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In this article, a bioinformatics pipeline is proposed that focuses on two types of elements, namely the mobile genetic elements (MGE) and Ribonucleic acids (RNAs). The MGEs are called insertion sequences (ISs) in the prokaryotic domain. The objective of this research work is to study the behaviour of RNAs and MGEs genes, and the effects of their presence around inversions in genome sequences. The proposed pipeline finds the relation between the transposase gene types (e.g., DDE and DEDD) located within insertion sequences according to their IS family and sub-family, and RNAs (tRNA and rRNA) on the one hand, and genomic inversion on the other hand. More precisely, we wonder whether the insertion sequences and RNAs have any effects on the occurrence of inversions. To investigate this, we start by extracting all inversions between every two close isolates of P. aeruginosa from a high supported phylo- genetic tree, and we investigate inversion boundaries, to determine the impact of IS elements and RNAs around these inversions. For RNAs genes, we noticed that a high occurrence of these elements are found in what we called InBoundary, while few to zero occurrences have been recorded in OutBoundary. We remark that the NCGM2.S1 genome has the largest amount of tRNAs and inversions comparing to other genomes. For RNA genes, the tRNAs genes (Arg, Ala, Leu) recorded the highest occurrences around the inversions, while there was few occurrences of rRNA genes, within all genomes. For IS elements, we noticed that the family of IS3 has the highest impact on inversions, but sometimes other type of IS elements can also be found with IS3 around the inversions. In this case, these types of ISs shared the same type of transposase gene (DDE). We finally conclude that the distribution of RNA and IS genes has a big impact on the inversions in bacterial genomes.

With the development of sequencing technology, microbiological genome sequencing analysis has attracted extensive attention. For inexperienced users without sufficient bioinformatics skills, making sense of sequencing data for microbial identification, especially for bacterial identification, through reads analysis is still challenging. In order to address the challenge of effectively analyzing genomic information, in this paper, we develop an effective approach and automatic bioinformatics pipeline called PBGI for bacterial genome identification, performing automatedly and customized bioinformatics analysis using short-reads or long-reads sequencing data produced by multiple platforms such as Illumina, PacBio and Oxford Nanopore. An evaluation of the proposed approach on the practical data set is presented, showing that PBGI provides a user-friendly way to perform bacterial identification through short or long reads analysis, and could provide accurate analyzing results. The source code of the PBGI is freely available at https://github.com/lyotvincent/PBGI.

Covering: up to 2021Terpenoids are a diverse group of chemicals used in a wide range of industries. Microbial terpenoid production has the potential to displace traditional manufacturing of these compounds with renewable processes, but further titre improvements are needed to reach cost competitiveness. This review discusses strategies to increase terpenoid titres in Escherichia coli with a focus on alternative metabolic pathways. Alternative pathways can lead to improved titres by providing higher orthogonality to native metabolism that redirects carbon flux, by avoiding toxic intermediates, by bypassing highly-regulated or bottleneck steps, or by being shorter and thus more efficient and easier to manipulate. The canonical 2-C-methyl-d-erythritol 4-phosphate (MEP) and mevalonate (MVA) pathways are engineered to increase titres, sometimes using homologs from different species to address bottlenecks. Further, alternative terpenoid pathways, including additional entry points into the MEP and MVA pathways, archaeal MVA pathways, and new artificial pathways provide new tools to increase titres. Prenyl diphosphate synthases elongate terpenoid chains, and alternative homologs create orthogonal pathways and increase product diversity. Alternative sources of terpenoid synthases and modifying enzymes can also be better suited for E. coli expression. Mining the growing number of bacterial genomes for new bacterial terpenoid synthases and modifying enzymes identifies enzymes that outperform eukaryotic ones and expand microbial terpenoid production diversity. Terpenoid removal from cells is also crucial in production, and so terpenoid recovery and approaches to handle end-product toxicity increase titres. Combined, these strategies are contributing to current efforts to increase microbial terpenoid production towards commercial feasibility.

Lanthipeptides are one of the largest groups of ribosomally synthesized and post-translationally modified peptides (RiPPs) and are characterized by the presence of lanthionine (Lan) or methyllanthionine residues (MeLan). Only very few lanthipeptides contain a C-terminal 2-aminovinyl-cysteine (AviCys) motif, but all of them show potent antibacterial activities. Recent advances of genome sequencing led to the rapid accumulation of new biosynthetic gene clusters (BGCs) for lanthipeptides. In this study, through our genome mining strategy, we found the AviCys containing lanthipeptides are widespread in the bacterial kingdom. A lanthipeptide-type biosynthetic gene cluster was identified from public bacterial genome database. Two new lanthipeptides, daspyromycins A and B (1 and 2) containing AviCys motif, along with two degraded products, daspyromycins C and D (3 and 4), were obtained after heterologous expression of the gene cluster in Streptomyces albus J1074. Daspyromycins A and B showed potent antimicrobial activity against a spectrum of Gram-positive and -negative bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE).

We have developed an efficient and inexpensive pipeline for streamlining large-scale collection and genome sequencing of bacterial isolates. Evaluation of this method involved a worldwide research collaboration focused on the model organism Salmonella enterica, the 10KSG consortium. By optimising a logistics pipeline that collected isolates as thermolysates, permitting shipment in ambient conditions, the project assembled a diverse collection of 10,419 clinical and environmental isolates from low- and middle-income countries in less than one year. The bacteria were obtained from 51 countries/territories dating from 1949 to 2017, with a focus on Africa and Latin-America. All isolates were collected in barcoded tubes and genome sequenced using an optimised DNA extraction method and the LITE pipeline for library construction. After Illumina sequencing, the total reagent cost was less than USD$10 per genome. Our method can be applied to genome-sequence other large bacterial collections at a relatively low cost, within a limited timeframe, to support global collaborations.

The development of base editing (BE) technology has opened a new avenue for research studies in bacteriology, particularly for bacterial species in which the DNA double-strand breaks (DSBs) introduced by CRISPR/Cas system would lead to cell death. However, a major limitation of BE-mediated gene editing is the restricted editable sites in the target bacterial genome due to highly diverse genomic compositions, such as GC content. Herein, we developed a broad-spectrum DNase-inactive Cpf1 (dCpf1) variant from Francisella novicida (bsdFnCpf1) through directed evolution. The resulting optimized mutant showed a substantially expanded targeting range, including previously non-canonical protospacer-adjacent motifs (PAMs), especially the GC-rich PAMs. Cytidine deaminase APOBEC1 and uracil DNA glycosylase inhibitor (UGI) were fused with bsdFnCpf1 to achieve specific C to T mutations at multiple target sites with canonical or non-canonical PAMs in the E. coli genome without compromising cell growth. We anticipate that bsdFnCpf1 could be applied for multiplex gene regulation and BE in species that have been reported to be suitable for Cpf1.

To improve the nitrogen fixation, legume crops are often inoculated with selected effective rhizobia. However, there is large variation in how well the inoculant strains compete with the indigenous microflora in soil. To assess the success of the inoculant, it is necessary to distinguish it from other, closely related strains. Methods used until now have generally been based either on fingerprinting methods or on the use of reporter genes. Nevertheless, these methods have their shortcomings, either because they do not provide sufficiently specific information on the identity of the inoculant strain, or because they use genetically modified organisms that need prior authorization to be applied in the field or other uncontained environments. Another possibility is to target a gene that is naturally present in the bacterial genomes. Here we have developed a method that is based on amplicon sequencing of the bacterial housekeeping gene rpoB, encoding the beta-subunit of the RNA polymerase, which has been proposed as an alternative to the 16S rRNA gene to study the diversity of rhizobial populations in soils. We evaluated the method under laboratory and field conditions. Peanut seeds were inoculated with various Bradyrhizobium strains. After nodule development, DNA was extracted from selected nodules and the nodulating rhizobia were analysed by amplicon sequencing of the rpoB gene. The analyses of the sequence data showed that the method reliably identified bradyrhizobial strains in nodules, at least at the species level, and could be used to assess the competitiveness of the inoculant compared to other bradyrhizobia.

Horizontal gene transfer (HGT) is arguably the most conspicuous feature of bacterial evolution. Evidence for HGT is found in most bacterial genomes. Although HGT can considerably alter bacterial genomes, not all transfer events may be biologically significant and may instead represent the outcome of an incessant evolutionary process that only occasionally has a beneficial purpose. When adaptive transfers occur, HGT and positive selection may result in specific, detectable signatures in genomes, such as gene-specific sweeps or increased transfer rates for genes that are ecologically relevant. In this Review, we first discuss the various mechanisms whereby HGT occurs, how the genetic signatures shape patterns of genomic variation and the distinct bioinformatic algorithms developed to detect these patterns. We then discuss the evolutionary theory behind HGT and positive selection in bacteria, and discuss the approaches developed over the past decade to detect transferred DNA that may be involved in adaptation to new environments.

Background To select the most complete, continuous, and accurate assembly for an organism of interest, comprehensive quality assessment of assemblies is necessary. We present a novel tool, called Evaluation of De Novo Assemblies (EvalDNA), which uses supervised machine learning for the quality scoring of genome assemblies and does not require an existing reference genome for accuracy assessment. Results EvalDNA calculates a list of quality metrics from an assembled sequence and applies a model created from supervised machine learning methods to integrate various metrics into a comprehensive quality score. A well-tested, accurate model for scoring mammalian genome sequences is provided as part of EvalDNA. This random forest regression model evaluates an assembled sequence based on continuity, completeness, and accuracy, and was able to explain 86% of the variation in reference-based quality scores within the testing data. EvalDNA was applied to human chromosome 14 assemblies from the GAGE study to rank genome assemblers and to compare EvalDNA to two other quality evaluation tools. In addition, EvalDNA was used to evaluate several genome assemblies of the Chinese hamster genome to help establish a better reference genome for the biopharmaceutical manufacturing community. EvalDNA was also used to assess more recent human assemblies from the QUAST-LG study completed in 2018, and its ability to score bacterial genomes was examined through application on bacterial assemblies from the GAGE-B study. Conclusions EvalDNA enables scientists to easily identify the best available genome assembly for their organism of interest without requiring a reference assembly. EvalDNA sets itself apart from other quality assessment tools by producing a quality score that enables direct comparison among assemblies from different species.

A subculture of S.epidermidis strain ATCC35984 that is amenable to genetically manipulate was occasionally found in our laboratory. This mutant exhibited susceptibility to methicillin in contrast to its parent strain. To unveil the underlying mechanism, whole-genome sequencing of the mutant was performed. A comparative analysis revealed that a large DNA fragment encompassing the CRISPR-Cas system, type I R-M system and the SCCmec element was deleted from the mutant. The large chromosomal deletion associated with CRISPR-Cas system was also observed to occur spontaneously in S. epidermidis in another independent laboratory, or artificially induced by introducing engineering crRNAs in other bacterial species. These findings imply the CRISPR-Cas systems can affect bacterial genome remodeling through deletion of the integrated MGEs (mobile genetic elements). Further bioinformatics analysis identified a higher carriage rate of SCCmec element in the S. epidermidis strains harboring the CRISPR-Cas system. MLST typing and phylogenetic analysis of those CRIPSR-Cas-positive S. epidermidis strains revealed multiple origins. In addition, distinct types of SCCmec carried in those strains suggested that acquisition of this MGE originated from multiple independent recombination events. Intriguingly, CRISPR-Cas systems are found to be always located in the vicinity of orfX gene among staphylococci. Allelic analysis of CRISPR loci flanking cas genes disclosed that the loci distal to the orfX gene are considerably stable and conserved, which probably serve as recombination hotspot between CRISPR-Cas system and phage or plasmid. Therefore, the findings generally support the notion that incomplete immune protection of CRISPR-Cas system can promote dissemination of its neighboring SCCmec element.

Background As carbapenem-resistant Acinetobacter baumannii is dominant in clinical settings, the old polymyxin antibiotic colistin has been revived as a therapeutic option. The development of colistin resistance during treatment is becoming a growing concern. Objectives To access low- to mid-level colistin-resistant A. baumannii blood isolates recovered from an outbreak in a tertiary care hospital from a national antimicrobial surveillance study. Methods The entire bacterial genome was sequenced through long-read sequencing methodology. Quantitative RT–PCR was carried out to determine the level of gene expression. Relative growth rates were determined to estimate fitness costs of each isolate caused by the genetic alterations. Results The A. baumannii isolates belonged to global clone 2 harbouring two intrinsic phosphoethanolamine transferases. Cumulative alterations continuing the colistin resistance were observed. PmrC overproduction caused by the PmrBA226T alteration was identified in A. baumannii isolates with low-level colistin resistance and an additional PmrCR109H substitution led to mid-level colistin resistance. Truncation of the PmrC enzyme by insertion of ISAba59 was compensated by ISAba10-mediated overproduction of EptA and, in the last isolate, the complete PmrAB two-component regulatory system was eliminated to restore the biological cost of the bacterial host. Conclusions During the in-hospital outbreak, a trajectory of genetic modification in colistin-resistant A. baumannii isolates was observed for survival in the harsh conditions imposed by life-threatening drugs with the clear purpose of maintaining drug resistance above a certain level with a reasonable fitness cost.

Made available in DSpace on 2022-04-29T08:34:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-03-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) The molecular bases for the symbiosis of the amphibian skin microbiome with its host are poorly understood. Here, we used the odor-producer Pseudomonas sp. MPFS and the treefrog Boana prasina as a model to explore bacterial genome determinants and the resulting mechanisms facilitating symbiosis. Pseudomonas sp. MPFS and its closest relatives, within a new clade of the P. fluoresens Group, have large genomes and were isolated from fishes and plants, suggesting environmental plasticity. We annotated 16 biosynthetic gene clusters from the complete genome sequence of this strain, including those encoding the synthesis of compounds with known antifungal activity and of odorous methoxypyrazines that likely mediate sexual interactions in Boana prasina. Comparative genomics of Pseudomonas also revealed that Pseudomonas sp. MPFS and its closest relatives have acquired specific resistance mechanisms against host antimicrobial peptides (AMPs), specifically two extra copies of a multidrug efflux pump and the same two-component regulatory systems known to trigger adaptive resistance to AMPs in P. aeruginosa. Subsequent molecular modeling indicated that these regulatory systems interact with an AMP identified in Boana prasina through the highly acidic surfaces of the proteins comprising their sensory domains. In agreement with a symbiotic relationship and a highly selective antibacterial function, this AMP did not inhibit the growth of Pseudomonas sp. MPFS but inhibited the growth of another Pseudomonas species and Escherichia coli in laboratory tests. This study provides deeper insights into the molecular interaction of the bacteria-amphibian symbiosis and highlights the role of specific adaptive resistance toward AMPs of the hosts. Departamento de Ciências Biomoleculares Faculdade de Ciências Farmacêuticas de Ribeirão Preto Universidade de São Paulo, SP Laboratorio de Genética Evolutiva Instituto de Biología Subtropical (CONICET – UNaM) Facultad de Ciencias Exactas Universidad Nacional de Misiones Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures Departamento de Biodiversidade e Centro de Aquicultura Instituto de Biociências Universidade Estadual Paulista, SP Departamento de Análises Clínicas Toxicológicas e Bromatológicas Faculdade de Ciências Farmacêuticas de Ribeirão Preto Universidade de São Paulo, SP IPEEC-CONICET Instituto Patagónico para el Estudio de los Ecosistemas Continentales Consejo Nacional de Investigaciones Científicas y Técnicas Mikrobiologie Technische Universität Braunschweig Departamento de Biodiversidade e Centro de Aquicultura Instituto de Biociências Universidade Estadual Paulista, SP FAPESP: 2013/50741-7 FAPESP: 2013/50954-0 FAPESP: 2014/20915-6 FAPESP: 2014/50265-3 FAPESP: 2017/23725-1 FAPESP: 2017/26162-8 CAPES: 88881.062205/2014-01 CAPES: 88887.464731/2019-00

Arr is an ADP-ribosyltransferase enzyme primarily reported in association with rifamycin resistance, which has been used to treat tuberculosis in addition to Gram-positive infections and, recently, pan-resistant Gram-negative bacteria. The arr gene was initially identified on the Mycolicibacterium smegmatis chromosome and later on Proteobacteria plasmids. This scenario raised concerns on the distribution and spread of arr, considering the Bacteria domain. Based on 198,082 bacterial genomes/metagenomes, we performed in silico analysis, including phylogenetic reconstruction of Arr in different genomic contexts. Besides, new arr alleles were evaluated by in vitro analysis to assess their association with rifampin resistance phenotype. The arr gene was prevalent in thousands of chromosomes and in hundreds of plasmids from environmental and clinical bacteria, mainly from the phyla Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes. Furthermore, this gene was identified in other and new genomic contexts. Interestingly, Arr sequences associated with rifampin resistance were distributed across all phylogeny, indicating that, despite the diversity, their association with rifampin resistance phenotype were maintained. In fact, we found that the key residues were highly conserved. In addition, other analyzes have raised evidence of another Arr function, which is related to guanidine metabolism. Finally, this scenario as a whole also suggested the Actinobacteria phylum as a potential ancestral source of arr within the Bacteria domain.

Background Long-read sequencing has shown its tremendous potential to address genome assembly challenges, e.g., achieving the first telomere-to-telomere assembly of a gapless human chromosome. However, many issues remain unresolved when leveraging error-prone long reads to characterize high-complexity metagenomes, for instance, complete/high-quality genome reconstruction from highly complex systems. Results Here, we developed an iterative haplotype-resolved hierarchical clustering-based hybrid assembly (HCBHA) approach that capitalizes on a hybrid (error-prone long reads and high-accuracy short reads) sequencing strategy to reconstruct (near-) complete genomes from highly complex metagenomes. Using the HCBHA approach, we first phase short and long reads from the highly complex metagenomic dataset into different candidate bacterial haplotypes, then perform hybrid assembly of each bacterial genome individually. We reconstructed 557 metagenome-assembled genomes (MAGs) with an average N50 of 574 Kb from a deeply sequenced, highly complex activated sludge (AS) metagenome. These high-contiguity MAGs contained 14 closed genomes and 111 high-quality (HQ) MAGs including full-length rRNA operons, which accounted for 61.1% of the microbial community. Leveraging the near-complete genomes, we also profiled the metabolic potential of the AS microbiome and identified 2153 biosynthetic gene clusters (BGCs) encoded within the recovered AS MAGs. Conclusion Our results established the feasibility of an iterative haplotype-resolved HCBHA approach to reconstruct (near-) complete genomes from highly complex ecosystems, providing new insights into “complete metagenomics”. The retrieved high-contiguity MAGs illustrated that various biosynthetic gene clusters (BGCs) were harbored in the AS microbiome. The high diversity of BGCs highlights the potential to discover new natural products biosynthesized by the AS microbial community, aside from the traditional function (e.g., organic carbon and nitrogen removal) in wastewater treatment.

Bacterial genomes harbour cryptic prophages that are mostly transcriptionally silent with many unannotated genes. Still, cryptic prophages may contribute to their host fitness and phenotypes. In Bacillus subtilis, the yqaF-yqaN operon belongs to the prophage element skin, and is tightly repressed by the Xre-like repressor SknR. This operon contains several small ORFs (smORFs) potentially encoding small-sized proteins. The smORF-encoded peptide YqaH was previously reported to bind to the replication initiator DnaA. Here, using a yeast two-hybrid assay, we found that YqaH binds to the DNA binding domain IV of DnaA and interacts with Spo0A, a master regulator of sporulation. We isolated single amino acid substitutions in YqaH that abolished the interaction with DnaA but not with Spo0A. Then, using a plasmid-based inducible system to overexpress yqaH WT and mutant derivatives, we studied in B. subtilis the phenotypes associated with the specific loss-of-interaction with DnaA (DnaA_LOI). We found that expression of yqaH carrying DnaA_LOI mutations abolished the deleterious effects of yqaH WT expression on chromosome segregation, replication initiation and DnaA-regulated transcription. When YqaH was induced after vegetative growth, DnaA_LOI mutations abolished the drastic effects of YqaH WT on sporulation and biofilm formation. Thus, YqaH inhibits replication, sporulation and biofilm formation mainly by antagonizing DnaA in a manner that is independent of the cell cycle checkpoint Sda.

The bacterial pan-genome is a relatively new concept that refers to the number of genes observed in a given set of bacterial genome sequences, either at the intra- or inter-species level. Determining the pan-genome of a given species of bacteria using a large number of strains allows one to compare multiple genes and to determine evolutionary links between isolates. This information can help to determine population structure, diversity in terms of prevalence in a given environment and pathogenicity of microorganisms. Within this review, we explain the most important issues related to pan-genome studies. We also include a brief description of some selected bacterial pan-genomes. Finally, we propose an easy-toperform workflow to study bacterial pan-genomes that will facilitate nonexperts in a pan-genome-based investigation.

Recent advances in next-generation sequencing, deep networks and other bioinformatic tools have enabled us to mine huge amount of genomic information about living organisms in the post-microarray era. However, these tools do not explicitly factor in the role of the underlying DNA biochemistry (particularly, DNA hybridization) essential to life processes. Here, we focus more precisely on the role that DNA hybridization plays in determining properties of biological organisms at the macro-level. We illustrate its role with solutions to challenging problems in human disease. These solutions are made possible by novel structural properties of DNA hybridization landscapes revealed by a metric model of oligonucleotides of a common length that makes them reminiscent of some planets in our solar system, particularly Earth and Saturn. They allow a judicious selection of so-called noncrosshybridizing (nxh) bases that offer substantial reduction of DNA sequences of arbitrary length into a few informative features. The quality assessment of the information extracted by them is high because of their very low Shannon Entropy, i.e. they minimize the degree of uncertainty in hybridization that makes results on standard microarrays irreproducible. For example, SNP classification (pathogenic/non-pathogenic) and pathogen identification can be solved with high sensitivity (~77%/100%) and specificity (~92%/100%, respectively) for combined taxa on a sample of over 264 fully coding sequences in whole bacterial genomes and fungal mitochondrial genomes using machine learning (ML) models. These methods can be applied to several other interesting research questions that could be addressed with similar genomic analyses.

Bacterial quantification by qPCR is considered the gold standard for microbial molecular diagnosis. However, a fundamental pre-requisite in this methodology is the designing of specific primers for the bacterium of interest. With the increase in bacterial genome sequencing data in the recent years, it has become possible to design specific primers that can be used to quantify different strains of the same bacterial species. To develop a real-time PCR (qPCR) protocol for the specific quantification of Azospirillum baldaniorum Sp245 strain (old Azospirillum brasilense), the Sp245 genome sequence was fragmented into small contigs with 500 base pairs each, and analyzed for similarity against the NCBI non-redundant database. A. baldaniorum-specific contigs were used to design the primers. The best pair of primers was used to quantify these bacteria after inoculation in different cultivars of Brachiaria, grown under field conditions. Our results showed that the primer pair Sp245p10 was highly specific for the Sp245 strain in the Brachiaria root and shoot field under different conditions. The qPCR assay using these primers showed differences among cultivars in the number of bacteria detected in plants after inoculation. Additionally, the number of bacteria observed in the roots was higher than that in the shoots. The qPCR methodology using a Sp245 strain-specific primer may be used to monitor A. baldaniorum inoculated into other plants and may find potential application in field experiments.

Introduction Because blaCTX-M is responsible for resistance of bacteria to the third generation cephalosporins, location of blaCTX-M could be a good indicator for classifying bacterial isolates harboring blaCTX-M in molecular epidemiology. However, determination of blaCTX-M location has been difficult when multiple copies of ISEcp1 were found on bacterial genome. We aimed to establish a high-throughput analytical method for upstream genetic structures (UGS) of ISEcp1 to facilitate determination of blaCTX-M location. Methods Extracted DNA samples obtained from 168 Escherichia coli isolates possessing blaCTX-M were digested by restriction enzyme, HaeIII, and the digested DNA fragments were ligated with homemade barcode adaptors. Then, DNA fragments containing UGS of ISEcp1 were amplified and subjected to the Nanopore sequencer. Results Nucleotide sequences and locations of 168 UGSs obtained from the examined E. coli isolates were determined. Among the 168 determined UGSs, 150 (89.3%) UGS were confirmed on plasmid and classified into eight types. Interestingly, coding sequence of ISEcp1 transposase gene in seven of the eight types were disrupted by IS26 insertion. The remaining 18 (10.7%) UGSs were observed in identical chromosomal region. The obtained nucleotide sequences the locations of UGSs were confirmed by conventional capillary sequencer and Southern blotting, respectively, and any discrepant result was not observed with these confirmation procedures. Conclusions Our results indicated that the established method was efficient for simultaneously determining at least 100 different UGS, and suggested that the determined UGSs of ISEcp1-blaCTX-M transposition unit was useful for classification of bacterial isolates harboring blaCTX-M.

Background Myxobacteria harbor numerous biosynthetic gene clusters that can produce a diverse range of secondary metabolites. Minicystis rosea DSM 24000T is a soil-dwelling myxobacterium belonging to the suborderSorangiineae and family Polyangiaceae and is known to produce various secondary metabolites as well as polyunsaturated fatty acids (PUFAs). Here, we use whole-genome sequencing to explore the diversity of biosynthetic gene clusters in M. rosea. Results Using PacBio sequencing technology, we assembled the 16.04 Mbp complete genome of M. rosea DSM 24000T, the largest bacterial genome sequenced to date. About 44% of its coding potential represents paralogous genes predominantly associated with signal transduction, transcriptional regulation, and protein folding. These genes are involved in various essential functions such as cellular organization, diverse niche adaptation, and bacterial cooperation, and enable social behavior like gliding motility, sporulation, and predation, typical of myxobacteria. A profusion of eukaryotic-like kinases (353) and an elevated ratio of phosphatases (8.2/1) in M. rosea as compared to other myxobacteria suggest gene duplication as one of the primary modes of genome expansion. About 7.7% of the genes are involved in the biosynthesis of a diverse array of secondary metabolites such as polyketides, terpenes, and bacteriocins. Phylogeny of the genes involved in PUFA biosynthesis (pfa) together with the conserved synteny of the complete pfa gene cluster suggests acquisition via horizontal gene transfer from Actinobacteria. Conclusion Overall, this study describes the complete genome sequence of M. rosea, comparative genomic analysis to explore the putative reasons for its large genome size, and explores the secondary metabolite potential, including the biosynthesis of polyunsaturated fatty acids.

Aim Characterization of biosurfactant produced by a carbazole degrading bacterium Roseomonas cervicalis and proteomic analysis of intracellular proteins of bacterium while growing on glucose and carbazole medium. Methods and results The bacterium R. cervicalis was isolated from a soil sample contaminated with crude petroleum oil. PCR amplification ascertained the existence of some hydrocarbon-degrading catabolic genes (alkB and PAH-RHDα, C12O, and C23O) in the bacterial genome. GC-MS and RP-HPLC analyses demonstrated 62% and 60% carbazole degradation, respectively, by R. cervicalis 144 h post-incubation at 37°C and pH 6.5. Due to the paucity of protein databases, expressions of only 29 and 14 intracellular proteins were explicitly recognized and quantitated by mass spectrometry analysis when R. cervicalis was grown in carbazole and glucose medium, respectively. FTIR, NMR, and HR-MS/MS analyses demonstrated the lipopeptide nature of the purified biosurfactant produced by R. cervicalis. The biosurfactant is also presumed to assist in the solubilization of carbazole. Conclusion The isolated R. cervicalis strain is a potential candidate for the bioremediation of carbazole in petroleum-oil contaminated sites. Significance and impact of the study This is the first report of the promising R. cervicalis strain proficient in carbazole biodegradation.

Prokaryote genomics started in earnest in 1995, with the complete sequences of two small bacterial genomes, those of Haemophilus influenzae and Mycoplasma genitalium. During the next quarter century, the prokaryote genome database has been growing exponentially, with no saturation in sight. For most of these 25 years, genome sequencing remained limited to cultivable microbes. Together with next-generation sequencing methods, advances in metagenomics and single-cell genomics have lifted this limitation, providing for an increasingly unbiased characterization of the global prokaryote diversity. Advances in computational genomics followed the progress of genome sequencing, even if occasionally lagging behind. Several major new branches of bacteria and archaea were discovered, including Asgard archaea, the apparent closest relatives of eukaryotes and expansive groups of bacteria and archaea with small genomes thought to be symbionts of other prokaryotes. Comparative analysis of numerous prokaryote genomes spanning a wide range of evolutionary distances changed the conceptual foundations of microbiology, supplanting the notion of species genomes with fixed gene sets with that of dynamic pangenomes and the notion of a single Tree of Life (ToL) with a statistical tree-like trend among individual gene trees. Strides were also made towards a theory and quantitative laws of prokaryote genome evolution.

Funder: National Institute for Health Research (NIHR), Recent years have seen a remarkable increase in the practicality of sequencing whole genomes from large numbers of bacterial isolates. The availability of this data has huge potential to deliver new insights into the evolution and epidemiology of bacterial pathogens, but the scalability of the analytical methodology has been lagging behind that of the sequencing technology. Here we present a step-by-step approach for such large-scale genomic epidemiology analyses, from bacterial genomes to epidemiological interpretations. A central component of this approach is the dated phylogeny, which is a phylogenetic tree with branch lengths measured in units of time. The construction of dated phylogenies from bacterial genomic data needs to account for the disruptive effect of recombination on phylogenetic relationships, and we describe how this can be achieved. Dated phylogenies can then be used to perform fine-scale or large-scale epidemiological analyses, depending on the proportion of cases for which genomes are available. A key feature of this approach is computational scalability and in particular the ability to process hundreds or thousands of genomes within a matter of hours. This is a clear advantage of the step-by-step approach described here. We discuss other advantages and disadvantages of the approach, as well as potential improvements and avenues for future research. This article is part of a discussion meeting issue 'Genomic population structures of microbial pathogens'.

Human gut microbiota modulates normal physiological functions, such as maintenance of barrier homeostasis and modulation of metabolism, as well as various chronic diseases including type 2 diabetes and gastrointestinal cancer. Despite decades of research, the composition of the gut microbiota remains poorly understood. Here, we established an effective extraction method to obtain high quality gut microbiota genomes, and analyzed them with third-generation sequencing technology. We acquired a large quantity of data from each sample and assembled large numbers of reliable contigs. With this approach, we constructed tens of completed bacterial genomes in which there were several new bacteria species. We also identified a new conditional pathogen, Enterococcus tongjius, which is a member of Enterococci. This work provided a novel and reliable approach to recover gut microbiota genomes, facilitating the discovery of new bacteria species and furthering our understanding of the microbiome that underlies human health and diseases.

Bacteria use carbohydrate-binding proteins (CBPs), such as lectins and carbohydrate-binding modules (CBMs), to anchor to specific sugars on host surfaces. CBPs in the gut microbiome are well studied, but their roles in the vagina microbiome and involvement in sexually transmitted infections, cervical cancer and preterm birth are largely unknown. We established a classification system for lectins and designed Hidden Markov Model (HMM) profiles for data mining of bacterial genomes, resulting in identification of >100,000 predicted bacterial lectins available at unilectin.eu/bacteria. Genome screening of 90 isolates from 21 vaginal bacterial species shows that those associated with infection and inflammation produce a larger CBPs repertoire, thus enabling them to potentially bind a wider array of glycans in the vagina. Both the number of predicted bacterial CBPs and their specificities correlated with pathogenicity. This study provides new insights into potential mechanisms of colonisation by commensals and potential pathogens of the reproductive tract that underpin health and disease states.

Horizontal gene transfer (HGT) is a major driving force for bacterial evolution. To avoid the deleterious effects due to the unregulated expression of newly acquired foreign genes, bacteria have evolved specific proteins named xenogeneic silencers to recognize foreign DNA sequences and suppress their transcription. As there is considerable diversity in genomic base compositions among bacteria, how xenogeneic silencers distinguish self- from nonself DNA in different bacteria remains poorly understood. This review summarizes the progress in studying the DNA binding preferences and the underlying molecular mechanisms of known xenogeneic silencer families, represented by H-NS of Escherichia coli, Lsr2 of Mycobacterium, MvaT of Pseudomonas, and Rok of Bacillus. Comparative analyses of the published data indicate that the differences in DNA recognition mechanisms enable these xenogeneic silencers to have clear characteristics in DNA sequence preferences, which are further correlated with different host genomic features. These correlations provide insights into the mechanisms of how these xenogeneic silencers selectively target foreign DNA in different genomic backgrounds. Furthermore, it is revealed that the genomic AT contents of bacterial species with the same xenogeneic silencer family proteins are distributed in a limited range and are generally lower than those species without any known xenogeneic silencers in the same phylum/class/genus, indicating that xenogeneic silencers have multifaceted roles on bacterial genome evolution. In addition to regulating horizontal gene transfer, xenogeneic silencers also act as a selective force against the GC to AT mutational bias found in bacterial genomes and help the host genomic AT contents maintained at relatively low levels.

Comparative sequence analysis methods are highly effective for uncovering novel classes of structured noncoding RNAs (ncRNAs) from bacterial genomic DNA sequence datasets. Previously, we developed a computational pipeline to more comprehensively identify structured ncRNA representatives from individual bacterial genomes. This search process exploits the fact that genomic regions serving as templates for the transcription of structured RNAs tend to be present in longer than average noncoding ‘intergenic regions’ (IGRs) that are enriched in G and C nucleotides compared to the remainder of the genome. In the present study, we apply this computational pipeline to identify structured ncRNA candidates from 26 diverse bacterial species. Numerous novel structured ncRNA motifs were discovered, including several riboswitch candidates, one whose ligand has been identified and others that have yet to be experimentally validated. Our findings support recent predictions that hundreds of novel ribo-switch classes and other ncRNAs remain undiscovered among the limited number of bacterial species whose genomes have been completely sequenced.

Labelling of Type IV pili (TFP) can greatly improve our understanding of the pivotal roles of TFP in a variety of bacterial activities including motility, surface sensing and DNA-uptake etc. Here we show a simple and switchable pili-labelling method by plasmid-based inducible replacement of PilA without genetic modification in bacterial genome employed by complicated methods. Using this method, we characterized pili morphology and twitching motility of Pseudomonas aeruginosa in details. More importantly, we demonstrate its application in studying the replenishment dynamics of pilin pool of P. aeruginosa. This article is protected by copyright. All rights reserved.

Background: Toxin-antitoxin systems (TAs) are two-component elements, which are extensive in the bacterial genome and have a regulatory role in many cellular activities including, growth arrest, survival, biofilm formation, and bacterial persistence. Objective: TAs have not well studied in Brucella spp. Methods: We evaluated the presence of different toxin-antitoxin systems, including relE- rhhlike, Fic- Phd, Cog- Rhh, and cogT- cogAT in 40 clinical Brucella melitensis isolates using PCRbased sequencing assay. Results: Our results showed the high presence of relE-rhh-like, Fic-Phd, Cog-rhh, and cogTcogAT s TAs genes in B. melitensis isolates that were 96.25%, 92.5%, 96.25%, and 95%, respectively. Conclusion: A high presence of TAs genes in clinical B. melitensis isolates revealed that the TA system could be an antibacterial target in B. melitensis but more investigation is necessitated to elucidate the exact roles of these genes.

We conducted global genome mining of 162,672 bacterial genomes and identified 829 cyclodipeptide (CDP) biosynthesis gene clusters (BGC) containing a cytochrome P450 gene. Heterologous expression of BGC from Saccharopolyspora hirsuta DSM 44795 led to the identification of two novel crownlike CDPs, cyctetryptomycin A (4) and B (5), which possess unprecedented complex macrocycle and show neuroprotective activity. The two cytochrome P450s found in the BGC catalyze sequential reactions leading to the cyclization of diketopiperazine dimers.

Single-molecule real-time DNA sequencing revealed that 4-methylcytosine (m4C) commonly exists in bacterial genomes. In this work, samples with different m4C methylation patterns were studied. Results reveal that m4C modification is a biochemical reaction with distance effect, and its distribution follows the power function in the positive, negative, and double strands of genomic DNA sequences of Geobacter sulfurreducens. Furthermore, the value of regression coefficient in the fitting formula for double strands was the sum of those in the fitting formulae for positive and negative strands. Meanwhile, the value of exponent coefficient was the average, implicating an interesting mathematical phenomenon about power function. Considering the potent role of m4C in gene expression and the present results being obtained from the same genomic DNA sequence, this work suggests that the patterns of m4C distribution may be served as a signal for G. sulfurreducens to rapidly identify the genes to respond to environmental stresses or signals. This study opens a new avenue to extend our knowledge about the modification mechanisms and the epigenetic information of m4C modification in prokaryotes.

Antimicrobial resistance has spread quickly on a worldwide scale, reducing therapeutic options for bacterial infections. CRISPR-Cas is an adaptive immune system found in many prokaryotes that can be designed to target bacterial genomes, leading to cell death. Repurposing the CRISPR-Cas system as a therapeutic strategy offers an attractive way to overcome antimicrobial resistance. However, this strategy requires efficient vectors for the CRISPR-Cas system to reach the bacterial genomes. Engineered phages offer an attractive option as cargo delivery vectors. In this review, we discuss the production of phage-based vectors and the relevance of using repurposed CRISPR-Cas systems as antimicrobials. We also discuss recent progress in phage engineering that can potentially overcome the limitations and increase the efficiency of CRISPR-Cas delivery.

Antibiotic resistance spreads among bacteria through horizontal transfer of antibiotic resistance genes (ARGs). Here, we set out to determine predictive features of ARG transfer among bacterial clades. We use a statistical framework to identify putative horizontally transferred ARGs and the groups of bacteria that disseminate them. We identify 152 gene exchange networks containing 22,963 bacterial genomes. Analysis of ARG-surrounding sequences identify genes encoding putative mobilisation elements such as transposases and integrases that may be involved in gene transfer between genomes. Certain ARGs appear to be frequently mobilised by different mobile genetic elements. We characterise the phylogenetic reach of these mobilisation elements to predict the potential future dissemination of known ARGs. Using a separate database with 472,798 genomes from Streptococcaceae, Staphylococcaceae and Enterobacteriaceae, we confirm 34 of 94 predicted mobilisations. We explore transfer barriers beyond mobilisation and show experimentally that physiological constraints of the host can explain why specific genes are largely confined to Gram-negative bacteria although their mobile elements support dissemination to Gram-positive bacteria. Our approach may potentially enable better risk assessment of future resistance gene dissemination., Antibiotic resistance spreads among bacteria through horizontal transfer of antibiotic resistance genes (ARGs). Here, Ellabaan et al. use a statistical approach to identify putative mobilisation elements and other features associated with ARG transfer among bacterial clades to predict the potential future dissemination of known ARGs.

Rapid bacterial growth depends on the speed at which ribosomes can translate mRNA into proteins. mRNAs that encode successive stretches of proline can cause ribosomes to stall, substantially reducing translation speed. Such stalling is especially detrimental for species that must grow and divide rapidly. Here we focus on di-prolyl motifs (XXPPX) and ask whether their incidence varies with growth rate. To find out we conducted a broad survey of such motifs in >3000 bacterial genomes across 36 phyla. Indeed, fast-growing species encode fewer motifs than slow-growing species, especially in highly expressed proteins. We also found many di-prolyl motifs within thermophiles, where prolines can help maintain proteome stability. Moreover, bacteria with complex, multicellular lifecycles also encode many di-prolyl motifs. This is especially evident in the slow-growing phylum Myxococcota. Bacteria in this phylum encode many serine-threonine kinases, and many di-prolyl motifs at potential phosphorylation sites within these kinases. Serine-threonine kinases are involved in cell signaling and help regulate developmental processes linked to multicellularity in the Myxococcota. Altogether, our observations suggest that weakened selection on translational rate, whether due to slow or thermophilic growth, may allow di-prolyl motifs to take on new roles in biological processes that are unrelated to translational rate.

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., A series of hapalosin analogues and several 1,2,3‐oxadiazine‐lactylate hybrids were discovered by exploiting the apparent inability of cyanobacteria to degrade fatty acids. When supplemented with perdeuterated fatty acids, cyanobacteria incorporate their residues into secondary metabolites, which can then be swiftly detected by LC‐MS analysis.

Antibiotic development based on natural products has faced a long lasting decline since the 1970s, while both the speed and the extent of antimicrobial resistance (AMR) development have been severely underestimated. The discovery of antimicrobial natural products of bacterial and fungal origin featuring new chemistry and previously unknown mode of actions is increasingly challenged by rediscovery issues. Natural products that are abundantly produced by the corresponding wild type organisms often featuring strong UV signals have been extensively characterized, especially the ones produced by extensively screened microbial genera such as streptomycetes. Purely synthetic chemistry approaches aiming to replace the declining supply from natural products as starting materials to develop novel antibiotics largely failed to provide significant numbers of antibiotic drug leads. To cope with this fundamental issue, microbial natural products science is being transformed from a ‘grind-and-find’ study to an integrated approach based on bacterial genomics and metabolomics. Novel technologies in instrumental analytics are increasingly employed to lower detection limits and expand the space of detectable substance classes, while broadening the scope of accessible and potentially bioactive natural products. Furthermore, the almost exponential increase in publicly available bacterial genome data has shown that the biosynthetic potential of the investigated strains by far exceeds the amount of detected metabolites. This can be judged by the discrepancy between the number of biosynthetic gene clusters (BGC) encoded in the genome of each microbial strain and the number of secondary metabolites actually detected, even when considering the increased sensitivity provided by novel analytical instrumentation. In silico annotation tools for biosynthetic gene cluster classification and analysis allow fast prioritization in BGC-to-compound workflows, which is highly important to be able to process the enormous underlying data volumes. BGC prioritization is currently accompanied by novel molecular biology-based approaches to access the so-called orphan BGCs not yet correlated with a secondary metabolite. Integration of metabolomics, in silico genomics and molecular biology approaches into the mainstream of natural product research will critically influence future success and impact the natural product field in pharmaceutical, nutritional and agrochemical applications and especially in anti-infective research., Antimicrobial resistance is a major public concern and novel antibiotics are largely based on natural products. We summarize recent analytical and genome based technological developments that gain increasing importance in the natural products field.

Non-ribosomal peptides are a group of structurally diverse natural products with various important therapeutic and agrochemical applications. Bacterial pyrrolizidine alkaloids (PAs), containing a scaffold of two fused five-membered ring system with a nitrogen atom at the bridgehead, have been found to originate from a multidomain non-ribosomal peptide synthetase to generate indolizidine intermediates, followed by multistep oxidation, catalysed by single Bayer-Villiger (BV) enzymes, to yield PA scaffolds. Although bacterial PAs are rare in natural product inventory, bioinformatics analysis suggested that the biosynthetic gene clusters (BGCs) that are likely to be responsible for the production of PA-like metabolites are widely distributed in bacterial genomes. However, most of the strains containing PA-like BGCs are not deposited in the public domain, therefore preventing further assessment of the chemical spaces of this group of bioactive metabolites. Here, we report a genomic scanning strategy to assess the potential of PA metabolites production in our culture collection without prior knowledge of genome information. Among the strains tested, we found fifteen contain the key BV enzymes that are likely to be involved in the last step of PA ring formation. Subsequently one-strain-many-compound (OSMAC) method, supported by a combination of HR-MS, NMR, SMART 2.0 technology, and GNPS analysis, allowed identification and characterization of a new [5 + 7] heterobicyclic carbamate, legoncarbamate, together with five known PAs, bohemamine derivatives, from Streptomyces sp. CT37, a Ghanaian soil isolate. The absolute stereochemistry of legoncarbamate was determined by comparison of measured and calculated ECD spectra. Legoncarbamate displays antibacterial activity against E. coli ATCC 25922 with an MIC value of 3.1 μg/mL. Finally, a biosynthetic model of legoncarbamate and other bohemamines was proposed based on the knowledge we have gained so far.

DNA methylation is an epigenetic mechanism that has been largely probed regarding eukaryotic nuclear genome and bacteria, and its role is especially crucial in the regulation of gene expression. In mammals, it is almost exclusively acting on a cytosine preceding a guanine (CpG), whereas it presents itself mainly in a non-CpG context in bacteria's DNA. Conversely to nuclear and bacterial genomes, the existence of methylation in the mitochondrial genome is still widely debated. This controversy has been attributed to structural differences between the nuclear and mitochondrial genomes, and to the techniques used to study methylation of cytosines, which were rather optimized for the study of nuclear DNA. However, novel studies suggest that cytosine methylation is truly existing in mitochondria, and that it is mostly found in a non-CpG context, just like in their evolutionary relative, the bacteria.

Genomic islands, defined as large clusters of genes mobilized through horizontal gene transfer, have a profound impact on evolution of prokaryotes. Recently, we developed a new program, IslandCafe, for identifying such large localized structures in bacterial genomes. A unique attribute of IslandCafe is its ability to decipher mosaic structures within genomic islands. Mosaic genomic islands have generated immense interest due to novel traits that have been attributed to such islands. To provide the Pseudomonas research community a catalogue of mosaic islands in Pseudomonas spp., we applied IslandCafe to decipher genomic islands in 224 completely sequenced genomes of Pseudomonas spp. We also performed comparative genomic analysis using BLAST to infer potential sources of distinct segments within genomic islands. Of the total 4271 genomic islands identified in Pseudomonas spp., 1036 were found to be mosaic. We also identified drug-resistant and pathogenic genomic islands and their potential donors. Our analysis provides a useful resource for Pseudomonas research community to further examine and interrogate mosaic islands in the genomes of interest and understand their role in the emergence and evolution of novel traits.

We determined the species composition of 219 isolates of potato blackleg or soft rot pathogens in Japan collected after 2000 from potato plants by species-specific PCR. Of the total, Pectobacterium wasabiae (Pw) and P. carotovorum subsp. brasiliense (Pcb) comprised 88% (Pw: 94 isolates, Pcb: 98 isolates) of the isolates and Dickeya dianthicola (Ddi) 12% (27 isolates). P. atrosepticum (Pa) was not detected. Genetic diversity of Pw, Pcb and Ddi was investigated using repetitive sequence-based PCR (rep-PCR) fingerprinting with three primer pairs complementary to repetitive sequences in the bacterial genome (BOX, ERIC and REP). One hundred and twenty-one Pw isolates, one hundred Pcb isolates and thirty-three Ddi isolates were assigned to twenty-nine, twenty-one and eleven fingerprint genotypes (FGs) based on the combination of fingerprint patterns for each rep-PCR experiment (BOX-, ERIC- and REP-PCR). The genetic diversity of Pw and Ddi isolates in Japan was considered high, with Simpson’s diversity index and Shannon’s index of 0.87 and 2.44 for Pw, and 0.84 and 2.05 for Ddi, respectively. Pcb isolates were less diversified genetically (diversity index: 0.57 and 1.58). The FGs of the isolates obtained from blackleg-affected potato plants in the seed tuber field agreed with those of the isolates in the field where the seeds produced were grown. These results support the occurrence of seed-borne transmission of the blackleg pathogen between the fields.

Hospital-associated infections are a major concern for global public health. Infections with antibiotic-resistant pathogens can cause empiric treatment failure, and for infections with multidrug-resistant bacteria which can overcome antibiotics of "last resort" there exists no alternative treatments. Despite extensive sanitization protocols, the hospital environment is a potent reservoir and vector of antibiotic-resistant organisms. Pathogens can persist on hospital surfaces and plumbing for months to years, acquire new antibiotic resistance genes by horizontal gene transfer, and initiate outbreaks of hospital-associated infections by spreading to patients via healthcare workers and visitors. Advancements in next-generation sequencing of bacterial genomes and metagenomes have expanded our ability to (1) identify species and track distinct strains, (2) comprehensively profile antibiotic resistance genes, and (3) resolve the mobile elements that facilitate intra- and intercellular gene transfer. This information can, in turn, be used to characterize the population dynamics of hospital-associated microbiota, track outbreaks to their environmental reservoirs, and inform future interventions. This review provides a detailed overview of the approaches and bioinformatic tools available to study isolates and metagenomes of hospital-associated bacteria, and their multi-layered networks of transmission.

The gut microbiota is closely associated with colorectal neoplasia. While most metagenomics studies utilized fecal samples, circulating bacterial DNA in colorectal neoplasia patients remained unexplored. This proof-of-concept study aims to characterize alterations of circulating bacterial DNA in colorectal neoplasia patients. We performed WGS of plasma samples from 25 colorectal cancer (CRC) patients, 10 colorectal adenoma (CRA) patients and 22 healthy controls (HC). Bacterial relative abundance was measured by removing the host genome and mapping reads into bacterial genomes. By diversity analysis, we found plasma samples required less sample size to approach saturation than fecal samples, and species diversity in HC was slightly higher compared with CRC/CRA patients. The majority of circulating bacterial DNA came from bacterial genera which commonly associated with gastrointestine and oral tract. By differential analysis, a total of 127 significant species between CRC patients and HC were identified, on which basis 28 species with top predictive ability were selected and showed promise in preliminary discrimination between CRC/CRA and HC. In CRA patients, relative abundance of the selected 28 species more closely resembled those in CRC patients than HC. By comparing with fecal metagenomics studies, we found there was moderate positive correlation between fold changes of the overlapped fecal and circulating bacterial DNA. Finally, species correlation analysis revealed that CRC and HC displayed distinct patterns of species association. In conclusion, this study demonstrated alterations of circulating bacterial DNA in colorectal neoplasia patients, which had the potential to become non-invasive biomarkers for colorectal neoplasia screening and early diagnosis.

Background The genomes of bacteria and archaea evolve by extensive loss and gain of genes which, for any group of related prokaryotic genomes, result in the formation of a pangenome with the universal, asymmetrical U-shaped distribution of gene commonality. However, the evolutionary factors that define the specific shape of this distribution are not thoroughly understood. Results We investigate the fit of simple models of genome evolution to the empirically observed gene commonality distributions and genome intersections for 33 groups of closely related bacterial genomes. A model with an infinite external gene pool available for gene acquisition and constant genome size (IGP-CGS model), and two gene turnover rates, one for slow- and the other one for fast-evolving genes, allows two approaches to estimate the parameters for gene content dynamics. One is by fitting the model prediction to the distribution of the number of genes shared by precisely k genomes (gene commonality distribution) and another by analyzing the distribution of the number of genes common for k genome sets (k-cores). Both approaches produce a comparable overall quality of fit, although the former significantly overestimates the number of the universally conserved genes, while the latter overestimates the number of singletons. We further explore the effect of dropping each of the assumptions of the IGP-CGS model on the fit to the gene commonality distributions and show that models with either a finite gene pool or unequal rates of gene loss and gain (greater gene loss rate) eliminate the overestimate of the number of singletons or the core genome size. Conclusions We examine the assumptions that are usually adopted for modeling the evolution of the U-shaped gene commonality distributions in prokaryote genomes, namely, those of infinitely many genes and constant genome size. The combined analysis of genome intersections and gene commonality suggests that at least one of these assumptions is invalid. The violation of both these assumptions reflects the limited ability of prokaryotes to gain new genes. This limitation seems to stem, at least partly, from the horizontal gene transfer barrier, i.e., the cost of accommodation of foreign genes by prokaryotes. Further development of models taking into account the complexity of microbial evolution is necessary for an improved understanding of the evolution of prokaryotes.

Genome analysis of Halomonas shambharensis, a novel species, was performed to understand the osmoprotectant strategies used by the strain to overcome the salinity stress and to explore the prospective industrial uses. It will also help to better understand the ecological roles of Halomonas species in hypersaline habitats. Ultrastructure of the cell was determined by using transmission electron microscopy. Standard microbiological methods were used to find out growth parameters and heterotrophic mode of nutrition. For Genome analysis, complete bacterial genome sequencing was performed using the Oxford Nanopore MinION DNA Sequencer. Assembly, annotation and finishing of the obtained sequence were done by using a Prokaryotic Genome Annotation Pipeline (PGAP) (SPAdes v. 3.10.1). Predicted Coading sequences (CDSs) obtained through the PGAP were used for functional annotation using Clusters of Orthologous Groups and Kyoto Encyclopedia of Genes and Genomes (KEGG) platforms. The H. shambharensis was found to be a Gram-stain-negative, rod-shaped bacterium, motile with a peritrichous flagella. The H. shambharensis bacterium can grow in a wide range of temperature (from 25 to 65 °C), pH (pH 4 to pH 12.0) and salt concentration (5.0% NaCl to 30.0% NaCl). After annotation and assembly, the total genome size obtained was 1,533,947 bp, which revealed 146 subsystems, 3847 coding sequences, and 19RNAs with G+C content of 63.6%. Gene annotation identified the genes related to various metabolic pathways, including carbohydrate metabolism, fatty acid metabolism and stress tolerance. The genomic dataset of H. shambharensis will be useful for analysis of protein-coding gene families and how these coding genes are significant for the survival and metabolism among the different species of Halomonas. The complete genome sequence presented here will help to unravel the biotechnological potential of H. shambharensis for production of the high-value products such as betaine, or as a source of gene-mining for individual enzymes.

Plasmids have a key role in bacterial ecology and evolution because they mobilize accessory genes by horizontal gene transfer. However, recent studies have revealed that the evolutionary impact of plasmids goes above and beyond their being mere gene delivery platforms. Plasmids are usually kept at multiple copies per cell, producing islands of polyploidy in the bacterial genome. As a consequence, the evolution of plasmid-encoded genes is governed by a set of rules different from those affecting chromosomal genes, and these rules are shaped by unusual concepts in bacterial genetics, such as genetic dominance, heteroplasmy or segregational drift. In this Review, we discuss recent advances that underscore the importance of plasmids in bacterial ecology and evolution beyond horizontal gene transfer. We focus on new evidence that suggests that plasmids might accelerate bacterial evolution, mainly by promoting the evolution of plasmid-encoded genes, but also by enhancing the adaptation of their host chromosome. Finally, we integrate the most relevant theoretical and empirical studies providing a global understanding of the forces that govern plasmid-mediated evolution in bacteria.

Bacterial endosymbiosis has been instrumental in eukaryotic evolution, and includes both mutualistic, dependent and parasitic associations. Here we characterize an intracellular bacterium inhabiting the flagellated protist Bodo saltans (Kinetoplastida). We present a complete bacterial genome comprising a 1.39 Mb circular chromosome with 40.6% GC content. Fluorescent in situ hybridisation confirms that the endosymbiont is located adjacent to the nuclear membrane, and a detailed model of its intracellular niche is generated using serial block-face scanning electron microscopy. Phylogenomic analysis shows that the endosymbiont belongs to the Holosporales, most closely related to other α-proteobacterial endosymbionts of ciliates and amoebae. Comparative genomics indicates that it has a limited metabolism and is nutritionally host-dependent. However, the endosymbiont genome does encode diverse symbiont-specific secretory proteins, including a type VI secretion system and three separate toxin-antitoxin systems. We show that these systems are actively transcribed and hypothesize they represent a mechanism by which B. saltans becomes addicted to its endosymbiont. Consistent with this idea, attempts to cure Bodo of endosymbionts led to rapid and uniform cell death. This study adds kinetoplastid flagellates to ciliates and amoebae as hosts of Paracaedibacter-like bacteria, suggesting that these antagonistic endosymbioses became established very early in Eukaryotic evolution.

Extracytoplasmic function σ factors (ECFs) represent one of the major bacterial signal transduction mechanisms in terms of abundance, diversity and importance, particularly in mediating stress responses. Here, we performed a comprehensive phylogenetic analysis of this protein family by scrutinizing all proteins in the NCBI database. As a result, we identified an average of ∼10 ECFs per bacterial genome and 157 phylogenetic ECF groups that feature a conserved genetic neighborhood and a similar regulation mechanism. Our analysis expands previous classification efforts ∼50-fold, enriches many original ECF groups with previously unclassified proteins and identifies 22 entirely new ECF groups. The ECF groups are hierarchically related to each other and are further composed of subgroups with closely related sequences. This two-tiered classification allows for the accurate prediction of common promoter motifs and the inference of putative regulatory mechanisms across subgroups composing an ECF group. This comprehensive, high-resolution description of the phylogenetic distribution of the ECF family, together with the massive expansion of classified ECF sequences and an openly accessible data repository called ‘ECF Hub’ (https://www.computational.bio.uni-giessen.de/ecfhub), will serve as a powerful hypothesis-generator to guide future research in the field.

The ability of CRISPR-Cas12a nucleases to function reliably in a wide range of species has been key to their rapid adoption as genome engineering tools. However, so far, Cas12a nucleases have been limited for use in organisms with growth temperatures up to 37 °C. Here, we biochemically characterize three Cas12a orthologs for their temperature stability and activity. We demonstrate that Francisella novicida Cas12a (FnCas12a) has great biochemical potential for applications that require enhanced stability, including use at temperatures >37°C. Furthermore, by employing the moderate thermophilic bacterium Bacillus smithii as our experimental platform, we demonstrate that FnCas12a is active in vivo at temperatures up to 43°C. Subsequently, we develop a single-plasmid FnCas12a-based genome editing tool for B. smithii, combining the FnCas12a targeting system with plasmid-borne homologous recombination (HR) templates that carry the desired modifications. Culturing of B. smithii cells at 45°C allows for the uninhibited realization of the HR-based editing step, while a subsequent culturing step at reduced temperatures induces the efficient counterselection of the non-edited cells by FnCas12a. The developed gene-editing tool yields gene-knockout mutants within 3 days, and does not require tightly controllable expression of FnCas12a to achieve high editing efficiencies, indicating its potential for other (thermophilic) bacteria and archaea, including those with minimal genetic toolboxes. Altogether, our findings provide new biochemical insights into three widely used Cas12a nucleases, and establish the first Cas12a-based bacterial genome editing tools for moderate thermophilic microorganisms.

Haemophilus influenzae has contributed to key bacterial genome sequencing hallmarks, as being not only the first bacterium to be genome-sequenced, but also starring the first genome-wide analysis of chromosomes directly transformed with DNA from a divergent genotype, and pioneering Tn-seq methodologies. Over the years, the phenomenal and constantly evolving development of –omic technologies applied to a whole range of biological questions of clinical relevance in the H. influenzae-host interplay, has greatly moved forward our understanding of this human-adapted pathogen, responsible for multiple acute and chronic infections of the respiratory tract. In this way, essential genes, virulence factors, pathoadaptive traits, and multi-layer gene expression regulatory networks with both genomic and epigenomic complexity levels are being elucidated. Likewise, the unstoppable increasing whole genome sequencing information underpinning H. influenzae great genomic plasticity, mainly when referring to non-capsulated strains, poses major challenges to understand the genomic basis of clinically relevant phenotypes and even more, to clearly highlight potential targets of clinical interest for diagnostic, therapeutic or vaccine development. We review here how genomic, transcriptomic, proteomic and metabolomic-based approaches are great contributors to our current understanding of the interactions between H. influenzae and the human airways, and point possible strategies to maximize their usefulness in the context of biomedical research and clinical needs on this human-adapted bacterial pathogen., N.L.-L. is funded by a PhD studentship from Regional Navarra Govern, Spain, reference 0011-1408-2017-000000. C.G.-C. is funded by a PhD studentship from Agencia Española de Investigación (AEI), Spain, reference PRE2019-088382. This work has been funded by grants from MINECO RTI2018-096369-B-I00, 875/2019 from SEPAR, and PC150-151–152 from Gobierno de Navarra to J.G. CIBER is an initiative from Instituto de Salud Carlos III (ISCIII), Madrid, Spain.