The TonB-dependent uptake of pyrroloquinoline-quinone (PQQ) and secretion of gluconate by Escherichia coli K-12.

Glucose is taken up by Escherichia coli through the phosphotransferase system (PTS) as the preferred carbon source. PTS mutants grow with glucose as a carbon source only in the presence of pyrroloquinoline quinone (PQQ), which is needed as a redox cofactor for the glucose dehydrogenase Gcd. The membrane-anchored Gcd enzyme oxidises glucose to gluconolactone in the periplasm. For this reaction to occur, external supply of PQQ is required as E. coli is unable to produce PQQ de novo. Growth experiments show that PqqU (previously YncD) is the TonB-ExbBD-dependent transporter for PQQ through the outer membrane. PQQ protected the cells from the PqqU-dependent phage IsaakIselin (Bas10) by competition for the receptor protein. As a high affinity uptake system, PqqU allows E. coli to activate Gcd even at surrounding PQQ concentrations of about 1 nmoL/L. At about 30-fold higher PQQ concentrations, the activation of Gcd gets PqqU independent. Due to its small size, Pqq may also pass the outer membrane through porins. The PQQ-dependent production of gluconate has been demonstrated in many plant growth-promoting bacteria that solubilise phosphate minerals in the soil by secreting this acid. Under phosphate limiting conditions also E. coli induces the glucose dehydrogenase and secretes gluconate, even in absence of PTS, that is, even when the bacterium is unable to grow on glucose without PQQ.

Transcription regulation of iron carrier transport genes by ECF sigma factors through signaling from the cell surface into the cytoplasm.

Bacteria are usually iron-deficient because the Fe3+ in their environment is insoluble or is incorporated into proteins. To overcome their natural iron limitation, bacteria have developed sophisticated iron transport and regulation systems. In gram-negative bacteria, these include iron carriers, such as citrate, siderophores, and heme, which when loaded with Fe3+ adsorb with high specificity and affinity to outer membrane proteins. Binding of the iron carriers to the cell surface elicits a signal that initiates transcription of iron carrier transport and synthesis genes, referred to as "cell surface signaling". Transcriptional regulation is not coupled to transport. Outer membrane proteins with signaling functions contain an additional N-terminal domain that in the periplasm makes contact with an anti-sigma factor regulatory protein that extends from the outer membrane into the cytoplasm. Binding of the iron carriers to the outer membrane receptors elicits proteolysis of the anti-sigma factor by two different proteases, Prc in the periplasm, and RseP in the cytoplasmic membrane, inactivates the anti-sigma function or results in the generation of an N-terminal peptide of ∼50 residues with pro-sigma activity yielding an active extracytoplasmic function (ECF) sigma factor. Signal recognition and signal transmission into the cytoplasm is discussed herein.

Compilation of Escherichia coli K-12 outer membrane phage receptors - their function and some historical remarks.

Many Escherichia coli phages have been sequenced, but in most cases their sequences alone do not suffice to predict their host specificity. Analysis of phage resistant E. coli K-12 mutants have uncovered a certain set of outer membrane proteins and polysaccharides as receptors. In this review, a compilation of E. coli K12 phage receptors is provided and their functional characterization, often driven by studies on phage resistant mutants, is discussed in the historical context. While great progress has been made in this field thus far, several proteins in the outer membrane still await characterization as phage receptors.

Novel Tat-Dependent Protein Secretion.

The transcription initiation signal elicited by the binding of ferric citrate to the outer membrane FecA protein is transmitted by the FecR protein across the cytoplasmic membrane to the FecI extracytoplasmic function (ECF) sigma factor. In this issue of Journal of Bacteriology, I. J. Passmore, J. M. Dow, F. Coll, J. Cuccui, et al. (J Bacteriol 202:e00541-19, 2020, https://doi.org/10.1128/JB.00541-19) report that the FecR sequence contains both the twin-arginine signal motif and the secretory (Sec) avoidance motif typical of proteins secreted by the twin-arginine translocation (TAT) system. The same study shows that FecR is indeed secreted by Tat and represents a new class of bitopic Tat-dependent membrane proteins.

Lipoproteins: Structure, Function, Biosynthesis.

The Lpp lipoprotein of Escherichia coli is the first identified protein with a covalently linked lipid. It is chemically bound by its C-terminus to murein (peptidoglycan) and inserts by the lipid at the N-terminus into the outer membrane. As the most abundant protein in E. coli (106 molecules per cell) it plays an important role for the integrity of the cell envelope. Lpp represents the type protein of a large variety of lipoproteins found in Gram-negative and Gram-positive bacteria and in archaea that have in common the lipid structure for anchoring the proteins to membranes but otherwise strongly vary in sequence, structure, and function. Predicted lipoproteins in known prokaryotic genomes comprise 2.7% of all proteins. Lipoproteins are modified by a unique phospholipid pathway and transferred from the cytoplasmic membrane into the outer membrane by a special system. They are involved in protein incorporation into the outer membrane, protein secretion across the cytoplasmic membrane, periplasm and outer membrane, signal transduction, conjugation, cell wall metabolism, antibiotic resistance, biofilm formation, and adhesion to host tissues. They are only found in bacteria and function as signal molecules for the innate immune system of vertebrates, where they cause inflammation and elicit innate and adaptive immune response through Toll-like receptors. This review discusses various aspects of Lpp and other lipoproteins of Gram-negative and Gram-positive bacteria and archaea.

CbrA is a flavin adenine dinucleotide protein that modifies the Escherichia coli outer membrane and confers specific resistance to Colicin M.

Colicin M (Cma) is a protein toxin produced by Escherichia coli that kills sensitive E. coli cells by inhibiting murein biosynthesis in the periplasm. Recombinant plasmids carrying cbrA (formerly yidS) strongly increased resistance of cells to Cma, whereas deletion of cbrA increased Cma sensitivity. Transcription of cbrA is positively controlled by the two-component CreBC system. A ΔcreB mutant was highly Cma sensitive because little CbrA was synthesized. Treatment of CbrA-overproducing cells by osmotic shock failed to render cells Cma sensitive because the cells were resistant to osmotic shock. In a natural environment with a growth-limiting nutrient supply, cells producing CbrA defend themselves against colicin M synthesized by competing cells. Isolated CbrA is a protein with noncovalently bound flavin adenine dinucleotide. Sequence comparison and structure prediction assign the closest relative of CbrA with a known crystal structure as digeranylgeranyl-glycerophospholipid reductase of Thermoplasma acidophilum. CbrA is found in Escherichia coli, Citrobacter, and Salmonella bongori but not in other enterobacteria. The next homologs with the highest identity (over 50%) are found in the anaerobic Clostridium botulinum group 1 and a few other Firmicutes.

The mechanisms of HAMP-mediated signaling in transmembrane receptors.

HAMP domains mediate signal transduction in over 7500 enzyme-coupled receptors represented in all kingdoms of life. The HAMP domain of the putative archaeal receptor Af1503 has a parallel, dimeric, four-helical coiled coil structure, but with unusual core packing, related to canonical packing by concerted axial rotation of the helices. This has led to the gearbox model for signal transduction, whereby the alternate packing modes correspond to signaling states. Here we present structures of a series of Af1503 HAMP variants. We show that substitution of a conserved small side chain within the domain core (A291) for larger residues induces a gradual transition in packing mode, involving both changes in helix rotation and bundle shape, which are most prominent at the C-terminal, output end of the domain. These are correlated with activity and ligand response in vitro and in vivo by incorporating Af1503 HAMP into mycobacterial adenylyl cyclase assay systems.

Recent insights into iron import by bacteria.

Bacteria are confronted with a low availability of iron owing to its insolubility in the Fe3+ form or its being bound to host proteins. The bacteria cope with the iron deficiency by using host heme or siderophores synthesized by themselves or other microbes. In contrast to most other nutrients, iron compounds are tightly bound to proteins at the cell surfaces, from which they are further translocated by highly specific proteins across the cell wall of gram-positive bacteria and the outer membrane of gram-negative bacteria. Once heme and iron siderophores arrive at the cytoplasmic membrane, they are taken up across the cytoplasmic membrane by ABC transporters. Here we present an outline of bacterial heme and iron siderophore transport exemplified by a few selected cases in which recent progress in the understanding of the transport mechanisms has been achieved.

Escherichia coli exports cyclic AMP via TolC.

In Escherichia coli more than 180 genes are regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. However, more than 90% of cAMP that is made by intracellular adenylyl cyclases is found in the culture medium. How is cAMP exported from E. coli? In a tolC mutant, 0.03 mM IPTG (isopropyl-ß-d-thiogalactopyranoside) was sufficient to induce ß-galactosidase compared to 0.1 mM IPTG in the parent strain. In a cya mutant unable to produce cAMP about 1 mM extracellular cAMP was required to induce ß-galactosidase, whereas in a cya tolC mutant 0.1 mM cAMP was sufficient. When cAMP in E. coli cya was generated intracellularly by a recombinant, weakly active adenylyl cyclase from Corynebacterium glutamicum, the critical level of cAMP necessary for induction of maltose degradation was only achieved in a tolC mutant and not in the parent strain. Deletion of a putative cAMP phosphodiesterase of E. coli, CpdA, resulted in a slightly similar, yet more diffuse phenotype. The data demonstrate that export of cAMP via TolC is a most efficient way of E. coli to lower high concentrations of cAMP in the cell and maintain its sensitivity in changing metabolic environments.

The interplay between siderophore secretion and coupled iron and copper transport in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120.

Iron uptake is essential for Gram-negative bacteria including cyanobacteria. In cyanobacteria, however, the iron demand is higher than in proteobacteria due to the function of iron as a cofactor in photosynthesis and nitrogen fixation, but our understanding of iron uptake by cyanobacteria stands behind the knowledge in proteobacteria. Here, two genes involved in this process in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 were identified. ORF all4025 encodes SchE, a putative cytoplasmic membrane-localized transporter involved in TolC-dependent siderophore secretion. Inactivation of schE resulted in an enhanced sensitivity to high metal concentrations and decreased secretion of hydroxamate-type siderophores. ORF all4026 encodes a predicted outer membrane-localized TonB-dependent iron transporter, IacT. Inactivation of iacT resulted in decreased sensitivity to elevated iron and copper levels. Expression of iacT from the artificial trc promoter (P(trc)) resulted in sensitization against tested metals. Further analysis showed that iron and copper effects are synergistic because a decreased supply of iron induced a significant decrease of copper levels in the iacT insertion mutant but an increase of those levels in the strain carrying P(trc)-iacT. Our results unravel a link between iron and copper homeostasis in Anabaena sp. PCC 7120.

Transmembrane signaling in chimeras of the Escherichia coli aspartate and serine chemotaxis receptors and bacterial class III adenylyl cyclases.

The Escherichia coli chemoreceptors for serine (Tsr) and aspartate (Tar) and several bacterial class III adenylyl cyclases (ACs) share a common molecular architecture; that is, a membrane anchor that is linked via a cytoplasmic HAMP domain to a C-terminal signal output unit. Functionality of both proteins requires homodimerization. The chemotaxis receptors are well characterized, whereas the typical hexahelical membrane anchor (6TM) of class III ACs, suggested to operate as a channel or transporter, has no known function beyond a membrane anchor. We joined the intramolecular networks of Tsr or Tar and two bacterial ACs, Rv3645 from Mycobacterium tuberculosis and CyaG from Arthrospira platensis, across their signal transmission sites, connecting the chemotaxis receptors via different HAMP domains to the catalytic AC domains. AC activity in the chimeras was inhibited by micromolar concentrations of l-serine or l-aspartate in vitro and in vivo. Single point mutations known to abolish ligand binding in Tar (R69E or T154I) or Tsr (R69E or T156K) abrogated AC regulation. Co-expression of mutant pairs, which functionally complement each other, restored regulation in vitro and in vivo. Taken together, these studies demonstrate chemotaxis receptor-mediated regulation of chimeric bacterial ACs and connect chemical sensing and AC regulation.

Absence of functional Hfe protects mice from invasive Salmonella enterica serovar Typhimurium infection via induction of lipocalin-2.

Mutations of HFE are associated with hereditary hemochromatosis, but their influence on host susceptibility to infection is incompletely understood. We report that mice lacking one or both Hfe alleles are protected from septicemia with Salmonella Typhimurium, displaying prolonged survival and improved control of bacterial replication. This increased resistance is paralleled by an enhanced production of the enterochelin-binding peptide lipocalin-2 (Lcn2), which reduces the availability of iron for Salmonella within Hfe-deficient macrophages. Accordingly, Hfe(-/-)Lcn2(-/-) macrophages are unable to efficiently control the infection or to withhold iron from intracellular Salmonella. Correspondingly, the protection conferred by the Hfe defect is abolished in Hfe(-/-) mice infected with enterochelin-deficient Salmonella as well as in Hfe(-/-)Lcn2(-/-) mice infected with wild-type bacteria. Thus, by induction of the iron-capturing peptide Lcn2, absence of functional Hfe confers host resistance to systemic infection with Salmonella, thereby providing an evolutionary advantage which may account for the high prevalence of genetic hemochromatosis.

Role of the twin-arginine translocation pathway in Staphylococcus.

In Staphylococcus, the twin-arginine translocation (Tat) pathway is present only in some species and is composed of TatA and TatC. The tatAC operon is associated with the fepABC operon, which encodes homologs to an iron-binding lipoprotein, an iron-dependent peroxidase (FepB), and a high-affinity iron permease. The FepB protein has a typical twin-arginine (RR) signal peptide. The tat and fep operons constitute an entity that is not present in all staphylococcal species. Our analysis was focused on Staphylococcus aureus and S. carnosus strains. Tat deletion mutants (DeltatatAC) were unable to export active FepB, indicating that this enzyme is a Tat substrate. When the RR signal sequence from FepB was fused to prolipase and protein A, their export became Tat dependent. Since no other protein with a Tat signal could be detected, the fepABC-tatAC genes comprise not only a genetic but also a functional unit. We demonstrated that FepABC drives iron import, and in a mouse kidney abscess model, the bacterial loads of DeltatatAC and Deltatat-fep mutants were decreased. For the first time, we show that the Tat pathway in S. aureus is functional and serves to translocate the iron-dependent peroxidase FepB.

Salmochelin, the long-overlooked catecholate siderophore of Salmonella.

Salmochelin is a C-glucosylated enterobactin produced by Salmonella species, uropathogenic and avian pathogenic Escherichia coli strains, and certain Klebsiella strains. It was the first glucosylated siderophore described. The glucosylation has been interpreted as a bacterial evasion mechanism against the mammalian catecholate siderophore-binding protein siderocalin (NGAL-lipocalin). The synthesis, excretion, and uptake of salmochelin requires five genes, iroBCDEN, and also the enterobactin biosynthesis and utilization system. Some salmochelin-producing strains also secrete microcins, which possess a C-terminal, linear glucosyl-enterobactin moiety. These microcins recognize the catecholate siderophore receptors IroN, Cir, Fiu, and FepA, and may inhibit the growth of competitors for catecholate siderophores.

Inhibition of growth of Shiga toxin-producing Escherichia coli by nonpathogenic Escherichia coli.

During routine quality control testing of diagnostic methods for Shiga toxin-producing Escherichia coli (STEC) using stool samples spiked with STEC, it was observed that the Shiga toxin could not be detected in 32 out of 82 samples tested. Strains of E. coli isolated from such stool samples were shown to be responsible for this inhibition. One particular isolate, named E. coli 1307, was intensively studied because of its highly effective inhibitory effect; this strain significantly reduced growth and Shiga toxin levels in coculture of several STEC strains regardless of serovar or Shiga toxin type. The probiotic E. coli Nissle 1917 inhibited growth and reduced Shiga toxin levels in STEC cultures to an extent similar to E. coli 1307, but commensal E. coli strains and several other known probiotic bacteria (enterococci, Bacillus sp., Lactobacillus acidophilus) showed no, or only small, inhibitory effects. Escherichia coli 1307 lacks obvious fitness factors, such as aerobactin, yersiniabactin, microcins and a polysaccharide capsule, that are considered to promote the growth of pathogenic bacteria. We therefore propose strain E. coli 1307 as a candidate probiotic for use in the prevention and treatment of infections caused by STEC.

Alr0397 is an outer membrane transporter for the siderophore schizokinen in Anabaena sp. strain PCC 7120.

Iron uptake in proteobacteria by TonB-dependent outer membrane transporters represents a well-explored subject. In contrast, the same process has been scarcely investigated in cyanobacteria. The heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 is known to secrete the siderophore schizokinen, but its transport system has remained unidentified. Inspection of the genome of strain PCC 7120 shows that only one gene encoding a putative TonB-dependent iron transporter, namely alr0397, is positioned close to genes encoding enzymes involved in the biosynthesis of a hydroxamate siderophore. The expression of alr0397, which encodes an outer membrane protein, was elevated under iron-limited conditions. Inactivation of this gene caused a moderate phenotype of iron starvation in the mutant cells. The characterization of the mutant strain showed that Alr0397 is a TonB-dependent schizokinen transporter (SchT) of the outer membrane and that alr0397 expression and schizokinen production are regulated by the iron homeostasis of the cell.

Periplasmic chaperone FkpA is essential for imported colicin M toxicity.

Chaperones facilitate correct folding of newly synthesized proteins. We show here that the periplasmic FkpA chaperone is required for killing Escherichia coli by colicin M entering cells from the outside. Highly active colicin M preparations were inactive against fkpA mutant cells; 10(4)-fold dilutions killed fkpA(+) cells. Three previously isolated spontaneous mutants tolerant to colicin M carried a stop codon or an IS1 insertion in the peptidyl-prolyl-cis-trans-isomerase (PPIase) domain (C-domain) of FkpA, which resulted in deletion of the domain. A randomly generated mutant carried a G148D mutation in the C-domain. A temperature-sensitive mutant tolerant to colicin M carried a Y25N mutation in the FkpA N-domain. Mutants transformed with wild-type fkpA were colicin M-sensitive. Isolated FkpA-His reduced colicin M-His cleavage by proteinase K and renatured denatured colicin M-His in vitro; renaturation was prevented by the PPIase inhibitor FK506. In both assays, periplasmic SurA-His had no effect. No other tested periplasmic chaperone could activate colicin M. Among the tested colicins, only colicin M required FkpA for activity. Colicin M bound to cells via FhuA was inactivated by trypsin; unbound colicin M retained activity. We propose that colicin M unfolds during import across the outer membrane, FkpA specifically assists in folding colicin M into an active toxin in the periplasm and PPIase is essential for colicin M activity. Colicin M is a suitable tool for the isolation of FkpA mutants used to elucidate the functions of the FkpA N- and C-domains.