A selective insecticidal protein from Pseudomonas mosselii for corn rootworm control

The coleopteran insect western corn rootworm (WCR, Diabrotica virgifera virgifera) is an economically important pest in North America and Europe. Transgenic corn plants producing Bacillus thuringiensis (Bt) insecticidal proteins have been useful against this devastating pest, but evolution of resistance has reduced their efficacy. Here, we report the discovery of a novel insecticidal protein, PIP‐47Aa, from an isolate of Pseudomonas mosselii. PIP‐47Aa sequence shows no shared motifs, domains or signatures with other known proteins. Recombinant PIP‐47Aa kills WCR, two other corn rootworm pests (Diabrotica barberi and Diabrotica undecimpunctata howardi) and two other beetle species (Diabrotica speciosa and Phyllotreta cruciferae), but it was not toxic to the spotted lady beetle (Coleomegilla maculata) or seven species of Lepidoptera and Hemiptera. Transgenic corn plants expressing PIP‐47Aa show significant protection from root damage by WCR. PIP‐47Aa kills a WCR strain resistant to mCry3A and does not share rootworm midgut binding sites with mCry3A or AfIP‐1A/1B from Alcaligenes that acts like Cry34Ab1/Cry35Ab1. Our results indicate that PIP‐47Aa is a novel insecticidal protein for controlling the corn rootworm pests.     

Biomineralization of calcium carbonate in the cell wall of Lithothamnion crispatum (Hapalidiales,Rhodophyta): correlation between the organic matrix and the mineral phase

Over the past few decades, progress has been made toward understanding the mechanisms of coralline algae mineralization. However, the relationship between the mineral phase and the organic matrix in coralline algae has not yet been thoroughly examined. The aim of this study was to describe the cell wall ultrastructure of Lithothamnion crispatum, a cosmopolitan rhodolith‐forming coralline algal species collected near Salvador (Brazil), and examine the relationship between the organic matrix and the nucleation and growth/shape modulation of calcium carbonate crystals. A nanostructured pattern was observed in L. crispatum along the cell walls. At the nanoscale, the crystals from L. crispatum consisted of several single crystallites assembled and associated with organic material. The crystallites in the bulk of the cell wall had a high level of spatial organization. However, the crystals displayed cleavages in the (104) faces after ultrathin sectioning with a microtome. This organism is an important model for biomineralization studies as the crystallographic data do not fit in any of the general biomineralization processes described for other organisms. Biomineralization in L. crispatum is dependent on both the soluble and the insoluble organic matrix, which are involved in the control of mineral formation and organizational patterns through an organic matrix‐mediated process. This knowledge concerning the mineral composition and organizational patterns of crystals within the cell walls should be taken into account in future studies of changing ocean conditions as they represent important factors influencing the physico‐chemical interactions between rhodoliths and the environment in coralline reefs.     

First Report of Pseudomonas lurida Causing Bacterial Leaf Spot on Miscanthus sinensis

Miscanthus spp. are large perennial wetland grasses that are receiving considerable attention as bioenergy crops. In late summer 2011, leaf spot symptoms were observed in a field of Miscanthus sinensis in Jeongseon, Gangwon province, Korea. Bacterial strains that belonged to the γ‐Proteobacteria genus Pseudomonas were isolated from the affected leaves. By phylogenetic analysis and phenotypic characterization, the representative strain MDM‐03 was identified as Pseudomonas lurida. Healthy M. sinensis leaves inoculated with MDM‐03 developed leaf spots similar to those observed in field. Bacteria re‐isolated from the leaf lesions were identical to the original strain MDM‐03 based on their cultural characteristics and 16S rDNA sequencing. This is the first report of bacterial leaf spot in Miscanthus sinensis.     

Intercropping System with Combined Application of Azospirillum and Pseudomonas fluorescens Reduces Root Rot Incidence Caused by Rhizoctonia bataticola and Increases Seed Cotton Yield

Root rot caused by Rhizoctonia bataticola is a serious threat in cotton. Field experiments were conducted to study the influences of intercropping system in cotton with inorganic fertilizer and two bioinoculants (Azospirillum and Pseudomonas) on root rot incidence and yield of cotton. The results revealed that among the intercropping systems, cotton intercropping with Sesbania aculeata (1 : 1 ratio) recorded the highest rhizosphere colonization of Pseudomonas fluorescens in the year 2007 and 2008 and the lowest root rot incidence of 1.40, 2.49 and 3.90; 1.02, 2.22 and 5.98% at the vegetative, flowering and maturity stages in the year 2007 and 2008, respectively. From nutrient management practices, integration of Azospirillum and Pseudomonas with 50% recommended dose of NPK recorded the highest rhizosphere colonization of P. fluorescens in both years and the lowest root rot incidence of 1.40, 2.32 and 3.36; 1.07, 2.01 and 5.25% at vegetative, flowering and maturity stages in 2007 and 2008, respectively. Cotton + S. aculeata recorded the maximum number of sympodial branches (23.5 and 20.62/plant in 2007 and 2008, respectively) and the highest seed cotton yield of 2010 and 1894 kg/ha. The highest cotton equivalent yield (CEY) of 2052 and 1895 kg/ha was recorded in cotton + onion system, which was closely followed by cotton + S. aculeata system that had the CEY of 2010 and 1894 kg/ha in 2007 and 2008, respectively. The increased CEY is due to increased cost of onion compared with S. aculeata. Combined application of 100% recommended dose of NPK and bioinoculants recorded the seed cotton yield of 2227 and 1983 kg/ha and CEY of 2460 and 2190 kg/ha in 2007 and 2008, respectively. The lowest root rot incidence and increased yield in cotton + S. aculeata combined with 50% NPK and bioinoculants could be due to synergistic effect among the bioinoculants and S. aculeata.     

A Pseudomonas aeruginosa TIR effector mediates immune evasion by targeting UBAP1 and TLR adaptors

Bacterial pathogens often subvert the innate immune system to establish a successful infection. The direct inhibition of downstream components of innate immune pathways is particularly well documented but how bacteria interfere with receptor proximal events is far less well understood. Here, we describe a Toll/interleukin 1 receptor (TIR) domain‐containing protein (PumA) of the multi‐drug resistant Pseudomonas aeruginosa PA7 strain. We found that PumA is essential for virulence and inhibits NF‐κB, a property transferable to non‐PumA strain PA14, suggesting no additional factors are needed for PumA function. The TIR domain is able to interact with the Toll‐like receptor (TLR) adaptors TIRAP and MyD88, as well as the ubiquitin‐associated protein 1 (UBAP1), a component of the endosomal‐sorting complex required for transport I (ESCRT‐I). These interactions are not spatially exclusive as we show UBAP1 can associate with MyD88, enhancing its plasma membrane localization. Combined targeting of UBAP1 and TLR adaptors by PumA impedes both cytokine and TLR receptor signalling, highlighting a novel strategy for innate immune evasion.     

Evaluation of small organic acids present in fast pyrolysis bio‐oil from lignocellulose as feedstocks for bacterial bioconversion

Small organic acids derived from fast pyrolysis of lignocellulosic biomass represent a significant proportion of microbially accessible carbon in bio‐oil. However, using bio‐oil for microbial cultivation is a highly challenging task due to its strong adverse effects on microbial growth as well as its complex composition. In this study, the main small organic acids present in bio‐oil as acetate, formate and propionate were evaluated with respect to their suitability as feedstocks for bacterial growth. For this purpose, the growth behavior of four biotechnological production hosts—Escherichia coli, Pseudomonas putida, Bacillus subtilis, and Corynebacterium glutamicum—was quantified and compared. The bacteria were cultivated on single acids and mixtures of acids in different concentrations and evaluated using common biotechnological efficiency parameters. In addition, cultivation experiments on pretreated fast pyrolysis‐derived bio‐oil fractions were performed with respect to the suitability of the bacterial strains to tolerate inhibitory substances. Results suggest that both P. putida and C. glutamicum metabolize acetate—the major small organic acid generated during fast pyrolysis of lignocellulosic biomass—as sole carbon source over a wide concentration range, are able to grow on mixtures of small organic acids present in bio‐oil and can, to a limited extent, tolerate the highly toxic inhibitory substances within bio‐oil. This work provides an important step in search of suitable bacterial strains for bioconversion of lignocellulosic‐based feedstocks and thus contributes to establishing efficient bioprocesses within a future bioeconomy.     

Incorporating germination‐induction into decontamination strategies for bacterial spores

Oenococcus oeni is the dominant species able to cope with a hostile environment of wines, comprising cumulative effects of low pH, high ethanol and SO₂ content, nonoptimal growth temperatures and growth inhibitory compounds. Ethanol tolerance is a crucial feature for the activity of O. oeni cells in wine because ethanol acts as a disordering agent of its cell membrane and negatively affects metabolic activity; it damages the membrane integrity, decreases cell viability and, as other stress conditions, delays the start of malolactic fermentation with a consequent alteration of wine quality. The cell wall, cytoplasmic membrane and metabolic pathways are the main sites involved in physiological changes aimed to ensure an adequate adaptive response to ethanol stress and to face the oxidative damage caused by increasing production of reactive oxygen species. Improving our understanding of the cellular impact of ethanol toxicity and how the cell responds to ethanol stress can facilitate the development of strategies to enhance microbial ethanol tolerance; this allows to perform a multidisciplinary endeavour requiring not only an ecological study of the spontaneous process but also the characterization of useful technological and physiological features of the predominant strains in order to select those with the highest potential for industrial applications.     

Acidic lipase Lip I.3 from a Pseudomonas fluorescens‐like strain displays unusual properties and shows activity on secondary alcohols

Aims

Identification, cloning, expression and characterization of a novel lipase – Lip I.3 – from strain Pseudomonas CR‐611.

Methods and Results

The corresponding gene was identified and isolated by PCR‐amplification, cloned and expressed in Escherichia coli, and purified by refolding from inclusion bodies. Analysis of the deduced amino acid sequence revealed high homology with members of the bacterial lipase family I.3, showing 97% identity to a putative lipase from Pseudomonas fluorescens Pf0‐1, and 93% identity to a crystallized extracellular lipase from Pseudomonas sp. MIS38. A typical C‐terminal type I secretion signal and several putative Ca²⁺ binding sites were also identified. Experimental data confirmed that Lip I.3 requires Ca²⁺ ions for correct folding and activity. The enzyme differs from the previously reported family I.3 lipases in optimal pH, being the first acidophilic lipase reported in this family. Furthermore, Lip I.3 shows a strong preference for medium chain fatty acid esters and does not display interfacial activation. When tested for activity on secondary alcohol hydrolysis, Lip I.3 displayed higher efficiency on aromatic alcohols rather than on alkyl alcohols.

Conclusions

A new family I.3 lipase with unusual properties has been isolated, cloned and described. This will contribute to a better knowledge of family I.3 lipases, a family that has been scarcely explored, and that might provide a novel source of biocatalysts.

Significance and Impact of the Study

The unusual properties shown by Lip I.3 and the finding of activity and enantioselectivity on secondary alcohol esters may contribute to the development of new enzymatic tools for applied biocatalysis.     

Genotypic and phenotypic analyses reveal distinct population structures and ecotypes for sugar beet‐associated Pseudomonas in Oxford and Auckland

Fluorescent pseudomonads represent one of the largest groups of bacteria inhabiting the surfaces of plants, but their genetic composition in planta is poorly understood. Here, we examined the population structure and diversity of fluorescent pseudomonads isolated from sugar beet grown at two geographic locations (Oxford, United Kingdom and Auckland, New Zealand). To seek evidence for niche adaptation, bacteria were sampled from three types of leaves (immature, mature, and senescent) and then characterized using a combination of genotypic and phenotypic analysis. We first performed multilocus sequence analysis (MLSA) of three housekeeping genes (gapA, gltA, and acnB) in a total of 152 isolates (96 from Oxford, 56 from Auckland). The concatenated sequences were grouped into 81 sequence types and 22 distinct operational taxonomic units (OTUs). Significant levels of recombination were detected, particularly for the Oxford isolates (rate of recombination to mutation (r/m) = 5.23 for the whole population). Subsequent ancestral analysis performed in STRUCTURE found evidence of six ancestral populations, and their distributions significantly differed between Oxford and Auckland. Next, their ability to grow on 95 carbon sources was assessed using the Biolog? GN2 microtiter plates. A distance matrix was generated from the raw growth data (A₆₆₀) and subjected to multidimensional scaling (MDS) analysis. There was a significant correlation between substrate utilization profiles and MLSA genotypes. Both phenotypic and genotypic analyses indicated presence of a geographic structure for strains from Oxford and Auckland. Significant differences were also detected for MLSA genotypes between strains isolated from immature versus mature/senescent leaves. The fluorescent pseudomonads thus showed an ecotypic population structure, suggestive of adaptation to both geographic conditions and local plant niches.     

Summer meeting 2013 – when the sleepers wake: the germination of spores of Bacillus species

Spores of Bacillus species can remain dormant and resistant for years, but can rapidly ‘come back to life’ in germination triggered by agents, such as specific nutrients, and non‐nutrients, such as CaDPA, dodecylamine and hydrostatic pressure. Major events in germination include release of spore core monovalent cations and CaDPA, hydrolysis of the spore cortex peptidoglycan (PG) and expansion of the spore core. This leads to a well‐hydrated spore protoplast in which metabolism and macromolecular synthesis begin. Proteins essential for germination include the GerP proteins that facilitate germinant access to spores' inner layers, germinant receptors (GRs) that recognize and respond to nutrient germinants, GerD important in rapid GR‐dependent germination, SpoVA proteins important in CaDPA release and cortex‐lytic enzymes that degrade cortex PG. Rates of germination of individuals in spore populations are heterogeneous, and methods have been developed recently to simultaneously analyse the germination of multiple individual spores. Spore germination heterogeneity is due primarily to large variations in GR levels among individual spores, with spores that germinate extremely slowly and termed superdormant having very low GR levels. These and other aspects of spore germination will be discussed in this review, and major unanswered questions will also be discussed.     

Bacillus probiotics: an alternative to antibiotics for livestock production

The use of probiotics as feed supplements in animal production has increased considerably over the last decade, particularly since the ban on antibiotic growth promoters in the livestock sector. Several Bacillus sp. are attractive for use as probiotic supplements in animal feed due to their ability to produce spores. Their heat stability and ability to survive the low pH of the gastric barrier represent an advantage over other probiotic micro‐organisms. This review discusses important characteristics required for selection of Bacillus probiotic strains and summarizes the beneficial effect of Bacillus‐based feed additives on animal production. Although the mechanism of action of Bacillus probiotics has not been fully elucidated, they are effective in improving the growth, survival and health status of terrestrial and aquatic livestock. Bacillus strains also have utility in bioremediation and can reduce nitrogenous waste, thereby improving environmental conditions and water quality. Finally, recent innovative approaches for using Bacillus spores in various applications are discussed.     

Biotransformations of Bile Acids with Bacteria from Cayambe Slaughterhouse (Ecuador): Synthesis of Bendigoles

The biotransformations of cholic acid ( 1a ), deoxycholic acid ( 1b ), and hyodeoxycholic acid ( 1c ) to bendigoles and other metabolites with bacteria isolated from the rural slaughterhouse of Cayambe (Pichincha Province, Ecuador) were reported. The more active strains were characterized, and belong to the genera Pseudomonas and Rhodococcus. Various biotransformation products were obtained depending on bacteria and substrates. Cholic acid ( 1a ) afforded the 3‐oxo and 3‐oxo‐4‐ene derivatives 2a and 3a (45% and 45%, resp.) with P. mendocina ECS10, 3,12‐dioxo‐4‐ene derivative 4a (60%) with Rh. erythropolis ECS25, and 9,10‐secosteroid 6 (15%) with Rh. erythropolis ECS12. Bendigole F ( 5a ) was obtained in 20% with P. fragi ECS22. Deoxycholic acid ( 1b ) gave 3‐oxo derivative 2b with P. prosekii ECS1 and Rh. erythropolis ECS25 (20% and 61%, resp.), while 3‐oxo‐4‐ene derivative 3b was obtained with P. prosekii ECS1 and P. mendocina ECS10 (22% and 95%, resp.). Moreover, P. fragi ECS9 afforded bendigole A ( 8b ; 80%). Finally, P. mendocina ECS10 biotransformed hyodeoxycholic acid ( 1c ) to 3‐oxo derivative 2c (50%) and Rh. erythropolis ECS12 to 6α‐hydroxy‐3‐oxo‐23,24‐dinor‐5β‐cholan‐22‐oic acid ( 9c , 66%). Bendigole G ( 5c ; 13%) with P. prosekii ECS1 and bendigole H ( 8c ) with P. prosekii ECS1 and Rh. erythropolis ECS12 (20% and 16%, resp.) were obtained.     

Survival of Phytophthora cinnamomi and Fusarium verticillioides in commercial potting substrates for ornamental plants

Live plants, particularly when accompanied by soil or potting substrates, are considered the main pathway for international spread of plant pathogens. Modern, rapid shipping technologies for international plant trade increase the probability of plant pathogen survival during transport and the subsequent chances of disease outbreaks in new locations. The survival of two model pathogens, an Oomycete, Phytophthora cinnamomi, and a filamentous fungus, Fusarium verticillioides, was studied in two different commercial potting substrates (peat and peat‐free) under glasshouse conditions in the absence of a plant host. Survival rates were analysed at 2, 7, 12 and 17 months after substrate inoculation. Fusarium verticillioides had the longest survival rate, and was still present at 17 months. In contrast, P. cinnamomi survived up to 7 months but was not recovered after 12 or 17 months. There was no significant difference in the number of colony‐forming units (CFUs) of either pathogen in the two substrates, except at 2 months, when higher numbers were recovered from peat substrates.     

Optimization of growth conditions and fatty acid analysis for three freshwater diatom isolates

Diatoms are a group of highly abundant and diverse aquatic algae species. They contain high lipid content along with many bioactive compounds that can be exploited for biotechnological applications. Despite these attractive attributes, diatoms are underrepresented in production projects due to difficulties in their cultivation. To optimize the growth of three freshwater diatom isolates, Cyclotella sp., Synedra sp. and Navicula sp., an orthogonal assay on N, P, Si and Fe, as well as temperature and pH, was designed using traditional single‐factor tests. We also studied the effect of using nanosilica as an alternate Si source on growth and found that the diatom isolates studied achieved their highest growth rates under different combinations of nutrient and environmental conditions. Silica had the greatest influence on growth, followed by phosphate and iron. The optimized growth conditions for Synedra sp. were N: 30 mg L^?1, P: 3 mg L^?1, Si: 14.8 mg L^?1, Fe: 0.448 mg L^?1, temperature 25°C and pH 8. For Navicula sp.: N: 20 mg L^?1, P: 2.5 mg L^?1, Si: 19.7 mg L^?1, Fe: 0.112 mg L^?1, temperature 30°C and pH 7.5–8. For Cyclotella sp.: N: 20 mg L^?1, P: 2.5 mg L^?1, Si: 19.7 mg L^?1, Fe: 0.448 mg L^?1, temperature 30°C and pH 7.5–8. Nano silica negatively affected growth in Navicula sp. and Cyclotella sp., but no such effect was observed in Synedra sp. Fatty acid profiling showed C16:0, C16:1(n ? 7), C18:0 and C20:5(n ? 3) as major fatty acids, with no significant differences in fatty acid methyl ester profiles between traditional and modified media. This work gives us a new insight into the growth requirements of freshwater diatom species, which are less studied than marine species.