Pseudomonas fluorescens are common soil bacteria that can improve plant health through nutrient cycling, pathogen antagonism and induction of plant defenses. The genome sequences of strains SBW25 and ...Pf0-1 were determined and compared to each other and with P. fluorescens Pf-5. A functional genomic in vivo expression technology (IVET) screen provided insight into genes used by P. fluorescens in its natural environment and an improved understanding of the ecological significance of diversity within this species.
Comparisons of three P. fluorescens genomes (SBW25, Pf0-1, Pf-5) revealed considerable divergence: 61% of genes are shared, the majority located near the replication origin. Phylogenetic and average amino acid identity analyses showed a low overall relationship. A functional screen of SBW25 defined 125 plant-induced genes including a range of functions specific to the plant environment. Orthologues of 83 of these exist in Pf0-1 and Pf-5, with 73 shared by both strains. The P. fluorescens genomes carry numerous complex repetitive DNA sequences, some resembling Miniature Inverted-repeat Transposable Elements (MITEs). In SBW25, repeat density and distribution revealed 'repeat deserts' lacking repeats, covering approximately 40% of the genome.
P. fluorescens genomes are highly diverse. Strain-specific regions around the replication terminus suggest genome compartmentalization. The genomic heterogeneity among the three strains is reminiscent of a species complex rather than a single species. That 42% of plant-inducible genes were not shared by all strains reinforces this conclusion and shows that ecological success requires specialized and core functions. The diversity also indicates the significant size of genetic information within the Pseudomonas pan genome.
Laccases are blue multicopper oxidases that catalyse the four-electron reduction of O2 to water coupled with the oxidation of small organic substrates. Secreted basidiomycete white-rot fungal ...laccases orchestrate this with high thermodynamic efficiency, making these enzymes excellent candidates for exploitation as industrial oxidants. However, these fungi are less tractable genetically than the ascomycetes, which predominantly produce lower-potential laccases. We address the state-of-play regarding expression of high reduction potential laccases in heterologous hosts, and issues regarding enzyme glycosylation status. We describe the synergistic role of structural biology, particularly in unmasking structure–function relationships following genetic modification and their collective impact on laccase yields. Such recent research draws closer the prospect of industrial quantities of designer, fit-for-purpose laccases.
Pseudomonas fluorescens F113 is a plant growth-promoting rhizobacterium (PGPR) that has biocontrol activity against fungal plant pathogens and is a model for rhizosphere colonization. Here, we ...present its complete genome sequence, which shows that besides a core genome very similar to those of other strains sequenced within this species, F113 possesses a wide array of genes encoding specialized functions for thriving in the rhizosphere and interacting with eukaryotic organisms.
The genome of the plant-colonizing bacterium Pseudomonas fluorescens SBW25 harbors a subset of genes that are expressed specifically on plant surfaces. The function of these genes is central to the ...ecological success of SBW25, but their study poses significant challenges because no phenotype is discernable in vitro. Here, we describe a genetic strategy with general utility that combines suppressor analysis with IVET (SPyVET) and provides a means of identifying regulators of niche-specific genes. Central to this strategy are strains carrying operon fusions between plant environment-induced loci (EIL) and promoterless 'dapB. These strains are prototrophic in the plant environment but auxotrophic on laboratory minimal medium. Regulatory elements were identified by transposon mutagenesis and selection for prototrophs on minimal medium. Approximately 10⁶ mutants were screened for each of 27 strains carrying 'dapB fusions to plant EIL and the insertion point for the transposon determined in approximately 2,000 putative regulator mutants. Regulators were functionally characterized and used to provide insight into EIL phenotypes. For one strain carrying a fusion to the cellulose-encoding wss operon, five different regulators were identified including a diguanylate cyclase, the flagella activator, FleQ, and alginate activator, AmrZ (AlgZ). Further rounds of suppressor analysis, possible by virtue of the SPyVET strategy, revealed an additional two regulators including the activator AlgR, and allowed the regulatory connections to be determined.
Summary
Erwinia herbicola
strain Eh1087 produces the broad‐spectrum phenazine antibiotic
D
‐alanylgriseoluteic acid (AGA). In this report, a cluster of 16
ehp
(
E
rwinia
h
erbicola
p
henazine) ...plasmid genes required for the production of AGA by Eh1087 is described. The extent of the gene cluster was revealed by the isolation of 82 different Eh1087 AGA
−
mutants, all found to possess single mini‐Tn
5lacZ2
insertions within a 14 kbp DNA region. Additional transposon insertions that did not affect antibiotic production by Eh1087 were created to define the boundaries of the gene cluster. The size and location of genes between these boundaries were derived from a combination of DNA sequence analyses, minicell protein analyses and the correlation between mutation position and the production of coloured AGA intermediates by many
ehp
mutants. Precursor‐feeding and complementation experiments resulted in 15
ehp
genes being assigned to one of four functional groups according to their role in the synthesis of AGA. Group 1 is required for the synthesis of the phenazine nucleus in the form of antibiotic precursor one (AP1, phenazine‐1,6‐dicarboxylic acid). Group 2 is responsible for conversion of AP1 to AP2, which is subsequently modified to AP3 (griseoluteic acid) and exported by the group 3 gene products. Group 4 catalyses the addition of
D
‐alanine to AP3 to create AGA, independently of groups 1, 2 and 3. A gene that is divergently transcribed from the 15 AGA synthesis
ehp genes confers resistance to AGA.
Abstract d -Alanylgriseoluteic acid (AGA) is a potent antimicrobial phenazine compound produced by Pantoea agglomerans ( Erwinia herbicola ) Eh1087. Susceptibility tests against a range of microbes ...indicated that AGA had a broad spectrum of antimicrobial activity and was particularly active against Gram-positive pathogens. Comparison of the in vitro efficacy of AGA with eight other antibiotics against 119 clinical isolates of Streptococcus pneumoniae demonstrated that all were inhibited by low concentrations of AGA (minimal inhibitory concentration range ≤0.06–0.75 μg/mL), regardless of their susceptibility to other antimicrobials. Investigations into the mode of action of AGA revealed that it induced the SOS response in Escherichia coli and slightly increased the frequency of GC–AT transition mutations. In cell-free assays, both AGA and griseoluteic acid reduced cytochrome c in the presence of a redox recycler (ferredoxin–NADP+ reductase), but AGA was six to seven times more readily reduced than griseoluteic acid in the absence of the reductase. The potency and broad spectrum of AGA activity suggest that AGA may warrant further investigation.
Summary
Stigma colonization by Erwinia amylovora is the crucial first step in the development of most fire blight infections in apple and pear trees. Suppression at this point of the disease process ...by antagonists of E. amylovora, such as Pantoea agglomerans (Erwinia herbicola) strain Eh1087, is a rational approach to control fire blight. We tested the hypothesis that the ability of E. amylovora to compete with Eh1087 for colonization of a stigma is reduced by the potential for Eh1087 to produce the phenazine antibiotic, d‐alanylgriseoluteic acid (AGA). In competition experiments on the stigmas of apple flowers, E. amylovora was significantly less successful against Eh1087 (AGA+) than against EhΔAGA (AGA‐). Further experiments to test the importance of pre‐emptive colonization of the stigma by either the pathogen or the antagonist suggested that AGA production significantly enhanced the competitiveness of Eh1087 when it was applied at the same time or 24 h before the pathogen. We also found that pre‐emptive stigma colonization by either the pathogen or the antagonist resulted in a population that was resilient to subsequent invasion by a second species suggesting that niche exclusion has a dominant influence on the dynamics of bacterial populations on stigmas.
The growth advantage in stationary phase (GASP) phenotype was shown to be present in two mutants lacking the antifungal phenotype (Af⁻ mutants) of Pseudomonas aureofaciens PA147-2. Complementation ...demonstrated a correlation between GASP and the antifungal defect in one strain but not in the second. Sequence analysis revealed the Af⁻ GASP strain had a mutation in a gene (finR) encoding a LysR-type regulator. Antifungal-minus mutants arose in starved cultures, and those aged cultures had increased fitness. Taken together, the results show that there are at least two paths to the GASP phenotype in P. aureofaciens, one of which results in a concomitant loss of the antifungal phenotype.
The rpoS gene of Serratia entomophila BC4B was cloned and used to create rpoS-mutant strain BC4BRS. Larvae of the New Zealand grass grub Costelytra zealandica infected with BC4BRS became amber ...colored but continued to feed, albeit to a lesser extent than infected larvae. Subsequently, we found that expression of the antifeeding gene anfA1 in trans was substantially reduced in BC4BRS relative to that in the parental strain BC4B. Our data show that a functional rpoS gene is vital for full expression of anfA1 and for development of the antifeeding component of amber disease.
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