Lipopeptide Production in Pseudomonas sp. Strain DSS73 Is Regulated by Components of Sugar Beet Seed Exudate via the Gac Two-Component Regulatory System

Pseudomonas sp. strain DSS73 isolated from the sugar beet rhizosphere produces the cyclic lipopeptide amphisin, which inhibits the growth of plant-pathogenic fungi. By Tn5::luxAB mutagenesis, we obtained two nonproducing mutant strains, DSS73-15C2 and DSS73-12H8. The gene interrupted by the transposon in strain DSS73-15C2 (amsY) encoded a protein with homology to peptide synthetases that was designated amphisin synthetase. DSS73-12H8 carried the transposon in a regulatory gene encoding a protein with homology to the sensor kinase GacS. Growth of strain DSS73-15C2 (amsY) was impaired during the transition to stationary phase in a minimal medium amended with an exudate of sugar beet seeds. This growth phenotype could be complemented by purified amphisin. Seed exudate further induced expression of bioluminescence from the amsY::luxAB reporter during the transition to stationary phase. This agreed with an increase in amphisin production by the DSS73 wild-type strain during early stationary phase. Amphisin synthesis in DSS73 was strictly dependent on GacS, and even induction by seed exudate depended on a functional gacS locus. Hence, a signal triggering the GacS/GacA two-component system appeared to be present in the seed exudate.     

Genes involved in cyclic lipopeptide production are important for seed and straw colonization by Pseudomonas sp. strain DSS73

Survival in natural bulk soil and colonization of sugar beet seeds and barley straw residues were determined for Pseudomonas sp. strain DSS73 and Tn5 mutants in amsY (encoding a peptide synthetase involved in production of the cyclic lipopeptide amphisin) and gacS (encoding the sensory kinase of the two-component GacA/GacS regulatory system). No differences in survival or growth in response to carbon amendment (citrate) were observed in bulk soil. However, both mutants were impaired in their colonization of sugar beet seeds and barley straw residues by an inoculum established in the bulk soil. The two mutants had comparable colonization phenotypes, suggesting that amphisin production is more important for colonization than other gacS-controlled traits.     

Genes Involved in Cyclic Lipopeptide Production Are Important for Seed and Straw Colonization by Pseudomonas sp. Strain DSS73

Survival in natural bulk soil and colonization of sugar beet seeds and barley straw residues were determined for Pseudomonas sp. strain DSS73 and Tn5 mutants in amsY (encoding a peptide synthetase involved in production of the cyclic lipopeptide amphisin) and gacS (encoding the sensory kinase of the two-component GacA/GacS regulatory system). No differences in survival or growth in response to carbon amendment (citrate) were observed in bulk soil. However, both mutants were impaired in their colonization of sugar beet seeds and barley straw residues by an inoculum established in the bulk soil. The two mutants had comparable colonization phenotypes, suggesting that amphisin production is more important for colonization than other gacS-controlled traits.     

Production of cyclic lipopeptides by Pseudomonas fluorescens strains in bulk soil and in the sugar beet rhizosphere

The production of cyclic lipopeptides (CLPs) with antifungal and biosurfactant properties by Pseudomonas fluorescens strains was investigated in bulk soil and in the sugar beet rhizosphere. Purified CLPs (viscosinamide, tensin, and amphisin) were first shown to remain highly stable and extractable (90%) when applied (ca. 5 microg g(-1)) to sterile soil, whereas all three compounds were degraded over 1 to 3 weeks in nonsterile soil. When a whole-cell inoculum of P. fluorescens strain DR54 containing a cell-bound pool of viscosinamide was added to the nonsterile soil, declining CLP concentrations were observed over a week. By comparison, addition of the strains 96.578 and DSS73 without cell-bound CLP pools did not result in detectable tensin or amphisin in the soil. In contrast, when sugar beet seeds were coated with the CLP-producing strains and subsequently germinated in nonsterile soil, strain DR54 maintained a high and constant viscosinamide level in the young rhizosphere for approximately 2 days while strains 96.578 and DSS73 exhibited significant production (net accumulation) of tensin or amphisin, reaching a maximum level after 2 days. All three CLPs remained detectable for several days in the rhizosphere. Subsequent tests of five other CLP-producing P. fluorescens strains also demonstrated significant production in the young rhizosphere. The results thus provide evidence that production of different CLPs is a common trait among many P. fluorescens strains in the soil environment, and further, that the production is taking place only in specific habitats like the rhizosphere of germinating sugar beet seeds rather than in the bulk soil.     

Production of Cyclic Lipopeptides by Pseudomonas fluorescens Strains in Bulk Soil and in the Sugar Beet Rhizosphere

The production of cyclic lipopeptides (CLPs) with antifungal and biosurfactant properties by Pseudomonas fluorescens strains was investigated in bulk soil and in the sugar beet rhizosphere. Purified CLPs (viscosinamide, tensin, and amphisin) were first shown to remain highly stable and extractable (90%) when applied (ca. 5 μg g⁻¹) to sterile soil, whereas all three compounds were degraded over 1 to 3 weeks in nonsterile soil. When a whole-cell inoculum of P. fluorescens strain DR54 containing a cell-bound pool of viscosinamide was added to the nonsterile soil, declining CLP concentrations were observed over a week. By comparison, addition of the strains 96.578 and DSS73 without cell-bound CLP pools did not result in detectable tensin or amphisin in the soil. In contrast, when sugar beet seeds were coated with the CLP-producing strains and subsequently germinated in nonsterile soil, strain DR54 maintained a high and constant viscosinamide level in the young rhizosphere for ~2 days while strains 96.578 and DSS73 exhibited significant production (net accumulation) of tensin or amphisin, reaching a maximum level after 2 days. All three CLPs remained detectable for several days in the rhizosphere. Subsequent tests of five other CLP-producing P. fluorescens strains also demonstrated significant production in the young rhizosphere. The results thus provide evidence that production of different CLPs is a common trait among many P. fluorescens strains in the soil environment, and further, that the production is taking place only in specific habitats like the rhizosphere of germinating sugar beet seeds rather than in the bulk soil.     

Construction of mini-Tn10luxABcam/Ptac-ATS and its use for developing a bacteriophage that transduces bioluminescence to Escherichia coli O157:H7

Mini-Tn10luxABcam/Ptac-ATS was constructed in order to develop a luciferase-transducing bacteriophage for detecting Escherichia coli O157:H7. The transposon was designed to deliver a 3.6-kb insertion that confers n-decanal-dependent bioluminescence and resistance to chloramphenicol and was constructed using mini-Tn10cam/Ptac-ATS in the plasmid pNK2884 and luxAB from Vibrio harveyi. PhiV10, a temperate bacteriophage infecting common phage types of Escherichia coli O157:H7, was mutagenized as a prophage in E. coli O157:H7 strain R508. PhiV10::luxABcamA1-23 was rescued from the strain by propagating it on a strain lacking the bacteriophage and the vector containing the transposon. The bacteriophage transduced n-decanal-dependent bioluminescence to E. coli O157:H7 strain R508 that was measurable approximately 1 h post infection.     

Structure, production characteristics and fungal antagonism of tensin - a new antifungal cyclic lipopeptide from Pseudomonas fluorescens strain 96.578

AIM: To study the antagonistic activity by Pseudomonas fluorescens strain 96.578 on the plant pathogenic fungus Rhizoctonia solani. METHODS AND RESULTS: Strain 96.578 produced a new cyclic lipopeptide, tensin. High tensin production per cell was detected in liquid media with glucose, mannitol or glutamate as growth substrate while fructose, sucrose and asparagine supported low production. Tensin production was nearly constant in media with different initial C levels, while low initial N contents reduced production. When applied to sugar beet seeds, strain 96.578 produced tensin during seed germination. When challenged with strain 96.578 or purified tensin, Rhizoctonia solani reduced radial mycelium extension but increased branching and rosette formation. CONCLUSION: The antagonistic activity of strain 96.578 towards Rhizoctonia solani was caused by tensin. SIGNIFICANCE AND IMPACT OF THE STUDY: When coated onto sugar beet seeds, tensin production by strain 96.578 could be of significant importance for inhibition of mycelial growth and seed infection by Rhizoctonia solani.     

Identification of cold shock gene loci in Sinorhizobium meliloti by using a luxAB reporter transposon

Using a luxAB reporter transposon, seven mutants of Sinorhizobium meliloti were identified as containing insertions in four cold shock loci. LuxAB activity was strongly induced (25- to 160-fold) after a temperature shift from 30 to 15 degrees C. The transposon and flanking host DNA from each mutant was cloned, and the nucleic acid sequence of the insertion site was determined. Unexpectedly, five of the seven luxAB mutants contained transposon insertions in the 16S and 23S rRNA genes of two of the three rrn operons of S. meliloti. Directed insertion of luxAB genes into each of the three rrn operons revealed that all three operons were similarly affected by cold shock. Two other insertions were found to be located downstream of a homolog of the major Escherichia coli cold shock gene, cspA. Although the cold shock loci were highly induced in response to a shift to low temperature, none of the insertions resulted in a statistically significant decrease in growth rate at 15 degrees C.     

cpmA, a Gene Involved in an Output Pathway of the Cyanobacterial Circadian System

We generated random mutations in Synechococcus sp. strain PCC 7942 to look for genes of output pathways in the cyanobacterial circadian system. A derivative of transposon Tn5 was introduced into the chromosomes of reporter strains in which cyanobacterial promoters drive the Vibrio harveyi luxAB genes and produce an oscillation of bioluminescence as a function of circadian gene expression. Among low-amplitude mutants, one mutant, tnp6, had an insertion in a 780-bp open reading frame. The tnp6 mutation produced an altered circadian phasing phenotype in the expression rhythms of psbAI::luxAB, psbAII::luxAB, and kaiA::luxAB but had no or little effect on those of psbAIII::luxAB, purF::luxAB, kaiB::luxAB, rpoD2::luxAB, ndhD::luxAB, and conII::luxAB. This suggests that the interrupted gene in tnp6, named cpmA (circadian phase modifier), is part of a circadian output pathway that regulates the expression rhythms of psbAI, psbAII, and kaiA.     

GacS sensor domains pertinent to the regulation of exoproduct formation and to the biocontrol potential of Pseudomonas fluorescens CHA0

In the root-colonizing biocontrol strain CHA0 of Pseudomonas fluorescens, cell density-dependent synthesis of extracellular, plant-beneficial secondary metabolites and enzymes is positively regulated by the GacS/GacA two-component system. Mutational analysis of the GacS sensor kinase using improved single-copy vectors showed that inactivation of each of the three conserved phosphate acceptor sites caused an exoproduct null phenotype (GacS-), whereas deletion of the periplasmic loop domain had no significant effect on the expression of exoproduct genes. Strain CHA0 is known to synthesize a solvent-extractable extracellular signal that advances and enhances the expression of exoproduct genes during the transition from exponential to stationary growth phase when maximal exoproduct formation occurs. Mutational inactivation of either GacS or its cognate response regulator GacA abolished the strain's response to added signal. Deletion of the linker domain of the GacS sensor kinase caused signal-independent, strongly elevated expression of exoproduct genes at low cell densities. In contrast to the wild-type strain CHA0, the gacS linker mutant and a gacS null mutant were unable to protect tomato plants from crown and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici in a soil-less microcosm, indicating that, at least in this plant-pathogen system, there is no advantage in using a signal-independent biocontrol strain.     

Characterization of devA, a gene required for the maturation of proheterocysts in the cyanobacterium Anabaena sp. strain PCC 7120.

Mutant M7, obtained by transposon mutagenesis of the cyanobacterium Anabaena sp. strain PCC 7120, is impaired in the development of mature heterocysts. Under aerobic conditions, the mutant is unable to fix N2 because of a deficiency of at least two components of the oxygen-protective mechanisms: a hemoprotein-coupled oxidative reaction and heterocyst-specific glycolipids. DNA contiguous with the inserted transposon was recovered from the mutant and sequenced. The transposon had inserted itself within a 732-bp open reading frame designated devA. The wild-type form of devA, obtained from a lambda-EMBL3 library of Anabaena sp. DNA, had the identical sequence. Directed mutagenesis of devA in the wild-type strain showed that the phenotype of the mutant was caused by insertion of the transposon. The wild-type form of devA on a shuttle vector complemented the mutation in M7. Expression of devA by whole filaments, monitored following nitrogen stepdown by using luxAB as the reporter, increased ca. eightfold during differentiation; the increase within differentiating cells was much greater. The deduced sequence of the DevA protein shows strong similarity to the ATP-binding subunit of binding protein-dependent transport systems. The product of devA may, therefore, be a component of a periplasmic permease that is required for the transition from a proheterocyst to a mature, nitrogen-fixing heterocyst.     

Helicotylenchus vulgaris and its association with damage to sugar beet

Severe and irregular stunting of sugar beet has been associated with Helicotylenchus vulgaris but little is known of the pathogenicity of this nematode. A survey was made at 10 sites in East Anglia on calcareous soils (Wantage series) on which the original damage was reported. Numbers were usually maintained under a variety of crops, despite fluctuations during the period. In field trials, aldicarb treatment did not improve the yield of either sugar beet or winter wheat on land infested with H. vulgaris. In pot tests growth of sugar beet was stimulated by low nematode inocula; this effect diminished as numbers inoculated increased. Sugar beet grown at 10 °C and 15 °C with H. vulgaris grew markedly better in sterilised soil than in untreated field soil, especially at the higher temperature early in the experiment. It is concluded that though H. vulgaris may damage sugar beet it requires an atypical combination of external conditions to cause significant yield loss.     

The use of water and some inorganic salt solutions to advance sugar beet seed. I. Laboratory studies

The emergence of sugar beet seedlings is often slow or irregular and insufficient plants may be established for the crop to yield fully. Treating the seed prior to sowing, such that the subsequent germination percentage is not reduced but germination is more rapid and better synchronised should be beneficial. A series of laboratory experiments was made to investigate seed treatment procedures involving water and various inorganic salt solutions. Many treatment combinations were identified which gave faster germination without decreasing germination percentage and left the seed dry and intact and suitable for sowing with conventional precision drills. No treatment was found which ‘primed’ the sugar beet seed and the term seed ‘advancement’ describes, more accurately, the effects observed. There was little difference in the performance of seeds optimally treated with water or some salt solutions generating an osmotic potential of between about –10 and –20 bars. However, the use of salt solutions, although more complicated, was preferred to water as inadvertent germination during treatment was less likely. The optimum treatment for the one bulk of sugar beet seed studied was, firstly, to wash the seeds for 3.5 h with water, then after air-drying, to moisten them on filter paper dampened with –15 bar (0–34 M) sodium chloride solution for 6 days at 15^oC followed by a final wash and air-drying.     

Identification and characterization of genes controlled by the sporulation-regulatory gene spo0H in Bacillus subtilis.

We describe a general strategy for the identification of genes that are controlled by a specific regulatory factor in vivo and the use of this strategy to identify genes in Bacillus subtilis that are controlled by spo0H, a regulatory gene required for the initiation of sporulation. The general strategy makes use of a cloned regulatory gene fused to an inducible promoter to control expression of the regulatory gene and random gene fusions to a reporter gene to monitor expression in the presence and absence of the regulatory gene product. spo0H encodes a sigma factor of RNA polymerase, sigma H, and is required for the extensive reprograming of gene expression during the transition from growth to stationary phase and during the initiation of sporulation. We identified 18 genes that are controlled by sigma H (csh genes) in vivo by monitoring expression of random gene fusions to lacZ, made by insertion mutagenesis with the transposon Tn917lac, in the presence and absence of sigma H. These genes had lower levels of expression in the absence of sigma H than in the presence of sigma H. Patterns of expression of the csh genes during growth and sporulation in wild-type and spo0H mutant cells indicated that other regulatory factors are probably involved in controlling expression of some of these genes. Three of the csh::Tn917lac insertion mutations caused noticeable phenotypes. One caused a defect in vegetative growth, but only in combination with a spo0H mutation. Two others caused a partial defect in sporulation. One of these also caused a defect in the development of genetic competence. Detailed characterization of some of the csh genes and their regulatory regions should help define the role of spo0H in the regulation of gene expression during the transition from growth to stationary phase and during the initiation of sporulation.     

Comparison of application methods to prolong the survival of potential biocontrol bacteria on stored sugar-beet seed

AIMS: To develop bacterial inoculation treatments on sugar-beet seed that will maintain a commercially acceptable degree of viability for a minimum of 4 months storage at ambient temperature. METHODS AND RESULTS: Single rifampicin-resistant (Rif(+)) strains of both Gram-positive and negative bacterial isolates (mostly pseudomonads) were applied in turn to sugar-beet seed in a comparative study by seed soaking, encapsulation in alginate, pelleting using an inoculated peat carrier or seed priming. The treated seed was assessed for bacterial survival over a time course by plating out homogenized samples onto a selective medium. Priming inoculation offered a significant improvement over all the other application strategies tested. After pelleting with fungicides and drying at 40 degrees C, Pseudomonas marginalis/putida P1W1 maintained populations of >6.6 log(10) CFU g(-1) seed during 4 months storage at 15 degrees C. Subsequent experiments verified a stabilized population under these storage conditions with commercial pellets at <7% moisture content. CONCLUSION: An inoculation method was established which allowed the survival on seed of a Gram-negative bacterium at ambient temperature with little loss in viability. SIGNIFICANCE AND IMPACT OF THE STUDY: This has promising implications for the delivery of beneficial bacteria, especially Gram-negative strains, on sugar beet.     

Growth promotion effects of the endophyte <Emphasis Type="Italic">Acinetobacter johnsonii</Emphasis> strain 3-1 on sugar beet

An endophytic bacteriumn identified as Acinetobacter johnsonii strain 3–1 was isolated from surface-sterilized roots of Beta vulgaris. Its effect on sugar beet seedling growth was studied using pot assays and field experiments. This strain promoted beet seedling growth following seed inoculation by seed dipping. Plant height and dry weight of beet increased by 19% and 69%, respectively, compared with controls. Strain 3–1 exhibited the ability to increase absorption of N, P, K, and Mg elements from soil and increase the content of vitamins B and C, and protein within beet. In addition, the strain also produced a phytohormone-auxin, produced nearly twice as much IAA as that produced by strain 2–2, and was able to solubilize phosphates. The concentration of dissolved P in the medium was 180.5 mg L⁻¹ after 4 days of incubation. In field experiments, strain 3–1 significantly increased the content of sucrose, fructose, and the yield of the beet. The growth-promoting properties of Acinetobacter johnsonii strain 3–1 indicates that this promising isolate merits further investigation into its symbiosis with beet plants and its potential application in agriculture.