Alginate beads as a storage, delivery and containment system for genetically modified PCB degrader and PCB biosensor derivatives of Pseudomonas fluorescens F113.

AIMS: Pseudomonas fluorescens F113Rifpcb is a genetically engineered rhizosphere bacterium with the potential to degrade polychlorinated biphenyls (PCBs). F113Rifpcbgfp and F113L::1180gfp are biosensor strains capable of detecting PCB bioavailability and biodegradation. The aim of this paper is to evaluate the use of alginate beads as a storage, delivery and containment system for use of these strains in PCB contaminated soils. METHODS AND RESULTS: The survival and release of Ps. fluorescens F113Rifpcb from alginate beads were evaluated. Two Ps. fluorescens F113-based biosensor strains were encapsulated, and their ability to detect 3-chlorobenzoate (3-CBA) and 3-chlorobiphenyl (3-CBP) degradation in soil was assessed. After 250 days of storage, 100% recovery of viable F113Rifpcb cells was possible. Amendments to the alginate formulation allowed for the timed release of the inoculant. Encapsulation of the F113Rifpcb cells provided a more targeted approach for the inoculation of plants and resulted in lower inoculum populations in the bulk soil, which may reduce the risk of unintentional spread of these genetically modified micro-organisms in the environment. Encapsulation of the biosensor strains in alginate beads did not interfere with their ability to detect either 3-CBA or 3-CBP degradation. In fact, detection of 3-CBP degradation was enhanced in encapsulated biosensors. CONCLUSIONS: Alginate beads are an effective storage and delivery system for PCB degrading inocula and biosensors. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas fluorescens F113Rifpcb and the F113 derivative PCB biosensor strains have excellent potential for detecting and bioremediation of PCB contaminated soils. The alginate bead delivery system could facilitate the application of these strains as biosensors.

Identification of Rhizobium plasmid sequences involved in recognition of Psophocarpus, Vigna, and other legumes.

Symbiotic DNA sequences involved in nodulation by Rhizobium must include genes responsible for recognizing homologous hosts. We sought these genes by mobilizing the symbiotic plasmid of a broad host-range Rhizobium MPIK3030 (= NGR234) that can nodulate Glycine max, Psophocarpus tetragonolobus, Vigna unguiculata, etc., into two Nod- Rhizobium mutants as well as into Agrobacterium tumefaciens. Subsequently, cosmid clones of pMPIK3030a were mobilized into Nod+ Rhizobium that cannot nodulate the chosen hosts. Nodule development was monitored by examining the ultrastructure of nodules formed by the transconjugants. pMPIK3030a could complement Nod- and Nif- deletions in R. leguminosarum and R. meliloti as well as enable A. tumefaciens to nodulate. Three non-overlapping sets of cosmids were found that conferred upon a slow-growing Rhizobium species, as well as on R. loti and R. meliloti, the ability to nodulate Psophocarpus and Vigna, thus pointing to the existence of three sets of host-specificity genes. Recipients harboring these hsn regions had truly broadened host-range since they could nodulate both their original hosts as well as MPIK3030 hosts.

Genetic analysis of a Pseudomonas locus encoding a pathway for biphenyl/polychlorinated biphenyl degradation.

The cistronic organization of the bph locus, encoding a biphenyl/polychlorinated biphenyl (PCB) degradation pathway in Pseudomonas sp. LB400, has been elucidated. Seven structural genes, encoding biphenyl dioxygenase (bphA1A2A3A4), biphenyl-2,3-dihydrodiol-2,3-dehydrogenase (bphB), biphenyl-2,3-diol-1,2-dioxygenase (bphC) and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (bphD), have been located. The complete sequences of bphB, bphC and bphD are reported. Taken together with the data of Erickson and Mondello [J. Bacteriol. 174 (1992) 2903-2912], Pseudomonas sp. LB400 is now the first strain for which the sequences of all genes encoding the catabolism from biphenyls to benzoates have been determined. Comparisons of the deduced amino acid (aa) sequences of BphB, BphC and BphD with those of related proteins led to predictions about catalytically important aa residues. Six Bph have been detected and identified. Five of them could be obtained as the most abundant proteins when their genes were expressed in Escherichia coli.

A DNA module encoding bph genes for the degradation of polychlorinated biphenyls (PCBs).

In this report we describe the development and construction of a DNA module which encodes bph genes for the metabolism of PCBs and which is capable of stable integration into the chromosome of Gram negative bacteria. Introduction of the bph-module into Pseudomonas putida KT2442, Pseudomonas sp. strain B13 and its genetically engineered derivative B13FR1 expanded the biodegradative ability of these strains to include biphenyl and 4-chlorobiphenyl. The bph operon was stably inherited under laboratory conditions. Behavior of the genetically engineered strains was evaluated under simulated natural habitat conditions in lake sediment microcosms with respect to survival and removal of 4-chlorobiphenyl. The genetically engineered strains persisted under these conditions and were effective in degrading 4-chlorobiphenyl over a five day incubation period.

Regulation of the iron uptake genes in Pseudomonas fluorescens M114 by pseudobactin M114: the pbrA sigma factor gene does not mediate the siderophore regulatory response.

The iron-regulated PbrA sigma factor dictates the production of the siderophore, pseudobactin M114, and its cognate outer membrane receptor, PbuA, in Pseudomonas fluorescens M114. However, the siderophore molecule also has a role in regulating the expression of the siderophore biosynthetic and siderophore receptor genes in P. fluorescens M114. This is based on the fact that beta-galactosidase levels from lacZ fusions of M114 siderophore promoters (biosynthetic and receptor) were reduced in M114 siderophore biosynthetic mutants compared to wild-type M114. Expression of both promoters was increased by the addition of pseudobactin M114 to the growth medium. This effect was widespread and applicable to all but one of the siderophore negative strains of M114 tested. Furthermore, it was demonstrated that transcription of the pbr A sigma factor gene was not reduced in the siderophore biosynthetic mutants. This excludes the possibility that reduced expression of the siderophore biosynthetic and receptor promoters in the siderophore biosynthetic mutants is mediated at the level of expression of the pbr A gene itself. In addition, it was noted that the siderophore regulated response was applicable to promoters with and without the DNA sequence motif, (G/C)CTAAATCCC, which is required for iron-regulated expression of some pseudomonad promoters.

Construction of a modified mini-Tn5 lacZY non-antibiotic marker cassette: ecological evaluation of a lacZY marked Pseudomonas strain in the sugarbeet rhizosphere.

In order to monitor the fate of genetically manipulated fluorescent pseudomonads following release into the environment, a lacZY transposable cassette, lacking antibiotic resistance genes, was constructed using a pUT suicide plasmid delivery system. The resulting plasmid, pUTLacZY, can be easily used to generate lacZY marked pseudomonads without having to use antibiotic resistance determinants. The lacZY transposon generates random, stable transcriptional/translational fusions on integration into the target genome. Pseudomonas fluorescens strain F113 was marked with lacZY and was unaltered with respect to ecological fitness in the rhizosphere. Although lateral gene transfer of the chromosomally integrated lacZY marker could be detected in vitro, it was not detected in rhizosphere microcosms.

Escherichia coli ferric uptake regulator (Fur) can mediate regulation of a pseudomonad iron-regulated promoter.

Introduction of a Pseudomonas iron-regulated promoter lacZ fusion (SP1) and a Pseudomonas transcriptional factor into Escherichia coli allowed expression of the promoter in this heterologous host. Evaluation of this promoter in wild-type and fur mutants of E. coli, by measuring beta-galactosidase activity, indicated that E. coli Fur can regulate the Pseudomonas promoter in response to iron starvation. Gel retardation assays suggested that purified Fur protein could interact with the SP1 promoter upstream of the transcriptional start. DNase I footprinting analysis established that Fur protected a primary 58-bp region (-50 to -106 bp). These protein/DNA interactions correlate with the observed in vivo regulation of the SP1 promoter in E. coli and indicate that Fur can functionally regulate a Pseudomonas iron-regulated promoter.

Plant protection by the recombinant, root-colonizing Pseudomonas fluorescens F113rifPCB strain expressing arsenic resistance: improving rhizoremediation.

AIMS: The present study was designed to evaluate the stable insertion and expression of an arsenic resistance operon in the rhizosphere competent, PCB degrading strain Pseudomonas fluorescens F113rifPCB (F113rifPCB) and to investigate its ability to protect plants from arsenic. METHODS AND RESULTS: Introduction of the clone pUM3 (arsRDABC) into F113rifPCB was carried out by triparental conjugation. The resultant arsenic resistant strain was screened through a number of phenotypic tests including ability to grow on biphenyl, its rhizosphere competence and plant protection potential. CONCLUSIONS: Insertion and expression of arsenic resistant operon arsRDABC (from plasmid R773) into F113rifPCB strain has allowed this strain to grow, colonize the root and degrade biphenyl (100 mmol l(-1)) in the presence of sodium arsenate concentrations of up to 11.5 mmol l(-1). The strain retains its ability to colonize the rhizosphere of plants and appears to provide seed germination protection to arsenic which is not seen by the wild type. SIGNIFICANCE AND IMPACT OF THE STUDY: Owing to the significantly improved growth characteristics of both this rhizobacterium and plant species, the use of F113rifPCB-ars endowed with arsenic resistance capabilities may be a promising strategy to remediate mixed organic metal-contaminated sites. These types of strain could be used in the inoculation of metal accumulation plants for phytoremediation.

Transcriptional regulation of the iron-responsive sigma factor gene pbrA.

In response to the intracellular iron concentration Pseudomonas fluorescens M114 coordinately regulates the production of pseudobactin M114, its cognate receptor PbuA, and a casein protease. Transcriptional initiation of this coordinate iron-stress response requires the sigma factor PbrA. PbrA is a member of the ECF (Extracytoplasmic function) subgroup of the sigma 70 family of eubacterial RNA polymerase sigma factors. Regulatory studies of the pbrA gene utilising promoter-lacZ transcriptional fusions demonstrate that expression of pbrA dictates the cellular response to iron. pbrA is transcribed in all phases of iron-limited growth but maximally at late-logarithmic to stationary phase. pbrA expression is independent of autoregulatory control but is strictly repressed in iron-rich conditions in a Fur-dependent fashion. Constitutive expression of pbrA from an inducible tac promoter permits the induction of PbrA-dependent transcription and pseudobactin M114 biosynthesis in high-iron conditions. A PbrA consensus sequences was derived from significant DNA sequence homologies observed within the "-25 bp" and "-16 bp" regions conserved among all PbrA-dependent promoters. The predicted PbrA target promoter consensus is homologous for the promoter recognition sites for other environmentally responsive ECF sigma factors.

Isolation of a gene (pbsC) required for siderophore biosynthesis in fluorescent Pseudomonas sp. strain M114.

An iron-regulated gene, pbsC, required for siderophore production in fluorescent Pseudomonas sp. strain M114 has been identified. A kanamycin-resistance cassette was inserted at specific restriction sites within a 7 kb genomic fragment of M114 DNA and by marker exchange two siderophore-negative mutants, designated M1 and M2, were isolated. The nucleotide sequence of approximately 4 kb of the region flanking the insertion sites was determined and a large open reading frame (ORF) extending for 2409 bp was identified. This gene was designated pbsC (pseudobactin synthesis C) and its putative protein product termed PbsC. PbsC was found to be homologous to a family of enzymes involved in the biosynthesis of secondary metabolites, including EntF of Escherichia coli. These enzymes are believed to act via ATP-dependent binding of AMP to their substrate. Several areas of high sequence homology between these proteins and PbsC were observed, including a conserved AMP-binding domain. The expression of pbsC is iron-regulated as revealed when a DNA fragment containing the upstream region was cloned in a promoter probe vector and conjugated into the wild-type strain, M114. The nucleotide sequence upstream of the putative translational start site contains a region homologous to previously defined -16 to -25 sequences of iron-regulated genes but did not contain an iron-box consensus sequence. It was noted that inactivation of the pbsC gene also affected other iron-regulated phenotypes of Pseudomonas M114.

Mutational Disruption of the Biosynthesis Genes Coding for the Antifungal Metabolite 2,4-Diacetylphloroglucinol Does Not Influence the Ecological Fitness of Pseudomonas fluorescens F113 in the Rhizosphere of Sugarbeets.

The ability of Pseudomonas fluorescens F113 to produce the antibiotic 2,4-diacetylphloroglucinol (DAPG) is a key factor in the biocontrol of the phytopathogenic fungus Pythium ultimum by this strain. In this study, a DAPG-producing strain (rifampin-resistant mutant F113Rif) was compared with a nearly isogenic DAPG-negative biosynthesis mutant (Tn5::lacZY derivative F113G22) in terms of the ability to colonize and persist in the rhizosphere of sugarbeets in soil microcosms during 10 plant growth-harvest cycles totaling 270 days. Both strains persisted similarly in the rhizosphere for 27 days, regardless of whether they had been inoculated singly onto seeds or coinoculated in a 1:1 ratio. In order to simulate harvest and resowing, the roots were removed from the soil and the pots were resown with uninoculated sugarbeet seeds for nine successive 27-day growth-harvest cycles. Strains F113Rif and F113G22 performed similarly with respect to colonizing the rhizosphere of sugarbeet, even after nine cycles without reinoculation. The introduced strains had a transient effect on the size of the total culturable aerobic bacterial population. The results indicate that under these experimental conditions, the inability to produce DAPG did not reduce the ecological fitness of strain F113 in the rhizosphere of sugarbeets.

Cloning of Rhizobium leguminosarum genes for competitive nodulation blocking on peas.

One type of competitive interaction among rhizobia is that between nonnodulating and nodulating strains of Rhizobium leguminosarum on primitive pea genotypes. Pisum sativum cv. Afghanistan nodulates effectively with R. leguminosarum TOM, and this can be blocked in mixed inoculations by R. leguminosarum PF2, which does not nodulate this cultivar. We termed this PF2 phenotype Cnb+, for competitive nodulation blocking. Strain PF2 contains three large plasmids including a 250-kilobase-pair symbiotic (Sym) plasmid. Transfer of this plasmid, pSymPF2, to nonblocking rhizobia conferred the Cnb+ phenotype on recipients in mixed inoculations on cultivar Afghanistan with TOM. A library of the PF2 genome constructed in the vector pMMB33 was used to isolate two cosmid clones which hybridize to pSymPF2. These cosmids, pDD50 and pDD58, overlapped to the extent of 23 kilobase pairs and conferred a Cnb+ phenotype on recipient Cnb- rhizobia, as did pSD1, a subclone from the common region.

Construction of a rhizosphere pseudomonad with potential to degrade polychlorinated biphenyls and detection of bph gene expression in the rhizosphere.

The genetically engineered transposon TnPCB, contains genes (bph) encoding the biphenyl degradative pathway. TnPCB was stably inserted into the chromosome of two different rhizosphere pseudomonads. One genetically modified strain, Pseudomonas fluorescens F113pcb, was characterized in detail and found to be unaltered in important parameters such as growth rate and production of secondary metabolites. The expression of the heterologous bph genes in F113pcb was confirmed by the ability of the genetically modified microorganism to utilize biphenyl as a sole carbon source. The introduced trait remained stable in laboratory experiments, and no bph-negative isolates were found after extensive subculture in nonselective media. The bph trait was also stable in nonselective rhizosphere microcosms. Rhizosphere competence of the modified F113pcb was assessed in colonization experiments in nonsterile soil microcosms on sugar beet seedling roots. F113pcb was able to colonize as efficiently as a marked wild-type strain, and no decrease in competitiveness was observed. In situ expression of the bph genes in F113pcb was found when F113pcb bearing a bph'lacZ reporter fusion was inoculated onto sugar beet seeds. This indicates that the bph gene products may also be present under in situ conditions. These experiments demonstrated that rhizosphere-adapted microbes can be genetically manipulated to metabolize novel compounds without affecting their ecological competence. Expression of the introduced genes can be detected in the rhizosphere, indicating considerable potential for the manipulation of the rhizosphere as a self-sustaining biofilm for the bioremediation of pollutants in soil. Rhizosphere bacteria such as fluorescent Pseudomonas spp. are ecologically adapted to colonize and compete in the rhizosphere environment. Expanding the metabolic functions of such pseudomonads to degrade pollutants may prove to be a useful strategy for bioremediation.

Evidence for signaling between the phytopathogenic fungus Pythium ultimum and Pseudomonas fluorescens F113: P. ultimum represses the expression of genes in P. fluorescens F113, resulting in altered ecological fitness.

There is increasing evidence that communication between members of the same species, as well as members of different species, is important for the survival of microorganisms in diverse ecological niches, such as the rhizosphere. To investigate whether the phytopathogen Pythium ultimum could alter gene expression in the biocontrol strain Pseudomonas fluorescens F113, which protects the roots of sugar beet from the fungus, a screening system was developed to detect differential expression of bacterial genes in the presence of P. ultimum. The transposon Tn5, containing a promoterless lacZ reporter gene, was used to generate a library of transcriptional gene fusions in P. fluorescens F113. By this screening procedure, five P. fluorescens F113 gene clusters were identified and shown to be repressed in the presence of P. ultimum. The ecological fitness of three of the five reporter mutants in the rhizosphere of seed-inoculated sugar beet was lower than that of the wild type. Furthermore, all five mutants were impaired in their ability to subsequently colonize the rhizosphere of uninoculated sugar beet sown repeatedly in the same soil. With the exception of reporter mutant SF10, which was impaired in nitrogen metabolism, the reporter mutants had growth requirements and biocontrol abilities similar to those of the wild type. This is the first reported case of a fungus repressing the expressing of bacterial genes.

Role of 2,4-Diacetylphloroglucinol in the Interactions of the Biocontrol Pseudomonad Strain F113 with the Potato Cyst Nematode Globodera rostochiensis.

The potato cyst nematode Globodera rostochiensis is an important pest of potato (Solanum tuberosum). Pseudomonas fluorescens F113, which produces 2,4-diacetylphloroglucinol (DAPG), was investigated as a potential biocontrol agent against G. rostochiensis. Exposure of nematode cysts to the pseudomonad, under in vitro conditions or in soil microcosms, almost doubled the ability of the eggs to hatch. The percentage of mobile juveniles was reduced threefold following their incubation in the presence of the pseudomonad, both in vitro and in soil. Results obtained with a transposon-induced DAPG-negative biosynthetic mutant of F113 and its complemented derivative with restored DAPG synthesis showed that the ability of strain F113 to produce DAPG was responsible for the increase in hatch ability and the reduction in juvenile mobility. Similar effects on egg hatch ability and juvenile mobility of G. rostochiensis were obtained in vitro by incubating nematode cysts and juveniles, respectively, in the presence of synthetic DAPG. DAPG-producing P. fluorescens F113 is proposed as a potential biocontrol inoculant for the protection of potato crops against the potato cyst nematode.

Iron-responsive gene expression in Pseudomonas fluorescens M114: cloning and characterization of a transcription-activating factor, PbrA.

In response to iron limitation. Pseudomonas fluorescens M114 induces a number of genes including an iron-scavenging siderophore termed pseudobactin M114, its cognate receptor, PbuA, and a casein protease. A Tn5lacZ-induced mutant (M114FA1) was isolated that exhibits a pleiotropic phenotype and lacks the ability to express these iron-regulated genes. A cosmid clone was identified which complements this mutation. This clone is capable of activating a number of iron-regulated promoter fusion constructs from P. fluorescens M114 and Pseudomonas putida WCS358 and can also promote expression of these fusions in Escherichia coli. A series of insertion mutants was constructed by homologous recombination which were unable to transcribe the promoter fusions. DNA sequence analysis of the complementing region identified one open reading frame (ORF) termed pbrA (pseudobactin regulation activation) and the deduced amino acid sequence shows domains with significant homology to a number of ECF (extracytoplasmic function) transcriptional regulators of the sigma 70 sigma factor family, including fecl required for expression of the ferric dicitrate outer-membrane receptor protein of E. coli. Sequences upstream of the pbrA gene suggest that transcription of pbrA may also be iron regulated.

Greenhouse and Field Evaluations of an Autoselective System Based on an Essential Thymidylate Synthase Gene for Improved Maintenance of Plasmid Vectors in Modified Rhizobium meliloti.

The stability of the thy autoselective system, based on an essential thymidylate synthase gene, for enhanced maintenance of plasmid vectors in Rhizobium meliloti was evaluated in the greenhouse and with field-grown alfalfa. The thy autoselective system consists of a free-replicating, broad-host-range plasmid vector containing a copy of the thyA gene from Lactococcus lactis subsp. lactis and a spontaneous mutant of R. meliloti deficient in thymidylate synthase (Thy(sup-)). Under greenhouse conditions, Thy(sup-) rhizobia did not persist in rooting solution alone unless supplemented with thymidine but survived in the presence of the host plant. Nodules formed on alfalfa plants grown in thymidine-free rooting solution and inoculated with Thy(sup-) rhizobia contained only Thy(sup+) revertants. In soil, Thy(sup-) rhizobia were compromised and failed to nodulate alfalfa. Thy(sup-) mutants containing a thy plasmid survived in the rhizosphere and nodulated alfalfa like the wild-type strain. The thy autoselective system was tested in the field with Thy(sup-) strain Rm24T and pPR602, a thy plasmid vector devoid of antibiotic resistance genes and marked with constitutively expressed lacZY. At 80 days after sowing, most rhizobia isolated from the nodules of field-grown alfalfa inoculated with Rm42T(pPR602) contained pPR602. The thy autoselective system proved useful to ensure maintenance of the plasmid vector under greenhouse and field conditions in R. meliloti.

Improved delivery of biocontrol Pseudomonas and their antifungal metabolites using alginate polymers.

Alginate polymer was evaluated as a carrier for seed inoculation with a genetically modified strain Pseudomonas fluorescens F113LacZY, which protects sugar-beet against Pythium-mediated damping-off. F113LacZY survived in alginate beads at 5 log10 CFU/bead or higher counts for 8 weeks of storage, regardless of the conditions of incubation. In plant inoculation experiments, colonisation of the growing area of the root by F113LacZY, derived from alginate beads placed in the soil next to the seed or from an alginate coating around the seeds, was improved compared with application of just free cells of the strain. F113LacZY trapped in alginate beads was an effective producer of antifungal phloroglucinols as indicated by direct HPLC quantification of phloroglucinols and in vitro inhibition of both the indicator bacterium Bacillus subtilis A1 and the pathogenic fungus Pythium ultimum. Alginate polymer represents a promising carrier for the delivery of biocontrol inoculants for root colonisation and production of antifungal metabolites.

Multiple host-specificity loci of the broad host-range Rhizobium sp. NGR234 selected using the widely compatible legume Vigna unguiculata.

Specificity in legume-Rhizobium symbiosis depends on plant and rhizobial genes. As our objective was to study broad host-range determinants of rhizobia, we sought a legume and a Rhizobium with the lowest possible specificity. By inoculating 12 different legumes with a heterogenous collection of 35 fast-growing rhizobia, we found Rhizobium sp. NGR234 to be the Rhizobium and Vigna unguiculata to be the plant with the lowest specificities. Transfer of cloned fragments of the Sym-plasmid pNGR234a into heterologous rhizobia, screening for extension of host-range of the transconjugants to include V. unguiculata, and restriction mapping of the Hsn- and overlapping clones, proved that there were at least three distinct Hsn-regions (HsnI, II, and III) on pNGR234a. HsnI is located next to nodD, HsnII is linked to nifKDH and HsnIII to nodC. In addition to nodulation of Vigna, HsnI conferred upon the transconjugants the ability to nodulate Glycine max, Macroptilium atropurpureum and Psophocarpus tetragonolobus. All three Hsn-regions, when transferred to the appropriate recipients, induced root-hair-curling on M. atropurpureum. Hsn-region III was able to complement a mutation in the host-range gene nodH of R. meliloti strain 2011. Homology to "nod-box"-sequences could be shown only for the sub-clones containing HsnII and HsnIII, thus suggesting different regulation mechanisms for HsnI and HsnII/III.