Transcriptional profiling of colicin-induced cell death of Escherichia coli MG1655 identifies potential mechanisms by which bacteriocins promote bacterial diversity

We report the transcriptional response of Escherichia coli MG1655 to damage induced by colicins E3 and E9, bacteriocins that kill cells through inactivation of the ribosome and degradation of chromosomal DNA, respectively. Colicin E9 strongly induced the LexA-regulated SOS response, while colicin E3 elicited a broad response that included the induction of cold shock genes, symptomatic of translational arrest. Colicin E3 also increased the transcription of cryptic prophage genes and other laterally acquired mobile elements. The transcriptional responses to both these toxins suggest mechanisms that may promote genetic diversity in E. coli populations, pointing to a more general role for colicins in adaptive bacterial physiology than has hitherto been realized

Dry and wet interfaces: Influence of solvent particles on molecular recognition

We present a coarse-grained lattice model to study the influence of water on the recognition process of two rigid proteins. The basic model is formulated in terms of the hydrophobic effect. We then investigate several modifications of our basic model showing that the selectivity of the recognition process can be enhanced by considering the explicit influence of single solvent particles. When the number of cavities at the interface of a protein-protein complex is fixed as an intrinsic geometric constraint, there typically exists a characteristic fraction that should be filled with water molecules such that the selectivity exhibits a maximum. In addition the optimum fraction depends on the hydrophobicity of the interface so that one has to distinguish between dry and wet interfaces.Comment: 11 pages, 7 figure

Phase separation in the outer membrane of Escherichia coli

Antimicrobial resistance is particularly prevalent in gram-negative bacteria, as antibiotics that act inside the cells must overcome their outer membrane. So far, technical limitations have prevented us from determining how outer-membrane proteins and lipids are organized to form this functional barrier. Here, we use nanoscale imaging of live bacteria to reveal that the most abundant outer-membrane proteins form a network that spans the entire bacterial surface, leaving only small gaps of phase-separated lipopolysaccharide. This tendency to phase separate is further emphasized by the formation of new domains when phospholipids are mislocated at the surface, rendering cells more susceptible to some antibiotics. Overall, the phase-separated nature of the outer membrane defines a perspective on its integrity and barrier function

Ferredoxin containing bacteriocins suggest a novel mechanism of iron uptake in <i>Pectobacterium spp</i>

In order to kill competing strains of the same or closely related bacterial species, many bacteria produce potent narrow-spectrum protein antibiotics known as bacteriocins. Two sequenced strains of the phytopathogenic bacterium &lt;i&gt;Pectobacterium carotovorum&lt;/i&gt; carry genes encoding putative bacteriocins which have seemingly evolved through a recombination event to encode proteins containing an N-terminal domain with extensive similarity to a [2Fe-2S] plant ferredoxin and a C-terminal colicin M-like catalytic domain. In this work, we show that these genes encode active bacteriocins, pectocin M1 and M2, which target strains of &lt;i&gt;Pectobacterium carotovorum&lt;/i&gt; and &lt;i&gt;Pectobacterium atrosepticum&lt;/i&gt; with increased potency under iron limiting conditions. The activity of pectocin M1 and M2 can be inhibited by the addition of spinach ferredoxin, indicating that the ferredoxin domain of these proteins acts as a receptor binding domain. This effect is not observed with the mammalian ferredoxin protein adrenodoxin, indicating that &lt;i&gt;Pectobacterium spp.&lt;/i&gt; carries a specific receptor for plant ferredoxins and that these plant pathogens may acquire iron from the host through the uptake of ferredoxin. In further support of this hypothesis we show that the growth of strains of &lt;i&gt;Pectobacterium carotovorum&lt;/i&gt; and &lt;i&gt;atrosepticum&lt;/i&gt; that are not sensitive to the cytotoxic effects of pectocin M1 is enhanced in the presence of pectocin M1 and M2 under iron limiting conditions. A similar growth enhancement under iron limiting conditions is observed with spinach ferrodoxin, but not with adrenodoxin. Our data indicate that pectocin M1 and M2 have evolved to parasitise an existing iron uptake pathway by using a ferredoxin-containing receptor binding domain as a Trojan horse to gain entry into susceptible cells

Structural and biophysical analysis of nuclease protein antibiotics

© 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society. Protein antibiotics (bacteriocins) are a large and diverse family of multidomain toxins that kill specific Gram-negative bacteria during intraspecies competition for resources. Our understanding of the mechanism of import of such potent toxins has increased significantly in recent years, especially with the reporting of several structures of bacteriocin domains. Less well understood is the structural biochemistry of intact bacteriocins and how these compare across bacterial species. Here, we focus on endonuclease (DNase) bacteriocins that target the genomes of Escherichia coli and Pseudomonas aeruginosa, known as E-Type colicins and S-Type pyocins, respectively, bound to their specific immunity (Im) proteins. First, we report the 3.2 Å structure of the DNase colicin ColE9 in complex with its ultra-high affinity Im protein, Im9. In contrast with Im3, which when bound to the ribonuclease domain of the homologous colicin ColE3 makes contact with the translocation (T) domain of the toxin, we find that Im9 makes no such contact and only interactions with the ColE9 cytotoxic domain are observed. Second, we report small-Angle X-ray scattering data for two S-Type DNase pyocins, S2 and AP41, into which are fitted recently determined X-ray structures for isolated domains. We find that DNase pyocins and colicins are both highly elongated molecules, even though the order of their constituent domains differs. We discuss the implications of these architectural similarities and differences in the context of the translocation mechanism of protein antibiotics through the cell envelope of Gram-negative bacteria

Structures of the Ultra-High-Affinity Protein-Protein Complexes of Pyocins S2 and AP41 and Their Cognate Immunity Proteins from Pseudomonas aeruginosa

© 2015 The Authors. Published by Elsevier Ltd. How ultra-high-affinity protein-protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins is an ultra-high-affinity interaction that is essential for the neutralisation of endogenous DNase catalytic activity and for protection against exogenous DNase bacteriocins. The colicin DNase-Im interaction is a model system for the study of high-affinity protein-protein interactions. However, despite the fact that closely related colicin-like bacteriocins are widely produced by Gram-negative bacteria, this interaction has only been studied using colicins from Escherichia coli. In this work, we present the first crystal structures of two pyocin DNase-Im complexes from Pseudomonas aeruginosa, pyocin S2 DNase-ImS2 and pyocin AP41 DNase-ImAP41. These structures represent divergent DNase-Im subfamilies and are important in extending our understanding of protein-protein interactions for this important class of high-affinity protein complex. A key finding of this work is that mutations within the immunity protein binding energy hotspot, helix III, are tolerated by complementary substitutions at the DNase-Immunity protein binding interface. Im helix III is strictly conserved in colicins where an Asp forms polar interactions with the DNase backbone. ImAP41 contains an Asp-to-Gly substitution in helix III and our structures show the role of a co-evolved substitution where Pro in DNase loop 4 occupies the volume vacated and removes the unfulfilled hydrogen bond. We observe the co-evolved mutations in other DNase-Immunity pairs that appear to underpin the split of this family into two distinct groups

The CcmC-CcmE interaction during cytochrome c maturation by System I is driven by protein-protein and not protein-heme contacts.

Cytochromes c are ubiquitous proteins, essential for life in most organisms. Their distinctive characteristic is the covalent attachment of heme to their polypeptide chain. This post-translational modification is performed by a dedicated protein system, which in many Gram-negative bacteria and plant mitochondria is a nine-protein apparatus (CcmA-I) called System I. Despite decades of study, mechanistic understanding of the protein-protein interactions in this highly complex maturation machinery is still lacking. Here, we focused on the interaction of CcmC, the protein that sources the heme cofactor, with CcmE, the pivotal component of System I responsible for the transfer of the heme to the apocytochrome. Using in silico analyses, we identified a putative interaction site between these two proteins (residues Asp &lt;sup&gt;47&lt;/sup&gt; , Gln &lt;sup&gt;50&lt;/sup&gt; , and Arg &lt;sup&gt;55&lt;/sup&gt; on CcmC; Arg &lt;sup&gt;73&lt;/sup&gt; , Asp &lt;sup&gt;101&lt;/sup&gt; , and Glu &lt;sup&gt;105&lt;/sup&gt; on CcmE), and we validated our findings by in vivo experiments in Escherichia coli Moreover, employing NMR spectroscopy, we examined whether a heme-binding site on CcmE contributes to this interaction and found that CcmC and CcmE associate via protein-protein rather than protein-heme contacts. The combination of in vivo site-directed mutagenesis studies and high-resolution structural techniques enabled us to determine at the residue level the mechanism for the formation of one of the key protein complexes for cytochrome c maturation by System I