalate (80:20, vol%) system which yielded 21-fold more 2-naphthol. More than 99% 2-naphthol was extracted to the dioctyl phthalate phase, dihydroxynaphthalene formation was prevented, 92% 2-naphthol was formed, and 12% naphthalene was converted. Similarly, using 50 vol% dioctyl phthalate, an initial concentration of 3.0 g l(-1) (39 mM), and wild-type T4MO, a 51+/-9% conversion of benzene was obtained and phenol was produced at a purity of 97%. Relative to the one-phase system, there was a 12-fold reduction in the formation of the byproduct catechol.

Denaturing gradient gel electrophoresis of amplified fragments of genes coding for 16S rRNA and for the largest subunit of multicomponent phenol hydroxylase (LmPH) was used to monitor the behaviour and relative abundance of mixed phenol-degrading bacterial populations (Pseudomonas mendocina PC1, P. fluorescens strains PC18, PC20 and PC24) during degradation of phenolic compounds in phenolic leachate- and oil-amended microcosms. The analysis indicated that specific bacterial populations were selected in each microcosm. The naphthalene-degrading strain PC20 was the dominant degrader in oil-amended microcosms and strain PC1 in phenolic leachate microcosms. Strain PC20 was not detectable after cultivation in phenolic leachate microcosms. Mixed bacterial populations in oil-amended microcosms aggregated and formed clumps, whereas the same bacteria had a planktonic mode of growth in phenolic leachate microcosms. Colony hybridisation data with catabolic gene specific probes indicated that, in leachate microcosms, the relative proportions of bacteria having meta (PC1) and ortho (PC24) pathways for degradation of phenol and p-cresol changed alternately. The shifts in the composition of mixed population indicated that different pathways of metabolism of aromatic compounds dominated and that this process is an optimised response to the contaminants present in microcosms.

The regiospecific oxidation of the polycyclic aromatic hydrocarbons naphthalene and fluorene was examined with Escherichia coli strains expressing wildtype toluene 4-monooxygenase (T4MO) from Pseudomonas mendocina KR1, toluene para-monooxygenase (TpMO) from Ralstonia pickettii PKO1, toluene ortho-monooxygenase (TOM) from Burkholderia cepacia G4, and toluene/ortho-xylene monooxygenase (ToMO) from P. stutzeri OX1. T4MO oxidized toluene (12.1 ± 0.8 nmol/min/mg protein at 109 μM), naphthalene (7.7 ± 1.5 nmol/min/mg protein at 5 mM), and fluorene (0.68 ± 0.04 nmol/min/mg protein at 0.2 mM) faster than the other wildtype enzymes (2–22-fold) and produced a mixture of 1-naphthol (52%) and 2-naphthol (48%) from naphthalene, which was successively transformed to a mixture of 2,3-, 2,7-, 1,7-, and 2,6-dihydroxynaphthalenes (7%, 10%, 20%, and 63%, respectively). TOM and ToMO made 1,7-dihydroxynaphthalene from 1-naphthol, and ToMO made a mixture of 2,3-, 2,6-, 2,7-, and 1,7-dihydroxynaphthalene (26%, 22%, 1%, and 44%, respectively) from 2-naphthol. TOM had no activity on 2-naphthol, and T4MO had no activity on 1-naphthol. To take advantage of the high activity of wildtype T4MO but to increase its regiospecificity on naphthalene, seven engineered enzymes containing mutations in T4MO alpha hydroxylase TmoA were examined; the selectivity for 2-naphthol by T4MO I100A, I100S, and I100G was enhanced to 88–95%, and the selectivity for 1-naphthol was enhanced to 87% and 99% by T4MO I100L and G103S/A107G, respectively, while high oxidation rates were maintained except for G103S/A107G. Therefore, the regiospecificity for naphthalene oxidation was altered to practically pure 1-naphthol or 2-naphthol. All four wildtype monooxygenases were able to oxidize fluorene to different monohydroxylated products; T4MO oxidized fluorene successively to 3-hydroxyfluorene and 3,6-dihydroxyfluorene, which was confirmed by gas chromatography – mass spectrometry and 1H nuclear magnetic resonance analysis. TOM and its variant TomA3 V106A oxidize fluorene to a mixture of 1-, 2-, 3-, and 4-hydroxyfluorene. This is the first report of using enzymes to synthesize 1-, 3-, and 4-hydroxyfluorene, and 3,6-dihydroxyfluorene from fluorene as well as 2-naphthol and 2,6-dihydroxynaphthalene from naphthalene. © 2005 Wiley Periodicals Inc.

The water contaminant N-nitrosodimethylamine (NDMA) is a probable human carcinogen whose appear- ance in the environment is related to the release of rocket fuel and to chlorine-based disinfection of water and waste- water. Although this compound has been shown to be biodegradable, there is minimal information about the or- ganisms capable of this degradation, and little is under- stood of the mechanisms or biochemistry involved. This study shows that bacteria expressing monooxygenase en- zymes functionally similar to those demonstrated to de- grade NDMA in eukaryotes have the capability to degrade NDMA. Specifically, induction of the soluble methane monooxygenase (sMMO) expressed by Methylosinus tri- chosporium OB3b, the propane monooxygenase (PMO) en- zyme of Mycobacterium vaccae JOB-5, and the toluene 4-monooxygenases found in Ralstonia pickettii PKO1 and Pseudomonas mendocina KR1 resulted in NDMA degrada- tion by these strains. In each of these cases, brief expo- sure to acetylene gas, a suicide substrate for certain monooxygenases, inhibited the degradation of NDMA. Further, Escherichia coli TG1/pBS(Kan) containing recom- binant plasmids derived from the toluene monooxygenases found in strains PKO1 and KR1 mimicked the behavior of the parent strains. In contrast, M. trichosporium OB3b ex- pressing the particulate form of MMO, Burkholderia cepacia G4 expressing the toluene 2-monooxygenase, and Pseu- domonas putida mt-2 expressing the toluene sidechain monooxygenase were not capable of NDMA degradation. In addition, bacteria expressing aromatic dioxygenases were not capable of NDMA degradation. Finally, Rhodo- coccus sp. RR1 exhibited the ability to degrade NDMA by an unidentified, constitutively expressed enzyme that, un- like the confirmed monooxygenases, was not inhibited by

The hydrolysis of p -nitrophenyl butyrate catalyzed by Pseudomonas mendocina 3121-1 lipase was strongly affected by guanidine hydrochloride indicating the importance of Arg residue. Since loss of the activity in the presence of urea was negligible, the inactivation could not be attributed to protein denaturation. The enzyme was unaffected by p -chlormercuribenzoic acid ( p -CMB), 2-mercaptoethanol and N -ethylmaleimide (NEM) at pH 7.0 indicating that Cys residue was essential neither to catalytic action nor to structural features of the lipase. The inactivation by K 3 Fe(CN) 6 implied that another oxidizable amino acid residue could be important. The inactivation by N -ethylmaleimide at pH 9.0 indicated the involvement of His in the catalysis. Phenylmethylsulfonyl fluoride (PMSF) strongly inhibited the enzyme pointing out the essential role of Ser residue.

The effect of carbon source on the regulation of the de novo pyrimidine biosynthetic enzymes in the bacterium Pseudomonas mendocina was studied. When glucose was the carbon source, orotic acid supplementation of P. mendocina cells produced the greatest depression of aspartate transcarbamoylase, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase activities while P. mendocina cells grown in the presence of uracil caused the maximal decrease in dihydroorotase and OMP decarboxylase activities. After the pyrimidine starvation of an orotate phosphoribosyltransferase mutant strain of P. mendocina grown on glucose, the pyrimidine biosynthetic pathway enzyme activities were generally diminished. With respect to pyrimidine starvation studies, the carbon source glucose appeared to lessen regulation at the level of enzyme synthesis compared to what has been observed when succinate served as the carbon source. The regulation of the pyrimidine biosynthetic pathway by carbon source in P. mendocina appeared to differ from how carbon source influenced the control of pyrimidine biosynthesis in the closely-related species Pseudomonas stutzeri.

The solvent-tolerant strain Pseudomonas putida DOT-T1E has been engineered for biotransformation of toluene into 4-hydroxybenzoate (4-HBA). P. putida DOT-T1E transforms toluene into 3-methylcatechol in a reaction catalyzed by toluene dioxygenase. The todC1C2 genes encode the α and β subunits of the multicomponent enzyme toluene dioxygenase, which catalyzes the first step in the Tod pathway of toluene catabolism. A DOT-T1EΔ todC mutant strain was constructed by homologous recombination and was shown to be unable to use toluene as a sole carbon source. The P. putida pobA gene, whose product is responsible for the hydroxylation of 4-HBA into 3,4-hydroxybenzoate, was cloned by complementation of a Pseudomonas mendocina pobA1 pobA2 double mutant. This pobA gene was knocked out in vitro and used to generate a double mutant, DOT-T1EΔ todCpobA , that was unable to use either toluene or 4-HBA as a carbon source. The tmo and pcu genes from P. mendocina KR1, which catalyze the transformation of toluene into 4-HBA through a combination of the toluene 4-monoxygenase pathway and oxidation of p- cresol into the hydroxylated carboxylic acid, were subcloned in mini-Tn 5Tc and stably recruited in the chromosome of DOT-T1EΔ todCpobA . Expression of the tmo and pcu genes took place in a DOT-T1E background due to cross-activation of the tmo promoter by the two-component signal transduction system TodST. Several independent isolates that accumulated 4-HBA in the supernatant from toluene were analyzed. Differences were observed in these clones in the time required for detection of 4-HBA and in the amount of this compound accumulated in the supernatant. The fastest and most noticeable accumulation of 4-HBA (12 mM) was found with a clone designated DOT-T1E-24.

Diversity of the functional genes encoding dissimilatory nitrite reductase was investigated for the first time in denitrifying halobenzoate degrading bacteria and in two 4-chlorobenzoate degrading denitrifying consortia. Nitrite reductase genes were PCR-amplified with degenerate primers (specific to the two different types of respiratory nitrite reductase, nirS and nirK), cloned and sequenced to determine which type of nitrite reductase was present in each isolate and consortium. Halobenzoate degrading isolates belonging to the genera Ochrobactrum, Ensifer and Mesorhizobium, as well as Pseudomonas mendocina CH91 were found to have nirK genes, which were closely related to the previously published nirK genes of Ochrobactrum anthropi, Achromobacter cycloclastes, Alcaligenes faecalis and Pseudomonas aureofaciens, respectively. The isolates assigned to the genera Acidovorax, Azoarcus and Thauera as well as all other species in the genera Thauera and Azoarcus contained nirS genes, which were closely related to the nirS genes from Pseudomonas stutzeri with some exceptions. In addition, only nirS genes were found in 4-chlorobenzoate degrading denitrifying consortia. Three different major terminal restriction fragments from the nirS genes were detected by terminal restriction fragment length polymorphism analysis of the consortia, and five different nirS genes were cloned from one consortium. Three nirS gene clones were closely related to nirS genes from Thauera chlorobenzoica, Azoarcus tolulyticus and Pseudomonas aeruginosa, respectively. The phylogeny of nir genes was not entirely congruent with the 16S rRNA phylogeny of the genera nor was it correlated with the ecological and geographical origins or isolation substrates used for isolation and enrichment of consortia.

The effects of addition of salicylate or glucose on enzyme activity and phenanthrene degradation by a new phenanthrene-degrading strain Pseudomonas mendocina were investigated. The phenanthrene degradation rate during the linear-decrease stage changed slightly with addition of salicylate or glucose. For intermediate metabolites, high level accumulation of 1-hydroxy-2-naphthoic acid (1H2N) and slight accumulation of 7,8-benzocoumarin were observed. Compared to the control (with only phenanthrene), the apparent accumulation rate of 1H2N increased in the presence of glucose or of a low concentration of salicylate. Dynamic profiles of activities of two key enzymes, polycyclic aromatic hydrocarbon dioxygenase (PDO) and catechol 2,3-dioxygenase (C23O), were also demonstrated. Salicylate addition led to a higher phenanthrene loading of PDO and a higher C23O activity, while glucose supplementation decreased the activities of PDO and C23O. Based on the kinetic data of phenanthrene degradation and PDO activity, PDO activity may not necessarily be the dominant factor affecting the biodegradation in the presence of salicylate or glucose.

Pseudomonas mendocina isolated from soil degraded an insecticide, Monocrotophos (MCP), by 67% and harbored a 7.4 kb plasmid, designated as pMCP424. On the basis of curing and transformation experiments, MCP degradation by Pseudomonas mendocinawas plasmid-borne and transferable to other bacteria.

This study describes a comparison of the polyhydroxyalkanoate (PHA) synthases PhaC1 and PhaC2 of Pseudomonas mendocina. The P. mendocina pha gene locus, encoding two PHA synthase genes [phaC1Pm and phaC2Pm flanking a PHA depolymerase gene (phaZ)], was cloned, and the nucleotide sequences of phaC1Pm (1,677 bp), phaZ (1,034 bp), and phaC2Pm (1,680 bp) were determined. The amino acid sequences deduced from phaC1Pm and phaC2Pm showed highest similarities to the corresponding PHA synthases from other pseudomonads sensu stricto. The two PHA synthase genes conferred PHA synthesis to the PHA-negative mutants P. putida GPp104 and Ralstonia eutropha PHB–4. In P. putida GPp104, phaC1Pm and phaC2Pm mediated PHA synthesis of medium-chain-length hydroxyalkanoates (C6–C12) as often reported for other pseudomonads. In contrast, in R. eutropha PHB–4, either PHA synthase gene also led to the incorporation of 3-hydroxybutyrate (3HB) into PHA. Recombinant strains of R. eutropha PHB–4 harboring either P. mendocina phaC gene even accumulated a homopolyester of 3HB during cultivation with gluconate, with poly(3HB) amounting to more than 80% of the cell dry matter if phaC2 was expressed. Interestingly, recombinant cells harboring the phaC1 synthase gene accumulated higher amounts of PHA when cultivated with fatty acids as sole carbon source, whereas recombinant cells harboring PhaC2 synthase accumulated higher amounts when gluconate was used as carbon source in storage experiments in either host. Furthermore, isogenic phaC1 and phaC2 knock-out mutants of P. mendocina provided evidence that PhaC1 is the major enzyme for PHA synthesis in P. mendocina, whereas PhaC2 contributes to the accumulation of PHA in this bacterium to only a minor extent, and then only when cultivated on gluconate.

An alginate lyase gene, algA, encoding a new poly beta-d-mannuronate (polyM)-specific alginate lyase AlgA, was cloned from Pseudomonas sp. E03. The recombinant AlgA with (His)(6)-tag, consisting of 364 amino acids (40.4 kDa),was purified using Ni-NTA Sepharose. The purified lyase had maximal activity (222 EU/mg) at pH 8 and 30 A degrees C and also maintained activity between pH 7-9 and below 45 A degrees C. It exclusively and endolytically depolymerized polyM by beta-elimination into oligosaccharides with degrees of polymerization (DP) of 2-5. Due to its high substrate specificity, AlgA could be a valuable tool for production of polyM oligosaccharides with low DP and for determining the fine structure of alginate.

The organophosphate pesticide chlorpyrifos (CP) has been used extensively since the 1960s for insect control. However, its toxic effects on mammals and persistence in environment necessitate its removal from contaminated sites, biodegradation studies of CP-degrading microbes are therefore of immense importance. Samples from a Pakistani agricultural soil with an extensive history of CP application were used to prepare enrichment cultures using CP as sole carbon source for bacterial community analysis and isolation of CP metabolizing bacteria. Bacterial community analysis (denaturing gradient gel electrophoresis) revealed that the dominant genera enriched under these conditions were Pseudomonas, Acinetobacter and Stenotrophomonas, along with lower numbers of Sphingomonas, Agrobacterium and Burkholderia. Furthermore, it revealed that members of Bacteroidetes, Firmicutes, α- and γ-Proteobacteria and Actinobacteria were present at initial steps of enrichment whereas β-Proteobacteria appeared in later steps and only Proteobacteria were selected by enrichment culturing. However, when CP-degrading strains were isolated from this enrichment culture, the most active organisms were strains of Acinetobacter calcoaceticus, Pseudomonas mendocina and Pseudomonas aeruginosa. These strains degraded 6-7.4 mg L(-1) day(-1) of CP when cultivated in mineral medium, while the consortium of all four strains degraded 9.2 mg L(-1) day(-1) of CP (100 mg L(-1)). Addition of glucose as an additional C source increased the degradation capacity by 8-14 %. After inoculation of contaminated soil with CP (200 mg kg(-1)) disappearance rates were 3.83-4.30 mg kg(-1) day(-1) for individual strains and 4.76 mg kg(-1) day(-1) for the consortium. These results indicate that these organisms are involved in the degradation of CP in soil and represent valuable candidates for in situ bioremediation of contaminated soils and waters.

The bioremediation of polluted groundwater and toxic waste sites requires that bacteria come into close physical contact with pollutants. This can be accomplished by chemotaxis. Five motile strains of bacteria that use five different pathways to degrade toluene were tested for their ability to detect and swim towards this pollutant. Three of the five strains ( Pseudomonas putida F1, Ralstonia pickettii PKO1, and Burkholderia cepacia G4) were attracted to toluene. In each case, the response was dependent on induction by growth with toluene. Pseudomonas mendocina KR1 and P. putida PaW15 did not show a convincing response. The chemotactic responses of P. putida F1 to a variety of toxic aromatic hydrocarbons and chlorinated aliphatic compounds were examined. Compounds that are growth substrates for P. putida F1, including benzene and ethylbenzene, were chemoattractants. P. putida F1 was also attracted to trichloroethylene (TCE), which is not a growth substrate but is dechlorinated and detoxified by P. putida F1. Mutant strains of P. putida F1 that do not oxidize toluene were attracted to toluene, indicating that toluene itself and not a metabolite was the compound detected. The two-component response regulator pair TodS and TodT, which control expression of the toluene degradation genes in P. putida F1, were required for the response. This demonstration that soil bacteria can sense and swim towards the toxic compounds toluene, benzene, TCE, and related chemicals suggests that the introduction of chemotactic bacteria into selected polluted sites may accelerate bioremediation processes.

Several toluene monooxygenase-producing organisms were tested for their ability to oxidize linear alkenes and chloroalkenes three to eight carbons long. Each of the wild-type organisms degraded all of the alkenes that were tested. Epoxides were produced during the oxidation of butene, butadiene, and pentene but not hexene or octadiene. A strain of Escherichia coli expressing the cloned toluene-4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 was able to oxidize butene, butadiene, pentene, and hexene but not octadiene, producing epoxides from all of the substrates that were oxidized. A T4MO-deficient variant of P. mendocina KR1 oxidized alkenes that were five to eight carbons long, but no epoxides were detected, suggesting the presence of multiple alkene-degrading enzymes in this organism. The alkene oxidation rates varied widely (ranging from 0.01 to 0.33 μmol of substrate/min/mg of cell protein) and were specific for each organism-substrate pair. The enantiomeric purity of the epoxide products also varied widely, ranging from 54 to >90% of a single epoxide enantiomer. In the absence of more preferred substrates, such as toluene or alkenes, the epoxides underwent further toluene monooxygenase-catalyzed transformations, forming products that were not identified.

Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by bacteria. In this study, the effect of C:N molar ratio on the monomer composition of PHAs was investigated, including medium chain length PHA produced by Pseudomonas mendocina 0806 and PHA blends consisting of monomers of 3-hydroxybutyrate and medium chain length hydroxyalkanoate produced by Pseudomonas pseudoalkaligenus YS1. It was observed that there were some fixed ranges of C:N molar ratio that affect the monomer composition of PHA independently of the substrate. For strain 0806, the ranges were C:N20, 20C:N200, and C:N200. The monomer composition was constant among these ranges when using glucose and octanoate as the sole substrate. For strain YS1, the ranges were C:N20, 20C:N45, and C:N45. These results are useful for controlling monomer composition in PHA production.

Pseudomonas mendocina P2d cells grown at room temperature in sodium benzoate as sole source of carbon, followed by storage on ice, form a viscous pellet on centrifugation. Such viscosity is not produced by cells grown on glucose or any other carbohydrates. Viscosity was found to be associated with the extracellular polysaccharide (EPS) of cells and not released into the supernatant fluid. A combination of sodium dodecyl sulphate-citrate buffer and homogenization was effective in releasing the EPS. The EPS is a heteropolysaccharide, consisting of rhamnose, fucose, glucose, ribose, arabinose and mannose, which has good emulsifying activity.

Growth of the Pseudomonas mendocina ymp strain on insoluble ferrihydrite is enhanced by exogenous reductants with concurrent increase in soluble iron concentrations. This shows that exogenous reductants play a substantial role in the overall microbial iron bioavailability. The exogenous reductants may work together with the siderophores, Fe-scavenging agents, to facilitate ferrihydrite dissolution.

Pulsed electric discharge (PED) and bioreme- diation were combined to create a novel two-stage sys- tem which dechlorinates the halogenated pollutants, 2,4- dichlorophenol and perchloroethylene, with repetitive (0.1-1 kHz), short pulse (~100 ns), low voltage (40-80 kV) discharges and then mineralizes the less chlorinated products with aerobic bacteria. A 6.1 mM aqueous di- chlorophenol sample was cycled through the PED reac- tor (60 kV of applied pulsed voltage and 300 Hz) 6 times, resulting in the release of 55% of the initial dichlorophe- nol chloride ions (1 mM Cl ˛ removed each cycle). The respective average specific efficiency is 0.4-0.6 keV/(Cl ˛ molecule). Pseudomonas mendocina KR1, which grows in minimal medium supplemented with phenol but not with dichlorophenol, increased in cell density in all cul- tures supplemented with the PED-treated DCP samples and yielded a maximum of two-fold additional Cl ˛ re- leased compared to the PED-related alone. The number of PED-treatment cycles, voltage, and frequency were also varied, showing that both cell densities and overall dichlorophenol dechlorination were highly dependent upon the number of PED-treatment cycles, rather than the tested voltages and frequencies. Using this two-stage treatment system, PED released 31% of the initial chlo- ride ions from dichlorophenol (after three cycles at 40-45 kV and 1.2 kHz) while P. mendocina KR1 in the second stage increased dechlorination to 90%. These results were corroborated by the 35% additional chloride release found with activated sludge cultures. Perchloroethylene (0.6 mM) was similarly treated in a first-stage PED reactor (80% chloride removal after four cycles) followed by bio- degradation of the dechlorinated products with a recom- binant toluene o-monooxygenase-expressing Pseudo- monas fluorescens strain. Gas chromatographic analysis showed that the PED reactor created less-chlori- nated byproducts (i.e., trichloroethylene) that were removed (74%) upon exposure to the recombinant bac- terium. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 438-444, 1998.

Pseudomonas mendocina MC2, able to use 9-fluorenone but not fluorene as its sole source of carbon and energy, was isolated. Identification of metabolites in growth media and washed cell suspensions indicated that strain MC2 metabolizes 9-fluorenone via angular dioxygenation of the ketone, to give 1,1a-dihydroxy-1-hydro-9-fluorenone, followed by the opening of the five-membered ring and further degradation of the resulting biphenyl derivative by reactions akin to those of biphenyl metabolism, which produce phthalate as an intermediate. The aim of this research was to study the biodegradation of fluorene by a co-culture of strain MC2 and Arthrobacter sp.strain F101, which grows on fluorene and simultaneously transforms a fraction of the substrate to 9-fluorenone, which accumulates as a dead-end product. Growing with 0.1 g fluorene/L, Arthrobacter sp. strain F101 caused the total removal of this compound from the cultures, but when this strain was grown with 1g fluorene/L, only 16% of the fluorene was used. The addition of 9-fluorenone to cultures growing on fluorene showed that 9-fluorenone inhibits fluorene degradation. Finally, when Pseudomonas mendocina MC2 and Arthrobacter sp. strain F101 were co-cultured with 1g fluorene/L as a sole source of carbon and energy, the growth of the strains completely removed fluorene in 2 days. 9-Fluorenone did not accumulate and the carbon assimilation into cell biomass was estimated as approximately 46%. Key words: microbial consortium, fluorene, 9-fluorenone, biodegradation.

Two microorganisms able to grow in mineral salt medium containing styrene (0.1–1.0%) as the sole carbon source were isolated under static culture conditions. The two strains, LF-1 and KO-8-1, belong to Pseudomonas mendocina . Both strains utilize not only styrene but also toluene and heptanol as carbon sources. Cell viability and total cell count of both strains were higher when they were grown in the presence of 0.1% styrene than in the presence of 1.0% styrene. In the presence of 0.1% styrene, cell viability reached 2.3 × 10 8 cells/ml for strain LF-1 and 7 × 10 8 cells/ml in strain KO8-1, and decreased gradually thereafter. Substrate utilization tests revealed that the substrate specificities of the strains differ. Styrene oxide was found as a degradation product in the culture.