ROS-Mediated Signalling in Bacteria: Zinc-Containing Cys-X-X-Cys Redox Centres and Iron-Based Oxidative Stress

Bacteria are permanently in contact with reactive oxygen species (ROS), both over the course of their life cycle as well that present in their environment. These species cause damage to proteins, lipids, and nucleotides, negatively impacting the organism. To detect these ROS molecules and to stimulate the expression of proteins involved in antioxidative stress response, bacteria use a number of different protein-based regulatory and sensory systems. ROS-based stress detection mechanisms induce posttranslational modifications, resulting in overall conformational and structural changes within sensory proteins. The subsequent structural rearrangements result in changes of protein activity, which lead to regulated and appropriate response on the transcriptional level. Many bacterial enzymes and regulatory proteins possess a conserved signature, the zinc-containing redox centre Cys-X-X-Cys in which a disulfide bridge is formed upon oxidative stress. Other metal-dependent oxidative modifications of amino acid side-chains (dityrosines, 2-oxo-histidines, or carbonylation) also modulate the activity of redox-sensitive proteins. Using molecular biology, biochemistry, biophysical, and structure biology tools, molecular mechanisms involved in sensing and response to oxidative stress have been elucidated in detail. In this review, we analyze some examples of bacterial redox-sensing proteins involved in antioxidative stress response and focus further on the currently known molecular mechanism of function

Deciphering the Transcriptional Response Mediated by the Redox-Sensing System HbpS-SenS-SenR from Streptomycetes

Busche T, Winkler A, Wedderhoff I, Rückert C, Kalinowski J, Lucana DO de O. Deciphering the Transcriptional Response Mediated by the Redox-Sensing System HbpS-SenS-SenR from Streptomycetes. PLOS ONE. 2016;11(8): e0159873.The secreted protein HbpS, the membrane-embedded sensor kinase SenS and the cytoplasmic response regulator SenR from streptomycetes have been shown to form a novel type of signaling pathway. Based on structural biology as well as different biochemical and biophysical approaches, redox stress-based post-translational modifications in the three proteins were shown to modulate the activity of this signaling pathway. In this study, we show that the homologous system, named here HbpSc-SenSc-SenRc, from the model species Streptomyces coelicolor A3(2) provides this bacterium with an efficient defense mechanism under conditions of oxidative stress. Comparative analyses of the transcriptomes of the Streptomyces coelicolor A3(2) wild-type and the generated hbpSc-senSc-senRc mutant under native and oxidative-stressing conditions allowed to identify differentially expressed genes, whose products may enhance the anti-oxidative defense of the bacterium. Amongst others, the results show an up-regulated transcription of genes for biosynthesis of cysteine and vitamin B-12, transport of methionine and vitamin B-12, and DNA synthesis and repair. Simultaneously, transcription of genes for degradation of an anti-oxidant compound is down-regulated in a HbpSc-SenSc-SenRc-dependent manner. It appears that HbpSc-SenSc-SenRc controls the non-enzymatic response of Streptomyces coelicolor A3(2) to counteract the hazardous effects of oxidative stress. Binding of the response regulator SenRc to regulatory regions of some of the studied genes indicates that the regulation is direct. The results additionally suggest that HbpSc-SenSc-SenRc may act in concert with other regulatory modules such as a transcriptional regulator, a two-component system and the Streptomyces B-12 riboswitch. The transcriptomics data, together with our previous in vitro results, enable a profound characterization of the HbpS-SenS-SenR system from streptomycetes. Since homologues to HbpS-SenS-SenR are widespread in different actinobacteria with ecological and medical relevance, the data presented here will serve as a basis to elucidate the biological role of these homologues

Elucidation of haem-binding sites in the actinobacterial protein HbpS

The extracellular haem-binding protein from Streptomyces reticuli (HbpS) has been shown to be involved in redox sensing and to bind haem. However, the residues involved in haem coordination are unknown. Structural alignments to distantly related haem-binding proteins from Mycobacterium tuberculosis were used to identify a candidate haem-coordinating residue, and site-directed mutagenesis with UV/Vis spectroscopy was used to assess haem binding in vivo and in vitro. We present strong evidence that HbpS belongs to the small set of proteins, which do not use histidine to coordinate the metal in the haem group. Further spectroscopic evidence strongly indicates that threonine 113 is actively involved in coordination of haem. Subsequent protein/haem titration experiments show a 1 : 2, protein/haem stoichiometry. We also present data showing the degradation of haem by HbpS in vivo. Because HbpS is conserved in many Actinobacteria, the presented results are applicable to related species

Data collection and refinement statistics of the HbpS-D₇₈XXD₈₁ crystal structure.

*<p>Values in parentheses are for the highest-resolution shell.</p

Iron-binding activities of HbpS proteins.

<p>The calculated relative iron-binding activities of the wild-type and indicated HbpS mutants regarding the three iron-binding motifs and neighbouring basic residues were measured after treatment with iron ions. The calculated value for the wild-type was set as 100%. The activity of the wild-type sample without previous incubation with iron is also shown (WT-).</p

D/EXXE motifs within the HbpS sequence and their position on the octameric structure.

<p>(A) Alignments of HbpS from <i>S. reticuli</i> (<i>S_ret</i>; GI:5834772; numbering according to PDB: 3FPV) and HbpS-like proteins from <i>S. kasugaensis</i> (<i>S_kas</i>; GI:157059904), <i>S. coelicolor</i> A3(2) (S_coe; GI: 8248773) <i>Nocardia cyriacigeorgica</i> GUH-2 (<i>N_cyr</i>; GI:379707916), <i>Leifsonia. xyli subsp. xyli str</i>. CTCB07 (<i>L_xyl</i>; GI: 50955378), <i>Thioalkalivibrio sulfidophilus</i> HL-EbGr7 (<i>T_sul</i>; GI: 220935915), <i>Paracoccus denitrificans</i> PD1222 (<i>P_den</i>; GI: 119383870), <i>Yersinia rohdei</i> ATCC 43380 (<i>Y_roh</i>; GI: 238750301) and <i>Vibrio sp</i>. MED222 (<i>V_sp</i>; GI: 86146209) are shown. Glutamates and aspartates from the studied D/EXXE motifs are marked with green background and their positions on HbpS are indicated. Conserved K83 and D143 are marked with yellow background. Neighbouring and conserved Lys and Arg are in red background. Conserved amino acid regions following E₇₈XXE₈₁ and preceding D₁₄₁XXE₁₄₄ are in grey background. (B) Positions of the exposed E₄₃XXE₄₆ (green box) and D₁₄₁XXE₁₄₄ (red box) as well as the internal E₇₈XXE₈₁ (blue box) on one 4-fold axis of the HbpS octamer are indicated (left). Enlargements of the marked boxes (right) are shown indicating the glutamates/aspartate from one subunit (E43, E46, E78, E81, D141 and E144 written in violet) and the adjacent subunit (Glu and Asp written in turquoise).</p

Structural rearrangements due to the E78D/E81D mutations.

<p>The side chains of the E/D78 and E/D81 residues in the wild-type and the mutated HbpS point towards the corresponding side chains from a neighboring monomer in the octameric assembly. The distance of the two E81 side chains in the wild-type protein (gray) is 6.7 Å, which would allow for iron coordination. Other nearby residues include Y77 and T63. In the mutated protein, this distance has increased to 8.6 Å. Furthermore, the side chain of D81 has turned to form a hydrogen bond with T63. The two monomers in the mutant HbpS are colored green and orange. The residues discussed are labeled, and the asterisk denotes the residues from the symmetry-related molecule in the crystal. The distances between the corresponding E/D81 residues as well as the hydrogen bond between D81 and T63 are marked with dotted lines.</p

Complete genome sequence of Streptomyces reticuli, an efficient degrader of crystalline cellulose

Wibberg D, Al-Dilaimi A, Busche T, et al. Complete genome sequence of Streptomyces reticuli, an efficient degrader of crystalline cellulose. JOURNAL OF BIOTECHNOLOGY. 2016;222:13-14.We report the complete, GC-rich genome sequence of the melanin producer Streptomyces reticuli Tu 45 (S. reticuli) that targets and degrades highly crystalline cellulose by the concerted action of a range of biochemically characterized proteins. It consists of a linear 8.3 Mb chromosome, a linear 0.8 Mb megaplasmid, a linear 94 kb plasmid and a circular 76 kb plasmid. Noteworthy, the megaplasmid is the second largest known Streptomyces plasmid. Preliminary analysis reveals, among others, 43 predicted gene clusters for the synthesis of secondary metabolites and 456 predicted genes for binding and degradation of cellulose, other polysaccharides and carbohydrate-containing compounds. (c) 2016 Elsevier B.V. All rights reserved

Electrostatic surfaces of the octameric HbpS.

<p>(A) and (B) show some of the exposed E43, E46 and D141 (red) residues within the predominantly positive charged (blue) HbpS surface. K108 as well as E43, E46 and D141 are indicated by arrows. (C) An enlargement of the octameric structure shows the roughly spherical and predominantly negative charged (red) core in which the indicated E78 and E81 from different subunits are shown.</p

Iron-based quenching of Trp fluorescence of HbpS proteins.

<p>(A–C) HbpS proteins were treated with increasing concentrations (up to 10 mM; marked by the arrow) of ferrous iron ions, as indicated in the Material and Methods section. The fluorescence spectra of the wild-type (WT) as well as of the mutants D141A and E78A/E81A were subsequently measured. (D) Differences in fluorescence (ΔF) were plotted against the concentrations of titrated ferrous iron for HbpS WT (▪), D141A (▴) and HbpS E78A/E81A (•).</p