The effect of oxygen on chemotaxis to naphthalene by Pseudomonas putida G7

Chemotactic bacteria can be attracted to electron donors they consume. In systems where donor is heterogeneously distributed, chemotaxis can lead to enhanced removal of donor relative to that achieved in the absence of chemotaxis. However, simultaneous consumption of an electron acceptor may result in the formation of an acceptor gradient to which the bacteria also respond, thus diminishing the positive effect of chemotaxis. Depletion of an electron acceptor can also reduce the rate of electron donor consumption in addition to its effect on chemotaxis. In this study, we examined the effect of oxygen on chemotaxis to naphthalene and on naphthalene consumption by Pseudomonas putida G7. The organism was able to move up an oxygen gradient when there was a naphthalene gradient in the opposite direction. In the absence of an oxygen gradient, low levels of oxygen attenuated chemotaxis to naphthalene but did not affect random motility. The rate of naphthalene consumption decreased at dissolved oxygen concentrations similar to those at which chemotaxis was attenuated. These results suggest that low dissolved oxygen concentrations can reduce naphthalene removal by P. putida G7 in systems where naphthalene is heterogeneously distributed by simultaneously attenuating chemotactic motion toward naphthalene and decreasing the rate of naphthalene degradation.     

趋化信号转导基因cheA突变对Pseudomonas putida DLL-1甲基对硫磷的趋化性及原位降解的影响

Pseudomonas putida DLL-1是一株甲基对硫磷(MP)高效降解菌株,同时对MP具有趋化性。cheA基因是菌株趋化信号转导过程中负责编码组氨酸激酶的基因,为了研究菌株趋化性在农药原位降解中的作用,通过基因打靶的方式使P.putida DLL-1染色体上单拷贝的cheA基因失活,成功地获得了MP的趋化突变株P.putida DAK,突变株与野生菌株生长能力没有显著差异。通过土壤盆钵试验(MP浓度为50mg/kg),发现在灭菌与未灭菌土壤中趋化突变株对MP的降解能力低于原始出发菌株DLL-1约20%~30%,说明菌株DLL-1趋化性的丧失会减慢其对农药的降解,趋化性在农药的原位降解过程中发挥重要作用。     

Biodegradation of benzene, toluene and naphthalene in soil-water slurry microcosms

Aerobic biodegradation of benzene, toluene andnaphthalene was studied in pre-equilibrated soil-waterslurry microcosms. The experiments were designed tosimulate biodegradation at waste sites where sorptionreaches equilibrium before biodegradation becomesimportant. Rates of biodegradation were reduced by thepresence of soil. For example, nearly completenaphthalene biodegradation (1.28 mg/L) by indigenoussoil bacteria occurred within 60 hours in aqueoussolution (soil-free) while it took two weeks todegrade the same amount in the presence of 0.47 kgsoil/L of water. The rate of biodegradation wasobserved to decrease with increasing organic compoundhydrophobicity, soil/water ratio, soil particle size,and soil organic carbon content. These resultsclearly indicate that the rate of biodegradation isaffected by both the extent and rate of sorption. Further analysis suggests that mass transfer couldcontrol the performance of in situ bioremediation forhighly hydrophobic organic contaminants which exhibita large extent of sorption and slow rate ofdesorption.     

Biodegradation of chlorophenol mixtures by Pseudomonas putida

The dynamic growth behavior of Pseudomonas putida has been studied when resting calls were inoculated into a growth medium containing inhibitory concentrations of mixtures of phenol and monochlorophenols. Resting cells inoculated into single carbon substrate media did not demonstrate enhanced cell lysis by any of the phenol substrates. The apprarent death rate was reduced as the concentrations of phenol or chlorophenols were increased. This behavior was modeled by employing a constant specific death rate (k(d) = 0.0075 h(-1)) and assuming all organic species result in a lag-phase, specific growth rate which may be larger or smaller than k(d).Logarithmic biomass growth on pure monochlorophenols did not occur within 2 weeks after inoculation. Logarithmic growth phases were only observed when the monochlorophenols were cometabolized with phenol. The delay time over which the lag phase exists increased exponentially with phenol concentration and linearly with monochlorophenol concentration. The log growth yield coefficient decreased linearly with monochlorophenol concentration.The lag-phase, specific growth rate was found to decrease exponentially with the concentration of monochlorophenols. This resulted in a 50% lag growth rate inhibition for both 3- and 4-chlorophenol of 9 ppm and for 2-chlorophenol of only 2 ppm. The new, empirical correlations are shown to closely model the complete lag and log growth behavior ot P. putida on phenol and chlorophenol mixtures. (c) 1992 John Wiley & Sons, Inc.     

Bacterial chemotaxis toward a NAPL source within a pore-scale microfluidic chamber

Chemotaxis toward chemical pollutants provides a mechanism for bacteria to migrate to locations of high contamination, which may improve the effectiveness of bioremediation. A microfluidic device was designed to mimic the dissolution of an organic-phase contaminant from a single pore into a larger macropore representing a preferred pathway for microorganisms that are carried along by groundwater flow. The glass windows of the microfluidic device allowed direct image analysis of bacterial distributions within the vicinity of the organic contaminant. Concentrations of chemotactic bacteria P. putida F1 near the organic/aqueous interface were 25% greater than those of a nonchemotactic mutant in the vicinity of toluene for a fluid velocity of 0.5 m/d. For E. coli responding to phenol, the bacterial concentrations were 60% greater than the controls, also at a velocity of 0.5 m/d. Velocities in the macropore were varied over a range from 0.5 to 10 m/d, the lower end of which is typical of groundwater velocities. The accumulation of chemotactic bacteria near the NAPL chemoattractant source decreased as the fluid velocity increased. Good agreement between computer-based simulations, generated using reasonable values of the model parameters, and the experimental data for P. putida strains confirmed the contribution due to chemotaxis. The experimental data for E. coli required a larger chemotactic sensitivity coefficient than that for P. putida, which was consistent with parameter values reported in the literature.     

细菌对环境污染物的趋化性及其在生物修复中的作用

细菌对有机化合物的降解能力是一种利用碳源和能源的优势,这种能力可以用来设计安全、有效和无二次污染的污染物的生物修复系统。趋化性是细菌适应外界化学环境变化而作出的行为反应,是一种寻找碳源和能源的优势。细菌的趋化性能够增强细菌在自然环境中的降解污染物的效果,细菌的趋化性与降解性之间的关系研究已经成为热点。介绍了细菌的趋化性的基本概念和趋化信号转导的机制,重点讨论了细菌对环境污染化合物的趋化性,从基因水平揭示了趋化性与降解性之间的紧密联系,认为趋化性可以有效地促进降解性细菌对污染物的生物修复作用。     

Influence of phenol on the biodegradation of pyridine by freely suspended and immobilized Pseudomonas putida MK1

AIMS: To study the effect of co-contaminants (phenol) on the biodegradation of pyridine by freely suspended and calcium alginate immobilized bacteria. METHODS AND RESULTS: Varying concentrations of phenol were added to free and calcium alginate immobilized Pseudomonas putida MK1 (KCTC 12283) to examine the effect of this pollutant on pyridine degradation. When the concentration of phenol reached 0.38 g l(-1), pyridine degradation by freely suspended bacteria was inhibited. The increased inhibition with the higher phenol levels was apparent in increased lag times. Pyridine degradation was essentially completely inhibited at 0.5 g l(-1) phenol. However, immobilized cells showed tolerance against 0.5 g l(-1) phenol and pyridine degradation by immobilized cell could be achieved. CONCLUSIONS: This works shows that calcium alginate immobilization of microbial cells can effectively increase the tolerance of P. putida MK1 to phenol and results in increased degradation of pyridine. SIGNIFICANCE AND IMPACT OF THE STUDY: Treatment of wastewater stream can be negatively affected by the presence of co-pollutants. This work demonstrates the potential of calcium alginate immobilization of microbes to protect cells against compound toxicity resulting in an increase in pollutant degradation.     

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     

Aerobic batch degradation of phenol using immobilized Pseudomonas putida

Alginate concentrations between 2 and 4% had little effect on the degradation rate of phenol by alginate-immobilized Pseudomonas putida. Ten-degree shifts from 25°C resulted in approximately 30% slower degradation. Maximal degradation rates were favored at pH 5.5–6.0. The response of degradation rate to increased air flow in the bubble column used was almost linear and an optimal higher than 16 vol vol⁻¹ was indicated, although free cells appeared in the reaction medium above 12 vol vol⁻¹. When the initial phenol concentration was raised, degradation rate was not significantly affected until levels higher than 1200 mg ml⁻¹ where performance was markedly reduced. Increasing the ratio of total bead volume to medium volume gave progressively smaller increases in degradation rate. At a medium volume to total bead volume ratio of 5:1, the maximum degradation rate was 250 mg L⁻¹ h⁻¹. Received 24 November 1998/ Accepted in revised form 27 January 1999     

Effects of the inoculant strain Pseudomonas putida KT2442 (pNF142) and of naphthalene contamination on the soil bacterial community

The naphthalene-degrading activity of a Pseudomonas sp. strain isolated from a creosote-contaminated soil was shown to be encoded by the IncP9 plasmid pNF142 by transfer to Pseudomonas putida KT2442. The effects of the inoculant strain KT2442 (pNF142) and of naphthalene contamination on the soil bacterial community were studied in microcosms with the following treatments: (I) soil, (II) soil with naphthalene, (III) soil with naphthalene and inoculated with KT2442 (pNF142). The inoculant became the dominant bacterial population in treatment (III) as evidenced by cultivation and denaturing gradient gel electrophoresis (DGGE) analysis. The bacterial DGGE profiles revealed drastically reduced complexity due to the numerical dominance of the inoculant. However, group-specific fingerprints (beta-proteobacteria, actinobacteria) that excluded KT2442 (pNF142) showed less severe changes in the bacterial community patterns. A major effect of naphthalene on the soil bacterial community was observed in treatment (II) after 21 days. Two dominant bands appeared whose sequences showed the highest similarity to those of Burkholderia sp. RP007 and Nocardia vinaceae based on 16S rRNA gene sequencing. These bands were less intense in treatment (III). The increased abundance of RP007-like populations due to naphthalene contamination was also confirmed by PCR amplification of the phnAc gene. The nahAc and nahH genes were detected in DNA and cDNA only in treatment III. Although the inoculant strain KT2442 (pNF142) showed good survival and expression of genes involved in naphthalene degradation, this study suggests that KT2442 (pNF142) suppressed the enrichment of indigenous naphthalene degraders.     

Genetic analysis of response regulator activation in bacterial chemotaxis suggests an intermolecular mechanism

Response regulator proteins of two-component systems are usually activated by phosphorylation. The phosphorylated response regulator protein CheY-P mediates the chemotaxis response in Escherichia coli. We performed random mutagenesis and selected CheY mutants that are constitutively active in the absence of phosphorylation. Although a single amino acid substitution can lead to constitutive activation, no single DNA base change can effect such a transition. Numerous different sets of mutations that activate in synergy were selected in several different combinations. These mutations were all located on the side of CheY defined by alpha4, beta5, alpha5, and alpha1. Our findings argue against the two-state hypothesis for response regulator activation. We propose an alternative intermolecular mechanism that involves a dynamic interplay between response regulators and their effector targets.     

Functional analysis of genes involved in biphenyl, naphthalene, phenanthrene, and m-xylene degradation by Sphingomonas yanoikuyae B1

Sphingomonas yanoikuyae B1 is able to utilize toluene, m-xylene, p-xylene, biphenyl, naphthalene, phenanthrene, and anthracene as sole sources of carbon and energy for growth. A forty kilobase region of DNA containing most of the genes for the degradation of these aromatic compounds was previously cloned and sequenced. Insertional inactivation of bphC results in the inability of B1 to grow on both polycyclic and monocyclic compounds. Complementation experiments indicate that the metabolic block is actually due to a polar effect on the expression of bphA3, coding for a ferredoxin component of a dioxygenase. Lack of the ferredoxin results in a nonfunctional polycyclic aromatic hydrocarbon dioxygenase and a nonfunctional toluate dioxygenase indicating that the electron transfer components are capable of interacting with multiple oxygenase components. Insertional inactivation of a gene for a dioxygenase oxygenase component downstream of bphA3 had no apparent effect on growth besides a polar effect on nahD which is only needed for growth of B1 on naphthalene. Insertional inactivation of either xylE or xylG in the meta-cleavage operon results in a polar effect on bphB, the last gene in the operon. However, insertional inactivation of xylX at the beginning of this cluster of genes does not result in a polar effect suggesting that the genes for the meta-cleavage pathway, although colinear, are organized in at least two operons. These experiments confirm the biological role of several genes involved in metabolism of aromatic compounds by S. yanoikuyae B1 and demonstrate the interdependency of the metabolic pathways for polycyclic and monocyclic aromatic hydrocarbon degradation. Received 13 May 1999/ Accepted in revised form 05 July 1999     

Rationally engineered biotransformation of p‐nitrophenol

An operon encoding enzymes responsible for degradation of the EPA priority contaminant para‐nitrophenol (PNP) from Pseudomonas sp. ENV2030 contains more genes than would appear to be necessary to mineralize PNP. To determine some necessary genes for PNP degradation, the genes encoding the proposed enzymes in the degradation pathway (pnpADEC) were assembled into a broad‐host‐range, BioBricks‐compatible vector under the control of a constitutive promoter. These were introduced into Escherichia coli DH10b and two Pseudomonas putida strains, one with a knockout of the aromatic transport TtgB and the parent with the native transporter. The engineered strains were assayed for PNP removal. E. coli DH10b harboring several versions of the refactored pathway was able to remove PNP from the medium up to a concentration of 0.2 mM; above which PNP was toxic to E. coli. A strain of P. putida harboring the PNP pathway genes was capable of removing PNP from the medium up to 0.5 mM. When P. putida harboring the native PNP degradation cluster was exposed to PNP, pnpADEC were induced, and the resulting production of β‐ketoadipate from PNP induced expression of its chromosomal degradation pathway (pcaIJF). In contrast, pnpADEC were expressed constitutively from the refactored constructs because none of the regulatory genes found in the native PNP degradation cluster were included. Although P. putida harboring the refactored construct was incapable of growing exclusively on PNP as a carbon source, evidence that the engineered pathway was functional was demonstrated by the induced expression of chromosomal pcaIJF. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

细菌趋化信号通路中的磷酸酯酶CheZ

细菌通过趋化系统获得在复杂环境中的生存优势。趋化性在细菌致病性、病菌定殖、固氮细菌与宿主共生、植物与微生物互作等方面有重要的作用。作为趋化信号适应中不可或缺的调节蛋白,对CheZ的深入研究具有重要意义。本文主要对CheZ的结构、作用机制、功能调节、蛋白定位以及进化地位等方面的研究现状进行了综述,旨在为其它细菌中趋化系统的研究提供有益参考。     

根际微生物组中细菌趋化系统的生态功能

在根际微环境中,特定的土壤微生物能够利用自身独特的趋化系统感应根系分泌物,响应植物的选择性招募。细菌的趋化系统介导了植物-微生物以及微生物间相互作用,在植物对根际微生物组的选择中发挥着关键的生态学功能。综述了根际微生物组中细菌趋化系统的研究进展,从生态学的角度提出了未来针对根际细菌趋化系统的研究方向,旨在阐明根际细菌趋化系统的生态学功能,为增进理解作物根际微生物组的募集过程,以及未来农业中根际微生物组的重组构建奠定理论基础。     

Multiple mutations at the active site of naphthalene dioxygenase affect regioselectivity and enantioselectivity

The importance of five amino acids at the active site of the multicomponent naphthalene dioxygenase (NDO) system was determined by generating site-directed mutations in various combinations. The substrate specificities of the mutant enzymes were tested with the substrates indole, indoline, 2-nitrotoluene (2NT), naphthalene, biphenyl, and phenanthrene. Transformation of these substrates measured the ability of the mutant enzymes to catalyze dioxygenation, monooxygenation, and desaturation reactions. In addition, the position of oxidation and the enantiomeric composition of products were characterized. All enzymes with up to three amino acid substitutions were able to catalyze dioxygenation reactions. A subset of these enzymes could also catalyze the monooxygenation of 2NT and desaturation of indoline. Single amino acid substitutions at positions 352 and 206 had the most profound effects on product formation. Of the single mutations made, only changes at position 352 affected the stereochemistry of naphthalene cis-dihydrodiol formed from naphthalene, but in the presence of the F352I mutation, changes at positions 206 and 295 also affected enantioselectivity. Major shifts in regioselectivity with biphenyl and phenanthrene resulted with several of the singly, doubly, and triply mutated enzymes. A new product not formed by the wild-type enzyme, phenanthrene cis-9,10-dihydrodiol, was formed as a major product from phenanthrene by enzymes with two (A206I/F352I) or three amino acid substitutions (A206I/F352I/H295I). The results indicate that a variety of amino acid substitutions are tolerated at the active site of NDO. Journal of Industrial Microbiology & Biotechnology (2001) 27, 94–103. Received 25 September 2000/ Accepted in revised form 29 June 2001     

Influence of fungal-bacterial interactions on bacterial conjugation in the residuesphere

Conjugal gene transfer among bacteria in the residuesphere (area between decaying plant material and soil) of leaves of barley straw was studied. The residuesphere was shown to be a hot-spot for conjugal gene transfer compared to conjugation in sterile sand and non-sterile bulk soil. Impact of fungal colonisation of the residuesphere on bacterial colonisation and conjugation was also investigated. The inhibition of fungal colonisation, due to the application of an eukaryotic inhibitor, increased bacterial colonisation of the residuesphere in soil microcosms compared to non-treated leaves. This treatment also had a transient, positive effect on conjugation. Bacterial conjugation in the residuesphere of leaves subjected to 17 days of fungal colonisation was significantly lower than in the residuesphere of non-colonised leaves. Fungal biomass, as measured by chitinase activity, was inversely related to the conjugation efficiency.     

Development of a molecular detection method for naphthalene degrading pseudomonads

Abstract: A combined polymerase chain reaction amplification and reverse dot blot assay was designed for the detection of bacterial genes from soil and sediments. Total nucleic acids were directly extracted from soil using a lysozyme/sodium dodecyl sulfate/freeze-thaw method followed by rapid purification through gel electrophoresis. DNA was amplified using a highly stringent polymerase chain reaction with primers directed against the nahR regulatory gene present in plasmid NAH7 of Pseudomonas putida G7. The resulting amplification product was detected colorimetrically by reverse dot blot with an improved sensitivity ten-fold greater than traditional ethidium bromide staining after gel electrophersis. A lower limit of 10³, P. putida G7 cfu (g soil)⁻¹ was detected. This method was successfully employed to detect indigenous naphthalene-degrading bacteria from subsurface sediment collected from different locations of a naphthalene-contaminated site. Similar approaches could be developed for other soil-borne genetic markers.     

CellShape: A user‐friendly image analysis tool for quantitative visualization of bacterial cell factories inside

Visualization of the intracellular constituents of individual bacteria while performing as live biocatalysts is in principle doable through more or less sophisticated fluorescence microscopy. Unfortunately, rigorous quantitation of the wealth of data embodied in the resulting images requires bioinformatic tools that are not widely extended within the community‐let alone that they are often subject to licensing that impedes software reuse. In this context we have developed CellShape, a user‐friendly platform for image analysis with subpixel precision and double‐threshold segmentation system for quantification of fluorescent signals stemming from single‐cells. CellShape is entirely coded in Python, a free, open‐source programming language with widespread community support. For a developer, CellShape enhances extensibility (ease of software improvements) by acting as an interface to access and use existing Python modules; for an end‐user, CellShape presents standalone executable files ready to open without installation. We have adopted this platform to analyse with an unprecedented detail the tridimensional distribution of the constituents of the gene expression flow (DNA, RNA polymerase, mRNA and ribosomal proteins) in individual cells of the industrial platform strain Pseudomonas putida KT2440. While the CellShape first release version (v0.8) is readily operational, users and/or developers are enabled to expand the platform further.     

Construction of a bacterial consortium for the biofiltration of benzene,toluene and xylene emissions

On equal parts of benzene, toluene and p-xylene (BTX), a stable bacterial consortium was enriched for removal of BTX vapours from air. As demonstrated by gas chromatographic monitoring, this consortium removed all three BTX components but was able to grow only on benzene and/or toluene. A Pseudomonas putida strain, PPO1, isolated from this consortium behaved in an identical manner. When immobilized on a porous peat/perlite column, both the consortium and the PPO1 isolated removed all three BTX components from metered air streams. However, due to the accumulation of products from the incompletely metabolized p-xylene, the removal rates were unsatisfactory and declined further with time. P. putida ATCC 33015 bearing the TOL plasmid was capable of growing on toluene, on para- and on meta- xylene isomers, but not on benzene. When the PPO1 and ATCC 33015 strains were immobilized, in equal parts, on peat/perlite columns a much improved and sustainable removal of all three BTX components was observed at the rate of 40–50 g/h. m3 filter bed. Due to the dominance of the ring-hydroxylating pathways over the TOL pathway, the classical enrichment approach did not result in a consortium capable of the sustained removal of all BTX components. However, a rationally formulated consortium consisting of members with complementary metabolic abilities was capable of this task and should be of use both in industrial emission control and in soil venting operations.