Enzymes are enriched in bacterial essential genes

Essential genes, those indispensable for the survival of an organism, play a key role in the emerging field, synthetic biology. Characterization of functions encoded by essential genes not only has important practical implications, such as in identifying antibiotic drug targets, but can also enhance our understanding of basic biology, such as functions needed to support cellular life. Enzymes are critical for almost all cellular activities. However, essential genes have not been systematically examined from the aspect of enzymes and the chemical reactions that they catalyze. Here, by comprehensively analyzing essential genes in 14 bacterial genomes in which large-scale gene essentiality screens have been performed, we found that enzymes are enriched in essential genes. Essential enzymes have overrepresented ligases (especially those forming carbon-oxygen bonds and carbon-nitrogen bonds), nucleotidyltransferases and phosphotransferases, while have underrepresented oxidoreductases. Furthermore, essential enzymes tend to associate with more gene ontology domains. These results, from the aspect of chemical reactions, provide further insights into the understanding of functions needed to support natural cellular life, as well as synthetic cells, and provide additional parameters that can be integrated into gene essentiality prediction algorithms.     

A complete collection of single‐gene deletion mutants of Acinetobacter baylyi ADP1

We have constructed a collection of single‐gene deletion mutants for all dispensable genes of the soil bacterium Acinetobacter baylyi ADP1. A total of 2594 deletion mutants were obtained, whereas 499 (16%) were not, and are therefore candidate essential genes for life on minimal medium. This essentiality data set is 88% consistent with the Escherichia coli data set inferred from the Keio mutant collection profiled for growth on minimal medium, while 80% of the orthologous genes described as essential in Pseudomonas aeruginosa are also essential in ADP1. Several strategies were undertaken to investigate ADP1 metabolism by (1) searching for discrepancies between our essentiality data and current metabolic knowledge, (2) comparing this essentiality data set to those from other organisms, (3) systematic phenotyping of the mutant collection on a variety of carbon sources (quinate, 2‐3 butanediol, glucose, etc.). This collection provides a new resource for the study of gene function by forward and reverse genetic approaches and constitutes a robust experimental data source for systems biology approaches.     

Characteristics of Plant Essential Genes Allow for within- and between-Species Prediction of Lethal Mutant Phenotypes

Essential genes represent critical cellular components whose disruption results in lethality. Characteristics shared among essential genes have been uncovered in fungal and metazoan model systems. However, features associated with plant essential genes are largely unknown and the full set of essential genes remains to be discovered in any plant species. Here, we show that essential genes in Arabidopsis thaliana have distinct features useful for constructing within- and cross-species prediction models. Essential genes in A. thaliana are often single copy or derived from older duplications, highly and broadly expressed, slow evolving, and highly connected within molecular networks compared with genes with nonlethal mutant phenotypes. These gene features allowed the application of machine learning methods that predicted known lethal genes as well as an additional 1970 likely essential genes without documented phenotypes. Prediction models from A. thaliana could also be applied to predict Oryza sativa and Saccharomyces cerevisiae essential genes. Importantly, successful predictions drew upon many features, while any single feature was not sufficient. Our findings show that essential genes can be distinguished from genes with nonlethal phenotypes using features that are similar across kingdoms and indicate the possibility for translational application of our approach to species without extensive functional genomic and phenomic resources.     

The genes and enzymes for the catabolism of galactitol,D-tagatose,and related carbohydrates in<Emphasis Type="Italic"> Klebsiella oxytoca</Emphasis> M5a1 and other enteric bacteria display convergent evolution

Enteric bacteria (Enteriobacteriaceae) carry on their single chromosome about 4000 genes that all strains have in common (referred to here as obligatory genes), and up to 1300 facultative genes that vary from strain to strain and from species to species. In closely related species, obligatory and facultative genes are orthologous genes that are found at similar loci. We have analyzed a set of facultative genes involved in the degradation of the carbohydrates galactitol, D-tagatose, D-galactosamine and N-acetyl-galactosamine in various pathogenic and non-pathogenic strains of these bacteria. The four carbohydrates are transported into the cell by phosphotransferase (PTS) uptake systems, and are metabolized by closely related or even identical catabolic enzymes via pathways that share several intermediates. In about 60% of Escherichia coli strains the genes for galactitol degradation map to a gat operon at 46.8 min. In strains of Salmonella enterica, Klebsiella pneumoniae and K. oxytoca, the corresponding gat genes, although orthologous to their E. coli counterparts, are found at 70.7 min, clustered in a regulon together with three tag genes for the degradation of D-tagatose, an isomer of D-fructose. In contrast, in all the E. coli strains tested, this chromosomal site was found to be occupied by an aga/kba gene cluster for the degradation of D-galactosamine and N-acetyl-galactosamine. The aga/kba and the tag genes were paralogous either to the gat cluster or to the fru genes for degradation of D-fructose. Finally, in more then 90% of strains of both Klebsiella species, and in about 5% of the E. coli strains, two operons were found at 46.8 min that comprise paralogous genes for catabolism of the isomers D-arabinitol (genes atl or dal) and ribitol (genes rtl or rbt). In these strains gat genes were invariably absent from this location, and they were totally absent in S. enterica. These results strongly indicate that these various gene clusters and metabolic pathways have been subject to convergent evolution among the Enterobacteriaceae. This apparently involved recent horizontal gene transfer and recombination events, as indicated by major chromosomal rearrangements found in their immediate vicinity.Communicated by A. Kondorosi     

Identification of essential alphaproteobacterial genes reveals operational variability in conserved developmental and cell cycle systems

The cell cycle of Caulobacter crescentus is controlled by a complex signalling network that co‐ordinates events. Genome sequencing has revealed many C. crescentus cell cycle genes are conserved in other Alphaproteobacteria, but it is not clear to what extent their function is conserved. As many cell cycle regulatory genes are essential in C. crescentus, the essential genes of two Alphaproteobacteria, Agrobacterium tumefaciens (Rhizobiales) and Brevundimonas subvibrioides (Caulobacterales), were elucidated to identify changes in cell cycle protein function over different phylogenetic distances as demonstrated by changes in essentiality. The results show the majority of conserved essential genes are involved in critical cell cycle processes. Changes in component essentiality reflect major changes in lifestyle, such as divisome components in A. tumefaciens resulting from that organism's different growth pattern. Larger variability of essentiality was observed in cell cycle regulators, suggesting regulatory mechanisms are more customizable than the processes they regulate. Examples include variability in the essentiality of divJ and divK spatial cell cycle regulators, and non‐essentiality of the highly conserved and usually essential DNA methyltransferase CcrM. These results show that while essential cell functions are conserved across varying genetic distance, much of a given organism's essential gene pool is specific to that organism.     

Bacterial Protein Interaction Networks: Connectivity is Ruled by Gene Conservation,Essentiality and Function

BackgroundProtein-protein interaction (PPI) networks are the backbone of all processes in living cells. In this work, we relate conservation, essentiality and functional repertoire of a gene to the connectivity k (i.e. the number of interactions, links) of the corresponding protein in the PPI network.MethodsOn a set of 42 bacterial genomes of different sizes, and with reasonably separated evolutionary trajectories, we investigate three issues: i) whether the distribution of connectivities changes between PPI subnetworks of essential and nonessential genes; ii) how gene conservation, measured both by the evolutionary retention index (ERI) and by evolutionary pressures, is related to the connectivity of the corresponding protein; iii) how PPI connectivities are modulated by evolutionary and functional relationships, as represented by the Clusters of Orthologous Genes (COGs).ResultsWe show that conservation, essentiality and functional specialisation of genes constrain the connectivity of the corresponding proteins in bacterial PPI networks. In particular, we isolated a core of highly connected proteins (connectivities k≥40), which is ubiquitous among the species considered here, though mostly visible in the degree distributions of bacteria with small genomes (less than 1000 genes).ConclusionThe genes that support this highly connected core are conserved, essential and, in most cases, belong to the COG cluster J, related to ribosomal functions and the processing of genetic information.     

Gene essentiality determines chromosome organisation in bacteria

In Escherichia coli and Bacillus subtilis, essentiality, not expressivity, drives the distribution of genes between the two replicating strands. Although essential genes tend to be coded in the leading replicating strand, the underlying selective constraints and the evolutionary extent of these findings have still not been subject to comparative studies. Here, we extend our previous analysis to the genomes of low G + C firmicutes and γ-proteobacteria, and in a second step to all sequenced bacterial genomes. The inference of essentiality by homology allows us to show that essential genes are much more frequent in the leading strand than other genes, even when compared with non- essential highly expressed genes. Smaller biases were found in the genomes of obligatory intracellular bacteria, for which the assignment of essentiality by homology from fast growing free-living bacteria is most problematic. Cross-comparisons used to assess potential errors in the assignment of essentiality by homology revealed that, in most cases, variations in the assignment criteria have little influence on the overall results. Essential genes tend to be more conserved in the leading strand than average genes, which is consistent with selection for this positioning and may impose a strong constraint on chromosomal rearrangements. These results indicate that essentiality plays a fundamental role in the distribution of genes in most bacterial genomes.     

Essentiality of threonylcarbamoyladenosine (t6A), a universal tRNA modification,in bacteria

Threonylcarbamoyladenosine (t⁶A) is a modified nucleoside universally conserved in tRNAs in all three kingdoms of life. The recently discovered genes for t⁶A synthesis, including tsaC and tsaD, are essential in model prokaryotes but not essential in yeast. These genes had been identified as antibacterial targets even before their functions were known. However, the molecular basis for this prokaryotic‐specific essentiality has remained a mystery. Here, we show that t⁶A is a strong positive determinant for aminoacylation of tRNA by bacterial‐type but not by eukaryotic‐type isoleucyl‐tRNA synthetases and might also be a determinant for the essential enzyme tRNA^Ile‐lysidine synthetase. We confirm that t⁶A is essential in Escherichia coli and a survey of genome‐wide essentiality studies shows that genes for t⁶A synthesis are essential in most prokaryotes. This essentiality phenotype is not universal in Bacteria as t⁶A is dispensable in Deinococcus radiodurans, Thermus thermophilus, Synechocystis PCC6803 and Streptococcus mutans. Proteomic analysis of t⁶A^? D. radiodurans strains revealed an induction of the proteotoxic stress response and identified genes whose translation is most affected by the absence of t⁶A in tRNAs. Thus, although t⁶A is universally conserved in tRNAs, its role in translation might vary greatly between organisms.     

Differential selection on gene translation efficiency between the filamentous fungus <Emphasis Type="Italic">Ashbya gossypii</Emphasis> and yeasts

Background  

The filamentous fungus Ashbya gossypii grows into a multicellular mycelium that is distinct from the unicellular morphology of its closely related yeast species. It has been proposed that genes important for cell cycle regulation play central roles for such phenotypic differences. Because A. gossypii shares an almost identical set of cell cycle genes with the typical yeast Saccharomyces cerevisiae, the differences might occur at the level of orthologous gene regulation. Codon usage patterns were compared to identify orthologous genes with different gene regulation between A. gossypii and nine closely related yeast species.     

Evolutionary origins of human apoptosis and genome-stability gene networks

Apoptosis is essential for complex multicellular organisms and its failure is associated with genome instability and cancer. Interactions between apoptosis and genome-maintenance mechanisms have been extensively documented and include transactivation-independent and -dependent functions, in which the tumor-suppressor protein p53 works as a ‘molecular node’ in the DNA-damage response. Although apoptosis and genome stability have been identified as ancient pathways in eukaryote phylogeny, the biological evolution underlying the emergence of an integrated system remains largely unknown. Here, using computational methods, we reconstruct the evolutionary scenario that linked apoptosis with genome stability pathways in a functional human gene/protein association network. We found that the entanglement of DNA repair, chromosome stability and apoptosis gene networks appears with the caspase gene family and the antiapoptotic gene BCL2. Also, several critical nodes that entangle apoptosis and genome stability are cancer genes (e.g. ATM, BRCA1, BRCA2, MLH1, MSH2, MSH6 and TP53), although their orthologs have arisen in different points of evolution. Our results demonstrate how genome stability and apoptosis were co-opted during evolution recruiting genes that merge both systems. We also provide several examples to exploit this evolutionary platform, where we have judiciously extended information on gene essentiality inferred from model organisms to human.     

Detecting clonemate pairs in multicellular diploid clonal species based on a shared heterozygosity index

Clonal reproduction, the formation of nearly identical individuals via mitosis in the absence of genetic recombination, is a very common reproductive mode across plants, fungi and animals. To detect clonal genetic structure, genetic similarity indices based on shared alleles are widely used, such as the Jaccard index, or identity by state. Here we propose a new pairwise genetic similarity index, the SH index, based on segregating genetic marker loci (typically single nucleotide polymorphisms) that are identically heterozygous for pairs of samples (N_SH). To test our method, we analyse two old seagrass clones (Posidonia australis, estimated to be around 8500 years old; Zostera marina, >750 years old) along with two young Z. marina clones of known age (17 years old). We show that focusing on shared heterozygosity amplifies the power to distinguish sample pairs belonging to different clones compared to methods focusing on all shared alleles. Our proposed workflow can successfully detect clonemates at a location dominated by a single clone. When the collected samples involve two or more clones, the SH index shows a clear gap between clonemate pairs and interclone sample pairs. Ideally N_SH should be on the order of approximately ≥3000, a number easily achievable via restriction-site associated DNA (RAD) sequencing or whole-genome resequencing. Another potential application of the SH index is to detect possible parent–descendant pairs under selfing. Our proposed workflow takes advantage of the availability of the larger number of genetic markers in the genomic era, and improves the ability to distinguish clonemates from nonclonemates in multicellular diploid clonal species.     

Caste‐biases in gene expression are specific to developmental stage in the ant Formica exsecta

Understanding how a single genome creates and maintains distinct phenotypes is a central goal in evolutionary biology. Social insects are a striking example of co‐opted genetic backgrounds giving rise to dramatically different phenotypes, such as queen and worker castes. A conserved set of molecular pathways, previously envisioned as a set of ‘toolkit’ genes, has been hypothesized to underlie queen and worker phenotypes in independently evolved social insect lineages. Here, we investigated the toolkit from a developmental point of view, using RNA‐Seq to compare caste‐biased gene expression patterns across three life stages (pupae, emerging adult and old adult) and two female castes (queens and workers) in the ant Formica exsecta. We found that the number of genes with caste‐biased expression increases dramatically from pupal to old adult stages. This result suggests that phenotypic differences between queens and workers at the pupal stage may derive from a relatively low number of caste‐biased genes, compared to higher number of genes required to maintain caste differences at the adult stage. Gene expression patterns were more similar among castes within developmental stages than within castes despite the extensive phenotypic differences between queens and workers. Caste‐biased expression was highly variable among life stages at the level of single genes, but more consistent when gene functions (gene ontology terms) were investigated. Finally, we found that a large part of putative toolkit genes were caste‐biased at least in some life stages in F. exsecta, and the caste‐biases, but not their direction, were more often shared between F. exsecta and other ant species than between F. exsecta and bees. Our results indicate that gene expression should be examined across several developmental stages to fully reveal the genetic basis of polyphenisms.     

Evolutionary analyses of non‐family genes in plants

There are a large number of ‘non‐family’ (NF) genes that do not cluster into families with three or more members per genome. While gene families have been extensively studied, a systematic analysis of NF genes has not been reported. We performed comparative studies on NF genes in 14 plant species. Based on the clustering of protein sequences, we identified ~94 000 NF genes across these species that were divided into five evolutionary groups: Viridiplantae wide, angiosperm specific, monocot specific, dicot specific, and those that were species specific. Our analysis revealed that the NF genes resulted largely from less frequent gene duplications and/or a higher rate of gene loss after segmental duplication relative to genes in both low‐copy‐number families (LF; 3–10 copies per genome) and high‐copy‐number families (HF; >10 copies). Furthermore, we identified functions enriched in the NF gene set as compared with the HF genes. We found that NF genes were involved in essential biological processes shared by all plant lineages (e.g. photosynthesis and translation), as well as gene regulation and stress responses associated with phylogenetic diversification. In particular, our analysis of an Arabidopsis protein–protein interaction network revealed that hub proteins with the top 10% most connections were over‐represented in the NF set relative to the HF set. This research highlights the roles that NF genes may play in evolutionary and functional genomics research.     

Comparative trace element and organic growth factor requirements of five hansenula species

A comparative response of specific trace elements and organic growth factors for the growth of five Hansenula species (H. anomala, H. beijerinckii, H. ciferrii, H. polymorpha and H. sydowiorum) has been studied. Out of twenty three trace elements tested, Fe, Zn, Mn and Cu were found to be essential for the growth of all yeast species studied here, whereas the rest of the elements exhibited variable essentiality. From fifteen organic growth factors tested, thiamine, biotin, pyridoxine and inositol are the most commonly required growth factors by the yeasts, whereas the rest of the organic growth factors showed variable essentiality. All species of yeasts investigated required different concentrations of trace elements and organic growth factors for their optimum growth. Concentrations higher than the optimum have been found to be inhibitory for the growth of all the yeasts studied.     

Outline of a unified approach to physics,biology and sociology

The theory of organismic sets, introduced by N. Rashevsky (Bulletin of Mathematical Biophysics,29, 139–152, 1967;30, 163–174, 1968), is developed further. As has been pointed out, a society is a set of individuals plus the products of their activities, which result in their interactions. A multicellular organism is a set of cells plus the products of their activities, while a unicellular organism is a set of genes plus the products of their activities. It is now pointed out that a physical system is a set of elementary particles plus the product of their activities, such as transitions from one energy level to another. Therefore physical, biological and sociological phenomena can be considered from a unified set-theoretical point of view. The notion of a “world set” is introduced. It consists of the union of physical and of organismic sets. In physical sets the formation of different structure is governed preponderantly by analytical functions, which are special type of relations. In organismic sets, which represent biological organisms and societies, the formation of various structures is governed preponderantly by requirements that some relations, which are not functions, be satisfied. This is called the postulate of relational forces. Inasmuch as every function is a relation (F-relation) but not every relation is a function (Q-relation), it has been shown previously (Rashevsky,Bulletin of Mathematical Biophysics,29, 643–648, 1967) that the physical forces are only a special kind of relational force and that, therefore, the postulate of relational forces applies equally to physics, biology and sociology. By developing the earlier theory of organismic sets, we deduce the following conclusions: 1) A cell in which the genes are completely specialized, as is implied by the “one gene—one enzyme” principle, cannot be formed spontaneously. 2) By introducing the notion of organismic sets of different orders so that the elements of an organismic set of ordern are themselves organismic sets of order (n−1), we prove that in multicellular organisms no cell can be specialized completely; it performs, in addition to its special functions, also a number of others performed by other cells. 3) A differentiated multicellular organism cannot form spontaneously. It can only develop from simpler, less differentiated organisms. The same holds about societies. Highly specialized contemporary societies cannot appear spontaneously; they gradually develop from primitive, non-specialized societies. 4) In a multicellular organism a specialization of a cell is practically irreversible. 5) Every organismic set of ordern>1, that is, a multicellular organism as well as a society, is mortal. Civilizations die, and others may come in their place. 6) Barring special inhibitory conditions, all organisms multiply. 7) In cells there must exist specially-regulatory genes besides the so-called structural genes. 8) In basically identically-built organisms, but which are built from different material (proteins), a substitution of a part of one organism for the homologous part of another impairs the normal functioning (protein specificity of different species). 9) Even unicellular organisms show sexual differentiation and polarization. 10) Symbiotic and parasitic phenomena are included in the theory of organismic sets. Finally some general speculations are made in regard to the possibility of discovering laws of physics by pure mathematical reasoning, something in which Einstein has expressed explicit faith. From the above theory, such a thing appears to be possible. Also the idea of Poincaré, that the laws of physics as we perceive them are largely due to our psychobiological structure, is discussed.     

A mutation in the essential and widely conserved DAMAGED DNA BINDING1‐Cullin4 ASSOCIATED FACTOR gene OZS3 causes hypersensitivity to zinc excess,cold and UV stress in Arabidopsis thaliana

The overly zinc sensitive Arabidopsis thaliana mutant ozs3 shows reduced growth of the primary root, which is exacerbated by an excess specifically of Zn ions. In addition, ozs3 plants display various subtle developmental phenotypes, such as longer petioles and early flowering. Also, ozs3 seedlings are completely but reversibly growth‐arrested when shifted to 4°C. The causal mutation was mapped to a gene encoding a putative substrate‐recognition receptor of cullin4 E3 ligases. OZS3 orthologous genes can be found in almost all eukaryotic genomes. Most species from Schizosaccharomyces pombe to Homo sapiens, and including A. thaliana, possess one ortholog. No functional data are available for these genes in any of the multicellular model systems. CRISPR‐Cas9‐mediated knockout demonstrated that a complete loss of OZS3 function is embryo‐lethal, indicating essentiality of OZS3 and its orthologs. The OZS3 protein interacts with the adaptor protein DAMAGED DNA BINDING1 (DDB1) in the nucleus. Thus, it is indeed a member of the large yet poorly characterized family of DDB1‐cullin4 associated factors in plants. Mutant phenotypes of ozs3 plants are apparently caused by the weakened DDB1–OZS3 interaction as a result of the exchange of a conserved amino acid near the conserved WDxR motif.     

A Statistical Framework for Improving Genomic Annotations of Prokaryotic Essential Genes

Large-scale systematic analysis of gene essentiality is an important step closer toward unraveling the complex relationship between genotypes and phenotypes. Such analysis cannot be accomplished without unbiased and accurate annotations of essential genes. In current genomic databases, most of the essential gene annotations are derived from whole-genome transposon mutagenesis (TM), the most frequently used experimental approach for determining essential genes in microorganisms under defined conditions. However, there are substantial systematic biases associated with TM experiments. In this study, we developed a novel Poisson model–based statistical framework to simulate the TM insertion process and subsequently correct the experimental biases. We first quantitatively assessed the effects of major factors that potentially influence the accuracy of TM and subsequently incorporated relevant factors into the framework. Through iteratively optimizing parameters, we inferred the actual insertion events occurred and described each gene’s essentiality on probability measure. Evaluated by the definite mapping of essential gene profile in Escherichia coli, our model significantly improved the accuracy of original TM datasets, resulting in more accurate annotations of essential genes. Our method also showed encouraging results in improving subsaturation level TM datasets. To test our model’s broad applicability to other bacteria, we applied it to Pseudomonas aeruginosa PAO1 and Francisella tularensis novicida TM datasets. We validated our predictions by literature as well as allelic exchange experiments in PAO1. Our model was correct on six of the seven tested genes. Remarkably, among all three cases that our predictions contradicted the TM assignments, experimental validations supported our predictions. In summary, our method will be a promising tool in improving genomic annotations of essential genes and enabling large-scale explorations of gene essentiality. Our contribution is timely considering the rapidly increasing essential gene sets. A Webserver has been set up to provide convenient access to this tool. All results and source codes are available for download upon publication at http://research.cchmc.org/essentialgene/.