“Terre Calde di Medolla” (TCM) (literally, “Hot Lands of Medolla”) refers to a farming area in Italy with anomalously high temperatures and diffuse emissions of biogenic CO₂, which has been linked to CH₄ oxidation processes from a depth of 0.7 m to the surface. We herein assessed the composition of the total bacterial community and diversity of methane-oxidizing bacteria (MOB) in soil samples collected at a depth at which the peak temperature was detected (0.6 m). Cultivation-independent methods were used, such as: i) a clone library analysis of the 16S rRNA gene and pmoA (coding for the α-subunit of the particulate methane monooxygenase) gene, and ii) Terminal Restriction Fragment Length Polymorphism (T-RFLP) fingerprinting. The 16S rRNA gene analysis assessed the predominance of Actinobacteria, Acidobacteria, Proteobacteria, and Bacillus in TCM samples collected at a depth of 0.6 m along with the presence of methanotrophs (Methylocaldum and Methylobacter) and methylotrophs (Methylobacillus). The phylogenetic analysis of pmoA sequences showed the presence of MOB affiliated with Methylomonas, Methylocystis, Methylococcus, and Methylocaldum in addition to as yet uncultivated and uncharacterized methanotrophs. Jaccard’s analysis of T-RFLP profiles at different ground depths revealed a similar MOB composition in soil samples at depths of 0.6 m and 0.7 m, while this similarity was weaker between these samples and those taken at a depth of 2.5 m, in which the genus Methylocaldum was absent. These results correlate the anomalously high temperatures of the farming area of “Terre Calde di Medolla” with the presence of microbial methane-oxidizing bacteria.

Knowledge on dynamic interactions in microbiota is pivotal for understanding the role of bacteria in the gut. We herein present comprehensive dynamic models of the horse cecal microbiota, which include short-chained fatty acids, carbohydrate metabolic networks, and taxonomy. Dynamic models were derived from time-series data in a crossover experiment in which four cecum-cannulated horses were fed a starch-rich diet of hay supplemented with barley (starch intake 2 g kg⁻¹ body weight per day) and a fiber-rich diet of only hay. Cecal contents were sampled via the cannula each h for 24 h for both diets. We observed marked differences in the microbial dynamic interaction patterns for Fibrobacter succinogenes, Lachnospiraceae, Streptococcus, Treponema, Anaerostipes, and Anaerovibrio between the two diet groups. Fluctuations and microbiota interactions were the most pronounced for the starch rich diet, with Streptococcus spp. and Anaerovibrio spp. showing the largest fluctuations. Shotgun metagenome sequencing revealed that diet differences may be explained by modular switches in metabolic cross-feeding between microbial consortia in which fermentation is linked to sugar alcohols and amino sugars for the starch-rich diet and monosaccharides for the fiber-rich diet. In conclusion, diet may not only affect the composition of the cecal microbiota, but also dynamic interactions and metabolic cross-feeding.

Legionella pneumophila is a pathogenic bacteria found in biofilms in freshwater. Iron is an essential nutrient for L. pneumophila growth. In this study, complex biofilms were developed using river water spiked with L. pneumophila, and the persistence of L. pneumophila in these complex biofilms was evaluated. In order to study the role of iron in the persistence of L. pneumophila, river water was supplied with either iron pyrophosphate or iron chelators (deferoxamine mesylate, DFX for ferric iron and dipyridyl, DIP for ferrous iron) to modulate iron availability. The addition of iron pyrophosphate and DFX did not markedly affect the persistence of L. pneumophila in the biofilms, whereas that of DIP had a beneficial effect. Since DIP specifically chelates ferrous iron, we hypothesized that DIP may protect L. pneumophila from the deleterious effects of ferrous iron. In conclusion, ferrous iron appears to be important for the persistence of L. pneumophila in complex biofilms. However, further studies are needed in order to obtain a better understanding of the role of ferrous iron in the behavior of this bacterium in the environment.

Acetobacter pasteurianus SKU1108 is a typical thermotolerant acetic acid bacterium. In this study, the complete genome sequence of the SKU1108 strain was elucidated, and information on genomic modifications due to the thermal adaptation of SKU1108 was updated. In order to obtain a clearer understanding of the genetic background responsible for thermotolerance, the SKU1108 genome was compared with those of two closely related complete genome strains, thermotolerant A. pasteurianus 386B and mesophilic A. pasteurianus NBRC 3283. All 24 “thermotolerant genes” required for growth at higher temperatures in the thermotolerant Acetobacter tropicalis SKU1100 strain were conserved in all three strains. However, these thermotolerant genes accumulated amino acid mutations. Some biased mutations, particularly those that occurred in xanthine dehydrogenase XdhA, may be related to thermotolerance. By aligning whole genome sequences, we identified ten SKU1108 strain-specific regions, three of which were conserved in the genomes of the two thermotolerant A. pasteurianus strains. One of the regions contained a unique paralog of the thermotolerant gene xdhA, which may also be responsible for conferring thermotolerance. Thus, comparative genomics of complete genome sequences may provide novel insights into the phenotypes of these thermotolerant strains.

Two thirds of Svalbard archipelago islands in the High Arctic are permanently covered with glacial ice and snow. Polar bacterial communities in the southern part of Svalbard were characterized using an amplicon sequencing approach. A total of 52,928 pyrosequencing reads were analyzed in order to reveal bacterial community structures in stream and lake surface water samples from the Fuglebekken and Revvatnet basins of southern Svalbard. Depending on the samples examined, bacterial communities at a higher taxonomic level mainly consisted either of Bacteroidetes, Betaproteobacteria, and Microgenomates (OP11) or Planctomycetes, Betaproteobacteria, and Bacteroidetes members, whereas a population of Microgenomates was prominent in 2 samples. At the lower taxonomic level, bacterial communities mostly comprised Microgenomates, Comamonadaceae, Flavobacteriaceae, Legionellales, SM2F11, Parcubacteria (OD1), and TM7 members at different proportions in each sample. The abundance of OTUs shared in common among samples was greater than 70%, with the exception of samples in which the proliferation of Planctomycetaceae, Phycisphaeraceae, and Candidatus Methylacidiphilum spp. lowered their relative abundance. A multi-variable analysis indicated that As, Pb, and Sb were the main environmental factors influencing bacterial profiles. We concluded that the bacterial communities in the polar aquatic ecosystems examined mainly consisted of freshwater and marine microorganisms involved in detritus mineralization, with a high proportion of zooplankton-associated taxa also being identified.

Crotalaria zanzibarica is an exotic and widely distributed leguminous plant in Taiwan. The relationship between C. zanzibarica and its rhizobial symbionts has been suggested to contribute to its successful invasion. A rhizobial strain (designed as CzR2) isolated from the root nodules of C. zanzibarica and cultivated in standard YEM medium displayed pleomorphism, with cells ranging between 2 and 10 μm in length and some branching. In the present study, we identified this rhizobial strain, investigated the causes of pleomorphism, and examined the nodules formed. The results of a multilocus sequence analysis of the atpD, dnaK, glnII, gyrB, recA, and rpoB genes revealed that CzR2 belongs to Bradyrhizobium arachidis, a peanut symbiont recently isolated from China. Cells of the strain were uniformly rod-shaped in basal HM medium, but displayed pleomorphism in the presence of yeast extract, mannitol, or fructose. These results indicate that the morphology of CzR2 in its free-living state is affected by nutrient conditions. Several highly pleomorphic bacteroids enclosed in symbiosomes were frequently detected in FM and TEM observations of sections of the indeterminate nodules induced by CzR2; however, no infection thread was identified. Flow cytometric analyses showed that CzR2 cells in YEM medium and in the nodules of C. zanzibarica had two or more than two peaks in relative DNA contents, respectively, suggesting that the elongated cells of CzR2 in its free-living state occur due to a cell cycle-delayed process, while those in its symbiotic state are from genomic endo-reduplication.

Suberin is a complex lipidic plant polymer found in various tissues including the potato periderm. The biological degradation of suberin is attributed to fungi. Soil samples from a potato field were used to inoculate a culture medium containing suberin as the carbon source, and a metaproteomic approach was used to identify bacteria that developed in the presence of suberin over a 60-d incubation period. The normalized spectral counts of predicted extracellular proteins produced by the soil bacterial community markedly decreased from day 5 to day 20 and then slowly increased, revealing a succession of bacteria. The population of fast-growing pseudomonads declined and was replaced by species with the ability to develop in the presence of suberin. The recalcitrance of suberin was demonstrated by the emergence of auxotrophic bacteria such as Oscillatoria on the last days of the assay. Nevertheless, two putative lipases from Rhodanobacter thiooxydans (I4WGM2) and Myxococcus xanthus (Q1CWS1) were detected in the culture supernatants, suggesting that at least some bacterial species degrade suberin. When grown in suberin-containing medium, R. thiooxydans strain LCS2 and M. xanthus strain DK 1622 both produced three lipases, including I4WGM2 and Q1CWS1. These strains also produced other proteins linked to lipid metabolism, including fatty acid and lipid transporters and β-oxidation enzymes, suggesting that they participate in the degradation of suberin. However, only the R. thiooxydans strain appeared to retrieve sufficient carbon and energy from this recalcitrant polymer in order to maintain its population over an extended period of time.

Potato peels consist of a tissue called phellem, which is formed by suberized cell layers. The degradation of suberin, a lipidic and recalcitrant polymer, is an ecological process attributed to soil fungal populations; however, previous studies have suggested that Streptomyces scabiei, the causal agent of potato common scab, possesses the ability to degrade suberin. In the present study, S. scabiei was grown in medium containing suberin-enriched potato phellem as the sole carbon source and its secretome was analyzed periodically (10- to 60-d-old cultures) with a special focus on proteins potentially involved in cell wall degradation. Although the amount and diversity of proteins linked to polysaccharide degradation remained high throughout the experiment, their abundance decreased over time. In contrast, proteins dedicated to lipid metabolism represented a small fraction of the secretome; however, their abundance increased during the experiment. The lipolytic enzymes detected may be involved in the degradation of the aliphatic fraction of suberin because the results of optical and transmission electron microscopy examinations revealed a loss in the integrity of suberized tissues exposed to S. scabiei cells. Chemical analyses identified a time period in which the concentration of aliphatic compounds in potato phellem decreased and the sugar concentration increased; at the end of the 60-d incubation period, the sugar concentration in potato phellem was significantly reduced. This study demonstrated the ability of S. scabiei to degrade the aliphatic portion of suberin.

A gene coding for a multicopper oxidase (BopA) was identified through the screening of a metagenomic library constructed from wastewater treatment activated sludge. The recombinant BopA protein produced in Escherichia coli exhibited oxidation activity toward 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in the presence of copper, suggesting that BopA is laccase. A bioinformatic analysis of the bopA gene sequence indicated that it has a phylogenetically bacterial origin, possibly derived from a bacterium within the phylum Deinococcus-Thermus. Purified BopA exhibited maximum activity at pH 7.5 with bilirubin as its substrate and was found to be active over a markedly broad pH range from 6 to 11. It also showed notable thermostability; its activity remained intact even after a heat treatment at 90°C for 60 min. This enzyme is a thermostable-bilirubin oxidase that exhibits markedly higher thermostability than that previously reported for laccases.

Members of the Marseilleviridae family are large DNA viruses with icosahedral particle structures that infect Acanthamoeba cells. The first Marseillevirus to be discovered was isolated in 2009. Since then, several other members of the Marseilleviridae family have been reported, including Lausannevirus, Senegalvirus, Cannes 8 virus, Insectomime virus, Tunisvirus, Melbournevirus, Port-Miou virus, and Brazilian Marseillevirus, which have been isolated from Europe, Africa, Australia, and South America. The morphological and genomic properties of a new Marseilleviridae family member, Tokyovirus, discovered in a water/soil sample from a Japanese river in Tokyo, were described in the present study. Tokyovirus possesses icosahedral particles of up to 200 nm in diameter, as revealed by a transmission electron microscopy (TEM) analysis, which form a giant virion factory in Acanthamoeba cells. A preliminary genome analysis predicted 487 coding sequences. A dot plot analysis and phylogenetic analysis using family B DNA polymerase, proliferating cell nuclear antigen (PCNA), and DNA-directed RNA polymerase alpha subunit genes revealed that Tokyovirus shares similarities with Marseillevirus, Melbournevirus, and Cannes 8 virus (Marseilleviridae subclade A), but not with Lausannevirus and Port-Miou virus (subclade B), Tunisvirus and Insectomime virus (subclade C), or Brazilian Marseillevirus (subclade D), suggesting that Tokyovirus has evolved separately from the previously described Marseilleviridae members.

The oxidation ditch process is one of the most economical approaches currently used to simultaneously remove organic carbon, nitrogen, and also phosphorus (P) from wastewater. However, limited information is available on biological P removal in this process. In the present study, microorganisms contributing to P removal in a full-scale oxidation ditch reactor were investigated using culture-dependent and -independent approaches. A microbial community analysis based on 16S rRNA gene sequencing revealed that a phylotype closely related to Dechloromonas spp. in the family Rhodocyclaceae dominated in the oxidation ditch reactor. This dominant Dechloromonas sp. was successfully isolated and subjected to fluorescent staining for polyphosphate, followed by microscopic observations and a spectrofluorometric analysis, which clearly demonstrated that the Dechloromonas isolate exhibited a strong ability to accumulate polyphosphate within its cells. These results indicate the potential key role of Dechloromonas spp. in efficient P removal in the oxidation ditch wastewater treatment process.

Standard cultivation fails to grow most microorganisms, whereas in situ cultivation allows for the isolation of comparatively diverse and novel microorganisms. Information on similarities and differences in the physiological properties of isolates obtained from in situ cultivation and standard cultivation is limited. Therefore, we used the arctic sediment samples and compared two culture collections obtained using standard and novel cultivation techniques. Even though there was no temperature selection at the isolation step, isolates from each method showed different reactions to temperature. The results of the present study suggest that isolates from in situ cultivation are more competitive in their natural environment.