Fine-Tuning Gene Expression in Bacteria by Synthetic Promoters.

Promoters are key genetic elements in the initiation and regulation of gene expression. A limited number of natural promoters has been described for the control of gene expression in synthetic biology applications. Therefore, synthetic promoters have been developed to fine-tune the transcription for the desired amount of gene product. Mostly, synthetic promoters are characterized using promoter libraries that are constructed via mutagenesis of promoter sequences. The strength of promoters in the library is determined according to the expression of a reporter gene such as gfp encoding green fluorescent protein. Gene expression can be controlled using inducers. The majority of the studies on gram-negative bacteria are conducted using the expression system of the model organism Escherichia coli while that of the model organism Bacillus subtilis is mostly used in the studies on gram-positive bacteria. Additionally, synthetic promoters for the cyanobacteria, which are phototrophic microorganisms, are evaluated, especially using the model cyanobacterium Synechocystis sp. PCC 6803. Moreover, a variety of algorithms based on machine learning methods were developed to characterize the features of promoter elements. Some of these in silico models were verified using in vitro or in vivo experiments. Identification of novel synthetic promoters with improved features compared to natural ones contributes much to the synthetic biology approaches in terms of fine-tuning gene expression.

The regulatory role of Fur-encoding SCLAV_3199 in iron homeostasis in Streptomyces clavuligerus.

Iron homeostasis is strictly regulated by complex cascades connected with secondary metabolism in bacteria. Ferric uptake regulators ('Fur's), siderophores, efflux systems, and two-component signal transduction systems are the leading players in response stimuli. However, these regulatory mechanisms remain to be elucidated in Streptomyces clavuligerus. Our study focused on unraveling a possible role of SCLAV_3199 which encodes a Fur family transcriptional regulator, particularly in iron regulation and at the global level in this species. We deleted the SCLAV_3199 gene in S. clavuligerus and compared gene expression differences with the wild-type strain based on iron availability by RNA-seq. We found a potential regulatory effect of SCLAV_3199 on many transcriptional regulators and transporters. Besides, the genes encoding iron sulfur binding proteins were overexpressed in the mutant in the presence of iron. Notably, catechol (SCLAV_5397), and hydroxamate-type (SCLAV_1952, SCLAV_4680) siderophore-related genes were upregulated in the mutant strain in iron scarcity. Concomitantly, S. clavuligerus Δ3199 produced 1.65 and 1.9 times more catechol and hydroxamate-type siderophores, respectively, than that of the wild type strain under iron depletion. Iron containing chemically defined medium did not favor antibiotic production in S. clavuligerus Δ3199 while fermentation in starch-asparagine medium led to improved cephamycin C (2.23-fold) and clavulanic acid (2.56-fold) production in the mutant compared to the control. However, better tunicamycin yield (2.64-fold) was obtained in trypticase soy broth-grown cultures of S. clavuligerus Δ3199. Our findings demonstrate that the SCLAV_3199 gene plays a significant role in regulating both iron homeostasis and secondary metabolite biosynthesis in S. clavuligerus.

Whole genome analysis of Flavobacterium aziz-sancarii sp. nov., isolated from Ardley Island (Antarctica), revealed a rich resistome and bioremediation potential.

Despite being one of the most isolated regions in the world, Antarctica is at risk of increased contamination with potentially toxic elements and other toxic chemicals through anthropogenic interventions. In this study, a psychrotolerant bacterium was isolated using the lake water collected from Ardley Island (Antarctica), which can grow at temperatures between 4 and 30 °C and pH values between 6.0 and 9.0. The isolate, named AC, had protease, amylase, and lipase activities with no NaCl tolerance and could degrade 1-5% diesel fuel. Multilocus sequence analysis (MLSA) using 16S rRNA, gyrB, tuf, and rpoD genes resulted in 92.91-98.6% sequence similarities between the isolate AC and other Flavobacterium spp. Whole genome analysis indicated that the genome length of Flavobacterium sp. AC is 5.8 Mbp with a GC content of 34.04% and 1274 genes predicted. The strain AC branched independently from other Flavobacterium spp. in the phylogenetic and phylogenomic trees and ranked a new species named Flavobacterium aziz-sancarii. Genome mining identified several cold-inducible genes, including stress-associated genes such as cold-shock proteins, chaperones, carotenoid biosynthetic genes, or oxidative-stress response genes. In addition, virulence, gliding motility, and biofilm-related genes were determined. Its genome contains 35 and 88 open-reading frames related to potentially toxic element and antibiotic resistance, respectively. F. aziz-sancarii showed a remarkable tolerance of Cr and Ni, with minimal inhibitory concentration values of 2.88 and 2.81 mM, respectively. Pb, Cu, and Zn exposure resulted in moderate toxicity (2.14-2.41 mM), while Cd showed the highest inhibitory effect in bacterial growth (0.74 mM). Antibiotic susceptibility testing indicated multidrug-resistant phenotype in correlation to in silico prediction of antibiotic resistance genes. Overall, our results contribute to biodiversity of Antarctica and provide new insights into resistome profile of Antarctic microorganisms. Additionally, the diesel degradation feature of F. aziz-sancarii offers potential use for the bioremediation of hydrocarbon-contaminated polar ecosystems.

Characterization of highly stable extracellular lipase from the extremely halophilic archaeon Halolamina sp

Enzymes of the archaea living in extreme environments are resistant to the challenging conditions. Lipase is among the important enzymes used in the industry and agriculture. In this study, the extracellular lipase from extremely halophilic archaeon Halolamina sp. was characterized for the first time. Optimum temperature for the enzyme activity was determined as 70oC, optimum pH was 7.0, and the optimum salt concentration was 3.6 M. Additionally, more than 70% of the enzyme activity was remained between pH 3.0-10.0 for 48 h as well as incubation of the enzyme at 70oC for 30 min increased its activity for 44%, and no activity loss was observed after incubation at 80oC. Also, presence of the metals increased the enzyme activity up to 88%. The enzyme was highly resistant to the organic solvents acetone, methanol, and DMSO while strong inhibition was caused by n-butanol. Among the detergents, the enzyme kept its activity substantially in the presence of SDS; however, other detergents caused inhibition of the enzyme activity. This characterization study showed that the lipase from the haloarchaeon Halolamina sp. is highly stable at the wide ranges of temperature and pH values as well as in the presence of diverse inhibitors. This enzyme is promising to be used in biotechnological applications.

Recombinant AhpC antigen from Mycobacterium bovis boosts BCG-primed immunity in mice.

Tuberculosis (TB) is still one of the most common infectious diseases around the world despite the widespread use of BCG (bacille Calmette-Guerin) strain of Mycobacterium bovis as a vaccine. This vaccine does not always protect people from TB, and, thus, new effective vaccines or vaccination strategies are being investigated. In this study, alkyl hydroperoxide reductase (AhpC) from M. bovis was evaluated as a new candidate vaccine antigen against TB in BALB/c mice model. The ahpC gene was amplified from M.bovis genome, cloned, and expressed in Escherichia coli. Vaccine antigen AhpC was formulated with Montanide ISA 61 VG, an oil-based emulsion adjuvant. Both IgG and IL-12 responses were observed in mice after administering the formulation both as a subunit vaccine alone and also as a booster vaccine for BCG immunization. However, a long-lasting response was observed when AhpC formulation was used as a booster (for BCG-primed immunization) as compared to being used as a subunit vaccine alone. In short, these findings suggested that AhpC has the potential to be used as a booster vaccine candidate for BCG-primed immunization.

Dynamic proteomic analysis of Phanerochaete chrysosporium under copper stress.

The model white rot fungus Phanerochaete chrysosporium is frequently preferred for heavy metal accumulation studies due to its high resistance to heavy metals, including copper (Cu). Here, the response of P. chrysosporium under Cu stress at different time points was investigated for the first time by a detailed proteomic analysis using 2DE MALDI-TOF/MS and nanoLC-MS/MS techniques. A total of 123 Cu-responsive protein spots were determined using 2DE approach, and 104 of them were corresponded to 73 distinct open reading frames (ORFs). Of identified ones, 88 spots were over-, and 16 spots were underrepresented. The majority of these proteins showed to the strongest response at 8th h of Cu exposure. Using nanoLC-MS/MS analysis, a total of 167 differentially produced proteins were identified from Cu-exposed cultures after enrichment of the membrane proteins followed by SILAC. Seventy four, 66, and 69 overrepresented, and 56, 71, and 64 underrepresented proteins were identified at 2 h, 4 h, and 8 h of Cu exposure, respectively. The bioinformatic analysis of these proteins revealed that intracellular trafficking proteins such as Ran GTPase and a p24 family protein, and certain proteins involved in posttranslational modification, protein turnover and folding were Cu-responsive. Three important transcription factors (TFs), NAC, BTF3, and homeobox TFs, 40S and 60S ribosomal proteins, chaperones such as Hsp26/Hsp42 and mortalin, as well as 20S proteasome, 14-3-3 proteins and Hsp90 involve in Cu-stress response of P. chrysosporium. Moreover, certain elements of translation machinery, the proteins related with aspartate, methionine, and pyruvate metabolisms, transketolase, and trehalase related with carbohydrate metabolism, citrate synthase, fumarase, V-ATPase, and F0F1-type ATPase playing role in energy production and conversion, transport proteins such as multidrug resistance and p24 family proteins as well as actin-related proteins involved in cytoskeleton remodeling were determined to be Cu-responsive. The present proteome analysis revealed that P. chrysosporium mainly regulates translational and posttranslational processes, certain transport processes, many metabolic pathways and cytoskeleton to overcome the Cu-induced oxidative stress.

Single-Molecule Characterization of Drug Delivery Systems.

Delivery of the drug to a desired point of body and controlled release of the therapeutic agent are important features, provided by drug delivery systems (DDSs), for development of today's effective medicines. A variety of nanomaterials or nanomolecules such as lipids/liposomes, nucleic acids, peptides/proteins, composites, polymers, or carbon nanotubes can be used as DDSs. Single-molecule characterization of these small materials in terms of their size, shape, surface, encapsulation efficiency, as well as interaction with the drug-receiving cell has importance for their efficiency. The loading, distribution, or leakage of the drug as well as its interaction with DDS should also be characterized. Although diverse techniques are present for characterization of specific DDS material, methods such as electron microscopy and fluorescence microscopy are widely used. In this review, the current methodologies utilized for the single-molecule characterization of mostly preferred DDS materials were presented.

Overexpression of Chitinase A Gene from Serratia marcescens in Bacillus subtilis and Characterization of Enhanced Chitinolytic Activity

Abstract Chitinase enzymes possess various usages in agriculture, biotechnology and medicine due to their chitin degrading property. Thus, efficient production of chitinase enzymes with desired properties has importance for its use. In this study, chitinase A (chiA) gene from Serratia marcescens Bn10 was cloned and heterologously overexpressed using pHT43 vector in Bacillus subtilis 168. The recombinant chitinase was characterized in terms of temperature, pH, and various effectors. The extracellular chitinase activity in recombinant B. subtilis was found 2.15-fold higher than the parental strain after 2 h of IPTG induction. Optimum temperature and pH for the extracellular chitinase activity in the recombinant B. subtilis were determined as 60 oC and pH 9.0, respectively. NaCl, Ca2+, Mn2+, Cu2+, Zn2+, sodium dodecyl sulfate (SDS), Tween-20, and ethanol increased the chitinase activity whereas Mg2+ caused an inhibition. The most notable increment on the chitinase activity was provided by Zn2+ (3.2 folds) and then by SDS (2.9 folds). The chitinase, overproduced by the recombinant B. subtilis 168 heterologously expressing chiA, was determined to have optimum activity at high temperature and alkaline conditions as well as various effectors increase its activity. The extracellular chitinase of recombinant B. subtilis might be a promising source for agricultural, biotechnological and medical applications.

Immune responses elicited by the recombinant Erp, HspR, LppX, MmaA4, and OmpA proteins from Mycobacterium tuberculosis in mice.

Immunogenic potency of the recombinant Erp, HspR, LppX, MmaA4, and OmpA proteins from Mycobacterium tuberculosis (MTB), formulated with Montanide ISA 720 VG adjuvant, was evaluated in BALB/c mice for the first time in this study. The five vaccine formulations, adjuvant, and BCG vaccine were subcutaneously injected into mice, and the sera were collected at days 0, 15, 30, 41, and 66. The humoral and cellular immune responses against vaccine formulations were determined by measuring serum IgG and serum interferon-gamma (IFN-γ) and interleukin-12 (IL-12) levels, respectively. All formulations significantly increased IgG levels post-vaccination. The highest increase in IFN-γ level was provided by MmaA4 formulation. The Erp, HspR, and LppX formulations were as effective as BCG in enhancement of IFN-γ level. The most efficient vaccine boosting the IL-12 level was HspR formulation, especially at day 66. Erp formulation also increased the IL-12 level more than BCG at days 15 and 30. The IL-12 level boosted by MmaA4 formulation was found to be similar to that by BCG. OmpA formulation was inefficient in enhancement of cellular immune responses. This study showed that MmaA4, HspR, and Erp proteins from MTB are successful in eliciting both humoral and cellular immune responses in mice.

Phenolic Profile, Essential Oil Composition and Bioactivity of Lasia spinosa (L) Thwaites

Abstract Lasia spinosa (L.) Thwaites is a widely used ethnomedicinal plant in Bangladesh. In this study, we investigated phenolic contents, volatile compounds and fatty acids, and essential oil components of extracts prepared from aerial parts of the plant. The main volatile compounds were methyl ester of oleic acid, palmitic acid and stearic acid as determined by GC/MS. Phenolic contents of the extracts were determined qualitatively and quantitatively by HPLC/TOF-MS. Six phenolic compounds (syringic acid, morin, gentistic acid, 4-hydroxybenzoic acid, cinnamic acid, and apigenin) were found in the extracts. GC/MS analysis of steam distilled essential oil showed camphor, α-pinene and δ-3-carene as the main constituents. In DPPH radical scavenging assay, the highest free radical scavenging activity was observed for the methanol extract with an IC50 value of 0.48 ± 0.04 mg/mL, whereas, in metal chelating activity on ferrous ions (Fe2+) assay, the highest chelating activity was observed for hexane extract (IC50 = 0.55 ± 0.08 mg/mL). The extracts and essential oil were tested against five severe human pathogenic bacteria using disc diffusion assay and subsequent MIC values were also determined. All the extracts (except methanol extract) and the essential oil were found to possess potential antimicrobial activity with corresponding inhibition zone and minimum inhibitory concentration (MIC) ranging from 9-23 mm and 62.5-500 µg/mL. This study has been explored the plant Lasia spinosa can be seen as a potential source of biologically active compounds.

Virtual Screening Approach of Bacterial Peptide Deformylase Inhibitors Results in New Antibiotics.

The increasing resistance of bacteria to antibacterial therapy poses an enormous health problem, it renders the development of new antibacterial agents with novel mechanism of action an urgent need. Peptide deformylase, a metalloenzyme which catalytically removes N-formyl group from N-terminal methionine of newly synthesized polypeptides, is an important target in antibacterial drug discovery. In this study, we report the structure-based virtual screening of ZINC database in order to discover potential hits as bacterial peptide deformylase enzyme inhibitors with more affinity as compared to GSK1322322, previously known inhibitor. After virtual screening, fifteen compounds of the top hits predicted were purchased and evaluated in vitro for their antibacterial activities against one Gram positive (Staphylococcus aureus) and three Gram negative (Escherichia coli, Pseudomonas aeruginosa and Klebsiella. pneumoniae) bacteria in different concentrations by disc diffusion method. Out of these, three compounds, ZINC00039650, ZINC03872971 and ZINC00126407, exhibited significant zone of inhibition. The results obtained were confirmed using the dilution method. Thus, these proposed compounds may aid the development of more efficient antibacterial agents.

Global transcriptome analysis of Halolamina sp. to decipher the salt tolerance in extremely halophilic archaea.

Extremely halophilic archaea survive in the hypersaline environments such as salt lakes or salt mines. Therefore, these microorganisms are good sources to investigate the molecular mechanisms underlying the tolerance to high salt concentrations. In this study, a global transcriptome analysis was conducted in an extremely halophilic archaeon, Halolamina sp. YKT1, isolated from a salt mine in Turkey. A comparative RNA-seq analysis was performed using YKT1 isolate grown either at 2.7M NaCl or 5.5M NaCl concentrations. A total of 2149 genes were predicted to be up-regulated and 1638 genes were down-regulated in the presence of 5.5M NaCl. The salt tolerance of Halolamina sp. YKT1 involves the up-regulation of genes related with membrane transporters, CRISPR-Cas systems, osmoprotectant solutes, oxidative stress proteins, and iron metabolism. On the other hand, the genes encoding the proteins involved in DNA replication, transcription, translation, mismatch and nucleotide excision repair were down-regulated. The RNA-seq data were verified for seven up-regulated genes as well as six down-regulated genes via qRT-PCR analysis. This comprehensive transcriptome analysis showed that the halophilic archaeon canalizes its energy towards keeping the intracellular osmotic balance minimizing the production of nucleic acids and peptides.

Functional Characterization of 4'OMT and 7OMT Genes in BIA Biosynthesis.

Alkaloids are diverse group of secondary metabolites generally found in plants. Opium poppy (Papaver somniferum L.), the only commercial source of morphinan alkaloids, has been used as a medicinal plant since ancient times. It produces benzylisoquinoline alkaloids (BIA) including the narcotic analgesic morphine, the muscle relaxant papaverine, and the anti-cancer agent noscapine. Though BIAs play crucial roles in many biological mechanisms their steps in biosynthesis and the responsible genes remain to be revealed. In this study, expressions of 3-hydroxy-N-methylcoclaurine 4'-methyltransferase (4'OMT) and reticuline 7-O-methyltransferase (7OMT) genes were subjected to manipulation to functionally characterize their roles in BIA biosynthesis. Measurements of alkaloid accumulation were performed in leaf, stem, and capsule tissues accordingly. Suppression of 4'OMT expression caused reduction in the total alkaloid content in stem tissue whereas total alkaloid content was significantly induced in the capsule. Silencing of the 7OMT gene also caused repression in total alkaloid content in the stem. On the other hand, over-expression of 4'OMT and 7OMT resulted in higher morphine accumulation in the stem but suppressed amount in the capsule. Moreover, differential expression in several BIA synthesis genes (CNMT, TYDC, 6OMT, SAT, COR, 4'OMT, and 7OMT) were observed upon manipulation of 4'OMT and 7OMT expression. Upon silencing and overexpression applications, tissue specific effects of these genes were identified. Manipulation of 4'OMT and 7OMT genes caused differentiated accumulation of BIAs including morphine and noscapine in capsule and stem tissues.

Comparative genome analysis of five Pasteurella multocida strains to decipher the diversification in pathogenicity and host specialization.

Pasteurella multocida is a Gram-negative bacterial pathogen causing economically important diseases in distinct animal species. Complete genome sequences of five P. multocida strains (Pm70, HB03, HN06, 3480, and 36950) isolated from poultry, swine or bovine, were retrieved from the GenBank database and compared with each other, for the first time. The missense mutations generating a dissimilar amino acid in the peptide chain, nonsense mutations, and insertion/deletions in the nucleotide sequence were identified due to the potential change in the protein function. A total of 500 putative mutant proteins were identified, and categorized into 10 groups including cellular compartments such as outer membrane, capsule and fimbria, and processes such as carbohydrate, energy, nucleic acid and amino acid metabolisms, transport, and drug resistance. The majority of the mutant proteins were associated with the outer compartments of the bacterial cell. Various mutations were also detected in the genes related with biosynthetic pathways. The highest and the lowest numbers of mutant proteins belonged to 36950 vs. HN06 and Pm70 vs. HB03 comparisons, respectively. The major impact on the diversification of P. multocida strains was observed to be conferred by the mutations related with pathogenicity. To exhibit the outcomes of the mutations in the peptide chains, three sample amino acid sequences belonging to AfuA, MetB, and d,d-heptose 1,7-bisphosphate phosphatase were aligned, and their phylogenetic relationships were shown. These comprehensive analyses improve the understanding of molecular pathogenicity and host specialization of P. multocida, and would have a contribution to the recombinant vaccine development against this pathogen.

Genome-wide fungal stress responsive miRNA expression in wheat.

MicroRNAs (miRNAs) are small non-coding class of RNAs. They were identified in many plants with their diverse regulatory roles in several cellular and metabolic processes. A number of miRNAs were involved in biotic and abiotic stress responses. Here, fungal stress responsive wheat miRNAs were analyzed by using miRNA-microarray strategy. Two different fungi (Fusarium culmorum and Bipolaris sorokiniana) were inoculated on resistant and sensitive wheat cultivars. A total of 87 differentially regulated miRNAs were detected in the 8 × 15 K array including all of the available plant miRNAs. Using bioinformatics tools, the target transcripts of responsive miRNAs were predicted, and related biological processes and mechanisms were assessed. A number of the miRNAs such as miR2592s, miR869.1, miR169b were highly differentially regulated showing more than 200-fold change upon fungal-inoculation. Some of the miRNAs were identified as fungal-inoculation responsive for the first time. The analyses showed that some of the differentially regulated miRNAs targeted resistance-related genes such as LRR, glucuronosyl transferase, peroxidase and Pto kinase. The comparison of the two miRNA-microarray analyses indicated that fungal-responsive wheat miRNAs were differentially regulated in pathogen- and cultivar-specific manners.

Transcriptome-wide identification of bread wheat WRKY transcription factors in response to drought stress.

The WRKY superfamily of transcription factors was shown to be involved in biotic and abiotic stress responses in plants such as wheat (Triticum aestivum L.), one of the major crops largely cultivated and consumed all over the world. Drought is an important abiotic stress resulting in a considerable amount of loss in agronomical yield. Therefore, identification of drought responsive WRKY members in wheat has a profound significance. Here, a total of 160 TaWRKY proteins were characterized according to sequence similarity, motif varieties, and their phylogenetic relationships. The conserved sequences of the TaWRKYs were aligned and classified into three main groups and five subgroups. A novel motif in wheat, WRKYGQR, was identified. To putatively determine the drought responsive TaWRKY members, publicly available RNA-Seq data were analyzed for the first time in this study. Through in silico searches, 35 transcripts were detected having an identity to ten known TaWRKY genes. Furthermore, relative expression levels of TaWRKY16/TaWRKY16-A, TaWRKY17, TaWRKY19-C, TaWRKY24, TaWRKY59, TaWRKY61, and TaWRKY82 were measured in root and leaf tissues of drought-tolerant Sivas 111/33 and susceptible Atay 85 cultivars. All of the quantified TaWRKY transcripts were found to be up-regulated in root tissue of Sivas 111/33. Differential expression of TaWRKY16, TaWRKY24, TaWRKY59, TaWRKY61 and TaWRKY82 genes was discovered for the first time upon drought stress in wheat. These comprehensive analyses bestow a better understanding about the WRKY TFs in bread wheat under water deficit, and increased number of drought responsive WRKYs would contribute to the molecular breeding of tolerant wheat cultivars.

Boron stress responsive microRNAs and their targets in barley.

Boron stress is an environmental factor affecting plant development and production. Recently, microRNAs (miRNAs) have been found to be involved in several plant processes such as growth regulation and stress responses. In this study, miRNAs associated with boron stress were identified and characterized in barley. miRNA profiles were also comparatively analyzed between root and leave samples. A total of 31 known and 3 new miRNAs were identified in barley; 25 of them were found to respond to boron treatment. Several miRNAs were expressed in a tissue specific manner; for example, miR156d, miR171a, miR397, and miR444a were only detected in leaves. Additionally, a total of 934 barley transcripts were found to be specifically targeted and degraded by miRNAs. In silico analysis of miRNA target genes demonstrated that many miRNA targets are conserved transcription factors such as Squamosa promoter-binding protein, Auxin response factor (ARF), and the MYB transcription factor family. A majority of these targets were responsible for plant growth and response to environmental changes. We also propose that some of the miRNAs in barley such as miRNA408 might play critical roles against boron exposure. In conclusion, barley may use several pathways and cellular processes targeted by miRNAs to cope with boron stress.

Nutrition metabolism plays an important role in the alternate bearing of the olive tree (Olea europaea L.).

The olive tree (Olea europaea L.) is widely known for its strong tendency for alternate bearing, which severely affects the fruit yield from year to year. Microarray based gene expression analysis using RNA from olive samples (on-off years leaves and ripe-unripe fruits) are particularly useful to understand the molecular mechanisms influencing the periodicity in the olive tree. Thus, we carried out genome wide transcriptome analyses involving different organs and temporal stages of the olive tree using the NimbleGen Array containing 136,628 oligonucleotide probe sets. Cluster analyses of the genes showed that cDNAs originated from different organs could be sorted into separate groups. The nutritional control had a particularly remarkable impact on the alternate bearing of olive, as shown by the differential expression of transcripts under different temporal phases and organs. Additionally, hormonal control and flowering processes also played important roles in this phenomenon. Our analyses provide further insights into the transcript changes between "on year" and "off year" leaves along with the changes from unrpipe to ripe fruits, which shed light on the molecular mechanisms underlying the olive tree alternate bearing. These findings have important implications for the breeding and agriculture of the olive tree and other crops showing periodicity. To our knowledge, this is the first study reporting the development and use of an olive array to document the gene expression profiling associated with the alternate bearing in olive tree.