Limits of Versatility of Versatile Peroxidase
. Applied and Environmental Microbiology 2016
, 4070. Publisher's VersionAbstract
Although Mn2+ is the most abundant substrate of versatile peroxidases (VPs), repression of Pleurotus ostreatus vp1 expression occurred in Mn2+-sufficient medium. This seems to be a biological contradiction. The aim of this study was to explore the mechanism of direct oxidation by VP1 under Mn2+-deficient conditions, as it was found to be the predominant enzyme during fungal growth in the presence of synthetic and natural substrates. The native VP1 was purified and characterized using three substrates, Mn2+, Orange II (OII), and Reactive Black 5 (RB5), each oxidized by a different active site in the enzyme. While the pH optimum for Mn2+ oxidation is 5, the optimum pH for direct oxidation of both dyes was found to be 3. Indeed, effective in vivo decolorization occurred in media without addition of Mn2+ only under acidic conditions. We have determined that Mn2+ inhibits in vitro the direct oxidation of both OII and RB5 while RB5 stabilizes both Mn2+ and OII oxidation. Furthermore, OII was found to inhibit the oxidation of both Mn2+ and RB5. In addition, we could demonstrate that VP1 can cleave OII in two different modes. Under Mn2+-mediated oxidation conditions, VP1 was able to cleave the azo bond only in asymmetric mode, while under the optimum conditions for direct oxidation (absence of Mn2+ at pH 3) both symmetric and asymmetric cleavages occurred. We concluded that the oxidation mechanism of aromatic compounds by VP1 is controlled by Mn2+ and pH levels both in the growth medium and in the reaction mixture. IMPORTANCE VP1 is a member of the ligninolytic heme peroxidase gene family of the white rot fungus Pleurotus ostreatus and plays a fundamental role in biodegradation. This enzyme exhibits a versatile nature, as it can oxidize different substrates under altered environmental conditions. VPs are highly interesting enzymes due to the fact that they contain unique active sites that are responsible for direct oxidation of various aromatic compounds, including lignin, in addition to the well-known Mn2+ binding active site. This study demonstrates the limits of versatility of P. ostreatus VP1, which harbors multiple active sites, exhibiting a broad range of enzymatic activities, but they perform differently under distinct conditions. The versatility of P. ostreatus and its enzymes is an advantageous factor in the fungal ability to adapt to changing environments. This trait expands the possibilities for the potential utilization of P. ostreatus and other white rot fungi.
Toward combined delignification and saccharification of wheat straw by a laccase-containing designer cellulosome
. Proceedings of the National Academy of Sciences 2016
, 10854. Publisher's VersionAbstract
Lignocellulosic biomass is a potential major resource for renewable energy production. Plant cell-wall deconstruction, however, remains an inefficient process, mainly due to the recalcitrant nature of the lignin and cellulosic components, that requires chemical pretreatment methods prior to degradation. This study aims to overcome this barrier by combining two paradigms into a single system, by using a synthetic biology approach. The designed system integrates an engineered laccase (an oxidizing enzyme that acts on lignin) into a multienzyme cellulosome complex, thereby producing enhanced decomposition of wheat straw. These findings demonstrate the potential of introducing complementary enzymes that fail to occur together in nature into designer cellulosomes for improved lignocellulose conversion.Efficient breakdown of lignocellulose polymers into simple molecules is a key technological bottleneck limiting the production of plant-derived biofuels and chemicals. In nature, plant biomass degradation is achieved by the action of a wide range of microbial enzymes. In aerobic microorganisms, these enzymes are secreted as discrete elements in contrast to certain anaerobic bacteria, where they are assembled into large multienzyme complexes termed cellulosomes. These complexes allow for very efficient hydrolysis of cellulose and hemicellulose due to the spatial proximity of synergistically acting enzymes and to the limited diffusion of the enzymes and their products. Recently, designer cellulosomes have been developed to incorporate foreign enzymatic activities in cellulosomes so as to enhance lignocellulose hydrolysis further. In this study, we complemented a cellulosome active on cellulose and hemicellulose by addition of an enzyme active on lignin. To do so, we designed a dockerin-fused variant of a recently characterized laccase from the aerobic bacterium Thermobifida fusca. The resultant chimera exhibited activity levels similar to the wild-type enzyme and properly integrated into the designer cellulosome. The resulting complex yielded a twofold increase in the amount of reducing sugars released from wheat straw compared with the same system lacking the laccase. The unorthodox use of aerobic enzymes in designer cellulosome machinery effects simultaneous degradation of the three major components of the plant cell wall (cellulose, hemicellulose, and lignin), paving the way for more efficient lignocellulose conversion into soluble sugars en route to alternative fuels production.
Universality of human microbial dynamics
, 259 - 262. Publisher's VersionAbstract
A new computational method to characterize the dynamics of human-associated microbial communities is applied to data from two large-scale metagenomic studies, and suggests that gut and mouth microbiomes of healthy individuals are subjected to universal (that is, host-independent) dynamics, whereas skin microbiomes are shaped by the host environment; the method paves the way to designing general microbiome-based therapies.
Correlation detection strategies in microbial data sets vary widely in sensitivity and precision
, 1669 - 1681. Publisher's VersionAbstract
Disruption of healthy microbial communities has been linked to numerous diseases, yet microbial interactions are little understood. This is due in part to the large number of bacteria, and the much larger number of interactions (easily in the millions), making experimental investigation very difficult at best and necessitating the nascent field of computational exploration through microbial correlation networks. We benchmark the performance of eight correlation techniques on simulated and real data in response to challenges specific to microbiome studies: fractional sampling of ribosomal RNA sequences, uneven sampling depths, rare microbes and a high proportion of zero counts. Also tested is the ability to distinguish signals from noise, and detect a range of ecological and time-series relationships. Finally, we provide specific recommendations for correlation technique usage. Although some methods perform better than others, there is still considerable need for improvement in current techniques.
Surveys, simulation and single-cell assays relate function and phylogeny in a lake ecosystem
16130. Publisher's VersionAbstract
Much remains unknown about what drives microbial community structure and diversity. Highly structured environments might offer clues. For example, it may be possible to identify metabolically similar species as groups of organisms that correlate spatially with the geochemical processes they carry out. Here, we use a 16S ribosomal RNA gene survey in a lake that has chemical gradients across its depth to identify groups of spatially correlated but phylogenetically diverse organisms. Some groups had distributions across depth that aligned with the distributions of metabolic processes predicted by a biogeochemical model, suggesting that these groups performed biogeochemical functions. A single-cell genetic assay showed, however, that the groups associated with one biogeochemical process, sulfate reduction, contained only a few organisms that have the genes required to reduce sulfate. These results raise the possibility that some of these spatially correlated groups are consortia of phylogenetically diverse and metabolically different microbes that cooperate to carry out geochemical functions.
Preferential interactions promote blind cooperation and informed defection
. Proceedings of the National Academy of Sciences 2016
, 13995. Publisher's VersionAbstract
Humans often behave in seemingly irrational ways. A common instance of such perplexing behavior is that we typically care about how and why people chose their actions, rather than caring only about the actions themselves. For example, when people agree to do us a favor, we prefer them to do so directly, rather than to first gather all the relevant information. Using game theory, we show that this preference may in fact be rational: The decision-making process often reveals hidden preferences of the decision maker, which can become relevant in a future interaction. This work elucidates the conditions that make caring about motivations beneficial and makes predictions regarding the real-world situations in which it is expected to occur.It is common sense that costs and benefits should be carefully weighed before deciding on a course of action. However, we often disapprove of people who do so, even when their actual decision benefits us. For example, we prefer people who directly agree to do us a favor over those who agree only after securing enough information to ensure that the favor will not be too costly. Why should we care about how people make their decisions, rather than just focus on the decisions themselves? Current models show that punishment of information gathering can be beneficial because it forces blind decisions, which under some circumstances enhances cooperation. Here we show that aversion to information gathering can be beneficial even in the absence of punishment, due to a different mechanism: preferential interactions with reliable partners. In a diverse population where different people have different—and unknown—preferences, those who seek additional information before agreeing to cooperate reveal that their preferences are close to the point where they would choose not to cooperate. Blind cooperators are therefore more likely to keep cooperating even if conditions change, and aversion to information gathering helps to interact preferentially with them. Conversely, blind defectors are more likely to keep defecting in the future, leading to a preference for informed defectors over blind ones. Both mechanisms—punishment to force blind decisions and preferential interactions—give qualitatively different predictions, which may enable experimental tests to disentangle them in real-world situations.
The vapB–vapC Operon of Acidovorax citrulli Functions as a Bona-fide Toxin–Antitoxin Module
. Frontiers in Microbiology 2016
1499. Publisher's VersionAbstract
Toxin–antitoxin systems are commonly found on plasmids and chromosomes of bacteria and archaea. These systems appear as biscystronic genes encoding a stable toxin and a labile antitoxin, which protects the cells from the toxin’s activity. Under specific, mostly stressful conditions, the unstable antitoxin is degraded, the toxin becomes active and growth is arrested. Using genome analysis we identified a putative toxin–antitoxin encoding system in the genome of the plant pathogen Acidovorax citrulli. The system is homologous to vapB–vapC systems from other bacterial species. PCR and phylogenetic analyses suggested that this locus is unique to group II strains of A. citrulli. Using biochemical and molecular analyses we show that A. citrulli VapBC module is a bona-fide toxin–antitoxin module in which VapC is a toxin with ribonuclease activity that can be counteracted by its cognate VapB antitoxin. We further show that transcription of the A. citrulli vapBC locus is induced by amino acid starvation, chloramphenicol and during plant infection. Due to the possible role of TA systems in both virulence and dormancy of human pathogenic bacteria, studies of these systems are gaining a lot of attention. Conversely, studies characterizing toxin–antitoxin systems in plant pathogenic bacteria are lacking. The study presented here validates the activity of VapB and VapC proteins in A. citrulli and suggests their involvement in stress response and host–pathogen interactions.
GAME9 regulates the biosynthesis of steroidal alkaloids and upstream isoprenoids in the plant mevalonate pathway
10654. Publisher's VersionAbstract
Steroidal glycoalkaloids (SGAs) are cholesterol-derived molecules produced by solanaceous species. They contribute to pathogen defence but are toxic to humans and considered as anti-nutritional compounds. Here we show that GLYCOALKALOID METABOLISM 9 (GAME9), an APETALA2/Ethylene Response Factor, related to regulators of alkaloid production in tobacco and Catharanthus roseus, controls SGA biosynthesis. GAME9 knockdown and overexpression in tomato and potato alters expression of SGAs and upstream mevalonate pathway genes including the cholesterol biosynthesis gene STEROL SIDE CHAIN REDUCTASE 2 (SSR2). Levels of SGAs, C24-alkylsterols and the upstream mevalonate and cholesterol pathways intermediates are modified in these plants. Δ(7)-STEROL-C5(6)-DESATURASE (C5-SD) in the hitherto unresolved cholesterol pathway is a direct target of GAME9. Transactivation and promoter-binding assays show that GAME9 exerts its activity either directly or cooperatively with the SlMYC2 transcription factor as in the case of the C5-SD gene promoter. Our findings provide insight into the regulation of SGA biosynthesis and means for manipulating these metabolites in crops.
Insights from the Genome Sequence of Acidovorax citrulli M6, a Group I Strain of the Causal Agent of Bacterial Fruit Blotch of Cucurbits
. Frontiers in Microbiology 2016
430. Publisher's VersionAbstract
Acidovorax citrulli is a seedborne bacterium that causes bacterial fruit blotch of cucurbit plants including watermelon and melon. A. citrulli strains can be divided into two major groups based on DNA fingerprint analyses and biochemical properties. Group I strains have been generally isolated from non-watermelon cucurbits, while group II strains are closely associated with watermelon. In the present study, we report the genome sequence of M6, a group I model A. citrulli strain, isolated from melon. We used comparative genome analysis to investigate differences between the genome of strain M6 and the genome of the group II model strain AAC00-1. The draft genome sequence of A. citrulli M6 harbors 139 contigs, with an overall approximate size of 4.85 Mb. The genome of M6 is ∼500 Kb shorter than that of strain AAC00-1. Comparative analysis revealed that this size difference is mainly explained by eight fragments, ranging from ∼35–120 Kb and distributed throughout the AAC00-1 genome, which are absent in the M6 genome. In agreement with this finding, while AAC00-1 was found to possess 532 open reading frames (ORFs) that are absent in strain M6, only 123 ORFs in M6 were absent in AAC00-1. Most of these M6 ORFs are hypothetical proteins and most of them were also detected in two group I strains that were recently sequenced, tw6 and pslb65. Further analyses by PCR assays and coverage analyses with other A. citrulli strains support the notion that some of these fragments or significant portions of them are discriminative between groups I and II strains of A. citrulli. Moreover, GC content, effective number of codon values and cluster of orthologs’ analyses indicate that these fragments were introduced into group II strains by horizontal gene transfer events. Our study reports the genome sequence of a model group I strain of A. citrulli, one of the most important pathogens of cucurbits. It also provides the first comprehensive comparison at the genomic level between the two major groups of strains of this pathogen.
Effects of nitrogen nutrition on disease development caused by Acidovorax citrulli on melon foliage
, 125 - 137. Publisher's VersionAbstract
Bacterial fruit blotch (BFB) of cucurbits, caused by the seed-borne bacterium Acidovorax citrulli, is a destructive disease that threatens the melon and watermelon industries worldwide. The available means to manage the disease are very limited and there are no reliable sources of BFB resistance. Mineral nutrition has marked effects on plant diseases. To the best of our knowledge, no studies reporting effects of mineral nutrition on BFB severity have been reported to date. In the present study we assessed the influence of nitrogen nutrition on BFB severity and A. citrulli establishment in the foliage of melon plants under greenhouse conditions. Our results show that nitrogen fertilization, based on nitrate only, led to reduced disease severity and bacterial numbers in melon leaves, as compared with two combinations of nitrate and ammonium. No consistent effect of nitrogen nutrition on expression of several plant defense-associated transcripts was found, except for hydroperoxide lyase (HPL), which upon inoculation was repressed to a greater extent under the “nitrate-only” nitrogen regime compared with combined nitrate and ammonium. Reducing BFB severity and A. citrulli establishment in the plant foliage are of particular importance since establishment of the pathogen during the growing season is assumed to increase the incidence of fruit infection, leading to serious yield losses. Further research is needed to elucidate the mechanisms by which nitrogen nutrition influences BFB development, and to assess the effects of nitrogen as well as other minerals on the disease under field conditions.
Plant phenolic acids affect the virulence of Pectobacterium aroidearum and P. carotovorum ssp. brasiliense via quorum sensing regulation
. Molecular Plant PathologyMolecular Plant PathologyMolecular Plant Pathology 2016
, 487 - 500. Publisher's VersionAbstract
Summary Several studies have reported effects of the plant phenolic acids cinnamic acid (CA) and salicylic acid (SA) on the virulence of soft rot enterobacteria. However, the mechanisms involved in these processes are not yet fully understood. Here, we investigated whether CA and SA interfere with the quorum sensing (QS) system of two Pectobacterium species, P.?aroidearum and P.?carotovorum ssp. brasiliense, which are known to produce N-acyl-homoserine lactone (AHL) QS signals. Our results clearly indicate that both phenolic compounds affect the QS machinery of the two species, consequently altering the expression of bacterial virulence factors. Although, in control treatments, the expression of QS-related genes increased over time, the exposure of bacteria to non-lethal concentrations of CA or SA inhibited the expression of QS genes, including expI, expR, PC1_1442 (luxR transcriptional regulator) and luxS (a component of the AI-2 system). Other virulence genes known to be regulated by the QS system, such as pecS, pel, peh and yheO, were also down-regulated relative to the control. In agreement with the low levels of expression of expI and expR, CA and SA also reduced the level of the AHL signal. The effects of CA and SA on AHL signalling were confirmed in compensation assays, in which exogenous application of N-(?-ketocaproyl)-l-homoserine lactone (eAHL) led to the recovery of the reduction in virulence caused by the two phenolic acids. Collectively, the results of gene expression studies, bioluminescence assays, virulence assays and compensation assays with eAHL clearly support a mechanism by which CA and SA interfere with Pectobacterium virulence via the QS machinery.
Plant phenolic volatiles inhibit quorum sensing in pectobacteria and reduce their virulence by potential binding to ExpI and ExpR proteins
38126. Publisher's VersionAbstract
Quorum sensing (QS) is a population density-dependent regulatory system in bacteria that couples gene expression to cell density through accumulation of diffusible signaling molecules. Pectobacteria are causal agents of soft rot disease in a range of economically important crops. They rely on QS to coordinate their main virulence factor, production of plant cell wall degrading enzymes (PCWDEs). Plants have evolved an array of antimicrobial compounds to anticipate and cope with pathogens, of which essential oils (EOs) are widely recognized. Here, volatile EOs, carvacrol and eugenol, were shown to specifically interfere with QS, the master regulator of virulence in pectobacteria, resulting in strong inhibition of QS genes, biofilm formation and PCWDEs, thereby leading to impaired infection. Accumulation of the signal molecule N-acylhomoserine lactone declined upon treatment with EOs, suggesting direct interaction of EOs with either homoserine lactone synthase (ExpI) or with the regulatory protein (ExpR). Homology models of both proteins were constructed and docking simulations were performed to test the above hypotheses. The resulting binding modes and docking scores of carvacrol and eugenol support potential binding to ExpI/ExpR, with stronger interactions than previously known inhibitors of both proteins. The results demonstrate the potential involvement of phytochemicals in the control of Pectobacterium.