Biotransformation of nicotine by microorganism: the case of Pseudomonas spp.

Several bacterial species are capable of using nicotine, the main alkaloid in tobacco plants, as a substrate for growth. The dominant species include members of two genera, Pseudomonas and Arthrobacter. The degradation pathway and genetic structure of nicotine catabolism in Arthrobacter nicotinovorans were recently reviewed (Brandsch Appl Microbiol Biotechnol 69:493–498, 2006). Here, we present up-to-date information on biodegradation of nicotine by Pseudomonas spp. Species in this genus capable of degrading nicotine are summarized and analyzed phylogenetically. Their metabolic intermediates and nicotine degradation-related genes were summarized, and the nicotine-biotransformation pathways were compared and discussed.     

Two closely related pathways of nicotine catabolism in <Emphasis Type="Italic">Arthrobacter nicotinovorans</Emphasis> and <Emphasis Type="Italic">Nocardioides</Emphasis> sp. strain JS614

A virtually identical nicotine catabolic pathway including the heterotrimeric molybdenum enzyme nicotine and 6-hydroxy-pseudo-oxynicotine dehydrogenase, 6-hydroxy-l-nicotine oxidase, 2,6-dihydroxy-pseudo-oxynicotine hydrolase, and 2,6-dihydroxypyridine hydroxylase have been identified in A. nicotinovorans and Nocardioides sp. JS614. Enzymes catalyzing the same reactions and similar protein antigens were detected in the extracts of the two microorganisms. Nicotine blue and methylamine, two end products of nicotine catabolism were detected in the growth medium of both bacterial species. Nicotine catabolic genes are clustered on pAO1 in A. nicotinovorans, but located chromosomally in Nocardioides sp. JS614.     

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。     

Nicotine degradation by two novel bacterial isolates of <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. TW and <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. TY and their responses in the presence of neonicotinoid insecticides

Two novel nicotine-degrading bacterial strains were isolated from tobacco waste and identified as Acinetobacter sp. TW and Sphingomonas sp. TY based on morphology, physiological and biochemical tests, Biolog analysis and 16S rDNA sequencing. The 16S rDNA sequences have been deposited in GenBank under the accession numbers FJ753401 for TW and FJ754274 for TY. The best culture conditions for nicotine degradation were 25–37°C and pH 7.0–8.0 for strain TW and 25–30°C and pH 6.0–7.0 for strain TY. Under the best conditions, the cell growth and nicotine-degradation kinetics of the two isolates were assessed, and 1.0 g/l nicotine was completely degraded within 12 and 18 h for TW and TY, respectively. Moreover, the presence of four widely-used commercial neonicotinoid insecticides in the medium had no effects on nicotine degradation by TW; among the four tested neonicotinoids, only thiamethoxam significantly delayed nicotine degradation by TY. TW and TY were also able to degrade selected neonicotinoids. This is the first report of nicotine degradation by Acinetobacter sp. and Sphingomonas sp. This study showed that these two newly isolated bacteria may be suitable for the disposal of tobacco waste and the reduction of nicotine in tobacco leaves.     

ndpT encodes a new protein involved in nicotine catabolism by Sphingomonas melonis TY

Sphingomonas melonis TY utilizes nicotine as a sole source of carbon, nitrogen, and energy to grow. One of the genes in its ndp catabolic cluster, ndpT, encodes a hypothetical transporter. Since no transporter for nicotine has been identified in microorganisms, we investigated whether NdpT is responsible for nicotine transport. ndpT was induced by nicotine, and gene knockout and complementation studies clearly indicated that ndpT is essential for the catabolism of nicotine in strain TY. NdpT-GFP was located at the periphery of the cells, suggesting that NdpT is a membrane protein. Uptake assays with L-[¹⁴C] nicotine illustrated that nicotine uptake in strain TY is mediated by a constitutively synthesized permease with a K_m of 0.362 ± 0.07 μM and a V_max of 0.762 ± 0.068 μmol min⁻¹ (mg cell dry weight)⁻¹ and that ndpT may play a role in nicotine exclusion. Hence, we consider NdpT a nicotine catabolism-related protein.

     

Effects of nicotine on the growth and protein expression of <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis>

Tobacco smoking is considered one of the most significant environmental risk factors for destructive periodontal disease. The effect of smoking on periodontopathic microbiota has not yet been elucidated, as previous studies failed to identify a concrete relationship between periodontopathic microorganisms and smoking. However, it is likely that smoking, as an environmental stress factor, may affect the behavior of dental plaque microorganisms, ultimately leading to alteration of the host-parasite interaction. The goal of this study was to examine the effect of nicotine, a major component of tobacco, on the growth and protein expression of the crucial periodontal pathogen Porphyromonas gingivalis. The growth of P. gingivalis 381 was measured after bacterial cells were cultivated in liquid broth containing various nicotine concentrations. First, P. gingivalis cells were allowed to grow in the presence of a single dose of nicotine (the single exposure protocol) at 0, 1, 2, 4, and 8 mg/L, respectively. Second, P. gingivalis cells were exposed to five consecutive doses of nicotine (the multiple exposure protocol) at 0, 1, 2, and 4 mg/L, respectively. Bacterial growth was measured by optical density and protein expression was analyzed by SDS-PAGE and 2-D gel electrophoresis. In the single nicotine exposure protocol, it was observed that the growth of P. gingivalis 381 was inhibited by nicotine in a dose-dependent manner. In the multiple nicotine exposure protocol, the growth rate of P. gingivalis increased with each subsequent nicotine exposure, even though bacterial growth was also inhibited in a dose dependent fashion. SDS-PAGE and 2-D gel electrophoresis analyses revealed a minor change in the pattern of protein expression, showing differences in proteins with low molecular weights (around 20 kDa) on exposure to nicotine. The results of this study suggest that nicotine exerts an inhibitory effect on the growth of P. gingivalis, and has a potential to modulate protein expression in P. gingivalis.     

Pathogen roid rage: Cholesterol utilization by Mycobacterium tuberculosis

Abstract

The ability of science and medicine to control the pathogen Mycobacterium tuberculosis (Mtb) requires an understanding of the complex host environment within which it resides. Pathological and biological evidence overwhelmingly demonstrate how the mammalian steroid cholesterol is present throughout the course of infection. Better understanding Mtb requires a more complete understanding of how it utilizes molecules like cholesterol in this environment to sustain the infection of the host. Cholesterol uptake, catabolism and broader utilization are important for maintenance of the pathogen in the host and it has been experimentally validated to contribute to virulence and pathogenesis. Cholesterol is catabolized by at least three distinct sub-pathways, two for the ring system and one for the side chain, yielding dozens of steroid intermediates with varying biochemical properties. Our ability to control this worldwide infectious agent requires a greater knowledge of how Mtb uses cholesterol to its advantage throughout the course of infection. Herein, the current state of knowledge of cholesterol metabolism by Mtb is reviewed from a biochemical perspective with a focus on the metabolic genes and pathways responsible for cholesterol steroid catabolism.     

Structural and molecular basis for the novel catalytic mechanism and evolution of DddP,an abundant peptidase‐like bacterial Dimethylsulfoniopropionate lyase: a new enzyme from an old fold

The microbial cleavage of dimethylsulfoniopropionate (DMSP) generates volatile dimethyl sulfide (DMS) and is an important step in global sulfur and carbon cycles. DddP is a DMSP lyase in marine bacteria, and the deduced dddP gene product is abundant in marine metagenomic data sets. However, DddP belongs to the M24 peptidase family according to sequence alignment. Peptidases hydrolyze C‐N bonds, but DddP is deduced to cleave C‐S bonds. Mechanisms responsible for this striking functional shift are currently unknown. We determined the structures of DMSP lyase RlDddP (the DddP from Ruegeria lacuscaerulensis ITI_1157) bound to inhibitory 2‐(N‐morpholino) ethanesulfonic acid or PO₄^3? and of two mutants of RlDddP bound to acrylate. Based on structural, mutational and biochemical analyses, we characterized a new ion‐shift catalytic mechanism of RlDddP for DMSP cleavage. Furthermore, we suggested the structural mechanism leading to the loss of peptidase activity and the subsequent development of DMSP lyase activity in DddP. This study sheds light on the catalytic mechanism and the divergent evolution of DddP, leading to a better understanding of marine bacterial DMSP catabolism and global DMS production.     

Genetics and biochemistry of phenol degradation byPseudomonas sp. CF600

Pseudomonas sp. strain CF600 is an efficient degrader of phenol and methylsubstituted phenols. These compounds are degraded by the set of enzymes encoded by the plasmid locateddmpoperon. The sequences of all the fifteen structural genes required to encode the nine enzymes of the catabolic pathway have been determined and the corresponding proteins have been purified. In this review the interplay between the genetic analysis and biochemical characterisation of the catabolic pathway is emphasised. The first step in the pathway, the conversion of phenol to catechol, is catalysed by a novel multicomponent phenol hydroxylase. Here we summarise similarities of this enzyme with other multicomponent oxygenases, particularly methane monooxygenase (EC 1.14.13.25). The other enzymes encoded by the operon are those of the well-knownmeta-cleavage pathway for catechol, and include the recently discoveredmeta-pathway enzyme aldehyde dehydrogenase (acylating) (EC 1.2.1.10). The known properties of thesemeta-pathway enzymes, and isofunctional enzymes from other aromatic degraders, are summarised. Analysis of the sequences of the pathway proteins, many of which are unique to themeta-pathway, suggests new approaches to the study of these generally little-characterised enzymes. Furthermore, biochemical studies of some of these enzymes suggest that physical associations betweenmeta-pathway enzymes play an important role. In addition to the pathway enzymes, the specific regulator of phenol catabolism, DmpR, and its relationship to the XylR regulator of toluene and xylene catabolism is discussed.     

Croissance,teneur en auxine et catabolisme auxinique chez Rhizobium

Summary Rhizobium is cultivated on a synthetic medium in presence of tryptophan. The exponential phase in the bacterial growth is over after three days. During the stationary phase, the IAA content of the medium continues to increase regularly after four days. The auxin degradation by cell-free preparations of 4, 8, 12-day old cultures decreases with time.These correlations between auxin catabolism, IAA content and bacterial growth are compared with those described in tissues of higher plants.     

Fatty acid metabolism by cutaneous bacteria and its role in axillary malodour

It is generally accepted that short (C₂-C₅) and medium (C₆-C₁₁) chain volatile fatty acids (VFAs) are among the primary causal molecules of axillary malodour. It is also widely acknowledged that malodour generation is attributable to the biotransformation of odourless natural secretions, into volatile odorous products, by cutaneous bacteria. However, little information is available on the biochemical origins of VFAs on axillary skin. In these studies, assay systems were developed to investigate the generation of VFAs from lipid substrates readily available to the bacteria resident on axillary skin. A major route to short and medium chain VFAs in the axilla was shown to be the partial catabolism of structurally unusual (e.g. methyl-branched) longer chain fatty acids by a previously uncharacterized sub-group of the Corynebacterium genus, corynebacteria (A). In contrast, corynebacteria (B) are incapable of growth on fatty acid. Structurally unusual fatty acids originate from the triacylglycerol component of sebum, and probably also apocrine sweat, by the action of bacterial lipases. Interestingly, VFA formation in the axilla is a dynamic process, with some cutaneous microorganisms, specifically micrococci and brevibacteria, capable of fully catabolizing these odorants. The results of these studies provide new understanding on the biochemical origins of VFA-based axillary malodour.     

Nicotine Exposure Does not Alter Plasma to Brain Choline Transfer

Acute and chronic nicotine exposure in rats is associated with an increase in brain acetylcholine (ACh) transmission. The acquisition of choline for neuronal ACh synthesis occurs primarily via two pathways; first, free choline is transported from the blood across the blood-brain barrier (BBB) and/or second, from synaptic choline generated by either hydrolysis of non-bound ACh or membrane phosphatidylcholine catabolism. To determine if nicotine-induced cholinergic demand is associated with increased choline transport rates into brain, we measured BBB choline transport in naïve and S-(−) nicotine exposed rats (acute and chronic, 4.5 mg/kg/d for 1, 14, 21 and 28 d; osmotic minipumps) using the in situ rat brain perfusion technique. No significant changes in choline uptake after acute or chronic nicotine exposure were observed in whole brain or cortex. Of considerable interest was a significant decrease in regional brain choline uptake measured in the hippocampus after chronic nicotine exposure (28 d). Our data suggest that the increased ACh transmission observed after nicotine exposure does not correlate with increased blood-to-brain transfer of choline. Considering these data and previous literature reports, we propose that the additional free choline required under conditions of nicotine exposure (for ACh synthesis) is primarily recruited from membrane phospholipid metabolism.     

Arginine catabolism: a new function of both octopine and nopaline Ti-plasmids of Agrobacterium.

Summary The oncogenic plasmids of Agrobacterium, the Ti-plasmids, carry genes that enable their bacterial host to catabolize opines. Opines are unusual amino acid derivatives that are only produced in crown gall tumours incited by oncogenic strains of Agrobacterium. The 2 opines, octopine and nopaline, are degraded by Agrobacterium strains carrying the octopine or the nopoline Ti-plasmid, respectively, to arginine and pyruvic acid, and to arginine and -ketoglutaric acid. In this paper it is shown that the Ti-plasmids carry gene(s) involved in the utilisation of arginine as a carbon source. Strains harbouring wild type octopine or nopaline Ti-plasmids in the chromosomal context of strain C58C1 do not grow on arginine as a carbon source. However, they are able to grow on arginine provided that they are induced, or constitutive for opine catabolism. The features of ornithine utilisation are identical. The gene(s) involved in arginine and ornithine utilization in C58C1 (pTi-oct) or C58C1 (pTi-nop) are under the control of the regulator gene that controls octopine or nopaline catabolism. A tentative pathway of octopine utilization is proposed, in which at least two steps are Ti-plasmid coded, and probably belong to the same operon: 1-scission of octopine into arginine and pyruvic acid 2-transformation of an arginine derivative (GSA?) to glutamic acid.Arginine utilization as a carbon source is therefore a new function of the Ti-plasmid. As this function is not inducible by arginine but by opines, it provides a method for selecting regulatory mutants of opine catabolism in the genetic background of strain C58.     

Detection of Deefgea chitinilytica in freshwater ornamental fish

Aim: To identify and characterize six chitinolytic bacterial strains isolated from ornamental fish. Methods and Results: Six different isolates of Deefgea chitinilytica were detected in healthy as well as diseased ornamental fish in Germany over a period of 2 years. Bacterial strains were identified using 16S rRNA partial gene sequencing and further characterized using different biochemical microtest systems and additional standard biochemical tests. Conclusion: We show that commercially available biochemical microtest systems are useful for identification of D. chitinilytica, supplemented by 16S rRNA partial gene sequencing. Furthermore, this study provides new information about the occurrence of D. chitinilytica, as this is the first isolation of D. chitinilytica from animals and first described isolation in Europe. Significance and Impact of the Study: Deefgea chitinilytica may be isolated regularly in fish diagnostic laboratories. Therefore, accurate identification of this bacterial species is important. Involvement of D. chitinilytica in opportunistic infections of aquatic organisms cannot be excluded and has to be further investigated.     

Xanthomonas campestris pv.parthenii pathovar nov. incitant of leaf blight of parthenium

A new bacterial leaf blight disease of parthenium (Parthenium hysterophorus L.) is described for the first time. The disease-causing bacterium was isolated and its morphological, physiological and biochemical characters were determined. The pathogenicity of bacterium is apparently limited only to parthenium. The pathogen was identified asXanthomonas campestris pv.parthenii pathovar nov. on the basis of morphological, physiological, biochemical and pathogenic characteristics.     

Genetic variation within the Chrna7 gene modulates nicotine reward‐like phenotypes in mice

Mortality from tobacco smoking remains the leading cause of preventable death in the world, yet current cessation therapies are only modestly successful, suggesting new molecular targets are needed. Genetic analysis of gene expression and behavior identified Chrna7 as potentially modulating nicotine place conditioning in the BXD panel of inbred mice. We used gene targeting and pharmacological tools to confirm the role of Chrna7 in nicotine conditioned place preference (CPP). To identify molecular events downstream of Chrna7 that may modulate nicotine preference, we performed microarray analysis of α7 knock‐out (KO) and wild‐type (WT) nucleus accumbens (NAc) tissue, followed by confirmation with quantitative polymerase chain reaction (PCR) and immunoblotting. In the BXD panel, we found a putative cis expression quantitative trait loci (eQTL) for Chrna7 in NAc that correlated inversely to nicotine CPP. We observed that gain‐of‐function α7 mice did not display nicotine preference at any dose tested, whereas conversely, α7 KO mice demonstrated nicotine place preference at a dose below that routinely required to produce preference. In B6 mice, the α7 nicotinic acetylcholine receptor (nAChR)‐selective agonist, PHA‐543613, dose‐dependently blocked nicotine CPP, which was restored using the α7 nAChR‐selective antagonist, methyllycaconitine citrate (MLA). Our genomic studies implicated a messenger RNA (mRNA) co‐expression network regulated by Chrna7 in NAc. Mice lacking Chrna7 demonstrate increased insulin signaling in the NAc, which may modulate nicotine place preference. Our studies provide novel targets for future work on development of more effective therapeutic approaches to counteract the rewarding properties of nicotine for smoking cessation .     

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.     

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.