Characterization of the beta-lactamase gene from {\it Enterococcus faecalis\/} strain HH22

The plasmid-encoded, constitutively produced $\beta$-lactamase gene from Enterococcus faecalis strain HH22 was genetically characterized. A restriction endonuclease map of the 5.1 kb EcoRI fragment encoding the enterococcal $\beta$-lactamase was prepared and compared with the restriction map of a cloned staphylococcal $\beta$-lactamase gene (from the naturally-occurring staphylococcal $\beta$-lactamase plasmid pI258). Comparison and hybridization studies showed that there were identical restriction sites in the region of the $\beta$-lactamase structural gene but not in the region surrounding this gene. Also the enterococcal $\beta$-lactamase plasmid did not encode resistance to mercury or cadmium which is encoded by the small, transducible staphylococcal $\beta$-lactamase plasmids. The nucleotide sequence of the enterococcal gene was shown to be identical to the published sequences of three of four staphylococcal type A $\beta$-lactamase genes; more differences were seen with the genes for staphylococcal type C and D enzymes. One hundred-forty nucleotides upstream of the $\beta$-lactamase start codon were also determined for the inducible staphylococcal $\beta$-lactamase gene on pI258; this sequence was identical to that of the constitutively expressed enterococcal gene indicating that the changes resulting in constitutive expression are not due to changes in the promoter or operator region. Moreover, complementation studies indicated that production of the enterococcal enzyme could be repressed. The gene for the enterococcal $\beta$-lactamase and an inducible staphylococcal $\beta$-lactamase were each cloned into a shuttle vector and then transformed into enterococcal and staphylococcal recipients. The major difference between the two host backgrounds was that more enzyme was produced by the staphylococcal host, regardless of the source of the gene but no qualitative difference was seen between the two genera. Also a difference in the level of resistance to ampicillin was seen between the two backgrounds with the cloned enzymes by MIC and time-kill studies. The location of the enzyme was found to be host dependent since each cloned gene generated extracellular (free) enzyme in the staphylococcus and cell bound enzyme in the enterococcus. Based on the identity of the enterococcal $\beta$-lactamase and several staphylococcal $\beta$-lactamases, these data suggest recent spread of $\beta$-lactamase to enterococci and also suggest loss of a functional repressor. ^

Anatomical organization of the memory underlying sensitization in the siphon-withdrawal reflex circuit of {\it Aplysia californica}

Previous studies have shown that short-term sensitization of the Aplysia siphon-withdrawal reflex circuit results in multiple sites of change in synaptic efficacy. In this dissertation I have used a realistic modeling approach (using an integrate-and-fire scheme), in conjunction with electrophysiological experiments, to evaluate the contribution of each site of plasticity to the sensitized response.^ This dissertation contains a detailed description of methodology for the construction of the model circuit, consisting of the LFS motor neurons and ten interneurons known to convey excitatory input to them. The model replicates closely the natural motor neuron firing response to a brief tactile stimulus.^ The various circuit elements have different roles for producing circuit output. For example, the sensory connections onto the motor neuron are important for the production of the phasic response, while the polysynaptic interneuronal connections are important for producing the tonic response.^ The multiple sites of plasticity that produce changes in circuit output also have specialized roles. Presynaptic facilitation of the sensory neuron to LFS connection enhances only the phasic component of the motor neuron firing response. The sensory neuron to interneuron connections primarily enhance the tonic component of the motor neuron firing response. Also, the L29 posttetanic potentiation and the L30 presynaptic inhibition primarily enhance the tonic component of the motor neuron firing response. Finally, the information content at the various sites of plasticity can shift with changes in stimulus intensity. This suggests that while the sites of plasticity encoding memory are fixed, the information content at these sites can be dynamic, shifting in anatomical location with changes in the intensity of the test stimulus.^ These sites of plasticity also produce specific changes in the behavioral response. Sensory-LFS plasticity selectively increases the amplitude of the behavioral response, and has no effect on the duration of the behavioral response. Interneuronal plasticity (L29 and L30) affects both the amplitude and duration of the behavioral response. Other sensory plasticity also affect both the amplitude and duration of the behavioral response, presumably by increasing the recruitment of the interneurons, which provide all of the effect on duration of the behavioral response. ^