Microbial Diversity in a Military Impacted Lagoon (Vieques, Puerto Rico) as Revealed by Metagenomics

The Anones Lagoon, located in the island municipality of Vieques, Puerto Rico (PR), received extensive bombing during military practices by the US Navy for decades. After military activities ceased in 2003, the bombing range was designated as part of a larger Superfund site by US EPA. Here, we employed shotgun metagenomic sequencing to investigate how microbial communities responded to pollution by heavy metals and explosives at this lagoon. Sediment samples (0-5 cm) from Anones were collected in 2005 and 2014 and compared to samples from two reference lagoons, i.e., Guaniquilla, Cabo Rojo (a natural reserve) and Condado, San Juan (PR’s capital city). Consistent with selection under low anthropogenic impacts, Guaniquilla exhibited the highest degree of diversity with lower frequency of genes related to xenobiotics metabolism among the three lagoons. Notably, a clear shift was observed in Anones, with Euryarchaeota becoming enriched (9% of total) and a concomitant increase in community diversity, by about one order of magnitude, after almost 10 years without bombing activities. In contrast, genes associated with explosives biodegradation and heavy metal transformation significantly decreased in abundance in Anones 2014 (by 91.5%). Five unique population genomes were recovered from the Anones 2005 sample that encoded genetic determinants implicated in biodegradation of contaminants. Collectively, these results provided new insights into the natural attenuation of explosive contaminants by the benthic microbial communities of the Anones lagoon and could serve as reference points to enhance bioremediation actions at this site and for assessing other similarly impacted sites. Importance This study represents the first assessment of the benthic microbial community in the Anones Lagoon in Vieques, Puerto Rico after the impact of intense pollution by bombs and unconventional weapons during military training exercises. Evaluating the microbial diversity of Anones, represents an opportunity to assess the microbial succession patterns during the active process of natural attenuation of pollutants. The culture-independent techniques employed to study these environmental samples allowed the recovery of almost complete genomes of several abundant species that were likely involved in the biodegradation of pollutants and thus, represented species responding to the strong selection pressure posed by military activities. Further, our results showed that natural attenuation has proceeded to a great extend ten years after the cease of military activities

Genomic determinants of organohalide-respiration in Geobacter lovleyi, an unusual member of the Geobacteraceae

Background Geobacter lovleyi is a unique member of the Geobacteraceae because strains of this species share the ability to couple tetrachloroethene (PCE) reductive dechlorination to cis-1,2-dichloroethene (cis-DCE) with energy conservation and growth (i.e., organohalide respiration). Strain SZ also reduces U(VI) to U(IV) and contributes to uranium immobilization, making G. lovleyi relevant for bioremediation at sites impacted with chlorinated ethenes and radionuclides. G. lovleyi is the only fully sequenced representative of this distinct Geobacter clade, and comparative genome analyses identified genetic elements associated with organohalide respiration and elucidated genome features that distinguish strain SZ from other members of the Geobacteraceae. Results Sequencing the G. lovleyi strain SZ genome revealed a 3.9 Mbp chromosome with 54.7% GC content (i.e., the percent of the total guanines (Gs) and cytosines (Cs) among the four bases within the genome), and average amino acid identities of 53–56% compared to other sequenced Geobacter spp. Sequencing also revealed the presence of a 77 kbp plasmid, pSZ77 (53.0% GC), with nearly half of its encoded genes corresponding to chromosomal homologs in other Geobacteraceae genomes. Among these chromosome-derived features, pSZ77 encodes 15 out of the 24 genes required for de novo cobalamin biosynthesis, a required cofactor for organohalide respiration. A plasmid with 99% sequence identity to pSZ77 was subsequently detected in the PCE-dechlorinating G. lovleyi strain KB-1 present in the PCE-to-ethene-dechlorinating consortium KB-1. Additional PCE-to-cis-DCE-dechlorinating G. lovleyi strains obtained from the PCE-contaminated Fort Lewis, WA, site did not carry a plasmid indicating that pSZ77 is not a requirement (marker) for PCE respiration within this species. Chromosomal genomic islands found within the G. lovleyi strain SZ genome encode two reductive dehalogenase (RDase) homologs and a putative conjugative pilus system. Despite the loss of many c-type cytochrome and oxidative-stress-responsive genes, strain SZ retained the majority of Geobacter core metabolic capabilities, including U(VI) respiration. Conclusions Gene acquisitions have expanded strain SZ’s respiratory capabilities to include PCE and TCE as electron acceptors. Respiratory processes core to the Geobacter genus, such as metal reduction, were retained despite a substantially reduced number of c-type cytochrome genes. pSZ77 is stably maintained within its host strains SZ and KB-1, likely because the replicon carries essential genes including genes involved in cobalamin biosynthesis and possibly corrinoid transport. Lateral acquisition of the plasmid replicon and the RDase genomic island represent unique genome features of the PCE-respiring G. lovleyi strains SZ and KB-1, and at least the latter signifies adaptation to PCE contamination

Genomic determinants of organohalide-respiration in Geobacter lovleyi, an unusual member of the Geobacteraceae

Background Geobacter lovleyi is a unique member of the Geobacteraceae because strains of this species share the ability to couple tetrachloroethene (PCE) reductive dechlorination to cis-1,2-dichloroethene (cis-DCE) with energy conservation and growth (i.e., organohalide respiration). Strain SZ also reduces U(VI) to U(IV) and contributes to uranium immobilization, making G. lovleyi relevant for bioremediation at sites impacted with chlorinated ethenes and radionuclides. G. lovleyi is the only fully sequenced representative of this distinct Geobacter clade, and comparative genome analyses identified genetic elements associated with organohalide respiration and elucidated genome features that distinguish strain SZ from other members of the Geobacteraceae. Results Sequencing the G. lovleyi strain SZ genome revealed a 3.9 Mbp chromosome with 54.7% GC content (i.e., the percent of the total guanines (Gs) and cytosines (Cs) among the four bases within the genome), and average amino acid identities of 53–56% compared to other sequenced Geobacter spp. Sequencing also revealed the presence of a 77 kbp plasmid, pSZ77 (53.0% GC), with nearly half of its encoded genes corresponding to chromosomal homologs in other Geobacteraceae genomes. Among these chromosome-derived features, pSZ77 encodes 15 out of the 24 genes required for de novo cobalamin biosynthesis, a required cofactor for organohalide respiration. A plasmid with 99% sequence identity to pSZ77 was subsequently detected in the PCE-dechlorinating G. lovleyi strain KB-1 present in the PCE-to-ethene-dechlorinating consortium KB-1. Additional PCE-to-cis-DCE-dechlorinating G. lovleyi strains obtained from the PCE-contaminated Fort Lewis, WA, site did not carry a plasmid indicating that pSZ77 is not a requirement (marker) for PCE respiration within this species. Chromosomal genomic islands found within the G. lovleyi strain SZ genome encode two reductive dehalogenase (RDase) homologs and a putative conjugative pilus system. Despite the loss of many c-type cytochrome and oxidative-stress-responsive genes, strain SZ retained the majority of Geobacter core metabolic capabilities, including U(VI) respiration. Conclusions Gene acquisitions have expanded strain SZ’s respiratory capabilities to include PCE and TCE as electron acceptors. Respiratory processes core to the Geobacter genus, such as metal reduction, were retained despite a substantially reduced number of c-type cytochrome genes. pSZ77 is stably maintained within its host strains SZ and KB-1, likely because the replicon carries essential genes including genes involved in cobalamin biosynthesis and possibly corrinoid transport. Lateral acquisition of the plasmid replicon and the RDase genomic island represent unique genome features of the PCE-respiring G. lovleyi strains SZ and KB-1, and at least the latter signifies adaptation to PCE contamination

imGLAD: accurate detection and quantification of target organisms in metagenomes

Accurate detection of target microbial species in metagenomic datasets from environmental samples remains limited because the limit of detection of current methods is typically inaccessible and the frequency of false-positives, resulting from inadequate identification of regions of the genome that are either too highly conserved to be diagnostic (e.g., rRNA genes) or prone to frequent horizontal genetic exchange (e.g., mobile elements) remains unknown. To overcome these limitations, we introduce imGLAD, which aims to detect (target) genomic sequences in metagenomic datasets. imGLAD achieves high accuracy because it uses the sequence-discrete population concept for discriminating between metagenomic reads originating from the target organism compared to reads from co-occurring close relatives, masks regions of the genome that are not informative using the MyTaxa engine, and models both the sequencing breadth and depth to determine relative abundance and limit of detection. We validated imGLAD by analyzing metagenomic datasets derived from spinach leaves inoculated with the enteric pathogen Escherichia coli O157:H7 and showed that its limit of detection can be comparable to that of PCR-based approaches for these samples (∼1 cell/gram)

Predominance of deterministic microbial community dynamics in salterns exposed to different light intensities

While the dynamics of microbial community assembly driven by environmental perturbations have been extensively studied, our understanding is far from complete, particularly for light‐induced perturbations. Extremely halophilic communities thriving in coastal solar salterns are mainly influenced by two environmental factors—salt concentrations and high sunlight irradiation. By experimentally manipulating light intensity through the application of shading, we showed that light acts as a deterministic factor that ultimately drives the establishment of recurrent microbial communities under near‐saturation salt concentrations. In particular, the stable and highly change‐resistant communities that established under high‐light intensities were dominated (>90% of metagenomic reads) by Haloquadratum spp. and Salinibacter spp. On the other hand, under 37‐fold lower light intensity, different, less stable and change‐resistant communities were established, mainly dominated by yet unclassified haloarchaea and relatively diverse photosynthetic microorganisms. These communities harboured, in general, much lower carotenoid pigment content than their high‐irradiation counterparts. Both assemblage types appeared to be highly resilient, re‐establishing when favourable conditions returned after perturbation (i.e. high‐irradiation for the former communities and low‐irradiation for the latter ones). Overall, our results revealed that stochastic processes were of limited significance to explain these patterns.This study was funded by the Spanish Ministry of Economy projects CGL2012-39627-C03-03 CLG2015_66686-C3-1-P and PGC2018-096956-B-C41 (to R.R.M.), CGL2015_66686-C3-3-P (to J.A.) and CGL2015_66686-C3-2-P (to J.E.G.P.), which were also supported with European Regional Development Fund (FEDER) funds. R.A. was funded by the Max Planck Society. KTK’s research was supported, in part, by the U.S. National Science Foundation (Award No. 1831582). T.V.P. received a pre-doctoral fellowship (No. BES-2013-064420) from the Spanish Government Ministry for Finance and Competition. R.R.M. acknowledges the financial support of the sabbatical stay at Georgia Tech supported by the Grant PRX18/00048 of the Ministry of Sciences, Innovation and Universities

The low diverse gastric microbiome of the jellyfish Cotylorhiza tuberculata is dominated by four novel taxa

Cotylorhiza tuberculata is an important scyphozoan jellyfish producing population blooms in the Mediterranean probably due to pelagic ecosystem's decay. Its gastric cavity can serve as a simple model of microbial–animal digestive associations, yet poorly characterized. Using state-of-the-art metagenomic population binning and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH), we show that only four novel clonal phylotypes were consistently associated with multiple jellyfish adults. Two affiliated close to Spiroplasma and Mycoplasma genera, one to chlamydial ‘Candidatus Syngnamydia’, and one to bacteroidetal Tenacibaculum, and were at least one order of magnitude more abundant than any other bacteria detected. Metabolic modelling predicted an aerobic heterotrophic lifestyle for the chlamydia, which were found intracellularly in Onychodromopsis-like ciliates. The Spiroplasma-like organism was predicted to be an anaerobic fermenter associated to some jellyfish cells, whereas the Tenacibaculum-like as free-living aerobic heterotroph, densely colonizing the mesogleal axis inside the gastric filaments. The association between the jellyfish and its reduced microbiome was close and temporally stable, and possibly related to food digestion and protection from pathogens. Based on the genomic and microscopic data, we propose three candidate taxa: ‘Candidatus Syngnamydia medusae’, ‘Candidatus Medusoplasma mediterranei’ and ‘Candidatus Tenacibaculum medusae’.This research from RRM’s group was partially supported by the Spanish Ministry of Economy projects CGL2012-39627-C03-03 and CLG2015_66686-C3-1-P, which were also supported with European Regional Development Fund (FEDER) funds. KTK’s research was supported, in part, by the U.S. National Science Foundation (Award No. 1241046). RRM acknowledges the economic support of grant PR2015-00008 included in the program Salvador de Madariaga of the Ministry of Education, Culture and Sports in order to undertake a research stay at the MPI-MM in Bremen. TVP acknowledges the predoctoral fellowship of the Ministerio de Economía y Competitividad of the Spanish Government for the FPI fellowship (Nr BES-2013-064420) supporting his research activities

Characterization of a Novel Gene, wosA, Regulating FlhDC Expression in Proteus mirabilis▿

In this study, we describe wosA, a Proteus mirabilis gene identified by its ability to increase swarming motility when overexpressed. At various times during the swarming cycle, the increased expression of wosA resulted in a 4- to 16-fold upregulation of the transcription of flhDC, encoding the master regulator of the flagellar cascade. In turn, the expression of flaA, encoding flagellin, was substantially increased in wosA-overexpressing strains. The overexpression of wosA also resulted in constitutive swarmer cell differentiation in liquid medium, a normally nonpermissive condition. However, in wosA-overexpressing strains, the onset of swarming was not altered. A null wosA allele resulted in a slight decrease in swarming motility. The expression of wosA was growth phase dependent during growth in liquid and on agar plates during swarmer cell differentiation. Increasing the viscosity of liquid medium by the addition of polyvinylpyrrolidone induced swarmer cell differentiation and resulted in a fourfold increase in wosA transcription. A fliL mutation that results in constitutive swarmer cell elongation also increased wosA transcription. In this study, we discuss the possible role of the wosA gene product in signal transduction from solid surfaces to induce swarmer cell differentiation, possibly via alterations in the motor switch complex. This study also suggests that despite constitutive swarmer cell differentiation in wosA-overexpressing strains, there are additional regulatory and/or environmental conditions that may control the onset of swarming migration

Similar Microbial Consortia and Genes Are Involved in the Biodegradation of Benzalkonium Chlorides in Different Environments

Benzalkonium chlorides (BACs) are emerging pollutants. Identification of microorganisms and the genes involved in the biodegradation of BACs is crucial for better understanding the fate of BACs in the environment and developing treatment strategies. Four microbial communities degrading BACs were developed from sewage (SEW), activated sludge (AS), soil (SOIL) and sea sediment (SEA) samples. According to 16S rRNA pyrosequencing and shotgun metagenome sequencing analyses, the most abundant species represented uncharacterized members of the <i>Pseudomonas</i> and <i>Achromobacter</i> genera. BAC biotransformation rates of the enriched microbial communities were 2.8, 3.2, 17.8, and 24.3 μM hr^–1 for SEA, AS, SOIL, and SEW, respectively, and were positively correlated with the relative abundance of a particular <i>Pseudomonas</i> sp. strain, BIOMIG1. The strain BIOMIG1 mineralizes BACs at a rate up to 2.40 μmol hr^–1 10^–11 cells. Genomes of four BAC degrading and nondegrading BIOMIG1 phenotypes were sequenced and differentially compared with each other. As a result, a gene cluster encoding for transporters, an integrase and a dioxygenase were involved in BAC biotransformation. Our results suggest that BIOMIG1 plays a key role on the fate of BACs in the environment and genes, other than those reported to date, are involved in BAC biotransformation in various habitats