scholarly journals Bacteria and Metabolic Potential in Karst Caves Revealed by Intensive Bacterial Cultivation and Genome Assembly

2021 ◽  
Vol 87 (6) ◽  
Author(s):  
Hai-Zhen Zhu ◽  
Zhi-Feng Zhang ◽  
Nan Zhou ◽  
Cheng-Ying Jiang ◽  
Bao-Jun Wang ◽  
...  
Keyword(s):  
Large Scale ◽  
Bacterial Species ◽  
Biogeochemical Cycling ◽  
Metagenomic Data ◽  
Karst Cave ◽  
Bacterial Genomes ◽  
Metabolic Potential ◽  
Karst Caves ◽  
Microbial Resources ◽  
Cave Ecosystems

ABSTRACT Karst caves are widely distributed subsurface systems, and the microbiomes therein are proposed to be the driving force for cave evolution and biogeochemical cycling. In past years, culture-independent studies on the microbiomes of cave systems have been conducted, yet intensive microbial cultivation is still needed to validate the sequence-derived hypothesis and to disclose the microbial functions in cave ecosystems. In this study, the microbiomes of two karst caves in Guizhou Province in southwest China were examined. A total of 3,562 bacterial strains were cultivated from rock, water, and sediment samples, and 329 species (including 14 newly described species) of 102 genera were found. We created a cave bacterial genome collection of 218 bacterial genomes from a karst cave microbiome through the extraction of 204 database-derived genomes and de novo sequencing of 14 new bacterial genomes. The cultivated genome collection obtained in this study and the metagenome data from previous studies were used to investigate the bacterial metabolism and potential involvement in the carbon, nitrogen, and sulfur biogeochemical cycles in the cave ecosystem. New N2-fixing Azospirillum and alkane-oxidizing Oleomonas species were documented in the karst cave microbiome. Two pcaIJ clusters of the β-ketoadipate pathway that were abundant in both the cultivated microbiomes and the metagenomic data were identified, and their representatives from the cultivated bacterial genomes were functionally demonstrated. This large-scale cultivation of a cave microbiome represents the most intensive collection of cave bacterial resources to date and provides valuable information and diverse microbial resources for future cave biogeochemical research. IMPORTANCE Karst caves are oligotrophic environments that are dark and humid and have a relatively stable annual temperature. The diversity of bacteria and their metabolisms are crucial for understanding the biogeochemical cycling in cave ecosystems. We integrated large-scale bacterial cultivation with metagenomic data mining to explore the compositions and metabolisms of the microbiomes in two karst cave systems. Our results reveal the presence of a highly diversified cave bacterial community, and 14 new bacterial species were described and their genomes sequenced. In this study, we obtained the most intensive collection of cultivated microbial resources from karst caves to date and predicted the various important routes for the biogeochemical cycling of elements in cave ecosystems.

scholarly journals Consistent Metagenome-Derived Metrics Verify and Delineate Bacterial Species Boundaries

mSystems ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Matthew R. Olm ◽  
Alexander Crits-Christoph ◽  
Spencer Diamond ◽  
Adi Lavy ◽  
Paula B. Matheus Carnevali ◽  
...  
Keyword(s):  
Bacterial Diversity ◽  
Ribosomal Proteins ◽  
Large Scale ◽  
Bacterial Species ◽  
Bacterial Genome ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Species Discrimination ◽  
Bacterial Genomes ◽  
Discrimination Power

ABSTRACT Longstanding questions relate to the existence of naturally distinct bacterial species and genetic approaches to distinguish them. Bacterial genomes in public databases form distinct groups, but these databases are subject to isolation and deposition biases. To avoid these biases, we compared 5,203 bacterial genomes from 1,457 environmental metagenomic samples to test for distinct clouds of diversity and evaluated metrics that could be used to define the species boundary. Bacterial genomes from the human gut, soil, and the ocean all exhibited gaps in whole-genome average nucleotide identities (ANI) near the previously suggested species threshold of 95% ANI. While genome-wide ratios of nonsynonymous and synonymous nucleotide differences (dN/dS) decrease until ANI values approach ∼98%, two methods for estimating homologous recombination approached zero at ∼95% ANI, supporting breakdown of recombination due to sequence divergence as a species-forming force. We evaluated 107 genome-based metrics for their ability to distinguish species when full genomes are not recovered. Full-length 16S rRNA genes were least useful, in part because they were underrecovered from metagenomes. However, many ribosomal proteins displayed both high metagenomic recoverability and species discrimination power. Taken together, our results verify the existence of sequence-discrete microbial species in metagenome-derived genomes and highlight the usefulness of ribosomal genes for gene-level species discrimination. IMPORTANCE There is controversy about whether bacterial diversity is clustered into distinct species groups or exists as a continuum. To address this issue, we analyzed bacterial genome databases and reports from several previous large-scale environment studies and identified clear discrete groups of species-level bacterial diversity in all cases. Genetic analysis further revealed that quasi-sexual reproduction via horizontal gene transfer is likely a key evolutionary force that maintains bacterial species integrity. We next benchmarked over 100 metrics to distinguish these bacterial species from each other and identified several genes encoding ribosomal proteins with high species discrimination power. Overall, the results from this study provide best practices for bacterial species delineation based on genome content and insight into the nature of bacterial species population genetics.

scholarly journals A Universal, Genomewide GuideFinder for CRISPR/Cas9 Targeting in Microbial Genomes

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Michelle Spoto ◽  
Changhui Guan ◽  
Elizabeth Fleming ◽  
Julia Oh
Keyword(s):  
Gene Function ◽  
Large Scale ◽  
Essential Gene ◽  
Bacterial Species ◽  
Bacterial Genome ◽  
Model Organisms ◽  
Design Parameters ◽  
Bacterial Genomes ◽  
Wide Range ◽  
User Friendly

ABSTRACT The CRISPR/Cas system has significant potential to facilitate gene editing in a variety of bacterial species. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) represent modifications of the CRISPR/Cas9 system utilizing a catalytically inactive Cas9 protein for transcription repression and activation, respectively. While CRISPRi and CRISPRa have tremendous potential to systematically investigate gene function in bacteria, few programs are specifically tailored to identify guides in draft bacterial genomes genomewide. Furthermore, few programs offer open-source code with flexible design parameters for bacterial targeting. To address these limitations, we created GuideFinder, a customizable, user-friendly program that can design guides for any annotated bacterial genome. GuideFinder designs guides from NGG protospacer-adjacent motif (PAM) sites for any number of genes by the use of an annotated genome and FASTA file input by the user. Guides are filtered according to user-defined design parameters and removed if they contain any off-target matches. Iteration with lowered parameter thresholds allows the program to design guides for genes that did not produce guides with the more stringent parameters, one of several features unique to GuideFinder. GuideFinder can also identify paired guides for targeting multiplicity, whose validity we tested experimentally. GuideFinder has been tested on a variety of diverse bacterial genomes, finding guides for 95% of genes on average. Moreover, guides designed by the program are functionally useful—focusing on CRISPRi as a potential application—as demonstrated by essential gene knockdown in two staphylococcal species. Through the large-scale generation of guides, this open-access software will improve accessibility to CRISPR/Cas studies of a variety of bacterial species. IMPORTANCE With the explosion in our understanding of human and environmental microbial diversity, corresponding efforts to understand gene function in these organisms are strongly needed. CRISPR/Cas9 technology has revolutionized interrogation of gene function in a wide variety of model organisms. Efficient CRISPR guide design is required for systematic gene targeting. However, existing tools are not adapted for the broad needs of microbial targeting, which include extraordinary species and subspecies genetic diversity, the overwhelming majority of which is characterized by draft genomes. In addition, flexibility in guide design parameters is important to consider the wide range of factors that can affect guide efficacy, many of which can be species and strain specific. We designed GuideFinder, a customizable, user-friendly program that addresses the limitations of existing software and that can design guides for any annotated bacterial genome with numerous features that facilitate guide design in a wide variety of microorganisms.

scholarly journals The Distribution of Tryptophan-Dependent Indole-3-Acetic Acid Synthesis Pathways in Bacteria Unraveled by Large-Scale Genomic Analysis

Molecules ◽  
2019 ◽  
Vol 24 (7) ◽  
pp. 1411 ◽  
Author(s):  
Pengfan Zhang ◽  
Tao Jin ◽  
Sunil Kumar Sahu ◽  
Jin Xu ◽  
Qiong Shi ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Large Scale ◽  
Growth Promotion ◽  
Plant Root ◽  
Genomic Analysis ◽  
Acid Synthesis ◽  
Metagenomic Data ◽  
Bacterial Genomes ◽  
Bacterial Phyla ◽  
Indole 3 Acetic Acid

Bacterial indole-3-acetic acid (IAA), an effector molecule in microbial physiology, plays an important role in plant growth-promotion. Here, we comprehensively analyzed about 7282 prokaryotic genomes representing diverse bacterial phyla, combined with root-associated metagenomic data to unravel the distribution of tryptophan-dependent IAA synthesis pathways and to quantify the IAA synthesis-related genes in the plant root environments. We found that 82.2% of the analyzed bacterial genomes were potentially capable of synthesizing IAA from tryptophan (Trp) or intermediates. Interestingly, several phylogenetically diverse bacteria showed a preferential tendency to utilize different pathways and tryptamine and indole-3-pyruvate pathways are most prevalent in bacteria. About 45.3% of the studied genomes displayed multiple coexisting pathways, constituting complex IAA synthesis systems. Furthermore, root-associated metagenomic analyses revealed that rhizobacteria mainly synthesize IAA via indole-3-acetamide (IAM) and tryptamine (TMP) pathways and might possess stronger IAA synthesis abilities than bacteria colonizing other environments. The obtained results refurbished our understanding of bacterial IAA synthesis pathways and provided a faster and less labor-intensive alternative to physiological screening based on genome collections. The better understanding of IAA synthesis among bacterial communities could maximize the utilization of bacterial IAA to augment the crop growth and physiological function.

scholarly journals The Distribution of Tryptophan-Dependent Indole-3-Acetic Acid Synthesis Pathways in Bacteria Unraveled by Large-Scale Genomic Analysis

2019 ◽  
Author(s):  
Pengfan Zhang ◽  
Tao Jin ◽  
Sunil Kumar Sahu ◽  
Jin Xu ◽  
Qiong Shi ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Large Scale ◽  
Growth Promotion ◽  
Plant Root ◽  
Genomic Analysis ◽  
Acid Synthesis ◽  
Metagenomic Data ◽  
Bacterial Genomes ◽  
Bacterial Phyla ◽  
Indole 3 Acetic Acid

Bacterial indole-3-acetic acid (IAA), an effector molecule in microbial physiology, plays an important role in plant growth-promotion. Here, we comprehensively analyzed about 7282 prokaryotic genomes representing diverse bacterial phyla, combined with root-associated metagenomic data to unravel the distribution of tryptophan-dependent IAA synthesis pathways and to quantify the IAA synthesis-related genes in the plant root environments. We found that 82.2% of the analyzed bacterial genomes were potentially capable of synthesizing IAA from tryptophan (Trp) or intermediates. Interestingly, several phylogenetically diverse bacteria showed a preferential tendency to utilize different pathways and tryptamine and indole-3-pyruvate pathways are most prevalent in bacteria. About 45.3% of the studied genomes displayed multiple coexisting pathways, constituting complex IAA synthesis systems. Furthermore, root-associated metagenomic analyses revealed that rhizobacteria mainly synthesize IAA via IAM and tryptamine (TMP) pathways and might possess stronger IAA synthesis abilities than bacteria colonizing other environments. The obtained results refurbished our understanding of bacterial IAA synthesis pathways and provided a faster and less labor-intensive alternative to physiological screening based on genome collections. The better understanding of IAA synthesis among bacterial communities could maximize the utilization of bacterial IAA to augment the crop growth and physiological function.

Study of the Effects of Karst Area Beneath the Tunnel on the Stability of Surrounding Rock

2014 ◽  
Vol 711 ◽  
pp. 402-405
Author(s):  
Li Wei Dong ◽  
Ping Cao ◽  
Jie Liu
Keyword(s):  
Large Scale ◽  
Surrounding Rock ◽  
Vertical Stress ◽  
Karst Area ◽  
Karst Cave ◽  
Karst Caves ◽  
Tunnel Floor ◽  
Stability Of Surrounding Rock ◽  
Karst Areas ◽  
The Stability

Geotechnical engineering in large scale is endangered by the karst areas which are close to the engineering. The stability of the tunnels is greatly affected by the sizes and locations of the karst caves. Based on a engineering example, a study of the effects of karst area beneath the tunnel on the stability of surrounding rock is conducted. It can be concluded that the displacement release of surrounding rocks and the distribution of the vertical stress in surrounding rocks are greatly affected by the sizes of the karst caves and the distance between the tunnel floor and the karst cave.

scholarly journals PaSiT: a novel approach based on short-oligonucleotide frequencies for efficient bacterial identification and typing

2020 ◽  
Vol 36 (8) ◽  
pp. 2337-2344 ◽  
Author(s):  
Gleb Goussarov ◽  
Ilse Cleenwerck ◽  
Mohamed Mysara ◽  
Natalie Leys ◽  
Pieter Monsieurs ◽  
...  
Keyword(s):  
Large Scale ◽  
Bacterial Species ◽  
Supplementary Information ◽  
Nucleotide Identity ◽  
Average Nucleotide Identity ◽  
Bacterial Genomes ◽  
Short Oligonucleotide ◽  
Novel Approach ◽  
Novel Method ◽  
Alignment Step

Abstract Motivation One of the most widespread methods used in taxonomy studies to distinguish between strains or taxa is the calculation of average nucleotide identity. It requires a computationally expensive alignment step and is therefore not suitable for large-scale comparisons. Short oligonucleotide-based methods do offer a faster alternative but at the expense of accuracy. Here, we aim to address this shortcoming by providing a software that implements a novel method based on short-oligonucleotide frequencies to compute inter-genomic distances. Results Our tetranucleotide and hexanucleotide implementations, which were optimized based on a taxonomically well-defined set of over 200 newly sequenced bacterial genomes, are as accurate as the short oligonucleotide-based method TETRA and average nucleotide identity, for identifying bacterial species and strains, respectively. Moreover, the lightweight nature of this method makes it applicable for large-scale analyses. Availability and implementation The method introduced here was implemented, together with other existing methods, in a dependency-free software written in C, GenDisCal, available as source code from https://github.com/LM-UGent/GenDisCal. The software supports multithreading and has been tested on Windows and Linux (CentOS). In addition, a Java-based graphical user interface that acts as a wrapper for the software is also available. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals A universal, genome-wide guide finder for CRISPR/Cas9 targeting in microbial genomes

2017 ◽  
Author(s):  
Michelle Spoto ◽  
Elizabeth Fleming ◽  
Julia Oh
Keyword(s):  
Large Scale ◽  
Essential Gene ◽  
Bacterial Species ◽  
Bacterial Genome ◽  
Design Parameters ◽  
Bacterial Genomes ◽  
Microbial Genomes ◽  
Genome Wide ◽  
Cas9 Protein ◽  
User Friendly

AbstractBackgroundThe CRISPR/Cas system has significant potential to facilitate gene editing in a variety of bacterial species. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) represent modifications of the CRISPR/Cas9 system utilizing a catalytically inactive Cas9 protein for transcription repression or activation, respectively. While CRISPRi and CRISPRa have tremendous potential to systematically investigate gene function in bacteria, no pan-bacterial, genome-wide tools exist for guide discovery. We have created Guide Finder: a customizable, user-friendly program that can design guides for any annotated bacterial genome.ResultsGuide Finder designs guides from NGG PAM sites for any number of genes using an annotated genome and fasta file input by the user. Guides are filtered according to user-defined design parameters and removed if they contain any off-target matches. Iteration with lowered parameter thresholds allows the program to design guides for genes that did not produce guides with the more stringent parameters, a feature unique to Guide Finder. Guide Finder has been tested on a variety of diverse bacterial genomes, on average finding guides for 95% of genes. Moreover, guides designed by the program are functionally useful—focusing on CRISPRi as a potential application—as demonstrated by essential gene knockdown in two staphylococcal species.ConclusionsThrough the large-scale generation of guides, this open-access software will improve accessibility to CRISPR/Cas studies for a variety of bacterial species.

Clin-mNGS: Automated Pipeline for Pathogen Detection from Clinical Metagenomic Data

2020 ◽  
Vol 15 ◽  
Author(s):  
Akshatha Prasanna ◽  
Vidya Niranjan
Keyword(s):  
Antimicrobial Resistance ◽  
High Performance ◽  
Pathogen Detection ◽  
Bacterial Species ◽  
Workflow Management ◽  
Metagenomic Data ◽  
Antimicrobial Resistance Genes ◽  
Culture Independent ◽  
Automated Pipeline ◽  
User Friendly

Background: Since bacteria are the earliest known organisms, there has been significant interest in their variety and biology, most certainly concerning human health. Recent advances in Metagenomics sequencing (mNGS), a culture-independent sequencing technology have facilitated an accelerated development in clinical microbiology and our understanding of pathogens. Objective: For the implementation of mNGS in routine clinical practice to become feasible, a practical and scalable strategy for the study of mNGS data is essential. This study presents a robust automated pipeline to analyze clinical metagenomic data for pathogen identification and classification. Method: The proposed Clin-mNGS pipeline is an integrated, open-source, scalable, reproducible, and user-friendly framework scripted using the Snakemake workflow management software. The implementation avoids the hassle of manual installation and configuration of the multiple command-line tools and dependencies. The approach directly screens pathogens from clinical raw reads and generates consolidated reports for each sample. Results: The pipeline is demonstrated using publicly available data and is tested on a desktop Linux system and a High-performance cluster. The study compares variability in results from different tools and versions. The versions of the tools are made user modifiable. The pipeline results in quality check, filtered reads, host subtraction, assembled contigs, assembly metrics, relative abundances of bacterial species, antimicrobial resistance genes, plasmid finding, and virulence factors identification. The results obtained from the pipeline are evaluated based on sensitivity and positive predictive value. Conclusion: Clin-mNGS is an automated Snakemake pipeline validated for the analysis of microbial clinical metagenomics reads to perform taxonomic classification and antimicrobial resistance prediction.

scholarly journals Comparative genomics suggests a taxonomic revision of the Staphylococcus cohnii species complex

2021 ◽  
Author(s):  
Anna Lavecchia ◽  
Matteo Chiara ◽  
Caterina De Virgilio ◽  
Caterina Manzari ◽  
Carlo Pazzani ◽  
...  
Keyword(s):  
Comparative Genomics ◽  
Species Complex ◽  
Large Scale ◽  
Genomic Sequence ◽  
Bacterial Species ◽  
Taxonomic Revision ◽  
Distinct Species ◽  
The Novel ◽  
Staphylococcus Cohnii ◽  
Taxonomic Assignments

Abstract Staphylococcus cohnii (SC), a coagulase-negative bacterium, was first isolated in 1975 from human skin. Early phenotypic analyses led to the delineation of two subspecies (subsp.), Staphylococcus cohnii subsp. cohnii (SCC) and Staphylococcus cohnii subsp. urealyticus (SCU). SCC was considered to be specific to humans whereas SCU apparently demonstrated a wider host range, from lower primates to humans. The type strains ATCC 29974 and ATCC 49330 have been designated for SCC and SCU, respectively. Comparative analysis of 66 complete genome sequences—including a novel SC isolate—revealed unexpected patterns within the SC complex, both in terms of genomic sequence identity and gene content, highlighting the presence of 3 phylogenetically distinct groups. Based on our observations, and on the current guidelines for taxonomic classification for bacterial species, we propose a revision of the SC species complex. We suggest that SCC and SCU should be regarded as two distinct species: SC and SU (Staphylococcus urealyticus), and that two distinct subspecies, SCC and SCB (SC subsp. barensis, represented by the novel strain isolated in Bari) should be recognized within SC. Furthermore, since large scale comparative genomics studies recurrently suggest inconsistencies or conflicts in taxonomic assignments of bacterial species, we believe that the approach proposed here might be considered for more general application.

Stability Analysis of Dam Foundation with Karst Cave

2011 ◽  
Vol 90-93 ◽  
pp. 2640-2643
Author(s):  
Wei Ma ◽  
Chong Shi ◽  
Wei Jing Pan
Keyword(s):  
Stability Analysis ◽  
Work Conditions ◽  
Karst Cave ◽  
Dam Foundation ◽  
Safety Coefficient ◽  
Karst Caves ◽  
Load Analysis ◽  
Dip Angle ◽  
The Stability ◽  
Structure Plane

Stability of dam foundation with Karst caves is analyzed in the presented work.Conceptual model is established according to load analysis such as dam gravity and water press. The curve of point safety coefficient on the structure plane is draw out by using stress checking method.With the comparison of curves under different work conditions, it shows that the most dangerous position of Karst cave is at the dam toe area,with the increasing of dip angle of the structure plane,the safety coefficient declines.Then the possible damage zones,mechanism and failure mode of the dam foundation with Karst caves are analyzed. The result can be used in evaluating the stability of dam foundation with Karst cave.

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