Network Relationships of Bacteria in a Stable Mixed Culture

We investigated the network relationships of bacteria in a structurally stable mixed culture degrading cellulose. The mixed culture consists of four bacterial strains (a cellulose-degrading anaerobe [strain S], a saccharide-utilizing anaerobe [strain F], a peptide- and acetate-utilizing aerobe [strain 3] and a peptide-, glucose-, and ethanol-utilizing aerobe [strain 5]). Interspecies interactions were examined by analyzing the effects of culture filtrates on the growth of the other strains and by comprehensively analyzing population dynamics in the mixed-culture systems with all possible combinations of the four bacterial strains. The persistence of strain S depends on the effects of strain 5. However, strain 5 is a disadvantaged strain because strain 3 has bacteriocidal activity on strain 5. The extinction of strain 5 is indirectly prevented by strain F that suppresses the growth of strain 3. Although strain F directly has suppressive effects on the growth of strain S, strain F is essential for the persistence of strain S, considering the indirect effects (maintaining strain 5, which is essential for the survival of strain S, by inhibiting strain 3). These indirect relationships form a bacterial network in which all the relationships including suppressive effects were well balanced to maintain the structural stability. In addition to direct metabolite interactions, such kind of indirect relationships could have a great impact on microbial community structure in the natural environment.     

Depth-dependent strain of patellofemoral articular cartilage in unconfined compression

This biomechanical study reports strain gradients in patellofemoral joint cross-sections of seven porcine specimens in response to 1% unconfined axial compression subsequent to specific amounts of off-set strain. Strain distributions were quantified with a customized laser-based electronic speckle pattern interferometry (ESPI) system in a non-contact manner, delivering high-resolution, high-sensitivity strain maps over entire patellofemoral cartilage cross-sections. Strain reports were evaluated to determine differences in strain magnitudes between the superficial, middle, and deep cartilage layers in femoral and patellar cartilage. In addition, the effect of 5%, 10%, 15%, and 20% off-set strain on depth-dependent strain gradients was quantified. Regardless of the amount of off-set strain, the superficial layer of femoral cartilage absorbed the most strain, and the deep layer absorbed the least strain. These depth-dependent strain gradients were most pronounced for 5% off-set strain, at which the superficial layer absorbed on average 5.7 and 23.7 times more strain as compared to the middle and deep layers, respectively. For increased off-set strain levels, strain gradients became less pronounced. At 20% off-set strain, differences in layer-specific strain were not statistically significant, with the superficial layer showing a 1.4 fold higher strain as the deep layer. Patellar cartilage exhibited similar strain gradients and effects of off-set strain, although the patellar strain was on average 19% larger as compared to corresponding femoral strain reports. This study quantified for the first time continuous strain gradients over patellofemoral cartilage cross-sections. Next to provision of a detailed functional characterization of normal diarthrodial joints, this novel experimental approach holds considerable attraction to investigate joint degenerative processes.     

Cloning and Characterization of the Genomic RNA Sequence of the Mumps Virus Strain Associated with a High Incidence of Aseptic Meningitis

cDNA clones of the mumps virus wild-type strain, associated with a high incidence of aseptic meningitis (ODATE-1 strain), were isolated and analyzed from genomic nucleotide position 22 to 8520 containing the NP, P, M., F, SH and HN protein coding region. The ODATE-1 strain exhibited a RFLP profile identical to that of the Urabe vaccine strain in spite of the fact that the virus was isolated from non-vaccinated cases. However, a comparison of nucleotide and amino acid sequences among the ODATE-1 strain, Urabe strain and Miyahara strain revealed that the ODATE-1 strain was not related to the Urabe strain.     

Coexistence of antibiotic-producing and antibiotic-sensitive bacteria in biofilms is mediated by resistant bacteria

Antibiotic-sensitive bacteria have been found to coexist with antibiotic-producing bacteria in biofilms, but little is known about how the former develop in such an environment. Here we isolated pyocyanin-sensitive bacteria belonging to the genus Brevibacillus from a biofilm derived from soil extract and based on the preestablished biofilm of a pyocyanin producer, Pseudomonas aeruginosa strain P1. In addition, pyocyanin-resistant strains belonging to the genus Raoultella were isolated from the same biofilm. Microbial relationships within biofilms were examined by using three strains, strain P1, Brevibacillus strain S1, and Raoultella strain R1, each of which individually formed a biofilm within 2 days in a flow cell. Strain S1 did not fully develop on the preestablished biofilm of strain P1 during 4 days of cultivation, whereas a mutant of strain P1 which was deficient in pyocyanin production allowed strain S1 to cocolonize within a biofilm. On the other hand, strain R1 developed on the biofilm of strain P1 regardless of pyocyanin production. When mixed 1:1 inocula of strains S1 and R1 were introduced into the strain P1 biofilm, all three species were found in the 4-day biofilm. In the mixed biofilm, strain S1 was surrounded by the layer of strain R1 and seemed to be separated from strain P1 and the outflow solution. However, strain S1 did not survive in a three-species mixed culture under planktonic conditions. These results indicate that the survival of sensitive bacteria in biofilm with a pyocyanin producer is achieved by covering them with a layer of resistant bacteria. We also evaluated the influence of antibiotic production on the producer.     

黑曲霉原生质体诱变选育β-葡萄糖苷酶高产菌株

本研究报道了以原生质体诱变技术选育高产β-葡萄糖苷酶的黑曲霉菌株,并研究了其发酵特性。以黑曲霉CGMCC3.316为出发菌株,通过紫外诱变得到突变株3-3M。然后以3-3M为供试菌株,研究了其原生质体制备与再生的条件。最后通过原生质体诱变,选育得到一株β-葡萄糖苷酶活力较高的突变株60B-3D。该菌株具有良好的遗传稳定性,酶活力平均达到23IU/mL,与出发菌株CGMCC3.316相比提高39%。此外,该菌株的木聚糖酶活力也有所增加。同时考察了黑曲霉60B-3D的发酵特性,并与3-3M和出发菌株进行比较,结果表明该菌株有较高的蛋白分泌能力。本研究为发酵生产β-葡萄糖苷酶提供了一株良好的供试菌株。     

Relationship between bone tissue strain and lattice strain of HAp crystals in bovine cortical bone under tensile loading

Cortical bone is a composite material composed of hydroxyapatite (HAp) and collagen. As HAp is a crystalline structure, an X-ray diffraction method is available to measure the strain of HAp crystals. However, HAp crystals in bone tissue have been known to have the low degree of crystallization. Authors have proposed an X-ray diffraction method to measure the lattice strain of HAp crystals from the diffusive intensity profile due to low crystallinity. The precision of strain measurement was greatly improved by this method. In order to confirm the possibility of estimating the bone tissue strain with measurements of the strain of HAp crystals, this work investigates the relationship between bone tissue strain on a macroscopic scale and the lattice strain of HAp crystals on a microscopic scale. The X-ray diffraction experiments were performed under tensile loading. Strip bone specimens of 40x6x0.8mm in size were cut from the cortical region of a shaft of bovine femur. A stepwise tensile load was applied in the longitudinal direction of the specimen. By detecting the diffracted X-ray beam transmitted through the specimen, the lattice strain was directly measured in the loading direction. As a result, the lattice strain of HAp crystals showed lower value than the bone tissue strain measured by a strain gage. The bone tissue strain was described with the mean lattice strain of the HAp crystals and the elastic modulus.     

Alternation of the Dissimilation Pathway for Glycerol and Amplification of Glycerol Dehydrogenase in Mutant Strains of Cellulomonas sp. NT3060

Mutant strain ME544, which is able to grow on glycerol slowly, was derived from glycerol-negative mutant strain G011, which is a derivative strain of Cellulomonas sp. NT3060 and is defective in both the enzyme activities of glycerol kinase and glycerol 3-phosphate dehydrogenase. The mutant strain still lacked both the enzyme activities involved in the dissimilation of glycerol and had the same level of glycerol dehydrogenase activity as the parent strain. Dihydroxyacetone kinase activity in mutant strain ME544 was inducibly formed, reaching 4-fold the level in mutant strain G011 in glycerol medium. Thus, the mutant strain seemed to dissimilate glycerol by means of glycerol dehydrogenase followed by an increase in dihydroxyacetone kinase. Subsequently, a mutant strain, GP1807, which was resistant to the inhibition of growth on glycerol by 1,2-propanediol, was derived from mutant strain ME544. Glycerol dehydrogenase activity of the mutant strain was amplified about 6-fold compared to that of the wild type strain.     

Mineralization of 2-chloro- and 2,5-dichlorobiphenyl by Pseudomonas sp. strain UCR2

Pseudomonas sp. strain UCR2 was isolated from a multi-chemostat mating experiment between a chlorobenzoate-degrader, Pseudomonas aeruginosa strain JB2, and a chlorobiphenyl-degrader, Arthrobacter sp. strain B1Barc. Strain UCR2 differed from either of the parental organisms in that it grew on both 2-chloro- and 2,5-dichlorobiphenyl with concomitant release of chloride. Phenotypic typing by the Biolog system indicated that strain UCR2 shared greater similarity with strain JB2 (88%) than strain B1Barc (3%). In DNA:DNA hybridization experiments, genomic DNA from strain UCR2 hybridized with both strain JB2 and strain B1Barc, with the former pairing yielding a much stronger signal than the latter. In contrast, no hybridization whatsoever was observed when the parental organisms strains JB2 and B1Barc were probed against each other.     

The effect of strain rate on the mechanical properties of human cortical bone

Bone mechanical properties are typically evaluated at relatively low strain rates. However, the strain rate related to traumatic failure is likely to be orders of magnitude higher and this higher strain rate is likely to affect the mechanical properties. Previous work reporting on the effect of strain rate on the mechanical properties of bone predominantly used nonhuman bone. In the work reported here, the effect of strain rate on the tensile and compressive properties of human bone was investigated. Human femoral cortical bone was tested longitudinally at strain rates ranging between 0.14-29.1 s(-1) in compression and 0.08-17 s(-1) in tension. Young's modulus generally increased, across this strain rate range, for both tension and compression. Strength and strain (at maximum load) increased slightly in compression and decreased (for strain rates beyond 1 s(-1)) in tension. Stress and strain at yield decreased (for strain rates beyond 1 s(-1)) for both tension and compression. In general, there seemed to be a relatively simple linear relationship between yield properties and strain rate, but the relationships between postyield properties and strain rate were more complicated and indicated that strain rate has a stronger effect on postyield deformation than on initiation of yielding. The behavior seen in compression is broadly in agreement with past literature, while the behavior observed in tension may be explained by a ductile to brittle transition of bone at moderate to high strain rates.     

Metabolism of thymineless mutants of Escherichia coli. I. Absence of thymidylate synthetase activity and growth characteristics of two sequential thymineless mutants

A study of optimal thymine and deoxythymidine (dThd) growth requirements of the thymineless mutants of Escherichia coli 15, E. coli 70-462 (strain 70), and a variant, E. coli 70V3-462 (strain 70V3), showed that for maximal turbidity (growth) strain 70 required 10-fold greater concentrations of thymine or dThd than did strain 70V3. On suboptimal concentrations of thymine or dThd, growth of strain 70 was greater on dThd than on thymine. In contrast, maximal growth of strain 70V3 was the same on equimolar concentrations of thymine and dThd. Growth rate of strain 70V3 was the same on equimolar concentrations of thymine and dThd up to 4 mum; at concentrations of 5 mum and greater, the "4-hr" growth was lower on dThd than on corresponding concentrations of thymine. Cultures of both thymineless mutants synthesized equal maximal amounts of DNA. Whereas strain 70V3 incorporated a maximum of 90% of the thymine or dThd in the media, strain 70 incorporated a maximum of only 10%. This poor utilization by strain 70 was neither a result of thymine or dThd conversion to a low-molecular-weight thymine derivative nor the production of a nonthymine inhibitory substance. Since strains 70 and 70V3 exhibited no thymidylate synthetase activity, the first mutation (strain 15 to strain 70) resulted in the loss of this activity. The second mutation (strain 70 to strain 70V3) probably brought about the loss of an enzyme(s) that catabolizes deoxyribose phosphate, permitting a greater net synthesis of dThd from thymine.     

Improvement of the quality control of thioglycol medium

The effect of thimerosal used at different concentrations on the growth of S. haemolyticus, strain Dick I, and A. faecalis, strain 415, has been studied. A. faecalis, strain 415, in contrast to S. haemolyticus, strain Dick I, has been shown to be highly sensitive to thimerosal. The growth and neutralizing properties of a number of lots of thyoglycol medium have been studied with the use of the above-mentioned strains. The advantages of A. faecalis, strain 415, over S. haemolyticus, strain Dick I, for the evaluation of these properties of thioglycol medium have been established. A. faecalis, strain 415, can be recommended for use as a test strain for evaluating the quality of thioglycol medium instead of S. haemolyticus, strain Dick I.     

Separation of a phenol carboxylating organism from a two-member, strict anaerobic co-culture

In a culture converting phenol to benzoic acid under anaerobic conditions and previously described as being constituted of only a Clostridium-like strain 6, another bacterium (strain 7) was observed. Each organism was enriched by centrifugation on a Percoll gradient. Strain 6 was purified by dilution and plating. Strain 7 did not grow on solid media, but a strain 7 culture, cleared of strain 6, was obtained by subculturing in the presence of ampicillin and by dilution. In fresh medium, phenol was transformed by the reconstituted co-culture but not by each strain alone. In a supernatant from a co-culture or from a strain 6 culture, strain 7 alone transformed phenol but not strain 6. Maintenance of an active strain 7 in fresh medium instead of co-culture supernatant became possible when phenol was replaced by 4-hydroxybenzoate (4-OHB), which is decarboxylated to phenol before being transformed to benzoate. Even with 4-OHB, the use of co-culture (or strain 6 culture) supernatant resulted in faster transformation activity and growth rate. A phylogenetic analysis placed strain 7 in a cluster of uncultivated or nonisolated bacteria (92-96% homology). Strain 7 is also related to Desulfotomaculum, Desulfitobacterium, Desulfosporosinus, Moorella, and Sporotomaculum genera (87-92% homology).     

Characterization of recombinant rabies virus carrying double glycoprotein genes

A recombinant rabies virus carrying double glycoprotein (G) genes, R(NPMGGL) strain, was generated by a reverse genetics system utilizing cloned cDNA of the RC-HL strain, and the biological properties of the virus were compared to those of the recombinant RC-HL (rRC-HL) strain. The extents of virus growth in cultured cells and virulence for adult mice of the R(NPMGGL) strain were almost same as those of the rRC-HL strain, while G protein content of the purified R(NPMGGL) virion and G protein expression level in R(NPMGGL)-infected cells were 1.5-fold higher than those of the rRC-HL strain. As a result of serial passages of the R(NPMGGL) strain in cultured cells, the expression level of G protein in cultured cells infected with the passaged R(NPMGGL) strain was maintained and virus titers rose with adaptation to the cultured cells. Furthermore, analysis of neutralization titers in mice immunized with UVinactivated virus suggested that the R(NPMGGL) strain had higher immunogenicity than that of the rRC-HL strain. The results suggest that the R(NPMGGL) strain carrying double G genes might be a useful candidate for development of a new inactivated rabies vaccine.     

A non-dechlorinating strain of Dehalospirillum multivorans: evidence for a key role of the corrinoid cofactor in the synthesis of an active tetrachloroethene dehalogenase

A strain of Dehalosprillum multivorans, designated strain N, was isolated from the same source as the formerly described tetrachloroethene (PCE)-dechlorinating D. multivorans, herein after referred to as strain K. Neither growing cells nor cell extracts of strain N were able to dechlorinate PCE. The pceA and pceB genes encoding for the PCE-reductive dehalogenase were detected in cells of strain N; and they were 100% homologous to the corresponding genes of strain K. Since the PCE dehalogenase of D. multivorans strain K contains a corrinoid cofactor, the corrinoids of strain N cells were extracted. Analysis of the corrinoids revealed the absence of the specific corrinoid, which is the cofactor of the PCE dehalogenase of strain K cells. RT-PCR of mRNA indicated that the pceA gene was transcribed in strain N cells to a far lower extent than the pceA gene of strain K under the same experimental conditions. Western blot analysis of crude extracts of strain N showed that, if at all, an insignificant amount of the apoprotein of the PCE dehalogenase was present. The results indicate that the inability of strain N to dechlorinate is due to the absence of the corrinoid cofactor of the enzyme mediating PCE dechlorination.     

微生物菌种保藏方法及保藏关键技术

菌种是国家的重要生物资源,也是生产、教学和科学研究的基本材料。为确保菌种的质量和活力,需要正确的菌种保藏方法。阐述了菌种保藏的重要性、菌种保藏常见方法及其存在的问题,探讨了常用菌种的保藏技术及关键点,好的保藏方法可延长菌种的保存时间,又可防止菌种退化。     

Comparison of cellular strain with applied substrate strain in vitro

Strain magnitudes within tenocytes undergoing substrate tensile strain are not well defined. It was hypothesized that strain magnitudes at the cellular level would reflect those of the applied substrate (equibiaxial or uniaxial) strain. A vacuum-operated device was used to apply equibiaxial or uniaxial tension to a flexible substrate upon which tenocytes were cultured in monolayer. Images of tenocytes labeled with Fura-2, to detect free intracellular calcium ions, and MitoFluor Green, to detect mitochondria, were taken prior to strain and for 20 min during application of static strain. A custom-written, texture correlation program computed strain magnitudes in the cell based on the change in pixel pattern displacements between images of non-strained and strained cells. On average, cellular strain was approximately 37+/-8% and 63+/-11% of the applied equibiaxial and uniaxial substrate strain, respectively. The largest cell strains were detected in cells oriented parallel to the direction of applied uniaxial tensile strain. However, strain magnitudes within a cell were heterogeneous. The variance in strain magnitude within and among tenocytes is dependent on cell orientation, cell stiffness, cytoskeleton organization, subcellular organelles, or placement and type of cell-substrate contacts. Results of the present study indicate that cultured tenocytes experience a moderate fraction of the applied substrate strain.     

Three-dimensional strain fields in a uniform osteotomy gap

Stable internal fixation usually results in a unique histological healing pattern which involves direct cortical reconstruction and an absence of periosteal bridging callus. While it has been suggested that longitudinal interfragmentary strain levels control this healing pattern, the complex, multiaxial strain fields in the interfragmentary region are not well understood. Based on an in-vivo study of gap healing in the sheep tibia by Mansmann et al., we used several finite element models of simplified geometry to: explore modeling assumptions on material linearity and deformation kinematics, and examine the strain distribution in a healing fracture gap subjected to known levels of interfragmentary strain. We found that a general nonlinear material, nonlinear geometric analysis is necessary to model an osteotomy gap subjected to a maximum longitudinal strain of 100 percent. The large displacement, large strain conditions which were used in the in-vivo study result in complex, multiaxial strain fields in the gap. Restricting the maximum longitudinal strain to 10 percent allows use of a linear geometric formulation without compromising the numerical results. At this reduced strain level a linear material model can be used to examine the extent of material yielding within a homogeneous osteotomy gap. Severe local strain variations occurred both through the thickness of the gap and radially from the endosteal to periosteal gap surfaces. The bone/gap interface represented a critical plane of high distortional and volumetric change and principal strain magnitudes exceeded the maximum longitudinal strains.     

Characteristics of Saccharomyces cerevisiae gal1 Delta and gal1 Delta hxk2 Delta mutants expressing recombinant proteins from the GAL promoter

Galactose can be used not only as an inducer of the GAL promoters, but also as a carbon source by Saccharomyces cerevisiae, which makes recombinant fermentation processes that use GAL promoters complicated and expensive. To overcome this problem during the cultivation of the recombinant strain expressing human serum albumin (HSA) from the GAL10 promoter, a gal1 Delta mutant strain was constructed and its induction kinetics investigated. As expected, the gal1 Delta strain did not use galactose, and showed high levels of HSA expression, even at extremely low galactose concentrations (0.05-0.1 g/L). However, the gal1 Delta strain produced much more ethanol, in a complex medium containing glucose, than the GAL1 strain. To improve the physiological properties of the gal1 Delta mutant strain as a host for heterologous protein production, a null mutation of either MIG1 or HXK2 was introduced into the gal1 Delta mutant strain, generating gal1 Delta mig1 Delta and gal1 Delta hxk2 Delta double strains. The gal1 Delta hxk2 Delta strain showed a decreased rate of ethanol synthesis, with an accelerated rate of ethanol consumption, compared to the gal1 Delta strain, whereas the gal1 Delta mig1 Delta strain showed similar patterns to the gal1 Delta strain. Furthermore, the gal1 Delta hxk2 Delta strain secreted much more recombinant proteins (HSA and HSA fusion proteins) than the other strains. The results suggest that the gal1 Delta hxk2 Delta strain would be useful for the large-scale production of heterologous proteins from the GAL10 promoter in S. cerevisiae.     

Effect on aflatoxin production of competition between wild-type and mutant strains of Aspergillus parasiticus

Co-cultivation of a strain of Aspergillus parasiticus, capable of making aflatoxins, with blocked mutant strains, capable of producing none or only a low level of aflatoxins, reduced the net yield of aflatoxins more than that expected based on spore recovery. Yields of aflatoxins were 8-fold less for a norsolorinic acid-producing strain, 14-fold less for an averantin-producing strain, 6-fold less for an averufin-producing strain, and 21-fold less for a versicolorin A-producing strain when co-cultured in equal amounts with a wild-type strain of Aspergillus parasiticus. Even when the wild-type strain was initially present in 100-fold excess, with two of the mutant strains, reduced aflatoxin production was still observed.