Ultra-small microorganisms are ubiquitous in Earth’s environments. Ultramicrobacteria, which are defined as having a cell volume of <0.1 μm³, are often numerically dominant in aqueous environments. Cultivated representatives among these bacteria, such as members of the marine SAR11 clade (e.g., “Candidatus Pelagibacter ubique”) and freshwater Actinobacteria and Betaproteobacteria, possess highly streamlined, small genomes and unique ecophysiological traits. Many ultramicrobacteria may pass through a 0.2-μm-pore-sized filter, which is commonly used for filter sterilization in various fields and processes. Cultivation efforts focusing on filterable small microorganisms revealed that filtered fractions contained not only ultramicrocells (i.e., miniaturized cells because of external factors) and ultramicrobacteria, but also slender filamentous bacteria sometimes with pleomorphic cells, including a special reference to members of Oligoflexia, the eighth class of the phylum Proteobacteria. Furthermore, the advent of culture-independent “omics” approaches to filterable microorganisms yielded the existence of candidate phyla radiation (CPR) bacteria (also referred to as “Ca. Patescibacteria”) and ultra-small members of DPANN (an acronym of the names of the first phyla included in this superphyla) archaea. Notably, certain groups in CPR and DPANN are predicted to have minimal or few biosynthetic capacities, as reflected by their extremely small genome sizes, or possess no known function. Therefore, filtered fractions contain a greater variety and complexity of microorganisms than previously expected. This review summarizes the broad diversity of overlooked filterable agents remaining in “sterile” (<0.2-μm filtered) environmental samples.

Filamentous anoxygenic photosynthetic bacteria grow by photosynthesis and aerobic respiration. The present study investigated the effects of light and O₂ on bacteriochlorophyll contents and the transcription levels of photosynthesis-related genes in Chloroflexus aurantiacus J-10-fl ^T. Under aerobic conditions, C. aurantiacus produced marked amounts of bacteriochlorophylls in the presence of light, although their production was strongly suppressed in the dark. The transcription levels of genes related to the synthesis of bacteriochlorophylls, photosystems, and chlorosomes: bchM, bchU, pufL, pufBA, and csmM, were markedly increased by illumination. These results suggest that C. aurantiacus continuously synthesizes ATP by photophosphorylation even in the presence of O₂.

Carbonyl sulfide (COS) is the most abundant sulfur compound in the atmosphere, and, thus, is important in the global sulfur cycle. Soil is a major sink of atmospheric COS and the numerical distribution of soil microorganisms that degrade COS is indispensable for estimating the COS-degrading potential of soil. However, difficulties are associated with counting COS-degrading microorganisms using culture-dependent approaches, such as the most probable number (MPN) method, because of the chemical hydrolysis of COS by water. We herein developed a two-step MPN method for COS-degrading microorganisms: the first step for chemoorganotrophic growth that supported a sufficient number of cells for COS degradation in the second step. Our new MPN analysis of various environmental samples revealed that the cell density of COS-degrading microorganisms in forest soils ranged between 10⁶ and 10⁸ MPN (g dry soil)^–1, which was markedly higher than those in volcanic deposit and water samples, and strongly correlated with the rate of COS degradation in environmental samples. Numerically dominant COS degraders that were isolated from the MPN-positive culture were related to bacteria in the orders Bacillales and Actinomycetales. The present results provide numerical evidence for the ubiquity of COS-degrading microbes in natural environments.

Interspecies interactions among oral microorganisms in the pathogenic biofilms causing dental caries have not yet been elucidated in detail. We herein demonstrated that indole and its derivatives induced biofilm formation by Streptococcus mutans. Indole is an intercellular signaling molecule that is produced by oral bacteria other than S. mutans. The amounts of biofilm and extracellular DNA were significantly increased by the addition of indole and 4-hydroxyindole (4-HI). An examination with quorum sensing mutants showed that the induction of biofilm formation by indole and 4-HI required a quorum sensing system. These results suggest that this intercellular signaling molecule plays a role in pathogenic biofilm formation.

Bacterial endosymbionts inhabit diverse fungal lineages. Although the number of studies on bacteria is increasing, the mechanisms by which bacteria affect their fungal hosts remain unclear. We herein examined the homothallic isolate, Mortierella sugadairana YTM39, harboring a Burkholderiaceae-related endobacterium, which did not produce sexual spores. We successfully eliminated the bacterium from fungal isolates using ciprofloxacin treatment and asexual spore isolation for germinated asexual spores. Sexual spore formation by the fungus was restored by eliminating the bacterium from isolates. These results indicate that sexual reproduction by the fungus was inhibited by the bacterium. This is the first study on the sexual spore infertility of fungal hosts by endofungal bacteria.

Atrazine is a triazine herbicide that is widely used to control broadleaf weeds. Its widespread use over the last 50 years has led to the potential contamination of soils, groundwater, rivers, and lakes. Its main route of complete degradation is via biological means, which is carried out by soil microbiota using a 6-step pathway. The aim of the present study was to investigate whether application of atrazine to soil changes the soil bacterial community. We used 16S rRNA gene sequencing and qPCR to elucidate the microbial community structure and assess the abundance of the atrazine degradation genes atzA, atzD, and trzN in a Brazilian soil. The results obtained showed that the relative abundance of atzA and trzN, encoding triazine-initiating metabolism in Gram-negative and -positive bacteria, respectively, increased in soil during the first weeks following the application of atrazine. In contrast, the abundance of atzD, encoding cyanuric acid amidohydrolase—the fourth step in the pathway—was not related to the atrazine treatment. Moreover, the overall soil bacterial community showed no significant changes after the application of atrazine. Despite this, we observed increases in the relative abundance of bacterial families in the 4^th and 8^th weeks following the atrazine treatment, which may have been related to higher copy numbers of atzA and trzN, in part due to the release of nitrogen from the herbicide. The present results revealed that while the application of atrazine may temporarily increase the quantities of the atzA and trzN genes in a Brazilian Red Latosol soil, it does not lead to significant and long-term changes in the bacterial community structure.

More than 3,000 isolates of fluorescent pseudomonads have been collected from plant roots in Japan and screened for the presence of antibiotic-synthesizing genes. In total, 927 hydrogen cyanide (HCN)-, 47 2,4-diacetylphloroglucinol (PHL)-, 6 pyoluteorin (PLT)-, 14 pyrrolnitrin (PRN)-, and 8 phenazine (PHZ)-producing isolates have been detected. A cluster analysis (≥99% identity) identified 10 operational taxonomic units (OTUs) in antibiotic biosynthesis gene-possessing pseudomonads. OTU HLR (PHL, PLT, and PRN) contained four antibiotics: HCN, PHL, PLT, and PRN, while OTU RZ (PRN and PHZ) contained three: HCN, PRN, and PHZ. OTU H1, H2, H3, H4, H5, H6, and H7 (PHL1-7) contained two antibiotics: HCN and PHL, while OTU H8 (PHL8) contained one: PHL. Isolates belonging to OTU HLR and RZ suppressed damping-off disease in cabbage seedlings caused by Rhizoctonia solani. Effective strains belonging to OTU HLR and RZ were related to Pseudomonas protegens and Pseudomonas chlororaphis, respectively. Antibiotic biosynthesis gene-possessing fluorescent pseudomonads are distributed among different geographical sites in Japan and plant species.

The nitrogen-fixing actinobacterium Frankia develops unique multicellular structures called vesicles, which are the site of nitrogen fixation. These vesicles are surrounded by a thick hopanoid lipid envelope that protects nitrogenase against oxygen inactivation. The phenotypes of five mutants that form smaller numbers of vesicles were investigated. The vesicles of these mutants were smaller than those of the wild type and had a phase dark appearance. They induced the expression of a glutamine synthetase gene in hyphae cells in response to ammonium starvation. These results suggest that genes impaired in the mutants do not function in global nitrogen regulation, but specifically function in vesicle differentiation.

Biofilm carriers have been used to remove ammonia in several wastewater treatment plants (WWTPs) in Japan. However, the abundance and species of ammonia oxidizers in the biofilms formed on the surface of carriers in full-scale operational WWTP tanks remain unclear. In the present study, we conducted quantitative PCR and PCR cloning of the amoA genes of ammonia-oxidizing bacteria and archaea (AOB and AOA) and a complete ammonia oxidizer (comammox) in the biofilm formed on the carriers in a full-scale WWTP. The quantification of amoA genes showed that the abundance of AOB and comammox was markedly greater in the biofilm than in the activated sludge suspended in a tank solution of the WWTP, while AOA was not detected in the biofilm or the activated sludge. A phylogenetic analysis of amoA genes revealed that as-yet-uncultivated comammox Nitrospira and uncultured AOB Nitrosomonas were predominant in the biofilm. The present results suggest that the biofilm formed on the surface of carriers enable comammox Nitrospira and AOB Nitrosomonas to co-exist and remain in the full-scale WWTP tank surveyed in this study.

Bradyrhizobium elkanii BLY3-8 does not form nodules on the roots of Rj₃-genotype soybean (cultivar D-51). This is a cultivar-specific nodulation restriction. The genes A6X20_40975 and A6X20_41030 in strain BLY3-8 were predicted to encode the transcriptional activator and apparatus of the type III secretion system (T3SS) (the proteins TtsI and RhcJ), respectively. Mutants disrupted in these genes overcame the nodulation restriction. These results suggest that an effector injected via T3SS into Rj₃-genotype soybean is involved in nodulation restriction by Rj₃-genotype soybean.

Vibrio parahaemolyticus is the leading cause of bacteria-associated foodborne diarrheal diseases and specifically causes early mortality syndrome (EMS), which is technically known as acute hepatopancreatic necrosis disease (AHPND), a serious threat to shrimp aquaculture. To investigate the genetic and evolutionary relationships of V. parahaemolyticus in China, 184 isolates from clinical samples (VPC, n=40), AHPND-infected shrimp (VPE, n=10), and various aquatic production sources (VPF, n=134) were collected and evaluated by a multilocus sequence analysis (MLST). Furthermore, the presence of potential virulence factors (tlh, tdh, and trh) and single nucleotide polymorphisms (SNPs) in V. parahaemolyticus isolates was assessed using genomic sequencing. Analyses of virulence factors revealed that the majority of VPC isolates (97.5%) possessed the tdh and/or trh genes, while most of the VPF isolates (83.58%) did not encode hemolysin genes. Therefore, we hypothesized that the environment is a potential reservoir that promotes horizontal DNA transfer, which drives evolutionary change that, in turn, leads to the emergence of novel, potentially pathogenic strains. Phylogenetic analyses identified VPF-112 as a non-pathogenic maternal strain isolated from aquatic products and showed that it had a relatively high evolutionary status. All VPE strains and some VPC strains were grouped into several small subgroups and evenly distributed on phylogenetic trees. Anthropogenic activities and environmental selective pressure may be important factors influencing the process of transforming strains from non-pathogenic to pathogenic bacteria.

To identify exoelectrogens involved in the generation of electricity from complex organic matter in coastal sediment (CS) microbial fuel cells (MFCs), MFCs were inoculated with CS obtained from tidal flats and estuaries in the Tokyo bay and supplemented with starch, peptone, and fish extract as substrates. Power output was dependent on the CS used as inocula and ranged between 100 and 600 mW m^–2 (based on the projected area of the anode). Analyses of anode microbiomes using 16S rRNA gene amplicons revealed that the read abundance of some bacteria, including those related to Shewanella algae, positively correlated with power outputs from MFCs. Some fermentative bacteria were also detected as major populations in anode microbiomes. A bacterial strain related to S. algae was isolated from MFC using an electrode plate-culture device, and pure-culture experiments demonstrated that this strain exhibited the ability to generate electricity from organic acids, including acetate. These results suggest that acetate-oxidizing S. algae relatives generate electricity from fermentation products in CS-MFCs that decompose complex organic matter.

Plant growth-promoting bacteria (PGPB) are beneficial microbes that increase plant growth and yield. However, limited information is currently available on PGPB in onion (Allium cepa Linn.). The aims of the present study were to isolate and identify PGPB in onion and examine the effects of isolated PGPB on germination and growth during the vegetative stage in onion, pak choy (Brassica chinensis), and sweet pepper (Capsicum annuum). Twenty-three strains of PGPB were isolated from the roots, bulbs, and rhizosphere soil of onion. All isolated bacterial strains showed one or more PGP traits, including indole acetic acid production, phosphate solubilization ability, and 1-aminocyclopropane-1-carboxylate deaminase and nitrogenase activities; most of these traits were derived from Bacillus sp., Microbacterium sp., and Pseudomonas sp. Eight bacteria that exhibited strong abilities to produce indole acetic acid were selected for a Petri dish trial, soil pot test, and vermiculate pot test. The Petri dish trial showed that strains ORE8 and ORTB2 simultaneously increased radicle and hypocotyl lengths in onion, but inhibited growth in sweet pepper after 7 d. The soil pot experiment on onion revealed that strains ORE5, ORE8, and ORTB2 strongly promoted growth during the vegetative stage with only a half dose of chemical fertilizer. The present results indicate that ORE8 (Bacillus megaterium) and ORTB2 (Pantoea sp.) are the most promising biofertilizers of onion and may simultaneously inhibit the seedling growth of other plants.

Bacteria change their metabolic states to increase survival by forming aggregates. Ammonia-oxidizing bacteria also form aggregates in response to environmental stresses. Nitrosomonas mobilis, an ammonia-oxidizing bacterium with high stress tolerance, often forms aggregates mainly in wastewater treatment systems. Despite the high frequency of aggregate formation by N. mobilis, its relationship with survival currently remains unclear. In the present study, aggregates were formed in the late stage of culture with the accumulation of nitrite as a growth inhibitor. To clarify the significance of aggregate formation in N. mobilis Ms1, a transcriptome analysis was performed. Comparisons of the early and late stages of culture revealed that the expression of stress response genes (chaperones and proteases) increased in the early stage. Aggregate formation may lead to stress avoidance because stress response genes were not up-regulated in the late stage of culture during which aggregates formed. Furthermore, comparisons of free-living cells with aggregates in the early stage of culture showed differences in gene expression related to biosynthesis (ATP synthase and ribosomal proteins) and motility and adhesion (flagella, pilus, and chemotaxis). Biosynthesis genes for growth were up-regulated in free-living cells, while motility and adhesion genes for adaptation were up-regulated in aggregates. These results indicate that N. mobilis Ms1 cells adapt to an unfavorable environment and grow through the division of labor between aggregates and free-living cells.

Invertebrates are a source of previously unknown RNA viruses that fill gaps in the viral phylogenetic tree. Although limited information is currently available on RNA viral diversity in the marine sponge, a primordial multicellular animal that belongs to the phylum Porifera, the marine sponge is one of the well-studied holobiont systems. In the present study, we elucidated the putative complete genome sequences of five novel RNA viruses from Hymeniacidon sponge using a combination of double-stranded RNA sequencing, called fragmented and primer ligated dsRNA sequencing, and a conventional transcriptome method targeting single-stranded RNA. We identified highly diverged RNA-dependent RNA polymerase sequences, including a potential novel RNA viral lineage, in the sponge and three viruses presumed to infect sponge cells.