Physiological and Biochemical Characterization of a Novel Nicotine-Degrading Bacterium Pseudomonas geniculata N1

Management of solid wastes with high nicotine content, such as those accumulated during tobacco manufacturing, poses a major challenge, which can be addressed by using bacteria such as Pseudomonas and Arthrobacter. In this study, a new species of Pseudomonas geniculata, namely strain N1, which is capable of efficiently degrading nicotine, was isolated and identified. The optimal growth conditions for strain N1 are a temperature of 30°C, and a pH 6.5, at a rotation rate of 120 rpm min⁻¹ with 1 g l⁻¹ nicotine as the sole source of carbon and nitrogen. Myosmine, cotinine, 6-hydroxynicotine, 6-hydroxy-N-methylmyosmine, and 6-hydroxy-pseudooxynicotine were detected as the five intermediates through gas chromatography-mass and liquid chromatography-mass analyses. The identified metabolites were different from those generated by Pseudomonas putida strains. The analysis also highlighted the bacterial metabolic diversity in relation to nicotine degradation by different Pseudomonas strains.     

Generalized plant defense: effects on multiple species

Summary Two species of lepidopteran herbivores, Manduca sexta (Sphingidae) and Trichoplusia ni (Noctuidae), were reared on synthetic diet containing either the alkaloid nicotine or the flavonoid rutin. Survival and pupal weight of the specialist M. sexta did not differ when larvae were reared on diet containing nicotine or rutin. In contrast, the generalist T. ni did not survive on diet containing 0.125% nicotine or greater, whereas larvae survived on all concentrations of rutin. These data demonstrate that the alkaloid nicotine is inhibitory toward generalist, but not specialist herbivores, whereas the flavonoid rutin has no effect on specialist herbivores and limited effects on generalist herbivores. Five species of Pseudomonas bacterial pathogens: P. syringae, P. syringae pv. angulata, P. syringae pv. tabaci, P. fluorescens, and P. solanacearum were grown on nutrient agar containing nicotine or rutin at concentrations ranging from 0.0 to 1.0% wet weight in 0.1% intervals. No species of Pseudomonas grew at concentrations greater than 0.5% nicotine when 10⁶ colony forming units (cfu) were used, but growth occurred at all concentrations of rutin when 10² cfu were used. These data indicate that nicotine was inhibitory to growth of both herbivores and pathogens, suggesting that certain plant secondary chemicals with high toxicity are of a generalized nature and affect multiple species. Differences in the sensitivity of organisms to allelochemicals such as generalist or specialist can make it appear that specific allelochemicals affect specific organisms, when in fact it is the tolerance of the organism to the plant chemical that is responsible. In four separate studies, the growth of M. sexta, T. ni and Helicoverpa zea was significantly lower on plants inoculated with P. solanacearum. Alteration in leaf quality by P. solanacearum was due to either reductions in leaf nutrients or increases in allelochemicals. We speculate that localized or systemic induction by both herbivores and pathogens can cause changes in leaf quality, effecting each other's subsequent colonization. The generalized nature of plant secondary compounds and potential reciprocal effects on induction by both species suggests that herbivores and pathogens may affect plant quality through induction and diffuse interactions of disparate species can alter the community of organisms colonizing a plant.     

Isolation and characterization of soil strains producing glutaryl-7-aminocephalosporanic acid acylase

A search was undertaken to screen microorganisms that produce an enzyme capable of deacylating glutaryl-7-aminocephalosporanic acid to 7-aminocephalosporanic acid in soil samples. The screening was carried out by preparing enrichment cultures containing glutaryl-7ACA and cephalosporin C as selective carbon sources. A non-β-lactam model compound, glutaryl-p-nitroanilide, was synthesized as a substrate suitable for the rapid screening of microorganisms isolated from the enrichment cultures. Two isolates exhibiting acylase activity, designated BY7.4 and BY8.1, were identified as strains ofPseudomonas species.Pseudomonas BY8.1 showed higher acylase activity toward Gl-7ACA thanPseudomonas BY7.4. Environmental conditions for the optimal acylase activity ofPseudomonas BY8.1 were shown to be pH 9 and 30°C.     

A <Emphasis Type="Italic">sirA</Emphasis>-like gene, <Emphasis Type="Italic">sirA2</Emphasis>, is essential for 3-succinoyl-pyridine metabolism in the newly isolated nicotine-degrading <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. HZN6 strain

A novel nicotine-degrading Pseudomonas sp. strain, HZN6, was isolated from a pesticide-wastewater treatment facility in Hangzhou. The strain could grow on nicotine as its sole source of carbon, nitrogen, and energy. The strain’s main intermediate metabolites were determined to be pseudooxynicotine, 3-succinoyl-pyridine (SP), and 6-hydroxy-3-succinoyl-pyridine (HSP). A Tn5 transposon mutant was generated in which the degradation pathway was blocked at the SP. A 4,583-bp DNA fragment flanking the transposon insertion site was obtained through self-formed adaptor PCR and analyzed. The mutant gene orfC displays 89% deduced amino acid sequence identity with the sirA-like gene (sirA2, a sulfurtransferase homologue gene) of Pseudomonas stutzeri A1501. The orfC-disrupted strain lost the ability to degrade SP, and the complementation strains with the orfC from the Pseudomonas sp. HZN6 and the sirA2 (PP_1233) from Pseudomonas putida KT2440 recovered the degradation ability. Though the orfC-disrupted strain also lost the xanthine dehydrogenase activity, the effects of tungsten on the degradation of SP and hypoxanthine revealed that the hydroxylation of SP to HSP was not a xanthine dehydrogenase type. These results demonstrated that the orfC gene was essential for the SP metabolism involved in the nicotine metabolic pathway in the Pseudomonas sp. HZN6 strain. This study might advance the understanding of the nicotine metabolic mechanism in Pseudomonas.     

Biodegradation of propoxur by Pseudomonas species

A bacterium capable of degrading propoxur (2-isopropoxyphenyl-N-methylcarbamate) was isolated from soil by enrichment cultures and was identified as a Pseudomonas species. The organism grew on propoxur at 2 g/l as sole source of carbon and nitrogen, and accumulated 2-isopropoxyphenol as metabolite in the culture medium. The cell free extract of Pseudomonas sp. grown on propoxur contained the activity of propoxur hydrolase. The results suggest that the organism degraded propoxur by hydrolysis to yield 2-isopropoxyphenol and methylamine, which was further utilized as carbon source.     

Degradation of cyanide by a bacterial mixture composed of new types of cyanide-degrading bacteria

Summary A mixed culture of bacteria capable of growth on cyanide was isolated from an activated sludge of coal tar wastewater by an enrichment culture technique. The predominant cyanide-degrading microorganisms found in this bacterial mixture were identified as species of the genera Klebsiella, Serratia, Moraxella, and Pseudomonas. Stoichiometric amounts of ammonia were released during the cyanide containing culture by microbial oxidation of cyanide.     

Biodegradation of nicotine by newly isolated Pseudomonas sp. CS3 and its metabolites

Aims: Isolation and characterization of nicotine‐degrading bacteria with advantages suitable for the treatment of nicotine‐contaminated water and soil and detection of their metabolites. Methods and Results: A novel nicotine‐degrading bacterial strain was isolated from tobacco field soil. Based on morphological and physiochemical properties and sequence of 16S rDNA, the isolate was identified as Pseudomonas sp., designated as CS3. The optimal culture conditions of strain CS3 for nicotine degradation were 30°C and pH 7·0. However, the strain showed broad pH adaptability with high nicotine‐degrading activity between pH 6·0 and 10·0. Strain CS3 could decompose nicotine nearly completely within 24 h in liquid culture (1000 mg L^?1 nicotine) or within 72 h in soil (1000–2500 mg kg^?1 nicotine) and could endure up to 4000 mg L^?1 nicotine in liquid media and 5000 mg kg^?1 nicotine in soil. Degradation tests in flask revealed that the strain had excellent stability and high degradation activity during the repetitive degradation processes. Additionally, three intermediates, 3‐(3,4‐dihydro‐2H‐pyrrol‐5‐yl) pyridine, 1‐methyl‐5‐(3‐pyridyl) pyrrolidine‐2‐ol and cotinine, were identified by GC/MS and NMR analyses. Conclusions: The isolate CS3 showed outstanding nicotine‐degrading characteristics such as high degradation efficiency, strong substrate endurance, broad pH adaptability, and stability and persistence in repetitive degradation processes and may serve as an excellent candidate for applications in the bioaugmentation process to treat nicotine‐contaminated water and soil. Also, detection of nicotine metabolites suggests that strain CS3 might decompose nicotine via a unique nicotine‐degradation pathway. Significance and Impact of the Study: The advantage of applying the isolated strain lies in broad pH adaptability and stability and persistence in repetitive use, the properties previously less focused in other nicotine‐degrading micro‐organisms. The strain might decompose nicotine via a nicotine‐degradation pathway different from those of other nicotine‐utilizing Pseudomonas bacteria reported earlier, another highlight in this study.     

Differential Habitat Use and Niche Partitioning by <Emphasis Type="Italic">Pseudomonas</Emphasis> Species in Human Homes

Many species of Pseudomonas have the ability to use a variety of resources and habitats, and as a result Pseudomonas are often characterized as having broad fundamental niches. We questioned whether actual habitat use by Pseudomonas species is equally broad. To do this, we sampled extensively to describe the biogeography of Pseudomonas within the human home, which presents a wide variety of habitats for microbes that live in close proximity to humans but are not part of the human flora, and for microbes that are opportunistic pathogens, such as Pseudomonas aeruginosa. From 960 samples taken in 20 homes, we obtained 163 Pseudomonas isolates. The most prevalent based on identification using the SepsiTest BLAST analysis of 16S rRNA () were Pseudomonas monteilii (42 isolates), Pseudomonas plecoglossicida, Pseudomonas fulva, and P. aeruginosa (approximately 25 each). Of these, all but P. fulva differed in recovery rates among evaluated habitat types (drains, soils, water, internal vertebrate sites, vertebrate skin, inanimate surfaces, and garbage/compost) and all four species also differed in recovery rates among subcategories of habitat types (e.g., types of soils or drains). We also found that at both levels of habitat resolution, each of these six most common species (the four above plus Pseudomonas putida and Pseudomonas oryzihabitans) were over- or under-represented in some habitats relative to their contributions to the total Pseudomonas collected across all habitats. This pattern is consistent with niche partitioning. These results suggest that, whereas Pseudomonas are often characterized as generalists with broad fundamental niches, these species in fact have more restricted realized niches. Furthermore, niche partitioning driven by competition among Pseudomonas species may be contributing to the observed variability in habitat use by Pseudomonas in this system.     

Aerobic biodegradation of nonylphenol by cold-adapted bacteria

Three strains capable of mineralizing nonylphenol as sole carbon source were isolated from a sample of contaminated soil and characterized as two Pseudomonas spp. and a Stenotrophomonas sp. The two Pseudomonas spp. expressed characteristics typical of psychrophiles growing optimally of 10 °C and capable of growing at 0 °C. The Stenotrophomonas sp. was more likely psychrotrophic because it had an optimal temperature between 14 and 22 °C although it was not capable of growing at 4 °C. At 14 °C, one of the Pseudomonas spp. exhibited the highest rate of degradation of nonylphenol (4.4 mg l^–1 d^–1), when compared with axenic or mixed cultures of the isolates. This study represents, to the best of our knowledge, the first reported case of cold-adapted microorganisms capable of mineralizing nonylphenol.     

Biodegradation of DDT by a <Emphasis Type="Italic">Pseudomonas</Emphasis> Species

A bacterial strain capable of degrading 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) was isolated from insecticide-contaminated soil by biphenyl enrichment culture and identified as a Pseudomonas species. The organism degraded DDT through the intermediate formation of 2,3-dihydroxy-DDT, which undergoes meta-ring cleavage, ultimately yielding 4-chlorobenzoic acid as a stable metabolite.     

Growth of Pseudomonas fluorescens in modified atmosphere packaged tofu

Aims: The objective was to study the growth of Pseudomonas in a food product (tofu) where it typically occurs as a spoilage organism, and when this product is stored under modified atmosphere. Methods and Results: A Pseudomonas strain was isolated from the endogenous microflora of tofu. Tofu was inoculated with the strain, packaged in different gas conditions (air, 100% N₂, 30% CO₂/70% N₂ or 100% CO₂) and stored under refrigerated conditions. Microbial loads and the headspace gas composition were monitored during storage. Conclusions: The strain was capable of growing in atmospheres containing no or limited amounts of oxygen and increased amounts of carbon dioxide. Even when 100% CO₂ was used, growth could not be inhibited completely. Significance and Impact of Study: In contrast to the general characteristics of the genus Pseudomonas (strictly aerobic, highly sensitive to CO₂), it should not be expected in the food industry that removing oxygen from the food package and increasing the carbon dioxide content, combined with cold storage, will easily avoid spoilage by Pseudomonas species. Guarantee of hygienic standards and combination of strategies with other microbial growth inhibiting measures should be implemented.     

尼古丁降解菌SCUEC1菌株的分离及其降解基因

【目的】分离并鉴定1株具有尼古丁降解能力的细菌,研究其尼古丁降解特性并对其降解基因进行分析,为尼古丁微生物降解提供基础。【方法】从烟草种植地土壤中分离1株具有尼古丁降解能力的细菌,通过16S r RNA基因和生理生化特性对该菌株进行鉴定,检测该菌株尼古丁降解率与生长量的关系,并进一步对该菌株进行尼古丁浓度耐受性测定,采用高通量测序技术对菌株进行全基因组测序,BLAST比对分析尼古丁降解相关基因。【结果】筛选到1株具有尼古丁降解能力的细菌,经鉴定命名为根癌土壤杆菌(Agrobacterium tumerficience)SCUEC1菌株,根癌土壤杆菌SCUEC1菌株尼古丁降解率可达到94.81%,该菌株在尼古丁浓度为0.50–5.00 g/L范围内生长良好且有较高的尼古丁降解能力。对根癌土壤杆菌SCUEC1菌株全基因组序列进行BLAST比对分析,推测该菌株的尼古丁降解代谢途径与中间苍白杆菌SYJ1菌株的尼古丁降解途径相似。【结论】本研究揭示了Agrobacterium tumerficienceSCUEC1菌株具备尼古丁降解特性,初步推测出尼古丁降解相关基因和降解代谢途径,为尼古丁微生物降解提供基础。     

Variation of Alkaloid Production in Nicotiana rustica Callus Cultures

Callus cultures have been established from the seed, root and leaf of Nicotiana rustica L. var. brasilia in a synthetic medium containing 1 μM 2,4-D and μM kinetin. These callus tissues behaved similarly not only in growth and organogenesis but also in nicotine production. The nicotine contents of callus cultures, which were in the order of 0.25–0.58% of dry weight during a few passages subsequent to callus induction, rapidly decreased to trace amounts in succeeding subcultures in association with the decline of the root-regenerating activity. On the other hand, free cells prepared from a callus tissue in the third passage developed into individual clones showing wide differences in growth and nicotine production. One of these clones gave rise to a relatively stable strain which is capable of producing nicotine at a high rate (0.29% of dry weight) in the absence of organization. The significance of these findings is discussed in connection with some results which have been reported for other callus cultures of Nicotiana species.     

Genetics of naphthalene and phenanthrene degradation by Comamonas testosteroni

Naphthalene and phenanthrene have long been used as model compounds to investigate the ability of bacteria to degrade polycyclic aromatic hydrocarbons. The catabolic pathways have been determined, several of the enzymes have been purified to homogeneity, and genes have been cloned and sequenced. However, the majority of this work has been performed with fast growing Pseudomonas strains related to the archetypal naphthalene-degrading P. putida strains G7 and NCIB 9816-4. Recently Comamonas testosteroni strains able to degrade naphthalene and phenanthrene have been isolated and shown to possess genes for polycyclic aromatic hydrocarbon degradation that are different from the canonical genes found in Pseudomonas species. For instance, C. testosteroni GZ39 has genes for naphthalene and phenanthrene degradation which are not only different from those found in Pseudomonas species but are also arranged in a different configuration. C. testosteroni GZ42, on the other hand, has genes for naphthalene and phenanthrene degradation which are arranged almost the same as those found in Pseudomonas species but show significant divergence in their sequences. Received 10 August 1997/ Accepted in revised form 15 August 1997     

Dehydrogenase activity of pseudomonas species

Single-strain cultures of Pseudomonas fragi, P. fluorescens, P. putrefaciens, and strains of two marine species, Pseudomonas type I and Pseudomonas type II, were found to be capable of reducing added acetaldehyde, proprionaldehyde, and butyraldehyde to the corresponding alcohols at 21 C. All species studied reduced propionaldehyde at 6 C. P. fragi, Pseudomonas type I, and Pseudomonas type II reduced butanone at 6 and 21 C. P. fragi and Pseudomonas type II reduced acetone at 21 C. Dehydrogenase activity was found in some cultures in which growth was not evident. Under aerobic conditions, a strain of P. fragi reduced added propionaldehyde to n-propanol quantitatively within 36 hr at 21 C.     

Recombineering facilitates the discovery of natural product biosynthetic pathways in Pseudomonas parafulva

Microbial natural products among other functions they play a vital role in the disease prevention in humans, animals and plants. Pseudomonas parafulva CRS01-1 is a broad-spectrum antagonistic bacterium present in plants. However, no natural products have been isolated from this strain till date. Corresponding biosynthetic gene clusters to natural products is an effective method for bioprospecting, for which, genome manipulation tools are essential. We previously developed Pseudomonas-specific phage-derived homologous recombination systems for genetic engineering in four Pseudomonas species. Herein, we report the application of these recombineering systems in Pseudomonas parafulva CRS01-1, along with structural elucidation and bioactivity evaluation of natural products. The Pseudomonas recombineering toolbox established before in different four species is efficient for genome mining and bioactive metabolite discovery from more distant species.     

A comparison of organochlorine removal from bleached kraft pulp and paper-mill effluents by dehalogenating Pseudomonas,Ancylobacter and Methylobacterium strains

The relative importance of each of three dechlorinating species to overall organochlorine removal from bleached kraft-mill effluents (BKME) was assessed. Ancylobacter aquaticus A7, Pseudomonas P1, and Methylobacterium CP13, strains indigenous to a BKME treatment system, were tested for growth on chlorinated acetic acids and alcohols, and for adsorbable organic halogen (AOX) reduction in batch cultures of sterile BKME from three sources. A. aquaticus A7 exhibited the broadest substrate range, but could only affect significant AOX reduction in softwood effluents. Methylobacterium CP13 exhibited a limited substrate range, but was capable of removing significant amounts of AOX from both hardwood and softwood effluents. By contrast, Pseudomonas sp. P1 exhibited a limited substrate range and poor to negligible reductions in AOX levels from both effluent types. Mixed inocula of all three species combined and inocula of sludge from mill treatment systems removed as much AOX from softwood effluents as did pure populations of Methylobacterium CP13. When BKME was hydrolysed prior to AOX analysis, the subsequent estimates of recalcitrant, or non-hydrolysable, AOX levels were far less variable than their counterpart total AOX measures. It is suggested that this is a relevant and useful measure of AOX for biodegradation studies.     

Elimination of volatile compounds of leaf tobacco from air emissions using biofiltration

The composition of the volatile organic compounds (VOCs) of various leaf tobacco brands and their blends has been studied. The differences in the content of nicotine, solanone, tetramethyl hexadecenol, megastigmatrienones, and other compounds, determining the specific tobacco smell, have been revealed. A microbial consortium, which is able to deodorize simulated tobacco emissions and decompose nicotine, has been formed by long-term adaptation to the VOCs of tobacco leaves in a laboratory reactor, functioning as a trickle-bed biofilter. Such a biofilter eliminates 90% of the basic toxic compound (nicotine) and odor-active compounds; the filtration efficiency does not change for tobacco brands with different VOC concentrations or in the presence of foreign substances. The main strains, isolated from the formed consortium and participating in the nicotine decomposition process, belong to the genera Pseudomonas, Bacillus, and Rhodococcus. An examination of the biofilter trickling fluid has shown full decomposition of nicotine and odor-active VOCs. The compounds, revealed in the trickling fluid, did not have any odor and were nontoxic. The obtained results make it possible to conduct scaling of the biofiltration process to eliminate odor from air emissions in the tobacco industry.     

Biodegradation and detoxification of nicotine in tobacco solid waste by a Pseudomonas sp.

A Pseudomonas sp. grew with nicotine optimally 3 g l^–1 and at 30 °C and pH 7. Nicotine was fully degraded within 10 h. The resting cells degraded nicotine in tobacco solid waste completely within 6 h in 0.02 m sodium phosphate buffer (pH 7) at maximally 56 mg nicotine h^–1 g dry cell^–1.