The preg\u3csup\u3ec \u3c/sup\u3estrain of N. crassa has abnormal vesicles when grown on both low- and high-P\u3csub\u3ei\u3c/sub\u3e media

The genetic and molecular mechanisms controlling the synthesis of de-repressible phosphatases in Neurospora crassa include four regulatory genes, nuc-2, preg, pgov, and nuc-1, involved in a hierarchical relationship (Metzenberg, 1979. Microbiol. Rev. 43: 361-383)

Mind the buffering capacity of citric acid

Many microbial cultures are buffered with citric acid over a pH range of 2.5 to 7.0 since the pKa values for this triprotic acid are 3.13, 4.76 and 6.40, as shown in The Merck Index (pp 330-331, 10th Edition, Martha Windholz, ed.). However, the information about the buffering range of this weak acid is controvertial since the pKa3 value may be 5.40, as specified in Day and Underwood (Quantitative Analysis, 6th Edition, 1991, p. 662. New Jersey: Prentice Hall). With this in mind, we determined the pKa values of citric acid at concentrations ranging from 5 mM to 50 mM, concentrations which are the most employed in buffers for the culture of many microorganisms

Toluene permeabilization differentially affects F- and P-type ATPase activities present in the plasma membrane of Streptococcus mutans

Streptococcus mutans membrane-bound P- and F-type ATPases are responsible for H+ extrusion from the cytoplasm thus keeping intracellular pH appropriate for cell metabolism. Toluene-permeabilized bacterial cells have long been used to study total membrane-bound ATPase activity, and to compare the properties of ATPase in situ with those in membrane-rich fractions. The aim of the present research was to determine if toluene permeabilization can significantly modify the activity of membrane-bound ATPase of both F-type and P-type. ATPase activity was assayed discontinuously by measuring phosphate release from ATP as substrate. Treatment of S. mutans membrane fractions with toluene reduced total ATPase activity by approximately 80% and did not allow differentiation between F- and P-type ATPase activities by use of the standard inhibitors vanadate (3 µM) and oligomycin (4 µg/mL). Transmission electron microscopy shows that, after S. mutans cells permeabilization with toluene, bacterial cell wall and plasma membrane are severely injured, causing cytoplasmic leakage. As a consequence, loss of cell viability and disruption of H+ extrusion were observed. These data suggest that treatment of S. mutans with toluene is an efficient method for cell disruption, but care should be taken in the interpretation of ATPase activity when toluene-permeabilized cells are used, because results may not reflect the real P- and F-type ATPase activities present in intact cell membranes. The mild conditions used for the preparation of membrane fractions may be more suitable to study specific ATPase activity in the presence of biological agents, since this method preserves ATPase selectivity for standard inhibitors.UNIUBECNPqCoordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES

A carbohydrate pulse experiment to demonstrate the sugar metabolization by S. mutans

Streptococcus mutans is a fast growing organism, of low cost and easily prepared culture medium. It has been  related  primarily to  an  elevated risk  of dental cavity development  in the host due  to the  acid-induced tooth demineralization. To prevent this disease, addition of fluoride can be required, promoting the mouth  hygiene. The  main  objective  of  this  experiment  is  to  show  the  influence  of  the  carbon  source  and fluoride on the acidogenic capacity of S.  mutans. The strain was cultivated in microaerophilia, at 37ºC for 12  hours  in  complete  medium  (stationary  phase).  The  cells  were  harvested  by  centrifugation  at  room temperature,  washed  with  saline  solution  and  suspended  in  the  same  solution.  The  absorbance  was adjusted  to  1  and  the  pH  to  7.3  using  0,1  mol/L  KOH  solution.  To  10  mL  of  the  cell  suspension,  distinct carbohydrates  (glucose,  xilose,  sucrose,  fructose  or  maltose)  were  added,  enough  to  establish  a  50 mMol/L final concentration. Fluoride was added (1 mmol/L final concentration) and the pH was monitored during  2 hours. In this  incubation  period,  the  suspension  was  kept  at  room  temperature  with  slow  stirring and  the  pH  was  monitored  each  7  minutes.  In  the  20  initial  minutes  of  incubation  with  glucose,  fructose, maltose  and  sucrose,  an  intense  and  very  similar  pH  decrease  (2.5  units)  can  be  observed.  This acidification reflects both the sugar uptake and anaerobic metabolization. After this initial acid liberation, a phase of slow pH decrease is observed, continuing up to 120 minutes of incubation. In presence of xilose, the  acidification  is  less  intense  and  reaches  a  similar  value  to  that  of  the  control  without  carbohydrate addition (decreasing  1.4 units  of pH). The  initial  acidification  in the presence of xilose  may  occur  due  to the mechanism of sugar uptake by this organism, which involves the antiport with H+. In media without the addition  of  carbohydrate,  the  acidification  may  be  due  to  the  metabolization  of  intracellular  reserves  of sugars. Fluoride affects negatively the acidogenic capacity of S. mutans for all metabolized sugars