Analysis of multipartite bacterial genomes using alignment free and alignment-based pipelines.

Since the discovery of second chromosome in Rhodobacter sphaeroides 2.4.1 in 1989, multipartite genomes have been reported in over three hundred bacterial species under nine different phyla. This has shattered the unipartite (single chromosome) genome dogma in bacteria. Since then, many questions on various aspects of multipartite genomes in bacteria have been addressed. However, our understanding of how multipartite genomes emerge and evolve is still lacking. Importantly, the knowledge of genetic factors underlying the differences in multipartite and single-chromosome genomes is lacking. In this work, we have performed comparative evolutionary and functional genomics analyses to identify molecular factors that discriminate multipartite from unipartite bacteria, with the goal to decipher taxon-specific factors, and those that are prevalent across the taxa, underlying these traits. We assessed the roles of evolutionary mechanisms, specifically gene gain, in driving the divergence of bacteria with single and multiple chromosomes. In addition, we performed functional genomic analysis to garner support for our findings from comparative evolutionary analysis. We found genes such as those encoding conserved hypothetical proteins in Deinococcus radiodurans R1, and putative phage phi-C31 gp36 major capsid like and hypothetical proteins in Rhodobacter sphaeroides 2.4.1, which are located on accessory chromosomes in these bacteria but were not found in the inferred ancestral sequences, and on the primary chromosomes, as well as were not found in their closest relatives with single chromosome within the same clade. Our study shines a new light on the potential roles of the secondary chromosomes in helping bacteria with multipartite genomes to adapt to specialized environments or growth conditions.

Prediction of Bacterial sRNAs Using Sequence-Derived Features and Machine Learning.

Small ribonucleic acid (sRNA) sequences are 50-500 nucleotide long, noncoding RNA (ncRNA) sequences that play an important role in regulating transcription and translation within a bacterial cell. As such, identifying sRNA sequences within an organism's genome is essential to understand the impact of the RNA molecules on cellular processes. Recently, numerous machine learning models have been applied to predict sRNAs within bacterial genomes. In this study, we considered the sRNA prediction as an imbalanced binary classification problem to distinguish minor positive sRNAs from major negative ones within imbalanced data and then performed a comparative study with six learning algorithms and seven assessment metrics. First, we collected numerical feature groups extracted from known sRNAs previously identified in Salmonella typhimurium LT2 (SLT2) and Escherichia coli K12 (E. coli K12) genomes. Second, as a preliminary study, we characterized the sRNA-size distribution with the conformity test for Benford's law. Third, we applied six traditional classification algorithms to sRNA features and assessed classification performance with seven metrics, varying positive-to-negative instance ratios, and utilizing stratified 10-fold cross-validation. We revisited important individual features and feature groups and found that classification with combined features perform better than with either an individual feature or a single feature group in terms of Area Under Precision-Recall curve (AUPR). We reconfirmed that AUPR properly measures classification performance on imbalanced data with varying imbalance ratios, which is consistent with previous studies on classification metrics for imbalanced data. Overall, eXtreme Gradient Boosting (XGBoost), even without exploiting optimal hyperparameter values, performed better than the other five algorithms with specific optimal parameter settings. As a future work, we plan to extend XGBoost further to a large amount of published sRNAs in bacterial genomes and compare its classification performance with recent machine learning models' performance.

Differential Selective Pressures Experienced by the Aurora Kinase Gene Family.

Aurora kinases (AKs) are serine/threonine kinases that are essential for cell division. Humans have three AK genes: AKA, AKB, and AKC. AKA is required for centrosome assembly, centrosome separation, and bipolar spindle assembly, and its mutation leads to abnormal spindle morphology. AKB is required for the spindle checkpoint and proper cytokinesis, and mutations cause chromosome misalignment and cytokinesis failure. AKC is expressed in germ cells, and has a role in meiosis analogous to that of AKB in mitosis. Mutation of any of the three isoforms can lead to cancer. AK proteins possess divergent N- and C-termini and a conserved central catalytic domain. We examined the evolution of the AK gene family using an identity matrix and by building a phylogenetic tree. The data suggest that AKA is the vertebrate ancestral gene, and that AKB and AKC resulted from gene duplication in placental mammals. In a nonsynonymous/synonymous rate substitution analysis, we found that AKB experienced the strongest, and AKC the weakest, purifying selection. Both the N- and C-termini and regions within the kinase domain experienced differential selection among the AK isoforms. These differentially selected sequences may be important for species specificity and isoform specificity, and are therefore potential therapeutic targets.

Professional Practices in Undergraduate Research Programs.

The undergraduate research experience (URE) is an important avenue within a college trajectory in which students enhance their critical thinking, learn about the scientific process, and develop the knowledge and values that will guide their future scientific and professional careers. Individual institutions, programs, departments, and faculty administer undergraduate research differently, but each should adhere to a common set of guidelines which govern the research mentoring process. Adherence to standard practices will enhance the research experience for both students and mentors. This article examines standards and guidelines for professional practices involving undergraduate research and scholarship, and will discuss lapses and limitations that students and faculty frequently confront. The growth, support, and proper management of undergraduate research programs (URPs) at primarily undergraduate institutions (PUIs) is important for maintaining a talented pool of young scientists, as students benefit greatly from direct interactions with faculty mentors that predominate at PUIs.

A Nonsynonymous/Synonymous Substitution Analysis of the B56 Gene Family Aids in Understanding B56 Isoform Diversity.

Gene duplication leads to the formation of gene families, wherein purifying or neutral selection maintains the original gene function, while diversifying selection confers new functions onto duplicated genes. The B56 gene family is highly conserved; it is encoded by one gene in protists and fungi, and five genes in vertebrates. B56 regulates protein phosphatase 2A (PP2A), an abundant heterotrimeric serine/threonine phosphatase that functions as a tumor suppressor and consists of a scaffolding "A" and catalytic "C" subunit heterodimer bound to a regulatory "B" subunit. Individual regulatory B56 subunits confer disparate functions onto PP2A in various cell-cell signaling pathways. B56 proteins share a conserved central core domain, but have divergent N- and C-termini which play a role in isoform specificity. We carried out a nonsynonymous/synonymous substitution analysis to better understand the divergence of vertebrate B56 genes. When five B56 paralogs from ten vertebrate species were analyzed, the gene family displayed purifying selection; stronger purifying selection was revealed when individual B56 isoforms were analyzed separately. The B56 core experienced stronger purifying selection than the N- and C-termini, which correlates with the presence of several contacts between the core and the AC heterodimer. Indeed, the majority of the contact points that we analyzed between B56 and the AC heterodimer experienced strong purifying selection. B56 subfamilies showed distinct patterns of selection in their N- and C-termini. The C-terminus of the B56-1 subfamily and the N-terminus of the B56-2 subfamily exhibited strong purifying selection, suggesting that these termini carry out subfamily-specific functions, while the opposite termini exhibited diversifying selection and likely carry out isoform-specific functions. We also found reduced synonymous substitutions at the N- and C-termini when grouping B56 genes by species but not by isoform, suggesting species-specific codon bias may have a role in regulating B56 gene expression.

Distribution and diversity of ribosome binding sites in prokaryotic genomes.

BACKGROUND: Prokaryotic translation initiation involves the proper docking, anchoring, and accommodation of mRNA to the 30S ribosomal subunit. Three initiation factors (IF1, IF2, and IF3) and some ribosomal proteins mediate the assembly and activation of the translation initiation complex. Although the interaction between Shine-Dalgarno (SD) sequence and its complementary sequence in the 16S rRNA is important in initiation, some genes lacking an SD ribosome binding site (RBS) are still well expressed. The objective of this study is to examine the pattern of distribution and diversity of RBS in fully sequenced bacterial genomes. The following three hypotheses were tested: SD motifs are prevalent in bacterial genomes; all previously identified SD motifs are uniformly distributed across prokaryotes; and genes with specific cluster of orthologous gene (COG) functions differ in their use of SD motifs. RESULTS: Data for 2,458 bacterial genomes, previously generated by Prodigal (PROkaryotic DYnamic programming Gene-finding ALgorithm) and currently available at the National Center for Biotechnology Information (NCBI), were analyzed. Of the total genes examined, ~77.0% use an SD RBS, while ~23.0% have no RBS. Majority of the genes with the most common SD motifs are distributed in a manner that is representative of their abundance for each COG functional category, while motifs 13 (5'-GGA-3'/5'-GAG-3'/5'-AGG-3') and 27 (5'-AGGAGG-3') appear to be predominantly used by genes for information storage and processing, and translation and ribosome biogenesis, respectively. CONCLUSION: These findings suggest that an SD sequence is not obligatory for translation initiation; instead, other signals, such as the RBS spacer, may have an overarching influence on translation of mRNAs. Subsequent analyses of the 5' secondary structure of these mRNAs may provide further insight into the translation initiation mechanism.

Evolutionary Analysis of the B56 Gene Family of PP2A Regulatory Subunits.

Protein phosphatase 2A (PP2A) is an abundant serine/threonine phosphatase that functions as a tumor suppressor in numerous cell-cell signaling pathways, including Wnt, myc, and ras. The B56 subunit of PP2A regulates its activity, and is encoded by five genes in humans. B56 proteins share a central core domain, but have divergent amino- and carboxy-termini, which are thought to provide isoform specificity. We performed phylogenetic analyses to better understand the evolution of the B56 gene family. We found that B56 was present as a single gene in eukaryotes prior to the divergence of animals, fungi, protists, and plants, and that B56 gene duplication prior to the divergence of protostomes and deuterostomes led to the origin of two B56 subfamilies, B56αßε and B56γδ. Further duplications led to three B56αßε genes and two B56γδ in vertebrates. Several nonvertebrate B56 gene names are based on distinct vertebrate isoform names, and would best be renamed. B56 subfamily genes lack significant divergence within primitive chordates, but each became distinct in complex vertebrates. Two vertebrate lineages have undergone B56 gene loss, Xenopus and Aves. In Xenopus, B56δ function may be compensated for by an alternatively spliced transcript, B56δ/γ, encoding a B56δ-like amino-terminal region and a B56γ core.

Evolutionary constraints and expression analysis of gene duplications in Rhodobacter sphaeroides 2.4.1.

BACKGROUND: Gene duplication is a major force that contributes to the evolution of new metabolic functions in all organisms. Rhodobacter sphaeroides 2.4.1 is a bacterium that displays a wide degree of metabolic versatility and genome complexity and therefore is a fitting model for the study of gene duplications in bacteria. A comprehensive analysis of 234 duplicate gene-pairs in R. sphaeroides was performed using structural constraint and expression analysis. RESULTS: The results revealed that most gene-pairs in in-paralogs are maintained under negative selection (ω ≤ 0.3), but the strength of selection differed among in-paralog gene-pairs. Although in-paralogs located on different replicons are maintained under purifying selection, the duplicated genes distributed between the primary chromosome (CI) and the second chromosome (CII) are relatively less selectively constrained than the gene-pairs located within each chromosome. The mRNA expression patterns of duplicate gene-pairs were examined through microarray analysis of this organism grown under seven different growth conditions. Results revealed that ~62% of paralogs have similar expression patterns (cosine ≥ 0.90) over all of these growth conditions, while only ~7% of paralogs are very different in their expression patterns (cosine < 0.50). CONCLUSIONS: The overall findings of the study suggest that only a small proportion of paralogs contribute to the metabolic diversity and the evolution of novel metabolic functions in R. sphaeroides. In addition, the lack of relationships between structural constraints and gene-pair expression suggests that patterns of gene-pair expression are likely associated with conservation or divergence of gene-pair promoter regions and other coregulation mechanisms.

Bacterial genome chimaerism and the origin of mitochondria.

Many studies have sought to determine the origin and evolution of mitochondria. Although the Alphaproteobacteria are thought to be the closest relatives of the mitochondrial progenitor, there is dispute as to what its particular sister group is. Some have argued that mitochondria originated from ancestors of the order Rickettsiales, or more specifically of the Rickettsiaceae family, while others believe that ancestors of the family Rhodospirillaceae are also equally likely the progenitors. To resolve some of these disputes, sequence similarity searches and phylogenetic analyses were performed against mitochondria-related proteins in Saccharomyces cerevisiae. The 86 common matches of 5 Alphaproteobacteria (Rickettsia prowazekii, Rhodospirillum rubrum, Rhodopseudomonas palustris, Rhodobacter sphaeroides, and Ochrobactrum anthropi) to yeast mitochondrial proteins were distributed fairly evenly among the 5 species when sorted by highest identity or score. Moreover, exploratory phylogenetic analyses revealed that among these common matches, 44.19% (38) had branched most closely with O. anthropi, while only 34.88% (30) corresponded with Rickettsia prowazekii. More detailed phylogenetic analyses with additional Alphaproteobacteria and including genes from the mitochondria of Reclinomonas americana found matches of mitochondrial genes to those of members of the Rickettsiaceae, Anaplasmataceae, and Rhodospirillaceae families. The results support the idea that notable bacterial genome chimaerism has occurred en route to the formation of mitochondria.

Complex prokaryotic genome structure: rapid evolution of chromosome II.

Although many bacteria with two chromosomes have been sequenced, the roles of such complex genome structuring are still unclear. To uncover levels of chromosome I (CI) and chromosome II (CII) sequence divergence, Mauve 2.2.0 was used to align the CI- and CII-specific sequences of bacteria with complex genome structuring in two sets of comparisons: the first set was conducted among the CI and CII of bacterial strains of the same species, while the second set was conducted among the CI and CII of species in Alphaproteobacteria that possess two chromosomes. The analyses revealed a rapid evolution of CII-specific DNA sequences compared with CI-specific sequences in a majority of organisms. In addition, levels of protein divergence between CI-specific and CII-specific genes were determined using phylogenetic analyses and confirmed the DNA alignment findings. Analysis of synonymous and nonsynonymous substitutions revealed that the structural and functional constraints on CI and CII genes are not significantly different. Also, horizontal gene transfer estimates in selected organisms demonstrated that CII in many species has acquired higher levels of horizontally transferred segments than CI. In summary, rapid evolution of CII may perform particular roles for organisms such as aiding in adapting to specialized niches.

The prevalence of gene duplications and their ancient origin in Rhodobacter sphaeroides 2.4.1.

BACKGROUND: Rhodobacter sphaeroides 2.4.1 is a metabolically versatile organism that belongs to α-3 subdivision of Proteobacteria. The present study was to identify the extent, history, and role of gene duplications in R. sphaeroides 2.4.1, an organism that possesses two chromosomes. RESULTS: A protein similarity search (BLASTP) identified 1247 orfs (~29.4% of the total protein coding orfs) that are present in 2 or more copies, 37.5% (234 gene-pairs) of which exist in duplicate copies. The distribution of the duplicate gene-pairs in all Clusters of Orthologous Groups (COGs) differed significantly when compared to the COG distribution across the whole genome. Location plots revealed clusters of gene duplications that possessed the same COG classification. Phylogenetic analyses were performed to determine a tree topology predicting either a Type-A or Type-B phylogenetic relationship. A Type-A phylogenetic relationship shows that a copy of the protein-pair matches more with an ortholog from a species closely related to R. sphaeroides while a Type-B relationship predicts the highest match between both copies of the R. sphaeroides protein-pair. The results revealed that ~77% of the proteins exhibited a Type-A phylogenetic relationship demonstrating the ancient origin of these gene duplications. Additional analyses on three other strains of R. sphaeroides revealed varying levels of gene loss and retention in these strains. Also, analyses on common gene pairs among the four strains revealed that these genes experience similar functional constraints and undergo purifying selection. CONCLUSIONS: Although the results suggest that the level of gene duplication in organisms with complex genome structuring (more than one chromosome) seems to be not markedly different from that in organisms with only a single chromosome, these duplications may have aided in genome reorganization in this group of eubacteria prior to the formation of R. sphaeroides as gene duplications involved in specialized functions might have contributed to complex genomic development.

Postgenomic adventures with Rhodobacter sphaeroides.

This review describes some of the recent highlights taken from the studies of Rhodobacter sphaeroides 2.4.1. The review is not intended to be comprehensive, but to reflect the bias of the authors as to how the availability of a sequenced and annotated genome, a gene-chip, and proteomic profile as well as comparative genomic analyses can direct the progress of future research in this system.

Construction and validation of the Rhodobacter sphaeroides 2.4.1 DNA microarray: transcriptome flexibility at diverse growth modes.

A high-density oligonucleotide DNA microarray, a genechip, representing the 4.6-Mb genome of the facultative phototrophic proteobacterium, Rhodobacter sphaeroides 2.4.1, was custom-designed and manufactured by Affymetrix, Santa Clara, Calif. The genechip contains probe sets for 4,292 open reading frames (ORFs), 47 rRNA and tRNA genes, and 394 intergenic regions. The probe set sequences were derived from the genome annotation generated by Oak Ridge National Laboratory after extensive revision, which was based primarily upon codon usage characteristic of this GC-rich bacterium. As a result of the revision, numerous missing ORFs were uncovered, nonexistent ORFs were deleted, and misidentified start codons were corrected. To evaluate R. sphaeroides transcriptome flexibility, expression profiles for three diverse growth modes--aerobic respiration, anaerobic respiration in the dark, and anaerobic photosynthesis--were generated. Expression levels of one-fifth to one-third of the R. sphaeroides ORFs were significantly different in cells under any two growth modes. Pathways involved in energy generation and redox balance maintenance under three growth modes were reconstructed. Expression patterns of genes involved in these pathways mirrored known functional changes, suggesting that massive changes in gene expression are the major means used by R. sphaeroides in adaptation to diverse conditions. Differential expression was observed for genes encoding putative new participants in these pathways (additional photosystem genes, duplicate NADH dehydrogenase, ATP synthases), whose functionality has yet to be investigated. The DNA microarray data correlated well with data derived from quantitative reverse transcription-PCR, as well as with data from the literature, thus validating the R. sphaeroides genechip as a powerful and reliable tool for studying unprecedented metabolic versatility of this bacterium.

DNA sequence duplication in Rhodobacter sphaeroides 2.4.1: evidence of an ancient partnership between chromosomes I and II.

The complex genome of Rhodobacter sphaeroides 2.4.1, composed of chromosomes I (CI) and II (CII), has been sequenced and assembled. We present data demonstrating that the R. sphaeroides genome possesses an extensive amount of exact DNA sequence duplication, 111 kb or approximately 2.7% of the total chromosomal DNA. The chromosomal DNA sequence duplications were aligned to each other by using MUMmer. Frequency and size distribution analyses of the exact DNA duplications revealed that the interchromosomal duplications occurred prior to the intrachromosomal duplications. Most of the DNA sequence duplications in the R. sphaeroides genome occurred early in species history, whereas more recent sequence duplications are rarely found. To uncover the history of gene duplications in the R. sphaeroides genome, 44 gene duplications were sampled and then analyzed for DNA sequence similarity against orthologous DNA sequences. Phylogenetic analysis revealed that approximately 80% of the total gene duplications examined displayed type A phylogenetic relationships; i.e., one copy of each member of a duplicate pair was more similar to its orthologue, found in a species closely related to R. sphaeroides, than to its duplicate, counterpart allele. The data reported here demonstrate that a massive level of gene duplications occurred prior to the origin of the R. sphaeroides 2.4.1 lineage. These findings lead to the conclusion that there is an ancient partnership between CI and CII of R. sphaeroides 2.4.1.

Whole-genome shotgun optical mapping of Rhodobacter sphaeroides strain 2.4.1 and its use for whole-genome shotgun sequence assembly.

Rhodobacter sphaeroides 2.4.1 is a facultative photoheterotrophic bacterium with tremendous metabolic diversity, which has significantly contributed to our understanding of the molecular genetics of photosynthesis, photoheterotrophy, nitrogen fixation, hydrogen metabolism, carbon dioxide fixation, taxis, and tetrapyrrole biosynthesis. To further understand this remarkable bacterium, and to accelerate an ongoing sequencing project, two whole-genome restriction maps (EcoRI and HindIII) of R. sphaeroides strain 2.4.1 were constructed using shotgun optical mapping. The approach directly mapped genomic DNA by the random mapping of single molecules. The two maps were used to facilitate sequence assembly by providing an optical scaffold for high-resolution alignment and verification of sequence contigs. Our results show that such maps facilitated the closure of sequence gaps by the early detection of nascent sequence contigs during the course of the whole-genome shotgun sequencing process.