Prokaryotic Genome Annotation Research Articles

Genome sequencing, annotation and application of a strain of Microbacterium paraoxydans – A bacterium with arsenic bioremediation and plant growth promoting potential

A heavy metal hypertolerant plant growth-promoting bacterium was isolated from the arsenic-contaminated garden soil of Bhagobangola I block (Murshidabad district). Metagenomic classification identified the bacterial isolate as a member of the genus Microbacterium. Taxonomic assessment of the isolated strain revealed its maximum average nucleotide identity (89.99%) with the Microbacterium paraoxydans strain DSM 15019. Prokaryotic genome annotation was performed by Prokka, DFAST and RAST. The entire genome consisted of 3365911 bases with 69.90% GC content. Prokka detected 3216 coding sequences (1461 hypothetical sequences), 3283 genes, 10 miscellaneous RNAs, 3 rRNAs, 53 tRNAs and 1 tmRNA in the bacterial genome. DFAST detected 3257 coding sequences (1217 hypothetical sequences), 3 rRNA sequences and 53 tRNA sequences in the bacterial genome. RAST detected 3285 coding sequences and 49 RNAs in the genome. Only 25% of these coding sequences could be categorised in the RAST subsystems. In RAST, 789 and 32 coding sequences were considered as non-hypothetical and hypothetical, respectively. The genes and gene clusters responsible for arsenic resistance (arsR, arsB, arsC, acr1, acr2 and acr3), tolerance to other heavy metals (copper, manganese, zinc, etc.) and plant growth promotion (auxin biosynthesis, siderophore-mediated iron acquisition, phosphate and polyphosphate metabolism, trehalose biosynthesis, etc.) were also identified in the Microbacterium paraoxydans genome. The bioremediation potential and plant growth-promoting nature of the bacterium were confirmed by silver diethyl dithio carbamate (SDDC) method and pot experiments, respectively. The aforementioned traits point towards potential of the bacterium as a bioremediation tool and biofertilizer for reducing arsenic toxicity and promoting plant growth.

Probiotic functional gene explorations in the genome of Limosilactobacillus fermentum GD5MG

Limosilactobacillus fermentum is an isolate obtained from oral gingival samples of healthy human individuals. The whole genome of Lb. fermentum GD5MG is composed of a circular DNA molecule containing 1,834,134 bp and exhibits a GC content of 52.80 %. The sequencing effort produced 38.6 million reads, each 150 bp in length, resulting in a sequencing depth of 2912.48x. Our examination unveiled a total of 1961 protein-coding genes, 27 rRNA genes, 24 tRNA genes, 3 non-coding RNA genes, and 63 pseudogenes with the use of gene annotations in NCBI Prokaryotic Genome Annotation tool. RAST revealed 1863 coding genes distributed across 209 subsystems, with a predominant involvement in amino acid, carbohydrate, and protein metabolism. Phylogenetic analysis infers that the Lb. fermentum GD5MG shares 281 gene clusters. Furthermore, the genome features showed a single CRISPR locus of 45 bp in length. Three genes associated with adhesion ability (strA, dltD, and dltA) and 26 genes related to acid tolerance, digestive enzyme secretion, and bile salt resistance were identified. Numerous genes associated with oral probiotic properties, comprising adhesion, acid and bile salt tolerance, oxidative stress tolerance, and sugar metabolism, were identified in the genome. Our findings shed light on the genomic characteristics of Lb. fermentum GD5MG, which are probable probiotics with functional benefits in humans.

Evaluating the use of smartphones for processing computational tasks: "A case study for the annotation of prokaryotic genomes"

Currently, there is a growing dependence on smartphones in the daily lives of thousands of people, noting that the number of users continues to rise, surpassing the number of computers in use globally. In addition, the performance of smartphone hardware has become an essential factor when choosing these devices, along with price, brand and operating system. Constant innovations in smartphones have made these devices increasingly powerful, even rivaling the performance of many computers. The example of the Samsung S23 5G, which has features comparable to a low-performance notebook, illustrates the advancement of the hardware in these devices. Despite the growing demand for advanced hardware in smartphones, many users may not fully understand the technical specifications of the processors. The article proposes an evaluation of the use of this hardware in the execution of specific tasks, with a focus on the annotation of prokaryotic genomes. The methodology of the study involved selecting popular smartphones from Samsung and Motorola, choosing the Android operating system due to its efficiency in resource management, using the Prokka tool for genomic annotation due to its minimal resource requirements and using Termux, a terminal emulator that allows users to run a full Linux distribution on Android devices. To evaluate performance, the article uses the Linux 'time' command to measure execution time, memory consumption, CPU utilization and other relevant metrics

Genomic analysis of a cAmpC (CMY-41)-producing Citrobacter freundii ST64 isolated from patient.

Antibiotic resistance in Citrobacter freundii is a public health concern. This study evaluated the closed genome of a C. freundii isolated from the stool of a hospitalized patient initially related to a Salmonella outbreak. Confirmation of the isolate was determined by whole-genome sequencing. Nanopore sequencing was performed using a MinION with a Flongle flow cell. Assembly using SPAdes and Unicycler yielded a closed genome annotated by National Center for Biotechnology Information Prokaryotic Genome Annotation Pipeline. Genomic analyses employed MLST 2.0, ResFinder4.1, PlasmidFinder2.1, and VFanalyzer. Phylogenetic comparison utilized the Center for Food Safety and Applied Nutrition (CFSAN)-single nucleotide polymorphism pipeline and Genetic Algorithm for Rapid Likelihood Inference. Antimicrobial susceptibility was tested by broth microdilution following Clinical and Laboratory Standards Institute criteria. Multi-locus sequence type in silico analysis assigned the C. freundii as sequence type 64 and the blaCMY-41 gene was detected in resistome investigation. The susceptibility to antibiotics, determined using Sensititre® plates, revealed resistance to aztreonam, colistin, cefoxitin, amoxicillin/clavulanic acid, sulfisoxazole, ampicillin, and streptomycin. The genetic relatedness of the C. freundii CFSAN077772 with publicly available C. freundii genomes revealed a close relationship to a C. freundii SRR1186659, isolated in 2009 from human stool in Tanzania. In addition, C. freundii CFSAN077772 is nested in the same cluster with C. freundii clinical strains isolated in Denmark, Mexico, Myanmar, and Canada, suggesting a successful intercontinental spread.

Draft Genome Sequence of Pantoea sp. Strain MHSD4, a Bacterial Endophyte With Bioremediation Potential.

Pantoea sp. strain MHSD4 is a bacterial endophyte isolated from the leaves of the medicinal plant Pellaea calomelanos. Here, we report on strain MHSD4 draft whole genome sequence and annotation. The draft genome size of Pantoea sp. strain MHSD4 is 4 647 677 bp with a G+C content of 54.2% and 41 contigs. The National Center for Biotechnology Information Prokaryotic Genome Annotation Pipeline tool predicted a total of 4395 genes inclusive of 4235 protein-coding genes, 87 total RNA genes, 14 non-coding (nc) RNAs and 70 tRNAs, and 73 pseudogenes. Biosynthesis pathways for naphthalene and anthracene degradation were identified. Putative genes involved in bioremediation such as copA, copD, cueO, cueR, glnGm, and trxC were identified. Putative genes involved in copper homeostasis and tolerance were identified which may suggest that Pantoea sp. strain MHSD4 has biotechnological potential for bioremediation of heavy metals.

Biotechnological Key Genes of the Rhodococcus erythropolis MGMM8 Genome: Genes for Bioremediation, Antibiotics, Plant Protection, and Growth Stimulation.

Anthropogenic pollution, including residues from the green revolution initially aimed at addressing food security and healthcare, has paradoxically exacerbated environmental challenges. The transition towards comprehensive green biotechnology and bioremediation, achieved with lower financial investment, hinges on microbial biotechnology, with the Rhodococcus genus emerging as a promising contender. The significance of fully annotating genome sequences lies in comprehending strain constituents, devising experimental protocols, and strategically deploying these strains to address pertinent issues using pivotal genes. This study revolves around Rhodococcus erythropolis MGMM8, an associate of winter wheat plants in the rhizosphere. Through the annotation of its chromosomal genome and subsequent comparison with other strains, its potential applications were explored. Using the antiSMASH server, 19 gene clusters were predicted, encompassing genes responsible for antibiotics and siderophores. Antibiotic resistance evaluation via the Comprehensive Antibiotic Resistance Database (CARD) identified five genes (vanW, vanY, RbpA, iri, and folC) that were parallel to strain CCM2595. Leveraging the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) for biodegradation, heavy metal resistance, and remediation genes, the presence of chlorimuron-ethyl, formaldehyde, benzene-desulfurization degradation genes, and heavy metal-related genes (ACR3, arsC, corA, DsbA, modA, and recG) in MGMM8 was confirmed. Furthermore, quorum-quenching signal genes, critical for curbing biofilm formation and virulence elicited by quorum-sensing in pathogens, were also discerned within MGMM8's genome. In light of these predictions, the novel isolate MGMM8 warrants phenotypic assessment to gauge its potential in biocontrol and bioremediation. This evaluation extends to isolating active compounds for potential antimicrobial activities against pathogenic microorganisms. The comprehensive genome annotation process has facilitated the genetic characterization of MGMM8 and has solidified its potential as a biotechnological strain to address global anthropogenic predicaments.

RefSeq and the prokaryotic genome annotation pipeline in the age of metagenomes.

The Reference Sequence (RefSeq) project at the National Center for Biotechnology Information (NCBI) contains over 315000 bacterial and archaeal genomes and 236 million proteins with up-to-date and consistent annotation. In the past 3 years, we have expanded the diversity of the RefSeq collection by including the best quality metagenome-assembled genomes (MAGs) submitted to INSDC (DDBJ, ENA and GenBank), while maintaining its quality by adding validation checks. Assemblies are now more stringently evaluated for contamination and for completeness of annotation prior to acceptance into RefSeq. MAGs now account for over 17000 assemblies in RefSeq, split over 165 orders and 362 families. Changes in the Prokaryotic Genome Annotation Pipeline (PGAP), which is used to annotate nearly all RefSeq assemblies include better detection of protein-coding genes. Nearly 83% of RefSeq proteins are now named by a curated Protein Family Model, a 4.7% increase in the past three years ago. In addition to literature citations, Enzyme Commission numbers, and gene symbols, Gene Ontology terms are now assigned to 48% of RefSeq proteins, allowing for easier multi-genome comparison. RefSeq is found at https://www.ncbi.nlm.nih.gov/refseq/. PGAP is available as a stand-alone tool able to produce GenBank-ready files at https://github.com/ncbi/pgap.

Characteristics of a ceftadine/avibatam resistance KPC-33-producing Klebsiella Pneumoniae strain with capsular serotype K19 belonging to ST15

The aim of this study was to characterize the blaKPC-33 in a ST15-K19 ceftazidime-avibactam (CAZ-AVI)-resistant Klebsiella pneumoniae strain after the antibiotic CAZ-AVI was approved for use in Wuxi No. 2 People's Hospital, China. Antimicrobial susceptibility testing was performed by the microdilution broth method. Whole genome sequencing (WGS) was performed using PacBio II and MiSeq sequencers. High-quality reads were assembled using the SOAPdenovo and GapCloser v1.12, and genome annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). Genomic characteristics were analysed by using bioinformatics methods. K. pneumoniae strain KPHRJ showed resistance to CAZ-AVI. WGS analysis showed that strain KPHRJ had one 5 536 506 bp chromosome (57.25% G+C content) and one plasmid (133 451 bp, G+C 54.29%). KPHRJ was classified as ST15 and K19 serotype. Resistome analysis showed that KPHRJ carries seven antimicrobial resistance genes (ARGs). WGS analysis and conjugation experiments demonstrated that the blaKPC-33 gene was carried by plasmid pKPHRJ, flanked by two copies of IS26 mobile elements (IS26-ISKpn27-blaKPC-33-ISKpn6-korC-TnAs1-tetR-tetA-Tn3-IS26). Besides these acquired resistance genes, mutations in porin protein-coding genes, such as OmpK36 and OmpK37, which may reduce susceptibility to the CAZ-AVI, were also identified from the genome. Here, we present the WGS of a CAZ-AVI resistant K. pneumoniae isolate, strain KPHRJ, with capsular serotype K19 and belonging to ST15. CAZ-AVI resistance is likely conferred by a KPC-2 variant, blaKPC-33 and mutations in porin-coding genes. We speculate that the approval of the CAZ-AVI in hospital could contribute to the emergence of these genomic features by providing a selective pressure leading to the emergence of CAZ-AVI resistant bacteria.

Complete genome sequence data of multidrug-resistant Aeromonas hydrophila Ah27 isolated from intussusception channel catfish (Ictalurus punctatus)

Aeromonas hydrophila Ah27, a multidrug-resistant bacterium, was separated from the liver of an intussusception channel catfish (Ictalurus punctatus) in Guizhou province, China. The genome of A. hydrophila Ah27 was sequenced using the PacBio RS and Illumina sequencing platforms. The complete genome of A. hydrophila Ah27 consists of a circular chromosome with 5,102,912 bp and a plasmid with 6162 bp. The chromosome's average GC content is 60.75 %. NCBI Prokaryotic Genome Annotation Pipeline (v1.02) was used to annotate the genome with the default parameters. The results showed that the chromosome contained 4726 coding genes, including 4505 protein coding sequences, 119 tRNA genes, 31 rRNA genes, 7 ncRNAs and 64 Pseudo Genes. According to the annotation analysis, strain Ah27 encodes β-lactams, aminoglycosides, quinolones, macrolides, sulfonamides, tetracyclines resistance genes, and four significant types of efflux pump genes. The Kirby-Bauer disk method was used to detect the sensitivity or resistance of Ah27. The results showed that strain Ah27 was resistant to 27 antibiotics, including spectinomycin, streptomycin, enrofloxacin, tetracycline, bacitracin, clarithromycin, erythromycin, trimethoprim, etc. At the same time, 30 mobile genetic elements were also obtained by comparison with the MobileElementFinder tool. The complete genome sequence of A. hydrophila Ah27 may also provide a fresh perspective on disease prevention and antimicrobial resistance mechanisms.

Mining the genome of Bacillus velezensis FS26 for probiotic markers and secondary metabolites with antimicrobial properties against aquaculture pathogens

Bacillus velezensis FS26 is a bacterium from the genus Bacillus that has been proven as a potential probiotic in aquaculture with a good antagonistic effect on Aeromonas spp. and Vibrio spp. Whole-genome sequencing (WGS) allows a comprehensive and in-depth analysis at the molecular level, and it is becoming an increasingly significant technique in aquaculture research. Although numerous probiotic genomes have been sequenced and investigated recently, there are minimal data on in silico analysis of B. velezensis as a probiotic bacterium isolated from aquaculture sources. Thus, this study aims to analyse the general genome characteristics and probiotic markers from the B. velezensis FS26 genome with secondary metabolites predicted against aquaculture pathogens. The B. velezensis FS26 genome (GenBank Accession: JAOPEO000000000) assembly proved to be of high quality, with eight contigs containing 3,926,371 bp and an average G + C content of 46.5%. According to antiSMASH analysis, five clusters of secondary metabolites from the B. velezensis FS26 genome showed 100% similarity. These clusters include Cluster 2 (bacilysin), Cluster 6 (bacillibactin), Cluster 7 (fengycin), Cluster 8 (bacillaene), and Cluster 9 (macrolactin H), which signify promising antibacterial, antifungal, and anticyanobacterial agents against pathogens in aquaculture. The probiotic markers of B. velezensis FS26 genome for adhesion capability in the hosts' intestine, as well as the acid and bile salt-tolerant genes, were also detected through the Prokaryotic Genome Annotation System (Prokka) annotation pipeline. These results are in agreement with our previous in vitro data, suggesting that the in silico investigation facilitates establishing B. velezensis FS26 as a beneficial probiotic for use in aquaculture.

Gymnodinialimonas phycosphaerae sp. nov., a phycosphere bacterium isolated from Karlodinium veneficum.

A facultative anaerobic, Gram-negative, non-motile, rod-shaped bacterial strain, designated N5T, was obtained from the phycosphere microbiota of the marine planktonic dinoflagellate, Karlodinium veneficum. Strain N5T showed growth on marine agar at 25 °C, pH 7 and 1 % (w/v) NaCl and produced a yellow colour. According to a phylogenetic study based on 16S rRNA gene sequences, strain N5T has a lineage within the genus Gymnodinialimonas. The G+C content in the genome of strain N5T is 62.9 mol% with a total length of 4 324 088 bp. The NCBI Prokaryotic Genome Annotation Pipeline revealed that the N5T genome contained 4230 protein-coding genes and 48 RNA genes, including a 5S rRNA, 16S rRNA, 23S rRNA, 42 tRNA, and three ncRNAs. Genome-based calculations (genome-to-genome distance, average nucleotide identity and DNA G+C content) clearly indicated that the isolate represents a novel species within the genus Gymnodinialimonas. The predominant fatty acids were C19 : 0 cyclo ω8c and feature 8 (comprising C18 : 1 ω6c and/or C18 : 1 ω7c). The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. The main respiratory quinone was Q-10. Based on its phenotypic, phylogenetic, genomic and chemotaxonomic features, strain N5T represents a novel species of the genus Gymnodinialimonas, for which the name Gymnodinialimonas phycosphaerae sp. nov. is proposed. The type strain is N5T (=KCTC 82362T=NBRC 114899T).

First report of Pseudomonas viridiflava causing fruit rot on sweet cherry trees in Chile.

Chile is an important producers of sweet cherry (Prunus avium L.), with a total of 356,385 t exported in the 2021 to 2022 season. The production area includes most of the country's regions. Bacterial samples were isolated in 2017 and 2018 from 18 commercial sweet cherry orchards with canker disease. From one of this samples collected in the spring of 2018, was isolated the strain A2M176 from buds of trees that presented canker and gomosis in Malloa locality (34°23' 46'' S 71°01' 39'' W). The strain produced fluorescent pigment on King's B agar medium. Is oxidase and arginine dihydrolase negative, potato soft rot positive and showed a slight degree of tobacco hypersensitivity. It was able to growth up to 0.8 mM (200 ppm) of CuSO4·5H2O. The strain A2M176 was deposited in the Colección Chilena de Recursos Genéticos Microbianos (CChRGM) under the no. RGM 3342. The DNA of this strain was extracted from a pure culture using silica spin columns (Epoch Life Science Inc., Sugar Land, USA). The complete DNA was sequenced using HiSeq with 150 bp paired-end at GENEWIZ (New York, USA). Raw data was checked using FASTQC and trimmed with BBDuk. The genome was assembled using Unicycler v0.4.9 with defaulf settings and annotated with Prokaryotic Genome Annotation Pipeline (PGAP) v4.3. The reads and genomes were uploaded to GenBank under the BioProyect no. PRJNA750090, BioSample no. SAMN26870984 and assembly no. GCA_022936465.1. The sequenced genome was compared through Average Nucleotide Identity algorithm (ANI) using FastANI v1.33 to compare with closest complete genome available on NCBI. The strain A2M176 was identified as P. viridiflava with ANI value of 98.06% with the strain p22.E7 (GCF_900585495). Maximum likelihood phylogenetic estimation clustered strain A2M176 with other P. viridiflava strains with 95% bootstrap. The pathogenicity of the strain was tested inoculating immature cherry fruits with a needle with a bacterial suspension (1x108 CFU/ml). The inoculated fruits were placed at room temperature in a humid chamber for 10 d. Soft rot lesions were observed, which appeared at 6 days post-inoculation (DPI). The control fruits treated with sterile water did not show symptoms. Further analyses in the genome of strain A2M176 led to identify genes related to pathogenicity, such as the effector gene avrE and the regulator gen HrpL, suggesting the pathogenic capacity of the strain. Also, there were identify genes of two known Pseudomonas copper resistance mechanisms, the cus and cop operon. These genes were found part of the copABCDns cluster similar to what was observed in Pseudomonas from Mango. Presence of P. viridiflava strains causing fruit rot in P. avium is not surprising, since P. viridiflava has a wide host range and causes a variety of symptoms in different plant parts, including stems, leaves, and blossoms. P. viridiflava represents one of the multiple phylogroups found within the P. syringae species complex. To our knowledge, this is the first report of a strain of P. viridiflava copper resistant causing infection on sweet cherries in Chile.

Genomic characterization of a multidrug-resistant Salmonella Rissen ST469 carrying blaCTX-M-55 gene and Tn6777 isolated from a paediatric patient in China

Here we report a complete genome sequence of a multidrug-resistant Salmonella Rissen, carrying blaCTX-M-55 and Tn6777, isolated from a Chinese paediatric patient. Whole genome of S. Rissen S1905 was sequenced using the Oxford Nanopore MinION and Illumina NovaSeq 6000 platforms. Unicycler was used to perform a de novo assembly of Illumina and Nanopore reads. The genome sequence was annotated using the NCBI Prokaryotic Genome Annotation Pipeline. In silico multilocus sequence typing, plasmid replicons, antimicrobial resistance genes and virulence factors were identified from the genome sequence by multiple bioinformatics tools. Core genome multilocus sequence typing analysis between S. Rissen S1905 and all retrieved from the NCBI GenBank database was performed using BacWGSTdb 2.0 server. Six contigs totaling 5 056 896 bp make up the complete genome sequence of S. Rissen S1905, which includes 1 chromosome and 5 plasmids. The blaCTX-M-55 was embedded in the ISEcp1-blaCTX-M-55-wbuC transposition unit located in an 85 991-bp IncI1 plasmid. However, the pco-sil operon and other eight antimicrobial resistance genes were carried by Tn6777 in the chromosome. There are 162 virulence genes in S1905. S. Rissen S1905 belongs to ST469; the closest relative was another isolate originating from a human faecal specimen in Shanghai, China, which differed by 60 core genome multilocus sequence type alleles. These data on the multidrug-resistant S. Rissen carrying blaCTX-M-55 and Tn6777 can provide a foundation for further studies on the molecular epidemiological characteristics, pathogenicity, antimicrobial resistance mechanisms, and dissemination mechanism of Salmonella.

StandEnA: a customizable workflow for standardized annotation and generating a presence-absence matrix of proteins.

Several genome annotation tools standardize annotation outputs for comparability. During standardization, these tools do not allow user-friendly customization of annotation databases; limiting their flexibility and applicability in downstream analysis. StandEnA is a user-friendly command-line tool for Linux that facilitates the generation of custom databases by retrieving protein sequences from multiple databases. Directed by a user-defined list of standard names, StandEnA retrieves synonyms to search for corresponding sequences in a set of public databases. Custom databases are used in prokaryotic genome annotation to generate standardized presence-absence matrices and reference files containing standard database identifiers. To showcase StandEnA, we applied it to six metagenome-assembled genomes to analyze three different pathways. StandEnA is an open-source software available at https://github.com/mdsufz/StandEnA. Supplementary data are available at Bioinformatics Advances online.

Complete genome sequencing of Bacillus cabrialesii TE3T: A plant growth-promoting and biological control agent isolated from wheat (Triticum turgidum subsp. durum) in the Yaqui Valley

Bacillus cabrialesii TE3T is a strictly aerobic and Gram-stain-positive plant growth-promoting bacterium, motile and catalase-positive. In addition, strain TE3T was also recently described as a biological control agent. Here, we present the complete circularized genome of this type strain, as well as a whole genome analysis identifying genes of agricultural interest. Thus, a hybrid assembly method was performed using short-read sequencing through the Illumina MiSeq platform, and long-read sequencing through the MinION sequencing technology by Oxford Nanopore Technology (ONT). This assembly method showed a closed circular chromosome of 4,125,766 bp and 44.2% G + C content. The strain TE3T genome annotation, based on the RAST platform, presented 4,282 Coding DNA sequences (CDS) distributed in 335 subsystems, from which 4 CDS are related to the promotion of plant growth and 28 CDS to biological control. Also, Prokka (Rapid Prokaryotic Genome Annotation) predicted a total of 119 RNAs composed of 87 tRNAs, 31 rRNA, and 1 tmRNA; and the PGAP (Prokaryotic Genome Annotation Pipeline) predicted a total of 4,212 genes (3,991 CDS). Additionally, seven putative biosynthetic gene clusters were identified by antiSMASH, such as Fengycin, Bacilysin, Subtilosin A, Bacillibactin, Bacillaene, Surfactin, and Rizocticin A, which are related to antimicrobial and antifungal properties, whose gene presence was further supported by the Prokaryotic Genome Annotation Pipeline (PGAP) annotation. Thus, the complete genome of Bacillus cabrialesii TE3T showed promising bioactivities for the use of this type strain to bioformulate bacterial inoculants for sustainable agriculture.

Saccharothrix luteola sp. nov., a novel cellulose-degrading actinobacterium isolated from soil and emended description of the genus Saccharothrix.

A novel cellulose-degrading actinobacterium, designated strain NEAU-S10T, was isolated from soil collected from Chifeng, Inner Mongolia Autonomous Region, PR China, and characterized using a polyphasic approach. Pairwise similarity of the 16S rRNA gene sequence showed that strain NEAU-S10T was a representative of Saccharothrix and was closely related to Saccharothrix carnea NEAU-yn17T (99.2 %), Saccharothrix saharensis SA152T (99.0 %), Saccharothrix texasensis DSM 44231T (98.5 %) and Saccharothrix xinjiangensis NBRC 101911T (98.5 %). Physiological and chemotaxonomic characteristics of the strain further supported its affiliation to the genus Saccharothrix. The whole-cell sugars contained galactose, ribose and mannose. The polar lipids contained diphosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. The predominant menaquinones were MK-9(H0), MK-9(H2), MK-9(H4) and MK-10(H4). The major fatty acids were iso-C16 : 0, C16 : 0, anteiso-C17 : 0, iso-C15 : 0 and iso-C17 : 0. The genomic DNA G+C content was 71.8 mol%. The levels of digital DNA-DNA hybridization between isolate and S. carnea NEAU-yn17T, S. saharensis SA152T and S. texasensis DSM 44231T were 40.1 % (37.6-42.6 %), 38.soap8 % (36.3-41.3 %) and 44.8 % (42.2-47.3 %) and the ANI values between them were determined to be 90.2, 89.8 and 91.7 %, the results indicated that strain NEAU-S10T could be distinguished from its reference strains. The assembled genome sequence of strain NEAU-S10T was found to be 10 305 394 bp long. The NCBI Prokaryotic Genome Annotation Pipeline (PGAP) revealed 8 994 protein-coding genes. Genomic analysis and Congo red staining test indicated that strain NEAU-S10T had the potential to degrade cellulose. The genomic and phenotypic results indicate that strain NEAU-S10T represents a novel species of the genus Saccharothrix, for which the name Saccharothrix luteola sp. nov. is proposed, with NEAU-S10T (=CCTCC AA 2020037T=JCM 34800T) as the type strain.

Genomic diversity and molecular epidemiology of a multidrug-resistant Pseudomonas aeruginosa DMC30b isolated from a hospitalized burn patient in Bangladesh

Objectives: Pseudomonas aeruginosa is a key opportunistic pathogen causing a wide range of community- and hospital-acquired infections in immunocompromised or catheterized patients. Here, we report the complete genome sequence of a multidrug-resistant (MDR) P. aeruginosa DMC30b to elucidate the genetic diversity, molecular epidemiology, and underlying mechanisms for antimicrobial resistance and virulence. Methods: P. aeruginosa DMC30b was isolated from septic wound swab of a severe burn patient. Whole-genome sequencing was performed under Ion Torrent platform. The genome was assembled using the SPAdes v. 3.12.01 in an integrated Genome Analysis Platform for Ion Torrent sequence data. The genome was annotated using the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline. In-silico predictions of antimicrobial resistance genes, virulence factor genes, and metabolic functional potentials were performed using different curated bioinformatics tools. Results: P. aeruginosa DMC30b was found as a MDR strain and belonged to sequence type 244 (ST244). The complete genome size is 6 994 756 bp with a coverage of 76.76x, guanine-cytosine content of 65.7% and a Benchmarking Universal Single-Copy Orthologs score of 100. The genome of P. aeruginosa DMC30b harboured two predicted plasmid replicons (e,g. IncP-6; 78 007 bp and ColRNAI; 9359 bp), 35 resistomes (antimicrobial resistance genes) conferring resistance to 18 different antibiotics (including four beta-lactam classes), and 214 virulence factor genes. It was identified as the 167th ST244 strain among ∼ 5800 whole-genome sequences of P. aeruginosa available in the NCBI database. Conclusion: The MDR P. aeruginosa DMC30b was identified as the 167th ST244 complete genome to be submitted to the NCBI, and the first ST244 isolate sequenced from Bangladesh. The complete genome data with high genetic diversity and underlying mechanisms for antimicrobial resistance and virulence of P. aeruginosa DMC30b will aid in understanding the evolution and phylogeny of such high-risk clones and provide a solid basis for further research on MDR or extensively drug resistant strains.

Genomic Characterization of a Clinical Listeria monocytogenes ST1 Isolate Recovered from the Blood Sample of a Woman with Third Trimester Stillbirth

BackgroundListeria monocytogenes is a foodborne gram-positive bacterium which causes adverse pregnancy outcomes. Here, the genomic and phylogenetic characteristics of a L. monocytogenes isolate obtained from blood sample of a third trimester pregnant woman with stillbirth are investigated.MethodsWhole genome DNA of L. monocytogenes ST1 was sequenced with HiSeq X Ten platform. The NCBI Prokaryotic Genome Annotation Pipeline was used to annotate the genome sequence. The sequence type (ST) and antimicrobial resistance genes were then identified. The core genome multilocus sequence typing (cgMLST) analysis with other closely related L. monocytogenes stored in the NCBI GenBank database was performed using BacWGSTdb 2.0.ResultsThe complete genome sequence of L. monocytogenes ST1 is made up of 20 contigs totaling 2,914,725 bp, with 2886 protein-coding sequences and a GC content of 37.9%. Fosfomycin [fosX], Lincosamide antibiotic [lin] and peptide antibiotic [mprF] were discovered as antimicrobial resistance genes. In silico serogroup typing prediction revealed that L. monocytogenes ST1 belonged to serotype IVb. The closest relative of L. monocytogenes ST1, obtained from Poland in 2015, differs by only 15 cgMLST alleles.ConclusionWe identified a L. monocytogenes ST1 strain from blood sample of a woman with third trimester stillbirth in China. These discoveries would aid in our understanding of the genomic characteristics, mechanisms of antimicrobial resistance, and epidemiological features of this pathogen.

ReNoteWeb – Web platform for the improvement of assembly result and annotation of prokaryotic genomes

The reduction in the cost of DNA sequencing and the total time to perform this process has resulted in a significant increase in the deposit of biological information in public databases such as the NCBI (National Center for Biotechnology Information). The production of large volumes of data per run has culminated in the need to develop algorithms capable of handling data with this new feature and assisting in analyses such as the assembly and annotation of prokaryotic genomes. Over the years, several pipelines and computational tools have been developed to automate this task and consequently reduce the total time to know the genetic content of a given organism, especially non-model organisms, collaborating with the identification of possible targets with biotechnological applicability. In the case of automatic annotation tools, the accuracy of the results is widely observed in the literature, however, this does not excludes the manual curation process, where the information inferred in the automatic process is verified and enriched by the curators. This task requires a time which is directly proportional to the number of gene products of the target organism under study. To assist in this process, we present the ReNoteWeb web tool, endowed with a simple and intuitive interface, to perform the assembly enhancement process, with the possibility of identifying the missing products in the original genomic sequence. In addition, ReNoteWeb is capable of performing the annotation process for all products, based on information obtained from highly accurate external databases. The engine responsible for performing the data processing was developed in JAVA and the web platform uses the resources of the Yii framework. The annotation produced by this platform aims to reduce the overall time in the manual curation process. Twenty-three organisms were used to validate the tool. The efficiency was verified by comparing the annotation of these same organisms available in the NCBI database and the annotation performed on the RAST platform. The tool is available at: http://biod.ufpa.br/renoteweb/.

Serratia nevei 9rpt1, a Potential Microorganism for Phosphorus Recovery

Aims: Phosphorus (P) is an essential element for life, finite and irreplaceable. Its constant exploration on a global scale has motivated frequent alerts regarding an eventual crisis due to the shortage of this nutrient. However, it is possible to recycle it and reintroduce it into the ecological cycle. One viable alternative is the microbial recovery of phosphate.
 Study design: This study is based on systematic bioprospection of bacteria in phosphate-deficient Amazon regions.
 Place and Duration of Study: Bacteria were isolated from black water samples, collected in the Rio Pretinho, located at Serra do Aracá, Barcelos, Amazonas, Brazil, from January to July 2019.
 Methodology: Microbial isolation was performed in Luria Bertani agar medium. For the genomic study, the isolate with the best performance in the phosphate uptake test was chosen. The WGS was carried out in a Illumina HiSeq 2500 System. The assembly of the draft genome was carried out with the SPAdes and the annotation by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP).
 Results: Serratia nevei 9rpt1 recovers 90% of the phosphate available in the culture medium. Its draft genome comprises 5.4 MB, the GC content is 59.52% and 4,922 coding sequences were identified, among these, two pst operons: one complete, containing the five pst genes and one missing pstS, pstC and phoU genes.
 Conclusion: Serratia nevei 9rpt1, isolated from an Amazonian environment poor in phosphate, is very efficient to uptake this nutrient in a Pi starvation condition. The genomic findings revealed this strain has an additional high affinity Pi uptake pst system containing the ATP-binding protein PstB, the canonical permease PstA, a putative permease other than PstC, upstream of the PstA and two essential enzymes in the polyphosphate metabolism: polyphosphate kinase 1 and exopolyphosphatase.