- Volume 139, Issue 9, 1993

SUMMARY: Fingerprinting by random amplification of polymorphic DNA (RAPD) was compared with existing molecular typing systems for Candida albicans. Fifteen isolates were chosen, including three from the same patient; these gave 14 distinct karyotypes by pulsed-field gel electrophoresis (PFGE) and 7 different DNA types by EcoRI-generated restriction fragment length polymorphisms (RFLPs). RAPD with primer I (5' GCT GGT GG3') gave 5 types, whereas primer II (5' GCG CAC GG3') yielded 11 types. Combining the results from both primers, all isolates were unique by RAPD with the exception of the three from the same patient. RAPD provided a fast, economical and reproducible means of typing C. albicans with a level of discrimination approaching that of PFGE.

SUMMARY: The ability of motile cells to remove small inanimate particles from solid substrata is well documented. We show here that motile cells will pick up and internalize infectious particles of the obligate intracellular parasite Chlamydia trachomatis when they are adherent to the substratum over which the host cells move. Two cell types were used to assess chlamydial uptake; a feeder independent human squamous cell carcinoma variant (AC3A cells) and the McCoy cell line. Purified chlamydial elementary bodies were attached to glass or collagen-coated glass by centrifugation. Suspensions of cells were then allowed to sediment on to the substrata to which chlamydiae had attached. Both types of cell picked up chlamydiae and transported them over their surface during the course of attachment and spreading. Stereoscopic images obtained by confocal microscopy demonstrated that chlamydiae were found mainly on the surface of non-spread cells. After the cells had spread on the substratum they began to move around forming tracks where the chlamydiae had been removed. Some cell-surface-attached chlamydiae were endocytosed and a proportion of these proliferated during the 48 h after plating. However, chlamydiae attached to the substratum lost infectivity by a simple exponential decay process within a few hours of incubation in the extracellular environment. Therefore, increasing numbers of non-viable organisms were probably endocytosed as the time of extracellular incubation increased. This mode of infection may be relevant to in vivo situations where cell migration occurs after damage to mucosal surfaces.

SUMMARY: HPLC analysis revealed that Yersinia enterocolitica WA-C produced two substances under iron-limiting conditions one of which was identified as 2,3-dihydroxybenzoyl-L-serine. The other compound had iron-complexing activity and was called yersiniabactin. The fur mutant H1852 was shown to produce yersiniabactin constitutively in an iron-independent manner. Yersiniabactin was isolated by ethyl acetate extraction from the spent medium of H1852, size-fractionation chromatography and preparative HPLC. A catechol function was demonstrated with different chemical assays and by UV-visible spectroscopy. The molecular mass of yersiniabactin was determined to be 482 Da. Purified yersiniabactin stimulated growth of Y. enterocolitica and Escherichia coli Ø under iron-limiting conditions and apparently served as an iron carrier. Transport of 55Fe-yersiniabactin was TonB-dependent, indicating a receptor-mediated uptake across the outer membrane. A pesticin-resistant mutant missing the receptor protein FyuA was unable to transport and use yersiniabactin as a siderophore.

SUMMARY: The sensitizing effect and the local and general toxicity related to membrane components of the archaeobacterium Sulfolobus solfataricus was studied. Cell envelope fragments were biologically active but this activity was lost upon separation of the lipid and protein components. The envelope fragments exerted lethal effects on mice sensitized with D-galactosamine that were prevented by pretreatment with anti-TNF-α serum. This lethal activity occurred in both LPS-responder (BALB/cByJ) and LPS-nonresponder (C3H/HeJ) mouse strains. In addition, Sulfolobus envelope fragments tested in rabbits caused a local Shwartzman reaction, and showed pyrogenic activity. In vitro, the envelope fragments that act on spleen lymphocytes of the LPS-responder (BALB/cByJ) and LPS-nonresponder (C3H/HeJ) mice caused an uptake of [3H]thymidine similar to that caused by concanavalin A. A similar toxic activity to that exerted by eubacteria is therefore exerted by this non-pathogenic archaeobacterium despite the difference in surface chemistry.

SUMMARY: Rabbit erythrocytes were agglutinated by 43.4 % of group B streptococci isolated from bovines but by none isolated from humans. Haemagglutination was enhanced by cultivation of the bacteria under microaerophilic conditions. Most of the haemagglutinating strains had protein type antigen X, either alone, or in combination with polysaccharide antigens. Heat and proteolytic treatment of the bacteria destroyed the haemagglutination activity. The haemagglutinin could be solubilized from the bacterial surface by mutanolysin treatment and isolated from culture supernatant fluid by ammonium sulphate precipitation. The isolated haemagglutinin did not cause direct agglutination of erythrocytes. However, binding of the haemagglutinin to rabbit erythrocytes could be visualized by agglutination of haemagglutinin-treated erythrocytes by specific antiserum obtained by absorption. Western blotting showed that the haemagglutinin obtained from erythrocyte lysates contained an antibody-reactive band with a molecular mass of 43 kDa. Haemagglutination-positive strains adhered to HeLa cells in higher numbers than did haemagglutination-negative strains. The HeLa cell adherence of Group B streptococci was inhibited in the presence of isolated haemagglutinin or of specific antiserum against the haemagglutinin. These observations suggest that the haemagglutinating adhesins of bovine group B streptococcal isolates are directly involved in the adherence mechanisms of these organisms.

SUMMARY: The macromolecular composition of Lactococcus was regulated by growth rate in the same general way as that of less fastidious bacteria such as Escherichia coli and Salmonella typhimurium. The ratios of RNA:DNA and RNA:protein increased approximately threefold over a 13.5-fold increase in growth rate, whereas the ratio of DNA:protein remained approximately constant. Using reporter genes fused to a DNA fragment of a cloned lactococcal rRNA operon, promoter activity was located upstream of the 16S rRNA structural gene. This DNA fragment had some characteristics typical of a rrn promoter in E. coli. Two consensus promoter sequences P1 and P2 were located 296 and 157 bp, respectively, upstream of the start of the 16S rRNA gene. Between P2 and the start of the 16S rRNA gene, sequences were identified with typical anti-termination motifs characteristic of E. coli rrn promoter regions. A putative transcription terminator sequence was identified downstream of the 5S rRNA gene and putative primary RNA transcript processing sites at both ends of the lactococcal rRNA operon were also noted.

SUMMARY: The Streptococcus mutans dexB gene, a member of the multiple sugar metabolism (msm) operon, encodes an intracellular glucan 1,6-α-glucosidase which releases glucose from the non-reducing terminus of α-1,6-linked isomaltosaccharides and dextran. Comparison of primary amino acid sequences showed strong homology to Bacillus oligo-1,6-glucosidases and, like these enzymes, DexB was able to release free glucose from the α-1,4,6-branch point in panose. This suggested a role for DexB in the metabolism of either starch or intracellular polysaccharide, which contain such branch points. However, purified intracellular polysaccharide from the wild-type S. mutans strain LT11 and a mutant deficient in dexB revealed no substantial differences in the extent of branching as demonstrated by iodine staining spectra and the degree of polymerization. Furthermore, thin layer chromatography of radiolabelled intracellular polysaccharide digested with S. mutans wild-type and mutant cell extracts showed no differences in the products obtained. The involvement of DexB in dietary starch metabolism was investigated using α-limit dextrins produced from the action of α-amylase on starch. These induced the msm operon, including dexB, and the DexB enzyme was able to act on the α-limit dextrins to give further fermentable substrates. The transport system encoded by the msm operon can also transport α-limit dextrin. DexB may therefore be important in the metabolism of extracellular starch.

SUMMARY: In monocultures of micro-organisms, growth is controlled by feedback mechanisms involving chemical factors such as limiting substrates and inhibitory metabolic products. The role of such feedback in the growth regulation of Escherichia coli O-124 was investigated by growing cells in batch culture using a medium containing glucose and mineral salts. In various phases of growth, portions of the native culture were diluted with culture filtrate, so that although cell density decreased, the chemical composition of the growth medium was unaltered. As the diluted cultures grew, variations in growth acceleration were calculated and compared with those of native (undiluted) cultures. Towards the end of the exponential phase and in the growth deceleration phase, the specific feedback level (FBL) was between −20 and −200 (h g 1-1)-1. The feedback components resulting from changes in glucose concentration were calculated using experimentally determined values of μ;max (0.55.0.05 h-1) and K s (2.5 ± 0.7 mg I-1). Only 0.1-40% of FBL could be accounted for by changes in glucose concentration, indicating the presence of additional growth regulators. The method developed may become a new tool for determination of growth-regulating cell-density factors in microbial cultures.

SUMMARY: β-Glucanase synthesis was temporally activated in Bacillus subtilis at the onset of stationary phase. This regulation was dependent upon a drop in the GTP concentration in response to nutrient limitation. The Spo0A and AbrB proteins were involved in the GTP-dependent temporal activation.

SUMMARY: In the budding yeast Saccharomyces cerevisiae cyclic AMP (cAMP) can influence the activity of key enzymes in carbohydrate metabolism through modulation of the activity of cAMP-dependent protein kinase. One of the components involved in cAMP production is the CDC25 gene product, which can activate the RAS/adenylate cyclase pathway by promoting the exchange of guanine nucleotides bound to RAS. In two yeast strains carrying different thermosensitive alleles of the CDC25 gene, cAMP levels respond differently to an increase in growth temperature from 23 °C (permissive) to 36 °C (restrictive). In strain OL86 (cdc25-5) the estimated intracellular concentration of cAMP dropped after transfer to restrictive temperature whereas in strain ts321 (cdc25-1) the cAMP level rose under the same conditions. Despite the differences in cAMP levels the glycolytic flux in the two mutants responded in a very similar way to the shift from permissive to restrictive temperature; after the increase in the incubation temperature, the specific glycolytic flux in both cdc25-1 and cdc25-5 initially increased from about 300 nmol min-1 (mg protein)-1 to about 500 nmol min-1 (mg protein)-1 (presumably mainly as a consequence of the increase in temperature), but then gradually fell to 100-200 nmol min-1 (mg protein)-1. A similar pattern of CO2 production to that found in the two cdc25 mutants was also observed for several other thermosensitive mutants displaying a Start-II type of G1 arrest. In contrast, in a wild-type strain and in strains giving a Start-I type of G1 arrest, CO2 production did not drop after a temperature shift. The specific activities of glycolytic enzymes in the two cdc25 mutants did not show much change after the temperature shift, indicating that the decrease in glycolytic flux was not caused by a decrease in the activity of any of the glycolytic enzymes. Our data show that, at least in long-term regulation, the cAMP levels per se are not likely to be a prime factor controlling glycolytic flux.

SUMMARY: Penicillium simplicissimum excreted more than 100 mmol citric acid l-1 [2-9 mmol (g dry wt)-1; 9 d] if an industrial filter dust (> 50 % ZnO) providing a high extracellular buffering capacity was present in the medium. A similar specific [2 mmol (g dry wt)-1], but lower absolute (26 mmol l-1), citric acid excretion occurred in the absence of an extracellular buffer and if amino acids or urea were used as nitrogen source. P. simplicissimum excreted no citric acid under conditions where Aspergillus niger produces citric acid (deficiency of trace elements, low pH and reduced biomass formation). Citric acid excretion by P. simplicissimum always paralleled biomass formation and occurred in a pH range between 4 and 7. This indicated that different imbalances of metabolism were responsible for citric acid excretion in A. niger and P. simplicissimum. However, provided a high extracellular buffering capacity was present, the response of the Penicillium system to different carbon and nitrogen sources was similar to the Aspergillus system. In contrast, the metals iron and copper had virtually no effect on citric acid excretion compared with A. niger. Estimation of intracellular citric acid, as well as the effects of the uncoupler 2,4-dinitrophenol, and the H+-ATPase inhibitor sodium orthovanadate, led to the conclusion that the buffer-stimulated citric acid efflux was dependent on metabolic energy and an energized plasma membrane, respectively. Despite similarities to the Aspergillus system, a different mechanism for buffer-stimulated citric acid excretion by P. simplicissimum seems probable.

SUMMARY: Three isolates of Phytophthora palmivora showing differing sensitivities to phosphonate were grown in the presence of a uniform concentration of the anion (1 mM), together with concentrations of phosphate which varied from zero to 3 mM. The phosphonate-sensitive isolate was inhibited by phosphonate at all levels of phosphate, but the resistant isolates were inhibited by phosphonate only when phosphate was limiting to growth and were able to exclude phosphonate more effectively than the sensitive isolate at higher concentrations of phosphate. The percentage inhibition in each strain was proportional to the internal concentration of phosphonate, but the slope of the plot of inhibition against internal phosphonate varied between strains. Differences in the capacity to discriminate between phosphate and phosphonate therefore provide part of, but not the whole explanation for differences in sensitivity between isolates. There was a significant increase in the internal concentration of orthophosphate in the mycelia which were inhibited by phosphonate.

SUMMARY: The closely related Frankia strains BR, S21 and Thr, isolated from the genus Casuarina, Allo2 and Dec from the genus Allocasuarina, and G80 from the genus Gymnostoma, could be distinguished by their esterase zymograms after ultra-low gelling point agarose-polyacrylamide electrophoresis. The kanamycin-resistant derivatives S21-kR and BR-kR could also be differentiated from their kanamycin-sensitive parental strains. Different patterns of intracellular esterases could be obtained by using β-naphthyl propionate or 3-indoxyl acetate as substrates. The substrate 5-bromo-4-chloro-3-indoxyl acetate proved particularly useful, revealing a variety of esterases from Gymnostoma isolates. Zymograms of aminopeptidases from all strains were found to be quite similar. Nevertheless, they allowed differentiation of Frankia strains BR and S21 from their kanamycin-resistant derivatives. Aminopeptidase and esterase zymograms obtained from Gymnostoma isolates were markedly different from all others. Zymograms of esterases and aminopeptidases may prove useful for the identification of some other Frankia strains.

SUMMARY: Thermus thermophilus B is one of the many Thermus strains able to utilize glutamate as a sole source of carbon. Sodium is an obligate requirement for glutamate transport by washed whole cells, but the affinity for sodium (K m = 23 mM) is low. At pH 7.6, uptake of glutamate is rapid in 50 mM-sodium at 65 °C and obeys saturation kinetics with an apparent K m of 0.23 μ;M-glutamate and a V max of 12 nmol glutamate min-1 (mg protein)-1. The transport system is insensitive to osmotic shock and is specific for glutamate, with both the L- and D-isomers being transported. Uptake is very sensitive to inhibitors that collapse the membrane potential (Δψ) or the chemical gradient of sodium ions (ΔpNa), but a transmembrane pH gradient (ΔpH) plays no role in the transport of glutamate. These results are therefore consistent with a membrane sodium/glutamate symport system.

SUMMARY: A laboratory study was conducted into the physiology of Skeletonema costatum grown under a simple sinusoidal and a fluctuating light regime. The latter simulated a light regime similar to that which could result from the vertical mixing caused by Langmuir circulation. It was shown that the culture simulating vertical mixing reacted by decreasing the photosynthetic unit (PSU) size and increasing the number of PSUs, and hence optimized the rate of maximal photosynthesis at high, saturating irradiances. This culture also showed some change in photosynthetic parameters during the light period, which was especially pronounced during the shift from a low to a higher irradiance. The effect of this on estimates of primary production in a water column is discussed. Further, it is speculated that the assimilation number is regulated by the maximum light intensity experienced during the day rather than the total daily light dose, because only the culture submitted to a fluctuating light regime showed a real change in the maximum rate of photosynthesis (P B max) upon transfer to higher light levels.

SUMMARY: Azospirillum brasilense in a motile Gram-negative bacterium that can adapt its flagellation to different environments. Cells growing in a liquid culture possess only a single polar flagellum; growth on a solid surface additionally induces multiple lateral flagella. The polar flagellum is primarily used for swimming, i.e. locomotion of the bacterium in a liquid environment, whereas the lateral flagella allow the bacteria to swarm over a solid surface. We have previously described a completely non-motile A. brasilense mutant (Sp7 p90D084), and shown that this mutant has a drastically reduced adsorption capacity to wheat roots. In the present work, we present several lines of evidence demonstrating that adsorption to wheat roots is mediated by the polar flagellum of A. brasilense. First, the non-adsorbing mutant Sp7 p90D084 forms no polar and no lateral flagella, but is otherwise undistinguishable from wild-type A. brasilense. Second, disintegration of the flagella by heat or acid eliminates adsorption. Third, using a polyclonal antiserum against the polar flagellum filament protein (Fla1), we have isolated out of a collection of 3000 Tn5-B30-induced mutants, three additional and genetically different non-flagellate mutants. Like Sp7 p90D084, these mutants show a severely reduced adsorption capacity to wheat roots. Finally, purified polar flagella bind to wheat roots in vitro.