Iron-siderophore Receptor Research Articles

Pacificimonas pallium sp. nov., an Isolated Bacterium from the Mantle of Pacific Oyster Crassostrea gigas in Germany, and Prediction of One-Carbon Metabolism

A yellow bacterium from marine agar, strain WHA3T, was isolated from the mantel of the Pacific oysters Crassostrea gigas in the Wilhelmshaven Sea in northern Germany. Based on the 16S rRNA gene sequence, strain WHA3T had a high similarity to Pacificimonas flava JLT2015T (95.80%) and 94.79% to Pacificimonas aurantium JLT2012T. Furthermore, the dDDH and ANI value analysis between WHA3T and other closest type strains were lower than 70% and 95%, respectively. The percentage of conserved proteins (POCP) and the average amino acid identity (AAI) value against Pacificimonas flava JLT2015T and Pacificimonas aurantium JLT2012T represented in the ranges of higher than 50% and 60%, respectively. Strain WHA3T contained ubiquinone-10 (Q-10) as the predominant quinone, and the major fatty acids were C16:1 ω7c and C18:1 ω7c. Granules of polyhydroxyalkanoates (PHAs) were absent. The main polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, several sphingoglycolipids, an unknown phospholipid, an unknown glycolipid, and an unknown polar lipid. The polyamines contained spermidine and spermine. The DNA G + C content of strain WHA3T was 61.69%. An analysis of the whole-genome sequence in the frame of genome mining strain WHA3T predicted the presence of genomes for one-carbon metabolism, TonB-dependent transporters, vitamin B12 transporter, iron siderophore receptor protein, and other genes, some of which play important roles against restricted nutrient sources. The extract of strain WHA3T moderately inhibited the growth of Candida albicans DSM 1665. The polyphasic taxonomic analysis results suggested that strain WHA3T could be separated from its closest type strains. Strain WHA3T represents a novel species in the genus Pacificimonas, for which we propose the name Pacificimonaspallium sp. nov., with the type strain WHA3T (= DSM 111825T = NCCB 100832T).

A pilot study using metagenomic sequencing of the sputum microbiome suggests potential bacterial biomarkers for lung cancer.

Lung cancer (LC) is the most prevalent cancer worldwide, and responsible for over 1.3 million deaths each year. Currently, LC has a low five year survival rates relative to other cancers, and thus, novel methods to screen for and diagnose malignancies are necessary to improve patient outcomes. Here, we report on a pilot-sized study to evaluate the potential of the sputum microbiome as a source of non-invasive bacterial biomarkers for lung cancer status and stage. Spontaneous sputum samples were collected from ten patients referred with possible LC, of which four were eventually diagnosed with LC (LC+), and six had no LC after one year (LC-). Of the seven bacterial species found in all samples, Streptococcus viridans was significantly higher in LC+ samples. Seven further bacterial species were found only in LC-, and 16 were found only in samples from LC+. Additional taxonomic differences were identified in regards to significant fold changes between LC+ and LC-cases, with five species having significantly higher abundances in LC+, with Granulicatella adiacens showing the highest level of abundance change. Functional differences, evident through significant fold changes, included polyamine metabolism and iron siderophore receptors. G. adiacens abundance was correlated with six other bacterial species, namely Enterococcus sp. 130, Streptococcus intermedius, Escherichia coli, S. viridans, Acinetobacter junii, and Streptococcus sp. 6, in LC+ samples only, which could also be related to LC stage. Spontaneous sputum appears to be a viable source of bacterial biomarkers which may have utility as biomarkers for LC status and stage.

Transport proteins promoting Escherichia coli pathogenesis

Escherichia coli is a genetically diverse species infecting hundreds of millions of people worldwide annually. We examined seven well-characterized E. coli pathogens causing urinary tract infections, gastroenteritis, pyelonephritis and haemorrhagic colitis. Their transport proteins were identified and compared with each other and a non-pathogenic E. coli K12 strain to identify transport proteins related to pathogenesis. Each pathogen possesses a unique set of protein secretion systems for export to the cell surface or for injecting effector proteins into host cells. Pathogens have increased numbers of iron siderophore receptors and ABC iron uptake transporters, but the numbers and types of low-affinity secondary iron carriers were uniform in all strains. The presence of outer membrane iron complex receptors and high-affinity ABC iron uptake systems correlated, suggesting co-evolution. Each pathovar encodes a different set of pore-forming toxins and virulence-related outer membrane proteins lacking in K12. Intracellular pathogens proved to have a characteristically distinctive set of nutrient uptake porters, different from those of extracellular pathogens. The results presented in this report provide information about transport systems relevant to various types of E. coli pathogenesis that can be exploited in future basic and applied studies.

Mechanism of bactericidal activity of microcin L in Escherichia coli and Salmonella enterica.

For the first time, the mechanism of action of microcin L (MccL) was investigated in live bacteria. MccL is a gene-encoded peptide produced by Escherichia coli LR05 that exhibits a strong antibacterial activity against related Enterobacteriaceae, including Salmonella enterica serovars Typhimurium and Enteritidis. We first subcloned the MccL genetic system to remove the sequences not involved in MccL production. We then optimized the MccL purification procedure to obtain large amounts of purified microcin to investigate its antimicrobial and membrane properties. We showed that MccL did not induce outer membrane permeabilization, which indicated that MccL did not use this way to kill the sensitive cell or to enter into it. Using a set of E. coli and Salmonella enterica mutants lacking iron-siderophore receptors, we demonstrated that the MccL uptake required the outer membrane receptor Cir. Moreover, the MccL bactericidal activity was shown to depend on the TonB protein that transduces the proton-motive force of the cytoplasmic membrane to transport iron-siderophore complexes across the outer membrane. Using carbonyl cyanide 3-chlorophenylhydrazone, which is known to fully dissipate the proton-motive force, we proved that the proton-motive force was required for the bactericidal activity of MccL on E. coli. In addition, we showed that a primary target of MccL could be the cytoplasmic membrane: a high level of MccL disrupted the inner membrane potential of E. coli cells. However, no permeabilization of the membrane was detected.

Dual Roles of the Central Domain of Colicin D tRNase in TonB-mediated Import and in Immunity

Colicin D import into Escherichia coli requires an interaction via its TonB box with the energy transducer TonB. Colicin D cytotoxicity is inhibited by specific tonB mutations, but it is restored by suppressor mutations in the TonB box. Here we report that there is a second site of interaction between TonB and colicin D, which is dependent upon a 45-amino acid region, within the uncharacterized central domain of colicin D. In addition, the 8th amino acids of colicin D (a glycine) and colicin B (a valine), adjacent to their TonB boxes, are also required for TonB recognition, suggesting that high affinity complex formation involves multiple interactions between these colicins and TonB. The central domain also contributes to the formation of the immunity complex, as well as being essential for uptake and thus killing. Colicin D is normally secreted in association with the immunity protein, and this complex involves the following two interactions: a major interaction with the C-terminal tRNase domain and a second interaction involving the central domain of colicin D and, most probably, the alpha4 helix of ImmD, which is on the opposite side of ImmD compared with the major interface. In contrast, formation of the immunity complex with the processed cytotoxic domain, the form expected to be found in the cytoplasm after colicin D uptake, requires only the major interaction. Klebicin D has, like colicin D, a ribonuclease activity toward tRNAArg and a central domain, which can form a complex with ImmD but which does not function in TonB-mediated transport.

Parasitism of Iron-siderophore Receptors of Escherichia Coli by the Siderophore-peptide Microcin E492m and its Unmodified Counterpart

Microcin E492 (MccE492) is an antibacterial peptide naturally secreted by Klebsiella pneumoniae RYC492. Initially described as an 84-residue unmodified peptide, it was also recently isolated in a posttranslationally modified form, MccE492m. The production of MccE492m is dependent on the synthesis of enterobactin and the mceABCDEFGHIJ gene cluster. The posttranslational modification was characterized as a trimer of N-(2,3-dihydroxybenzoyl)-L-serine (DHBS) linked to the Ser84-carboxylate via a beta-D-glucose moiety. MccE492m was shown to bind ferric ions through the trimer of DHBS. This is the first example of a novel type of antibacterial peptide termed siderophore-peptide. Recognition of MccE492m, but also of the unmodified MccE492, was shown to be mediated by the catecholate siderophore receptors FepA, Cir and Fiu at the outer membrane of E. coli. The siderophore-type modification was shown to be responsible for a significant enhancement of the microcin antibacterial activity. Therefore, we propose that MccE492 and MccE492m use iron-siderophore receptors for uptake into the target bacteria and that improvement of MccE492 antimicrobial activity upon modification results from an increase in the microcin/receptor affinity.

Identification of an Essential Cleavage Site in ColE7 Required for Import and Killing of Cells

Colicin E7 (ColE7), a nuclease toxin released from Escherichia coli, kills susceptible bacteria under environmental stress. Nuclease colicins are processed during translocation with only the cytotoxic nuclease domains traversing the inner membrane to cleave tRNA, rRNA, or DNA in the cytoplasm of target cells. In this study, we show that the E. coli periplasmic extract cleaves ColE7 between Lys(446) and Arg(447) in the presence or absence of its inhibitor Im7 protein. Several residues near cleavage sites were mutated, but only mutants of Arg(447) completely lost in vivo cell-killing activity. Both the full-length and the nuclease domain of Arg(447) mutants retained their nuclease activities, indicating that failure to kill cells was not a consequence of damage to the endonuclease activity of the enzyme. Moreover, the R447E ColE7 mutant was not cleaved at its 447 site by periplasmic extracts or transported into the cytoplasm of target cells. Collectively, these results suggest that ColE7 is cleaved at Arg(447) during translocation and that cleavage is an essential step for ColE7 import into the cytoplasm of target cells and its cell-killing activity. Conserved basic residues aligned with Arg(447) have also been found in other nuclease colicins, implying that the processing at this position may be common to other colicins during translocation.

Import of the Transfer RNase Colicin D Requires Site-Specific Interaction with the Energy-Transducing Protein TonB

The transfer RNase colicin D and ionophoric colicin B appropriate the outer membrane iron siderophore receptor FepA and share a common translocation requirement for the TonB pathway to cross the outer membrane. Despite the almost identical sequences of the N-terminal domains required for the translocation of colicins D and B, two spontaneous tonB mutations (Arg158Ser and Pro161Leu) completely abolished colicin D toxicity but did not affect either the sensitivity to other colicins or the FepA-dependent siderophore uptake capacity. The sensitivity to colicin D of both tonB mutants was fully restored by specific suppressor mutations in the TonB box of colicin D, at Ser18(Thr) and Met19(Ile), respectively. This demonstrates that the interaction of colicin D with TonB is critically dependent on certain residues close to position 160 in TonB and on the side chains of certain residues in the TonB box of colicin D. The effect of introducing the TonB boxes from other TonB-dependent receptors and colicins into colicins D and B was studied. The results of these and other changes in the two TonB boxes show that the role of residues at positions 18 and 19 in colicin D is strongly modulated by other nearby and/or distant residues and that the overall function of colicin D is much more dependent on the interaction with TonB involving the TonB box than is the function of colicin B.

Microcin E492 antibacterial activity: evidence for a TonB-dependent inner membrane permeabilization on Escherichia coli.

The mechanism of action of microcin E492 (MccE492) was investigated for the first time in live bacteria. MccE492 was expressed and purified to homogeneity through an optimized large-scale procedure. Highly purified MccE492 showed potent antibacterial activity at minimal inhibitory concentrations in the range of 0.02-1.2 microM. The microcin bactericidal spectrum of activity was found to be restricted to Enterobacteriaceae and specifically directed against Escherichia and Salmonella species. Isogenic bacteria that possessed mutations in membrane proteins, particularly of the TonB-ExbB-ExbD complex, were assayed. The microcin bactericidal activity was shown to be TonB- and energy-dependent, supporting the hypothesis that the mechanism of action is receptor mediated. In addition, MccE492 depolarized and permeabilized the E. coli cytoplasmic membrane. The membrane depolarization was TonB dependent. From this study, we propose that MccE492 is recognized by iron-siderophore receptors, including FepA, which promote its import across the outer membrane via a TonB- and energy-dependent pathway. MccE492 then inserts into the inner membrane, whereupon the potential becomes destabilized by pore formation. Because cytoplasmic membrane permeabilization of MccE492 occurs beneath the threshold of the bactericidal concentration and does not result in cell lysis, the cytoplasmic membrane is not hypothesized to be the sole target of MccE492.

Pyoverdine-Mediated Regulation of FpvA Synthesis in Pseudomonas aeruginosa : Involvement of a Probable Extracytoplasmic-Function Sigma Factor, FpvI

A search of the pvd pyoverdine biosynthesis locus of Pseudomonas aeruginosa identified an open reading frame, PA2387, whose product exhibited a sequence similar to those of a number of so-called extracytoplasmic- function sigma factors responsible for siderophore-dependent expression of iron-siderophore receptors in Escherichia coli and Pseudomonas putida. Deletion of this gene, dubbed fpvI, compromised pyoverdine-dependent FpvA ferric pyoverdine receptor production and fpvA gene expression, while the cloned gene stimulated fpvA expression. A Fur-binding site was identified immediately upstream of fpvI, consistent with the observed iron-regulated expression of fpvI and fpvA.

Iron acquisition and its control in Pseudomonas aeruginosa: many roads lead to Rome.

Iron plays an important role in the pathogenesis and rhizosphere competence of the fluorescent group of pseudomonads and it is, thus, fitting that the characteristic fluorescence of these organisms is attributable to an iron-chelating molecule, pyoverdine. Pseudomonas aeruginosa is likely the best-studied member of this group, and while it synthesizes two siderophores, pyochelin and pyoverdine, it is also able to use a number of heterologous siderophores of fungal and bacterial origin and its genome is rich with homologues of iron-siderophore receptor genes, reflecting the enormous flexibility of the organism vis-a-vis iron carriers that it can use in nature. The ability to utilize a variety of heterologous siderophores is shared by other fluorescent pseudomonads and likely reflects both the importance of this vital nutrient for growth and survival and the need to compete with other microorganisms in the aquatic and terrestrial environments that they inhabit. Expression of the various receptors is, however, regulated, with receptor production responding positively to available siderophores only, and selection from multiple available siderophores based on their successful chelation of iron and subsequent transport. Thus, the superior siderophore in a given environment will upregulate the cognate receptor at the expense of other receptors. Such siderophore-dependent regulation of receptor gene expression is common in bacteria, particularly the fluorescent pseudomonads, and typically requires a signal transduction cascade that involves the receptor itself, whose binding to the siderophore initiates the cascade, as well as a regulatory protein pair that includes an environmentally-responsive so-called extracytoplasmic function (ECF) sigma factor, which activates receptor gene expression, and an anti-sigma factor that controls sigma factor activity. Despite the plethora of ferric siderophore receptors in P. aeruginosa, its genome sequence reveals a striking lack of obvious periplasmic and cytoplasmic membrane transport components capable of accommodating these molecules. Unlike e.g. Escherichia coli, then, where ferric siderophore permeases providing transport to the cytoplasm are clearly in evidence, iron-siderophore complexes in P. aeruginosa may be dissociated in the periplasm, with a common iron carrier then responsible for iron uptake into the cell interior.

Localization of the virulence-associated genes pilA, pilR, rpoN, fliA, fliC, ent, and fbp on the physical map of Pseudomonas aeruginosa PAO1 by pulsed-field electrophoresis.

Seven virulence-associated genes have been placed on a genomic map of Pseudomonas aeruginosa PAO1, using pulsed-field electrophoresis, on the basis of the previous physical maps of Romling et al. (U. Romling, M. Duchene, D. Essar, D. Galloway, C. Guidi-Rontani, D. Hill, A. Lazdunski, R. Miller, K. Schleifer, D. Smith, H. Toschka, and B. Tummler, J. Bacteriol. 174:327-330, 1992; U. Romling, D. Grothues, W. Bautsch, and B. Tummler, EMBO J. 8:4081-4089, 1989) and Ratnaningsih et al. (E. Ratnaningsih, S. Dharmsthiti, V. Krishnapillai, A. Morgan, M. Sinclair, and B. W. Holloway, J. Gen. Microbiol. 136:2351-2357, 1990). The new locations for the outer membrane enterobactin iron-siderophore receptor ent gene (41 to 42 min) and the fliA gene (59 to 61 min), which encodes a minor sigma factor of RNA polymerase, are given. The pilA (the pilin structural gene), pilR (a pilin regulatory gene), and rpoN (encoding another minor sigma factor of RNA polymerase) genes map together at 71 to 75 min, locations correcting the previously reported values (V. Shortridge, M. Pato, A. Vasil, and M. Vasil, Infect. Immun. 59:3596-3603, 1990). The fbp gene (28 to 29 min), which encodes an outer membrane ferripyochelin-binding protein of low molecular weight, and the fliC gene (64 to 66 min), the flagellin structural gene, were determined to lie in the previously reported locations.

Iron uptake and detection of an outer membrane iron-siderophore receptor protein from Pseudomanas aeruginosa PA01

Under iron-restricted conditions, Pseudomonas aeruginosa PA01 produced large amounts of the fluorescent pigment, pyoverdine, which is a siderophore of this organism. Maximum iron uptake was found in P. aeruginosa PA01 only in the presence of its own pyoverdine and not in those from other Pseudomonas species, suggesting species specificity of pyoverdine for ion uptake. An iron-binding protein (60 000 mol.wt.) present in outer membranes of iron-restricted cultures was absent from iron-supplemented cultures.

Iron uptake and detection of an outer membrane iron-siderophore receptor protein from Pseudomanas aeruginosa PA01

Under iron-restricted conditions, Pseudomonas aeruginosa PA01 produced large amounts of the fluorescent pigment, pyoverdine, which is a siderophore of this organism. Maximum iron uptake was found in P. aeruginosa PA01 only in the presence of its own pyoverdine and not in those from other Pseudomonas species, suggesting species specificity of pyoverdine for ion uptake. An iron-binding protein (60 000 mol.wt.) present in outer membranes of iron-restricted cultures was absent from iron-supplemented cultures.