Diego Romero

d-Amino Acids Trigger Biofilm Disassembly

Biofilm Today, Gone Tomorrow Most bacteria can form complex, matrix-containing multicellular communities known as biofilms, which protect residents from environmental stresses such as antibiotic exposure. However, as biofilms age, nutrients become limiting and waste products accumulate, and biofim disassembly is triggered. Now Kolodkin-Gal et al. (p. 627) have found that d-amino acids found in conditioned medium from mature biofilms of Bacillus subtilis prevent biofilm formation and trigger existing biofilm disassembly. Bacteria secrete an unusual form of amino acids to escape from aging communities by dissolving the surrounding matrix. Bacteria form communities known as biofilms, which disassemble over time. In our studies outlined here, we found that, before biofilm disassembly, Bacillus subtilis produced a factor that prevented biofilm formation and could break down existing biofilms. The factor was shown to be a mixture of d-leucine, d-methionine, d-tyrosine, and d-tryptophan that could act at nanomolar concentrations. d-Amino acid treatment caused the release of amyloid fibers that linked cells in the biofilm together. Mutants able to form biofilms in the presence of d-Amino acids contained alterations in a protein (YqxM) required for the formation and anchoring of the fibers to the cell. d-Amino acids also prevented biofilm formation by Staphylococcus aureus and Pseudomonas aeruginosa. d-amino acids are produced by many bacteria and, thus, may be a widespread signal for biofilm disassembly.

Amyloid fibers provide structural integrity to Bacillus subtilis biofilms

Bacillus subtilis forms biofilms whose constituent cells are held together by an extracellular matrix. Previous studies have shown that the protein TasA and an exopolysaccharide are the main components of the matrix. Given the importance of TasA in biofilm formation, we characterized the physicochemical properties of this protein. We report that purified TasA forms fibers of variable length and 10–15 nm in width. Biochemical analyses, in combination with the use of specific dyes and microscopic analyses, indicate that TasA forms amyloid fibers. Consistent with this hypothesis, TasA fibers required harsh treatments (e.g., formic acid) to be depolymerized. When added to a culture of a tasA mutant, purified TasA restored wild-type biofilm morphology, indicating that the purified protein retained biological activity. We propose that TasA forms amyloid fibers that bind cells together in the biofilm.

The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca

Podosphaera fusca is the main causal agent of cucurbit powdery mildew in Spain. Four Bacillus subtilis strains, UMAF6614, UMAF6619, UMAF6639, and UMAF8561, with proven ability to suppress the disease on melon in detached leaf and seedling assays, were subjected to further analyses to elucidate the mode of action involved in their biocontrol performance. Cell-free supernatants showed antifungal activities very close to those previously reported for vegetative cells. Identification of three lipopeptide antibiotics, surfactin, fengycin, and iturin A or bacillomycin, in butanolic extracts from cell-free culture filtrates of these B. subtilis strains pointed out that antibiosis could be a major factor involved in their biocontrol ability. The strong inhibitory effect of purified lipopeptide fractions corresponding to bacillomycin, fengycin, and iturin A on P. fusca conidia germination, as well as the in situ detection of these lipopeptides in bacterial-treated melon leaves, provided interesting evidence of their putative involvement in the antagonistic activity. Those results were definitively supported by site-directed mutagenesis analysis, targeted to suppress the biosynthesis of the different lipopeptides. Taken together, our data have allowed us to conclude that the iturin and fengycin families of lipopeptides have a major role in the antagonism of B. subtilis toward P. fusca.

Plant protection and growth stimulation by microorganisms: biotechnological applications of Bacilli in agriculture

The increasing demand for a steady, healthy food supply requires an efficient control of the major pests and plant diseases. Current management practices are based largely on the application of synthetic pesticides. The excessive use of agrochemicals has caused serious environmental and health problems. Therefore, there is a growing demand for new and safer methods to replace or at least supplement the existing control strategies. Biological control, that is, the use of natural antagonists to combat pests or plant diseases has emerged as a promising alternative to chemical pesticides. The Bacilli offer a number of advantages for their application in agricultural biotechnology. Several Bacillus-based products have been marketed as microbial pesticides, fungicides or fertilisers. Bacillus-based biopesticides are widely used in conventional agriculture, by contrast, implementation of Bacillus-based biofungicides and biofertilizers is still a pending issue.

Antibiotics as Signal Molecules

Antibiotics have been extensively used in the treatment of infectious diseases. The lethality of these compounds has been exploited in clinical and laboratory approaches and their specific targets in bacterial physiology elucidated. However, along with this development of drugs has come the problem of increasing resistance in microbes, resulting in dramatically reduced therapeutic effectiveness 1. Pathogenic microbes have rapidly evolved efficient mechanisms of resistance, including increased efflux, enzymatic inactivation, target modification, or biofilm formation 2. The concentrations of these molecules required to achieve an antimicrobial effect are likely extremely high compared to the concentrations in which these molecules can be found in natural environments. While we know their effect at lethal concentrations, the activities of these molecules at concentrations below the inhibitory limit needs deeper investigation 3. The findings of the nineteenth-century pharmacologist Hugo Schulz, who noted that certain disinfectants could have stimulatory effect on yeast growth at low concentrations, could be considered the first evidence that the action of an antimicrobial can cause a differential response depending on the concentration. His observation was the first example of what it would be later called hormesis. This term was coined by Chester Southam and John Ehrlich in the mid-1920s and it is used to refer to the ability of certain molecules to induce diverse responses depending on the concentration used 3. The vast amount of information related to the lethal concentration of antibiotics, targets, or side effects, contrasts dramatically with the relatively few studies focused on their effect at concentrations below the MIC (minimal inhibitory concentration). As pointed by Davies, only a small fraction of natural products that have antimicrobial activity have been extensively studied, and their role in natural settings is poorly understood. Thus it is possible that many of these molecules formerly considered antibiotics might have a different function in nature. It is now believed that many of these compounds might act as signaling molecules that modulate gene expression in microbial populations, or physiological functions such as motility, pigmentation, and production of metabolites, and thus facilitate inter- and intra-species communication 4. This fundamental lack of understanding may be rooted in our conception of the microbial world as single and separated species, as they are usually studied under laboratory conditions. However, in nature, each niche is complex, and to different extents, variable in microbial community composition 5,6–8. Therefore it is conceivable that molecules fluctuate in concentration and diversity, thus facilitating communication among species 9. Many interesting lines of research are currently focused on understanding how some antibiotics may affect, either positively or negatively, cell-cell communication systems, and the physiological responses that are affected as a result. In some cases, natural products can influence the ability of bacteria to transition from a planktonic state to complex multicellular aggregates attached to surfaces known as biofilms. Cells in bioflms are encased in an extracellular matrix which can serve as a barrier for antibiotics 10. One example is the biofilm produced by the gram-negative pathogen, Pseudomonas aeruginosa, which is resistant to antibiotics produced by gram-positive competitors 11. Studies on many model microorganisms from the genera Bacillus, Streptomyces, and Pseudomonas are shedding new light on the fascinating world of cell signaling and communication in microbial world 5,12,13. We will discuss in this review several examples of the dual functions of some well-known antibiotics, including those that when used at sub-inhibitory concentrations (SIC) promote an interesting response in bacterial populations and the ecological implications of such varied responses. Also, the role of other naturally synthesized antibiotics will be discussed in the context of cell communication in natural environments, including one of the most exploited environmental niches for antibiotic discovery, the soil.

Isolation and characterization of antagonistic Bacillus subtilis strains from the avocado rhizoplane displaying biocontrol activity

Aim:  This study was undertaken to isolate Bacillus subtilis strains with biological activity against soil‐borne phytopathogenic fungi from the avocado rhizoplane.

An accessory protein required for anchoring and assembly of amyloid fibres in B. subtilis biofilms

Cells within Bacillus subtilis biofilms are held in place by an extracellular matrix that contains cell‐anchored amyloid fibres, composed of the amyloidogenic protein TasA. As biofilms age they disassemble because the cells release the amyloid fibres. This release appears to be the consequence of incorporation of d‐tyrosine, d‐leucine, d‐tryptophan and d‐methionine into the cell wall. Here, we characterize the in vivo roles of an accessory protein TapA (TasA anchoring/assembly protein; previously YqxM) that serves both to anchor the fibres to the cell wall and to assemble TasA into fibres. TapA is found in discrete foci in the cell envelope and these foci disappear when cells are treated with a mixture of d‐amino acids. Purified cell wall sacculi retain a functional form of this anchoring protein such that purified fibres can be anchored to the sacculi in vitro. In addition, we show that TapA is essential for the proper assembly of the fibres. Its absence results in a dramatic reduction in TasA levels and what little TasA is left produces only thin fibres that are not anchored to the cell.

The powdery mildew fungus Podosphaera fusca (synonym Podosphaera xanthii), a constant threat to cucurbits

UNLABELLED Numerous vegetable crops are susceptible to powdery mildew, but cucurbits are arguably the group most severely affected. Podosphaera fusca (synonym Podosphaera xanthii) is the main causal agent of cucurbit powdery mildew and one of the most important limiting factors for cucurbit production worldwide. Although great efforts have been invested in disease control, by contrast, many basic aspects of the biology of P. fusca remain unknown. TAXONOMY Podosphaera fusca (Fr.) Braun & Shishkoff. Kingdom Fungi; Phylum Ascomycota; Subdivision Pezizomycotina; Class Leotiomycetes; Order Erysiphales; Family Erysiphaceae; genus Podosphaera; species fusca. IDENTIFICATION Superficial persistent mycelium. Conidia in chains, hyaline, ellipsoid to ovoid or doliform, about 24-40 x 15-22 microm, with cylindrical or cone-shaped fibrosin bodies, which often germinate from a lateral face and produce a broad, clavate germ tube and cylindrical foot-cells. Unbranched erect conidiophores. Cleistothecia globose, mostly 70-100 microm in diameter, dark brown/black. One ascus per cleistothecium with eight ascospores. HOST RANGE Angiosperm species that include several families, such as Asteracea, Cucurbitaceae, Lamiaceae, Scrophulariaceae, Solanaceae and Verbenaceae. DISEASE SYMPTOMS White colonies develop on leaf surfaces, petioles and stems. Under favourable environmental conditions, the colonies coalesce and the host tissue becomes chlorotic and usually senesces early. CONTROL Chemical control and the use of resistant cultivars. Resistance has been documented in populations of P. fusca to some of the chemicals registered for control.

Effect of lipopeptides of antagonistic strains of Bacillus subtilis on the morphology and ultrastructure of the cucurbit fungal pathogen Podosphaera fusca

Aims:  To analyse the morphological and ultrastructural effects of lipopeptides of cell‐free liquid cultures from the antagonistic Bacillus subtilis strains, UMAF6614 and UMAF6639, on the cucurbit powdery mildew fungus, Podosphaera fusca, conidial germination.

The antagonistic strain Bacillus subtilis UMAF6639 also confers protection to melon plants against cucurbit powdery mildew by activation of jasmonate- and salicylic acid-dependent defence responses

Biological control of plant diseases has gained acceptance in recent years. Bacillus subtilis UMAF6639 is an antagonistic strain specifically selected for the efficient control of the cucurbit powdery mildew fungus Podosphaera fusca, which is a major threat to cucurbits worldwide. The antagonistic activity relies on the production of the antifungal compounds iturin and fengycin. In a previous study, we found that UMAF6639 was able to induce systemic resistance (ISR) in melon and provide additional protection against powdery mildew. In the present work, we further investigated in detail this second mechanism of biocontrol by UMAF6639. First, we examined the signalling pathways elicited by UMAF6639 in melon plants, as well as the defence mechanisms activated in response to P. fusca. Second, we analysed the role of the lipopeptides produced by UMAF6639 as potential determinants for ISR activation. Our results demonstrated that UMAF6639 confers protection against cucurbit powdery mildew by activation of jasmonate‐ and salicylic acid‐dependent defence responses, which include the production of reactive oxygen species and cell wall reinforcement. We also showed that surfactin lipopeptide is a major determinant for stimulation of the immune response. These results reinforce the biotechnological potential of UMAF6639 as a biological control agent.