The Secreted Esterase of Propionibacterium freudenreichii Has a Major Role in Cheese Lipolysis

Free fatty acids are important flavor compounds in cheese. Propionibacterium freudenreichii is the main agent of their release through lipolysis in Swiss cheese. Our aim was to identify the esterase(s) involved in lipolysis by P. freudenreichii. We targeted two previously identified esterases: one secreted esterase, PF#279, and one putative cell wall-anchored esterase, PF#774. To evaluate their role in lipolysis, we constructed overexpression and knockout mutants of P. freudenreichii CIRM-BIA1^T for each corresponding gene. The sequences of both genes were also compared in 21 wild-type strains. All strains were assessed for their lipolytic activity on milk fat. The lipolytic activity observed matched data previously reported in cheese, thus validating the relevance of the method used. The mutants overexpressing PF#279 or PF#774 released four times more fatty acids than the wild-type strain, demonstrating that both enzymes are lipolytic esterases. However, inactivation of the pf279 gene induced a 75% reduction in the lipolytic activity compared to that of the wild-type strain, whereas inactivation of the pf774 gene did not modify the phenotype. Two of the 21 wild-type strains tested did not display any detectable lipolytic activity. Interestingly, these two strains exhibited the same single-nucleotide deletion at the beginning of the pf279 gene sequence, leading to a premature stop codon, whereas they harbored a pf774 gene highly similar to that of the other strains. Taken together, these results clearly demonstrate that PF#279 is the main lipolytic esterase in P. freudenreichii and a key agent of Swiss cheese lipolysis.     

Identification of a Novel Alkaliphilic Esterase Active at Low Temperatures by Screening a Metagenomic Library from Antarctic Desert Soil

A novel esterase was identified through functional screening of a metagenomic library in Escherichia coli obtained from Antarctic desert soil. The 297-amino-acid sequence had only low (<29%) similarity to a putative esterase from Burkholderia xenovorans. The enzyme was active over a temperature range of 7 to 54°C and at alkaline pH levels and is a potential candidate for industrial application.The cold deserts of the McMurdo Dry Valleys, South Victoria Land, Eastern Antarctica, are widely acknowledged as having the harshest soil environments on Earth (6, 8, 26). Despite the apparent hostility of the environment, we and others have reported both unexpectedly high biomass (9) and phylogenetic diversity (1, 19, 24, 29) in Antarctic soils. The presence of numerous novel taxa suggests that these soils might prove to be valuable sources of genetic material for mining novel industrial enzymes active at low temperatures (9, 23).Esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3) catalyze the hydrolysis and synthesis of ester compounds. Their applications in industry cover a broad spectrum, including as detergent additives, in food processing, in environmental bioremediation, and in biomass and plant waste degradation for the production of useful organocompounds (3, 16).In this study, a novel esterase was isolated from a metagenomic fosmid library obtained from Antarctic soil. The enzyme displays activity over a wide temperature range and has very low similarity (<29% amino acid identity) to esterases in the GenBank database. The study demonstrates the value of Antarctic metagenomes as a source of novel industrial products.     

Cloning and Characterization of an Intracellular Esterase from the Wine-Associated Lactic Acid Bacterium Oenococcus oeni

We report the cloning and characterization of EstB28, the first esterase to be so characterized from the wine-associated lactic acid bacterium, Oenococcus oeni. The published sequence for O. oeni strain PSU-1 was used to identify putative esterase genes and design PCR primers in order to amplify the corresponding region from strain Ooeni28, an isolate intended for inoculation of wines. In this way a 912-bp open reading frame (ORF) encoding a putative esterase of 34.5 kDa was obtained. The amino acid sequence indicated that EstB28 is a member of family IV of lipolytic enzymes and contains the GDSAG motif common to other lactic acid bacteria. This ORF was cloned into Escherichia coli using an appropriate expression system, and the recombinant esterase was purified. Characterization of EstB28 revealed that the optimum temperature, pH, and ethanol concentration were 40°C, pH 5.0, and 28% (vol/vol), respectively. EstB28 also retained marked activity under conditions relevant to winemaking (10 to 20°C, pH 3.5, 14% [vol/vol] ethanol). Kinetic constants were determined for EstB28 with p-nitrophenyl (pNP)-linked substrates ranging in chain length from C₂ to C₁₈. EstB28 exhibited greatest specificity for C₂ to C₄ pNP-linked substrates.The quality of fermented foods and beverages is affected in part by their composition of aroma compounds. In winemaking, the malolactic fermentation is used to deacidify wine and is typically carried out by Lactobacillus spp., Pediococcus spp., and particularly Oenococcus oeni (10, 7). Numerous reports clearly show that outside of this core function, lactic acid bacteria (LAB) can also bring about significant changes of sensorial importance (2, 5, 12, 32, 40, 49, 55). Such studies typically examined the action of active cultures or whole cells; however, the promise of LAB as a source of purified enzymes for use as additives in winemaking has recently been highlighted (43).Oenococcus oeni is acidophilic and indigenous to wine and similar environments. While the genome of the commercial PSU-1 strain has been sequenced and analyzed (44), there is limited information on the genes or their potential contribution to food and beverage aroma. The only such genes which have been cloned and partially characterized are alsS and alsD (24), which are thought to be responsible for the production of diacetyl, the principle compound conferring “buttery” aroma and flavor in wine (reviewed in reference 4). Analogous characterization of other flavor-related genes and enzymes not only may have practical implications for processes using LAB but also may be of fundamental interest.As a group, esters are a quantitatively significant constituent of beverages such as wine (total of >100 mg·liter⁻¹) (15). Included in this group are the C₄ to C₁₀ ethyl esters of organic acids, ethyl esters of straight-chain fatty acids (and branched-chain fatty acids to a lesser degree), and acetates of higher alcohols which are largely, if not exclusively, responsible for the fruity aroma of wine (13, 14). Some volatile esters are frequently found in fermented beverages in only trace amounts, often below threshold concentrations (3, 21, 25, 29, 50). However, they are extremely important for the flavor profile of these products, with different esters often having a synergistic effect to collectively affect aroma when their individual threshold concentrations are not exceeded. The fact that most esters are present in wine at concentrations around the threshold value implies that minor concentration changes might have a dramatic effect on the wine''s flavor (3, 21, 25, 29, 50). For this reason, an understanding of the hydrolysis and synthesis of esters in winemaking and how these may be manipulated is essential.A large amount of esters is formed during the primary fermentation by yeast; after this, LAB can contribute by increasing and decreasing the ester concentration (2, 5, 12, 40, 49, 55). Ester hydrolysis and synthesis can be catalyzed by esterases (6, 35, 38, 54). These enzymes commonly contain a catalytic triad composed of Ser, His, and Asp/Glu residues and a nucleophilic elbow structural motif (GXSXG), which contains the active-site serine residue (1, 31, 36, 48). They also contain an oxyanion hole, of which two residues donate their backbone amide protons to stabilize the substrate in the transition state. The oxyanion hole residues (in bold) have been divided into two groups termed GX and GGGX, with the glycine and a hydrophobic residue (X) being highly conserved (48).While extensive research has been carried out on the enzymes responsible for ester formation by wine strains of Saccharomyces cerevisiae (22, 23, 45, 51), esterase activity for wine-related LAB is not well documented. Most characterization of esterases in LAB has focused on dairy isolates (9, 16-18, 20). Parallel work in a wine context is limited despite general acceptance of the importance of esters in wine. Until recently, most evidence that wine LAB possess esterase activity came from wine volatile profiling studies which investigated the changes in concentration of individual esters during malolactic fermentation (12, 40, 55). Such changes in ester concentration were strain specific and had the potential to greatly affect the final aroma of wine.Our survey of the esterase activities of whole LAB cells found variations within species and even greater variation between the genera (42), with O. oeni showing greatest activity toward the p-nitrophenyl (pNP)-linked substrates tested. More recently (41), the esterase activities of whole O. oeni, lactobacillus, and pediococcus cells was determined under conditions with some relevance to wine. At least partial resistance to the harsh conditions used was observed, thereby demonstrating a necessary requirement of any enzyme intended for application in analogous environments. To more completely characterize esterases of LAB, the enzymes and their structural genes must be fully investigated. This study represents an effort to dissect the complex array of ester synthesis and hydrolysis activities in whole cells by cloning, heterologous expression, partial purification, and biochemical characterization of a single esterase from O. oeni. With a view to applying such an esterase under conditions found in wine and perhaps other industrial settings, enzyme function under the harsh physicochemical conditions frequently encountered in wine was examined.     

Selection of a Bacillus pumilus Strain Highly Active against Ceratitis capitata (Wiedemann) Larvae

Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), the Mediterranean fruit fly (medfly), is one of the most important fruit pests worldwide. The medfly is a polyphagous species that causes losses in many crops, which leads to huge economic losses. Entomopathogenic bacteria belonging to the genus Bacillus have been proven to be safe, environmentally friendly, and cost-effective tools to control pest populations. As no control method for C. capitata based on these bacteria has been developed, isolation of novel strains is needed. Here, we report the isolation of 115 bacterial strains and the results of toxicity screening with adults and larvae of C. capitata. As a result of this analysis, we obtained a novel Bacillus pumilus strain, strain 15.1, that is highly toxic to C. capitata larvae. The toxicity of this strain for C. capitata was related to the sporulation process and was observed only when cultures were incubated at low temperatures before they were used in a bioassay. The mortality rate for C. capitata larvae ranged from 68 to 94% depending on the conditions under which the culture was kept before the bioassay. Toxicity was proven to be a special characteristic of the newly isolated strain, since other B. pumilus strains did not have a toxic effect on C. capitata larvae. The results of the present study suggest that B. pumilus 15.1 could be considered a strong candidate for developing strategies for biological control of C. capitata.The Mediterranean fruit fly (medfly), Ceratitis capitata, is considered a highly invasive agricultural and economically important pest throughout the world. In less than 200 years the range of this species has expanded from its native habitat in sub-Saharan Africa, and it has become a cosmopolitan species (26) that is present on five continents (14, 46). The wide distribution of the medfly is attributed, among other things, to its remarkably polyphagous behavior (more than 300 host plants have been reported) (43), to its resistance to cold climates (65), and to successful establishment after multiple introductions (30, 49) as a result of the increasing frequency of global trade (46).Medfly infestations cause serious economic losses and sometimes result in complete loss of crops (76). Numerous methods have been tried to control medfly populations, including chemical products, such as malathion and other organophosphate insecticides (4, 8), classic biological control programs based on the release of some of parasitoids and predators (38, 41, 44), toxic baits (2, 13, 31, 32, 35, 56), mass trapping systems (24, 51), the sterile insect technique (7, 34, 61, 63, 72, 73), and development of integrated strategies of management (71). In spite of all of these attempts, control of Mediterranean fruit fly populations has been ineffective, and losses associated with this pest worldwide are constantly increasing (21, 46).Insecticides based on microbial agents (bacteria, fungi, and viruses) are a promising alternative that has received a great deal of attention for control of C. capitata (5, 13, 18, 40, 55), but so far no such insecticide has reached a commercial stage. Among the microbial insecticides, bacteria are very successful agents in biological control programs (17, 29). The entomopathogenic bacteria belonging to the genus Bacillus are natural agents used for biological control of invertebrate pests and are the basis of many commercial insecticides. Three species of the genus Bacillus have been mass produced and commercialized: Bacillus sphaericus, Bacillus thuringiensis, and Paenibacillus popilliae (formerly Bacillus popilliae) (29, 54). These organisms have different spectra and levels of activity that are correlated with the nature of the toxins, which are very frequently produced during sporulation (16, 17). B. thuringiensis was the first Bacillus species used in biological control programs for pests and human vector disease insects (17, 62). During its stationary phase, this Gram-positive, aerobic, ubiquitous, endospore-forming bacterium produces parasporal crystalline inclusions composed mainly of two types of insecticidal proteins (Cry and Cyt toxins) (62) that are toxic to a variety of insects, in some cases at the species level.There have been some reports of B. thuringiensis strains active against other fruit flies (3, 37, 58, 59, 67), but there has been no report of any Bacillus strain with activity against C. capitata.The aim of this study was to search for novel bacteria belonging to the genus Bacillus, specifically B. thuringiensis, with activity against adults and larvae of C. capitata that could be used as biological control agents. Isolation of 115 bacterial strains, evaluation of the insecticidal activities of these strains, and identification of a novel strain of Bacillus pumilus that is highly toxic to C. capitata larvae are reported here. In addition, we found that toxicity was observed only when cultures of B. pumilus strain 15.1 were exposed to low temperatures. The isolation of this novel pathogenic strain could be important for future development of biotechnological strategies aimed at reducing the economic losses caused by C. capitata.     

Zoosporic Tolerance to pH Stress and Its Implications for Phytophthora Species in Aquatic Ecosystems

Phytophthora species, a group of destructive plant pathogens, are commonly referred to as water molds, but little is known about their aquatic ecology. Here we show the effect of pH on zoospore survival of seven Phytophthora species commonly isolated from irrigation reservoirs and natural waterways and dissect zoospore survival strategy. Zoospores were incubated in a basal salt liquid medium at pH 3 to 11 for up to 7 days and then plated on a selective medium to determine their survival. The optimal pHs differed among Phytophthora species, with the optimal pH for P. citricola at pH 9, the optimal pH for P. tropicalis at pH 5, and the optimal pH for the five other species, P. citrophthora, P. insolita, P. irrigata, P. megasperma, and P. nicotianae, at pH 7. The greatest number of colonies was recovered from zoospores of all species plated immediately after being exposed to different levels of pH. At pH 5 to 11, the recovery rate decreased sharply (P ≤ 0.0472) after 1-day exposure for five of the seven species. In contrast, no change occurred (P ≥ 0.1125) in the recovery of any species even after a 7-day exposure at pH 3. Overall, P. megasperma and P. citricola survived longer at higher rates in a wider range of pHs than other species did. These results are generally applicable to field conditions as indicated by additional examination of P. citrophthora and P. megasperma in irrigation water at different levels of pH. These results challenge the notion that all Phytophthora species inhabit aquatic environments as water molds and have significant implications in the management of plant diseases resulting from waterborne microbial contamination.Phytophthora species, a group of oomycetes in the kingdom of Stramenopila and well-known plant pathogens, were first listed as “water molds” by Blackwell in 1944 (5), and this notion has since been generally accepted. These species are phylogenetically close to golden-brown algae, although morphologically and physiologically, they resemble fungi. Most algae are aquatic in nature. Phytophthora species produce flagellate zoospores as their primary dispersal structure (35-37, 39). Zoospores can travel in aquatic environments actively on their own locomotion and passively through water movement (12, 13, 41).More than 20 species of Phytophthora, including P. ramorum, the sudden oak death pathogen, have been isolated from irrigation reservoirs and natural waterways (20-22, 30, 40, 43), and a number of previously unknown taxa also have been documented in aquatic environments (8, 24). These pathogens pose a threat to agricultural sustainability and natural ecosystems, as agriculture increasingly depends on recycled water for irrigation in light of rapidly spreading global water scarcity (19, 22). Recycling irrigation systems provide an efficient means of pathogen dissemination from a single point of infection to an entire farm and from a single farm to other farms sharing the same water resources (22, 24).A search of science-based solutions to this crop health issue reveals a surprising lack of information on the aquatic ecology of Phytophthora species. For instance, hydrogen ion concentration (pH) is among the most important water quality parameters which influence sporangium production and germination (1-3, 6, 32, 34, 38), survival of thick-walled chlamydospores and oospores in the soil environment, and disease development (2, 4, 33, 44). However, the effect of pH on the survival of zoospores and growth of germlings in aquatic environments is not known. As motile zoospores lack cell walls and encysted spores or cysts have thin walls, they are presumably more vulnerable to pH stress than chlamydospores and oospores are. On the other hand, the pH level is likely to fluctuate more regularly and at a greater range in aquatic systems, such as irrigation reservoirs, than in soil systems. pH can change diurnally due to respiration of aquatic plants and seasonally due to rain, oxidation of sulfide-containing sediments through the production of sulfuric acid, algal blooms, and released bases or acids from residues of fertilizer and pesticides. Thus, zoospores and aquatic systems are more prone to the influence of wide pH changes than chlamydospores/oospores in soil systems are. The aim of this study was to determine the impact of pH on zoospore survival and understand the aquatic ecology of different Phytophthora species.     

Evolution of Lactococcus lactis Phages within a Cheese Factory

We have sequenced the double-stranded DNA genomes of six lactococcal phages (SL4, CB13, CB14, CB19, CB20, and GR7) from the 936 group that were isolated over a 9-year period from whey samples obtained from a Canadian cheese factory. These six phages infected the same two industrial Lactococcus lactis strains out of 30 tested. The CB14 and GR7 genomes were found to be 100% identical even though they were isolated 14 months apart, indicating that a phage can survive in a cheese plant for more than a year. The other four genomes were related but notably different. The length of the genomes varied from 28,144 to 32,182 bp, and they coded for 51 to 55 open reading frames. All five genomes possessed a 3′ overhang cos site that was 11 nucleotides long. Several structural proteins were also identified by nano-high-performance liquid chromatography-tandem mass spectrometry, confirming bioinformatic analyses. Comparative analyses suggested that the most recently isolated phages (CB19 and CB20) were derived, in part, from older phage isolates (CB13 and CB14/GR7). The organization of the five distinct genomes was similar to the previously sequenced lactococcal phage genomes of the 936 group, and from these sequences, a core genome was determined for lactococcal phages of the 936 group.The manufacture of cheeses requires the inoculation of carefully selected bacterial cultures, known as starter cultures, at concentrations of at least 10⁷ live bacteria per ml of heat-treated milk. The purpose of this process is to control the fermentation and to obtain high-quality fermented products (29). Starter cultures are a combination of lactic acid bacteria (LAB), of which one of the most important species is Lactococcus lactis. L. lactis is a low-GC gram-positive bacterium used to metabolize lactose into lactic acid during the production of several cheese varieties. Because large amounts of lactococcal cells are cultivated each day in large-scale fermentation vats and because these cells are susceptible to bacteriophage infection, it is not surprising that most cheese factories have experienced problems with phage contamination (13). Even a single phage infecting a starter strain is enough to begin a chain reaction that can eventually inhibit bacterial growth and cause production delays, taste and texture variations, and even complete fermentation failures (1, 29).Phage infections are unpredictable in food fermentations. Their presence and persistence in a dairy factory can be explained in many ways. First, raw milk can introduce new phages into an industrial plant (25). Madera et al. (22) also reported that newly isolated lactococcal phages were more resistant to pasteurization. Whey, a liquid by-product of cheese manufacturing, is another reservoir that can spread phages in a factory environment (25). Airborne phage dissemination may also be important since concentrations of up to 10⁶ PFU/m³ have been observed close to a functional whey separation tank (32).For decades, the dairy industry has been working to curtail the propagation of virulent phages using a variety of practical strategies, including, among others, sanitation, optimized factory design, air filtration units, rotation of bacterial strains, and the use of phage resistance systems (13). Yet new virulent phages emerge on a regular basis. Indeed, large-scale industrial milk fermentation processes can be slowed down by virulent phages of the Caudovirales order. Members of three lactococcal phage groups, namely, 936, c2, and P335, are mostly found in dairy plants. The 936-like phages are by far the most predominant worldwide (3, 18, 22, 27).Phages of the 936 group have a double-stranded DNA genome and possess a long noncontractile tail connected to a capsid with icosahedral symmetry characteristic of the Siphoviridae family. Currently, six complete phage genomes of the lactococcal 936 group are available in public databases, including sk1 (6), bIL170 (10), jj50, 712, P008 (23), and bIBB29 (16). Their comparative analysis revealed a conserved gene organization despite being isolated from different countries. Most of the differences have been observed in the early gene module, where insertions, deletions, and point mutations likely occurred (16, 23). Moreover, it is assumed that these phages can also exchange DNA through recombination with other bacterial viruses present in the same ecosystem.Because new members of this lactococcal phage group are regularly isolated, a better understanding of their evolution is warranted to better control them. A cheese factory is a particular man-made niche where rapidly growing bacterial strains encounter ubiquitous phages. Such active environments provide ample opportunities for phage evolution, especially to dodge phage resistance mechanisms that may be present in host cells. Nonetheless, the evolutionary dynamics that shape the diversity of lactococcal phage populations are still not well understood.In this study, we analyzed the genome and structural proteome of six 936-group phages (SL4, CB13, CB14, CB19, CB20, and GR7) that infected the same L. lactis strains and were isolated over a 9-year period from a cheese factory.     

Vegetation and Soil Environment Influence the Spatial Distribution of Root-Associated Fungi in a Mature Beech-Maple Forest

Although the level of diversity of root-associated fungi can be quite high, the effect of plant distribution and soil environment on root-associated fungal communities at fine spatial scales has received little attention. Here, we examine how soil environment and plant distribution affect the occurrence, diversity, and community structure of root-associated fungi at local patch scales within a mature forest. We used terminal restriction fragment length polymorphism and sequence analysis to detect 63 fungal species representing 28 different genera colonizing tree root tips. At least 32 species matched previously identified mycorrhizal fungi, with the remaining fungi including both saprotrophic and parasitic species. Root fungal communities were significantly different between June and September, suggesting a rapid temporal change in root fungal communities. Plant distribution affected root fungal communities, with some root fungi positively correlated with tree diameter and herbaceous-plant coverage. Some aspects of the soil environment were correlated with root fungal community structure, with the abundance of some root fungi positively correlated with soil pH and moisture content in June and with soil phosphorous (P) in September. Fungal distribution and community structure may be governed by plant-soil interactions at fine spatial scales within a mature forest. Soil P may play a role in structuring root fungal communities at certain times of the year.In temperate forests, most trees form relationships with ectomycorrhizal (ECM) fungi, and the diversity of this fungal group alone can approach 100 species within a forest stand (17, 20, 60). The ECM mutualism may be necessary for the success of some native plant species, as approximately 90% of roots of some tree species are colonized by ECM fungi (65). Nevertheless, we still know surprisingly little about what controls the community structure and distribution of root-associated fungi in forest systems (44, 46). The occurrence of root-associated fungi may broadly reflect soil environmental conditions and the presence of preferred plant hosts (28, 61), but how these factors interact to influence the diversity, distribution, and community structure of these fungi within forest habitat patches at a local scale is uncertain.The distribution of root-associated fungi may be primarily a species response to local soil environmental conditions. For example, both the quality (i.e., nutrient content) and the quantity of soil organic matter are known to influence the diversity of ECM communities (18, 20, 32). ECM fungi also vary in drought tolerance (14, 36), resistance to fire (61, 65), and tolerance to soil acidity (19) and temperature (56). Changes in soil chemistry, especially as they relate to pH and the availability of nitrogen (N) and phosphorous (P), might favor selection of fungi most capable of tolerating environmental extremes (2, 28, 29).Plant distribution and identity may, however, play the strongest role in structuring the below-ground diversity of root-associated fungi. Many ECM fungi can colonize a wide range of plant species, and plant species can be host to a large number of ECM fungi (63), especially those in the families Russulaceae and Thelephoraceae (34, 35, 62). Moreover, some ECM fungi are also specific to certain tree species (e.g., Suillus and Rhizopogon species are specific to species in the family Pinaceae [38, 39]). At the local scale, fungal distribution and richness might be influenced by differences in root growth and architecture (30, 42), by the distance to the bole of the tree (11, 42, 49), or by the presence of neighboring trees (29, 64). Temporal changes in ECM communities could be associated with seasonal changes in plant physiology and phenology (3, 8, 17).An often overlooked factor influencing root-associated fungi of tree roots is the occurrence of herbaceous plant species within forest stands. Many species of parasitic, achlorophyllous angiosperms obtain carbon (C) from ECM fungi that colonize tree roots (43), and some autotrophic plants could also obtain C from ECM fungi during certain times of the year (58). Herbaceous plants also influence the cycling of nutrients, including N, P, and K (potassium) (31, 50), within forests, which could affect the distribution of root-associated fungi. Herbaceous plants can also produce secondary compounds that inhibit colonization of tree roots (68).In this study, we examine the effect of soil environment and plant distribution on root-associated fungi of tree roots in a mature beech-maple forest at two points in the growing season. We predict that plant distribution, both the distribution of host trees and that of herbaceous plants, influences fungi associated with tree roots in terms of both community structure and diversity. Molecular typing protocols, including a site-specific database of fungal sequences and fingerprints, were used to identify fungi on tree roots (i.e., beech or maple trees) to the species level.     

Insulin Regulates Adipocyte Lipolysis via an Akt-Independent Signaling Pathway

After a meal, insulin suppresses lipolysis through the activation of its downstream kinase, Akt, resulting in the inhibition of protein kinase A (PKA), the main positive effector of lipolysis. During insulin resistance, this process is ineffective, leading to a characteristic dyslipidemia and the worsening of impaired insulin action and obesity. Here, we describe a noncanonical Akt-independent, phosphoinositide-3 kinase (PI3K)-dependent pathway that regulates adipocyte lipolysis using restricted subcellular signaling. This pathway selectively alters the PKA phosphorylation of its major lipid droplet-associated substrate, perilipin. In contrast, the phosphorylation of another PKA substrate, hormone-sensitive lipase (HSL), remains Akt dependent. Furthermore, insulin regulates total PKA activity in an Akt-dependent manner. These findings indicate that localized changes in insulin action are responsible for the differential phosphorylation of PKA substrates. Thus, we identify a pathway by which insulin regulates lipolysis through the spatially compartmentalized modulation of PKA.The storage and mobilization of nutrients from specialized tissues requires the spatial organization of both signaling functions and energy stores. Nowhere is this more evident than in mammalian adipose tissue, which maintains the most efficient repository for readily available energy. Here, fuel is segregated into lipid droplets, once thought to be inert storehouses but now recognized as complex structures that represent a regulatable adaptation of a ubiquitous organelle (5, 40). The synthesis and maintenance of functional lipid droplets requires numerous proteins, not only fatty acid binding proteins and enzymes of lipid synthesis but also molecules critical to constitutive and specialized membrane protein trafficking (23).During times of nutritional need, triglycerides within the adipocyte lipid droplet are hydrolyzed into their components, fatty acids, acyl-glycerides, and, ultimately, glycerol. This process, termed lipolysis, is controlled dynamically by multiple hormonal signals that respond to the nutrient status of the organism. During fasting, catecholamines such as norepinephrine stimulate lipolysis via beta-adrenergic receptor activation, promoting adenylyl cyclase activity and the production of cyclic AMP (cAMP) (17). cAMP binds to the regulatory subunits of its major effector, protein kinase A (PKA), triggering the dissociation of these subunits and the subsequent activation of the catalytic subunits (62, 63). PKA is frequently sequestered into multiple parallel, intracellular signaling complexes, though such structures have not been studied in hormone-responsive adipocytes (68). Two targets of activated PKA important for lipolysis are hormone-sensitive lipase (HSL) and perilipin, the major lipid droplet coat protein (17). The phosphorylation of HSL on Ser 559/660 is crucial for its activation and translocation to the lipid droplet, where HSL catalyzes the hydrolysis of diglycerides to monoglycerides (26, 55). Another lipase, adipose triglyceride lipase (ATGL), carries out the initial cleavage of triglycerides to diglycerides and most likely is rate limiting for lipolysis, but it does not appear to be regulated directly via PKA phosphorylation (24, 73). Perilipin under basal conditions acts as a protective barrier against lipase activity; upon stimulation, the phosphorylation of least six PKA consensus sites triggers a conformational change in perilipin, permitting access to the lipid substrates in the droplet, the recruitment of HSL, and possibly the activation of ATGL (7, 8, 21, 41, 46, 58, 60, 61). Perilipin, therefore, possesses dual functions, both blocking lipolysis in the basal state as well as promoting lipolysis upon its phosphorylation (5, 58, 60).Following the ingestion of a meal, insulin stimulates the uptake of nutrients such as glucose into specialized tissues and also potently inhibits lipolysis in adipocytes (17). Insulin signaling in the adipocyte involves the activation of the insulin receptor tyrosine kinase, the phosphorylation of insulin receptor substrates, the activation of PI3K, and the subsequent production of specific phosphoinositides at the plasma membrane (59). These phosphoinositides then recruit Akt, via its pleckstrin homology domain, to the plasma membrane, where Akt becomes phosphorylated and activated by two upstream kinases. Akt stimulates the translocation of the glucose transporter GLUT4 to the plasma membrane, thereby promoting the uptake of glucose into the cell (2). The mechanism by which insulin inhibits lipolysis has been proposed to involve the reduction of cAMP levels and thus PKA activity. In this model, insulin signaling activates phosphodiesterase 3b (PDE3b) via the Akt-mediated phosphorylation of Ser273 (14, 32). Upon activation by Akt, PDE3b catalyzes the hydrolysis of cAMP to 5′AMP, thereby attenuating PKA activity and lipolysis. Recent studies of PDE3b knockout mice have highlighted the importance of PDE3b activity in the regulation of lipolysis but were uninformative regarding the mechanism of insulin action (12). Adipocytes isolated from these mice exhibit reduced responses to insulin with respect to lipolysis, but it is not clear whether this is due to the loss of the critical target enzyme or a normal mechanism being overwhelmed by supraphysiological concentrations of cAMP (12). Biochemical studies using dominant-inhibitory Akt have demonstrated that Akt can regulate PDE3b activity, and other studies also have suggested that Akt interacts directly with PDE3b, implying a direct connection to lipolysis regulation (1, 32). Nevertheless, the actual requirement for Akt in insulin action with regard to the lipolysis itself has not been demonstrated directly in, for example, genetic loss-of-function experiments.There now is substantial evidence implicating elevated free fatty acid levels as a consequence of inappropriate lipolysis as a major etiological factor for insulin resistance and type 2 diabetes mellitus (T2DM) (51). Conditions such as obesity and diabetes are characterized by a pathophysiological state in which these tissues become unresponsive to insulin, which contribute to the adverse long-term sequelae of diseases such as T2DM and the metabolic syndrome (4, 44). Thus, understanding in detail the mechanism by which insulin suppresses fat cell lipolysis is critical to identifying the underlying defect in resistant adipose tissue and ultimately developing effective therapeutics. In the present study, we investigated both Akt-dependent and -independent modes of insulin action toward lipolysis. We found the latter to predominate at low, physiological levels of adrenergic stimulation, acting via a pathway dependent on the preferential phosphorylation of downstream PKA substrates.     

Expression of a Synthesized Gene Encoding Cationic Peptide Cecropin B in Transgenic Tomato Plants Protects against Bacterial Diseases

The cationic lytic peptide cecropin B (CB), isolated from the giant silk moth (Hyalophora cecropia), has been shown to effectively eliminate Gram-negative and some Gram-positive bacteria. In this study, the effects of chemically synthesized CB on plant pathogens were investigated. The S₅₀s (the peptide concentrations causing 50% survival of a pathogenic bacterium) of CB against two major pathogens of the tomato, Ralstonia solanacearum and Xanthomonas campestris pv. vesicatoria, were 529.6 μg/ml and 0.29 μg/ml, respectively. The CB gene was then fused to the secretory signal peptide (sp) sequence from the barley α-amylase gene, and the new construct, pBI121-spCB, was used for the transformation of tomato plants. Integration of the CB gene into the tomato genome was confirmed by PCR, and its expression was confirmed by Western blot analyses. In vivo studies of the transgenic tomato plant demonstrated significant resistance to bacterial wilt and bacterial spot. The levels of CB expressed in transgenic tomato plants (∼0.05 μg in 50 mg of leaves) were far lower than the S₅₀ determined in vitro. CB transgenic tomatoes could therefore be a new mode of bioprotection against these two plant diseases with significant agricultural applications.Bacterial plant diseases are a source of great losses in the annual yields of most crops (5). The agrochemical methods and conventional breeding commonly used to control these bacterially induced diseases have many drawbacks. Indiscriminate use of agrochemicals has a negative impact on human, as well as animal, health and contributes to environmental pollution. Conventional plant-breeding strategies have limited scope due to the paucity of genes with these traits in the usable gene pools and their time-consuming nature. Consequently, genetic engineering and transformation technology offer better tools to test the efficacies of genes for crop improvement and to provide a better understanding of their mechanisms. One advance is the possibility of creating transgenic plants that overexpress recombinant DNA or novel genes with resistance to pathogens (36). In particular, strengthening the biological defenses of a crop by the production of antibacterial proteins with other origins (not from plants) offers a novel strategy to increase the resistance of crops to diseases (35, 39, 41). These antimicrobial peptides (AMPs) include such peptides as cecropins (2, 15, 20, 23-24, 27, 31, 42, 50), magainins (1, 9, 14, 29, 47), sarcotoxin IA (35, 40), and tachyplesin I (3). The genes encoding these small AMPs in plants have been used in practice to enhance their resistance to bacterial and fungal pathogens (8, 22, 40). The expression of AMPs in vivo (mostly cecropins and a synthetic analog of cecropin and magainin) with either specific or broad-spectrum disease resistance in tobacco (14, 24, 27), potato (17, 42), rice (46), banana (9), and hybrid poplar (32) have been reported. The transgenic plants showed considerably greater resistance to certain pathogens than the wild types (4, 13, 24, 27, 42, 46, 50). However, detailed studies of transgenic tomatoes expressing natural cecropin have not yet been reported.The tomato (Solanum lycopersicum) is one of the most commonly consumed vegetables worldwide. The annual yield of tomatoes, however, is severely affected by two common bacterial diseases, bacterial wilt and bacterial spot, which are caused by infection with the Gram-negative bacteria Ralstonia solanacearum and Xanthomonas campestris pv. vesicatoria, respectively. Currently available pesticides are ineffective against R. solanacearum, and thus bacterial wilt is a serious problem.Cecropins, one of the natural lytic peptides found in the giant silk moth, Hyalophora cecropia (25), are synthesized in lipid bodies as proteins consisting of 31 to 39 amino acid residues. They adopt an α-helical structure on interaction with bacterial membranes, resulting in the formation of ion channels (12). At low concentrations (0.1 μM to 5 μM), cecropins exhibit lytic antibacterial activity against a number of Gram-negative and some Gram-positive bacteria, but not against eukaryotic cells (11, 26, 33), thus making them potentially powerful tools for engineering bacterial resistance in crops. Moreover, cecropin B (CB) shows the strongest activity against Gram-negative bacteria within the cecropin family and therefore has been considered an excellent candidate for transformation into plants to improve their resistance against bacterial diseases.The introduction of genes encoding cecropins and their analogs into tobacco has been reported to have contradictory results regarding resistance against pathogens (20). However, subsequent investigations of these tobacco plants showed that the expression of CB in the plants did not result in accumulation of detectable levels of CB, presumably due to degradation of the peptide by host peptidases (20, 34). Therefore, protection of CB from cellular degradation is considered to be vital for the exploitation of its antibacterial activity in transgenic plants. The secretory sequences of several genes are helpful, because they cooperate with the desired genes to enhance extracellular secretion (24, 40, 46). In the present study, a natural CB gene was successfully transferred into tomatoes. The transgenic plants showed significant resistance to the tomato diseases bacterial wilt and bacterial spot, as well as with a chemically synthesized CB peptide.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Calcium Oxalate Biomineralization by Piloderma fallax in Response to Various Levels of Calcium and Phosphorus

Piloderma fallax is an ectomycorrhizal fungus commonly associated with several conifer and hardwood species. We examined the formation of calcium oxalate crystals by P. fallax in response to calcium (0.0, 0.1, 0.5, 1, and 5 mM) and phosphorus (0.1 and 6 mM) additions in modified Melin-Norkrans agar medium. Both calcium and phosphorus supplementation significantly affected the amount of calcium oxalate formed. More calcium oxalate was formed at high P levels. Concentrations of soluble oxalate in the fungus and medium were higher at low P levels. There was a strong positive linear relationship between Ca level and calcium oxalate but only under conditions of phosphorus limitation. Calcium oxalate crystals were identified as the monohydrate form (calcium oxalate monohydrate [COM] whewellite) by X-ray diffraction analysis. Prismatic, styloid, and raphide forms of the crystals, characteristic COM, were observed on the surface of fungal hyphae by scanning electron microscopy. P. fallax may be capable of dissolving hyphal calcium oxalate under conditions of limited Ca. The biomineralization of calcium oxalate by fungi may be an important step in the translocation and cycling of Ca and P in soil.Many fungi from forest litter, including ectomycorrhizal fungi, exhibit calcium oxalate (CaOx) crystals on their hyphae. The ubiquity of CaOx crystals on fungal hyphae suggests that their formation may provide a selective advantage to the organism (4). CaOx formation is hypothesized to regulate intracellular pH and levels of oxalate and Ca and, hence, serves as a major sink for toxic amounts of Ca in soil and other environments (52, 53, 61). In plants, CaOx crystals have also been proposed to serve as a calcium source under conditions of calcium limitation (14, 18, 41), but such a process has yet to be established among fungi.CaOx on fungal hyphae is formed from soil-derived calcium and biologically synthesized oxalate. Oxalate released by ectomycorrhizae has been correlated with increased phosphorus bioavailability in the rhizosphere (V. Casarin, cited by Hinsinger in reference 25). The ability of oxalate to chelate metal ions makes it important in the solubilization and transport of metals in soil, the weathering and diagenesis of rocks and soil minerals (9, 23, 31, 57), and, consequently, the transport of nutrients. It is generally presumed that CaOx crystals form on the surface of fungal hyphae as a result of precipitation when released oxalic acid interacts with calcium cations (23, 43). However, the regularity of the CaOx crystals suggests that their formation is regulated and that they may be formed within the fungal hyphae at specific sites of origin (3, 5, 7).CaOx crystals vary in morphology, ranging from plates to raphides, druses, tetragonal bipyramids, and prisms. This variation in morphology can be seen among fungal genera and species (4). The crystals also usually occur either as CaOx monohydrate (COM; whewellite) (29) or CaOx dihydrate (weddellite) (3, 5, 28, 35, 60). Either crystal form or both may be present on fungal hyphae at the same time.In earlier studies (8, 9), we reported that Piloderma fallax is one of the major species of ectomycorrhizal fungi in subboreal forests. In addition, Piloderma sp. is found in temperate forest soils in association with conifer and hardwood species (34). Piloderma influences nutrient uptake and modifies mineral transformation in rock and soil systems (3, 33). In this study, we chose P. fallax because of (i) its ability to produce oxalate and form CaOx crystals (8, 56), (ii) its presence in many types of forest ecosystems, and (iii) its significant role in the breakdown and formation of soil minerals (9).The objective of this study was to quantify and characterize the formation of CaOx by P. fallax in response to various P and Ca levels in agar medium. We tested the hypothesis that P limitation will induce the production of oxalate and that increased concentrations of Ca will result in greater CaOx formation. This study also examined the dissolution of CaOx on P. fallax when it is grown on Ca-deficient medium and determined whether CaOx can serve as temporary Ca storage. Our study was conducted to add to knowledge of the ecological significance of CaOx, especially of its influence in biogeochemical cycling of P and Ca in soils.     

Syntrophic Degradation of Cadaverine by a Defined Methanogenic Coculture

A novel, strictly anaerobic, cadaverine-oxidizing, defined coculture was isolated from an anoxic freshwater sediment sample. The coculture oxidized cadaverine (1,5-diaminopentane) with sulfate as the electron acceptor. The sulfate-reducing partner could be replaced by a hydrogenotrophic methanogenic partner. The defined coculture fermented cadaverine to acetate, butyrate, and glutarate plus sulfide or methane. The key enzymes involved in cadaverine degradation were identified in cell extracts. A pathway of cadaverine fermentation via 5-aminovaleraldehyde and crotonyl-coenzyme A with subsequent dismutation to acetate and butyrate is suggested. Comparative 16S rRNA gene analysis indicated that the fermenting part of the coculture belongs to the subphylum Firmicutes but that this part is distant from any described genus. The closest known relative was Clostridium aminobutyricum, with 95% similarity.Cadaverine is a biogenic primary aliphatic amine. Together with other biogenic amines, like putrescine or spermidine, it is formed during oxygen-limited decomposition of protein-rich organic matter by decarboxylation of amino acids or by amination of aldehydes and ketones (8, 27, 30, 42, 53, 54). These putrid-smelling and, at higher concentrations (100 to 400 mg per kg), often toxic compounds play a major role in food microbiology, e.g., as flavoring constituents in the ripening of cheese or as contaminants of fish and meat products, wine, and beer (24, 29, 49).Little is known about the degradation of primary amines. Mono- and diamine oxidases of higher organisms and bacteria (23, 41, 64) initiate aerobic degradation, leading to the respective formation of aldehyde, ammonia, and hydrogen peroxide as products (28). Alternatively, in a putrescine-degrading mutant of Escherichia coli, putrescine is degraded by a putrescine-2-oxoglutarate transaminase and a subsequent dehydrogenase to form 4-aminobutyrate, which is further metabolized via succinate (43).Anaerobic degradation of primary amines could follow basically similar pathways. The released reducing equivalents can be disposed of in a manner similar to that described for primary alcohols (9, 15, 16). In the absence of external electron acceptors, such as sulfate or nitrate, incomplete oxidation of cadaverine to fatty acids or dicarboxylic acids could be coupled to syntrophic methane production, homoacetogenesis, or reductive synthesis of long-chain fatty acids (1, 25, 31).In the present study, we describe a new isolate of strictly anaerobic bacteria which oxidizes cadaverine syntrophically with the methanogen Methanospirillum hungatei and forms acetate, butyrate, glutarate, and methane as products. The enzymes involved in the degradation of cadaverine were identified, and a catabolic pathway is proposed.     

YjhS (NanS) Is Required for Escherichia coli To Grow on 9-O-Acetylated N-Acetylneuraminic Acid

The nanATEK-yhcH, yjhATS, and yjhBC operons in Escherichia coli are coregulated by environmental N-acetylneuraminic acid, the most prevalent sialic acid in nature. Here we show that YjhS (NanS) is a probable 9-O-acetyl N-acetylneuraminic acid esterase required for E. coli to grow on this alternative sialic acid, which is commonly found in mammalian host mucosal sites.The coregulated nanATEK-yhcH, yjhATS, and yjhBC operons involved in sialic acid catabolism in Escherichia coli are thought to be induced by the most common sialic acid, N-acetylneuraminic acid (Neu5Ac), through reversible inactivation of the NanR repressor encoded by nanR mapping immediately upstream of nanA (15, 27, 28; http://vetmed.illinois.edu/path/sialobiology/). Sialic acids are a family of over 40 naturally occurring 9-carbon keto sugar acids found mainly in metazoans of the deuterostome (starfish to human) developmental lineage and in some, mostly pathogenic, bacteria, where sialic acids expressed at the microbial cell surface inhibit host innate immunity (27). By contrast, most bacterial commensals and pathogens catabolize sialic acids as sole carbon and nitrogen sources, indicating exploitation of the sialic acid-rich host mucosal environment by a wide range of species (2, 27, 28). Interestingly, in vivo experimental evidence further indicates that sialic acid catabolism functions directly (nutrition) or indirectly (surface decoration and cell signaling) in host-microbe commensal and pathogenic interactions in organisms such as E. coli, Haemophilus influenzae, Pasteurella multocida, Salmonella enterica serovar Typhi, Streptococcus pneumoniae, Vibrio vulnificus, and Vibrio cholerae (1, 3, 5, 6, 10, 14, 23, 24, 26, 29). The animal species used for these studies include rodent models and natural hosts such as cattle and turkeys. The structural diversity of sialic acids at the terminal positions on glycoconjugates (glycoproteins and glycolipids) of mucosal surfaces of these hosts requires sialidases, acetyl esterases, and probably other enzymes that convert alternative or at least minor sialic acids to the more digestible Neu5Ac form (8, 9). We have previously demonstrated that E. coli has an epicurean propensity for metabolizing alternative sialic acids (30, 31). In the current communication, we show that YjhS is required for growth of E. coli on 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac₂).Because most sialic acids are bound to other sugars, including other sialic acids, as part of the oligosaccharide chains on glycoconjugates, either microbial or endogenous (host) sialidases (NanH, or N-acylneuraminate hydrolases) are needed to release free sugar, which is then transported by NanT in E. coli (15, 16, 26, 31). Once internalized, sialic acid is cleaved by an nanA-encoded aldolase or lyase to yield the 6-carbon hexosamine, N-acetylmannosamine (ManNAc), and pyruvate, with the latter entering the tricarboxylic acid cycle or gluconeogenesis. ManNAc is converted to its 6-phosphate derivative by a specific kinase encoded by nanK and epimerized by NanE to yield N-acetylglucosamine 6-phosphate, which is converted to fructose 6-phosphate by products of the nag operon (15, 17, 31, 32). The functions of the coregulated yjhS, yjhB, yjhC, and yhcH gene products are unknown but are not required for growth on Neu5Ac (15). However, YjhA (NanC) is an outer membrane porin required for diffusion of Neu5Ac in the absence of the major porins (7), while YjhT (NanM) is a mutarotase that catalyzes the conversion of the alpha sialic acid isomer to the more thermodynamically stable beta form (21). Neither nanC nor nanM is required for growth on Neu5Ac (15), suggesting that yjhS, yjhBC, and yhcH are involved in reactions that convert alternative sialic acids to Neu5Ac (22, 23). YhcH was crystallized and has been suggested to be an isomerase or epimerase involved in processing N-glycolylneuraminic acid (Neu5Gc) (25), but deletion of yhcH did not affect growth on this sialic acid as a sole carbon source (16).Computer-assisted analysis indicated that YjhB is a permease similar to NanT (16) whereas YjhC is a likely oxidoreductase or dehydrogenase. Orthologs of yhcH, nanC, nanM, and yjhBC are found in most bacterial species with intact Neu5Ac utilization systems, while yjhS is confined to E. coli and shigellae, either as part of the chromosomes in these strains or integrated with phages or phage remnants. However, a significant match (E value = 0.0007) was found between YjhS and AxeA in Rhodopirellula baltica, where AxeA is an acetyl xylan esterase (11), suggesting YjhS might be a sialate esterase. We propose that YjhS should be designated NanS to indicate its direct participation in utilization of an alternative sialic acid.