Determinants of associations between codon and amino acid usage patterns of microbial communities and the environment inferred based on a cross-biome metagenomic analysis.

Codon and amino acid usage were associated with almost every aspect of microbial life. However, how the environment may impact the codon and amino acid choice of microbial communities at the habitat level is not clearly understood. Therefore, in this study, we analyzed codon and amino acid usage patterns of a large number of environmental samples collected from diverse ecological niches. Our results suggested that samples derived from similar environmental niches, in general, show overall similar codon and amino acid distribution as compared to samples from other habitats. To substantiate the relative impact of the environment, we considered several factors, such as their similarity in GC content, or in functional or taxonomic abundance. Our analysis demonstrated that none of these factors can fully explain the trends that we observed at the codon or amino acid level implying a direct environmental influence on them. Further, our analysis demonstrated different levels of selection on codon bias in different microbial communities with the highest bias in host-associated environments such as the digestive system or oral samples and the lowest level of selection in soil and water samples. Considering a large number of metagenomic samples here we showed that microorganisms collected from similar environmental backgrounds exhibit similar patterns of codon and amino acid usage irrespective of the location or time from where the samples were collected. Thus our study suggested a direct impact of the environment on codon and amino usage of microorganisms that cannot be explained considering the influence of other factors.

Exploring Potential Signals of Selection for Disordered Residues in Prokaryotic and Eukaryotic Proteins.

Intrinsically disordered proteins (IDPs) are an important class of proteins in all domains of life for their functional importance. However, how nature has shaped the disorder potential of prokaryotic and eukaryotic proteins is still not clearly known. Randomly generated sequences are free of any selective constraints, thus these sequences are commonly used as null models. Considering different types of random protein models, here we seek to understand how the disorder potential of natural eukaryotic and prokaryotic proteins differs from random sequences. Comparing proteome-wide disorder content between real and random sequences of 12 model organisms, we noticed that eukaryotic proteins are enriched in disordered regions compared to random sequences, but in prokaryotes such regions are depleted. By analyzing the position-wise disorder profile, we show that there is a generally higher disorder near the N- and C-terminal regions of eukaryotic proteins as compared to the random models; however, either no or a weak such trend was found in prokaryotic proteins. Moreover, here we show that this preference is not caused by the amino acid or nucleotide composition at the respective sites. Instead, these regions were found to be endowed with a higher fraction of protein-protein binding sites, suggesting their functional importance. We discuss several possible explanations for this pattern, such as improving the efficiency of protein-protein interaction, ribosome movement during translation, and post-translational modification. However, further studies are needed to clearly understand the biophysical mechanisms causing the trend.

Genome-wide analysis of horizontally acquired genes in the genus Mycobacterium.

Horizontal gene transfer (HGT) was attributed as a major driving force for the innovation and evolution of prokaryotic genomes. Previously, multiple research endeavors were undertaken to decipher HGT in different bacterial lineages. The genus Mycobacterium houses some of the most deadly human pathogens; however, the impact of HGT in Mycobacterium has never been addressed in a systematic way. Previous initiatives to explore the genomic imprints of HGTs in Mycobacterium were focused on few selected species, specifically among the members of Mycobacterium tuberculosis complex. Considering the recent availability of a large number of genomes, the current study was initiated to decipher the probable events of HGTs among 109 completely sequenced Mycobacterium species. Our comprehensive phylogenetic analysis with more than 9,000 families of Mycobacterium proteins allowed us to list several instances of gene transfers spread across the Mycobacterium phylogeny. Moreover, by examining the topology of gene phylogenies here, we identified the species most likely to donate and receive these genes and provided a detailed overview of the putative functions these genes may be involved in. Our study suggested that horizontally acquired foreign genes had played an enduring role in the evolution of Mycobacterium genomes and have contributed to their metabolic versatility and pathogenicity.

A protocol for culturing environmental strains of the Buruli ulcer agent, Mycobacterium ulcerans.

Contaminations and fastidiousness of M. ulcerans may have both hamper isolation of strains from environmental sources. We aimed to optimize decontamination and culture of environmental samples to circumvent both limitations. Three strains of M. ulcerans cultured onto Middlebrook 7H10 at 30 °C for 20 days yielded a significantly higher number of colonies in micro-aerophilic atmosphere compared to ambient atmosphere, 5% CO2 and anaerobic atmosphere. In a second step, we observed that M. ulcerans genome uniquely encoded chitinase, fucosidase and A-D-GlcNAc-diphosphoryl polyprenol A-3-L-rhamnosyl transferase giving M. ulcerans the potential to metabolize chitine, fucose and N-acetyl galactosamine (NAG), respectively. A significant growth-promoting effect of 0.2 mg/mL chitin (p < 0.05), 0.01 mg/mL N-acetyl galactosamine (p < 0.05), 0.01 mg/mL fucose (p < 0.05) was observed with M. ulcerans indicating that NAG alone or combined with fucose and chitin could complement Middlebrook 7H10. Finally, the protocol combining 1% chlorhexidine decontamination with micro-aerophilic incubation on Middlebrook 7H10 medium containing chitin (0.2%), NAG (0.01%) and fucose (0.01%) medium and auto-fluorescence detection of colonies allowed for the isolation of one mycolactone-encoding strain from Thryonomys swinderianus (aulacode) feces specimens collected near the Kossou Dam, Côte d'Ivoire. We propose that incubation of chlorhexidine-decontaminated environmental specimens on Middlebrook 7H10-enriched medium under micro-aerophilic atmosphere at 30 °C may be used for the tentative isolation of M. ulcerans strains from potential environmental sources.

Phylogenomic Analysis of ß-Lactamase in Archaea and Bacteria Enables the Identification of Putative New Members.

ß-lactamases are enzymes which are commonly produced by bacteria and which degrade the ß-lactam ring of ß-lactam antibiotics, namely penicillins, cephalosporins, carbapenems, and monobactams, and inactivate these antibiotics. We performed a rational and comprehensive investigation of ß-lactamases in different biological databases. In this study, we constructed hidden Markov model profiles as well as the ancestral sequence of four classes of ß-lactamases (A, B, C, and D), which were used to identify potential ß-lactamases from environmental metagenomic (1206), human microbiome metagenomic (6417), human microbiome reference genome (1310), and NCBI's nonredundant databases (44101). Our analysis revealed the existence of putative ß-lactamases in the metagenomic databases, which appeared to be similar to the four different molecular classes (A-D). This is the first report on the large-scale phylogenetic diversity of new members of ß-lactamases, and our results revealed that metagenomic database dark-matter contains ß-lactamase-like antibiotic resistance genes.

Insights into human intrinsically disordered proteins from their gene expression profile.

Expression level provides important clues about gene function. Previously, various efforts have been undertaken to profile human genes according to their expression level. Intrinsically disordered proteins (IDPs) do not adopt any rigid conformation under physiological conditions, however, are considered as an important functional class in all domains of life. Based on a human tissue-averaged gene expression level, previous studies showed that IDPs are expressed at a lower level than ordered globular proteins. Here, we examined the gene expression pattern of human ordered and disordered proteins in 32 normal tissues. We noticed that in most of the tissues, ordered and disordered proteins are expressed at a similar level. Moreover, in a number of tissues IDPs were found to be expressed at a higher level than ordered proteins. Rigorous statistical analyses suggested that the lower tissue-averaged gene expression level of IDPs (reported earlier) may be the consequence of their biased gene expression in some specific tissues and higher protein length. When we considered the gene repertory of each tissue we noticed that a number of human tissues (brain, testes, etc.) selectively express a higher fraction of disordered proteins, which help them to maintain higher protein connectivity by forming disordered binding motifs and to sustain their functional specificities. Our results demonstrated that the disordered proteins are indispensable in these tissues for their functional advantages.

MimiLook: A Phylogenetic Workflow for Detection of Gene Acquisition in Major Orthologous Groups of Megavirales.

With the inclusion of new members, understanding about evolutionary mechanisms and processes by which members of the proposed order, Megavirales, have evolved has become a key area of interest. The central role of gene acquisition has been shown in previous studies. However, the major drawback in gene acquisition studies is the focus on few MV families or putative families with large variation in their genetic structure. Thus, here we have tried to develop a methodology by which we can detect horizontal gene transfers (HGTs), taking into consideration orthologous groups of distantly related Megavirale families. Here, we report an automated workflow MimiLook, prepared as a Perl command line program, that deduces orthologous groups (OGs) from ORFomes of Megavirales and constructs phylogenetic trees by performing alignment generation, alignment editing and protein-protein BLAST (BLASTP) searching across the National Center for Biotechnology Information (NCBI) non-redundant (nr) protein sequence database. Finally, this tool detects statistically validated events of gene acquisitions with the help of the T-REX algorithm by comparing individual gene tree with NCBI species tree. In between the steps, the workflow decides about handling paralogs, filtering outputs, identifying Megavirale specific OGs, detection of HGTs, along with retrieval of information about those OGs that are monophyletic with organisms from cellular domains of life. By implementing MimiLook, we noticed that nine percent of Megavirale gene families (i.e., OGs) have been acquired by HGT, 80% OGs were Megaviralespecific and eight percent were found to be sharing common ancestry with members of cellular domains (Eukaryote, Bacteria, Archaea, Phages or other viruses) and three percent were ambivalent. The results are briefly discussed to emphasize methodology. Also, MimiLook is relevant for detecting evolutionary scenarios in other targeted phyla with user defined modifications. It can be accessed at following link 10.6084/m9.figshare.4653622.

Expression Pattern Similarities Support the Prediction of Orthologs Retaining Common Functions after Gene Duplication Events.

The identification of functionally equivalent, orthologous genes (functional orthologs) across genomes is necessary for accurate transfer of experimental knowledge from well-characterized organisms to others. This frequently relies on automated, coding sequence-based approaches such as OrthoMCL, Inparanoid, and KOG, which usually work well for one-to-one homologous states. However, this strategy does not reliably work for plants due to the occurrence of extensive gene/genome duplication. Frequently, for one query gene, multiple orthologous genes are predicted in the other genome, and it is not clear a priori from sequence comparison and similarity which one preserves the ancestral function. We have studied 11 organ-dependent and stress-induced gene expression patterns of 286 Arabidopsis lyrata duplicated gene groups and compared them with the respective Arabidopsis (Arabidopsis thaliana) genes to predict putative expressologs and nonexpressologs based on gene expression similarity. Promoter sequence divergence as an additional tool to substantiate functional orthology only partially overlapped with expressolog classification. By cloning eight A. lyrata homologs and complementing them in the respective four Arabidopsis loss-of-function mutants, we experimentally proved that predicted expressologs are indeed functional orthologs, while nonexpressologs or nonfunctionalized orthologs are not. Our study demonstrates that even a small set of gene expression data in addition to sequence homologies are instrumental in the assignment of functional orthologs in the presence of multiple orthologs.

Overlapping genes: A significant genomic correlate of prokaryotic growth rates.

Elucidating the genomic features influencing prokaryotic growth rates has always been a study of interest. Previously, it was observed that overlapping genes (OGs) play a crucial role in the prokaryotic genome size reduction. This study is focused to explore whether OGs act as a potential correlate of prokaryotic growth rates. For this purpose, we compiled a dataset of 25 archaeal and 117 eubacterial genomes and analyzed the inter-correlation between the proportion of overlapping regions in these genomes with their growth rates. Here, we observed that the proportion of overlapping region holds a significant negative correlation with generation time in archaeal domain, whereas no correlation was observed in the eubacterial domain. However, after masking the effect of tRNA, rRNA multiplicity and environmental diversity, OGs show an independent effect over growth rates in the eubacterial domain as well as in the archaeal domain. Moreover, the influence of OGs on prokaryotic growth rates provides different delineations in archaeal and eubacterial domains. In archaea, both long overlap frequency (LOF) and short overlap frequency (SOF) influence the growth rates by increasing the degree of operonization. On the contrary, in the case of bacteria, neither SOF nor LOF plays any significant role in achieving faster growth rates.

Exploring the evolutionary rate differences between human disease and non-disease genes.

Comparisons of evolutionary features between human disease and non-disease genes have a wide implication to understand the genetic basis of human disease genes. However, it has not yet been resolved whether disease genes evolve at slower or faster rate than the non-disease genes. To resolve this controversy, here we integrated human disease genes from several databases and compared their protein evolutionary rates with non-disease genes in both housekeeping and tissue-specific group. We noticed that in tissue specific group, disease genes evolve significantly at a slower rate than non-disease genes. However, we found no significant difference in evolutionary rates between disease and non-disease genes in housekeeping group. Tissue specific disease genes have a higher protein complex number, elevated gene expression level and are also associated with conserve biological processes. Finally, our regression analysis suggested that protein complex number followed by protein multifunctionality independently modulates the evolutionary rate of human disease genes.

Elucidating evolutionary features and functional implications of orphan genes in Leishmania major.

Orphan genes are protein coding genes that lack recognizable homologs in other organisms. These genes were reported to comprise a considerable fraction of coding regions in all sequenced genomes and thought to be allied with organism's lineage-specific traits. However, their evolutionary persistence and functional significance still remain elusive. Due to lack of homologs with the host genome and for their probable lineage-specific functional roles, orphan gene product of pathogenic protozoan might be considered as the possible therapeutic targets. Leishmania major is an important parasitic protozoan of the genus Leishmania that is associated with the disease cutaneous leishmaniasis. Therefore, evolutionary and functional characterization of orphan genes in this organism may help in understanding the factors prevailing pathogen evolution and parasitic adaptation. In this study, we systematically identified orphan genes of L. major and employed several in silico analyses for understanding their evolutionary and functional attributes. To trace the signatures of molecular evolution, we compared their evolutionary rate with non-orphan genes. In agreement with prior observations, here we noticed that orphan genes evolve at a higher rate as compared to non-orphan genes. Lower sequence conservation of orphan genes was previously attributed solely due to their younger gene age. However, here we observed that together with gene age, a number of genomic (like expression level, GC content, variation in codon usage) and proteomic factors (like protein length, intrinsic disorder content, hydropathicity) could independently modulate their evolutionary rate. We considered the interplay of all these factors and analyzed their relative contribution on protein evolutionary rate by regression analysis. On the functional level, we observed that orphan genes are associated with regulatory, growth factor and transport related processes. Moreover, these genes were found to be enriched with various types of interaction and trafficking motifs, implying their possible involvement in host-parasite interactions. Thus, our comprehensive analysis of L. major orphan genes provided evidence for their extensive roles in host-pathogen interactions and virulence.

Overlapping genes: a new strategy of thermophilic stress tolerance in prokaryotes.

Overlapping genes (OGs) draw the focus of recent day's research. However, the significance of OGs in prokaryotic genomes remained unexplored. As an adaptation to high temperature, thermophiles were shown to eliminate their intergenic regions. Therefore, it could be possible that prokaryotes would increase their OG content to adapt to high temperature. To test this hypothesis, we carried out a comparative study on OG frequency of 256 prokaryotic genomes comprising both thermophiles and non-thermophiles. It was found that thermophiles exhibit higher frequency of overlapping genes than non-thermophiles. Moreover, overlap frequency was found to correlate with optimal growth temperature (OGT) in prokaryotes. Long overlap frequency was found to hold a positive correlation with OGT resulting in an abundance of long overlaps in thermophiles compared to non-thermophiles. On the other hand, short overlap (1-4 nucleotides) frequency (SOF) did not yield any direct correlation with OGT. However, the correlation of SOF with CAIavg (extent of variation of codon usage bias measured as the mean of codon adaptation index of all genes in a given genome) and IG% (proportion of intergenic regions) indicate that they might upregulate the aforementioned factors (CAIavg and IG%) which are already known to be vital forces for thermophilic adaptation. From these evidences, we propose that the OG content bears a strong link to thermophily. Long overlaps are important for their genome compaction and short overlaps are important to uphold high CAIavg. Our findings will surely help in better understanding of the significance of overlapping gene content in prokaryotic genomes.

GC-made protein disorder sheds new light on vertebrate evolution.

At the emergence of endothermic vertebrates, GC rich regions of the ectothermic ancestral genomes underwent a significant GC increase. Such an increase was previously postulated to increase thermodynamic and structural stability of proteins through selective increase of protein hydrophobicity. Here, we found that, increase in GC content promotes a higher content of disorder promoting amino acid in endothermic vertebrates proteins and that the increase in hydrophobicity is mainly due to a higher content of the small disorder promoting amino acid alanine. In endothermic vertebrates, prevalence of disordered residues was found to promote functional diversity of proteins encoded by GC rich genes. Higher fraction of disordered residues in this group of proteins was also found to minimize their aggregation tendency. Thus, we propose that the GC transition has favored disordered residues to promote functional diversity in GC rich genes, and to protect them against functional loss by protein misfolding.

Prevalent structural disorder carries signature of prokaryotic adaptation to oxic atmosphere.

Microbes have adopted efficient mechanisms to contend with environmental changes. The emergence of oxygen was a major event that led to an abrupt change in Earth's atmosphere. To adjust with this shift in environmental condition ancient microbes must have undergone several modifications. Although some proteomic and genomic attributes were proposed to facilitate survival of microorganisms in the presence of oxygen, the process of adaptation still remains elusive. Recent studies have focused that intrinsically disordered proteins play crucial roles in adaptation to a wide range of ecological conditions. Therefore, it is likely that disordered proteins could also play indispensable roles in microbial adaptation to the aerobic environment. To test this hypothesis we measured the disorder content of 679 prokaryotes from four oxygen requirement groups. Our result revealed that aerobic proteomes are endowed with the highest protein disorder followed by facultative microbes. Minimal disorder was observed in anaerobic and microaerophilic microbes with no significant difference in their disorder content. Considering all the potential confounding factors that can modulate protein disorder, here we established that the high protein disorder in aerobic microbe is not a by-product of adaptation to any other selective pressure. On the functional level, we found that the high disorder in aerobic proteomes has been utilized for processes that are important for their aerobic lifestyle. Moreover, aerobic proteomes were found to be enriched with disordered binding sites and to contain transcription factors with high disorder propensity. Based on our results, here we proposed that the high protein disorder is an adaptive opportunity for aerobic microbes to fit with the genomic and functional complexities of the aerobic lifestyle.

Insights into the evolutionary features of human neurodegenerative diseases.

Comparative analyses between human disease and non-disease genes are of great interest in understanding human disease gene evolution. However, the progression of neurodegenerative diseases (NDD) involving amyloid formation in specific brain regions is still unknown. Therefore, in this study, we mainly focused our analysis on the evolutionary features of human NDD genes with respect to non-disease genes. Here, we observed that human NDD genes are evolutionarily conserved relative to non-disease genes. To elucidate the conserved nature of NDD genes, we incorporated the evolutionary attributes like gene expression level, number of regulatory miRNAs, protein connectivity, intrinsic disorder content and relative aggregation propensity in our analysis. Our studies demonstrate that NDD genes have higher gene expression levels in favor of their lower evolutionary rates. Additionally, we observed that NDD genes have higher number of different regulatory miRNAs target sites and also have higher interaction partners than the non-disease genes. Moreover, miRNA targeted genes are known to have higher disorder content. In contrast, our analysis exclusively established that NDD genes have lower disorder content. In favor of our analysis, we found that NDD gene encoded proteins are enriched with multi interface hubs (party hubs) with lower disorder contents. Since, proteins with higher disorder content need to adapt special structure to reduce their aggregation propensity, NDD proteins found to have elevated relative aggregation propensity (RAP) in support of their lower disorder content. Finally, our categorical regression analysis confirmed the underlined relative dominance of protein connectivity, 3'UTR length, RAP, nature of hubs (singlish/multi interface) and disorder content for such evolutionary rates variation between human NDD genes and non-disease genes.