Charles F. Earhart, Jr.

Title:	Professor	Video - PC  Mac
Education:	Ph.D.: 1967, Purdue University; B.A.: 1962, Knox College
Postdoc.:	Purdue University; Tufts University School of Medicine
Research:	Microbial physiology and genetics; biotechnology
Office:	ESB 505A
Phone:	(512) 471-1561
E-mail:	earhart@mail.utexas.edu
Postal Address:	The University of Texas at Austin Molecular Genetics & Microbiology 1 University Station  A5000 Austin TX 78712-0162
Courses taught:	BIO 392 "Problems in Host-Parasite Biology: Bacterial Envelopes" BIO 396 "Membranes and Walls of Bacteria"

To obtain iron in environments such as body fluids that are low in readily-available iron, most aerobic and facultatively aerobic bacteria synthesize and secrete small iron-binding molecules termed siderophores. Siderophores form a complex with extracellular iron and, in a process requiring at least six bacterial envelope proteins, the ferrisiderophore complex is transported into the cell. Once in the cytoplasm, iron is released from the complex enzymatically. The iron acquisition process is precisely regulated, and the genes necessary for siderophore biosynthesis and transport are expressed only under iron-deficient conditions.

The siderophore produced by E. coli, Salmonella typhimurium, and several other enteric bacteria is termed enterobactin (Ent). Seven genes (entA-G) are necessary for Ent biosynthesis, at least five genes (fepA-D,G) are specifically required for ferriEnt transport, and the product of the fes gene functions to remove iron from Ent in the cytoplasm (see figure). These genes are organized into five operons located at approximately 13 min on the E. coli map. The 21Kb of DNA that contains the Ent cluster genes has been isolated and sequenced. Specific areas of research include (i) localizing enterobactin synthase, a putative enzyme complex (EntD, E, F, G) responsible for the last steps in Ent biosynthesis, (ii) characterizing the process by which Ent is secreted from cells, (iii) determining when, how and to what extent Ent-related compounds function in providing iron to cells and (iv) isolating and characterizing FepB, the periplasmic protein that binds ferriEnt complexes. Regulation of fepB is also being studied; the transcript for fepB has a 213 base leader sequence that can be drawn as a stable stem and loop structure and that includes an open reading frame for a 68 amino acid polypeptide.

Also, our efforts at elucidating the molecular mechanisms by which iron transport is accomplished and regulated lead us into the related fields of global iron regulation, environmentally-induced alterations in the bacterial cell surface, periplasmic transport systems, outer membrane receptor proteins, bacterial protein export, and the relationship between iron availability and virulence of certain pathogenic bacteria.

Our second research area involves a genetic construct that permits the targeting of normally soluble proteins to the E. coli surface. A fusion protein vector consisting of sequences of the major lipoprotein (for anchoring to the outer membrane) and five transmembrane sequences from OmpA (for surface exposure of passenger polypeptide) is used. When their genes have been added in-frame downstream of the plasmid vector ompA sequence, several proteins including periplasmic protein betaB-lactamase and authentic cytoplasmic protein thioredoxin have been localized to the outer membrane and oriented so as to react with molecules in the environment. Current work concerns optimizing the system and extending it to include large soluble proteins normally located in the cytoplasm. This system appears to have broad biotechnology applications.

Earhart, C. F. (2004). Iron uptake via the enterobactin system, p. 133-146. In J. Crosa, A. Mey and S. Payne (eds.) Iron Transport in Bacteria, ASM Press, Washington, D.C.

Earhart, C. F. (2003). Iron Metabolism, p. 637-644. In Moselio Schaechter, (ed.), Desk Encyclopedia of Microbiology, Elsevier Science (USA), San Diego, CA.

Earhart, C. F. (2000). Use of the LppOmpA vehicle for surface display, p. 506-516. In J. N. Abelson, S. D. Emr, and J. Thorner (eds.), Chimeric proteins, parts A and B, Methods in Enzymology, v. 236. Academic Press, San Diego, CA.

Membrane association of the Escherichia coli enterobactin synthase proteins EntB/G, EntE, and EntF.
Hantash F.M. and C.F. Earhart
The Journal of Bacteriology 182 (2000): 1768-1773

ABSTRACT: The cytosolic proteins EntE, EntF, and EntB/G, which are Escherichia coli enzymes necessary for the final stage of enterobactin synthesis, are released by osmotic shock. Here, consistent with the idea that cytoplasmic proteins found in shockates have an affinity for membranes, a small fraction of each was found in membrane preparations. Two procedures demonstrated that the enzymes were enriched in a minor membrane fraction of buoyant density intermediate between that of cytoplasmic and outer membranes, providing indirect support for the notion that these proteins have a role in enterobactin excretion as well as synthesis.

ABSTRACT: The terminal reactions in the synthesis of the siderophore enterobactin (Ent) by Escherichia coli require the EntD, E, F and B/G polypeptides. The idea that these molecules form a complex (Ent synthase) that is membrane-associated was re-evaluated. In vitro results provided no evidence in support of the proposal: (i) Ent synthase activity occurred normally under conditions where membrane was either absent or disrupted by high concentrations of neutral detergents, and (ii) immunoprecipitation experiments conducted on extracts engaged in Ent synthesis failed to detect any association among the Ent polypeptides. However, Western blot analyses showed that EntE, F and B/G were released from cells by osmotic shock and freeze/thaw treatment but not by conversion of cells to spheroplasts. These results demonstrated that EntE, F and B/G belong to the Beacham group D class of proteins. The shockability of a given group D Ent protein was unaffected by the absence of either EntB/G or EntD and, for EntB/G, the N-terminus was sufficient for release by osmotic shock. The behaviour of group D proteins is generally attributed to their association (partial, loose or transient) with cytoplasmic membrane; therefore, the results are indirect evidence that Ent synthase interacts with membrane in vivo. At the very least, the data indicate that EntE, F and B/G are compartmentalized in E. coli and, because other biosynthetic enzymes for siderophores and surfactants are related to these Ent proteins, suggest that this entire protein class may be sequestered in vivo.

Uptake and metabolism of iron and molybdenum.
Earhart, C. F.
in: "Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology", 2nd ed., F. C. Neidhardt (ed.) Washington, DC: American Society for Microbiology (1996): 1075-1090.

ABSTRACT: The Lpp'OmpA(46-159) hybrid protein can serve as an efficient targeting vehicle for localizing a variety of procaryotic and eucaryotic soluble proteins onto the E. coli surface, thus providing a system for several possible biotechnology applications. Here we show that fusion between Lpp'OmpA(46-159) and bacterial alkaline phosphatase (PhoA), a normally periplasmic dimeric enzyme, are also targeted to the outer membrane. However, protease accessibility experiments and immunoelectron microscopy revealed that, unlike other periplasmic proteins, the PhoA domain of these fusions is not exposed on the cell surface in cells having an intact outer membrane. Conditions that affect the formation of disulfide bonds and the folding of the PhoA domain in the periplasm not only did not facilitate targeting to the cell surface but led to lethality when the fusion was expressed from a high-copy-number plasmid. Furthermore, E. coli expressing the Lpp'OmpA(46-159)-PhoA fusion exhibited strain- and temperature-dependent alterations in outer-membrane permeability. Our results are consistent with previous studies with other vehicles indicating that PhoA is not displayed on the surface when fused to cell-surface expression vectors. Presumably, the enzyme rapidly assumes a tightly folded dimeric conformation that cannot be transported across the outer membrane. The large size and quaternary structure of PhoA may define a limitation of the Lpp'OmpA(46-159) fusion system for the display of periplasmic proteins on the cell surface. Alkaline phosphatase is a unique protein among a group of five periplasmic proteins (beta-lactamase, alkaline phosphatase, Cex cellulase Cex cellulose-binding domain, and a single-chain Fv antibody fragment), which have been tested as passengers for the Lpp'OmpA(46-159) expression system to date, since it was the only protein not displayed on the surface.

ABSTRACT: Bacterial cell-surface exposure of foreign peptides and soluble proteins has been achieved recently by employing a fusion protein methodology. An Lpp'-OmpA(46-159)-Bla fusion protein has been shown previously to display the normally periplasmic enzyme beta-lactamase (Bla) on the cell surface of the Gram-negative bacterium Escherichia coli. Here, we have investigated the role of the OmpA domain of the tripartite fusion protein in the surface display of the passenger domain (Bla) and have characterized the effects of the fusion proteins on the integrity and permeability of the outer membrane. We show that in addition to OmpA(46-159), a second OmpA segment, consisting of amino acids 46-66, can also mediate the display of Bla on the cell surface. Other OmpA domains of various lengths (amino acids 46-84, 46-109, 46-128, 46-141 and 46-145) either anchored the Bla domain on the periplasmic face of the outer membrane or caused a major disruption of the outer membrane, allowing the penetration of antibodies into the cell. Detergent and antibiotic sensitivity and periplasmic leakage assays showed that changes in the permeability of the outer membrane are an unavoidable consequence of displaying a large periplasmic protein on the surface of E. coli. This is the first systematic report on the effects that cell surface engineering may have on the integrity and permeability properties of bacterial outer membranes.