The bacA gene of Escherichia coli encodes an undecaprenyl pyrophosphate phosphatase activity

The bacA gene, the overexpression of which results in bacitracin resistance, was inactivated and shown to be non-essential for growth of Escherichia coli. It was proposed earlier that the bacA gene product may confer resistance to the antibiotic by phosphorylation of undecaprenol (Cain, B. D., Norton, P. J., Eubanks, W., Nick, H. S., and Allen, C. M. (1983) J. Bacteriol. 175, 3784-3789). In the present work, this extremely hydrophobic membrane protein was overproduced and purified to near homogeneity. The analysis of its catalytic properties clearly demonstrated that the purified BacA protein exhibited undecaprenyl pyrophosphate phosphatase activity but not undecaprenol phosphokinase activity. This finding was perfectly consistent with the mechanism of action of bacitracin that consists in the sequestration of undecaprenyl pyrophosphate, the BacA enzyme substrate. The level of undecaprenyl pyrophosphate phosphatase was increased by 280-fold in cells carrying bacA on a multicopy expression plasmid. It was decreased by approximately 75% but was not completely abolished in a bacA disruption mutant, suggesting that BacA is the main E. coli undecaprenyl pyrophosphate phosphatase but that other protein(s) exhibiting such an activity should exist to account for the residual activity and viability of the mutant strain. This is the first gene encoding undecaprenyl pyrophosphate phosphatase identified to date. Considering its newly identified function, we propose to rename the bacA gene uppP.     

Substrate specificity and membrane topology of Escherichia coli PgpB, an undecaprenyl pyrophosphate phosphatase

The synthesis of the lipid carrier undecaprenyl phosphate (C(55)-P) requires the dephosphorylation of its precursor, undecaprenyl pyrophosphate (C(55)-PP). The latter lipid is synthesized de novo in the cytosol and is also regenerated after its release from the C(55)-PP-linked glycans in the periplasm. In Escherichia coli the dephosphorylation of C(55)-PP was shown to involve four integral membrane proteins, BacA, and three members of the type 2 phosphatidic acid phosphatase family, PgpB, YbjG, and YeiU. Here, the PgpB protein was purified to homogeneity, and its phosphatase activity was examined. This enzyme was shown to catalyze the dephosphorylation of C(55)-PP with a relatively low efficiency compared with diacylglycerol pyrophosphate and farnesyl pyrophosphate (C(15)-PP) lipid substrates. However, the in vitro C(55)-PP phosphatase activity of PgpB was specifically enhanced by different phospholipids. We hypothesize that the phospholipids are important determinants to ensure proper conformation of the atypical long axis C(55) carrier lipid in membranes. Furthermore, a topological analysis demonstrated that PgpB contains six transmembrane segments, a large periplasmic loop, and the type 2 phosphatidic acid phosphatase signature residues at a periplasmic location.     

Organization of genes encoding membrane proteins of the Escherichia coli ferrienterobactin permease

Transposon mutagenesis and plasmid complementation studies have identified two genes, fepD and fepG, which are essential for ferrienterobactin transport in Escherichia coli. These genes mapped in the enterobactin gene cluster and genetic evidence indicated that they are transcribed as part of an operon (fepD, fepG, fepC). The nucleotide sequence of fepD was determine; it could encode a hydrophobic 33.8 kDa protein with sequence homologies to other iron and vitamin B12 transport proteins. Also identified, between fepD and fepB, was an open reading frame (ORF43) with no detectable function; its 43 kDa protein product (P43) was seen on polyacrylamide gels. The fepD-C operon and ORF43 were divergently transcribed from a 110bp region containing a binding site for the repressor protein Fur.     

Product distribution and pre-steady-state kinetic analysis of Escherichia coli undecaprenyl pyrophosphate synthase reaction

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes the condensation of eight molecules of isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate C(55) undecaprenyl pyrophosphate. We investigated the kinetics and mechanism of this reaction pathway using Escherichia coli UPPs. With a variety of different ratios of enzyme to substrate and FPP to IPP in the presence or absence of Triton, different product distributions were found. In the presence of excess FPP, the intermediates (C(25)-C(50)) accumulated. Under a condition with enzyme and FPP in excess of IPP, instead of C(20)-geranylgeranyl pyrophosphate, C(20), C(25), and C(30) were the major products. The UPPs steady-state k(cat) value (2.5 s(-1)) in the presence of 0.1% Triton was 190-fold larger than in the absence of Triton (0.013 s(-1)). The k(cat) value matched the rate constant of each IPP condensation obtained from the enzyme single-turnover experiments. This suggested that the IPP condensation rather than product release was the rate-limiting step in the presence of Triton. In the absence of Triton, the intermediates formed and disappeared in a similar manner under enzyme single turnover in contrast to the slow steady-state rate, which indicated a step after product generation was rate limiting. This was further supported by a burst product formation. Judging from the accumulation level of C(55), C(60), and C(65), their dissociation from the enzyme cannot be too slow and an even slower enzyme conformational change with a rate of 0.001 s(-1) might govern the UPPs reaction rate under the steady-state condition in the absence of Triton.     

Molecular insights into substrate binding mechanism of undecaprenyl pyrophosphate with membrane integrated phosphatidyl glycerophosphate phosphatase B (PgpB) using molecular dynamics simulation approach

Undecaprenyl phosphate (C55-P) acts as carrier lipid in the synthesis of peptidoglycan, which is de novo synthesized from dephosphorylation of undecaprenyl pyrophosphate (C55-PP). The phosphatidylglycerol phosphate phosphatase B (PgpB) catalyzes the dephosphorylation of C55-PP and forms C55-P. As no structural study has been made regarding the binding of C55-PP to PgpB, in the current study, in silico molecular docking, followed by 150 ns molecular dynamics simulation of the putative binding complex in membrane/solvent environment has been performed to understand conformational dynamics. Results are compared with simulated apo form and PE inhibitor-bound form. Analysis of correlated residual fluctuation network in apo form, C55-PP bound and PE inhibitor-bound form suggests that difference in dynamic coupling between TM domain and α2 and α3 helix of periplasmic domain provides ligand binding to facilitate catalysis or to show inhibitory activity. Distance distribution in catalytic residual pair, H207-R104; H207-R201 and H207-D211 which stabilizes phosphate-enzyme intermediate shows a narrow peak in 2.4–3.6 Å in substrate-bound compared to apo form. Binding interactions and binding free energy analyses complement the partial inhibition of PE where PE has less binding free energy compared to the C55-PP substrate as well as the difference in binding interaction with catalytic pocket. Thus, the present study provides how substrate binding couples the movement in TM domain and periplasmic domain which might help in the understanding of active site communication in PgpB. C55-PP phosphatase interactions with a catalytic pocket of PgpB provide new insight for designing drugs against bacterial infection.     

Identification of genes encoding the folate- and thiamine-binding membrane proteins in Firmicutes

Genes encoding high-affinity folate- and thiamine-binding proteins (FolT, ThiT) were identified in the Lactobacillus casei genome, expressed in Lactococcus lactis, and functionally characterized. Similar genes occur in many Firmicutes, sometimes next to folate or thiamine salvage genes. Most thiT genes are preceded by a thiamine riboswitch.     

Cloning of genes encoding coli-surface (CS) antigens in enterotoxigenic Escherichia coli

Abstract Sequences encoding the CS6 antigen of colonisation factor antigen (CFA)IV were cloned on a 3kb Cla I fragment. The recombinant plasmid pDEP5 coded for surface expression of CS6 measured by ELISA and production of CS6 polypeptides was detected in E. coli minicells. The genes for the CS1, CS2 and CS3 components of colonisation factor antigen CFA/II were cloned together on a length of DNA corresponding to about 17kb. CS3 was always expressed but production of either CS1 or CS2 depended on the serotype and biotype of the host strain. Separate subclones were obtained that expressed CS3 or CS1 and CS2.     

Identification of three genes controlling production of new outer membrane pore proteins in Escherichia coli K-12.

Escherichia coli K-12 strains carrying mutations in the ompB gene or double mutations in the tolF and par genes lack the major outer membrane proteins 1a and 1b. These strains are deficient in the transport of small hydrophylic compounds and are multiply colicin resistant. When revertants of these strains were sought, a number of extragenic pseudorevertants were obtained which produced new outer membrane proteins. These new proteins could be divided into three classes by differences in electrophoretic mobility on polyacrylamide gels, by differing specificities for transport of small molecules, and by the identification of three different genetic loci for genes controlling their production. These genetic loci are designated as nmpA (at approximately 82.5 min on the E. coli K-12 genetic map), nmpB (8.6 min), and nmpC (12 min). The new proteins produced in strains carrying nmpA, nmpB, or nmpC mutations did not cross-react with antiserum against a mixture of proteins 1a and 1b, or with antiserum against phage-directed protein 2. Production of the new membrane proteins restored sensitivity to some of the colicins.     

Identification and antibody-therapeutic targeting of chloramphenicol-resistant outer membrane proteins in Escherichia coli

Bacterial resistance to an antibiotic may result from survival in a suddenly strong antibiotic or in sub-minimum inhibitory concentration of the drug. Their shared proteins responsible for the resistance should be potential targets for designing new drugs to inhibit the growth of the antibiotic-resistant bacteria. In the current study, comparative proteomic methodologies were used for identification of sharedly altered outer membrane proteins (OM proteins) that are responsible for chloramphenical (CAP)-resistant Escherichia coli and for survival in medium with suddenly strong CAP treatment. Six differential OM proteins and another protein with unknown location were determined to be sharedly CAP-resistant-related proteins with the use of 2-DE/MS, Western blotting and gene mutant methods, in which TolC, OmpT, OmpC, and OmpW were critically altered proteins and potential targets for designing of the new drugs. Furthermore, a novel method of specific antibody combating bacterial growth was developed on these OM proteins. Only anti-TolC showed a very significant inhibition on bacterial growth in medium with CAP when antisera to TolC, OmpC, OmpT, and OmpW were separately utilized. The growth of CAP-resistant E. coli and its original strain was completely inhibited when they bound with anti-TolC and survived in 1/8 MIC of CAP. This observed result is basically the same to the finding that DeltatolC was survived in the same concentration of the antibiotic. Our study demonstrates that the enhancement of expression of antibody target with antibiotic could be very effective approach compared to using a drug alone, which highlights a potential way for treatment of infection by antibiotic-resistant bacteria.     

Phosphoprotein phosphatase activity associated with the cytoplasmic membrane of Salmonella typhimurium and Escherichia coli.

Membrane-associated phosphoprotein phosphatase activity was demonstrated in extracts of Salmonella typhimurium and Escherichia coli. The active protein could be extracted from the membrane as a large water-soluble complex (Mr greater than 150,000). Maximal activity was observed at pH 6 to 7 in the presence of a divalent cation. The enzyme appears to be distinct from previously described phosphatases.     

Identification and network of outer membrane proteins regulating streptomysin resistance in Escherichia coli

Bacterial Outer membrane (OM) proteins involved in antibiotic resistance have been reported. However, little is known about the OM proteins and their interaction network regulating streptomycin (SM) resistance. In the present study, a subproteomic approach was utilized to characterize OM proteins of Escherichia coli with SM resistance. TolC, OmpT and LamB were found to be up-regulated, and FadL, OmpW and a location-unknown protein Dps were down-regulated in the SM-resistant E. coli strain. These changes at the level of protein expression were validated using Western blotting. The possible roles of the altered proteins involved in the SM resistance were investigated using genetic modified strains with the deletion of these altered genes. It is found that decreased and elevated minimum inhibitory concentrations and survival capabilities of the gene deleted strains and their resistant strains, Delta tolC, Delta ompT, Delta dps, Delta tolC-R, Delta ompT-R, Delta dps-R and Delta fadL-R, were correlated with the changes of TolC, OmpT, Dps and FadL at the protein expression levels detected by 2-DE gels, respectively. The results may suggest that these proteins are the key OM proteins and play important roles in the regulation of SM resistance in E. coli. Furthermore, an interaction network of altered OM proteins involved in the SM resistance was proposed in this report. Of the six altered proteins, TolC may play a central role in the network. These findings may provide novel insights into mechanisms of SM resistance in E. coli.     

Analysis of six new genes encoding lysis proteins and coat proteins in Escherichia coli group A RNA phages

Group A RNA phages consist of four genes-maturation protein, coat protein, lysis protein and replicase genes. We analyzed six plasmids containing lysis protein genes and coat protein genes of Escherichia coli group A RNA phages and compared their amino acid sequences with the known proteins of E. coli(group A), Pseudomonas aeruginosa(PP7) RNA phages and Rg-lysis protein from Qbeta phage. The size of lysis proteins was different by the groups but the coat proteins were almost the same size among phages. The phylogenetic analysis shows that the sub-groups A-I and A-II of E. coli RNA phages were clearly dispersed into two clusters.     

Biogenesis of inner membrane proteins in Escherichia coli

For a long time, it was generally assumed that the biogenesis of inner membrane proteins in Escherichia coli occurs spontaneously, and that only the translocation of large periplasmic domains requires the aid of a protein machinery, the Sec translocon. However, evidence obtained in recent years indicates that most, if not all, inner membrane proteins require the assistance of protein factors to reach their native conformation in the membrane. Here, we review and discuss recent advances in our understanding of the biogenesis of inner membrane proteins in E. coli.     

Biogenesis of inner membrane proteins in Escherichia coli

The inner membrane proteome of the model organism Escherichia coli is composed of inner membrane proteins, lipoproteins and peripherally attached soluble proteins. Our knowledge of the biogenesis of inner membrane proteins is rapidly increasing. This is in particular true for the early steps of biogenesis - protein targeting to and insertion into the membrane. However, our knowledge of inner membrane protein folding and quality control is still fragmentary. Furthering our knowledge in these areas will bring us closer to understand the biogenesis of individual inner membrane proteins in the context of the biogenesis of the inner membrane proteome of Escherichia coli as a whole. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

Cloning and expression of multiple integral membrane proteins from Mycobacterium tuberculosis in Escherichia coli

Seventy integral membrane proteins from the Mycobacterium tuberculosis genome have been cloned and expressed in Escherichia coli. A combination of T7 promoter-based vectors with hexa-His affinity tags and BL21 E. coli strains with additional tRNA genes to supplement sparsely used E. coli codons have been most successful. The expressed proteins have a wide range of molecular weights and number of transmembrane helices. Expression of these proteins has been observed in the membrane and insoluble fraction of E. coli cell lysates and, in some cases, in the soluble fraction. The highest expression levels in the membrane fraction were restricted to a narrow range of molecular weights and relatively few transmembrane helices. In contrast, overexpression in insoluble aggregates was distributed over a broad range of molecular weights and number of transmembrane helices.     

Construction and application of the vectors to identify genes encoding exported proteins of Escherichia coli

In order to clone genes having signal sequences of Escherichia coli, four vectors with or without Lac or Ara promoter were constructed using a leaderless β-lactamase as reporter. Fragments of tetracycline resistance gene (Tet) with or without promoter were used to confirm the vectors’ ability to clone and report signal sequences. The minimum inhibitory concentration of ampicillin of the transformants was measured to detect the expression and secretion efficiency of the vectors. The results showed that the β-lactamase could be co-expressed and secreted with Tet protein. The Lac or Ara promoter in the vectors could be regulated by different inducers, and the Ara promoter showed higher regulative efficiency than the Lac. The best induction dose of l-arabinose for the Ara promoter is 1.25 %. All the four vectors were stably maintained in host after being inoculated for 20 passages in antibiotics-free media. Genomic library of an avian pathogenic strain, E. coli O₂, was constructed using the pMB-Ara-T vector we developed. 318 clones were obtained from the genomic library of E. coli strain O₂, and the inserts in these clones represented 276 genes based on sequence analysis. Among the 276 cloned fragments, only 128 had complete promoter sequence. For the 128 fragments with promoter, only 27 could be expressed under LB culture condition without inducer, the other 101 were only expressed under induction. The results showed our constructed vectors could efficiently capture all kinds of exported protein genes in vitro, including the ones without promoter or with inactive promoter.     

Molecular cloning in Escherichia coli of Erwinia chrysanthemi genes encoding multiple forms of pectate lyase.

The phytopathogenic enterobacterium Erwinia chrysanthemi excretes multiple isozymes of the plant tissue-disintegrating enzyme, pectate lyase (PL). Genes encoding PL were cloned from E. chrysanthemi CUCPB 1237 into Escherichia coli HB101 by inserting Sau3A-generated DNA fragments into the BamHI site of pBR322 and then screening recombinant transformants for the ability to sink into pectate semisolid agar. Restriction mapping of the cloned DNA in eight pectolytic transformants revealed overlapping portions of a 9.8-kilobase region of the E. chrysanthemi genome. Deletion derivatives of these plasmids were used to localize the pectolytic genotype to a 2.5-kilobase region of the cloned DNA. PL gene expression in E. coli was independent of vector promoters, repressed by glucose, and not induced by galacturonan. PL accumulated largely in the periplasmic space of E. coli. An activity stain used in conjunction with ultrathin-layer isoelectric focusing resolved the PL in E. chrysanthemi culture supernatants and shock fluids of E. coli clones into multiple forms. One isozyme with an apparent pI of 7.8 was produced at a far higher level in E. coli and was common to all of the pectolytic clones. Activity staining of renatured PL in sodium dodecyl sulfate-polyacrylamide gels revealed that this isozyme comigrated with the corresponding isozyme produced by E. chrysanthemi. The PL isozyme profiles produced by different clones and deletion derivative subclones suggest that the cloned region contains at least two PL isozyme structural genes. Pectolytic E. coli clones possessed a limited ability to macerate potato tuber tissues.     

Characterization of multiple fumarase proteins in Escherichia coli

Two different types of fumarase were found in sonic extracts of Escherichia coli; one required Fe-S for the enzyme activity, and the other did not. When the cells were grown without aeration, the Fe-S-independent enzyme occupied over 80% of the overall fumarase activity. Highly purified Fe-S-independent enzyme was suggested to be composed of four subunits (Mr = 48 kDa) by SDS-polyacrylamide gel electrophoresis and gel filtration. Amino acid and N-terminal sequence analyses supported the possibility that the enzyme is a product of fumC gene (FUMC). In aerobically grown cells, however, the content of FUMC was low and the Fe-S-dependent fumarase occupied over 80% of the overall activity. The Fe-S-dependent enzyme appeared to be labile and the activity was rapidly lost during purification. Although the spontaneous inactivation was previously ascribed to thermal lability (S.A. Woods & J.R. Guest (1987) FEMS Microbiol. Lett. 48, 219), the activity could be restored by anaerobic incubation with ferrous ions and SH-compounds.