Uptake of extracellular biotin by Escherichia coli biotin prototrophs.

Uptake of exogenous biotin by two Escherichia coli biotin prototroph strains, K-12 and Crookes, appeared to involve incorporation at a fixed number of binding sites located at the cell membrane. Incorporation was characterized as a binding process specific for biotin, not requiring energy, and stimulated by acidic pH. Constant saturation quantities of exogenous biotin were incorporated by these cells, and the amounts, which were titrated, depended on whether the cells were resting or dividing. Resting cells incorporated exogenous biotin amounting to 2% of their total intracellular biotin content. Fifty percent of the exogenous biotin was incorporated into their free biotin fraction, and 50% was incorporated into their bound biotin fraction. On the other hand, dividing cells incorporated exogenous biotin into all of their intracellular sites, 88% going into the intracellular-bound biotin fraction, and 12% going into the free biotin fraction. Calculations suggested that each cell contained approximately 3,000 binding sites for biotin. It was postulated that biotin incorporation sites might have been components of acetyl coenzyme A carboxylase located at or near the membrane.     

Repression of biotin biosynthesis in Escherichia coli during growth on biotin vitamers.

A strain of Escherichia coli in which the lacZ gene was fused to the bioA promoter was constructed. Colonies of this strain formed Lac(+) colonies on low-biotin agar (1.6 to 4.1 nM) and Lac(-) colonies on high-biotin agar (41 nM). This lac-bio fusion strain was used to study the question of whether cells growing on the biotin vitamers d-biotin-d-sulfoxide (BDS) and dethiobiotin (DTB) generate enough biotin to give maximal repression of beta-galactosidase synthesis. Repression by high concentrations (400 nM) of BDS was almost maximal (about 96%), whereas DTB repression reached a saturation level of about 80% with increasing DTB concentrations. The levels of repression obtained with both vitamers were sufficient to cause the colonies to appear Lac(-). When the lac-bio fusion was transduced into lines carrying mutations (bis) that prevent reduction of BDS to biotin, the transductants were not repressed by added BDS. Repression by BDS is unlikely to result from accumulation of extracellular biotin-related substances because (i) washed bis(+) cells were not detectably derepressed when transferred into medium containing BDS and (ii) washed bis cells were not detectably repressed when transferred into medium in which bis(+) cells had grown. Lactose agar plates containing high concentrations of DTB or BDS comprise an efficient selective medium for bioB or bis mutants and were used to isolate spontaneous mutations of these genes. This method should be adaptable to the selection of mutations in any biosynthetic pathway subject to end-product repression.     

The biotin carboxylase-biotin carboxyl carrier protein complex of Escherichia coli acetyl-CoA carboxylase

Escherichia coli acetyl-CoA carboxylase (ACC) is composed of four different protein molecules. These proteins form a large but very unstable complex. Hints of a sub-complex between the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) subunits have been reported in the literature, but the complex was not isolated and thus the protein stoichiometry could not be determined. We report isolation of the BC.BCCP complex. By use of affinity chromatography using two different affinity tags it was shown that the complex consists of a two BCCP molecules per BC molecule. The molar ratio in the complex is the same as the ratio of the subunit proteins synthesized in vivo. We conclude that the complex consists of a dimer of BC plus four BCCP molecules instead of the 2BC.2BCCP complex previously assumed. This subunit ratio allows two conflicting models of the ACC mechanism to be rectified. We also report that the N-terminal 30 or so residues of BCCP are responsible for the interaction of BCCP with BC and that the BC.BCCP complex is a substrate for biotinylation in vitro.     

Molybdenum cofactor requirement for biotin sulfoxide reduction in Escherichia coli.

The bisC gene of Escherichia coli is tentatively identified as the structural gene for biotin sulfoxide reductase by the isolation of bisC(Ts) mutants that make thermolabile enzyme. The products of four other E. coli genes (chlA, chlB, chlE and chlG) are also needed for enzymatic activity. Mutations previously assigned to the bisA, bisB, and bisD genes belong to genes chlA, chlE, and chlG, respectively. The biotin sulfoxide reductase deficiency of a chlG, mutant is partially reversed by the addition of 10 mM molybdate to the growth medium. Mutational inactivation of the chlD gene reduces the specific activity of biotin sulfoxide reductase about twofold. This effect is reversed by the addition of 1 mM molybdate to the growth medium. The specific activity of biotin sulfoxide reductase is decreased about 30-fold by the presence of tungstate in the growth medium, an effect that has been observed previously with nitrate reductase and other molybdoenzymes. The specific activity of biotin sulfoxide reductase is not elevated in a lysate prepared by derepressing a lambda cI857 chlG prophage. Whereas biotin sulfoxide reductase prepared by sonic extraction of growing cells is almost completely dependent on the presence of a small heat-stable protein resembling thioredoxin, much of the enzyme obtained from lysates of thermoinduced lambda cI857 lysogens does not require this factor.     

Genes of the Escherichia coli pur regulon are negatively controlled by a repressor-operator interaction.

Fusions of lacZ were constructed to genes in each of the loci involved in de novo synthesis of IMP. The expression of each pur-lacZ fusion was determined in isogenic purR and purR+ strains. These measurements indicated 5- to 17-fold coregulation of genes purF, purHD, purC, purMN, purL, and purEK and thus confirm the existence of a pur regulon. Gene purB, which encodes an enzyme involved in synthesis of IMP and in the AMP branch of the pathway, was not regulated by purR. Each locus of the pur regulon contains a 16-base-pair conserved operator sequence that overlaps with the promoter. The purR product, purine repressor, was shown to bind specifically to each operator. Thus, binding of repressor to each operator of pur regulon genes negatively coregulates expression.     

Conversion of dethiobiotin to biotin in cell-free extracts of Escherichia coli.

We constructed the plasmid pTTB151 in which the E. coli bioB gene was expressed under the control of the tac promoter. Conversion of dethiobiotin to biotin was demonstrated in cell-free extracts of E. coli carrying this plasmid. The requirements for this biotin-forming reaction included fructose-1,6-bisphosphate, Fe2+, S-adenosyl-L-methionine, NADPH, and KCl, as well as dethiobiotin as the substrate. The enzymes were partially purified from cell-free extracts by a procedure involving ammonium sulfate fractionation. Our results suggest that an unidentified enzyme(s) besides the bioB gene product is obligatory for the conversion of dethiobiotin to biotin.     

Bioassay of biotin concentration with a Escherichia coli bio deletion mutant.

Biotin concentration was determined unequivocally with the E. coli bio mutant. The results demonstrate that this simple and efficient method can determine biotin concentration in the range of 10 pg to 50 ng/ml. The present method can also clearly distinguish biotin from its precursor and analog, dethiobiotin.     

Comparing native and irradiated E. coli lactose repressor-operator complex by molecular dynamics simulation

The function of the E. coli lactose operon requires the binding of the tetrameric repressor protein to the operator DNA. We have previously shown that γ-irradiation destabilises the repressor-operator complex because the repressor gradually loses its DNA-binding ability (Radiat Res 170:604–612, 2008). It was suggested that the observed oxidation of tyrosine residues and the concomitant structural changes of irradiated headpieces (DNA-binding domains of repressor monomers) could be responsible for the inactivation. To unravel the mechanisms that lead to repressor-operator complex destabilisation when tyrosine oxidation occurs, we have compared by molecular dynamic simulations two complexes: (1) the native complex formed by two headpieces and the operator DNA, and (2) the damaged complex, in which all tyrosines are replaced by their oxidation product 3,4-dihydroxyphenylalanine (DOPA). On a 20 ns time scale, MD results show effects consistent with complex destabilisation: increased flexibility, increased DNA bending, modification of the hydrogen bond network, and decrease of the positive electrostatic potential at the protein surface and of the global energy of DNA-protein interactions.     

Biotin uptake in biotin regulatory mutant of Escherichia coli

A transport system for biotin in Escherichia coli is regulated by biotin and is not affected by the mutation (bioR) that causes the constitutive synthesis of the bio operon enzymes.     

Binding characteristics of Escherichia coli type 1 fimbriae in the human kidney

Binding of the Escherichia coli type 1 fimbriae to frozen sections of human kidney was examined by indirect immunofluorescence. The purified fimbriae bound specifically to the luminal and cytoplasmic aspects of proximal tubules and to the connective tissue layer of veins and arteries. Distal tubules, collecting ducts, glomeruli and vascular endothelium were not stained by the fimbriae. The results do not support a role for type 1 fimbriae in invasion of E. coli into the renal parenchyma.     

Promiscuous protein biotinylation by Escherichia coli biotin protein ligase

Biotin protein ligases (BPLs) are enzymes of extraordinary specificity. BirA, the BPL of Escherichia coli biotinylates only a single cellular protein. We report a mutant BirA that attaches biotin to a large number of cellular proteins in vivo and to bovine serum albumin, chloramphenicol acetyltransferase, immunoglobin heavy and light chains, and RNAse A in vitro. The mutant BirA also self biotinylates in vivo and in vitro. The wild type BirA protein is much less active in these reactions. The biotinylation reaction is proximity-dependent in that a greater extent of biotinylation was seen when the mutant ligase was coupled to the acceptor proteins than when the acceptors were free in solution. This approach may permit facile detection and recovery of interacting proteins by existing avidin/streptavidin technology.     

The gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase.

We report the molecular cloning and DNA sequence of the gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase. The biotin carboxylase gene encodes a protein of 449 residues that is strikingly similar to amino-terminal segments of two biotin-dependent carboxylase proteins, yeast pyruvate carboxylase and the alpha-subunit of rat propionyl-CoA carboxylase. The deduced biotin carboxylase sequence contains a consensus ATP binding site and a cysteine-containing sequence preserved in all sequenced bicarbonate-dependent biotin carboxylases that may play a key catalytic role. The gene encoding the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase is located upstream of the biotin carboxylase gene and the two genes are cotranscribed. As previously reported by others, the BCCP sequence encoded a protein of 16,688 molecular mass. However, this value is much smaller than that (22,500 daltons) obtained by analysis of the protein. Amino-terminal amino acid sequencing of the purified BCCP protein confirmed the deduced amino acid sequence indicating that BCCP is a protein of atypical physical properties. Northern and primer extension analyses demonstrate that BCCP and biotin carboxylase are transcribed as a single mRNA species that contains an unusually long untranslated leader preceding the BCCP gene. We have also determined the mutational alteration in a previously isolated acetyl-CoA carboxylase (fabE) mutant and show the lesion maps within the BCCP gene and results in a BCCP species defective in acceptance of biotin. Translational fusions of the carboxyl-terminal 110 or 84 (but not 76) amino acids of BCCP to beta-galactosidase resulted in biotinated beta-galactosidase molecules and production of one such fusion was shown to result in derepression of the biotin biosynthetic operon.     

Binding of lysozyme to common pili of Escherichia coli.

Common pili from Escherichia coli were found to bind hen egg white lysozyme. The binding was highly dependent on ionic strength, and the maximum binding occurred near an ionic strength of 0.02. The pili were aggregated by lysozyme, and this process could be followed by optical turbidity, electron microscopy, and coprecipitation. Near the maximum saturation of binding, one lysozyme molecule was bound by two pilus protein subunits. Electron micrographs of this aggregate indicated that they were paracrystalline structures. Piliated bacteria were more readily agglutinated by lysozyme than were nonpiliated bacteria. Since lysozyme is considered to be an antibacterial humoral factor and since pili are considered to be a colonization factor, the binding of lysozyme may represent an important bacterium-host interaction     

Characterization of the cofactor composition of Escherichia coli biotin synthase

The cofactor content of in vivo, as-isolated, and reconstituted forms of recombinant Escherichia coli biotin synthase (BioB) has been investigated using the combination of UV-visible absorption, resonance Raman, and M?ssbauer spectroscopies along with parallel analytical and activity assays. In contrast to the recent report that E. coli BioB is a pyridoxal phosphate (PLP)-dependent enzyme with intrinsic cysteine desulfurase activity (Ollagnier-deChoudens, S., Mulliez, E., Hewitson, K. S., and Fontecave, M. (2002) Biochemistry 41, 9145-9152), no evidence for PLP binding or for PLP-induced cysteine desulfurase or biotin synthase activity was observed with any of the forms of BioB investigated in this work. The results confirm that BioB contains two distinct Fe-S cluster binding sites. One site accommodates a [2Fe-2S](2+) cluster with partial noncysteinyl ligation that can only be reconstituted in vitro in the presence of O(2). The other site accommodates a [4Fe-4S](2+,+) cluster that binds S-adenosylmethionine (SAM) at a unique Fe site of the [4Fe-4S](2+) cluster and undergoes O(2)-induced degradation via a distinct type of [2Fe-2S](2+) cluster intermediate. In vivo M?ssbauer studies show that recombinant BioB in anaerobically grown cells is expressed exclusively in an inactive form containing only the as-isolated [2Fe-2S](2+) cluster and demonstrate that the [2Fe-2S](2+) cluster is not a consequence of overexpressing the recombinant enzyme under aerobic growth conditions. Overall the results clarify the confusion in the literature concerning the Fe-S cluster composition and the in vitro reconstitution and O(2)-induced cluster transformations that are possible for recombinant BioB. In addition, they provide a firm foundation for assessing cluster transformations that occur during turnover and the catalytic competence of the [2Fe-2S](2+) cluster as the immediate S-donor for biotin biosynthesis.     

Reductive cleavage of S-adenosylmethionine by biotin synthase from Escherichia coli

Biotin synthase (BioB) catalyzes the insertion of a sulfur atom between the C6 and C9 carbons of dethiobiotin. Reconstituted BioB from Escherichia coli contains a [4Fe-4S](2+/1+) cluster thought to be involved in the reduction and cleavage of S-adenosylmethionine (AdoMet), generating methionine and the reactive 5'-deoxyadenosyl radical responsible for dethiobiotin H-abstraction. Using EPR and M?ssbauer spectroscopy as well as methionine quantitation we demonstrate that the reduced S = 1/2 [4Fe-4S](1+) cluster is indeed capable of injecting one electron into AdoMet, generating one equivalent of both methionine and S = 0 [4Fe-4S](2+) cluster. Dethiobiotin is not required for the reaction. Using site-directed mutagenesis we show also that, among the eight cysteines of BioB, only three (Cys-53, Cys-57, Cys-60) are essential for AdoMet reductive cleavage, suggesting that these cysteines are involved in chelation of the [4Fe-4S](2+/1+) cluster.     

Binding of basement-membrane laminin by Escherichia coli

An invasive Escherichia coli (EIEC) isolate was found to bind basement-membrane laminin in a saturable and time-dependent manner. Excess of unlabelled laminin inhibited the binding of the radioactively labelled protein. Non-invasive E. coli K-12 exhibited only low-level laminin binding but introduction of the virulence-associated plasmid from the EIEC isolate led to high-level binding. Expression of a receptor for laminin on the bacteria was therefore associated with the presence of the virulence plasmid. Scatchard plot analysis indicated approximately 1000 receptors per bacterial cell, and a Kd of high-affinity binding of 0.5 pM. A laminin-binding protein which correlated with the presence of the plasmid was isolated and characterized. Its sequence of the eight amino-terminal amino acids was identical to that of the LamB protein of E. coli, although the molecular mass of the two in sodium dodecyl sulphate/polyacrylamide gel (SDS-PAGE) appeared to be slightly different. Both proteins reacted in immunoblot assays with polyclonal antisera raised against either protein, and both proteins bound laminin. Southern-blot hybridization analysis established that both the EIEC strain and the K-12 strains with or without the virulence plasmid contained one lamB gene only, and no laminin-binding protein appeared when the virulence plasmid was introduced into bacteria deleted for the lamB gene. On the basis of these results we suggest that native LamB protein of E. coli or a modified variant of it serves as a major receptor for laminin binding and is present at an increased level in invasive E. coli containing the virulence plasmid.