Isolation and some properties of extracellular glucan-producing strains of human oral Streptococcus salivarius

A total of eighteen strains of Streptococcus salivarius, which formed rough gelatinous, rough mucoid or smooth mucoid colonies on sucrose agar media, were isolated from the saliva and tongue dorsum of adults. All of the isolates produced glucans as well as fructans from sucrose. The bulk of the glucans was synthesized by the extracellular enzyme fraction and was water insoluble, whereas most of the fructans were synthesized by the cell-associated enzyme fraction and were water soluble. All strains formed microbial deposits on wire and glass surfaces when cultured in sucrose broth, but their sucrose-dependent adhesion was apparently looser than that produced by a cariogenic S. sobrinus strain. The rough gelatinous colony forming strains possessed a greater ability to synthesize water-insoluble glucans and produced heavier deposits with higher cohesion. Preliminary studies showed that the S. salivarius of such characteristic forms of colony were detected primarily in the saliva and tongue dorsum: the smooth mucoid colony formers appeared to predominate in the tongue coat and the rough mucoid and rough gelatinous colony formers were prominent in saliva. Isolation of these S. salivarius from dental plaques was low.     

Multiple Streptococcus mutans Genes Are Involved in Biofilm Formation

Streptococcus mutans has been strongly implicated as the principal etiological agent in dental caries. One of the important virulence properties of these organisms is their ability to form biofilms known as dental plaque on tooth surfaces. Since the roles of sucrose and glucosyltransferases in S. mutans biofilm formation have been well documented, we focused our attention on sucrose-independent factors. We have initially identified several mutants that appear to be defective in biofilm formation on abiotic surfaces by an insertional inactivation mutagenesis strategy applied to S. mutans. A total of 27 biofilm-defective mutants were isolated and analyzed in this study. From these mutants, three genes were identified. One of the mutants was defective in the Bacillus subtilis lytR homologue. Another of the biofilm-defective mutants isolated was a yulF homologue, which encodes a hypothetical protein of B. subtilis whose function in biofilm formation is unknown. The vast majority of the mutants were defective in the comB gene required for competence. We therefore have constructed and examined comACDE null mutants. These mutants were also found to be attenuated in biofilm formation. Biofilm formation by several other regulatory gene mutants were also characterized using an in vitro biofilm-forming assay. These results suggest that competence genes as well as the sgp and dgk genes may play important roles in S. mutans biofilm formation.     

Role of Bacteriocin During Plaque Formation by Streptococcus salivarius and Streptococcus sanguis on a Tooth in an Artificial Mouth

Bacteriocinogenic strains of Streptococcus salivarius antagonized Strep. sanguis on blood agar and in Todd-Hewitt broth with, but not without, sucrose. Each organism produced plaque in vitro but, after a mixed inoculum with both organisms, the numbers of Strep. sanguis rapidly fell to <0.01% plaque organisms. A non-bacterio-cinogenic mutant of Strep. salivarius was itself inhibited by Strep. sanguis in the plaque-producing system; derivatives of Strep. sanguis partially resistant to bacteriocin in the plate test nevertheless failed to co-habit plaque with bacteriocinogenic Strep. salivarius. The latter could suppress Strep. sanguis in established monoculture plaque but only if sucrose were continuously supplied. It was concluded that the effect of bacteriocin in plaque formation by these streptococci is linked to other as yet unknown properties which may account for the absence of Strep. salivarius from plaque in vivo .     

Virulence of Streptococcus mutans: biochemical and pathogenic characteristics of mutant isolates.

The in vitro dextran-sucrase activities and adherence to glass of S. mutans 6715 and PS14 wild types and mutants were quantitated and compared with their in vivo cariogenicity in young, gnotobiotic rats. In general, S. mutans PS14 mutants B414 and B421 and 6715 mutant C4 demonstrated less dextran-sucrase activity and adherence than parental strains and caused fewer carious lesions in gnotobiotic rats. Rats monoinfected with either PS14 mutants B414 or B421 had less plaque and viable S. mutans in plaque than rats infected with parental strain. Both S. mutans 6715 mutants C211 and C229, demonstrated greater enzyme activity and adherence than the parental strain and produced more carious lesions.     

M13 vectors for selective cloning of sequences specifying initiation of DNA synthesis on single-stranded templates

M13 cloning vectors have been developed for the selection of DNA sequences capable of directing initiation of DNA synthesis on single-stranded templates. These vectors are derived from viable M13 mutants containing large deletions in the region of the complementary strand origin. The deletion mutants are defective in the conversion of viral single strands to the duplex replicative form (SS leads to RF) both in vivo and in vitro, give a reduced phage yield and form turbid plaques. A receptor site for foreign single strand initiation determinants has been introduced into the mutants by the insertion of EcoRI linker sequences at the deletion sites. Specific cloned sequences from bacteriophage G4 RF and from Co1E1 DNA restore a clear plaque type and normal phage growth. Selection of clear-plaque isolates obtained by transfection with RF from one of these vectors, M13 delta E101, carrying inserted Co1E1 HaeIII fragments resulted in the selective cloning of one specific fragment, the HaeIII-E fragment. Insertion of either the H or L strand of the HaeIII-E fragment into the M13 delta E101 viral strand gives a clear plaque phenotype, indicating the presence of initiation determinants on both the H- and L-strands of the Co1E1 HaeIII-E fragment. These cloning vectors provide a new means for the functional dissection of replication origins and for the identification of DNA sequences that determine the enzymatic mechanism of discontinuous synthesis along the length of the bacterial chromosome. The ability to assess initiation capability on the basis of plaque morphology also provides a means for rapid genetic analysis of initiation determinants.     

Hemagglutinin-neuraminidase glycoprotein as a determinant of pathogenicity in mumps virus hamster encephalitis: analysis of mutants selected with monoclonal antibodies.

With the aid of monoclonal antibodies directed against a specific site on the hemagglutinin-neuraminidase surface glycoprotein, four mutants of the Kilham neurotropic strain of mumps virus were isolated. All four mutants had increased neuraminidase activity. Two mutants (M10 and M12) lost their hemagglutination capacity with human O erythrocytes but retained their ability to agglutinate guinea pig erythrocytes at 4 degrees C. A third mutant (M11) showed a change in the molecular weight of the hemagglutinin-neuraminidase glycoprotein. These three mutants (M10, M11, and M12) showed unaltered capacity to infect tissue cultures and to cause encephalitis in newborn hamsters. A fourth mutant (M13) retained its hemagglutination activity and capacity to infect Vero cell cultures but showed significantly lower neurovirulence in the suckling hamster brain than did the parental Kilham strain and the other three mutants. Both the number of infected neurons and the amount of infectious virus in the brain was reduced. On the other hand, there were no apparent differences in the occurrence of viral antigen in ependymal cells, indicating a selective change in affinity for neurons in the brain. These results suggest that certain changes in the hemagglutinin-neuraminidase glycoprotein may lead to an alteration of the neuropathogenicity of the Kilham strain of mumps virus.     

Vitrification of in vitro produced bovine embryos: in vitro and in vivo evaluations

The efficacy of different vitrification solutions to cryopreserve in vitro produced bovine blastocysts was evaluated based upon in vitro development of embryos in culture and on in vivo development of embryos transferred into recipients. In the first experiment, ethylene glycol + glycerol (Eg + Gly) + different sucrose concentrations were evaluated. There were no significant differences in development rates among solutions. As for hatching, the Eg + Gly + 0.1 M sucrose group had a greater rate as compared with Eg + Gly + 0 M sucrose and Eg + Gly + 0.5 M sucrose groups in the evaluations of Day 6, Day 7 and Day 6 + Day 7 embryos; and, Eg + Gly + 0.3 M sucrose group had a greater rate as compared with the Eg + Gly + 0 M sucrose and Eg + Gly + 0.5 M sucrose groups in evaluations of Day 6 and Day 6 + Day 7 embryos. There were no significant differences in development and hatching rates between Day 6 and 7 in in vitro produced bovine embryos within each treatment group. There were significant differences in nuclei number after vitrification between Eg + Gly + 0.1 M and Eg + Gly + 0 M sucrose groups and the Eg + Gly + 0.5 M sucrose group. Pregnancy after 60 days of transfer and calving rates showed a difference between in vivo produced embryos freshly transferred and in vitro produced embryos vitrified with Eg + Gly + 0.3 M. There were no significant differences in gestation length and sex ratio between treatments. As for birth weight, there were significant differences between fresh in vivo produced embryos and all treatments of in vitro produced embryos. There were significant differences in dystocial parturition between in vivo produced embryos and all treatments with in vitro produced embryos. These results demonstrate that vitrification can be used successfully in the cryopreservation of in vitro produced bovine embryos, and that it might be considered for use in commercial programs.     

Association between M. pneumoniae hemolysis, attachment, and pulmonary pathogenicity

Three different groups of hemolysis mutants were produced by treatment of the M. pneumoniae FH-P24 strain with N-methyl-N-nitro-nitrosoguanidine. The first group of mutants, strains P24-L1, L2, and L11, showed wide and clear hemolytic zones, and attaching ability to erythrocytes and to hamster lung cells were the same as the properties of the parent strain and produced significant microscopic lung lesions. Mutant P24-S1 showed non-hemolysis and non-hemadsorption, yet retained the attaching ability to lung cells and produced milder lung lesions. Mutant P24-S11 showed none of those activities, did not cause any lung lesion, and was never recovered from the lungs of hamsters. A close relationship between the hemolytic ability of M. pneumoniae and the histopathogenicity in the hamster lung is suggested in this study. The attaching ability of organisms seems to be an important factor at the initial stage of infection.     

Characterization and Pathogenicity of Hemolysis Mutants of Mycoplasma pneumoniae

Hemolysis mutants were produced by treating Mycoplasma pneumoniae FH-P24 strain with N-methyl-N-nitro-nitrosoguanidine and were classified into three different groups. The first group of mutants, strains P24-L1, L2, and L11, showed wide and clear hemolytic zones. Their attachment ability to erythrocytes of various animals and to hamster lung cells were the same as those of the parent strain. The second group, strain P24-S1, showed non-hemolysis and non-hemadsorption, but retained the attachment ability to lung cells, although not to erythrocytes. The third group, strain P24-S11, was non-hemolytic, had completely lost the attaching ability, and did not proliferate in vivo. Strains in the first group produced significant microscopic pneumonic lesions in hamsters while strain P24-S1 produced milder lung lesions. Strain P24-S11 did not cause any lung lesions, and organisms were not recovered from the lungs of hamsters. The attachment of M. pneumoniae to respiratory epithelium as a cause of infection and the existence of a relationship between the hemolytic abilities of the organisms and histopathogenicity in the hamster lung tissue were further supported by the present data. It was also shown that the use of hemolysis mutants is useful for the elucidation of pathogenesis in mycoplasmal infections.     

Temperature-Sensitive Cell Division Mutants of Escherichia coli with Thermolabile Penicillin-Binding Proteins

The thermostability of the penicillin-binding proteins (PBPs) of 31 temperature-sensitive cell division mutants of Escherichia coli has been examined. Two independent cell division mutants have been found that have highly thermolabile PBP3. Binding of [(14)C]benzylpenicillin to PBP3 (measured in envelopes prepared from cells grown at the permissive temperature) was about 30% of the normal level at 30 degrees C, and the ability to bind [(14)C]benzylpenicillin was rapidly lost on incubation at 42 degrees C. The other PBPs were normal in both mutants. At 30 degrees C both mutants were slightly longer than their parents and on shifting to 42 degrees C they ceased dividing, but cell mass and deoxyribonucleic acid synthesis continued and long filaments were formed. At 42 degrees C division slowly recommenced, but at 44 degrees C this did not occur. The inhibition of division at 42 degrees C was suppressed by 0.35 M sucrose, and in one of the mutants it was partially suppressed by 10 mM MgCl(2). PBP3 was not stabilized in vitro at 42 degrees C by these concentrations of sucrose or MgCl(2). Revertants that grew as normal rods at 42 degrees C regained both the normal level and the normal thermostability of PBP3. The results provide extremely strong evidence that the inactivation of PBP3 at 42 degrees C in the mutants is the cause of the inhibition of cell division at this temperature and identify PBP3 as an essential component of the process of cell division in E. coli. It is the inactivation of this protein by penicillins and cephalosporins that results in the inhibition of division characteristic of low concentrations of many of these antibiotics.     

Genetic assay of misincorporation

A system to characterize mutations arising from in vitro nucleotide misincorporation, which avoids the effects of in vivo mismatch repair on recovery of mutants, was constructed and evaluated. The lacI gene of Escherichia coli was inserted into phage M13 and the M13-lacI recombinant was introduced into a strain of E. coli lacking a resident lacI gene. In this system the function of the M13-bearing lacI gene can be detected by plaque color. Mutants in the 5'-region of the lacI gene (encoding operator-binding domain) are seen as blue plaques when the host strain is grown in the presence of chromogenic substrate, X-gal, in the absence of inducer. The use of uracil-containing single stranded DNA from M13-lacI as template for DNA synthesis avoids the contribution of mismatch repair (in transfection recipients) on the recovery of mutants. To demonstrate the usefulness of the M13-lacI system we produced nucleotide misincorporations by in vitro DNA synthesis in the N-terminal region of the lacI template in the presence of only 3 deoxynucleoside triphosphates (dNTPs). Such mutagenic reactions were conducted in the absence of dATP with 4 different primers and in the absence of dGTP with 2 primers. The type of mutants produced by these reactions were identified through sequencing of DNA from progeny phage after screening for i- (blue plaque) phenotype. Mutations recovered in this system consisted of single and multiple base substitutions in the region of the template near the 3'-terminus of the primer. Nearly all of the mutants induced by '-A' conditions were T----C base substitutions, and those induced by '-G' conditions were C----T transitions. In general, the results were consistent with the spectrum of spontaneous mutants produced in strains deficient in mismatch repair, although some differences were noted. Several new base substitutions within the lacI gene (producing i- phenotype and unobserved by others) were isolated by the procedures described in this paper.     

Continuous Monitoring of pH and Eh in Bacterial Plaque Grown on a Tooth in an Artificial Mouth

Apparatus which enables the simultaneous continuous monitoring of pH and Eh of bacterial dental plaque as it develops on a tooth surface in an artificial mouth is described. Details of the electrodes used, monitoring equipment, and culture conditions are given. Preliminary results are given showing the Eh and pH values of plaque produced in vitro to be in close agreement with readings reported for plaque in vivo. The effect of the incorporation of 1 per cent sucrose in the medium on these parameters is reported and a distinct inverse relationship between pH and Eh recorded.     

Mutants of simian virus 40 differing in plaque size, oncogenicity, and heat sensitivity

Takemoto, K. K. (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), R. L. Kirschstein, and K. Habel. Mutants of simian virus 40 differing in plaque size, oncogenicity, and heat sensitivity. J. Bacteriol. 92:990-994. 1966.-Three mutants of simian virus 40 were isolated on the basis of the type of plaques produced in primary cultures of African green monkey kidney cells and designated as L (large), S (small), and M (minute) strains. Significant differences in oncogenicity for hamsters were observed, with the 50% oncogenic dose being 10(4.5) for the L, 10(5.2) for the S, and 10(5.8) for the M strains. All three strains were capable of transforming human diploid cells (W138 strain). At temperatures up to 41 C, the S and M mutants were capable of multiplying to titers almost equivalent to those obtained at 37 C. In contrast, infectious virus was not produced when cells were infected with the L mutant and were incubated at temperatures above 39 C, although complement-fixing viral and tumor antigens were formed. The temperature-sensitive phase of replication of the L strain was shown to be a late stage in viral maturation or assembly.     

Circadian organization of tau mutant hamsters: aftereffects and splitting

Homozygous tau mutant (tau(ss)) hamsters show an extremely short (20 h) circadian period (tau) that is attributable to altered enzymatic activity of casein kinase 1epsilon. It has been proposed that coupling of constituent circadian oscillators is strengthened in tau(ss) hamsters, explaining their tendency to show strong resetting after prolonged exposure to constant darkness. To evaluate further the circadian organization of tau(ss) hamsters, the authors assessed the extent of shortening of period as an aftereffect of exposure to light:dark cycles whose period (T) is 91% of tau and the ability of constant light to induce splitting. They find that tau(ss) hamsters show aftereffects comparable to wild types, indicating that normal CK1epsilon activity is not required for T cycles to shorten tau. This finding also contradicts the proposal that circadian period is homeostatically conserved. However, the authors find that tau(ss) hamsters rarely show splitting in constant light. Furthermore, LL does not induce lengthening of tau or reduction of activity duration (alpha) in these mutants. The authors' findings support the conclusion that the tau mutation alters the coupling between constituent circadian oscillators.     

The role of the maltodextrin-binding site in determining the transport properties of the LamB protein

We have examined by the liposome swelling technique the permeability properties of the modified LamB proteins isolated from mutants of Escherichia coli K12 with altered affinities toward starch and/or maltose (Ferenci, T., and Lee, K-S. (1982) J. Mol. Biol. 160, 431-444). The results revealed the following. A mutant strain exhibiting a markedly lowered affinity toward starch produced a LamB protein that has lost the ability to permeate longer maltodextrins. This protein retained a nonspecific pore for a wide variety of small sugars. A mutant strain with partially reduced affinity for starch produced a LamB protein which still permeated maltodextrins, maltose, and non-maltose sugars but had also gained an ability to permit the diffusion of sucrose and raffinose; in this strain sucrose and raffinose could now compete for the starch-binding site. A mutant with enhanced affinity for both maltose and starch produced a protein which exhibited elevated rates of diffusion for longer maltodextrins but still permeated other small sugars. Two other mutants with altered affinities showed relatively minor changes in the diffusion of maltose and non-maltose sugars. It could be concluded from these studies that the LamB proteins form pores allowing the diffusion of a wide variety of monosaccharides irrespective of the presence or the absence of affinity of a binding site for maltodextrins. However, the presence of a sugar-binding site is crucial in determining the rate of the diffusion of maltodextrins or other oligosaccharides.     

Single-strand breakage in DNA of Escherichia coli exposed to Cd2+.

When a growing culture of Escherichia coli was exposed to 3 X 10(-6) M Cd2+, 85 to 95% of the cells lost their ability to form colonies on agar plates. Loss of viability was accompanied by considerable single-strand breakage in the DNA, with no detectable increase in double-strand breaks. A direct correlation appeared to exist between the number of single-strand breaks and the concentrations of Cd2+ to which the cells were exposed. Exposure of DNA in vitro to a Cd2+ concentration of 3 X 10(-6) M or higher, followed by sedimentation in alkaline sucrose gradients, demonstrated no single-strand breaks. Cadmium-exposed cells recovered viability when incubated in Cd2+-free liquid medium containing 10 mM hydroxyurea. During the early period of recovery, there was a lag in the incorporation of labeled thymidine, but cellular DNA, at least in part, appeared to be repaired.     

Characterization of qa-2 mutants of Neurospora crassa by genetic, enzymatic, and immunological techniques.

Genetic and complementation mapping studies using 20 qa-2 mutants defective for catabolic dehydroquinase indicate that the qa-2 gene encodes a single polypeptide chain and is the structural gene for catabolic dehydroquinase, a 220,000-molecular-weight protein composed of identical 10,000-molecular-weight subunits. Many qa-2 mutants are capable of reversion, but no evidence has yet been obtained for nonsense mutations in this gene. The biochemical consequences of the mutations in two complementing qa-2 strains (M239 and M204) have been determined. Both mutants have extremely low levels of catalytic activity and form a heterocaryon with about 4% of the wild-type activity. As assayed by immunological cross-reactivity, mutant M239 and the heterocaryon have nearly wild-type levels of native-molecular-weight catabolic dehydroquinase protein, whereas M204 has no detectable amount of this protein. Thus it is concluded that M239 has a mutation at or near the catalytic site which reduces the activity 10,000-fold but has little or no influence on the formation of the native multimeric structure. In contrast, M204 apparently has a mutation that severely inhibits aggregation and may have only a minor effect on the inherent potential for catalytic conversion at the reactive site. The heterocaryon would appear to form a mixed multimer with the monomeric subunits from M239 providing the aggregated structure and those from M204, the catalytically active moiety.     

Differentiation of cariogenic streptococci by fluorescent antibody

Jablon, J. M. (University of Miami, Miami, Fla.), and D. D. Zinner. Differentiation of cariogenic streptococci by fluorescent antibody. J. Bacteriol. 92:1590-1596. 1966.-Eight strains of streptococci were isolated from human carious lesions by the fluorescent-antibody (FA) technique. Seven of these strains produced experimental caries in hamsters or rats maintained on a high sucrose diet. The eighth strain was noncariogenic in animals but possessed some antigenic components in common with the cariogenic strains. On the basis of antigen-antibody reactions by microprecipitin and agar-gel diffusion patterns, the strains were divided into four groups; these groups differed with regard to their cariogenic activity in hamsters. Fluorescein-conjugated antisera, prepared against the human strains, showed some cross-reactions which interfered with the efficacy of the FA technique in differentiating between the related streptococcal groups. To eliminate these cross-reactions, a small amount of related-strain antisera was added to the fluorescein-conjugated antisera to the cariogenic strains. This technique is effective in blocking cross-reactions and should be tried wherever cross-reactions are encountered in the FA technique.     

Changes in structure of starch and enzyme activities affected by sugary mutations in developing rice endosperm. Possible role of starch debranching enzyme (R-enzyme) in amylopectin biosynthesis

In maturing endosperms of a variety of sugary mutants of rice, phytoglycogen-like polysaccharides with highly branched a -glucans were accumulated instead of amylopectin. while the amylose content greatly decreased. Measurement of activities per endosperm of the 10 major enzymes involved in starch and sucrose metabolism revealed that the activity of starch debranching enzyme (R-enzyme) was specifically reduced in the sugary mutants. The activity of starch branching enzyme I (Q-enzyme I) was also significantly decreased, but less so than the R-enzyme, in the mutants, suggesting some coordination of the expression of the genes coding for R-enzyme and Q-enzyme I. Western blot analysis showed that the sugary mutations of rice resulted in a decrease in the amount of R-enzyme protein, but not in major modification of the enzyme. These findings strongly suggest that R-enzyme plays a critical role in determining the amylopectin fine structure, since at the extremely low level of R-enzyme activity as compared with Q-enzyme activity, as found in sugary mutants, the rice endosperm produced phytoglycogen. We hypothesize that balance of activities or interaction between Q-enzyme and R-enzyme may be responsible for the fine structure of a -polyglucans in plant tissues.     

The mitogen-activated protein kinase gene MAF1 is essential for the early differentiation phase of appressorium formation in Colletotrichum lagenarium

Colletotrichum lagenarium, the causal agent of cucumber anthracnose, invades host plants by forming a specialized infection structure called an appressorium. In this fungus, the mitogen-activated protein kinase (MAPK) gene CMK1 is involved in several steps of the infection process, including appressorium formation. In this study, the goal was to investigate roles of other MAPKs in C. lagenarium. The MAPK gene MAF1, related to Saccharomyces cerevisiae MPK1 and Magnaporthe grisea MPS1, was isolated and functionally characterized. The maf1 gene replacement mutants grew normally, but there was a significant reduction in conidiation and fungal pathogenicity. The M. grisea mps1 mutant forms appressoria, but conidia of the C. lagenarium maf1 mutants produced elongated germ tubes without appressoria on both host plant and glass, on which the wild type forms appressoria, suggesting that MAF1 has an essential role in appressorium formation on inductive surfaces. On a nutrient agar, wild-type conidia produced elongated germ tubes without appressoria. The morphological phenotype of the wild type on the nutrient agar was similar to that of the maf1 mutants on inductive surfaces, suggesting repression of the MAF1-mediated appressorium differentiation on the nutrient agar. The cmk1 mutants failed to form normal appressoria but produced swollen, appressorium-like structures on inductive surfaces, which is morphologically different from the maf1 mutants. These findings suggest that MAF1 is required for the early differentiation phase of appressorium formation, whereas CMK1 is involved in the maturation of appressoria.