Thermus oshimai JL-2 and T. thermophilus JL-18 genome analysis illuminates pathways for carbon,nitrogen, and sulfur cycling

The complete genomes of Thermus oshimai JL-2 and T. thermophilus JL-18 each consist of a circular chromosome, 2.07 Mb and 1.9 Mb, respectively, and two plasmids ranging from 0.27 Mb to 57.2 kb. Comparison of the T. thermophilus JL-18 chromosome with those from other strains of T. thermophilus revealed a high degree of synteny, whereas the megaplasmids from the same strains were highly plastic. The T. oshimai JL-2 chromosome and megaplasmids shared little or no synteny with other sequenced Thermus strains. Phylogenomic analyses using a concatenated set of conserved proteins confirmed the phylogenetic and taxonomic assignments based on 16S rRNA phylogenetics. Both chromosomes encode a complete glycolysis, tricarboxylic acid (TCA) cycle, and pentose phosphate pathway plus glucosidases, glycosidases, proteases, and peptidases, highlighting highly versatile heterotrophic capabilities. Megaplasmids of both strains contained a gene cluster encoding enzymes predicted to catalyze the sequential reduction of nitrate to nitrous oxide; however, the nitrous oxide reductase required for the terminal step in denitrification was absent, consistent with their incomplete denitrification phenotypes. A sox gene cluster was identified in both chromosomes, suggesting a mode of chemolithotrophy. In addition, nrf and psr gene clusters in T. oshmai JL-2 suggest respiratory nitrite ammonification and polysulfide reduction as possible modes of anaerobic respiration.     

Integrated transcriptomic and proteomic analysis of the global response of Wolbachia to doxycycline-induced stress

The bacterium Wolbachia (order Rickettsiales), representing perhaps the most abundant vertically transmitted microbe worldwide, infects arthropods and filarial nematodes. In arthropods, Wolbachia can induce reproductive alterations and interfere with the transmission of several arthropod-borne pathogens. In addition, Wolbachia is an obligate mutualist of the filarial parasites that cause lymphatic filariasis and onchocerciasis in the tropics. Targeting Wolbachia with tetracycline antibiotics leads to sterilisation and ultimately death of adult filariae. However, several weeks of treatment are required, restricting the implementation of this control strategy. To date, the response of Wolbachia to stress has not been investigated, and almost nothing is known about global regulation of gene expression in this organism. We exposed an arthropod Wolbachia strain to doxycycline in vitro, and analysed differential expression by directional RNA-seq and label-free, quantitative proteomics. We found that Wolbachia responded not only by modulating expression of the translation machinery, but also by upregulating nucleotide synthesis and energy metabolism, while downregulating outer membrane proteins. Moreover, Wolbachia increased the expression of a key component of the twin-arginine translocase (tatA) and a phosphate ABC transporter ATPase (PstB); the latter is associated with decreased susceptibility to antimicrobials in free-living bacteria. Finally, the downregulation of 6S RNA during translational inhibition suggests that this small RNA is involved in growth rate control. Despite its highly reduced genome, Wolbachia shows a surprising ability to regulate gene expression during exposure to a potent stressor. Our findings have general relevance for the chemotherapy of obligate intracellular bacteria and the mechanistic basis of persistence in the Rickettsiales.     

A genetic approach of wine yeast fermentation capacity in nitrogen-starvation reveals the key role of nitrogen signaling

Background

In conditions of nitrogen limitation, Saccharomyces cerevisiae strains differ in their fermentation capacities, due to differences in their nitrogen requirements. The mechanisms ensuring the maintenance of glycolytic flux in these conditions are unknown. We investigated the genetic basis of these differences, by studying quantitative trait loci (QTL) in a population of 133 individuals from the F2 segregant population generated from a cross between two strains with different nitrogen requirements for efficient fermentation.

Results

By comparing two bulks of segregants with low and high nitrogen requirements, we detected four regions making a quantitative contribution to these traits. We identified four polymorphic genes, in three of these four regions, for which involvement in the phenotype was validated by hemizygote comparison. The functions of the four validated genes, GCN1, MDS3, ARG81 and BIO3, relate to key roles in nitrogen metabolism and signaling, helping to maintain fermentation performance.

Conclusions

This study reveals that differences in nitrogen requirement between yeast strains results from a complex allelic combination. The identification of three genes involved in sensing and signaling nitrogen and specially one from the TOR pathway as affecting nitrogen requirements suggests a role for this pathway in regulating the fermentation rate in starvation through unknown mechanisms linking nitrogen signaling to glycolytic flux.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-495) contains supplementary material, which is available to authorized users.     

Genomic versatility and functional variation between two dominant heterotrophic symbionts of deep-sea Osedax worms

An unusual symbiosis, first observed at ∼3000 m depth in the Monterey Submarine Canyon, involves gutless marine polychaetes of the genus Osedax and intracellular endosymbionts belonging to the order Oceanospirillales. Ecologically, these worms and their microbial symbionts have a substantial role in the cycling of carbon from deep-sea whale fall carcasses. Microheterogeneity exists among the Osedax symbionts examined so far, and in the present study the genomes of the two dominant symbionts, Rs1 and Rs2, were sequenced. The genomes revealed heterotrophic versatility in carbon, phosphate and iron uptake, strategies for intracellular survival, evidence for an independent existence, and numerous potential virulence capabilities. The presence of specific permeases and peptidases (of glycine, proline and hydroxyproline), and numerous peptide transporters, suggests the use of degraded proteins, likely originating from collagenous bone matter, by the Osedax symbionts. ¹³C tracer experiments confirmed the assimilation of glycine/proline, as well as monosaccharides, by Osedax. The Rs1 and Rs2 symbionts are genomically distinct in carbon and sulfur metabolism, respiration, and cell wall composition, among others. Differences between Rs1 and Rs2 and phylogenetic analysis of chemotaxis-related genes within individuals of symbiont Rs1 revealed the influence of the relative age of the whale fall environment and support possible local niche adaptation of ‘free-living'' lifestages. Future genomic examinations of other horizontally-propogated intracellular symbionts will likely enhance our understanding of the contribution of intraspecific symbiont diversity to the ecological diversification of the intact association, as well as the maintenance of host diversity.     

Continuous,high-resolution biospeckle imaging reveals a discrete zone of activity at the root apex that responds to contact with obstacles

Background and Aims

Shining a laser onto biological material produces light speckles termed biospeckles. Patterns of biospeckle activity reflect changes in cell biochemistry, developmental processes and responses to the environment. The aim of this work was to develop methods to investigate the biospeckle activity in roots and to characterize the distribution of its intensity and response to thigmostimuli.

Methods

Biospeckle activity in roots of Zea mays, and also Jatropha curcas and Citrus limonia, was imaged live and in situ using a portable laser and a digital microscope with a spatial resolution of 10 μm per pixel and the ability to capture images every 0·080 s. A procedure incorporating a Fujii algorithm, image restoration using median and Gaussian filters, image segmentation using maximum-entropy threshold methods and the extraction of features using a tracing algorithm followed by spline fitting were developed to obtain quantitative information from images of biospeckle activity. A wavelet transform algorithm was used for spectral decomposition of biospeckle activity and generalized additive models were used to attribute statistical significance to changes in patterns of biospeckle activity.

Key Results

The intensity of biospeckle activity was greatest close to the root apex. Higher frequencies (3–6 Hz) contributed most to the total intensity of biospeckle activity. When a root encountered an obstacle, the intensity of biospeckle activity decreased abruptly throughout the root system. The response became attenuated with repeated thigmostimuli.

Conclusions

The data suggest that at least one component of root biospeckle activity resulted from a biological process, which is located in the zone of cell division and responds to thigmostimuli. However, neither individual cell division events nor root elongation is likely to be responsible for the patterns of biospeckle activity.     

Long-range RNA pairings contribute to mutually exclusive splicing

Mutually exclusive splicing is an important means of increasing the protein repertoire, by which the Down''s syndrome cell adhesion molecule (Dscam) gene potentially generates 38,016 different isoforms in Drosophila melanogaster. However, the regulatory mechanisms remain obscure due to the complexity of the Dscam exon cluster. Here, we reveal a molecular model for the regulation of the mutually exclusive splicing of the serpent pre-mRNA based on competition between upstream and downstream RNA pairings. Such dual RNA pairings confer fine tuning of the inclusion of alternative exons. Moreover, we demonstrate that the splicing outcome of alternative exons is mediated in relative pairing strength-correlated mode. Combined comparative genomics analysis and experimental evidence revealed similar bidirectional structural architectures in exon clusters 4 and 9 of the Dscam gene. Our findings provide a novel mechanistic framework for the regulation of mutually exclusive splicing and may offer potentially applicable insights into long-range RNA–RNA interactions in gene regulatory networks.     

Lineage-specific molecular probing reveals novel diversity and ecological partitioning of haplosporidians

Haplosporidians are rhizarian parasites of mostly marine invertebrates. They include the causative agents of diseases of commercially important molluscs, including MSX disease in oysters. Despite their importance for food security, their diversity and distributions are poorly known. We used a combination of group-specific PCR primers to probe environmental DNA samples from planktonic and benthic environments in Europe, South Africa and Panama. This revealed several highly distinct novel clades, novel lineages within known clades and seasonal (spring vs autumn) and habitat-related (brackish vs littoral) variation in assemblage composition. High frequencies of haplosporidian lineages in the water column provide the first evidence for life cycles involving planktonic hosts, host-free stages or both. The general absence of haplosporidian lineages from all large online sequence data sets emphasises the importance of lineage-specific approaches for studying these highly divergent and diverse lineages. Combined with host-based field surveys, environmental sampling for pathogens will enhance future detection of known and novel pathogens and the assessment of disease risk.     

Several deep-sea mussels and their associated symbionts are able to live both on wood and on whale falls

Bathymodiolin mussels occur at hydrothermal vents and cold seeps, where they thrive thanks to symbiotic associations with chemotrophic bacteria. Closely related genera Idas and Adipicola are associated with organic falls, ecosystems that have been suggested as potential evolutionary 'stepping stones' in the colonization of deeper and more sulphide-rich environments. Such a scenario should result from specializations to given environments from species with larger ecological niches. This study provides molecular-based evidence for the existence of two mussel species found both on sunken wood and bones. Each species specifically harbours one bacterial phylotype corresponding to thioautotrophic bacteria related to other bathymodiolin symbionts. Phylogenetic patterns between hosts and symbionts are partially congruent. However, active endocytosis and occurrences of minor symbiont lineages within species which are not their usual host suggest an environmental or horizontal rather than strictly vertical transmission of symbionts. Although the bacteria are close relatives, their localization is intracellular in one mussel species and extracellular in the other, suggesting that habitat choice is independent of the symbiont localization. The variation of bacterial densities in host tissues is related to the substrate on which specimens were sampled and could explain the abilities of host species to adapt to various substrates.     

Sensitivity of growth and biomass allocation patterns to increasing nitrogen: a comparison between ephemerals and annuals in the Gurbantunggut Desert,north-western China

Background and Aims

Biomass accumulation and allocation patterns are critical to quantifying ecosystem dynamics. However, these patterns differ among species, and they can change in response to nutrient availability even among genetically related individuals. In order to understand this complexity further, this study examined three ephemeral species (with very short vegetative growth periods) and three annual species (with significantly longer vegetative growth periods) in the Gurbantunggut Desert, north-western China, to determine their responses to different nitrogen (N) supplements under natural conditions.

Methods

Nitrogen was added to the soil at rates of 0, 0·5, 1·0, 3·0, 6·0 and 24·0 g N m⁻² year⁻¹. Plants were sampled at various intervals to measure relative growth rate and shoot and root dry mass.

Key Results

Compared with annuals, ephemerals grew more rapidly, increased shoot and root biomass with increasing N application rates and significantly decreased root/shoot ratios. Nevertheless, changes in the biomass allocation of some species (i.e. Erodium oxyrrhynchum) in response to the N treatment were largely a consequence of changes in overall plant size, which was inconsistent with an optimal partitioning model. An isometric log shoot vs. log root scaling relationship for the final biomass harvest was observed for each species and all annuals, while pooled data of three ephemerals showed an allometric scaling relationship.

Conclusions

These results indicate that ephemerals and annuals differ observably in their biomass allocation patterns in response to soil N supplements, although an isometric log shoot vs. log root scaling relationship was maintained across all species. These findings highlight that different life history strategies behave differently in response to N application even when interspecific scaling relationships remain nearly isometric.     

Community Structure of Plant-parasitic Nematodes Related to Soil Types in Illinois and Indiana Soybean Fields

Comparisons of plant parasitic nematode populations using a resemblance equation and community ordination showed that community structure tends to be similar on dark-colored, highly productive soybean soils throughout Indiana and Illinois. On lighter-colored soils community structures differed somewhat from those of darker soils and from each other.     

Leaf trait co-ordination in relation to construction cost, carbon gain and resource-use efficiency in exotic invasive and native woody vine species

Background and Aims

Success of invasive plant species is thought to be linked with their higher leaf carbon fixation strategy, enabling them to capture and utilize resources better than native species, and thus pre-empt and maintain space. However, these traits are not well-defined for invasive woody vines.

Methods

In a glass house setting, experiments were conducted to examine how leaf carbon gain strategies differ between non-indigenous invasive and native woody vines of south-eastern Australia, by investigating their biomass gain, leaf structural, nutrient and physiological traits under changing light and moisture regimes.

Key Results

Leaf construction cost (CC), calorific value and carbon : nitrogen (C : N) ratio were lower in the invasive group, while ash content, N, maximum photosynthesis, light-use efficiency, photosynthetic energy-use efficiency (PEUE) and specific leaf area (SLA) were higher in this group relative to the native group. Trait plasticity, relative growth rate (RGR), photosynthetic nitrogen-use efficiency and water-use efficiency did not differ significantly between the groups. However, across light resource, regression analyses indicated that at a common (same) leaf CC and PEUE, a higher biomass RGR resulted for the invasive group; also at a common SLA, a lower CC but higher N resulted for the invasive group. Overall, trait co-ordination (using pair-wise correlation analyses) was better in the invasive group. Ordination using 16 leaf traits indicated that the major axis of invasive-native dichotomy is primarily driven by SLA and CC (including its components and/or derivative of PEUE) and was significantly linked with RGR.

Conclusions

These results demonstrated that while not all measures of leaf resource traits may differ between the two groups, the higher level of trait correlation and higher revenue returned (RGR) per unit of major resource need (CC) and use (PEUE) in the invasive group is in line with their rapid spread where introduced.     

Genome analysis reveals three genomospecies in Mycobacterium abscessus

Background

Mycobacterium abscessus complex, the third most frequent mycobacterial complex responsible for community- and health care-associated infections in developed countries, comprises of M. abscessus subsp. abscessus and M. abscessus subsp. bolletii reviously referred as Mycobacterium bolletii and Mycobacterium massiliense. The diversity of this group of opportunistic pathogens is poorly described.

Results

In-depth analysis of 14 published M. abscessus complex genomes found a pan-genome of 6,153 proteins and core-genome of 3,947 (64.1%) proteins, indicating a non-conservative genome. Analysing the average percentage of amino-acid sequence identity (from 94.19% to 98.58%) discriminates three main clusters C1, C2 and C3: C1 comprises strains belonging to M. abscessus, C2 comprises strains belonging to M. massiliense and C3 comprises strains belonging to M. bolletii; and two sub-clusters in clusters C2 and C3. The phylogenomic network confirms these three clusters. The genome length (from 4.8 to 5.51-Mb) varies from 5.07-Mb in C1, 4.89-Mb in C2A, 5.01-Mb in C2B and 5.28-Mb in C3. The mean number of prophage regions (from 0 to 7) is 2 in C1; 1.33 in C2A; 3.5 in C2B and five in C3. A total of 36 genes are uniquely present in C1, 15 in C2 and 15 in C3. These genes could be used for the detection and identification of organisms in each cluster. Further, the mean number of host-interaction factors (including PE, PPE, LpqH, MCE, Yrbe and type VII secretion system ESX3 and ESX4) varies from 70 in cluster C1, 80 in cluster C2A, 74 in cluster C2B and 93 in clusters C3A and C3B. No significant differences in antibiotic resistance genes were observed between clusters, in contrast to previously reported in-vitro patterns of drug resistance. They encode both penicillin-binding proteins targeted by β-lactam antibiotics and an Ambler class A β-lactamase for which inhibitors exist.

Conclusions

Our comparative analysis indicates that M. abscessus complex comprises three genomospecies, corresponding to M. abscessus, M. bolletii, and M. massiliense. The genomics data here reported indicate differences in virulence of medical interest; and suggest targets for the refined detection and identification of M. abscessus.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-359) contains supplementary material, which is available to authorized users.     

Effects and Dynamics of a Nematode Community on Maize

Relationships between nematode density and yield and between final and preplant population levels were examined in small maize plots on sandy soils in north-central Florida. Plant-parasitic nematodes present in the community included Belonolaimus longicaudatus, Criconemella sphaerocephala, Meloidogyne incognita, Paratrichodorus minor, Pratylenchus brachyurus, and a Xiphinema sp. Plant growth--including stand count, grain yield, stalk weight, and size of young plants--often was inversely correlated (P ≤ 0.05) with densities of B. longicaudatus and occasionally with P. brachyurus, but not with densities of other species or with a range of soil variables. More severe losses in grain yields from B. longicaudatus occurred in 1987 than in 1988, although mean preplant nematode densities in February were similar in both years (4.4 vs. 3.9/100 cm³ soil). Final population densities of most nematode species were linearly related (P ≤ 0.05) to densities measured at planting or earlier. These relationships were stronger (higher r²) with the ectoparasites B. longicaudatus and C. sphaerocephala than with the endoparasites M. incognita and P. brachyurus. No significant correlations were found between population densities of different nematode species.     

Effects and Dynamics of a Nematode Community on Soybean

The relationships between densities of all members of a plant-parasitic nematode community and yield of ''Davis'' soybean and between final and preplant population levels were examined in small plots on sandy soils in north-central Florida. Plant-parasitic nematodes present in the community included Belonolaimus longicaudatus, Criconemella sphaerocephala, Meloidogyne incognita, Paratrichodorus minor, Pratylenchus brachyurus, and Xiphinema sp. Plant growth, including stand count, soybean yield (kg/ha), and size of young plants, was occasionally inversely correlated (P ≤ 0.05) with densities of B. longicaudatus or P. brachyurus, but not with densities of other species or with a range of soil variables. The nature of this relationship varied with season, with more severe stand losses noted during 1987 than in 1988. Final population densities (Pf) of most nematode species showed significant (P ≤ 0.05) linear relationships to densities measured at planting or earlier (Pi). These relationships were stronger (higher r²) with the ectoparasite B. longicaudatus than with the endoparasites M. incognita and P. brachyurus. Criconemella sphaerocephala declined under soybean cultivation, reaching levels near zero after two seasons. A quadratic model showed an improvement (P ≤ 0.05) over the linear model in describing the relationship between Pf and Pi measured at planting for B. longicaudatus, and gave a better indication of the leveling off of Pf at high values of Pi.     

Community genomic analysis of an extremely acidophilic sulfur-oxidizing biofilm

Highly acidic (pH 0–1) biofilms, known as ‘snottites'', form on the walls and ceilings of hydrogen sulfide-rich caves. We investigated the population structure, physiology and biogeochemistry of these biofilms using metagenomics, rRNA methods and lipid geochemistry. Snottites from the Frasassi cave system (Italy) are dominated (>70% of cells) by Acidithiobacillus thiooxidans, with smaller populations including an archaeon in the uncultivated ‘G-plasma'' clade of Thermoplasmatales (>15%) and a bacterium in the Acidimicrobiaceae family (>5%). Based on metagenomic evidence, the Acidithiobacillus population is autotrophic (ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), carboxysomes) and oxidizes sulfur by the sulfide–quinone reductase and sox pathways. No reads matching nitrogen fixation genes were detected in the metagenome, whereas multiple matches to nitrogen assimilation functions are present, consistent with geochemical evidence, that fixed nitrogen is available in the snottite environment to support autotrophic growth. Evidence for adaptations to extreme acidity include Acidithiobacillus sequences for cation transporters and hopanoid synthesis, and direct measurements of hopanoid membrane lipids. Based on combined metagenomic, molecular and geochemical evidence, we suggest that Acidithiobacillus is the snottite architect and main primary producer, and that snottite morphology and distributions in the cave environment are directly related to the supply of C, N and energy substrates from the cave atmosphere.     

Responses of the coastal bacterial community to viral infection of the algae Phaeocystis globosa

The release of organic material upon algal cell lyses has a key role in structuring bacterial communities and affects the cycling of biolimiting elements in the marine environment. Here we show that already before cell lysis the leakage or excretion of organic matter by infected yet intact algal cells shaped North Sea bacterial community composition and enhanced bacterial substrate assimilation. Infected algal cultures of Phaeocystis globosa grown in coastal North Sea water contained gamma- and alphaproteobacterial phylotypes that were distinct from those in the non-infected control cultures 5 h after infection. The gammaproteobacterial population at this time mainly consisted of Alteromonas sp. cells that were attached to the infected but still intact host cells. Nano-scale secondary-ion mass spectrometry (nanoSIMS) showed ∼20% transfer of organic matter derived from the infected ¹³C- and ¹⁵N-labelled P. globosa cells to Alteromonas sp. cells. Subsequent, viral lysis of P. globosa resulted in the formation of aggregates that were densely colonised by bacteria. Aggregate dissolution was observed after 2 days, which we attribute to bacteriophage-induced lysis of the attached bacteria. Isotope mass spectrometry analysis showed that 40% of the particulate ¹³C-organic carbon from the infected P. globosa culture was remineralized to dissolved inorganic carbon after 7 days. These findings reveal a novel role of viruses in the leakage or excretion of algal biomass upon infection, which provides an additional ecological niche for specific bacterial populations and potentially redirects carbon availability.     

Single cell genomic study of Dehalococcoidetes species from deep-sea sediments of the Peruvian Margin

The phylum Chloroflexi is one of the most frequently detected phyla in the subseafloor of the Pacific Ocean margins. Dehalogenating Chloroflexi (Dehalococcoidetes) was originally discovered as the key microorganisms mediating reductive dehalogenation via their key enzymes reductive dehalogenases (Rdh) as sole mode of energy conservation in terrestrial environments. The frequent detection of Dehalococcoidetes-related 16S rRNA and rdh genes in the marine subsurface implies a role for dissimilatory dehalorespiration in this environment; however, the two genes have never been linked to each other. To provide fundamental insights into the metabolism, genomic population structure and evolution of marine subsurface Dehalococcoidetes sp., we analyzed a non-contaminated deep-sea sediment core sample from the Peruvian Margin Ocean Drilling Program (ODP) site 1230, collected 7.3 m below the seafloor by a single cell genomic approach. We present for the first time single cell genomic data on three deep-sea Chloroflexi (Dsc) single cells from a marine subsurface environment. Two of the single cells were considered to be part of a local Dehalococcoidetes population and assembled together into a 1.38-Mb genome, which appears to be at least 85% complete. Despite a high degree of sequence-level similarity between the shared proteins in the Dsc and terrestrial Dehalococcoidetes, no evidence for catabolic reductive dehalogenation was found in Dsc. The genome content is however consistent with a strictly anaerobic organotrophic or lithotrophic lifestyle.     

Tylenchulus semipenetrans Alters the Microbial Community in the Citrus Rhizosphere

Infection of citrus seedlings by Tylenchulus semipenetrans was shown to reduce subsequent infection of roots by Phytophthora nicotianae and to increase plant growth compared to plants infected by only the fungus. Hypothetical mechanisms by which the nematode suppresses fungal development include nutrient competition, direct antibiosis, or alteration of the microbial community in the rhizosphere to favor microorganisms antagonistic to P. nicotianae. A test of the last hypothesis was conducted via surveys of five sites in each of three citrus orchards infested with both organisms. A total of 180 2-cm-long fibrous root segments, half with a female T. semipenetrans egg mass on the root surface and half without, were obtained from each orchard site. The samples were macerated in water, and fungi and bacteria in the suspensions were isolated, quantified, and identified. No differences were detected in the numbers of microorganism species isolated from nematode-infected and uninfected root segments. However, nematode-infected root segments had significantly more propagules of bacteria at all orchard sites. Bacillus megaterium and Burkholderia cepacia were the dominant bacterial species recovered. Bacteria belonging to the genera Arthrobacter and Stenotrophomonas were encountered less frequently. The fungus community was dominated by Fusarium solani, but Trichoderma, Verticillum, Phythophthora, and Penicillium spp. also were recovered. All isolated bacteria equally inhibited the growth of P. nicotianae in vitro. Experiments using selected bacteria, T. semipenetrans, and P. nicotianae, alone or in combination, were conducted in both the laboratory and greenhouse. Root and stem fresh weights of P. nicotianae-infected plants treated with T. semipenetrans, B. cepacia, or B. megaterium were greater than for plants treated only with the fungus. Phytophthora nicotianae protein in roots of fungus-infected plants was reduced by nematodes (P ≤ 0.001), either alone or in combination with either bacterium. However, treatment with bacteria did not affect P. nicotianae development in roots. The results suggest different mechanisms by which T. semipenetrans, B. cepacia, and B. megaterium may mitigate virulence of P. nicotianae.