Bacillus sp.: A Remarkable Source of Bioactive Lipopeptides.

Surfactin, one of the best lipopeptide surfactants, was first isolated from Bacillus sp. in 1969. Since then, Bacillus sp. has been a remarkable source of bioactive lipopeptides, with a huge natural biodiversity. Lipopeptides from Bacillus sp. are now divided into three main families: surfactin, fengycin, and iturin. The peptide moiety of these lipopeptides is synthesised by huge multi-enzymatic proteins called nonribosomal peptide synthetases, which are responsible for the peptide biodiversity of these lipopeptides. Moreover, the fatty acid chain also encompasses a high diversity with different ß-hydroxy or ß-amino fatty acid chains of different lengths, isomery, or saturation, which can be incorporated. After describing the mode of synthesis of the different families of lipopeptides produced by Bacillus sp. and their biodiversity, this chapter describes how this lipopeptide biodiversity can be increased using genetic engineering and how the lipopeptides can be overproduced and purified. The high biodiversity of lipopeptides induces a broad range of physicochemical properties, which can be linked to multiple biological activities with many applications in different sectors. The increasing understanding of the mode of biosynthesis of these lipopeptides should lead to the development of novel compounds with increased properties and applications.

Insights into the defense-related events occurring in plant cells following perception of surfactin-type lipopeptide from Bacillus subtilis.

Multiple strains of Bacillus subtilis were demonstrated to stimulate plant defense responses, and cyclic lipopeptides may be involved in the elicitation of this induced systemic resistance phenomenon. Here, we further investigated molecular events underlying the interaction between such lipopeptides and plant cells. Addition of surfactin but not fengycin or iturin in the micromolar range to tobacco cell suspensions induced defense-related early events such as extracellular medium alkalinization coupled with ion fluxes and reactive oxygen species production. Surfactin also stimulated the defense enzymes phenylalanine ammonia lyase and lipoxygenase and modified the pattern of phenolics produced by the elicited cells. The occurrence of these surfactin-elicited early events is closely related to Ca(2+) influx and dynamic changes in protein phosphorylation but is not associated with any marked phytotoxicity or adverse effect on the integrity and growth potential of the treated tobacco cells. Reduced activity of some homologues also indicates that surfactin perception is dictated by structural clues in both the acyl moiety and cyclic peptide part. Our results suggest that these molecules could interact without irreversible pore formation but in a way sufficient to induce disturbance or transient channeling in the plasma membrane that can, in turn, activate a biochemical cascade of molecular events leading to defensive responses. The present study sheds new light not only on defense-related events induced following recognition of amphiphilic lipopeptides from Bacillus spp. but also more globally on the way elicitors from beneficial bacteria can be perceived by host plant cells.

Bacillus-based biocontrol of fusarium disease on tomato cultures in Burundi.

The biocontrol potential of a Plant Growth-Promoting Rhizobacterium (PGPR) Bacillus subtilis S499 on tomato was studied in open field sites in low attitude area of the plain of Imbo in Burundi. Application of the PGPR strain on seed before sowing have significantly increased growth and fruit yield of tomato plants in addition to its remarkable control of a local important disease caused by a fungus type-Fusarium.. This pathogen causing large tosses in Burundi tomato plantings is closely related to Fusarium semitectum based on a preliminary identification. Results obtained in open field assays from two successive years on the same site demonstrate that treatment with B. subtilis S499 strain suspensions significantly increase growth and fruit-yield and provided a high level of protection of tomato plantings against the new fungal disease apparently uncontrolled by routine chemical pesticides.

Effect of monopropylene glycol and gamma irradiation on Yarrowia lipolytica lipase stabilization.

This work investigated the effects of monopropylene glycol, protease inhibitor, and gamma irradiation on Yarrowia lipolytica lipase stability during storage. Enzyme liquid stabilization was achieved by addition of monopropylene glycol (MPG) at respective concentrations of 50, 75, and 90%, the protease inhibitors (P2714 and P8215) at 0.1%, and the gamma irradiation with 10kGy, 15kGy, and 25kGy doses. The results showed that monopropylene glycol limited the microorganism growth and decreased the enzymatic activity at high concentration (up to 50%), at two temperatures (20 and 4 degrees C). Enzyme stored at 20 degrees C lost its activity by 80% after two months. This loss was attributed to the protease's effect. At this temperature, the protease's activities have been limited by the specific inhibitors. The gamma irradiations improve microbial safety of liquid enzyme.

Role of lipopeptides produced by Bacillus subtilis GA1 in the reduction of grey mould disease caused by Botrytis cinerea on apple.

AIM: Test of Bacillus subtilis strain GA1 for its potential to control grey mould disease of apple caused by Botrytis cinerea. METHODS AND RESULTS: GA1 was first tested for its ability to antagonize in vitro the growth of a wide variety of plant pathogenic fungi responsible for diseases of economical importance. The potential of strain GA1 to reduce post-harvest infection caused by B. cinerea was tested on apples by treating artificially wounded fruits with endospore suspensions. Strain GA1 was very effective at reducing disease incidence during the first 5 days following pathogen inoculation and a 80% protection level was maintained over the next 10 days. Treatment of fruits with an extract of GA1 culture supernatant also exerted a strong preventive effect on the development of grey mould. Further analysis of this extract revealed that strain GA1 produces a wide variety of antifungal lipopeptide isomers from the iturin, fengycin and surfactin families. A strong evidence for the involvement of such compounds in disease reduction arose from the recovery of fengycins from protected fruit sites colonized by bacterial cells. CONCLUSIONS: The results presented here demonstrate that, despite unfavourable pH, B. subtilis endospores inoculated on apple pulp can readily germinate allowing significant cell populations to establish and efficient in vivo synthesis of lipopeptides which could be related to grey mould reduction. SIGNIFICANCE AND IMPACT OF THE STUDY: This work enables for the first time to correlate the strong protective effect of a particular B. subtilis strain against grey mould with in situ production of fengycins in infected sites of apple fruits.

Fluorescent Pseudomonas mainly produce the dihydro form of pyoverdine at low specific growth rate.

AIMS: To analyse the influence of cell growth rate and iron concentration on the production of pyoverdines (PVDs) and of their reduced dihydro forms by three fluorescent Pseudomonas strains (P. putida BTP16, P. fluorescens BTP7 and P. aeruginosa 7NSK2). METHODS: PVD and dihydropyoverdine (DHPVD) productions were determined by LC ESI-MS and spectrophotometry during batch and chemostat culture at different dilution rates. SIGNIFICANCE: The relatively high PVD-to-DHPVD ratio (0.57) observed in pH-controlled batch cultures suggested that a base-catalysed chemical oxidation of the dihydroform is not the prime mechanism involved in generating PVDs. Interestingly, in chemostat cultures the PVD-to-DHPVD ratio was significantly reduced at low specific growth rate. Our results suggest that the oxidation of DHPVD to PVD is catalysed by an iron-dependent enzymatic reaction rather than a chemical oxidation.

Influence of moderate temperatures on myristoyl-CoA metabolism and acyl-CoA thioesterase activity in the psychrophilic antarctic yeast Rhodotorula aurantiaca.

The inability of psychrophilic microorganisms to grow at moderate temperatures (>20 degrees C) presently represents an unresolved thermodynamic paradox. Here we report for the psychrophilic yeast Rhodotorula aurantiaca A19, isolated from Antarctic ice, that the inability to grow at temperatures close to 20 degrees C is associated with profound alterations in cell morphology and integrity. High performance liquid chromatography analysis of the intracellular acyl-CoA esters revealed an abnormal accumulation of myristoyl-CoA (C14-CoA) in cells cultivated close to the nonpermissive temperature. Its concentration (500 microm) was found to be 28-fold higher than in cells cultivated at 0 degrees C. If one considers its ability to disrupt membrane bilayers and to inhibit many cellular enzymes and functions, intracellular myristoyl-CoA accumulation in the psychrophile R. aurantiaca represents one of the principal causes of growth arrest at moderate temperatures. Intracellular acyl-CoA concentrations are believed to be regulated by thioesterase activity. Thus in an attempt to explore the mechanism by which temperature disrupts myristoyl-CoA metabolism, we isolated and characterized a long chain acyl-CoA thioesterase. The monomeric 80-kDa thioesterase from the psychrophilic yeast shows a very strong specificity for myristoyl-CoA. The affinity for substrate and the catalytic efficiency of the thioesterase are optimal below 5 degrees C (temperatures habitually experienced by the strain) and dramatically decrease with increasing temperature. The loss of affinity for substrate is related to the intracellular increase of myristoyl-CoA concentration. Our observations reveal one of the probable mechanisms by which temperature fixes the limit of growth for this psychrophilic yeast.

Structure and characterization of isopyoverdin from Pseudomonas putida BTP1 and its relation to the biogenetic pathway leading to pyoverdins.

Pyoverdin type siderophores produced by six fluorescent Pseudomonas strains isolated from different rhizospheres were purified and characterized. The purified ferri-pyoverdins were tested for their ability to promote the growth of other strains grown under iron deficiency conditions. Only the one obtained from Pseudomonas putida BTP1 did not act as a growth promoter. The structure of the BTP1 siderophore was elucidated by spectroscopic methods and degradation studies. It turned out that it contains a chromophore which differs from the one typical for pyoverdins insofar as it carries the carboxyl group in 3- rather than in 1-position ((3S)-5-amino-1,2-dihydro-8,9-dihydroxy-3H-pyrimido[1,2a]quinoline-3- carboxylic acid). The amino group of the chromophore is substituted with the 5-carboxyl group of L-glutamic acid and its carboxyl group with the N-terminus of the peptide L-Asp-L-Ala-L-Asp-D-N5-Ac-N5-OH-Orn-L-Ser-L-c-N5-OH-Orn. This isopyoverdin fits into the biogenetic scheme which postulates ferribactins as the precursors of pyoverdins.