Hypersensitivity to Catabolite Repression in the l-Arabinose Operon of Escherichia coli B/r Is trans Acting

Mutations causing hypersensitivity to catabolite repression have been assigned to gene araC (activator protein) by complementation analysis. The araO (operator region) is non-essential for catabolite repression.     

大肠埃希菌trp operon的克隆与表达

色氨酸操纵子所表达酶的高效表达和酶活性的提高,从而构建高产色氨酸菌株.利用PCR的方法从大肠埃希菌基因组中直接克隆色氨酸操纵子,并将其连接到原核表达载体pBV220中,得到重组质粒pBV220-trp operon,转化大肠埃希菌DH5α,温度诱导重组蛋白表达,表达产物经SDS-PAGE分析并用比色法测定其活性.通过凝胶电泳观察PCR扩增产物大小约为7 kb.SDS-PAGE鉴定目的蛋白得到了高效表达,邻氨基苯甲酸合成酶和色氨酸合成酶的活性分别比对照提高了3.4倍和2.5倍.成功构建了重组质粒pBV220-trp operon,邻氨基苯甲酸合成酶和色氨酸合成酶的表达量和表达活性在大肠埃希菌中得到了提高,为高产色氨酸基因工程菌的构建奠定基础.     

Amount of Colicin Release in Escherichia coli Is Regulated by Lysis Gene Expression of the Colicin E2 Operon

The production of bacteriocins in response to worsening environmental conditions is one means of bacteria to outcompete other microorganisms. Colicins, one class of bacteriocins in Escherichia coli, are effective against closely related Enterobacteriaceae. Current research focuses on production, release and uptake of these toxins by bacteria. However, little is known about the quantitative aspects of these dynamic processes. Here, we quantitatively study expression dynamics of the Colicin E2 operon in E. coli on a single cell level using fluorescence time-lapse microscopy. DNA damage, triggering SOS response leads to the heterogeneous expression of this operon including the cea gene encoding the toxin, Colicin E2, and the cel gene coding for the induction of cell lysis and subsequent colicin release. Advancing previous whole population investigations, our time-lapse experiments reveal that at low exogenous stress levels all cells eventually respond after a given time (heterogeneous timing). This heterogeneous timing is lost at high stress levels, at which a synchronized stress response of all cells 60 min after induction via stress can be observed. We further demonstrate, that the amount of colicin released is dependent on cel (lysis) gene expression, independent of the applied exogenous stress level. A heterogeneous response in combination with heterogeneous timing can be biologically significant. It might enable a bacterial population to endure low stress levels, while at high stress levels an immediate and synchronized population wide response can give single surviving cells of the own species the chance to take over the bacterial community after the stress has ceased.     

The Cytochrome c Maturation Operon Is Involved in Manganese Oxidation in Pseudomonas putida GB-1

A Pseudomonas putida strain, strain GB-1, oxidizes Mn²⁺ to Mn oxide in the early stationary growth phase. It also secretes a siderophore (identified as pyoverdine) when it is subjected to iron limitation. After transposon (Tn5) mutagenesis several classes of mutants with differences in Mn²⁺ oxidation and/or secretion of the Mn²⁺-oxidizing activity were identified. Preliminary analysis of the Tn5 insertion site in one of the nonoxidizing mutants suggested that a multicopper oxidase-related enzyme is involved in Mn²⁺ oxidation. The insertion site in another mutant was preliminarily identified as a gene involved in the general protein secretion pathway. Two mutants defective in Mn²⁺-oxidizing activity also secreted porphyrins into the medium and appeared to be derepressed for pyoverdine production. These strains were chosen for detailed analysis. Both mutants were shown to contain Tn5 insertions in the ccmF gene, which is part of the cytochrome c maturation operon. They were cytochrome oxidase negative and did not contain c-type cytochromes. Complementation with part of the ccm operon isolated from the wild type restored the phenotype of the parent strain. These results indicate that a functional ccm operon is required for Mn²⁺ oxidation in P. putida GB-1. A possible relationship between porphyrin secretion resulting from the ccm mutation and stimulation of pyoverdine production is discussed.In a number of studies during the last three decades it has been shown that various microbial species are able to stimulate the oxidation of Mn²⁺ through direct catalysis. These organisms produce proteinaceous macromolecules which catalyze the oxidation reaction. Manganese oxidations by a soil Arthrobacter species (24), Oceanospirillum and Vibrio strains (2, 3), Pseudomonas putida MnB1 (22, 30), Leptothrix discophora SS-1 (1, 11), and marine Bacillus strain SG-1 (23) are examples in which enzymes are most likely involved in the process. P. putida MnB1 produces a soluble protein which catalytically oxidizes Mn²⁺ in cell extracts (22). Manganese-oxidizing proteins from L. discophora SS-1 (1, 11) and from the spore coats of Bacillus strain SG-1 (43) have been identified on polyacrylamide gels. The oxidizing proteins have not been quantitatively purified or analyzed so far. In Bacillus strain SG-1, an operon containing seven genes appears to be involved in Mn²⁺ oxidation (46). One of these genes encodes a 137-kDa protein related to the family of multicopper oxidases (47). In a previous study we reported the isolation of a structural gene and its promoter postulated to be involved in Mn²⁺ oxidation in L. discophora (19). The encoded protein also contains the copper-binding signatures of multicopper oxidases. The oxidase-related proteins may represent Mn²⁺-oxidizing enzymes (44), but evidence supporting this hypothesis is still lacking.In this paper we describe a genetic analysis of Mn²⁺ oxidation in a freshwater Pseudomonas strain, strain GB-1. In a previous study (32) this strain was preliminarily identified as a Pseudomonas fluorescens strain, but more recent data (see Materials and Methods) indicate that it should be identified as a P. putida strain. When supplied with Mn²⁺ ions, the cells deposit manganese oxide around the outer membrane in the early stationary growth phase (32). They form brown colonies on Mn²⁺-containing agar. Experiments performed with cell extracts indicated that Mn²⁺ oxidation is catalyzed by a protein. The Mn²⁺-oxidizing factor was partially purified, and electrophoresis on an acrylamide gradient gel revealed oxidizing proteins with apparent molecular weights of ca. 250,000 and 180,000 (32). An additional oxidizing factor with a lower molecular weight (ca. 130,000) was identified in another study by using different isolation and electrophoretic procedures (16). It has been suggested that the Mn²⁺-oxidizing protein isolated is part of a larger complex which disintegrates into smaller fragments that retain activity (32). The protein is supposed to be located in the outer membrane of the bacteria. It has not been chemically characterized, and nothing is known about its cellular function or about the possible involvement of other cellular components, such as electron carriers, in Mn²⁺ oxidation.We used transposon mutagenesis to identify genes relevant to the Mn²⁺-oxidizing process in P. putida GB-1. One of these genes appeared to be part of the cytochrome c maturation operon. Transposon insertion in this gene not only abolished Mn²⁺ oxidation but also led to secretion of siderophores and porphyrins.An accompanying report on the involvement of the cytochrome c maturation operon in Mn²⁺ oxidation in P. putida MnB1 (14) supports our findings.     

MocA Is a Specific Cytidylyltransferase Involved in Molybdopterin Cytosine Dinucleotide Biosynthesis in Escherichia coli

We have purified and characterized a specific CTP:molybdopterin cytidylyltransferase for the biosynthesis of the molybdopterin (MPT) cytosine dinucleotide (MCD) cofactor in Escherichia coli. The protein, named MocA, shows 22% amino acid sequence identity to E. coli MobA, the specific GTP:molybdopterin guanylyltransferase for molybdopterin guanine dinucleotide biosynthesis. MocA is essential for the activity of the MCD-containing enzymes aldehyde oxidoreductase YagTSR and the xanthine dehydrogenases XdhABC and XdhD. Using a fully defined in vitro assay, we showed that MocA, Mo-MPT, CTP, and MgCl₂ are required and sufficient for MCD biosynthesis in vitro. The activity of MocA is specific for CTP; other nucleotides such as ATP and GTP were not utilized. In the defined in vitro system a turnover number of 0.37 ± 0.01 min⁻¹ was obtained. A 1:1 binding ratio of MocA to Mo-MPT and CTP was determined to monomeric MocA with dissociation constants of 0.23 ± 0.02 μm for CTP and 1.17 ± 0.18 μm for Mo-MPT. We showed that MocA was also able to convert MPT to MCD in the absence of molybdate, however, with only one catalytic turnover. The addition of molybdate after one turnover gave rise to a higher MCD production, revealing that MCD remains bound to MocA in the absence of molybdate. This work presents the first characterization of a specific enzyme involved in MCD biosynthesis in bacteria.The biosynthesis of the molybdenum cofactor (Moco)² is an ancient, ubiquitous, and highly conserved pathway leading to the biochemical activation of molybdenum. In Moco the molybdenum atom is coordinated to the dithiolene group of the 6-alkyl side chain of a pterin called molybdopterin (MPT). Moco biosynthesis has been extensively studied in Escherichia coli by using a combination of biochemical, genetic, and structural approaches (1, 2). The biosynthesis of Moco has been divided into four major steps in Escherichia coli: (i) formation of precursor Z (3, 4), (ii) formation of MPT from precursor Z (5, 6), (iii) insertion of molybdenum to form Moco via an adenylylated MPT intermediate (7–9), and (iv) additional modification by covalent addition of GMP to the C4′ phosphate of MPT via a pyrophosphate bond, forming the molybdopterin guanine dinucleotide (MGD) cofactor (10, 11). In E. coli, GMP attachment to Moco is catalyzed by the MobA and MobB proteins (12). Although MobA was shown to be essential for this reaction and acts as a GTP:molybdopterin guanylyltransferase (11), the role of MobB still remains uncertain. From the crystal structure, it was postulated that MobB is an adapter protein acting in concert with MobA to achieve the efficient biosynthesis and utilization of MGD (13). Although MobA was shown to bind MPT, Mo-MPT, and MGD (14), investigations of in vitro studies using purified MobA, MgCl₂, GTP, and either MPT or Mo-MPT showed that MGD was only formed by MobA when the molybdenum atom was already ligated to MPT (15). The formation of bis-MGD is one of the most enigmatic steps in Moco biosynthesis in E. coli. It is still not known whether the two MGD molecules assemble on MobA or instead after the insertion into the respective target proteins like DMSO reductase or nitrate reductase A. In other bacteria like Arthrobacter nicotinovorans, Veillonella atypica, or Oligotropha carboxidovorans, Moco can be further modified by the attachment of CMP to the C4′ phosphate of MPT forming the molybdopterin cytosine dinucleotide (MCD) cofactor (16–18). A specific enzyme catalyzing the CTP:molybdopterin cytidylyltransferase reaction has not been identified so far. For A. nicotinovorans nicotine dehydrogenase and ketone dehydrogenase the involvement of a MobA homologous protein for MCD formation was reported (16); however, it was not shown whether the MobA protein was specifically required for MCD biosynthesis or whether it was also involved in the biosynthesis of MGD in this bacterium. Furthermore, enzymes binding MCD in bacteria usually contain an additional modification at the molybdenum site of Moco, where a terminal oxo-ligand is exchanged by a sulfido ligand, forming sulfurated or mono-oxo Moco (19). Recently, the MCD-containing protein YagTSR was identified and characterized in E. coli as a periplasmic aldehyde oxidoreductase which oxidizes a broad spectrum of aldehydes using ferredoxin as electron acceptor (20). It was shown that for the production of an active form of YagTSR, the YagQ protein was required, which is believed to be a MCD binding chaperone involved in the sulfuration of the Mo site and the insertion of sulfurated MCD into apoYagTSR (20). The majority of the other molybdoenzymes in E. coli were shown to bind the bis-MGD form of Moco, in which molybdenum is coordinated to two MGD moieties. The other exception is the YedY protein, being so far the only E. coli protein binding the Mo-MPT form of Moco (21). However, the physiological role of this protein still remains unclear.Investigations on YagTSR showed that MCD was inserted into YagR independent of the function of MobA, indicating that a so-far unidentified protein is involved in MCD biosynthesis in E. coli (20). Here, we report the identification of the specific CTP:molybdopterin cytidylyltransferase, which we named MocA (formerly named YgfJ by the E. coli nomenclature of genes with unknown function). Purified MocA was shown to catalyze the formation of MCD from Mo-MPT and CTP in vitro. Additionally, we report that a disruption in the mocA gene impaired MCD biosynthesis in E. coli, resulting in an inactive YagTSR protein devoid of Moco.     

Conserved Structural Regions Involved in the Catalytic Mechanism of Escherichia coli K-12 WaaO (RfaI)

Escherichia coli K-12 WaaO (formerly known as RfaI) is a nonprocessive α-1,3 glucosyltransferase, involved in the synthesis of the R core of lipopolysaccharide. By comparing the amino acid sequence of WaaO with those of 11 homologous α-glycosyltransferases, four strictly conserved regions, I, II, III, and IV, were identified. Since functionally related transferases are predicted to have a similar architecture in the catalytic sites, it is assumed that these four regions are directly involved in the formation of α-glycosidic linkage from α-linked nucleotide diphospho-sugar donor. Hydrophobic cluster analysis revealed a conserved domain at the N termini of these α-glycosyltransferases. This domain was similar to that previously reported for β-glycosyltransferases. Thus, this domain is likely to be involved in the formation of β-glycosidic linkage between the donor sugar and the enzyme at the first step of the reaction. Site-directed mutagenesis analysis of E. coli K-12 WaaO revealed four critical amino acid residues.     

The Listeria monocytogenes Homolog of the Escherichia coli era Gene Is Involved in Adhesion to Inert Surfaces

Two transposon-insertional mutants of Listeria monocytogenes showing smaller viable surface-attached cell populations after disinfection with N,N-didecyl-N,N-dimethylammonium chloride were identified. In both mutants, transposon Tn917-lac was found to be inserted into the same gene, lmo1462, which is homologous to the essential Escherichia coli era gene. Both L. monocytogenes lmo1462-disrupted mutants displayed lower growth rates, as was also shown for several E. coli era mutants, and the lmo1462 gene was able to complement the growth defect of an E. coli era mutant. We showed that the disruption of lmo1462 decreased the ability of L. monocytogenes cells to adhere to stainless steel. Our results suggest that this era-like gene is involved in adhesion and contributes to the presence of L. monocytogenes on surfaces.     

The Escherichia coli Lpt Transenvelope Protein Complex for Lipopolysaccharide Export Is Assembled via Conserved Structurally Homologous Domains

Lipopolysaccharide is a major glycolipid component in the outer leaflet of the outer membrane (OM), a peculiar permeability barrier of Gram-negative bacteria that prevents many toxic compounds from entering the cell. Lipopolysaccharide transport (Lpt) across the periplasmic space and its assembly at the Escherichia coli cell surface are carried out by a transenvelope complex of seven essential Lpt proteins spanning the inner membrane (LptBCFG), the periplasm (LptA), and the OM (LptDE), which appears to operate as a unique machinery. LptC is an essential inner membrane-anchored protein with a large periplasm-protruding domain. LptC binds the inner membrane LptBFG ABC transporter and interacts with the periplasmic protein LptA. However, its role in lipopolysaccharide transport is unclear. Here we show that LptC lacking the transmembrane region is viable and can bind the LptBFG inner membrane complex; thus, the essential LptC functions are located in the periplasmic domain. In addition, we characterize two previously described inactive single mutations at two conserved glycines (G56V and G153R, respectively) of the LptC periplasmic domain, showing that neither mutant is able to assemble the transenvelope machinery. However, while LptCG56V failed to copurify any Lpt component, LptCG153R was able to interact with the inner membrane protein complex LptBFG. Overall, our data further support the model whereby the bridge connecting the inner and outer membranes would be based on the conserved structurally homologous jellyroll domain shared by five out of the seven Lpt components.     

Characterization of Two Homologous Disulfide Bond Systems Involved in Virulence Factor Biogenesis in Uropathogenic Escherichia coli CFT073

Disulfide bond (DSB) formation is catalyzed by disulfide bond proteins and is critical for the proper folding and functioning of secreted and membrane-associated bacterial proteins. Uropathogenic Escherichia coli (UPEC) strains possess two paralogous disulfide bond systems: the well-characterized DsbAB system and the recently described DsbLI system. In the DsbAB system, the highly oxidizing DsbA protein introduces disulfide bonds into unfolded polypeptides by donating its redox-active disulfide and is in turn reoxidized by DsbB. DsbA has broad substrate specificity and reacts readily with reduced unfolded proteins entering the periplasm. The DsbLI system also comprises a functional redox pair; however, DsbL catalyzes the specific oxidative folding of the large periplasmic enzyme arylsulfate sulfotransferase (ASST). In this study, we characterized the DsbLI system of the prototypic UPEC strain CFT073 and examined the contributions of the DsbAB and DsbLI systems to the production of functional flagella as well as type 1 and P fimbriae. The DsbLI system was able to catalyze disulfide bond formation in several well-defined DsbA targets when provided in trans on a multicopy plasmid. In a mouse urinary tract infection model, the isogenic dsbAB deletion mutant of CFT073 was severely attenuated, while deletion of dsbLI or assT did not affect colonization.Disulfide bonds bridging cysteine pairs impart structural stability and protease resistance to secreted and membrane-associated proteins. Most organisms contain specific mechanisms for the formation of disulfide bonds in proteins, a process called oxidative protein folding. In bacteria, this folding process is catalyzed by the disulfide bond family of proteins (18, 22). The best-characterized bacterial disulfide bond machinery is the Escherichia coli K-12 oxidative system, which consists of two enzymes, the periplasmic DsbA and the inner-membrane DsbB (25, 35). DsbA is a monomeric protein comprising a thioredoxin (TRX) domain with an embedded helical insertion and a redox-active CPHC motif (34). This highly oxidizing protein introduces disulfide bonds into unfolded polypeptides by donating its redox-active disulfide (2, 4, 5), and as a result, the two cysteines contained in the CPHC catalytic motif become reduced. DsbB reoxidizes this cysteine pair and restores the oxidizing activity of DsbA, enabling it to assist the folding of a new substrate protein (21).The DsbAB oxidative protein folding system plays a well-documented part in bacterial virulence. Several studies have demonstrated a direct role for both enzymes, particularly DsbA, in the biogenesis of virulence factors utilized by bacterial pathogens in various stages of the infection process (19). The protein forming the P-ring of E. coli flagella, FlgI, was one of the first DsbA substrates identified (10) and flagellum-mediated motility was subsequently demonstrated to require the presence of functional DsbA in several gram-negative pathogens, including Salmonella enterica (1), Proteus mirabilis (8), Erwinia carotovora subsp. atroseptica (9), Burkholderia cepacia (17), and Campylobacter jejuni (42). In Yersinia pestis, S. enterica, Shigella flexneri, and enteropathogenic E. coli, deletion of dsbA results in defective type III secretion, a major virulence mechanism employed by these enteric pathogens to manipulate the host during infection. The defect was shown in each case to involve the outer membrane secretin (YscC, SpiA, Spa32, and EscC, respectively), which requires a single intramolecular disulfide bond to adopt a functional conformation (23, 36, 37, 49). Fimbria-mediated adhesion is a crucial first step of the infection process as it allows host colonization by mucosal pathogens. DsbA is required for functional assembly of several types of fimbriae, including P fimbriae of uropathogenic E. coli (UPEC) (24), bundle-forming pili (Bfp) of enteropathogenic E. coli (55), mannose-resistant Proteus-like (MR/P) fimbriae of Proteus mirabilis (8), plasmid-encoded fimbriae (Pef) of Salmonella enterica (6), type IV pili of Neisseria meningitidis (47), and toxin-coregulated pili (Tcp) of Vibrio cholerae (41). A number of studies have reported that dsbA and/or dsbB mutants are attenuated in infection models (9, 16, 41, 48, 52).The recent exponential increase in sequenced genomes has offered a first glimpse at the diversity of disulfide bond systems present in bacteria (13). In addition, it is now evident that several bacterial species encode multiple DsbA paralogues, often with demonstrated differences in substrate specificity. Neisseria meningitidis, for example, encodes three DsbA oxidoreductases: two inner membrane-associated lipoproteins (DsbA1 and DsbA2) and one periplasmic enzyme (DsbA3). While redundancy was observed in the oxidative folding of virulence-associated proteins by DsbA1 and DsbA2, DsbA3 alone was unable to restore important meningococcal virulence traits, such as type IV pilus-mediated adhesion to human endothelial cells (47). Recently, a second E. coli disulfide bond system (DsbLI) was identified in the genome-sequenced UPEC strain CFT073 and was demonstrated to be a functional paralogue of the prototypic DsbAB system (14). The oxidoreductase DsbL has the strongest oxidizing potential of all DsbA homologues characterized to date. Although the crystal structure of DsbL revealed a similar overall fold and domain architecture to DsbA, DsbL contains a longer helical insertion and deletions in the TRX domain that result in a truncated peptide binding groove. Moreover, DsbL shows different surface properties, including a distinct basic patch around the active site, which was suggested to allow stricter substrate specificity than the highly hydrophobic surface surrounding the active site of DsbA. Grimshaw and colleagues (14) demonstrated the specificity of the DsbLI system for the periplasmic enzyme arylsulfate sulfotransferase (ASST) encoded by assT, a gene found immediately upstream of dsbL and dsbI on the CFT073 chromosome. ASST belongs to a group of poorly characterized large bacterial ASSTs that are proposed to mediate detoxification of phenolic substances by catalyzing the transfer of sulfuryl groups from phenolic sulfates to phenol (26-28, 30). A reason for the specificity of DsbLI for ASST folding could be the presence of an allosteric disulfide bond, recently revealed by the enzyme''s crystal structure (33). This class of disulfide bond forms between C_α atoms of cysteines in unusually close proximity (3.8 Å in the case of ASST) and has higher steric strain energy than catalytic or structural disulfide bonds, thus explaining the requirement for the stronger DsbL oxidase for its formation (33). The activity of DsbL and DsbI was studied using plasmids introduced into E. coli K-12 strains with the native DsbAB system deleted. As yet, the role of the DsbLI system in UPEC virulence has not been investigated.E. coli CFT073 is a prototypic UPEC strain isolated from a female patient with acute pyelonephritis (38). UPEC strains are the causative agent of >80% of community-acquired urinary tract infections (UTIs) and >30% of nosocomial infections (7). The uropathogenic lifestyle of UPEC CFT073 is reflected in its genome, which contains several factors with an established role in urovirulence, including the well-studied type 1 and P fimbriae (50). Genomic comparison of CFT073—and other recently sequenced UPEC strains—with E. coli strains with distinct lifestyles (gut commensals, enteric pathogens, and avian pathogens) allows the discovery of genes unique to genomes of uropathogenic bacteria that are potentially novel urovirulence factors. One such UPEC-specific gene is assT, the gene located upstream of dsbL and dsbI in the chromosome of CFT073 (32).Here we characterize the DsbLI system in its native genetic background of UPEC CFT073 and compare and contrast the contribution of each of the two paralogous disulfide bond systems of CFT073 in the production of UPEC-associated virulence factors and in vivo uropathogenesis. Using isogenic dsbAB and dsbLI deletion mutants of CFT073, we demonstrate that the recently identified DsbLI oxidative protein folding machinery of UPEC CFT073 plays a secondary role in the production of urovirulence factors and does not appear to contribute to virulence in the mouse infection model used in this study. We also show that in the same infection model, an isogenic assT deletion mutant of CFT073 is not attenuated.