Degradation of o-chloronitrobenzene as the sole carbon and nitrogen sources by Pseudomonas putida OCNB-1

A bacterial strain that utilized o-chloronitrobenzene （o-CNB） as the sole carbon, nitrogen and energy sources was isolated from an activated sludge collected from an industrial waste treatment plant. It was identified as Pseudomonas putida based on its morphology, physiological, and biochemical characteristics with an automatic biometrical system and the 16S rRNA sequence analysis. Microcosm study showed that the biodegradation of o-CNB was optimized at culture medium pH 8.0 and 32℃. At these conditions, the strain degraded 85% of o-CNB at a starting concentration of 1.1 mmol/L in 42 h. o-Chloroaniline was identified as the major metabolite with high performance liquid chromatography （HPLC） and gas chromatography-mass spectrometry （GC-MS）. The study showed that o-CNB degradation by Pseudomonas putida OCNB-1 was initiated by aniline dioxyenase, nitrobenzene reductase and catechol-l,2- dioxygenase.

Enhanced Biological Phosphorus Removal with Pseudomonas putida GM6 from Activated Sludge

The enhanced biological phosphorus removal （EBPR） method is widely adopted for phosphorus removal from wastewater, yet little is known about its microbiological and molecular mechanisms. Therefore, it is difficult to predict and control the deterioration of the EBPR process in a large-scale municipal sewage treatment plant. This study used a novel strain isolated in the laboratory, Pseudomonas putida GM6, which had a high phosphate accumulating ability and could recover rapidly from the deteriorated system and enhance the capability of phosphorus removal in activated sludge. Strain GM6 marked with gfp gene, which was called GMTR, was delivered into a bench-scale sequencing batch reactor （SBR） of low efficiency, to investigate the colonization of GMTR and removal of phosphorus. After 21 days, the proportion of GMTR in the total bacteria of the sludge reached 9.2%, whereas the phosphorus removal rate was 96%, with an effluent concentration of about 0.2 mg L^-1. In the reactor with the addition of GMTR, phosphorus was removed quickly, in 1 h under anaerobic conditions, and in 2 h under aerobic conditions. These evidences were characteristic of EBPR processes. Field testing was conducted at a hospital sewage treatment facility with low phosphorus removal capability. Twentyone days after Pseudomonas putida GM6 was added, effluent phosphorus concentration remained around 0.3 mg L^-1, corresponding to a removal rate of 96.8%. It was therefore demonstrated that Pseudomonas putida GM6 could be used for a quick startup and enhancement of wastewater biological phosphorus removal, which provided a scientific basis for potential large-scale engineering application.

Production of D-p-Hydroxyphenylglycine from DL-5-p-Hydroxyphenyl Hydantoin by Immobilized Pseudomonas Putida Cells in a Hollow Fiber Membrane Bioreactor

An immobilized cell membrane bioreaction system was developed to promote cell stability. The hollow fiber membrane bioreactor with immobilized Pseudomonas putida cells, operating in continual repeated batch operation mode was used for producing D-p-hydroxyphenylglycine from DL-5-p-hydroxyphenyl hydantoin. The concentration of N-carbamyl-D-p-hydroxyphenylglycine and D-p-hydroxyphenylglycine in the efflux was analyzed by high performance liquid chromatography at different intervals.

Strain Pseudomonas putida PAO-1 Isolate with Polyphosphate Accumulating and Elongation Ability

Filamentous bacteria(FB)overgrowth is an important cause of sludge bulking in wastewater treatment plants(WWTPs).However,to date,methods for the cultivation and preservation of isolated FB in the laboratory have not been completely described.Furthermore,research on whether FB can function as phosphorus accumulating organisms(PAOs)is limited.In this study,a pure strain,a Pseudomonas putida PAO-1(P.putida PAO-1)isolate with phosphorus removal functions was isolated from the biofilm of an alternating anaerobic/aerobic biofilter(AABF),and its physiological characteristics were studied.Nitrate or nitrite could be used by the strain P.putida PAO-1 as electron acceptors for denitrification during phosphorus anoxic uptake,and 0.63 mg NO-3-N was consumed to reduce 1 mg soluble orthophosphate(SOP)by P.putida PAO-1.The strain P.putida PAO-1 consumed phosphorus within the optimal pH range of 6 to 8 and the temperature range of 25℃to 35℃.Cell deformity was a main morphological trait of the strain P.putida PAO-1,and it could elongate(with an elongation rate of 300%-500%)when it was subjected to oligotrophic or high-salt stress(15 g·L-1 NaCl).The findings in this study provide a microbiological reference for understanding the special characteristics of a denitrifying PAO.

Fundamentals and application in phytoremediation of an efficient arsenate reducing bacterium Pseudomonas putida ARS1

Arsenic is a ubiquitous environmental pollutant.Microbe-mediated arsenic biotransformations significantly infuence arsenic mobility and toxicity.Arsenic transformations by soil and aquatic organisms have been well documented,while little is known regarding effects due to endophytic bacteria.An endophyte Pseudomonas putida ARS1 was isolated from rice grown in arsenic contaminated soil.P.putida ARS1 shows high tolerance to arsenite(As(Ⅲ))and arsenate(As(V)),and exhibits efficient As(V)reduction and As(Ⅲ)effux activities.When exposed to 0.6 mg/L As(V),As(V)in the medium was completely converted to As(Ⅲ)by P.putida ARS1 within 4 hr.Genome sequencing showed that P.putida ARS1 has two chromosomal arsenic resistance gene clusters(arsRCBH)that contribute to efficient As(V)reduction and As(Ⅲ)effux,and result in high resistance to arsenicals.Wolffia globosa is a strong arsenic accumulator with high potential for arsenic phytoremediation,which takes up As(Ⅲ)more efficiently than As(V).Co-culture of P.putida ARS1 and W.globosa enhanced arsenic accumulation in W.globosa by 69%,and resulted in 91%removal of arsenic(at initial concentration of 0.6 mg/L As(V))from water within 3 days.This study provides a promising strategy for in situ arsenic phytoremediation through the cooperation of plant and endophytic bacterium.

Electricity generation by Pseudomonas putida B6-2 in microbial fuel cells using carboxylates and carbohydrate as substrates

Microbial fuel cells(MFCs)employing Pseudomonas putida B6-2(ATCC BAA-2545)as an exoelectrogen have been developed to harness energy from various conventional substrates,such as acetate,lactate,glucose,and fructose.Owing to its metabolic versatility,P.putida B6-2 demonstrates adaptable growth rates on diverse,cost-effective carbon sources within MFCs,exhibiting distinct energy production characteristics.Notably,the anode chamber’s pH rises with carboxylates’(acetate and lactate)consumption and decreases with carbohydrates’(glucose and fructose)utilization.The MFC utilizing fructose as a substrate achieved the highest power density at 411 mW m^(−2).Initial analysis revealed that P.putida B6-2 forms biofilms covered with nanowires,contributing to bioelectricity generation.These microbial nanowires are likely key players in direct extracellular electron transport through physical contact.This study established a robust foundation for producing valuable compounds and bioenergy from common substrates in bioelectrochemical systems(BESs)utilizing P.putida as an exoelectrogen.

Rapid generation of recombinant Pseudomonas putida secondary metabolite producers using yTREX

Microbial secondary metabolites represent a rich source of valuable compounds with a variety of applications in medicine or agriculture.Effective exploitation of this wealth of chemicals requires the functional expression of the respective biosynthetic genes in amenable heterologous hosts.We have previously established the TREX system which facilitates the transfer,integration and expression of biosynthetic gene clusters in various bacterial hosts.Here,we describe the yTREX system,a new tool adapted for one-step yeast recombinational cloning of gene clusters.We show that with yTREX,Pseudomonas putida secondary metabolite production strains can rapidly be constructed by random targeting of chromosomal promoters by Tn5 transposition.Feasibility of this approach was corroborated by prodigiosin production after yTREX cloning,transfer and expression of the respective biosynthesis genes from Serratia marcescens.Furthermore,the applicability of the system for effective pathway rerouting by gene cluster adaptation was demonstrated using the violacein biosynthesis gene cluster from Chromobacterium violaceum,producing pathway metabolites violacein,deoxyviolacein,prodeoxyviolacein,and deoxychromoviridans.Clones producing both prodigiosin and violaceins could be readily identified among clones obtained after random chromosomal integration by their strong color-phenotype.Finally,the addition of a promoter-less reporter gene enabled facile detection also of phenazine-producing clones after transfer of the respective phenazine-1-carboxylic acid biosynthesis genes from Pseudomonas aeruginosa.All compounds accumulated to substantial titers in the mg range.We thus corroborate here the suitability of P.putida for the biosynthesis of diverse natural products,and demonstrate that the yTREX system effectively enables the rapid generation of secondary metabolite producing bacteria by activation of heterologous gene clusters,applicable for natural compound discovery and combinatorial biosynthesis.

Bacteriostatic effect of lipopeptides from Bacillus subtilis N-2 on Pseudomonas putida using soybean meal by solid-state fermentation

Bacillus subtilis N-2 which was isolated from natto,produced lipopeptides using soybean meal as a substrate.This work aimed to purify,identify,and determine the antibacterial mechanism of lipopeptides produced by B.subtilis N-2.The fermented product obtained by solid-state fermentation was subjected to water extraction,acid precipitation,and methanol extraction.Fractions were separated and collected using a two-step ultrafiltration method and then identified by LC-MS/MS.Mass spectrometry characterization revealed the presence of four variants of iturin A that differed according to the p-amino fatty acid chain from C14 to C17 as well as the amino acid positions.A new lipopeptide(m/z 1070.3)was identified and its structure was different from the previously reported lipopeptides.The lipopeptides were shown to inhibit the growth of an isolate of Pseudomonas putida,a common pathogen in decaying fish,by changing membrane permeability.These results suggest that the lipopeptides from B.subtilis N-2 could be used as a biocontrol agent in aquaculture.

Volatiles of Shiraia fruiting body-associated Pseudomonas putida No.24 stimulate fungal hypocrellin production

Hypocrellins are major bioactive perylenequinones from Shiraia fruiting bodies and have been developed as efficient photosensitizers for photodynamic therapy.Pseudomonas is the second dominant genus inside Shiraia fruiting bodies,but with less known actions on the host fungus.In this work,the effects of bacterial volatiles from the Shiraia-associated Pseudomonas on fungal hypocrellin production were investigated.Pseudomonas putida No.24 was the most active to promote significantly accumulation of Shiraia perylenequinones including hypocrellin A(HA),HC,elsinochrome A(EA)and EC.Headspace analysis of the emitted volatiles revealed dimethyl disulfide as one of active compounds to promote fungal hypocrellin production.The bacterial volatiles induced an apoptosis in Shiraia hyphal cell,which was associated with the generation of reactive oxygen species(ROS).ROS generation was proved to mediate the volatile-induced membrane permeability and up-regulation of gene expressions for hypocrellin biosynthesis.In the submerged volatile co-culture,the bacterial volatiles stimulated not only HA content in mycelia,but also HA secretion into the medium,leading to the enhanced HA production to 249.85 mg/L,about 2.07-fold over the control.This is the first report on the regulation of Pseudomonas volatiles on fungal perylenequinone production.These findings could be helpful to understand the roles of bacterial volatiles in fruiting bodies and also provide new elicitation method using bacterial volatiles to stimulate fungal secondary metabolite production.

Improvement of the activity and thermostability of l‑threonine aldolase from Pseudomonas putida via tailoring of the active sites lining the binding pocket

l-threonine aldolases catalyze the conversion of glycine and aldehydes to synthesizeβ-hydroxy-α-amino acids with unsatisfactory enzyme activity.Here,we expressed the l-threonine aldolase from Pseudomonas putida KT2440(l-PpTA)in Escherichia coli BL21(DE3)and improved the activity and thermostability by protein engineering.Five amino acid residues(Ser10,His89,Asp93,Arg177,and Arg321)located in the substrate-binding pocket were selected and for mutation.Eight mutants(D93A,D93G,D93M,D93F,D93S,D93Q,D93Y and D93H)with increased enzyme activity were identified and their k_(cat)/K_(M)values showed about 1-7-fold higher than wild-type.Among all the variants,D93H showed the highest catalytic efficiency with 2925 and 4515 s^(−1)mM^(−1)of k_(cat)/K_(M)values toward l-threonine and l-allo-threonine,respectively.In addition,circular dichroism spectrum exhibited that the melting temperature of D93H(54.2℃)was 5℃higher than wild-type(49.2℃).Molecular dynamics simulations illustrated that the D93H variant shortens the distance between the imidazole group of H93 and the hydroxyl group of substrate,which facilitated the proton extraction and promote the enzymatic reaction.This work affords a candidate for the synthesis ofβ-hydroxy-α-amino acids with improved catalytic efficiency and thermostability and provides structural insights into the l-TA family by protein engineering.

A case report of acute pediatric bacterial meningitis due to the rare isolate,Pseudomonas putida

Acute bacterial meningitis(ABM)is the medical emergency which warrants an early diagnosis and an aggressive therapy.Despite the availability of the potent newer antibiotics,the mortality caused by ABM and its complications remain high in India,ranging from 16%to 32%.The aim of this case report is to present the rare isolation of Pseudomonas putida from cerebrospinal fluid sample.Besides this,the author also emphasizes the importance of correctly identifying the organism and thus the selection of the most accurate antibiotic from the susceptibility profile to allow for early recovery and to improve the patient outcome and survival.

秸秆还田及磷细菌对土壤微生态及豆角产量的影响

为研究秸秆还田及外源添加磷细菌对土壤微生态及农作物产量的影响,以玉米秸秆和具有解磷能力的恶臭假单胞菌(Pseudomonas putida)为研究对象,通过在设施大棚中栽培豆角,研究了不同处理对土壤和豆角根际解磷类细菌数量、土壤有效磷含量、解磷能力以及豆角产量的影响。研究结果显示,玉米秸秆(处理1)、玉米秸秆+磷细菌(处理2)、磷细菌(处理3)三个处理的土壤中解磷类细菌数量有较大差异,其中处理2数量最高,比对照区高31.89%,差异显著(P<0.05);三个处理均能明显增加豆角根际解磷类细菌数量,差异达到显著水平(P<0.05),其中处理2最高,比对照高86.30%,差异达到极显著水平(P<0.01);三个处理土壤有效磷含量均明显高于对照,差异显著(P<0.05),其中处理2最高,比对照高9.8%;三个处理土壤解磷能力差异较大,处理1和处理2可明显提高土壤解磷能力,分别比对照高50.2%和65.2%,差异极显著(P<0.01);三个处理对豆角均有增产效果,但差异较大,处理2比处理1、处理3产量增加明显,分别增产6.8%、10.3%,差异达到显著水平(P<0.05);处理2比对照增产15.5%,差异达极显著水平(P<0.01)。研究结果表明,玉米秸秆配合外源添加磷细菌恶臭假单胞菌,较单一使用玉米秸秆和磷细菌可显著增加土壤及豆角根际解磷类细菌数量、增加土壤有效磷含量、提高土壤解磷能力、促进豆角增产,说明玉米秸秆和磷细菌有相互促进作用。本研究可为秸秆还田和磷细菌田间施用方法的研究和应用提供参考。

Acclimation and Characterization of a Pseudomonas Strain for Improved Phosphorus Removal

A Pseudomonas strain(named as P.PAO-1) with phosphorous removal function was isolated and characterized.A new method of two-stage cultivation was applied to the strain to induce the synthesis of intracellular polyhydroxylbutyrate(PHB) in the cells.In the first stage,bacterial cells were enriched under a high carbon source condition;in the second stage,the bacteria cells were cultivated under the nutrient-imbalanced conditions with the carbon source-regulated medium.As a result,the PHB content of strain P.PAO-1 reached 22.8% compared with the normal 7.41% in the non-acclimated strain.This change had led to the rising of P uptake from 1 to 25 mg·L^(-1).When added the strain P.PAO-1into the activated sludge(with the addition proportion of 1:1),the ratio of biological phosphorus removal increased by 14.9%under the normal alternating anaerobic/aerobic conditions with low-carbon consumption.The results demonstrated that the isolated pure culture strain P.PAO-1 belonged to the functional group of poly-P accumulating microorganism.When cultured by the two-stage cultivation method,the initial accumulation of PHB in the strain cells could be achieved and the phosphorous removal capacity of strain P.PAO-1 could be induced subsequently.When applied to the wastewater,strain P.PAO-1 performed phosphorus removal from the wastewater with or without the addition of activated sludge.

Rs-198液体有机菌肥制备及其促生性能研究

为简化液体有机菌肥的制备工艺,将液体有机肥和植物根际促生菌Pseudomonas putida Rs-198复配,分析Rs-198液体有机菌肥的制备工艺、稳定性及其对萝卜幼苗促生能力的影响。结果表明:液体有机肥的最佳稀释倍数为5倍;Rs-198在稀释液体有机肥中的最佳培养条件为30℃、140 r/min、装液量100 mL;摇瓶发酵36 h时,菌体密度最大可达4.6×10^(9)CFU/mL;制备的Rs-198液体有机菌肥室温储存28 d后有效活菌数为2×10^(7)CFU/mL;膨润土作为其最佳营养助剂,有效活菌数提高了60.2%。液体有机菌肥可显著提高萝卜幼苗生物量,其中株高、鲜重和干重分别增加19.6%、22.2%和17.6%,萝卜叶片中可溶性糖和可溶性蛋白含量分别提高49.4%和12.4%,Rs-198液体有机菌肥应用前景良好。

Comparative Analysis of Various Strains of Plant Growth Promoting Rhizobacteria on the Physiology of Garlic (Allium sativum)

Garlic is a most important medicinal herb belonging to the family Liliaceae. Both its leaves and bulb are edible. The current study was based on evaluating the growth promoting potential of plant growth promoting rhizobacteria (PGPR) on garlic (Allium sativum L.) growth and biochemical contents. Garlic cloves were inoculated with 3 kinds of PGPRs, Pseudomonas putida (KX574857), Pseudomonas stutzeri (Kx574858) and Bacillus cereus (ATCC14579) at 108 cells/mL prior to sowing. Under natural conditions, plants were grown in the net house. The PGPR significantly enhanced % germination, leaf and root growth and their biomass also increased the diameter of bulb and fresh and dry weight. The flavonoids, phenolics, chlorophyll, protein and sugar content were also significantly increased due to PGPR inoculation. The Pseudomonas stutzeri was found most effective for producing longer leaves with moderate sugar, high flavonoids (129%) and phenolics (263%) in bulb over control (Tap). The Pseudomonas putida exhibited a maximum increase in bulb diameter and bulb biomass with maximum phenolics and flavonoid contents.

调控细胞形态相关基因表达提高P.putidaKT2440突变株KTU-U13生产PHA的能力

聚羟基脂肪酸酯(PHA)是多种细菌都能合成的一类多元聚酯,它具有类似于合成塑料的物化特性,同时又具备了合成塑料所不具备的生物可降解性、生物相容性、光学活性、气体相隔性等诸多特性,被认为是最有前途的生物塑料之一.P.putida KT2440突变株KTU-U13是本实验室之前构建的生理性能好.PHA产量高的优良底盘细胞,而据文献报道,细胞体积越大越有利于积累更多的包涵体,通过扩展细胞大小,包括长度和宽度,减少PHA颗粒的空间障碍,增加每个细胞的PHA积累,可以提高产量.本实验希望通过调控细胞形态相关基因mreB,ftsZ及sulA的表达进一步提高突变株KTU-U13的PHA产量,结果显示无论对mreB和ftsZ基因进行质粒过表达还是sRNA抑制都没能提高PHA产量,而质粒过表达sul.A基因使PHA产率提高了10.21%.该结果为在突变株KTU-U13的基础上进一步构建PHA高产菌株提供了可参考的策略.

鹦鹉鱼恶臭假单胞菌的分离鉴定及致病性研究

为有助于防控恶臭假单胞菌引起的鱼病,从天津市某养殖场发病血鹦鹉(Vieja synspila♀×Amphilophus citrinellus♂)的中肾、肝脏处提取到一株细菌,并对其进行病原的分离鉴定、人工回归感染试验以及药敏试验。病灶处分离出的病原菌命名为AC4,经人工回归感染后证明AC4具有致病性,临床症状与其自然发病症状一致,半致死浓度为3×10^(8) CFU/mL。对提取分离出来的致病菌生化鉴定后再进行16S rRNA测序,分离菌革兰氏染色为阴性杆状,该株分离菌为假单胞菌属(Pseudomonas)。药敏试验结果显示,该菌株对氨曲南、头孢他啶、头孢曲松、左氧氟沙星、头孢吡肟、链霉素、多黏菌素B、卡那霉素、阿米卡星等敏感。

Preparation of slow-release regulated Rs-198 bilayer microcapsules and application of its lyophilized bacterial inoculant on Capsicum annuum L.under salt stress

The survival adaptation of bacteria in saline soil is poor.The bilayer microcapsules were prepared by secondary embedding of monolayer sodium alginate(NaAlg)-bentonite(Bent)-sodium carboxy-methylcellulose(CMC)microcapsules wrapped with plant growth promoting rhizobacteria(PGPR)Pseudomonas putida Rs-198 by chitosan solution to promote the synergistic effect of bilayer microen-capsulation and PGPR.The characterization of the Rs-198 bilayer microcapsules showed that the amino and carboxyl groups were cross-linked and a thin layer of chitosan was formed on the outside of the microcapsule.The bilayer microcapsule(Ch-d)with a chitosan concentration of 0.8 wt%and pH 6 showed a slow release of bacteria with a maximum release of 6.06 × 10^(9) cfu/g on the 7th day.The viable bacteria of Ch-d increased by 4.42%after 60 days of storage compared with monolayer microcapsules.The 0.9 wt%L-cysteine,10 wt%glycerinum,10 wt%trehalose and 12 wt%soluble starch were added as bacterial protective agents during the process of preparing the Ch-d lyophilized bacterial inoculant(Ch-d LBI).Pot experiments showed that Ch-d LBI exhibited better growth promotion of Capsicum annuum L.under salt stress.Therefore,the bilayer microcapsule as slow-release bacterial inoculant is a potential alternative for sustainable agriculture.