Gene expression profiles of Escherichia coli K-12 W3110 were compared as a function of steady-state external pH. Cultures were grown to an optical density at 600 nm of 0.3 in potassium-modified Luria-Bertani medium buffered at pH 5.0, 7.0, and 8.7. For each of the three pH conditions, cDNA from RNA of five independent cultures was hybridized to Affymetrix E. coli arrays. Analysis of variance with an ␣ level of 0.001 resulted in 98% power to detect genes showing a twofold difference in expression. Normalized expression indices were calculated for each gene and intergenic region (IG). Differential expression among the three pH classes was observed for 763 genes and 353 IGs. Hierarchical clustering yielded six well-defined clusters of pH profiles, designated Acid High (highest expression at pH 5.0), Acid Low (lowest expression at pH 5.0), Base High (highest at pH 8.7), Base Low (lowest at pH 8.7), Neutral High (highest at pH 7.0, lower in acid or base), and Neutral Low (lowest at pH 7.0, higher at both pH extremes). Flagellar and chemotaxis genes were repressed at pH 8.7 (Base Low cluster), where the cell's transmembrane proton potential is diminished by the maintenance of an inverted pH gradient. High pH also repressed the proton pumps cytochrome o (cyo) and NADH dehydrogenases I and II. By contrast, the proton-importing ATP synthase F 1 F o and the microaerophilic cytochrome d (cyd), which minimizes proton export, were induced at pH 8.7. These observations are consistent with a model in which high pH represses synthesis of flagella, which expend proton motive force, while stepping up electron transport and ATPase components that keep protons inside the cell. Acid-induced genes, on the other hand, were coinduced by conditions associated with increased metabolic rate, such as oxidative stress. All six pH-dependent clusters included envelope and periplasmic proteins, which directly experience external pH. Overall, this study showed that (i) low pH accelerates acid consumption and proton export, while coinducing oxidative stress and heat shock regulons; (ii) high pH accelerates proton import, while repressing the energy-expensive flagellar and chemotaxis regulons; and (iii) pH differentially regulates a large number of periplasmic and envelope proteins.

Fish is a scaffolding protein and Src substrate. It contains an amino-terminal Phox homology (PX) domain and five Src homology 3 (SH3) domains, as well as multiple motifs for binding both SH2 and SH3 domaincontaining proteins. We have determined that the PX domain of Fish binds 3-phosphorylated phosphatidylinositols (including phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate). Consistent with this, a fusion protein of green fluorescent protein and the Fish PX domain localized to punctate structures similar to endosomes in normal fibroblasts. However, the full-length Fish protein was largely cytoplasmic, suggesting that its PX domain may not be able to make intermolecular interactions in unstimulated cells. In Src-transformed cells, we observed a dramatic re-localization of some Fish molecules to actin-rich structures called podosomes; the PX domain was both necessary and sufficient to effect this translocation. We used a phage display screen with the fifth SH3 domain of Fish and isolated ADAM19 as a binding partner. Subsequent analyses in mammalian cells demonstrated that Fish interacts with several members of the ADAMs family, including ADAMs 12, 15, and 19. In Src-transformed cells, ADAM12 co-localized with Fish in podosomes. Because members of the ADAMs family have been implicated in growth factor processing, as well as cell adhesion and motility, Fish could be acting as an adaptor molecule that allows Src to impinge on these processes.

Acetate and formate are major fermentation products of Escherichia coli. Below pH 7, the balance shifts to lactate; an oversupply of acetate or formate retards growth. E. coli W3110 was grown with aeration in potassium-modified Luria broth buffered at pH 6.7 in the presence or absence of added acetate or formate, and the protein profiles were compared by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Acetate increased the steady-state expression levels of 37 proteins, including periplasmic transporters for amino acids and peptides (ArtI, FliY, OppA, and ProX), metabolic enzymes (YfiD and GatY), the RpoS growth phase regulon, and the autoinducer synthesis protein LuxS. Acetate repressed 17 proteins, among them phosphotransferase (Pta). An ackA-pta deletion, which nearly eliminates interconversion between acetate and acetyl-coenzyme A (acetyl-CoA), led to elevated basal levels of 16 of the acetate-inducible proteins, including the RpoS regulon. Consistent with RpoS activation, the ackA-pta strain also showed constitutive extreme-acid resistance. Formate, however, repressed 10 of the acetate-inducible proteins, including the RpoS regulon. Ten of the proteins with elevated basal levels in the ackA-pta strain were repressed by growth of the mutant with formate; thus, the formate response took precedence over the loss of the ackA-pta pathway. The similar effects of exogenous acetate and the ackA-pta deletion, and the opposite effect of formate, could have several causes; one possibility is that the excess buildup of acetyl-CoA upregulates stress proteins but excess formate depletes acetyl-CoA and downregulates these proteins.

The 49-residue functional upstream domain (FUD) of Streptococcus pyogenes F1 adhesin interacts with fibronectin (FN) in a heretofore unknown manner that prevents assembly of a FN matrix. Biotinylated FUD (b-FUD) bound to adsorbed FN or its recombinant N-terminal 70-kDa fibrin- and gelatin-binding fragment (70K). Binding was blocked by FN or 70K, but not by fibrin- or gelatin-binding subfragments of 70K. Isothermal titration calorimetry showed that FUD binds with Kd values of 5.2 and 59 nm to soluble 70K and FN, respectively. We tested sets of FUD mutants and epitope-mapped monoclonal antibodies (mAbs) for ability to compete with b-FUD for binding to FN or to block FN assembly by cultured fibroblasts. Deletions or alanine substitutions throughout FUD caused loss of both activities. mAb 4D1 to the 2FNI module had little effect, whereas mAb 7D5 to the 4FNI module in the fibrin-binding region, 5C3 to the 9FNI module in the gelatin-binding region, or L8 to the G-strand of 1FNIII module adjacent to 9FNI caused loss of binding of b-FUD to FN and decreased FN assembly. Conversely, FUD blocked binding of 7D5, 5C3, or L8, but not of 4D1, to FN. Circular dichroism indicated that FUD binds to 70K by β-strand addition, a possibility supported by modeling based on crystal structures of peptides bound to 2FNI-5FNI of the fibrin-binding domain and 8FNI-9FNI of the gelatin-binding domain. Thus, the interaction likely involves an extensive anti-parallel β-zipper in which FUD interacts with the E-strands of 2FNI-5FNI and 8FNI-9FNI.

Fibronectin is a large vertebrate glycoprotein that is found in soluble and insoluble forms and involved in diverse processes. Protomeric fibronectin is a dimer of subunits, each of which comprises 29 to 31 modules—12 type I, two type II, and 15-17 type III. Plasma fibronectin is secreted by hepatocytes and circulates in a compact conformation before it binds to cell surfaces, converts to an extended conformation, and is assembled into fibronectin fibrils. Here we review biophysical and structural studies that have shed light on how plasma fibronectin transitions from the compact to the extended conformation. The three types of modules each have a well-organized secondary and tertiary structure as defined by NMR and crystallography and have been likened to “beads on a string”. There are flexible sequences in the N-terminal tail, between the fifth and sixth type I modules, between the first two and last two of the type III modules, and at the C-terminus. Several specific module-module interactions have been identified that likely maintain the compact quaternary structure of circulating fibronectin. The quaternary structure is perturbed in response to binding events, including binding of fibronectin to the surface of vertebrate cells for fibril assembly and to bacterial adhesins.

We describe the identification and characterization of two genes and their gene products responsible for determining receptor binding and pilus length in type 1-piliated Escherichia coli. One gene, pilE, conferred the ability of piliated cells to agglutinate guinea pig erythrocytes. The other gene, pilF, determined pilus length, in that mutants having lesions in pilF had very long pili. The two genes were detected after TnS mutagenesis of a cloned segment of DNA that normally complemented a pilE lesion in the chromosome. Thus, lesions in pilE or pilF on the cloned segment resulted in mutants having the PilE-phenotype (piliated but unable to agglutinate erythrocytes). Introduction of the plasmid-encoded mutant alleles of pilE and pilF into the chromosome followed by electron microscopic examination of the mutants showed that only lesions in pilF conferred the striking increase in pilus length. Mutations in pilF could be complemented in trans by the original cloned segment to produce cells with parental-length pili. Minicell transcription and translation of the cloned pilE and pilF genes having representative TnS insertion mutations showed that the pilE gene product was a protein of ca. 31 kilodaltons and that the pilF gene product was a protein of ca. 18 kilodaltons. We believe that the pilF gene product may act as a competititve inhibitor of pilus polymerization. Thus, pilus length may be controlled by the ratio of pilin to pilF gene product present within the cell.Type 1 pili of Escherichia coli provide a convenient model for studying the control and assembly of supramolecular extracellular structures. Also, they provide a model for cell-cell interaction, since type 1 pili are required to mediate attachment of E. coli to a variety of eucaryotic cells through a mannose-sensitive interaction with a receptor on the eucaryotic cell (4).One interesting facet of the assembly of type 1 pili concerns how the cell monitors the number of pili it has and how long those pili are. These two considerations would seem to be important for the cell to conserve energy. However, since pili are on the outside of the cell, it is unclear how the cell would keep track of these two parameters. It is apparent from recent work that at least one mechanism exists for controlling the number of pili per cell. This fact is indicated by the existence of hyperpiliated mutants (13). Since hyperpiliated mutants also have longer pili (13), one might infer that the cell has a way to monitor pilus length also. However, no such pilus length mutants have been isolated.Another interesting feature of type 1 pili concerns their involvement in receptor-ligand interactions. Recently, it has become evident that while type 1 pili are required for receptor binding, their presence is not sufficient for binding (8). This fact is indicated by the existence of fully piliated mutants that fail to agglutinate guinea pig erythrocytes (8).

Fibronectin (FN) is a glycoprotein recognized originally in the 1940s as a contaminant of fibrinogen in Cohn fraction I of plasma. Decades of research demonstrated FN synthesis by a variety of cells and defined FN as an essential component of the extracellular matrix with roles in embryogenesis, development, and wound healing. More recently, FN has emerged as player in platelet thrombus formation and diseases associated with thrombosis including vascular remodeling, atherosclerosis, and cardiac repair following a myocardial infarct. We discuss the mechanisms by which this might occur and conclude that FN may have a unique role in thrombosis without affecting normal hemostasis and therefore may be a reasonable therapeutic target for the prevention of thrombotic diseases.

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