TonB-dependent receptors (TBDRs) allow Gram-negative bacteria to uptake scarce resources from competitive environments with very high affinity. Early reports on TBDRs focused on the uptake of siderophore-iron complexes but recent studies have showed that the spectrum of ligands includes sugars, vitamins, heme, and other non-ferrous cations. To investigate the possible roles of TBDRs in nitrogen-fixing, nodulating bacteria, a bioinformatics approach was adopted to identify their presence in the genome of 13 selected rhizobacteria. The number of TBDR-like genes ranged from 1 (Mesorhizobium loti MAFF303099) to 14 (Azorhizobium caulinodans ORS 571 and Methylobacterium nodulans ORS 2060). These TBDRs can be largely grouped into two clusters, the ‘heme’ cluster and the ‘iron-siderophores’ cluster. The only exceptions are a putative nickel-specific TBDR (bll6948) in Bradyrhizobium japonicum USDA110 and two putative sugar-specific TBDRs in B. japonicum USDA110 and A. caulinodans ORS571 genomes, respectively. No TBDR-like sequences lie in the ‘Vitamin B12’ or ‘Non-Fe cations’ clusters. A model of the biological roles of TBDRs in free-living and symbiotic states is proposed for B. japonicum.
Staphylococcus aureus, a major opportunistic pathogen responsible for a broad spectrum of infections, naturally inhabits the human nasal cavity in about 30% of the population. The unique adaptive potential displayed by S. aureus has made it one of the major causes of nosocomial infections today, emphasized by the rapid emergence of multiple antibiotic-resistant strains over the past few decades. The uncanny ability to adapt to harsh environments is essential for staphylococcal persistence in infections or as a commensal, and a growing body of evidence has revealed critical roles in this process for cellular structural dynamics, and population heterogeneity. These two exciting areas of research are now being explored to identify new molecular mechanisms governing these adaptational strategies.
The effect of four amino acids, unutilizable by Ralstonia solanacearum, on tomato bacterial wilt (TBW) was tested in three Japanese soils. Results confirmed our previous findings that the addition (2.5 mg g−1 soil) of lysine and serine suppressed TBW, but that of tyrosine and valine did not. The number of the pathogen in non-rhizosphere soil, rhizosphere soil and the rhizoplane was markedly lower in the lysine and serine treatments than in the tyrosine and valine treatments, while the opposite result was obtained for the total bacterial population. Substrate-induced respiration analysis revealed that bacteria became more predominant in the amino acid treatments, especially in lysine. There were no apparent correlations between the microbial activities in soil and the disease index. PCR-DGGE targeting the 16S rRNA gene of the rhizoplane samples from lysine-added Nagoya, Fuchu and Iwate soils revealed 10, 1 and 2 unique bands, respectively, indicating shifts in bacterial community. DGGE patterns in the suppressive treatments belonged to the same cluster while the not suppressive treatments clustered differently. The lower disease incidence of TBW following lysine treatment is likely related to a specific bacterial community in the rhizoplane that developed on the addition of lysine.
Tetraheme cytochrome c-554 is a physiological electron acceptor of hydroxylamine oxidoreductase (HAO), a core enzyme of ammonia oxidation in chemoautotrophic nitrifiers. Here we report the purification of cytochrome c-554 from Nitrosococcus oceani strain NS58, a marine gammaproteobacterial ammonia-oxidizing bacterium. The NS58 cytochrome is a 25 kDa-protein having four hemes c. The absorption spectrum of the cytochrome showed peaks at 420 nm, 523 nm, and 554 nm, with shoulders at around 430 nm and 580 nm in the reduced state. In contrast to the highly basic counterpart from the betaproteobacterium Nitrosomonas europaea, the NS58 cytochrome c-554 was an acidic protein whose isoelectric point was 4.6. HAO was also purified, and the reaction with the NS58 cytochrome was found to be salt-tolerant. Compared with the activity observed in a non-salt solution, 60% of the activity remained in a saline concentration comparable to that of seawater.
A newly-developed molecular method, hierarchical oligonucleotide primer extension (HOPE), was used to analyze various groups within the species Cylindrospermopsis raciborskii. PCR-amplified internally transcribed spacer sequences of 16S-23S from C. raciborskii in reservoir samples of Taiwan and Kinmen were examined. One of eight sequevars in the clone libraries was closely related to strains obtained from the European continent, while the others, designated of Taiwan (TW) type, belonged to a novel group. Optimized HOPE analyses showed that C. raciborskii distributed in different reservoirs with a relative abundance of 0.5% to 76.4% in the cyanobacterial communities. They further detected the concurrence of three C. raciborskii subpopulations, in which European and TW groups were predominant. The TW sequevars accounted for greater than 87.5% of C. raciborskii in the reservoirs Taihu, Yangmin, Jinsha, and Mudan, while this decreased to 55.4-58.1%, accompanied by a proportional increase of the European group, in reservoirs Lantan and Renyi. These findings revealed the complex subspecies structure within C. raciborskii and the subspecies dynamics associated with geographic locations.
The relationship between the activity and community structure of microbes associated with the oxidation of ammonia in a full-scale rockwool biofilter was examined by kinetic, denaturing gradient gel electrophoresis (DGGE), and sequence analyses. The packing materials were sampled from two different depths at 3 sites. Estimated Km values were similar among depths at same sampling sites, while Vmax differed in the mid-point sample. The lower depth of this site had the highest Vmax. A correspondence analysis showed the DGGE profile of ammonia-oxidizing bacterial amoA of the lower depth of the mid-point sample to be distinguishable from the others. Banding patterns at other sites were similar among depths. Banding patterns of ammonia-oxidizing archaeal amoA of the mid-point sample were also similar among depths. The results suggested an association between the ammonia-oxidizing bacterial community’s composition and ammonium oxidation kinetics in samples. Sequence analysis indicated that the ammonia-oxidizing bacterial community mainly belonged to the Nitrosomonas europaea lineage and Nitrosospira cluster 3. The ammonia-oxidizing archaeal amoA-like sequences were related to those belonging to soil and sediment groups, including one with 84% nucleotide similarity with Nitrosopumilus maritimus.
Many Gram-negative bacteria naturally produce membrane vesicles (MVs) to the extracellular milieu. The Pseudomonas quinolone signal (PQS), a quorum-sensing signal of Pseudomonas aeruginosa, is a positive regulator of MV production. In this study, we investigated its effects on MV production in other Gram-negative and -positive bacterial species. The addition of PQS to an Escherichia coli K12 culture resulted in increased MV production and enlarged MVs. An excessive amount of MgCl2 repressed E. coli MV production either with or without PQS, suggesting that an anionic repulsion of cellular surfaces increases MV production. PQS was found in the cellular membrane and MVs in E. coli. The enhancement of MV production by PQS occurred in other Gram-negative bacteria, including Burkholderia and Pseudomonas species. Moreover, PQS induced MV production in a Gram-positive bacterium, Bacillus subtilis 168, which does not normally produce MV under laboratory conditions. An excessive amount of MgCl2 did not repress B. subtilis MV production in the presence of PQS, suggesting the production mechanism to be different from that in Gram-negative bacteria. Together, these results indicated that PQS enhances MV production in Gram-negative bacteria and induces it in Gram-positive bacteria.
A transect from the Tomoe River Mouth through Shimizu Port to Suruga Bay, Japan, was examined between 2005 and 2009 to reveal the population dynamics of Crenarchaeota and Euryarchaeota in an estuary environment. Crenarchaeota tended to increase in abundance in waters deeper than 100 m compared with Euryarchaeota, and comprised 11% of total direct counts. Archaeal abundance was highest in the Tomoe River Mouth, with a strong negative correlation between surface euryarchaeal abundance and salinity (P<0.001). The diversity index for the phylotypic archaeal community in the mouth was three times higher than that at sites St1-1m and St1-10m in the estuary, and OTUs represented most of the OTU groups at the sites. Three of the seven total OTUs, which comprised 83.6% of the 140 sequenced clones in the estuary, were related to the OTUs in the mouth with similarities higher than 97%. A significant proportion of the archaeal community appears to be derived from the Tomoe River. The two dominant phylotypes of the archaeal community in Shimizu Port, belonging to MGI and MGII, occurred ubiquitously.
To isolate a biocatalytic enzyme, metagenomic libraries were constructed in fosmids from samples of activated sludge used to treat coke plant wastewater. Six indigo-producing clones were isolated from approximately 40,000 metagenomic clones in the search for the oxygenase responsible. In vitro mutagenesis and whole-sequencing revealed one open reading frame to be responsible for the production of indigo in the fosmid clones. The deduced sequence of the gene product showed 60% identity with 2-naphthoate monooxygenase from Burkholderia sp. JT1500. Subclones carrying this open reading frame (icpA) retained indigo production, and indigo-producing enzymes expressed from subclones catalyzed the oxidization of 4-nitrotoluene to form 4-nitrobenzyl alcohol. These results suggested that the icp product is an enzyme involved in catalyzing 4-nitrotoluene’s oxygenation.
Sulfur-turf microbial mats develop in sulfide-containing hot spring water dominated by chemolithoautotrophic sulfur-oxidizing bacteria. The sulfur-turf mat that developed at a source of hot water (72°C, pH 6.8) exhibited a growth rate of 0.48±0.04 h−1 and biomass production of 4.6±1.0 mg of C h−1. On a per-cell basis, this biomass production was at least an order of magnitude higher than the CO2 uptake rate calculated for a photosynthetic mat dominated by thermophilic Synechococcus spp. at 70°C. The sulfur-turf-associated microbial community likely contributes to carbon fixation and primary production in this geothermal habitat.