Choice of microbial host for the naphthalene dioxygenase bioconversion

The use of whole cell biotransformations for single and multistep enzyme conversions is gaining widespread application. In this study the naphthalene dioxygenasenah A gene was transferred intoPseudomonas aeruginosa PAC 1R,Escherichia coli JM107 andPseudomonas putida PpG 277. The effect of ethanol on these genetically engineered Gram-negative bacteria was studied by measurement of enzyme activity, stability and cell integrity. Ethanol has been used in biotransformations as a co-substrate carbon source for co-factor recycling and as a co-solvent increasing dissolved substrate and product levels. Ethanol increased the dissolved substrate (naphthalene) concentration slightly and dissolved product ((+)-cis-(1R, 2S)-dihydroxy-1,2-dihydronaphthalene) by approximately 30% at 4% (w/v) ethanol. BothP. aeruginosa PAC 1R andP. putida PpG 277 showed decreased activity with increasing ethanol concentration whilstE. coli enzyme activity increased with increasing ethanol concentration being comparable to that when glucose was used as a carbon source. This project highlighted the many factors involved in the selection of microbial hosts for whole cell biotransformation processes.     

Use of an Exotic Carbon Source To Selectively Increase Metabolic Activity and Growth of Pseudomonas putida in Soil

Respiration and growth of Pseudomonas putida PpG7, containing catabolic plasmid NAH7, was determined in three agricultural field soils amended with the carbon source salicylate. The addition of salicylate to soil significantly increased the population of PpG7. However, there was a lack of relationship between microbial numbers and activity as determined by evolution of CO₂. In soils containing 30 to 1,500 μg of salicylate per g, metabolic activities of PpG7 peaked between 18 and 42 h and population densities increased approximately 10¹-to 10⁵-fold. However, the metabolic activity of PpG7 rapidly declined after salicylate was utilized, whereas peak population densities were maintained for the duration of the experiments (5 to 7 days). Thus, elevated population densities of PpG7 were represented by inactive cells. Soil type had only minor effects on respiration rates or growth curves of PpG7 when amended with comparable concentrations of salicylate. Respiration and growth rates were optimal at concentrations between 300 and 1,000 μg of salicylate per g in the test soils. At 1,500 to 2,500 μg/g, respiration and growth of PpG7 were initially suppressed, but after a short lag time both attained levels similar to or greater than those resulting from the use of lower concentrations of salicylate. The culturing of PpG7 on a salicylate-amended medium to induce salicylate-degradative enzymes did not affect the lag time before utilization of salicylate in soil. Although PpG7 competed well with fungi for the substrate, suppression of fungal populations with cycloheximide resulted in significantly increased population densities of PpG7 in two of three soils amended with salicylate. The beneficial activities of bacteria in soil are discussed in relation to population density, population metabolic activity, and selective carbon source utilization.     

Expression of Pseudomonas aeruginosa transposable phages in Pseudomonas putida cells. I. The establishment of a lysogenic state and the effectiveness of lytic development

Expression of transposable phages (TP) of Pseudomonas aeruginosa in the cells of P. putida was studied. The high efficiency of phage lytic development was shown both as a consequence of zygotic induction after transfer of the RP4::TPc+ plasmid into nonlysogenic recipients, and as a result of heat induction of lysogens PpG1 (D3112cts15). The high phage yield (20-25 particles of D3112cts phage per one cell of P. putida) is an evidence for a high level of transposition in the cells of this bacterial species. Plasmids RP4::TP are transferred into cells of PpG1 and PAO1 with similar frequency. However, the efficiency of establishment of the lysogenic state is lower in PpG1. Transposable phages of P. aeruginosa can integrate into the chromosome of PpG1 producing stable inducible lysogens. The presence of RP4 in the P. putida cells is not necessary for expression of transposable phages. The transposable phage D3112cts15 can be used in experiments of interspecies transduction of plasmids and chromosomal genes.     

The T7-related Pseudomonas putida phage φ15 displays virion-associated biofilm degradation properties

Formation of a protected biofilm environment is recognized as one of the major causes of the increasing antibiotic resistance development and emphasizes the need to develop alternative antibacterial strategies, like phage therapy. This study investigates the in vitro degradation of single-species Pseudomonas putida biofilms, PpG1 and RD5PR2, by the novel phage ϕ15, a ‘T7-like virus’ with a virion-associated exopolysaccharide (EPS) depolymerase. Phage ϕ15 forms plaques surrounded by growing opaque halo zones, indicative for EPS degradation, on seven out of 53 P. putida strains. The absence of haloes on infection resistant strains suggests that the EPS probably act as a primary bacterial receptor for phage infection. Independent of bacterial strain or biofilm age, a time and dose dependent response of ϕ15-mediated biofilm degradation was observed with generally a maximum biofilm degradation 8 h after addition of the higher phage doses (10⁴ and 10⁶ pfu) and resistance development after 24 h. Biofilm age, an in vivo very variable parameter, reduced markedly phage-mediated degradation of PpG1 biofilms, while degradation of RD5PR2 biofilms and ϕ15 amplification were unaffected. Killing of the planktonic culture occurred in parallel with but was always more pronounced than biofilm degradation, accentuating the need for evaluating phages for therapeutic purposes in biofilm conditions. EPS degrading activity of recombinantly expressed viral tail spike was confirmed by capsule staining. These data suggests that the addition of high initial titers of specifically selected phages with a proper EPS depolymerase are crucial criteria in the development of phage therapy.     

Physiological function of the Pseudomonas putida PpG6 (Pseudomonas oleovorans) alkane hydroxylase: monoterminal oxidation of alkanes and fatty acids.

Pseudomonas putida PpG6 is able to utilize purified n-alkanes of six to ten carbon atoms for growth. It can also grow on the primary terminal oxidation products of these alkanes and on 1-dodecanol but not on the corresponding 2-ketones or 1,6-hexanediol, adipic acid, or pimelic acid. Revertible point mutants can be isolated which have simultaneously lost the ability to grow on all five n-alkane growth substrates but which can still grow on octanol or nonanol. An acetate-negative mutant defective in isocitrate lysase activity is unable to grow on even-numbered alkanes and fatty acids. Analysis of double mutants defective in acetate and propionate or in acetate and glutarate metabolism shows that alkane carbon is assimilated only via acetyl-coenzyme A and propionyl-coenzyme A. These results support the following conclusions: (i) The n-alkane growth specificity of P. putida PpG6 is due to the substrate specificity of whole-cell alkane hydroxylation; (ii) there is a single alkane hydroxylase enzyme complex; (iii) the physiological role of this complex is to initiate the monoterminal oxidation of alkane chains; and (iv) straight-chain fatty acids from butyric through nonanoic are degraded exclusively by beta-oxidation from the carboxyl end of the molecule.     

Walking the tightrope of bioavailability: growth dynamics of PAH degraders on vapour‐phase PAH

Microbial contaminant degradation may either result in the utilization of the compound for growth or act as a protective mechanism against its toxicity. Bioavailability of contaminants for nutrition and toxicity has opposite consequences which may have resulted in quite different bacterial adaptation mechanisms; these may particularly interfere when a growth substrate causes toxicity at high bioavailability. Recently, it has been demonstrated that a high bioavailability of vapour‐phase naphthalene (NAPH) leads to chemotactic movement of NAPH‐degrading Pseudomonas putida (NAH7) G7 away from the NAPH source. To investigate the balance of toxic defence and substrate utilization, we tested the influence of the cell density on surface‐associated growth of strain PpG7 at different positions in vapour‐phase NAPH gradients. Controlled microcosm experiments revealed that high cell densities increased growth rates close (< 2 cm) to the NAPH source, whereas competition for NAPH decreased the growth rates at larger distances despite the high gas phase diffusivity of NAPH. At larger distance, less microbial biomass was likewise sustained by the vapour‐phase NAPH. Such varying growth kinetics is explained by a combination of bioavailability restrictions and NAPH‐based inhibition. To account for this balance, a novel, integrated ‘Best Equation’ describing microbial growth influenced by substrate availability and inhibition is presented.     

Plasmid control of the Pseudomonas aeruginosa and Pseudomonas putida phenotypes and of linalool and p-cymene oxidation.

Two Pseudomonas strains (PpG777 and PaG158) were derived from the parent isolate Pseudomonas incognita (putida). Strain PpG777 resembles the parental culture in growth on linalool as a source of carbon and slight growth on p-cymene, whereas PaG158 grows well on p-cymene, but not on linalool or other terpenes tested, and has a P. aeruginosa phenotype. Curing studies indicate that linalool metabolism is controlled by an extrachromosomal element whose loss forms a stable strain PaG158 with the p-cymene growth and P. aeruginosa phenotype characters. The plasmid can be transferred by PpG777 to both P. putida and P. aeruginosa strains. Surprisingly, the latter assume the P. putida phenotype. We conclude that the genetic potential to oxidize p-cymene is inherent in PpG777 but expression is repressed. Similarly, this observation implies that support of linalool oxidation effectively conceals the P. aeruginosa character.     

Plasmid-coded degradation of salicylate using the catechol cleavage pathway of the host

Degradation rates of salicylate and phenol by Pseudomonas putida PpG1064 carrying the nahG gene on a multicopy plasmid were compared with those in NAH-carrying P. putida. Degradation rates of salicylate and phenol and the growth rate of the recombinant were higher than those in NAH-carrying P. putida in SP medium. The catechol 1,2 oxygenase activity of the recombinant in Sp medium was about twice that of the catechol 2,3 oxygenase and catechol 1,2 oxygenase activities of NAH-carrying P. putida. It was suggested that in simultaneous degradation of phenol and salicylate, the recombinant stimulated its ortho cleavage pathway and attained the higher degradation rates and growth rate.     

Expression of Pseudomonas aeruginosa phage transposons in Pseudomonas putida PpG1 cells. II. Zygotic induction--a necessary condition for the formation of defective lysogens

The transfer of hybrid plasmid RP4::PT (where PT is the genome of a transposable phage specific for Pseudomonas aeruginosa) into recipient cells of P. putida strain PpG1 occurs with the same frequency as into P. aeruginosa, the homologous host for PT. Approximately 1/3 of all PpG1 exconjugants carrying RP4 markers lost the capability to produce viable PT phage. In contrast, in a cross with homologous recipient P. aeruginosa all exconjugant clones contained nondefective prophages in the hybrid plasmids. Zygotic induction is an obligatory condition for detection of PpG1 exconjugants with defective phages. The defective prophages in RP4::PT hybrid plasmids have deletions of different size; the other carry mutations indistinguishable from point mutations in an essential phage gene. Some of deletions also cover plasmid genes. At least some of the defective prophages, including deleted ones, have arisen in the recipient cells of P. putida after transfer of the hybrid plasmid.     

Phage-stable mutants of Pseudomonas putida: new types of stability

More than 170 phage-resistant mutants (PRM) of the first order of Pseudomonas putida strain PpG1 were obtained using newly isolated and previously described bacteriophages specific for this strain. According to the results of analysis of resistance of the mutants to each of 31 phages of PpG1 strain and 8 phages of the PpN strain, the PRM strains were distributed into 20 groups. In most cases, the reason for resistance is loss of absorption capacity of bacteria. However, no direct relation between the level of absorption and efficiency of phage plating was detected. It was shown that some of the PRM of P. putida PpG1 strains acquired the ability to maintain the growth of phages specific for the other P. putida strain, PpN. Frequencies of isolating mutants of various resistance types depend on the concrete phage used. In accordance with their absorption specificity, all phages were distributed into 23 groups, and a tridimensional formal scheme of receptor sites for these phages on the PpG1 strain was drawn. In the process of selection of the PpG1 clones resistant to non-lysogenizing mutant of temperate PP71 phage, a variant of this strain manifesting the phenomenon of "auto-plaquing" was found. These results support the mutational origin of this phenomenon in some cases.     

Maintenance requirements: energy supply from simultaneous endogenous respiration and substrate consumption

Abstract A model is presented that describes energy for maintenance purposes (ATP) as being obtained simultaneously from biomass degradation as well as from substrate degradation in excess of growth requirements. The ratio between both catabolic processes was taken to be growth rate dependent. As such, this approach is intermediate between established models; its significant features are negative growth and the absence of substrate consumption at zero substrate concentration, and the attainability of the maximum specific growth rate (the model parameter μ _max) at elevated substrate concentrations. As a simple case, the amounts of ATP obtained from direct substrate catabolism or from the degradation of an equivalent amount of biomass were taken as identical. Also, the maintenance demand in terms of ATP per unit time and biomass was taken to be constant. True growth rate dependency of maintenance can be implemented by relaxing either of these assumptions.     

Evidence of indigenous NAH plasmid of naphthalene degrading Pseudomonas putida PpG7 strain implicated in limonin degradation

A well characterized naphthalene-degrading strain, Pseudomonas putida PpG7 was observed to utilize limonin, a highly-oxygenated triterpenoid compound as a sole source of carbon and energy. Limonin concentrations evidenced a 64% reduction over 48 h of growth in batch cultures. Attempts were made to acquire a plasmid-less derivative via various methods (viz. Ethidium Bromide, SDS, elevated temperature & mitomycin C), among which the method involving mitomycin C (20 ug/ml) proved successful. Concomitant with the loss of plasmid in P. putida PpG7 strain, the cured derivative was identified as a lim- phenotype. The lim+ phenotype could be conjugally transferred to the cured derivative. Based on the results of curing with mitomycin C, conjugation studies and presence of ndo gene encoding naphthalene 1,2 dioxygenase, it was demonstrated that genes for the limonin utilization were encoded on an 83 kb indigenous transmissible Inc. P9 NAH plasmid in Pseudomonas putida PpG7 strain.     

XYL, a nonconjugative xylene-degradative plasmid in Pseudomonas Pxy.

Pseudomanas Pxy metabolizes p- or m-xylene through intermediate formation of the corresponding methylbenzyl alcohol and toluic acid via the meta pathway. The strain Pseudomonas Pxy spontaneously loses its ability to grow with xylene or toluate, and the rate of loss of this ability is greatly enhanced by treatment of the cells with mitomycin C. The assay of enzymes involved in xylene degradation in xylene-negative Pxy cells indicates the loss of the entire enzyme complement of the pathway. The genes specifying all the xylene-degradative enzymes, including those of the meta pathway, appear to be borne on a nonconjugative plasmid and can be transferred to xylene-negative Pxy or P. putida strain PpG1 cells only in the presence of a transfer plasmid termed factor K. When transferred to strain PpG1, the xylene-degradative plasmid, termed XYL, coexists stably with factor K, but transduction of XYL is not accompanied by a cotransfer of factor K. XYL appears to be compatible wit- all the other known degradative plasmids in P. putida. The xylene pathway is inducible in wild-type Pxy as well as in Pxy and PpG1 exconjugants, suggesting the cotransfer of regulatory genes along with the plasmid. The enzymes converting xylene to toluate are induced by xylene, methylbenzyl alcohol, or the aldehyde derivatives but not significantly by toluate, whereas catechol dioxygenase and other enzymes are induced by toluates and presumable by xylene as well.     

Toluene degradation kinetics for planktonic and biofilm-grown cells of Pseudomonas putida 54G

Toluene degradation kinetics by biofilm and planktonic cells of Pseudomonas putida 54G were compared in this study. Batch degradation of (14)C toluene was used to evaluate kinetic parameters for planktonic cells. The kinetic parameters determined for toluene degradation were: specific growth rate, mu(max) = 10.08 +/- 1.2/day; half-saturation constant, K(S) = 3.98 +/- 1.28 mg/L; substrate inhibition constant, K(I) = 42.78 +/- 3.87 mg/L. Biofilm cells, grown on ceramic rings in vapor phase bioreactors, were removed and suspended in batch cultures to calculate (14)C toluene degradation rates. Specific activities measured for planktonic and biofilm cells were similar based on toluene degrading cells and total biomass. Long-term toluene exposure reduced specific activities that were based on total biomass for both biofilm and planktonic cells. These results suggest that long-term toluene exposure caused a large portion of the biomass to become inactive, even though the biofilm was not substrate limited. Conversely, specific activities based on numbers of toluene-culturable cells were comparable for both biofilm and planktonically grown cultures. Planktonic cell kinetics are often used in bioreactor models to model substrate degradation and growth of bacteria in biofilms, a procedure we found to be appropriate for this organism. For superior bioreactor design, however, changes in cellular activity that occur during biofilm development should be investigated under conditions relevant to reactor operation before predictive models for bioreactor systems are developed. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 535-546, 1997.     

Continuous limonin degradation by immobilizedRhodococcus fascians cells in K-carrageenan

Limonin can be effectively degraded byRhodococcus fascians cells. These bacteria can be entraped in -carrageenan, and used in a continuous stirred tank reactor to degrade limonin in a continuous process. The effects of temperature limonin concentration, dilution rate, and aeration on the reactor behaviour have been tested, and the results correlated with changes in limonin conversion, substrate degradation rate, and free and immobilized biomass. Results showed that the immobilized cells were able to debitter limonin-containing media and the immobilized biomass was quite stable throughout the operational conditions tested. A population of free biomass was present in the reactor, the quantity of which was dependent on dilution rate. The immobilized bacteria increased its limonin-degrading capability when the substrate concentration was increased. The aeration was not strictly necessary for limonin degradation. Additionally, the immobilized cells were active and stable for more than 2 months of continuous operation, and were able to recover their limonin-degrading capability when used intermittently. Finally, none of the main components of a juice was noticeably altered during limonin degradation, so the reactor response was good enough to consider its application.     

Inhibition of substance P degradation in rat brain preparations by peptide hydroxamic acids

A peptidase activity of rat diencephalon membranes, which acts on the C-terminal hexapeptide sequence of substance P, was characterized using the radiolabeled substrate N alpha-[( 125I]iododesaminotyrosyl)-substance P (6-11)-hexapeptide. This activity presents certain characteristics similar to those of the substance-P-degrading enzyme purified from human brain by Lee et al. [Eur. J. Biochem. 114, 315-327 (1981)]. It is inhibited by metal chelators and some thiol reagents, but is insensitive to inhibitors of serine proteases and aminopeptidases. The activity is different from angiotensin-converting enzyme and enkephalinase, since it is not affected by specific inhibitors of these enzymes. Substance P and substance P C-terminal fragments longer than the pentapeptide inhibited the degradation of the radiolabeled substrate with inhibition constants around 200 microM. Short fragments of the substance P sequence, such as Boc-Phe-Phe-OMe and Boc-Phe-Phe-Gly-OEt, were also found to inhibit the degradation of the substrate. When the metal-chelating hydroxamic acid moiety was attached to the carboxyl terminus of these short peptides, potent inhibitors of the substance-P-degrading activity were obtained, with inhibition constants in the micromolar range. The most potent of these compounds, iododesaminotyrosyl-Phe-Phe-Gly-NHOH (IBH-Phe-Phe-Gly-NHOH), is a competitive inhibitor, with a Ki value of 1.9 microM. The degradation of substance P by rat diencephalon slices was inhibited to the same extent (40-50%) by IBH-Phe-Phe-Gly-NHOH (20 microM) and by phosphoramidon (1 microM). A combination of both reagents reduced the degradation rate by 75-80%, suggesting that both enkephalinase and the substance-P-degrading activity are involved in the metabolism of substance P in this preparation. IBH-Phe-Phe-Gly-NHOH seems to be quite specific for the latter enzyme, since at a high concentration (0.1 mM) it did not affect the degradation of the radiolabeled substrate by alpha-chymotrypsin, papain, or thermolysin.     

内蒙古草甸草原土壤理化性质和微生物学特性对刈割与氮添加的响应

频繁的刈割和氮输入增加是导致草地生态系统退化的重要原因.土壤微生物学特性作为评估土壤质量的重要生物学指标,对草地刈割和氮输入增加的响应规律仍不十分明确.本研究依托内蒙古呼伦贝尔草原刈割复合氮添加野外实验平台,分析了土壤理化性质、土壤微生物生物量、土壤呼吸和土壤酶对刈割、氮添加的响应及其生长季动态变化.结果表明: 刈割显著降低了土壤微生物生物量碳、氮、磷和土壤呼吸(基础呼吸和底物诱导呼吸),与刈割后导致的水分限制及碳限制有关.刈割显著降低了氮磷获取酶(N-乙酰-β-D-葡萄糖苷酶和酸性磷酸单酯酶)的活性,符合“资源分配假说”.氮添加显著降低土壤pH值,但土壤微生物生物量对氮添加和pH降低均无显著响应,表明氮输入增加引起的土壤酸化不是影响微生物生物量的主要因素.氮添加对土壤呼吸和酶活性也无显著影响,与以往在典型草原的大多数研究结果不一致.刈割和氮添加复合处理显著降低了土壤微生物生物量磷,但提高了土壤中有效磷含量,降低了酸性磷酸酶活性.微生物生物量碳、氮、磷和土壤呼吸等的相关参数均在7月最高,这与夏季高温多雨有关.土壤酶活性在春夏季较高,生长季末期较低.这表明在该草甸草原,刈割将导致土壤碳氮磷养分失衡,从而加剧草原退化;而氮添加在短期内并未对土壤微生物生物量和活性产生显著影响.     

Protein components of a cytochrome P-450 linalool 8-methyl hydroxylase

The cytochrome P-450 heme-thiolate monooxygenases that hydroxylate monoterpene hydrocarbon groups are effective models for the cytochrome P-450 family. We have purified and characterized the three proteins from a P-450-dependent linalool 8-methyl hydroxylase in Pseudomonas putida (incognita) strain PpG777. The proteins resemble the camphor 5-exohydroxylase components in chemical and physical properties; however, they show neither immunological cross-reactivity nor catalytic activity in heterogenous recombination. These two systems provide an excellent model to probe more deeply the heme-thiolate reaction center, molecular domains of substrate specificity, redox-pair interactions, and the regulation of the reaction cycle.