Development and characterization of microbial biosensors for evaluating low biochemical oxygen demand in rivers.

Five microorganisms were used to construct a biosensor for the evaluation of low biochemical oxygen demand (BOD) in rivers. Characterization and comparison of BOD biosensors were performed using two standard solutions: glucose and glutamic acid (GGA) and artificial wastewater (AWW). Pseudomonas putida SG10 demonstrated the best response when using AWW. Trichosporon cutaneum IFO10466, however, had an extremely poor response. When evaluating the biosensor response to each component of AWW, all of the microorganisms except T. cutaneum displayed the highest response to tannic acid. In a comparison of the two standard solutions for all the microorganisms, the biosensor responses of GGA were approximately three times higher than those of AWW were. In the BOD determination of environmental samples, the biosensor BOD values evaluated using AWW were slightly lower or equivalent to BOD5 values, whereas the biosensor BOD values evaluated using GGA were considerably lower. These results suggest that GGA is suitable for the detection of high BOD in industrial wastewaters and factory effluents, while AWW is suitable for the detection of low BOD in rivers.

A deeply branching thermophilic bacterium with an ancient acetyl-CoA pathway dominates a subsurface ecosystem.

A nearly complete genome sequence of Candidatus 'Acetothermum autotrophicum', a presently uncultivated bacterium in candidate division OP1, was revealed by metagenomic analysis of a subsurface thermophilic microbial mat community. Phylogenetic analysis based on the concatenated sequences of proteins common among 367 prokaryotes suggests that Ca. 'A. autotrophicum' is one of the earliest diverging bacterial lineages. It possesses a folate-dependent Wood-Ljungdahl (acetyl-CoA) pathway of CO(2) fixation, is predicted to have an acetogenic lifestyle, and possesses the newly discovered archaeal-autotrophic type of bifunctional fructose 1,6-bisphosphate aldolase/phosphatase. A phylogenetic analysis of the core gene cluster of the acethyl-CoA pathway, shared by acetogens, methanogens, some sulfur- and iron-reducers and dechlorinators, supports the hypothesis that the core gene cluster of Ca. 'A. autotrophicum' is a particularly ancient bacterial pathway. The habitat, physiology and phylogenetic position of Ca. 'A. autotrophicum' support the view that the first bacterial and archaeal lineages were H(2)-dependent acetogens and methanogenes living in hydrothermal environments.

Biodegradation analyses of trichloroethylene (TCE) by bacteria and its use for biosensing of TCE.

Trichloroethylene (TCE) is a toxic, recalcitrant groundwater pollutant. TCE-degrading microorganisms were isolated from various environments. The aerobic bacteria isolated from toluene- and tryptophan-containing media were Pseudomonas sp. strain ASA86 and Burkholderia sp. strain TAM17, respectively; these are necessary for inducing TCE biodegradation in a selective medium. The half-degradation time of TCE to a concentration of 1mg/L was 18 h for strain ASA86 and 7 days for strain TAM17. While identifying toluene/TCE degradation genes, we found that in strain ASA86, the gene was the same as the todC1 gene product encoding toluene dioxygenase identified in Pseudomonas putida F1, and that in strain TAM17, the gene was similar to the tecA1 gene product encoding chlorobenzene dioxygenase identified in Burkholderia sp. PS12. A novel TCE biosensor was developed using strain ASA86 as the inducer of toluene under aerobic conditions. The TCE biosensor exhibited a linear relationship below 3 ppm TCE. Detection limit of the biosensor was 0.05 ppm TCE. The response time of the biosensor was less than 10 min. The biosensor response displayed a constant level during a 2 day period. The TCE biosensor displayed sufficient sensitivity for monitoring TCE in environmental systems.

Alternative splicing by participation of the group II intron ORF in extremely halotolerant and alkaliphilic Oceanobacillus iheyensis.

Group II introns inserted into genes often undergo splicing at unexpected sites, and participate in the transcription of host genes. We identified five copies of a group II intron, designated Oi.Int, in the genome of an extremely halotolerant and alkaliphilic bacillus, Oceanobacillus iheyensis. The Oi.Int4 differs from the Oi.Int3 at four bases. The ligated exons of the Oi.Int4 could not be detected by RT-PCR assays in vivo or in vitro although group II introns can generally self-splice in vitro without the involvement of an intron-encoded open reading frame (ORF). In the Oi.Int4 mutants with base substitutions within the ORF, ligated exons were detected by in vitro self-splicing. It was clear that the ligation of exons during splicing is affected by the sequence of the intron-encoded ORF since the splice sites corresponded to the joining sites of the intron. In addition, the mutant introns showed unexpected multiple products with alternative 5' splice sites. These findings imply that alternative 5' splicing which causes a functional change of ligated exons presumably has influenced past adaptations of O. iheyensis to various environmental changes.

Insights into the evolution of Archaea and eukaryotic protein modifier systems revealed by the genome of a novel archaeal group.

The domain Archaea has historically been divided into two phyla, the Crenarchaeota and Euryarchaeota. Although regarded as members of the Crenarchaeota based on small subunit rRNA phylogeny, environmental genomics and efforts for cultivation have recently revealed two novel phyla/divisions in the Archaea; the 'Thaumarchaeota' and 'Korarchaeota'. Here, we show the genome sequence of Candidatus 'Caldiarchaeum subterraneum' that represents an uncultivated crenarchaeotic group. A composite genome was reconstructed from a metagenomic library previously prepared from a microbial mat at a geothermal water stream of a sub-surface gold mine. The genome was found to be clearly distinct from those of the known phyla/divisions, Crenarchaeota (hyperthermophiles), Euryarchaeota, Thaumarchaeota and Korarchaeota. The unique traits suggest that this crenarchaeotic group can be considered as a novel archaeal phylum/division. Moreover, C. subterraneum harbors an ubiquitin-like protein modifier system consisting of Ub, E1, E2 and small Zn RING finger family protein with structural motifs specific to eukaryotic system proteins, a system clearly distinct from the prokaryote-type system recently identified in Haloferax and Mycobacterium. The presence of such a eukaryote-type system is unprecedented in prokaryotes, and indicates that a prototype of the eukaryotic protein modifier system is present in the Archaea.

Stopped-flow system with ozonizer for the estimation of low biochemical oxygen demand in environmental samples.

The stopped-flow system with an ozonizer was developed to estimate low biochemical oxygen demand (BOD) in rivers. Rivers contain many biopersistent organic compounds such as humic acid, lignin, and gum arabic. Free radicals generated by self-decomposition of ozone were used as powerful oxidants to split organic compounds. Ozonysis of the samples was carried out by 42.4 gN(-1)m(-3) ozone for 3 min at pH 7.0. Artificial wastewater (AWW) solutions were employed as standard solutions for the calibrations of the BOD sensor. At a BOD of 1 mgl(-1), the sensor response after ozonation was 1.6-fold higher than that before ozonation. The response time of the BOD sensor was only 5min, being independent of the concentrations, and the lower detection limit was 0.5 mgl(-1) BOD. The degradations of lignin and tannic acid by ozonation were 54.1 and 42.3%, respectively. In the biosensor responses by ozonation, lignin, gum arabic, and surfactant increased by double or more compared with previous responses. BOD in rivers was estimated using the stopped-flow system. Environmental samples pretreated with ozone gave high responses to the biosensor that were similar to those of the conventional BOD(5) method. Accordingly, a good correlation between the sensor and the conventional BOD(5) was obtained (r=0.989). The system has to evolve the highly sensitive BOD determination.

Housekeeping recA gene interrupted by group II intron in the thermophilic Geobacillus kaustophilus.

Most of group II introns are found in intergenes and CDSs with unknown functions, but not in housekeeping genes. In particular, no group II intron within the housekeeping recA gene has been reported either in eukaryotic genomes or in prokaryotic genomes. In this study, we found that the recA gene of the thermophilic Geobacillus kaustophilus genome is interrupted by a group II intron (Gk. Int1), and that Gk.Int1 can splice in temperatures above 70 degrees C in vivo. Here, we report the first prokaryotic group II intron to be found in a housekeeping gene, the characteristics of its self-splicing in vivo and in vitro, and our conclusion that the recA gene functions through the self-splicing of Gk.Int1. It is suggested that the amelioration of Gk.Int1 intron has occurred recently, and that it is still in the process of evolution to the recipient genome.

Development of photocatalytic biosensor for the evaluation of biochemical oxygen demand.

The photocatalytic biosensor of flow system using semiconductor TiO2 was developed to evaluate biochemical oxygen demand (BOD) levels in river water. Photocatalysis of sample was carried out in a photoreactor with TiO2 and a 6W black-light blue fluorescent tube as light source. Sample from a photoreactor outlet was measured by an oxygen electrode with a biofilm. The sensor response of photocatalytic biosensor was between 5 and 10 min depending on concentration of biochemical in the samples. At BOD of 1 mgl-1, the sensor response increased 1.33-fold in comparison with that without photocatalysis. The degradation of tannic acid and humic acid with photocatalysis were 51.8 and 38.4%, respectively. Gum arabic and linear alkylbenzene sulfonate (LAS) were degraded a little, but gave the responses of more than double to the sensor. Free radicals yielded by photocatalysis in a photoreactor did not affect the sensor response because their lifetime is extremely short. Fairly good correlation (r=0.983) between the sensor method and the conventional method was obtained for test samples. This biosensor using photocatalytic pretreatment improved the sensitivity.

Thermoadaptation trait revealed by the genome sequence of thermophilic Geobacillus kaustophilus.

We present herein the first complete genome sequence of a thermophilic Bacillus-related species, Geobacillus kaustophilus HTA426, which is composed of a 3.54 Mb chromosome and a 47.9 kb plasmid, along with a comparative analysis with five other mesophilic bacillar genomes. Upon orthologous grouping of the six bacillar sequenced genomes, it was found that 1257 common orthologous groups composed of 1308 genes (37%) are shared by all the bacilli, whereas 839 genes (24%) in the G.kaustophilus genome were found to be unique to that species. We were able to find the first prokaryotic sperm protamine P1 homolog, polyamine synthase, polyamine ABC transporter and RNA methylase in the 839 unique genes; these may contribute to thermophily by stabilizing the nucleic acids. Contrasting results were obtained from the principal component analysis (PCA) of the amino acid composition and synonymous codon usage for highlighting the thermophilic signature of the G.kaustophilus genome. Only in the PCA of the amino acid composition were the Bacillus-related species located near, but were distinguishable from, the borderline distinguishing thermophiles from mesophiles on the second principal axis. Further analysis revealed some asymmetric amino acid substitutions between the thermophiles and the mesophiles, which are possibly associated with the thermoadaptation of the organism.

Identification and distribution of new insertion sequences in the genome of the extremely halotolerant and alkaliphilic Oceanobacillus iheyensis HTE831.

Six kinds of new insertion sequences (ISs), IS667 to IS672, a group II intron (Oi.Int), and an incomplete transposon (Tn852loi) were identified in the 3,630,528-bp genome of the extremely halotolerant and alkaliphilic Oceanobacillus iheyensis HTE831. Of 19 ISs identified in the HTE831 genome, 7 were truncated, indicating the occurrence of internal rearrangement of the genome. All ISs except IS669 generated a 4- to 8-bp duplication of the target site sequence, and these ISs carried 23- to 28-bp inverted repeats (IRs). Sequence analysis revealed that four ISs (IS669, IS670, IS671, and IS672) were newly identified as belonging to separate IS families (IS200/IS605, IS30, IS5, and IS3, respectively). IS667 and IS668 were also characterized as new members of the ISL3 family. Tn8521oi, which belongs to the Tn3 family as a new member, generated a 5-bp duplication of the target site sequence and carried complete 38-bp IRs. Of the eight protein-coding sequences (CDSs) identified in Tn8521oi, three CDSs (OB481, OB482, and OB483) formed a ger gene cluster, and two other paralogous gene clusters were found in the HTE831 genome. Most of the ISs and the group II intron widely distributed throughout the genome were inserted in noncoding regions, while two ISs (IS667-08 and IS668-02) and Oi.Int-04 were inserted in the coding regions.