Fluorescent Antibody Study of the Gram-Positive Anaerobic Cocci

Fluorescent antibody conjugates were prepared from five species of anaerobic cocci commonly isolated from human infections. When tested with homologous and heterologous cells these conjugates were found to be highly specific. There was no evidence of a common genus antigen. Peptococcus magnus conjugates detected a species-specific antigen; cross-reactions with Peptostreptococcus anaerobius were readily eliminated by absorption. The conjugates from Peptococcus asaccharolyticus, Peptococcus prevotii, Peptostreptococcus, anaerobius, and Peptostreptococcus intermedius displayed a high degree of strain specificity. Occasional cross-reactions were detected with homologous strains, suggesting the presence of common antigens, but no attempt was made to determine the number of different serotypes in these species.     

Analysis of EDTA-soluble cell surface components of gram-positive anaerobic cocci

The protein content of EDTA extracts from 76 strains of Gram-positive anaerobic cocci was examined using SDS-PAGE. Strains of Peptostreptococcus anaerobius produced almost identical profiles; greater heterogeneity was observed within the species Peptococcus magnus, Peptococcus prevotii and Peptococcus asaccharolyticus, but several strains within each biotype produced similar patterns. Serological investigation of these extracts by ELISA revealed numerous cross-reactions among the different biotypes. Immunoblot transfers from polyacrylamide gels demonstrated two common antigens within strains of the species, Ps. anaerobius, but these were not species-specific.     

An rRNA approach for assessing the role of obligate amino acid-fermenting bacteria in ruminal amino acid deamination.

Ruminal amino acid degradation is a nutritionally wasteful process that produces excess ruminal ammonia. Monensin inhibited the growth of monensin-sensitive, obligate amino acid-fermenting bacteria and decreased the ruminal ammonia concentrations of cattle. 16S rRNA probes indicated that monensin inhibited the growth of Peptostreptococcus anaerobius and Clostridium sticklandii in the rumen. Clostridium aminophilum was monensin sensitive in vitro, but C. aminophilum persisted in the rumen after monensin was added to the diet. An in vitro culture system was developed to assess the competition of C. aminophilum, P. anaerobius, and C. sticklandii with predominant ruminal bacteria (PRB). PRB were isolated from a 10(8) dilution of ruminal fluid and maintained as a mixed population with a mixture of carbohydrates. PRB did not hybridize with the probes to C. aminophilum, P. anaerobius, or C. sticklandii. PRB deaminated Trypticase in continuous culture, but the addition of C. aminophilum, P. anaerobius, and C. sticklandii caused a more-than-twofold increase in the steady-state concentration of ammonia. C. aminophilum, P. anaerobius, and C. sticklandii accounted for less than 5% of the total 16S rRNA and microbial protein. Monensin eliminated P. anaerobius and C. sticklandii from continuous cultures, but it could not inhibit C. aminophilum. The monensin resistance of C. aminophilum was a growth rate-dependent, inoculum size-independent phenomenon that could not be maintained in batch culture. On the basis of these results, we concluded that the feed additive monensin cannot entirely counteract the wasteful amino acid deamination of obligate amino acid-fermenting ruminal bacteria.     

Polyclonal antibodies against Staphylococcus aureus ATCC 12 600 catalase do not recognize any protein in cellular extracts from S. aureus subsp. anaerobius

Rabbits were immunized with electrophoretically pure catalase from Staphylococcus aureus ATCC 12 600. The antiserum was used to study whether S. aureus subsp. anaerobius was able to synthesize the apoprotein of catalase. Proteins were separated on polyacrylamide gels (SDS-PAGE) and transferred to nitrocellulose membranes and were detected by immunoblotting. No protein reacting with the purified immunoglobulins against S. aureus ATCC 12,600 catalase could be detected in crude and partially purified cellular extracts from S. aureus subsp. anaerobius or its aerotolerant mutants.     

Description of strains of Peptostreptococcus anaerobius isolated from subcutaneous abscesses in cats.

Strains of Peptostreptococcus, Streptococcus and of a Gram-positive coccus, which was initially isolated as an anaerobe but grew subsequently as a facultative organism, were isolated from subcutaneous abscesses in cats. The cat strains of Peptostreptococcus gave metabolic fermentation products in combinations described for P. anaerobius. The Streptococcus strains conformed to the group S. intermedius. The facultative organism described had the metabolic products of P. anaerobius but the distinctly different biochemical characteristics of S. intermedius and fits neither of the genera strictly.     

Extracellular Deoxyribonuclease Production by Anaerobic Bacteria

The production of extracellular deoxyribonuclease was examined with anaerobic organisms isolated from clinical specimens. Nuclease activity was extraordinarily common. All strains of Fusobacterium, including eight species, as well as Bacteroides fragilis and B. melaninogenicus, displayed enzyme activity. Whereas the gram-positive bacteria were generally less productive, all strains of Clostridium perfringens, Peptostreptococcus intermedius, and P. anaerobius specifically produced deoxyribonuclease. The test is taxonomically valuable, particularly in the characterization of gram-positive cocci, since a deoxyribonuclease-producing coccus indicates P. intermedius or P. anaerobius. Additionally, possession of the enzyme may prove to be a useful correlate of the potential pathogenicity of anaerobes.     

Modifying action of oxytocin on the biological properties of the causative agents of anaerobic non-clostridial infection

In a number of in vitro experiments the effect of oxytocin on the antilysozyme and anticomplemental activity of Propiobacterium propionicum, Bacteroides fragilis, Prevotella melaninogenica and Peptostreprtococcus anaerobius, isolated from patients with acute pyoinflammatory pleuropulmonary diseases, was studied. Antibiotic resistance dynamics of the infective agents under study to lincomycin, clindamycin, thienam, vancomycin was also detected. The inhibiting activity of oxytocin on the persistence properties of B. fragilis, P. melanogenica and P. anaerobius was noted. Under the influence of the preparations used changes in the sensitivity of the strains to a number of antibiotics of the lincosamide, carbapenem and glycopeptide groups were found to occur. The data thus obtained were indicative of the possible mechanisms of action of oxytocin in the treatment of acute pyoinflammatory pleuropulmonary diseases of anaerobic nonclostridial etiology.     

A phylogenetic analysis of staphylococci, Peptococcus saccharolyticus and Micrococcus mucilaginosus

The intra- and intergeneric relationships of the genus Staphylococcus, and the phylogenetic position of Peptococcus saccharolyticus and Micrococcus (Staphylococcus salivarius), were investigated by comparative oligonucleotide cataloguing of 16S rRNA. All the staphylococci investigated form a phylogenetically coherent group at the genus level that, in addition, contains the anaerobic species Peptococcus saccharolyticus. The genus Staphylococcus belongs to the broad Bacillus-Lactobacillus-Streptococcus cluster that is defined by Gram-positive bacteria with a low DNA G+C content. Micrococcus mucilaginosus is not a genuine member of the genus Micrococcus. The binary matching coefficients between the 16S rRNA of Micrococcus mucilaginosus and those representatives of the Arthrobacter/Micrococcus group and related genera indicate that Micrococcus mucilaginosus should be regarded as a member of a new genus.     

Selenophosphate synthetase 2 is essential for selenoprotein biosynthesis

Selenophosphate synthetase (SelD) generates the selenium donor for selenocysteine biosynthesis in eubacteria. One homologue of SelD in eukaryotes is SPS1 (selenophosphate synthetase 1) and a second one, SPS2, was identified as a selenoprotein in mammals. Earlier in vitro studies showed SPS2, but not SPS1, synthesized selenophosphate from selenide, whereas SPS1 may utilize a different substrate. The roles of these enzymes in selenoprotein synthesis in vivo remain unknown. To address their function in vivo, we knocked down SPS2 in NIH3T3 cells using small interfering RNA and found that selenoprotein biosynthesis was severely impaired, whereas knockdown of SPS1 had no effect. Transfection of SPS2 into SPS2 knockdown cells restored selenoprotein biosynthesis, but SPS1 did not, indicating that SPS1 cannot complement SPS2 function. These in vivo studies indicate that SPS2 is essential for generating the selenium donor for selenocysteine biosynthesis in mammals, whereas SPS1 probably has a more specialized, non-essential role in selenoprotein metabolism.     

Accumulation of porphyrins and pyrrole pigments by Staphylococcus aureus ssp. anaerobius and its aerobic mutant

Abstract The anaerobic, Gram-positive coccus Staphylococcus aureus ssp. anaerobius and its aerobic mutant MVF-SR, when kept under anaerobic conditions, excreted coproporphyrin (mainly type III) into the medium and enriched uroporphyrin (mainly type I) within the cells.
The rate of porphyrin synthesis stayed practically unaltered when the growth medium was supplemented with 50 μ g/ml 5-aminolevulinic acid (ALA), but was significantly enhanced upon supplementation with hemin (0.5 μ g/ml). When hemin and ALA were given simultaneously, a more than two-fold increase in porphyrin production compared to normal growth medium was observed. These observations indicate a stimulation of porphyrin synthesis in S. aureus by hemin.
An as yet unidentified violet pigment with an intense red-violet fluorescence under UV light ( λ = 366 nm) was found to be present in considerable amounts in cells of S. aureus ssp. anaerobius , whereas the supernatant medium of aerobically grown cells of the mutant MVF-SR contained an equally unidentified blue, non-fluorescing pigment.     

蔗糖磷酸合酶功能、结构与催化机制的研究进展

蔗糖是自然界中广泛存在的一种天然产物.在植物等生命体中,蔗糖磷酸合酶(Sucrose phosphate synthase,SPS)是蔗糖合成的限速酶.SPS催化合成蔗糖-6-磷酸;蔗糖磷酸酶(Sucrose Phosphatase,SPP)进一步把蔗糖-6-磷酸上的磷酸根水解下来而形成蔗糖.近几十年来关于SPS的研究...     

Enumeration and isolation of anaerobic microbiota of piggery wastes.

Media for enumeration of the microbiota of anaerobically stored piggery wastes were tested. Highest colony counts were obtained with 80 to 100% farm slurry supernatant included in the anaerobic roll tube media. Colony counts with these media numbered 2 X 10(9) to 12 X 10(9)/g (wet weight), which represents about 20% of the microscopic counts. Lower percentages of slurry supernatant in the media gave lower colony counts. Addition of glucose, cellobiose, and starch or of Trypticase to media with 20% slurry supernatant did not increase colony counts. Higher values were obtained when hemicellulose preparations were added to these media. Incubation at 25 degrees C gave the highest numbers. Incubation at 15 to 37 degrees C gave counts of about 70 and 10%, respectively, of those at 25 degrees C. Of the colonies picked for isolation, about 20% were obtained in pure culture. The isolates apparently belonged to the genera Peptococcus, Ruminococcus, Peptococcus, Ruminococcus, Pepostreptococcus, and Bacteroides.     

Selective antibacterial action of 2-mercaptoethanol on propionibacteria in skin cultures.

2-Mercaptoethanol applied to the surface of agar medium had a selective antibacterial effect on Propionibacterium acnes and Propionibacterium granulosum without interfering with the growth of Peptococcus saccharolyticus or staphylococci in anaerobic cultures of skin or in pure cultures. In aerobic broth culture, 2-mercaptoethanol inhibited aerobes and stimulated anaerobes, consistent with its action as a reducing agent.     

Identification and subcellular localization of two solanesyl diphosphate synthases from Arabidopsis thaliana

Two solanesyl diphosphate synthases, designated SPS1 and SPS2, which are responsible for the synthesis of the isoprenoid side chain of either plastoquinone or ubiquinone in Arabidopsis thaliana, were identified. Heterologous expression of either SPS1 or SPS2 allowed the generation of UQ-9 in a decaprenyl diphosphate synthase-defective strain of fission yeast and also in wild-type Escherichia coli. SPS1-GFP was found to localize in the ER while SPS2-GFP localized in the plastid of tobacco BY-2 cells. These two different subcellular localizations are thought to be the reflection of their roles in solanesyl diphosphate synthesis in two different parts: presumably SPS1 and SPS2 for the side chains of ubiquinone and plastoquinone, respectively.     

Expression of a cyanobacterial sucrose-phosphate synthase from Synechocystis sp. PCC 6803 in transgenic plants

Sucrose-phosphate synthase (SPS) from the cyanobacterium Synechocystis sp. PCC 6803 lacks all of the Ser residues known to be involved in the regulation of higher plant SPS by protein phosphorylation. The Synechocystis SPS is also not allosterically regulated by glucose 6-phosphate or orthophosphate. To investigate the effects of expressing a potentially unregulated SPS in plants, the Synechocystis sps gene was introduced into tobacco, rice and tomato under the control of constitutive promoters. The Synechocystis SPS protein was expressed at a high level in the plants, which should have been sufficient to increase overall SPS activity 2-8-fold in the leaves. However, SPS activities and carbon partitioning in leaves from transgenic and wild-type plants were not significantly different. The maximal light-saturated rates of photosynthesis in leaves from tomato plants expressing the Synechocystis SPS were the same as those from wild-type plants. Tomato plants expressing the maize SPS showed 2-3-fold increases in SPS activity, increased partitioning of photoassimilate to sucrose and up to 58% higher maximal rates of photosynthesis. To investigate the apparent inactivity of the Synechocystis SPS the enzyme was purified from transgenic tobacco and rice plants. Surprisingly, the purified enzyme was found to have full catalytic activity. It is proposed that some other protein in plant cells binds to the Synechocystis SPS resulting in inhibition of the enzyme.     

Role of sucrose-phosphate synthase in sucrose metabolism in leaves

Sucrose is formed in the cytoplasm of leaf cells from triose phosphates exported from the chloroplast. Flux control is shared among key enzymes of the pathway, one of which is sucrose-phosphate synthase (SPS). Regulation of SPS by protein phosphorylation is important in vivo and may explain diurnal changes in SPS activity and carbon partitioning. The signal transduction pathway mediating the light activation of SPS in vivo appears to involve metabolites and novel “coarse” control of the protein phosphatase that dephosphorylates and activates SPS. Regulation of the phosphorylation of SPS may provide a general mechanism whereby sucrose formation is coordinated with the rate of photosynthesis and the rate of nitrate assimilation. There are apparent differences among species in the properties of SPS that may reflect different strategies for the control of carbon partitioning. The SPS gene has recently been cloned from maize; results of preliminary studies with transgenic tomato plants expressing high levels of maize SPS support the postulate that SPS activity can influence the partitioning of carbon between starch and sucrose.     

Sucrose phosphate synthase, a key enzyme for sucrose biosynthesis in plants: protein purification from corn leaves and immunological detection

We have purified the protein for the enzyme sucrose phosphate synthase (SPS) from corn (Zea mays) leaves. Partially purified SPS protein was used to generate specific monoclonal antibodies. The following immunoaffinity chromatography allowed the isolation of pure SPS protein. The apparent molecular mass of the SPS polypeptide is 138 kilodaltons. By immunoblot, an SPS antigen was found to accumulate in mature leaves. SPS protein levels remain constant during the day/night cycle. The observed diurnal fluctuation of extractable enzyme activity, therefore, must be caused by modification of the specific activity of SPS in vivo.