Comparison of Direct Plating Media for the Isolation and Enumeration of Enterococci in Certain Frozen Foods

A total of 15 agar media were examined for their yield, selectivity, readability, and simplicity of preparation and use. A thallium medium of Barnes was selected as the better of the high yield-fair selectivity type of medium and an azide-citrate medium of Reinbold appeared to be the better of the low yield-high selectivity type of medium. Sodium carbonate (optimal concentration, 0.20%) was found to increase recovery substantially when added to certain media, especially in the presence of 0.05% Tween 80. When these two ingredients were incorporated into a medium modified after Slanetz and Bartley, the resultant medium was superior to other media for the isolation and enumeration of enterococci in certain frozen foods, such as peas and hamburger, by the direct plating method.     

Microbiology of Ensiled High-Moisture Corn

The effects of gaseous environment and temperature on the microbial populations of ensiled high-moisture corn were investigated. Molds, coliform bacteria, mesophilic aerobic bacteria, and yeasts were enumerated at intervals during ensiling. The numbers of aerobic bacteria were similar in structures containing different concentrations of gas and held at different temperatures. Coliform bacteria could not be detected after 10 days of ensiling. Mold numbers were relatively low, but were important in the deterioration of corn at the surface of the silos. Yeast and bacterial numbers increased rapidly following an initial aeration period, but no increase was observed after a second aeration series. Lack of a growth response to the second aeration series is believed related to the depletion of an assimilable carbon source.     

Isolation of amylolytic strains of Thermoactinomyces vulgaris and production of thermophilic actinomycete amylases

Kuo, M. J. (Iowa State University, Ames), and P. A. Hartman. Isolation of amylolytic strains of Thermoactinomyces vulgaris and production of thermophilic actinomycete amylases. J. Bacteriol. 92:723-726. 1966.-Of 759 isolates obtained from dung, compost, and soil samples, a culture of Thermoactinomyces vulgaris (strain 5) was selected for further study on the basis of quantities of amylase produced in synthetic and nonsynthetic media, rapid growth and sporulation, culture stability upon prolonged storage at 5 C, and growth temperature range. Inoculum preparation, temperature optimum for amylase formation, and the effects of various kinds and levels of carbon and nitrogen sources on amylase production were studied with T. vulgaris strain 5. An optimal procedure for production of T. vulgaris amylases is proposed.     

Characterization of crystals of genetically engineered human manganese superoxide dismutase

The genetically engineered human manganese superoxide dismutase crystallizes in space group P2(1)2(1)2 with a = 75.51 A, b = 79.00 A, c = 67.95 A. At room temperature the crystals are not stable against radiation, so we cooled them to 90 K and collected a data set to 3 A resolution at this temperature.     

Classical plant taxonomic ambiguities extend to the molecular level

Summary The molecular evolution of cytochrome c from angiosperms is compared to that from vertebrates. On the basis of a cladistic analysis from 26 plant species, compared to that from 27 vertebrate species, we find that although the vertebrate sequences yield reasonably well-defined minimal trees that are congruent with the biological tree, the plant sequences yield multiple minimal trees that are not only highly incongruent with each other, but none of which is congruent with any reasonably biological tree. That is, the plant sequence set is much more homoplastic than that of the animal. However, as judged by the relative rate test, the extent of divergence, and degree of functional constraint, cytochrome c evolution in plants does not appear to differ from that of vertebrates.     

Enumeration of anaerobic oxalate-degrading bacteria in the ruminal contents of sheep

Abstract Concentrations of oxalate-degrading anaerobes in ruminal contents of sheep were determined from counts of colonies producing clear zones on a calcium oxalate medium (D agar with 7 mM CaCl₂). Viable counts of oxalate degraders from a 55-kg sheep fed a diet containing 32% halogeton (4.6% oxalate) averaged 2.6 × 10⁶/ g (dry weight). When the halogeton concentration in the diet was reduced to 16%, counts of oxalate degraders decreased nearly 300-fold. Oxalate-degrading isolates from this sheep were similar to OxB, the type strain of Oxalobacter formigenes . When a 45-kg sheep was fed diets containing 2.2, 1.5, and 0.8% oxalate, viable counts of oxalate degraders (enumerated on D agar with 14 mM CaCl₂ and 20% filter-sterilized ruminal fluid) represented 0.85, 0.52, and 0.06% of the total viable population, respectively; total viable counts were essentially unchanges by these concentrations of dietary oxalate. Similar percentages of oxalate degraders were also observed when a 23-kg sheep was fed diets containing 1.5 or 0.8% oxalate. This report presents the first direct measurements of the concentrations of oxalate-degrading bacteria in the rumen and supports the concept that the availability of oxalate in the diet influences the proportion of oxalate-degrading bacteria in the rumen     

Catalytic site of rat liver and bovine heart fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Identification of fructose 6-phosphate binding site

Fructose-6-P binding sites of rat liver and bovine heart Fru-6-P,2-kinase:Fru-2,6-bisphosphatase were investigated with an affinity labeling reagent, N-bromoacetylethanolamine phosphate. The rat liver enzyme was inactivated 97% by the reagent in 60 min, and the rate of inactivation followed pseudo-first order kinetics. The bovine heart enzyme was inactivated 90% within 60 min, but the inactivation rate followed pseudo-first order up to 80% inactivation and then became nonlinear. The presence of fructose-6-P retarded the extent of the inactivation to approximately 40% in 60 min. In order to determine the amino acid sequence of the fructose-6-P binding site, both enzymes were reacted with N-bromo[14C]acetylethanolamine-P and digested with trypsin; radiolabeled tryptic peptides were isolated and sequenced. A single 14C-labeled peptide was isolated from the rat liver enzyme, and the amino acid sequence of the peptide was determined as Lys-Gln-Cys-Ala-Leu-Ala-Leu-Lys. A major and two minor peptides were isolated from bovine heart enzyme whose amino acid sequences were Lys-Gln-Cys-Ala-Leu-Val-Ala-Leu-Lys, Arg-Ile-Glu-Cys-Tyr-Lys, and Ile-Glu-Cys-Tyr-Lys, respectively. In all cases, N-bromoacetylethanolamine-P had alkylated the cysteine residues. The amount of bromo[14C]acetylethanolamine-P incorporated into rat liver and beef heart was 1.3 mol/mol of subunit and 2.1 mol/mol of subunit, respectively, and the incorporations in the presence of Fru-6-P were reduced to 0.34 mol/mol of subunit and 0.9 mol/mol of subunit, respectively. Thus, the main fructose-6-P binding site of rat liver and bovine heart enzymes was identical except for a single amino acid substitution of valine for alanine in the latter enzyme. This peptide corresponded to residues 105 to 113 from the N terminus of the known amino acid sequence of rat liver enzyme, but since the complete sequence of bovine heart enzyme is not known, the location of the same peptide in the heart enzyme cannot be assigned.     

Affinity labeling of spinach phosphoribulokinase subsequent toS-methylation at Cys16

The chloroplast enzyme phosphoribulokinase is reversibly deactivated by oxidation of Cys16 and Cys55 to a disulfide. Although not required for catalysis, Cys16 is an active-site residue positioned at the nucleotide-binding domain (Porter and Hartman, 1988). The hyperreactivity of Cys16 has heretofore limited further active-site characterization by chemical modification. To overcome this limitation, the partially active enzyme,S-methylated at Cys16, has been probed with a potential affinity reagent. Treatment of methylated enzyme with bromoacetylethanolamine phosphate results in essentially complete loss of catalytic activity. Inactivation follows pseudo-first-order kinetics and exhibits a rate saturation with an apparentK _d of 3–4 mM. ATP, but not ribulose 5-phosphate, affords substantial protection. Complete inactivation correlates with incorporation of 1 mol of [¹⁴C]reagent per mole of enzyme subunit. Amino acid analysis of the [¹⁴C]-labeled enzyme demonstrates that only cysteine is modified, and mapping of tryptic digests shows that Cys55 is a major site of alkylation. These results indicate that Cys55 is also located in the ATP-binding domain of the active-site.     

Syncytium-inducing (SI) phenotype suppression at seroconversion after intramuscular inoculation of a non-syncytium-inducing/SI phenotypically mixed human immunodeficiency virus population.

Two distinct biological phenotypes of human immunodeficiency virus (HIV) have been described: the non-syncytium-inducing (NSI) phenotype, best characterized by the inability to infect MT-2 cells, and the syncytium-inducing (SI) phenotype, with the ability to infect MT-2 cells. The earliest virus population observed following HIV transmission is generally of the NSI phenotype, even after exposure to inocula of mixed NSI/SI phenotype. In this study, the issue of intrapatient selection of virus phenotype following transmission was addressed by studying two cases of accidental transmission. A comparison of the sequences of the V1-V2 and the V3 coding regions of the envelope gene and the p17 region of the gag gene showed that the donor-recipient pairs were tightly clustered in all gene segments, but away from local and published transmission controls. The intrasample variation of the p17 sequence was greater in the recipients and smaller in the donors than that of the V3 region sequence, indicating selection of V3 at transmission. In these transmission cases, the effects of an intravenous inoculation of a small quantity of blood containing predominantly SI V3 sequences (6 of 8 clonal sequences) were compared with those of an intramuscular inoculation of a large quantity of blood containing predominantly NSI viruses (14 of 16 clonal sequences). Both SI and NSI V3 regions were demonstrated to be phenotypic expressions of genetically related viral strains. The inoculation of the predominantly SI virus population resulted in the persistence of an SI virus population in the recipient and a rapid CD4+ T-cell decline. The inoculation of the predominantly NSI population resulted in a selective amplification of SI viruses before seroconversion, followed by a suppression of SI viruses at seroconversion and a rapid decline of CD4+ T-cell numbers. These data suggest that the suppression of SI viruses can be accomplished following the development of HIV-specific immunity and that the ability to suppress SI viruses does not prevent the development of immunodeficiency.