Bacterial Phylogeny Research Articles

Microbial symbiosis and coevolution of an entire clade of ancient vertebrates: the gut microbiota of sea turtles and its relationship to their phylogenetic history

BackgroundThe microbiota plays a critical role in host homeostasis and has been shown to be a major driving force in host evolution. However, our understanding of these important relationships is hampered by a lack of data for many species, and by significant gaps in sampling of the evolutionary tree. In this investigation we improve our understanding of the host-microbiome relationship by obtaining samples from all seven extant species of sea turtle, and correlate microbial compositions with host evolutionary history.ResultsOur analysis shows that the predominate phyla in the microbiota of nesting sea turtles was Proteobacteria. We also demonstrate a strong relationship between the bacterial phyla SR1 and sea turtle phylogeny, and that sea turtle microbiotas have changed very slowly over time in accordance with their similarly slow phenotypic changes.ConclusionsThis is one of the most comprehensive microbiota studies to have been performed in a single clade of animals and further improves our knowledge of how microbial populations have influenced vertebrate evolution.

Abstract PR05: Elucidating role of bacteria during pancreatic ductal adenocarcinoma (PDAC)

Abstract Long-term survival in pancreatic cancer (PC) is rare as only a minority of all resected patients survive for >5 years. Interestingly, somatic mutations do not predict long-term survivorship, highlighting the potential role of extrinsic factors. Recent studies have reported the role of the gut and tumor microbiome in pancreatic tumorigenesis and PC treatment responses. Since intratumoral bacteria have been detected in human PC tumors, possibly arising from relocation of intestinal bacteria, it is possible that intratumoral T cells get primed with microbial antigens within the tumor microenvironment. To reconstruct bacterial phylogenies, we performed prokaryotic 16S ribosomal RNA sequencing and immunoprofiling of PC tumors from “Long-Term Survivors-LTS” (>5y post-resection) and “Short-Term Survivors-STS” (<5y post-resection). Strikingly, we found that LTS from both discovery and validation cohorts had higher microbial diversity and LTS from both cohorts were enriched for a tumor microbial signature: Pseudoxanthomonas, Streptomyces, Saccharopolyspora, and Bacillus clausii. Moreover, the most overrepresented species in LTS tumors strongly correlated with enhanced immunoactivation of the tumor microenvironment, suggestive of bacterial-mediated immune cell recruitment and activation. Based on these findings, we hypothesized that gut bacteria affect PC by triggering immune responses either locally by migrating to the tumor or systemically through responses originating in the gut. To test this, we performed in vitro studies and found that single bacterial species identified from LTS tumor microbial signature can induce activation of cytotoxic T lymphocytes (CD8+ CD69+ IFNg+) only when in direct contact with antigen-presenting cells, suggesting that bacterial antigens need to be processed and presented to intratumoral T cells to induce their activation. Furthermore, through preliminary in vivo studies, we have found that orally administered single bacterial species can be delivered to the pancreas via the gut. Further work needs to determine whether single/specific LTS bacteria play a causative role in regulating tumor immunity in vivo. If the beneficial modulatory role of specific bacteria is established, then bacteriotherapy as single therapy or in combination with fecal microbial transplants could be used as innovative strategies for treatment of pancreatic cancer. This abstract is also being presented as Poster B27. Citation Format: Vidhi Chandra, Olivereen Le Roux, Erick Riquelme, Yu Zhang, Anirban Maitra, James. R White, Florencia McAllister. Elucidating role of bacteria during pancreatic ductal adenocarcinoma (PDAC) [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr PR05.

Reconstructing the Phylogeny of Corynebacteriales while Accounting for Horizontal Gene Transfer.

Horizontal gene transfer is a common mechanism in Bacteria that has contributed to the genomic content of existing organisms. Traditional methods for estimating bacterial phylogeny, however, assume only vertical inheritance in the evolution of homologous genes, which may result in errors in the estimated phylogenies. We present a new method for estimating bacterial phylogeny that accounts for the presence of genes acquired by horizontal gene transfer between genomes. The method identifies and corrects putative transferred genes in gene families, before applying a gene tree-based summary method to estimate bacterial species trees. The method was applied to estimate the phylogeny of the order Corynebacteriales, which is the largest clade in the phylum Actinobacteria. We report a collection of 14 phylogenetic trees on 360 Corynebacteriales genomes. All estimated trees display each genus as a monophyletic clade. The trees also display several relationships proposed by past studies, as well as new relevant relationships between and within the main genera of Corynebacteriales: Corynebacterium, Mycobacterium, Nocardia, Rhodococcus, and Gordonia. An implementation of the method in Python is available on GitHub at https://github.com/UdeS-CoBIUS/EXECT (last accessed April 2, 2020).

A Systematic Approach to Bacterial Phylogeny Using Order Level Sampling and Identification of HGT Using Network Science.

Reconstructing and visualizing phylogenetic relationships among living organisms is a fundamental challenge because not all organisms share the same genes. As a result, the first phylogenetic visualizations employed a single gene, e.g., rRNA genes, sufficiently conserved to be present in all organisms but divergent enough to provide discrimination between groups. As more genome data became available, researchers began concatenating different combinations of genes or proteins to construct phylogenetic trees believed to be more robust because they incorporated more information. However, the genes or proteins chosen were based on ad hoc approaches. The large number of complete genome sequences available today allows the use of whole genomes to analyze relationships among organisms rather than using an ad hoc set of genes. We present a systematic approach for constructing a phylogenetic tree based on simultaneously clustering the complete proteomes of 360 bacterial species. From the homologous clusters, we identify 49 protein sequences shared by 99% of the organisms to build a tree. Of the 49 sequences, 47 have homologous sequences in both archaea and eukarya. The clusters are also used to create a network from which bacterial species with horizontally-transferred genes from other phyla are identified.

Phylogenomic Analyses of Members of the Widespread Marine Heterotrophic Genus Pseudovibrio Suggest Distinct Evolutionary Trajectories and a Novel Genus, Polycladidibacter gen. nov.

Bacteria belonging to the Pseudovibrio genus are widespread, metabolically versatile, and able to thrive as both free-living and host-associated organisms. Although more than 50 genomes are available, a comprehensive comparative genomics study to resolve taxonomic inconsistencies is currently missing. We analyzed all available genomes and used 552 core genes to perform a robust phylogenomic reconstruction. This in-depth analysis revealed the divergence of two monophyletic basal lineages of strains isolated from polyclad flatworm hosts, namely, Pseudovibrio hongkongensis and Pseudovibrio stylochi These strains have reduced genomes and lack sulfur-related metabolisms and major biosynthetic gene clusters, and their environmental distribution appears to be tightly associated with invertebrate hosts. We showed experimentally that the divergent strains are unable to utilize various sulfur compounds that, in contrast, can be utilized by the type strain Pseudovibrio denitrificans Our analyses suggest that the lineage leading to these two strains has been subject to relaxed purifying selection resulting in great gene loss. Overall genome relatedness indices (OGRI) indicate substantial differences between the divergent strains and the rest of the genus. While 16S rRNA gene analyses do not support the establishment of a different genus for the divergent strains, their substantial genomic, phylogenomic, and physiological differences strongly suggest a divergent evolutionary trajectory and the need for their reclassification. Therefore, we propose the novel genus Polycladidibacter gen. nov.IMPORTANCE The genus Pseudovibrio is commonly associated with marine invertebrates, which are essential for ocean health and marine nutrient cycling. Traditionally, the phylogeny of the genus has been based on 16S rRNA gene analysis. The use of the 16S rRNA gene or any other single marker gene for robust phylogenetic placement has recently been questioned. We used a large set of marker genes from all available Pseudovibrio genomes for in-depth phylogenomic analyses. We identified divergent monophyletic basal lineages within the Pseudovibrio genus, including two strains isolated from polyclad flatworms. These strains showed reduced sulfur metabolism and biosynthesis capacities. The phylogenomic analyses revealed distinct evolutionary trajectories and ecological adaptations that differentiate the divergent strains from the other Pseudovibrio members and suggest that they fall into a novel genus. Our data show the importance of widening the use of phylogenomics for better understanding bacterial physiology, phylogeny, and evolution.

The translucens group of Xanthomonas translucens: Complicated and important pathogens causing bacterial leaf streak on cereals.

Xanthomonas translucens is a group of gram‐negative bacteria that can cause important diseases in cereal crops and forage grasses. Different pathovars have been defined according to their host ranges, and molecular and biochemical characteristics. Pathovars have been placed into two major groups: translucens and graminis. The translucens group contains the pathovars causing bacterial leaf streak (BLS) on cereal crops such as wheat, barley, triticale, rye, and oat. In recent years, BLS has re‐emerged as a major problem for many wheat‐ and barley‐producing areas worldwide. The biology of the pathogens and the host–pathogen interactions in cereal BLS diseases were poorly understood. However, recent genome sequence data have provided an insight into the bacterial phylogeny and identification and pathogenicity/virulence. Furthermore, identification of sources of resistance to BLS and mapping of the resistance genes have been initiated.TaxonomyKingdom Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Xanthomonadales; Family Xanthomonadacea e; Genus Xanthomonas; Species X. translucens; translucens group pathovars: undulosa, translucens, cerealis, hordei, and secalis; graminis group pathovars: arrhenatheri, graminis, poae, phlei; newly established pathovar: pistaciae.Host range X. translucens mainly infects plant species in the Poaceae with the translucens group on cereal crop species and the graminis group on forage grass species. However, some strains have been isolated from, and are able to infect, ornamental asparagus and pistachio trees. Most pathovars have a narrow host range, while a few can infect a broad range of hosts.GenomeThe complete genome sequence is available for two X. translucens pv. undulosa strains and one pv. translucens strain. A draft genome sequence is also available for at least one strain from each pathovar. The X. translucens pv. undulosa strain Xt4699 was the first to have its complete genome sequenced, which consists of 4,561,137 bp with total GC content approximately at 68% and 3,528 predicted genes.Virulence mechanismsLike most xanthomonads, X. translucens utilizes a type III secretion system (T3SS) to deliver a suite of T3SS effectors (T3Es) inside plant cells. Transcription activator‐like effectors, a special group of T3Es, have been identified in most of the X. translucens genomes, some of which have been implicated in virulence. Genetic factors determining host range virulence have also been identified.

Phylogenetic tree analysis based on the 16S sequence alignment for Klebsiella spp. isolated from different sources

16S ribosomal RNA (16S rRNA) gene sequences used to study bacterial phylogeny and taxonomy have been by far the most common housekeeping genetic marker utilized for identification and ancestor determination. This study aimed to investigate, for the first time, the relationship between Klebsiella spp. isolated from clinical and environmental samples in Iraq.
 Fifty Klebsiella spp. isolates were isolated from clinical and environmental sources. Twenty-five isolates were collected from a fresh vegetable (Apium graveolens) and 25 from clinical samples (sputum, wound swab, urine). Enteric bacteria were isolated on selective and differential media and identified by an automatic identification system, vitek-2. The total DNA was extracted and PCR amplified for selected isolates. The 16S rRNA gene was amplified by using the universal primer 27F (5'- AGAGTTTGATCCTGGCTCAG- 3') and 1492R (5'- GGTTACCTTGTTACGACTT- 3’). The 16SrRNA gene sequence was analysed among some local isolates, and the results were compared with the standard data of similar registered strains in NCBI. The most common species of Klebsiella was Klebsiella pneumoniae pneumoniae (Kpp), followed by Klebsiella pneumoniae ozaenae (Kpo) and Klebsiella oxytoca (Ko). The results of the identification of species and sub species by using the biochemical test (vitek-2) were more precise than those obtained by the use of the universal primer.Phylogenetic tree strategies have clearly indicated a relatively close similarity amongst all analysed Klebsiella isolates and revealed the intra-species genetic distance between the individual isolates of the Klebsiella spp. In conclusion, our results revealed the main advantage of using universal primers for the identification of Klebsiella spp. and their root from nature.

Bacterial Tradeoffs in Growth Rate and Extracellular Enzymes.

Like larger organisms, bacteria possess traits, or phenotypic characteristics, that influence growth and impact ecosystem processes. Still, it remains unclear how these traits are organized across bacterial lineages. Using 49 bacterial strains isolated from leaf litter in Southern California, we tested the hypothesis that bacterial growth rates trade off against extracellular enzyme investment. We also tested for phylogenetic conservation of these traits under high and low resource conditions represented, respectively, by Luria broth (LB) and a monomer-dominated medium extracted from plant litter. In support of our hypotheses, we found a negative correlation between the maximum growth rate and the total activity of carbon-, nitrogen-, and phosphorus-degrading extracellular enzymes. However, this tradeoff was only observed under high resource conditions. We also found significant phylogenetic signal in maximum growth rate and extracellular enzyme investment under high and low resource conditions. Driven by our bacterial trait data, we proposed three potential life history strategies. Resource acquisition strategists invest heavily in extracellular enzyme production. Growth strategists invest in high growth rates. Bacteria in a third category showed lower potential for enzyme production and growth, so we tentatively classified them as maintenance strategists that may perform better under conditions we did not measure. These strategies were related to bacterial phylogeny, with most growth strategists belonging to the phylum Proteobacteria and most maintenance and resource acquisition strategists belonging to the phylum Actinobacteria. By accounting for extracellular enzyme investment, our proposed life history strategies complement existing frameworks, such as the copiotroph-oligotroph continuum and Grime’s competitor-stress tolerator-ruderal triangle. Our results have biogeochemical implications because allocation to extracellular enzymes versus growth or stress tolerance can determine the fate and form of organic matter cycling through surface soil.

Host identity determines plant associated resistomes

Plant microbiome, as the second genome of plant, and the interface between human and environmental microbiome, represents a potential pathway of human exposure to environmental pathogens and resistomes. However, the impact of host identity on the profile of resistomes in plant phyllosphere is unclear and this knowledge is vital for establishing a framework to evaluate the dissemination of antibiotic resistance via the plant microbiome. Here, we explored the phyllosphere microbiome and resistomes in 12 selected plant species. By using High-throughput quantitative PCR, we identified a total of 172 unique resistance genes in plant phyllosphere microbiome, which was significantly divergent from the profile of resistomes in associated soils (Adonis, P < 0.01). Host identity had a significant effect on the plant resistome, which was mainly attributed to the dissimilarity of phyllosphere bacterial phylogeny across different plants. We identified a core set of plant resistomes shared in more than 80% of samples, which accounted for more than 64% of total resistance genes. These plant core resistomes conferred resistance to antibiotics that are commonly administered to humans and animals. Our findings extend our knowledge regarding the resistomes in plant phyllosphere microbiome and highlight the role of host identity in shaping the plant associated antibiotic resistance genes.

Dynamic Meta-Storms enables comprehensive taxonomic and phylogenetic comparison of shotgun metagenomes at the species level.

An accurate and reliable distance (or dissimilarity) among shotgun metagenomes is fundamental to deducing the beta-diversity of microbiomes. To compute the distance at the species level, current methods either ignore the evolutionary relationship among species or fail to account for unclassified organisms that cannot be mapped to definite tip nodes in the phylogenic tree, thus can produce erroneous beta-diversity pattern. To solve these problems, we propose the Dynamic Meta-Storms (DMS) algorithm to enable the comprehensive comparison of metagenomes on the species level with both taxonomy and phylogeny profiles. It compares the identified species of metagenomes with phylogeny, and then dynamically places the unclassified species to the virtual nodes of the phylogeny tree via their higher-level taxonomy information. Its high speed and low memory consumption enable pairwise comparison of 100 000 metagenomes (synthesized from 3688 bacteria) within 6.4 h on a single computing node. An optimized implementation of DMS is available on GitHub (https://github.com/qibebt-bioinfo/dynamic-meta-storms) under a GNU GPL license. It takes the species-level profiles of metagenomes as input, and generates their pairwise distance matrix. The bacterial species-level phylogeny tree and taxonomy information of MetaPhlAn2 have been integrated into this implementation, while customized tree and taxonomy are also supported. Supplementary data are available at Bioinformatics online.

Bacterial phylogeny in the Cayley graph

Many models of genome rearrangement involve operations that are self-inverse, and hence generate a group acting on the space of genomes. This gives a correspondence between genome arrangements and the elements of a group, and consequently, between evolutionary paths and walks on the Cayley graph. Many common methods for phylogenetic reconstruction rely on calculating the minimal distance between two genomes; this omits much of the other information available from the Cayley graph. In this paper, we begin an exploration of some of this additional information, in particular describing the phylogeny as a Steiner tree within the Cayley graph, and exploring the “interval” between two genomes. While motivated by problems in systematic biology, many of these ideas are of independent group-theoretic interest.

Phylogenetic tree analysis based on the 16S sequence alignment for Klebsiella spp. isolated from different sources

16S ribosomal RNA (16S rRNA) gene sequences used to study bacterial phylogeny and taxonomy have been by far the most common housekeeping genetic marker utilized for identification and ancestor determination. This study aimed to investigate, for the first time, the relationship between Klebsiella spp. isolated from clinical and environmental samples in Iraq.&#x0D; Fifty Klebsiella spp. isolates were isolated from clinical and environmental sources. Twenty-five isolates were collected from a fresh vegetable (Apium graveolens) and 25 from clinical samples (sputum, wound swab, urine). Enteric bacteria were isolated on selective and differential media and identified by an automatic identification system, vitek-2. The total DNA was extracted and PCR amplified for selected isolates. The 16S rRNA gene was amplified by using the universal primer 27F (5'- AGAGTTTGATCCTGGCTCAG- 3') and 1492R (5'- GGTTACCTTGTTACGACTT- 3’). The 16SrRNA gene sequence was analysed among some local isolates, and the results were compared with the standard data of similar registered strains in NCBI. The most common species of Klebsiella was Klebsiella pneumoniae pneumoniae (Kpp), followed by Klebsiella pneumoniae ozaenae (Kpo) and Klebsiella oxytoca (Ko). The results of the identification of species and sub species by using the biochemical test (vitek-2) were more precise than those obtained by the use of the universal primer.Phylogenetic tree strategies have clearly indicated a relatively close similarity amongst all analysed Klebsiella isolates and revealed the intra-species genetic distance between the individual isolates of the Klebsiella spp. In conclusion, our results revealed the main advantage of using universal primers for the identification of Klebsiella spp. and their root from nature.

Post-translational Protein Acetylation: An Elegant Mechanism for Bacteria to Dynamically Regulate Metabolic Functions.

Post-translational modifications (PTM) decorate proteins to provide functional heterogeneity to an existing proteome. The large number of known PTMs highlights the many ways that cells can modify their proteins to respond to diverse stimuli. Recently, PTMs have begun to receive increased interest because new sensitive proteomics workflows and structural methodologies now allow researchers to obtain large-scale, in-depth and unbiased information concerning PTM type and site localization. However, few PTMs have been extensively assessed for functional consequences, leaving a large knowledge gap concerning the inner workings of the cell. Here, we review understanding of N-𝜀-lysine acetylation in bacteria, a PTM that was largely ignored in bacteria until a decade ago. Acetylation is a modification that can dramatically change the function of a protein through alteration of its properties, including hydrophobicity, solubility, and surface properties, all of which may influence protein conformation and interactions with substrates, cofactors and other macromolecules. Most bacteria carry genes predicted to encode the lysine acetyltransferases and lysine deacetylases that add and remove acetylations, respectively. Many bacteria also exhibit acetylation activities that do not depend on an enzyme, but instead on direct transfer of acetyl groups from the central metabolites acetyl coenzyme A or acetyl phosphate. Regardless of mechanism, most central metabolic enzymes possess lysines that are acetylated in a regulated fashion and many of these regulated sites are conserved across the spectrum of bacterial phylogeny. The interconnectedness of acetylation and central metabolism suggests that acetylation may be a response to nutrient availability or the energy status of the cell. However, this and other hypotheses related to acetylation remain untested.

In search of microbial indicator taxa: shifts in stream bacterial communities along an urbanization gradient.

A majority of environmental studies describe microbiomes at coarse scales of taxonomic resolution (bacterial community, phylum), ignoring key ecological knowledge gained from finer-scales and microbial indicator taxa. Here, we characterized the distribution of 940 bacterial taxa from 41 streams along an urbanization gradient (0%-83% developed watershed area) in the Raleigh-Durham area of North Carolina (USA). Using statistical approaches derived from macro-organismal ecology, we found that more bacterial taxa were classified as intolerant than as tolerant to increasing watershed urbanization (143 vs 48 OTUs), and we identified a threshold of 12.1% developed watershed area beyond which the majority of intolerant taxa were lost from streams. Two bacterial families strongly decreased with urbanization: Acidobacteriaceae (Acidobacteria) and Xanthobacteraceae (Alphaproteobacteria). Tolerant taxa were broadly distributed throughout the bacterial phylogeny, with members of the Comamonadaceae family (Betaproteobacteria) presenting the highest number of tolerant taxa. Shifts in microbial community structure were strongly correlated with a stream biotic index, based on macroinvertebrate composition, suggesting that microbial assemblages could be used to establish biotic criteria for monitoring aquatic ecosystems. In addition, our study shows that classic methods in community ecology can be applied to microbiome datasets to identify reliable microbial indicator taxa and determine the environmental constraints on individual taxa distributions along environmental gradients.

A preliminary examination of bacterial, archaeal, and fungal communities inhabiting different rhizocompartments of tomato plants under real-world environments

Plant microbiota is a key determinant of plant health and productivity. The composition and structure of plant microbiota varies according to plant tissue and compartment, which are specific habitats for microbial colonization. To investigate the structural composition of the microbiome associated with tomato roots under natural systems, we characterized the bacterial, archaeal, and fungal communities of three belowground compartments (rhizosphere, endosphere, and bulk soil) of tomato plants collected from 23 greenhouses in 7 geographic locations of South Korea. The microbial diversity and structure varied by rhizocompartment, with the most distinctive community features found in the endosphere. The bacterial and fungal communities in the bulk soil and rhizosphere were correlated with soil physicochemical properties, such as pH, electrical conductivity, and exchangeable cation levels, while this trend was not evident in the endosphere samples. A small number of core bacterial operational taxonomic units (OTUs) present in all samples from the rhizosphere and endosphere represented more than 60% of the total relative abundance. Among these core microbes, OTUs belonging to the genera Acidovorax, Enterobacter, Pseudomonas, Rhizobium, Streptomyces, and Variovorax, members of which are known to have beneficial effects on plant growth, were more relatively abundant in the endosphere samples. A co-occurrence network analysis indicated that the microbial community in the rhizosphere had a larger and more complex network than those in the bulk soil and endosphere. The analysis also identified keystone taxa that might play important roles in microbe-microbe interactions in the community. Additionally, profiling of predicted gene functions identified many genes associated with membrane transport in the endospheric and rhizospheric communities. Overall, the data presented here provide preliminary insight into bacterial, archaeal, and fungal phylogeny, functionality, and interactions in the rhizocompartments of tomato roots under real-world environments.

Shedding light on a Group IV (ECF11) alternative σ factor.

SummaryThis year marks the 50th anniversary of the discovery of σ70 as a protein factor that was needed for bacterial RNA polymerase to accurately transcribe a promoter in vitro. It was 25 years later that the Group IV alternative σs were described as a distinct family of proteins related to σ70. In the intervening time, there has been an ever‐growing list of Group IV σs, numbers of genes they transcribe, insight into the diverse suite of processes they control, and appreciation for their impact on bacterial lifestyles. This work summarizes knowledge of the Rhodobacter sphaeroides σE‐ChrR pair, a member of the ECF11 subfamily of Group IV alternative σs, in protecting cells from the reactive oxygen species, singlet oxygen. It describes lessons learned from analyzing ChrR, a zinc‐dependent anti‐σ factor, that are generally applicable to Group IV σs and relevant to the response to single oxygen. This MicroReview also illustrates insights into stress responses in this and other bacteria that have been acquired by analyzing or modeling the activity of the σE‐ChrR across the bacterial phylogeny.

Robust Stoichiometry of FliW-CsrA Governs Flagellin Homeostasis and Cytoplasmic Organization in Bacillus subtilis.

Flagellin (Hag) is one of the most abundant proteins in Bacillus subtilis Here we show that each flagellar filament is assembled from ∼12,000 Hag monomers and that there is a cytoplasmic pool of Hag that is restricted to 5% of the total. Hag is thought to be restricted at the level of translation by a partner-switching mechanism involving FliW and the homodimeric RNA-binding protein CsrA (CsrAdimer). We further show that the mechanism of translation inhibition is hypersensitive due to a 1:1 ratio of Hag to FliW, a 1:1 inhibitory ratio of FliW to CsrAdimer, and a nearly 1:1 ratio of CsrAdimer to hag transcripts. Equimolarity of all components couples single-molecule detection of Hag export to compensatory translation and causes cytoplasmic Hag concentrations to oscillate around the level of FliW. We found that stoichiometry is ensured by genetic architecture, translational coupling, and the ability of CsrAdimer to restrict hag transcript accumulation. We further show that homeostasis prevents Hag hyperaccumulation that would otherwise cause severe defects in intracellular architecture, perhaps due to increased molecular crowding. We note that FliW-CsrA-mediated structural homeostasis has similarities to that seen with some toxin-antitoxin systems.IMPORTANCE The intracellular concentration of flagellar filament protein Hag is restricted by the Hag-FliW-CsrA system in B. subtilis Here we show that the Hag-FliW-CsrAdimer system functions at nearly 1:1:1 stoichiometry and that the system is both robust with respect to perturbation and hypersensitive to the Hag intracellular concentration. Moreover, restriction of cytoplasmic Hag levels is important for maintaining proper intracellular architecture, as artificial Hag hyperaccumulation led to generalized spatial defects and a high frequency of minicell production. The Hag-FliW-CsrA system is conserved in the deeper branches of bacterial phylogeny, and we note that the Hag-FliW-CsrA "homeostasis module" resembles a toxin-antitoxin system where, by analogy, CsrA is the "toxin," FliW is the "antitoxin," and Hag is the target.

Using the Open-Source MALDI TOF-MS IDBac Pipeline for Analysis of Microbial Protein and Specialized Metabolite Data

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, we developed IDBac-a low-cost and high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bioinformatics pipeline. IDBac software is designed for non-experts, is freely available, and capable of analyzing a few to thousands of bacterial colonies. Here, we present procedures for the preparation of bacterial colonies for MALDI-TOF MS analysis, MS instrument operation, and data processing and visualization in IDBac. In particular, we instruct users how to cluster bacteria into dendrograms based on protein MS fingerprints and interactively create Metabolite Association Networks (MANs) from specialized metabolite data.

AnnoTree: visualization and exploration of a functionally annotated microbial tree of life.

Bacterial genomics has revolutionized our understanding of the microbial tree of life; however, mapping and visualizing the distribution of functional traits across bacteria remains a challenge. Here, we introduce AnnoTree—an interactive, functionally annotated bacterial tree of life that integrates taxonomic, phylogenetic and functional annotation data from over 27 000 bacterial and 1500 archaeal genomes. AnnoTree enables visualization of millions of precomputed genome annotations across the bacterial and archaeal phylogenies, thereby allowing users to explore gene distributions as well as patterns of gene gain and loss in prokaryotes. Using AnnoTree, we examined the phylogenomic distributions of 28 311 gene/protein families, and measured their phylogenetic conservation, patchiness, and lineage-specificity within bacteria. Our analyses revealed widespread phylogenetic patchiness among bacterial gene families, reflecting the dynamic evolution of prokaryotic genomes. Genes involved in phage infection/defense, mobile elements, and antibiotic resistance dominated the list of most patchy traits, as well as numerous intriguing metabolic enzymes that appear to have undergone frequent horizontal transfer. We anticipate that AnnoTree will be a valuable resource for exploring prokaryotic gene histories, and will act as a catalyst for biological and evolutionary hypothesis generation. AnnoTree is freely available at http://annotree.uwaterloo.ca

Abundance of antibiotic resistance genes and their association with bacterial communities in activated sludge of wastewater treatment plants: Geographical distribution and network analysis

Wastewater treatment plants (WWTPs) are deemed reservoirs of antibiotic resistance genes (ARGs). Bacterial phylogeny can shape the resistome in activated sludge. However, the co-occurrence and interaction of ARGs abundance and bacterial communities in different WWTPs located at continental scales are still not comprehensively understood. Here, we applied quantitative PCR and Miseq sequence approaches to unveil the changing profiles of ARGs (sul1, sul2, tetW, tetQ, tetX), intI1 gene, and bacterial communities in 18 geographically distributed WWTPs. The results showed that the average relative abundance of sul1and sul2 genes were 2.08 × 10−1 and 1.32 × 10−1 copies/16S rRNA copies, respectively. The abundance of tetW gene was positively correlated with the Shannon diversity index (H′), while both studied sul genes had significant positive relationship with the intI1gene. The highest average relative abundances of sul1, sul2, tetX, and intI1 genes were found in south region and oxidation ditch system. Network analysis found that 16 bacterial genera co-occurred with tetW gene. Co-occurrence patterns were revealed distinct community interactions between aerobic/anoxic/aerobic and oxidation ditch systems. The redundancy analysis model plot of the bacterial community composition clearly demonstrated that the sludge samples were significant differences among those from the different geographical areas, and the shifts in bacterial community composition were correlated with ARGs. Together, these findings from the present study will highlight the potential risks of ARGs and bacterial populations carrying these ARGs, and enable the development of suitable technique to control the dissemination of ARGs from WWTPs into aquatic environments.