General Stress Response Genes Research Articles

Expression of virulence and stress response genes in Aeromonas hydrophila under various stress conditions.

Aeromonas hydrophila has emerged as an important human pathogen as it causes gastroenteritis and extra-intestinal infections. Information regarding the influence of environmental stresses on gene expression profile of A. hydrophila is lacking. The impact of nutrient replenishment, nutrient deprivation, acid stress, and cold shock on housekeeping, general stress-response, and virulence genes was studied using quantitative real-time PCR in two A. hydrophila strains, CECT 839T , and A331. These sub-lethal stresses invoked significant changes in the expression of these genes in a strain-dependent manner. Overall, nutrient replenishment and deprivation significantly induced the expression of housekeeping (rpoD), general stress regulators (uspA and rpoS), and virulence (aer) genes, indicating their importance in regulating the survival and virulence of A. hydrophila under these stress conditions. rpoS gene was significantly induced under cold shock; whereas, acid stress significantly induced the expression of uspA gene. This is the first study to investigate the effect of environmental parameters on the expression of stress-response and virulence genes in A. hydrophila strains.

Heat-tolerance in Cotton Is Correlated with Induced Overexpression of Heat-Shock Factors, Heat-Shock Proteins, and General Stress Response Genes

Breeding for heat tolerance is complicated by the polygenic nature of the trait and genetic engineering awaits delineation of the molecular mechanisms. To understand heat-tolerance mechanisms in cotton (Gossypum hirsutum L.), the world’s most important natural fiber crop, this study compared expression of selected heat-stress response genes between heat-tolerant (VH260 and MNH456) and heat-sensitive (ST213 and ST4288) cotton genotypes. Cotton orthologs of selected Arabidopsis heat-stress response genes included two heat-stress transcription factors, three heat-shock proteins, and the general stress response genes: ascorbate peroxidase and the calcium-dependent stress responder, ANNAT8. The seedlings and flowering plants of each genotype were exposed to heat (38°C) for 1 to 3 hours or days, respectively, followed by RNA isolation from the seedlings and ovaries of flowering plants for gene expression analysis by real-time quantitative polymerase chain reactions. All genes, except the heat-shock protein GHSP26, were found to be induced exclusively in the heat-tolerant lines. The genotype VH260, which reportedly is tolerant to extreme heat (45°C), was found to display much higher induction of these genes than MNH456, the heat-tolerance level of which has not been thoroughly studied. Further, VH260 showed high gene induction in both seedlings and ovaries, whereas MNH456 showed lower induction levels. Strong heat tolerance in VH260 most likely is based on early induction of multiple mechanisms coordinately functioning to defend the plants against membrane damage, protein denaturation, and oxidative stress, in addition to rapid sensing of heat stress to signal the defense processes at the flowering stage towards minimizing yield losses and increasing boll retention during heat stress.

Characterization of the general stress response in Bartonella henselae

Bacteria utilize a general stress response system to combat stresses from their surrounding environments. In alpha-proteobacteria, the general stress response uses an alternate sigma factor as the main regulator and incorporates it with a two-component system into a unique regulatory circuit. This system has been described in several alpha-proteobacterial species, including the pathogens Bartonella quintana and Brucella abortus. Most of the studies have focused on characterizing the PhyR anti-anti-sigma factor, the NepR anti-sigma factor, and the alternate sigma factor. However, not enough attention is directed toward studying the role of histidine kinases in the general stress response. Our study identifies the general stress response system in Bartonella henselae, where the gene synteny is conserved and both the PhyR and alternate sigma factor have similar sequence and domain structures with other alpha-proteobacteria. Our data showed that the general stress response genes are up-regulated under conditions that mimic the cat flea vector. Furthermore, we showed that both RpoE and PhyR positively regulate this system and that RpoE also affects transcription of genes encoding heme-binding proteins and the gene encoding the BadA adhesin. Finally, we identified a histidine kinase, annotated as BH13820 that can potentially phosphorylate PhyR.

RNA-seq analysis reveals genetic response and tolerance mechanisms to ozone exposure in soybean.

BackgroundOxidative stress caused by ground level ozone is a contributor to yield loss in a number of important crop plants. Soybean (Glycine max) is considered to be ozone sensitive, and current research into its response to oxidative stress is limited. To better understand the genetic response in soybean to oxidative stress, an RNA-seq analysis of two soybean cultivars was performed comparing an ozone intolerant cultivar (Mandarin-Ottawa) and an ozone resistant cultivar (Fiskeby III) following exposure to ozone.ResultsAnalysis of the transcriptome data revealed cultivar-specific expression level differences of genes previously implicated in oxidative stress responses, indicating unique cultivar-specific responses. Both Fiskeby III and Mandarin (Ottawa) exhibit an increased expression of oxidative response genes as well as glutathiones, phenylpropanoids, and phenylalanine ammonia-lyases. Mandarin (Ottawa) exhibited more general stress response genes whereas Fiskeby III had heightened expression of metabolic process genes. An examination of the timing of gene responses over the course of ozone exposure identified significantly more differentially expressed genes across all time points in Mandarin (Ottawa) than in Fiskeby III. The timing of expression was also considered to identify genes that may be indicative of a delayed response to ozone stress in Fiskeby III, We found that Mandarin (Ottawa) exhibits an higher level of expression in early time points for oxidative and general stress response genes while Fiskeby III seems to maintain expression of defense and stress response genes. Of particular interest was the expression of wax and cutin biosynthetic genes that we found to be expressed in Mandarin (Ottawa) in all sampled time points, whereas the expression of this pathway is only in the first time point for Fiskeby III.ConclusionsWe were able to identify differentially expressed genes that correspond to each of the known or expected categories of genes previously implicated in other species for ozone stress. Our study shows evidence that at least part of the observed ozone tolerance of Fiskeby III may be due to its thicker, denser leaves providing passive resistance thereby limiting the degree of ozone exposure. The observed diminished genetic response is then likely a consequence of this reduced exposure.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1637-7) contains supplementary material, which is available to authorized users.

Decreased in vivo virulence and altered gene expression by a Brucella melitensis light-sensing histidine kinase mutant.

Brucella species utilize diverse virulence factors. Previously, Brucella abortus light-sensing histidine kinase was identified as important for cellular infection. Here, we demonstrate that a Brucella melitensis LOV-HK (BM-LOV-HK) mutant strain has strikingly different gene expression than wild type. General stress response genes including the alternative sigma factor rpoE1 and its anti-anti-sigma factor phyR were downregulated, while flagellar, quorum sensing (QS), and type IV secretion system genes were upregulated in the ΔBM-LOV-HK strain vs. wild type. Contextually, expression results agree with other studies of transcriptional regulators involving ΔrpoE1, ΔphyR, ΔvjbR, and ΔblxR (ΔbabR) Brucella strains. Additionally, deletion of BM-LOV-HK decreases virulence in mice. During C57BL/6 mouse infection, the ΔBM-LOV-HK strain had 2 logs less CFUs in the spleen 3 days postinfection, but similar levels 6 days post infection compared to wild type. Infection of IRF-1(-/-) mice more specifically define ΔBM-LOV-HK strain attenuation with fewer bacteria in spleens and significantly increased survival of mutant vs. wild-type infected IRF-1(-/-) mice. Upregulation of flagella, QS, and VirB genes, along with downregulation of rpoE1 and related sigma factor, rpoH2 (BMEI0280) suggest that BM-LOV-HK modulates both QS and general stress response regulatory components to control Brucella gene expression on a global level.

Distinct roles for mitogen-activated protein kinase signaling and CALMODULIN-BINDING TRANSCRIPTIONAL ACTIVATOR3 in regulating the peak time and amplitude of the plant general stress response.

To survive environmental challenges, plants have evolved tightly regulated response networks, including a rapid and transient general stress response (GSR), followed by well-studied stress-specific responses. The mechanisms underpinning the GSR have remained elusive, but a functional cis-element, the rapid stress response element (RSRE), is known to confer transcription of GSR genes rapidly (5 min) and transiently (peaking 90-120 min after stress) in vivo. To investigate signal transduction events in the GSR, we used a 4xRSRE:LUCIFERASE reporter in Arabidopsis (Arabidopsis thaliana), employing complementary approaches of forward and chemical genetic screens, and identified components regulating peak time versus amplitude of RSRE activity. Specifically, we identified a mutant in CALMODULIN-BINDING TRANSCRIPTIONAL ACTIVATOR3 (CAMTA3) with reduced RSRE activation, verifying this transcription factor's role in activation of the RSRE-mediated GSR. Furthermore, we isolated a mutant in MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) KINASE KINASE1 (mekk1-5), which displays increased basal and an approximately 60-min earlier peak of wound-induced RSRE activation. The double mekk1/camta3 mutant positioned CAMTA3 downstream of MEKK1 and verified their distinct roles in GSR regulation. mekk1-5 displays programmed cell death and overaccumulates reactive oxygen species and salicylic acid, hallmarks of the hypersensitive response, suggesting that the hypersensitive response may play a role in the RSRE phenotype in this mutant. In addition, chemical inhibition studies suggest that the MAPK network is required for the rapid peak of the RSRE response, distinguishing the impact of chronic (mekk1-5) from transient (chemical inhibition) loss of MAPK signaling. Collectively, these results reveal underlying regulatory components of the plant GSR and further define their distinct roles in the regulation of this key biological process.

Effects of Indole-3-Acetic Acid on the Transcriptional Activities and Stress Tolerance of Bradyrhizobium japonicum

A genome-wide transcriptional profile of Bradyrhizobium japonicum, the nitrogen-fixing endosymbiont of the soybean plant, revealed differential expression of approximately 15% of the genome after a 1 mM treatment with the phytohormone indole-3-acetic acid (IAA). A total of 1,323 genes were differentially expressed (619 up-regulated and 704 down-regulated) at a two-fold cut off with q value ≤ 0.05. General stress response genes were induced, such as those involved in response to heat, cold, oxidative, osmotic, and desiccation stresses and in exopolysaccharide (EPS) biosynthesis. This suggests that IAA is effective in activating a generalized stress response in B. japonicum. The transcriptional data were corroborated by the finding that stress tolerance of B. japonicum in cell viability assays was enhanced when pre-treated with 1 mM IAA compared to controls. The IAA treatment also stimulated biofilm formation and EPS production by B. japonicum, especially acidic sugar components in the total EPS. The IAA pre-treatment did not influence the nodulation ability of B. japonicum. The data provide a comprehensive overview of the potential transcriptional responses of the symbiotic bacterium when exposed to the ubiquitous hormone of its plant host.

Stress resistance and C1 metabolism involved in plant colonization of a methanotroph Methylosinus sp. B4S

Methanotrophs are widespread and have been isolated from various environments including the phyllosphere. In this study, we characterized the plant colonization by Methylosinus sp. B4S, an α-proteobacterial methanotroph isolated from plant leaf. The gfp-tagged Methylosinus sp. B4S cells were observed to colonize Arabidopsis leaf surfaces by forming aggregates. We cloned and sequenced the general stress response genes, phyR, nepR and ecfG, from Methylosinus sp. B4S. In vitro analysis showed that the phyR expression level was increased after heat shock challenge, and phyR was shown to be involved in resistance to heat shock and UV light. In the phyllospheric condition, the gene expression level of phyR as well as mmoX and mxaF was found to be relatively high, compared with methane-grown liquid cultures. The phyR-deletion strain as well as the wild-type strain inoculated on Arabidopsis leaves proliferated at the initial phase and then gradually decreased during plant colonization. These results have shed light firstly on the importance of general stress resistance and C1 metabolism in methanotroph living in the phyllosphere.

Metabolomic analysis of the selection response of Drosophila melanogaster to environmental stress: are there links to gene expression and phenotypic traits?

We investigated the global metabolite response to artificial selection for tolerance to stressful conditions such as cold, heat, starvation, and desiccation, and for longevity in Drosophila melanogaster. Our findings were compared to data from other levels of biological organization, including gene expression, physiological traits, and organismal stress tolerance phenotype. Overall, we found that selection for environmental stress tolerance changes the metabolomic (1)H NMR fingerprint largely in a similar manner independent of the trait selected for, indicating that experimental evolution led to a general stress selection response at the metabolomic level. Integrative analyses across data sets showed little similarity when general correlations between selection effects at the level of the metabolome and gene expression were compared. This is likely due to the fact that the changes caused by these selection regimes were rather mild and/or that the dominating determinants for gene expression and metabolite levels were different. However, expression of a number of genes was correlated with the metabolite data. Many of the identified genes were general stress response genes that are down-regulated in response to selection for some of the stresses in this study. Overall, the results illustrate that selection markedly alters the metabolite profile and that the coupling between different levels of biological organization indeed is present though not very strong for stress selection at this level. The results highlight the extreme complexity of environmental stress adaptation and the difficulty of extrapolating and interpreting responses across levels of biological organization.

Genomic resources for a model in adaptation and speciation research: characterization of the Poecilia mexicana transcriptome

BackgroundElucidating the genomic basis of adaptation and speciation is a major challenge in natural systems with large quantities of environmental and phenotypic data, mostly because of the scarcity of genomic resources for non-model organisms. The Atlantic molly (Poecilia mexicana, Poeciliidae) is a small livebearing fish that has been extensively studied for evolutionary ecology research, particularly because this species has repeatedly colonized extreme environments in the form of caves and toxic hydrogen sulfide containing springs. In such extreme environments, populations show strong patterns of adaptive trait divergence and the emergence of reproductive isolation. Here, we used RNA-sequencing to assemble and annotate the first transcriptome of P. mexicana to facilitate ecological genomics studies in the future and aid the identification of genes underlying adaptation and speciation in the system.DescriptionWe provide the first annotated reference transcriptome of P. mexicana. Our transcriptome shows high congruence with other published fish transcriptomes, including that of the guppy, medaka, zebrafish, and stickleback. Transcriptome annotation uncovered the presence of candidate genes relevant in the study of adaptation to extreme environments. We describe general and oxidative stress response genes as well as genes involved in pathways induced by hypoxia or involved in sulfide metabolism. To facilitate future comparative analyses, we also conducted quantitative comparisons between P. mexicana from different river drainages. 106,524 single nucleotide polymorphisms were detected in our dataset, including potential markers that are putatively fixed across drainages. Furthermore, specimens from different drainages exhibited some consistent differences in gene regulation.ConclusionsOur study provides a valuable genomic resource to study the molecular underpinnings of adaptation to extreme environments in replicated sulfide spring and cave environments. In addition, this study adds to the increasing number of genomic resources in the family Poeciliidae, which are widely used in comparative analyses of behavior, ecology, evolution, and medical genetics.

Mechanisms underlying toxicity induced by CdTe quantum dots determined in an invertebrate model organism

A systematic and thorough quantitative analysis of the in vivo effects of inorganic nanoparticles is extremely important for the design of functional nanomaterials for diagnostic and therapeutic applications, better understanding of their non-specificity toward tissues and cell types, and for assessments of their toxicity. This study was undertaken to examine the impact of CdTe quantum dots (QDs) on an invertebrate freshwater model organism, Hydra vulgaris, for assessment of long term toxicity effects. The continuous exposure of living polyps to sub-lethal doses of QDs caused time and dose dependent morphological damages more severe than Cd 2+ ions at the same concentrations, impaired both reproductive and regenerative capability, activated biochemical and molecular responses. Of remarkable interest, low QD doses, apparently not effective, caused early changes in the expression of general stress responsive and apoptotic genes. The occurrence of subtle genetic variations, in the absence of morphological damages, indicates the importance of genotoxicity studies for nanoparticle risk assessment. The versatility in morphological, cellular, biochemical and molecular responses renders Hydra a perfect model system for high-throughput screening of toxicological and ecotoxicological impact of nanomaterials on human and environmental health.

Adaptive Evolution of a Novel Drosophila Lectin Induced by Parasitic Wasp Attack

Drosophila melanogaster has long been used as a model for the molecular genetics of innate immunity. Such work has uncovered several immune receptors that recognize bacterial and fungal pathogens by binding unique components of their cell walls and membranes. Drosophila also act as hosts to metazoan pathogens such as parasitic wasps, which can infect a majority of individuals in natural populations, but many aspects of their immune responses against these more closely related pathogens are poorly understood. Here, we present data describing the transcriptional induction and molecular evolution of a candidate Drosophila anti-wasp immunity gene, lectin-24A. Lectin-24A has a secretion signal sequence and its lectin domain suggests a function in sugar group binding. Transcript levels of lectin-24A were induced significantly stronger and faster following wasp attack than following wounding or bacterial infection, demonstrating lectin-24A is not a general stress response or defense response gene but is instead part of a specific response against wasps. The major site of lectin-24A transcript production is the fat body, the main humoral immune tissue of flies. Interestingly, lectin-24A is a new gene of the D. melanogaster/Drosophila simulans clade, displaying very little homology to any other Drosophila lectins. Population genetic analyses of lectin-24A DNA sequence data from African and North American populations of D. melanogaster and D. simulans revealed gene length polymorphisms segregating at high frequencies as well as strong evidence of repeated and recent selective sweeps. Thus, lectin-24A is a rapidly evolving new gene that has seemingly developed functional importance for fly resistance against infection by parasitic wasps.

Antibacterial Activity and Mechanism of Action of Zinc Oxide Nanoparticles against Campylobacter jejuni

The antibacterial effect of zinc oxide (ZnO) nanoparticles on Campylobacter jejuni was investigated for inhibition and inactivation of cell growth. The results showed that C. jejuni was extremely sensitive to treatment with ZnO nanoparticles. The MIC of ZnO nanoparticles for C. jejuni was determined to be 0.05 to 0.025 mg/ml, which is 8- to 16-fold lower than that for Salmonella enterica serovar Enteritidis and Escherichia coli O157:H7 (0.4 mg/ml). The action of ZnO nanoparticles against C. jejuni was determined to be bactericidal, not bacteriostatic. Scanning electron microscopy examination revealed that the majority of the cells transformed from spiral shapes into coccoid forms after exposure to 0.5 mg/ml of ZnO nanoparticles for 16 h, which is consistent with the morphological changes of C. jejuni under other stress conditions. These coccoid cells were found by ethidium monoazide-quantitative PCR (EMA-qPCR) to have a certain level of membrane leakage. To address the molecular basis of ZnO nanoparticle action, a large set of genes involved in cell stress response, motility, pathogenesis, and toxin production were selected for a gene expression study. Reverse transcription-quantitative PCR (RT-qPCR) showed that in response to treatment with ZnO nanoparticles, the expression levels of two oxidative stress genes (katA and ahpC) and a general stress response gene (dnaK) were increased 52-, 7-, and 17-fold, respectively. These results suggest that the antibacterial mechanism of ZnO nanoparticles is most likely due to disruption of the cell membrane and oxidative stress in Campylobacter.

Investigating transcriptional regulation of Plasmodium falciparum upon drug perturbation

Gene regulation of the malaria parasite Plasmodium falciparum has proven to be complex with evidence supporting both transcriptional [1,2] and post-transcriptional level control [3,4]. Transcriptional profiling of environmentally perturbed malaria parasites can reveal functionally related genes with common regulatory mechanisms that are responsive to external stimuli [2]. By interrogating microarray data derived from P. falciparum populations treated with several drug classes, a subset of four genes potentially involved with the parasite's survival mechanisms following drug perturbation were identified, i.e. general stress response genes. In addition, data from two independent functional genomics investigations on the response of P. falciparum parasites to polyamine biosynthesis inhibitors (DFMO/MDL73811 co-inhibition [5] and cyclohexylamine [6]), revealed four genes of which the transcript abundance appear to be uniquely affected upon polyamine depletion only [5,6], i.e. perturbation-specific stress response genes. The regulation of this generalized versus perturbation-specific transcriptional responses was subsequently investigated. In order to investigate transcriptional regulation of P. falciparum, stable transfection methodology was established through the use of green fluorescent protein and luciferase as transgene expression markers [7]. Subsequently, the regions 1500 bp upstream of the coding sequence (presumably containing the promoters) of the eight genes were inserted into the transfection vector driving luciferase expression, with the heat shock protein 86 promoter as control. Parasite transfection was performed and transfectants were selected for resistance against the antifolate WR99210. Stable lines of the P. falciparum transfectants were exposed to a variety of compounds from different antimalarial drug classes including two polyamine biosynthesis inhibitors (i.e. artemisinin, mefloquine,doxycycline, DFMO and cyclohexylamine) and luciferase activity was quantified as a measure of promoter activity. The altered luciferase activity was used to determine whether the observed transcriptional response upon drug perturbation were indeed the result of true transcriptional control at the promoter level as opposed to alteration of mRNA stability or other post-transcriptional means.

Arachidonic Acid: An Evolutionarily Conserved Signaling Molecule Modulates Plant Stress Signaling Networks

Fatty acid structure affects cellular activities through changes in membrane lipid composition and the generation of a diversity of bioactive derivatives. Eicosapolyenoic acids are released into plants upon infection by oomycete pathogens, suggesting they may elicit plant defenses. We exploited transgenic Arabidopsis thaliana plants (designated EP) producing eicosadienoic, eicosatrienoic, and arachidonic acid (AA), aimed at mimicking pathogen release of these compounds. We also examined their effect on biotic stress resistance by challenging EP plants with fungal, oomycete, and bacterial pathogens and an insect pest. EP plants exhibited enhanced resistance to all biotic challenges, except they were more susceptible to bacteria than the wild type. Levels of jasmonic acid (JA) were elevated and levels of salicylic acid (SA) were reduced in EP plants. Altered expression of JA and SA pathway genes in EP plants shows that eicosapolyenoic acids effectively modulate stress-responsive transcriptional networks. Exogenous application of various fatty acids to wild-type and JA-deficient mutants confirmed AA as the signaling molecule. Moreover, AA treatment elicited heightened expression of general stress-responsive genes. Importantly, tomato (Solanum lycopersicum) leaves treated with AA exhibited reduced susceptibility to Botrytis cinerea infection, confirming AA signaling in other plants. These studies support the role of AA, an ancient metazoan signaling molecule, in eliciting plant stress and defense signaling networks.

Dynamic Transcriptional and Metabolic Responses in Yeast Adapting to Temperature Stress

Understanding the response processes in cellular systems to external perturbations is a central goal of large-scale molecular profiling experiments. We investigated the molecular response of yeast to increased and lowered temperatures relative to optimal reference conditions across two levels of molecular organization: the transcriptome using a whole yeast genome microarray and the metabolome applying the gas chromatography/mass spectrometry (GC/MS) technology with in vivo stable-isotope labeling for accurate relative quantification of a total of 50 different metabolites. The molecular adaptation of yeast to increased or lowered temperatures relative control conditions at both the metabolic and transcriptional level is dominated by temperature-inverted differential regulation patterns of transcriptional and metabolite responses and the temporal response observed to be biphasic. The set of previously described general environmental stress response (ESR) genes showed particularly strong temperature-inverted response patterns. Among the metabolites measured, trehalose was detected to respond strongest to the temperature stress and with temperature-inverted up- and downregulation relative to control, midtemperature conditions. Although associated with the same principal environmental parameter, the two different temperature regimes caused very distinct molecular response patterns at both the metabolite and the transcript level. While pairwise correlations between different transcripts and between different metabolites were found generally preserved under the various conditions, substantial differences were also observed indicative of changed underlying network architectures or modified regulatory relationships. Gene and associated gene functions were identified that are differentially regulated specifically under the gradual stress induction applied here compared to abrupt stress exposure investigated in previous studies, including genes of as of yet unidentified function and genes involved in protein synthesis and energy metabolism.

Identification of Components Associated with Thermal Acclimation of Photosystem II in Synechocystis sp. PCC6803

BackgroundPhotosystem II (PSII) is the most thermally sensitive component of photosynthesis. Thermal acclimation of this complex activity is likely to be critically important to the ability of photosynthetic organisms to tolerate temperature changes in the environment.Methodology/FindingsWe have analysed gene expression using whole-genome microarrays and monitored alterations in physiology during acclimation of PSII to elevated growth temperature in Synechocystis sp. PCC 6803. PSII acclimation is complete within 480 minutes of exposure to elevated temperature and is associated with a highly dynamic transcriptional response. 176 genes were identified and classified into seven distinct response profile groups. Response profiles suggest the existence of an early transient phase and a sustained phase to the acclimation response. The early phase was characterised by induction of general stress response genes, including heat shock proteins, which are likely to influence PSII thermal stability. The sustained phase consisted of acclimation-specific alterations that are involved in other cellular processes. Sustained responses included genes involved in phycobillisome structure and modification, photosynthesis, respiration, lipid metabolism and motility. Approximately 60% of genes with sustained altered expression levels have no known function. The potential role of differentially expressed genes in thermotolerance and acclimation is discussed. We have characterised the acclimation physiology of selected gene ‘knockouts’ to elucidate possible gene function in the response.Conclusions/SignificanceAll mutants show lower PSII rates under normal growth conditions. Basal PSII thermotolerance was affected by mutations in clpB1, cpcC2, hspA, htpG and slr1674. Final PSII thermotolerance was affected by mutations in cpcC2, hik34, hspA and hypA1, suggesting that these gene products play roles in long-term thermal acclimation of PSII.

Gene expression profiling of Dunaliella sp. acclimated to different salinities

SUMMARYTo investigate which genes may be important for growth under extreme conditions such as very low or high salinities, a survey of the Dunaliella sp. transcriptome was performed with a cDNA microarray which had been generated previously representing 778 expressed sequence tags. The comparative microarray analysis indicated that 142 genes differed in expression levels by more than twofold in cells grown at extreme salinities (0.08 M and 4.5 M NaCl) when compared with cells grown at intermediate salinity (1.5 M NaCl). Of these genes, 28 had increased expression and 57 were suppressed in cells grown at low salinity. In cells grown at high salinity, 43 genes showed increased expression and 69 genes showed suppressed expression. However, we did observe a large overlap in the expression of extreme salinity‐responsive genes based on Venn diagram analysis, which found 55 genes that responded to both of the two extreme salinity conditions. Further, we found that several genes had similar expression levels under low and high salinities, including some general stress response genes that were upregulated in both extreme salinity conditions. For confirmation of the validity of the cDNA microarray analysis, expression of several genes was independently confirmed by the use of gene‐specific primers and real‐time polymerase chain reaction. The present study is the first large‐scale comparative survey of the transcriptome from the microalga Dunaliella sp. acclimated to extreme salinities, thus providing a platform for further functional investigation of differentially expressed genes in Dunaliella.

A Transcriptional “Scream” Early Response of E. coli Prey to Predatory Invasion by Bdellovibrio

We have transcriptionally profiled the genes differentially expressed in E. coli prey cells when predatorily attacked by Bdellovibrio bacteriovorus just prior to prey cell killing. This is a brief, approximately 20–25 min period when the prey cell is still alive but contains a Bdellovibrio cell in its periplasm or attached to and penetrating its outer membrane. Total RNA was harvested and labelled 15 min after initiating a semi-synchronous infection with an excess of Bdellovibrio preying upon E. coli and hybridised to a macroarray spotted with all predicted ORFs of E. coli. SAM analysis and t-tests were performed on the resulting data and 126 E. coli genes were found to be significantly differentially regulated by the prey upon attack by Bdellovibrio. The results were confirmed by QRT-PCR. Amongst the prey genes upregulated were a variety of general stress response genes, potentially “selfish” genes within or near prophages and transposable elements, and genes responding to damage in the periplasm and osmotic stress. Essentially, the presence of the invading Bdellovibrio and the resulting damage to the prey cell elicited a small “transcriptional scream”, but seemingly no specific defensive mechanism with which to counter the Bdellovibrio attack. This supports other studies which do not find Bdellovibrio resistance responses in prey, and bodes well for its use as a “living antibiotic”.

Intracellular biology and virulence determinants ofFrancisella tularensisrevealed by transcriptional profiling inside macrophages

Summary The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen, whose virulence requires proliferation inside host cells, including macrophages. Here we have performed a global transcriptional profiling of the highly virulent F. tularensis ssp. tularensis Schu S4 strain during its intracellular cycle within primary murine macrophages, to characterize its intracellular biology and identify pathogenic determinants based on their intracellular expression profiles. Phagocytosed bacteria rapidly responded to their intracellular environment and subsequently altered their transcriptional profile. Differential gene expression profiles were revealed that correlated with specific intracellular locale of the bacteria. Upregulation of general and oxidative stress response genes was a hallmark of the early phagosomal and late endosomal stages, while induction of transport and metabolic genes characterized the cytosolic replication stage. Expression of the Francisella Pathogenicity Island (FPI) genes, which are required for intracellular proliferation, increased during the intracellular cycle. Similarly, 27 chromosomal loci encoding putative hypothetical, secreted, outer membrane proteins or transcriptional regulators were identified as upregulated. Among these, deletion of FTT0383, FTT0369c or FTT1676 abolished the ability of Schu S4 to survive or proliferate intracellularly and cause lethality in mice, therefore identifying novel determinants of Francisella virulence from their intracellular expression profile.