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Staphylococcus aureus is a common colonizer of the human gut and in doing so it must be able to resist the actions of the host’s innate defences. Bile salts are a class of molecules that possess potent antibacterial activity that control growth. Bacteria that colonize and survive in that niche must be able to resist the action of bile salts, but the mechanisms by which S. aureus does so are poorly understood. Here we show that FadB is a bile-induced oxidoreductase which mediates bile salt resistance and when heterologously expressed in Escherichia coli renders them resistant. Deletion of fadB attenuated survival of S. aureus in a model of the human distal colon.

Wajiha Imtiaz,1,2,* Zainab Syed,1,* Zara Rafaque,3 Simon Colin Andrews,2 Javid Iqbal Dasti1 1Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan; 2School of Biological Sciences, Whiteknights, University of Reading, Reading RG6 6AJ, UK; 3Department of Microbiology, Hazara University, Mansehra, Pakistan*These authors contributed equally to this workCorrespondence: Javid Iqbal DastiDepartment of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, PakistanTel +92-51-90644175Email Iqbal78@hushmail.comBackground: The emergence of carbapenem-resistant and hypervirulent hypermucoviscous Klebsiella pneumoniae strains poses a significant public health challenge. We determined the MDR profiles, antibiotic resistance factors, virulence gene complement, and hypermucoviscous features of 200 clinical K. pneumoniae isolates from two major tertiary care hospitals in Islamabad and Rawalpindi, Pakistan.Methods: Susceptibility profiling and phenotypic analysis were performed according to the CLSI guidelines. Genetic determinants of antibiotic resistance and virulence were detected by PCR. Biofilm formation analysis was performed by microtiter plate assay.Results: The isolates displayed a high degree of antibiotic resistance: 36% MDR-CRKP; 38% carbapenem resistance; 55% gentamicin resistance; 53% ciprofloxacin resistance; and 59% aztreonam resistance. In particular, the level of resistance against fosfomycin (22%) and colistin (15%) is consistent with previous reports of increased resistance levels. Combined resistance to carbapenem and colistin was 7%. Genetic factors associated with colistin resistance (mcr-1 and mcr-2 genes) were detected in 12 and 9% of the isolates, respectively. Significant differences in resistance to gentamicin and levofloxacin were observed between the 200 isolates. Many of the isolates harbored genes specifying extended-spectrum and/or carbapenem-resistant β-lactamases: blaCTX-M-15 (46%), blaNDM-1 (39%), and blaOXA-48 (24%). The prevalence of the hypermucoviscous phenotype was 22% and 13% of the MDR isolates carried the rmpA gene (regulator for mucoid phenotype). Key virulence factor genes detected include those encoding: porins (ompK35 and ompK36; at 56 and 55% prevalence, respectively); adhesins (fimH, mrkD, and ycfM; at 19, 18, and 22% prevalence, respectively); and the polysaccharide regulator, bss, at 16% prevalence.Conclusion: This report highlights carbapenem-resistant K. pneumoniae (CRKP) prevalence, emerging resistance to fosfomycin, and the presence of mcr-1 and mcr-2 in colistin-resistant isolates. Further, the detection of rmpA signifies the prevalence of the hypermucoviscous trait in CRKP clinical isolates from Pakistan.Keywords: Klebsiella pneumoniae, multidrug resistance, carbapenemases, colistin resistance, hypermucoviscous K. pneumoniae

EfeUOB/M has been characterised in Pseudomonas syringae pathovar. syringae as a novel type of ferrous-iron transporter, consisting of an inner-membrane protein (EfeUPsy) and three periplasmic proteins (EfeOPsy, EfeMPsy and EfeBPsy). The role of an iron permease and peroxidase function has been identified for the EfeU and EfeB proteins, respectively, but the role of EfeO/M remains unclear. EfeMPsy is an ‘M75-only’ EfeO-like protein with a C-terminal peptidase-M75 domain (EfeOII/EfeM family). Herein, we report the 1.6 Å resolution crystal structure of EfeMPsy, the first structural report for an EfeM component of P. syringae pv. syringae. The structure possesses the bi-lobate architecture found in other bacterial periplasmic substrate/solute binding proteins. Metal binding studies, using SRCD and ICP-OES, reveal a preference of EfeMPsy for copper, iron and zinc. This work provides detailed knowledge of the structural scaffold, the metal site geometry, and the divalent metal binding potential of EfeM. This work provides crucial underpinning for a more detailed understanding of the role of EfeM/EfeO proteins and the peptidase-M75 domains in EfeUOB/M iron uptake systems in bacteria.

IntroductionTuberculosis (TB) caused by Mycobacterium tuberculosis is a common infectious disease associated with significant morbidity and mortality, especially in low and middle-income countries. Successful treatment of the disease requires prolonged intake (6 – 8 months) of multiple antibiotics with potentially detrimental consequences on the composition and functional potential of the human microbiome. The protocol described in the current study aims to identify microbiome (oral and gut) signatures associated with TB pathogenesis, treatment response and, outcome in humans.Methods and analysisFour hundred and fifty, newly diagnosed TB patients from three district levels (Peshawar, Mardan, Swat) TB diagnosis and treatment centers will be recruited in this non-interventional, prospective cohort study and will be followed and monitored until treatment completion. Demographic and dietary intake data, anthropometric measurement and blood, stool and salivary rinse samples will be collected at baseline, day 15, month-2 and end of the treatment. Additionally, we will recruit age (± 3 years) and sex-matched healthy controls (n=30). Blood sampling will allow monitoring of the immune response during the treatment, while salivary rinse and fecal samples will allow monitoring of dynamic changes in oral and gut microbiome diversity. Within this prospective cohort study, a nested case-control study design will be conducted to assess perturbations in oral and gut microbiome diversity (microbial dysbiosis) and immune response and compare between the patients groups (treatment success vs failure).Ethics and disseminationThe study has received ethics approval from the Ethic Board of Khyber Medical University Peshawar, and administrative approval from Provincial TB Control Program of Khyber Pakhtunkhwa, Pakistan. The study results will be presented in national and international conferences and published in peer-reviewed journals.Trial registration numberNCT04985994; pre-resultsStrengths and limitations of the studyProspective study design.Large sample size, several sampling time points, and samples taken before, during and after anti-tuberculosis treatment will ensure capture of an accurate picture of any emerging microbial dysbiosis and immune modulation; this will provide new understanding of how these two factors are mutually influenced and how they are impacted by treatment and outcome in TB patients.In addition to microbial signatures, collection of socio-demographic, anthropometric, nutrition and co-morbidities data will permit further subgroup analysis and meaningful results that will be shared with the scientific community.A potential weakness is that not all patients may be able to provide samples for all of the specified time points.Another weakness is the raised likelihood that some participants will withdraw since the study is carried out in resource poor communities

Malnutrition continues to threaten the lives of millions across the world, with children being hardest hit. Although inadequate access to food and infectious disease are the primary causes of childhood malnutrition, the gut microbiota may also contribute. This review considers the evidence on the role of diet in modifying the gut microbiota, and how the microbiota impacts childhood malnutrition. It is widely understood that the gut microbiota of children is influenced by diet, which, in turn, can impact child nutritional status. Additionally, diarrhoea, a major contributor to malnutrition, is induced by pathogenic elements of the gut microbiota. Diarrhoea leads to malabsorption of essential nutrients and reduced energy availability resulting in weight loss, which can lead to malnutrition. Alterations in gut microbiota of severe acute malnourished (SAM) children include increased Proteobacteria and decreased Bacteroides levels. Additionally, the gut microbiota of SAM children exhibits lower relative diversity compared with healthy children. Thus, the data indicate a link between gut microbiota and malnutrition in children, suggesting that treatment of childhood malnutrition should include measures that support a healthy gut microbiota. This could be of particular relevance in sub-Saharan Africa and Asia where prevalence of malnutrition remains a major threat to the lives of millions.

Pakistan has hosted millions of Afghan refugees over the last several decades. Due to poor socioeconomic status, food insecurity and inadequate access to health care, these refugees are considered to be at high risk of malnutrition. Previous studies on nutritional assessment of high-risk populations (refugees) have focused mainly on women and children (0–59 months). The current study aims to assess nutritional status of adolescent Afghan refugees, the population who are equally vulnerable to malnutrition and its consequences. In this cross sectional study, the nutritional status of 206 adolescent (10–19 years old) Afghans boys and girls living in a refugee camp in Peshawar, Pakistan was assessed using standard methods. The results indicate a prevalence of stunting, thinness, and overweight and obesity at 35.3%, 4.4% and 14.8%, respectively. Furthermore, there was a significantly high prevalence of micronutrient deficiencies (vitamin D, 80.5%, vitamin B12, 41.9%, and folate, 28.2%), and anemia (10.1%). Together, these findings indicate that this vulnerable population group suffers from the double burden of malnutrition and are thus at serious risk of impaired psychosocial cognitive development, general ill-health and diminished wellbeing. This study therefore highlights the urgent need to include adolescents in regular screening and intervention programs of such at-risk populations.

Antibiotic resistance in urinary tract infections is a major global challenge and improved cost-effective and high throughput antibiotic susceptibility tests (AST) are urgently needed to inform correct antibiotic selection. We evaluated a high throughput microfluidic test strip for AST and minimum inhibitory concentration (MIC) determination in 20 urinary pathogenic E. coli (UPEC) isolates using six commonly prescribed or therapeutically beneficial antibiotics. The microfluidic MIC performs broth microdilution in 1 microliter volume capillaries, 100 X smaller than standard broth microdilution. Each test strip contains 10 parallel capillaries which are dipped into a single well of a 96 well plate, significantly increasing throughput over a microtitre plate. When tested with clinical UPEC isolates at standardised inoculum density, these devices gave 100% essential agreement (+/- 1 doubling dilution of antibiotic) to the gold standard microplate broth microdilution method described by CLSI. Although for some antibiotic/isolate combinations an earlier endpoint readout reduced accuracy, MIC test strips read at a 6h endpoint still gave 69 – 100 % essential agreement depending on the antibiotic. Growth could be detected significantly earlier than 6h, but with a trade-off between speed vs accuracy. These high-throughput, multiplexed test strips could be used to increase throughput and give faster results than microplates while retaining the core broth microdilution methodology of gold standard techniques for AST and MIC determination.

Eggs are a whole food which affordably support human nutritional requirements worldwide.\ud Eggs strongly resist bacterial infection due to an arsenal of defensive systems, many of which\ud reside in the egg white. However, despite improved control of egg production and distribution,\ud eggs remain a vehicle for foodborne transmission of Salmonella enterica serovar Enteritidis, which\ud continues to represent a major public health challenge. It is generally accepted that iron deficiency,\ud mediated by the iron-chelating properties of the egg-white protein ovotransferrin, has a key role\ud in inhibiting infection of eggs by Salmonella. Ovotransferrin has an additional antibacterial activity\ud beyond iron-chelation, which appears to depend on direct interaction with the bacterial cell surface,\ud resulting in membrane perturbation. Current understanding of the antibacterial role of ovotransferrin\ud is limited by a failure to fully consider its activity within the natural context of the egg white, where a\ud series relevant environmental factors (such as alkalinity, high viscosity, ionic composition, and egg\ud white protein interactions) may exert significant influence on ovotransferrin activity. This review\ud provides an overview of what is known and what remains to be determined regarding the antimicrobial\ud activity of ovotransferrin in egg white, and thus enhances understanding of egg safety through\ud improved insight of this key antimicrobial component of eggs.

International audience; A recent work revealed that egg white (EW) at 45°C exhibits powerful bactericidal activity against S. enterica serovar Enteritidis, which is surprisingly little affected by removal of the >10 kDa EW proteins. Here, we sought to identify the major EW factors responsible for this bactericidal activity by fractionating EW using ultrafiltration and nanofiltration and by characterizing the physicochemical and antimicrobial properties of the resulting fractions. In particular, 22 peptides were identified by nano-LC/MS-MS and the bactericidal activities of representative peptides (with predicted antimicrobial activity) were further assessed. Two peptides (FVPPVQR and GDPSAWSWGAEAHS) were found to be bactericidal against S. enterica serovar Enteritidis at 45°C when provided in an EW environment. Nevertheless, these peptides contribute only part of this bactericidal activity, suggesting other, yet to be determined, antimicrobial factors.

A recent work revealed that egg white (EW) at 45 °C exhibits powerful bactericidal activity against

IntroductionTuberculosis (TB) caused by Mycobacterium tuberculosis is a common infectious disease associated with significant morbidity and mortality, especially in low-income and middle-income countries. Successful treatment of the disease requires prolonged intake (6–8 months) of multiple antibiotics with potentially detrimental consequences on the composition and functional potential of the human microbiome. The protocol described in the current study aims to identify microbiome (oral and gut) signatures associated with TB pathogenesis, treatment response and outcome in humans.Methods and analysisFour hundred and fifty, newly diagnosed patients with TB from three district levels (Peshawar, Mardan and Swat) TB diagnosis and treatment centres, will be recruited in this non-interventional, prospective cohort study and will be followed and monitored until treatment completion. Demographic and dietary intake data, anthropometric measurement and blood, stool and salivary rinse samples will be collected at baseline, day 15, month-2 and end of the treatment. Additionally, we will recruit age (±3 years) and sex-matched healthy controls (n=30). Blood sampling will allow monitoring of the immune response during the treatment, while salivary rinse and faecal samples will allow monitoring of dynamic changes in oral and gut microbiome diversity. Within this prospective cohort study, a nested case–control study design will be conducted to assess perturbations in oral and gut microbiome diversity (microbial dysbiosis) and immune response and compare between the patients groups (treatment success vs failure).Ethics and disseminationThe study has received ethics approval from the Ethic Board of Khyber Medical University Peshawar, and administrative approval from Provincial TB Control Programme of Khyber Pakhtunkhwa, Pakistan. The study results will be presented in national and international conferences and published in peer-reviewed journals.Trial registration numberNCT04985994.

Aims: To isolate and characterize the bacteriocin activity identified by the genomic analysis of a novel enterococcal isolate, Enterococcus faecium W1 Methods and Results: Culture fermentates of a novel E. faecium isolate, W1 showed high levels of antimicrobial activity against Listeria monocytogenesisand a variety of other Gram+ve and Gram-ve bacteria. Ammonium sulphate precipitation of the fermentates followed by hydrophobic interaction chromatography showed bactericidal activity correlated with fractions containing only low molecular weight proteins. The same fractions had activity on human cancer cell line HT-29 measured by cytotoxicity assay (MTT) and apoptosis induction. Whole-genome sequencing of E. faecium W1 revealed five bacteriocin related gene clusters, a lack of virulence factors and lack of antibiotic resistance genes. To confirm that antimicrobial activity was correlated with the bacteriocin genes identified, selected bacteriocin genes were cloned and expressed as His-tagged proteins in E. coli. Bactericidal activity against L. monocytogenes was associated with protein purified by metal affinity chromatography. A structure-function analysis to investigate the role of key residues in activity is ongoing. Conclusion: E. faecium W1 is a promising candidate for use as a probiotic supplement or in food preservation. The bacteriocin related genes are functional and could be improved for future use as antimicrobial and cancer cell therapeutics. Significance and Impact of the Study: There is a need for new approaches to the growing problem of antibiotic resistance. Expression of bacteriocins identified in E. faecium W1 could contribute to this endeavour in addition to its use as a probiotic organism.

A growing body of research evidence suggests that elevated homocysteine level (hyperhomocysteinemia) is an independent risk factor for cardiovascular diseases. The current study aimed to investigate the prevalence and associated risk factors for hyperhomocysteinemia among adolescent Afghan refugees aged 10–19 years. In total, 206 healthy adolescent boys and girls were randomly recruited from a refugee village in Peshawar, Pakistan, in 2020. Socio-demographic data, anthropometric assessment, and blood sample collection were performed following standard methods. Serum homocysteine was assessed using a chemiluminescent microparticle immunoassay, with hyperhomocysteinemia defined as levels ≥ 15 µmol/L. The overall prevalence of hyperhomocysteinemia was 25%, with mean homocysteine levels significantly (p = 0.004) higher among boys (14.1 µmol/L) than girls (11.8 µmol/L). Multivariate logistic regression analysis revealed a significant association between hyperhomocysteineimia and serum levels of vitamin B12 (OR 0.29; 95% CI of 0.14 to 0.62; p < 0.01) and folate (OR 0.1; 95% CI of 0.03 to 0.27; p < 0.001). Overall, our study findings indicate high prevalence of hyperhomocysteinemia among adolescent Afghan refugees who are potentially at high risk of developing cardiovascular diseases in future. There is a dire need to develop and implement nutritional and public health strategies to control hyperhomocysteinemia, protect against related diseases and complications in future, and ensure healthy lives and well-being among these vulnerable populations.

Objectives Enterococci are Gram-positive lactic acid bacteria and common inhabitants of the gastrointestinal tract of mammals, including humans. They are also widely distributed in diverse environments such as soil, water, vegetables and food. Enterococcus faecium is able to produce antimicrobial compounds (enterocins) and thus can act as a probiotic. E. faecium SP15 is a newly identified enterocin-producing strain from spring water that has been subjected to genome sequence analysis to provide understanding of its antimicrobial and probiotic properties. Data description The draft genome of E. faecium SP15 comprises of 2,783,033 bp with a G+C content of 38.08%. Five genetic loci predicted to specify enterocin production were identified, but no virulence factors could be detected and only two potential antibiotic resistance genes were noted.

Enterococcus faecalis 24FS is a bacteriocin-producing, multiply antibiotic-resistant, and potentially virulent bacterium isolated from healthy infant feces. The draft 2.9-Mb genome sequence revealed 2,968 protein-encoding genes; 11 antibiotic resistance, 8 virulence, and 3 bacteriocin genes; and 2 plasmids, 4 prophages, 30 insertion sequence (IS) elements, 1 transposon, and 1 integron.

Aims To characterise the genome and enterocin content of Enterococcus faecium SP15, a new isolate from natural spring water. Methods and Results Enterococcus faecium strain SP15 was isolated from natural spring water and its identity confirmed using 16S rDNA sequence. The bacterial strain produced antimicrobial compounds (enterocins) against pathogenic bacteria including Listeria monocytogenesis as determined by the agar spot method. In addition, an anti-cancer activity was observed on human cancer cell line HT-29 by cytotoxicity assay (MTT) and apoptosis study. A draft genome sequence revealed the presence of several enterocin genes capable of activity against a panel of pathogenic bacteria including L. monocytogenesis. The active production of enterocins was also supported by the presence of peptides in part purified fermentates following trypsin digest and mass spectrometry. The genome showed no classical virulence factors or hemolysins and was free of antibiotic resistance genes. To confirm enterocin related killing, select enterocin genes were cloned and expressed as His-tagged proteins in E. coli. Purified enterocin retained activity in L. monocytogenes overlay assay. Conclusion E. faecium SP15 is a promising strain for probiotic use and/or food preservation. Purified enterocins retain their activity suggesting them a template for structure-function studies and future improvements as antimicrobial and cancer cell therapeutics. Significance and Impact of the Study: New antibacterial and anticancer agents are required to combat antibiotic resistance and a limited drug repertoire. Enterocins from Enterococcus faecium SP15 could fulfil these goals in addition to use of the strain as a whole organism probiotic.

Summary Background How specific nutrients influence adaptive immunity is of broad interest. Iron deficiency is the most common micronutrient deficiency worldwide and imparts a significant burden of global disease; however, its effects on immunity remain unclear. Methods We used a hepcidin mimetic and several genetic models to examine the effect of low iron availability on T cells in vitro and on immune responses to vaccines and viral infection in mice. We examined humoral immunity in human patients with raised hepcidin and low serum iron caused by mutant TMPRSS6. We tested the effect of iron supplementation on vaccination-induced humoral immunity in piglets, a natural model of iron deficiency. Findings We show that low serum iron (hypoferremia), caused by increased hepcidin, severely impairs effector and memory responses to immunizations. The intensified metabolism of activated lymphocytes requires the support of enhanced iron acquisition, which is facilitated by IRP1/2 and TFRC. Accordingly, providing extra iron improved the response to vaccination in hypoferremic mice and piglets, while conversely, hypoferremic humans with chronically increased hepcidin have reduced concentrations of antibodies specific for certain pathogens. Imposing hypoferremia blunted the T cell, B cell, and neutralizing antibody responses to influenza virus infection in mice, allowing the virus to persist and exacerbating lung inflammation and morbidity. Conclusions Hypoferremia, a well-conserved physiological innate response to infection, can counteract the development of adaptive immunity. This nutrient trade-off is relevant for understanding and improving immune responses to infections and vaccines in the globally common contexts of iron deficiency and inflammatory disorders. Funding Medical Research Council, UK, Graphical Abstract, Highlights Low serum iron caused by hepcidin impairs primary and memory immune responses Activated T-cells demand iron and iron scarcity inhibits mitochondrial metabolism Patients with mutant TMPRSS6 have high hepcidin and lower IgG against pathogens High hepcidin during viral infection inhibits T- and B-cells and inflames disease, Context and Significance Iron deficiency is very common in humans and animals. Frost et al demonstrate that low concentrations of iron in serum, caused by the hormone hepcidin, inhibit the body’s response to vaccines and infections; conversely, increasing iron can boost immunity., Iron deficiency is very common in humans and animals. Frost et al. demonstrate that low concentrations of iron in serum, caused by the hormone hepcidin, inhibit the body’s response to vaccines and infections; conversely, increasing iron can boost immunity.

Conference: 11th International Symposium on Biometals (Biometals) Location: Ottawa, CANADA Date: 2018; Salmonella enterica serovar Enteritidis (SE) is the most frequently-detected Salmonella in foodborne outbreaks in the European Union. Among such outbreaks, egg and egg products were identifiedas the most common vehicles of infection. Possibly, the major antibacterial property of egg white is ironrestriction, which results from the presence of the ironbinding protein, ovotransferrin. To circumvent ironrestriction, SE synthesise catecholate siderophores (i.e. enterobactin and salmochelin) that can chelateiron from host iron-binding proteins. Here, we highlight the role of lipocalin-like proteins found in eggwhite that could enhance egg-white iron restriction through sequestration of certain siderophores, includingenterobactin. Indeed, it is now apparent that the egg-white lipocalin, Ex-FABP, can inhibit bacterialgrowth via its siderophore-binding capacity in vitro. However, it remains unclear whether Ex-FABP performssuch a function in egg white or during bird infection. Regarding the two other lipocalins of eggwhite (Cal-c and a-1-glycoprotein), there is currently no evidence to indicate that they sequestersiderophores.

Skin conductivity (i.e., sweat) forms the basis of many physiology-based emotion and stress detection systems. However, such systems typically do not detect the biomarkers present in sweat, and thus do not take advantage of the biological information in the sweat. Likewise, such systems do not detect the volatile organic components (VOC’s) created under stressful conditions. This work presents a review into the current status of human emotional stress biomarkers and proposes the major potential biomarkers for future wearable sensors in affective systems. Emotional stress has been classified as a major contributor in several social problems, related to crime, health, the economy, and indeed quality of life. While blood cortisol tests, electroencephalography and physiological parameter methods are the gold standards for measuring stress; however, they are typically invasive or inconvenient and not suitable for wearable real-time stress monitoring. Alternatively, cortisol in biofluids and VOCs emitted from the skin appear to be practical and useful markers for sensors to detect emotional stress events. This work has identified antistress hormones and cortisol metabolites as the primary stress biomarkers that can be used in future sensors for wearable affective systems.

The RIC (repair of iron clusters) protein of Escherichia coli is a di-iron hemerythrin-like protein that has a proposed function in repairing stress-damaged iron-sulfur clusters. In this work, we performed a bacterial two-hybrid screening to search for RIC-protein interaction partners in E. coli. As a result, the DNA-binding protein from starved cells (Dps) was identified, and its potential interaction with RIC was tested by bacterial adenylate cyclase-based two-hybrid (BACTH) system, bimolecular fluorescence complementation, and pulldown assays. Using the activity of two Fe-S-containing enzymes as indicators of cellular Fe-S cluster damage, we observed that strains with single deletions of ric or dps have significantly lower aconitase and fumarase activities. In contrast, the ric dps double mutant strain displayed no loss of aconitase and fumarase activity with respect to that of the wild type. Additionally, while complementation of the ric dps double mutant with ric led to a severe loss of aconitase activity, this effect was no longer observed when a gene encoding a di-iron site variant of the RIC protein was employed. The dps mutant exhibited a large increase in reactive oxygen species (ROS) levels, but this increase was eliminated when ric was also inactivated. Absence of other iron storage proteins, or of peroxidase and catalases, had no impact on RIC-mediated redox stress induction. Hence, we show that RIC interacts with Dps in a manner that serves to protect E. coli from RIC protein-induced ROS. IMPORTANCE The mammalian immune system produces reactive oxygen and nitrogen species that kill bacterial pathogens by damaging key cellular components, such as lipids, DNA, and proteins. However, bacteria possess detoxifying and repair systems that mitigate these deleterious effects. The Escherichia coli RIC (repair of iron clusters) protein is a di-iron hemerythrin-like protein that repairs stress-damaged iron-sulfur clusters. E. coli Dps is an iron storage protein of the ferritin superfamily with DNA-binding capacity that protects cells from oxidative stress. This work shows that the E. coli RIC and Dps proteins interact in a fashion that counters RIC protein-induced reactive oxygen species (ROS). Altogether, we provide evidence for the formation of a new bacterial protein complex and reveal a novel contribution for Dps in bacterial redox stress protection.

Objectives Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). The pandemic sequence type 131 (ST131) clonal group is associated with multidrug resistance and virulence. Here we report the first draft genome of a ST131-O25b- H 30 strain from Pakistan, isolated from a patient with community-acquired UTI. Methods Next-generation sequencing was performed using MiSeq and HiSeq 2500 platforms. De novo assembly of the reads was performed using SPAdes v.3.11. Genomic features were determined with PATRIC and RAST tool kits. Results The 5 327 975-bp draft genome sequence has 5433 coding sequences and 82 tRNAs, an array of antimicrobial resistance genes [ bla CTX-M-15 , bla OXA-1 , bla CMY-2 , sul2 , catB , dfrA17 , mph ( A )], a class 1 integron, 77 insertion sequence (IS) elements, a Tn 3 -like transposon, multiple virulence markers and 7 intact prophage loci. Conclusion In conclusion, the genome sequence of this new UPEC isolate from Pakistan provides a novel insight into the genetic attributes of an epidemic clone associated with a high level of resistance and virulence.

Lactobacillus plantarum 10CH is a bacteriocin-producing potential probiotic lactic acid bacterium (LAB) strain isolated from cheese. Its complete nucleotide sequence shows a single circular chromosome of 3.3 Mb, with a G+C content of 44.51%, a 25-gene plantaricin bacteriocin gene cluster, and the absence of recognized virulence factors.

Lactobacillus plantarum 10CH is a bacteriocin-producing potential probiotic lactic acid bacterium (LAB) strain isolated from cheese. Its complete nucleotide sequence shows a single circular chromosome of 3.3 Mb, with a G+C content of 44.51%, a 25-gene plantaricin bacteriocin gene cluster, and the absence of recognized virulence factors.

Sponsored by the Friends of Israel Educational Foundation (FIEF)\ud and Ben-Gurion University of the Negev and supported by the EU COST\ud action Zinc-Net (COST TD1304), a three-day collaborative UK-Israel\ud workshop was organized by Drs Assaf Rudich, Imre Lengyel and Arie\ud Moran. Participants from the UK and Israel met at the Desert Iris Hotel,\ud Yeruham, Israel between the 28-30th of November 2016 for in-depth\ud discussions, rather than a lecture series, to set the stage for future\ud collaborative grants and projects on diabetes and zinc. Two days of\ud formal scientific sessions with dynamic and wide-ranging discussions\ud was followed by a day of touring and informal networking in the Negev\ud area. This format was previously recognized by our sponsors as both\ud effective and enjoyable and all participants agreed at the end of the\ud meeting that the 3-days provided an excellent basis for future scientific\ud collaboration. The discussions were centered on diabetes and obesity,\ud already at pandemic levels, and zinc homeostasis which is related to the\ud clinical issues and themes of the meeting. The free-flowing discussions\ud were based on short presentations setting the scene for the six main\ud topics: ‘Diabetes and zinc transporters’, ‘Nutrition related factors’,\ud ‘Biomarkers’, ‘Clinical epidemiology’, ‘the Microbiome and diabetes’,\ud and ‘Related diseases’. The abstract style summary of the sessions is\ud followed by the major discussion points raised by the Authors and other\ud participants (UK: Patrik Rorsman, Oxford University; Alan Stewart,\ud University of St Andrews and Israel: Assaf Rudich, Idit Liberty, Rahel\ud Gol, Guy Las and Amos Katz, Ben-Gurion University; Sarah Zangen,\ud Haddassa University). We hope that readers will find this discourse\ud stimulating and some of the ideas might make their way into their research\ud efforts.

Sequence type 38 is considered a uropathogenic Escherichia coli/enteroaggregative E. coli hybrid associated with multidrug resistance and urinary tract infections. The draft genome sequence of UEC59 from a woman in Pakistan revealed a 5 324 938 bp genome with 5386 coding sequences (CDS), 86 transfer RNA genes and multiple antibiotic resistance genes (blaTEM-1, CMY-2, sul1, sul2, dfrA17, tetA, mphA) and mobile elements (int1, two transposons, 30 insertion sequence elements, one integrative conjugative element, four plasmids, five prophages), along with many virulence genes. Keywords: Draft genome, MDR, Pakistan, ST38, UPEC, virulence

At least three ferritins are found in the bacterium Escherichia coli: the heme-containing bacterioferritin (EcBFR) and two nonheme bacterial ferritins (EcFtnA and EcFtnB). In addition to the conserved A and B sites of the diiron ferroxidase center, EcFtnA has a third iron-binding site (the C site) of unknown function that is nearby the diiron site. In the present work, the complex chemistry of iron oxidation and deposition in EcFtnA was further defined through a combination of oximetry, pH stat, stopped-flow and conventional kinetics, UV–vis, fluorescence, and EPR spectroscopic measurements on both the wild-type protein and site-directed variants of the A, B, and C sites. The data reveal that although H2O2 is a product of dioxygen reduction in EcFtnA and oxidation occurs with a stoichiometry of Fe2+/O2 ∼ 3:1 most of the H2O2 produced is consumed in subsequent reactions with a 2:1 Fe2+/H2O2 stoichiometry, thus suppressing hydroxyl-radical formation. Although the A and B sites are essential for rapid iron o...

Chicken egg white protects the embryo from bacterial invaders by presenting an assortment of antagonistic activities that combine together to both kill and inhibit growth. The key features of the egg-white anti-bacterial system are iron restriction, high pH, antibacterial peptides and proteins, and viscosity. Salmonella enterica serovar Enteritidis is the major pathogen responsible for egg-borne infection in humans, which is partly explained by its exceptional capacity for survival under the harsh conditions encountered within egg white. However, at temperatures up to 42 ˚C, egg white exerts a much stronger bactericidal effect on S. Enteritidis than at lower tempertaures, although the mechanism of egg-white-induced killing is only partly understood. Here, for the first time, the impact of exposure of S. Enteritidis to egg white under bactericidal conditions (45 ˚C) is explored by global-expression analysis. A large-scale (18.7% of genome) shift in transcription is revealed suggesting major changes in specific aspects of S. Enteritidis physiology: induction of egg-white related stress-responses (envelope damage, exposure to heat and alkalinity, and translation shutdown); shift in energy metabolism from respiration to fermentation; and enhanced micronutrient provision (due to iron and biotin restriction). Little evidence of DNA damage or redox stress was obtained. Instead, data are consistent with envelope damage resulting in cell death by lysis. A surprise was the high degree of induction of hexonate/hexuronate utilisation genes, despite no evidence indicating the presence of these substrates in egg white.

Verocytotoxigenic Escherichia coli (VTEC) can contaminate crop plants, potentially using them as secondary hosts, which can lead to food-borne infection. Currently, little is known about the influence of the specific plant species on the success of bacterial colonization. As such, we compared the ability of the VTEC strain, E. coli O157:H7 'Sakai,' to colonize the roots and leaves of four leafy vegetables: spinach (Spinacia oleracea), lettuce (Lactuca sativa), vining green pea (Pisum sativum), and prickly lettuce (Lactuca serriola), a wild relative of domesticated lettuce. Also, to determine the drivers of the initial response on interaction with plant tissue, the whole transcriptome of E. coli O157:H7 Sakai was analyzed following exposure to plant extracts of varying complexity (spinach leaf lysates or root exudates, and leaf cell wall polysaccharides from spinach or lettuce). Plant extracts were used to reduce heterogeneity inherent in plant-microbe interactions and remove the effect of plant immunity. This dual approach provided information on the initial adaptive response of E. coli O157:H7 Sakai to the plant environment together with the influence of the living plant during bacterial establishment and colonization. Results showed that both the plant tissue type and the plant species strongly influence the short-term (1 h) transcriptional response to extracts as well as longer-term (10 days) plant colonization or persistence. We show that propagation temperature (37 vs. 18°C) has a major impact on the expression profile and therefore pre-adaptation of bacteria to a plant-relevant temperature is necessary to avoid misleading temperature-dependent wholescale gene-expression changes in response to plant material. For each of the plant extracts tested, the largest group of (annotated) differentially regulated genes were associated with metabolism. However, large-scale differences in the metabolic and biosynthetic pathways between treatment types indicate specificity in substrate utilization. Induction of stress-response genes reflected the apparent physiological status of the bacterial genes in each extract, as a result of glutamate-dependent acid resistance, nutrient stress, or translational stalling. A large proportion of differentially regulated genes are uncharacterized (annotated as hypothetical), which could indicate yet to be described functional roles associated with plant interaction for E. coli O157:H7 Sakai.

Verocytotoxigenic Escherichia coli (VTEC) can contaminate crop plants, potentially using them as secondary hosts, which can lead to food-borne infection. Currently, little is known about the influence of the specific plant species on the success of bacterial colonisation. As such, we compared the ability of the VTEC strain, E. coli O157:H7 ‘Sakai’, to colonise the roots and leaves of four leafy vegetables: spinach (Spinacia oleracea), lettuce (Lactuca sativa), vining green pea (Pisum sativum) and prickly lettuce (L. serriola), a wild relative of domesticated lettuce. Also, to determine the drivers of the initial response on interaction with plant tissue, the whole transcriptome of E. coli O157:H7 Sakai was analysed following exposure to plant extracts of varying complexity (spinach leaf lysates or root exudates, and leaf cell wall polysaccharides from spinach or lettuce). Plant extracts were used to reduce heterogeneity inherent in plant-microbe interactions and remove the effect of plant immunity. This dual approach provided information on the initial adaptive response of E. coli O157:H7 Sakai to the plant environment together with the influence of the living plant during bacterial establishment and colonisation. Results showed that both the plant tissue type and the plant species strongly influence the short-term (1 hour) transcriptional response to extracts as well as longer-term (10 days) plant colonisation or persistence. We show that propagation temperature (37 versus 18 oC) has a major impact on the expression profile and therefore pre-adaptation of bacteria to a plant-relevant temperature is necessary to avoid misleading temperature-dependent wholescale gene-expression changes in response to plant material. For each of the plant extracts tested, the largest group of (annotated) differentially regulated genes were associated with metabolism. However, large-scale differences in the metabolic and biosynthetic pathways between treatment types indicate specificity in substrate utilisation. Induction of stress-response genes reflected the apparent physiological status of the bacterial genes in each extract, as a result of glutamate-dependent acid resistance, nutrient stress or translational stalling. A large proportion of differentially regulated genes are uncharacterised (annotated as hypothetical), which could indicate yet to be described functional roles associated with plant interaction for E. coli O157:H7 Sakai.

The health benefits of garlic have been proven by epidemiological and experimental studies. Diallyl disulphide (DADS), the major organosulfur compound found in garlic oil, is known to lower the incidence of breast cancer both in vitro and in vivo. The studies reported here demonstrate that DADS induces apoptosis in the MCF-7 breast-cancer cell line through interfering with cell-cycle growth phases in a way that increases the sub-G0 population and substantially halts DNA synthesis. DADS also induces phosphatidylserine (PS) translocation from the inner to the outer leaflet of the plasma membrane and activates caspase-3. Further studies revealed that DADS modulates the cellular levels of Bax, Bcl-2, Bcl-xL and Bcl-w in a dose-dependent manner, suggesting the involvement of Bcl-2 family proteins in DADS induced apoptosis. Histone deacetylation inhibitors (HDACi) are known to suppress cancer growth and induce apoptosis in cancer cells. Here it is shown that DADS has HDACi properties in MCF-7 cells as it lowers the removal of an acetyl group from an acetylated substrate and induces histone-4 (H4) hyper-acetylation. The data thus indicate that the HDACi properties of DADS may be responsible for the induction of apoptosis in breast cancer cells.

Iron is an essential cofactor for both mycobacterial growth during infection and for a successful protective immune response by the host. The immune response partly depends on the regulation of iron by the host, including the tight control of expression of the iron-storage protein, ferritin. BCG vaccination can protect against disease following Mycobacterium tuberculosis infection, but the mechanisms of protection remain unclear. To further explore these mechanisms, splenocytes from BCG-vaccinated guinea pigs were stimulated ex vivo with purified protein derivative from M. tuberculosis and a significant down-regulation of ferritin light- and heavy-chain was measured by reverse-transcription quantitative-PCR ( P ≤ 0.05 and ≤0.01, respectively). The mechanisms of this down-regulation were shown to involve TNFα and nitric oxide. A more in depth analysis of the mRNA expression profiles, including genes involved in iron metabolism, was performed using a guinea pig specific immunological microarray following ex vivo infection with M. tuberculosis of splenocytes from BCG-vaccinated and naive guinea pigs. M. tuberculosis infection induced a pro-inflammatory response in splenocytes from both groups, resulting in down-regulation of ferritin ( P ≤ 0.05). In addition, lactoferrin ( P ≤ 0.002), transferrin receptor ( P ≤ 0.05) and solute carrier family 11A1 ( P ≤ 0.05), were only significantly down-regulated after infection of the splenocytes from BCG-vaccinated animals. The results show that expression of iron-metabolism genes is tightly regulated as part of the host response to M. tuberculosis infection and that BCG-vaccination enhances the ability of the host to mount an iron-restriction response which may in turn help to combat invasion by mycobacteria.

Colon cancer is a leading and expanding cause of death worldwide. A major contributory factor to this disease is diet composition; some components are beneficial (e.g, dietary fiber), whereas others are detrimental (e.g., alcohol). Garlic oil is a prominent dietary constituent that prevents the development of colorectal cancer. This effect is believed to be mainly due to diallyl disulphide (DADS), which selectively induces redox stress in cancerous (rather than normal) cells that leads to apoptotic cell death. However, the detailed mechanism by which DADS causes apoptosis remains unclear. We show that DADS treatment of colonic adenocarcinoma cells (HT-29) initiates a cascade of molecular events characteristic of apoptosis. These include a decrease in cellular proliferation, translocation of phosphatidylserine to the plasma-membrane outer-layer, activation of caspase-3 and -9, genomic DNA fragmentation, and G(2)/M phase cell-cycle arrest. Short-chain fatty acids (SCFAs), particularly butyrate (abundantly produced in the gut by bacterial fermentation of dietary polysaccharides), enhance colonic cell integrity but, in contrast, inhibit colonic cancer cell growth. Combining DADS with butyrate augmented the apoptotic effect of butyrate on HT-29 cells. These results suggest that the anticancerous properties of DADS afford greater benefit when supplied with other favorable dietary factors (short chain fatty acids/polysaccharides) that likewise reduce colonic tumor development.

Ribonucleotide reductases supply cells with their deoxyribonucleotides. Three enzyme types are known, classes I, II and III. Class II enzymes are anaerobic whereas class I enzymes are aerobic, and so class I and II enzymes are often produced by the same organism under opposing oxygen regimes. Escherichia coli contains two types of class I enzyme (Ia and Ib) with the Fe-dependent Ia enzyme (NrdAB) performing the major role aerobically, leaving the purpose of the Ib enzyme (NrdEF) unclear. Several papers have recently focused on the class Ib enzymes showing that they are Mn (rather than Fe) dependent and suggesting that the E. coli NrdEF may function under redox-stress conditions. A paper published in this issue of Molecular Microbiology from James Imlay's group confirms that this unexplained NrdEF Ib enzyme is Mn-dependent, but shows that it does not substitute for NrdAB during redox stress. Instead, a role during iron restriction is demonstrated. Thus, the purpose of NrdEF (and possibly other class Ib enzymes) is to enhance growth under aerobic, low-iron conditions, and to functionally replace the Fe-dependent NrdAB when iron is unavailable. This finding reveals a new mechanism by which bacteria adjust to life under iron deprivation.

Bacterioferritin (BFR) from Escherichia coli is a member of the ferritin family of iron storage proteins and has the capacity to store very large amounts of iron as an Fe(3+) mineral inside its central cavity. The ability of organisms to tap into their cellular stores in times of iron deprivation requires that iron must be released from ferritin mineral stores. Currently, relatively little is known about the mechanisms by which this occurs, particularly in prokaryotic ferritins. Here we show that the bis-Met-coordinated heme groups of E. coli BFR, which are not found in other members of the ferritin family, play an important role in iron release from the BFR iron biomineral: kinetic iron release experiments revealed that the transfer of electrons into the internal cavity is the rate-limiting step of the release reaction and that the rate and extent of iron release were significantly increased in the presence of heme. Despite previous reports that a high affinity Fe(2+) chelator is required for iron release, we show that a large proportion of BFR core iron is released in the absence of such a chelator and further that chelators are not passive participants in iron release reactions. Finally, we show that the catalytic ferroxidase center, which is central to the mechanism of mineralization, is not involved in iron release; thus, core mineralization and release processes utilize distinct pathways.

The induction of apoptosis in mammalian cells by bacteria is well reported. This process may assist infection by pathogens whereas for non-pathogens apoptosis induction within carcinoma cells protects against colon cancer. Here, apoptosis induction by a major new gut bacterium, Atopobium minutum, was compared with induction by commensal (Escherichia coli K-12 strains), probiotic (Lactobacillus rhamnosus, Bifidobacterium latis) and pathogenic (E. coli: EPEC and VTEC) gut bacteria within the colon cancer cell line, Caco-2. The results show a major apoptotic effect for the pathogens, mild effects for the probiotic strains and A. minutum, but no effect for commensal E. coli. The mild apoptotic effects observed are consistent with the beneficial roles of probotics in protection against colon cancer and suggest, for the first time, that A. minutum possesses similar advantageous, anti-cancerous activity. Although bacterial infection increased Caco-2 membrane FAS levels, caspase-8 was not activated indicating that apoptosis is FAS independent. Instead, in all cases, apoptosis was induced through the mitochondrial pathway as indicated by BAX translocation, cytochrome c release, and caspase-9 and -3 cleavage. This suggests that an intracellular stimulus initiates the observed apoptosis responses.

FtnA is the major iron-storage protein of Escherichia coli accounting for < or = 50% of total cellular iron. The FtnA gene (ftnA) is induced by iron in an Fe(2+)-Fur-dependent fashion. This effect is reportedly mediated by RyhB, the Fe(2+)-Fur-repressed, small, regulatory RNA. However, results presented here show that ftnA iron induction is independent of RyhB and instead involves direct interaction of Fe(2+)-Fur with an 'extended' Fur binding site (containing five tandem Fur boxes) located upstream (-83) of the ftnA promoter. In addition, H-NS acts as a direct repressor of ftnA transcription by binding at multiple sites (I-VI) within, and upstream of, the ftnA promoter. Fur directly competes with H-NS binding at upstream sites (II-IV) and consequently displaces H-NS from the ftnA promoter (sites V-VI) which in turn leads to derepression of ftnA transcription. It is proposed that H-NS binding within the ftnA promoter is facilitated by H-NS occupation of the upstream sites through H-NS oligomerization-induced DNA looping. Consequently, Fur displacement of H-NS from the upstream sites prevents cooperative H-NS binding at the downstream sites within the promoter, thus allowing access to RNA polymerase. This direct activation of ftnA transcription by Fe(2+)-Fur through H-NS antisilencing represents a new mechanism for iron-induced gene expression.

The EfeUOB system of Escherichia coli is a tripartite, low pH, ferrous iron transporter. It resembles the high-affinity iron transporter (Ftr1p-Fet3p) of yeast in that EfeU is homologous to Ftr1p, an integral-membrane iron-permease. However, EfeUOB lacks an equivalent of the Fet3p component--the multicopper oxidase with three cupredoxin-like domains. EfeO and EfeB are periplasmic but their precise roles are unclear. EfeO consists primarily of a C-terminal peptidase-M75 domain with a conserved 'HxxE' motif potentially involved in metal binding. The smaller N-terminal domain (EfeO-N) is predicted to be cupredoxin (Cup) like, suggesting a previously unrecognised similarity between EfeO and Fet3p. Our structural modelling of the E. coli EfeO Cup domain identifies two potential metal-binding sites. Site I is predicted to bind Cu(2+) using three conserved residues (C41 and 103, and E66) and M101. Of these, only one (C103) is conserved in classical cupredoxins where it also acts as a Cu ligand. Site II most probably binds Fe(3+) and consists of four well conserved surface Glu residues. Phylogenetic analysis indicates that the EfeO-Cup domains form a novel Cup family, designated the 'EfeO-Cup' family. Structural modelling of two other representative EfeO-Cup domains indicates that different subfamilies employ distinct ligand sets at their proposed metal-binding sites. The ~100 efeO homologues in the bacterial sequence databases are all associated with various iron-transport related genes indicating a common role for EfeO-Cup proteins in iron transport, supporting a new copper-iron connection in biology.

Escherichia coli possesses iron transporters specific for either Fe2+ or Fe3+. Although Fe2+ is far more soluble than Fe3+, it rapidly oxidizes aerobically at pH >= 7. Thus, FeoAB, the major Fe2+ transporter of E. coli, operates anaerobically. However, Fe2+ remains stable aerobically under acidic conditions, although a low-pH Fe2+ importer has not been previously identified. Here we show that ycdNOB (efeUOB) specifies the first such transporter. efeUOB is repressed at high pH by CpxAR, and is Fe2+-Fur repressed. EfeU is homologous to the high-affinity iron permease, Ftr1p, of Saccharomyces cerevisiae and other fungi. EfeO is periplasmic with a cupredoxin N-terminal domain; EfeB is also periplasmic and is haem peroxidase-like. All three Efe proteins are required for Efe function. The efeU gene of E. coli K-12 is cryptic due to a frameshift mutation - repair of the single-base-pair deletion generates a functional EfeUOB system. In contrast, the efeUOB operon of the enterohaemorrhagic strain, O157:1147, lacks any frameshift and is functional. A 'wild-type' K-12 strain bearing a functional EfeUOB displays a major growth advantage under aerobic, low-pH, low-iron conditions when a competing metal is provided. Fe-55 transport assays confirm the ferrous iron specificity of EfeUOB.

Salmonella enterica serovar Enteritidis is the prevalent egg-product-related food-borne pathogen. The egg-contamination capacity of S. Enteritidis includes its exceptional survival capability within the harsh conditions provided by egg white. Egg white proteins, such as lysozyme and ovotransferrin, are well known to play important roles in defence against bacterial invaders. Indeed, several additional minor proteins and peptides have recently been found to play known or potential roles in protection against bacterial contamination. However, although such antibacterial proteins are well studied, little is known about their efficacy under the environmental conditions prevalent in egg white. Thus, the influence of factors such as temperature, alkalinity, nutrient restriction, viscosity and cooperative interactions on the activities of antibacterial proteins in egg white remains unclear. This review critically assesses the available evidence on the antimicrobial components of egg white. In addition, mechanisms employed by S. Enteritidis to resist egg white exposure are also considered along with various genetic studies that have shed light upon egg white resistance systems. We also consider how multiple, antibacterial proteins operate in association with specific environmental factors within egg white to generate a lethal protective cocktail that preserves sterility.

The elaC gene of Escherichia coli encodes a binuclear zinc phosphodiesterase (ZiPD). ZiPD homologs from various species act as3' tRNA processing endoribonucleases, and although the homologous gene in Bacillus subtilis is essential for viability [EMBO J. 22(2003) 4534], the physiological function of E. coli ZiPD has remained enigmatic. In order to investigate the function of E. coli ZiPDwe generated and characterized an E. coli elaC deletion mutant. Surprisingly, the E. coli elaC deletion mutant was viable and had wild-type like growth properties. Microarray-based transcriptional analysis indicated expression of the E. coli elaC gene at basal levels during aerobic growth. The elaC gene deletion had no effect on the expression of genes coding for RNases or amino-acyl tRNA synthetases or any other gene among a total of > 1300 genes probed. 2D-PAGE analysis showed that the elaC mutation, like-wise, had no effect on the proteome. These results strengthen doubts about the involvement of E. coli ZiPD in tRNA maturation and suggest functional diversity within the ZiPD/ElaC1 protein family. In addition to these unexpected features of the E. coli elaC deletion mutant, a sequence comparison of ZiPD (ElaC1) proteins revealed specific regions for either enterobacterial or mammalian ZiPD (ElaC1) proteins.

The DcuS-DcuR system of Escherichia coli is a two-component sensor-regulator that controls gene expression in response to external C 4 -dicarboxylates and citrate. The DcuS protein is particularly interesting since it contains two PAS domains, namely a periplasmic C 4 -dicarboxylate-sensing PAS domain (PASp) and a cytosolic PAS domain (PASc) of uncertain function. For a study of the role of the PASc domain, three different fragments of DcuS were overproduced and examined: they were PASc-kinase, PASc, and kinase. The two kinase-domain-containing fragments were autophosphorylated by [γ- 32 P]ATP. The rate was not affected by fumarate or succinate, supporting the role of the PASp domain in C 4 -dicarboxylate sensing. Both of the phosphorylated DcuS constructs were able to rapidly pass their phosphoryl groups to DcuR, and after phosphorylation, DcuR dephosphorylated rapidly. No prosthetic group or significant quantity of metal was found associated with either of the PASc-containing proteins. The DNA-binding specificity of DcuR was studied by use of the pure protein. It was found to be converted from a monomer to a dimer upon acetylphosphate treatment, and native polyacrylamide gel electrophoresis suggested that it can oligomerize. DcuR specifically bound to the promoters of the three known DcuSR-regulated genes ( dctA , dcuB , and frdA ), with apparent K D s of 6 to 32 μM for untreated DcuR and ≤1 to 2 μM for the acetylphosphate-treated form. The binding sites were located by DNase I footprinting, allowing a putative DcuR-binding motif [tandemly repeated (T/A)(A/T)(T/C)(A/T)AA sequences] to be identified. The DcuR-binding sites of the dcuB , dctA , and frdA genes were located 27, 94, and 86 bp, respectively, upstream of the corresponding +1 sites, and a new promoter was identified for dcuB that responds to DcuR.

Organisms generally respond to iron deficiency by increasing their capacity to take up iron and by consuming intracellular iron stores. Escherichia coli, in which iron metabolism is particularly well understood, contains at least 7 iron-acquisition systems encoded by 35 iron-repressed genes. This Fe-dependent repression is mediated by a transcriptional repressor, Fur (ferric uptake regulation), which also controls genes involved in other processes such as iron storage, the Tricarboxylic Acid Cycle, pathogenicity, and redox-stress resistance. Our macroarray-based global analysis of iron- and Fur-dependent gene expression in E. coli has revealed several novel Fur-repressed genes likely to specify at least three additional iron-transport pathways. Interestingly, a large group of energy metabolism genes was found to be iron and Fur induced. Many of these genes encode iron-rich respiratory complexes. This iron- and Fur-dependent regulation appears to represent a novel iron-homeostatic mechanism whereby the synthesis of many iron-containing proteins is repressed under iron-restricted conditions. This mechanism thus accounts for the low iron contents of fur mutants and explains how E. coli can modulate its iron requirements. Analysis of 55Fe-labeled E. coli proteins revealed a marked decrease in iron-protein composition for the fur mutant, and visible and EPR spectroscopy showed major reductions in cytochrome b and d levels, and in iron-sulfur cluster contents for the chelator-treated wild-type and/or fur mutant, correlating well with the array and quantitative RT-PCR data. In combination, the results provide compelling evidence for the regulation of intracellular iron consumption by the Fe2+-Fur complex.