Search

Computational models of gene regulations help to understand regulatory mechanisms and are extensively used in a wide range of areas, e.g., biotechnology or medicine, with significant benefits. Unfortunately, there are only a few computational gene regulatory models of whole genomes allowing static and dynamic analysis due to the lack of sophisticated tools for their reconstruction. Here, we describe Augusta, an open-source Python package for Gene Regulatory Network (GRN) and Boolean Network (BN) inference from the high-throughput gene expression data. Augusta can reconstruct genome-wide models suitable for static and dynamic analyses. Augusta uses a unique approach where the first estimation of a GRN inferred from expression data is further refined by predicting transcription factor binding motifs in promoters of regulated genes and by incorporating verified interactions obtained from databases. Moreover, a refined GRN is transformed into a draft BN by searching in the curated model database and setting logical rules to incoming edges of target genes, which can be further manually edited as the model is provided in the SBML file format. The approach is applicable even if information about the organism under study is not available in the databases, which is typically the case for non-model organisms including most microbes. Augusta can be operated from the command line and, thus, is easy to use for automated prediction of models for various genomes. The Augusta package is freely available at github.com/JanaMus/Augusta. Documentation and tutorials are available at augusta.readthedocs.io.

The taxonomic status of strain P5891T, isolated from an Adélie penguin beak swab, was investigated. Based on the 16S rRNA gene sequence, the strain was identified as a potentially novel Corynebacterium species, with the highest sequence similarities to Corynebacterium rouxii FRC0190T (96.7%) and Corynebacterium epidermidicanis DSM 45586T (96.6%). The average nucleotide identity values between strain P5891T and C. rouxii FRC0190T and C. epidermidicanis DSM 45586T were 68.2 and 69.2%, respectively. The digital DNA–DNA hybridization values between strain P5891T and C. rouxii FRC0190T and C. epidermidicanis DSM 45586T were 23.7 and 21.4%, respectively. Phylogenetic trees based on the 16S rRNA sequence placed strain P5891T in a separate branch with Corynebacterium canis 1170T and Corynebacterium freiburgense 1045T, while a phylogenomic tree based on the Corynebacterium species core genome placed the strain next to Corynebacterium choanae 200CHT. Extensive phenotyping and genomic analyses clearly confirmed that strain P5891T represents a novel species of the genus Corynebacterium, for which the name Corynebacterium mendelii sp. nov. is proposed, with the type strain P5891T (=CCM 8862T=LMG 31627T).

Purple photosynthetic bacteria (PPB) are versatile microorganisms capable of producing various value-added chemicals, e.g., biopolymers and biofuels. They employ diverse metabolic pathways, allowing them to adapt to various growth conditions and even extreme environments. Thus, they are ideal organisms for the Next Generation Industrial Biotechnology concept of reducing the risk of contamination by using naturally robust extremophiles. Unfortunately, the potential of PPB for use in biotechnology is hampered by missing knowledge on regulations of their metabolism. Although Rhodospirillum rubrum represents a model purple bacterium studied for polyhydroxyalkanoate and hydrogen production, light/chemical energy conversion, and nitrogen fixation, little is known regarding the regulation of its metabolism at the transcriptomic level. Using RNA sequencing, we compared gene expression during the cultivation utilizing fructose and acetate as substrates in case of the wild-type strain R. rubrum DSM 467T and its knock-out mutant strain that is missing two polyhydroxyalkanoate synthases PhaC1 and PhaC2. During this first genome-wide expression study of R. rubrum, we were able to characterize cultivation-driven transcriptomic changes and to annotate non-coding elements as small RNAs.

The RNA-Seq method has become a key tool in transcriptomics, providing deep insight into the genetic functioning of organisms. It is essential for understanding how genes are expressed, for discovering new gene variants and for understanding pathologies at the genetic level. RNA-Seq simulators are great for testing and validating bioinformatics tools, they offer a way to compare different computational algorithms and allow examining different sequencing protocols, parameter settings and sample sizes to ensure the most efficient use of resources. This study introduces BacSeqer, an RNA-Seq simulator specifically designed for bacterial transcriptomes with the aim of producing data that faithfully resembles those obtained in real experiments. It allows to take into account the specificities of the bacterial genome, especially an existence of only exons and their organization in operons that are not normally present in eukaryotes.

Bacterial small RNAs (sRNAs) are regulatory elements often mentioned in the context of stress response. So are polyhydroxyalkanoates (PHAs), a possible replacement for conventional plastics, are frequently produced by bacteria facing stress conditions. PHAs then serve as the source of the carbon as well as the defense mechanism against further stress. Here, we present sRNA analysis of PHA-producing bacterium Rhodospirillum rubrum, especially the co-expression analysis of inferred sRNA candidates from RNA-Seq data. Weighted correlation network analysis (WGCNA) was used to compute clusters of genes with similar expression profiles, resulting in 28 modules. Further, the functional annotation of genes in each module was applied and the overall functional profiles were mentioned using a ‘guilt by association’ approach. Additionally, functional enrichment of modules was obtained through overrepresentation analysis (ORA). These results provide an overview of regulatory elements and their predicted functions in R. rubrum, which further unveil its dynamic properties and can help with the assessment of its industrial application.

This study compares the efficiency of Pacific Biosciences technology (PacBio) and Oxford Nanopore Technology (ONT) in sequencing, assembling, and annotating the Aneurinibacillus sp AFn2 bacterium. We aim to evaluate the performance based on contiguity, depth, and functional annotation of the resulting genome. Using ONT we generated 152,047 long reads assembling into 2 contigs with total base count of 0.4 billion which provided us efficient assembly while PacBio produced 139,701 reads, assembling into 21 contigs with a total base count of 1.4 billion. Functional annotation revealed differences in the number of coding sequences, with PacBio detecting more comprehensive gene sets than ONT. The comparative analysis done in this research shows the strengths and limitations of both the platforms, with ONT providing higher assembly contiguity and PacBio offering greater detail in genetic content. We aim to offer insights of both the sequencing technologies, guiding researchers in selecting the appropriate technology.

The complete genome sequence of thermophilic type strain Tepidimonas tawanensis LMG 22826T confirmed the previous statement of high biotechnological potential of this bacterium that is besides reported alkaline protease production capable of polyhydroxyalkanoates (PHA) synthesis. Here, we present further descriptive analysis based on pathways detection and consequently understanding of some significant properties that can be used for possible modeling purposes. In total, 108 biological pathways were detected, phaCAB operon and granule-associated proteins - phasins were found and butanoate pathway comparison with other PHA producing bacteria was provided as well as simplified PHA synthesis pathway was introduced.

The study of bacterial organisms is given great emphasis in scientific research. By studying the bacterial genome, scientists can gain knowledge of various infectious diseases. Nowadays, it is even possible to edit genes and thus advancing the field of tailored medicine by treating many genetic diseases. Furthermore, it is becoming the norm to utilize bacteria in the industry for their ability to produce important products such as plastics, or fuels. It is therefore essential to extract the necessary information about its genes and other features. This paper focuses mainly on improving the functional annotation of bacterial genomes, classification of protein-coding sequences into clusters of orthologous groups, and visualization of the final annotated genome. For this task, the python package was developed with several functions for working with the outputs of multiple annotation tools, namely eggNOG-mapper, Operon-mapper, and Batch CD-Search. The package was tested on Clostridium diolis DSM 15410 and Clostridium beijerinckii DSM 791.

Tepidimonas taiwanensis is a moderately thermophilic, Gram-negative, rod-shaped, chemoorganoheterotrophic, motile bacterium. The alkaline protease producing type strain Tepidimonas taiwanensis LMG 22826T was recently reported to also be a promising producer of polyhydroxyalkanoates (PHAs) – renewable and biodegradable polymers representing an alternative to conventional plastics. Here, we present its first complete genome sequence which is also the first complete genome sequence of the whole species. The genome consists of a single 2,915,587bp long circular chromosome with GC content of 68.75%. Genome annotation identified 2,764 genes in total while 2,634 open reading frames belonged to protein-coding genes. Although functional annotation of the genome and division of genes into Clusters of Orthologous Groups (COGs) revealed a relatively high number of 694 genes with unknown function or unknown COG, the majority of genes were assigned a function. Most of the genes, 406 in total, were involved in energy production and conversion, and amino acid transport and metabolism. Moreover, particular key genes involved in the metabolism of PHA were identified. Knowledge of the genome in connection with the recently reported ability to produce bioplastics from the waste stream of wine production makes T. taiwanensis LMG 22826T an ideal candidate for further genome engineering as a bacterium with high biotechnological potential.

Genotyping methods are used to distinguish bacterial strains from one species. Thus, distinguishing bacterial strains on a global scale, between countries or local districts in one country is possible. However, the highly selected bacterial populations (e.g. local populations in hospital) are typically closely related and low diversified. Therefore, currently used typing methods are not able to distinguish individual strains from each other. Here, we present a novel pipeline to detect highly variable genetic segments for genotyping a closely related bacterial population. The method is based on a degree of disorder in analyzed sequences that can be represented by sequence entropy. With the identified variable sequences, it is possible to find out transmission routes and sources of highly virulent and multiresistant strains. The proposed method can be used for any bacterial population, and due to its whole genome range, also noncoding regions are examined.

Clostridium beijerinckii is a relatively widely studied, yet non-model, bacterium. While 246 genome assemblies of its various strains are available currently, the diversity of the whole species has not been studied, and it has only been analyzed in part for a missing genome of the type strain. Here, we sequenced and assembled the complete genome of the type strain Clostridium beijerinckii DSM 791T, composed of a circular chromosome and a circular megaplasmid, and used it for a comparison with other genomes to evaluate diversity and capture the evolution of the whole species. We found that strains WB53 and HUN142 were misidentified and did not belong to the Clostridium beijerinckii species. Additionally, we filtered possibly misassembled genomes, and we used the remaining 237 high-quality genomes to define the pangenome of the whole species. By its functional annotation, we showed that the core genome contains genes responsible for basic metabolism, while the accessory genome has genes affecting final phenotype that may vary among different strains. We used the core genome to reconstruct the phylogeny of the species and showed its great diversity, which complicates the identification of particular strains, yet hides possibilities to reveal hitherto unreported phenotypic features and processes utilizable in biotechnology.

Actinobacteria belonging to the genus Rubrobacter are known for their multi-extremophilic growth conditions-they are highly radiation-resistant, halotolerant, thermotolerant or even thermophilic. This work demonstrates that the members of the genus are capable of accumulating polyhydroxyalkanoates (PHA) since PHA-related genes are widely distributed among Rubrobacter spp. whose complete genome sequences are available in public databases. Interestingly, all Rubrobacter strains possess both class I and class III synthases (PhaC). We have experimentally investigated the PHA accumulation in two thermophilic species, R. xylanophilus and R. spartanus. The PHA content in both strains reached up to 50% of the cell dry mass, both bacteria were able to accumulate PHA consisting of 3-hydroxybutyrate and 3-hydroxyvalerate monomeric units, none other monomers were incorporated into the polymer chain. The capability of PHA accumulation likely contributes to the multi-extremophilic characteristics since it is known that PHA substantially enhances the stress robustness of bacteria. Hence, PHA can be considered as extremolytes enabling adaptation to extreme conditions. Furthermore, due to the high PHA content in biomass, a wide range of utilizable substrates, Gram-stain positivity, and thermophilic features, the Rubrobacter species, in particular Rubrobacter xylanophilus, could be also interesting candidates for industrial production of PHA within the concept of Next-Generation Industrial Biotechnology.

Gene expression analysis through reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) depends on correct data normalization by reference genes with stable expression. Although Clostridium beijerinckii NRRL B-598 is a promising Gram-positive bacterium for the industrial production of biobutanol, validated reference genes have not yet been reported. In this study, we selected 160 genes with stable expression based on an RNA sequencing (RNA-Seq) data analysis, and among them, seven genes (zmp, rpoB1, rsmB, greA, rpoB2, topB2, and rimO) were selected for experimental validation by RT-qPCR and gene ontology enrichment analysis. According to statistical analyses, zmp and greA were the most stable and suitable reference genes for RT-qPCR normalization. Furthermore, our methodology can be useful for selection of the reference genes in other strains of Clostridium beijerinckii and it also suggests that the RNA-Seq data can be used for the initial selection of novel reference genes, however their validation is required.

Routinely used typing methods including MLST, rep-PCR and whole genome sequencing (WGS) are time-consuming, costly, and often low throughput. Here, we describe a novel mini-MLST scheme for Eschericha coli as an alternative method for rapid genotyping. Using the proposed mini-MLST scheme, 10,946 existing STs were converted into 1,038 Melting Types (MelTs). To validate the new mini-MLST scheme, in silico analysis was performed on 73,704 strains retrieved from EnteroBase resulting in discriminatory power D = 0.9465 (CI 95% 0.9726-0.9736) for mini-MLST and D = 0.9731 (CI 95% 0.9726-0.9736) for MLST. Moreover, validation on clinical isolates was conducted with a significant concordance between MLST, rep-PCR and WGS. To conclude, the great portability, efficient processing, cost-effectiveness, and high throughput of mini-MLST represents immense benefits, even when accompanied with a slightly lower discriminatory power than other typing methods. This study proved mini-MLST is an ideal method to screen and subgroup large sets of isolates and/or quick strain typing during outbreaks. In addition, our results clearly showed its suitability for prospective surveillance monitoring of emergent and high-risk E. coli clones'.

Schlegelella thermodepolymerans is a moderately thermophilic bacterium capable of producing polyhydroxyalkanoates (PHA) – biodegradable polymers representing an alternative to conventional plastics. Here, we present the first complete genome of the type strain S. thermodepolymerans DSM 15344 that was assembled by hybrid approach using both, long (Oxford Nanopore) and short (Illumina) reads. The genome consists of a single 3,858,501bp long circular chromosome with GC content of 70.3%. Genome annotation identified 3,650 genes in total while 3,598 open reading frames belonged to protein coding genes. Functional annotation of the genome and division of genes into clusters of orthologous groups (COG) revealed a relatively high number of 1,013 genes with unknown function or unknown COG, which reflects the fact that only a little is known about thermophilic PHA producing bacteria on a genome level. On the other hand, 270 genes involved in energy conversion and production were detected. This group covers genes involved in catabolic processes which suggests capability of S. thermodepolymerans DSM 15344 to utilize and biotechnologically convert various substrates such as lignocellulose-based saccharides, glycerol, or lipids. Based on the knowledge of its genome, it can be stated that S. thermodepolymerans DSM 15344 is a very interesting, metabolically versatile bacterium with great biotechnological potential.

A group of 11 bacterial strains was isolated from streams and lakes located in a deglaciated northern part of James Ross Island, Antarctica. They were rod-shaped, Gram-stain-negative, motile, and catalase-positive and produced blue-violet-pigmented colonies on R2A agar. A polyphasic taxonomic approach based on 16S rRNA gene sequencing, whole-genome sequencing, automated ribotyping, repetitive element sequence-based PCR (rep-PCR), MALDI-TOF MS, fatty acid profile, chemotaxonomy analyses, and extensive biotyping was applied in order to clarify the taxonomic position of these isolates. Phylogenetic analysis based on the 16S rRNA gene indicated that all the isolates constituted a coherent group belonging to the genus Rugamonas. The closest relatives to the representative isolate P5900T were Rugamonas rubra CCM 3730T, Rugamonas rivuli FT103WT, and Rugamonas aquatica FT29WT, exhibiting 99.2%, 99.1%, and 98.6% 16S rRNA pairwise similarity, respectively. The average nucleotide identity and digital DNA-DNA hybridization values calculated from the whole-genome sequencing data clearly proved that P5900T represents a distinct Rugamonas species. The G+C content of genomic DNAs was 66.1mol%. The major components in fatty acid profiles were summed feature 3 (C16:1 7c/C16:1 6c), C 16:0, and C12:0. The cellular quinone content contained exclusively ubiquinone Q-8. The predominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The polyamine pattern was composed of putrescine, 2-hydroxputrescine, and spermidine.

Clostridium diolis DSM 15410 is a type strain of solventogenic clostridium capable of conducting isopropanol-butanol-ethanol fermentation. By studying its growth on different carbohydrates, we verified its ability to utilize glycerol and produce 1,3-propanediol and discovered its ability to produced isopropanol. Complete genome sequencing showed that its genome is a single circular chromosome and belongs to the cluster I (sensu scricto) of the genus Clostridium. By cultivation analysis we highlighted its specific behavior in comparison to two selected closely related strains. Despite the fact that several CRISPR loci were found, 16 putative prophages showed the ability to receive foreign DNA. Thus, the strain has the necessary features for future engineering of its 1,3-propanediol biosynthetic pathway and for the possible industrial utilization in the production of biofuels.

N-Butanol, a valuable solvent and potential fuel extender, can be produced via acetone-butanol-ethanol (ABE) fermentation. One of the main drawbacks of ABE fermentation is the high toxicity of butanol to producing cells, leading to cell membrane disruption, low culture viability and, consequently, low produced concentrations of butanol. The goal of this study was to obtain mutant strains of Clostridium beijerinckii NRRL B-598 with improved butanol tolerance using random chemical mutagenesis, describe changes in their phenotypes compared to the wild-type strain and reveal changes in the genome that explain improved tolerance or other phenotypic changes. Nine mutant strains with stable improved features were obtained by three different approaches and, for two of them, ethidium bromide (EB), a known substrate of efflux pumps, was used for either selection or as a mutagenic agent. It is the first utilization of this approach for the development of butanol-tolerant mutants of solventogenic clostridia, for which generally there is a lack of knowledge about butanol efflux or efflux mechanisms and their regulation. Mutant strains exhibited increase in butanol tolerance from 36 % up to 127 % and the greatest improvement was achieved for the strains for which EB was used as a mutagenic agent. Additionally, increased tolerance to other substrates of efflux pumps, EB and ethanol, was observed in all mutants and higher antibiotic tolerance in some of the strains. The complete genomes of mutant strains were sequenced and revealed that improved butanol tolerance can be attributed to mutations in genes encoding typical stress responses (chemotaxis, autolysis or changes in cell membrane structure), but, also, to mutations in genes X276_07980 and X276_24400, encoding efflux pump regulators. The latter observation confirms the importance of efflux in butanol stress response of the strain and offers new targets for rational strain engineering.

Clostridium beijerinckii NRRL B-598 is a sporulating, butanol and hydrogen producing strain that utilizes carbohydrates by the acetone-butanol-ethanol (ABE) fermentative pathway. The pathway consists of two metabolic phases, acidogenesis and solventogenesis, from which the latter one can be coupled with sporulation. Thorough transcriptomic profiling during a complete life cycle and both metabolic phases completed with flow cytometry, microscopy and a metabolites analysis helped to find out key genes involved in particular cellular events. The description of genes/operons that are closely involved in metabolism or the cell cycle is a necessary condition for metabolic engineering of the strain and will be valuable for all C. beijerinckii strains and other Clostridial species. The study focused on glucose transport and catabolism, hydrogen formation, metabolic stress response, binary fission, motility/chemotaxis and sporulation, which resulted in the composition of the unique image reflecting clostridial population changes. Surprisingly, the main change in expression of individual genes was coupled with the sporulation start and not with the transition from acidogenic to solventogenic metabolism. As expected, solvents formation started at pH decrease and the accumulation of butyric and acetic acids in the cultivation medium.

In-depth knowledge of cell metabolism and nutrient uptake mechanisms can lead to the development of a tool for improving acetone-butanol-ethanol (ABE) fermentation performance and help to overcome bottlenecks in the process, such as the high cost of substrates and low production rates. Over 300 genes potentially encoding transport of amino acids, metal ions, vitamins and carbohydrates were identified in the genome of the butanol-producing strain Clostridium beijerinckii NRRL B-598, based on similarity searches in protein function databases. Transcriptomic data of the genes were obtained during ABE fermentation by RNA-Seq experiments and covered acidogenesis, solventogenesis and sporulation. The physiological roles of the selected 81 actively expressed transport genes were established on the basis of their expression profiles at particular stages of ABE fermentation. This article describes how genes encoding the uptake of glucose, iron, riboflavin, glutamine, methionine and other nutrients take part in growth, production and stress responses of C. beijerinckii NRRL B-598. These data increase our knowledge of transport mechanisms in solventogenic Clostridium and may be used in the selection of individual genes for further research.

Studying bacterial population diversity is important to understand healthcare associated infections’ epidemiology and has a significant impact on dealing with multidrug resistant bacterial outbreaks. We characterised the extended-spectrum beta-lactamase producing K. pneumoniae (ESBLp KPN) population in our hospital using mini-MLST. Then we used whole genome sequencing (WGS) to compare selected isolates belonging to the most prevalent melting types (MelTs) and the colonization/infection pair isolates collected from one patient to study the ESBLp KPN population’s genetic diversity. A total of 922 ESBLp KPN isolates collected between 7/2016 and 5/2018 were divided into 38 MelTs using mini-MLST with only 6 MelTs forming 82.8% of all isolates. For WGS, 14 isolates from the most prominent MelTs collected in the monitored period and 10 isolates belonging to the same MelTs collected in our hospital in 2014 were randomly selected. Resistome, virulome and ST were MelT specific and stable over time. A maximum of 23 SNV per core genome and 58 SNV per core and accessory genome were found. To determine the SNV relatedness cut-off values, 22 isolates representing colonization/infection pair samples obtained from 11 different patients were analysed by WGS with a maximum of 22 SNV in the core genome and 40 SNV in the core and accessory genome within pairs. The mini-MLST showed its potential for real-time epidemiology in clinical practice. However, for outbreak evaluation in a low diversity bacterial population, mini-MLST should be combined with more sensitive methods like WGS. Our findings showed there were only minimal differences within the core and accessory genome in the low diversity hospital population and gene based SNV analysis does not have enough discriminatory power to differentiate isolate relatedness. Thus, intergenic regions and mobile elements should be incorporated into the analysis scheme to increase discriminatory power.

Background One of the main obstacles preventing solventogenic clostridia from achieving higher yields in biofuel production is the toxicity of produced solvents. Unfortunately, regulatory mechanisms responsible for the shock response are poorly described on the transcriptomic level. Although the strain Clostridium beijerinckii NRRL B-598, a promising butanol producer, has been studied under different conditions in the past, its transcriptional response to a shock caused by butanol in the cultivation medium remains unknown. Results In this paper, we present a transcriptional response of the strain during a butanol challenge, caused by the addition of butanol to the cultivation medium at the very end of the acidogenic phase, using RNA-Seq. We resequenced and reassembled the genome sequence of the strain and prepared novel genome and gene ontology annotation to provide the most accurate results. When compared to samples under standard cultivation conditions, samples gathered during butanol shock represented a well-distinguished group. Using reference samples gathered directly before the addition of butanol, we identified genes that were differentially expressed in butanol challenge samples. We determined clusters of 293 down-regulated and 301 up-regulated genes whose expression was affected by the cultivation conditions. Enriched term “RNA binding” among down-regulated genes corresponded to the downturn of translation and the cluster contained a group of small acid-soluble spore proteins. This explained phenotype of the culture that had not sporulated. On the other hand, up-regulated genes were characterized by the term “protein binding” which corresponded to activation of heat-shock proteins that were identified within this cluster. Conclusions We provided an overall transcriptional response of the strain C. beijerinckii NRRL B-598 to butanol shock, supplemented by auxiliary technologies, including high-pressure liquid chromatography and flow cytometry, to capture the

Many studies correlate changes in human gut microbiome with the onset of various diseases, mostly by 16S rRNA gene sequencing. Setting up the optimal sampling and DNA isolation procedures is crucial for robustness and reproducibility of the results. We performed a systematic comparison of several sampling and DNA isolation kits, quantified their effect on bacterial gDNA quality and the bacterial composition estimates at all taxonomic levels. Sixteen volunteers tested three sampling kits. All samples were consequently processed by two DNA isolation kits. We found that the choice of both stool sampling and DNA isolation kits have an effect on bacterial composition with respect to Gram-positivity, however the isolation kit had a stronger effect than the sampling kit. The proportion of bacteria affected by isolation and sampling kits was larger at higher taxa levels compared to lower taxa levels. The PowerLyzer PowerSoil DNA Isolation Kit outperformed the QIAamp DNA Stool Mini Kit mainly due to better lysis of Gram-positive bacteria while keeping the values of all the other assessed parameters within a reasonable range. The presented effects need to be taken into account when comparing results across multiple studies or computing ratios between Gram-positive and Gram-negative bacteria.