Eric R. Olson

Influence of pH on bacterial gene expression

Bacteria respond to changes in internal and external pH by adjusting the activity and synthesis of proteins associated with many different processes, including proton translocation, amino acid degradation, adaptation to acidic or basic conditions and virulence. While, for many of these examples, the physiological and biological consequence of the pH‐induced response is clear, the mechanism by which the transcription/translation machinery is signalled is not. These examples are discussed along with several others in which the function of the gene or protein remains a mystery.

Altered pH lysine signalling mutants of cadC, a gene encoding a membrane‐bound transcriptional activator of the Escherichia coli cadBA operon

The Escherichia coli CadC protein is required for activation of cadBA transcription under conditions of low external pH and exogenous lysine. cadBA encodes proteins involved in the decarboxylation of lysine to cadaverine, and cadaverine excretion. Sequence analysis suggested that CadC contains a single transmembrane segment separating a DNA‐binding domain in the amino terminus from a periplasmic domain. Western analysis of subcellular fractions demonstrated that CadC is expressed and concentrated in the cytoplasmic membrane in cells grown either at an inducing pH (pH5.8) or at a non‐inducing pH (pH7.6.). Eight cadC mutants were isolated based on their ability to confer expression of a cadA‐lacZ fusion independent of low external pH or exogenous lysine. Five of these mutants expressed the cadA‐lacZ fusion at both pH 5.8 and pH 7.6, but retained the requirement for the lysine signal while the other three mutants displayed pH independence in the presence of lysine, and lysine independence at pH 5.8 but not at pH 7.6. These results support a model in which CadC is a membrane‐bound transcriptional activator of the cadBA operon capable of sensing (directly or indirectly) signals generated outside the cytoplasmic membrane as a consequence of acidic pH and lysine.

Functional and Biochemical Characterization of Escherichia coli Sugar Efflux Transporters

A family of bacterial transporters, the SET (sugar efflux transporter) family, has been recently reported (Liu, J. Y., Miller, P. F., Gosink, M., and Olson, E. R. (1999) Mol. Microbiol. 31, 1845–1851). In this study, the biochemical and cell biological properties of the three Escherichia coli members (SetA, SetB, and SetC) of the family are characterized. We show that both SetA and SetB can transport lactose and glucose. In addition, SetA has broad substrate specificity, with preferences for glucosides or galactosides with alkyl or aryl substituents. Consistent with the observed in vitro substrate specificities, strains that hyperexpress SetA or SetB are desensitized to lactose analogues as measured by induction of the lac operon. In addition, strains that hyperexpress SetA are resistant to the growth inhibitory sugar analogueo-nitrophenyl-β-d-thiogalactoside. Strains disrupted for any one or all of the set genes are viable and show no defects in lactose utilization nor increased sensitivity to inducers of the lac operon and nonmetabolizable sugar analogues. The data suggest that the set genes are either poorly expressed under normal laboratory growth conditions or are redundant with other cellular gene products.

The identification of a new family of sugar efflux pumps in Escherichia coli.

Using a functional cloning strategy with an Escherichia coli genomic plasmid library, we have identified a new family of sugar efflux proteins with three highly homologous members in the E. coli genome. In addition, two open reading frames, one present in Yersinia pestis and the other in Deinococcus radioduransappear to encode closely related proteins. An in vitro transport assay using inside‐out membrane vesicles prepared from overproducing strains was used to demonstrate that members of this new family can efflux [14C]‐lactose. As sugar efflux phenomena have been reported previously in several bacterial species including E. coli, the identification of a new family of sugar efflux proteins may help to reveal the physiological role of sugar efflux in metabolism. It is proposed that the E. coli members of this family, whose functions were previously unknown, be given the gene family designation SET for sugar efflux transporter.

Application of two-dimensional protein gels in biotechnology

The optimal use of biological systems for technologically developed products will not be achieved until biological systems are completely defined in biochemical terms. Two-dimensional polyacrylamide gel electrophoresis, 2-D gels, are contributing to this goal. These gels separate complex mixtures of proteins into individual polypeptide species. The ultimate use of 2-D gels is the construction of cellular 2-D gel databases which identify the proteins on the gels and catalog their responses to different environmental conditions. In addition to these global analyses, many applications for 2-D gels in basic, applied and clinical research have been shown.

Bacterial Two-Component Signalling as a Therapeutic Target in Drug Design

Bacterial resistance to the currently available antibiotics is a worldwide problem with catastrophic potential (Kunin, 1993; Berkowitz, 1995; Domagala and Sanchez, 1997). In the US, overall mortality from infectious diseases has increased 58% with deaths from respiratory infections and septicemia up 20 and 83% respectively (Pinner et al., 1996). Increased resistance has been documented for every major group of pathogens with methicillin resistant Staphyloccus aureus (MRSA), vancomycin resistant enterococci (VRE) and β-lactam resistant streptococci causing the greatest alarm (Cormican and Jones, 1996; Howe et al., 1996). In fact, VRE has been described as the most feared nosocomial pathogen today (Hagman and Strausbaugh, 1996). The fear of vancomycin resistant MRS A has sparked calls for stringent preemptive controls (Edmund et al., 1996). Equally alarming is the dramatic increase in resistance among the gram negative organisms (Jones, 1996). Nosocomial pneumonias (often with resistant organisms) occur in 0.5–5% of all hospitalized patients with a mortality of 20–70%, and an added annual cost of

Global analysis of proteins synthesized during phosphorus restriction in Escherichia coli.

2.5 billion for increased care and treatment (Gaynes and Lynch, 1991; Swartz, 1994). So great and urgent is the problem that the prestigious journal Science has devoted two issues to bacterial resistance (Travis, 1994; Koshland, 1992), and the American Society for Microbiology formed a Task Force to define the problem and make recommendations. Their insightful report, issued in 1995 (Report 1995), called for an increased effort in the discovery of new drugs and new drug targets in bacteria.