Environmental regulation of Bacillus subtilis sigma(D)-dependent gene expression

The sigma(D) regulon of Bacillus subtilis is composed of genes encoding proteins for flagellar synthesis, motility, and chemotaxis. Concurrent analyses of sigma(D) protein levels and flagellin mRNA demonstrate that sigD expression and sigma(D) activity are tightly coupled during growth in both complex and minimal media, although they exhibit different patterns of expression. We therefore used the sigma(D)-dependent flagellin gene (hag) as a model gene to study the effects of different nutritional environments on sigma(D)-dependent gene expression. In complex medium, the level of expression of a hag-lacZ fusion increased exponentially during the exponential growth phase and peaked early in the transition state. In contrast, the level of expression of this reporter remained constant and high throughout growth in minimal medium. These results suggest the existence of a nutritional signal(s) that affects sigD expression and/or sigma(D) activity. This signal(s) allows for nutritional repression early in growth and, based on reconstitution studies, resides in the complex components of sporulation medium, as well as in a mixture of mono-amino acids. However, the addition of Casamino Acids to minimal medium results in a dose-dependent decrease in hag-lacZ expression throughout growth and the postexponential growth phase. In work by others, CodY has been implicated in the nutritional repression of several genes. Analysis of a codY mutant bearing a hag-lacZ reporter revealed that flagellin expression is released from nutritional repression in this strain, whereas mutations in the transition state preventor genes abrB, hpr, and sinR failed to elicit a similar effect during growth in complex medium. Therefore, the CodY protein appears to be the physiologically relevant regulator of hag nutritional repression in B. subtilis.     

Induction of polymyxin resistance in Pseudomonas fluorescens by phosphate limitation

Shift of Pseudomonas fluorescens NCMB 129 from a phosphate rich into a phosphate limited medium results in a reduction of the membrane phospholipids phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. Concomitantly a positively charged ornithine amide lipid is synthesized. The gradual increase of this lipid is paralleled by an increasing resistance to polymyxin B. The binding capacities of intact cells, and isolated inner and outer membranes for the antibiotic are reduced in the resistant organisms. It is discussed that the observed effect could be circumstantial evidence that the positively charged polymyxin B needs negatively charged receptors in biological membranes in order to exert its antibiotic activity.List of Abbreviations PE phosphatidylethanolamine - PG phosphatidylglycerol - CL cardiolipin - PX polymyxin B     

Elevated zinc induces siderophore biosynthesis genes and a zntA-like gene in Pseudomonas fluorescens

Zinc-regulated genes were analyzed in Pseudomonas fluorescens employing mutagenesis with a reporter gene transposon. Six mutants responded with increased gene expression to elevated concentrations of zinc. Genetic and biochemical analysis revealed that in four of the six mutants the transposon had inserted into genes essential for the biosynthesis of the siderophore pyoverdine. The growth of one of the mutants was severely impaired in the presence of elevated concentrations of cadmium and zinc ions. In this mutant, the transposon had inserted in a gene with high similarity to P-type ATPases involved in zinc and cadmium ion transport. Four mutants reacted with reduced gene expression to elevated concentrations of zinc. One of these mutants was sensitive to zinc, cadmium and copper ions. The genetic region targeted in this mutant did not show similarity to any known gene.     

Survival ofalginate-ecapsulated Pseudomonas fluorescens cells in soil

Alginate-encapsulated and unencapsulated cells of Pseudomonas fluorescens Rsf were introduced into soil microcosms with and without wheat plants to evaluate bacterial survival and colonization of the rhizoplane and rhizosphere. Encapsualtion of cells in alginate amended with skim milk or with skim milk plus bentonite clay significantly enchanced long-term survival of the cells. There was a negligible effect on long-term bacterial survival when cells were encapsulated in alginate amended with TY medium or soil extract, as compared to water. Drying of beads resulted in a significant reduction in bacterial viability. After addition to soil, cells in dried beads increased in numbers and exhibited stable population densities, whereas cells added in moist beads showed stable dynamics at a higher level. Cells encapsulated in dried beads or fresh beads survived better than unencapsulated cells added to soil. Both cells in moist and dried alginate beads also survide a dry/wet cycle in soil, whereas unencapsulated cells were sensitive to these moisture fluctuations. Shortly after inoculation and 63 days after this, cells from moist beads colonized wheat roots at significantly higher levels than unencapsulated cells, whereas cells in dried beads did so at levels similat to unencapsulated cells. Cells in beads initially placed at different distance from developing root mat were able to move towards and colonize the rhizosphere, at levels of roughly 10⁴ to 10⁶ colony-forming units fo P. fluorescens R2f per gram of dry soil. Correspondence to: J. T. Trevors or J. D. van Elsas     

Cadmium-regulated gene fusions in Pseudomonas fluorescens

To study the mechanisms soil bacteria use to cope with elevated concentrations of heavy metals in the environment, a mutagenesis with the lacZ-based reporter gene transposon Tn5B20 was performed. Random gene fusions in the genome of the common soil bacterium Pseudomonas fluorescens strain ATCC 13525 were used to create a bank of 5,000 P. fluorescens mutants. This mutant bank was screened for differential gene expression in the presence of the toxic metal cadmium. Fourteen mutants were identified that responded with increased or reduced gene expression to the presence of cadmium. The mutants were characterized with respect to their metal-dependent gene expression and their metal tolerance. Half the identified mutants reacted with differential gene expression specifically to the metal cadmium, whereas some of the other mutants also responded to elevated concentrations of copper and zinc ions. One of the mutants, strain C8, also showed increased gene expression in the presence of the solvent ethanol, but otherwise no overlap between cadmium-induced gene expression and general stress response was detected. Molecular analysis of the corresponding genetic loci was performed using arbitrary polymerase chain reaction (PCR), DNA sequencing and comparison of the deduced protein products with sequences deposited in genetic databases. Some of the genetic loci targeted by the transposon did not show any similarities to any known genes; thus, they may represent 'novel' loci. The hypothesis that genes that are differentially expressed in the presence of heavy metals play a role in metal tolerance was verified for one of the mutants. This mutant, strain C11, was hypersensitive to cadmium and zinc ions. In mutant C11, the transposon had inserted into a genetic region displaying similarity to genes encoding the sensor/regulator protein pairs of two-component systems that regulate gene expression in metal-resistant bacteria, including czcRS of Ralstonia eutropha, czrRS of Pseudomonas aeruginosa and copRS of Pseudomonas syringae. Although the P. fluorescens strain used in this study had not been isolated from a metal-rich environment, it nevertheless contained at least one genetic region enabling it to cope with elevated concentrations of heavy metals.     

RpoN (sigma54) controls production of antifungal compounds and biocontrol activity in Pseudomonas fluorescens CHA0

Pseudomonas fluorescens CHA0 is an effective biocontrol agent of root diseases caused by fungal pathogens. The strain produces the antibiotics 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT) that make essential contributions to pathogen suppression. This study focused on the role of the sigma factor RpoN (sigma54) in regulation of antibiotic production and biocontrol activity in P. fluorescens. An rpoN in-frame-deletion mutant of CHAO had a delayed growth, was impaired in the utilization of several carbon and nitrogen sources, and was more sensitive to salt stress. The rpoN mutant was defective for flagella and displayed drastically reduced swimming and swarming motilities. Interestingly, the rpoN mutant showed a severalfold enhanced production of DAPG and expression of the biosynthetic gene phlA compared with the wild type and the mutant complemented with monocopy rpoN+. By contrast, loss of RpoN function resulted in markedly lowered PLT production and plt gene expression, suggesting that RpoN controls the balance of the two antibiotics in strain CHA0. In natural soil microcosms, the rpoN mutant was less effective in protecting cucumber from a root rot caused by Pythium ultimum. Remarkably, the mutant was not significantly impaired in its root colonization capacity, even at early stages of root infection by Pythium spp. Taken together, our results establish RpoN for the first time as a major regulator of biocontrol activity in Pseudomonas fluorescens.     

ppGpp accumulation in Pseudomonas aeruginosa and Pseudomonas fluorescens subjected to nutrient limitation and biocide exposure

The effect of a commonly used biocide, 1,2-benzisothiazolin-3-one (BIT) on ppGpp accumulation in the pathogen, Pseudomonas aeruginosa PAO1, and an environmental isolate, Ps. fluorescens, was examined. It is concluded that BIT is able to induce a stringent response in Ps. aeruginosa and Ps. fluorescens, determined by the rapid accumulation of ppGpp following addition of BIT to exponentially-growing cells. Western blot analysis of whole-cell extracts with anti-RelA antibody demonstrated that both species contain a RelA homologue. This is the first report of a RelA-like protein in pseudomonads.     

Siderophore cooperation of the bacterium Pseudomonas fluorescens in soil

While social interactions play an important role for the evolution of bacterial siderophore production in vitro, the extent to which siderophore production is a social trait in natural populations is less clear. Here, we demonstrate that siderophores act as public goods in a natural physical environment of Pseudomonas fluorescens: soil-based compost. We show that monocultures of siderophore producers grow better than non-producers in soil, but non-producers can exploit others'' siderophores, as shown by non-producers'' ability to invade populations of producers when rare. Despite this rare advantage, non-producers were unable to outcompete producers, suggesting that producers and non-producers may stably coexist in soil. Such coexistence is predicted to arise from the spatial structure associated with soil, and this is supported by increased fitness of non-producers when grown in a shaken soil–water mix. Our results suggest that both producers and non-producers should be observed in soil, as has been observed in marine environments and in clinical populations.     

Cloning and expression of a novel lipase gene from Pseudomonas fluorescens B52

Here we describe an advanced polymerase chain reaction (PCR) technique, the compatible ends ligation inverse PCR (CELI-PCR) for chromosome walking. In CELI-PCR, several restriction enzymes, which produce compatible cohesive ends, were used to digest target DNA simultaneously or sequentially to produce DNA fragments of suitable size. DNA fragments were then easily circularized and PCR amplification could be carried out efficiently. The previous limitations of inverse PCR were overcome, such as unavailable restriction sites, poor template DNA circularization, and low amplification efficiency. Therefore, successive chromosome walking was performed successfully. Our work, isolating a 11,395-bp fragment from Gossypium hirsutum, was presented as an example to describe how CELI-PCR was carried out.     

Survival of rifampin-resistant mutants of Pseudomonas fluorescens and Pseudomonas putida in soil systems

The fate of spontaneous chromosomal rifampin-resistant (Rifr) mutants of Pseudomonas putida and Pseudomonas fluorescens in sterile and live organic soil from which they were isolated was studied. In sterile native-soil assays, a Rifr mutant of P. putida showed no decrease in competitive fitness when compared with the wild-type parent. However, mutants of P. fluorescens were of two general categories. Group 1 showed no difference from the wild type in terms of growth rate, competitive fitness, and membrane protein composition. Group 2 showed a slower growth rate in both minimal and enriched media and an altered membrane protein profile. These mutants also demonstrated decreased competitive fitness compared with the wild-type strain. In live soil, the Rifr P. putida strain persisted throughout the 38-day test period with a decay rate of 0.7 log10 CFU/g of soil per 10 days. A group 1 Rifr P. fluorescens mutant maintained its inoculated titer for 7 to 10 days and then decayed at a rate of 0.2 to 0.4 log10 CFU/g of soil per 10 days. A group 2 Rifr P. fluorescens mutant remained at its titer for 1 to 5 days before decaying at a two- to threefold-faster rate. These findings indicate that rifampin resistance may not be an innocuous mutation in some pseudomonads and that marked strains should be compared with wild-type parents before being used as monitors of parental strain survival. Colonization of sterile soil with either the wild-type or mutant strain precluded normal colonization of the second added strain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Simultaneous expression of an arylacetonitrilase from Pseudomonas fluorescens and a (S)-oxynitrilase from Manihot esculenta in Pichia pastoris for the synthesis of (S)-mandelic acid

The arylacetonitrilase of Pseudomonas fluorescens EBC191 catalyzes the conversion of (S)-mandelonitrile to (S)-mandelic acid and (S)-mandeloamide. This biotransformation is optimally performed under acidic pH values because (S)-mandelonitrile rapidly decomposes under neutral conditions. Therefore, the gene encoding the arylacetonitrilase of P. fluorescens EBC191 was integrated and expressed under the control of the AOX1 promoter in the methylotrophic yeast Pichia pastoris which was supposed to act as an acidotolerant expression system. These recombinant strains hydrolyzed (R,S)-mandelonitrile at pH values >or=3 to mandelic acid and mandeloamide and were more acidotolerant than previously constructed Escherichia coli whole cell catalysts synthesizing the same nitrilase activity. Subsequently, recombinant P. pastoris strains were constructed which simultaneously expressed the (S)-oxynitrilase of Manihot esculenta and the arylacetonitrilase of P. fluorescens EBC191 each under the control of individual AOX1 promoters in order to obtain a whole cell catalyst for the synthesis of (S)-mandelic acid from benzaldehyde and cyanide. Resting cells of the recombinant strains converted under acidic conditions benzaldehyde and cyanide initially to mandelonitrile which was immediately converted to mandelic acid and mandeloamide. The chiral analysis of the products formed revealed a high enantiomeric excess for the (S)-enantiomers.     

Natural transformation of Pseudomonas fluorescens and Agrobacterium tumefaciens in soil

Little information is available concerning the occurrence of natural transformation of bacteria in soil, the frequency of such events, and the actual role of this process on bacterial evolution. This is because few bacteria are known to possess the genes required to develop competence and because the tested bacteria are unable to reach this physiological state in situ. In this study we found that two soil bacteria, Agrobacterium tumefaciens and Pseudomonas fluorescens, can undergo transformation in soil microcosms without any specific physical or chemical treatment. Moreover, P. fluorescens produced transformants in both sterile and nonsterile soil microcosms but failed to do so in the various in vitro conditions we tested. A. tumefaciens could be transformed in vitro and in sterile soil samples. These results indicate that the number of transformable bacteria could be higher than previously thought and that these bacteria could find the conditions necessary for uptake of extracellular DNA in soil.     

Reliable protein production in a Pseudomonas fluorescens expression system

A bottleneck to product development can be reliable expression of active target protein. A wide array of recombinant proteins in development, including an ever growing number of non-natural proteins, is being expressed in a variety of expression systems. A Pseudomonas fluorescens expression platform has been developed specifically for recombinant protein production. The development of an integrated molecular toolbox of expression elements and host strains, along with automation of strain screening is described. Examples of strain screening and scale-up experiments show rapid development of expression strains producing a wide variety of proteins in a soluble active form.