Bacilysin from Bacillus amyloliquefaciens FZB42 Has Specific Bactericidal Activity against Harmful Algal Bloom Species

Harmful algal blooms, caused by massive and exceptional overgrowth of microalgae and cyanobacteria, are a serious environmental problem worldwide. In the present study, we looked for Bacillus strains with sufficiently strong anticyanobacterial activity to be used as biocontrol agents. Among 24 strains, Bacillus amyloliquefaciens FZB42 showed the strongest bactericidal activity against Microcystis aeruginosa, with a kill rate of 98.78%. The synthesis of the anticyanobacterial substance did not depend on Sfp, an enzyme that catalyzes a necessary processing step in the nonribosomal synthesis of lipopeptides and polyketides, but was associated with the aro gene cluster that is involved in the synthesis of the sfp-independent antibiotic bacilysin. Disruption of bacB, the gene in the cluster responsible for synthesizing bacilysin, or supplementation with the antagonist N-acetylglucosamine abolished the inhibitory effect, but this was restored when bacilysin synthesis was complemented. Bacilysin caused apparent changes in the algal cell wall and cell organelle membranes, and this resulted in cell lysis. Meanwhile, there was downregulated expression of glmS, psbA1, mcyB, and ftsZ—genes involved in peptidoglycan synthesis, photosynthesis, microcystin synthesis, and cell division, respectively. In addition, bacilysin suppressed the growth of other harmful algal species. In summary, bacilysin produced by B. amyloliquefaciens FZB42 has anticyanobacterial activity and thus could be developed as a biocontrol agent to mitigate the effects of harmful algal blooms.     

Expression of the lantibiotic mersacidin in Bacillus amyloliquefaciens FZB42

Lantibiotics are small peptide antibiotics that contain the characteristic thioether amino acids lanthionine and methyllanthionine. As ribosomally synthesized peptides, lantibiotics possess biosynthetic gene clusters which contain the structural gene (lanA) as well as the other genes which are involved in lantibiotic modification (lanM, lanB, lanC, lanP), regulation (lanR, lanK), export (lanT(P)) and immunity (lanEFG). The lantibiotic mersacidin is produced by Bacillus sp. HIL Y-85,54728, which is not naturally competent.

Methodology/Principal Findings

The aim of these studies was to test if the production of mersacidin could be transferred to a naturally competent Bacillus strain employing genomic DNA of the producer strain. Bacillus amyloliquefaciens FZB42 was chosen for these experiments because it already harbors the mersacidin immunity genes. After transfer of the biosynthetic part of the gene cluster by competence transformation, production of active mersacidin was obtained from a plasmid in trans. Furthermore, comparison of several DNA sequences and biochemical testing of B. amyloliquefaciens FZB42 and B. sp. HIL Y-85,54728 showed that the producer strain of mersacidin is a member of the species B. amyloliquefaciens.

Conclusions/Significance

The lantibiotic mersacidin can be produced in B. amyloliquefaciens FZB42, which is closely related to the wild type producer strain of mersacidin. The new mersacidin producer strain enables us to use the full potential of the biosynthetic gene cluster for genetic manipulation and downstream modification approaches.     

Erratum to: genomic comparison of 93 Bacillus phages reveals 12 clusters, 14 singletons and remarkable diversity

Background

The Bacillus genus of Firmicutes bacteria is ubiquitous in nature and includes one of the best characterized model organisms, B. subtilis, as well as medically significant human pathogens, the most notorious being B. anthracis and B. cereus. As the most abundant living entities on the planet, bacteriophages are known to heavily influence the ecology and evolution of their hosts, including providing virulence factors. Thus, the identification and analysis of Bacillus phages is critical to understanding the evolution of Bacillus species, including pathogenic strains.

Results

Whole genome nucleotide and proteome comparison of the 83 extant, fully sequenced Bacillus phages revealed 10 distinct clusters, 24 subclusters and 15 singleton phages. Host analysis of these clusters supports host boundaries at the subcluster level and suggests phages as vectors for genetic transfer within the Bacillus cereus group, with B. anthracis as a distant member. Analysis of the proteins conserved among these phages reveals enormous diversity and the uncharacterized nature of these phages, with a total of 4,442 protein families (phams) of which only 894 (20%) had a predicted function. In addition, 2,583 (58%) of phams were orphams (phams containing a single member). The most populated phams were those encoding proteins involved in DNA metabolism, virion structure and assembly, cell lysis, or host function. These included several genes that may contribute to the pathogenicity of Bacillus strains.

Conclusions

This analysis provides a basis for understanding and characterizing Bacillus and other related phages as well as their contributions to the evolution and pathogenicity of Bacillus cereus group bacteria. The presence of sparsely populated clusters, the high ratio of singletons to clusters, and the large number of uncharacterized, conserved proteins confirms the need for more Bacillus phage isolation in order to understand the full extent of their diversity as well as their impact on host evolution.     

Collagen-Like Proteins (ClpA,ClpB, ClpC,and ClpD) Are Required for Biofilm Formation and Adhesion to Plant Roots by Bacillus amyloliquefaciens FZB42

The genes of collagen-like proteins (CLPs) have been identified in a broad range of bacteria, including some human pathogens. They are important for biofilm formation and bacterial adhesion to host cells in some human pathogenic bacteria, including several Bacillus spp. strains. Interestingly, some bacterial CLP-encoding genes (clps) have also been found in non-human pathogenic strains such as B. cereus and B. amyloliquefaciens, which are types of plant-growth promoting rhizobacteria (PGPR). In this study, we investigated a putative cluster of clps in B. amyloliquefaciens strain FZB42 and a collagen-related structural motif containing glycine-X-threonine repeats was found in the genes RBAM_007740, RBAM_007750, RBAM_007760, and RBAM_007770. Interestingly, biofilm formation was disrupted when these genes were inactivated separately. Scanning electron microscopy and hydrophobicity value detection were used to assess the bacterial cell shape morphology and cell surface architecture of clps mutant cells. The results showed that the CLPs appeared to have roles in bacterial autoaggregation, as well as adherence to the surface of abiotic materials and the roots of Arabidopsis thaliana. Thus, we suggest that the CLPs located in the outer layer of the bacterial cell (including the cell wall, outer membrane, flagella, or other associated structures) play important roles in biofilm formation and bacteria-plant interactions. This is the first study to analyze the function of a collagen-like motif-containing protein in a PGPR bacterium. Knocking out each clp gene produced distinctive morphological phenotypes, which demonstrated that each product may play specific roles in biofilm formation. Our in silico analysis suggested that these four tandemly ranked genes might not belong to an operon, but further studies are required at the molecular level to test this hypothesis. These results provide insights into the functions of clps during interactions between bacteria and plants.     

Glycanase activities associated with cell walls ofCicer arietinum L. epicotyls

The effect of the proteins extracted with 1 M LiCl from theCicer arietinum etiolated epicotyl cell walls on the degradation of polysaccharides extracted with alkali was studied. The hemicellulosic polysaccharides were fractionated into three fractions extracted with 4 % KOH, 4 % KOH containing 8 M urea, and 24 % KOH. The protein extract showed exo-glycanase activity against all three hemicellulosic fractions whilst endo-glycanase activity was shown mainly against the hemicellulosic polysaccharides extracted with 4% KOH.     

Effects of Bacillus amyloliquefaciens FZB42 on Lettuce Growth and Health under Pathogen Pressure and Its Impact on the Rhizosphere Bacterial Community

The soil-borne pathogen Rhizoctonia solani is responsible for crop losses on a wide range of important crops worldwide. The lack of effective control strategies and the increasing demand for organically grown food has stimulated research on biological control. The aim of the present study was to evaluate the rhizosphere competence of the commercially available inoculant Bacillus amyloliquefaciens FZB42 on lettuce growth and health together with its impact on the indigenous rhizosphere bacterial community in field and pot experiments. Results of both experiments demonstrated that FZB42 is able to effectively colonize the rhizosphere (7.45 to 6.61 Log ₁₀ CFU g⁻¹ root dry mass) within the growth period of lettuce in the field. The disease severity (DS) of bottom rot on lettuce was significantly reduced from severe symptoms with DS category 5 to slight symptom expression with DS category 3 on average through treatment of young plants with FZB42 before and after planting. The 16S rRNA gene based fingerprinting method terminal restriction fragment length polymorphism (T-RFLP) showed that the treatment with FZB42 did not have a major impact on the indigenous rhizosphere bacterial community. However, the bacterial community showed a clear temporal shift. The results also indicated that the pathogen R. solani AG1-IB affects the rhizosphere microbial community after inoculation. Thus, we revealed that the inoculant FZB42 could establish itself successfully in the rhizosphere without showing any durable effect on the rhizosphere bacterial community.     

Characterization of a Chitin-Binding Protein from Bacillus thuringiensis HD-1

Strains of Bacillus thuringiensis produce insecticidal proteins. These strains have been isolated from diverse ecological niches, such as soil, phylloplane, insect cadavers and grain dust. To effectively propagate, these strains produce a range of molecules that facilitate its multiplication in a competing environment. In this report, we have examined synthesis of a chitin-binding protein and evaluated its effect on fungi encountered in environment and its interaction with insecticidal proteins synthesized by B. thuringiensis. The gene encoding chitin-binding protein has been cloned and expressed. The purified protein has been demonstrated to interact with Cry insecticidal protein, Cry1Ac by Circular Dichrosim spectroscopy (CD) and in vitro pull down assays. The chitin-binding protein potentiates insecticidal activity of bacillar insecticidal protein, Cry1Ac. Further, chitin-binding protein was fungistatic against several soil fungi. The chitin binding protein is expressed in spore mother cell and deposited along with insecticidal protein, Cry1Ac. It interacts with Cry1Ac to potentiate its insecticidal activity and facilitate propagation of Bacillus strain in environment by inhibiting growth of certain fungi.     

Application of Bacillus sp. strain VT-8 for decontamination of TNT-polluted sites

Bacterial strain Bacillus sp. VT-8 was shown to use trinitrotoluene (TNT) as the sole source of carbon, nitrogen, and energy, as well as to carry out its co-oxidation. Resistance of Bacillus sp. VT-8 to high TNT concentrations was shown. Efficient detoxification of TNT-contaminated soil and water samples was demonstrated. This strain may be recommended for TNT biodegradation at concentrations of up to 140 mg/L due to its high degradation activity and the absence of toxic effect of TNT on Bacillus sp. VT-8.     

Prof. Dr. H. C. Władysław Węgorek 16 February 1918-16 July 2001

Abstract

In 2004, two field trials with cotton were conducted at two locations in Tajikistan to estimate the effectiveness of FZB 24® Bacillus subtilis (ABiTEP GmbH Berlin) as a biofertilizer for increasing the cotton yield in comparison to the utilization of the conventional fertilizer containing nitrogen (N), phosphorus (P) and potassium (K), with an application rate per ha 180 kg N, 120 kg P, 60 kg K. The following treatments of cotton cultivated as test variants each on 1 ha field under the same conditions were carried out and the effect on the plant growth, the development of number of bolls/plant and mainly the cotton fibre yield were calculated: (i) Seed treatment with water and only crop application of the NPK fertilizer; (ii) Only seed treatment with spore suspension of FZB 24 WG Bacillus subtilis, without application of the NPK fertilizer; (iii) Seed treatment with spore suspension of FZB 24 WG Bacillus subtilis plus crop application of the NPK fertilizer; and (iv) Only seed treatment with the comparable plant-growth-promoting rhizobacteria product Extrasol 55 (ARRIAM St Petersburg), without application of the fertilizer. Comparing cotton growth and yield of the variants in both field tests, there appeared a remarkable yield and growth increase (up to 30%) by the use of FZB 24 alone compared to the application of the NPK fertilizer. The additional application of the NPK fertilizer (Variant: FZB 24 + NPK) could not enlarge this plant productivity-promoting effect. Apparently under the test conditions the increase of the root system in size and capacity as well as the additional enzymatic mobilizing of organic bound phosphorus for the plant as activities from the Bacillus subtilis introduction was more important for the plant system than the limited direct input with N, P and K. The plant treatment alone with Extrasol 55 resulted in similar promotion of cotton growth and yield as FZB 24 when compared with the NPK plant fertilizing, showing that the activity of FZB 24 was not very specific. However the cotton field tests generally demonstrate for FZB 24 Bacillus subtilis a high ability to improve plant growth and yield based on increasing the capacity of roots to mobilize and take up nutrients and substances for overall reproductive plant fitness. The use and value for part-substitutions of some environmentally problematical mineral fertilizers in other and intensive plant production systems is discussed.     

Genomic and Enzymatic Results Show Bacillus cellulosilyticus Uses a Novel Set of LPXTA Carbohydrases to Hydrolyze Polysaccharides

Background

Alkaliphilic Bacillus species are intrinsically interesting due to the bioenergetic problems posed by growth at high pH and high salt. Three alkaline cellulases have been cloned, sequenced and expressed from Bacillus cellulosilyticus N-4 (Bcell) making it an excellent target for genomic sequencing and mining of biomass-degrading enzymes.

Methodology/Principal Findings

The genome of Bcell is a single chromosome of 4.7 Mb with no plasmids present and three large phage insertions. The most unusual feature of the genome is the presence of 23 LPXTA membrane anchor proteins; 17 of these are annotated as involved in polysaccharide degradation. These two values are significantly higher than seen in any other Bacillus species. This high number of membrane anchor proteins is seen only in pathogenic Gram-positive organisms such as Listeria monocytogenes or Staphylococcus aureus. Bcell also possesses four sortase D subfamily 4 enzymes that incorporate LPXTA-bearing proteins into the cell wall; three of these are closely related to each other and unique to Bcell. Cell fractionation and enzymatic assay of Bcell cultures show that the majority of polysaccharide degradation is associated with the cell wall LPXTA-enzymes, an unusual feature in Gram-positive aerobes. Genomic analysis and growth studies both strongly argue against Bcell being a truly cellulolytic organism, in spite of its name. Preliminary results suggest that fungal mycelia may be the natural substrate for this organism.

Conclusions/Significance

Bacillus cellulosilyticus N-4, in spite of its name, does not possess any of the genes necessary for crystalline cellulose degradation, demonstrating the risk of classifying microorganisms without the benefit of genomic analysis. Bcell is the first Gram-positive aerobic organism shown to use predominantly cell-bound, non-cellulosomal enzymes for polysaccharide degradation. The LPXTA-sortase system utilized by Bcell may have applications both in anchoring cellulases and other biomass-degrading enzymes to Bcell itself and in anchoring proteins other Gram-positive organisms.     

The highly modified microcin peptide plantazolicin is associated with nematicidal activity of Bacillus amyloliquefaciens FZB42

Bacillus amyloliquefaciens FZB42 has been shown to stimulate plant growth and to suppress the growth of plant pathogenic organisms including nematodes. However, the mechanism underlying its effect against nematodes remains unknown. In this study, we screened a random mutant library of B. amyloliquefaciens FZB42 generated by the mariner transposon TnYLB-1 and identified a mutant strain F5 with attenuated nematicidal activity. Reversible polymerase chain reaction revealed that three candidate genes RAMB_007470, yhdY, and prkA that were disrupted by the transposon in strain F5 potentially contributed to its decreased nematicidal activity. Bioassay of mutants impaired in the three candidate genes demonstrated that directed deletion of gene RBAM_007470 resulted in loss of nematicidal activity comparable with that of the F5 triple mutant. RBAM_007470 has been reported as being involved in biosynthesis of plantazolicin, a thiazole/oxazole-modified microcin with hitherto unknown function. Electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) analyses of surface extracts revealed that plantazolicin bearing a molecular weight of 1,354 Da was present in wild-type B. amyloliquefaciens FZB42, but absent in the ΔRABM_007470 mutant. Furthermore, bioassay of the organic extract containing plantazolicin also showed a moderate nematicidal activity. We conclude that a novel gene RBAM_007470 and its related metabolite are involved in the antagonistic effect exerted by B. amyloliquefaciens FZB42 against nematodes.     

A novel secreted metzincin metalloproteinase from Bacillus intermedius

The mprBi gene from Bacillus intermedius 3-19 encoding a novel secreted metalloproteinase was identified. The mpriBi gene was expressed in an extracellular proteinase-deficient Bacillus subtilis BG 2036 strain and the corresponding protein was characterized biochemically. The 19 kDa MprBi protein was purified to homogeneity and sequenced by mass spectroscopy and Edman degradation methods. Amino acid sequence analysis of MprBi identified an active site motif HEYGHNFGLPHD and a conserved structural component Met-turn, both of which are unique features of the metzincin clan. Furthermore, MprBi harbors a number of distinct sequence elements characteristic of proteinase domains in eukaryotic adamalysins. We conclude that MprBi and similar proteins from other Bacillus species form a novel group of metzincin metalloproteinases in prokaryotes.     

Genomic comparison of 93 Bacillus phages reveals 12 clusters, 14 singletons and remarkable diversity

Background

The Bacillus genus of Firmicutes bacteria is ubiquitous in nature and includes one of the best characterized model organisms, B. subtilis, as well as medically significant human pathogens, the most notorious being B. anthracis and B. cereus. As the most abundant living entities on the planet, bacteriophages are known to heavily influence the ecology and evolution of their hosts, including providing virulence factors. Thus, the identification and analysis of Bacillus phages is critical to understanding the evolution of Bacillus species, including pathogenic strains.

Results

Whole genome nucleotide and proteome comparison of the 93 extant Bacillus phages revealed 12 distinct clusters, 28 subclusters and 14 singleton phages. Host analysis of these clusters supports host boundaries at the subcluster level and suggests phages as vectors for genetic transfer within the Bacillus cereus group, with B. anthracis as a distant member of the group. Analysis of the proteins conserved among these phages reveals enormous diversity and the uncharacterized nature of these phages, with a total of 4,922 protein families (phams) of which only 951 (19%) had a predicted function. In addition, 3,058 (62%) of phams were orphams (phams containing a gene product from a single phage). The most populated phams were those encoding proteins involved in DNA metabolism, virion structure and assembly, cell lysis, or host function. These included several genes that may contribute to the pathogenicity of Bacillus strains.

Conclusions

This analysis provides a basis for understanding and characterizing Bacillus phages and other related phages as well as their contributions to the evolution and pathogenicity of Bacillus cereus group bacteria. The presence of sparsely populated clusters, the high ratio of singletons to clusters, and the large number of uncharacterized, conserved proteins confirms the need for more Bacillus phage isolation in order to understand the full extent of their diversity as well as their impact on host evolution.     

Extracellular hydrolases of strain <Emphasis Type="Italic">Bacillus</Emphasis> sp. 739 and their involvement in the lysis of micromycete cell walls

The mycolytic bacterial strain Bacillus sp. 739 produces extracellular enzymes which degrade in vitro the cell walls of a number of phytopathogenic and saprophytic fungi. When Bacillus sp. 739 was cultivated with Bipolaris sorokiniana, a cereal root-rot pathogen, the fungus degradation process correlated with the levels of the β-1,3-glucanase and protease activity. The comparative characteristic of Bacillus sp. 739 enzymatic preparations showed that efficient hydrolysis of the fungus cell walls was the result of the action of the complex of enzymes produced by the strain when grown on chitin-containing media. Among the enzymes of this complex, chitinases and β-1,3-glucanases hydrolyzed most actively the disintegrated cell walls of B. sorokiniana. However, only β-1,3-glucanases were able to degrade the cell walls of native fungal mycelium in the absence of other hydrolases, which is indicative of their key role in the mycolytic activity of Bacillus sp. 739.     

Sequence Analysis and Comparative Bioinformatics Study of Camelysin Gene (<Emphasis Type="Italic">calY</Emphasis>) Isolated from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Bacillus strains have been widely used for the production of fibrinolytic enzymes having role in the treatment of cardiovascular disorders. Purification and overproduction of such enzymes has increased their usage in medical fields including metalloproteinases with the ability to degrade extracellular matrix (ECM). Camelysin, a neutral metalloproteinase has been isolated from different species of bacteria like Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis with fibrinolytic, collagenolytic and actin degradation activity. This project successfully demonstrated the presence of 734-bp coding DNA sequence (CDS) encoding a 20.72331 kDa camelysin gene in local strain of Bacillus thuringiensis containing a signal peptide with cleavage site between residues 19 and 20. The sequence was submitted to GenBank (KT023597) and the sequence showed high homology with the camelysin protein of closely related Bacillus species. The alignment of related proteins through ClustalW displayed difference of four amino acids (“Q” replaced by “P” at position 169 and at position 182–184, “NQE” replaced by “HLK”) in the isolated protein. Comparison including structural and functional analysis of camelysin sequences isolated from different Bacillus species was carried out using different bioinformatics tools and software. The information would help in better understanding the properties of camelysin protein and its role in pathogenicity and clinical treatments.     

Influence of a Cell-Wall-Associated Protease on Production of α-Amylase by Bacillus subtilis

AmyL, an extracellular α-amylase from Bacillus licheniformis, is resistant to extracellular proteases secreted by Bacillus subtilis during growth. Nevertheless, when AmyL is produced and secreted by B. subtilis, it is subject to considerable cell-associated proteolysis. Cell-wall-bound proteins CWBP52 and CWBP23 are the processed products of the B. subtilis wprA gene. Although no activity has been ascribed to CWBP23, CWBP52 exhibits serine protease activity. Using a strain encoding an inducible wprA gene, we show that a product of wprA, most likely CWBP52, is involved in the posttranslocational stability of AmyL. A construct in which wprA is not expressed exhibits an increased yield of α-amylase. The potential role of wprA in protein secretion is discussed, together with implications for the use of B. subtilis and related bacteria as hosts for the secretion of heterologous proteins.The cell envelope of the gram-positive bacterium Bacillus subtilis consists of a single (cytoplasmic) membrane surrounded by a relatively thick cell wall consisting of similar proportions of peptidoglycan and covalently attached anionic polymers. The absence of an outer membrane means that there is no equivalent of the membrane-enclosed periplasm found in gram-negative bacteria. However, by virtue of its thickness and high density of negative charge, the cell wall may perform some of the roles of the periplasm in gram-positive bacteria.The absence of an outer membrane in gram-positive bacteria also simplifies the secretion pathway, and, consequently, B. subtilis and its close relatives have the potential to secrete proteins directly into the growth medium, at concentrations in excess of 5 grams per liter (4). Despite its extensive use in the production of commercially important Bacillus enzymes (e.g., α-amylases and alkaline proteases), attempts to exploit B. subtilis for the production of heterologous proteins at high concentrations have proved disappointing (8). One reason for this failure is the production and release into the culture medium of several extracellular proteases (24, 28, 37). Although native Bacillus proteins are generally resistant to these proteases, heterologous proteins are often rapidly degraded in their presence. As a result, strains of B. subtilis that are multiply deficient in extracellular proteases have been developed (11, 37). The more developed of these strains have less than 1% of the proteolytic activity of the wild type (37). To date, efforts have concentrated mainly on the proteases which reside in a truly extracellular location, while those which remain cell associated have been largely overlooked.Although strains deficient in extracellular proteases have improved the productivity of B. subtilis for the production of heterologous proteins, they have only partially overcome problems of unexpectedly low yields. We and others have recently shown (22, 31) that significant amounts of secretory protein are degraded within minutes of being synthesized. This degradation is observed even for Bacillus proteins that are highly resistant to proteases released into the culture medium, suggesting that a component of this degradation is cell associated.Margot and Karamata recently reported the identification of a cell-wall-associated protease encoded by the wprA gene (21). The primary product of this gene is a 96-kDa polypeptide that is processed into two previously identified cell wall proteins, namely, CWBP52 and CWBP23. The processing of the WprA precursor during secretion accompanies the targeting of CWBP52 and CWBP23 to the cell wall and is analagous to the processing of another B. subtilis cell-wall-bound protein, namely, WapA (5). The amino acid sequence of CWBP52 shows a high degree of similarity with serine proteases of the subtilisin family, and phenylmethylsulfonile fluoride (PMSF)-sensitive protease activity was detected in proteins extracted from the cell wall of a wprA⁺ strain, but not one in which this gene had been insertionally inactivated (21). In the absence of homology to proteins in the databases, the N-terminal CWBP23 moiety was presumed to function as a chaperone-like propeptide that is proteolytically processed on the trans side of the membrane. In this paper, we report on a potential role of products of wprA in the integrity of secretory proteins during late stages in the secretion pathway. We also discuss the potential of wprA mutants to increase the productivity of B. subtilis for secretory proteins.