Genetic relationships among the oral streptococci.

Genetic relationships and species limits among the oral streptococci were determined by an analysis of electrophoretically demonstrable variation in 16 metabolic enzymes. Fifty isolates represented 40 electrophoretic types, among which the mean genetic diversity per locus was 0.857. Mannitol-1-phosphate dehydrogenase was not detected in isolates of the sanguis species complex, and glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were absent in species of the mutans complex. Clustering from a matrix of Gower's coefficient of genetic similarity placed the 40 electrophoretic types in 10 well-defined groups corresponding to the Streptococcus species S. mutans, S. sobrinus, S. cricetus, S. rattus, S. ferus, S. oralis (mitior), two distinct assemblages of S. sanguis strains, and two subdivisions of "S. milleri." The assignments of isolates to these groups were the same as those indicated by DNA hybridization experiments, and the coefficient of correlation between genetic distance estimated by multilocus enzyme electrophoresis and genetic similarity indexed by DNA hybridization was -0.897 (P less than 0.001) for 50 pairwise combinations of isolates. S. ferus, which is widely believed to be a member of the mutans complex, was shown to be phylogenetically closer to species of the sanguis complex.     

The activation of the sodium pump in pig red blood cells by internal and external cations

A study has been made with pig red blood cells of the activation of the sodium pump by internal and external cations. Cell Na and K concentrations were altered using a PCMBS cation loading procedure. The procedure was characterised for resultant ionic conditions, maintenance of ATP levels and fragility. The activation of the sodium pump by external K was measured in cells suspended in choline (Na-free) solutions. External Cs was used as a substitute for K and elicited lower rates of pump activity. Both the Vmax and apparent Km for 42K influx and 134Cs influx increased as internal Na concentration was raised (within the non-saturating range). Vmax/apparent Km ratios for cation influx were constant. Raising external Cs concentration exerted a similar influence on pump activation by internal Na: both the maximum pump velocity and the apparent Na-site dissociation constant (K'Na) increased. The results provide evidence for a transmembrane connection between cation binding sites on opposite faces of the membrane and are consistent with a consecutive model for the sodium pump in pig red blood cells.     

Enzyme polymorphism and genetic population structure in Escherichia coli and Shigella

Electrophoretically demonstrable variation in 12 enzymes was studied in more than 1 600 isolates of Escherichia coli from human and animal sources and in 123 strains of the four species of Shigella. All 12 enzymes were polymorphic; and the number of allozymes (mobility variants), which were equated with alleles, averaged 9.3 per locus in E. coli. For Shigella species, the mean number of alleles was 2.9 per locus. Some 77% of the allozymes recorded in Shigella were shared with E. coli. A total of 302 unique genotypic combinations of alleles over the 12 loci (electrophoretic types, ETs) was distinguished, of which 279 represented E. coli and 23 were Shigella. Among electrophoretic types, mean allelic diversity per locus was 0.52 for E. coli and 0.29 for Shigella. It was estimated that there are, on the average, about 0.3 detectable codon differences per locus between pairs of strains of E. coli and Shigella, which is roughly equivalent to 1.2 amino acid differences per enzyme. Evidence that the enzyme loci studied are a random sample of the genome is provided by a significant positive correlation between estimates of genetic divergence between pairs of strains obtained by DNA reassociation tests and estimates of genetic distance between the same strains based on electrophoresis. A principal components analysis of allozyme profiles revealed that the 302 ETs fall into three overlapping clusters, reflecting strong non-random associations of alleles, largely at four loci. Each of the four ETs of E. coli that have been most frequently recovered from natural populations has an allozyme profile that is very similar to, or identical with, the hypothetical modal ET of one of the groups. ETs of Shigella fall into two of the groups. No biological significance can at present bbe attributed to the genetic structure revealed by Multilocus electrophoretic techniques. The electrophoretic data are fully compatible with other molecular and more conventional evidence of a close affinity between E. coli and Shigella, and they raise questions regarding the present assignments of certain strains to species. In support of evidence from DNA reassociation tests and serotyping, the present study suggests that S. sonnei is homogeneous in chromosomal genotype.     

The influence of external sodium ions on the sodium pump in erythrocytes

1. A study has been made of the interaction between Na(+) and K(+) on the adenosine triphosphatase activity of erythrocyte ;ghosts', and on the K(+) influx and Na(+) efflux of intact erythrocytes. The adenosine triphosphatase activity and the ion movements were greater at a low external K(+) concentration in the absence of Na(+) than they were in the presence of 150mm-Na(+). The inhibition by external Na(+) of K(+) influx had an inhibitory constant of 5-10mm. 2. Activation by K(+) of kidney microsomal adenosine triphosphatase was retarded by Na(+), and activation by Na(+) was retarded by K(+). Fragmented erythrocyte membranes behaved similarly. 3. These observations suggest that there is competition between Na(+) and K(+) at the K(+)-sensitive site of the membrane.     

The Efflux of Potassium from Electroplaques of Electric Eels

1. The movement of labeled potassium ions has been measured across the innervated membranes of single isolated electroplaques, obtained from the organ of Sachs of Electrophorus electricus, mounted in an apparatus which allowed a separate washing of the two membranes. 2. Equations have been derived for a 3 compartment system in series in which tracer from a large pool in one outer compartment is collected in the other outer compartment. The amount of unlabeled ion in the middle compartment may be calculated and also the fluxes across the two membranes. 3. The flux of potassium across the innervated membranes of resting cells in a steady state was between 700 to 1000 µµmoles/cm.²/sec. and was unaffected by d-tubocurarine. 4. Direct stimulation of electroplaques with external electrodes caused an increase in the efflux of potassium from the innervated membrane of 5 to 8 µµmoles/cm.²/impulse, which was unaffected by d-tubocurarine; no change occurred in the efflux across the non-innervated membrane. 5. It is concluded that the discharge of electroplaques is accompanied by a small outward movement of potassium ions across the innervated membrane of the same order of magnitude as that found on excitation of squid giant axons. The data show a basic similarity of potassium movements across these two entirely different types of conducting membranes and suggest that this phenomenon may be a general feature of bioelectric currents propagating an action potential.     

Effects of sodium and potassium ions on oxidative phosphorylation in relation to respiratory control by a cell-membrane adenosine triphosphatase

1. A study has been made of the oxygen consumption of kidney homogenates in relation to the ADP concentration as regulated by the cell-membrane adenosine triphosphatase. Stimulation of this enzymic activity by Na(+) and K(+) caused parallel increases in oxygen consumption and ADP concentration. Similarly, inhibition with ouabain caused a parallel fall. The membrane adenosine triphosphatase concerned in active transport therefore appears to regulate respiration through its control of ADP concentration. 2. The respiration of homogenates and mitochondria was also stimulated by K(+) in a way independent of adenosine-triphosphatase activity. It was shown that K(+) facilitates oxidative phosphorylation and the respiratory response to ADP. A K(+) concentration of 25-50mm was needed for maximum oxidative phosphorylation in the presence of physiological concentration of Na(+). Na(+) counteracted K(+) in the effects on mitochondria. It is concluded that K(+) regulates cellular respiration at two structures, one directly in mitochondria, and the second indirectly through control of ADP production at the cell membrane.     

Occurrence of Shiga-like toxin-producing Escherichia coli in retail fresh seafood, beef, lamb, pork, and poultry from grocery stores in Seattle, Washington.

Fresh meat, poultry, and seafood purchased from Seattle area grocery stores were investigated for the presence of Shiga-like toxin-producing Escherichia coli by using DNA probes for Shiga-like toxin (SLT) genes I and II. Of the 294 food samples tested, 17% had colonies with sequence homology to SLT I and/or SLT II genes.     

RAPD (arbitrary primer) PCR is more sensitive than multilocus enzyme electrophoresis for distinguishing related bacterial strains.

The RAPD (random amplified polymorphic DNA) fingerprinting method, which utilizes low stringency PCR amplification with single primers of arbitrary sequence to generate strain-specific arrays of anonymous DNA fragments, was calibrated relative to the widely used, protein-based multilocus enzyme electrophoretic (MLEE) typing method. RAPD fingerprinting was carried out on five isolates from each of 15 major groups of Escherichia coli strains that cause diarrheal disease worldwide (75 isolates in all). Each group consisted of isolates that were not distinguishable from one another by MLEE typing using 20 diagnostic enzyme markers. In our RAPD tests, three or more distinct subgroups in each MLEE group were distinguished with each of five primers, and 74 of the 75 isolates were distinguished when data obtained with five primers were combined. Thus, RAPD typing is far more sensitive than MLEE typing for discriminating among related strains of a species. Despite their different sensitivities, the same general relationships among strains were inferred from MLEE and RAPD data. Thus, our results recommend use of the RAPD method for studies of bacterial population genetic structure and evolution, as well as for epidemiology.