Effects of calcium and magnesium ions and host viability on growth of bdellovibrios

Bdellovibrio spp. strains 6-5-S, 100, 109 (Davis), and A3.12 multiply in the presence of viable but non-proliferating or heat-killed (70 or 100 C, 10 min; 121 C, 5 min) cells ofSpirillum serpens strain VHL suspended in buffers supplemented with Ca⁺⁺ and/or Mg⁺⁺. Ca⁺⁺ (optimal, 2 × 10⁻³ m) and Mg⁺⁺ (optimal, 2 × 10⁻⁵ m) independently stimulate the groth of bdellovibrios: additive effects are noted. Multiplication ofBdellovibrio in the presence of Ca⁺⁺ and Mg⁺⁺ is associated with the release into the culture supernatant solution of UV-absorbing materials and of amino sugars (presumably by activating or stabilizing lytic enzymes). The growth rate ofBdellovibrio strain 6-5-S in suspensions of heat-killed host cells is lower than in living but non-proliferating host cells. Bdellovibrio spp. strains 100, 109 (Davis), 109 (Jerusalem), A3.12, and 6-5-S all require added Ca⁺⁺ for growth in cell suspensions of homologous or heterologous host bacteria which have been grown in minimal medium.Bdellovibrio sp. strain 109 (Jerusalem) is capable of growing in the presence of the low level of Ca⁺⁺ boundin situ to the cells of its host,E. coli B, when the host cells had been cultivated in a complex medium but not when the host cells had been grown in a Ca⁺⁺-depleted minimal medium (except when Ca⁺⁺ is added). Addition of ethylenediaminetetraacetic acid (0.01m) preventsBdellovibrio growth, which is restored by addition of Ca⁺⁺ and Mg⁺⁺. The nonparasitic growth ofBdellovibrio spp. strains 100, 109, A3.12, and 6-5-S in heat-killed cell suspensions only in the presence of added cations indicates that, in this system, the cations are essential for activity of bacteriolytic and other enzymes and that they might also directly affectBdellovibrio growth rather than — as may be the case in other systems of live host cells plusBdellovibrio — only indirectly by affecting attachment to the host cell, maintaining integrity of the host spheroplasts, and increasing the burst size.     

Wirtskreis und Infektionscyclus eines neu isolierten Bdellovibrio bacteriovorus-Stammes

Zusammenfassung Der neu isolierte Stamm W von Bdellovibio bacteriovorus infiziert und lysiert Rhodospirillum rubrum F und alle anderen untersuchten Athiorhodaceae, nicht aber Pseudomonas aeruginosa und Spirillum serpens. Er befällt auch zahlreiche Enterobacteriaceae und von den grampositiven Bakterien Streptococcus faecalis und Lactobacillus plantarum.Nach dem Festheften an der Zellwand wird diese in 3–20 min durchdrungen. In 10–60 min ist Bdellovibrio vollständig in die Zelle eingedrungen und hat sich im Raum zwischen Zellwand und cytoplasmatischer Membran angesiedelt.In 3–5 Std wird der gesamte Zellinhalt bis auf die Membranen aufgelöst. In dieser Phase erfolgt die Vermehrung von Bdellovibrio. In den ghosts sind die Parasiten in lebhafter Bewegung. Die Geißel hat einen Gesamtdurchmesser von 29 m und eine Länge von etwa 3 . Sie ist von einer Geißelscheide umgeben, die in Verbindung zur Zellwand steht. Der Durchmesser der Geißel ohne Scheide beträgt etwa 18 m. Bdellovibrio kann oberhalb eines Sauerstoffpartialdruckes von 4–5 mm Hg infizieren und sich vermehren. Der Titer von Bdellovibrio nimmt bei Aufbewahrung in lysierten Kulturen in 36 Tagen von 10⁸ auf 10¹ pfu (plaque forming units) je ml ab. Bei Aufbewahrung in Nährkultur sinkt der Titer nur auf 10⁴ pfu/ml ab. Die Zahl der Plaques im Verhältnis zum Titer der Impfsuspension von Bdellovibrio schwankt in Abhängigkeit vom Wirtsstamm. Wenn man die Plaque-Bildungsrate bei R. rubrum gleich 1 setzt, beträgt sie bei Serratia marcescens 0,0001, bei Proteus vulgaris 10. Bd. bacteriovorus, Stamm W wächst nicht in synthetischer Nährlösung oder Lysaten. Ein geringes Wachstum ohne Zellteilung findet in Zellextrakten von R. rubrum statt. Der Stamm vermehrt sich jedoch in hitzeinaktiviertem R. rubrum. Die Plaque-Bildungsrate ist unter diesen Bedingungen aber sehr niedrig.In Lysaten treten encystierte Dauerformen von Bdellovibrio bacteriovorus auf.

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The host range and the infectious cycle of a new isolated, on gram-positive and gram-negative bacteria parasiting Bdellovibrio bacteriovorus strain

Summary Rhodospirillum rubrum and all other investigated Athiorhodaceae are infected and lysed by the new isolated strain W of Bdellovibrio bacteriovorus. This strain W parasites on numerous Enterobacteriaceae and the gram-positive bacteria Streptococcus faecalis and Lactobacillus plantarum, but not on Pseudomonas aeruginosa and Spirillum serpens.After attachment of Bdellovibrio to the host, the cell wall is penetrated in 3 to 20 min. In 10 to 60 min Bdellovibrio has completely entered the host cell. He remains in the space between cell wall and cytoplasmic membrane of the host.The host cell is completely lysed within 3 to 5 hours. During this phase the size and cell number of Bdellovibrio are increased and a new flagellum is likely to be formed. In the ghosts of the host cell a strong movement is observed. The single polar flagellum of Bdellovibrio has a diameter of 29 m. The flagellum consists of an inner core ( 18 m) and an outer sheath which is continued into the cell wall. Bdellovibrio is able to grow and to infect only under aerobic or semiaerobic conditions (oxygen partial pressure 4 to 5 mm Hg and more). The titer of Bdellovibrio is gradually decreased from 10⁸ to 10¹ plaque forming units (pfu) per ml, when kept in the lysate for 36 days. In a synthetic medium there is a diminution of 10⁴ pfu/ml only. The plating efficiency is dependent of the host strain. If the plating efficiency of Bdellovibrio with Rhodospirillum rubrum is 1.0, the rate varies from 0.0001 with Serratia marcescens to 10 with Proteus vulgaris. Bdellovibrio bacteriovorus strain W does not grow in a synthetic medium. However, it grows but does not multiply in cell free extracts of Rhodospirillum rubrum. The parasite is also able to infect and lyse heat inactivated R. rubrum. But the plating efficiency in this case is very low.It has been observed that in lysed cells of R. rubrum certain amount of Bdellovibrio is encysted. The morphology and fine structure of these cells is quite different from the normal virulent type.

     

Isolation of Bdellovibrio sp. from Wastewater and Their Potential Application in Control of Salmonella paratyphi in Water

The problem of the increasing resistance of bacteria to conventional antibiotics gives the bacteria Bdellovibrio a great utility as a potential alternative source of antibiotics. Therefore, the preliminary goal of the present study was isolation and identification of antibiotic-resistant bacteria used as prey organisms for isolated Bdellovibrio sp., by xylose lysine desoxycholate (XLD) agar from different types of water in the Taif area, Saudi Arabia, and also to investigate water quality. Four antibiotic-resistant isolates of Salmonella sp. which were susceptible to Bdellovibrio were identified by morphological, biochemical and 16S rRNA characterization as Salmonella paratyphi. Seventeen strains of Bdellovibrio sp. were isolated from sewage wastewater using isolated S. paratyphi as prey bacteria by a double-layer plate. Only one of them causing a large plaque after 48 h of incubation at 37°C was designated Bdellovibrio AOA12. The shape of Bdellovibrio was confirmed by morphological characterization and electron microscopy. Bdellovibrio could lyse four antibiotic-resistant Gram-negative bacterial strains of Salmonella paratyphi but could not lyse Gram-positive bacteria such as Staphylococcus aureus and Bacillus cereus. The kinetic lysis of the Bdellovibrio as predator to four isolates of antibiotic-resistant Salmonella paratyphi as prey organisms was demonstrated. The results suggest that it may be possible to utilize Bdellovibrio to control antibiotic-resistant S. paratyphi in water.     

Relationships among the bdellovibrios revealed by partial sequences of 16S ribosomal RNA

Members of the genusBdellovibrio possess the unifying phenotypic trait of attacking and preying upon other Gram-negative bacteria. It has been suggested that this common lifestyle arose by convergent evolution. Physiological and G + C studies have led to the notion that bdellovibrios are a heterogeneous group of loosely related bacteria. We have inferred the phylogenetic relatedness of 12 strains ofBdellovibrio through the analysis of partial 16S ribosomal RNA sequences. Similarity and degree of homology were assessed, and a phylogenetic tree was constructed by the distance matrix method. One branch of the two-branched tree consisted ofB. bacteriovorus and related strains (W, 6-5-S, 109, 109D, 109J, 114, HI Ox9-2, and HI Ox9-3). The other branch was itself branched, withB. starrii, B. stolpii, and marine strain BM4 in separate sub-branches. AllBdellovibrio strains in turn clustered with representatives of the delta division of theProteobacteria. The results indicate that there are at least two subdivisions of the genusBdellovibrio and that present-day bdellovibrios arose from a common ancestor. The placement of the genusBdellovibrio within the delta division of theProteobacteria was confirmed.     

The outer cyst wall ofBdellovibrio bdellocysts is made de novo and not from preformed units from the prey wall

Bdellovibrio sp. strain W bdellocysts were produced inEscherichia coli using three sources of³H-diaminopimelic acid (DAP) for incorporation into the cyst wall peptidoglycan: (a) labeledE. coli peptidoglycan, (b) labeledBdellovibrio peptidoglycan, and (c) exogenous³H-DAP in the encystment medium. After cysts were produced, they were either sonicated to remove the prey cell wall, or germinated to solubilize the cyst wall. The results show that label was incorporated into the cyst wall preferentially from the exogenous DAP in the medium, and not from the bdellovibrio or bdelloplast peptidoglycan. The encysting bdellovibrio does not therefore incorporate existing peptidoglycan units from the bdelloplast for synthesis of the cyst wall.     

Formation of Stable Bdelloplasts as a Starvation-Survival Strategy of Marine Bdellovibrios

Several wild-type isolates of marine bdellovibrios formed stable bdelloplasts when they infected gram-negative bacterial prey under certain culture conditions. Synchronous predator-prey cultures and low nutrient concentrations increased the yield of stable bdelloplasts. The bdellovibrio cells retained in the stable bdelloplasts showed a high survival capacity in nutrient-depleted saline solution (10% viable Bdellovibrio cells after 3 months at 25°C), whereas Bdellovibrio attack-phase cells kept under the same starvation conditions lost viability more quickly (1% viable cells after 48 h). The addition of yeast extract to a stable bdelloplast suspension induced lysis of the bdelloplasts and release of motile infecting attack-phase Bdellovibrio cells. Other substances, such as free amino acids, protein hydrolysates, NH₄⁺, carbohydrates, and organic amines, did not induce such a release. Stable bdelloplasts were highly hydrophobic and had a lower endogenous respiration rate than attack-phase cells. In general, stable bdelloplasts were almost as sensitive to temperature changes, desiccation, sonication, tannic acid, and Triton X-100 treatment as attack-phase cells. Electron microscopy of stable bdelloplasts did not reveal any extra cell wall layer, either in the bdelloplast envelope or in the retained Bdellovibrio cells, unlike the bdellocysts of the soil bacterium Bdellovibrio sp. strain W. We propose that formation of stable bdelloplasts is a survival strategy of marine bdellovibrios which occurs in response to nutrient- and prey-poor seawater habitats.     

Alterations in the cell wall of Spirillum serpens VHL early in its association with Bdellovibrio bacteriovorus 109D

In both freeze-etched and critical-point dried preparations examined by transmission and scanning electron microscopy, respectively, the outer surfaces of the cells of Spirillum serpens VHL assume a wrinkled appearance 10–15 min after challenge by Bdellovibrion bacteriovorus 109D. This wrinkling effect is believed (on circumstantial evidence) to be caused by the bdellovibrio's disruption of the cell wall lipoprotein of the Spirillum. With the exception of those topological changes caused by wrinkling, the outer membrane of the Spirillum cell wall retains a normal appearance as viewed in freeze-etched preparations, even after the Spirillum cell has been converted into a bdelloplast. Although the peptidoglycan layer of the Spirillum cell presumably is weakened somewhat by the invading Bdellovibrio, evidence obtained from freeze-fractured preparations of Spirillum bdelloplasts suggests that the peptidoglycan remains as a discrete cell wall layer, even though the Spirillum cell wall apparently has lost much of its rigidity. That the peptidoglycan backbone remains essentially intact, even after the Spirillum cell has been entered by the Bdellovibrio, is supported by the observation that the soluble amino sugar content of the culture medium, as determined by chemical analysis, does not rise even 5.0 h after the association of the Bdellovibrio with the Spirillum has begun.     

Factors affecting growth of Bdellovibrio on Rhizobium

The extent of decline in the population density of Rhizobium sp. exposed to Bdellovibrio was markedly reduced in the presence of montmorillonite, kaolinite or vermiculite but not by a soil clay fraction. Increasing levels of montmorillonite reduced the numbers of vibrios that appeared in a two-membered culture and allowed for greater survival of the rhizobia. Bdellovibrio and not Rhizobium sp. was retained when mixed with the three clay minerals, but no appreciable retention was evident with the soil clay fraction. Suspensions of colloidal soil organic matter protected the hosts from parasitism, although aqueous extracts of soil did not affect the relationship. Cells from old Rhizobium sp. cultures were attacked only after a lag phase, but rhizobia that had been stored were more rapidly lysed than cells tested immediately after removal from the growth medium. The possible significance of these findings to the survival of rhizobia in soils containing Bdellovibrio is discussed.     

The maintenance ofBdellovibrio at low prey density

A mathematical model for the interaction ofBdellovibrio and its prey predicted that a relatively high prey density (7×10⁵ cells ml^–1) would be required for the establishment of an equilibrium in a mixed population [8]. The present report shows thatBdellovibrio can be maintained in a continuous culture when the prey cell density is much lower (2–5×10⁴ cells ml^–1), and closer to that of naturally occurring bacterial populations in sea waters.     

Growth-substrate dependent dechlorination of 1,2-dichloroethane by a homoacetogenic bacterium

A rod shaped, gram positive, non sporulating Acetobacterium strain was isolated that dechlorinated 1,2-dichloroethane (1,2-DCA) to ethene at a dechlorination rate of up to 2 nmol Cl^- min^-1 mg^-1 of protein in the exponential growth phase with formate (40 mM) as the substrate. Although with other growth substrates such as pyruvate, lactate, H₂/CO₂, and ethanol higher biomass productions were obtained,the dechlorination rate with these substrates was more than 10-fold lower compared with formate growing cells. Neither cell extracts nor autoclaved cells of the isolatedAcetobacterium strain mediated the dechlorination of 1,2-DCA at significant rates. The addition of 1,2-DCA to the media did not result in increased cell production. No significant differences in corrinoid concentrations could be measured in cells growing on several growth-substrates. However, these measurements indicated that differences in corrinoid structure might cause the different dechlorination activity. The Acetobacterium sp. strain gradually lost its dechlorination ability during about 10 transfers in pure culture, probably due to undefined nutritional requirements. 16S rDNA analysis of the isolate revealed a 99.7% similarity with Acetobacterium wieringae. However, the type strains of A. wieringae and A. woodii did not dechlorinate 1,2-DCA.     

Cloning, sequencing and expression of a Bacillus bacteriolytic enzyme in Escherichia coli

Summary Several hundred bacterial isolates were screened for bacteriolytic activity by growing them on agar medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells as the substrate. A Bacillus sp. producing the largest lytic zone was selected. A genomic bank of this selected bacterium was constructed in the multi-functional vector pTZ18R, with partial SauIIIA DNA fragments inserted at the SalI restriction site. Screening of 800 colonies of this bank for cell lysis gave 5 recombinants exhibiting lytic activity, as detected by analysis of extracts of sonicated Escherichia coli cells on denaturing polyacrylamide gels containing autoclaved, lyophilized M. lysodeikticus cells as the substrate. One clone (pBH2500), expressed inE. coli strain NM522, was found to code for a lytic enzyme corresponding, in molecular weight, to the 27 kDa Bacillus sp hydrolase. This clone with an insertion of 2.5 kb was then subcloned as a 929 bp EcoRI-SauIIIA fragment in pTZ18R (pBH929) and showed higher cell lytic activity. A unique open reading frame for a protein of 251 amino acids, followed by a putative terminator sequence, was found after a consensus ribosome binding site. A putative leader sequence was identified in the first 37 amino acids. One truncated subclone (pBH703), corresponding to 196 out of 251 residues from the protein N-terminal end, still possessed lytic activity.     

Prey Range Characterization, Ribotyping, and Diversity of Soil and Rhizosphere Bdellovibrio spp. Isolated on Phytopathogenic Bacteria

Thirty new Bdellovibrio strains were isolated from an agricultural soil and from the rhizosphere of plants grown in that soil. Using a combined molecular and culture-based approach, we found that the soil bdellovibrios included subpopulations of organisms that differed from rhizosphere bdellovibrios. Thirteen soil and seven common bean rhizosphere Bdellovibrio strains were isolated when Pseudomonas corrugata was used as prey; seven and two soil strains were isolated when Erwinia carotovora subsp. carotovora and Agrobacterium tumefaciens, respectively, were used as prey; and one tomato rhizosphere strain was isolated when A. tumefaciens was used as prey. In soil and in the rhizosphere, depending on the prey cells used, the concentrations of bdellovibrios were between 3 × 10² to 6 × 10³ and 2.8 × 10² to 2.3 × 10⁴ PFU g⁻¹. A prey range analysis of five soil and rhizosphere Bdellovibrio isolates performed with 22 substrate species, most of which were plant-pathogenic and plant growth-enhancing bacteria, revealed unique utilization patterns and differences between closely related prey cells. An approximately 830-bp fragment of the 16S rRNA genes of all of the Bdellovibrio strains used was obtained by PCR amplification by using a Bdellovibrio-specific primer combination. Soil and common bean rhizosphere strains produced two and one restriction patterns for this PCR product, respectively. The 16S rRNA genes of three soil isolates and three root-associated isolates were sequenced. One soil isolate belonged to the Bdellovibrio stolpii-Bdellovibrio starrii clade, while all of the other isolates clustered with Bdellovibrio bacteriovorus and formed two distantly related, heterogeneous groups.     

Cellular defense in an Arthropod in response to infection with aSalmonella typhimurium strain

Larvae ofGalleria mellonella L. when injected with cells ofSalmonella typhimurium strain LT 2 responded by cellular defense: their hemocytes gathered and formed a pseudo-tissue by which the bacteria were encapsulated. In contrast,S. typhimurium strain 7 Suc LL caused lysis of about 64% of the hemocytes and cellular defense against this strain was lacking.This explains the difference in the mortality rate of the larvae which was 10% after intracoelomic injection with 2×10⁴ cells of strain LT 2 and 48% with the same number of cells of strain 7 Suc LL.After injection of strain 7 Suc LL, lysis of hemocytes preceded proliferation of bacteria; moreover such lysis also occurred after injection of cell-free culture filtrate of this strain. This suggests that lysis is due to either a toxic substance or a proteolytic enzyme produced by strain 7 Suc LL.These investigations were supported by the Medical Research Council of Canada Grant MA-2385 and the National Research Council of Canada Grant A-3746.     

Cloning, purification, and characterization of chitosanase from Bacillus sp. DAU101

A chitosanase-producing Bacillus sp. DAU101 was isolated from Korean traditional food. This strain was identified on the basis of phylogenetic analysis of the 16S rDNA sequence, gyrA gene, and phenotypic analysis. The gene encoding chitosanase (csn) was cloned and sequenced. The csn gene consisted of an open reading frame of 837 nucleotides and encodes 279 amino acids with a deduced molecular weight of 31,420 Da. The deduced amino acid sequence of the chitosanase from Bacillus sp. DAU101 exhibits 88 and 30 % similarity to those from Bacillus subtilis and Pseudomonas sp., respectively. The chitosanase was purified by glutathione S-transferase fusion purification system. The molecular weight of purified enzyme was about 27 kDa, which suggests the deletion of a signal peptide by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The pH and temperature optima of the enzyme were 7.5 and 50 °C, respectively. The enzyme activity was increased by about 1.6-fold by the addition of 5 or 10 mM Ca²⁺. However, Hg²⁺ and Ni⁺ ions strongly inhibited the enzyme. The enzyme produced, GlcN_2–4, were the major products from a soluble chitosan.     

Production of Lytic Exoenzymes in Casamino Acids Media by Myxococcus virescens

The production of proteolytic enzymes and enzymes active on autoclaved aerobacter cells by Myxococcus virescens, strain B2, was studied In Casamino acids media. By modification of the N III–B media of Norén to a higher concentration of casamino acids and to a lower content of sodium chloride, the maximal production of lytic exoenzymes could be increased at least four times. Too high a concentration of casamino acids (more than 17 g/1) and sodium chloride (30 mM), as well as too low a concentration of sodium chloride, inhibited the enzyme production.     

Cloning,expression, and characterization of <Emphasis Type="Italic">β</Emphasis>-glucosidase from <Emphasis Type="Italic">Exiguobacterium</Emphasis> sp. DAU5 and transglycosylation activity

A gram-negative bacterium, designated strain DAU5, was isolated from shrimp shell samples because it demonstrated high β-glucosidase activity. Through 16S rDNA gene sequence analysis the strain was identified as belonging to the genus Exiguobacterium. The β-glucosidase gene of Exiguobacterium sp. DAU5 was successfully cloned by the shotgun method. Nucleotide sequence determination by sodium dodecyl sulfate-ployacrylamide gel electrophoresis indicated that the gene for the enzyme contained 1,350 bp, was coded by 450 amino acids, and was 52 kDa. The polypeptide exhibits significant homology with other bacterial β-glucosidases and belongs to the Glycoside Hydrolase Family 1. The β-glucosidase was purified by a His-fusion purification system. The optimal pH and temperature of the enzyme were 7.0 and 45°C, respectively. The enzyme activity was strongly inhibited by Ca²⁺, and Li⁺, K⁺, Zn²⁺, Mg²⁺, Na²⁺, Ni²⁺, and EDTA partially inhibited the enzyme activity. The BglA showed the highest activity with p-NPG and MUG. However, strain DAU5 β-glucosidase, which is for degradation of oligosaccharides, is expected to be useful for the fermentation of cellulose degradation and the transglycosylation of saccharides.     

A membrane filter direct count technique for enumeratingBdellovibrio

A membrane filter direct count method was devised for enumeratingBdellovibrio cells in “clean” suspensions. The procedure involves filtering a specified volume of a diluted, Trisbuffered suspension ofBdellovibrio cells through a known area of a 100-nm-pore-size Millipore brand membrane filter. A clarification solvent was used to render the filter transparent, so that the bdeloyvibrios on the filter could be photomicrographed and counted either visually or by means of a Quantimet 720 Image Analyzing Computer. The number ofBdellovibrio cells per milliliter in the undituted suspension could be calculated from the mean number of cells per unit area of the filter, the dilution factor, and the volume of diluted suspension filtered. TheBdellovibrio cells were distributed on the filters in a Poisson manner when there were not more than about 3.5 cells per 100 μm² of filter surface. The membrane filter direct counts correlated well with direct counts obtained by the Petroff-Hausser method. The correlation of direct counts with plaque (“viable”) counts showed that 80 to 95% of the direct-countedBdellovibrio cells in the clean suspensions were capable of forming plaques on lawns of suitable substrate bacteria. *** DIRECT SUPPORT *** A01R4002 00007     

Purification and Characterization of a Thermostable Alkaline Protease from Alkalophilic Thermoactinomyces sp. H8682

Protease secreted into the culture medium by alkalophilic Thermoactinomyces sp. HS682 was purified to an electrophoretically homogeneous state through only two chromatograhies using Butyl-Toyopearl 650M and SP-Toyopearl 650S columns. The purified enzyme has an apparent relative molecular mass of 25, 000 according to gel filtration on a Sephadex G-75 column and SDS-PAGE and an isoelectric point above 11.0.

Its proteolytic activity was inhibited by active-site inhibitors of serine protease, DFP and PMSF, and metal ions, Cu²⁺ and Hg²⁺. The enzyme was stable toward some detergents, sodium perborate, sodium triphosphate, sodium-n-dodecylbenzenesulfonate, and sodium dodecyl sulfate, at a concentration of 0.1% and pH 11.5 and 37°C for 60 min. The optimum pH was pH 11.5–13.0 at 37°C and the optimum temperature was 70°C at pH 11.5. Calcium divalent cation raised the pH and heat stabilities of the enzyme. In the presence of 5 mM CaCl₂, it showed maximum proteolytic activity at 80°C and stability from pH 4–12.5 at 60°C and below 75°C at pH 11.5. The stabilization by Ca²⁺ was observed in secondary conformation deduced from the circular dichroic spectrum of the enzyme. The protease hydrolyzed the ester bond of benzoyl leucine ester well. The amino acid terminal sequence of the enzyme showed high homology with those of Microbiol serine protease, although alanine of the NH₂-terminal amino acid was deleted.     

Peculiarities of Ethanol Metabolism in an Acinetobacter sp. Mutant Strain Defective in Exopolysaccharide Synthesis

Activities of the key enzymes of ethanol metabolism were assayed in ethanol-grown cells of an Acinetobacter sp. mutant strain unable to synthesize exopolysaccharides (EPS). The original EPS-producing strain could not be used for enzyme analysis because its cells could not to be separated from the extremely viscous EPS with a high molecular weight. In Acinetobacter sp., ethanol oxidation to acetaldehyde proved to be catalyzed by the NAD⁺-dependent alcohol dehydrogenase (EC 1.1.1.1.). Both NAD⁺ and NADP⁺ could be electron accepters in the acetaldehyde dehydrogenase reaction. Acetate is implicated in the Acinetobacter sp. metabolism via the reaction catalyzed by acetyl-CoA-synthetase (EC 6.2.1.1.). Isocitrate lyase (EC 4.1.3.1.) activity was also detected, indicating that the glyoxylate cycle is the anaplerotic mechanism that replenishes the pool of C₄-dicarboxylic acids in Acinetobacter sp. cells. In ethanol metabolism by Acinetobacter sp., the reactions involving acetate are the bottleneck, as evidenced by the inhibitory effect of sodium ions on both acetate oxidation in the intact cells and on acetyl-CoA-synthetase activity in the cell-free extracts, as well as by the limitation of the C₂-metabolism by coenzyme A. The results obtained may be helpful in developing a new biotechnological procedure for obtaining ethanol-derived exopolysaccharide ethapolan.     

Biodegradation of nicotine by a newly isolated Agrobacterium sp. strain S33

Aims: To isolate and characterize bacteria capable of degrading nicotine from the rhizospheric soil of a tobacco plant and to use them to degrade the nicotine in tobacco solid waste. Methods and Results: A bacterium, strain S33, was newly isolated from the rhizospheric soil of a tobacco plant, and identified as Agrobacterium sp. based on morphology, physiological tests, Biolog MicroLog3 4·20 system and 16S rRNA gene sequence. Using nicotine as the sole source of carbon and nitrogen in the medium, it grew optimally with 1·0 g l^?1 of nicotine at 30°C and pH 7·0, and nicotine was completely degraded within 6 h. The resting cells prepared from the glucose‐ammonium medium or LB medium could not degrade nicotine within 10 h, while those prepared from the nicotine medium could completely degrade 3 g l^?1 of nicotine in 1·5 h at a maximal rate of 1·23 g nicotine h^?1 g^?1 dry cell. Using the medium containing nicotine, glucose and ammonium simultaneously to cultivate strain S33, the resting cells could degrade 98·87% of nicotine in tobacco solid waste with the concentration as 30 mg nicotine g^?1 dry weight tobacco solid waste within 7 h at a maximal rate of 0·46 g nicotine h^?1 g^?1 dry cell. Conclusions: This is the first report that Agrobacterium sp. has the ability to degrade nicotine. Agrobacterium sp. S33 could use nicotine as the sole source of carbon and nitrogen. The use of resting cells of the strain S33 prepared from the nicotine–glucose–ammonium medium was an effective method to degrade nicotine and detoxify tobacco solid waste. Significance and Impact of the Study: Nicotine in tobacco wastes is both toxic and harmful to human health and the environment. This study showed that Agrobacterium sp. S33 may be suitable for the disposal of tobacco wastes and reducing the nicotine content in tobacco leaves.