Detection and Enumeration of a Tagged Pseudomonas fluorescens Strain by Using Soil with Markers Associated with an Engineered Catabolic Pathway

Previously we described a novel gene tagging method, using the moc (mannityl opine catabolism) region from the Agrobacterium tumefaciens Ti plasmid pTi15955, to identify microorganisms destined for release into the environment. Here, we used the engineered strain Pseudomonas fluorescens PF5MT12 carrying the moc region integrated into the bacterial chromosome to demonstrate the usefulness of the markers for detection and direct selection of marked organisms present in soil samples. Using this system, we routinely detected population levels as low as 10(sup2) CFU per g of soil sampled. In addition to direct selection, we developed an immunologically based assay using MOP cyclase, a unique enzyme associated with moc, as the epitope for detecting the tagged organism. The colony immunoblot assay proved to be highly specific and without any false-positive signals when used to identify organisms cultured from soil on nonselective medium. The numbers of colonies that were immunoreactive with the anti-MOP cyclase antibody were essentially equal to those that grew out on selection plates. This indicates that MOP cyclase can be used as a marker and that we can use nonselective medium to retrieve the marked genetically engineered microorganisms and then identify them by using colony immunoblot assays. These direct selection and colony immunoblot methods provide a sensitive and accurate strategy for identifying and enumerating marked organisms recovered from soil samples. We also developed a rapid assay for MOP cyclase that does not require cell permeabilization with toluene. This assay can be used to verify tagged organisms isolated by other methods or to screen large numbers of colonies for the tag following nonselective isolation.     

Effect of Humic Fractions and Clay on Biodegradation of Phenanthrene by a Pseudomonas fluorescens Strain Isolated from Soil

The mineralization of phenanthrene in pure cultures of a Pseudomonas fluorescens strain, isolated from soil, was measured in the presence of soil humic fractions and montmorillonite. Humic acid and clay, either separately or in combination, shortened the acclimation phase. A higher mineralization rate was measured in treatments with humic acid at 100 μg/ml. Humic acid at 10 μg/ml stimulated the transformation only in the presence of 10 g of clay per liter. We suggest that sorption of phenanthrene to these soil components may result in a higher concentration of substrate in the vicinity of the bacterial cells and therefore may increase its bioavailability.     

Detection of Plasmid Transfer from Pseudomonas fluorescens to Indigenous Bacteria in Soil by Using Bacteriophage phiR2f for Donor Counterselection

The transfer of a genetically marked derivative of plasmid RP4, RP4p, from Pseudomonas fluorescens to members of the indigenous microflora of the wheat rhizosphere was studied by using a bacteriophage that specifically lyses the donor strain and a specific eukaryotic marker on the plasmid. Transfer of RP4p to the wheat rhizosphere microflora was observed, and the number of transconjugants detected was approximately 10 transconjugants per g of soil when 10 donor cells per g of soil were added; transfer in the corresponding bulk soil was slightly above the limit of detection. All of the indigenous transconjugants which we analyzed contained a 60-kb plasmid and were able to transfer this plasmid to a Nx RpP. fluorescens recipient strain. The indigenous transconjugants were identified as belonging to Pseudomonas spp., Enterobacter spp., Comamonas spp., and Alcaligenes spp.     

Survival and Ecological Fitness of Pseudomonas fluorescens Genetically Engineered with Dual Biocontrol Mechanisms

The antibiotic 2,4-diacetylphloroglucinol (Phl) is produced by a range of naturally occurring fluorescent pseudomonads. One isolate, Pseudomonas fluorescens F113, protects pea plants from the pathogenic fungus Pythium ultimum by reducing the number of pathogenic lesions on plant roots, but with a concurrent reduction in the emergence of plants such as pea. The genes responsible for Phl production have been shown to be functionally conserved between the wild-type (wt) P. fluorescens strains F113 and Q2-87. In this study the genes from F113 were isolated using an optimized long PCR method and a 6.7-kb gene cluster inserted into the chromosome of the non-Phl-producing P. fluorescens strain SBW25 EeZY6KX. This strain is a lacZY, km^R marked derivative of the wt SBW25 which effects biological control against the plant pathogen Pythium ultimum by competitive exclusion as a result of its strong rhizosphere-colonizing ability. We describe here the integration of the Phl antifungal and competitive exclusion mechanisms into a single strain, and the impact this has on survival and plant emergence in microcosms. The insertion of the Phl biosynthetic genes from the F113 into the SBW25 chromosome gave a Phl-producing transformant (strain Pa21) able to suppress P. ultimum through antibiotic production. The growth of Pa21 was not reduced in flask culture at 20°C compared with its parent strain. When inoculated on pea seedlings, the strain containing the Phl operon behaved similarly to the SBW25 EeZY6KX parent but did not show the tendency of the wt Phl producer F113 to cause lower pea seed emergence. Pea roots inoculated with SBW25 EeZY6KX have significantly lower indigenous populations than with F113 and the control. This is indicative of this strains strong colonising presence. Pa21, the Phl-modified strain, is able to exclude the resident population from roots to the same degree as the SBW25 EeZY6KX from which it is derived. This suggests that it has maintained its competitiveness around the root systems of plants even with the introduction of the Phl locus. Thus, strain Pa21 possesses the qualities necessary to provide effective integrated biocontrol, through maintaining both its wt trait of competitive exclusion on the plant roots, while also expressing the genes from the F113 biocontrol strain for Phl production. Interestingly, however, an additional beneficial trait appears to emerge with the strain Pa21s lowered survival competence compared with SBW25 EeZY6KX in the rhizosphere soil. With fears of the spread of genetically modified organisms and persistence in the soil, this trait may be of some ecological and commercial benefit and becomes a candidate for further investigation and possible exploitation.     

Production of Cellobionate from Cellulose Using an Engineered Neurospora crassa Strain with Laccase and Redox Mediator Addition

We report a novel production process for cellobionic acid from cellulose using an engineered fungal strain with the exogenous addition of laccase and a redox mediator. A previously engineered strain of Neurospora crassa (F5∆ace-1∆cre-1∆ndvB) was shown to produce cellobionate directly from cellulose without the addition of exogenous cellulases. Specifically, N. crassa produces cellulases, which hydrolyze cellulose to cellobiose, and cellobiose dehydrogenase (CDH), which oxidizes cellobiose to cellobionate. However, the conversion of cellobiose to cellobionate is limited by the slow re-oxidation of CDH by molecular oxygen. By adding low concentrations of laccase and a redox mediator to the fermentation, CDH can be efficiently oxidized by the redox mediator, with in-situ re-oxidation of the redox mediator by laccase. The conversion of cellulose to cellobionate was optimized by evaluating pH, buffer, and laccase and redox mediator addition time on the yield of cellobionate. Mass and material balances were performed, and the use of the native N. crassa laccase in such a conversion system was evaluated against the exogenous Pleurotus ostreatus laccase. This paper describes a working concept of cellobionate production from cellulose using the CDH-ATBS-laccase system in a fermentation system.     

Importance of Preferential Flow and Soil Management in Vertical Transport of a Biocontrol Strain of Pseudomonas fluorescens in Structured Field Soil

The large-scale release of wild-type or genetically modified bacteria into the environment for control of plant diseases or for bioremediation entails the potential risk of groundwater contamination by these microorganisms. For a model study on patterns of vertical transport of bacteria under field conditions, the biocontrol strain Pseudomonas fluorescens CHA0, marked with a spontaneous resistance to rifampin (CHA0-Rif), was applied to a grass-clover ley plot (rotation grassland) and a wheat plot. Immediately after bacterial application, heavy precipitation was simulated by sprinkling, over a period of 8 h, 40 mm of water containing the mobile tracer potassium bromide and the dye Brilliant Blue FCF to identify channels of preferential flow. One day later, a 150-cm-deep soil trench was dug and soil profiles were prepared. Soil samples were extracted at different depths of the profiles and analyzed for the number of CHA0-Rif cells and the concentration of bromide and Brilliant Blue FCF. Dye coverage in the soil profiles was estimated by image analysis. CHA0 was present at 10(sup8) CFU/g in the surface soil, and 10(sup6) to 10(sup7) CFU/g of CHA0 was detected along macropores between 10 and 150 cm deep. Similarly, the concentration of the tracer bromide along the macropores remained at the same level below 20 cm deep. Dye coverage in lower soil layers was higher in the ley than in the wheat plot. In nonstained parts of the profiles, the number of CHA0-Rif cells was substantially smaller and the bromide concentration was below the detection limit in most samples. We conclude that after heavy rainfall, released bacteria are rapidly transported in large numbers through the channels of preferential flow to deeper soil layers. Under these conditions, the transport of CHA0-Rif is similar to that of the conservative tracer bromide and is affected by cultural practice.     

Isolation and Characterization of a Pseudomonas fluorescens Strain Suppressive to Bipolaris maydis

Strain PJ0210, identified as Pseudomonas fluorescens, inhibited Bipolaris maydis both in vitro. The absence of detectable inhibitory substances assayed by a variety of methods indicated nutrient competition as the operative component of antagonism. This was further supported by observations of reduced inhibition by addition of glucose both in vitro and in vivo. Strain PJ0210 was adaptecd to the phyllosphere showing good survivability and colonizing ability both inplant house and field trials. Disease suppression was generally correlated with population sizes of strain PJ0210. Population sizes in the field were lower than those recorded in the plant house with a concomitant reduction in disease suppressive ability, of strain PJ0210.     

Identification,Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain

A gene, pfa1, encoding an autotransporter was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased fish. The expression of pfa1 is enhanced during infection and is regulated by growth phase and growth conditions. Mutation of pfa1 significantly attenuates the overall bacterial virulence of TSS and impairs the abilities of TSS in biofilm production, interaction with host cells, modulation of host immune responses, and dissemination in host blood. The putative protein encoded by pfa1 is 1,242 amino acids in length and characterized by the presence of three functional domains that are typical for autotransporters. The passenger domain of PfaI contains a putative serine protease (Pap) that exhibits apparent proteolytic activity when expressed in and purified from Escherichia coli as a recombinant protein. Consistent with the important role played by PfaI in bacterial virulence, purified recombinant Pap has a profound cytotoxic effect on cultured fish cells. Enzymatic analysis showed that recombinant Pap is relatively heat stable and has an optimal temperature and pH of 50°C and pH 8.0. The domains of PfaI that are essential to autotransporting activity were localized, and on the basis of this, a PfaI-based autodisplay system (named AT1) was engineered to facilitate the insertion and transport of heterologous proteins. When expressed in E. coli, AT1 was able to deliver an integrated Edwardsiella tarda immunogen (Et18) onto the surface of bacterial cells. Compared to purified recombinant Et18, Et18 displayed by E. coli via AT1 induced significantly enhanced immunoprotection.Protein secretion plays important roles in bacterial life, as many of the secreted proteins are involved in biological processes that are fundamental to the survival and environmental adaptations of the cells. Gram-negative bacteria have evolved a number of secretion systems that utilize different secretion apparatus and mechanisms (9). The classical autotransporter secretion pathway belongs to the type V secretion system (8, 12, 13). Compared to other types of secretion mechanisms, the autotransporter system is unique in that all the components that are required for protein translocation are contained within a single polypeptide. Structurally, autotransporters are characterized by three domains: (i) an N-terminal signal sequence that is recognized by the Sec translocon; (ii) a central passenger domain (or α-domain) that contains the effector molecule and is highly variable; and (iii) a C-terminal translocation domain (or β-/autotransporter domain) that is conserved in length (250 to 300 amino acids) but varies in primary structure (7, 12, 55). In most cases, the β-domain contains 12 antiparallel strands of 9 to 12 residues that, upon integration into the outer membrane, form a β-barrel conformation (27, 57). Another conserved feature of the β-domain is the presence at the C terminus of a sequence motif, (Y/V/I/F/W)-X-(F/W), that is characterized by alternating hydrophobic and hydrophilic residues and ends with either a tryptophan or a phenylalanine (12, 16, 27). The integrity of this end motif seems to be required for protein translocation, as deletion of certain residues in this sequence impairs protein secretion. The secretion process of the autotransporter is initiated by the signal sequence, which directs the translocation of the protein precursor across the inner membrane into the periplasmic space via the Sec system. Once inside the periplasm, the β-domain inserts into the outer membrane and adopts the structure of a β-barrel through which the passenger domain is translocated to the cell surface, where it may exist as a membrane-anchored protein covalently linked to the β-domain or be cleaved from the β-domain as a result of proteolysis (7).Since the discovery of the gonococcal immunoglobulin A1 protease (35), the first autotransporter, and especially with the advent of genome sequencing technology, autotransporters have been identified in many bacterial species (18, 19, 28, 33). Functions assigned to autotransporters are mostly associated with bacterial pathogenicity, which includes adhesion and invasion into host cells, biofilm formation, and cytotoxicity (11, 54). In the present study, we identified and analyzed an autotransporter, PfaI, from a pathogenic Pseudomonas fluorescens strain isolated from diseased fish. We found that, like many of the autotransporters identified in other pathogens, PfaI is a virulence factor that is involved in interactions with host cells and modulation of host immune responses via a protease effector. In addition, we found that the autotransporter property of PfaI could be exploited for the delivery and surface display of an immunoprotective antigen.     

Detection of Plasmid Transfer from Pseudomonas fluorescens to Indigenous Bacteria in Soil by Using Bacteriophage φR2f for Donor Counterselection

The transfer of a genetically marked derivative of plasmid RP4, RP4p, from Pseudomonas fluorescens to members of the indigenous microflora of the wheat rhizosphere was studied by using a bacteriophage that specifically lyses the donor strain and a specific eukaryotic marker on the plasmid. Transfer of RP4p to the wheat rhizosphere microflora was observed, and the number of transconjugants detected was approximately 10³ transconjugants per g of soil when 10⁷ donor cells per g of soil were added; transfer in the corresponding bulk soil was slightly above the limit of detection. All of the indigenous transconjugants which we analyzed contained a 60-kb plasmid and were able to transfer this plasmid to a Nx^r Rp^rP. fluorescens recipient strain. The indigenous transconjugants were identified as belonging to Pseudomonas spp., Enterobacter spp., Comamonas spp., and Alcaligenes spp.     

Rhizoremediation of Trichloroethylene by a Recombinant, Root-Colonizing Pseudomonas fluorescens Strain Expressing Toluene ortho-Monooxygenase Constitutively

Trichloroethylene (TCE) was removed from soils by using a wheat rhizosphere established by coating seeds with a recombinant, TCE-degrading Pseudomonas fluorescens strain that expresses the tomA⁺ (toluene o-monooxygenase) genes from Burkholderia cepacia PR1₂₃(TOM_23C). A transposon integration vector was used to insert tomA⁺ into the chromosome of P. fluorescens 2-79, producing a stable strain that expressed constitutively the monooxygenase at a level of 1.1 nmol/min · mg of protein (initial TCE concentration, 10 μM, assuming that all of the TCE was in the liquid) for more than 280 cell generations (36 days). We also constructed a salicylate-inducible P. fluorescens strain that degraded TCE at an initial rate of 2.6 nmol/min · mg of protein in the presence of 10 μM TCE [cf. B. cepacia G4 PR1₂₃(TOM_23C), which degraded TCE at an initial rate of 2.5 nmol/min · mg of protein]. A constitutive strain, P. fluorescens 2-79TOM, grew (maximum specific growth rate, 0.78 h⁻¹) and colonized wheat (3 × 10⁶ CFU/cm of root) as well as wild-type P. fluorescens 2-79 (maximum specific growth rate, 0.77 h⁻¹; level of colonization, 4 × 10⁶ CFU/cm of root). Rhizoremediation of TCE was demonstrated by using microcosms containing the constitutive monooxygenase-expressing microorganism, soil, and wheat. These closed microcosms degraded an average of 63% of the initial TCE in 4 days (20.6 nmol of TCE/day · plant), compared to the 9% of the initial TCE removed by negative controls consisting of microcosms containing wild-type P. fluorescens 2-79-inoculated wheat, uninoculated wheat, or sterile soil.     

Curium(III) Speciation Studies with Cells of a Groundwater Strain of Pseudomonas fluorescens

Ubiquitous Pseudomonads have great potential to influence the speciation and mobility of actinides in the environment. This study explores the unknown interaction between curium(III) and cell-suspensions of Pseudomonas fluorescens (CCUG 32456) isolated from the Äspö site, Sweden. The interaction between curium(III) and P. fluorescens cells was studied at trace curium(III) concentrations (0.3 μM) using time-resolved laser-induced fluorescence spectroscopy. Extraction studies have shown that the biosorption of curium(III) is a reversible process. Two Cm³⁺?P. fluorescens (CCUG 32456) species were identified, R?O?PO₃H?Cm²⁺ and R?COO?Cm²⁺, having emission maxima at 599.6 and 601.9 nm, respectively. The corresponding surface complexation constants were determined to be log β₁₁₁ = 12.7 ± 0.6 and log β₁₁₀ = 6.1 ± 0.5, respectively.     

Involvement of Nitrate Reductase and Pyoverdine in Competitiveness of Pseudomonas fluorescens Strain C7R12 in Soil

Involvement of nitrate reductase and pyoverdine in the competitiveness of the biocontrol strain Pseudomonas fluorescens C7R12 was determined, under gnotobiotic conditions, in two soil compartments (bulk and rhizosphere soil), with the soil being kept at two different values of matric potential (−1 and −10 kPa). Three mutants affected in the synthesis of either the nitrate reductase (Nar⁻), the pyoverdine (Pvd⁻), or both (Nar⁻ Pvd⁻) were used. The Nar⁻ and Nar⁻ Pvd⁻ mutants were obtained by site-directed mutagenesis of the wild-type strain and of the Pvd⁻ mutant, respectively. The selective advantage given by nitrate reductase and pyoverdine to the wild-type strain was assessed by measuring the dynamic of each mutant-to-total-inoculant (wild-type strain plus mutant) ratio. All three mutants showed a lower competitiveness than the wild-type strain, indicating that both nitrate reductase and pyoverdine are involved in the fitness of P. fluorescens C7R12. The double mutant presented the lowest competitiveness. Overall, the competitive advantages given to C7R12 by nitrate reductase and pyoverdine were similar. However, the selective advantage given by nitrate reductase was more strongly expressed under conditions of lower aeration (−1 kPa). In contrast, the selective advantage given by nitrate reductase and pyoverdine did not differ in bulk and rhizosphere soil, indicating that these bacterial traits are not specifically involved in the rhizosphere competence but rather in the saprophytic ability of C7R12 in soil environments.     

Isolation and Antifungal and Antioomycete Activities of Aerugine Produced by Pseudomonas fluorescens Strain MM-B16

The bacterial strain MM-B16, which showed strong antifungal and antioomycete activity against some plant pathogens, was isolated from a mountain forest soil in Korea. Based on the physiological and biochemical characteristics and 16S ribosomal DNA sequence analysis, the bacterial strain MM-B16 was identical to Pseudomonas fluorescens. An antibiotic active against Colletotrichum orbiculare and Phytophthora capsici in vitro and in vivo was isolated from the culture filtrates of P. fluorescens strain MM-B16 using various chromatographic procedures. The molecular formula of the antibiotic was deduced to be C₁₀H₁₁NO₂S (M⁺, m/z 209.0513) by analysis of electron impact mass spectral data. Based on the nuclear magnetic resonance and infrared spectral data, the antibiotic was confirmed to have the structure of a thiazoline derivative, aerugine [4-hydroxymethyl-2-(2-hydroxyphenyl)-2-thiazoline]. C. orbiculare, P. capsici, and Pythium ultimum were most sensitive to aerugine (MICs for these organisms were approximately 10 μg ml⁻¹). However, no antimicrobial activity was found against yeasts and bacteria even at concentrations of more than 100 μg ml⁻¹. Treatment with aerugine exhibited a significantly high protective activity against development of phytophthora disease on pepper and anthracnose on cucumber. However, the control efficacy of aerugine against the diseases was in general somewhat less than that of the commercial fungicides metalaxyl and chlorothalonil. This is the first study to isolate aerugine from P. fluorescens and demonstrate its in vitro and in vivo antifungal and antioomycete activities against C. orbiculare and P. capsici.     

The Semi Large-scale Production, Extraction, Purification and Properties of an Antibiotic Produced by Pseudomonas fluorescens Strain 175

S ummary : Production, extraction, purification and properties of an antibiotic produced by Pseudomonas fluorescens , strain 175, are described. Extraction efficiency was c. 57% with 3–5 g of product/350 1 batch culture, the product having an activity of 7900 units/mg (0· 12 μg/ml) against Staphylococcus aureus Paler. The antibiotic had the properties, of a weak acid and was active against both Gram positive and Gram negative bacteria; it was stable over a wide pH range, was bactericidal, inactivated by serum, had low toxicity and was haemolytic at relatively high concentrations.     

Mineralization of Paraoxon and Its Use as a Sole C and P Source by a Rationally Designed Catabolic Pathway in Pseudomonas putida

Organophosphate compounds, which are widely used as pesticides and chemical warfare agents, are cholinesterase inhibitors. These synthetic compounds are resistant to natural degradation and threaten the environment. We constructed a strain of Pseudomonas putida that can efficiently degrade a model organophosphate, paraoxon, and use it as a carbon, energy, and phosphorus source. This strain was engineered with the pnp operon from Pseudomonas sp. strain ENV2030, which encodes enzymes that transform p-nitrophenol into β-ketoadipate, and with a synthetic operon encoding an organophosphate hydrolase (encoded by opd) from Flavobacterium sp. strain ATCC 27551, a phosphodiesterase (encoded by pde) from Delftia acidovorans, and an alkaline phosphatase (encoded by phoA) from Pseudomonas aeruginosa HN854 under control of a constitutive promoter. The engineered strain can efficiently mineralize up to 1 mM (275 mg/liter) paraoxon within 48 h, using paraoxon as the sole carbon and phosphorus source and an inoculum optical density at 600 nm of 0.03. Because the organism can utilize paraoxon as a sole carbon, energy, and phosphorus source and because one of the intermediates in the pathway (p-nitrophenol) is toxic at high concentrations, there is no need for selection pressure to maintain the heterologous pathway.     

Isolation and Characterization of a Stilbene-degrading Strain of Pseudomonas fluorescens,and Production of Antioxidant Compounds by Stilbene Metabolism

In this study, we consider the use of hydrocarbon-degrading bacteria that degrade trans-stilbene as a novel approach for synthesizing potentially bioactive hydroxylated stilbenes. A trans-stilbene-degrading bacterium, MN2, was isolated from activated sludge through enrichment culture, and identified as Pseudomonas fluorescens using conventional techniques. Degradation of trans-stilbene by this strain yielded two metabolites that had significant antioxidant activity.     

Production of Aromatic Compounds by Metabolically Engineered Escherichia coli with an Expanded Shikimate Pathway

Escherichia coli was metabolically engineered by expanding the shikimate pathway to generate strains capable of producing six kinds of aromatic compounds, phenyllactic acid, 4-hydroxyphenyllactic acid, phenylacetic acid, 4-hydroxyphenylacetic acid, 2-phenylethanol, and 2-(4-hydroxyphenyl)ethanol, which are used in several fields of industries including pharmaceutical, agrochemical, antibiotic, flavor industries, etc. To generate strains that produce phenyllactic acid and 4-hydroxyphenyllactic acid, the lactate dehydrogenase gene (ldhA) from Cupriavidus necator was introduced into the chromosomes of phenylalanine and tyrosine overproducers, respectively. Both the phenylpyruvate decarboxylase gene (ipdC) from Azospirillum brasilense and the phenylacetaldehyde dehydrogenase gene (feaB) from E. coli were introduced into the chromosomes of phenylalanine and tyrosine overproducers to generate phenylacetic acid and 4-hydroxyphenylacetic acid producers, respectively, whereas ipdC and the alcohol dehydrogenase gene (adhC) from Lactobacillus brevis were introduced to generate 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, respectively. Expression of the respective introduced genes was controlled by the T7 promoter. While generating the 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, we found that produced phenylacetaldehyde and 4-hydroxyphenylacetaldehyde were automatically reduced to 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol by endogenous aldehyde reductases in E. coli encoded by the yqhD, yjgB, and yahK genes. Cointroduction and cooverexpression of each gene with ipdC in the phenylalanine and tyrosine overproducers enhanced the production of 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol from glucose. Introduction of the yahK gene yielded the most efficient production of both aromatic alcohols. During the production of 2-phenylethanol, 2-(4-hydroxyphenyl)ethanol, phenylacetic acid, and 4-hydroxyphenylacetic acid, accumulation of some by-products were observed. Deletion of feaB, pheA, and/or tyrA genes from the chromosomes of the constructed strains resulted in increased desired aromatic compounds with decreased by-products. Finally, each of the six constructed strains was able to successfully produce a different aromatic compound as a major product. We show here that six aromatic compounds are able to be produced from renewable resources without supplementing with expensive precursors.     

Cloning and Sequencing of the Gene of Tryptophan-7-Halogenase from Pseudomonas fluorescens Strain CHA0

The gene of tryptophan-7-halogenase from the Pseudomonas fluorescens strain CHA0, a producer of the halogenated antibiotic pyrrolnitrin, has been cloned and sequenced.