Functional Diversity of TonB-Like Proteins in the Heterocyst-Forming Cyanobacterium Anabaena sp. PCC 7120

The genomes of many organisms encode more than one TonB protein, and their number does not necessarily correlate with that of TonB-dependent outer membrane transporters. Consequently, specific as well as redundant functions of the different TonB proteins have been identified.

The Esterase PfeE, the Achilles’ Heel in the Battle for Iron between Pseudomonas aeruginosa and Escherichia coli

Bacteria access iron, a key nutrient, by producing siderophores or using siderophores produced by other microorganisms. The pathogen Pseudomonas aeruginosa produces two siderophores but is also able to pirate enterobactin (ENT), the siderophore produced by Escherichia coli. ENT-Fe complexes are imported across the outer membrane of P. aeruginosa by the two outer membrane transporters PfeA and PirA. Iron is released from ENT in the P. aeruginosa periplasm by hydrolysis of ENT by the esterase PfeE. We show here that pfeE gene deletion renders P. aeruginosa unable to grow in the presence of ENT because it is unable to access iron via this siderophore. Two-species co-cultures under iron-restricted conditions show that P. aeruginosa strongly represses the growth of E. coli as long it is able to produce its own siderophores. Both strains are present in similar proportions in the culture as long as the siderophore-deficient P. aeruginosa strain is able to use ENT produced by E. coli to access iron. If pfeE is deleted, E. coli has the upper hand in the culture and P. aeruginosa growth is repressed. Overall, these data show that PfeE is the Achilles’ heel of P. aeruginosa in communities with bacteria producing ENT.

Structural insight into toxin secretion by contact-dependent growth inhibition transporters

Bacterial contact-dependent growth inhibition (CDI) systems use a type Vb secretion mechanism to export large CdiA toxins across the outer membrane by dedicated outer membrane transporters called CdiB. Here, we report the first crystal structures of two CdiB transporters from Acinetobacter baumannii and Escherichia coli. CdiB transporters adopt a TpsB fold, containing a 16-stranded transmembrane β-barrel connected to two periplasmic domains. The lumen of the CdiB pore is occluded by an N-terminal α-helix and the conserved extracellular loop 6; these two elements adopt different conformations in the structures. We identified a conserved DxxG motif located on strand β1 that connects loop 6 through different networks of interactions. Structural modifications of DxxG induce rearrangement of extracellular loops and alter interactions with the N-terminal α-helix, preparing the system for α-helix ejection. Using structural biology, functional assays, and molecular dynamics simulations, we show how the barrel pore is primed for CdiA toxin secretion.

Iron acquisition system of Sphingobium sp. strain SYK-6, a degrader of lignin-derived aromatic compounds

Iron, an essential element for all organisms, acts as a cofactor of enzymes in bacterial degradation of recalcitrant aromatic compounds. The bacterial family, Sphingomonadaceae comprises various degraders of recalcitrant aromatic compounds; however, little is known about their iron acquisition system. Here, we investigated the iron acquisition system in a model bacterium capable of degrading lignin-derived aromatics, Sphingobium sp. strain SYK-6. Analyses of SYK-6 mutants revealed that FiuA (SLG_34550), a TonB-dependent receptor (TBDR), was the major outer membrane iron transporter. Three other TBDRs encoded by SLG_04340, SLG_04380, and SLG_10860 also participated in iron uptake, and tonB2 (SLG_34540), one of the six tonB comprising the Ton complex which enables TBDR-mediated transport was critical for iron uptake. The ferrous iron transporter FeoB (SLG_36840) played an important role in iron uptake across the inner membrane. The promoter activities of most of the iron uptake genes were induced under iron-limited conditions, and their regulation is controlled by SLG_29410 encoding the ferric uptake regulator, Fur. Although feoB, among all the iron uptake genes identified is highly conserved in Sphingomonad strains, the outer membrane transporters seem to be diversified. Elucidation of the iron acquisition system promises better understanding of the bacterial degradation mechanisms of aromatic compounds.

Iron acquisition system of Sphingobium sp. strain SYK-6, a degrader of lignin-derived aromatic compounds

Iron, an essential element for all organisms, acts as a cofactor of enzymes in bacterial degradation of recalcitrant aromatic compounds. The bacterial family, Sphingomonadaceae comprises various degraders of recalcitrant aromatic compounds; however, little is known about their iron acquisition system. Here, we investigated the iron acquisition system in a model bacterium capable of degrading lignin-derived aromatics, Sphingobium sp. strain SYK-6. Analyses of SYK-6 mutants revealed that FiuA (SLG_34550), a TonB-dependent receptor (TBDR), was the major outer membrane iron transporter. Three other TBDRs encoded by SLG_04340, SLG_04380, and SLG_10860 also participated in iron uptake, and tonB2 (SLG_34550), one of the six tonB comprising the Ton complex which enables TBDR-mediated transport was critical for iron uptake. The ferrous iron transporter FeoB (SLG_36840) played an important role in iron uptake across the inner membrane. The promoter activities of most of the iron uptake genes were induced under iron-limited conditions, and their regulation is controlled by SLG_29410 encoding the ferric uptake regulator, Fur. Although feoB, among all the iron uptake genes identified is highly conserved in Sphingomonad strains, the outer membrane transporters seem to be diversified. Elucidation of the iron acquisition system promises better understanding of the bacterial degradation mechanisms of aromatic compounds.

Going Outside the TonB Box: Identification of Novel FepA-TonB Interactions In Vivo

ABSTRACT In Gram-negative bacteria, the cytoplasmic membrane protein TonB transmits energy derived from proton motive force to energize transport of important nutrients through TonB-dependent transporters in the outer membrane. Each transporter consists of a beta barrel domain and a lumen-occluding cork domain containing an essential sequence called the TonB box. To date, the only identified site of transporter-TonB interaction is between the TonB box and residues ∼158 to 162 of TonB. While the mechanism of ligand transport is a mystery, a current model based on site-directed spin labeling and molecular dynamics simulations is that, following ligand binding, the otherwise-sequestered TonB box extends into the periplasm for recognition by TonB, which mediates transport by pulling or twisting the cork. In this study, we tested that hypothesis with the outer membrane transporter FepA using in vivo photo-cross-linking to explore interactions of its TonB box and determine whether additional FepA-TonB interaction sites exist. We found numerous specific sites of FepA interaction with TonB on the periplasmic face of the FepA cork in addition to the TonB box. Two residues, T32 and A33, might constitute a ligand-sensitive conformational switch. The facts that some interactions were enhanced in the absence of ligand and that other interactions did not require the TonB box argued against the current model and suggested that the transport process is more complex than originally conceived, with subtleties that might provide a mechanism for discrimination among ligand-loaded transporters. These results constitute the first study on the dynamics of TonB-gated transporter interaction with TonB in vivo. IMPORTANCE The TonB system of Gram-negative bacteria has a noncanonical active transport mechanism involving signal transduction and proteins integral to both membranes. To achieve transport, the cytoplasmic membrane protein TonB physically contacts outer membrane transporters such as FepA. Only one contact between TonB and outer membrane transporters has been identified to date: the TonB box at the transporter amino terminus. The TonB box has low information content, raising the question of how TonB can discriminate among multiple different TonB-dependent transporters present in the bacterium if it is the only means of contact. Here we identified several additional sites through which FepA contacts TonB in vivo, including two neighboring residues that may explain how FepA signals to TonB that ligand has bound.

Transposon Mutagenesis Paired with Deep Sequencing of Caulobacter crescentus under Uranium Stress Reveals Genes Essential for Detoxification and Stress Tolerance

ABSTRACTThe ubiquitous aquatic bacteriumCaulobacter crescentusis highly resistant to uranium (U) and facilitates U biomineralization and thus holds promise as an agent of U bioremediation. To gain an understanding of howC. crescentustolerates U, we employed transposon (Tn) mutagenesis paired with deep sequencing (Tn-seq) in a global screen for genomic elements required for U resistance. Of the 3,879 annotated genes in theC. crescentusgenome, 37 were found to be specifically associated with fitness under U stress, 15 of which were subsequently tested through mutational analysis. Systematic deletion analysis revealed that mutants lacking outer membrane transporters (rsaFaandrsaFb), a stress-responsive transcription factor (cztR), or a ppGpp synthetase/hydrolase (spoT) exhibited a significantly lower survival rate under U stress. RsaFaand RsaFb, which are homologues of TolC inEscherichia coli, have previously been shown to mediate S-layer export. Transcriptional analysis revealed upregulation ofrsaFaandrsaFbby 4- and 10-fold, respectively, in the presence of U. We additionally show thatrsaFamutants accumulated higher levels of U than the wild type, with no significant increase in oxidative stress levels. Our results suggest a function for RsaFaand RsaFbin U efflux and/or maintenance of membrane integrity during U stress. In addition, we present data implicating CztR and SpoT in resistance to U stress. Together, our findings reveal novel gene targets that are key to understanding the molecular mechanisms of U resistance inC. crescentus.IMPORTANCECaulobacter crescentusis an aerobic bacterium that is highly resistant to uranium (U) and has great potential to be used in U bioremediation, but its mechanisms of U resistance are poorly understood. We conducted a Tn-seq screen to identify genes specifically required for U resistance inC. crescentus. The genes that we identified have previously remained elusive using other omics approaches and thus provide significant insight into the mechanisms of U resistance byC. crescentus. In particular, we show that outer membrane transporters RsaFaand RsaFb, previously known as part of the S-layer export machinery, may confer U resistance by U efflux and/or by maintaining membrane integrity during U stress.

Identification of New Candidate Pathogenicity Factors in the Xylem-Invading Pathogen Xanthomonas albilineans by Transposon Mutagenesis

Xanthomonas albilineans is a xylem-invading pathogen that produces the toxin albicidin that blocks chloroplast differentiation, resulting in disease symptoms of sugarcane leaf scald. In contrast to other xanthomonads, X. albilineans does not possess a hypersensitive response and pathogenicity type III secretion system and does not produce xanthan gum. Albicidin is the only previously known pathogenicity factor in X. albilineans, yet albicidin-deficient mutant strains are still able to efficiently colonize sugarcane. To identify additional host adaptation or pathogenicity factors, sugarcane ‘CP80-1743’ was inoculated with 1,216 independently derived Tn5 insertions in X. albilineans XaFL07-1 from Florida. Sixty-one Tn5 mutants were affected in development of leaf symptoms or in stalk colonization. The Tn5 insertion sites of these mutants were determined and the interrupted genes were identified using the recently available genomic DNA sequence of X. albilineans GPE PC73 from Guadeloupe. Several pathogenicity-related loci that were not previously reported in Xanthomonas spp. were identified, including loci encoding hypothetical proteins, a membrane fusion protein conferring resistance to novobiocin, transport proteins, TonB-dependent outer-membrane transporters, and an OmpA family outer-membrane protein.