Ribosomal RNA (rRNA) sequence-based molecular techniques emerged in the late 1980s, which completely changed our general view of microbial life. Coincidentally, the Japanese Society of Microbial Ecology (JSME) was founded, and its official journal “Microbes and Environments (M&E)” was launched, in 1985. Thus, the past 25 years have been an exciting and fruitful period for M&E readers and microbiologists as demonstrated by the numerous excellent papers published in M&E. In this minireview, recent progress made in microbial ecology and related fields is summarized, with a special emphasis on 8 landmark areas; the cultivation of uncultured microbes, in situ methods for the assessment of microorganisms and their activities, biofilms, plant microbiology, chemolithotrophic bacteria in early volcanic environments, symbionts of animals and their ecology, wastewater treatment microbiology, and the biodegradation of hazardous organic compounds.

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The actinorhizal symbiosis is a major contributor to the global nitrogen budget, playing a dominant role in ecological successions following disturbances. The mechanisms involved are still poorly known but there emerges the vision that on the plant side, the kinases that transmit the symbiotic signal are conserved with those involved in the transmission of the Rhizobium Nod signal in legumes. However, on the microbial side, complementation with Frankia DNA of Rhizobium nod mutants failed to permit identification of symbiotic genes. Furthermore, analysis of three Frankia genomes failed to permit identification of canonical nod genes and revealed symbiosis-associated genes such as nif, hup, suf and shc to be spread around the genomes. The present review explores some recently published approaches aimed at identifying bacterial symbiotic determinants.

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Over the past few decades, degradative plasmids have been isolated from bacteria capable of degrading a variety of both natural and man-made compounds. Degradative plasmids belonging to three incompatibility (Inc) groups in Pseudomonas (IncP-1, P-7, and P-9) have been well studied in terms of their replication, maintenance, and capacity for conjugative transfer. The host ranges of these plasmids are determined by replication or conjugative transfer systems. The host range of IncP-1 is broad, that of IncP-9 is intermediate, and that of IncP-7 is narrow. To understand the behavior of these plasmids and their hosts in various environments, the survivability of inocula, stability or transferability, and efficiency of biodegradation in environments and microcosms have been monitored. The biodegradation and plasmid transfer in various environments have been observed for all three groups, although the kinds of transconjugants differed with the Inc groups. In some cases, the deletion and amplification of catabolic genes acted to reduce the production of toxic catabolic intermediates, or to increase the activity on a particular catabolic pathway. The combination of degradative genes, the plasmid backbone of each Inc group, and the host of the plasmids is key to the degraders adapting to various hosts or to heterogeneous environments.

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Genetic typing of Pseudomonas aeruginosa isolated from the open ocean has revealed that marine strains form unique clusters. To clarify whether this genetic variation reflects differences in pattern of culturability and survival, a marine strain was compared with a freshwater strain and a clinical strain in microcosms with different levels of NaCl (0 to 7% [w/v]), pH (4.0 to 9.0) and temperature (−20, 0, 4, 25 and 37°C) in both artificial seawater (ASW) and distilled water (DW). The viable but non-culturable (VBNC) state of P. aeruginosa was also monitored. The marine strain 1200 grew better at high NaCl and pH, whereas the freshwater strain 1030 did better at 0 to 3% NaCl and a pH of less than 7.0. The clinical strain 1564 grew best at neutral pH and 0% NaCl. No significant differences were observed among the strains in culturability at different temperatures. Like other bacteria, P. aeruginosa enters a VBNC state under stressful conditions. The marine P. aeruginosa isolate exhibits a unique pattern of culturability and survival which demonstrates a physiological adaptation to the ocean environment.

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Sinorhizobium sp. BL3 is a salt-tolerant strain that can fix atmospheric nitrogen in symbiosis with leguminous host plants under salt-stress conditions. Since cell membranes are the first barrier to environmental change, it is interesting to explore the membrane proteins within this protective barrier under salt stress. The protein contents of membrane-enriched fractions obtained from BL3 were analyzed by nanoflow liquid chromatography interfaced with electrospray ionization tandem mass spectrometry. A total of 105 membrane proteins were identified. These proteins could be classified into 17 functional categories, the two biggest of which were energy production and conversion, and proteins not in clusters of orthologous groups (COGs). In addition, a comparative analysis of membrane proteins between salt-stressed and non-stressed BL3 cells was conducted using a membrane enrichment method and off-line SCX fractionation coupled to nanoLC-MS/MS. These techniques would be useful for further comparative analysis of membrane proteins that function in the response to environmental stress.

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We evaluated the fungal specificity and detection bias of four fungal 18S rRNA gene (18S rDNA) primer sets for denaturing-gradient gel electrophoresis (DGGE). We constructed and compared clone libraries amplified from upland and paddy field soils with each primer set (1, NS1/GCFung; 2, FF390/FR1-GC; 3, NS1/FR1-GC; and 4, NS1/EF3 for the first PCR and NS1/FR1-GC for the second PCR). Primer set 4 (for nested PCR) showed the highest specificity for fungi but biased specific sequences. Sets 1, 2, and 3 (for single PCR) amplified non-fungal eukaryotic sequences (from 7 to 16% for upland soil and from 20 to 31% for paddy field soil) and produced libraries with similar distributions of fungal 18S rDNA sequences at both the phylum and the class level. Set 2 tended to amplify more diverse fungal sequences, maintaining higher specificity for fungi. In addition, clone analyses revealed differences among primer sets in the frequency of chimeras. In upland field soil, the libraries amplified with primer sets 3 and 4, which targeted long fragments, contained many chimeric 18S rDNA sequences (18% and 48%, respectively), while the libraries obtained with sets 1 and 2, which targeted short fragments, contained fewer chimeras (5% and 10%, respectively).

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The galatheid crab, Shinkaia crosnieri (Decapoda: Galatheidae), forms dense colonies in the Iheya North and Hatoma Knoll deep-sea hydrothermal fields and has numerous setae covered with filamentous epibiotic microorganisms. Molecular phylogenetic analyses revealed that the epibiotic communities in S. crosnieri consisted mainly of yet-uncultivated phylotypes within Epsilonproteobacteria and Gammaproteobacteria in both hydrothermal vent fields. Uptake experiments using ¹³C-labeled tracers clearly demonstrated that both H¹³CO₃⁻ and ¹³CH₄ were assimilated into not only the epibiotic microbial communities associated with the setae, but also the epibiont-free tissue of living S. crosnieri. In addition, the incorporation of H¹³CO₃⁻ into the microbial cells was strongly stimulated by the presence of reduced sulfur compounds but not by H₂. In conclusion, the uptake experiments suggested that sulfur-oxidizing chemolithoautotrophic and methanotrophic production by the epibionts provides the nutrition for S. crosnieri.

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The cyanophage Ma-LMM01, specifically-infecting Microcystis aeruginosa, has an insertion sequence (IS) element that we named IS607-cp showing high nucleotide similarity to a counterpart in the genome of the cyanobacterium Cyanothece sp. We tested 21 strains of M. aeruginosa for the presence of IS607-cp using PCR and detected the element in strains NIES90, NIES112, NIES604, and RM6. Thermal asymmetric interlaced PCR (TAIL-PCR) revealed each of these strains has multiple copies of IS607-cp. Some of the ISs were classified into three types based on their inserted positions; IS607-cp-1 is common in strains NIES90, NIES112 and NIES604, whereas IS607-cp-2 and IS607-cp-3 are specific to strains NIES90 and RM6, respectively. This multiplicity may reflect the replicative transposition of IS607-cp. The sequence of IS607-cp in Ma-LMM01 showed robust affinity to those found in M. aeruginosa and Cyanothece spp. in a phylogenetic tree inferred from counterparts of various bacteria. This suggests the transfer of IS607-cp between the cyanobacterium and its cyanophage. We discuss the potential role of Ma-LMM01-related phages as donors of IS elements that may mediate the transfer of IS607-cp; and thereby partially contribute to the genome plasticity of M. aeruginosa.

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The microbial communities of the rhizoplane, the surface part of roots, in aquatic plants are not understood at all. In this study, we analyzed microbial communities in the rhizoplane of a floating aquatic plant, giant duckweed (Spirodela polyrrhiza), based on cultivation-dependent and independent analyses. The cultivation-based analysis using agar and gellan gum plates revealed that the rhizoplane isolates were affiliated with four bacterial lineages; the Alphaproteobacteria, Betaproteobacteria, Bacteroidetes and Verrucomicrobia. Interestingly, microbes belonging to the phylum Verrucomicrobia accounted for 24% of all the isolates, suggesting that the rhizoplane of S. polyrrhiza forms a specific habitat for the organisms within this phylum. Culture-independent 16S rRNA gene cloning showed that the clonal sequences were affiliated with eight bacterial classes and phyla: the classes Alphaproteobacteria (14% total clones), Betaproteobacteria (45%), Gammaproteobacteria (2%) and Deltaproteobacteria (2%), and the phyla Bacteroidetes (11%), Verrucomicrobia (2%), Planctomycetes (2%) and Cyanobacteria (22%). Comparative analysis of the microbial communities in the rhizoplane between culture-dependent and independent methods revealed that 33% of the taxonomic groups of bacterial species detected in the molecular analysis were cultivable. Our findings suggest that the microbes in the rhizoplane of giant duckweed are comprised of a diverse array of readily cultured organisms including a variety of strains within the Verrucomicrobia, a well-known phylum that contains a number of yet-to-be cultivated organisms.

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To elucidate how temperature affects bradyrhizobial ecology, long-term incubations of Bradyrhizobium japonicum USDA 6^T, 38, and 123 and of Bradyrhizobium elkanii USDA 76^T were conducted under various temperature conditions. Proliferative traits in liquid culture and population occupancies in soil microcosms were compared. The occupancies of USDA 76^T and USDA 123 in soil microcosms during long-term incubation changed with the temperature conditions. These results suggest that temperature is an environmental factor affecting the ecology and occupancy of bradyrhizobia in soils.

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The nuclear ribosomal RNA gene operon is interrupted by internal transcribed spacer (ITS) 1 and ITS2. Although the secondary structure of ITS2 has been widely investigated, less is known about ITS1 and its structure. In this study, the secondary structure of ITS1 sequences for Paramecium and other ciliates was predicted. Each Paramecium ITS1 forms an open loop with three helices, A through C. Helix B was highly conserved among Paramecium, and similar helices were found in other ciliates. A phylogenetic analysis using the ITS1 sequences showed high-resolution, implying that ITS1 is a good tool for species-level analyses.

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We focused on bacterial interspecies relationships at the air-liquid interface where the formation of pellicles by aerobes was observed. Although an obligate aerobe (Brevibacillus sp. M1-5) was initially dominant in the pellicle population, a facultative aerobe (Pseudoxanthomonas sp. M1-3) emerged and the viability of M1-5 rapidly decreased due to severe competition for oxygen. Supplementation of the medium with carbohydrates allowed the two species to coexist at the air-liquid interface. These results indicate that the population dynamics within pellicles are primarily governed by oxygen utilization which was affected by a combination of carbon sources.

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Microscopic and molecular analyses showed the presence of endobacteria inside the mycelia of four out of twelve nitrous oxide (N₂O)-producing fungal isolates identified as Mortierella elongata. The 16S rRNA gene was successfully amplified with DNA extracted directly from the endobacterium-containing fungal strains and all sequences were related to that of Candidatus Glomeribacter gigasporarum in the family Burkholderiaceae. Bacterial endotoxin was detected in the endobacterium-positive fungal strains but only trace levels were found in endobacterium-negative strains. No significant relationship was found between the fungal N₂O-producing activity and the presence of endobacteria.

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Volume 24, no. 2, Page 105-112, 2009.
In Table3, TSC1 isolate was shown nodulating the four cowpea cultivars with an acetylene-reducing activity. However, by further purification of the TSC1 culture on AIE agar plates, it is likely that it was contaminated with compatible bradyrhizobia which certainly had caused the nodulation when TSC1 was inoculated to cowpea. In addition, repetitive inoculations tests of this purified isolate (TSC1) to cowpea have indicated that Ralstonia sp. TSC1 has no nodulation and no nitrogen fixation capability. As a result, we decide to delete the previous Table 3, and edited the text as follows:
Page 110, line 13 to 19 of left column (Cross-inoculation with TSC1........Dan IIa, Tv-7778): Cross-inoculation with TSC1 (data not shown) indicated that this strain didn’t react with any of the four host cultivars and had no nodulation and no nitrogen fixation capability.
Page 111, line 17 to 19 of left column (Further mbrolecular analysis........symbiotic characteristic): Further analysis should be carried out on the novel species isolated from cowpea (Ralstonia sp. TSC1) to better understand its features.
The paper has scientific significance in that it provides valuable information on the diversity and phylogeny of cowpea nodulating-bacteria in the West-South area of Japan. Furthermore, among the isolates described in the paper, strains with high nitrogen-fixing potential were identified.

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