John J. Kendall’s research while affiliated with The University of Sheffield and other places

Table S1. Microarray data and RT‐PCR comparing the 150% and 40% aerobiosis steady states and at time points during the transition from 150% to 40% aerobiosis. In the ‘All significant (p < 0.05) genes’ tab, the fold‐changes of genes which showed a greater than twofold change in expression with a significance of p < 0.05 at any one of the time points or in a steady‐state are listed in a simple coloured heat map format (shades of red indicate > 2.0‐fold up‐regulation and shades of blue indicate > 2.0‐fold down‐regulation). Fold‐changes less than twofold are shown as greyed out values. A few values were not significant at p < 0.05 and are highlighted in red. The ‘summary’ tab just shows the significant twofold changes in either direction. The ‘RT‐PCR’ tab, shows the results of RT‐PCR on the same steady‐state samples as used for the microarray, for the mfrA, peb1a, cstA, putA and aspA genes, using the primers shown.

Fig. S1. Physiological data for the transition experiment from 150% to 40% aerobiosis. At time zero, the input gas composition was changed from 7.5% v/v oxygen (150% aerobiosis) to 1.88% oxygen (40% aerobiosis). A. Change in optical density at 600 nm. B. Corresponding increase in the specific acetate excretion rate. C. Dry weight at the high and low aerobiosis conditions and at 300 min after the transition. D. Cell viability measured by plate counts. SS; steady‐state. The data represent the means of at least three determinations; error bars are standard deviation from the mean. Fig. S2. Summary of gene expression changes during the 150–40% aerobiosis transition. Genes were divided into up‐ or downregulated based on a twofold or more change in expression and their functions classified using the TIGR main roles categories. The full data set from which this figure is derived is given in Supporting Information Table S1. Fig. S3. Measurements of substrate dependent respiratory activities. Cells from the steady‐states indicated were harvested from the chemostat, washed and resuspended in 20 mM phosphate buffer pH 7.4 and incubated with the substrates indicated in A–E (10 mM final concentration for all except sulphite which was used at 0.5 mM final concentration) at 37°C in a Clark type oxygen electrode. For formate oxidation (A), cells were grown to steady‐state either in media without added selenate (standard medium used for all other experiments in this study) or with 10 μM sodium selenate as indicated. In (F), the activity of the cytochrome c oxidase CcoNOQP was assayed as oxygen consumption in the presence of 1 mM sodium ascorbate and 0.25 mM N,N,N′,N′‐ tetramethyl‐p‐phenylenediamine (TMPD). The histograms represent the means of three determinations; error bars are standard deviation from the mean. **** p < 0.0001; *, p < 0.05 by Students t‐test. ns, not significant. Fig. S4. Comparison of c‐type cytochrome abundance at 40% and 150% aerobiosis. Cell‐free extracts (∼200 μg protein) were denatured in Laemmli sample buffer without mercaptoethanol, run on SDS‐PAGE gels which were either stained with Coomassie Blue (A) or blotted onto nitrocellulose where c‐type cytochromes were detected by their haem‐associated peroxidase activity using enhanced chemiluminescence with CCD imaging (B). The designations of those c‐type cytochromes shown are based on their molecular weights and comparison with the pattern of cytochromes in the gels published previously (Liu and Kelly, 2015). Lanes 1–3 for each aerobiosis condition are independent replicate steady‐state samples. Fig. S5. Modelling of RacR activity. A. Inferred activity of RacR during the 150–40% aerobiosis shift. A connectivity matrix for RacR (see Experimental Procedures) was used as the input for TFINFER, which interrogated the microarray dataset for the transition. RacR activity is predicted to show a transient decrease up to 60 min after the transition. B. Histogram of the relative regulatory strengths of target genes in the RacR regulon. Fig. S6. Pattern of glycosylated proteins at 40% and 150% aerobiosis. Glycosylated proteins were detected after SDS‐PAGE and electroblotting onto nitrocellulose membranes by reaction with Soybean agglutinin lectin conjugated with horseradish peroxidase (SBA‐HRP), followed by enhanced chemiluminescence and CCD imaging. A. Coomassie blue stained gel with ∼200 μg protein loaded per lane. B. Corresponding blot after reaction with SBA‐HRP. Lanes 1–3 for each aerobiosis condition are independent replicate steady‐state samples.

Table S2. Label‐free LC‐MS/MS analysis of proteins at 40% and 150% aerobiosis. Samples from three independent steady‐states at 1.88% v/v oxygen or 40% aerobiosis (LF1‐3) and three at 7.5% v/v oxygen or 150% aerobiosis (LF‐4‐6) were analysed by LC‐MS/MS as described in Experimental Procedures. In the tables shown, A,B,C are gel lane replicates of each independent steady‐state sample. The emPAI values shown in these columns are the average for triplicated Orbitrap injections for each gel lane replicate. The ‘normalised emPAI HB sorted’ tab sorts the data according to significance based on the Hochberg‐Benjamini correction, with p values <0.02. The ‘normalised emPAI Cj sorted’ tab sorts the data based on Cj locus tag/gene number. The ‘normalised emPAI ratio sorted’ tab sorts the data on the basis of the ratio of the means of the LF1A‐LF3C data and the LF4A‐LF6C data to give a fold‐change of protein abundance between 40% and 150% aerobiosis. In each case, the identified proteins are grouped according to whether they are highest at 40% or 150% aerobiosis. Data for proteins detected by LC‐MS/MS but showing no statistically significant change and proteins not detected but predicted from the genome sequence, are also shown. The ‘Venn Diagram’ tab shows a simple Venn diagram to illustrate the numbers of differentially expressed proteins under each condition.

Table S3. 2D‐gel analysis of proteins from cells grown at all steady‐states from 25% to 345% aerobiosis. In the ‘All steady‐state raw data’ tab, the normalised spot volumes from image analysis for a given protein are shown for multiple gels run with a minimum of 1 steady‐state per oxygen input condition (50% and 75% aerobiosis) or with two or more independent steady‐states (25%, 40%, 88%, 100%, 150%, 200% and 345% aerobiosis). CV refers to coefficient of variation. The data are sorted by the ANOVA value. Note that some proteins appeared as multiple spots and so may have more than one entry. For each aerobiosis condition the spot volumes for a given protein on each gel were averaged and these values are shown in the ‘mean spot volumes’ tab. These mean values were used to construct the graphs of changes in selected protein abundance shown in the paper.

Campylobacter jejuni, the most frequent cause of food-borne bacterial gastroenteritis worldwide, is a microaerophile that has to survive high environmental oxygen tensions, adapt to oxygen limitation in the intestine and resist host oxidative attack. Here, oxygen-dependent changes in C. jejuni physiology were studied at constant growth rate using carbon (serine)-limited continuous chemostat cultures. We show that a perceived aerobiosis scale can be calibrated by the acetate excretion flux, which becomes zero when metabolism is fully aerobic (100% aerobiosis). Transcriptome changes in a downshift experiment from 150% to 40% aerobiosis revealed many novel oxygen-regulated genes and highlighted re-modelling of the electron transport chains. A label-free proteomic analysis showed that at 40% aerobiosis, many proteins involved in host colonisation (e.g. PorA, CadF, FlpA, CjkT) became more abundant. PorA abundance increased steeply below 100% aerobiosis. In contrast, several citric-acid cycle enzymes, the peptide transporter CstA, PEB1 aspartate/glutamate transporter, LutABC lactate dehydrogenase and PutA proline dehydrogenase became more abundant with increasing aerobiosis. We also observed a co-ordinated response of oxidative stress protection enzymes and Fe-S cluster biogenesis proteins above 100% aerobiosis. Our approaches reveal key virulence factors that respond to restricted oxygen availability and specific transporters and catabolic pathways activated with increasing aerobiosis. This article is protected by copyright. All rights reserved.

Aims: Carbon monoxide (CO)-releasing molecules (CORMs) are candidates for animal and antimicrobial therapeutics. We aimed to probe the antimicrobial potential of a novel manganese CORM. Results: [Mn(CO)4S2CNMe(CH2CO2H)], CORM-401, inhibits growth of Escherichia coli and several antibiotic-resistant clinical pathogens. CORM-401 releases CO that binds oxidases in vivo but is an ineffective respiratory inhibitor. Extensive CORM accumulation (assayed as intracellular manganese) accompanies antimicrobial activity. CORM-401 stimulates respiration, polarizes the cytoplasmic membrane in an uncoupler-like manner and elicits loss of intracellular potassium and zinc. Transcriptomics and mathematical modeling of transcription factor activities reveal a multifaceted response characterized by elevated expression of genes encoding potassium uptake, efflux pumps, and envelope stress responses. Regulators implicated in stress responses (CpxR), respiration (Arc, Fnr), methionine biosynthesis (MetJ) and iron homeostasis (Fur) are significantly disturbed. Although CORM-401 reduces bacterial growth in combination with cefotaxime and trimethoprim, fractional inhibition studies reveal no interaction. Innovation: We present the most detailed microbiological analysis yet of a CORM that is not a ruthenium carbonyl. We demonstrate CO-independent, striking effects on the bacterial membrane and global transcriptomic responses. Conclusions: CORM-401, contrary to our expectations of a CO delivery vehicle, does not inhibit respiration. It accumulates in the cytoplasm, acts like an uncoupler in disrupting cytoplasmic ion balance, and triggers multiple effects including osmotic stress and futile respiration. Rebound Track: This work was rejected during standard peer review and rescued by Rebound Peer Review (Antioxid Redox Signal 16:293-296, 2012) with the following serving as open reviewers: Miguel Aon, Giancarlo Biagini, James Imlay, Nigel Robinson.