Imp/OstA is required for cell envelope biogenesis in Escherichia coli

In Gram-negative bacteria, all components of the outer membrane are synthesized in the cytoplasm or the cytoplasmic leaflet of the inner membrane and must thus traverse the inner membrane and the periplasm on the way to their final destination. In this study, we show Imp/OstA to have characteristics typical for proteins involved in envelope biogenesis. Imp is essential and forms a high-molecular-weight disulphide-bonded complex in the outer membrane. Upon depletion of Imp, lipids and outer membrane proteins appear in a novel membrane fraction with higher density than the outer membrane. We propose Imp to be part of a targeting/usher system for components of the outer membrane.     

The chitin disaccharide, N,N'-diacetylchitobiose, is catabolized by Escherichia coli and is transported/phosphorylated by the phosphoenolpyruvate:glycose phosphotransferase system

We have previously reported that wild type strains of Escherichia coli grow on the chitin disaccharide N,N'-diacetylchitobiose, (GlcNAc)(2), as the sole source of carbon (Keyhani, N. O., and Roseman, S. (1997) Proc. Natl. Acad. Sci., U. S. A. 94, 14367-14371). A nonhydrolyzable analogue of (GlcNAc)(2,) methyl beta-N, N'-[(3)H]diacetylthiochitobioside ([(3)H]Me-TCB), was used to characterize the disaccharide transport process, which was found to be mediated by the phosphoenolpyruvate:glycose phosphotransferase system (PTS). Here and in the accompanying papers (Keyhani, N. O., Boudker, O., and Roseman, S. (2000) J. Biol. Chem. 275, 33091-33101; Keyhani, N. O., Bacia, K., and Roseman, S. (2000) J. Biol. Chem. 275, 33102-33109; Keyhani, N. O., Rodgers, M., Demeler, B., Hansen, J., and Roseman, S. (2000) J. Biol. Chem. 275, 33110-33115), we report that transport of [(3)H]Me-TCB and (GlcNAc)(2) involves a specific PTS Enzyme II complex, requires Enzyme I and HPr of the PTS, and results in the accumulation of the sugar derivative as a phosphate ester. The phosphoryl group is linked to the C-6 position of the GlcNAc residue at the nonreducing end of the disaccharide. The [(3)H]Me-TCB uptake system was induced only by (GlcNAc)(n), n = 2 or 3. The apparent K(m) of transport was 50-100 micrometer, and effective inhibitors of uptake included (GlcNAc)(n), n = 2 or 3, cellobiose, and other PTS sugars, i.e. glucose and GlcNAc. Presumably the PTS sugars inhibit by competing for PTS components. Kinetic properties of the transport system are described.     

ZipA is required for targeting of DMinC/DicB, but not DMinC/MinD, complexes to septal ring assemblies in Escherichia coli

The MinC division inhibitor is required for accurate placement of the septal ring at the middle of the Escherichia coli cell. The N-terminal domain of MinC ((Z)MinC) interferes with FtsZ assembly, while the C-terminal domain ((D)MinC) mediates both dimerization and complex formation with either MinD or DicB. Binding to either of these activators greatly enhances the division-inhibitory activity of MinC in the cell. The MinD ATPase plays a crucial role in the rapid pole-to-pole oscillation of MinC that is proposed to force FtsZ ring formation to midcell. DicB is encoded by one of the cryptic prophages on the E. coli chromosome (Qin) and is normally not synthesized. Binding of MinD or DicB to (D)MinC produces complexes that have high affinities for one or more septal ring-associated targets. Here we show that the FtsZ-binding protein ZipA is required for both recruitment of the (D)MinC/DicB complex to FtsZ rings and the DicB-inducible division block normally seen in MinC(+) cells. In contrast, none of the known FtsZ-associated factors, including ZipA, FtsA, and ZapA, appear to be specifically required for targeting of the (D)MinC/MinD complex to rings, implying that the two MinC/activator complexes must recognize distinct features of FtsZ assemblies. MinD-dependent targeting of MinC may occur in two steps of increasing topological specificity: (i) recruitment of MinC from the cytoplasm to the membrane, and (ii) specific targeting of the MinC/MinD complex to nascent septal ring assemblies on the membrane. Using membrane-tethered derivatives of MinC, we obtained evidence that both of these steps contribute to the efficiency of MinC/MinD-mediated division inhibition.     

Escherichia coli parA is an allele of the gyrB gene

Summary A thermosensitive (ts) parA mutant, MFT110, of Escherichia coli carried at least two ts mutations. The major ts defect, resulting from a mutation mapped originally at 95 min and complemented by pLC8-47, was most probably due to psd. A plasmid carrying the 1.6 kb BamHI-PvuII fragment recloned from pLC8-47 complemented the major ts mutation in MFT110 and psd(ts) in two mutants, but did not correct the Par phenotype of MFT110. The second ts mutation was salt-repairable and mapped at 83 min close to recF and tnaA. This mutation was linked with the Par phenotype as shown unambiguously by 4,6-diamidino-2-phenylindole stained nucleoids in parA mutant cells with the W3110 genetic background. Both salt-repairable ts and Par traits were corrected concomitantly by a plasmid carrying the chromosomal region solely for the gyrB gene. This strongly suggests that parA is an allele of gyrB.     

YodA from Escherichia coli is a metal-binding,lipocalin-like protein

We have determined the crystal structure of YodA, an Escherichia coli protein of unknown function. YodA had been identified under conditions of cadmium stress, and we confirm that it binds metals such as cadmium and zinc. We have also found nickel bound in one of the crystal forms. YodA is composed of two domains: a main lipocalin/calycin-like domain and a helical domain. The principal metal-binding site lies on one side of the calycin domain, thus making YodA the first metal-binding lipocalin known. Our experiments suggest that YodA expression may be part of a more general stress response. From sequence analogy with the C-terminal domain of a metal-binding receptor of a member of bacterial ATP-binding cassette transporters, we propose a three-dimensional model for this receptor and suggest that YodA may have a receptor-type partner in E. coli.     

Escherichia coli RecQ is a rapid, efficient, and monomeric helicase

RecQ family helicases play a key role in chromosome maintenance. Despite extensive biochemical, biophysical, and structural studies, the mechanism by which helicase unwinds double-stranded DNA remains to be elucidated. Using a wide array of biochemical and biophysical approaches, we have previously shown that the Escherichia coli RecQ helicase functions as a monomer. In this study, we have further characterized the kinetic mechanism of the RecQ-catalyzed unwinding of duplex DNA using the fluorometric stopped-flow method based on fluorescence resonance energy transfer. Our results show that RecQ helicase binds preferentially to 3'-flanking duplex DNA. Under the pre-steady-state conditions, the burst amplitude reveals a 1:1 ratio between RecQ and DNA substrate, suggesting that an active monomeric form of RecQ helicase is involved in the catalysis. Under the single-turnover conditions, the RecQ-catalyzed unwinding is independent of the 3'-tail length, indicating that functional interactions between RecQ molecules are not implicated in the DNA unwinding. It was further determined that RecQ unwinds DNA rapidly with a step size of 4 bp and a rate of approximately 21 steps/s. These kinetic results not only further support our previous conclusion that E. coli RecQ functions as a monomer but also suggest that some of the Superfamily 2 helicases may function through an "inchworm" mechanism.     

Hypoxanthine incorporation is nonmutagenic in Escherichia coli

Endonuclease V, encoded by the nfi gene, initiates removal of the base analogs hypoxanthine and xanthine from DNA, acting to prevent mutagenesis from purine base deamination within the DNA. On the other hand, the RdgB nucleotide hydrolase in Escherichia coli is proposed to prevent hypoxanthine and xanthine incorporation into DNA by intercepting the noncanonical DNA precursors dITP and dXTP. Because many base analogs are mutagenic when incorporated into DNA, it is intuitive to think of RdgB as acting to prevent similar mutagenesis from deaminated purines in the DNA precursor pools. To test this idea, we used a set of Claire Cupples' strains to detect changes in spontaneous mutagenesis spectra, as well as in nitrous acid-induced mutagenesis spectra, in wild-type cells and in rdgB single, nfi single, and rdgB nfi double mutants. We found neither a significant increase in spontaneous mutagenesis in rdgB and nfi single mutants or the double mutant nor any changes in nitrous acid-induced mutagenesis for rdgB mutant strains. We conclude that incorporation of deaminated purines into DNA is nonmutagenic.     

Bleomycin sensitivity in Escherichia coli is medium-dependent

Bleomycin (BLM) is a glycopeptide antibiotic and anti-tumor agent that targets primarily the furanose rings of DNA and in the presence of ferrous ions produces oxidative damage and DNA strand breaks. Escherichia coli cells growing in broth medium and exposed to low concentrations of BLM contain double-strand breaks and require homologous recombination to survive. To a lesser extent, the cells also require the abasic (AP) endonucleases associated with base excision repair, presumably to repair oxidative damage. As expected, there is strong induction of the SOS system in treated cells. In contrast, E. coli cells growing in glucose or glycerol minimal medium are resistant to the lethal action of BLM and do not require either homologous recombination functions or AP-endonucleases for survival. DNA ligase activity, however, is needed for cells growing in minimal medium to resist the lethal effects of BLM. There is weak SOS induction in such treated cells.     

The inner membrane protein, YhiM, is necessary for Escherichia coli (E. coli) survival in acidic conditions

Escherichia coli must be able to survive extreme acidic conditions. We were interested in determining the role of the inner membrane protein YhiM in survival in acidic conditions. Previous data demonstrated that the yhiM gene was upregulated in acidic conditions (Tucker et al. in J Bacteriol. 184:6551-6558, 2002). We therefore tested tn10 insertions into the yhiM gene for their ability to survive at low pH (pH 2.5). We show that YhiM was required for survival at pH 2.5. We also tested the YhiM dependence of the different acid resistance pathways. YhiM was required for the RpoS, glutamine and lysine-dependent acid resistance pathways. In contrast, YhiM was not required for the arginine-dependent acid resistance pathway.     

A predicted ABC transporter, FtsEX, is needed for cell division in Escherichia coli

FtsE and FtsX have homology to the ABC transporter superfamily of proteins and appear to be widely conserved among bacteria. Early work implicated FtsEX in cell division in Escherichia coli, but this was subsequently challenged, in part because the division defects in ftsEX mutants are often salt remedial. Strain RG60 has an ftsE::kan null mutation that is polar onto ftsX. RG60 is mildly filamentous when grown in standard Luria-Bertani medium (LB), which contains 1% NaCl, but upon shift to LB with no NaCl growth and division stop. We found that FtsN localizes to potential division sites, albeit poorly, in RG60 grown in LB with 1% NaCl. We also found that in wild-type E. coli both FtsE and FtsX localize to the division site. Localization of FtsX was studied in detail and appeared to require FtsZ, FtsA, and ZipA, but not the downstream division proteins FtsK, FtsQ, FtsL, and FtsI. Consistent with this, in media lacking salt, FtsA and ZipA localized independently of FtsEX, but the downstream proteins did not. Finally, in the absence of salt, cells depleted of FtsEX stopped dividing before any change in growth rate (mass increase) was apparent. We conclude that FtsEX participates directly in the process of cell division and is important for assembly or stability of the septal ring, especially in salt-free media.     

Loop X/XI, the largest cytoplasmic loop in the membrane-bound melibiose carrier of Escherichia coli, is a functional re-entrant loop

The melibiose carrier of Escherichia coli is a membrane-bound sugar-cation cotransporter consisting of 12 transmembrane helices connected by cytoplasmic and periplasmic loops, with both N- and C-terminus on the cytoplasmic side. Using a functional cysteine-less carrier, cysteine was substituted individually for residues 347-378 that comprise the largest cytoplasmic loop X/XI. The majority of the cysteine mutants have good protein expression levels. The cysteine mutants were studied for their transport activities, and the inhibitory effects of two sulfhydryl reagents, PCMBS (7-A long) and BM (29-A long). Cysteine substitution resulted in substantial loss of transport in 12 mutants. While PCMBS caused significant inhibition in only two mutants, T373C and V376C, from the periplasmic side (in a substrate-protective manner), more extensive inhibition pattern was observed from the cytoplasmic side, in seven mutants: V353C, Y358C, V371C, Q372C, T373C, V376C and G378C, suggesting that these residues are along the sugar pathway in the aqueous channel, close to the cytoplasmic side. Furthermore, the inhibitory effect of BM on the inside-out vesicles of the above mutants was clearly less than that of PCMBS, suggesting channel space limitation to large molecules, consistent with those residues being inside the channel. Three second-site revertants (A350C/F268L, A350C/I22S, and A350C/I22N) were selected. They may suggest proximities between loop X/XI and helices I and VIII, in agreement with a re-entrant loop structure. Self thiol cross-linkings of the cysteine mutants on loop X/XI failed to form dimers, suggesting that most of the loop is not surface-exposed from cytoplasmic side. Together, these results strongly indicated a functional re-entrant loop mechanistically important in Na+-coupled transporters.     

Arg276 of GseA, a Chlamydiatrachomatis Kdo transferase, is required for the synthesis of the chlamydial genus-specific epitope in Escherichia coli

GseA is an enzyme from Chlamydia trachomatis that can catalyse the addition of three 3-deoxy-D-manno-octulosonic acid (Kdo) residues onto lipid A precursors. GseA is similar, and in a few stretches identical, in its amino acid sequence to KdtA, an Escherichia coli Kdo transferase. In this study we altered an amino acid of GseA in a region that is identical between GseA and KdtA to test its importance in the structure or catalytic activity of GseA. We found that when Arg276 was changed to Lys, Ile or Ser, GseA activity was lost, suggesting an enzymatic role for this amino acid residue.     

HpaII methyltransferase is mutagenic in Escherichia coli.

A genetic reversion assay to study C-to-T mutations within CG sites in DNA is described. It was used to demonstrate that the presence of HpaII methyltransferase (MTase) in Escherichia coli causes a substantial increase in C-to-T mutations at CG sites. This is similar to the known mutagenic effects of E. coli MTase Dcm within its own recognition sequence. With this genetic system, a homolog of an E. coli DNA repair gene in Haemophilus parainfluenzae was tested for antimutagenic activity. Unexpectedly, the homolog was found to have little effect on the reversion frequency. The system was also used to show that HpaII and SssI MTases can convert cytosine to uracil in vitro. These studies define 5-methylcytosine as an intrinsic mutagen and further elaborate the mutagenic potential of cytosine MTases.     

The Escherichia coli btuE gene, encodes a glutathione peroxidase that is induced under oxidative stress conditions

Most aerobic organisms are exposed to oxidative stress. Looking for enzyme activities involved in the bacterial response to this kind of stress, we focused on the btuE-encoded Escherichia coli BtuE, an enzyme that shares homology with the glutathione peroxidase (GPX) family. This work deals with the purification and characterization of the btuE gene product.Purified BtuE decomposes in vitro hydrogen peroxide in a glutathione-dependent manner. BtuE also utilizes preferentially thioredoxin A to decompose hydrogen peroxide as well as cumene-, tert-butyl-, and linoleic acid hydroperoxides, confirming that its active site confers non-specific peroxidase activity. These data suggest that the enzyme may have one or more organic hydroperoxide as its physiological substrate.The btuE gene was induced when cells were exposed to oxidative stress elicitors that included potassium tellurite, menadione and hydrogen peroxide, among others, suggesting that BtuE could participate in the E. coli response to reactive oxygen species. To our knowledge, this is the first report describing a glutathione peroxidase in E. coli.     

Sodium/proton antiport is required for growth of Escherichia coli at alkaline pH

Evidence is presented indicating that Escherichia coli requires the Na+/H+ antiporter and external sodium (or lithium) ion to grow at high pH. Cells were grown in plastic tubes containing medium with a very low Na+ content (5-15 microM). Normal cells grew at pH 7 or 8 with or without added Na+, but at pH 8.5 external Na was required for growth. A mutant with low antiporter activity failed to grow at pH 8.5 with or without Na+. On the other hand, another mutant with elevated antiporter activity grew at a higher pH than normal (pH 9) in the presence of added Na+ or Li+. Amiloride, an inhibitor of the antiporter, prevented cells from growing at pH 8.5 (plus Na+), although it had no effect on growth in media of lower pH values.     

Phosphatidylethanolamine Deficiency Impairs Escherichia coli Adhesion by Downregulating Lipopolysaccharide Synthesis,Which is Reversible by High Galactose/Lactose Cultivation

As the initiation step of bacterial infection or biofouling, bacterial adhesion on cells or substrates is generally an optimal target for antibacterial design. Phosphatidylethanolamine (PE) is the principal phospholipid in bacteria, and its function in bacterial adhesion remains unclear. In this study, four E. coli strains including two PE-deficient mutants (PE^?PC^? and PE^?PC^+?strains) and two PE-containing wild-type controls (PE?⁺?PC^? strains) were recruited to investigate the influence of PE deficiency on bacterial adhesion. We found that PE deficiency could impair E. coli adhesion on macrophages (human THP-1-derived and mouse RAW264.7 macrophages) or glass coverslips by downregulating lipopolysaccharide (LPS) biosynthesis, which could be reversible by high galactose/lactose but not glucose cultivation. The data imply that PE play important role in bacterial adhesion probably via affecting LPS biosynthesis and suggest that targeting PE biosynthesis is also a potential antibacterial strategy.