4. Discussion

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As a potent plant growth-promoting rhizobacteria, the cellular functions of B. amyloliquefaciens FZB42 are important to be studied. Several modes of action such as phytase production, auxin synthesis and production of antibiotics which support its beneficial activity towards the growth of the plant have been elucidated (Idriss et al.  2002; Idriss et al. 2007; Koumutsi et al. 2004 and Chen et al. 2009b). However, not all of the molecular mechanisms are completely understood. In this work, I presented additional information regarding molecular mechanisms of plant growth promotion activity of B. amyloliquefaciens FZB42 generated by transposon mutagenesis.

4.1  Himar1 transposon mutagenesis of  B. amyloliquefaciens FZB42 

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Transposon mutagenesis is a powerful tool in molecular biology used to create random insertion mutagenesis. It has been applied to and established in vitro and in vivo in most scientifically and industrially relevant organisms ranging from prokaryote to eukaryote (Petzke and Luzhetskyy, 2009). Among many transposons used, mariner -based transposons gain much attention recent time. Himar1 has been useful for in vitro and in vivo transposon mutagenesis of a variety of bacteria due to its advantage features in transposition which does not require host-specific factors and inserts more randomly into the chromosome, since the mariner  transposon requires only the dinucleotide TA (Akerley et al. 1996; Ashour and Hondalus 2003; Zhang et al. 2000). In the present work, I applied the mariner transposable element Himar1 (TnYLB-1) transposon that has been previously used in B. subtilis (Le Breton et al. 2006) to create a transposon library in B. amyloliquefaciens FZB42.

Three different plasmids containing a mariner-based Himar1 tranposon (pMarA, pMarB and pMarC) were used in transposon mutagenesis of B. amyloliquefaciens FZB42. Both pMarA and pMarB contained the Himar1 transposase gene; however they have different promoters that drive the expression of transposase. The pMarA plasmid is under the control of the housekeeping σ factor σA of B.subtilis, whereas pMarB employes σ factor σB. The pMarC does not contain the transposase gene as well as its promoter. I have been successfully transformed plasmids pMarA and pMarC into B. amyloliquefaciens FZB42, but due to unknown reasons I failed to transform pMarB. However, this failure might be due to the different promoter used in the plasmid.

The frequency of transposition of TnYLB-1 in B. amyloliquefaciens FZB42 in this work (10-2) is similar to the frequency of transposition in B. subtilis, indicating that TnYLB-1 is inserted in the rate to the genome B. amyloliquefaciens FZB42 as in B. subtilis. Interestingly, the Kanr clones of B. amyloliquefaciens FZB42 which represent the transposition events were recovered at an efficiency of 2.6 x 107 which is higher than in B. subtilis (6.2 x 106). In order to examine the utility of TnYLB-1 for mutant isolation and to prove that it is an efficient tool to generate transposon library in B. amyloliquefaciens FZB42, the temperature-resistant Kanr clones were transferred to a glucose minimum medium to screen for auxotrophic mutations. From 778 Kanr colonies, I observed seven mutants that have failed to grow under this condition that corresponded to 0.89% recovery of auxotrophs. In contrast, lower frequency of auxotrophy (0.15%) was obtained in a screen of Francisella tularensis transposon mutants using Himar1 transposon on Chamberlain’s chemically defined medium (Maier et al. 2006). However, similar frequency of auxotrophy of transposon mutants was reported in the screening of Burkholderia pseudomallei on M9 minimal-glucose kanamycin media (0.72%) and Bacillus subtilis on glucose-minimal media (1%) (Rholl et al. 2008 and Le Breton et al. 2006). Moreover, the result obtained here is comparable to the isolation of auxotrophs in other bacteria using other transposon system which often results in 1 to 2% recovery rate (Petzke and Luzhetskyy 2009).

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Further characterization of two out of seven auxotroph mutants by Southern Blot analysis and mapping of genomic insertion sites showed a single transposon insertion in the genome. The insertion sites of the two auxotroph mutants were located in the pabB and hisJ genes, respectively. These genes encode para-aminobenzoate synthase (subunit A) and histidinol phosphate phosphatase involved in folate and histidine biosynthetic pathways, respectively. The respective mutants are therefore para-aminobenzoate (pabB) and histidine (hisJ) auxotroph. These auxotrophies were experimentally confirmed since the growth of the pabB and hisJ mutants in glucose minimal medium was restored by the addition of para-aminobenzoic acid (PABA) and histidine, respectively. These results have demonstrated the utility of TnYLB-1 for transposon mutagenesis in B. amyloliquefaciens FZB42. After examining that the TnYLB-1 has been transposed in the genome of B. amyloliquefaciens FZB42, I began to implement transposon mutagenesis strategy to generate a transposon library for the desired phenotype.

4.2 Identification of genes involved in swarming motility and biofilm formation in B. amyloliquefaciens FZB42 genome 

In order to detect genes that may be potentially involved in plant-associated lifestyle, especially in rizhosphere competence, I applied the TnYLB-1 transposon to a generate transposon library for screening the transposants with swarming and biofilm mutant phenotype. Swarming is the fastest known bacterial mode of surface translocation that enables the rapid colonization of a nutrient-rich environment and host tissues. This complex multicellular behavior requires the integration of chemical and physical signals, which leads to the physiological and morphological differentiation of the bacteria into swarmer cells (Verstraeten et al. 2009). They become multinucleate, elongate, synthesize large numbers of flagella, secrete surfactants and advance across the surface in coordinated packs (Berg 2005). Bacteria capable of swarming motility produce highly organized communities initially consisting of vegetative cells, i.e. the swimmer cells, which undergo a co-ordinated surface induced differentiation process characterized by the production of hyperflagellated, elongated, multinucleate cells, i.e. the swarm cells (Senesi et al. 2002). Swarming motility as a flagellum-dependent behavior has been implicated in biofilm formation, including colonization and bacterial virulence (Kirov et al. 2002; Merritt et al. 2007). In Pseudomonas aeruginosa, Salmonella typhimurium and Proteus mirabilis, swarming ability as a part of motility behavior is associated with their pathogenesis due to the level of specific virulence factors production that is higher during their swarm-cell state (Allison et al. 1994; Wang et al. 2004; Yeung et al. 2009). In addition, swarmer cells of P. aeruginosa also exhibits elevated adaptive antibiotic resistance to ciprofloxacin, gentamicin and polymyxin B, thus swarming is part of an alternative growth state and is a complex adaptation process that occurs in response to specific stimuli (Overhage et al. 2008).

There are many genetic determinants that govern the swarming motility. In P. aeruginosa the swarming associated genes functioned not only in flagellar or type IV pilus biosynthesis but also in transport, secretion and metabolism process. As much as 35 transcriptional regulators were found to be involved in swarming, including a variety of two-component sensors and response regulators (Yeung et al. 2009). In my screening for swarming mutant, one swarming-deficient mutant with transposon insertion was found in the degU gene. Interestingly, this mutant also showed impairing in biofilm production. This result is in contrast to the finding of Mireles et al. (2001) where a swarming mutant of Salmonella enterica defective in lipopolysaccharide (LPS) synthesis promotes biofilm formation. In that research, they found that O-antigen mutants of S. enterica, which are defective in swarming, were generally more proficient in biofilm formation than the wild type. The O-antigen has been postulated to provide a surfactant or wettability function that allows spreading of the swarmer colony. Surfactin, a cyclic lipopeptide from B. subtilis which rescued the swarming defect of the LPS mutants, inhibited biofilm formation. They have concluded that the absence of surfactants inhibits swarming but promotes biofilm formation and vice versa.

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The degU gene which is involved in two-component response regulation has a key role in regulating several post-exponential phase processes in B. subtilis, including the activation and the inhibition of genetic competence, the inhibition of flagellar-based-motility, the activation of degradative enzyme production and activation of poly-γ-glutamic acid production (Verhamme et al. 2007). As swarming motility is a flagella-dependent behaviour, the inhibition of the genes required for the assembly of the flagella will also affect the swarming behaviour as shown in B. amyloliquefaciens FZB42 degU mutant. The degU mutant and retransformation of the degU showed that both mutants failed to colonize the ‘swarm plates’ (LB medium with 0.7% agar) after 24 hours incubation, whereas the wild type and complementation of degU colonized the entire swarm plates (Fig. 8). Amati et al. 2004 have reported that phosphorylated DegU (DegU~P) acts as a repressor of transcription of the fla-che operon which comprises the majority of the genes whose products are structural components of the flagellum, in addition to several genes involved in chemotaxis. Hence, repression of fla-che operon prevents the expression of genes coding for the hook and basal body components of the flagellum and for the σD transcription factor. A repressive role of DegU~P on the fla-che operon is supported by the fact that of this operon genes were among those that were down-regulated by the overproduction of degU (Maeder et al. 2002). In B. subtilis, very low levels of DegU~P activate swarming, while high levels of DegU~P inhibit swarming, however, the genes required for swarming motility that are regulated by very low levels of DegU~P are currently unknown (Verhamme et al. 2007).

Biofilms are structured communities of cells that are adherent to a surface, an interface, or each other are encased in a self-produced polymeric matrix (Stanley et al. 2003). Initiation of biofilm formation is characterized by interaction of cells with a surface or an interface as well as with each other. Once enough cells have aggregated, the biofilm begins to mature through the production of an extracellular matrix, which contributes greatly to the final architecture of the community (Branda et al. 2005). The biofilm offers many advantages for the bacteria. The first, bacteria experience a certain degree of shelter and homeostatis when residing within biofilm and one of the key components of this microniche is the surrounding extrapolymeric substance matrix. This matrix is composed of a mixture of components, such as exopolysaccharide (EPS), protein, nucleic acids and other substances. The EPS provides protection from a variety of environmental stresses, such as UV radiation, pH shift, osmotic shock, desiccation and also antibiotic (Davey and O’toole 2000; Fleming 1993). The second, highly permeable water channels interspersed throughout the biofilm in the areas surrounding microcolonies gives an effective means of exchanging nutrients and metabolites with the bulk aqueous phase, enhancing nutrient availability as well as removal of potentially toxic metabolites (Costerton et al. 1995). And lastly, biofilm is also a place where gene transfer takes place. Hence, it helps acquisition of new genetic traits (Davey and O’toole 2000).

In general, there are four universal systems routinely used to study the biofilm formation. Depending on the aims of the experiment, these system can be roughly categorized as flow cells biofilm which are usually analyzed using CSLM, ‘Microtiter’ biofilms which are formed on the surfaces of microtiter dish wells under standing culture conditions, Floating biofilms or pellicles which are formed at the air–liquid interface of a standing culture and finally, the bacterial colonies which grow on the surface of agar-solidified media (Branda et al. 2005). In my biofilm screening, I have applied screening of floating or pellicles formation in standing culture. Three biofilm mutants that were found in this screening showed defects in biofilm formation compared with the wild type. These mutants were unable to build wrinkled-upper layer of pellicles in the MSgg medium.

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The biofilm degU mutant was impaired in its ability to form robust pellicles when incubated in screening medium. This mutant formed a thin membrane in the upper layer of the medium with little granules located in the center of surface (Fig. 9 B). The complementation using intact degU gene from the wild type restored the ability of mutant to form robust pellicle (Fig. 9 C) confirming the role of degU gene in formation of biofilm. The response regulator DegU controls the production of exoenzymes such as proteases, levan-sucrase, and α-amylase and it is also involved in competence development and motility. DegU is the second element of a two-component signaling system that is phosphorylated by the first component, DegS (Amati et al. 2004). The finding that the degU gene is involved in biofilm formation is in accordance with the result of Kobayashi (2007) and Verhamme et al. (2007) who investigated the role of degU in pellicle formation. In B. subtilis a low level of phosphorylated degU (DegU~P) is needed to activate the yvcA gene, coding for a membrane-bound lipoprotein, which is required for complex colony architecture. How yvcA functions to control this process is still unclear, although it is possible that it could form a structural component of the extracellular matrix or could be involved in a signaling process. However, the yvcA gene is not present in the genome of B. amyloliquefaciens FZB42 (Verhamme et al. 2007). Kobayashi (2007) found the yuaB gene which had a role in pellicle formation under regulation of degU. It is required for a later stage of pellicle formation, such as floating of cell clusters to the surface of the medium. The degU mutation abolishes both flagellum and pellicle formation, suggesting that regulation of DegU activity may be a key to the transition between the two cell states.

The phenotype of biofilm yusV and pabB mutants showed almost the same appearances where both mutants formed flat and thin pellicles on the surface of MSgg media (Fig. 13 B and 17 B). These two mutants were not able to form robust and wrinkled pellicles as the wild type. Insertion of the intact yusV and pabB genes from the wild type restored pellicle formation in each mutant (Fig. 13 C and 17 C), confirming that both genes might be essential in biofilm formation. The function of the yusV gene is still unknown, however, it shows similarity to iron (III) dicitrate transport permease. In addition, the yusV nucleotide sequence shows 97% similarity to iron (III)-siderophore transporter (ATP binding component) of Bacillus amyloliquefaciens DSM7. The production of low-molecular-weight Fe (III) siderophores enables microorganisms to efficiently scavenge iron even in aerobic environments where iron exists primarily as insoluble hydroxides (Wandersman and Delepelaire 2004). As iron is an essential nutrient for nearly all living organisms, the transport systems for the uptake of iron and iron-chelation complexes are therefore critical for growth (Ollinger et al. 2006). Hence, disruption of transposon TnYLB-1 in yusV gene which caused defect in pellicles formation of B. amyloliquefaciens FZB42 might be due to the growth defects. However, the precise explanation how this gene is involved in pellicle formation is still unclear.

The pabB gene encodes for para-aminobenzoate (PABA) synthase. In E. coli PABA is made from chorismate in two steps. First, pabA and pabB interact to catalyze transfer of the amide nitrogen of glutamine to chorismate, forming 4-amino-4-deoxychorismate (ADC). Association of pabA and pabB with one another to form the ADC synthase are commonly called PABA synthase. Second, the PabC protein then mediates elimination of pyruvate and aromatization of ADC to give PABA (Basset et al. 2003). PABA is a substrate of 7,8-dihydropteroate synthase (DHPS) in the de novo biosynthesis of folate which is important for the formation of purines, thymidylate, serine, methionine, glycine and formylmethionyl-tRNA (James et al. 2002). Disruption of pabB gene with TnYLB-1 transposon leads to inhibition of ADC synthase, as the ammonia that is generated from glutamine by pabA gene can not be used to aminate chorismate, producing ADC. Since there is no production of ADC, PABA will not be produced. Beside complementation of intact pabB gene which restored pellicles formation in this mutant, addition of 0.1 mM para-aminobenzoic acid into the MSgg medium also repaired pellicles formation (Fig. 17 E). Hence, the role of pabB gene in biofilm formation is indirect as it affects the growth of the mutant.

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4.3 Identification of genes involved in plant growth promotion and colonization of the mutants in the root of A. thaliana

PGPR, which belong to diverse genera such as genera Pseudomonas and Bacillus, are rhizosphere-inhabiting bacteria that have drawn much attention in recent years because of their contribution to the biological control of plant pathogens and the improvement of plant growth (Persello-Cartieux et al. 2003). Several studies have already described the utility of Bacillus PGPR species for promoting plant growth. Determination of the mechanism of action of such bacteria revealed that they are able to solubilize phosphate (Idriss et al. 2002), produce plant hormone (IAA & gibberellins) (Idriss et al. 2007 and Chakraborty et al. 2005), siderophore and antifungal as well as antibacterial metabolite (Chen 2009c, Joo et al. 2005 and Danielsson et al. 2006). Nonetheless, the mechanisms used by Bacillus spp. to stimulate plant growth are not fully understood.

In this study, I used a miniaturized L. minor biotest system which has been proven to compare the phytostimulatory effects exerted by FZB42 wild-type and mutant strains (Idriss et al. 2007). Duckweeds (Lemnaceae) possess physiological properties (small size, high multiplication rates, and vegetative propagation), which make them an ideal test system. Particularly interesting is the mechanism of propagation: under favourable environmental conditions both frond primordia grow out, thus forming new fronds by clonal propagation and producing a population of genetically homogeneous plants (Naumann et al. 2006). Hence L. minor is an attractive subject for investigating plant-microbe interactions (Lockhart et al. 1989). In this system, a selected clone from duck weed, L. minor ST, was cultured in Steinberg medium and it yielded reproducible results in plant growth test assays. By screening using the L. minor biotest system, I found three mutants which showed deficient in plant growth promotion activity. After sequencing and searching with BLAST, I identified that the TnYLB-1 disrupted nfraA, abrB and RBAM_017410 genes. In order to validate, whether or not these genes were also responsible for the plant growth promotion phenotype in higher plant, I used A. thaliana to determine the effect of the mutants to its growth. The experiment with A. thaliana also offered a different system as in L. minor biotest system which used liquid media, whereas A. thaliana was grown in solid media.

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Effective colonization of plant roots by PGPR plays an important role in growth promotion, irrespective of the mechanism of action (production of metabolites, production of antibiotics against pathogens, nutrient uptake effects, or induced plant resistance) (Bolwerk et al. 2003). Bacterial colonization in natural environment is mainly facilitated by biofilm formation. Bacterial biofilms established on plant roots could protect the colonization sites and act as a sink for the nutrients in the rhizosphere, hence reducing the availability of root exudate nutritional elements for pathogen stimulation or subsequent colonization on the root (Haggag and Timmusk 2007). The ability to colonize plants is a multifactorial process requiring resistance to plant defence systems as well as the ability to initiate growth on plant surfaces, invade tissues and develop within the plant. In return for a safe and nutrient rich environment, the bacterium may provide the host with resistance to plant pathogens through the synthesis of antibiotics and enzymes and promote plant growth (Reva et al. 2004). As rhizosphere competence of biocontrol agents comprises effective root colonization combined with the ability to survive and proliferate along growing plant roots over a considerable time period, in this study I also examined the colonization of FZB42 and its mutants in A. thaliana roots using SEM and CLSM. The PGPR nfrA mutants exhibited reduction in plant growth promoting activity either in Lemna system (26%) or A. thaliana (40%). Complementation of the gene restored the growth promoting activity, whereas retransformation from the mutant recuded the growth promoting effect (Fig. 21-24). In B. subtilis, nfrA gene is a putative essential oxidoreductase which is induced under heat shock and oxidative stress conditions. This gene belongs to the class III heat shock genes and its transcription is induced in a σD-dependent manner at the onset of the stationary phase and also by heat stress from a σA-dependent promoter overlapping the σD promoter. Sigma factor σD is usually necessary to transcribe genes involved in motility and cell division (Moch et al. 2000; Moch et al. 1998). DNA microarray analysis revealed that nfrA is induced by superoxide stress and H2O2 stress. These results indicate that the putative essential NADPH-dependent oxidoreductase nfrA (ywcH) might play an important role in the oxidative stress response (Mostertz, et al. 2004). The nfrA was postulated to be an essential protein in B. subtilis but not in S. aureus. However, the enzyme could have a significant function in the bacterial stress response during phases of oxidative stress in infections caused by S. aureus (Streker et al. 2005). The precise role how nfrA gene involves in growth promoting activity is still unclear. However, Rudrappa et al. 2007 have proposed that oxidative stress results in down-regulating of biofilm formation of B. subtilis on A. thaliana roots. In his study, biofilm formation on A. thaliana NahG plants was suppressed by the presence of catechol on the root surface (and in the surrounding area), resulting in ROS (reactive oxygen species)-mediated downregulation of genes required for biofilm formation by B. subtilis. It has been demonstrated in rice and Arabidopsis systems that higher catechol levels result in the production of superoxides and H2O2 leading to increased ROS generation (Yang et al. 2004; van Wees and Glazebrook 2003). By SEM examination, a defect in biofilm formation and change in the shape of the cell were shown, when the nfrA mutant colonized A. thaliana roots (Fig. 46). Distribution of nfrA mutant cells on the A. thaliana roots was limited on border cells and root surface (Fig. 45). These impairments could be the causal of the reduction growth promoting activity in this mutant.

The second mutant which exhibited loss of plant growth activity was the abrB gene. Disruption of the abrB gene by TnYLB-1 transposon reduced the growth promoting activity in Lemna (26%) as well as in A. thaliana (17%) (Fig. 28-31). The abrB gene is a DNA-binding global regulator of a plethora of functions that are expressed during the transition from exponential growth to stationary phase and under suboptimal growth conditions (Strauch et al. 2005). During this period, the bacterial cells adopt for growth and survival in an altered, nutrient-deprived environment. It is supposed to be one of the most important transition state regulators, because it is involved in the regulation of various cellular functions, such as antibiotic production, competence development, expression of dipeptide transport proteins, proteases, and degradative enzymes, like histidase. AbrB displays its regulatory function by acting as a repressor, activator, or preventer (Klein et al. 2000). It is unclear how this gene is involved in plant growth stimulation as the pleiotropic abrB protein is known to play a role in regulating many antimicrobial metabolites in addition to numerous other genes expressed by postexponential-phase cells (Strauch et al. 2007). Hence, we suggest that the loss of growth-promoting activity by abrB mutant is more likely due to termination of the metabolic pathway involved in growth stimulation. Through CLSM we observed the limited distribution of the cells when colonizing the root. Most of abrB mutant cells resided mostly in border cells of the A. thaliana root (Fig. 47) accompanied by defects in biofilm formation within the cells (Fig. 48).

The PGPR RBAM_017410 mutant showed reduction of plant growth promoting activity both in Lemna (42%) and in A. thaliana (33%). The complementation of this gene not fully restored the growth promoting ability; however it grew better than control and mutant (Fig. 34-37). The RBAM_017410 is a small peptide with unknown function. Nevertheless, using BLASTX search analyses, the amino sequence of this small peptide shows 76% similarity to ribonucleoside-diphosphate reductase small subunit from B. subtilis. In E. coli, ribonucleoside diphosphate reductase (NDP reductase) is the only specific enzyme catalyzing the reduction of ribonucleotides to deoxyribonucleotides (dNTPs), the precursors of DNA synthesis, and thus essential to all known life (Guarino et al. 2007; Jiang et al. 2008). In the B. amyloliquefaciens FZB42 genome, RBAM_017410 gene is located in the region where the gene cluster has still unknown function (Fig. 32). The examination of colonization of this mutant on the root of A. thaliana showed no cells appearing on the root (Fig. 49). An alternative explanation for that would be a mishandling during the preparation of the root when monitoring the colonization or may be to the low fluorescence emittion from the cells. Using SEM, we found that the cells are impaired in their ability to form biofilm when colonizing the root (Fig. 50). In addition, the shape of the cell was slim rod which was different form the wild type.

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In this research, three PGPR mutants showed the same characteristics that were limited distribution and impairment in biofilm formation when colonizing on the root. Reva et al. 2004 found that the ability effective initiation of colonization of the root was associated with the ability to form biofilms. The bacterium had to attach to the seed surface, primary roots and root hairs, and survived from the bacteriocidal substances released by roots. An intriguing finding in this study was the capability of the biofilm mutants to exhibit growth promotion as well as the wild type, even when the colonization on the root was limited. Further investigation is required to reveal this phenomenon. Wang et al. 2005 discovered that quinolinate phosphoribosyltransferase (QAPRTase) was involved in plant growth-promoting activity in Burkholderia sp. strain PsJN. However, how QAPRTase participates in the growth promotion signal pathway is still unknown. Another study by Choi et al. 2008 showed the involvement of pyrroloquinoline quinone (PQQ) in plant growth promotion by P. fluorescens B16 and suggested that PQQ acts as an antioxidant in plants.

An interesting result which was observed from analyzing the metabolites released by B. amyloliquefaciens FZB42 when interacting with L. minor in the Steinberg medium was the production of surfactin. This finding confirmed the study of Bais et al. (2004) who demonstrated the role of surfactin from undomesticated B. subtilis for biofilm formation and colonization of A. thaliana roots. Other lipopeptides and polyketides produced by FZB42 grown in the Landy medium (Chen et al. 2006; Chen et al. 2007; Koumoutsi et al. 2007), were not detected by MALDI-TOF mass spectrometry performed with the plant extract from Lemna minor and the growth medium, inoculated with FZB42. This implies an important role of surfactin in colonizing plant roots by plant associated B. amyloliquefaciens strains. In contrast to bacillomycin D and fengycin, and the polyketides bacillaene, difficidin and macrolactin, which are all produced by FZB42 under laboratory conditions, antimicrobial action of surfactin is relatively weak. However, in B. subtilis 6051 the biocontrol ability toward P. syringae is related with surfactin formation (Bais et al. 2004). In addition, together with fengycin, surfactin of B. subtilis strain S449 elicits induced systemic resistance in plants (Ongena et al. 2007).

4.4 Identification of genes involved in production of antibiotic and nematocidal 

Generating mutants in B. amyloliquefaciens FZB42 strain CH5 by employing the transposon TnYLB-1 led to discover two new ribosomally synthesized secondary metabolites named amylocyclicin and plantazolicin. Both antibiotics do not depend on phosphopantetheinyl transferase, like cyclic lipopeptides and polyketides. Amylocyclicin A could be identified as the substance responsible for the reported strong activity of FZB42 against Bacillus subtilis HB0042 and also has a strong antibacterial activity against other gram-positive bacteria. This antibiotic belongs to the disparate group I of circular bacteroicins like CclA, lactocyclicin Q, AS-48, circularin A, and uberolysin, with a high pI. On the basis of the predicted secondary structure, amylocyclicin A also contains helical structures und presumably has a gobular structure. It could interact with the membrane of sensitive gram-positive bacteria and form nonselective pores in lipid bilayers, allowing the free diffusion of ions and low molecular weight solutes across the membrane like AS-48 and in a similar way gassericin A and reutericin 6 (Galvez et al. 1991; Kawai et al. 2004) leading to cell death or forms anion selective channels in lipid bilayers like CclAI (Gong et al. 2009). Amylocyclicin A is the first representative of circular bacteriocins of the genus Bacillus belonging to class 2d/IV of bacteriocins (Scholz et al. 2011). Plantazolicin is a novel, antibacterial, microcin B17/streptolysin-like compound that was identified in the culture supernatant and cell surface extract from B. amyloliquefaciens FZB42. The genetic and biochemical conservation within this particular natural product family has led to a new classification of small, highly modified bacteriocins, the thiazole/oxazole-modified microcins (TOMMs) (Lee et al. 2008; Haft et al. 2010).

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Another interesting result from the screening based on transposon TnYLB-1 mutants of B. amyloliquefaciens FB42 was the finding of four mutants which showed reduction in nematocidal effectiveness (Table. 7). Complementation and characterization of the genes and metabolites involved in nematocidal is still in progress conducted in Yunan University – China.


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