The Smallest Known Genomes of Multicellular and Toxic Cyanobacteria: Comparison,Minimal Gene Sets for Linked Traits and the Evolutionary Implications

Cyanobacterial morphology is diverse, ranging from unicellular spheres or rods to multicellular structures such as colonies and filaments. Multicellular species represent an evolutionary strategy to differentiate and compartmentalize certain metabolic functions for reproduction and nitrogen (N₂) fixation into specialized cell types (e.g. akinetes, heterocysts and diazocytes). Only a few filamentous, differentiated cyanobacterial species, with genome sizes over 5 Mb, have been sequenced. We sequenced the genomes of two strains of closely related filamentous cyanobacterial species to yield further insights into the molecular basis of the traits of N₂ fixation, filament formation and cell differentiation. Cylindrospermopsis raciborskii CS-505 is a cylindrospermopsin-producing strain from Australia, whereas Raphidiopsis brookii D9 from Brazil synthesizes neurotoxins associated with paralytic shellfish poisoning (PSP). Despite their different morphology, toxin composition and disjunct geographical distribution, these strains form a monophyletic group. With genome sizes of approximately 3.9 (CS-505) and 3.2 (D9) Mb, these are the smallest genomes described for free-living filamentous cyanobacteria. We observed remarkable gene order conservation (synteny) between these genomes despite the difference in repetitive element content, which accounts for most of the genome size difference between them. We show here that the strains share a specific set of 2539 genes with >90% average nucleotide identity. The fact that the CS-505 and D9 genomes are small and streamlined compared to those of other filamentous cyanobacterial species and the lack of the ability for heterocyst formation in strain D9 allowed us to define a core set of genes responsible for each trait in filamentous species. We presume that in strain D9 the ability to form proper heterocysts was secondarily lost together with N₂ fixation capacity. Further comparisons to all available cyanobacterial genomes covering almost the entire evolutionary branch revealed a common minimal gene set for each of these cyanobacterial traits.     

Asymmetry Indices for Analysis and Prediction of Replication Origins in Eukaryotic Genomes

DNA replication was recently shown to induce the formation of compositional skews in the genomes of the yeasts Saccharomyces cerevisiae and Kluyveromyces lactis. In this work, I have characterized further GC and TA skew variations in the vicinity of S. cerevisiae replication origins and termination sites, and defined asymmetry indices for origin analysis and prediction. The presence of skew jumps at some termination sites in the S. cerevisiae genome was established. The majority of S. cerevisiae replication origins are marked by an oriented consensus sequence called ACS, but no evidence could be found for asymmetric origin firing that would be linked to ACS orientation. Asymmetry indices related to GC and TA skews were defined, and a global asymmetry index I_GC,TA was described. I_GC,TA was found to strongly correlate with origin efficiency in S. cerevisiae and to allow the determination of sets of intergenes significantly enriched in origin loci. The generalized use of asymmetry indices for origin prediction in naive genomes implies the determination of the direction of the skews, i.e. the identification of which strand, leading or lagging, is enriched in G and which one is enriched in T. Recent work indicates that in Candida albicans and in several related species, centromeres contain early and efficient replication origins. It has been proposed that the skew jumps observed at these positions would reflect the activity of these origins, thus allowing to determine the direction of the skews in these genomes. However, I show here that the skew jumps at C. albicans centromeres are not related to replication and that replication-associated GC and TA skews in C. albicans have in fact the opposite directions of what was proposed.     

Gene Structure Prediction Using an Orthologous Gene of Known Exon-Intron Structure

Given the availability of complete genome sequences from related organisms, sequence conservation can provide important clues for predicting gene structure. In particular, one should be able to leverage information about known genes in one species to help determine the structures of related genes in another. Such an approach is appealing in that high-quality gene prediction can be achieved for newly sequenced species, such as mouse and puffer fish, using the extensive knowledge that has been accumulated about human genes. This article reports a novel approach to predicting the exon-intron structures of mouse genes by incorporating constraints from orthologous human genes using techniques that have previously been exploited in speech and natural language processing applications. The approach uses a context-free grammar to parse a training corpus of annotated human genes. A statistical training procedure produces a weighted recursive transition network (RTN) intended to capture the general features of a mammalian gene. This RTN is expanded into a finite state transducer (FST) and composed with an FST capturing the specific features of the human orthologue. This model includes a trigram language model on the amino acid sequence as well as exon length constraints. A final stage uses the free software package ClustalW to align the top n candidates in the search space. For a set of 98 orthologous human-mouse pairs, we achieved 96% sensitivity and 97% specificity at the exon level on the mouse genes, given only knowledge gleaned from the annotated human genome.     

真核生物基因组转录诱导融合基因的研究进展

真核生物的基因组由基因和基因间区组成.基因转录时,从转录起始点开始到该基因的转录终止点结束,形成独立的转录单元.然而有少量的文献表明,转录有时会通读基因间区,产生包含上游基因、基因间区和下游基因的融合基因转录本.融合转录本经基因间剪接而成为有功能的成熟转录本.对真核生物转录诱导融合基因的基因间剪接方式、产生机制和意义进行了综述.     

Automated Prediction and Annotation of Small Open Reading Frames in Microbial Genomes

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Evidence That Inconsistent Gene Prediction Can Mislead Analysis of Dinoflagellate Genomes

Comparative algal genomics often relies on predicted genes from de novo assembled genomes. However, the artifacts introduced by different gene-prediction approaches, and their impact on comparative genomic analysis remain poorly understood. Here, using available genome data from six dinoflagellate species in the Symbiodiniaceae, we identified methodological biases in the published genes that were predicted using different approaches and putative contaminant sequences in the published genome assemblies. We developed and applied a comprehensive customized workflow to predict genes from these genomes. The observed variation among predicted genes resulting from our workflow agreed with current understanding of phylogenetic relationships among these taxa, whereas the variation among the previously published genes was largely biased by the distinct approaches used in each instance. Importantly, these biases affect the inference of homologous gene families and synteny among genomes, thus impacting biological interpretation of these data. Our results demonstrate that a consistent gene-prediction approach is critical for comparative analysis of dinoflagellate genomes.     

RGAAT: A Reference-based Genome Assembly and Annotation Tool for New Genomes and Upgrade of Known Genomes

The rapid development of high-throughput sequencing technologies has led to a dramatic decrease in the money and time required for de novo genome sequencing or genome resequencing projects, with new genome sequences constantly released every week. Among such projects, the plethora of updated genome assemblies induces the requirement of version-dependent annotation files and other compatible public dataset for downstream analysis. To handle these tasks in an efficient manner, we developed the reference-based genome assembly and annotation tool (RGAAT), a flexible toolkit for resequencing-based consensus building and annotation update. RGAAT can detect sequence variants with comparable precision, specificity, and sensitivity to GATK and with higher precision and specificity than Freebayes and SAMtools on four DNA-seq datasets tested in this study. RGAAT can also identify sequence variants based on cross-cultivar or cross-version genomic alignments. Unlike GATK and SAMtools/BCFtools, RGAAT builds the consensus sequence by taking into account the true allele frequency. Finally, RGAAT generates a coordinate conversion file between the reference and query genomes using sequence variants and supports annotation file transfer. Compared to the rapid annotation transfer tool (RATT), RGAAT displays better performance characteristics for annotation transfer between different genome assemblies, strains, and species. In addition, RGAAT can be used for genome modification, genome comparison, and coordinate conversion. RGAAT is available at https://sourceforge.net/projects/rgaat/ and https://github.com/wushyer/RGAAT_v2 at no cost.     

仿生模式识别在细菌基因组水平转移基因预测中的应用

水平转移基因的预测对于生物进化过程的理解和物种之间遗传物质进行定性和定量的估计都有重要的意义.本文提出一种利用仿生模式识别原理来对细茵基因组水平转移基因进行预测的方法.仿生模式识别是基于同调连续性原理一特征空间中同类样本的连续性特性,强调用"认识"模式取代传统的模式"分类"与划分,它更接近于人类"认识"事物的特性.仿生模式识别理论已经成功应用于多镜头人脸身份确认,人脸识别,图像复原,语音识别等领域.我们采用超香肠神经元网络对水平转移基因进行识别,结果显示,仿生模式识别方法优于目前预测结果最好的八联核苷酸频率的打分算法,和基于支撑向量机的识别算法.特别是在对大肠杆菌(Eschefichia coli K12)基因组,识别率分别提高了42.3%和30.5%.     

Conservation and Periodicity of DNA Bend Sites in Eukaryotic Genomes

DNA bend sites appear every 680 bp on average in the human -and ß-globin gene regions. Although most of theirmolecular nature has not been unraveled, a potential bend coresequence A₂N₈A₂N₈A₂ (A/A/A) and its complementary T₂N₈T₂N₈T₂(T/T/T) appeared preferentially either in or very close to mostof the bend sites, whereas other combinations of A₂ and T₂ dinucleotides,A/T/T + A/A/T, T/T/A + T/A/A and A/T/A + T/A/T, did not. Thedistances between any two of the core sequences in the entireß-globin locus showed a strong bias to a length of701–800 bp and multiples thereof, suggesting that thereis periodicity throughout the locus. This bias was not foundfor other combinations of A₂ and T₂. Again, this periodicitywas identified in many eukaryotic genes, whereas the tendencywas absent in mRNAs and prokaryotic as well as viral genomes.     

基于支持向量机的细菌基因组水平转移基因预测

随着各种生物基因组序列测定工作的完成,大量的DNA序列数据涌现出来,为研究在基因组中寻找水平转移基因提供了极大的便利.将基因序列特征分析和支持向量机技术结合起来,通过分析基因序列的特征差异发现水平转移基因.依据以前研究工作的基础,选取了绝对密码子使用频率(FCU)作为序列特征,主要因为它既包含了基因密码子使用偏性的信息,也包含了基因所编码蛋白的氨基酸组成信息,支持向量机利用这些信息进行水平转移基因分析和预测,可以提高预测的准确性.另外,提出了基于分链的水平转移基因预测新方法,即将细菌基因组前导链和滞后链上的基因区别对待,分别进行水平转移基因预测.结果显示,基本预测方法要优于目前预测结果最好的Tsirigos等提出的基于八联核苷酸频率的打分算法,命中率的相对提高率最高达31.47%,而基于分链的方法对水平转移基因的预测取得了更好的结果.     

Production and Characterization of Novel Recombinant Adeno-Associated Virus Replicative-Form Genomes: A Eukaryotic Source of DNA for Gene Transfer

Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV) inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or “CELiD”, DNA). CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5×10⁹ Sf9 cells, and 1–15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA.     

The Smallest Known Tetrapod Footprints: Batrachichnus Salamandroides from the Carboniferous of Joggins,Nova Scotia,Canada

A new specimen of Batrachichnus salamandroides from the classic Carboniferous section at Joggins, Nova Scotia, is the smallest set of tetrapod footprints known in the fossil record. The trackmaker was a juvenile, quadrupedal temnospondyl or microsaur with a trunk length of 3.55 mm and an estimated body length of 8 mm (skull, presacral vertebrae, and caudal vertebrae). The 48 mm-long trackway preserves a high degree of extramorphological variation along its course, including a gait change associated with a change in direction along with an increased stride and pace, and the appearance of overstepped imprints, in the latter portion of the trackway. These morphological changes suggest the tetrapod changed from a walking gait to a running gait.     

Extra-Large G Proteins Expand the Repertoire of Subunits in Arabidopsis Heterotrimeric G Protein Signaling

Heterotrimeric G proteins, consisting of Gα, Gβ, and Gγ subunits, are a conserved signal transduction mechanism in eukaryotes. However, G protein subunit numbers in diploid plant genomes are greatly reduced as compared with animals and do not correlate with the diversity of functions and phenotypes in which heterotrimeric G proteins have been implicated. In addition to GPA1, the sole canonical Arabidopsis (Arabidopsis thaliana) Gα subunit, Arabidopsis has three related proteins: the extra-large GTP-binding proteins XLG1, XLG2, and XLG3. We demonstrate that the XLGs can bind Gβγ dimers (AGB1 plus a Gγ subunit: AGG1, AGG2, or AGG3) with differing specificity in yeast (Saccharomyces cerevisiae) three-hybrid assays. Our in silico structural analysis shows that XLG3 aligns closely to the crystal structure of GPA1, and XLG3 also competes with GPA1 for Gβγ binding in yeast. We observed interaction of the XLGs with all three Gβγ dimers at the plasma membrane in planta by bimolecular fluorescence complementation. Bioinformatic and localization studies identified and confirmed nuclear localization signals in XLG2 and XLG3 and a nuclear export signal in XLG3, which may facilitate intracellular shuttling. We found that tunicamycin, salt, and glucose hypersensitivity and increased stomatal density are agb1-specific phenotypes that are not observed in gpa1 mutants but are recapitulated in xlg mutants. Thus, XLG-Gβγ heterotrimers provide additional signaling modalities for tuning plant G protein responses and increase the repertoire of G protein heterotrimer combinations from three to 12. The potential for signal partitioning and competition between the XLGs and GPA1 is a new paradigm for plant-specific cell signaling.The classical heterotrimeric G protein consists of a GDP/GTP-binding Gα subunit with GTPase activity bound to an obligate dimer formed by Gβ and Gγ subunits. In the signaling paradigm largely elucidated from mammalian systems, the plasma membrane-associated heterotrimer contains Gα in its GDP-bound form. Upon receiving a molecular signal, typically transduced by a transmembrane protein (e.g. a G protein-coupled receptor), Gα exchanges GDP for GTP and dissociates from the Gβγ dimer. Both Gα and Gβγ interact with intracellular effectors to initiate downstream signaling cascades. The intrinsic GTPase activity of Gα restores Gα to the GDP-bound form, which binds Gβγ, thereby reconstituting the heterotrimer (McCudden et al., 2005; Oldham and Hamm, 2008).Signal transduction through a heterotrimeric G protein complex is an evolutionarily conserved eukaryotic mechanism common to metazoa and plants, although there are distinct differences in the functional intricacies between the evolutionary branches (Jones et al., 2011a, 2011b; Bradford et al., 2013). The numbers of each subunit encoded within genomes, and therefore the potential for combinatorial complexity within the heterotrimer, is one of the most striking differences between plants and animals. For example, the human genome encodes 23 Gα (encoded by 16 genes), five Gβ, and 12 Gγ subunits (Hurowitz et al., 2000; McCudden et al., 2005; Birnbaumer, 2007). The Arabidopsis (Arabidopsis thaliana) genome, however, only encodes one canonical Gα (GPA1; Ma et al., 1990), one Gβ (AGB1; Weiss et al., 1994), and three Gγ (AGG1, AGG2, and AGG3) subunits (Mason and Botella, 2000, 2001; Chakravorty et al., 2011), while the rice (Oryza sativa) genome encodes one Gα (Ishikawa et al., 1995), one Gβ (Ishikawa et al., 1996), and either four or five Gγ subunits (Kato et al., 2004; Chakravorty et al., 2011; Botella, 2012). As expected, genomes of polyploid plants have more copies due to genome duplication, with the soybean (Glycine max) genome encoding four Gα, four Gβ (Bisht et al., 2011), and 10 Gγ subunits (Choudhury et al., 2011). However, Arabidopsis heterotrimeric G proteins have been implicated in a surprisingly large number of phenotypes, which is seemingly contradictory given the relative scarcity of subunits. Arabidopsis G proteins have been implicated in cell division (Ullah et al., 2001; Chen et al., 2006) and morphological development in various tissues, including hypocotyls (Ullah et al., 2001, 2003), roots (Ullah et al., 2003; Chen et al., 2006; Li et al., 2012), leaves (Lease et al., 2001; Ullah et al., 2001), inflorescences (Ullah et al., 2003), and flowers and siliques (Lease et al., 2001), as well as in pathogen responses (Llorente et al., 2005; Trusov et al., 2006; Cheng et al., 2015), regulation of stomatal movement (Wang et al., 2001; Coursol et al., 2003; Fan et al., 2008) and development (Zhang et al., 2008; Nilson and Assmann, 2010), cell wall composition (Delgado-Cerezo et al., 2012), responses to various light stimuli (Warpeha et al., 2007; Botto et al., 2009), responses to multiple abiotic stimuli (Huang et al., 2006; Pandey et al., 2006; Trusov et al., 2007; Zhang et al., 2008; Colaneri et al., 2014), responses to various hormones during germination (Ullah et al., 2002), and postgermination development (Ullah et al., 2002; Pandey et al., 2006; Trusov et al., 2007). Since the Gγ subunit appeared to be the only subunit that provides diversity in heterotrimer composition in Arabidopsis, it was proposed that all functional specificity in heterotrimeric G protein signaling was provided by the Gγ subunit (Trusov et al., 2007; Chakravorty et al., 2011; Thung et al., 2012, 2013). This allowed for only three heterotrimer combinations to account for the wide range of G protein-associated phenotypes.In addition to the above typical G protein subunits, the plant kingdom contains a conserved protein family of extra-large GTP-binding proteins (XLGs). XLGs differ from typical Gα subunits in that they possess a long N-terminal extension of unknown function, but they are similar in that they all have a typical C-terminal Gα-like region, with five semiconserved G-box (G1–G5) motifs. The XLGs also possess the two sequence features that differentiate heterotrimeric G protein Gα subunits from monomeric G proteins: a helical region between the G1 and G2 motifs and an Asp/Glu-rich loop between the G3 and G4 motifs (Lee and Assmann, 1999; Ding et al., 2008; Heo et al., 2012). The Arabidopsis XLG family comprises XLG1, XLG2, and XLG3, and all three have demonstrated GTP-binding and GTPase activities, although they differ from GPA1 in exhibiting a much slower rate of GTP hydrolysis, with a Ca²⁺ cofactor requirement instead of an Mg²⁺ requirement, as for canonical Gα proteins (Heo et al., 2012). All three Arabidopsis XLGs were observed to be nuclear localized (Ding et al., 2008). Although much less is known about XLGs than canonical Gα subunits, XLG2 positively regulates resistance to the bacterial pathogen Pseudomonas syringae and was immunoprecipitated with AGB1 from tissue infected with P. syringae (Zhu et al., 2009). xlg3 mutants, like agb1 mutants, are impaired in root-waving and root-skewing responses (Pandey et al., 2008). During the preparation of this report, Maruta et al. (2015) further investigated XLG2, particularly focusing on the link between XLG2 and Gβγ in pathogen responses. Based on symptom progression in xlg mutants, they found that XLG2 is a positive regulator of resistance to both bacterial and fungal pathogens, with a minor contribution from XLG3 in resistance to Fusarium oxysporum. XLG2 and XLG3 are also positive regulators of reactive oxygen species (ROS) production in response to pathogen-associated molecular pattern elicitors. The resistance and pathogen-associated molecular pattern-induced ROS phenotypes of the agg1 agg2 and xlg2 xlg3 double mutants were not additive in an agg1 agg2 xlg2 xlg3 quadruple mutant, indicating that these two XLGs and the two Gγ subunits function in the same, rather than parallel, pathways. Unfortunately, the close proximity of XLG2 and AGB1 on chromosome 4 precluded the generation of an agb1 xlg2 double mutant; therefore, direct genetic evidence of XLG2 and AGB1 interaction is still lacking, but physical interactions between XLG2 and the Gβγ dimers were shown by yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescence complementation (BiFC) assays (Maruta et al., 2015). Localization of all three XLGs was also reexamined, indicating that XLGs are capable of localizing to the plasma membrane in addition to the nucleus (Maruta et al., 2015).Interestingly, several other plant G protein-related phenotypes, in addition to pathogen resistance, have been observed only in Gβ and Gγ mutants, with opposite phenotypes observed in Gα (gpa1) mutants. Traditionally, the observation of opposite phenotypes in Gα versus Gβγ mutants in plants and other organisms has mechanistically been attributed to signaling mediated by free Gβγ, which increases in abundance in the absence of Gα. However, an intriguing alternative is that XLG proteins fulfill a Gα-like role in forming heterotrimeric complexes with Gβγ and function in non-GPA1-based G protein signaling processes. If XLGs function like Gα subunits, the corresponding increase in subunit diversity could potentially account for the diversity of G protein phenotypes. In light of this possibility, we assessed the heterotrimerization potential of all possible XLG and Gβγ dimer combinations, XLG localization and its regulation by Gβγ, and the effect of xlg mutation on selected known phenotypes associated with heterotrimeric G proteins. Our results provide compelling evidence for the formation of XLG-Gβγ heterotrimers and reveal that plant G protein signaling is substantially more complex than previously thought.     

The Evolution and Structure Prediction of Coiled Coils across All Genomes

Coiled coils are α-helical interactions found in many natural proteins. Various sequence-based coiled-coil predictors are available, but key issues remain: oligomeric state and protein-protein interface prediction and extension to all genomes. We present SpiriCoil (http://supfam.org/SUPERFAMILY/spiricoil), which is based on a novel approach to the coiled-coil prediction problem for coiled coils that fall into known superfamilies: hundreds of hidden Markov models representing coiled-coil-containing domain families. Using whole domains gives the advantage that sequences flanking the coiled coils help. SpiriCoil performs at least as well as existing methods at detecting coiled coils and significantly advances the state of the art for oligomer state prediction. SpiriCoil has been run on over 16 million sequences, including all completely sequenced genomes (more than 1200), and a resulting Web interface supplies data downloads, alignments, scores, oligomeric state classifications, three-dimensional homology models and visualisation. This has allowed, for the first time, a genomewide analysis of coiled-coil evolution. We found that coiled coils have arisen independently de novo well over a hundred times, and these are observed in 16 different oligomeric states. Coiled coils in almost all oligomeric states were present in the last universal common ancestor of life. The vast majority of occasions that individual coiled coils have arisen de novo were before the last universal common ancestor of life; we do, however, observe scattered instances throughout subsequent evolutionary history, mostly in the formation of the eukaryote superkingdom. Coiled coils do not change their oligomeric state over evolution and did not evolve from the rearrangement of existing helices in proteins; coiled coils were forged in unison with the fold of the whole protein.     

The Structure and Gene Repertoire of an Ancient Red Algal Plastid Genome

Photosynthetic eukaryotes can, according to features of their chloroplasts, be divided into two major groups: the red and the green lineage of plastid evolution. To extend the knowledge about the evolution of the red lineage we have sequenced and analyzed the chloroplast genome (cp-genome) of Cyanidium caldarium RK1, a unicellular red alga (AF022186). The analysis revealed that this genome shows several unusual structural features, such as a hypothetical hairpin structure in a gene-free region and absence of large repeat units. We provide evidence that this structural organization of the cp-genome of C. caldarium may be that of the most ancient cp-genome so far described. We also compared the cp-genome of C. caldarium to the other known cp-genomes of the red lineage. The cp-genome of C. caldarium cannot be readily aligned with that of Porphyra purpurea, a multicellular red alga, or Guillardia theta due to a displacement of a region of the cp-genome. The phylogenetic tree reveals that the secondary endosymbiosis, through which G. theta evolved, took place after the separation of the ancestors of C. caldarium and P. purpurea. We found several genes unique to the cp-genome of C. caldarium. Five of them seem to be involved in the building of bacterial cell envelopes and may be responsible for the thermotolerance of the chloroplast of this alga. Two additional genes may play a role in stabilizing the photosynthetic machinery against salt stress and detoxification of the chloroplast. Thus, these genes may be unique to the cp-genome of C. caldarium and may be required for the endurance of the extreme living conditions of this alga. Received: 3 June 2000 / Accepted: 18 July 2000     

Geptop: A Gene Essentiality Prediction Tool for Sequenced Bacterial Genomes Based on Orthology and Phylogeny

Integrative genomics predictors, which score highly in predicting bacterial essential genes, would be unfeasible in most species because the data sources are limited. We developed a universal approach and tool designated Geptop, based on orthology and phylogeny, to offer gene essentiality annotations. In a series of tests, our Geptop method yielded higher area under curve (AUC) scores in the receiver operating curves than the integrative approaches. In the ten-fold cross-validations among randomly upset samples, Geptop yielded an AUC of 0.918, and in the cross-organism predictions for 19 organisms Geptop yielded AUC scores between 0.569 and 0.959. A test applied to the very recently determined essential gene dataset from the Porphyromonas gingivalis, which belongs to a phylum different with all of the above 19 bacterial genomes, gave an AUC of 0.77. Therefore, Geptop can be applied to any bacterial species whose genome has been sequenced. Compared with the essential genes uniquely identified by the lethal screening, the essential genes predicted only by Gepop are associated with more protein-protein interactions, especially in the three bacteria with lower AUC scores (<0.7). This may further illustrate the reliability and feasibility of our method in some sense. The web server and standalone version of Geptop are available at http://cefg.uestc.edu.cn/geptop/ free of charge. The tool has been run on 968 bacterial genomes and the results are accessible at the website.