The 2,160,837-base pair genome sequence of an isolate of Streptococcus pneumoniae, a Gram-positive pathogen that causes pneumonia, bacteremia, meningitis, and otitis media, contains 2236 predicted coding regions; of these, 1440 (64%) were assigned a biological role. Approximately 5% of the genome is composed of insertion sequences that may contribute to genome rearrangements through uptake of foreign DNA. Extracellular enzyme systems for the metabolism of polysaccharides and hexosamines provide a substantial source of carbon and nitrogen for S. pneumoniae and also damage host tissues and facilitate colonization. A motif identified within the signal peptide of proteins is potentially involved in targeting these proteins to the cell surface of low-guanine/cytosine (GC) Gram-positive species. Several surface-exposed proteins that may serve as potential vaccine candidates were identified. Comparative genome hybridization with DNA arrays revealed strain differences in S. pneumoniae that could contribute to differences in virulence and antigenicity.
Staphylococci that acquire the mecA gene are usually resistant to -lactam antibiotics (methicillin or oxacillin resistance). mecA encodes a penicillin-binding protein (PBP 2a) that has a reduced affinity for -lactams. In some isolates with methicillin or oxacillin resistance, only a small proportion (<0.1%) of the population expresses resistance to >10 g of oxacillin per ml (heterotypic resistance [HeR]), while in other isolates most of the population expresses resistance (homotypic resistance [HoR]). In the present study, growth of Staphylococcus aureus or Staphylococcus epidermidis strains with HeR in concentrations of oxacillin (0.3 to 0.7 g/ml) that produced a fall or a lag in optical density converted the strains from the HeR to the HoR phenotype. The conversion from the HeR to the HoR phenotype appeared to be due to the selection of a highly resistant mutant population, as determined by fluctuation analysis and the failure of populations with HoR to revert to HeR after 60 generations of growth in antibiotic-free media. The frequencies of conversion were as high as 10 ؊3 to 10 ؊2 . Conversion to HoR required an intact mecA gene and an increase in the level of mecA transcription since no highly resistant subpopulation could be selected after growth in oxacillin when mecA transcription was constitutively repressed or when mecA had been inactivated. In addition, in both S. epidermidis and S. aureus the level of resistance to vancomycin, which also acts directly on the staphylococcal cell wall, was greater among convertants with HoR than their isogenic parents. The conversion of a population from HeR to HoR involves the selection of a mutation(s) that occurs at a high frequency and most likely requires abundant PBP 2a.
Methicillin resistance in staphylococci is mediated by PBP2a, a penicillin binding protein with low affinity for β-lactam antibiotics. The gene encoding PBP2a, mecA, is transcriptionally regulated in some clinical isolates bymecR1 and mecI, genes divergently transcribed from mecA that encode a signal transducer and repressor, respectively. The biochemical basis of MecI-mediatedmecA transcriptional repression was investigated by using purified MecI. In DNase I protection studies, MecI protected a 30-bp palindrome encompassing the predicted mecA −10 and themecR1 −35 promoter sequences. The larger palindrome contained 15 bp of dyad symmetry within which was a smaller 6-bp palindrome. Electrophoretic mobility shift assays established a requirement for the entire 15-bp half-site for initial repressor binding. Fragments containing the 30-bp palindrome and the entire mecA-mecR1 intergenic region were retarded in gels as multiple discrete bands varying in molecular size, characteristic of cooperative DNA binding. Glutaraldehyde cross-linking confirmed oligomerization of repressor in solution. A naturally occurring MecI mutant (MecI*; D39G) repressed mecAtranscription sixfold less well than the wild type in vivo. Although MecI* protected the same target sequences and exhibited similar gel shift patterns to MecI, 5- to 10-fold more protein was required. MecI* exhibited defective oligomerization in solution, suggesting that the MecI amino terminus is important in protein-protein interactions and that protein oligomerization is necessary for optimum repression.
The MICs for many oxacillin-resistant (OR) Staphylococcus epidermidis (ORSE) strains are below the Staphylococcus aureus methicillin or oxacillin resistance breakpoint. The difficulty detecting the OR phenotype in S. epidermidis may be due to extreme heterotypy in resistance expression and/or transcriptional repression of mecA, the OR gene, by MecI. To determine the role of these factors in the phenotypic expression of ORSE, 17 geographically diverse mecI ؉ ORSE isolates representing 14 distinct pulsed-field gel electrophoresis pulse types (>3 band differences) were investigated. Thirteen of the 14 types contained mecI and mecA promoteroperator sequences known to be associated with maximal mecA repression, and in all isolates, mecA transcription was repressed. All 17 were heterotypic in their resistance expression. Oxacillin MICs ranged from 1 to 128 g/ml and increased for 16 of 17 isolates after -lactam induction. Allelic replacement inactivation of mecI in three isolates similarly resulted in a four-to sevenfold increase in MIC. In the two of these three isolates producing -lactamase, mecA transcription was regulated by both mecI and -lactamase regulatory sequences. Heterotypic expression of resistance in these three isolates was unaffected by either -lactam induction or mecI inactivation. However, prolonged incubation in concentrations of oxacillin just sufficient to produce a lag in growth (0.5 to 1.0 g/ml) converted the population resistance expression from heterotypic to homotypic. Homotypic conversion could also be demonstrated in microtiter wells during MIC determinations in one isolate for which the MIC was high. We conclude that the phenotypic expression of S. epidermidis OR in broth can be affected both by mecA transcriptional regulation and by subpopulation resistance expression.
When commercial vacuum tubes are used to amplify small low-frequency voltages it is found that random disturbances of the order of 100 to 1000 microvolts are present in the anode circuit. These disturbances exist almost entirely in the range below 100 cycles per second, and therefore fix the minimum voltage which can be measured over this low frequency band from 10 to 100 microvolts. These disturbances are shown to be caused by any or all of the following: (1) insulating material in or near electron path; (2) irregularity of filament emission; (3) gas; (4) positive ions emitted by filament; (5) insulating or foreign deposits on grid wires. A tube has been developed in which the above effects are removed or reduced to a point where the disturbances are nearly that of the shot effect of the electrons, as limited by space charge. This allows the amplification of low-frequency voltages of less than 1 microvolt over the entire frequency band below 100 cycles.
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