The Aer protein and the serine chemoreceptor Tsr independently sense intracellular energy levels and transduce oxygen, redox, and energy signals for Escherichia coli behavior.

We identified a protein, Aer, as a signal transducer that senses intracellular energy levels rather than the external environment and that transduces signals for aerotaxis (taxis to oxygen) and other energy-dependent behavioral responses in Escherichia coli. Domains in Aer are similar to the signaling domain in chemotaxis receptors and the putative oxygen-sensing domain of some transcriptional activators. A putative FAD-binding site in the N-terminal domain of Aer shares a consensus sequence with the NifL, Bat, and Wc-1 signal-transducing proteins that regulate gene expression in response to redox changes, oxygen, and blue light, respectively. A double mutant deficient in aer and tsr, which codes for the serine chemoreceptor, was negative for aerotaxis, redox taxis, and glycerol taxis, each of which requires the proton motive force and/or electron transport system for signaling. We propose that Aer and Tsr sense the proton motive force or cellular redox state and thereby integrate diverse signals that guide E. coli to environments where maximal energy is available for growth.

A general method for detecting and sizing large plasmids.

We have devised a method for detecting and estimating the sizes of large bacterial plasmids in the presence of genomic DNA by pulsed-field gel electrophoresis (PFGE). Bacteria harboring plasmids were embedded in agarose and lysed using a rapid protocol. Plugs were incubated with S1 nuclease and subjected to PFGE in agarose gels. S1 nuclease converted supercoiled plasmids into full-length linear molecules. Large plasmids migrated as discrete bands that were readily observed after ethidium staining. Their sizes were reliably estimated by comparison with linear DNA markers. Without S1 digestion, supercoiled plasmids migrated at rates that were not a simple function of their molecular weights, making size determinations problematic. S1-PFGE detected megaplasmids up to 609 kilobases (kb) in six genera of bacteria (Agrobacterium, Escherichia, Klebsiella, Pseudomonas, Salmonella, and Staphylococcus). The procedure gave size values consistent with previous estimates for characterized megaplasmids. Eight new plasmids between 102 and 316 kb were discovered in Klebsiella and Staphylococcus. S1-PFGE avoids the difficulties of plasmid isolation, eliminates the preparation of probes, and does not require knowledge of restriction enzyme cleavage sites. It detects multiple large plasmids up to the limits of PFGE and can be used to screen for megaplasmids in many strains simultaneously.

Diversity and stability of restriction enzyme profiles of plasmid DNA from methicillin-resistant Staphylococcus aureus.

Nosocomial infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a significant epidemiological problem. Detecting the sources of epidemic strains and preventing their access to patients, however, depend upon the availability of techniques to reliably distinguish among MRSA strains. We evaluated restriction enzyme analysis of plasmid DNA for use as an epidemiological marker of MRSA strains. The diversity of plasmid types was assessed by examining 120 clinical and environmental MRSA isolates from five southern California hospitals and from the American Type Culture Collection. Thirty-seven distinctive EcoRI digestion patterns were observed. We characterized each strain by the number of plasmids it contained and the sizes of the fragments that were generated by EcoRI. Very few of the isolates (4.2%) lacked plasmids, and some (6.7%) contained DNA that was not digested by EcoRI. Several isolates (12.5%) contained two or more plasmids. We were able to assess the stability of MRSA plasmid types by tracking epidemic strains over a 2-year period. We also examined successive isolates from 10 individual patients during their hospitalization. In all but one case, the patient's plasmid profiles remained unchanged. We conclude that the diversity and stability of MRSA plasmid types make them excellent epidemiological markers. In support of this conclusion, we found that our data provided significant epidemiological insights. Two epidemic strains, accounting for more than half of the infections, were identified in the five hospitals. The remaining cases were sporadic, caused by MRSA strains that appeared very infrequently and that may have originated from sources outside the hospitals.