Background Cellulolytic, hemicellulolytic, and amylolytic (CHA) enzyme-producing halophiles are understudied. The recently defined taxon Iocasia fonsfrigidae consists of one well-described anaerobic bacterial strain: NS-1 T . Prior to characterization of strain NS-1 T , an isolate designated Halocella sp. SP3-1 was isolated and its genome was published. Based on physiological and genetic comparisons, it was suggested that Halocella sp. SP3-1 may be another isolate of I. fronsfrigidae . Despite being geographic variants of the same species, data indicate that strain SP3-1 exhibits genetic, genomic, and physiological characteristics that distinguish it from strain NS-1 T . In this study, we examine the halophilic and alkaliphilic nature of strain SP3-1 and the genetic substrates underlying phenotypic differences between strains SP3-1 and NS-1 T with focus on sugar metabolism and CHA enzyme expression. Methods Standard methods in anaerobic cell culture were used to grow strains SP3-1 as well as other comparator species. Morphological characterization was done via electron microscopy and Schaeffer-Fulton staining. Data for sequence comparisons ( e.g. , 16S rRNA) were retrieved via BLAST and EzBioCloud. Alignments and phylogenetic trees were generated via CLUTAL_X and neighbor joining functions in MEGA (version 11). Genomes were assembled/annotated via the Prokka annotation pipeline. Clusters of Orthologous Groups (COGs) were defined by eegNOG 4.5. DNA-DNA hybridization calculations were performed by the ANI Calculator web service. Results Cells of strain SP3-1 are rods. SP3-1 cells grow at NaCl concentrations of 5-30% (w/v). Optimal growth occurs at 37 °C, pH 8.0, and 20% NaCl (w/v). Although phylogenetic analysis based on 16S rRNA gene indicates that strain SP3-1 belongs to the genus Iocasia with 99.58% average nucleotide sequence identity to Iocasia fonsfrigida NS-1 T , strain SP3-1 is uniquely an extreme haloalkaliphile. Moreover, strain SP3-1 ferments D-glucose to acetate, butyrate, carbon dioxide, hydrogen, ethanol, and butanol and will grow on L-arabinose, D-fructose, D-galactose, D-glucose, D-mannose, D-raffinose, D-xylose, cellobiose, lactose, maltose, sucrose, starch, xylan and phosphoric acid swollen cellulose (PASC). D-rhamnose, alginate, and lignin do not serve as suitable culture substrates for strain SP3-1. Thus, the carbon utilization profile of strain SP3-1 differs from that of I. fronsfrigidae strain NS-1 T . Differences between these two strains are also noted in their lipid composition. Genomic data reveal key differences between the genetic profiles of strain SP3-1 and NS-1 T that likely account for differences in morphology, sugar metabolism, and CHA-enzyme potential. Important to this study, I. fonsfrigidae SP3-1 produces and extracellularly secretes CHA enzymes at different levels and composition than type strain NS-1 T . The high salt tolerance and pH range of SP3-1 makes it an ideal candidate for salt and pH tolerant enzyme discovery.
more »
« less
Sodalis ligni Strain 159R Isolated from an Anaerobic Lignin-Degrading Consortium
ABSTRACT Novel bacterial isolates with the capabilities of lignin depolymerization, catabolism, or both, could be pertinent to lignocellulosic biofuel applications. In this study, we aimed to identify anaerobic bacteria that could address the economic challenges faced with microbial-mediated biotechnologies, such as the need for aeration and mixing. Using a consortium seeded from temperate forest soil and enriched under anoxic conditions with organosolv lignin as the sole carbon source, we successfully isolated a novel bacterium, designated 159R. Based on the 16S rRNA gene, the isolate belongs to the genus Sodalis in the family Bruguierivoracaceae . Whole-genome sequencing revealed a genome size of 6.38 Mbp and a GC content of 55 mol%. To resolve the phylogenetic position of 159R, its phylogeny was reconstructed using (i) 16S rRNA genes of its closest relatives, (ii) multilocus sequence analysis (MLSA) of 100 genes, (iii) 49 clusters of orthologous groups (COG) domains, and (iv) 400 conserved proteins. Isolate 159R was closely related to the deadwood associated Sodalis guild rather than the tsetse fly and other insect endosymbiont guilds. Estimated genome-sequence-based digital DNA-DNA hybridization (dDDH), genome percentage of conserved proteins (POCP), and an alignment analysis between 159R and the Sodalis clade species further supported that isolate 159R was part of the Sodalis genus and a strain of Sodalis ligni . We proposed the name Sodalis ligni str. 159R (=DSM 110549 = ATCC TSD-177). IMPORTANCE Currently, in the paper industry, paper mill pulping relies on unsustainable and costly processes to remove lignin from lignocellulosic material. A greener approach is biopulping, which uses microbes and their enzymes to break down lignin. However, there are limitations to biopulping that prevent it from outcompeting other pulping processes, such as requiring constant aeration and mixing. Anaerobic bacteria are a promising alternative source for consolidated depolymerization of lignin and its conversion to valuable by-products. We presented Sodalis ligni str. 159R and its characteristics as another example of potential mechanisms that can be developed for lignocellulosic applications.
more »
« less
- Award ID(s):
- 1832210
- NSF-PAR ID:
- 10389632
- Author(s) / Creator(s):
- ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; more »
- Editor(s):
- Rotaru, Amelia-Elena
- Date Published:
- Journal Name:
- Microbiology Spectrum
- Volume:
- 10
- Issue:
- 3
- ISSN:
- 2165-0497
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
more » « less
Abstract Strain KD21T, isolated from the fecal sample of a healthy female volunteer, is a strictly anaerobic, non-motile, Gram-staining-positive, saccharolytic small rod that does not produce spores. Strain KD21T was able to grow in the range of temperature 28°C–37°C (optimum, 37 °C), pH 6.0–8.0 (optimum, pH 7.0), and with 0–5.0 g/l NaCl (optimum, 0 g/l NaCl). Bacteria cells reduced nitrates to nitrites. Its major fatty acids were C18:1ω9c, C16:0, C18:0, and summed in feature 8 (C18:1ω7c and/or C18:1ω6c). 16S rRNA gene phylogenetic analysis revealed that KD21T is a member of the genus Tractidigestivibacter and is distinct from any species with validly published names. The sequence showed 98.48% similarity with T. scatoligenes SK9K4T. The DNA G + C content of strain KD21T was 62.6 mol%. The DNA–DNA hybridization and OrthoANI values between strain KD21T and T. scatoligenes SK9K4T were 40.2% and 90.2%, respectively. Differences in phenotypic, phylogenetic, chemotaxonomic, and genomic characteristics indicated that strain KD21T represents a novel species within the genus Tractidigestivibacter. The name T. montrealensis sp. nov. is proposed and the type strain is KD21T (= CSUR Q8103T = DSM 115111T).
-
more » « less
Abstract A polyphasic taxonomic approach, incorporating analysis of phenotypic features, cellular fatty acid profiles, 16S rRNA gene sequences, and determination of average nucleotide identity (ANI) plus digital DNA–DNA hybridization (dDDH), was applied to characterize an anaerobic bacterial strain designated KD22Tisolated from human feces. 16S rRNA gene-based phylogenetic analysis showed that strain KD22Twas found to be most closely related to species of the genus
Gabonibacter. At the 16S rRNA gene level, the closest species from the strain KD22Tcorresponded withGabonibacter massiliensis GM7T, with a similarity of 97.58%. Cells of strain KD22T were Gram-negative coccobacillus, positive for indole and negative for catalase, nitrate reduction, oxidase, and urease activities. The fatty acid analysis demonstrated the presence of a high concentration of iso-C15: 0(51.65%). Next, the complete whole-genome sequence of strain KD22T was 3,368,578 bp long with 42 mol% of DNA G + C contents. The DDH and ANI values between KD22T and type strains of phylogenetically related species were 67.40% and 95.43%, respectively. These phylogenetic, phenotypic, and genomic results supported the affiliation of strain KD22Tas a novel bacterial species within the genusGabonibacter. The proposed name isGabonibacter chumensis and the type strain is KD22T(= CSUR Q8104T = DSM 115208 T). -
Senko, John M. (Ed.)more » « less
In commercial large-scale aquaria, controlling levels of nitrogenous compounds is essential for macrofauna health. Naturally occurring bacteria are capable of transforming toxic nitrogen species into their more benign counterparts and play important roles in maintaining aquaria health. Nitrification, the microbially-mediated transformation of ammonium and nitrite to nitrate, is a common and encouraged process for management of both commercial and home aquaria. A potentially competing microbial process that transforms ammonium and nitrite to dinitrogen gas (anaerobic ammonium oxidation [anammox]) is mediated by some bacteria within the phylum Planctomycetes. Anammox has been harnessed for nitrogen removal during wastewater treatment, as the nitrogenous end product is released into the atmosphere rather than in aqueous discharge. Whether anammox bacteria could be similarly utilized in commercial aquaria is an open question. As a first step in assessing the viability of this practice, we (i) characterized microbial communities from water and sand filtration systems for four habitats at the Tennessee Aquarium and (ii) examined the abundance and anammox potential of Planctomycetes using culture-independent approaches. 16S rRNA gene amplicon sequencing revealed distinct, yet stable, microbial communities and the presence of Planctomycetes (~1–15% of library reads) in all sampled habitats. Preliminary metagenomic analyses identified the genetic potential for multiple complete nitrogen metabolism pathways. However, no known genes diagnostic for the anammox reaction were found in this survey. To better understand the diversity of this group of bacteria in these systems, a targeted Planctomycete-specific 16S rRNA gene-based PCR approach was used. This effort recovered amplicons that share <95% 16S rRNA gene sequence identity to previously characterized Planctomycetes, suggesting novel strains within this phylum reside within aquaria.
-
Ravel, Jacques (Ed.)ABSTRACT Invertebrates, particularly sponges, have been a dominant source of new marine natural products. For example, lasonolide A (LSA) is a potential anticancer molecule isolated from the marine sponge Forcepia sp., with nanomolar growth inhibitory activity and a unique cytotoxicity profile against the National Cancer Institute 60-cell-line screen. Here, we identified the putative biosynthetic pathway for LSA. Genomic binning of the Forcepia sponge metagenome revealed a Gram-negative bacterium belonging to the phylum Verrucomicrobia as the candidate producer of LSA. Phylogenetic analysis showed that this bacterium, here named “ Candidatus Thermopylae lasonolidus,” only has 88.78% 16S rRNA identity with the closest relative, Pedosphaera parvula Ellin514, indicating that it represents a new genus. The lasonolide A ( las ) biosynthetic gene cluster (BGC) was identified as a trans -acyltransferase (AT) polyketide synthase (PKS) pathway. Compared with its host genome, the las BGC exhibits a significantly different GC content and pentanucleotide frequency, suggesting a potential horizontal acquisition of the gene cluster. Furthermore, three copies of the putative las pathway were identified in the candidate producer genome. Differences between the three las repeats were observed, including the presence of three insertions, two single-nucleotide polymorphisms, and the absence of a stand-alone acyl carrier protein in one of the repeats. Even though the verrucomicrobial producer shows signs of genome reduction, its genome size is still fairly large (about 5 Mbp), and, compared to its closest free-living relative, it contains most of the primary metabolic pathways, suggesting that it is in the early stages of reduction. IMPORTANCE While sponges are valuable sources of bioactive natural products, a majority of these compounds are produced in small quantities by uncultured symbionts, hampering the study and clinical development of these unique compounds. Lasonolide A (LSA), isolated from marine sponge Forcepia sp., is a cytotoxic molecule active at nanomolar concentrations, which causes premature chromosome condensation, blebbing, cell contraction, and loss of cell adhesion, indicating a novel mechanism of action and making it a potential anticancer drug lead. However, its limited supply hampers progression to clinical trials. We investigated the microbiome of Forcepia sp. using culture-independent DNA sequencing, identified genes likely responsible for LSA synthesis in an uncultured bacterium, and assembled the symbiont’s genome. These insights provide future opportunities for heterologous expression and cultivation efforts that may minimize LSA’s supply problem.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1128/spectrum.02346-21
