A rapid method for estimating the population densities of uncultured bacteria in the environment was developed. This method consists of (i) addition of particular bacterial cells of a known cell number to an environmental sample, (ii) PCR amplification of partial 16S rDNA fragments with a set of eubacterial universal primers, (iii) temperature gradient gel electrophoresis (TGGE) analysis of the PCR products, and (iv) densitometry of the TGGE pattern. The utility of this competitive PCR/TGGE (cPCR/TGGE) method was demonstrated in the analysis of dominant bacterial populations in seawater sampled from the Heita sound (Kamaishi, Iwate, Japan). The nucleotide-sequence analysis of the TGGE bands revealed that the dominant populations were closely related to previously characterized uncultured marine bacteria.
Specificity of a DNA probe developed for some species of a Vibrio core group was examined using a fluorescence resonance energy transfer system. The probe was designed based on the sequence of a 16S rDNA fragment of Vibrio parahaemolyticus cloned in a plasmid, pTPR1. The 5'-and 3'-end of the probe was ligated with fluorogenic dyes. The specificity was examined against clonal 16S rDNA fragments and chromosomal DNAs extracted from different species of Vibrio. The 16S rDNA fragments, cloned from V. parahaemolyticus, Vibrio natriegens, Vibrio fischeri, Vibrio mediterranei, and Vibrio tubiashii, had from one to four nucleotide substitutions compared with pTPR1 in the probe region. Probe hybridization with the clonal or chromosomal DNA fragments was monitored by determining the increase in fluorescence during PCR amplification. At the highest stringent condition, the probe hybridized only to identical sequences, but not to the sequences with substitution.
The fine structure and spatial arrangement of Frankia vesicles in the root nodules of Alnus hirsuta, Myrica rubra, Elaeagnus pungens, and Coriaria japonica growing naturally in Japan were compared by electron micrography. The shape and spatial arrangement of the vesicles in the nodules varied with the species. The vesicles of A. hirsuta and E. pungens were spherical and internally compartmentalized into multiple small segments by septa, and those in M. rubra were ellipsoidal and internally septate. Mesosomes which appeared to be connected to septa were often observed in the vesicles of A. hirsuta and M. rubra. However, clear mesosomes were not observed in the vesicles of E. pungens. The vesicles of C. japonica were club-shaped and arranged peripherally around the central vacuole, and unseparated by septa. Clear lamella mesosomes were observed in the hyphae and appeared to be associated with an electron-translucent nucleoid region.
We isolated endophytic Frankia from the root nodules of Elaeagnus macrophylla and Alnus sieboldiana and examined the infectivity of the isolates to the host plants. One strain from E. macrophylla and 13 strains from A. sieboldiana were isolated by the simple method of suspending the nodule homogenate directly in liquid Qmod medium. Hyphae, sporangia and vesicles, which are morphological characteristics of Frankia, were observed in the E. macrophylla isolate. However, only hyphae and sporangia were observed in the A. sieboldiana isolate. The E. macrophylla isolate formed nodules on the roots of the host plant and the average acetylene reduction activity (ARA) was 17.7±6.9μmol C2H4h-1 (g fresh wt nod.)-1. Eleven strains isolated from A. sieboldiana formed nodules on the root of the host plants, but 2 strains did not nodulate. The average ARA was 4.22±3.73μmol C2H4h-1 (g fresh wt nod.)-1.
The protein phosphatase inhibition assay was used to quantify low levels of microcystin in lake water polluted by cyanobacterial blooms of Microcystis, and toxin concentrations in cultures of 15 strains of Microcystis. This method was highly sensitive permitting measurement of microcystin in concentrations of the order of μg1-1. The concentrations of microcystin in the surface water of Lake Kasumigaura in the summer of 1995 were in the range of 0.16 to 2.7μg1-1 microcystin-LR equivalents. On the other hand, markedly high levels of microcystin (160 to 1, 429μg1-1) were measured in the culture fluids of M. aeruginosa TAC 192-2, M. ichthyoblabe TAC 69, M. ichthyoblabe TAC 113-1, M. viridis TAC 45-1, M. viridis TAC 64 and M. viridis TAC 92. There were strains which did not produce microcystin in the culture fluid within the same species of Microcystis. Therefore, the toxin productivity in the culture fluid is not a useful criterion for discrimination of the species of Microcystis.
Enhanced biological phosphorus removal in the processes utilizing anaerobic/aerobic conditions is established by the increase of polyphosphate content in the activated sludge and the withdrawal of the sludge. For stable operation to achieve efficient phosphorus removal, better understanding of the microorganisms including polyphosphate-accumulating bacteria in activated sludge of these processes is required. However, physiology and ecology of bacteria which have an important role in these processes are still unclear. In this paper, the characteristics of important bacteria constructing phosphorus removal processes and the progress of microbial population analysis based on 16S rDNA sequences were introduced.
Volatile aliphatic chlorinated compounds such as trichloroethylene (TCE), 1, 1, 1-trichloroethane (TCA), and tetrachloroethylene have been detected in groundwater throughout Japan. Various soil and groundwater cleanup technologies are now being developed, studied, and evaluated in Japan. For chlorinated compounds, soil vapor extraction, pumping up and treatment, and digging up and drying are very common approaches. Physical and chemical methods for remediation are expensive. Therefore, less expensive but more complete pollutant destruction technology is required. Bioremediation could be one of the most promising new technologies for cleaning up groundwater contamination, because of its low cost and the complete destruction of pollutants. In this paper, we reported TCE and TCA degrading bacteria and determined their fundamental characteristics for bioremediation.
It has been understood as a common sense that petroleum can be oxidized by the microorganisms in the presence of molecular oxygen. In contrast, we isolated an extremely interesting bacterium strain HD-1 which could assimilate petroleum under anaerobic condition. The bacterium could grow on CO2 as a sole carbon source, and produced n-alkane/alkene, major components of petroleum.
Bacterial chromosomes have genes for transport of inorganic nutrient cations (such as NH4+, K+, Mg2+, Co2+, Fe3+, Mn2+, Zn2+ and other trace cations) and oxyanions (such as PO43- and SO42- and less abundant oxyanions). Together these account for sometimes several hundred genes in many bacteria. Bacterial plasmids encode resistance systems for toxic metal and metalloid ions including Ag+, AsO2-, AsO43-, Cd2+, Co2+, CrO42-, Cu2+, Hg2+, Ni2+, Pb2+, Sb3+, TeO32-, Tl+, and Zn2+. Most resistance systems function by energy-dependent efflux of toxic ions. A few involve enzymatic (mostly redox) transformants. Some of the efflux resistance systems are ATPases and others are chemiosmotic ion/proton exchangers. Mercury resistance is due to enzymatic detoxification with organomercurial lyase (cutting the C-Hg bond of compounds such as methylmercury and phenylmercury) and mercuric reductase (Hg2+→Hg0). The Cd2+-resistance cation P-type ATPases of Gram-positive bacteria drives Cd2+ (and Zn2+) efflux from resistant cells. The genes defective in the human hereditary diseases of copper metabolism, Menkes syndrome and Wilson's disease, encode Cu-specific P-type ATPases that are similar to bacterial Cd2+ ATPases. The arsenic resistance system transports arsenite [As(III)], alternatively with the ArsB protein functioning as a chemiosmotic efflux transporter or with two proteins, ArsB and ArsA, functioning as an ATPase transporter. The third protein of the arsenic resistance system is an enzyme that reduces intracellular arsenate [As(V)] to arsenite [As(III)], the substrate of the efflux system. In Gram negative cells, a three polypeptide complex functions as a chemiosmotic cation/proton exchanger to efflux Cd2+, Zn2+, and Co2+. This pump consists of an inner membrane (CzcA), an outer membrane (CzcC) and a membrane-spanning (CzcB) protein that function together.