Identification and characterization of VapBC toxin-antitoxin system in Bosea sp. PAMC 26642 isolated from Arctic lichens.

Toxin-antitoxin (TA) systems are genetic modules composed of a toxin interfering with cellular processes and its cognate antitoxin, which counteracts the activity of the toxin. TA modules are widespread in bacterial and archaeal genomes. It has been suggested that TA modules participate in the adaptation of prokaryotes to unfavorable conditions. The Bosea sp. PAMC 26642 used in this study was isolated from the Arctic lichen Stereocaulon sp. There are 12 putative type II TA loci in the genome of Bosea sp. PAMC 26642. Of these, nine functional TA systems have been shown to be toxic in Escherichia coli The toxin inhibits growth, but this inhibition is reversed when the cognate antitoxin genes are coexpressed, indicating that these putative TA loci were bona fide TA modules. Only the BoVapC1 (AXW83_01405) toxin, a homolog of VapC, showed growth inhibition specific to low temperatures, which was recovered by the coexpression of BoVapB1 (AXW83_01400). Microscopic observation and growth monitoring revealed that the BoVapC1 toxin had bacteriostatic effects on the growth of E. coli and induced morphological changes. Quantitative real time polymerase chain reaction and northern blotting analyses showed that the BoVapC1 toxin had a ribonuclease activity on the initiator tRNAfMet, implying that degradation of tRNAfMet might trigger growth arrest in E. coli Furthermore, the BoVapBC1 system was found to contribute to survival against prolonged exposure at 4°C. This is the first study to identify the function of TA systems in cold adaptation.

Lipid A from Oligotropha carboxidovorans Lipopolysaccharide That Contains Two Galacturonic Acid Residues in the Backbone and Malic Acid A Tertiary Acyl Substituent.

The free-living Gram-negative bacterium Oligotropha carboxidovorans (formerly: Pseudomonas carboxydovorans), isolated from wastewater, is able to live in aerobic and, facultatively, in autotrophic conditions, utilizing carbon monoxide or hydrogen as a source of energy. The structure of O. carboxidovorans lipid A, a hydrophobic part of lipopolysaccharide, was studied using NMR spectroscopy and high-resolution mass spectrometry (MALDI-ToF MS) techniques. It was demonstrated that the lipid A backbone is composed of two d-GlcpN3N residues connected by a ß-(1→6) glycosidic linkage, substituted by galacturonic acids (d-GalpA) at C-1 and C-4' positions. Both diaminosugars are symmetrically substituted by 3-hydroxy fatty acids (12:0(3-OH) and 18:0(3-OH)). Ester-linked secondary acyl residues (i.e., 18:0, and 26:0(25-OH) and a small amount of 28:0(27-OH)) are located in the distal part of lipid A. These very long-chain hydroxylated fatty acids (VLCFAs) were found to be almost totally esterified at the (ω-1)-OH position with malic acid. Similarities between the lipid A of O. carboxidovorans and Mesorhizobium loti, Rhizobium leguminosarum, Caulobacter crescentus as well as Aquifex pyrophylus were observed and discussed from the perspective of the genomic context of these bacteria.

AidB, a Novel Thermostable N-Acylhomoserine Lactonase from the Bacterium Bosea sp.

Many Gram-negative bacteria employ N-acylhomoserine lactones (AHLs) as quorum-sensing (QS) signal molecules to regulate virulence expression in a density-dependent manner. Quorum quenching (QQ) via enzymatic inactivation of AHLs is a promising strategy to reduce bacterial infections and drug resistance. Herein, a thermostable AHL lactonase (AidB), which could degrade different AHLs, with or without a substitution of carbonyl or hydroxyl at the C-3 position, was identified from the soil bacterium Bosea sp. strain F3-2. Ultrahigh-performance liquid chromatography analysis demonstrated that AidB is an AHL lactonase that hydrolyzes the ester bond of the homoserine lactone (HSL) ring. AidB was thermostable in the range 30 to 80°C and showed maximum activity after preincubation at 60°C for 30 min. The optimum temperature of AidB was 60°C, and the enzyme could be stably stored in double-distilled water (ddH2O) at 4°C or room temperature. AidB homologs were found only in Rhizobiales and Rhodospirillales of the Alphaproteobacteria AidB from Agrobacterium tumefaciens and AidB from Rhizobium multihospitium (with amino acid identities of 50.6% and 52.8% to AidB, respectively) also showed thermostable AHL degradation activity. When introduced into bacteria, plasmid-expressed AidB attenuated pyocyanin production by Pseudomonas aeruginosa PAO1 and the pathogenicity of Pectobacterium carotovorum subsp. carotovorum Z3-3, suggesting that AidB is a potential therapeutic agent by degrading AHLs.IMPORTANCE A quorum-sensing system using AHLs as the signal in many bacterial pathogens is a critical virulence regulator and an attractive target for anti-infective drugs. In this work, we identified a novel AHL lactonase, AidB, from a soil bacterial strain, Bosea sp. F3-2. The expression of aidB reduced the production of AHL signals and QS-dependent virulence factors in Pseudomonas aeruginosa and Pectobacterium carotovorum The homologs of AidB with AHL-degrading activities were found only in several genera belonging to the Alphaproteobacteria Remarkably, AidB is a thermostable enzyme that retained its catalytic activity after treatment at 80°C for 30 min and exhibits reliable storage stability at both 4°C and room temperature. These properties might make it more suitable for practical application.

Characterization of the antimonite- and arsenite-oxidizing bacterium Bosea sp. AS-1 and its potential application in arsenic removal.

Arsenic (As) and antinomy (Sb) usually coexist in natural environments where both of them pollute soils and water. Microorganisms that oxidize arsenite [As(III)] and tolerate Sb have great potential in As and Sb bioremediation, In this study, a Gram-negative bacterial strain, Bosea sp. AS-1, was isolated from a mine slag sample collected in Xikuangshan Sb mine in China. AS-1 could tolerate 120 mM of As(III) and 50 mM of antimonite [Sb(III)]. It could also oxidize 2 mM of As(III) or Sb(III) completely under heterotrophic and aerobic conditions. Interestingly, strain AS-1 preferred to oxidize As(III) with yeast extract as the carbon source, whereas Sb(III) oxidation was favored with lactate in the medium. Genomic analysis of AS-1 confirmed the presence of several gene islands for As resistance and oxidation. Notably, a system of AS-1 and goethite was found to be able to remove 99% of the As with the initial concentration of 500 µg/L As(III) and 500 µg/L Sb(III), which suggests the potential of this approach for As removal in environments especially with the presence of high Sb.

Spatio-Temporal Variations in the Abundance and Structure of Denitrifier Communities in Sediments Differing in Nitrate Content.

Spatial and temporal variations related to hydric seasonality in abundance and diversity of denitrifier communities were examined in sediments taken from two sites differing in nitrate concentration along a stream Doñana National Park during a 3-year study. We found a positive relationship between the relative abundance of denitrifiers, determined as narG, napA, nirK, nirS and nosZ denitrification genes, and sediment nitrate content, with similar spatial and seasonal variations. However, we did not find association between denitrification activity and the community structure of denitrifiers. Because nosZ showed the strongest correlation with the content of nitrate in sediments, we used this gene as a molecular marker to construct eight genomic libraries. Analysis of these genomic libraries revealed that diversity of the nosZ-bearing communities was higher in the site with higher nitrate content. Regardless of nitrate concentration in the sediments, the Bradyrhizobiaceae and Rhodocyclaceae were the most abundant families. On the contrary, Rhizobiaceae was exclusively present in sediments with higher nitrate content. Results showed that differences in sediment nitrate concentration affect the composition and diversityof nosZ-bearing communities.

Structure of O-specific polysaccharide of Oligotropha carboxidovorans OM5 - a wastewater bacterium.

Oligotropha carboxidovorans strain OM5 (previously known as Pseudomonas carboxydovorans OM5) is a rod-shaped Gram-negative bacterium isolated from wastewater. This bacterium is able to live in aerobic and, facultatively, in autotrophic conditions. For autotrophic growth, the bacteria can utilize carbon monoxide or hydrogen as a source of energy. The O-specific polysaccharide isolated from O. carboxidovorans OM5 lipopolysaccharide was structurally characterized using chemical analyses, 1D and 2D NMR spectroscopy, and MALDI-TOF mass spectrometry techniques. The polysaccharide was found to be a homopolymer built up of 3-O-methyl-α-d-mannose residues linked by (1 â†’ 2)-glycosidic bonds. The degree of polymerization of high-molecular-weight polysaccharide was estimated at approximately 35-40 units. The structure of the homopolymer is depicted below: [Formula: see text].

Transformation of galena to pyromorphite produces bioavailable sulfur for neutrophilic chemoautotrophy.

The aqueous concentration of lead [Pb(II)] in geochemical environments is controlled by the solubility of Pb-bearing minerals and their weathering products. In contaminated soils, a common method for in situ stabilization of Pb(II) is the addition of phosphate to convert more redox sensitive sulfide minerals into sparingly soluble pyromorphite [Pb5 (PO4 )3 X]. In this study, we conducted experimental studies to investigate the fate of reduced sulfur during the conversion of galena [PbS] to chloropyromorphite [Pb5 (PO4 )3 Cl]. Powder X-ray diffraction analysis indicated that the reaction of phosphate with galena under oxic conditions resulted in the oxidation of sulfide and formation of elemental sulfur [S8 ]. Under oxic abiotic conditions, the S8 was retained in the solid phase, and negligible concentrations of sulfur as sulfide and thiosulfate were detected in the aqueous phase and only a small amount of sulfate. When PbS reacted in the presence of the chemoautotrophic organism Bosea sp. WAO, the S8 in the secondary mineral was oxidized to sulfate. Strain WAO produced significantly more sulfate from the secondary S8 than from the primary galena. Microscopic analysis of mineral-microbe aggregates on mineral-embedded slide cultures showed that the organism was colocalized and increased in biomass over time on the secondary mineral surface supporting a microbial role. The results of this study indicate that stimulation of sulfur-oxidizing activity may be a direct consequence of phosphate amendments to Pb(II)-contaminated soils.

Biosorption of Cr(VI) ions from aqueous solutions by a newly isolated Bosea sp. strain Zer-1 from soil samples of a refuse processing plant.

The adsorption behavior of Cr(VI) ions from aqueous solution by a chromium-tolerant strain was studied through batch experiments. An isolate designated Zer-1 was identified as a species of Bosea on the basis of 16S rRNA results. It showed a maximum resistance to 550 mg·L(-1) Cr(VI). The effects of 3 important operating parameters, initial solution pH, initial Cr(VI) concentration, and biomass dose, were investigated by central composite design. On the basis of response surface methodology results, maximal removal efficiency of Cr(VI) was achieved under the following conditions: pH, 2.0; initial concentration of metal ions, 55 mg·L(-1); and biomass dose, 2.0 g·L(-1). Under the optimal conditions, the maximum removal efficiency of Cr(VI) ions was found to be nearly 98%. The experimental data exhibited a better fit with the Langmuir model than the Freundlich model. The biosorption mechanisms were investigated with pseudo-first-order, pseudo-second-order, and intraparticle diffusion kinetics models. These results revealed that biosorption of Cr(VI) onto bacterial biomass could be an alternative method for the removal of metal ions from aqueous solution.

[Influence of iron on siderophore and photosynthetic pigments biosynthesis by siderophore-producing Rhodopesudomonnas palustris].

OBJECTIVE: To explore the regulation of iron on siderophore production, cell growth and photosynthetic pigments biosynthesis by siderophore-producing anoxygenic phototrophic bacteria. METHODS: Siderophore production was determined using Chrome Azurol S (CAS) assay. The siderophore types were determined by Arnow method, Csaky test and Shenker test. The compositions and contents of photosynthetic pigments were determined by spectrophotometry and HPLC analysis. RESULTS: Rhodopseudomonas palustris (Rps. palustris) CQV97 was capable of producing hydroxamate-type of siderophore. Siderophore production reached the highest yield in the absence of ferric chloride. With increasing ferric chloride concentrations, the lag phase of cell growth was shortened, and the cell growth rate, final biomass and the total amounts of carotenoid and bacteriochlorophyll a were increased significantly. The characteristic absorption maxima of carotenoids from pigment extracts were blueshifted. Iron concentration had little effect on the compositions and relative contents of bacteriochlorophylls a, whereas predominately affected carotenoid compositions, rhodopin was present as major carotenoid component instead of spirillxanthin. Culture tends to accumulate the Cars having shorter conjugated double bonds at the expense of longer conjugated double bonds as the ferric chloride concentration increased. The changes in carotenoid composition were consistent with those of the blue shift of absorption spectra of pigment extracts. CONCLUSION: Rps. palustris CQV97 can produce siderophore and the changes in microbial growth, siderophore production and photosynthetic pigments accumulation of anoxygenic phototrophic bacteria are related to the iron concentration in the medium.

[Identification of a new heterotrophic nitrobacterium strain Colloides sp. JZ1-1 and its nitrifying capability].

A heterotrophic nitrobacterium strain JZ1-1 with higher nitrifying capability was isolated and mutagenized from an acclimated activated sludge. The JZ1-1 was identified as Colloides sp., according to its morphological and physiological features. The factors affecting the nitrifying capability of JZ1-1 were investigated, including medium carbon source, C/N ratio, pH value, dissolved oxygen, temperature, and ammonium nitrogen concentration. The optimal carbon source was sodium citrate, and the nitrification was favored when the C/N ratio was from 10 to 14, temperature was 30 degrees C, and pH value was 6-9 when cultured at a rotating speed of more than 150 r x min(-1). JZ1-1 could degrade ammonium nitrogen effectively when the initial concentration of ammonium nitrogen was from 100 mg x L(-1) to 500 mg x L(-1). JZ1-1 was stable after 5 generations of subculture.

[Dynamic changes in functional genes for nitrogen bioremediation of petroleum-contaminated soil cycle during].

Microorganisms in nitrogen cycle serve as an important part of the ecological function of soil. The aim of this research was to monitor the abundance of nitrogen-fixing, denitrifying and nitrifying bacteria during bioaugmentation of petroleum-contaminated soil using real-time polymerase chain reaction (real-time PCR) of nifH, narG and amoA genes which encode the key enzymes in nitrogen fixation, nitrification and ammoniation respectively. Three different kinds of soils, which are petroleum-contaminated soil, normal soil, and remediated soil, were monitored. It was shown that the amounts of functional microorganisms in petroleum-contaminated soil were far less than those in normal soil, while the amounts in remediated soil and normal soil were comparable. Results of this experiment demonstrate that nitrogen circular functional bacteria are inhibited in petroleum-contaminated soil and can be recovered through bioremediation. Furthermore, copies of the three functional genes as well as total petroleum hydrocarbons (TPH) for soils with six different treatments were monitored. Among all treatments, the one, into which both E. cloacae as an inoculant and wheat straw as an additive were added, obtained the maximum copies of 2.68 x 10(6), 1.71 x 10(6) and 8.54 x 10(4) per gram dry soil for nifH, narG and amoA genes respectively, companying with the highest degradation rate (48% in 40 days) of TPH. The recovery of functional genes and removal of TPH were better in soil inoculated with E cloacae and C echinulata collectively than soil inoculated with E cloacae only. All above results suggest that the nitrogen circular functional genes could be applied to monitor and assess the bioremediation of petroleum-contaminated soil.

Phylogeny and photoheterotrophy in the acidophilic phototrophic purple bacterium Rhodoblastus acidophilus.

Norbert Pfennig isolated the first acidophilic purple bacterium over 40 years ago and named the organism Rhodopseudomonas acidophila (now Rhodoblastusacidophilus). Since the original work of Pfennig, no systematic study has been conducted on the phylogeny and carbon nutrition of a collection of strains of Rbl. acidophilus. We have isolated six new strains of Rbl. acidophilus from a Canadian peat bog. These strains, three of the original Pfennig strains and two additional putative R. acidophilus strains isolated several years ago in this laboratory,were characterized as to their pigments, phylogeny, and carbon sources supporting photoheterotrophic growth. Phototrophic cultures were either purple or orange in color,and the color of a particular strain was linked to phylogeny. As for the Pfennig strains of Rbl. acidophilus, all new strains grew photoheterotrophically at pH 5 on a variety of organic and fatty acids. However, in addition to methanol and ethanol, the new strains as well as the Pfennig strains grew on several other primary alcohols, results not reported in the original species description. Our work shows that some phylogenetic and physiological diversity exists within the species Rbl. acidophilus and supports the observation that few species of acidophilic purple bacteria appear to exist in nature.

Proteome and membrane fatty acid analyses on Oligotropha carboxidovorans OM5 grown under chemolithoautotrophic and heterotrophic conditions.

Oligotropha carboxidovorans OM5 T. (DSM 1227, ATCC 49405) is a chemolithoautotrophic bacterium able to utilize CO and H(2) to derive energy for fixation of CO(2). Thus, it is capable of growth using syngas, which is a mixture of varying amounts of CO and H(2) generated by organic waste gasification. O. carboxidovorans is capable also of heterotrophic growth in standard bacteriologic media. Here we characterize how the O. carboxidovorans proteome adapts to different lifestyles of chemolithoautotrophy and heterotrophy. Fatty acid methyl ester (FAME) analysis of O. carboxidovorans grown with acetate or with syngas showed that the bacterium changes membrane fatty acid composition. Quantitative shotgun proteomic analysis of O. carboxidovorans grown in the presence of acetate and syngas showed production of proteins encoded on the megaplasmid for assimilating CO and H(2) as well as proteins encoded on the chromosome that might have contributed to fatty acid and acetate metabolism. We found that adaptation to chemolithoautotrophic growth involved adaptations in cell envelope, oxidative homeostasis, and metabolic pathways such as glyoxylate shunt and amino acid/cofactor biosynthetic enzymes.

Rhizobium rosettiformans sp. nov., isolated from a hexachlorocyclohexane dump site, and reclassification of Blastobacter aggregatus Hirsch and Muller 1986 as Rhizobium aggregatum comb. nov.

A Gram-negative, rod-shaped, motile, aerobic bacterial strain, W3(T), was isolated from hexachlorocyclohexane (HCH)-contaminated groundwater from Lucknow, India, and its taxonomic position was determined using a polyphasic approach. Strain W3(T) shared highest 16S rRNA gene sequence similarity of 97.8 % with Rhizobium selenitireducens B1(T), followed by Rhizobium daejeonense L61(T) (97.7 %), Rhizobium radiobacter ATCC 19358(T) (97.5 %) and Blastobacter aggregatus IFAM 1003(T) (97.2 %). Strain W3(T) formed a monophyletic clade with Blastobacter aggregatus IFAM 1003(T) ( = DSM 1111(T)) in the cluster of species of the genus Rhizobium. Phylogenetic analyses of strain W3(T) using atpD and recA gene sequences confirmed the phylogenetic arrangements obtained by using 16S rRNA gene sequences. Hence, the taxonomic characterization of B. aggregatus DSM 1111(T) was also undertaken. Strains W3(T) and B. aggregatus DSM 1111(T) contained summed feature 8 (18 : 1ω7c and/or 18 : 1ω6c; 65.4 and 70.8 %, respectively) as the major fatty acid, characteristic of the genus Rhizobium. DNA-DNA relatedness of strain W3(T) with Rhizobium selenitireducens LMG 24075(T), Rhizobium daejeonense DSM 17795(T), Rhizobium radiobacter DSM 30147(T) and B. aggregatus DSM 1111(T) was 42, 34, 30 and 34 %, respectively. The DNA G+C contents of strain W3(T) and B. aggregatus DSM 1111(T) were 62.3 and 62.7 mol%, respectively. A nifH gene encoding dinitrogenase reductase was detected in strain W3(T) but not in B. aggregatus DSM 1111(T). Based on the results obtained by phylogenetic and chemotaxonomic analyses, phenotypic characterization and DNA-DNA hybridization, it is concluded that strain W3(T) represents a novel species of the genus Rhizobium, for which the name Rhizobium rosettiformans sp. nov. is proposed (type strain W3(T)  = CCM 7583(T)  = MTCC 9454(T)). It is also proposed that Blastobacter aggregatus Hirsch and Müller 1986 be transferred to the genus Rhizobium as Rhizobium aggregatum comb. nov. (type strain IFAM 1003(T)  = DSM 1111(T)  = ATCC 43293(T)).

Interaction of CO dehydrogenase with the cytoplasmic membrane monitored by fluorescence correlation spectroscopy.

We have investigated the interaction of carbon monoxide dehydrogenase (CODH), an enzyme that catalyses the oxidation of CO in the aerobic eubacterium Oligotropha carboxidovorans, with the cytoplasmic membrane by using fluorescence correlation spectroscopy (FCS). Our results reveal that in vitro this interaction of CODH is specific for cytoplasmic membranes from CO-grown bacteria.

Description of Microvirga aerophila sp. nov. and Microvirga aerilata sp. nov., isolated from air, reclassification of Balneimonas flocculans Takeda et al. 2004 as Microvirga flocculans comb. nov. and emended description of the genus Microvirga.

Two bacterial strains, 5420S-12(T) and 5420S-16(T), isolated from air samples, were characterized using a polyphasic approach. 16S rRNA gene sequence analysis showed that strain 5420S-12(T) was related phylogenetically to Microvirga subterranea FaiI4(T) (97.4 % sequence similarity) and Microvirga guangxiensis 25B(T) (97.1 %) and that strain 5420S-16(T) was closely related to Balneimonas flocculans TFB(T) (98.0 %) and Microvirga guangxiensis 25B(T) (97.2 %). The G+C content of the genomic DNA was 62.2 mol% for strain 5420S-12(T) and 61.5 mol% for strain 5420S-16(T). The major fatty acid was C(18 : 1)ω7c. The results of DNA-DNA hybridization and the phenotypic data showed that strains 5420S-12(T) and 5420S-16(T) could be distinguished from phylogenetically related species and represent two novel species within the genus Microvirga, for which the names Microvirga aerophila sp. nov. (type strain 5420S-12(T) =KACC 12743(T) =NBRC 106136(T)) and Microvirga aerilata sp. nov. (type strain 5420S-16(T) =KACC 12744(T) =NBRC 106137(T)) are proposed. Furthermore, the reclassification of Balneimonas flocculans as Microvirga flocculans comb. nov. (type strain TFB(T) =JCM 11936(T) =KCTC 12101(T) =IAM 15034(T) =ATCC BAA-817(T)) is proposed and an emended description of the genus Microvirga is provided.

[Redox zones and its functional bacteria in a contaminated landfill site].

A column filled with sandy soil was constructed to investigate biogeochemical process of leachate polluted zones. Experimental results demonstrated that four sequent redox zones appeared in pollution plume. The zones can be named sulfate reduction zone, iron reduction zone, nitrate reduction zone and oxygen reduction zone, ranges of them were 0-27 cm, 27-62 cm, 47-74 cm, 74-91 cm. In the redox zones bacterial community structure changed, and the preponderant bacteria were sulfate reduction bacteria (SRB), iron reduction bacteria (IRB), nitrate reduction bacteria (NRB) correspondingly, however there were other bacteria in the redox zones. Distribution of redox zones and functional bacteria means that there is not a significant boundary between redox zones, moreover one redox zone overlaps others. Evolvement of functional bacterial community brings the redox zones substitute.

[Investigation of nitrogen removal performance with membrane sequencing batch reactor process].

Nitrogen removal performance was investigated using synthetic wastewater as feed without sludge discharge in an anaerobic/aerobic membrane sequencing batch reactor (MSBR) during 300 days operation. The results showed that MLSS in reactor was retained up to about 18 g x L(-1), sludge size larger than 100 microm was 96%, and aerobic granular sludge was developed. The bacterial community observation of AOB and NOB by FISH-CLSM for sludge revealed that they were existed in larger numbers. When influent NH(4+) -N concentration was about 50 mg x L(-1), effluent NH(4+) -N concentration was lower than to 1 mg x L(-1), and nitrification could complete in 180-210 min. There was a good correlation between nitrification reaction and aeration strength. When aeration strength was 100 m3 x (m2 x h)(-1), NH(4+) -N degradation rate 24.25 mg x (L x h)(-1), and nitrification reaction in MSBR was stable. The main factor determining nitrogen removal of the system was denitrification rate, which was optimal at aeration 69 m3 x (m2 x h)(-1), when the nitrification rate of NO(3-) -N 10.98 mg x (L x h)(-1), effluent NO(3-) -N 4.4 mg x L(-1), and NO(3-) -N in the beginning of anaerobic phase 3.5 mg x L(-1). The denitrification performance was not benefited by excessive aeration or deficient aeration. Bigger volumetric exchange ratio was helpful for nitrogen removal and the system treatment capacity. The C/N ratio 2 was suitable to good denitrification rate, while there was a NO(2-) -N accumulation if C/N ratio was larger than 2.

Dynamics of viable nitrifier community, N-mineralization and nitrification in seasonally dry tropical forests and savanna.

The study was conducted in Vindhyan region, to assess the N-mineralization, nitrification and size of viable community of ammonium- and nitrite-oxidizing bacteria as affected by different sites and seasons. Six different ecosystems (four forests and two savannas), which differ in terms of topography, vegetation and moisture status, were selected for the present study. The soils of the study sites differ significantly in its physico-chemical properties. The savanna site had significantly higher pH (7.2), bulk density (1.37 g cm(-3)) and silt content (67.80%) but lower water holding capacity (1.37%), total-C (16,356 microg g(-1) dry soil), N (1090 microg g(-1) dry soil) and P (213 microg g(-1) dry soil) than forest sites. The soil moisture content, N-mineralization, nitrification rates and numbers of ammonium- and nitrite-oxidizing bacteria were highest in the wet season and lowest in dry season, while the size of mineral-N (NH4(+)-N and NO3(-)-N) showed a reverse trend at the sites. The N-mineralization, nitrification and nitrifier population size differ significantly across the site and season. The numbers of free-living cells of ammonium- and nitrite-oxidizing bacteria were significantly related to each other and to N-mineralization, nitrification, soil moisture and mineral-N components. The N-mineralization, nitrification and the viable number of nitrifying cells were consistently higher for forest soils compared to savanna sites. It was concluded that soil microbial process (N-mineralization and nitrification) and nitrifier population size were dependent on site topography, vegetation cover and soil moisture status.

Isolation of novel bacteria and actinomycetes using soil-extract agar medium.

Novel bacteria were discovered using an isolation technique consisting of (i) selection of microorganisms that grew on soil-extract agar medium, but not on conventional media, and (ii) detection of small microbial colonies with a microscope. Three bacterial strains thus isolated were provisionally designated Shinshu-th1, -th2, -th 3, and five actinomycete strains, Shinshu-MS-01, -02, -03, -04, -05, respectively. Sequence analysis of their 16S rDNA showed that th1 had 95--96% homology with three unculturable bacteria, and th2 had 96% similarity to Bradyrhizobium sp., one unculturable and one unidentified bacterial strain. A phylogenetic study indicated that both strains were alpha-Proteobacteria belonging to the order Rhizobiales and the family Bradyrhizobiaceae. Since they had low homology (96%) with their close relatives, it is possible that th1 and th2 belong to a new genus. The actinomycetes Shinshu-MS-02 and -03 had 95--96% homology with four strains of Actinomadura, -04 had 95--96% similarity to Streptosporangium and Microbispora, and -05 had 97--98% homology with three strains of Acrocarpospora, Herbidospora and Planotetraspora. According to the phylogenetic study, both 02 and 03 are possibly new species of Actinomadura, -04 of Streptosporangium, and -05 of Acrocarpospora. Shinshu-th 3 and -MS-01 were identified as Mycobacterium cookii and Frankia sp., respectively, having 99% homology with these species.