Unique chromosomal regions associated with virulence of an avian pathogenic Escherichia coli strain.

The avian pathogenic Escherichia coli strain (chi)7122 (serotype O78:K80:H9) causes airsacculitis and colisepticemia in chickens. To identify genes associated with avian disease, a genomic subtraction technique was performed between strain (chi)7122 and the E. coli K-12 strain (chi)289. The DNA isolated using this method was found only in strain (chi)7122 and was used to identify cosmid clones carrying unique DNA from a library of (chi)7122 that were then used to map the position of unique DNA on the E. coli chromosome. A total of 12 unique regions were found, 5 of which correspond to previously identified positions for unique DNA sequence in E. coli strains. To assess the role each unique region plays in virulence, mutants of (chi)7122 were constructed in which a segment of unique DNA was replaced with E. coli K-12 DNA by cotransduction of linked transposon insertions in DNA flanking the unique sequence. The resulting replacement mutants were assessed for inability to colonize the air sac and cause septicemia in 2-week-old white Leghorn chickens. Two mutants were found to be avirulent when injected into the right caudal air sac of 2-week-old chickens. One avirulent mutant, designated (chi)7145, carries a replacement of the rfb locus at 44 min, generating a rough phenotype. The second mutant is designated (chi)7146, and carries a replacement at position 0.0 min on the genetic map. Both mutants could be complemented to partial virulence by cosmids carrying sequences unique to (chi)7122.

The anti-angiogenic agent fumagillin covalently modifies a conserved active-site histidine in the Escherichia coli methionine aminopeptidase

Methionine aminopeptidase (MetAP) exists in two forms (type I and type II), both of which remove the N-terminal methionine from proteins. It previously has been shown that the type II enzyme is the molecular target of fumagillin and ovalicin, two epoxide-containing natural products that inhibit angiogenesis and suppress tumor growth. By using mass spectrometry, N-terminal sequence analysis, and electronic absorption spectroscopy we show that fumagillin and ovalicin covalently modify a conserved histidine residue in the active site of the MetAP from Escherichia coli, a type I enzyme. Because all of the key active site residues are conserved, it is likely that a similar modification occurs in the type II enzymes. This modification, by occluding the active site, may prevent the action of MetAP on proteins or peptides involved in angiogenesis. In addition, the results suggest that these compounds may be effective pharmacological agents against pathogenic and resistant forms of E. coli and other microorganisms.

Multiple independent origins of Shigella clones of Escherichia coli and convergent evolution of many of their characteristics

The evolutionary relationships of 46 Shigella strains representing each of the serotypes belonging to the four traditional Shigella species (subgroups), Dysenteriae, Flexneri, Boydii, and Sonnei, were determined by sequencing of eight housekeeping genes in four regions of the chromosome. Analysis revealed a very similar evolutionary pattern for each region. Three clusters of strains were identified, each including strains from different subgroups. Cluster 1 contains the majority of Boydii and Dysenteriae strains (B1–4, B6, B8, B10, B14, and B18; and D3–7, D9, and D11–13) plus Flexneri 6 and 6A. Cluster 2 contains seven Boydii strains (B5, B7, B9, B11, B15, B16, and B17) and Dysenteriae 2. Cluster 3 contains one Boydii strain (B12) and the Flexneri serotypes 1–5 strains. Sonnei and three Dysenteriae strains (D1, D8, and D10) are outside of the three main clusters but, nonetheless, are clearly within Escherichia coli. Boydii 13 was found to be distantly related to E. coli. Shigella strains, like the other pathogenic forms of E. coli, do not have a single evolutionary origin, indicating convergent evolution of Shigella phenotypic properties. We estimate the three main Shigella clusters to have evolved within the last 35,000 to 270,000 years, suggesting that shigellosis was one of the early infectious diseases of humans.

Complementation of an Escherichia coli adhE mutant by the Entamoeba histolytica EhADH2 gene provides a method for the identification of new antiamebic drugs.

The pathogenic protozoan parasite Entamoeba histolytica, the cause of amebic dysentery and amebic liver abscess, is an obligate anaerobe, and derives energy from the fermentation of glucose to ethanol with pyruvate and acetyl coenzyme A as intermediates. We have isolated EhADH2, a key enzyme in this pathway, that is a NAD+- and Fe2+-dependent bifunctional enzyme with acetaldehyde dehydrogenase and alcohol dehydrogenase activities. EhADH2 is the only known eukaryotic member of a newly defined family of prokaryotic multifunctional enzymes, which includes the Escherichia coli AdhE enzyme, an enzyme required for anaerobic growth of E. coli. Because of the critical role of EhADH2 in the amebic fermentation pathway and the lack of known eukaryotic homologues of the EhADH2 enzyme, EhADH2 represents a potential target for antiamebic chemotherapy. However, screening of compounds for antiamebic activity is hampered by the cost of large scale growth of Ent. histolytica, and difficulties in quantitating drug efficacy in vitro. To approach this problem, we expressed the EhADH2 gene in a mutant strain of E. coli carrying a deletion of the adhE gene. Expression of EhADH2 restored the ability of the mutant E. coli strain to grow under anaerobic conditions. By screening compounds for the ability to inhibit the anaerobic growth of the E. coli/EhADH2 strain, we have developed a rapid assay for identifying compounds with anti-EhADH2 activity. Using bacteria to bypass the need for parasite culture in the initial screening process for anti-parasitic agents could greatly simplify and reduce the cost of identifying new therapeutic agents effective against parasitic diseases.

Escherichia coli exhibits negative chemotaxis in gradients of hydrogen peroxide, hypochlorite, and N-chlorotaurine: products of the respiratory burst of phagocytic cells.

Escherichia coli can respond to gradients of specific compounds, moving up gradients of attractants and down gradients of repellents. Stimulated phagocytic leukocytes produce H2O2, OCl-, and N-chlorotaurine in a response termed the respiratory burst. E. coli is actively repelled by these compounds. Catalase in the suspending medium eliminated the effect of H2O2. Repulsion by H2O2 could be demonstrated with 1 microM H2O2, which is far below the level that caused overt toxicity. Strains with defects in the biosynthesis of glutathione or lacking hydroperoxidases I and II retained this response to H2O2, and 2.0 mM CN- did not interfere with it. Mutants with defects in any one of the four known methyl-accepting chemotaxis proteins also retained the ability to respond to H2O2, but a "gutted" mutant that was deleted for all four methyl-accepting chemotaxis proteins, as well as for CheA, CheW, CheR, CheB, CheY, and CheZ, did not respond to H2O2. Hypochlorite and N-chlorotaurine were also strongly repellent. Chemotaxis down gradients of H2O2, OCl-, and N-chlorotaurine may contribute to the survival of commensal or pathogenic microorganisms.

Control of cell division in Escherichia coli: regulation of transcription of ftsQA involves both rpoS and SdiA-mediated autoinduction.

The conditioning of culture medium by the production of growth-regulatory substances is a well-established phenomenon with eukaryotic cells. It has recently been shown that many prokaryotes are also capable of modulating growth, and in some cases sensing cell density, by production of extracellular signaling molecules, thereby allowing single celled prokaryotes to function in some respects as multicellular organisms. As Escherichia coli shifts from exponential growth to stationary growth, many changes occur, including cell division leading to formation of short minicells and expression of numerous genes not expressed in exponential phase. An understanding of the coordination between the morphological changes associated with cell division and the physiological and metabolic changes is of fundamental importance to understanding regulation of the prokaryotic cell cycle. The ftsQA genes, which encode functions required for cell division in E. coli, are regulated by promoters P1 and P2, located upstream of the ftsQ gene. The P1 promoter is rpoS-stimulated and the second, P2, is regulated by a member of the LuxR subfamily of transcriptional activators, SdiA, exhibiting features characteristic of an autoinduction (quorum sensing) mechanism. The activity of SdiA is potentiated by N-acyl-homoserine lactones, which are the autoinducers of luciferase synthesis in luminous marine bacteria as well as of pathogenesis functions in several pathogenic bacteria. A compound(s) produced by E. coli itself during growth in Luria Broth stimulates transcription from P2 in an SdiA-dependent process. Another substance(s) enhances transcription of rpoS and (perhaps indirectly) of ftsQA via promoter P1. It appears that this bimodal control mechanism may comprise a fail-safe system, such that transcription of the ftsQA genes may be properly regulated under a variety of different environmental and physiological conditions.

Expression of Batis maritima methyl chloride transferase in Escherichia coli

Methyl chloride transferase, a novel enzyme found in several fungi, marine algae, and halophytic plants, is a biological catalyst responsible for the production of atmospheric methyl chloride. A previous paper reports the purification of this methylase from Batis maritima and the isolation of a cDNA clone of the gene for this enzyme. In this paper, we describe the isolation of a genomic clone of the methylase gene and the expression of recombinant methyl chloride transferase in Escherichia coli and compare the kinetic behavior of the wild-type and recombinant enzyme. The recombinant enzyme is active and promotes the production of methyl chloride by E. coli under in vivo conditions. The kinetic data indicate that the recombinant and wild-type enzymes have similar halide (Cl−, Br−, and I−)-binding capacities. Both the recombinant and wild-type enzymes were found to function well in high NaCl concentrations. This high salt tolerance resembles the activity of halobacterial enzymes rather than halophytic plant enzymes. These findings support the hypothesis that this enzyme functions in the control and regulation of the internal concentration of chloride ions in halophytic plant cells.

A single-headed dimer of Escherichia coli ribosomal protein L7/L12 supports protein synthesis

During protein synthesis, the two elongation factors Tu and G alternately bind to the 50S ribosomal subunit at a site of which the protein L7/L12 is an essential component. L7/L12 is present in each 50S subunit in four copies organized as two dimers. Each dimer consists of distinct domains: a single N-terminal (“tail”) domain that is responsible for both dimerization and binding to the ribosome via interaction with the protein L10 and two independent globular C-terminal domains (“heads”) that are required for binding of elongation factors to ribosomes. The two heads are connected by flexible hinge sequences to the N-terminal domain. Important questions concerning the mechanism by which L7/L12 interacts with elongation factors are posed by us in response to the presence of two dimers, two heads per dimer, and their dynamic, mobile properties. In an attempt to answer these questions, we constructed a single-headed dimer of L7/L12 by using recombinant DNA techniques and chemical cross-linking. This chimeric molecule was added to inactive core particles lacking wild-type L7/L12 and shown to restore activity to a level approaching that of wild-type two-headed L7/L12.

DnaA-stimulated transcriptional activation of oriλ: Escherichia coli RNA polymerase β subunit as a transcriptional activator contact site

We present evidence that Escherichia coli RNA polymerase β subunit may be a transcriptional activator contact site. Stimulation of the activity of the pR promoter by DnaA protein is necessary for replication of plasmids derived from bacteriophage λ. We found that DnaA activates the pR promoter in vitro. Particular mutations in the rpoB gene were able to suppress negative effects that certain dnaA mutations had on the replication of λ plasmids; this suppression was allele-specific. When a potential DnaA-binding sequence located several base pairs downstream of the pR promoter was scrambled by in vitro mutagenesis, the pR promoter was no longer activated by DnaA both in vivo and in vitro. Therefore, we conclude that DnaA may contact the β subunit of RNA polymerase during activation of the pR promoter. A new classification of prokaryotic transcriptional activators is proposed.

Interacting helical faces of subunits a and c in the F1Fo ATP synthase of Escherichia coli defined by disulfide cross-linking

Subunits a and c of Fo are thought to cooperatively catalyze proton translocation during ATP synthesis by the Escherichia coli F1Fo ATP synthase. Optimizing mutations in subunit a at residues A217, I221, and L224 improves the partial function of the cA24D/cD61G double mutant and, on this basis, these three residues were proposed to lie on one face of a transmembrane helix of subunit a, which then interacted with the transmembrane helix of subunit c anchoring the essential aspartyl group. To test this model, in the present work Cys residues were introduced into the second transmembrane helix of subunit c and the predicted fourth transmembrane helix of subunit a. After treating the membrane vesicles of these mutants with Cu(1,10-phenanthroline)2SO4 at 0°, 10°, or 20°C, strong a–c dimer formation was observed at all three temperatures in membranes of 7 of the 65 double mutants constructed, i.e., in the aS207C/cI55C, aN214C/cA62C, aN214C/cM65C, aI221C/cG69C, aI223C/cL72C, aL224C/cY73C, and aI225C/cY73C double mutant proteins. The pattern of cross-linking aligns the helices in a parallel fashion over a span of 19 residues with the aN214C residue lying close to the cA62C and cM65C residues in the middle of the membrane. Lesser a–c dimer formation was observed in nine other double mutants after treatment at 20°C in a pattern generally supporting that indicated by the seven landmark residues cited above. Cross-link formation was not observed between helix-1 of subunit c and helix-4 of subunit a in 19 additional combinations of doubly Cys-substituted proteins. These results provide direct chemical evidence that helix-2 of subunit c and helix-4 of subunit a pack close enough to each other in the membrane to interact during function. The proximity of helices supports the possibility of an interaction between Arg210 in helix-4 of subunit a and Asp61 in helix-2 of subunit c during proton translocation, as has been suggested previously.

Heteroduplex joint formation in Escherichia coli recombination is initiated by pairing of a 3′-ending strand

The formation of heteroduplex joints in Escherichia coli recombination is initiated by invasion of double-stranded DNA by a single-stranded homologue. To determine the polarity of the invasive strand, linear molecules with direct terminal repeats were released by in vivo restriction of infecting chimeric phage DNA and heteroduplex products of intramolecular recombination were analyzed. With this substrate, the invasive strand is expected to be incorporated into the circular crossover product and the complementary strand is expected to be incorporated into the reciprocal linear product. Strands of both polarities were incorporated into heteroduplex structures, but only strands ending 3′ at the break were incorporated into circular products. This result indicates that invasion of the 3′-ending strand initiates the heteroduplex joint formation and that the complementary 5′-ending strand is incorporated into heteroduplex structures in the process of reciprocal strand exchange. The polarity of the invasive strand was not affected by recD, recJ, or xonA mutations. However, xonA and recJ mutations increased the proportion of heteroduplexes containing 5′-ending strands. This observation suggests that RecJ exonuclease and exonuclease I may enhance recombination by degrading the displaced strands during branch migration and thereby causing strand exchange to be unidirectional.

Quorum sensing in Escherichia coli and Salmonella typhimurium

Escherichia coli and Salmonella typhimurium strains grown in Luria–Bertani medium containing glucose secrete a small soluble heat labile organic molecule that is involved in intercellular communication. The factor is not produced when the strains are grown in Luria–Bertani medium in the absence of glucose. Maximal secretion of the substance occurs in midexponential phase, and the extracellular activity is degraded as the glucose is depleted from the medium or by the onset of stationary phase. Destruction of the signaling molecule in stationary phase indicates that, in contrast to other quorum-sensing systems, quorum sensing in E. coli and S. typhimurium is critical for regulating behavior in the prestationary phase of growth. Our results further suggest that the signaling factor produced by E. coli and S. typhimurium is used to communicate both the cell density and the metabolic potential of the environment. Several laboratory and clinical strains of E. coli and S. typhimurium were screened for production of the signaling molecule, and most strains make it under conditions similar to those shown here for E. coli AB1157 and S. typhimurium LT2. However, we also show that E. coli strain DH5α does not make the soluble factor, indicating that this highly domesticated strain has lost the gene(s) or biosynthetic machinery necessary to produce the signaling substance. Implications for the involvement of quorum sensing in pathogenesis are discussed.

Riboregulation in Escherichia coli: DsrA RNA acts by RNA:RNA interactions at multiple loci

DsrA is an 87-nt untranslated RNA that regulates both the global transcriptional silencer and nucleoid protein H-NS and the stationary phase and stress response sigma factor RpoS (σs). We demonstrate that DsrA acts via specific RNA:RNA base pairing interactions at the hns locus to antagonize H-NS translation. We also give evidence that supports a role for RNA:RNA interactions at the rpoS locus to enhance RpoS translation. Negative regulation of hns by DsrA is achieved by the RNA:RNA interaction blocking translation of hns RNA. In contrast, results suggest that positive regulation of rpoS by DsrA occurs by formation of an RNA structure that activates a cis-acting translational operator. Sequences within DsrA complementary to three additional genes, argR, ilvIH, and rbsD, suggest that DsrA is a riboregulator of gene expression that acts coordinately via RNA:RNA interactions at multiple loci.

The Escherichia coli SOS mutagenesis proteins UmuD and UmuD′ interact physically with the replicative DNA polymerase

The Escherichia coli umuDC operon is induced in response to replication-blocking DNA lesions as part of the SOS response. UmuD protein then undergoes an RecA-facilitated self-cleavage reaction that removes its N-terminal 24 residues to yield UmuD′. UmuD′, UmuC, RecA, and some form of the E. coli replicative DNA polymerase, DNA polymerase III holoenzyme, function in translesion synthesis, the potentially mutagenic process of replication over otherwise blocking lesions. Furthermore, it has been proposed that, before cleavage, UmuD together with UmuC acts as a DNA damage checkpoint system that regulates the rate of DNA synthesis in response to DNA damage, thereby allowing time for accurate repair to take place. Here we provide direct evidence that both uncleaved UmuD and UmuD′ interact physically with the catalytic, proofreading, and processivity subunits of the E. coli replicative polymerase. Consistent with our model proposing that uncleaved UmuD and UmuD′ promote different events, UmuD and UmuD′ interact differently with DNA polymerase III: whereas uncleaved UmuD interacts more strongly with β than it does with α, UmuD′ interacts more strongly with α than it does with β. We propose that the protein–protein interactions we have characterized are part of a higher-order regulatory system of replication fork management that controls when the umuDC gene products can gain access to the replication fork.

Structural kinetics of transcription activation at the malT promoter of Escherichia coli by UV laser footprinting

We have studied the kinetics of transcriptional initiation and activation at the malT and malTp1 promoters of Escherichia coli using UV laser footprinting. Contrary to previous studies and because of the very rapid signal acquisition by this technique, we can obtain structural information about true reaction intermediates of transcription initiation. The consequences of adding a transcriptional activator, the cAMP receptor protein/cAMP complex (CRP), are monitored in real time, permitting us to assign specific interactions to the activation of discrete steps in transcription initiation. Direct protein–protein contacts between CRP and the RNA polymerase appeared very rapidly, followed by DNA melting around the −10 hexamer. CRP slightly increased the rate of this isomerization reaction but, more importantly, favored the establishment of additional contacts between the DNA upstream of the CRP binding site and RNA polymerase subsequent to open complex formation. These contacts make a major contribution to transcriptional activation by stabilizing open forms of the promoter complex, thereby indirectly accelerating promoter escape. The ensemble of the kinetic, structural signals demonstrated directly that CRP exerts most of its activating effects on the late stages of transcriptional initiation at the malT promoter.