Ben F. Luisi

Staphylococcus aureus requires iron for proliferation during infection and acquires it mainly from host hemoglobin (Hb) through the iron-regulated surface determinant (Isd) system. The hemophores IsdB and IsdH mediate the initial steps of Hb recognition. Notably, IsdH also recognizes the hemoglobin:haptoglobin (HbHp) complex, although the molecular determinants and physiological relevance of this interaction remain unclear. Here, combining cryo-electron microscopy and biochemical and cellular assays, we define the basis of HbHp recognition by IsdH. The structure reveals how IsdH engages HbHp and captures the intrinsic conformational flexibility of the HbHp assembly. Functional analyses demonstrate that, despite retaining the ability to extract heme from HbHp, IsdH does not support S. aureus growth under iron-restricted conditions when HbHp is the sole iron source. These findings suggest that HbHp recognition by IsdH may serve functions beyond nutrient iron acquisition, contributing to modulation of host-pathogen interactions.

Summary YicC is a conserved protein, recently discovered to have hydrolytic endoribonuclease activity, with a phylogenetic distribution implicating an ancestry deeper than that for most extant ribonucleases central to RNA metabolism. We present evidence that Escherichia coli YicC can cleave the small regulatory RNAs (sRNAs), RyhB and RprA, even when they are sequestered in otherwise protective complexes with the RNA chaperone Hfq. Nonetheless, YicC activity is markedly diminished when RyhB and other sRNAs are paired with cognate mRNA. Our cryoEM structures of catalytically inactive YicC in complex with RyhB and in the apo state reveal quaternary and tertiary structural switches, triggered by substrate engagement, that engulf and ratchet a stem loop element of the sRNA into an internal channel, where metal-assisted hydrolytic action occurs. Based on these findings, we propose that the enzyme favours specific stem-loop structures and may discriminate between pools of active, target-engaged sRNAs and those that are inactive. The mechanism for substrate-triggered conformational switching could represent an ancient strategy for selective RNA degradation.

Staphylococcus aureus is a human pathogen whose virulence depends on iron acquisition. The bacterium expresses the hemophores IsdB and IsdH that enable heme capture from host hemoglobin (Hb). Unlike IsdB, IsdH can bind both free Hb and Hb:haptoglobin (Hb:Hp) complexes. Here, we present a comprehensive structural analysis of full-length IsdH in complex with free Hb, overcoming the limitations of previous studies based on truncated IsdH constructs. Cryo-EM revealed a previously unobserved oligomeric state and a unique binding pose of the N-terminal Hb-binding domain, likely representing the initial step of Hb engagement. Time-resolved and single-molecule force spectroscopy experiments delineated the sequential steps and mechanical aspects of Hb binding and heme extraction. Together, these findings provide an integrated structural and functional view of the IsdH–Hb interaction in the absence of Hp, as may occur during hemolysis, and offer insights into S. aureus heme scavenging and potential avenues for therapeutic inhibition.

The outer membrane protein TolC from Escherichia coli belongs to an extensive superfamily whose members are found throughout the didermal, Gram-negative bacterial lineages. The protein serves as an activated exit duct in multi-drug efflux pumps and protein secretion machinery. Many TolC homologues bear a lipid modification on the N-terminus that embeds into the inner leaflet of the outer membrane and appears to have been a conserved feature; however, the moiety is absent entirely in the E. coli TolC. We have discovered that the E. coli lipoprotein YbjP interacts extensively with the periplasmic surface of TolC and its N-terminal lipid moiety is embedded in the membrane, mimicking the intramolecular and modification-membrane interactions seen in TolC homologues. Here, we present cryo-EM structures of the MacA-MacB-TolC and AcrA-AcrB-TolC tripartite pumps complexed to YbjP. Although the association occurs spontaneously both in vitro and in vivo, the YbjP-TolC interaction is not required for efflux activity under standard laboratory conditions. YbjP may contribute to stabilising the orientation and distribution of TolC in the outer membrane, as well as the expression of transporters for tryptophan and cyclic peptide toxins.

The outer-membrane protein TolC from Escherichia coli belongs to an extensive superfamily whose members are found throughout the didermal, Gram-negative bacterial lineages. The protein serves as an activated exit duct in multi-drug efflux pumps and protein secretion machinery. Many TolC homologs bear a lipid modification on the N-terminus that embeds into the inner leaflet of the outer membrane and appears to have been a conserved feature; however, the moiety is absent entirely in the E. coli TolC. We have discovered that the E. coli lipoprotein YbjP interacts extensively with the periplasmic surface of TolC and its N-terminal lipid moiety is embedded in the membrane, mimicking the intramolecular and modification-membrane interactions seen in TolC homologs. Here, we present cryo-EM structures of the MacA-MacB-TolC and AcrA-AcrB-TolC tripartite pumps complexed to YbjP. Although the association occurs spontaneously both in vitro and in vivo, the YbjP-TolC interaction is not required for efflux activity under standard laboratory conditions. YbjP may contribute stabilising the orientation and distribution of TolC in the outer membrane as well as the expression of transporters for tryptophan and cyclic peptide toxins.

Transport of proteins and small molecules across cellular membrane is crucial for bacterial interaction with the environment and survival against antibiotics. In Gram-negative bacteria that possess two layers of membranes, specialized macromolecular machines are required to transport substrates across the cell envelope, often via an indirect stepwise process. The major facilitator superfamily (MFS)-type tripartite efflux pumps use proton electrochemical gradient to extrude drugs in diverse bacterial species, but the architecture of the assembly and structural mechanisms remain elusive. A representative MFS-type tripartite efflux pump, EmrAB-TolC, mediates resistance to multiple antimicrobial drugs through proton-coupled EmrB, a member of the DHA2 transporter family. Here, we report the high-resolution (3.13 Å) structure of the EmrAB-TolC pump, revealing a distinct, asymmetric architecture emerging from the assembly of TolC:EmrA:EmrB with a ratio of 3:6:1 and contacts that are essential for the pump assembly. Key residues involved in drug transport are identified and corroborated by mutagenesis and antibiotic sensitivity assays. The structural and functional data support a model for one-step drug transport by the MFS pump across the entire envelope of Gram-negative bacteria.

In many bacterial species, transcription and translation can be coupled physically, with potential impact on the rates and efficiency of gene expression. Here, we present structural evidence from cryo-EM demonstrating that a bacterial RNA polymerase that is paused proximally to the promoter can associate with the pioneering 30S translation initiation complex (30S IC). These findings suggest that the physical link between transcription and translation can be established prior to commitment to protein synthesis. Although the mRNA is embedded in this ‘early expressome’ complex, it can nonetheless interact with small regulatory RNA (sRNA) and be targeted for cleavage in the protein-coding region by the RNA degradosome assembly in vitro. The potential tagging of transcripts with sRNA during pioneering and subsequent stages of translation initiation, when the 30S IC is at the 5′ end of a polyribosome, may in principle contribute to efficient and rapid termination of gene expression in response to regulatory signals.

In the model gram-negative bacterium Escherichia coli, three of the four Type VII ATP-binding cassette (ABC) transporter systems have been well characterized structurally and functionally. A new study reports the cryo-EM structures of the fourth Type VII ABC system, YbbAP-TesA, and provides evidence for its role in extracting hydrophobic compounds from the bacterial inner membrane and their subsequent hydrolytic transformation.

In Escherichia coli and numerous other bacteria, two of the principal enzymes mediating messenger RNA decay and RNA processing—RNase E, an endoribonuclease, and polynucleotide phosphorylase (PNPase), an exoribonuclease—assemble into a multi-enzyme complex known as the RNA degradosome. While RNase E forms a homotetramer and PNPase a homotrimer, it remains unclear how these two enzymes interact within the RNA degradosome to potentially satisfy all mutual recognition sites. In this study, we used cryo-EM, biochemistry, and biophysical studies to discover and characterize a new binding mode for PNPase encompassing two or more motifs that are necessary and sufficient for strong interaction with RNase E. While a similar interaction is seen in Salmonella enterica, a different recognition mode arose for Pseudomonas aeruginosa, illustrating the evolutionary drive to maintain physical association of the two ribonucleases. The data presented here suggest a model for the quaternary organization of the RNA degradosome of E. coli, where one PNPase trimer interacts with one RNase E protomer. Conformational transitions are predicted to facilitate substrate capture and transfer to catalytic centres. The model suggests how the endo- and exo-ribonucleases might cooperate in cellular RNA turnover and recruitment of regulatory RNA by the degradosome assembly.

Bedaquiline, an antitubercular drug that targets ATP-synthase, is a key component of a new oral drug regimen that has revolutionized the treatment of multidrug-resistant tuberculosis. Clinical bedaquiline resistance in Mycobacterium tuberculosis has rapidly emerged, primarily due to mutations in the transcriptional repressor Rv0678 that result in upregulation of the resistance-nodulation-division (RND) efflux pump MmpS5/MmpL5 (MmpS5L5). Here, to understand how MmpS5L5 effluxes bedaquiline, we determined the structure of the MmpS5L5 complex using cryo-electron microscopy, revealing a trimeric architecture distinct from the canonical tripartite RND efflux pumps of gram-negative bacteria. Structure prediction modeling in conjunction with functional genetic analysis indicates that it uses a periplasmic coiled-coil tube to transport molecules across the cell wall. Structure-guided genetic approaches identify MmpL5 mutations that alter bedaquiline transport; these mutations converge on a region in MmpL5 located in the lower portion of the periplasmic cavity, proximal to the outer leaflet of the inner membrane, suggesting a route for bedaquiline entry into the pump. While currently known clinical resistance to bedaquiline is due to pump upregulation, our findings that several MmpL5 variants increase bedaquiline efflux may presage the emergence of additional modes of clinical resistance.