Methylocystis silviterrae sp.nov., a high-affinity methanotrophic bacterium isolated from the boreal forest soil

A novel species is proposed for a high-affinity methanotrophic representative of the genus Methylocystis . Strain FST was isolated from a weakly acidic (pH 5.3) mixed forest soil of the southern Moscow area. Cells of FST are aerobic, Gram-negative, non-motile, curved coccoids or short rods that contain an intracytoplasmic membrane system typical of type-II methanotrophs. Only methane and methanol are used as carbon sources. FST grew at a temperature range of 4–37 °C (optimum 25–30 °C) and a pH range of 4.5 to 7.5 (optimum pH 6.0–6.5). The major fatty acids were C18 : 1ω8c, C18 : 1ω7c and C18 : 0; the major quinone as Q-8. FST displays 16S rRNA gene sequences similarity to other taxonomically recognized members of the genus Methylocystis, with Methylocystis hirsuta CSC1T (99.6 % similarity) and Methylocystis rosea SV97T (99.3 % similarity) as its closest relatives. The genome comprises 3.85 Mbp and has a DNA G+C content of 62.6 mol%. Genomic analyses and DNA–DNA relatedness with genome-sequenced members of the genus Methylocystis demonstrated that FST could be separated from its closest relatives. FST possesses two particulate methane monooxygenases (pMMO): low-affinity pMMO1 and high-affinity pMMO2. In laboratory experiments, it was demonstrated that FST might oxidize methane at atmospheric concentration. The genome contained various genes for nitrogen fixation, polyhydroxybutyrate synthesis, antibiotic resistance and detoxification of arsenic, cyanide and mercury. On the basis of genotypic, phenotypic and chemotaxonomic characteristics, it is proposed that the isolate represents a novel species, Methylocystis silviterrae sp. nov. The type strain is FST (=KCTC 82935T=VKM B-3535T).

Broad Ultrastructural and Transcriptomic Changes Underlie the Multinucleated Giant Hemocyte Mediated Innate Immune Response against Parasitoids

Multinucleated giant hemocytes (MGHs) represent a novel type of blood cell in insects that participate in a highly efficient immune response against parasitoid wasps involving isolation and killing of the parasite. Previously, we showed that circulating MGHs have high motility and the interaction with the parasitoid rapidly triggers encapsulation. However, structural and molecular mechanisms behind these processes remained elusive. Here, we used detailed ultrastructural analysis and live cell imaging of MGHs to study encapsulation in <i>Drosophila ananassae</i> after parasitoid wasp infection. We found dynamic structural changes, mainly driven by the formation of diverse vesicular systems and newly developed complex intracytoplasmic membrane structures, and abundant generation of giant cell exosomes in MGHs. In addition, we used RNA sequencing to study the transcriptomic profile of MGHs and activated plasmatocytes 72 h after infection, as well as the uninduced blood cells. This revealed that differentiation of MGHs was accompanied by broad changes in gene expression. Consistent with the observed structural changes, transcripts related to vesicular function, cytoskeletal organization, and adhesion were enriched in MGHs. In addition, several orphan genes encoding for hemolysin-like proteins, pore-forming toxins of prokaryotic origin, were expressed at high level, which may be important for parasitoid elimination. Our results reveal coordinated molecular and structural changes in the course of MGH differentiation and parasitoid encapsulation, providing a mechanistic model for a powerful innate immune response.

Feeding experiments of the seep-associated foraminifer Nonionellina labradorica with a marine methanotroph from the Arctic

&lt;p&gt;Foraminifera on the seafloor are known to have species-specific feeding habits. Among those are deposit feeders, eating organic detritus and bacteria. Little is known about the feeding habits of foraminifera from Arctic seep environments. That is, in particular, of interest as variable &amp;#948;&lt;sup&gt;13&lt;/sup&gt;C values in the tests of foraminifera have been suggested to be partly linked with a diet rich in bacteria, themselves lighter in &amp;#948;&lt;sup&gt;13&lt;/sup&gt;C values. As there is little information on the ecology of the foraminifer &lt;em&gt;Nonionellina labradorica&lt;/em&gt; (Dawson, 1860), this study examined feeding habits on bacteria and compared them to in situ collected specimens, using Transmission Electron microscopy (TEM). As bacterial food, the marine methane-oxidizing bacterium &lt;em&gt;Methyloprofundus sedimenti&lt;/em&gt; was chosen, which is an important representative of methanotrophs in the marine environment near methane seeps. Sediment samples containing living N. labradorica specimens collected in close vicinity(approx. 5 m) from an active methane seep in Storfjordrenna, Barents Sea (382-m water depth).&amp;#160; We performed a feeding experiment on &lt;em&gt;N. labradorica &lt;/em&gt;(n=17 specimen), which were incubated in the dark at in situ temperature. Specimens were fed at the beginning of the experiment, except the un-fed controls, and incubations terminated after 4, 8 and 20 h. After fixation in epoxy resin the ultrastructure of all specimens and their food vacuoles was observed and compared using a TEM. All examined specimens were living at the time of fixation, based on observation of intact mitochondrial membranes. In all specimens, inorganic detritus was preserved inside food vacuoles. Closer observation of food vacuoles also revealed that in addition to inorganic debris, such as clay, occasionally bacteria were visible. This led us to conclude that our &lt;em&gt;N. labradorica &lt;/em&gt;can&amp;#160; generally be classified as a deposit feeder, which is rather a generalist than a specialist. Regarding uptake of &lt;em&gt;M. sedimenti&lt;/em&gt;, the timing of the experimentation seemed to be critical. We did not observe methanotrophs preserved in the resin at the 4 and 8 h incubations, but found two putative methanotrophs near the apertural region after the 20-h incubation. After closer observation, we could identify one of those two putative specimen as the menthanothroph &lt;em&gt;M. sedimenti&lt;/em&gt; near the foraminiferal aperture, based on presence of a typical type I stacked intracytoplasmic membrane (ICM) and storage granules (SC). We concluded that &lt;em&gt;N. labradorica&lt;/em&gt; may ingest &lt;em&gt;M. sedimenti&lt;/em&gt; via &amp;#8220;untargeted grazing&amp;#8221; in seeps. Further studies must examine the exact relationship between diet and &amp;#948;&lt;sup&gt;13&lt;/sup&gt;C in foraminiferal test on several different paleo-oceanographically relevant species.&lt;/p&gt;

Ammonia oxidation at pH 2.5 by a new gammaproteobacterial ammonia-oxidizing bacterium

Ammonia oxidation was considered impossible under highly acidic conditions, as the protonation of ammonia leads to decreased substrate availability and formation of toxic nitrogenous compounds. Recently, some studies described archaeal and bacterial ammonia oxidizers growing at pH as low as 4, while environmental studies observed nitrification at even lower pH values. In this work, we report on the discovery, cultivation, and physiological, genomic, and transcriptomic characterization of a novel gammaproteobacterial ammonia-oxidizing bacterium enriched via continuous bioreactor cultivation from an acidic air biofilter that was able to grow and oxidize ammonia at pH 2.5. This microorganism has a chemolithoautotrophic lifestyle, using ammonia as energy source. The observed growth rate on ammonia was 0.196 day−1, with a doubling time of 3.5 days. The strain also displayed ureolytic activity and cultivation with urea as ammonia source resulted in a growth rate of 0.104 day−1 and a doubling time of 6.7 days. A high ammonia affinity (Km(app) = 147 ± 14 nM) and high tolerance to toxic nitric oxide could represent an adaptation to acidic environments. Electron microscopic analysis showed coccoid cell morphology with a large amount of intracytoplasmic membrane stacks, typical of gammaproteobacterial ammonia oxidizers. Furthermore, genome and transcriptome analysis showed the presence and expression of diagnostic genes for nitrifiers (amoCAB, hao, nor, ure, cbbLS), but no nirK was identified. Phylogenetic analysis revealed that this strain belonged to a novel bacterial genus, for which we propose the name “Candidatus Nitrosacidococcus tergens” sp. RJ19.

Isolation of a member of the candidate phylum ‘Atribacteria’ reveals a unique cell membrane structure

A key feature that differentiates prokaryotic cells from eukaryotes is the absence of an intracellular membrane surrounding the chromosomal DNA. Here, we isolate a member of the ubiquitous, yet-to-be-cultivated phylum ‘Candidatus Atribacteria’ (also known as OP9) that has an intracytoplasmic membrane apparently surrounding the nucleoid. The isolate, RT761, is a subsurface-derived anaerobic bacterium that appears to have three lipid membrane-like layers, as shown by cryo-electron tomography. Our observations are consistent with a classical gram-negative structure with an additional intracytoplasmic membrane. However, further studies are needed to provide conclusive evidence for this unique intracellular structure. The RT761 genome encodes proteins with features that might be related to the complex cellular structure, including: N-terminal extensions in proteins involved in important processes (such as cell-division protein FtsZ); one of the highest percentages of transmembrane proteins among gram-negative bacteria; and predicted Sec-secreted proteins with unique signal peptides. Physiologically, RT761 primarily produces hydrogen for electron disposal during sugar degradation, and co-cultivation with a hydrogen-scavenging methanogen improves growth. We propose RT761 as a new species, Atribacter laminatus gen. nov. sp. nov. and a new phylum, Atribacterota phy. nov.

Methylococcus geothermalis sp. nov., a methanotroph isolated from a geothermal field in the Republic of Korea

A Gram-stain-negative, aerobic, non-motile and coccoid methanotroph, strain IM1T, was isolated from hot spring soil. Cells of strain IM1T were catalase-negative, oxidase-positive and displayed a characteristic intracytoplasmic membrane arrangement of type I methanotrophs. The strain possessed genes encoding both membrane-bound and soluble methane monooxygenases and grew only on methane or methanol. The strain was capable of growth at temperatures between 15 and 48 °C (optimum, 30–45 °C) and pH values between pH 4.8 and 8.2 (optimum, pH 6.2–7.0). Based on phylogenetic analysis of 16S rRNA gene and PmoA sequences, strain IM1T was demonstrated to be affiliated to the genus Methylococcus . The 16S rRNA gene sequence of this strain was most closely related to the sequences of an uncultured bacterium clone FD09 (100 %) and a partially described cultured Methylococcus sp. GDS2.4 (99.78 %). The most closely related taxonomically described strains were Methylococcus capsulatus TexasT (97.92 %), Methylococcus capsulatus Bath (97.86 %) and Methyloterricola oryzae 73aT (94.21 %). Strain IM1T shared average nucleotide identity values of 85.93 and 85.62 % with Methylococcus capsulatus strains TexasT and Bath, respectively. The digital DNA–DNA hybridization value with the closest type strain was 29.90 %. The DNA G+C content of strain IM1T was 63.3 mol% and the major cellular fatty acids were C16 : 0 (39.0 %), C16 : 1 ω7c (24.0 %), C16 : 1 ω6c (13.6 %) and C16 : 1 ω5c (12.0 %). The major ubiquinone was methylene-ubiquinone-8. On the basis of phenotypic, genetic and phylogenetic data, strain IM1T represents a novel species of the genus Methylococcus for which the name Methylococcus geothermalis sp. nov. is proposed, with strain IM1T (=JCM 33941T=KCTC 72677T) as the type strain.

Rhodobacter flagellatus sp. nov., a thermophilic bacterium isolated from a hot spring

A thermophilic bacterium, designated SYSU G03088T, was isolated from Moincer hot spring, Tibet, PR China. Polyphasic taxonomic analyses and whole-genome sequencing were used to determine the taxonomic position and genomic profiles of the strain. Phylogenetic analysis using 16S rRNA gene sequence indicated that SYSU G03088T showed highest sequence similarity to Rhodobacter blasticus CGMCC 1.3365T (96.0 % sequence identity). The strain could be differentiated from most recognized species of the genus Rhodobacter by its slightly purple colony colour, distinct phenotypic characters and low ANI values. Cells were Gram-staining negative, and oval-to-rod shaped. Poly-β-hydroxybutyrate and vesicular intracytoplasmic membrane structures were formed inside cells. Growth occurred optimally at 45 °C and pH 7.0. Ubiquinone 10 was the only respiratory quinone. The major fatty acids (>10 %) were C18 : 0, C18 : 1ω7c 11-methyl and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The detected polar lipids of SYSU G03088T included diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylmethylethanolamine. The DNA G+C content of SYSU G03088T was 67.7 % (genome). On the basis of the differences in the phenotypic characteristics and phylogenetic analyses, SYSU G03088T is considered to represent a novel species of the genus Rhodobacter , for which the name Rhodobacter flagellatus sp. nov. is proposed. The type strain is SYSU G03088T (=CGMCC 1.16876T=KCTC 72354T).

Membrane-bounded nucleoid discovered in a cultivated bacterium of the candidate phylum ‘Atribacteria’

A key feature that differentiates prokaryotes from eukaryotes is the absence of an intracellular membrane surrounding the chromosomal DNA. Here, we report isolation of an anaerobic bacterium that possesses an additional intracytoplasmic membrane surrounding a nucleoid, affiliates with the yet-to-be-cultivated ubiquitous phylum ‘Ca. Atribacteria’, and possesses unique genomic features likely associated with organization of complex cellular structure. Exploration of the uncharted microorganism overturned the prevailing dogma of prokaryotic cell structure.

Restricted Localization of Photosynthetic Intracytoplasmic Membranes (ICMs) in Multiple Genera of Purple Nonsulfur Bacteria

ABSTRACTIn bacteria and eukaryotes alike, proper cellular physiology relies on robust subcellular organization. For the phototrophic purple nonsulfur bacteria (PNSB), this organization entails the use of a light-harvesting, membrane-bound compartment known as the intracytoplasmic membrane (ICM). Here we show that ICMs are spatially and temporally localized in diverse patterns among PNSB. We visualized ICMs in live cells of 14 PNSB species across nine genera by exploiting the natural autofluorescence of the photosynthetic pigment bacteriochlorophyll (BChl). We then quantitatively characterized ICM localization using automated computational analysis of BChl fluorescence patterns within single cells across the population. We revealed that while many PNSB elaborate ICMs along the entirety of the cell, species across as least two genera restrict ICMs to discrete, nonrandom sites near cell poles in a manner coordinated with cell growth and division. Phylogenetic and phenotypic comparisons established that ICM localization and ICM architecture are not strictly interdependent and that neither trait fully correlates with the evolutionary relatedness of the species. The natural diversity of ICM localization revealed herein has implications for both the evolution of phototrophic organisms and their light-harvesting compartments and the mechanisms underpinning spatial organization of bacterial compartments.IMPORTANCEMany bacteria organize their cellular space by constructing subcellular compartments that are arranged in specific, physiologically relevant patterns. The purple nonsulfur bacteria (PNSB) utilize a membrane-bound compartment known as the intracytoplasmic membrane (ICM) to harvest light for photosynthesis. It was previously unknown whether ICM localization within cells is systematic or irregular and if ICM localization is conserved among PNSB. Here we surveyed ICM localization in diverse PNSB and show that ICMs are spatially organized in species-specific patterns. Most strikingly, several PNSB resolutely restrict ICMs to regions near the cell poles, leaving much of the cell devoid of light-harvesting machinery. Our results demonstrate that bacteria of a common lifestyle utilize unequal portions of their intracellular space to harvest light, despite light harvesting being a process that is intuitively influenced by surface area. Our findings therefore raise fundamental questions about ICM biology and evolution.