Vallitalea longa sp. nov., an anaerobic bacterium isolated from marine sediment.

A novel bacterium, strain SH18-1T, was isolated from marine sediment collected near Sado Island in the Sea of Japan. This strain was strictly anaerobic, Gram-stain-negative, non-spore-forming, rod-shaped, motile, and mesophilic. It grew at 15-40 °C (optimum, 30-35 °C), at a NaCl concentration of 0.2-5.0 % (w/v; optimum, 1.5-2.5 %), and at pH 5.5-8.5 (optimum, pH 7.0). Results of 16S rRNA gene phylogenetic analysis showed a similarity value of 97.49 % between strain SH18-1T and Vallitalea guaymasensis Ra1766G1T, which was the most closely related species. The genome size of strain SH18-1T was 5.71 Mb and its G+C content was 30.2 mol%. Genome sequence analyses for comparison between strain SH18-1T and V. guaymasensis Ra1766G1T showed values lower than the threshold for species demarcation determined using the Genome-to-Genome Distance Calculator and the Average Nucleotide Identity Calculator. Elemental sulphur, sulphate, thiosulphate, sulphite, fumarate, nitrate, and nitrite were not used as terminal electron acceptors. The major fatty acids in strain SH18-1T were iso-C15 : 0, anteiso-C15 : 0, and C16 : 0, and the detected polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphoglycolipid, glycolipid, three unidentified phospholipids, and one unidentified polar lipid. From these results, strain SH18-1T (=NBRC 115488T=DSM 114058T) is suggested to represent a novel species of the genus Vallitalea and the name Vallitalea longa sp. nov. is proposed.

A novel amylolytic/xylanolytic/cellulolytic multienzyme complex from Clostridium manihotivorum that hydrolyzes polysaccharides in cassava pulp.

Some anaerobic bacteria, particularly Clostridium species, produce extracellular cellulolytic and xylanolytic enzymes as multienzyme complexes (MECs). However, an amylolytic/xylanolytic/cellulolytic multienzyme complex (AXC-MEC) from anaerobic bacteria is rarely found. In this work, the glycoprotein AXC-MEC, composed of subunits of amylolytic, xylanolytic, and cellulolytic enzymes, was isolated from crude extracellular enzyme of the mesophilic anaerobic bacterium Clostridium manihotivorum CT4, grown on cassava pulp, using a milled cassava pulp column and Sephacryl S-500 gel filtration chromatography. The isolated AXC-MEC showed a single band upon native-polyacrylamide gel electrophoresis (native-PAGE). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed at least eight protein bands of the multienzyme complex which predominantly exhibited amylolytic enzyme activity, followed by xylanolytic and cellulolytic enzyme activities. The AXC-MEC is highly capable of degrading starch and non-starch polysaccharides present in cassava pulp into glucose and oligosaccharides, without conventional pretreatment. Base on the genomic analysis of C. manihotivorum CT4, we found no evidence of the known structural components of the well-known multienzyme complexes from Clostridium species, cellulosomes such as scaffoldin, cohesin, and dockerin, indicating that AXC-MEC from strain CT4 exhibit a different manner of assembly from the cellulosomes. These results suggest that AXC-MEC from C. manihotivorum CT4 is a new MEC capable of hydrolyzing cassava pulp into value-added products, which will benefit the starch industry. KEY POINTS: • Glycoprotein AXC-MEC was first reported in Clostridium manihotivorum. • Unlike cellulosomes, AXC-MEC consists of amylase, xylanase, and cellulase. • Glucose and oligosaccharides were hydrolysis products from cassava pulp by AXC-MEC.

Proteiniclasticum sediminis sp. nov., an obligate anaerobic bacterium isolated from anaerobic sludge.

An obligate anaerobic bacterial BAD-10 T was isolated from anaerobic acetochlor-degrading sludge. The strain was Gram-stain negative, curved rod-shaped, non-motile and non-spore-forming. Growth was observed in PYT medium at pH 6.0-9.0 (optimum, pH 7.5), at 25-47 °C (37 °C) and with 0-1.0% NaCl (w/v, 0%). Strain BAD-10 T could degrade acetochlor. The major fermentation products from peptone-yeast (PY) medium were acetate and butyrate. The predominant cellular fatty acids were iso-C15:0 FAME, anteiso-C15:0 FAME and C16:0 FAME. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain BAD-10 T showed closest affiliation to Proteiniclasticum ruminis D3RC-2 T, with a sequence similarity of 97.6%. Genome sequencing revealed a genome size of 2,983,986 bp, a G + C content of 51.4 mol% and protein-coding genes of 3,102. The average nucleotide identity and in silico DNA-DNA hybridization values between strain BAD-10 T and Proteiniclasticum ruminis D3RC-2 T were 71.0% and 20.4%, respectively, which were below the standard thresholds for species differentiation. On the basis of phenotypic, physiological and phylogenetic evidence, strain BAD-10 T represents a novel species in the genus Proteiniclasticum, for which the name Proteiniclasticum sediminis sp. nov. is proposed. Strain BAD-10 T (= CCTCC AB 2021091 T = KCTC 25288 T) is the type strain of the proposed novel species.

Aquipluma nitroreducens gen. nov. sp. nov., a novel facultatively anaerobic bacterium isolated from a freshwater lake.

A novel facultatively anaerobic, nitrate-reducing bacterium, designated MeG22T, was isolated from a freshwater lake in Japan. Cells of the strain were straight rods (0.8×2.5-10 µm), motile, and Gram-stain-negative. For growth, the optimum NaCl concentration was 0 % and the optimum temperature was 30 °C. Under anoxic conditions, strain MeG22T reduced nitrate to nitrite. Major cellular fatty acids were C15 : 1 ω6c (13.6 %), C17 : 0 (11.9 %), anteiso-C15 : 0 (10.6 %) and iso-C15 : 0 (10.6 %). The major respiratory quinone was menaquinone-7. The genome sequence of strain MeG22T consists of 5 712 279 bp with a G+C content of 40.3 mol%. Phylogenetic analysis based on the 16S rRNA gene revealed that the novel strain belonged to the family Prolixibacteraceae within the phylum Bacteroidetes. The closest relative of strain MeG22T was Sunxiuqinia faeciviva strain JAM-BA0302T with a 16S rRNA gene sequence similarity of 90.9 %. On the basis of phylogenetic and phenotypic characterization, Aquipluma nitroreducens, gen. nov., sp. nov., belonging to the family Prolixibacteraceae is proposed with the type strain MeG22T (=NBRC 112896T=DSM 106262T).

Desulfobotulus mexicanus sp. nov., a novel sulfate-reducing bacterium isolated from the sediment of an alkaline crater lake in Mexico.

A novel Gram-negative, non-spore-forming, vibrio-shaped, anaerobic, alkaliphilic, sulfate-reducing bacterium, designated strain PAR22NT, was isolated from sediment samples collected at an alkaline crater lake in Guanajuato (Mexico). Strain PAR22NT grew at temperatures between 15 and 37 °C (optimum, 32 °C), at pH between pH 8.3 and 10.1 (optimum, pH 9.0-9.6), and in the presence of NaCl up to 10 %. Pyruvate, 2-methylbutyrate and fatty acids (4-18 carbon atoms) were used as electron donors in the presence of sulfate as a terminal electron acceptor and were incompletely oxidized to acetate and CO2. Besides sulfate, both sulfite and elemental sulfur were also used as terminal electron acceptors and were reduced to sulfide. The predominant fatty acids were summed feature 10 (C18 : 1 ω7c and/or C18 : 1 ω9t and/or C18 : 1 ω12t), C18 : 1 ω9c and C16 : 0. The genome size of strain PAR22NT was 3.8 Mb including 3391 predicted genes. The genomic DNA G+C content was 49.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that it belongs to the genus Desulfobotulus within the class Deltaproteobacteria. Its closest phylogenetic relatives are Desulfobotulus alkaliphilus (98.4 % similarity) and Desulfobotulus sapovorans (97.9 % similarity). Based on phylogenetic, phenotypic and chemotaxonomic characteristics, we propose that the isolate represents a novel species of the genus Desulfobotulus with the name Desulfobotulus mexicanus sp. nov. The type strain is PAR22NT (=DSM 105758T=JCM 32146T).

Synergistic Activity of Nitroimidazole-Oxazolidinone Conjugates against Anaerobic Bacteria.

The introductions of the bicyclic 4-nitroimidazole and the oxazolidinone classes of antimicrobial agents represented the most significant advancements in the infectious disease area during the past two decades. Pretomanid, a bicyclic 4-nitroimidazole, and linezolid, an oxazolidinone, are also part of a combination regimen approved recently by the US Food and Drug Administration for the treatment of pulmonary, extensively drug resistant (XDR), treatment-intolerant or nonresponsive multidrug-resistant (MDR) Mycobacterium tuberculosis (TB). To identify new antimicrobial agents with reduced propensity for the development of resistance, a series of dual-acting nitroimidazole-oxazolidinone conjugates were designed, synthesized and evaluated for their antimicrobial activity. Compounds in this conjugate series have shown synergistic activity against a panel of anaerobic bacteria, including those responsible for serious bacterial infections.

Whole-genome sequence of the strictly anaerobic bacterial strain SANA belonging to the family Gottschalkiaceae, isolated from a xenic culture of an anaerobic protist.

An anaerobic bacterial strain SANA was isolated from a xenic culture of an anaerobic heterolobosean protist which was obtained from a saline lake in Japan. Its draft genome comprises 1 circular chromosome (3,490,293 bp), harboring 3,275 predicted protein-coding and 73 tRNA-encoding genes and 8 rRNA operons.

Genomics and cellulolytic, hemicellulolytic, and amylolytic potential of Iocasia fonsfrigidae strain SP3-1 for polysaccharide degradation.

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-1T. Prior to characterization of strain NS-1T, 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-1T. 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-1T 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-1T, 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-1T. 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-1T 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-1T. The high salt tolerance and pH range of SP3-1 makes it an ideal candidate for salt and pH tolerant enzyme discovery.

Emphysematous liver abscess with Edwardsiella tarda infection.

This report presents the case of a 51-year-old woman on an immunosuppressant drug and steroids, who presented with general fatigue and was admitted to the intensive care unit. Her serum procalcitonin, lactate, aspartate aminotransferase, and alanine aminotransferase levels and white blood cell counts were elevated. Computed tomography revealed gas formation in her liver, and her culture results revealed Edwardsiella tarda and Escherichia coli infections. She underwent percutaneous transhepatic abscess drainage in addition to antimicrobial administration. She was discharged after 40 days. Cases of emphysematous liver abscess with Edwardsiella tarda infections are rarely reported in the literature and may present in patients with poorly controlled type 2 diabetes. The fatality rate associated with the condition is markedly high.

Clostridium manihotivorum sp. nov., a novel mesophilic anaerobic bacterium that produces cassava pulp-degrading enzymes.

BACKGROUND: Cassava pulp is a promising starch-based biomasses, which consists of residual starch granules entrapped in plant cell wall containing non-starch polysaccharides, cellulose and hemicellulose. Strain CT4T, a novel mesophilic anaerobic bacterium isolated from soil collected from a cassava pulp landfill, has a strong ability to degrade polysaccharides in cassava pulp. This study explored a rarely described species within the genus Clostridium that possessed a group of cassava pulp-degrading enzymes. METHODS: A novel mesophilic anaerobic bacterium, the strain CT4T, was identified based on phylogenetic, genomic, phenotypic and chemotaxonomic analysis. The complete genome of the strain CT4T was obtained following whole-genome sequencing, assembly and annotation using both Illumina and Oxford Nanopore Technology (ONT) platforms. RESULTS: Analysis based on the 16S rRNA gene sequence indicated that strain CT4T is a species of genus Clostridium. Analysis of the whole-genome average amino acid identity (AAI) of strain CT4T and the other 665 closely related species of the genus Clostridium revealed a separated strain CT4T from the others. The results revealed that the genome consisted of a 6.3 Mb circular chromosome with 5,664 protein-coding sequences. Genome analysis result of strain CT4T revealed that it contained a set of genes encoding amylolytic-, hemicellulolytic-, cellulolytic- and pectinolytic enzymes. A comparative genomic analysis of strain CT4T with closely related species with available genomic information, C. amylolyticum SW408T, showed that strain CT4T contained more genes encoding cassava pulp-degrading enzymes, which comprised a complex mixture of amylolytic-, hemicellulolytic-, cellulolytic- and pectinolytic enzymes. This work presents the potential for saccharification of strain CT4T in the utilization of cassava pulp. Based on phylogenetic, genomic, phenotypic and chemotaxonomic data, we propose a novel species for which the name Clostridium manihotivorum sp. nov. is suggested, with the type strain CT4T (= TBRC 11758T = NBRC 114534T).

Genome sequence and description of Bacteroides bouchesdurhonensis sp. nov., a new anaerobic bacterium isolated from the human gut.

Bacteroides bouchesdurhonensis sp. nov., strain Marseille-P2653T (= CSUR; P2653=DSM103120) is a new bacterial species belonging to the Firmicutes phylum in the family Bacteroidaceae that was isolated from the human gut microbiota.

Massilicoli timonensis sp. nov., a new bacterium isolated from the human microbiota.

Massilicoli timonensis sp. nov., strain Marseille-P3755T (= CSUR P3755 = DSM 103513) is a new bacterial species from the phylum Firmicutes and the family Clostridiales which was isolated from the human gut microbiota.

Analysis of a Lys-specific serine endopeptidase secreted via the type IX secretion system in Porphyromonas gingivalis.

Porphyromonas gingivalis, a significant causative agent of adult periodontitis, possesses a novel secretion system called the type IX secretion system (T9SS). A number of virulence factors, such as Arg-gingipain (Rgp), are translocated onto the cell surface and into the extracellular milieu via the T9SS. In this study, we found that the PGN_1416 90- to 120-kDa diffuse protein bands were located in the outer membrane fraction and that the presence of the bands was dependent on genes involved in the T9SS and the formation of anionic lipopolysaccharide (A-LPS). These data strongly suggest that the PGN_1416 protein is secreted by the T9SS and anchored onto the cell surface by binding to A-LPS. Enzymatic analysis using outer membrane fractions suggested that the PGN_1416 protein has a Lys-specific serine endopeptidase activity and that its activation requires processing by Rgp. Homologues of the gene encoding PGN_1416, which is referred to as pepK, were found in bacteria belonging to the phyla Bacteroidetes and Proteobacteria, whereas homologues encoding the C-terminal domain, which is essential for T9SS-mediated secretion, and the catalytic domain were only observed in bacteria belonging to the Bacteroidetes phylum.