Interaction of Salivary alpha-Amylase and Amylase-Binding-Protein A (AbpA) of <Emphasis Type="Italic">Streptococcus gordonii</Emphasis> with Glucosyltransferase of <Emphasis Type="Italic">S. gordonii</Emphasis> and <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

Background  

Glucosyltransferases (Gtfs), enzymes that produce extracellular glucans from dietary sucrose, contribute to dental plaque formation byStreptococcus gordoniiandStreptococcus mutans. The alpha-amylase-binding protein A (AbpA) ofS. gordonii, an early colonizing bacterium in dental plaque, interacts with salivary amylase and may influence dental plaque formation by this organism. We examined the interaction of amylase and recombinant AbpA (rAbpA), together with Gtfs ofS. gordoniiandS. mutans.     

The transcriptional landscape of the deep-sea bacterium Photobacterium profundum in both a toxR mutant and its parental strain

Background

Mutans streptococci are a group of gram-positive bacteria including the primary cariogenic dental pathogen Streptococcus mutans and closely related species. Two component systems (TCSs) composed of a signal sensing histidine kinase (HK) and a response regulator (RR) play key roles in pathogenicity, but have not been comparatively studied for these oral bacterial pathogens.

Results

HKs and RRs of 8 newly sequenced mutans streptococci strains, including S. sobrinus DSM20742, S. ratti DSM20564 and six S. mutans strains, were identified and compared to the TCSs of S. mutans UA159 and NN2025, two previously genome sequenced S. mutans strains. Ortholog analysis revealed 18 TCS clusters (HK-RR pairs), 2 orphan HKs and 2 orphan RRs, of which 8 TCS clusters were common to all 10 strains, 6 were absent in one or more strains, and the other 4 were exclusive to individual strains. Further classification of the predicted HKs and RRs revealed interesting aspects of their putative functions. While TCS complements were comparable within the six S. mutans strains, S. sobrinus DSM20742 lacked TCSs possibly involved in acid tolerance and fructan catabolism, and S. ratti DSM20564 possessed 3 unique TCSs but lacked the quorum-sensing related TCS (ComDE). Selected computational predictions were verified by PCR experiments.

Conclusions

Differences in the TCS repertoires of mutans streptococci strains, especially those of S. sobrinus and S. ratti in comparison to S. mutans, imply differences in their response mechanisms for survival in the dynamic oral environment. This genomic level study of TCSs should help in understanding the pathogenicity of these mutans streptococci strains.     

A Highly Arginolytic Streptococcus Species That Potently Antagonizes Streptococcus mutans

The ability of certain oral biofilm bacteria to moderate pH through arginine metabolism by the arginine deiminase system (ADS) is a deterrent to the development of dental caries. Here, we characterize a novel Streptococcus strain, designated strain A12, isolated from supragingival dental plaque of a caries-free individual. A12 not only expressed the ADS pathway at high levels under a variety of conditions but also effectively inhibited growth and two intercellular signaling pathways of the dental caries pathogen Streptococcus mutans. A12 produced copious amounts of H₂O₂ via the pyruvate oxidase enzyme that were sufficient to arrest the growth of S. mutans. A12 also produced a protease similar to challisin (Sgc) of Streptococcus gordonii that was able to block the competence-stimulating peptide (CSP)–ComDE signaling system, which is essential for bacteriocin production by S. mutans. Wild-type A12, but not an sgc mutant derivative, could protect the sensitive indicator strain Streptococcus sanguinis SK150 from killing by the bacteriocins of S. mutans. A12, but not S. gordonii, could also block the XIP (comX-inducing peptide) signaling pathway, which is the proximal regulator of genetic competence in S. mutans, but Sgc was not required for this activity. The complete genome sequence of A12 was determined, and phylogenomic analyses compared A12 to streptococcal reference genomes. A12 was most similar to Streptococcus australis and Streptococcus parasanguinis but sufficiently different that it may represent a new species. A12-like organisms may play crucial roles in the promotion of stable, health-associated oral biofilm communities by moderating plaque pH and interfering with the growth and virulence of caries pathogens.     

Antifouling activity of seaweed extracts on the green alga Enteromorpha prolifera and the mussel Mytilus edulis

Twenty-seven species of common seaweeds from the coast of Korea havebeen screened for antifouling activity. The seaweed extracts were tested inlaboratory assays against the marine fouling green alga Enteromorphaprolifera and the blue mussel Mytilus edulis. Tissue growth, sporesettlement, zygote formation and germlings of the E. prolifera wereinhibited by methanol extracts of the seaweed Ishige sinicola (= I. foliacea) and Sargassum horneri. Spore settlement was stronglyinhibited by using extract concentrations as low as 30 g mL^-1with I. sinicola and 120 g mL^-1 with S. horneri. The repulsive activity of the foot of the mussel was completely inhibited bymethanol extracts of I. sinicola and Scytosiphon lomentaria atconcentrations of 40 g per 10 L drop supplied to eachmussel. These extracts also showed strong antifouling activities onlarval settlement with, respectively, no or only 6% of the spat settlingwhen a test concentration of 0.8 mg mL^-1 was used. This work isthe first stage towards the development of novel antifouling agents frommarine macroalgae.     

SelectingRhizobium phaseoli strains for use with beans (Phaseolus vulgaris L.) in Kenya: Infectiveness and tolerance of acidity and aluminium

Forty strains ofRhizobium phaseoli, isolated from Kenyan soils, were tested for infectiveness on common bean (Phaseolus vulgaris L.). 28 strains were infective and a cultivar × Rhizobium interaction was observed. 48 strains were screened for tolerance of acidity and Al in liquid culture. Assessment of visible turbidity after 14 days indicated 34 strains tolerant of pH 4.5 but none tolerant of pH 3.5. No strain was tolerant of 50 M Al at pH 5.5. Three strains were tolerant of 20 M Al at pH 5.5 and 10 M Al at pH 4.5. Screening on a solid medium identified strains tolerant of 20 and 50 M Al at pH 5.5 and 4.5 which were sensitive to these treatments in liquid culture. Those strains tolerant to 20 M Al at pH 4.5 and 5.5 in liquid culture were correctly identified on the solid medium.     

Oral Streptococci Exhibit Diverse Susceptibility to Human β-Defensin-2: Antimicrobial Effects of hBD-2 on Oral Streptococci

We examined the antimicrobial effects of human -defensin-2 (hBD-2) on 17 species of oral streptococci to investigate the involvement of antimicrobial peptide activity in oral microflora development and the clinical use of the antimicrobial peptide for oral microflora control. Oral streptococci exhibit diverse levels of susceptibility to human -defensin-2 (hBD-2). Two major cariogenic bacterial species, Streptococcus mutans (S. mutans) and S. sobrinus, were found to be susceptible to the peptide, indicating that it is a potential therapeutic agent for preventing dental caries. S. mitis exhibited the lowest susceptibility to the peptide. S. mitis is a major indigenous bacterium in the oral microflora, and our results suggest that it might possess a certain resistance mechanism against hBD-2.     

Mechanism of arginine transport in Chlorella

The incubation of Chlorella cells with glucose causes the induction of an uptake system, which is specific for the basic amino acids arginine and lysine. Both amino acids are taken up in the positively charged form and with high affinity (K _m values 2 M and 7 M, respectively). The transport of arginine depolarizes the membrane by 20–30 mV. The charge compensation is achieved within a few seconds after arginine addition by the proton pump, later on K⁺ efflux serves for charge compensation. No evidence for arginine-proton symport was found, neither by inhibitor studies nor by use of other Chlorella strains which have a slower-responding proton pump. The accumulation of arginine is appreciably higher than it should be according to the thermodynamic force of the membrane potential. There is, however, some evidence that a large proportion of arginine is trapped by intracellular compartments and is therefore not in equilibrium with the outside arginine.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - FCCP p-trifluoromethoxycarbonylcyanide phenylhydrazone     

Intracellular pH inSchizosaccharomyces pombe — Comparison withSaccharomyces cerevisiae

We examined cytoplasmic pH regulation inSchizosaccharomyces pombe andSaccharomyces cerevisiae using pH-sensitive fluorescent dyes. Of several different fluorescent compounds tested, carboxy-seminaphthorhodafluor-1 (C.SNARF-1) was the most effective. Leakage of C.SNARF-1 fromS. pombe was much slower than leakage fromC. cerevisiae. Using the pH-dependent fluorescence of C.SNARF-1 we showed that at an external pH of 7, mean resting internal pH was 7.0 forS. pombe and 6.6 forS. cerevisiae. We found that internal pH inS. pombe was maintained over a much narrower range in response to changes in external pH, especially at acidic pH. The addition of external glucose caused an intracellular alkalinization in both species, although the effect was much greater inS. cerevisiae than inS. pombe. The plasma membrane H⁺-ATPase inhibitor diethylstilbestrol reduced both the rate and extent of alkalinisation, with an IC₅₀ of approximately 35 M in both species. Amiloride also inhibited internal alkalinisation with IC₅₀'s of 745 M forS. cerevisiae and 490 M forS. pombe.Abbreviations C.SNARF-1 carboxy-seminaphthorhodafluor-1 (-AM-acetoxy-methylester) - DES diethylstilbestrol - IC₅₀ apparent inhibitory constant - BCECF 2,7-bis-(carboxyethyl)-5(6)-carboxyfluorescein (-AM--pentaacetoxymethyl ester) - FDA fluorescein diacetate     

Phosphomannomutase activity in wild-type and alginate-producing strains ofPseudomonas aeruginosa

Phosphomannomutase (PMM) activity was detected in the soluble cytoplasmic fraction of crude extracts of both mucoid (alginate-producing) and nonmucoid strains ofPseudomonas aeruginosa. The enzyme activity was concentrated and partially purified from cell extracts of mucoid strain V388 by precipitation with ammonium sulfate and by molecular exclusion chromatography. These preparations catalyzed the conversion of mannose 1-phosphate to mannose 6-phosphate in a coupled assay system that contained commercial phosphomannoisomerase, phosphoglucoisomerase, and glucose 6-phosphate dehydrogenase. Catalytic activity in this system was strictly dependent on the presence of glucose 1,6-diphosphate (apparent Km, 150 M) and exhibited a pH optimum of around 9 in Bicine-NaOH buffer. PMM exhibited an apparent Km of 60 M for mannose 1-phosphate, but concentrations greater than 150 M caused significant inhibition. Specific activities of PMM were consistently higher in the soluble fractions of mucoid strains (1.2–3.6 nmol/min/mg protein) than of nonmucoid strains (0.2–0.6 nmol/min/mg protein).     

Flexibility of myosin in pyrophosphate and NaCl solutions

The orientational relaxation time of myosin has been reported as 38 s when measured in pyrophosphate media at elevated pH (Hvidt et al. 1984) and as 17 s when measured in 0.3 M KCl at pH 7.3 (Bernengo and Cardinaud 1982). This discrepancy, which is reexamined in the present report, suggests that in KCl solution the rod portion of the myosin molecule is bent with an average angle close to 110°, whereas in pyrophosphate at elevated pH it assumes a nearly straight and rigid conformation. Electric birefringence shows that the amount of dimeric and polymeric species in pyrophosphate media at pH's 8.0 and 8.5 is certainly greater than usually thought. In these media, relaxation times can be measured correctly at pH 9.0. A comparative analysis of the influence of protein concentration, field strength, medium composition and concentration, pH and temperature showed that a high relaxation time is associated with the presence of pyrophosphate and that the myosin tail is significantly stiffened in the presence of this anion. Offprint requests to: R. Cardinaud     

The effects of high field DC pulse and liquid medium conductivity on survivability of Lactobacillus brevis

Survivability of Lactobacillus brevis cells in suspensions of phosphate buffer solutions of different conductivities (170 S/cm to 2230 S/cm) using electric pulse application has been investigated under varied test conditions. Survivability decreased rapidly with the application of the first few pulses (approx. 25 to 50 depending on the test conditions). However, the destruction performance decreased with increased number of pulse applications. Hence to obtain a maximum reduction in survivability, the electrical conditions should be so selected that effective killing is achieved with the fewest number of pulses applied. The maximum reduction in survivability (N/N₀, approx. 10^–7) was obtained in liquid possessing the lowest conductivity (170 S/cm) with an application of 150 pulses of 160-s pulse width. Despite the increase in liquid medium temperature during pulse treatment, the killing was significantly due to pulse as the maximum temperature rise (22° C) during treatment was insufficient to cause any synergistic effect of temperature and pulse treatment. In this work we have shown for the first time that if the pulse width is kept constant, the higher reduction in survivabilities observed in liquids with lower conductivities was primarily due to conductivity influencing the membrane permeability. The small change in test liquid pH (<0.5) indicated that the killing of cells was affected primarily by high field pulses rather than by-products of electrolysis in the medium of different conductivities. Correspondence to: A. Margaritis     

The endogenous development of two new species of Isospora (Apicomplexa: Eimeriidae) from skinks

Two new species of Isospora are described from skinks, I. cryptoblephari n. sp. in Cryptoblepharus virgatus and I. delmae n. sp. in Delma nasuta, both collected in Australia. I. cryptoblephari oöcysts are ellipsoidal to subspherical, 17.5–22.5 × 25.0–30.0 m with two ovoid sporocysts, 9.0–10.0 × 12.5–14.0 m. I. delmae oöcysts are spherical to subspherical, 16.5–19.0 × 16.5–20.0 m with two ovoid sporocysts, 5.0–6.5 × 9.0–12.5 m. These species of Isospora had two sporocysts, each containing four sporozoites and a characteristic Stieda body. A study of endogenous stages in the host's intestine revealed that I. cryptoblephari develops in the nucleus and I. delmae in the cytoplasm of the host's gut epithelial cell. In the former, both merogony and gamogony occurred in the nucleus.     

Chimeric Antimicrobial Peptides Exhibit Multiple Modes of Action

Pyrrhocoricin and drosocin, representatives of the short, proline-rich antimicrobial peptide family kill bacteria by inactivating the bacterial heat shock protein DnaK and inhibiting chaperone-assisted protein folding. The molecular architecture of these peptides features an N-terminal DnaK-binding half and a C-terminal delivery unit, capable of crossing bacterial membranes. Cell penetration is enhanced if multiple copies of pyrrhocoricin are conjugated. To obtain drug leads with improved antimicrobial properties, and possible utility as therapeutic agents, we synthesized chimeric dimers, in which pyrrhocoricins potent DnaK-binding domain was connected to drosocins superior cell penetrating module. Indeed, the new constructs not only exhibited enhanced in vitro antibacterial properties against the originally sensitive strains Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium, but also showed activity against Staphylococcus aureus, a bacterial strain resistant to native pyrrhocoricin and drosocin. The improved antimicrobial profile could be demonstrated with assays designed to distinguish intracellular or membrane activities. While a novel mixed pyrrhocoricin–drosocin dimer and the purely pyrrhocoricin-based old dimer bound E. coli DnaK with an identical 4 M K_d, the mixed dimers penetrated a significantly larger number of E. coli and S. aureus cells than the previous analogs and destroyed a larger percentage of bacterial membrane structures. Toxicity to human red blood cells could not be observed up to the highest peptide concentration tested, 640 M. In addition, repetitive reculturing of E. coli or S. aureus cells with sublethal concentrations of the mixed dimer did not result in resistance induction to the novel peptide antibiotic. The new concept of pyrrhocoricin–drosocin mixed dimers yields antibacterial peptide derivatives acting with a multiple mode of action, and can serve as a useful addition to the current antimicrobial therapy repertoire.     

A novel antifungal from an Actinomadurae with preferential activity against the mycelial phase ofCandida albicans

Summary Sch 40873, a novel antifungal compound isolated from the fermentation broth of anActinomadura spp. was discovered in an assay designed to detect compounds with preferential activity against the invasive mycelial form ofCandida albicans. The geometric mean MIC of Sch 40873 against sevenCandida spp. in Sabouraud dextrose broth (yeast phase) was 58 g/ml and in Eagles minimum essential medium (mycelial phase) was <0.03 g/ml. Sch 40873 demonstrated slight in vivo topical activity in a hamster vaginal model.     

A Role for Lewis a antigens on salivary agglutinin in binding to Streptococcus mutans

Streptococcus mutans is a major etiological agent in dental caries. Salivary agglutinin is one of the main salivary components binding to S.mutans. To learn more about the interaction of salivary agglutinin with S.mutans, parotid, submandibular, sublingual and palatal saliva samples were incubated with S. mutans suspension. Both depleted saliva samples and bacterial extracts were analyzed by SDS-PAGE and immunoblotting. Salivary agglutinin was present in all types of glandular saliva and in all cases bound to S.mutans, also to PC337C, a P1^– mutant of S.mutans. Agglutinin was separated by SDS-PAGE under reducing and non-reducing conditions and then transferred to nitrocellulose. Non-reduced agglutinin bound S.mutans, but reduced agglutinin did not. Adhesion of S.mutans to agglutinin-coated microplates was inhibited by amine-containing components, 1 M NaCl or KCl and EDTA. Adhesion decreased with decreasing pH with no adhesion below pH 5.0. These data suggest that calcium-dependent electrostatic interactions play a role in binding. By immunoblotting was demonstrated that blood group antigens and Lewis antigens were present on agglutinin. Synthetic blood group antigens and Lewis antigens covalently coupled to polyacrylamide were tested for binding to S.mutans. Only Le^a(Gal1,3(Fuc1,4)GlcNAc) bound to S.mutans, whereas the blood group antigens Le^b, Le^x, Le^y, H1, H2, A, B and sialylated Le^a did not. Le^a without galactose (Fuc1,4GlcNAc) still bound to S. mutans, but Le^a without fucose (Gal1,3GlcNAc) did not. Binding of agglutinin to S. mutans was not inhibited by Le^a. In conclusion, S. mutans can bind to Le^a carbohydrate epitopes in which the fucose is an essential residue. Le^a carbohydrate epitopes are present on salivary agglutinin but play no major role in binding.     

Xylitol and Erythritol Decrease Adherence of Polysaccharide-Producing Oral Streptococci

Xylitol consumption decreases counts of mutans streptococci. However, the mechanism behind this decrease is not well understood. We studied not only type strains and clinical isolates of mutans streptococci, but also other polysaccharide-forming oral streptococci. Growth inhibition and adherence of cells to a smooth glass surface—reflecting synthesis of water-insoluble polysaccharides were studied in the presence of 2% (0.13 mol/l) and 4% (0.26 mol/l) xylitol. The effect of xylitol was compared to a novel polyol sweetener, erythritol. Except for Streptococcus mutans 10449 and S. sobrinus OMZ 176, the glass surface adhesion of most polysaccharide-forming streptococci was reduced by the presence of both 4% xylitol and erythritol. For the S. mutans and S. sobrinus type strains, the growth inhibition with 4% xylitol and erythritol was 36–77% and for the clinical S. mutans isolates 13–73%. Of the other oral streptococci, only S. sanguinis was inhibited with 4% xylitol (45–55%). For both polyols, the magnitude of the growth inhibition observed was not associated with the magnitude of the decrease in adherence (xylitol: r = −0.18; erythritol: r = 0.49). In conclusion, both xylitol and erythritol can decrease polysaccharide-mediated cell adherence contributing to plaque accumulation through a mechanism not dependent on growth inhibition.     

Role of Intracellular Polysaccharide in Persistence of Streptococcus mutans

Intracellular polysaccharide (IPS) is accumulated by Streptococcus mutans when the bacteria are grown in excess sugar and can contribute toward the cariogenicity of S. mutans. Here we show that inactivation of the glgA gene (SMU1536), encoding a putative glycogen synthase, prevented accumulation of IPS. IPS is important for the persistence of S. mutans grown in batch culture with excess glucose and then starved of glucose. The IPS was largely used up within 1 day of glucose starvation, and yet survival of the parental strain was extended by at least 15 days beyond that of a glgA mutant; potentially, some feature of IPS metabolism distinct from providing nutrients is important for persistence. IPS was not needed for persistence when sucrose was the carbon source or when mucin was present.Streptococcus mutans is a facultative colonizer of the human dental plaque, the microbial pellicle that covers the surface of the teeth. It is the major etiological agent of dental caries (17). Sugar metabolism is central to the behavior of S. mutans (4, 7). It can use a variety of sugars. The sugars are fermented by glycolysis with production of organic acids, particularly lactic acid (4, 7). In addition to providing energy, sucrose is used to produce extracellular polysaccharides to form the biofilm matrix that aids in the association of S. mutans with the dental plaque. Once the S. mutans biofilm becomes part of the dental plaque, the acidic by-products of sugar fermentation dissolve tooth enamel, eventually resulting in dental caries (17). The presence of sugars in the dental plaque is periodic and reflects the intake of dietary sugars. If there is excess sugar available, in addition to producing organic acids and matrix, intracellular (iodophilic) polysaccharide (IPS; glycogen) is formed.The IPS of S. mutans is a polymer of the glycogen-amylopectin type, with α-(1, 4)- and α-(1, 6)-linked glucose, and is stored as intracellular granules (10). Intracellular glycogen storage reserves in various bacterial species are synthesized from glucose-1-P via ADP-glucose (1). The synthesis involves at least three enzymes: glycogen synthase, glucose-1-phosphate pyrophosphorylase, and branching enzyme. The genes encoding these enzymes are commonly found in a glg operon, although the order of genes differs between species. In two gram-positive species, Bacillus subtilis and Bacillus stearothermophilus, the gene order is glgB-glgC-glgD-glgA-glgP (15, 29): glgA encodes glycogen synthase, glgB encodes glucan branching enzyme, and glgC and glgD encode subunits of glucose-1-phosphate pyrophosphorylase. The glgP gene encodes glycogen phosphorylase, which is unlikely to be involved in glycogen synthesis (29). Genes putatively encoding similar enzymes are present in the same order in the genome of S. mutans (29); they are thought also to form an operon.The IPS can be used as a source of carbohydrate for fermentation upon nutrient depletion (11, 13). In planktonic cultures, IPS reserves are largely consumed within 12 h of the imposition of sugar starvation (11, 13, 32). In S. mutans, IPS utilization may prolong acid production and hence the period of lowered pH of the resting (between meals) plaque, a factor that contributes to the incidence of caries. Indeed, IPS is implicated in dental caries: a mutant that synthesized elevated levels of IPS was hypercariogenic in germfree rats (27). Strains isolated from human carious lesions were nearly all stable IPS producers, whereas most strains from caries-inactive persons were variable IPS producers (13, 33).Since S. mutans deep in the dental plaque may not have access to nutrients because of competition with the bacteria at the surface of the plaque, the bacteria may need to survive longer periods of nutrient starvation. Previous studies in our laboratory showed that S. mutans can survive under sugar starvation conditions, provided that the pH remains above ∼5.5 (22). The presence of spent medium and mucin significantly prolonged survival of sugar-starved biofilms and batch cultures (22; also unpublished observations). Here we examine the role of IPS.The role of IPS (glycogen) in bacterial survival has been tested for several other bacterial species. It was found to extend survival of Aerobacter aerogenes (8) and Escherichia coli (28). Intracellular glycogen was also shown to support the survival of Streptococcus mitis during stationary-phase starvation (32). In contrast, glycogen-rich Sarcina lutea died at a higher rate during starvation than did bacteria without glycogen (2).In order to test the role of IPS in S. mutans survival, we constructed an IPS-deficient mutant by inactivating glgA (GenBank SMU.1536) (http://www.oralgen.lanl.gov/), putatively encoding the glycogen synthase. We also constructed a mutant potentially altered in IPS metabolism by inactivating the putative pullulanase structural gene, pul (SMU.1541). Pullulanases are responsible for hydrolyzing α-(1,6) linkages (and in some cases 1,4 linkages) in pullulan and in other polysaccharides (35) and may be important in determining the branching in IPS and/or affecting the catabolism of IPS. We studied the persistence of bacteria under conditions of sugar limitation and of sugar excess in both batch cultures and biofilms. We found that IPS can play a role in supporting S. mutans persistence in batch cultures but found no role for IPS in survival in biofilms.     

Micropropagation ofPinus sylvestris

Plantlet regeneration from axillary buds, induced on seedling apices ofPinus sylvestris, is described. The influence of medium containing 1, 5, 10 or 50 M BA on the initiation of buds on 3, 6 and 9-week-old seedlings was investigated. With higher BA concentrations the number of apices that formed buds and the total number of buds increased, wheres their length decreased. Soaking the explants for 2 h in 111 M BA and for 5 h in 44 M BA gave better results, best expressed in longer buds. After 30 days, axillary buds were separated and transferred to a medium with 2% sucrose. Separated buds exposed to far-red light during the first week elongated better in comparison with the control. 64% of shoots rooted after a 24 h period of treatment with 53.8 M NAA incorporated in 0.6% water agar and transferred to 1/16-strength Murashige & Skoog medium as modified by Cheng supplemented with 1% sucrose.     

Effect of mercuric chloride on carbon mineralization in soils

Summary The short and long-term effects of mercuric chloride amendment of five surface soils from southeastern Montana on carbon mineralization was studied. Short-term radiorespirometric studies utilizing a glucose substrate indicated Hg levels greater than 40 g/g soil were required for significant inhibition in all soils tested. Under chronic exposure conditions, levels from 0.1 to greater than 100 g Hg/g soil proved necessary for inhibition.