Annotation Of Bacterial Genomes Research Articles

Small proteins in Gram-positive bacteria.

Small proteins comprising less than 100 amino acids have been often ignored in bacterial genome annotations. About 10 years ago, focused efforts started to investigate whole peptidomes, which resulted in the discovery of a multitude of small proteins, but only a number of them have been characterized in detail. Generally, small proteins can be either membrane or cytosolic proteins. The latter interact with larger proteins, RNA or even metal ions. Here, we summarize our current knowledge on small proteins from Gram-positive bacteria with a special emphasis on the model organism Bacillus subtilis. Our examples include membrane-bound toxins of type I toxin-antitoxin systems, proteins that block the assembly of higher order structures, regulate sporulation or modulate the RNA degradosome. We do not consider antimicrobial peptides. Furthermore, we present methods for the identification and investigation of small proteins.

Intrinsic tet(L) sub-class in Bacillus velezensis and Bacillus amyloliquefaciens is associated with a reduced susceptibility toward tetracycline.

Annotations of non-pathogenic bacterial genomes commonly reveal putative antibiotic resistance genes and the potential risks associated with such genes is challenging to assess. We have examined a putative tetracycline tet(L) gene (conferring low level tetracycline resistance), present in the majority of all publicly available genomes of the industrially important operational group Bacillus amyloliquefaciens including the species B. amyloliquefaciens, Bacillus siamensis and Bacillus velezensis. The aim was to examine the risk of transfer of the putative tet(L) in operational group B. amyloliquefaciens through phylogenetic and genomic position analysis. These analyses furthermore included tet(L) genes encoded by transferable plasmids and other Gram-positive and -negative bacteria, including Bacillus subtilis. Through phylogenetic analysis, we could group chromosomally and plasmid-encoded tet(L) genes into four phylogenetic clades. The chromosomally encoded putative tet(L) from operational group B. amyloliquefaciens formed a separate phylogenetic clade; was positioned in the same genomic region in the three species; was not flanked by mobile genetic elements and was not found in any other bacterial species suggesting that the gene has been present in a common ancestor before species differentiation and is intrinsic. Therefore the gene is not considered a safety concern, and the risk of transfer to and expression of resistance in other non-related species is considered negligible. We suggest a subgrouping of the tet(L) class into four groups (tet(L)1.1, tet(L)1.2 and tet(L)2.1, tet(L)2.2), corresponding with the phylogenetic grouping and tet(L) from operational group B. amyloliquefaciens referred to as tet(L)2.2. Phylogenetic analysis is a useful tool to correctly differentiate between intrinsic and acquired antibiotic resistance genes.

Hidden in plain sight: challenges in proteomics detection of small ORF-encoded polypeptides.

Genomic studies of bacteria have long pointed toward widespread prevalence of small open reading frames (sORFs) encoding for short proteins, <100 amino acids in length. Despite the mounting genomic evidence of their robust expression, relatively little progress has been made in their mass spectrometry-based detection and various blanket statements have been used to explain this observed discrepancy. In this study, we provide a large-scale riboproteogenomics investigation of the challenging nature of proteomic detection of such small proteins as informed by conditional translation data. A panel of physiochemical properties alongside recently developed mass spectrometry detectability metrics was interrogated to provide a comprehensive evidence-based assessment of sORF-encoded polypeptide (SEP) detectability. Moreover, a large-scale proteomics and translatomics compendium of proteins produced by Salmonella Typhimurium (S. Typhimurium), a model human pathogen, across a panel of growth conditions is presented and used in support of our in silico SEP detectability analysis. This integrative approach is used to provide a data-driven census of small proteins expressed by S. Typhimurium across growth phases and infection-relevant conditions. Taken together, our study pinpoints current limitations in proteomics-based detection of novel small proteins currently missing from bacterial genome annotations.

Bakta: rapid and standardized annotation of bacterial genomes via alignment-free sequence identification.

Command-line annotation software tools have continuously gained popularity compared to centralized online services due to the worldwide increase of sequenced bacterial genomes. However, results of existing command-line software pipelines heavily depend on taxon-specific databases or sufficiently well annotated reference genomes. Here, we introduce Bakta, a new command-line software tool for the robust, taxon-independent, thorough and, nonetheless, fast annotation of bacterial genomes. Bakta conducts a comprehensive annotation workflow including the detection of small proteins taking into account replicon metadata. The annotation of coding sequences is accelerated via an alignment-free sequence identification approach that in addition facilitates the precise assignment of public database cross-references. Annotation results are exported in GFF3 and International Nucleotide Sequence Database Collaboration (INSDC)-compliant flat files, as well as comprehensive JSON files, facilitating automated downstream analysis. We compared Bakta to other rapid contemporary command-line annotation software tools in both targeted and taxonomically broad benchmarks including isolates and metagenomic-assembled genomes. We demonstrated that Bakta outperforms other tools in terms of functional annotations, the assignment of functional categories and database cross-references, whilst providing comparable wall-clock runtimes. Bakta is implemented in Python 3 and runs on MacOS and Linux systems. It is freely available under a GPLv3 license at https://github.com/oschwengers/bakta. An accompanying web version is available at https://bakta.computational.bio.

Philympics 2021: Prophage Predictions Perplex Programs

Background Most bacterial genomes contain integrated bacteriophages—prophages—in various states of decay. Many are active and able to excise from the genome and replicate, while others are cryptic prophages, remnants of their former selves. Over the last two decades, many computational tools have been developed to identify the prophage components of bacterial genomes, and it is a particularly active area for the application of machine learning approaches. However, progress is hindered and comparisons thwarted because there are no manually curated bacterial genomes that can be used to test new prophage prediction algorithms. Methods We present a library of gold-standard bacterial genome annotations that include manually curated prophage annotations, and a computational framework to compare the predictions from different algorithms. We use this suite to compare all extant stand-alone prophage prediction algorithms to identify their strengths and weaknesses. We provide a FAIR dataset for prophage identification, and demonstrate the accuracy, precision, recall, and f1 score from the analysis of seven different algorithms for the prediction of prophages. Results We identified different strengths and weaknesses between the prophage prediction tools. Several tools exhibit exceptional f1 scores, while others have better recall at the expense of more false positives. The tools vary greatly in runtime performance with few exhibiting all desirable qualities for large-scale analyses. Conclusions Our library of gold-standard prophage annotations and benchmarking framework provide a valuable resource for exploring strengths and weaknesses of current and future prophage annotation tools. We discuss caveats and concerns in this analysis, how those concerns may be mitigated, and avenues for future improvements. This framework will help developers identify opportunities for improvement and test updates. It will also help users in determining the tools that are best suited for their analysis.

Discovery of Cyanobacterial Natural Products Containing Fatty Acid Residues*.

In recent years, extensive sequencing and annotation of bacterial genomes has revealed an unexpectedly large number of secondary metabolite biosynthetic gene clusters whose products are yet to be discovered. For example, cyanobacterial genomes contain a variety of gene clusters that likely incorporate fatty acid derived moieties, but for most cases we lack the knowledge and tools to effectively predict or detect the encoded natural products. Here, we exploit the apparent absence of a functional β‐oxidation pathway in cyanobacteria to achieve efficient stable‐isotope‐labeling of their fatty acid derived lipidome. We show that supplementation of cyanobacterial cultures with deuterated fatty acids can be used to easily detect natural product signatures in individual strains. The utility of this strategy is demonstrated in two cultured cyanobacteria by uncovering analogues of the multidrug‐resistance reverting hapalosin, and novel, cytotoxic, lactylate‐nocuolin A hybrids—the nocuolactylates.

Discovery of Cyanobacterial Natural Products Containing Fatty Acid Residues**

AbstractIn recent years, extensive sequencing and annotation of bacterial genomes has revealed an unexpectedly large number of secondary metabolite biosynthetic gene clusters whose products are yet to be discovered. For example, cyanobacterial genomes contain a variety of gene clusters that likely incorporate fatty acid derived moieties, but for most cases we lack the knowledge and tools to effectively predict or detect the encoded natural products. Here, we exploit the apparent absence of a functional β‐oxidation pathway in cyanobacteria to achieve efficient stable‐isotope‐labeling of their fatty acid derived lipidome. We show that supplementation of cyanobacterial cultures with deuterated fatty acids can be used to easily detect natural product signatures in individual strains. The utility of this strategy is demonstrated in two cultured cyanobacteria by uncovering analogues of the multidrug‐resistance reverting hapalosin, and novel, cytotoxic, lactylate‐nocuolin A hybrids—the nocuolactylates.

Simple, Reliable, and Time-Efficient Manual Annotation of Bacterial Genomes with MAISEN.

Over the last 15years, the costs of DNA sequencing have sharply fallen, effectively shifting the costs of DNA analysis from sequencing to bioinformatic curation and storage. A huge number of available DNA sequences (including genomes and metagenomes) resulted in the development of various tools for sequence annotation. While much effort has been invested into the development of automatic annotation pipelines, manual curation of their results is still necessary in order to obtain a reliable and strictly validated data. Unfortunately, due to its time-consuming nature, manual annotation is now rarely used.In this chapter, a protocol for efficient manual annotation of prokaryotic DNA sequences using a novel bioinformatic tool-MAISEN ( http://maisen.ddlemb.com ), is presented. MAISEN is a free, web-based tool designed to accelerate manual annotation, by providing the user with simple interface and precomputed alignments for each predicted feature. It was designed to be available for every scientist, regardless of their bioinformatic proficiency.

1207. Combining standard bacterial vaginosis treatment with cystine uptake inhibitors to block growth of Lactobacillus iners is a potential a target for shifting the cervicovaginal microbiota towards health-associated Lactobacillus crispatus-dominant communities

BackgroundCervicovaginal microbiota domination by Lactobacillus crispatus is associated with beneficial health outcomes, whereas L. iners dominance has more adverse associations. However bacterial vaginosis (BV) treatment with metronidazole (MTZ) typically leads to domination by L. iners rather than L. crispatus. L. iners differs from other lactobacilli by its inability to grow in MRS media. We hypothesized that exploring this growth difference would identify targets for selective L. iners inhibition.MethodsBacteria were grown anaerobically. Nutrient uptake and metabolism were assessed using UPLC-MS/MS and isotopically labeled substrates. Bacterial genome annotation employed Prodigal, Roary, and EggNOG. Competition experiments with mock mixed communities were analyzed by 16S rRNA gene sequencing. We confirmed result generalizability using a diverse collection of South African and North American strains and genomes.ResultsSupplementing MRS broth with L-cysteine (Cys) or L-cystine permitted robust L. iners growth, while L. crispatus grew without Cys supplementation. Despite their different growth requirements, neither species could synthesize Cys via canonical pathways. Adding the cystine uptake inhibitors S-methyl-L-cysteine (SMC, Fig 1) or seleno-DL-cystine (SDLC) blocked growth of L. iners but not other lactobacilli, suggesting L. iners lacks mechanisms other lactobacilli use to exploit complex exogenous Cys sources. Notably, cydABCD, an operon with Cys/glutathione transport and redox homeostasis activities, is absent from L. iners but present in non-iners Lactobacillus species. Consistent with possible roles for cydABCD in explaining the observed phenotypes, (1) L. iners failed to take up exogenous glutathione and (2) supplementing MRS with reducing agents permitted L. iners growth, which could be blocked by SMC or SDLC. In growth competitions testing L. iners and L. crispatus within mock BV-like communities, SMC plus MTZ outperformed MTZ alone in promoting L. crispatus dominance (Figs 2&3).Figure 1: S-methyl-L-cysteine (SMC) selectively blocks growth of L. iners but not other cervicovaginal Lactobacillus species in cysteine-supplemented MRS broth. Growth was measured by optical density and inhibition calculated relative to Cys-supplemented no-inhibitor control during exponential growth. Values displayed are median (+/- maximum/minimum) for 3 replicates from a single experiment. In all panels, representative data are shown from 1 of >=2 independent experiments for each bacterial strain and media condition. Results are representative of multiple strains for L. iners (n = 16), L. crispatus (n = 7), and L. jensenii (n = 2). Figure 2: Relative abundance of L. crispatus, L. iners, or various BV-associated bacteria in mock bacterial communities grown in rich, non-selective media with or without metronidazole (MTZ) and/or SMC. Relative abundance was determined by bacterial 16S rRNA gene sequencing. Data are shown for three representative mock communities with 5 replicates per media condition. Figure 3: Ratio of L. crispatus to other species in the mock bacterial communities depicted in Figure 2. Statistical significance determined via 1-way ANOVA of log10-transformed ratios with post-hoc Tukey test; selected pairwise comparisons are shown (***, p < 0.001). Conclusion L. iners has unique requirements for exogenous cysteine/cystine or a reduced environment for growth. Targeting cystine uptake to inhibit L. iners is a potential strategy for shifting cervicovaginal microbiota towards L. crispatus-dominant communities.Disclosures Douglas S. Kwon, MD, PhD, Day Zero Diagnostics (Consultant, Shareholder, Other Financial or Material Support, co-founder)

Combinatorial Glycomic Analyses to Direct CAZyme Discovery for the Tailored Degradation of Canola Meal Non-Starch Dietary Polysaccharides.

Canola meal (CM), the protein-rich by-product of canola oil extraction, has shown promise as an alternative feedstuff and protein supplement in poultry diets, yet its use has been limited due to the abundance of plant cell wall fibre, specifically non-starch polysaccharides (NSP) and lignin. The addition of exogenous enzymes to promote the digestion of CM NSP in chickens has potential to increase the metabolizable energy of CM. We isolated chicken cecal bacteria from a continuous-flow mini-bioreactor system and selected for those with the ability to metabolize CM NSP. Of 100 isolates identified, Bacteroides spp. and Enterococcus spp. were the most common species with these capabilities. To identify enzymes specifically for the digestion of CM NSP, we used a combination of glycomics techniques, including enzyme-linked immunosorbent assay characterization of the plant cell wall fractions, glycosidic linkage analysis (methylation-GC-MS analysis) of CM NSP and their fractions, bacterial growth profiles using minimal media supplemented with CM NSP, and the sequencing and de novo annotation of bacterial genomes of high-efficiency CM NSP utilizing bacteria. The SACCHARIS pipeline was used to select plant cell wall active enzymes for recombinant production and characterization. This approach represents a multidisciplinary innovation platform to bioprospect endogenous CAZymes from the intestinal microbiota of herbivorous and omnivorous animals which is adaptable to a variety of applications and dietary polysaccharides.

Lost and Found: Re-searching and Re-scoring Proteomics Data Aids Genome Annotation and Improves Proteome Coverage.

Prokaryotic genome annotation is heavily dependent on automated gene annotation pipelines that are prone to propagate errors and underestimate genome complexity. We describe an optimized proteogenomic workflow that uses ribosome profiling (ribo-seq) and proteomic data for Salmonella enterica serovar Typhimurium to identify unannotated proteins or alternative protein forms. This data analysis encompasses the searching of cofragmenting peptides and postprocessing with extended peptide-to-spectrum quality features, including comparison to predicted fragment ion intensities. When this strategy is applied, an enhanced proteome depth is achieved, as well as greater confidence for unannotated peptide hits. We demonstrate the general applicability of our pipeline by reanalyzing public Deinococcus radiodurans data sets. Taken together, our results show that systematic reanalysis using available prokaryotic (proteome) data sets holds great promise to assist in experimentally based genome annotation.IMPORTANCE Delineation of open reading frames (ORFs) causes persistent inconsistencies in prokaryote genome annotation. We demonstrate that by advanced (re)analysis of omics data, a higher proteome coverage and sensitive detection of unannotated ORFs can be achieved, which can be exploited for conditional bacterial genome (re)annotation, which is especially relevant in view of annotating the wealth of sequenced prokaryotic genomes obtained in recent years.

Bacterial riboproteogenomics: the era of N-terminal proteoform existence revealed.

With the rapid increase in the number of sequenced prokaryotic genomes, relying on automated gene annotation became a necessity. Multiple lines of evidence, however, suggest that current bacterial genome annotations may contain inconsistencies and are incomplete, even for so-called well-annotated genomes. We here discuss underexplored sources of protein diversity and new methodologies for high-throughput genome reannotation. The expression of multiple molecular forms of proteins (proteoforms) from a single gene, particularly driven by alternative translation initiation, is gaining interest as a prominent contributor to bacterial protein diversity. In consequence, riboproteogenomic pipelines were proposed to comprehensively capture proteoform expression in prokaryotes by the complementary use of (positional) proteomics and the direct readout of translated genomic regions using ribosome profiling. To complement these discoveries, tailored strategies are required for the functional characterization of newly discovered bacterial proteoforms.

ANNOgesic: a Swiss army knife for the RNA-seq based annotation of bacterial/archaeal genomes.

To understand the gene regulation of an organism of interest, a comprehensive genome annotation is essential. While some features, such as coding sequences, can be computationally predicted with high accuracy based purely on the genomic sequence, others, such as promoter elements or noncoding RNAs, are harder to detect. RNA sequencing (RNA-seq) has proven to be an efficient method to identify these genomic features and to improve genome annotations. However, processing and integrating RNA-seq data in order to generate high-resolution annotations is challenging, time consuming, and requires numerous steps. We have constructed a powerful and modular tool called ANNOgesic that provides the required analyses and simplifies RNA-seq-based bacterial and archaeal genome annotation. It can integrate data from conventional RNA-seq and differential RNA-seq and predicts and annotates numerous features, including small noncoding RNAs, with high precision. The software is available under an open source license (ISCL) at https://pypi.org/project/ANNOgesic/.

REPARATION: ribosome profiling assisted (re-)annotation of bacterial genomes.

Prokaryotic genome annotation is highly dependent on automated methods, as manual curation cannot keep up with the exponential growth of sequenced genomes. Current automated methods depend heavily on sequence composition and often underestimate the complexity of the proteome. We developed RibosomeE Profiling Assisted (re-)AnnotaTION (REPARATION), a de novo machine learning algorithm that takes advantage of experimental protein synthesis evidence from ribosome profiling (Ribo-seq) to delineate translated open reading frames (ORFs) in bacteria, independent of genome annotation (https://github.com/Biobix/REPARATION). REPARATION evaluates all possible ORFs in the genome and estimates minimum thresholds based on a growth curve model to screen for spurious ORFs. We applied REPARATION to three annotated bacterial species to obtain a more comprehensive mapping of their translation landscape in support of experimental data. In all cases, we identified hundreds of novel (small) ORFs including variants of previously annotated ORFs and >70% of all (variants of) annotated protein coding ORFs were predicted by REPARATION to be translated. Our predictions are supported by matching mass spectrometry proteomics data, sequence composition and conservation analysis. REPARATION is unique in that it makes use of experimental translation evidence to intrinsically perform a de novo ORF delineation in bacterial genomes irrespective of the sequence features linked to open reading frames.

Assessment of genome annotation using gene function similarity within the gene neighborhood

BackgroundFunctional annotation of bacterial genomes is an obligatory and crucially important step of information processing from the genome sequences into cellular mechanisms. However, there is a lack of computational methods to evaluate the quality of functional assignments.ResultsWe developed a genome-scale model that assigns Bayesian probability to each gene utilizing a known property of functional similarity between neighboring genes in bacteria.ConclusionsOur model clearly distinguished true annotation from random annotation with Bayesian annotation probability >0.95. Our model will provide a useful guide to quantitatively evaluate functional annotation methods and to detect gene sets with reliable annotations.

Comparative Genomics and Transcriptional Analysis of Flavobacterium columnare Strain ATCC 49512.

Flavobacterium columnare is a Gram-negative fish pathogen causing columnaris disease in wild and cultured fish species. Although the pathogen is widespread in aquatic environments and fish worldwide, little is known about biology of F. columnare and mechanisms of columnaris disease pathogenesis. Previously we presented the complete genome sequence of F. columnare strain ATCC 49512. Here we present a comparison of the strain ATCC 49512 genome to four other Flavobacterium genomes. In this analysis, we identified predicted proteins whose functions indicate F. columnare is capable of denitrification, which would enable anaerobic growth in aquatic pond sediments. Anaerobic growth of F. columnare ATCC 49512 with nitrate supplementation was detected experimentally. F. columnare ATCC 49512 had a relatively high number of insertion sequences and genomic islands compared to the other Flavobacterium species, suggesting a larger degree of horizontal gene exchange and genome plasticity. A type VI subtype III secretion system was encoded in F. columnare along with F. johnsoniae and F. branchiophilum. RNA sequencing proved to be a valuable technique to improve annotation quality; 41 novel protein coding regions were identified, 16 of which had a non-traditional start site (TTG, GTG, and CTT). Candidate small noncoding RNAs were also identified. Our results improve our understanding of F. columnare ATCC 49512 biology, and our results support the use of RNA sequencing to improve annotation of bacterial genomes, particularly for type strains.

Whole-Genome Sequence and Fosfomycin Resistance of Bacillus sp. Strain G3(2015) Isolated from Seawater off the Coast of Malaysia.

ABSTRACTBacillus sp. is a Gram-positive bacterium that is commonly found in seawater. In this study, the genome of marine Bacillus sp. strain G3(2015) was sequenced using MiSeq. The fosfomycin resistant gene fosB was identified upon bacterial genome annotation.

Identification of Unannotated Small Genes in Salmonella

Increasing evidence indicates that many, if not all, small genes encoding proteins ≤100 aa are missing in annotations of bacterial genomes currently available. To uncover unannotated small genes in the model bacterium Salmonella enterica Typhimurium 14028s, we used the genomic technique ribosome profiling, which provides a snapshot of all mRNAs being translated (translatome) in a given growth condition. For comprehensive identification of unannotated small genes, we obtained Salmonella translatomes from four different growth conditions: LB, MOPS rich defined medium, and two infection-relevant conditions low Mg2+ (10 µM) and low pH (5.8). To facilitate the identification of small genes, ribosome profiling data were analyzed in combination with in silico predicted putative open reading frames and transcriptome profiles. As a result, we uncovered 130 unannotated ORFs. Of them, 98% were small ORFs putatively encoding peptides/proteins ≤100 aa, and some of them were only expressed in the infection-relevant low Mg2+ and/or low pH condition. We validated the expression of 25 of these ORFs by western blot, including the smallest, which encodes a peptide of 7 aa residues. Our results suggest that many sequenced bacterial genomes are underannotated with regard to small genes and their gene annotations need to be revised.

Messages from the first International Conference on Clinical Metagenomics (ICCMg)

Metagenomics is recently entering in the clinical microbiology and an increasing number of diagnostic laboratories are now proposing the sequencing & annotation of bacterial genomes and/or the analysis of clinical samples by direct or PCR-based metagenomics with short time to results. In this context, the first International Conference on Clinical Metagenomics (ICCMg) was held in Geneva in October 2016 and several key aspects have been discussed including: i) the need for improved resolution, ii) the importance of interpretation given the common occurrence of sequence contaminants, iii) the need for improved bioinformatic pipelines, iv) the bottleneck of DNA extraction, v) the importance of gold standards, vi) the need to further reduce time to results, vii) how to improve data sharing, viii) the applications of bacterial genomics and clinical metagenomics in better adapting therapeutics and ix) the impact of metagenomics and new sequencing technologies in discovering new microbes. Further efforts in term of reduced turnaround time, improved quality and lower costs are however warranted to fully translate metagenomics in clinical applications.

Genix: a new online automated pipeline for bacterial genome annotation.

Next-generation sequencing has significantly reduced the cost of genome-sequencing projects, resulting in an expressive increase in the availability of genomic data in public databases. The cheaper and easier is to sequence new genomes, the more accurate the annotation steps have to be to avoid both the loss of information and the accumulation of erroneous features that may affect the accuracy of further analysis. In the case of bacteria genomes, a range of web annotation software has been developed; however, many applications have yet to incorporate the steps required to improve their result, including the removal of false-positive/spurious and a more complete identification of non-coding features. We present Genix, a new web-based bacterial genome annotation pipeline. A comparison of the results generated by Genix for four reference genomes against those generated by other annotation tools indicated that our pipeline is able to provide results that are closer to the reference genome annotation, with a smaller amount of false-positive proteins and missing functional annotated proteins. Additionally, the metrics obtained by Genix were slightly better than those obtained by Prokka, a state-of-art standalone annotation system. Our results indicate that Genix is a useful tool that is able to provide a more refined result, and may be a user-friendly way to obtain high-quality results.