Fungi transcriptionally upregulate expression of azole efflux pumps and ergosterol biosynthesis pathway genes when exposed to antifungal agents that target ergosterol biosynthesis. To date, these transcriptional responses have been shown to be dependent on the presence of the azoles and/or depletion of ergosterol. Using an inducible promoter to regulate , which encodes the major azole target, sterol 14α-demethylase, we were able to demonstrate that the CDR4 azole efflux pump can be transcriptionally activated by ergosterol biosynthesis inhibition even in the absence of azoles. By analyzing ergosterol deficient mutants, we demonstrate that the transcriptional responses by and, unexpectedly, genes encoding ergosterol biosynthesis enzymes ( genes) that are responsive to azoles, are not dependent on ergosterol depletion. Nonetheless, deletion of , which encodes C-8 sterol isomerase, also induced expression of . Deletion of also induced the expression of , the gene encoding C-14 sterol reductase, but not other tested genes which were responsive to inactivation. This indicates that inhibition of specific steps of ergosterol biosynthesis can result in different transcriptional responses, which is further supported by our results obtained using different ergosterol biosynthesis inhibitors. Together with the sterol profiles, these results suggest that the transcriptional responses by and genes are associated with accumulation of specific sterol intermediate(s). This was further supported by the fact that when the mutant was treated with ketoconazole, upstream inhibition overrode the effects by downstream inhibition on ergosterol biosynthesis pathway. Even though expression is associated with the accumulation of sterol intermediates, intra- and extracellular sterol analysis by HPLC-MS indicated that the transcriptional induction of did not result in efflux of the accumulated intermediate(s). This study demonstrates, by detailed genetic and chemical analysis, that transcriptional responses by a major efflux pump and genes of the ergosterol biosynthesis pathway to ergosterol biosynthesis inhibitors can be independent of the presence of the drugs and are linked with the accumulation of ergosterol intermediate(s).

Fourteen Trichoderma (Hypocreales) species were identified during a survey of the genus in South Africa. These include T. afroharzianum, T. asperelloides, T. asperellum, T. atrobrunneum, T. atroviride, T. camerunense, T. gamsii, T. hamatum, T. koningii, T. koningiopsis, T. saturnisporum, T. spirale, T. virens, and T. viride. Ten of these species were not known to occur in South Africa prior to this investigation. Five additional species were novel and are described here as T. beinartii, T. caeruleimontis, T. chetii, T. restrictum, and T. undulatum. These novel Trichoderma species display morphological traits that are typical of the genus. Based on molecular identification using calmodulin, endochitinase, nuc rDNA internal transcribed spacers (ITS1-5.8S-ITS2), RNA polymerase II subunit B, and translation elongation factor 1-α gene sequence data, T. beinartii, T. caeruleimontis, and T. chetii were found to belong to the Longibrachiatum clade, whereas T. restrictum is a member of the Hamatum clade. Trichoderma undulatum occupies a distinct lineage distantly related to other Trichoderma species. Strains of T. beinartii and T. chetii were isolated previously in Hawaii and Israel; however, T. caeruleimontis, T. restrictum, and T. undulatum are so far known only from South Africa.

The Neurospora crassa Mps One Binder (MOB) proteins MOB2A and MOB2B physically interact with the Nuclear Dbf2 Related (NDR) kinase COT1 and have been shown to have overlapping functions in various aspects of asexual development. Here, we identified two N. crassa MOB2A residues, Tyr117 and Tyr119, which are potentially phosphorylated. Using phosphomimetic mob-2a mutants we have been able to establish that apart from their previously described roles, MOB2A/B are involved in additional developmental processes. Enhanced conidial germination, accompanied by conidial agglutination, in the phosphomimetic mutants indicated that MOB2A is a negative regulator of germination. Thick-section imaging of perithecia revealed slow maturation and a lack of asci alignment in the mutant strains demonstrating a role for MOB2A in sexual development. We demonstrate that even though MOB2A and MOB2B have some overlapping functions, MOB2B cannot compensate for the roles MOB2A has in conidiation and germination. Altering Tyr residues 117 and 119 impaired the physical interactions between MOB2A and COT1, most likely contributing to some of the observed effects. As cot-1 and the phosphomimetic mutants share an extragenic suppressor (gul-1), we concluded that at least some of the effects imposed by altering Tyr117 and Tyr119 are mediated by the NDR kinase.

There is a growing appreciation for the important roles microorganisms play in association with plants. Microorganisms are drawn to distinct plant surfaces by the nutrient-rich microenvironment, and in turn some of these colonizing microbes provide mutualistic benefits to their host. The development of plant probiotics to increase crop yield and provide plant resistance against biotic and abiotic stresses, while minimizing chemical inputs, would benefit from a deeper mechanistic understanding of plant-microbe interaction. Technological advances in molecular biology and high-throughput -omics provide stepping stones to the elucidation of critical microbiome gene functions that aid in improving plant performance. Here, we review -omics-based approaches that are propelling forward the current understanding of plant-associated bacterial gene functions, and describe how these technologies have helped unravel key bacterial genes and pathways that mediate pathogenic, beneficial, and commensal host interactions.

Plants intimately associate with diverse bacteria. Plant-associatedbacteria have ostensibly evolved genes that enable them to adapt to plantenvironments. However, the identities of such genes are mostly unknown, and theirfunctions are poorly characterized. We sequenced 484 genomes of bacterial isolatesfrom roots of Brassicaceae, poplar, and maize. We then compared 3,837 bacterialgenomes to identify thousands of plant-associated gene clusters. Genomes ofplant-associated bacteria encode more carbohydrate metabolism functions and fewermobile elements than related non-plant-associated genomes do. We experimentallyvalidated candidates from two sets of plant-associated genes: one involved in plantcolonization, and the other serving in microbe–microbe competition betweenplant-associated bacteria. We also identified 64 plant-associated protein domainsthat potentially mimic plant domains; some are shared with plant-associated fungiand oomycetes. This work expands the genome-based understanding of plant–microbeinteractions and provides potential leads for efficient and sustainable agriculturethrough microbiome engineering.

This study examined the effects of environmental conditions on the distribution of marine sponges. We measured the abundance of the sponge Batzella inops (Topsent, 1891) in two contrasting habitats: inside submerged caves and on the surfaces of submerged boulders. We hypothesised that caves are a preferred habitat for B. inops over the boulder surfaces, and tested this by descriptive (quadrate sampling) and manipulative (reciprocal transplantation) experiments. In addition, we tested B. inops in situ for the presence of photosynthetic activity. We found that B. inops is more abundant inside the caves (mean ± s.e.m., 1.2 ± 0.6 individuals m–2) than on the outside boulder surfaces (0.15 ± 0.19 individuals m–2). We also detected photosynthetic activity in B. inops in both habitats. The results of transplantation experiments suggested that the sponge prefers the transfer from inside to outside the cave rather than vice versa. Therefore, we conclude that although B. inops is more abundant in sheltered habitats, such as submerged caves, adult individuals of this sponge can survive transfer to exposed conditions. Altogether, our findings point to the plasticity of B. inops habitat preferences and may aid further research into conservation or mariculture of this and possibly other sponge species.

Background:The Mediterranean fruit fly Ceratitis capitata is a major pest in horticulture. The development of fly larvae is mediated by bacterial decay in the fruit tissue. Despite the importance of bacteria on larval development, very little is known about the interaction between bacteria and larvae in their true ecological context. Understanding their relationship and inter-dependence in the host fruit is important for the development of new pest control interfaces to deal with this pest.Results:We find no negative effects on egg hatch or larval development brought about by the bacterial isolates tested. The various symbionts inhabiting the fly’s digestive system differ in their degree of contribution to the development of fly larvae depending on the given host and their sensitivity to induced inhibition caused by female produced antimicrobial peptides. These differences were observed not only at the genus or species level but also between isolates of the same species. We demonstrate how the microbiota from the mother’s gut supports the development of larvae in the fruit host and show that larvae play a major role in spreading the bacterial contagion in the infected fruit itself. In addition, we present (for the first time) evidence for horizontal transfer of bacteria between larvae of different maternal origin that develop together in the same fruit.Conclusions:Larvae play a major role in the spread and shaping of the microbial population in the fruit. The transfer of bacteria between different individuals developing in the same fruit suggests that the infested fruit serves as a microbial hub for the amplification and spread of bacterial strains between individuals.

Finding optimal markers for microorganisms important in the medical, agricultural, environmental or ecological fields is of great importance. Thousands of complete microbial genomes now available allow us, for the first time, to exhaustively identify marker proteins for groups of microbial organisms. In this work, we model the biological task as the well-known mathematical “hitting set” problem, solving it based on both greedy and randomized approximation algorithms. We identify unique markers for 17 phenotypic and taxonomic microbial groups, including proteins related to the nitrite reductase enzyme as markers for the non-anammox nitrifying bacteria group, and two transcription regulation proteins, nusG and yhiF, as markers for the Archaea and Escherichia/Shigella taxonomic groups, respectively. Additionally, we identify marker proteins for three subtypes of pathogenic E. coli, which previously had no known optimal markers. Practically, depending on the completeness of the database this algorithm can be used for identification of marker genes for any microbial group, these marker genes may be prime candidates for the understanding of the genetic basis of the group's phenotype or to help discover novel functions which are uniquely shared among a group of microbes. We show that our method is both theoretically and practically efficient, while establishing an upper bound on its time complexity and approximation ratio; thus, it promises to remain efficient and permit the identification of marker proteins that are specific to phenotypic or taxonomic groups, even as more and more bacterial genomes are being sequenced.

ABSTRACTForensic implementation of soil bacterial DNA profiling is limited by the potential for temporal mismatch of DNA profiles, e.g. after storage or seasonal changes. We compared profiles of samples retrieved at one location over 14 years after air-drying, freeze-drying and ?80 °C freezing storage. Sample mismatch in freeze-dried and air-dried samples was significant after two years and continued to increase yearly, whereas profiles after ?80 °C freezing remained unchanged for many years. In an attempt to mitigate inter-seasonal temporal mismatches, e.g. when months pass between crime and seizure of evidence, soils sampled in winter and summer were exposed to artificial ?summer? and ?winter? conditions, respectively, and their DNA profiles were compared. Differences were small between soil types, larger between seasons and largest between ?natural? and ?artificial? seasons. Understanding sources of temporal variations is critical for storage of forensics samples and for developing mitigation procedures that could help overcome these time-induced limitations.

Plasmids harboring qnr genes confer resistance to low fluoroquinolone concentrations. These genes are of significant clinical, evolutionary and environmental importance, since they are widely distributed in a diverse array of natural and clinical environments. We previously extracted and sequenced a large (∼185 Kbp) qnrB-harboring plasmid, and several small (∼8 Kbp) qnrS-harboring plasmids, from Klebsiella pneumoniae isolates from municipal wastewater biosolids, and hypothesized that these plasmids provide host bacteria a selective advantage in wastewater treatment plants (WWTPs) that often contain residual concentrations of fluoroquinolones. The objectives of this study were therefore to determine the effect of residual fluoroquinolone concentrations on the growth kinetics of qnr plasmid-harboring bacteria; and on the copy number of qnr plasmids and expression of qnr genes. Electrotransformants harboring either one of the two types of plasmids could grow at ciprofloxacin concentrations exceeding 0.5 μg ml(-1), but growth was significantly decreased at concentrations higher than 0.1 μg ml(-1). In contrast, plasmid-free strains failed to grow even at 0.05 μg ml(-1). No differences were observed in plasmid copy number under the tested ciprofloxacin concentrations, but qnr expression increased incrementally from 0 to 0.4 μg ml(-1), suggesting that the transcription of this gene is regulated by antibiotic concentration. This study reveals that wastewater-derived qnr plasmids confer a selective advantage in the presence of residual fluoroquinolone concentrations and provides a mechanistic explanation for this phenomenon.

2-Aminoacetophneone (2-AA) is a volatile molecule produced in high amounts by the opportunistic pathogen Pseudomonas aeruginosa. We have previously shown that 2-AA activates the quorum sensing (QS) LuxR receptor of Aliivibrio fischeri. In the present study we were able to improve LuxR's affinity and detection limit for 2-AA by genetic modification of three amino acids within the binding pocket of the receptor. Expression of the modified LuxR receptor in a luminescent bacterial biosensor provided an efficient detection assay of 2-AA in clinical P. aeruginosa strains isolated from blood and lung infections, as well as in phlegm samples obtained from subjects suffering from lung infections.

Improved easy-to-use diagnostic tools for infections are in strong demand worldwide. Yet, despite dramatic advances in diagnostic technologies, the gold-standard remains culturing. Here we offer an alternative tool demonstrating that a bacterial biosensor can efficiently detect Pseudomonas aeruginosa infections in patients suffering from otitis externa. Detection was based on specific binding between the biosensor and 2-aminoacetophenone (2-AA), a volatile produced by P. aeruginosa in high amounts. We collected pus samples from ears of 26 subjects exhibiting symptoms of otitis externa. Detection of P. aeruginosa using the biosensor was compared to detection using gold-standard culturing assay and to gas-chromatograph-mass-spectrometry (GC-MS) analyses of 2-AA. The biosensor strain test matched the culture assay in 24 samples (92%) and the GC-MS analyses in 25 samples (96%). With this result in hand, we designed a device containing a whole-cell luminescent biosensor combined with a photo-multiplier tube. This device allowed detection of 2-AA at levels as low as 2 nmol, on par with detection level of GC-MS. The results of the described study demonstrate that the volatile 2-AA serves as an effective biomarker for P. aeruginosa in ear infections, and that activation of the biosensor strain by 2-AA provides a unique opportunity to design an easy-to-use device that can specifically detect P. aeruginosa infections.

During the process of bioethanol production, cellulose is hydrolyzed into its monomeric soluble units. For efficient hydrolysis, a chemical and/or mechanical pretreatment step is required. Such pretreatment is designed to increase enzymatic digestibility of the cellulose chains inter alia by de-crystallization of the cellulose chains and by removing barriers, such as lignin from the plant cell wall. Biological pretreatment, in which lignin is decomposed or modified by white-rot fungi, has also been considered. One disadvantage in biological pretreatment, however, is the consumption of the cellulose by the fungus. Thus, fungal species that attack lignin with only minimal cellulose loss are advantageous. The secretomes of white-rot fungi contain carbohydrate-active enzymes (CAZymes) including lignin-modifying enzymes. Thus, modification of secretome composition can alter the ratio of lignin/cellulose degradation.

: Pleurotus eryngii is recognized for its prominent nutritional and medicinal value. In our study, we tested the effect of glucans on lipopolysaccharide (LPS)-induced production of TNF-α. We demonstrated that glucan extracts are more effective than mill mushroom preparations. Additionally, the effectiveness of stalk-derived glucans were slightly more pronounced than of caps. Cap and stalk glucans from mill or isolated glucan competed dose-dependently with anti-Dectin-and anti-CR-3 antibodies, indicating that they contain β-glucans recognized by these receptors. Using the dextran sulfate sodium (DSS)-inflammatory bowel disease mice model, intestinal inflammatory response to the mill preparations was measured and compared to extracted glucan fractions from caps and stalks. We found that mill and glucan extracts were very effective in downregulating IFN-γ and MIP-2 levels and that stalk-derived preparations were more effective than from caps. The tested glucans were equally effective in regulating the number of CD14/CD16 monocytes and upregulating the levels of fecal-released IgA to almost normal levels. In conclusion, the most effective glucans in ameliorating some IBD-inflammatory associated symptoms induced by DSS treatment in mice were glucan extracts prepared from the stalk of P. eryngii. These spatial distinctions may be helpful in selecting more effective specific anti-inflammatory mushrooms-derived glucans.

ABSTRACTSeveral composts were tested for their capacity to moderate the effect of Verticillium dahliae Kleb. (VCG B4, VD) on eggplant (Solanum melongena) under greenhouse conditions. Eggplants plantlets were inoculated by immersing their roots in conidial suspension and then planted in pots filled with mixtures of compost or peat moss, mixed with perlite. Six composts and peat moss mixtures were tested, of which tomato waste compost suppressed V. dahliae, and turkey litter compost partially suppressed it. Reduced levels of symptoms and lower fungal colonization were detected in the xylem of eggplants planted in tomato waste compost, and these plants accumulated more dry matter and had higher chlorophyll content compared to other media. However, survival of conidia in tomato waste compost showed only a moderate decrease compared with a sharp decrease in other media, suggesting that conidial eradication cannot be proposed as the suppressiveness mechanism. ? irradiation of tomato waste compost and peat at 2.5 Mrad reduced microorganism density by four orders of magnitude, but irradiation of tomato waste compost did not reduce its suppressiveness of V. dahliae. Composts properties affected progress rate of VD in the xylem tissue of eggplant seedling. These properties could indicate both biotic and abiotic factors affecting the process.

Anthropogenic activities alter the structure and function of a bacterial community. Furthermore, bacterial communities structured by the conditions the anthropogenic activities present may consequently reduce their stability in response to an unpredicted acute disturbance. The present mesocosm-scale study exposed soil bacterial communities to different irrigation water types, including freshwater, fertilized freshwater, treated wastewater, and artificial wastewater, and evaluated their response to a disturbance caused by heat. These effectors may be considered deterministic and stochastic forces common in agricultural operations of arid and semiarid regions. Bacterial communities under conditions of high mineral and organic carbon availability (artificial wastewater) differed from the native bacterial community and showed a proteobacterial dominance. These bacterial communities had a lower resistance to the heat treatment disturbance than soils under conditions of low resource availability (high-quality treated wastewater or freshwater). The latter soil bacterial communities showed a higher abundance of operational taxonomic units (OTUs) classified as Bacilli. These results were elucidated by soil under conditions of high resource availability, which lost higher degrees of functional potential and had a greater bacterial community composition change. However, the functional resilience, after the disturbance ended, was higher under a condition of high resource availability despite the bacterial community composition shift and the decrease in species richness. The functional resilience was directly connected to the high growth rates of certain Bacteroidetes and proteobacterial groups. A high stability was found in samples that supported the coexistence of both resistant OTUs and fast-growing OTUs.IMPORTANCE This report presents the results of a study employing a hypothesis-based experimental approach to reveal the forces involved in determining the stability of a soil bacterial community to disturbance. The resultant postdisturbance bacterial community composition dynamics and functionality were analyzed. The paper demonstrates the relatedness of community structure and stability under cultivation conditions prevalent in an arid area under irrigation with water of different qualities. The use of common agricultural practices to demonstrate these features has not been described before. The combination of a fundamental theoretical issue in ecology with common and concerning disturbances caused by agricultural practice makes this study unique. Furthermore, the results of the present study have applicable importance regarding soil conservation, as it enables a better characterization and monitoring of stressed soil bacterial communities and possible intervention to reduce the stress. It will also be of valued interest in coming years, as fresh water scarcity and the use of alternative water sources are expected to rise globally.

Waterborne pathogens are a major health threat and must be eliminated to guarantee safe usage of water for potable purposes. For this purpose, a new carbon-based nanomaterial composed of single-walled carbon nanotubes (SWCNTs) and iron oxides was constructed for bacterial inactivation. Owing to its magnetic properties, the SWCNT–iron oxide nanocomposite may serve as a reusable antimicrobial agent. The nanocomposite material exhibited high antimicrobial activity against Escherichia coli. Successful reuse of the nanocomposite material was achieved by washing with calcium chloride and distilled water, which restored its performance for several successive cycles. To investigate the cytotoxicity mechanisms of the nanocomposite material, we exposed it to single-gene knockout mutant strains of E. coli. Mutants bearing shorter lipopolysaccharide (LPS) layers in the outer membrane (ΔrfaC and ΔrfaG) demonstrated an increased sensitivity in comparison to the wildtype strain, exemplified in enhanced removal by the nanocomposite material. This finding suggests that the LPS acts as a protective shield against the nanocomposite material. Inactivation of mutants impaired in specific oxidative stress defense mechanisms (ΔsodA, ΔkatG and ΔsoxS) emphasized that oxidative stress plays a significant role in the inactivation mechanism of the nanocomposite. This study sheds light on the mechanisms of bacterial inactivation by carbon-based nanomaterials and advances their potential implementation for water disinfection.

Fecal microbiota transplantation (FMT) from healthy donor to patient is a treatment for microbiome-associated diseases. Although the success of FMT requires donor bacteria to engraft in the patient's gut, the forces governing engraftment in humans are unknown. Here we use an ongoing clinical experiment, the treatment of recurrent Clostridium difficile infection, to uncover the rules of engraftment in humans. We built a statistical model that predicts which bacterial species will engraft in a given host, and developed Strain Finder, a method to infer strain genotypes and track them over time. We find that engraftment can be predicted largely from the abundance and phylogeny of bacteria in the donor and the pre-FMT patient. Furthermore, donor strains within a species engraft in an all-or-nothing manner and previously undetected strains frequently colonize patients receiving FMT. We validated these findings for metabolic syndrome, suggesting that the same principles of engraftment extend to other indications.

Based on molecular data, previous studies have suggested a high overall diversity and co-infection rates of Bartonella bacteria in wild rodents and their fleas. However, partial genetic characterization of uncultured co-infecting bacteria limited sound conclusions concerning intra- and inter-specific diversity of the circulating Bartonella. To overcome this limitation, Bartonella infections of wild populations of two sympatric gerbil species and their fleas were explored by multiple isolations of Bartonella organisms. Accordingly, 448 pure Bartonella isolates, obtained from 20 rodent blood and 39 flea samples, were genetically characterized to the genotype and species levels. Results revealed a remarkable diversity and co-infection rates of Bartonella among these sympatric rodents and their associated fleas. Specifically, 38 genotypes, classified into four main Bartonella species, were identified. Co-infection was confirmed in 56% of the samples, which contained two to four Bartonella genotypes per sample, belonging to up to three different species. Recombination within and between these species was demonstrated, serving as a direct evidence of the frequent bacteria-bacteria interactions. Moreover, despite the noticeable interchange of common Bartonella genotypes between rodents and fleas, the co-occurrence of genotypes was not random and differences in the overall diversity, and the ecological and phylogenetic similarities of the infection compositions were significantly associated with the carrier type (rodent vs. flea) and the rodent species. Thus, comprehensive identification of the co-infecting organisms enabled the elucidation of ecological factors affecting the Bartonella distribution among reservoirs and vectors. This study may serve as a model for the investigation of other vector-borne organisms and their relationships with Bartonella.