Transcriptional landscape of small non-coding RNAs reveals diversity of categories and functions in molluscs.

Small non-coding RNAs (sncRNAs) are non-coding RNA molecules that play various roles in metazoans. Among the sncRNAs, microRNAs (miRNAs) guide post-translational gene regulation during cellular development, proliferation, apoptosis, and differentiation, while PIWI-interacting RNAs (piRNAs) suppress transposon activity to safeguard the genome from detrimental insertion mutagenesis. While an increasing number of piRNAs are being identified in the soma and germlines of various organisms, they are scarcely reported in molluscs. To unravel the small RNA (sRNA) expression patterns and genomic function in molluscs, we generated a comprehensive sRNA dataset by sRNA sequencing (sRNA-seq) of eight mollusc species. Abundant miRNAs were identified and characterized in all investigated molluscs, and ubiquitous piRNAs were discovered in both somatic and gonadal tissues in six of the investigated molluscs, which are more closely associated with transposon silencing. Tens of piRNA clusters were also identified based on the genomic mapping results, which varied among different tissues and species. Our dataset serves as important reference data for future genomic and genetic studies on sRNAs in these molluscs and related species, especially in elucidating the ancestral state of piRNAs in bilaterians.

In Silico Screening of Bacteriocin Gene Clusters within a Set of Marine Bacillota Genomes.

Due to their potential application as an alternative to antibiotics, bacteriocins, which are ribosomally synthesized antimicrobial peptides produced by bacteria, have received much attention in recent years. To identify bacteriocins within marine bacteria, most of the studies employed a culture-based method, which is more time-consuming than the in silico approach. For that, the aim of this study was to identify potential bacteriocin gene clusters and their potential producers in 51 marine Bacillota (formerly Firmicutes) genomes, using BAGEL4, a bacteriocin genome mining tool. As a result, we found out that a majority of selected Bacillota (60.78%) are potential bacteriocin producers, and we identified 77 bacteriocin gene clusters, most of which belong to class I bacteriocins known as RiPPs (ribosomally synthesized and post-translationally modified peptides). The identified putative bacteriocin gene clusters are an attractive target for further in vitro research, such as the production of bacteriocins using a heterologous expression system.

Pilot study of a comprehensive resource estimation method from environmental DNA using universal D-loop amplification primers.

Many studies have investigated the ability of environmental DNA (eDNA) to identify the species. However, when individual species are to be identified, accurate estimation of their abundance using traditional eDNA analyses is still difficult. We previously developed a novel analytical method called HaCeD-Seq (haplotype count from eDNA by sequencing), which focuses on the mitochondrial D-loop sequence for eels and tuna. In this study, universal D-loop primers were designed to enable the comprehensive detection of multiple fish species by a single sequence. To sequence the full-length D-loop with high accuracy, we performed nanopore sequencing with unique molecular identifiers (UMI). In addition, to determine the D-loop reference sequence, whole genome sequencing was performed with thin coverage, and complete mitochondrial genomes were determined. We developed a UMI-based Nanopore D-loop sequencing analysis pipeline and released it as open-source software. We detected 5 out of 15 species (33%) and 10 haplotypes out of 35 individuals (29%) among the detected species. This study demonstrates the possibility of comprehensively obtaining information related to population size from eDNA. In the future, this method can be used to improve the accuracy of fish resource estimation, which is currently highly dependent on fishing catches.

Zebrafish Danio rerio myotomal muscle structure and growth from a spatial transcriptomics perspective.

Fish exhibit different muscle structures and growth characteristics compared with mammals. We used a spatial transcriptomics approach and examined myotomal muscle sections from zebrafish. Adult muscles were divided into eight regions according to spatial gene expression characteristics. Slow muscle was located in the wedge-shaped region near the lateral line and at the base of the dorsal fin, intermediate muscle was located in a ribbon-shaped region adjacent to slow muscle, and fast muscle was located in the deep region of the trunk, surrounded by intermediate muscle; the interior of fast muscle was further divided into 6 parts by their transcriptomic features. Combined analysis of adult and larval data revealed that adult muscles contain specific regions similar to larval muscles. These regions showed active myogenesis and a high expression of genes associated with muscle hyperplasia. This is the first study to apply spatial transcriptomics to fish myotomal muscle structure and growth.

Induction of endoplasmic reticulum stress markers in an acromegaly model.

Acromegaly is a growth hormone (GH) excess pathological condition in humans. Acromegaly is associated with somatic disfigurement and a wide range of systemic manifestations such as arthritis, neuropathy, carpal tunnel syndrome, reproductive disorders, metabolic disorders, and gastrointestinal complications. The influence of excess GH on the cellular level could aid in understanding the root causes of acromegaly-related health complications. Previously, we found that GH excess induces DNA damage to somatic cells and reduces the stem cells number and causes premature aging. In this study, an in-depth analysis of the acromegaly RNAseq data revealed the disruption of important biological cellular processes. Gene set enrichment analysis, heatmap, and enrichment analysis of acromegaly RNAseq data revealed induction of endoplasmic reticulum (ER) stress markers in various organs. Interestingly, the induction of ER stress was even more apparent than in aged zebrafish. Splicing of box-binding protein-1 (XBP1) mRNA is a hallmark of ER stress. Therefore, we quantified spliced XBP1 mRNA in different organs of our acromegaly model. Thus, our study emphasizes the importance of ER stress in GH oversecretion, which is important for understanding the health complications of acromegaly.

A model explaining mRNA level fluctuations based on activity demands and RNA age.

Cellular RNA levels typically fluctuate and are influenced by different transcription rates and RNA degradation rates. However, the understanding of the fundamental relationships between RNA abundance, environmental stimuli, RNA activities, and RNA age distributions is incomplete. Furthermore, the rates of RNA degradation and transcription are difficult to measure in transcriptomic experiments in living organisms, especially in studies involving humans. A model based on activity demands and RNA age was developed to explore the mechanisms of RNA level fluctuations. Using single-cell time-series gene expression experimental data, we assessed the transcription rates, RNA degradation rates, RNA life spans, RNA demand, accumulated transcription levels, and accumulated RNA degradation levels. This model could also predict RNA levels under simulation backgrounds, such as stimuli that induce regular oscillations in RNA abundance, stable RNA levels over time that result from long-term shortage of total RNA activity or from uncontrollable transcription, and relationships between RNA/protein levels and metabolic rates. This information contributes to existing knowledge.

Environmental DNA study on aquatic ecosystem monitoring and management: Recent advances and prospects.

Environmental DNA (eDNA) is organismal DNA that can be detected in the environment and is derived from cellular material of organisms shed into aquatic or terrestrial environments. It can be sampled and monitored using molecular methods, which is important for the early detection of invasive and native species as well as the discovery of rare and cryptic species. While few reviews have summarized the latest findings on eDNA for most aquatic animal categories in the aquatic ecosystem, especially for aquatic eDNA processing and application. In the present review, we first performed a bibliometric network analysis of eDNA studies on aquatic animals. Subsequently, we summarized the abiotic and biotic factors affecting aquatic eDNA occurrence. We also systematically discussed the relevant experiments and analyses of aquatic eDNA from various aquatic organisms, including fish, molluscans, crustaceans, amphibians, and reptiles. Subsequently, we discussed the major achievements of eDNA application in studies on the aquatic ecosystem and environment. The application of eDNA will provide an entirely new paradigm for biodiversity conservation, environment monitoring, and aquatic species management at a global scale.

Physiological RNA dynamics in RNA-Seq analysis.

Physiological RNA dynamics cause problems in transcriptome analysis. Physiological RNA accumulation affects the analysis of RNA quantification, and physiological RNA degradation affects the analysis of the RNA sequence length, feature site and quantification. In the present article, we review the effects of physiological degradation and accumulation of RNA on analysing RNA sequencing data. Physiological RNA accumulation and degradation probably led to such phenomena as incorrect estimations of transcription quantification, differential expressions, co-expressions, RNA decay rates, alternative splicing, boundaries of transcription, novel genes, new single-nucleotide polymorphisms, small RNAs and gene fusion. Thus, the transcriptomic data obtained up to date warrant further scrutiny. New and improved techniques and bioinformatics software are needed to produce accurate data in transcriptome research.

Discovery and functional understanding of MiRNAs in molluscs: a genome-wide profiling approach.

Small non-coding RNAs play a pivotal role in gene regulation, repression of transposable element and viral activity in various organisms. Among the various categories of these small non-coding RNAs, microRNAs (miRNAs) guide post-translational gene regulation in cellular development, proliferation, apoptosis, oncogenesis, and differentiation. Here, we performed a genome-wide computational prediction of miRNAs to improve the understanding of miRNA observation and function in molluscs. As an initial step, hundreds of conserved miRNAs were predicted in 35 species of molluscs through genome scanning. Afterwards, the miRNAs' population, isoforms, organization, and function were characterized in detail. Furthermore, the key miRNA biogenesis factors, including AGO2, DGCR8, DICER, DROSHA, TRABP2, RAN, and XPO5, were elucidated based on homologue sequence searching. We also summarized the miRNAs' function in biomineralization, immune and stress response, as well as growth and development in molluscs. Because miRNAs play a vital role in various lifeforms, this study will provide insight into miRNA biogenesis and function in molluscs, as well as other invertebrates.

A Review of Discovery Profiling of PIWI-Interacting RNAs and Their Diverse Functions in Metazoans.

PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs (sncRNAs) that perform crucial biological functions in metazoans and defend against transposable elements (TEs) in germ lines. Recently, ubiquitously expressed piRNAs were discovered in soma and germ lines using small RNA sequencing (sRNA-seq) in humans and animals, providing new insights into the diverse functions of piRNAs. However, the role of piRNAs has not yet been fully elucidated, and sRNA-seq studies continue to reveal different piRNA activities in the genome. In this review, we summarize a set of simplified processes for piRNA analysis in order to provide a useful guide for researchers to perform piRNA research suitable for their study objectives. These processes can help expand the functional research on piRNAs from previously reported sRNA-seq results in metazoans. Ubiquitously expressed piRNAs have been discovered in the soma and germ lines in Annelida, Cnidaria, Echinodermata, Crustacea, Arthropoda, and Mollusca, but they are limited to germ lines in Chordata. The roles of piRNAs in TE silencing, gene expression regulation, epigenetic regulation, embryonic development, immune response, and associated diseases will continue to be discovered via sRNA-seq.

Gene expression profiles at different stages for formation of pearl sac and pearl in the pearl oyster Pinctada fucata.

BACKGROUND: The most critical step in the pearl formation during aquaculture is issued to the proliferation and differentiation of outer epithelial cells of mantle graft into pearl sac. This pearl sac secretes various matrix proteins to produce pearls by a complex physiological process which has not been well-understood yet. Here, we aimed to unravel the genes involved in the development of pearl sac and pearl, and the sequential expression patterns of different shell matrix proteins secreted from the pearl sac during pearl formation by pearl oyster Pinctada fucata using high-throughput transcriptome profiling. RESULTS: Principal component analysis (PCA) showed clearly different gene expression profiles between earlier (before 1 week) and later stages (1 week to 3 months) of grafting. Immune-related genes were highly expressed between 0 h - 24 h (donor dependent) and 48 h - 1 w (host dependent), and in the course of wound healing process pearl sac was developed by two weeks of graft transplantation. Moreover, for the first time, we identified some stem cell marker genes including ABCG2, SOX2, MEF2A, HES1, MET, NRP1, ESR1, STAT6, PAX2, FZD1 and PROM1 that were expressed differentially during the formation of pearl sac. The expression profiling of 192 biomineralization-related genes demonstrated that most of the shell matrix proteins (SMPs) involved in prismatic layer formation were first up-regulated and then gradually down-regulated indicating their involvement in the development of pearl sac and the onset of pearl mineralization. Most of the nacreous layer forming SMPs were up-regulated at 2 weeks after the maturation of pearl sac. Nacrein, MSI7 and shematrin involved in both layer formation were highly expressed during 0 h - 24 h, down-regulated up to 1 week and then up-regulated again after accomplishment of pearl sac formation. CONCLUSIONS: Using an RNA-seq approach we unraveled the expression pattern of the key genes involved in the development of pearl sac and pearl as a result of host immune response after grafting. These findings provide valuable information in understanding the molecular mechanism of pearl formation and immune response in P. fucata.

Changes in free amino acid concentrations and associated gene expression profiles in the abdominal muscle of kuruma shrimp (Marsupenaeus japonicus) acclimated at different salinities.

Shrimps inhabiting coastal waters can survive in a wide range of salinity. However, the molecular mechanisms involved in their acclimation to different environmental salinities have remained largely unknown. In the present study, we acclimated kuruma shrimp (Marsupenaeus japonicus) at 1.7%, 3.4% and 4.0% salinities. After acclimating for 6, 12, 24 and 72 h, we determined free amino acid concentrations in their abdominal muscle, and performed RNA sequencing analysis on this muscle. The concentrations of free amino acids were clearly altered depending on salinity after 24 h of acclimation. Glutamine and alanine concentrations were markedly increased following the increase of salinity. In association with such changes, many genes related to amino acid metabolism changed their expression levels. In particular, the increase of the expression level of the gene encoding glutamate-ammonia ligase, which functions in glutamine metabolism, appeared to be associated with the increased glutamine concentration at high salinity. Furthermore, the increased alanine concentration at high salinity was likely associated with the decrease in the expression levels of the the gene encoding alanine-glyoxylate transaminase. Thus, there is a possibility that changes in the concentration of free amino acids for osmoregulation in kuruma shrimp are regulated by changes in the expression levels of genes related to amino acid metabolism.

Lactomycins A-C, Dephosphorylated Phoslactomycin Derivatives that Inhibit Cathepsin B, from the Marine-derived Streptomyces sp. ACT232.

Three new polyketides, lactomycins A (1)-C (3), were isolated from the culture broth of a marine-derived Streptomyces sp. ACT232 as cathepsin B inhibitors. Their structures were determined by a combination of NMR and MS data analyses to be the dephosphorylated derivatives of a phoslactomycin class of metabolites. Lactomycins exhibited cathepsin B inhibitory activity (IC50 0.8 to 4.5 µg/mL). Even though the biosynthetic gene clusters found in the genome of the current strain have high similarity to those of phoslactomycin, neither phoslactomycins nor leustroducsins were detected by LC-MS analyses of the crude extract.

Promoter analysis of the fish gene of slow/cardiac-type myosin heavy chain implicated in specification of muscle fiber types.

Vertebrate skeletal muscles consist of heterogeneous tissues containing various types of muscle fibers, where specification of the fiber type is crucial for muscle development. Fish are an attractive experimental model to study the mechanisms of such fiber type specification because of the separated localization of slow and fast muscles in the trunk myotome. We examined regulation of expression of the torafugu gene of slow/cardiac-type myosin heavy chain, MYH M5 , and isolated an operational promoter in order to force its tissue-specific expression across different fish species via the transgenic approach in zebrafish and medaka. This promoter activity was observed in adaxial cell-derived superficial slow muscle fibers under the control of a hedgehog signal. We also uncovered coordinated expression of MYH M5 and Sox6b, which is an important transcriptional repressor for specification of muscle fiber types and participates in hedgehog signaling. Sequence comparison in the 5'-flanking region identified three conserved regions, CSR1-CSR3, between torafugu MYH M5 and its zebrafish ortholog. Analysis of deletion mutants showed that CSR1 significantly stimulates gene expression in slow muscle fibers. In contrast, deletion of CSR3 resulted in ectopic expression of a reporter gene in fast muscle fibers. CSR3 was found to contain a putative Sox family protein-binding site. These results indicate that the dual mechanism causing inhibition in fast muscle fibers and activation in slow muscle fibers is essential for slow muscle fiber-specific gene expression in fish.

Deep sequencing, profiling and detailed annotation of microRNAs in Takifugu rubripes.

BACKGROUND: microRNAs (miRNAs) in fish have not been as extensively studied as those in mammals. The fish species Takifugu rubripes is an intensively studied model organism whose genome has been sequenced. The T. rubripes genome is approximately eight times smaller than the human genome, but has a similar repertoire of protein-coding genes. Therefore, it is useful for identifying non-coding genes, including miRNA genes. To identify miRNA expression patterns in different organs of T. rubripes and give fundamental information to aid understanding of miRNA populations in this species, we extracted small RNAs from tissues and performed deep sequencing analysis to profile T. rubripes miRNAs. These data will be of assistance in functional studies of miRNAs in T. rubripes. RESULTS: After analyzing a total of 139 million reads, we found miRNA species in nine tissues (fast and slow muscles, heart, eye, brain, intestine, liver, ovaries, and testes). We identified 1420 known miRNAs, many of which were strongly expressed in certain tissues with expression patterns similar to those described for other animals in previous reports. Most miRNAs were expressed in tissues other than the ovaries or testes. However, some miRNA families were highly abundant in the gonads, but expressed only at low levels in somatic tissue, suggesting specific function in germ cells. The most abundant isomiRs (miRNA variants) of many miRNAs had identical sequences in the 5' region. However, isomiRs of some miRNAs, including fru-miR-462-5p, varied in the 5' region in some tissues, suggesting that they may target different mRNA transcripts. Longer small RNAs (26-31 nt), which were abundant in the gonads, may be putative piRNAs because of their length and their origin from repetitive elements. Additionally, our data include possible novel classes of small RNAs. CONCLUSIONS: We elucidated miRNA expression patterns in various organs of T. rubripes. Most miRNA sequences are conserved in vertebrates, indicating that the basic functions of vertebrate miRNAs share a common evolution. Some miRNA species exhibit different distributions of isomiRs between tissues, suggesting that they have a broad range of functions.

Characterization of the torafugu (Takifugu rubripes) immunoglobulin heavy chain gene locus.

In this study, we investigated the immunoglobulin heavy (IGH) gene locus of torafugu (Takifugu rubripes) from publicly available assembly sequences and presented an annotated locus map, including the IGHV genes, pseudogenes, and IGHC genes. Three new IGHV gene families (IGHV3-IGHV5) were discovered. We observed the interspersion of IGHV1 and IGHV2 family members and that they often intermingled with each other, while other family members were further interspersed. Conservation of the promoter and recombination signal sequences (RSS) was observed in a family-specific manner. In addition to known variable region genes present on chromosome 5 (current torafugu genome assembly), we found 34 additional IGHV genes on scaffold 287 and three novel potentially functional IGHD genes on scaffold 483. In total, the variable region of the torafugu IGH locus consists of at least 48 IGHV genes, seven IGHD genes, and six IGHJ genes. IGHC genes have also been mapped in this study, with three genes encoding immunoglobulin classes: IgT, IgM, and IgD. We confirmed the expression of newly identified IGHV3 family sequences in the spleen and kidney of adult torafugu and found a favorable IGHV segment usage by IgM and IgT. Possible structural variation in the IGHδ locus was observed based on the current torafugu assembly. The complete characterization of the torafugu IGH locus will facilitate detailed studies of large-scale mechanisms associated with the recombination of the variable region genes and will offer insights into the genetic basis of the potential diversity in the antibody response observed in torafugu.

A novel mouse model for Down syndrome that harbor a single copy of human artificial chromosome (HAC) carrying a limited number of genes from human chromosome 21.

Down syndrome (DS), also known as Trisomy 21, is the most common chromosome aneuploidy in live-born children and displays a complicated symptom. To date, several kinds of mouse models have been generated to understand the molecular pathology of DS, yet the gene dosage effects and gene(s)-phenotype(s) correlation are not well understood. In this study, we established a novel method to generate a partial trisomy mice using the mouse ES cells that harbor a single copy of human artificial chromosome (HAC), into which a small human DNA segment containing human chromosome 21 genes cloned in a bacterial artificial chromosome (BAC) was recombined. The produced mice were found to maintain the HAC carrying human genes as a mini-chromosome, hence termed as a Trans-Mini-Chromosomal (TMC) mouse, and HAC was transmitted for more than twenty generations independent from endogenous mouse chromosomes. The three human transgenes including cystathionine ß-synthase, U2 auxiliary factor and crystalline alpha A were expressed in several mouse tissues with various expression levels relative to mouse endogenous genes. The novel system is applicable to any of human and/or mouse BAC clones. Thus, the TMC mouse carrying a HAC with a limited number of genes would provide a novel tool for studying gene dosage effects involved in the DS molecular pathogenesis and the gene(s)-phenotype(s) correlation.

Stimulatory and inhibitory mechanisms of slow muscle-specific myosin heavy chain gene expression in fish: transient and transgenic analysis of torafugu MYH(M86-2) promoter in zebrafish embryos.

The myosin heavy chain gene, MYHM86-2, exhibited restricted expression in slow muscle fibers of torafugu embryos and larvae, suggesting its functional roles for embryonic and larval muscle development. However, the transcriptional mechanisms involved in its expression are still ambiguous. The present study is the first extensive analysis of slow muscle-specific MYHM86-2 promoter in fish for identifying the cis-elements that are crucial for its expression. Combining both transient transfection and transgenic approaches, we demonstrated that the 2614bp 5'-flanking sequences of MYHM86-2 contain a sufficient promoter activity to drive gene expression specific to superficial slow muscle fibers. By cyclopamine treatment, we also demonstrated that the differentiation of such superficial slow muscle fibers depends on hedgehog signaling activity. The deletion analyses defined an upstream fragment necessary for repressing ectopic MYHM86-2 expression in the fast muscle fibers. The transcriptional mechanism that prevents MYHM86-2 expression in the fast muscle fibers is mediated through Sox6 binding elements. We also demonstrated that Sox6 may function as a transcriptional repressor of MYHM86-2 expression. We further discovered that nuclear factor of activated T cells (NFAT) binding elements plays a key role and myocyte enhancer factor-2 (MEF2) binding elements participate in the transcriptional regulation of MYHM86-2 expression.

Transcriptional responses of a bicarbonate-tolerant monocot, Puccinellia tenuiflora, and a related bicarbonate-sensitive species, Poa annua, to NaHCO3 stress.

Puccinellia tenuiflora is an alkaline salt-tolerant monocot found in saline-alkali soil in China. To identify the genes which are determining the higher tolerance of P. tenuiflora compared to bicarbonate sensitive species, we examined the responses of P. tenuiflora and a related bicarbonate-sensitive Poeae plant, Poa annua, to two days of 20 mM NaHCO3 stress by RNA-seq analysis. We obtained 28 and 38 million reads for P. tenuiflora and P. annua, respectively. For each species, the reads of both unstressed and stressed samples were combined for de novo assembly of contigs. We obtained 77,329 contigs for P. tenuiflora and 115,335 contigs for P. annua. NaHCO3 stress resulted in greater than two-fold absolute expression value changes in 157 of the P. tenuiflora contigs and 1090 of P. annua contigs. Homologs of the genes involved in Fe acquisition, which are important for the survival of plants under alkaline stress, were up-regulated in P. tenuiflora and down-regulated in P. annua. The smaller number of the genes differentially regulated in P. tenuiflora suggests that the genes regulating bicarbonate tolerance are constitutively expressed in P. tenuiflora.

Transcriptome analysis of Edwardsiella piscicida during intracellular infection reveals excludons are involved with the activation of a mitochondrion-like energy generation program.

Phylogenetic evidence suggests a shared ancestry between mitochondria and modern Proteobacteria, a phylum including several genera of intracellular pathogens. Studying these diverse pathogens, particularly during intracellular infection of their hosts, can reveal characteristics potentially representative of the mitochondrial-Proteobacterial ancestor by identifying traits shared with mitochondria. While transcriptomic approaches can provide global insights into intracellular acclimatization by pathogens, they are often limited by excess host RNAs in extracts. Here, we developed a method employing magnetic nanoparticles to enrich RNA from an intracellular Gammaproteobacterium, Edwardsiella piscicida, within zebrafish, Danio rerio, fin fibroblasts, enabling comprehensive exploration of the bacterial transcriptome. Our findings revealed that the intracellular E. piscicida transcriptome reflects a mitochondrion-like energy generation program characterized by the suppression of glycolysis and sugar transport, coupled with upregulation of the tricarboxylic acid (TCA) cycle and alternative import of simple organic acids that directly flux into TCA cycle intermediates or electron transport chain donors. Additionally, genes predicted to be members of excludons, loci of gene pairs antagonistically co-regulated by overlapping antisense transcription, are significantly enriched in the set of all genes with perturbed sense and antisense transcription, suggesting a general but important involvement of excludons with intracellular acclimatization. Notably, genes involved with the activation of the mitochondrion-like energy generation program, specifically with metabolite import and glycolysis, are also members of predicted excludons. Other intracellular Proteobacterial pathogens appear to employ a similar mitochondrion-like energy generation program, suggesting a potentially conserved mechanism for optimized energy acquisition from hosts centered around the TCA cycle.IMPORTANCEPhylogenetic evidence suggests that mitochondria and Proteobacteria, a phylum encompassing various intracellular pathogens, share a common ancestral lineage. In this study, we developed a novel method employing magnetic nanoparticles to explore the transcriptome of an aquatic Gammaproteobacterium, Edwardsiella piscicida, during intracellular infection of host cells. We show that the strategy E. piscicida uses to generate energy strikingly mirrors the function of mitochondria-energy generators devoid of glycolytic processes. Notably, several implicated genes are members of excludons-gene pairs antagonistically co-regulated by overlapping antisense transcription. Other intracellular Proteobacterial pathogens appear to adopt a similar mitochondrion-like energy generation program, indicating a possibly conserved strategy for optimized energy acquisition from hosts centered around the tricarboxylic acid cycle.