Ichiro Suzuki

Purification and Properties of an Enzyme Capable of Degrading the Sheath of Sphaerotilus natans

ABSTRACT Microorganisms which can degrade and grow on the purified sheath of a sheathed bacterium Sphaerotilus natans were collected from soil and river water. Two bacterial strains were isolated from the soil and designated strains TB and TK. Both strains are rod shaped, negatively stained by gram staining, facultatively anaerobic, and formed ellipsoidal endospores. These characteristics suggested that the isolates belong to the genus Paenibacillus, according to Ash et al. (C. Ash, F. G. Priest, and M. D. Collins, Antonie Leeuwenhoek 64:253–260, 1993). Phylogenetic analysis based on the 16S rDNA supported this possibility. Purification of the sheath-degrading enzyme was carried out from the culture broth of strain TB. The molecular weight of the enzyme was calculated to be 78,000 and 50,000 by sodium dodecyl sulfate-polyacrylamide electrophoresis and gel filtration chromatography, respectively. Enzyme activity was optimized at pH 6.5 to 7.0 and 30 to 40°C. The reaction was accelerated by the addition of Mg2+, Ca2+, Fe3+, and iodoacetamide, whereas it was inhibited by the addition of Cu2+, Mn2+, and dithiothreitol. The enzyme acted on the polysaccharide moiety of the sheath, producing an oligosaccharide the size of which was between the sizes of maltopentaose and maltohexaose. As the reaction proceeded, the absorbance at 235 nm of the reaction mixture increased, suggesting the generation of unsaturated sugars. Incorporation of unsaturated sugars was also suggested by the thiobarbituric acid reaction. It is possible that the enzyme is not a hydrolytic enzyme but a kind of polysaccharide eliminase which acts on the basic polysaccharide.

Cloning and Expression of the Gene Encoding Thermostable Poly(3-Hydroxybutyrate) Depolymerase.

The gene encoding extracellular poly(3-hydroxybutyrate) depolymerase from a thermophilic poly(3-hydroxybutyrate)-degrading bacterium, strain HS, was cloned and intracellularly expressed in Escherichia coli. The gene was found to consist of 1485-bp nucleotide sequence coding for a 22-amino-acid signal peptide and a 473-amino-acid mature protein. Phylogenetic analysis and domain structure showed that the enzyme was clustered with type II PHB depolymerases. The gene was expressed in E. coli under the control of the tac promoter. A 46-kDa protein was detected in the cell extract by SDS-PAGE. The N-terminal sequence of the protein agreed with that of the original enzyme. The crude enzyme was able to degrade PHB particles at 70 degrees C.

Prosthecobacter fluviatilis sp. nov., which lacks the bacterial tubulin btubA and btubB genes.

Leptothrix cholodnii is a sheathed bacterium often found in metal-rich and oligotrophic aquatic environments. A bacterial strain that is able to degrade the NaOH-treated sheath of L. cholodnii was isolated. The isolate was a Gram-negative, aerobic and prosthecate bacterium. The optimum growth temperature and pH were 30 degrees C and pH 7.0, respectively. The DNA G+C content was 62.9 mol%. The major respiratory quinone was MK-6. A phylogenetic analysis based on the 16S rRNA gene indicated that the isolate is a member of the genus Prosthecobacter. The nearest relative was the type strain of Prosthecobacter vanneervenii, with a similarity of 97.1 %. However, the isolate does not possess the bacterial tubulin genes, btubA and btubB, unique to known species of the genus Prosthecobacter. It is proposed that the isolate represents a novel species, Prosthecobacter fluviatilis sp. nov. The type strain is HAQ-1(T) (=JCM 14805(T) =KACC 12649(T) =KCTC 22182(T)).

Balneomonas flocculans gen. nov., sp. nov., A New Cellulose-producing Member of the α-2 subclass of Proteobacteria

A new bacterial strain capable of producing cellulose was isolated from a hot spring. The isolate was Gram-negative, aerobic, and rod-shaped. The optimum temperature for growth was 40-45 degrees C. Methanol, glucose and other common carbohydrates were not utilized as sole growth substrates. Thiosulfate was not oxidized. The G+C content of the DNA was determined to be 64.0 mol%. Comparative 16S rDNA analysis indicated that Bosea thiooxidans and some strains of the genus Methylobacterium were the nearest relatives. The isolate can be distinguished from these relatives by its defectiveness in methanol utilization and thiosulfate oxidation. On the basis of its phenotypic properties and phylogeny, it is proposed that the isolate be designated Balneomonas flocculans gen. nov., sp. nov. The type strain is TFBT (= JCM 11936T, = KCTC 12101T, = IAM 15034T, = ATCC BAA-817T).

Adsorption and abiotic oxidation of arsenic by aged biofilter media: equilibrium and kinetics.

Removal of arsenic from groundwater by biological adsorptive filtration depends largely on its interaction with biogenic iron and manganese oxides surfaces. In the present study we investigated the arsenic adsorption and abiotic oxidation capacities of an aged biofilter medium (BM2) collected from a long time established groundwater treatment plant for removal of iron and manganese by biological filtration. Batch oxidation/adsorption kinetic experiments indicated that BM2 can easily oxidize As(III) to As(V) with the rate of oxidation less affected by pH-variations from 4 to 8.5. The adsorption capacity of the biofilter medium for the produced or added As(V), however, depends strongly on the pH of the solution. The kinetics results have shown that As(III) sorption followed pseudo-second order kinetics, whereas the sorption of As(V) was best described by the intra-particle diffusion model, indicating that adsorptions of As(III) and As(V) onto BM2 were governed by different mechanisms. Adsorption isotherms at 25 degrees C were measured for a range of arsenite and arsenate initial concentrations of 0.67-20 micromol/L and the pH range from 4 to 9. Adsorption maxima were highest at pH 4 and decrease steadily as the pH increases. The equilibrium data for both As(III) and As(V) fitted very well to the Freundlich and Sips isotherm equations and, in most cases, the two isotherms overlapped with the same correlation coefficients, indicating sorption to be multilayer on the heterogeneous surface of BM2. The implication of the data for arsenic removal from water by biological filtration has been discussed.

Identification and Characterization of a Mycobacterial (2R,3R)-2,3-Butanediol Dehydrogenase

Bacterial strain B-009, capable of using racemic 1,2-propanediol (PD), was identified as a rapid-growing member of the genus Mycobacterium. The strain is phylogenetically related to M. gilvum, but has slightly different physiological characteristics. An NAD+-dependent enantioselective alcohol dehydrogenase, which acts on R-PD, was purified from the strain. The enzyme was a homodimer of a peptide coded by a 1047-bp gene (mbd1). A highly conserved sequence for medium-chain dehydrogenase/reductases with a preference for secondary alcohols was found in the gene. Hydroxyacetone was produced from R-PD by an enzymatic reaction, indicating that position 2 of the substrate was oxidized. The enzyme activity was highest for (2R,3R)-2,3-butanediol (R,R-BD), enabling the enzyme to be identified as (2R,3R)-2,3-butanediol dehydrogenase (R,R-BD-DH). A homology search revealed M. gilvum, M. vanbaalenii, and M. semegmatis to have ORFs similar to mbd1, suggesting the widespread distribution of genes encoding R,R-BD-DH among mycobacterial strains.

Comparison of Arsenate, Lead, and Cadmium Adsorption onto Aged Biofilter Media

Many biological water treatment plants for removal of iron and manganese from groundwater are in place for quite a long time, and thus their filters are aged—naturally coated with metal oxides and associated biomass. The particular reactivity and high adsorption capacity of these biogenic surface coatings make them potentially applicable for cost effective removal of arsenic and other heavy metals from contaminated water. However, the nature of interaction between various toxic elements and the composite materials in biological filters is not well understood. This study combines macroadsorption experiments with electron probe analysis to evaluate the adsorption properties of the biogenic surface coatings of an aged biofilter medium (BFM) for cationic lead and cadmium as well as arsenate anion. Results of batch adsorption showed that BFM has higher adsorption capacity for lead and cadmium as compared to arsenate anion. At pH 5.5, the maximum adsorption capacities of the medium for As(V), Pb(II), and Cd(II)...

Commensal relationship between a sheath-forming bacterium, Sphaerotilus natans, and a sheath-degrading bacterium, Paenibacillus sp.

Paenibacillus sp. strain TB is capable of degrading the sheath prepared from a sheathed bacterium, Sphaerotilus natans. S. natans was able to grow alone on casamino acids but strain TB was not. Cocultivation of strain TB and S. natans was examined in a medium supplemented with casamino acids as a growth substrate. The growth of strain TB was observed when the sheath was supplied to the medium or in cocultivation with S. natans. The phospholipid amount reached a maximum after 24 h of cocultivation and subsequently kept almost the same level for 96 h. The sheath amount also reached a maximum after 24 h and then gradually declined. The cell concentration of strain TB increased throughout the cocultivation. By competitive PCR targeted for amplification of a part of 16S rDNA, the abundance ratio (S. natans/strain TB) of 6.7 was obtained at 72 h. Almost no growth of strain TB was detected in a coculture with a sheath-less mutant of S. natans. The evidence allows the conclusion that strain TB grew by utilizing the intact sheath in coculture with S. natans.

Characterization of Streptomyces D3 derived from protoplast fusion between Streptomyces cyaneus 190-1 and Streptomyces griseoruber 42-9

Streptomyces D3, derived from protoplast fusion between Streptomyces cyaneus 190-1 and Streptomyces griseoruber 42-9, has the ability to produce high levels of xylose isomerase when grown on hemicellulosic materials such as xylan as the carbon source. Comparison between the partial nucleotide sequences of the 16S ribosomal RNA genes from S. cyaneus 190-1, S. griseoruber 42-9, and fusant D3 showed that the 16S rRNA gene of fusant D3 was identical to that of S. cyaneus 190-1. Partial sequence analysis of the xylose isomerase genes also indicated that the gene of fusant D3 was identical to that of S. cyaneus 190-1. The partial DNA fragments for the xylanase genes (xlnA and xlnB) of fusant D3 were amplified by PCR, and subjected to Southern hybridization analysis. The results revealed that the xlnB gene of fusant D3 was similar to that of S. cyaneus 190-1, but that the xlnA gene of fusant D3 was similar to that of S. griseoruber 42-9. These results suggest that the majority of the genome of fusant D3 may be derived from S. cyaneus 190-1.

Lagging strand replication of rolling-circle plasmids in Streptomyces lividans: an RNA polymerase-independent primer synthesis

The rolling circle (RC) mechanism of DNA replication generating single-stranded DNA (ssDNA) intermediates is common in various high-copy circular plasmids in Streptomyces, and the ssDNA released after leading strand synthesis is converted to its double-stranded form (dsDNA) by the host proteins. The in vivo and in vitro lagging strand syntheses from ssDNA replicative intermediates of RC plasmid pSN22 in Streptomyces lividans was characterized. The presence or absence of the single-strand origin (sso), the replication initiation site of lagging strand synthesis, did not significantly affect the copy numbers of pSN22 derivatives. In vivo lagging strand synthesis was not affected by the rifampicin inhibition of S. lividans RNA polymerase. Likewise, in vitro lagging strand synthesis using cell-free extracts revealedsso-independent, rifampicin-resistant lagging strand synthesis in S. lividans. Although all four dNTPs are usually required for the initiation of such synthesis, the presence of only one NTP was sufficient to carry outlagging strand synthesis in vitro. Interestingly, the cell-free extract of exponential-phase cells required less ATP than that of stationary-phase cells. These results reveal a predominant RNA polymerase-independent priming system in S. lividans that may be a result of the stabilization of RC plasmids lacking sso in S. lividans.