Robert E. Marquis

High-Pressure Microbial Physiology

Publisher Summary This particular chapter focuses on the barophysiology of micro-organisms, primarily bacteria, with the aim of developing some feeling for the molecular aspects of the pressure responses. The vacuoles of blue-green algae proved to be somewhat more resistant to collapse, but still one can envisage a situation in which these vacuoles would be collapsed by pressure in relatively shallow waters, and that this collapse would decrease the tendency of the organisms to raise to the surface where the light for photosynthesis is most intense. The effects of pressure on some specific microbial cell functions such as permeability and transport reactions, catabolic processes, biopolymer synthesis, cell division and morphological differentiation, regulatory functions, and motility are discussed in this chapter. Much of the current perspective of the molecular aspects of barophysiology is because of their efforts, particularly their application of the theory of absolute reaction rates to pressure–temperature studies of biological processes. The long-term survival of any organism depends on its adaptive capacities. Free-living bacteria have highly developed adaptive mechanisms, many of which are related to the need of small unicellular organisms to cope with limited and varying nutrient supply in highly competitive natural situations. Many bacteria grow under pressure to become elongate forms, and so it appears that cell division of these organisms are slowed by pressure more than is cell growth. However, other bacteria undergo no such change in morphology and division does not appear to be more barosensitive than is growth. There is a general feeling in the field of microbial barophysiology that the inhibitory effects of pressure on the growth of microorganisms are because of the pressure inhibition of polymer synthesis.

Reduction of Acidurance of Streptococcal Growth and Glycolysis by Fluoride and Gramicidin

The acidurance of glycolysis by intact cells of Streptococcus mutans GS-5, Streptococcus salivarius ATCC 25925, and Streptococcus sanguis NCTC 10904 was found to be highly dependent on membrane functions affected by gramicidin, which increases the proton permeability of cell membranes. Plots of % glucose utilized during two hours against suspension pH values for cells suspended in 100 mM phosphate buffer plus 1 mM MgCl 2 plus 13.9 mM glucose indicated, for 50% glucose utilization, pH values of 5.0 for S. mutans, 5.7 for S. salivarius, and 6.2 for S. sanguis. Gramicidin treatment shifted these values to 6.0, 6.3, and 6.9, respectively. Growth of S. mutans and S. salivarius in complex media proved to be more acid-sensitive than was glycolysis, and in batch cultures, there was a well-defined, post-growth phase of glycolysis. Minimum pH values for growth and for glycolysis in medium with excess glucose were approximately 4.8 and 4.4, respectively, for S. mutans, and 4.9 and 4.3 for S. salivarius. S. sanguis was less aciduric and showed little differential acid sensitivity, with minimum pH values of about 5.2 for both growth and glycolysis. Fluoride acted to eliminate the differences in acidurance of growth and glycolysis for S. mutans or S. salivarius and to render both processes more acid-sensitive. Thus, glycolysis was more fluoride-sensitive than was growth. Growth was found to be acid-limited in media with initial glucose levels greater than 0.2, 0.3, and 0.5% (weight/volume) for S. sanguis, S. mutans, and S. salivarius, respectively, and to be glucose-limited at lower levels. When 1.0 mM NaF was added to the media, the acid-sensitizing effect of fluoride was evident in major shifts in the initial glucose levels for transition from glucose-limited to acid-limited growth to 0.1 and 0.2% for S. mutans and S. salivarius, respectively. S. sanguis was less severely affected by fluoride. Fluoride treatment of glycolyzing cells of S. mutans at a pH value of 5 resulted in little change in intracellular levels of phosphoenolpyruvate but increased levels of 2-phosphoglycerate, indicative of inhibition of intracellular enolase. However, there were also large decreases in levels of early glycolytic intermediates, to about the levels found in starved cells, presumably due to fluoride inhibition of glucose uptake.

Elastic, flexible peptidoglycan and bacterial cell wall properties

The peptidoglycan sacculus serves as a mechanical framework for the cell walls of most eubacteria and largely determines cell shape. The notion that the structure is a rigid shell is contradicted by findings that peptidoglycan can expand and contract. Thus, the sacculus functions as an elastic, flexible, polyionic, amphoteric, restraining network.

Inhibition of proton-translocating ATPases of Streptococcus mutans and Lactobacillus casei by fluoride and aluminum

One of the major effects of fluoride on oral bacteria is a reduction in acid tolerance, and presumably also in cariogenicity. The reduction appears to involve transport of protons across the cell membrane by the weak acid HF to dissipate the pH gradient, and also direct inhibition of the F1F0, proton-translocating ATPases of the organisms, especially for Streptococcus mutans. This direct inhibition by fluoride was found to be dependent on aluminum. The dependence on aluminum was indicated by the protection against fluoride inhibition afforded by the Al-chelator deferoxamine and by loss of protection after addition of umolar levels of Al3+, which were not inhibitory for the enzyme in the absence of fluoride. The F1 form of the enzyme dissociated from the cell membrane previously had been found to be resistant to fluoride in comparison with the F1F0 membrane-associated form. However, this difference appeared to depend on less aluminum in the F1 preparation in that the sensitivity of the F1 enzyme to fluoride could be increased by addition of umolar levels of Al3+. The effects of Al on fluoride inhibition were apparent when enzyme activity was assayed in terms of phosphate release from ATP or with an ATP-regenerating system containing phosphoenolpyruvate, pyruvate kinase, NADH and lactic dehydrogenase. Also, Be2+ but not other metal cations, e.g. Co2+, Fe2+, Fe3+, Mn2, Sn2+, and Zn2+, served to sensitize the enzyme to fluoride inhibition. The differences in sensitivities of enzymes isolated from various oral bacteria found previously appeared also to be related to differences in levels of Al. Even the fluoride-resistant enzyme of isolated membranes of Lactobacillus casei ATCC 4646 could be rendered fluoride-sensitive through addition of Al3+. Thus, the F1F0 ATPases of oral bacteria were similar to E1E2 ATPases of eukaryotes in being inhibited by Al-F complexes, and the inhibition presumably involved formation of ADP-Al-Finf3sup-complexes during catalysis at the active sites of the enzymes.

Inhibitory and cidal antimicrobial actions of electrically generated silver ions

One promising alternative to antibiotics in the treatment of localized infections is the generation of antimicrobial silver ions by the use of low intensity direct current from a pure silver anode implanted at the site of an infection. This study investigates the in vitro bacteriostatic and bactericidal properties of this system on a variety of organisms.

Diminished Acid Tolerance of Plaque Bacteria Caused by Fluoride

Fluoride acts to reduce acid tolerances of plaque bacteria by upsetting normal proton currents across cell membranes. Streptococcus mutans was found to be unusually sensitive to fluoride, in part because its F1F0, proton-translocating ATPase is directly inhibited by fluoride at plaque levels. Thus, not only does fluoride serve in the HF form to bring extruded protons back into the cell, but it also reduces the capacity of the cell to extrude protons. Reductions in acid tolerance caused by fluoride would be expected to result in concomitant reductions in cariogenic potential.

Physiologic homeostasis and stress responses in oral biofilms.

Studies performed since the early, 1970s have yielded tremendous amounts of information about the physiology, genetics, and interactions of oral bacteria. This pioneering work has provided a solid foundation to begin to apply the knowledge and technologies developed using suspended populations for studying oral bacteria under conditions that more closely mimic conditions in the oral cavity, in biofilms. Our current understanding of phenotypic capabilities of individual and complex mixtures of adherent oral bacteria is in its infancy. There is ample evidence that oral streptococci have different patterns of gene expression than planktonic cells, but we have little understanding of the basis for these observations. Even in biofilmforming bacteria with very well-developed genetic systems it is only very recently that genetic loci involved in biofilm formation and responses to surface growth have been identified. A comprehensive study of the physiology and gene expression characteristics of adherent oral bacteria not only will enhance our abilities to control oral diseases, but it will provide critical information that can be applied to a variety of other pathogenic microorganisms.

Membrane-associated and Solubilized ATPases of Streptococcus mutans and Streptococcus sanguis

The proton-translocating, membrane ATPases of oral streptococci have been implicated in cytoplasmic pH regulation, acidurance, and cariogenicity. Membranes were isolated from Streptococcus mutans GS-5 and Streptococcus sanguis NCTC 10904 following salt-induced lysis of cells treated with lysozyme and mutanolysin. The ATPase activities of these membranes were 1.8 and 1.1 units per mg membrane protein, respectively. F1 ATPases were washed free from the membranes and purified by fast protein liquid chromatography (FPLC). Hydrolytic activities of the F 1 ATPases were maximal at pH values between 6.0 and 6.6, whereas the membrane-bound enzymes had pH maxima of 7.5 (S. sanguis) and 6.0 (S. mutans). The F1 ATPases of the streptococci were similar to the well-characterized enzyme of Escherichia coli; they consisted of five different polypeptides and had apparent, aggregate molecular weights of from 335 to 350 Kd. The membrane-bound ATPases were characterized biochemically and found to be similar to those of proton-translocating ATPases of E. coli and Streptococcus faecalis. K m values for the membranes with respect to ATP were found to be 0.9 and 1.0 mmol/L for S. mutans and S. sanguis, respectively. Both enzymes had specificities for purine triphosphates and were active with a variety of divalent cations, although optimal activity occurred with ATP and Mg. The membrane-associated enzymes were sensitive to the inhibitors dicyclohexylcarbodiimide (DCCD) and azide, but insensitive to ouabain and vanadate. Overall, it appears that the membrane-associated ATPases of S. mutans and S. sanguis are similar to the extensively studied proton-translocating ATPases of E. coli and S. faecalis, and that differences in pH sensitivities of the enzymes from the oral bacteria are greater for the membrane-bound, proton-translocating forms than for the soluble, F, forms.

Protocols to Study the Physiology of Oral Biofilms

The oral cavity harbors several hundred different bacterial species that colonize both hard (teeth) and soft tissues, forming complex populations known as microbial biofilms. It is widely accepted that the phenotypic characteristics of bacteria grown in biofilms are substantially different from those grown in suspensions. Because biofilms are the natural habitat for the great majority of oral bacteria, including those contributing to oral diseases, a better understanding of the physiology of adherent populations is clearly needed to control oral microbes in health and disease. In this chapter, we use oral streptococci as examples for studying the physiology of oral biofilms.

Inhibition and Killing of Oral Bacteria by Silver Ions Generated with Low Intensity Direct Current

Silver cations generated by passing low intensity direct current through pure silver electrodes were found to be sufficiently antibacterial to cause sterilization of samples of infected dentin. The optimal procedure involved a 5 μA current applied for 20 minutes with the anode then left in contact with the sample. Minimal inhibitory concentrations of electrically generated silver ions for representative oral bacteria were essentially equal to those for silver ions added as nitrate or fluoride salts, and medium constituents, including sodium thioglycolate, antagonized antibacterial action. A major advantage to the use of the electrode method is that it allows for continuous, focal application of antibacterial silver cations.