Mycosubtilin overproduction by Bacillus subtilis BBG100 enhances the organism's antagonistic and biocontrol activities

A Bacillus subtilis derivative was obtained from strain ATCC 6633 by replacement of the native promoter of the mycosubtilin operon by a constitutive promoter originating from the replication gene repU of the Staphylococcus aureus plasmid pUB110. The recombinant strain, designated BBG100, produced up to 15-fold more mycosubtilin than the wild type produced. The overproducing phenotype was related to enhancement of the antagonistic activities against several yeasts and pathogenic fungi. Hemolytic activities were also clearly increased in the modified strain. Mass spectrometry analyses of enriched mycosubtilin extracts showed similar patterns of lipopeptides for BBG100 and the wild type. Interestingly, these analyses also revealed a new form of mycosubtilin which was more easily detected in the BBG100 sample. When tested for its biocontrol potential, wild-type strain ATCC 6633 was almost ineffective for reducing a Pythium infection of tomato seedlings. However, treatment of seeds with the BBG100 overproducing strain resulted in a marked increase in the germination rate of seeds. This protective effect afforded by mycosubtilin overproduction was also visualized by the significantly greater fresh weight of emerging seedlings treated with BBG100 compared to controls or seedlings inoculated with the wild-type strain.     

Improved production of carotenoid-free welan gum in a genetic-engineered <Emphasis Type="Italic">Alcaligenes</Emphasis> sp. ATCC31555

Objective

To improve the production of welan gum and obtain a carotenoid-free strain while reducing the fermentation and post-treatment costs.

Results

The vitreoscilla globin (vgb) gene combined with the β-galactosidase (lacZ) promoter was inserted into the phytoene synthase (crtB) gene region of the chromosome in Alcaligenes sp. ATCC31555. When the recombinant strain was grown in a 5 l fermentor, welan gum was produced at 24 ± 0.4 g l^?1 compared to 21 g ± 0.4 g l^?1 in the wild type. Furthermore, the carotenoid-free welan gum produced using Alcaligenes sp. ATCC31555 VHb strain was less expensive with improved properties.

Conclusions

Alcaligenes sp. ATCC31555 VHb strain was a better neutral welan-producing strain with a higher production than the wild-type strain.

     

Mycosubtilin and surfactin are efficient,low ecotoxicity molecules for the biocontrol of lettuce downy mildew

The use of surfactin and mycosubtilin as an eco-friendly alternative to control lettuce downy mildew caused by the obligate pathogen Bremia lactucae was investigated. Preliminary ecotoxicity evaluations obtained from three different tests revealed the rather low toxicity of these lipopeptides separately or in combination. The EC50 (concentration estimated to cause a 50 % response by the exposed test organisms) was about 100 mg L^?1 in Microtox assays and 6 mg L^?1 in Daphnia magna immobilization tests for mycosubtilin and 125 mg L^?1 and 25 mg L^?1 for surfactin, respectively. The toxicity of the mixture mycosubtilin/surfactin (1:1, w/w) was close to that obtained with mycosubtilin alone. In addition, the very low phytotoxic effect of these lipopeptides has been observed on germination and root growth of garden cress Lepidium sativum L. While a surfactin treatment did not influence the development of B. lactucae on lettuce plantlets, treatment with 100 mg L^?1 of mycosubtilin produced about seven times more healthy plantlets than the control samples, indicating that mycosubtilin strongly reduced the development of B. lactucae. The mixture mycosubtilin/surfactin (50:50 mg L^?1) gave the same result on B. lactucae development as 100 mg L^?1 of mycosubtilin. The results of ecotoxicity as well as those obtained in biocontrol experiments indicated that the presence of surfactin enhances the biological activities of mycosubtilin. Mycosubtilin and surfactin were thus found to be efficient compounds against lettuce downy mildew, with low toxicity compared to the toxicity values of chemical pesticides. This is the first time that Bacillus lipopeptides have been tested in vivo against an obligate pathogen and that ecotoxic values have been given for surfactin and mycosubtilin.     

Quinolobactin, a New Siderophore of Pseudomonas fluorescens ATCC 17400, the Production of Which Is Repressed by the Cognate Pyoverdine

Transposon mutant strain 3G6 of Pseudomonas fluorescens ATCC 17400 which was deficient in pyoverdine production, was found to produce another iron-chelating molecule; this molecule was identified as 8-hydroxy-4-methoxy-quinaldic acid (designated quinolobactin). The pyoverdine-deficient mutant produced a supplementary 75-kDa iron-repressed outer membrane protein (IROMP) in addition to the 85-kDa IROMP present in the wild type. The mutant was also characterized by substantially increased uptake of ⁵⁹Fe-quinolobactin. The 75-kDa IROMP was produced by the wild type after induction by quinolobactin-containing culture supernatants obtained from the pyoverdine-negative mutant or by purified quinolobactin. Conversely, adding purified wild-type pyoverdine to the growth medium resulted in suppression of the 75-kDa IROMP in the pyoverdine-deficient mutant; however, suppression was not observed when Pseudomonas aeruginosa PAO1 pyoverdine, a siderophore utilized by strain 3G6, was added to the culture. Therefore, we assume that the quinolobactin receptor is the 75-kDa IROMP and that the quinolobactin-mediated iron uptake system is repressed by the cognate pyoverdine.     

Endophytic Colonization and In Planta Nitrogen Fixation by a Herbaspirillum sp. Isolated from Wild Rice Species

Nitrogen-fixing bacteria were isolated from the stems of wild and cultivated rice on a modified Rennie medium. Based on 16S ribosomal DNA (rDNA) sequences, the diazotrophic isolates were phylogenetically close to four genera: Herbaspirillum, Ideonella, Enterobacter, and Azospirillum. Phenotypic properties and signature sequences of 16S rDNA indicated that three isolates (B65, B501, and B512) belong to the Herbaspirillum genus. To examine whether Herbaspirillum sp. strain B501 isolated from wild rice, Oryza officinalis, endophytically colonizes rice plants, the gfp gene encoding green fluorescent protein (GFP) was introduced into the bacteria. Observations by fluorescence stereomicroscopy showed that the GFP-tagged bacteria colonized shoots and seeds of aseptically grown seedlings of the original wild rice after inoculation of the seeds. Conversely, for cultivated rice Oryza sativa, no GFP fluorescence was observed for shoots and only weak signals were observed for seeds. Observations by fluorescence and electron microscopy revealed that Herbaspirillum sp. strain B501 colonized mainly intercellular spaces in the leaves of wild rice. Colony counts of surface-sterilized rice seedlings inoculated with the GFP-tagged bacteria indicated significantly more bacterial populations inside the original wild rice than in cultivated rice varieties. Moreover, after bacterial inoculation, in planta nitrogen fixation in young seedlings of wild rice, O. officinalis, was detected by the acetylene reduction and ¹⁵N₂ gas incorporation assays. Therefore, we conclude that Herbaspirillum sp. strain B501 is a diazotrophic endophyte compatible with wild rice, particularly O. officinalis.     

Involvement of luxS in Biofilm Formation by Capnocytophaga ochracea

Capnocytophaga ochracea is present in the dental plaque biofilm of patients with periodontitis. Biofilm cells change their phenotype through quorum sensing in response to fluctuations in cell-population density. Quorum sensing is mediated by auto-inducers (AIs). AI-2 is involved in intercellular signaling, and production of its distant precursor is catalyzed by LuxS, an enzyme involved in the activated methyl cycle. Our aim was to clarify the role of LuxS in biofilm formation by C. ochracea. Two luxS-deficient mutants, TmAI2 and LKT7, were constructed from C. ochracea ATCC 27872 by homologous recombination. The mutants produced significantly less AI-2 than the wild type. The growth rates of these mutants were similar to that of the wild-type in both undiluted Tryptic soy broth and 0.5 × Tryptic soy broth. However, according to crystal violet staining, they produced significantly less biofilm than the wild type. Confocal laser scanning microscopy and scanning electron microscopy showed that the biofilm of the TmAI2 strain had a rougher structure than that of the wild type. Complementation of TmAI-2 with extrinsic AI-2 from the culture supernatant of wild-type strain did not restore biofilm formation by the TmAI2 strain, but complementation of LKT7 strain with luxS partially restored biofilm formation. These results indicate that LuxS is involved in biofilm formation by C. ochracea, and that the attenuation of biofilm formation by the mutants is likely caused by a defect in the activated methyl cycle rather than by a loss of AI-2.     

Bacillus subtilis and Enterobacter cloacae endophytes from healthy Theobroma cacao L. trees can systemically colonize seedlings and promote growth

Clonal genotypes resistant to fungal diseases are an important component of the cocoa production system in southeastern Bahia state (Brazil), so that technologies for faster production of stronger and healthier plantlets are highly desirable. In this study, the effects of inoculated bacterial endophytes isolated from healthy adult cacao plants on seedlings, and aspects related to inoculation methods, colonization patterns, and photosynthesis were investigated. Sequencing of 16S rRNA, hsp-60, and rpo-B genes placed the wild-type isolates within the species Enterobacter cloacae (isolates 341 and 344) and Bacillus subtilis (isolate 629). Spontaneous rifampicin-resistant (rif^R) variants for 344 were also produced and tested. Endophytic application was either by immersion of surface sterilized seeds in bacterial suspensions or direct inoculation into soil, 20 days after planting non-inoculated seeds into pots. Results from in vitro recovery of inoculated isolates showed that the wild-type endophytes and rif^R variants systemically colonized the entire cacao seedlings in 15–20 days, regardless of the inoculation method. Some endophytic treatments showed significant increases in seedlings’ height, number of leaves, and dry matter. Inoculation methods affected the combined application of endophytes, which maintained the growth-promotion effects, but not in the same manner as in single applications. Interestingly, the 344-3.2 rif^R variant showed improved performance in relation to both the wild type and another related variant. Photosynthetic rates and stomatal conductance increased significantly for some endophytic treatments, being partially associated with effects on growth and affected by the inoculation method. The results suggest that E. cloacae and B. subtilis endophytes from healthy adult plants (not transmitted by seeds) were able to promote vegetative growth on cacao seedlings. The development of products for large-scale use in seedlings/plantlets production systems was discussed.     

Photoproduction of H2 from Cellulose by an Anaerobic Bacterial Coculture

Cellulomonas sp. strain ATCC 21399 is a facultatively anaerobic, cellulose-degrading microorganism that does not evolve hydrogen but produces organic acids during cellulose fermentation. Rhodopseudomonas capsulata cannot utilize cellulose, but grows photoheterotrophically under anaerobic conditions on organic acids or sugars. This report describes an anaerobic coculture of the Cellulomonas strain with wild-type R. capsulata or a mutant strain lacking uptake hydrogenase, which photoevolves molecular hydrogen by the nitrogenase system of R. capsulata with cellulose as the sole carbon source. In coculture, the hydrogenase-negative mutant produced 4.6 to 6.2 mol of H₂ per mol of glucose equivalent, compared with 1.2 to 4.3 mol for the wild type.     

The modified rice αAmy8 promoter confers high-level foreign gene expression in a novel hypoxia-inducible expression system in transgenic rice seedlings

Expression of α-amylase genes in rice is induced not only by sugar starvation and gibberellin (GA) but also by O₂ deficiency. Promoters of two rice α-amylase genes, αAmy3 and αAmy8, have been shown to direct high-level production of recombinant proteins in rice suspension cells and germinated seeds. In the present study, we modified the cis-acting DNA elements within the sugar/GA response complex (SRC/GARC) of αAmy8 promoter. We found that addition of a G box and duplicated TA box leads to high-level expression of αAmy8 SRC/GARC and significantly enhances αAmy8 promoter activity in transformed rice cells and germinated transgenic rice seeds. We also show that these modifications have drastically increased the activity of αAmy8 promoter in rice seedlings under hypoxia. Our results reveal that the G box and duplicated TA box may play important roles in stimulating promoter activity in response to hypoxia in rice. The modified αAmy8 promoter was used to produce the recombinant human epidermal growth factor (hEGF) in rice cells and hypoxic seedlings. We found that the bioactive recombinant hEGF are stably produced and yields are up to 1.8 % of total soluble protein (TSP) in transformed rice cells. The expression level of synthetic hEGF containing preferred rice codon usage comprises up to 7.8 % of TSP in hypoxic transgenic seedlings. Our studies reveal that the modified αAmy8 promoter can be applicable in establishing a novel expression system for the high-level production of foreign proteins in transgenic rice cells and seedlings under hypoxia.     

Combined gene cluster engineering and precursor feeding to improve gougerotin production in Streptomyces graminearus

Gougerotin is a peptidyl nucleoside antibiotic produced by Streptomyces graminearus. It is a specific inhibitor of protein synthesis and exhibits a broad spectrum of biological activities. Generation of an overproducing strain is crucial for the scale-up production of gougerotin. In this study, the natural and engineered gougerotin gene clusters were reassembled into an integrative plasmid by λ-red-mediated recombination technology combined with classic cloning methods. The resulting plasmids pGOU and pGOUe were introduced into S. graminearus to obtain recombinant strains Sgr-GOU and Sgr-GOUe, respectively. Compared with the wild-type strain, Sgr-GOU led to a maximum 1.3-fold increase in gougerotin production, while Sgr-GOUe resulted in a maximum 2.1-fold increase in gougerotin production. To further increase the yield of gougerotin, the effect of different precursors on its production was investigated. All precursors, including cytosine, serine, and glycine, had stimulatory effect on gougerotin production. The maximum gougerotin yield was achieved with Sgr-GOUe in the presence of glycine, and it was approximately 2.5-fold higher than that of the wild-type strain. The strategies used in this study can be extended to other Streptomyces for improving production of industrial important antibiotics.     

Amino acid replacement in the protein S5 from a spectinomycin resistant mutant of Bacillus subtilis

Summary Ribosomal protein S5 was isolated from wild type Bacillus subtilis ATCC 6633 and from a spectinomycin resistant mutant (BSPC 111) derived from spectinomycin sensitive to resistance is accomtrypsin and all the tryptic peptides were isolated by column- and paper-chromatography. By comparative amino acid analyses of the peptides, it was demonstrated that the S5 from the mutant differs from the wild type S5 by a replacement of one amino acid, namely lysine by isoleucine in the peptide T9. The results are compared with E. coli spectinomycin resistant mutants.     

Isolation and partial characterization of carotenoid underproducing and overproducing mutants from an extremely thermophilic Thermus thermophilus HB27

Twenty-two carotenoid underproducing and thirteen overproducing mutants were obtained from Thermus thermophilus HB27. The strain HB27 was found to produce at least seven colored carotenoids, believed to be identical to those produced by Thermus aquaticus YT1. Based on the results of the genetic analyses performed on twelve carotenoid underproducing mutants, they were classified into three groups; groups 1, 2 and 3. No colored carotenoid was extracted from the cells of mutants belonging to groups 2 and 3, although the accumulation of phytoene, a colorless carotenoid, was observed in group 2 strains. Group 1 was subdivided into groups 1-a and 1-b, where 1-a strains produced neither colored carotenoids nor phytoene and 1-b strains produced two polar colored carotenoids. All of the overproducing mutants produced about twelve times as much seven colored carotenoid mixtures as the parental strain. The mutation loci among all the overproducing mutants were very close to one another, possibly in the same gene. Carotenoid overproducing mutants showed an extensive resistancy to UV-irradiation and showed poorer growth at higher temperatures. Carotenoid underproducing mutants were slightly more UV-sensitive but they grew almost normally at higher temperatures. These results suggest that carotenoids are secondary metabolites which are not essential for growth of T. thermophilus.     

Possible Role of Xanthobaccins Produced by Stenotrophomonas sp. Strain SB-K88 in Suppression of Sugar Beet Damping-Off Disease

Three antifungal compounds, designated xanthobaccins A, B, and C, were isolated from the culture fluid of Stenotrophomonas sp. strain SB-K88, a rhizobacterium of sugar beet that suppresses damping-off disease. Production of xanthobaccin A in culture media was compared with the disease suppression activities of strain SB-K88 and less suppressive strains that were obtained by subculturing. Strain SB-K88 was applied to sugar beet seeds, and production of xanthobaccin A in the rhizosphere of seedlings was confirmed by using a test tube culture system under hydroponic culture conditions; 3 μg of xanthobaccin A was detected in the rhizosphere on a per-plant basis. Direct application of purified xanthobaccin A to seeds suppressed damping-off disease in soil naturally infested by Pythium spp. We suggest that xanthobaccin A produced by strain SB-K88 plays a key role in suppression of sugar beet damping-off disease.     

Mutant Strain of Bradyrhizobium japonicum with Increased Symbiotic N2 Fixation Rates and Altered Mo Metabolism Properties

Mutant strains of Bradyrhizobium japonicum that required higher levels of molybdate than the wild-type strain for growth on NO₃-containing medium were obtained after transposon Tn5 mutagenesis of the wild-type strain. The mutant strains expressed more than fivefold-greater nitrate reductase activities in the range of 0.1 to 1.0 mM added molybdate compared with activities expressed upon incubation in non-Mo-supplemented medium, whereas the nitrate reductase activity of the wild-type strain (JH) was not markedly influenced by Mo supplementation. In free-living culture, mutant strains JH310 and JH359 expressed substantial nitrogenase activity, even in medium treated to remove molybdate, and nitrogenase activity was influenced little by Mo supplementation, whereas the wild-type strain required 100 nM added Mo for highest nitrogenase activity. Double-reciprocal plots of Mo uptake rates versus Mo concentration showed that both bacteroids and free-living cells of mutant strain JH359 had about the same affinity for Mo as did the parent strain. Bacteroids of both the mutants and the wild type also exhibited similar Mo accumulation rates over a 9-min period under very-low-Mo (4 nM) conditions. Nitrogenase activities for strain JH359 and for the wild-type strain in free-living culture were both strongly inhibited by tungsten; thus, the nitrogenase activities of both strains are probably the result of a “conventional” Mo-containing nitrogenase. Soybeans inoculated with strain JH359 and grown under either Mo-supplemented or Mo-deficient conditions had greater specific acetylene reduction rates and significantly greater plant fresh weight than those inoculated with the wild-type strain. Under Mo-deficient conditions, the acetylene reduction rates and plant fresh weights were up to 35 and 58% greater, respectively, for mutant-nodulated plants compared with wild-type-strain-nodulated plants.     

Oxygen-Limiting Growth Conditions and Deletion of the Transition State Regulator Protein Abrb in Bacillus subtilis 6633 Result in an Increase in Subtilosin Production and a Decrease in Subtilin Production

It has been recently shown, that certain strains/isolates of Bacillus subtilis can be used as a probiotic for humans. The production of the macrocyclic sactibiotic subtilosin in B. subtilis ATCC 6633 is highly regulated. To improve the subtilosin productivity of B. subtilis, different growth conditions were compared for maximal expression of the sbo promoter that regulates the expression of the subtilosin biosynthetic gene cluster. Oxygen-limiting conditions led to a strong increase of sbo promoter activities compared to aerobic conditions, and accordingly, the subtilosin amount determined by reversed phase HPLC (7.8 mg/L) was 15-fold superior to the amount of aerobic grown cultures (0.5 mg/L). A further promising enhancement of the subtilosin yield was achieved using a deletion mutant that is avoiding the general transition state regulator protein AbrB. The subtilosin titer of 42 mg/L produced by ΔabrB cells grown under oxygen-limiting conditions corresponds to an 84-fold increase compared to the subtilosin titer obtained from B. subtilis wild type cells propagated in aerobic conditions. Furthermore, evidence is provided that oxygen-limiting conditions led to a strong decrease in the productivity of the lantipeptide subtilin suggesting contrary regulatory mechanisms for the B. subtilis antimicrobials subtilin and subtilosin.