David N. Dowling

Metabolites of Pseudomonas involved in the biocontrol of plant disease

Abstract There is increasing commercial and environmental interest in the use of microbe-based agents as alternatives to, or in combination with, chemicals for controlling the spread and severity of a range of crop diseases. The identification of specific microbial metabolites that are able to control certain plant diseases has led to the development of strategies for improving the performance and predictability of the microbial strains that produce these metabolites for application in the agricultural industry. This article focuses on antimicrobial metabolites produced by fluorescent pseudomonads, discusses their role in suppressing fungal diseases of important crops and reviews the prospects of genetically manipulating the producer organisms to improve the efficacy of these biocontrol agents.

Biological control of Pythium ultimum by Stenotrophomonas maltophilia W81 is mediated by an extracellular proteolytic activity

Stenotrophomonas maltophilia strain W81, isolated from the rhizosphere of field-grown sugar beet, produced the extracellular enzymes chitinase and protease and inhibited the growth of the phytopathogenic fungus Pythium ultimum in vitro. The role of these lytic enzymes in the interaction between W81 and P. ultimum was investigated using Tn5 insertion mutants of W81 incapable of producing extracellular protease (W81M1), extracellular chitinase (W81M2) or the two enzymes (W81A1). Lytic enzyme activity was restored in W81A1 following introduction of a 15 kb cosmid-borne fragment of W81 genomic DNA. Incubation of P. ultimum in the presence of commercial purified protease or cell-free supernatants from cultures of wild-type W81, the chitinase-negative mutant W81M2 or the complemented derivative W81A1 (pCU800) resulted in hyphal lysis and loss of subsequent fungal growth ability once re-inoculated onto fresh plates. In contrast, commercial purified chitinase or cell-free supernatants from cultures of the protease-negative mutant WS1M1 or the chitinase- and protease-negative mutant W81A1 had no effect on integrity of the essentially chitin-free Pythium mycelium, and did not prevent subsequent growth of the fungus. In soil microcosms containing soil naturally infested by Pythium spp., strains W81, W81M2 and W81A1(pCU800) reduced the ability of Pythium spp. to colonize the seeds of sugar beet and improved plant emergence compared with the untreated control, whereas W81A1 and W21M1 failed to protect sugar beet from damping-off. Wild-type W81 and its mutant derivatives colonized the rhizosphere of sugar beet to similar extents, it was concluded that the ability of S. maltophilia W81 to protect sugar beet from Pythium -mediated damping-off was due to the production of an extracellular protease.

The Behavior of Bacteria Designed for Biodegradation

Mineralization of organic molecules by microbes is essential for the carbon cycle to operate. The massive mobilization of compounds stored in natural resources, or the introduction of xenobiotics into the biosphere, leads to unidirectional fluxes, which result in the persistance of a number of chemicals in the biosphere, and thus constitute a source of pollution. Molecular biology offers the tools to optimize the biodegradative capacities of microorganisms, accelerate the evolution of “new” activities, and construct totally “new” pathways through the assemblage of catabolic segments from different microbes. Although the number of genetically engineered microbes (GEMs) for potential use in biodegradation is not large, these recombinant microbes function in microcosms according to their design. The survival and fate of recombinant microbes in different ecological niches under laboratory conditions is similar to what has been observed for the unmodified parental strains. rDNA, both on plasmids and on the host chromosome, is usually stably inherited by GEMs. The potential lateral transfer of rDNA from the GEMs to other microbes is significantly diminished, though not totally inhibited, when rDNA is incorporated on the host chromosome. The behavior and fate of GEMs can be predicted more accurately through the coupling of regulatory circuits that control the expression of catabolic pathways to killing genes, so that the GEMs survive in polluted environments, but die when the target chemical is eliminated.

Improved delivery of biocontrolPseudomonas and their antifungal metabolites using alginate polymers

Alginate polymer was evaluated as a carrier for seed inoculation with a genetically modified strainPseudomonas fluorescens F113LacZY, which protects sugar-beet againstPythium-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 bacteriumBacillus subtilis A1 and the pathogenic fungusPythium ultimum. Alginate polymer represents a promising carrier for the delivery of biocontrol inoculants for root colonisation and production of antifungal metabolites.

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 (pseuciobactin regulation activation) and the deduced amino acid sequence shows domains with significant homology to a number of ECF (extracytoplasmic function) transcriptional regulators of the σ70 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.

Transcriptional regulation of the iron-responsive sigma factor genepbrA

In response to the intracellular iron concentrationPseudomonas 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 (Extracytoplasmicfunction) subgroup of the σ70 family of eubacterial RNA polymerase sigma factors. Regulatory studies of thepbrA gene utilising promoter-lacZ transcriptional fusions demonstrate that expression ofpbrA 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 ofpbrA from an inducibletac promoter permits the induction of PbrA-dependent transcription and pseudobactin M114 biosynthesis in high-iron conditions. A PbrA consensus sequence 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 by 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.

A chromosomal genetic map of Rhizobium sp. NGR234 generated with Tn5-Mob

SummaryRhizobium sp. NGR234 in a fast-growing Rhizobium strain with a broad host range. The location and role of chromosomal genes involved in cellular metabolism or in the legume symbioses is unknown. We isolated a series of auxotrophic and antibiotic resistant mutants of NGR234 and utilized a chromosome mobilization system based on Tn5-Mob and pJB3JI; Tn5-Mob donor strains behaved like Hfr strains, transferring the chromosome polarly at high frequency from a fixed point of insertion. The use of four different strains with Tn5-Mob located at different nutritional loci in crosses with double auxotrophic recipients, allowed us to build up a circular linkage map of NGR234 based on relative recombination frequencies. Also, symbiotically important genes identified by site-directed mutagenesis, such as hemA and ntrA, could be located and mapped on the chromosome.

A versatile most probable number system to quantify lacZY‐marked pseudomonads present at low cell numbers in the rhizosphere

Quantitation of introduced genetically modified micro‐organisms (GMMs) in the rhizosphere is a key issue when their release in the environment is planned. An improved most probable number (MPN) system, using a titration plate for incubation of rhizosphere extracts and two microcomputer programmes made recently available, MPNES (Woomer et al. 1990) and MPN 2.80 (Klee 1993) to generate the MPNs, is described. The MPN system was compared with colony counts to assess colonization of sugarbeet roots by an introduced lacZY‐modified rhizosphere pseudomonad. The MPNs displayed wider confidence intervals compared with drop‐plate counts but allowed the quantitation limit to be lowered to 2.30 log10 cfu per root system. The MPN system proved useful to quantify GMMs present at low cell numbers in the rhizosphere of sugarbeet.