Helper Strain Research Articles

Facemask acne attenuation through modulation of indirect microbiome interactions

During the COVID-19 pandemic, facemasks played a pivotal role in preventing person-person droplet transmission of viral particles. However, prolonged facemask wearing causes skin irritations colloquially referred to as ‘maskne’ (mask + acne), which manifests as acne and contact dermatitis and is mostly caused by pathogenic skin microbes. Previous studies revealed that the putative causal microbes were anaerobic bacteria, but the pathogenesis of facemask-associated skin conditions remains poorly defined. We therefore characterized the role of the facemask-associated skin microbiota in the development of maskne using culture-dependent and -independent methodologies. Metagenomic analysis revealed that the majority of the facemask microbiota were anaerobic bacteria that originated from the skin rather than saliva. Previous work demonstrated direct interaction between pathogenic bacteria and antagonistic strains in the microbiome. We expanded this analysis to include indirect interaction between pathogenic bacteria and other indigenous bacteria classified as either ‘pathogen helper (PH)’ or ‘pathogen inhibitor (PIn)’ strains. In vitro screening of bacteria isolated from facemasks identified both strains that antagonized and promoted pathogen growth. These data were validated using a mouse skin infection model, where we observed attenuation of symptoms following pathogen infection. Moreover, the inhibitor of pathogen helper (IPH) strain, which did not directly attenuate pathogen growth in vitro and in vivo, functioned to suppress symptom development and pathogen growth indirectly through PH inhibitory antibacterial products such as phenyl lactic acid. Taken together, our study is the first to define a mechanism by which indirect microbiota interactions under facemasks can control symptoms of maskne by suppressing a skin pathogen.

Rhodotorula mucilaginosa A8, a potential helper strain in a vitamin C microbial fermentation process.

In the vitamin C microbial fermentation system, oxidative stress limits the growth and 2-keto-l-gulonic acid (2-KLG, the precursor of vitamin C) production of Ketogulonicigenium vulgare. Most Bacillus strains, as helper strains, have been reported to release key biomolecules to reduce oxidative stress and promote the growth and 2-KLG production of K. vulgare. To understand the specific mechanism by which the helper strain and K. vulgare interact to reduce oxidative stress, a novel helper strain, Rhodotorula mucilaginosa A8, was used to construct a consortium in the co-culture fermentation system. Based on the activities of the antioxidant enzymes and quantitative polymerase chain reaction (qPCR) analysis, R. mucilaginosa A8 could reduce oxidative stress and increase 2-KLG production in K. vulgare by upregulating antioxidant enzyme activities and related gene-expression levels. In addition, the carotenoids of R. mucilaginosa promoted 2-KLG production in K. vulgare. Coculture of R. mucilaginosa with K. vulgare increased the yield of carotenoids. This study suggested that helper strains with the ability to reduce oxidative stress in K. vulgare would likely act as potential helper strains for facilitating 2-KLG biosynthesis. This work could provide a theoretical basis for the search for potential helper strains for vitamin C microbial fermentation and for the construction of synthetic microbial communities to produce valuable products.

A bottom-up approach to select microbes from textile wastewater for detoxification of aromatic amine and azo dye in a single stage

Biological processes show great promise for treating textile wastewater, but the complexity and high energy needs limit their industrial applications. Complete dye degradation involves physiologically distinct bacteria in a two-stage process. First, anaerobic bacteria cleave azo dyes into hazardous aromatic amines, whereas its further degradation requires aerobic bacteria. We propose strategic microbial selection based on their physiological capabilities while developing a bioremediation design to detoxify aromatic amines and dyes in a single stage. Bacillus firmus PM1, the amine degrader, and Serratia liquefaciens PM2, the helper strain, were isolated from textile wastewater. B. firmus PM1 completely degraded 4-Nitroaniline (4-NA), a common dye intermediate, under a range of physicochemical conditions prevalent in textile effluent viz. varying pH (7–11), oxygen availability (static/shaking), temperature (30–40 °C), and 4-NA concentrations (200–1000 μM). Along with 4-NA degradation (100 %), B. firmus PM1 could also efficiently degrade co-contaminants, aniline (35 %), and phenols (45 %). UPLC, FTIR, and LC-MS analysis suggested that when B. firmus PM1 is used in co-culture with S. liquefaciens PM2, it efficiently mineralizes 4-NA (95 %) and reduces Acid Black 1 (AB1) (80 %) dye both in synthetic and the real textile effluent. Further, the co-culture could also significantly reduce 4-NA and AB1 mixture induced phytotoxicity/cytogenotoxicty in Vigna mungo and Allium cepa. Thus, the study demonstrates a novel bottom-up approach that relies on isolating bacteria degrading the penultimate toxic dye metabolite and developing a co-culture for degrading the parent dye. The study demonstrates that detoxification-centric use of specialized microbes has potential to evolve sustainable industrial practices.

Defining Paenibacillus azoreducens (P8) and Acetobacter pasteurianus (UMCC 2951) strains performances in producing acetic acid.

In this study, spore-forming bacteria isolated from saccharified rice were selected for producing acetic acid. From the screening of 15 strains, P8 strain was chosen as a candidate. The strain was identified as Paenibacillus azoreducens by 16S rRNA analysis (99.85% similarity with P. azoreducens CM1T). Acetic acid is the main component of vinegar but also an industrial commodity produced by chemical synthesis. Sustainable routes for obtaining acetic acid are of great interest for decreasing the environmental impact generated by chemical syntheses. Biological acetic acid production is effective for vinegar production by acetic acid bacteria, but it cannot economically compete with the chemical synthesis for producing it as a pure commodity. Considering the need to improve the yield of pure acetic acid produced by microbial conversions, in this study, P8 strain was chosen for designing processes in different fermentation conditions. Tests were conducted in single and semi-continuous systems, using rice wine as substrate. Acetic acid produced by P8 strain was compared with that of Acetobacter pasteurianus (UMCC 2951), a strain known for producing acetic acid from rice wine. Even though the fermentation performances of P. azoreducens P8 were slightly lower than those of acetic acid bacteria usually used for vinegar production, results highlight its suitability for producing acetic acid. The final acetic acid produced by P. azoreducens P8 was 73 g/L, in a single stage fermentation, without losses. In nine cycles of semi-continuous regime the average of acetification rate was 0.814 (g/L/days). Two main attributes of P. azoreducens P8 are of relevance for producing acetic acid, namely the ability to grow at temperature higher (+ 37°C), than mesophilic acetic acid bacteria, and the absence of cytoplasmic assimilation of acetic acid. These features allow to design multiple strains cultures, in which P. azoreducens can acts as a helper strain. Based on our results, the new isolate P. azoreducens P8 can be propagated in fermenting broths for boosting acetic acid production, under the selected conditions, and used in combination with acetic acid bacteria to produce biological acetic acid, as a non-food grade commodity.

Siderophores of Bacillus pumilus promote 2-keto-L-gulonic acid production in a vitamin C microbial fermentation system.

The precursor of vitamin C, 2-keto-l-gulonic acid (2-KLG), is bio-converted from l-sorbose by a microbial consortium of Ketogulonicigenium vulgare and a helper strain (Bacillus spp.). Most helper strains produce siderophores. To understand the effects of siderophores on promoting 2-KLG yield, the siderophores of Bacillus pumilus SY-A9 were purified and added to a monoculture fermentation system of K. vulgare 25B-1. The results revealed that the titer of 2-KLG reached 7.18 g/L within 60 h and increased by 71.45% when the added concentration of siderophores was 500 µg/L. Moreover, the increased production of 2-KLG was accompanied by the overexpressed iron uptake system-related genes, electron transfer chain-related genes, ATP synthesis enzyme-related genes, antioxidant enzyme-related genes, and 2-KLG producing enzyme-related genes, which reduced oxidative stress and ensured the normal energy metabolism of K. vulgare 25B-1. This study demonstrated that siderophores of the helper strain play a key role in the enhancement of 2-KLG biosynthesis.

Construction of synthetic microbial consortia for 2-keto-L-gulonic acid biosynthesis

Currently, the establishment of synthetic microbial consortia with rational strategies has gained extensive attention, becoming one of the important frontiers of synthetic biology. Systems biology can offer insights into the design and construction of synthetic microbial consortia. Taking the high-efficiency production of 2-keto-l-gulonic acid (2-KLG) as an example, we constructed a synthetic microbial consortium “Saccharomyces cerevisiae-Ketogulonigenium vulgare” based on systems biology analysis. In the consortium, K. vulgare was the 2-KLG producing strain, and S. cerevisiae acted as the helper strain. Comparative transcriptomic analysis was performed on an engineered S. cerevisiae (VTC2) and a wild-type S. cerevisiae BY4741. The results showed that the up-regulated genes in VTC2, compared with BY4741, were mainly involved in glycolysis, TCA cycle, purine metabolism, and biosynthesis of amino acids, B vitamins, and antioxidant proteases, all of which play important roles in promoting the growth of K. vulgare. Furthermore, Vitamin C produced by VTC2 could further relieve the oxidative stress in the environment to increase the production of 2-KLG. Therefore, VTC2 would be of great advantage in working with K. vulgare. Thus, the synthetic microbial consortium "VTC2-K. vulgare" was constructed based on transcriptomics analyses, and the accumulation of 2-KLG was increased by 1.49-fold compared with that of mono-cultured K. vulgare, reaching 13.2 ± 0.52 g/L. In addition, the increased production of 2-KLG was accompanied by the up-regulated activities of superoxide dismutase and catalase in the medium and the up-regulated oxidative stress-related genes (sod, cat and gpd) in K. vulgare. The results indicated that the oxidative stress in the synthetic microbial consortium was efficiently reduced. Thus, systems analysis confirmed a favorable symbiotic relationship between microorganisms, providing guidance for further engineering synthetic consortia.

Coculture method for in vitro cultivation of uncultured oral bacteria.

Purpose:The purpose of the study is to culture uncultured oral bacteria with helper strains using the coculture method from the subgingival plaque samples of chronic periodontitis patients.Materials and Methods:The samples were processed and inoculated on a blood agar medium enriched with hemin and Vitamin K. A helper strain Propionibacterium acnes (ATCC 6919) was cross-streaked across the inoculums to facilitate coculture. The plates were then incubated for 7 days with subsequent subculturing and further incubation.Results:Satellite colonies around helper strain showed one colony type of Porphyromonas gingivalis, one was of nonpigmented Prevotella, three were of Fusobacterium nucleatum and five isolates remained unidentified.Conclusions:Coculture could be used effectively as one of the methods in the isolation and in vitro cultivation of oral bacteria. Incubation using the anaerobic jar technique was found to be economical and efficient for the growth of anaerobic oral bacteria.

Enhanced 2-keto-L-gulonic acid production by a mixed culture of Ketogulonicigenium vulgare and Bacillus megaterium using three-stage temperature control strategy.

As a key precursor of vitamin C, 2-keto-L-gulonic acid (2-KLG) was mainly produced from L-sorbose by mixed fermentation of Ketogulonicigenium vulgare and a helper strain (Bacillus spp.) with a low conversion rate for decades. The aim of this study was to enhance the 2-KLG production by co-culturing K. vulgare and Bacillus megaterium using three-stage temperature control (TSTC) strategy. By investigating the temperature effect on the 2-KLG fermentation, the optimum temperatures for the growths of K. vulgare and B. megaterium were 32°C and 29°C, respectively, while the optimum temperature for 2-KLG production was 35°C. We developed a TSTC process: the temperature was kept at 32°C during the first 16h of fermentation, then decreased to 29°C for the following 14h, and maintained at 35°C to the end of fermentation. By using this new process, the productivity and yield of 2-KLG from L-sorbose were obtained at 2.19 ± 0.19g/L/h and 92.91 ± 1.02g/L in 20-L fermentors for 5 batches, respectively, which were 22.35% and 6.02% higher than that of the control treatment (the single temperature of 29°C). The increased cell density of K. vulgare during the exponential phase and the enhanced SDH activity (increased by 25.18% at 36h, 17.14% at 44h) in the production stage might be the reasons for enhanced 2-KLG conversion rate and yield. Our results demonstrated the feasibility of the TSTC strategy for 2-KLG production.

Bioproduction of pure, kilobase-scale single-stranded DNA

Scalable production of kilobase single-stranded DNA (ssDNA) with sequence control has applications in therapeutics, gene synthesis and sequencing, scaffolded DNA origami, and archival DNA memory storage. Biological production of circular ssDNA (cssDNA) using M13 addresses these needs at low cost. However, one unmet goal is to minimize the essential protein coding regions of the exported DNA while maintaining its infectivity and production purity to produce sequences less than 3,000 nt in length, relevant to therapeutic and materials science applications. Toward this end, synthetic miniphage with inserts of custom sequence and size offers scalable, low-cost synthesis of cssDNA at milligram and higher scales. Here, we optimize growth conditions using an E. coli helper strain combined with a miniphage genome carrying only an f1 origin and a β-lactamase-encoding (bla) antibiotic resistance gene, enabling isolation of pure cssDNA with a minimum sequence genomic length of 1,676 nt, without requiring additional purification from contaminating DNA. Low-cost scalability of isogenic, custom-length cssDNA is demonstrated for a sequence of 2,520 nt using a bioreactor, purified with low endotoxin levels (<5 E.U./ml). We apply these exonuclease-resistant cssDNAs to the self-assembly of wireframe DNA origami objects and to encode digital information on the miniphage genome for biological amplification.

Delivering "Chromatic Bacteria" Fluorescent Protein Tags to Proteobacteria Using Conjugation.

Recently, we published a large and versatile set of plasmids, the chromatic bacteria toolbox, to deliver eight different fluorescent protein genes and four combinations of antibiotic resistance genes to Gram-negative bacteria. Fluorescent tags are important tools for single-cell microbiology, synthetic community studies, biofilm, and host-microbe interaction studies. Using conjugation helper strain E. coli S17-1 as a donor, we show how plasmid conjugation can be used to deliver broad host range plasmids, Tn5 transposons delivery plasmids, and Tn7 transposon delivery plasmids into species belonging to the Proteobacteria. To that end, donor and recipient bacteria are grown under standard growth conditions before they are mixed and incubated under non-selective conditions. Then, transconjugants or exconjugant recipients are selected on selective media. Mutant colonies are screened using a combination of tools to ensure that the desired plasmids or transposons are present and that the colonies are not containing any surviving donors. Through conjugation, a wide range of Gram-negative bacteria can be modified without prior, often time-consuming, establishment of competent cell and electroporation procedures that need to be adjusted for every individual strain. The here presented protocol is not exclusive for the delivery of Chromatic bacteria plasmids and transposons, but can also be used to deliver other mobilizable plasmids to bacterial recipients.

Enhanced 2-keto-l-gulonic acid production by applying l-sorbose-tolerant helper strain in the co-culture system

2-Keto-l-gulonic acid (the precursor of vitamin C) is bio-converted from l-sorbose by mixed fermentation of Ketogulonicigenium vulgare and a helper strain. The helper strain promotes the conversion of 2-KLG by enhancing the growth of K. vulgare, but its growth is greatly inhibited by high concentration of l-sorbose, which consequently influence the 2-KLG production. The aim of this study is to obtain l-sorbose-tolerant helper strain (LHS) by experimental evolution for reduced l-sorbose-inhibition-effect and enhanced 2-KLG productivity in high concentration of l-sorbose. After three steps screening by using our devised screening strategy, three strains (i.e., Bc 21, Bc 47, Bc 50) with high resistance to high concentration of l-sorbose were obtained. The fermentation tests by co-culturing Bc 21 and K. vulgare 418 showed that the production of 2-KLG was increased by 17.9% in 11% l-sorbose medium than that in 8% after 55 h of fermentation and the conversion rate was 89.5%. The results suggested that Bc 21 could be an ideal helper strain for 2-KLG production under high concentration of l-sorbose and demonstrated the feasibility of using experimental evolution to breed LHS for vitamin C production.

Highly efficient and inducible DNA excision in transgenic silkworms using the FLP/FRT site-specific recombination system

Efficient and inducible recombinase-mediated DNA excision is an optimal technology for automatically deleting unwanted DNA sequences, including selection marker genes. However, this methodology has yet to be established in transgenic silkworms. To achieve efficient and inducible FLP recombinase-mediated DNA excision in transgenic silkworms, one transgenic target strain (TTS) containing an FRT-flanked silkworm cytoplasmic actin 3 gene promoter (A3)-enhanced green fluorescent protein (EGFP) expression cassette, as well as two different types of FLP recombinase expression helper strains were generated. Then, the FLP recombinase was introduced into the TTS silkworms by pre-blastoderm microinjection and sexual hybridization. Successful recombinase-mediated deletion of the A3-EGFP expression cassette was observed in the offspring of the TTS, and the excision efficiencies of the FLP expression vector and FLP mRNA pre-blastoderm microinjection were 2.38 and 13.3%, respectively. The excision efficiencies resulting from hybridization between the TTS and the helper strain that contained a heat shock protein 70 (Hsp70)-FLP expression cassette ranged from 32.14 to 36.67% after heat shock treatment, while the excision efficiencies resulting from hybridization between the TTS and the helper strain containing the A3-FLP expression cassette ranged from 97.01 to 100%. These results demonstrate that the FLP/FRT system can be used to achieve highly efficient and inducible post-integration excision of unwanted DNA sequences in transgenic silkworms in vivo. Our present study will facilitate the development and application of the FLP/FRT system for the functional analysis of unknown genes, and establish the safety of transgenic technologies in the silkworm and other lepidopteran species.

L-sorbose is not only a substrate for 2-keto-L-gulonic acid production in the artificial microbial ecosystem of two strains mixed fermentation.

The co-culture system of the fermentation process of vitamin C can be regarded as an artificial microbial ecosystem (AME). To extend our understanding of this AME, an investigation of the relationship between strains, substrate and product was carried out in this study. The results showed that both Ketogulonicigenium vulgare and 2-keto-L-gulonic acid (2-KLG, the precursor of vitamin C) can inhibit the growth of the helper strain, while the helper strain promoted the growth of K. vulgare and 2-KLG production. Moreover, L-sorbose is not only a substrate for 2-KLG production in the AME, but also a promoter of K. vulgare and an inhibitor of the helper strain. In the earlier stage of fermentation, the inhibition of L-sorbose on the helper strain's growth is a key factor for ensuring an efficient fermentation. In the condition of adding the extra helper strain (OD: 0.57, ratio of inoculation: 2%), the yields of 2-KLG is increased by 9% in the 14% L-sorbose medium. To the best of our knowledge, this is the first report about the inhibition of substrate in the AME of 2-KLG production.

The Unculturables: targeted isolation of bacterial species associated with canine periodontal health or disease from dental plaque.

BackgroundThe current inability to culture the entirety of observed bacteria is well known and with the advent of ever more powerful molecular tools, that can survey bacterial communities at previously unattainable depth, the gap in our capacity to culture and define all of these species increases exponentially. This gap has essentially become the rate limiting step in determining how the knowledge of which species are present in a sample can be applied to understand the role of these species in an ecosystem or disease process. A case in point is periodontal disease, which is the most widespread oral disease in dogs. If untreated the disease results in significant pain, eventual loss of the dentition and potentially an increased risk of systemic diseases. Previous molecular based studies have identified the bacterial species associated with periodontal disease in dogs; however without cultured strains from many of these species it has not been possible to study whether they play a role in the disease process.ResultsUsing a quantitative polymerase chain reaction (qPCR) directed approach a range of microbiological media were screened and optimized to enrich for previously uncultivated target species. A systematic screening methodology was then employed to isolate the species of interest. In cases where the target species were not cultivable in isolation, helper strains grown underneath a nitrocellulose membrane were used to provide the necessary growth factors. This guided media optimization approach enabled the purification of 14 species, 8 of which we had previously been unable to cultivate in isolation. It is also applicable to the targeted isolation of isolates from species that have previously been cultured (for example to study intra-species variation) as demonstrated by the successful isolation of 6 targeted isolates of already cultured species.ConclusionsTo our knowledge this is the first time this combination of qPCR guided media optimization, strategic screening and helper strain support has been used successfully to isolate previously uncultured bacteria. This approach can be applied to any uncultured bacterial species where knowledge of their nutritional requirements or low relative abundance impedes their isolation.

Transgenic rice lines constitutively co-expressing tlp-D34 and chi11 display enhancement of sheath blight resistance

Transgenic rice (Oryza sativa L. subsp. indica cv. White Ponni) constitutively expressing the rice thaumatin-like protein gene (tlp-D34, PR-5) individually or in combination with the rice chitinase gene (chi11, PR-3) was generated using an Agrobacterium vir helper strain with multiple copies of pTiBo542 virB and virG. Transformation with the tlp-D34 gene alone and tlp-D34 + chi11 genes yielded five and seven single-copy transgenic lines, respectively. Southern blot analysis with two probes, one flanking the right T-DNA border and the second flanking the left T-DNA border, confirmed that all transgenic plants harboured single and complete T-DNA copies. Homozygous transgenic lines were first identified in the T1 generation by Southern blot analysis and were subsequently confirmed by segregation analysis of T2 plants. Accumulation of transcripts encoded by the transgenes was confirmed in T0 plants and homozygous T2 plants by Northern blot analysis. The homozygous T2 plants harbouring tlp-D34 + chi11 genes showed 2.8- to 4.2-fold higher chitinase activity. Western blot analysis revealed the accumulation of thaumatin-like protein and chitinase in the respective transgenic plants. Upon infection with Rhizoctonia solani, the disease index reduced from 100 % in control plants to 65 % in a T3 homozygous transgenic line T4 expressing the tlp-D34 gene alone. In a T2 homozygous transgenic line CT22 co-expressing tlp-D34 and chi11 genes, the disease index reduced to 39 %.

Hitchhikers on the fungal highway: The helper effect for bacterial migration via fungal hyphae

Previous work in our laboratory showed that several bacterial strains, either singly or in association with other bacteria (community migration), were capable of migrating together with the saprotrophic fungus Lyophyllum sp. strain Karsten through soil microcosms. A possible involvement of the type III secretion system (TTSS) in migration was indicated. In this study, we addressed the basis of the community migration, which might lie in a migration helper effect exerted by particular single-strain migrators on other members of the community. Different culturing (plating) as well as culture-independent (PCR-DGGE) methods were applied to assess the effects of putative bacterial helpers in the migration. We used, as a model, the migration-proficient Burkholderia terrae BS001 as the canonical helper strain. PCR-DGGE analysis of the soil system with or without added strain BS001, revealed that the latter consistently stimulated the migration of different bacterial species through the soil. This was observed both following introduction of the organism to a bacterial community from soil and on the basis of a similar organism that was naturally present. One strain, Dyella japonica BS003, was identified as an avid comigrator with B. terrae BS001, although it appeared to lag behind the latter strain in its migration speed. Further examination of the B. terrae BS001/D. japonica BS003 interaction at Lyophyllum sp. strain Karsten hyphae showed that the presence of the D. japonica strain did not negatively affect the growth and migration of B. terrae BS001 with the fungus. A biofilm of B. terrae BS001 was formed on the fungal hyphal front, and we postulate a role for this biofilm in the migration helper effect.

Salmonella entericaSerovar Typhimurium Binds to HeLa Cells via Fim-Mediated Reversible Adhesion and Irreversible Type Three Secretion System 1-Mediated Docking

The food-borne pathogen Salmonella enterica serovar Typhimurium invades mammalian epithelial cells. This multistep process comprises bacterial binding to the host cell, activation of the Salmonella type three secretion system 1 (T1), injection of effector proteins, triggering of host cell actin rearrangements, and S. Typhimurium entry. While the latter steps are well understood, much less is known about the initial binding step. Earlier work had implicated adhesins (but not T1) or T1 (but not other adhesins). We have studied here the Salmonella virulence factors mediating S. Typhimurium binding to HeLa cells. Using an automated microscopy assay and isogenic S. Typhimurium mutants, we analyzed the role of T1 and of several known adhesins (Fim, Pef, Lpf, Agf, and Shd) in host cell binding. In wild-type S. Typhimurium, host cell binding was mostly attributable to T1. However, in the absence of T1, Fim (but not Pef, Lpf, Agf, and Shd) also mediated HeLa cell binding. Furthermore, in the absence of T1 and type I fimbriae (Fim), we still observed residual binding, pointing toward at least one additional, unidentified binding mechanism. Dissociation experiments established that T1-mediated binding was irreversible ("docking"), while Fim-mediated binding was reversible ("reversible adhesion"). Finally, we show that noninvasive bacteria docking via T1 or adhering via Fim can efficiently invade HeLa cells, if actin rearrangements are triggered in trans by a wild-type S. Typhimurium helper strain. Our data show that binding to HeLa cells is mediated by at least two different mechanisms and that both can lead to invasion if actin rearrangements are triggered.

Role of fungal trehalose and bacterial thiamine in the improved survival and growth of the ectomycorrhizal fungus Laccaria bicolor S238N and the helper bacterium Pseudomonas fluorescens BBc6R8

The mycorrhiza helper bacterial strain Pseudomonas fluorescens BBc6R8 enhances the establishment of Laccaria bicolor S238N ectomycorrhizae by improving the pre-symbiotic growth and survival of the fungus. Nothing is known about the effect of the ectomycorrhizal fungus on the helper bacteria or the molecules that are involved in the interaction. In this study, we have monitored the population density of the helper strain P. fluorescens BBc6R8 in soils inoculated with L. bicolor and in control soils and found that the ectomycorhizal fungus improves the survival of the helper bacteria. We investigated the identity of the fungal and bacterial metabolites involved in this reciprocal growth-promoting effect using a combination of growth measurements, chemoattractant assays, HPLC and in silico genome analyses. We showed that trehalose, a disaccharide that accumulates to high levels in the fungal hyphae, chemoattracted and promoted the growth of the helper bacteria. Meanwhile, P. fluorescens BBc6R8 produced thiamine at concentrations that enhanced the fungal growth in vitro. Altogether our data indicate that the interaction between the two microorganisms is beneficial for both species and relies, at least in part, on trophic mutualism.

Siderophores from Neighboring Organisms Promote the Growth of Uncultured Bacteria

The majority of bacterial species do not grow on synthetic media. Many non-growers require growth factors from other bacteria, but the nature of these compounds is largely unknown. We show here that previously uncultured isolates from marine sediment biofilm grow on a Petri dish in the presence of cultured organisms from the same environment. The growth factors produced by one cultured helper strain were identified as new acyl-desferrioxamine siderophores. A panel of previously uncultured isolates exhibited a range of siderophore promiscuity for growth promotion. This siderophore-based approach has enabled the culturing of organisms only distantly related to previously cultured microbes. The lack of growth in the laboratory for many strains from this habitat stems from an inability to autonomously produce siderophores, and the resulting chemical dependence on other microorganisms regulates community establishment in the environment.

Production of the first transgenic cassava in Africa via direct shoot organogenesis from friable embryogenic calli and germination of maturing somatic embryos

The impact of cassava transformation technologies for agricultural development in Africa will depend largely on how successfully these capabilities are transferred and adapted to the African environment and local needs. Here we report on the first successful establishment of cassava regeneration and transformation capacity in Africa via organogenesis, somatic embryogenesis and friable embryogenic callus (FEC). As a prerequisite for genetic engineering, we evaluated six African cassava genotypes for the ability of a) induction of FEC b) hygromycin sensitivity and c) T-DNA integration potential by different Agrobacterium strains. FEC was induced in genotypes TMS 60444, TME 1 and TMS 91/02327. Potential tissues for FEC formation were induced in TMS 91/02324, TME 12 and TME 13. Pure and proliferating FEC was obtained and maintained only in TMS 60444. FEC growth and shoot organogenesis were completely suppressed when hygromycin was used at a concentration of 20 mg/l in all tissue types and genotypes. With somatic cotyledons, statistically significant differences (p
0.05) were observed between Agrobacterium strains and genotypes with respect to T-DNA transfer efficiency. Using somatic cotyledons, TME 8 was found to be the most amenable to transformation with maximum blue spots per GUS-positive explants, and Agrobacterium GV3101 proved to be superior to EHA105, LBA4404, and AGl-1 for T-DNA transfer based on transient assays with a reporter gene (GUS). With FEC, Agrobacterium LBA4404 was superior to other strains. This study also identified EHA105 as a new vir helper strain to recover transgenic cassava plants. PCR and Southern hybridization of genomic DNA of the hygromycin-resistant cassava plants to a hpt probe confirmed the integration of hpt with integration events varying between 1 and 2 insertions. The benefit of combining the FEC and shoot organogenesis systems for recovering transgenic cassava plants is described. The contributions of this report to enhancing the development and deployment of genetic engineering of cassava for agricultural biotechnology development in Africa are discussed.