Development of enterobacterium-specific oligonucleotide probes based on the surface-exposed regions of outer membrane proteins

Outer membrane proteins of members of the family Enterobacteriaceae consist of conserved membrane-spanning segments and hypervariable, surface-exposed regions. We demonstrate that the hypervariable DNA segments corresponding to the surface-exposed regions of these proteins can be used to develop specific DNA probes for the identification of members of the family Enterobacteriaceae.     

The psychrotrophic bacterium Yersinia enterocolitica requires expression of pnp, the gene for polynucleotide phosphorylase, for growth at low temperature (5°C)

The psychrotrophic bacterium Yersinia enterocolitica is characterized by temperature-dependent adaptations. To investigate Y . enterocolitica genes involved in cold adaptation, a mutant restricted in its ability to grow at 5°C was isolated from a transposon mutant library. The transposon insertion site in this psychrotrophy-defective (PD) mutant mapped 16 bp upstream of an open reading frame whose predicted amino acid sequence showed 93% similarity with the Escherichia coli exoribonuclease polynucleotide phosphorylase (PNPase), encoded by pnp . Expression of this gene was blocked in the PD mutant. However, the introduction of a second copy of pnp , including 0.33 kbp sequences upstream of its coding region, into the chromosome of the PD mutant restored pnp expression as well as the ability to grow at 5°C. Furthermore, the expression of pnp appeared to be temperature dependent: in the parental Y . enterocolitica strain, the levels of both pnp mRNA and PNPase were 1.6-fold higher at 5°C compared with 30°C. A similarly enhanced level of PNPase at 5°C was observed in the merodiploid recombinant strain, which indicates that the 0.33 kbp region upstream of pnp harboured a cold-inducible promoter. A putative cold shock promoter motif (ATTGG) was observed in this region.     

Corresponding increase in long-chain acyl-CoA and acylcarnitine after exercise in muscle from VLCAD mice

Long-chain acylcarnitines accumulate in long-chain fatty acid oxidation defects, especially during periods of increased energy demand from fat. To test whether this increase in long-chain acylcarnitines in very long-chain acyl-CoA dehydrogenase (VLCAD(-/-)) knock-out mice correlates with acyl-CoA content, we subjected wild-type (WT) and VLCAD(-/-) mice to forced treadmill running and analyzed muscle long-chain acyl-CoA and acylcarnitine with tandem mass spectrometry (MS/MS) in the same tissues. After exercise, long-chain acyl-CoA displayed a significant increase in muscle from VLCAD(-/-) mice [C16:0-CoA, C18:2-CoA and C18:1-CoA in sedentary VLCAD(-/-): 5.95 +/- 0.33, 4.48 +/- 0.51, and 7.70 +/- 0.30 nmol x g(-1) wet weight, respectively; in exercised VLCAD(-/-): 8.71 +/- 0.42, 9.03 +/- 0.93, and 14.82 +/- 1.20 nmol x g(-1) wet weight, respectively (P < 0.05)]. Increase in acyl-CoA in VLCAD-deficient muscle was paralleled by a significant increase in the corresponding chain length acylcarnitine. Exercise resulted in significant lowering of the free carnitine pool in VLCAD(-/-) muscle. This is the first study demonstrating that acylcarnitines and acyl-CoA directly correlate and concomitantly increase after exercise in VLCAD-deficient muscle.     

Comparison of p-Nitrophenol Biodegradation in Field and Laboratory Test Systems

Acclimation of microbial communities exposed to p-nitrophenol (PNP) was measured in laboratory test systems and in a freshwater pond. Laboratory tests were conducted in shake flasks with water, shake flasks with water and sediment, eco-cores, and two sizes of microcosm. The sediment and water samples used in the laboratory experiments were obtained from the pond. After a 6-day acclimation period, PNP was biodegraded rapidly in the pond. When the pond was treated with PNP a second time, biodegradation began immediately. The acclimation periods in laboratory test systems that contained sediment were similar to that in the pond. The acclimation period was threefold longer in shake flasks without sediment. PNP was biodegraded more slowly by microbial communities acclimated in the laboratory than it was in the pond, and the rate of biodegradation varied with the type of test. The number of bacteria able to mineralize PNP increased by 3 orders of magnitude in the pond during the acclimation period. Similar increases accompanied acclimation in the laboratory systems.     

The type antigen III of group F streptococci separation of group and type antigens and partial characterization of type III antigen

Polysaccharides extracted from Z₃III streptococci either with formamide or with dilute hydrochloric acid or isolated from the growth medium could be fractionated in type III- and group Z₃ antigens by alcohol precipitation. No good separation could be obtained from TCA extracts. When the same extractions and fractionations were applied to streptococci carrying type III antigen, but different group antigens, good separations were again obtained of all formamide extracts, but not of all hydrochloric acid extracts. The group antigens showed a rhamnose content of at least 50% and contained hexosamines. Type III antigens contained mainly rhamnose, glucose and galactose in relative amounts of approximately 1:2:3. Analysis of the methylated type III antigen suggests it to be a polysaccharide with a linear structure. Type III antigen isolated from the medium was characterized not only by a different sugar ratio, but also by its fucose content of 20%. In some cases the purified polysaccharides contained considerable amounts of glycogen-like material. Partial acid hydrolysis of the type III antigen extracted with formamide yielded a great number of oligosaccharides. Analyses, inhibition reactions and methylation studies gave indications that the most probable structure of a determinant group of type III antigen is β-glucosyl-(1-6)-galactosyl-(1-6)-galactosyl-(1-3)-rhamnose. The possibility of the existence of a second determinant group is discussed.     

Analysis of Streptococcal Cell Wall Fractions by Curie-Point Pyrolysis Gas-Liquid Chromatography

A streptococcal strain, classified as Z(3)III was differentiated from its mutant strain, Z(3), lacking the type III polysaccharide antigen, by Curie-point pyrolysis gas-liquid chromatography. Differences observed in pyrograms of whole cells or cell envelopes of both strains could be directly related to the pyrolysis pattern of the purified type III antigen. The same results were obtained when streptococcus F III and its mutant were analyzed. Whereas the pyrolysis patterns of the type III antigen extracted from Z(3)III and F III bacteria were identical, marked differences were found in pyrograms of the serologically identical type III antigen isolated from the culture medium. Type III antigen was also easily differentiated from the purified type I, II and IV antigens. From the above findings it was concluded that pyrolysis gas-liquid chromatography can be used as a tool for the quality control and identification of streptococcal cell wall components.     

Isolation and Characterization of Chloroplast DNA from the Duckweed Spirodela oligorrhiza

Chloroplast DNA of the duckweed Spirodela oligorrhiza, isolated by CsCl gradient centrifugation, was characterized by its buoyant density, guanine + cytosine content, melting behavior, circularity, and contour length. In all these characteristics, chloroplast DNA of S. oligorrhiza is similar to the chloroplast genomes of other higher plants, except that it has a significantly larger size.     

Streptococcus mutans and dental caries in humans: a bacteriological and immunological study

Plaque samples from caries-active subjects showed a higher incidence of S. mutans than plaque samples from caries-free subjects. This was especially evident in approximal incisor plaque. S. mutans serotype d was almost exclusively present in approximal plaque obtained from caries-active subjects. Tooth surfaces infected with S. mutans still harbored this micro-organism 10 months later, while uninfected tooth surfaces remained free of S. mutans.Caries development predominantly occurs on those tooth surfaces which harbor relatively high percentages of S. mutans (> 5%). It is unlikely that serum or saliva antibodies against S. mutans play a major role in the protection against dental caries in these caries-free subjects since subjects with the greatest number of decayed surfaces showed the highest antibody titre as measured by haemagglutination or by the enzyme-linked immuno sorbent assay (ELISA).The present investigation shows that serum or salivary antibodies against S. mutans do not seem to play a role in caries resistance in young adults. However, this does not preclude a role of antibodies, prior to infection with S. mutans, in man.     

Random amplified polymorphic DNA and restriction enzyme analysis of PCR amplified rDNA in taxonomy: two identification techniques for food-borne yeasts

The random amplified polymorphic DNA (RAPD) assay and the restriction enzyme analysis of PCR amplified rDNA are compared for the identification of the common spoilage yeasts Zygosaccharomyces bailii, Z. rouxii, Saccharomyces cerevisiae, Candida valida and C. lipolytica. Both techniques proved to be adequate tools for yeast identification. Since the RAPD does provide less stable patterns than restriction enzyme analysis of PCR amplified rDNA, and a large amount of data had to be compared without data reduction, Principal Component Analysis (PCA) was applied successfully for clustering the RAPD patterns. The success of PCA is highly influenced by the primer used in RAPD and the amount of reference samples. A large amount of reference samples improves the performance of clustering in PCA. The primer of choice was shown to be important with respect to the discriminatory power of the RAPD method. Some primers used enabled discrimination on the subspecies level. The results collected with both typing methods justify the conclusion that the present typing system can be applied for taxonomical purposes.