Identification of the monooxygenase gene clusters responsible for the regioselective oxidation of phenol to hydroquinone in mycobacteria

Mycobacterium goodii strain 12523 is an actinomycete that is able to oxidize phenol regioselectively at the para position to produce hydroquinone. In this study, we investigated the genes responsible for this unique regioselective oxidation. On the basis of the fact that the oxidation activity of M. goodii strain 12523 toward phenol is induced in the presence of acetone, we first identified acetone-induced proteins in this microorganism by two-dimensional electrophoretic analysis. The N-terminal amino acid sequence of one of these acetone-induced proteins shares 100% identity with that of the protein encoded by the open reading frame Msmeg_1971 in Mycobacterium smegmatis strain mc(2)155, whose genome sequence has been determined. Since Msmeg_1971, Msmeg_1972, Msmeg_1973, and Msmeg_1974 constitute a putative binuclear iron monooxygenase gene cluster, we cloned this gene cluster of M. smegmatis strain mc(2)155 and its homologous gene cluster found in M. goodii strain 12523. Sequence analysis of these binuclear iron monooxygenase gene clusters revealed the presence of four genes designated mimABCD, which encode an oxygenase large subunit, a reductase, an oxygenase small subunit, and a coupling protein, respectively. When the mimA gene (Msmeg_1971) of M. smegmatis strain mc(2)155, which was also found to be able to oxidize phenol to hydroquinone, was deleted, this mutant lost the oxidation ability. This ability was restored by introduction of the mimA gene of M. smegmatis strain mc(2)155 or of M. goodii strain 12523 into this mutant. Interestingly, we found that these gene clusters also play essential roles in propane and acetone metabolism in these mycobacteria.     

Interactions of bacteria with different mechanisms for chitin degradation result in the formation of a mixed-species biofilm

In this study, interactions between bacteria possessing either released or cell-associated enzymes for polymer degradation were investigated. For this, a co-culture of Aeromonas hydrophila strain AH-1N as an enzyme-releasing bacterium and of Flavobacterium sp. strain 4D9 as a bacterium with cell-associated enzymes was set up with chitin embedded into agarose beads to account for natural conditions, under which polymers are usually embedded in organic aggregates. In single cultures, strain AH-1N grew with embedded chitin, while strain 4D9 did not. In co-cultures, strain 4D9 grew and outcompeted strain AH-1N in the biofilm fraction. Experiments with cell-free culture supernatants containing the chitinolytic enzymes of strain AH-1N revealed that growth of strain 4D9 in the co-culture was based on intercepting N-acetylglucosamine from chitin degradation. For this, strain 4D9 had to actively integrate into the biofilm of strain AH-1N. This study shows that bacteria using different chitin degradation mechanisms can coexist by formation of a mixed-species biofilm.     

Modification of the pathogenicity of Schistosoma mattheei for sheep by passage of the parasite in hamsters

Border Leicester X Suffolk sheep infected with a strain of S. mattheei maintained in hamsters do not develop the same pathological changes as Romney Marsh sheep infected with the same strain of parasite before hamster passage. To determine the cause of this reduced pathogenicity, five Romney Marsh sheep were each infected with 10 000 cercariae of the hamster-passaged parasite and five with 10 000 cercariae of a S. mattheei strain from Onderstepoort, South Africa, passaged exclusively through sheep. Striking pathological and parasitological differences were found between the two strains. Infection with the "sheep" strain was lethal, whereas infection with the "hamster" strain produced little evidence of clinical disease. By 13 weeks post-infection the mean body weight of the sheep infected with the sheep strain had declined by 15% compared with both the uninfected controls and the sheep infected with the hamster strain, and the mean PCV was lowered to 20% in the sheep strain infected animals. Egg production began at seven weeks with the sheep strain, faecal counts rising to more than 300 e.p.g., whereas only two of the sheep infected with the hamster strain passed eggs in the faeces (at nine weeks) and the maximum egg count was 50 e.p.g. Twice as many adult worms of the sheep strain were recovered, and, although the number of eggs found in the tissues "per worm pair" was not significantly different, overall egg production was higher for the sheep strain; also more of the sheep strain eggs were deposited in the intestines. Similar parasite differences were seen in a supplementary study in mice and it seemed that "attenuation" of the parasite had occurred, presumably due to its maintenance in hamsters. Histopathological observations and faecal egg counts both indicated an inability of hamster strain eggs to penetrate the intestinal lumen; this was probably important in reducing the pathogenicity of the hamster strain.     

Two oscillators might control the locomotor activity rhythm of the high-altitude Himalayan strain of Drosophila helvetica

The properties of the pacemaker controlling the adult locomotor activity rhythm of the high-altitude Himalayan (haH) strain (Hemkund Sahib, 4121 m above sea level) of Drosophila helvetica are strikingly different from those of the low-altitude Himalayan (laH) strain (Birahi, 1132 m above sea level) of the same species. The haH strain has a unimodal activity pattern with a delayed peak occurring about 4.5 h after lights-on of the entraining light-dark (LD) cycle, while the laH strain has a bimodal activity pattern with the morning and evening peaks. It is rather unusual for a wild type strain of any Drosophila species to have a unimodal activity pattern during entrainment as observed in the haH strain. The single activity peak of the haH strain is regarded as a consequence of delayed morning peak merging with the evening one. Three experiments were performed to test this hypothesis. The first experiment examined whether the single activity peak could be dissociated into two components by LD cycles in which photoperiods varied from 10 to 16 h per 24 h. The haH strain again exhibited a unimodal activity pattern with a delayed peak in 10, 12, and 14 h photoperiods but a bimodal activity pattern in 16 h photoperiod. The laH strain had bimodality in 10 and 12 h photoperiods, unimodality in a 14 h photoperiod, but complete arrhythmicity in a 16 h photoperiod. In the second experiment, the haH flies were transferred from LD 16:8 to LL at 5 lux to confirm whether the bimodality of this strain in LD 16:8 cycles was not the result of masking by the long photoperiod of 16 h. Bimodality of the haH strain persisted in LL too; moreover, the morning component free-ran with period (tau) <24 h, while the evening component free-ran with tau>24 h. The third experiment examined the LL-induced splitting of activity peak of the haH strain. Flies were transferred from LD 12:12 cycles to LL at 0, 1, 5, and 15 lux. The haH strain was rhythmic in LL at 0 and 1 lux with a unimodal activity pattern. It was also rhythmic in LL at 5 lux, but the single activity peak was split into two discrete components; the morning component free-ran with tau<24 h, while the evening component free-ran with tau>24 h. This strain, however, was completely arrhythmic in LL at 15 lux. The laH strain was uniformly arrhythmic in LL at all levels of light intensity. These results suggest that the single but late activity component of the haH strain during entrainment appears to be the consequence of merging the delayed morning peak with the evening one as an adaptation to the environmental conditions at the altitude of origin of this strain, where these flies begin activity in the forenoon owing to non-permissible low temperature in the morning.     

A comparative study of the inhibitory effect of ethanol and substrates on the fermentation rate of parent and a respiratory-deficient mutant

The inhibitory effect of alcohol and substrates on the fermentation rate of one strain of Candida pseudotropicalis and of a respiratory-deficient mutant of this strain is investigated. For the parent strain maximum fermentative activity is identical in the presence of glucose or lactose. For a respiratory-deficient mutant, the fermentation rate is always higher than that of the parent strain. The inhibitory effect of alcohol and substrate is always less with the respiratory-deficient mutant than with the parent strain.     

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.     

Distribution of capsular materials on the cell wall surface of strain Smith diffuse of Staphylococcus aureus.

The fine structure of the capsule of Staphylococcus aureus Smith diffuse was examined by the technique of freeze-substitution and immunoelectron microscopy. The cell surface was covered with a thick layer consisting of fine fibrous structures which were absent from an unencapsulated strain, Smith compact. Anti-teichoic acid antibody did not react with this surface layer but reacted with the surface of strain Smith compact. Anti-capsular antibody, made from the serum of a rabbit immunized with strain Smith diffuse and specific absorption with unencapsulated strain Wood 46, reacted with the fibrous layer of the Smith diffuse strain. Since the anti-teichoic acid antibody did not react with the encapsulated strain Smith diffuse, the capsular layer acts as a barrier to penetration of the anti-teichoic acid antibody through the capsular layer. A portion of a few cell surfaces of the encapsulated strain remained accessible to the anti-teichoic acid antibody. The capsular layer in this portion of the cell surface was thin, and this surface seemed to be a new cell wall surface created by the cell separation.     

Effects of changes in the mitochondrial genome on the performance of baking yeasts

Modifications to the mitochondria in baking yeasts affect their performance in doughraising tests significantly. Conversion of a respiratory-competent baking strain, sensitive to glucose repression, to the petite mutant, yielded a strain which was released from this effect, as shown by the increased development of the cytochrome c peak in the cytochrome spectrum, and which showed a comparably improved dough-raising performance, approaching the levels found for a respiratory-competent strain which was fully derepressed. Replacement of the mitochrondria of one strain by those from another, in some cases, improved the performance of the recipient strain, especially if the latter was sensitive to glucose repression. In addition reduction of glucose repression in single-spore clones derived from the composite strains, as determined by the increased size of the cytochrome c peak, usually segregated 2∶2 both for this character and for improved dough-raising capability as well. Presented in part at the European Biotechnology Symposium, May 8–10, 1986, Como, Italy.     

The relationship of the antigenic characteristics of the tick-borne encephalitis virus to the level of the protective activity of inactivated cultured vaccines]

As shown in this study, the immunization of animals with killed vaccines against tick-borne encephalitis (TBE) leads to the formation of specific immunity, depending on the antigenic structure of the vaccine strain and the test strains used for challenge. Vaccines obtained on the basis of the TBE virus strain of the Eastern antigenic variant induced the development of a wider spectrum of specific protective activity than vaccines obtained on the basis of the TBE virus strain of the Western antigenic variant.     

Superproduction and rapid purification of Escherichia coli aspartate transcarbamylase and its catalytic subunit under extreme derepression of the pyrimidine pathway

A strain of Escherichia coli has been constructed which greatly overproduces the enzyme aspartate transcarbamylase. This strain has a deletion in the pyrB region of the chromosome and also carries a leaky mutation in pyrF. Although this strain is a pyrimidine auxotroph, it will grow very slowly without pyrimidines if a plasmid containing the pyrB gene is introduced into it. Derepression occurs when this strain exhausts its uracil supply during exponential growth. Under extreme derepression, aspartate transcarbamylase can account for as much as 60% of the total cellular protein. This host strain/plasmid system can be utilized for the rapid purification of wild-type aspartate transcarbamylase or plasmid-born mutant versions of the enzyme. This system is particularly well-suited for analysis of the latter since the control of overproduction resides exclusively on the bacterial chromosome. Therefore, any plasmid bearing the pyrBI operon can be made to overproduce aspartate transcarbamylase in this host strain. Based on this system, a rapid purification procedure has been developed for E. coli aspartate transcarbamylase. The purification scheme involves an ammonium sulfate fractionation followed by a single precipitation of the enzyme at its isoelectric point. In a similar fashion, this strain can also be employed to produce exclusively the catalytic subunit of the enzyme if the plasmid only carries the pyrB gene. This system may be adapted to overproduce other proteins as well by using this host strain and the strong pyrB promoter linked to another gene.     

Ascorbic acid might play a role in the sclerotial differentiation of Sclerotium rolfsii

Certain phytopathogenic fungi differentiate by forming sclerotia by an unclear biochemical mechanism. We have proposed that sclerotial differentiation might be regulated by fungal antioxidant defense. Part of this defense might be ascorbic acid, which in its reduced form is a well-known antioxidant. This natural antioxidant was studied in Sclerotium rolfsii in relation to oxidative-growth conditions, developmental stages and strain-differentiating ability. The transition of a sclerotial strain from the undifferentiated to the differentiated stage was accompanied by a sharp shift in the ratio of reduced/oxidized ascorbate toward the oxidized form. Ascorbate profiles and lipid peroxidation levels were different between the sclerotial strain grown under high- and low-oxidative stress conditions, as well as between a nonsclerotial S. rolfsii strain grown under high-oxidative stress conditions. In addition, the ratio of reduced/oxidized ascorbate in the nonsclerotial strain remained unchanged throughout growth. Lipid peroxidation under high-oxidative stress conditions in sclerotial S. rolfsii colonies one day before differentiation was 3.6-fold higher than in same-day colonies of this strain grown under low-oxidative stress conditions and 2.5-fold higher than in similar-day colonies of the nonsclerotial strain grown under high-oxidative stress conditions. Exogenous ascorbate caused a concentration-dependent reduction of lipid peroxidation and a proportional inhibition of the degree of sclerotial differentiation in the sclerotial strain grown under high-oxidative stress conditions by lowering its lipid peroxidation before differentiation to levels similar to the strain grown under low-oxidative stress conditions and to the nonsclerotial strain. Ascorbic acid might be produced by the sclerotial strain to reduce oxidative stress, although less efficiently than the nondifferenting strain. The data of this study support our theory that oxidative stress might be the triggering factor of sclerotial differentiation in phytopathogenic fungi.     

The role of the vacB gene in the pathogenesis of Brucella abortus

Brucella species are important zoonotic pathogens affecting a wide variety of mammals. Therefore, the identification of new Brucella virulence factors is of great interest in understanding bacterial pathogenesis and immune evasion. In this study, we have identified Brucella abortus vacB gene that presents 2343 nucleotides and 781 amino acids and it shows 39% identity with Shigella flexneri vacB gene that encodes an exoribonuclease RNase R involved in bacterial virulence. Further, we have inactivated Brucella vacB by gene replacement strategy generating a deletion mutant strain. In order to test the role of Brucella vacB in pathogenesis, BALB/c and interferon regulatory factor-1 (IRF-1) knockout (KO) mice received Brucella vacB mutant, the virulent parental strain 2308 or the vaccine strain RB51 and the bacterial CFU numbers in spleens and mous survival were monitored. Our results demonstrated that the B. abortus DeltavacB mutant and the wild type strain 2308 showed similar CFU numbers in BALB/c mice. Additionally, IRF-1 KO mice that received either the vacB mutant or S2308 strain died in 12-14 days postinfection; in contrast, all animals that received the RB51 vaccine strain survived for 30 days postinoculation. In summary, this study reports that the vacB gene in B. abortus has no impact on bacterial pathogenesis.     

Measurement of the circumferential mechanical properties of the umbilical vein: experimental and numerical analyses

Coronary artery disease is responsible for almost 30% of all deaths worldwide. The saphenous vein and umbilical vein (UV) are the most common veins using for treatment as a coronary artery bypass graft (CABG). The mechanical properties of UV belonging to its long-term patency for CABG are very important. However, there is a lack of knowledge on the linear elastic and nonlinear hyperelastic mechanical properties of the UV. In this study, three stress definitions (second Piola–Kichhoff stress, engineering stress and true stress) and four strain definitions (Almansi–Hamel strain, Green–St Venant strain, engineering strain and true strain) are used to determine the elastic modulus, maximum stress and strain of eight human UVs under circumferential loading. The nonlinear mechanical behaviour of the UV is computationally investigated using Mooney–Rivlin hyperelastic model. A numerical finite element analysis is also carried out to simulate the constitutive modelling versus its numerical results. The results show that the Almansi–Hamel strain definition overestimates the elastic modulus while Green–St Venant strain definition underestimates the elastic modulus at different stress definitions. The true stress–true strain definition, which gives more accurate measurements of the tissue's response using the instantaneous values, reveals the Young's modulus and maximum stress of 2.18 and 6.01 MPa, respectively. The Mooney–Rivlin material model is well represented by the nonlinear mechanical behaviour of the UV. The findings of this study could have implications not only for understanding the extension and rupture mechanism of UV but also for interventions and surgeries, including balloon angioplasty, bypass and stenting.     

The effect of loading rate on the development of early damage in articular cartilage

Experimental reports suggest that cartilage damage depends on strain magnitude. Additionally, because of its poro-viscoelastic nature, strain magnitude in cartilage can depend on strain rate. The present study explores whether cartilage damage may develop dependent on strain rate, even when the presented damage numerical model is strain-dependent but not strain-rate-dependent. So far no experiments have been distinguished whether rate-dependent cartilage damage occurs in the collagen or in the non-fibrillar network. Thus, this research presents a finite element analysis model where, among others, collagen and non-fibrillar matrix are incorporated as well as a strain-dependent damage mechanism for these components. Collagen and non-fibrillar matrix stiffness decrease when a given strain is reached until complete failure upon reaching a maximum strain. With such model, indentation experiments at increasing strain rates were simulated on cartilage plugs and damage development was monitored over time. Collagen damage increased with increasing strain rate from 21 to 42 %. In contrast, damage in the non-fibrillar matrix decreased with increasing strain rates from 72 to 34 %. Damage started to develop at a depth of approximately 20 % of the sample height, and this was more pronounced for the slow and modest loading rates. However, the most severe damage at the end of the compression step occurred at the surface for the plugs subjected to 120 mm/min strain rate. In conclusion, the present study confirms that the location and magnitude of damage in cartilage may be strongly dependent on strain rate, even when damage occurs solely through a strain-dependent damage mechanism.     

The effect of aerosolization on the changes in major biological properties of the vaccine strain of Francisella tularensis

The exposure of F. tularensis vaccine strain 15/10 in the form of aerosol for more than 10 minutes results in the decrease of its virulence, immunogenicity and the content of species-specific antigen and in the increase of the dissociation level. Proceeding from this fact, during aerosol immunization with this vaccine strain exposure must not exceed 5 minutes for unstabilized aerosol and 10 minutes for aerosol stabilized with 5% glycerin. Under these conditions the properties of F. tularensis strain 15/10 are retained practically on the initial level.     

A novel mechanical loading model for studying the distributions of strain and mechano-growth factor expression

Mechano-growth factor (MGF), an insulin-like growth factor-I (IGF-I) splice variant, often serves as an important local tissue repair factor in response to the mechanical environment. However, there is no model for exhibiting the MGF expression in a series of strain distribution up to now. In this study, a novel mechanical loading model containing different stresses and strains simultaneously was developed to examine the MGF expression. The strain distributions were predicted by finite element modeling. The MC3T3-E1 cells on a silicone membrane with a central circular hole were exposed to a variable strain environment through stretching. The finite element analysis showed that, when the strain reached the magnitude of 10%, the strain concentration near the circular hole displayed along with the vertical stretch direction, while the minimum strain appeared in the parallel stretch direction. Furthermore, the results showed that MGF expression decreased gradually from high to low strain regions by immunocytochemistry. Meanwhile, the proliferation of osteoblasts increased significantly in the high strain region. In conclusion, this mechanical loading model can present the different distributions of the strain of osteoblasts in vitro. MGF expression and osteoblast proliferation have a high correlation with the levels of strain.     

Mechanisms underlying modifications in the severity of audiogenic convulsions

Catecholamine levels were selectively decreased in peripheral sites or in brain of mice by intravenous or intraventricular injections of 6-hydroxydopamine. Brain dopamine alone was decreased by administering i.p. desmethylimipramine prior to 6 hydroxydopamine injections. A brain noradrenaline decrease does not induce mortality in a tonic strain of mice but can transform clonic audiogenic seizures into tonic seizures in a clonic mouse strain, while a dopamine depletion alone has no effect. However, a noradrenaline decrease at peripheral sites increases the number of lethal seizures in a tonic strain without modifying clonic seizures in a clonic strain. Catecholamine metabolism is neither involved in the onset of seizures in a non-sensitive strain nor involved in the change of clonus into tonus in unilaterally deafened tonic strain animals, which in this case present only clonic seizures.     

Temperature sensitivity of the cdc9-1 allele of Saccharomyces cerevisiae DNA ligase is dependent on specific combinations of amino acids in the primary structure of the expressed protein.

Summary In this study we present the characterization of the temperature-sensitive mutant allele cdc9-1 encoding DNA ligase, of Saccharomyces cerevisiae strain A364A by DNA sequencing. Comparison with the published wild-type sequence from strain SKI revealed 13 nucleotide exchanges between these two sequences, which are derived from non-isogenic genetic backgrounds. Only four of these changes, distributed over the whole coding region, lead to amino acid exchanges in the protein chain. Our analysis of the sequence of the wild-type CDC9 allele from strain A364A revealed differences from the isogenic cdc9-1 allele in only two nucleotides: one silent change and one leading to a single amino acid exchange. The latter is therefore responsible for the temperature-sensitive phenotype. A mosaic protein, in which a region carrying this amino acid exchange has been inserted in place of the corresponding part of CDC9 from the non-isogenic strain SKI, is not temperature sensitive. The exchange of a longer stretch of DNA leading to atteration of three amino acids of the protein compared with the original sequence of SKI is required to obtain a temperature-sensitive DNA ligase in this strain, while in strain A364A a single amino acid change is sufficient for expression of a temperature-sensitive protein.