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Life can be pretty stressful, can't it? I'm sure that you, like me, sometimes wish for nothing more than to briefly transform into a roll of toilet tissue. Imagine; rolling around in 2-3 ply bliss, temporarily free of all the cares and worries that come with the human condition. Well, great news – Prey allows you to simulate this!! It's not too long before you can unlock the right power and, once you find some of that magical absorption material to emulate, off you go.
Oh, also there are some aliens and guns and stuff.
Prey is a science fantasy game set in an alternate reality. Although the bulk of the explaining is rather oddly done in the loading screens, this imagines a world where America and the USSR formed a short alliance during the space race upon discovery of a powerful and unfriendly alien race; the Typhon. Eventually, the decision is taken to store and study a large number of these mysterious, supernaturally-powered, shape-shifting, rapidly asexually reproducing lifeforms on a space station close to Earth. What could possibly go wrong?
Luckily for us things go spectacularly wrong, and you're right in the middle of it. After a brief, surprising, and imaginative intro, you find yourself trapped on the aforementioned space station – Talos 1 – and of course virtually all the alien nasties have escaped and multiplied. You begin with nothing more than a wrench to defend yourself, but as you progress you'll have access to a wide range of weapons and abilities. The first projectile weapon you get is the Gloo gun. Despite dealing precisely no damage, this will prove a vital bit of kit throughout your journey.
Gloo can be used to temporarily incapacitate any enemy (with one spoilery exception), but it may well be the second use that you rely most heavily upon. This stuff will adhere to any surface except glass in big chunks that bear an uncanny resemblance to popcorn. You can in this way plug pipes spitting out fire or gas, but more interestingly it allows you to build your own little platforms. This allows you to make your own shortcuts or reach otherwise inaccessible places and, as the demo proved before the game even released, even give you access to areas the developers never intended (or at the least, much earlier than they intended).
"Bioshock in space" isn't quite right, but it gives you a good idea of the atmosphere and gameplay that await you. In place of Plasmids we have here Neuromods, one-time consumables used to unlock abilities within several skill trees (including, after a while, alien powers). This is a game crammed full of secret stashes, entirely optional rooms and missions, alternate routes, and that kind of malarkey. So for example, do you want a strong hacking ability to get through doors and safes? Or build up those muscles so you can move heavy objects blocking doorways, and hurl them at enemies? Maybe you just want more inventory space, or the ability to fully upgrade weapons, or maximise your ability to morph into objects that you find, or nab every offensive upgrade possible, or some mix of all this. Prey is keen to encourage you to personalise your experience.
As part of this, Talos 1 is an open-world-ish environment. It's possible – likely, in fact – that you'll finish the game without seeing everything, and without even exploring certain areas. I have to say in fact that I'm genuinely impressed with what was clearly a painstakingly designed game world. I can't remember exploring one before that felt more like a real place (despite the shapeshifting aliens, etc.). What I mean by that is, while you're obviously poked in the right direction in various places as the story demands, there are big chunks of game world that you only have cause to briefly pass through, if at all. With rare exceptions, you can make your way to your next destination as slowly or as quickly as you like, and toddle off elsewhere if you get distracted without consequence. The environment dictates events rather than the other way around.
It's also great how completing side missions can sometimes have further-reaching consequences than you might expect. It's not a simple case of "thanks for saving me, here's a medkit. Now I'm going to glue my backside to this chair and spout the same two lines of dialogue if you try talking to me again for the rest of the game". Help a survivor out, and hours later you might be rewarded with items and/or yet more optional quests that would've otherwise been blocked off to you. Maybe you'll be rewarded with another way to finish a mission, or even the opportunity for an alternate game ending. Or, perhaps, altruism will be punished…
At first, enemies are primarily Mimics; the spider-like beings that can take the form of virtually any small-to-medium object not nailed down. Chairs, weapons, medkits, mugs, boxes; anything and everything could be a Mimic. Expect to get ambushed often. The tension doesn't so much come from creeping into a new area blind, as it does when you know that there's definitely a Mimic (or two) somewhere – because it's already attacked you before scuttling off, or you saw movement at the edge of your vision – but you don't know what it is. You might attack a banana peel in a blind panic, only to have a nearby crate reveal itself to be a monster determined to attach itself to your face.
Mimics don't take too many hits (although there's a stronger version that pops up later), but the camouflage combined with speed and agility means fighting them is far from easy. It's fairly early on in the game that the focus of combat switches to Phantoms, Telepaths, Technopaths, and a few other types. Phantoms are anthropomorphic teleporters with projectiles, split into normal, electric, and fire types. They don't go down easily, and can be annoying to fight in the early stages if you don't have much in the way of health or weaponry. Telepaths can mind-control any nearby humans (except you for some reason) and send them after you. As a nice touch, the heads of said humans explode if they get too close to you. Technopaths can hack nearby turrets and 'operators' (hovering robots) to turn them against you and, frustratingly, they also have an attack that works at both short and long range which temporarily disables your guns. They keep you on your toes, but to be honest it's much better when it's just you versus Mimics.
The videogame norm of pictorial hacking….
Sadly, this isn't the most technically stable game. More than once, I experienced an extended period of terrible frame rate for no obvious reason. The game crashed on me during what was supposed to be a dramatic late-game twist. I once accidentally glitched through a door. Actually, I gratefully quick-saved immediately after that glitch, because I'd spent about twenty minutes trying to work out a way into the room. Maybe the game just felt sorry for me.
Prey, the game where you can be a toilet Roll.
Depending on your playstyle and chosen difficulty, Prey could last you anything from 15-25 hours. However long it ends up lasting you, it's going to be about two hours too long, because toward the end of the story there's some cheap forced backtracking. There's a regular supply of surprises and neat ideas to prevent this from being too painful, but it's still disappointing to see. Overall, though, this is a memorable experience crammed full of good ideas that I encourage you to try for yourself.
What's the bottom line? Handling freedom of choice better than most games, Prey makes close to two dozen hours on and around a space station more fun than it should be.
Review by the always amazing Luke Kemp (editor of Criticalgamer.co.uk and contributor to other sites), and the thanks to Xbox for providing the review code!


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Here are three key aspects that the companies can help you with:
• wroclaw airport transfers – not lots of men and ladies are aware that in Poland here are about ten airports. They are placed by the ocean like Gdańsk, in the capital of Poland – Warsaw (here are 2 flight terminals) or in the west areas of Poland – Wrocław, Katowice and Cracow. Nevertheless, Wroclaw is a perfect location to start the travelling in Poland – it is placed by Odra lake and has lots bridges likewise to Amsterdam. The company will transfer you from the Wroclaw airport to the hotel. It is very practical and the safest way – you will not miss the way and you can obtain many helpful information from our cab drivers. The transfer is carried out in the luxurious and air-conditioned cars what will make the journey more enjoyable. Read about this service: • Accommodation– in Wroclaw there are lots locations to stay for a night. It is clear that the best places are situated in the center of the town – in the town centre. Generally there are also the most luxurious accommodations and youth hostels. Nonetheless, everyone can find something which will be right for the budgets.
• Sightseeing tours – every individual should try sightseeing trips that can help to get the area better. The corporation will provide the sightseeing warsawcitytours.info/tours/one-day-cracow-and-wieliczka-tour tours in purple Double Decker vehicles like those which can be viewed in London's roads. Wroclaw is a great city for every individual who enjoys new, Eastern Europe location. Everyone who enjoys to visit Polish cities should start the journey from this city to get the Polish customs and history better.

subset: commoncrawl


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Often cybercriminals use thousands to millions of infected devices and control them remotely to attack websites at a large scale. Known as the 'botnet' attacks or bot attacks, these are difficult to trace and monitor as they use computing power and resources from trusted networks.
Cybercriminals create bot armies to launch DDoS attacks, spam campaigns and other types of attacks that can be used to bring your website down or/and steal data. Surprisingly, these types of attacks are easy to carry out too with botnet armies available on a rental basis. Full-service attacks are available for as little as $5 per hour, and the interested party can easily stretch their hour with a monthly plan that averages $38.
Such botnet attacks are motivated by revenge, politics, trolling and terrorism, money is frequently involved. According to security analysts, ransom and blackmail are the most common motives behind bot attacks. Hackers disrupt web services and hold it against a ransom. Hackers ask for heavy ransom amount, which when paid, leads to the attack being stopped and services made available to customers.
Similarly, competition motivates people to bring down better-performing rivals while revenge from disgruntled employees leaves a similar impact. So, if you have a website, you will have bots crawling your site too. Differentiating between good and bad bots is critical and that's exactly what we will show you in our upcoming webinar.
Venkatesh (Venky) Sundar, web security expert and founder & CMO of Indusface, will show you the good, the bad, and the ugly of bots, what they mean for your business, and how to keep your website safe from them.
Founder & Chief Marketing Officer, Indusface
Venky has played multiple roles within Indusface for the past 6 years. He was instrumental in building the product/service and technology team from scratch and grew it from ideation to getting initial customers with a proven/validated business model poised for scale. He has proven experience (10+ years) in the security industry and has held various mgmt/leadership roles in Product Development, Professional Services, and Sales during his time at Entrust Data card.


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I do like to reflect ideas and inspirations from my working life (and sometimes from my family life) into my work Blog. But how can I find inspiration when the first act of the working week is met with:
'Train cancelled'
due to;
'shortage of train crew availability'.
I managed to get up and out of bed but it appears that someone else either did not want to or couldn't. It was a big shame for the train load of people that then had to squash into the next train. Like the person who breaks down on the motorway and holds up thousands of people while they are towed off. Clearly it's tough for the individuals concerned but the impact is out of all proportion to their difficulty or illness.
What can 'brittle' transport systems teach us about decision-making?
What happens at work if a lot of people are waiting on a decision? A re-location, or a pay deal or an investment decision? What pressure does that create on the decision-maker? How does that pressure aid or impeded their ability to decide? Sometimes the pressure means they go with the 'popular' option or go without the full analysis being done. Sometimes the pressure goes the other way and they might retreat and say 'no' if they feel boxed in.
So how do we avoid the;
'cancellation of decision due to lack of small bit of information'
How about we:
- Look ahead….
- Plan the effort and activity required to support the decision
- Work out who can deputise and when (keep those trains running…)
- Design an explicit evaluation framework – so we know what information or aspects are we waiting for?
- Rehearse the 'Three Treatments' of risk;
- Remove the risk
- Mitigate the risk
- Accept the risk
So the next time your week starts with an unwelcome delay (possibly tomorrow!) – you could do worse that use that time to reflect on how to lift the performance bar in your own organisational processes around decision-making.
Happy strike dodging.


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Species group: Uromastyx, Dabb and Spiny-Tailed Lizards
Other common names: Rainbow Uromastyx, South Arabian Spiny-tailed Lizard
Scientific name: Uromastyx yemenensis
Active little lizards.
We had two that bounce around their cage, chasing and head bobing and hunting. They are very entertaining to watch and easily handled..
From Kacie Bingham Sep 28 2017 9:51PM


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Yes, Firefox Quantum and Firefox Nightly can run together. Here's how.
How to Transfer the Contents of User Profiles in Firefox
If you are trying out a new Firefox-based browser, you can transfer the content of your old user profiles so all your items are intact. Here's how to do it.
How To Install Firefox 3.1 On Ubuntu Without Affecting The Default Setting
If you are someone who always like to test out bleeding edge products and applications, then Firefox 3.1 is something that you definitely don't want to miss. Not only does it comes with more improved features, it is also much faster than its predecessor and other browsers. The current version is at beta 2 and […]


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To investigate in pregnant women the adverse pulmonary effects of magnesium sulfate (MgSO4),
a commonly used drug for tocolysis or arrest of labor.
Preterm labor is a major public health concern in the United States. The delay of labor,
particularly for idiopathic preterm labor, is important because it allows further fetal
development and the opportunity to administer steroids which improve fetal lung maturity. It
is now common for MgSO4 to be administered to delay labor. The most dangerous adverse event
with MgSO4 treatment is the development of pulmonary edema which may lead to maternal adult
respiratory distress syndrome and death with associated fetal morbidity and mortality. The
estimates of the frequency of this complication vary from 0 to 8 percent. Therefore,
improved understanding of the pulmonary toxicity of this therapeutic agent is clinically
There were three phases in the study. The first, a retrospective cohort study, examined the
incidence rate of pulmonary edema when MgSO4 was administered as a tocolytic agent. The
second, a case-control study nested within the cohort, examined the relationship between the
patient and other clinical factors and the incidence of pulmonary edema in patients treated
with MgSO4. The third phase used the results of the case-control study to develop and
validate a predictive index to define a group of patients at increased risk of developing
pulmonary edema associated with MgSO4.
Patients were identified using ICD-9-CM codes for preterm labor and delivery. The medical
record was reviewed to determine exposure to MgSO4 as a tocolytic agent, excluding patients
who received MgSO4 for other indications. Cases were defined as probable when patients met
the clinical definition for pulmonary edema, including chest X-ray findings. The medical
charts of both cases and a random sample of non cases were then reviewed in detail and the
data from these groups were compared. The primary analysis of these case-control data
included unadjusted analysis and multivariable explanatory models to provide insight into
the risk factors for pulmonary edema associated with MgSO4. The data from the case-control
study were used to develop a clinical predictive index. The predictive rule was then
validated in a separate patient population.
No eligibility criteria
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Philadelphia, Pennsylvania 19104United StatesThere is no listed contact information for this specific location.Site Status: N/A
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Dave House will compose the score for An Official Guide to Surviving The Invasion, a short horror anthology based on a poem by writer and editor Dr. Russell Jones.
Scotland is on the cusp of an epidemic. A media frenzy calls for imperative action as strange abductions and attacks are reported. Who, if anybody have the full details? Through shifting perspectives might we find the real cause for concern? Are all incidents connected? Are they acts of racism? Terrorism? Hacking? Or perhaps indeed…an invasion?
The music is based on an outtake from House's 2017 album which was considered too dark for the final cut. What better subject matter than epidemics and terror to bring the piece back from the dead! Listen to the working concept below:


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A group of us were sharing at our Bible study about losing friendships. People that were in our lives for a period of time just seem to move on. Sometimes they enrich our lives and sometimes they leave tears behind. When one is divorced, couples that you knew don't know which side to take so they just pull away. Friends at church who spoke to you all the time, suddenly feel you are a threat to their husbands and back off. People you helped financially and emotionally, finally show their true colors as takers, and you have to sever the relationship. So many shared that after a death, whether a parent or another relative, that there is a battle royal for who gets what and the family is fractured. Things and possessions are more important than relationships. It is a sad thing when those you have loved, and thought loved you in return, turn away. My son-in-law had a cousin he spent much time with as a boy. He occupied himself building balsa airplanes, carefully fitting them together and painting them so they looked authentic. They hung by string from the ceiling of the bedroom he used. When the cousin died unexpectedly, before anyone else knew, a relative took a U-haul truck, cleared out the house and threw all the airplanes in the trash. My son-in-law would have gladly gone and retrieved them, or paid to ship them to him. He was devastated. Another friend, whose mother died, thought that she had been given the house, which her mother had paid for, saving her stepfather from foreclosure. She had to pay two stepsisters for their share of her mother's house and it took almost everything she had saved. Another told about a cookie jar a friend's mother kept full of cookies for her four kids when they were young. When the friend gave the cookie jar to one of her daughters, the other three were incensed! How do you split up a cookie jar?
When I was married the first time, we were used to 35 to 40 relatives at Thanksgiving and Christmas. Unfortunately, they were my husband's family and after the divorce it was only my three children, my mother and me, quite an emotional adjustment. Death or divorce splits up families. The loss of ones I loved for years takes getting used to, but the grief remains.


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A loyal TWN reader sent me this short clip of Ambassador John Bolton's appearance on the Jon Stewart Show Tuesday night — and it's well worth watching.
At about 4:45 in the 5 minute and 23 second clip, Bolton agrees with Jon Stewart that President Bush should stop with oversimplified black/white pandering of "freedom is good" narratives vs. "people who hate liberty are trying to kill us" rhetoric.
While I would debate John Bolton's underlying current of anti-internationalism, he was very reasonable in this interview and poked at "the idealists." Who are the idealists Bolton is referring to?
Well, of the idealists that matter in US foreign policy circles today, liberal interventionists and neoconservatives stand out — and it seems to me that the idealism of the Bush administration has been driven more by the neocon crowd, many of who are John Bolton's colleagues at the American Enterprise Institute.
I wish John Bolton had been at a dinner the New America Foundation/Middle East Policy Initiative hosted last night for former Israel Shin Bet Chief Ami Ayalon — a leading star in Israel's Labor Party in the Knesset who narrowly lost a race recently against Ehud Barak to head the party.
Ayalon revealed that in discussions he had with former Deputy Secretary of Defense Paul Wolfowitz before the March 2003 Iraq invasion, Wolfowitz and he were discussing how "democracy" could be achieved in the Middle East. Ayalon reported that Wolfowitz said that "when tyranny and tyrants are removed, democracy will just burst out."
I think Bolton would have concurred with Ayalon's skepticism of Paul Wolfowitz's alleged view. To be fair to Wolfowitz, I happen to know that his views on nation building are substantially more complex — but in the heady times in the Bush administration between the 9/11 attacks and the decision to invade Iraq, it's easy to imagine Wolfowitz and other Bush administration idealists/neoconservatives making such statements.
Bolton's reasonableness may in fact be spreading to other quarters inside AEI. Jacob Heilbrunn, author of They Knew They Were Right: The Rise of the Neocons is speaking at an AEI lunch on Friday.
I have already heard of internal angst inside AEI over Heilbrunn being invited — but I commend those at AEI who did pull this lunch together and think that it's a testament to any institution that it can entertain speakers and ideas that may run at odds with the general milieu of an organization. I might even go.
More later.
— Steve Clemons


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Streptococcus agalactiae is a major neonatal pathogen whose infectious route involves septicemia. This pathogen does not synthesize heme, but scavenges it from blood to activate a respiration metabolism, which increases bacterial cell density and is required for full virulence. Factors that regulate heme pools in S. agalactiae are unknown. Here we report that one main strategy of heme and protoporphyrin IX (PPIX) homeostasis in S. agalactiae is based on a regulated system of efflux using two newly characterized operons, gbs1753 gbs1752 (called pefA pefB), and gbs1402 gbs1401 gbs1400 (called pefR pefC pefD), where pef stands for 'porphyrin-regulated efflux'. In vitro and in vivo data show that PefR, a MarR-superfamily protein, is a repressor of both operons. Heme or PPIX both alleviate PefR-mediated repression. We show that bacteria inactivated for both Pef efflux systems display accrued sensitivity to these porphyrins, and give evidence that they accumulate intracellularly. The ΔpefR mutant, in which both pef operons are up-regulated, is defective for heme-dependent respiration, and attenuated for virulence. We conclude that this new efflux regulon controls intracellular heme and PPIX availability in S. agalactiae, and is needed for its capacity to undergo respiration metabolism, and to infect the host.
Author Summary
The infectious route of numerous bacterial pathogens includes septicemia, where bacteria are exposed to heme-rich blood. Heme (iron protoporphyrin IX) is generally considered a bacterial iron source. However, while some pathogens do not biosynthesize heme, they use environmental heme to activate key functions. For example, incorporation of heme by the major neonatal pathogen Streptococcus agalactiae activates a latent respiration chain. Respiration metabolism stimulates S. agalactiae growth and survival in blood, and is needed for virulence. While the importance of heme in S. agalactiae behavior is documented, how it manages its intracellular heme pools remains unknown. We discovered a novel regulon, called Pef for "porphyrin-regulated efflux", that modulates S. agalactiae intracellular availability of heme and protoporphyrin IX. A single transcriptional regulator, PefR, represses two distinct efflux transport operons. Regulator-mediated repression is alleviated by heme or protoporphyrin IX. Importantly, over-expression of Pef efflux transporters led to intracellular heme insufficiency, and consequent respiration and virulence defects. Inversely, when Pef efflux transporters were inactivated, results indicated an increased intracellular accumulation of heme and protoporphyrin IX. These studies point to the important role of regulated efflux transport systems in bacterial pathogens for maintaining intracellular heme at levels sufficient to stimulate growth and promote infection.
Citation: Fernandez A, Lechardeur D, Derré-Bobillot A, Couvé E, Gaudu P, Gruss A (2010) Two Coregulated Efflux Transporters Modulate Intracellular Heme and Protoporphyrin IX Availability in Streptococcus agalactiae. PLoS Pathog 6(4): e1000860. https://doi.org/10.1371/journal.ppat.1000860
Editor: Michael R. Wessels, Children's Hospital Boston, United States of America
Received: November 23, 2009; Accepted: March 16, 2010; Published: April 22, 2010
Copyright: © 2010 Fernandez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Research was funded by the Institut National de la Recherche Agronomique, and a grant, for the StrepRespire project, from the French research agency 'Agence Nationale de la Recherche'. A.F. was a recipient of a post-doctoral fellowship from the ANR StrepRespire Project. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Heme (iron protoporphyrin IX) is a redox-active molecule, and a cofactor for numerous cell functions used in oxygen sensing and signal transmission, metabolism, and metal homeostasis [1],[–],[3]. In addition to its varied activities as a cofactor, heme promotes toxic oxygen radical production [4]. The duality between heme as a multifunctional cofactor, and a potentially toxic molecule, suggests the need for strict limitation of its intracellular levels. Metal-free protoporphyrin IX (PPIX) is also found intracellularly, as a heme precursor in bacteria that synthesize heme, and as an intermediate during iron recovery from heme, as shown in Escherichia coli [5]. Thus, cells might have to deal with both heme and PPIX intracellular pools.
While most studied organisms synthesize heme to ensure activity of hemoproteins under the appropriate conditions, numerous bacteria lack heme biosynthesis genes, making heme-catalyzed processes fully dependent upon external heme supplies. For example, Haemophilus influenzae, Bacteroides sp., and several Firmicutes, including Lactococcus lactis, Enterococcus faecalis, Streptococcus agalactiae, and numerous Lactobacillus sp., require heme to activate a respiration metabolic pathway [6],[18]. Some of these bacteria (e.g., H. influenzae, E. faecalis, Bacteroides sp., Lactobacillus brevis and Lactobacillus plantarum) also encode heme-dependent catalases, which rely on exogenous heme for both their stability and activity [19]. Thus, heme supplied by the environment can have a determining effect on the metabolic and enzymatic capacities in organisms lacking the heme biosynthetic pathway.
All bacteria, regardless of their heme biosynthesis capacities, need to manage their intracellular heme pools. Regulation may be exerted at the levels of biosynthesis, uptake, degradation, and possibly efflux, which is in some cases coordinated with the cell iron status [20],[25]. The use of efflux as a means of heme homeostasis remains in question. The sole candidate, HrtAB, a heme-regulated transporter, was first reported as having a role in heme toxicity in Staphylococcus aureus; orthologs of this system were also described in L. lactis and Bacillus anthracis [12], [26],[–],[28]. HrtAB in S. aureus, B. anthracis, and likely several Gram-positive pathogens responds to an extracytoplasmic heme sensor, HssS (part of HssSR two-component system) to activate expression [27], [28].
S. agalactiae is an important human pathogen that does not synthesize its own heme. Nevertheless, infection by this bacterium involves compulsory passage through the bloodstream, causing septicemia and subsequent meningitis [29], [30]. We showed previously that although S. agalactiae generally grows by a fermentation metabolism, it can also use heme, present in blood, to activate the terminal cytochrome bd quinol oxidase for respiration metabolism. Thus, beyond the potential use of heme uptake to acquire iron, the use of heme to activate the energetically favorable respiration metabolism confers a significant gain in cell density, and is required for full bacterial virulence in a septicemia model [17], [18].
Despite the importance of growth in heme-rich blood as an initial step of S. agalactiae infection, the functions involved in regulating intracellular heme levels are unknown. Homologs of HrtAB and HssSR exist in S. agalactiae, and might participate in modulating heme toxicity upon sensing of extracellular heme. However, the mechanism by which HrtAB modulates heme toxicity is unknown, and the question remains whether other systems are needed to regulate heme pools. Here we report the existence of a novel regulon comprising two efflux operons and a single repressor that senses intracellular heme and PPIX, and is needed to maintain their homeostasis. This newly described system is shown to impact on S. agalactiae respiration capacity and virulence.
Identification of gbs1753 gbs1752 as a putative heme- and PPIX- induced locus in S. agalactiae
Transcriptome studies, comparing S. agalactiae gene expression under respiration (i.e., aerobic growth in the presence of exogenous heme and menaquinone) versus aerobic fermentation (i.e., no addition) conditions, initially revealed that gbs1753 gbs1752 was 2.4 to 5.1 fold higher under respiration conditions (AD, PG, EC, and P. Glaser [Pasteur Institute]; Table S1). The gbs1753 ORF encodes an integral membrane protein of the drug:H+ antiporter family, belonging to the major facilitator superfamily (MFS) [31]. The gbs1752 ORF encodes an unknown protein with 2 transmembrane domains. We chose this locus for study, as it was conserved among Gram-positive bacteria that lack a complete heme biosynthesis pathway, but encode cytochrome bd quinol oxidases, indicating a capacity for respiration metabolism (Fig. 1 and Table S2).
A, gbs1753 gbs1752 (pefA pefB) and B, gbs1402 gbs1401 gbs1400 (pefR pefC pefD) loci. Bent arrows and lollipops indicate the mapped or putative promoters, and rho-independent terminators, respectively.
The +1 transcriptional start of gbs1753 gbs1752 was located by 5′-RACE PCR mapping at 134 nucleotides upstream of the gbs1753 start codon at a cytosine (Fig. 2A). The gbs1753 promoter region was fused to a lacZ reporter, referred to as Pgbs1753-lacZ, and expressed on a low copy number plasmid (Fig. 2A) [32]. In the absence of added heme, Pgbs1753-lacZ displayed basal level expression in static and aerobic growth conditions (possibly reflecting trace amounts of heme in BHI medium). Cultures grown with added heme (from 0.1 µM to 10 µM) displayed up to 9-fold higher Pgbs1753-lacZ expression compared to controls without heme, even in non-respiration-permissive conditions (Fig. 2B), indicating that heme, and not the state of respiration, was the inducing factor for the gbs1753 promoter. Significant Pgbs1753-lacZ induction was observed at heme concentrations of 0.3 µM and above. Similarly, PPIX, gallium protoporphyrin (GaPPIX), and zinc mesoporphyrin (ZnMPIX) also induced Pgbs1753-lacZ, while free iron had no inducing effect. These initial results indicate that gbs1753 expression is induced by different porphyrin molecules, regardless of their metal core status.
A. Schematic map of the transcriptional fusion PpefA-lacZ (same as Pgbs1753-lacZ). Sequence of the pefAB promoter region is displayed. The +1 transcriptional start and −10 and −35 motifs are in bold blue characters. B. Expression analysis of PpefA-lacZ by determination of β-galactosidase activity in early stationary phase. S. agalactiae was grown in BHI liquid medium in the presence of the indicated amount of candidate inducers. Strains harboring the pTCV-lac vector (promoterless negative control) did not show β-galactosidase activity during growth (0.4±0.1 Miller Units). Measurements were performed at least three times.
Identification of Gbs1402 as a putative regulator of gbs1753 gbs1752 expression
A random mutagenesis approach was used to identify genetic factors that affect gbs1753 gbs1752 expression. A transposon-generated mutant library was screened for clones in which expression of the Pgbs1753-lacZ fusion was up-regulated, as detected by deep blue colony color. Several mutations mapped in a single ORF, gbs1402. The gbs1402 gene encodes a multiple antibiotic resistance MarR-like regulator (for review see [33]), and is located just upstream of gbs1401 gbs1400, encoding a putative ABC-type multidrug transport complex (Fig. 1B). Sequence analysis predicted that the Gbs1401 and Gbs1400 proteins both contain a transmembrane domain and an ATPase signature. These observations led us to formulate a working hypothesis that both gbs1753 gbs1752 and gbs1401 gbs1400 loci are involved in PPIX and metalloporphyrin efflux, and are regulated by a single protein, Gbs1402. The genes were renamed as pef genes, for porphyrin-regulated efflux. gbs1753 gbs1752 are renamed pefA pefB; gbs1401 gbs1400 are renamed pefC pefD; and the gbs1402 gene for the potential regulator is renamed pefR (Fig. 1).
PefR binds specifically to pefAB and pefRCD promoter regions and represses their transcription
Proteins of the MarR family characteristically bind to DNA inverted repeat motifs (IR) [33]. Sequence analysis revealed the presence of a near-perfect 18-nucleotide IR within a 23 bp consensus, (5′-TAAAATAGTTCTCACGATAACTA-3′) present once upstream of pefA pefB, and twice (one identical sequence, and one inexact copy with 3 nucleotide substitutions in italics) upstream of pefR pefC pefD (Fig. 3A). The IR is not present elsewhere on the S. agalactiae genome. This 23-nucleotide sequence contains the −10 region (TAAAAT) of a putative promoter and constituted a candidate target site for PefR binding to pefAB and pefRCD promoter regions. Mobility shift assays were performed using a purified PefR His-fusion protein, in combination with DNA fragments comprising the IRs of pefAB or pefRCD promoter regions, plus a fragment without an IR as negative internal control (Fig. 3B). PefR caused a mobility shift of both the pefAB and pefRCD fragments in a protein concentration-dependent manner. No shift was observed with the control DNA fragment.
A. A conserved 23-nucleotide motif is present once upstream of pefAB, and twice upstream of pefRCD and is highlighted. The IR present in each of the motifs is marked by arrows. The −10 and −35 motifs, and the mRNA pefAB and putative pefRCD start sites are in blue. Start codons and RBS sequence are in italics. One motif upstream of pefRCD (dotted arrows) differs from the other motifs by 3 nucleotides, which are in gray italics. B. PefR binds the pefAB and pefRCD promoter regions in gel shift assays. Two pmoles of pefAB or pefRCD promoter fragment were incubated in the presence of 0, 4, 17, 34, 63 pmoles of PefR (lanes 1 to 5) and 4.6 pmoles control fragment, corresponding to an 116-bp fragment of the pefA gene. C. Northern blot analyses of pefAB and pefRCD mRNA in the WT and pefR strains, using locus-specific probes (see Materials and Methods). As probe efficiencies and times of exposure differ for each target RNA, differences between pefAB and pefRCD levels are not comparable. ldhL mRNA ('ctrl') was used to control for RNA quantity.
To determine whether PefR impacts on pefA pefB and pefR pefC pefD expression in vivo, Northern blot experiments were performed on the WT strain and an in-frame ΔpefR mutant, using pefAB and pefC as probes (Fig. 3C). The deduced transcript sizes were compatible with organization of the pefA and pefB genes as one operon, and of pefR, pefC, and pefD genes as another. Compared to the WT strain, expression of both pefAB and pefRCD operons was strongly increased in the ΔpefR mutant. We also evaluated expression of the PpefA-lacZ transcriptional fusion in the WT and ΔpefR mutant strains. β-galactosidase expression in the ΔpefR background was 12 times higher than in the WT strain (Miller Units were 62.9±9.2 in ΔpefR versus 5.1±1.7 in the WT strain).
The above in vitro and in vivo results indicate that PefR is a transcriptional repressor of both the pefAB and the pefRCD operons, by binding directly to the operator regions.
PefR is an intracellular heme and PPIX sensor and regulator of pef expression
A characteristic of MarR family regulators is their binding to effector molecules, which leads to induction or repression of their target genes [34]–[36]. Transcriptional fusions showed that the pefAB operon was induced in the presence of metalloporphyrins or PPIX (Fig. 2B). As PefR binds the two pef promoter regions, and appears to repress transcription, we hypothesized that PefR binding and repression are modulated by heme and PPIX. To test this in vitro, mobility shift assays between PefR and the pefAB or pefRCD promoter region DNA fragments were performed with and without heme or PPIX. For both DNA targets, PefR–DNA binding was alleviated in a heme or PPIX concentration-dependent manner (Fig. 4A, Fig. S1A). Iron had no effect on PefR-DNA binding (Fig. S1B).
A. Gel mobility shift analysis of the effect of heme on PefR binding to pefAB (right) and pefRCD (left) promoter regions. 2 pmoles of PpefAB or PpefRCD fragments and 30 pmoles of PefR were mixed. Increased concentrations of heme were added in ratios indicated (keeping PefR constant). Assays were also performed with PPIX (Fig. S1A), giving similar results. On the two top panels, lanes 1–3 were juxtaposed to lanes 4–5, which were separated on the initial same gel. B. Northern blot analyses of pefAB and pefRCD expression in the presence of heme or PPIX in WT NEM316. Cultures were grown in the presence of 0, 1, 5 and 10 µM heme or PPIX, and harvested for total RNA extraction in early stationary phase. As probe efficiencies and times of exposure differ for each target RNA, differences between pefAB and pefRCD levels are not comparable. ldhL (gbs0947; 'ctrl') mRNA was used to control for RNA quantities.
Transcriptional fusion data showed that heme and PPIX up-regulate pefAB expression. To further confirm these results, we performed Northern blot experiments in the absence and presence of these molecules, using pefAB and pefC DNA as probes. Both heme and PPIX induced expression of the two pef operons (Fig. 4B). These in vivo results are in keeping with in vitro gel shift data showing that PefR repression is alleviated in the presence of these porphyrins. We conclude that heme and PPIX both modulate PefR repression of the pefAB and pefRCD operons.
Expression of hrtAB as a function of heme and PPIX levels in S. agalactiae
Genomic studies of S. agalactiae revealed the existence of an analog of HrtAB, a heme-regulated transport system initially characterized in S. aureus and shown to be involved in heme toxicity [37]. Gbs0119 and Gbs0120 showed 45% and 29% identity with HrtB and HrtA of S. aureus respectively. We used Northern blot experiments (Fig. 5A) and a lacZ promoter fusion, Pgbs0119-lacZ (Fig. 5B), to assess the heme and PPIX concentrations needed to induce the hrtAB locus. Interestingly, hrtAB expression was low at heme concentrations below 1 µM, with strong induction at 10 µM; PPIX did not induce its expression, as was observed in S. aureus. Importantly, no hrtAB induction was observed at heme levels where pefAB was induced, i.e., between 0.1 and 0.5 µM heme (Fig. 2B). We conclude that the pef regulon is induced at heme concentrations below those needed for hrtAB induction, indicating that these functions are active under different conditions.
A. Northern blot analyses of gbs0119 expression in the presence of heme or PPIX in WT NEM316. Cultures were grown in the presence of 0, 1, 5 and 10 µM heme or PPIX, and harvested for total RNA extraction in early stationary phase. ldhL (gbs0947; 'crtl') mRNA was used as RNA quantity control. The hybridization was performed on the same membrane as that used in Fig. 4B. B. Expression analysis of Pgbs0119-lacZ by determination of β-galactosidase activity in early stationary phase. S. agalactiae was grown in BHI liquid medium in the presence of different heme concentrations. Measurements were performed three or more times.
Role of pefAB and pefRCD operons in porphyrin sensitivity
The above results led us to ask whether pefAB and pefRCD loci are involved in porphyrin efflux. Several in vivo approaches were developed to explore this question. We constructed in-frame pefA (encoding a putative drug:H+ antiporter) and pefB (encoding a membrane protein of unknown function) deletion mutants; as the two components of pefCD are predicted to encode a single ATP-dependent transporter, we generated a deletion removing both ORFs. Mutations were combined to inactivate both pef operons. Tests were also performed with the ΔpefR deletion mutant, in which expression of both pef loci were highly induced (Fig. 3C).
Sensitivity of ΔpefR, ΔpefA ΔpefCD, and ΔpefB ΔpefCD mutants to metalloporphyrins was evaluated by plate inhibition tests (Fig. S2). Both ΔpefA ΔpefCD and ΔpefB ΔpefCD mutants showed greater sensitivity than the WT strain to 1 nmole heme (Fig. S2A). Similar results were obtained using Gallium PPIX (GaPPIX; tested at 50 nmoles, Fig. S2B). We noted that ΔpefA ΔpefCD was more sensitive than ΔpefB ΔpefCD; this phenotypic difference might suggest an accessory role of PefB in PefA PefB function. The single ΔpefA, ΔpefB, ΔpefC or ΔpefD mutants gave little or no inhibition by these porphyrins (data not shown), suggesting a functional redundancy between the efflux systems encoded by these loci. Sensitivity of the ΔpefR mutant to heme and GaPPIX did not differ significantly from the WT. We further tested heme-mediated inhibition in liquid medium by growing WT and pef single and double mutants, and the ΔpefR mutant, in 0 or 1 µM heme (Fig. 6A and data not shown). The ΔpefA ΔpefCD and ΔpefB ΔpefCD mutants, but not the other tested strains, displayed a slight growth inhibition in the presence of heme. Addition of 2 µM or 4 µM heme exacerbates growth retardation of these mutants (data not shown). These results indicate the need for at least one of the Pef efflux systems to avoid heme toxicity.
A. Growth curves of WT and mutant strains in the presence of heme. Cells were grown aerobically in M17G medium supplemented with 0 or 1 µM heme. WT (black circle), WT +1 µM heme (black square), ΔpefA ΔpefCD (red circle), ΔpefA ΔpefCD +1 µM heme (red square), ΔpefB ΔpefCD (gray circle, dashed line) and ΔpefB ΔpefCD +1 µM heme (gray square, dashed line). Results shown are representative of 3 experiments. Growth retardation of ΔpefA ΔpefCD or ΔpefB ΔpefCD was more pronounced with 2 and 4 µM heme. B. Photosensitivity of WT and mutant S. agalactiae strains grown in the presence of PPIX. Cells were grown until early stationary phase in M17G medium supplemented or not with 10 µM PPIX. Serial 10-fold dilutions (exponent is indicated) were exposed to 0, 10, or 50 minutes visible light. Plates were photographed after 24 h incubation.
To evaluate PPIX accumulation in S. agalactiae WT and mutant cells, we exploited its reactivity upon exposure to visible light; when excited by light, PPIX generates reactive oxygen species [38],[39]. The WT strain, and ΔpefAB ΔpefCD single and combined mutants, and the ΔpefR mutant, were grown with 10 µM PPIX and exposed to visible light for 0, 10 or 50 minutes. Viability of cells grown without PPIX and exposed to light was equivalent for all strains (Fig. 6B). Strikingly, inactivation of both putative pumps led to total mortality upon short light exposure (Fig. 6B). These results suggested that PPIX accumulates in S. agalactiae when pefAB and pefCD systems are inactivated. There were also marked differences between WT and ΔpefR sensitivity to PPIX after a longer (50 minute) light treatment. The ΔpefR mutant, which is up-regulated for pefAB and pefCD expression, showed essentially full viability, compared to a >10-fold drop for the WT strain. These data, showing heme and PPIX sensitivities of ΔpefAB ΔpefCD mutants, and lower PPIX sensitivity of the ΔpefR mutant give strong evidence for a role of the pef regulon in porphyrin efflux and intracellular homeostasis.
Intracellular porphyrin availability in S. agalactiae WT and ΔpefA ΔpefCD strains
We used two approaches to evaluate differences in intracellular porphyrin in the WT versus ΔpefA ΔpefCD strain. First, we exploited the fact that heme- and PPIX- induce PpefA-lacZ (same as Pgbs1753-lacZ above), to compare induction levels in the WT and ΔpefA ΔpefCD double mutant strains (Fig. 7A). In response to heme and PPIX addition, β-galactosidase activity was respectively about 2-fold and 3-fold increased in the ΔpefA ΔpefCD mutant compared to WT, suggesting that more heme and PPIX are available to activate the pefA (gbs1753) promoter in this mutant.
A. Expression analysis of PpefA-lacZ by β-galactosidase activity determinations was performed in early stationary phase cells of WT and ΔpefA ΔpefCD strains. S. agalactiae and derivatives were grown in BHI liquid medium. Results represent the mean ± standard deviation from triplicate experiments. Asterisks denote statistically significant differences as determined by Student's t-test (p≤0.05). B. PPIX- or heme-dependent production of the E. faecalis catalase KatA in S. agalactiae WT, and ΔpefA ΔpefCD mutant strains. Cells were grown in M17G supplemented or not with 1 µM of PPIX or heme. The loading of equivalent amounts of protein was verified by Coomassie stained gels performed in parallel. KatA was detected in total S. agalactiae protein extracts by immunoblot assays. Results shown are representative of 3 experiments.
Second, we used an in vivo "heme and PPIX sensor": the E. faecalis catalase KatA is degraded if not bound to a porphyrin molecule [19]. Intracellular availability of heme or PPIX was evaluated by comparing the relative stability of the E. faecalis KatA expressed in the WT strain and ΔpefA ΔpefCD and ΔpefR mutants (Fig. 7B and data not shown). Cells were grown without porphyrins or in 1 µM heme or PPIX, and KatA levels were followed in Western blots on cell lysates. Amounts of KatA in the WT and ΔpefR mutant strains were not significantly different (data not shown), which might reflect the limits of this reporter system. However, in the ΔpefA ΔpefCD mutant to which heme or PPIX was added, KatA showed pronounced stabilization, as expected for higher intracellular porphyrin levels in that strain. Results of both in vivo systems used above point to greater availability of porphyrins in the ΔpefA ΔpefCD mutant.
Physiological impact of pefAB and pefRCD activities on S. agalactiae respiration and virulence
S. agalactiae takes up exogenous heme, which activates its membrane cytochrome bd quinol oxidase, and is needed for respiration metabolism; the shift to respiration increases cell density by at least 20% compared to aerobic fermentation growth [17]. We compared WT, ΔpefR, ΔpefA ΔpefCD, ΔpefB ΔpefCD, and the ΔpefR strain complemented by a plasmid-carried pefR gene (ppefR) for their capacity to grow in respiration conditions (Fig. 8A). The WT, ΔpefA ΔpefCD, ΔpefB ΔpefCD, and the complemented ΔpefR strain showed increased growth densities, indicative of respiration growth in these conditions. In contrast, the ΔpefR mutant was not augmented, suggesting a respiration defect of this strain. The pefR::ISS1 insertional mutant gave the same results as the ΔpefR deletion strain (data not shown).
A. Impact on respiration metabolism of deregulating pefAB pefCD expression by pefR inactivation. S. agalactiae WT, ΔpefA ΔpefCD, ΔpefB ΔpefCD, ΔpefR and ΔpefR transformed with vector containing a wild-type copy of pefR (ΔpefR + ppefR) strains were grown in respiration-permissive conditions (i.e., 1 µM heme and 10 µM menaquinone). S. agalactiae respiration growth is characterized by a gain in cell density [17]. Absorbance (OD600) is shown on cultures after 22 h growth. Errors bars represent the standard deviation of three independent experiments. B. Impact on virulence of deregulating pefAB pefCD expression by pefR inactivation. Survival curves in adult mice infected with S. agalactiae WT (black square), ΔpefA ΔpefCD (red diamond), ΔpefB ΔpefCD (gray triangle, dashed line) or ΔpefR (blue triangle) strains. Differences in mortality between mice infected with the WT versus the ΔpefR mutant were statistically significant (p<0.001).
To confirm that the growth difference observed above was due to respiration metabolism, we performed the same growth experiments in the presence of 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), which inhibits respiration but allows fermentation growth (data not shown; [17]). All tested strains except ΔpefR displayed lower growth densities in HQNO, which were comparable to those in aerobic fermentation; HQNO had no effect on cell density of the ΔpefR mutant. These results confirm that all strains tested above (Fig. 8A), except ΔpefR, activated respiration growth.
Respiration growth provokes an increase in oxygen consumption [17]. This property was examined to confirm the respiration-defect of ΔpefR. Oxygen consumption in aerobic fermentation growth by the WT, ΔpefR, and ΔpefR (ppefR) strains was measured as 20.3, 21.0, and 31.1 µM.min−1 per OD600 = 1 cells (Table 1), which in these conditions reflects cytoplasmic NADH oxidase activity [17]. In respiration-permissive conditions, oxygen consumption by the WT and ΔpefR (ppefR) strain was markedly higher (44.2 and 51.6 µM.min−1 respectively), but only moderately higher for the ΔpefR mutant (30.2 µM.min−1) (Table 1), further indicating the respiration-defect in the ΔpefR mutant. These results, together with the above studies, indicate that high PefAB and PefCD efflux activities impact on respiration by diminishing heme availability.
WT, ΔpefAB ΔpefCD and ΔpefR strains were tested in a mouse infection model (Fig. 8B). Virulence of the parental strain, and the ΔpefA ΔpefCD or ΔpefB ΔpefCD mutants was essentially the same. In contrast, the ΔpefR mutant (as well as the pefR::ISS1 insertional mutant; data not shown) showed markedly attenuated virulence. Earlier findings showed that a S. agalactiae respiration-defective mutant is attenuated for virulence [17]. Reduced intracellular heme of pefR leads to a respiration deficiency, which can explain virulence attenuation of this mutant.
The present study uncovers a previously unknown system of porphyrin homeostasis in S. agalactiae, comprising two operons encoding distinct efflux pumps, controlled by a single intracellular regulator. Our results indicate that PefR, like numerous MarR-type proteins, acts as an intracellular sensor to regulate efflux of its ligands [40], [33], in this case, porphyrins. The range of PefR regulation is confined to the pefAB and pefRCD operons, as deduced from the presence of an IR within a 23 bp sequence uniquely upstream of these operons. This configuration is typical of MarR binding sites [33], [41], [42]. Expression of the pefAB and pefCD efflux loci limits S. agalactiae intracellular porphyrin availability; at high pef expression levels, S. agalactiae may be unable to activate respiration metabolism, and to cause septicemic infection in the animal host. The management of intracellular porphyrin levels by efflux pumps, and regulation by an intracellular sensor in S. agalactiae constitutes new information on heme and PPIX homeostasis strategies.
Homology searches revealed that 9 out of 10 species carrying pefAB genes also encode cytochrome bd quinol oxidases, and thus are genetically equipped (like S. agalactiae) to activate a respiration metabolism in the presence of heme, or heme and a menaquinone (Table S2). In contrast, the pefRCD operon seems to be specific to the Streptococcus genus, and is also present in species lacking the respiration genes. Among conditionally respiring bacteria, most characterized S. agalactiae isolates [43], and Streptococcus uberis encode the complete pef regulon. Since members of both these species share the same ecological niche (both are responsible for bovine intramammary infection [44], [45]), the presence of the complete pef regulon might conceivably arise from a genetic transfer event. However, this possibility is unlikely, as the identity between their pef ORFs (73% to 49%) is not higher than the 72% identity between these species at the genome level (Average Nucleotide Identity; calculated as in [46]). Conservation of the pef regulon may indicate a role in survival and/or infection in the particular biotopes of both these species.
Results of this study add heme and PPIX to the inventory of MarR interactants. Several MarR proteins were previously shown to bind to, and regulate efflux of lipophilic and planar molecules, such as antibiotics, aromatic aldehydes or fatty acids, usually as a means to limit cellular toxicity [47],[51]. PefR activity would expectedly be tuned to retain sufficient intracellular heme for functions such as respiration. Indeed, pefAB expression in cells grown with porphyrins is induced to only half the levels compared to a fully induced ΔpefR mutant (data not shown). This, and the fact that the ΔpefR mutant is respiration defective, supports the need for basal levels of intracellular heme. Moreover, the presence of two putative PefR binding sites upstream of pefRCD (versus one upstream of pefAB) might influence the differential regulation of the two loci; possibly, lower levels of pefCD expression could participate in limiting intracellular heme depletion.
PefAB and PefCD add to a handful of other systems described as being involved in porphyrin efflux. The PefAB and PefCD systems share common features with two efflux systems in eukaryotes that control porphyrin pools: Feline leukemia virus receptor C, FLVRC, is an MFS-family protein (like PefA) that exports cytoplasmic metalloporphyrins across an ion-proton gradient [52], [53]. The breast cancer resistance protein Bcrp/ABCG2 in humans is an ABC family transporter (like PefCD) that regulates eukaryotic intracellular heme levels under hypoxic conditions [54], [55]. Just two possible porphyrin efflux systems were described in bacteria, neither of which is an analog of PefAB or PefCD: In E. coli, TolC effluxes PPIX, possibly to facilitate its turnover after iron extraction from heme; neither its regulation, nor its capacity to efflux heme have been reported [5], [56]. In S. aureus, the hrtAB locus is regulated by heme, but not PPIX, and its inactivation provokes accrued heme sensitivity [28], [27]. The S. agalactiae hrtAB homologous system (gbs0120 gbs0119) is likely to have a similar role in limiting heme toxicity. In contrast, the pef regulon is activated by PPIX, and at heme concentrations that do not induce hrtAB.
As PefAB efflux relies on proton motive force (PMF) to export substrates, we speculate that activity would be stimulated by the greater PMF generated under respiration conditions, as shown in L. lactis [57], [17], [58]. Indeed, the PefAB system is conserved in streptococci and lactobacilli having the capacity to activate a respiration metabolism. As PefCD requires ATPase activity, its activity likely varies according to growth conditions [59], [60]. The need for both PefAB and PefCD activities in S. agalactiae may accommodate the different metabolic states encountered in vivo, e.g., anaerobic fermentation in abscess, aerobic fermentation in lungs, and respiration in sepsis.
A model for heme and PPIX homeostasis in S. agalactiae, based on the newly characterized Pef operons, is proposed (Fig. 9): PefR represses expression of pefAB and pefRCD operons. Heme and PPIX are assimilated by unknown mechanisms. Once intracellular, these molecules interact with PefR, which detaches from its binding sites to activate PefAB and PefCD efflux pumps. Activities of the two Pef systems may assure rapid adjustment of intracellular heme levels under different cell physiological conditions. Whether PPIX efflux by pefAB pefCD has a biological role in S. agalactiae as an enzymatic byproduct of iron capture as in E. coli [5] remains to be determined. At higher heme concentrations, HrtAB homologs Gbs0120 and Gbs0119 could be activated via the putative two-component system Gbs0122 (heme sensor) and Gbs0121 (regulator), as was demonstrated for HssRS in S. aureus [27]. This system would have a dominant role in protecting cells in a heme-rich environment, e.g., in conditions of massive degradation of host red blood cells; such a role is consistent with the strong induction of hrtAB (>200-fold) when heme levels are high (Fig. 5). Inactivation of pefAB and pefCD loci results in increased intracellular heme and PPIX pools, with consequences on growth and on heme-dependent enzyme activities; constitutive activity of these operons by pefR inactivation depletes these intracellular pools, resulting in respiration and virulence defects.
Extracellular heme and PPIX may be internalized by as yet unknown transporters. By homology with a recently described system in S. aureus [27], heme, but not PPIX, may bind to a putative external two-component receptor protein Gbs0122, the homolog of S. aureus HssS, resulting in gbs0119 gbs0120 (hrtAB) induction. Once inside the cell, free porphyrin molecules may encounter different binding proteins, including PefR. Apo-PefR binds to pefAB and pefRCD promoter regions to repress their expression. PefR-porphyrin binding releases PefR from DNA, to enable pefAB and pefRCD expression. The PefAB and PefCD loci mediate efflux of free heme and PPIX to avoid toxicity. Overexpression of PefAB and PefCD leads to heme depletion and respiration and virulence defects. Phenotypes of ΔpefR versus ΔpefAB ΔpefCD mutants are shown in the Table below.
A role for respiration functions in virulence has been reported for diverse bacterial pathogens, including Brucella abortus, Shigella flexnei, S. aureus and Mycobacterium tuberculosis; unlike S. agalactiae, these bacteria biosynthesize heme and are autonomous for respiration [61],[64]. In S. agalactiae, respiration is part of the metabolic reprogramming that occurs during the requisite septicemic phase of infection, in heme-rich blood [17]. We suggest that a correlation between unavailability of heme due to high pefAB and pefCD activity, and the respiration defect explains virulence attenuation in the ΔpefR mutant. The use of efflux to regulate metabolic activity of a major bacterial pathogen has not been previously reported, and will lead to a better understanding of how bacteria deal with heme and other porphyrins in the variable conditions they encounter during infection.
Materials and Methods
Ethics statement
All animal experiments were performed in strict accordance with INRA institutional guidelines of good animal practice (Jouy-en-Josas, France), and approved by the Direction des Services Vétérinaires (accreditation number 78-63).
Bacterial strains, growth conditions and plasmids
Strains and plasmids used in this study are described in Tables 2 and 3. NEM316, a S. agalactiae capsular serotype III strain was used as wild type (referred to as WT) and was isolated from a fatal case of septicemia [65]. S. agalactiae and its derivatives were cultivated at 37°C in M17 medium (Oxoid) supplemented with glucose (0.2% for pre-cultures and 1% for test cultures [referred to as M17G]). For β-galactosidase assays, S. agalactiae was grown in BHI (brain heart infusion, Difco Laboratories) supplemented with 0.8% glucose. Aerated cultures were grown under agitation at 200 rpm in a ratio of air space to liquid of approximately 5/1. Respiring cultures were grown under agitation in the presence of 1 µM hemin (from a stock solution of 10 mM hemin chloride, Fluka) and 10 µM vitamin K2 (a menaquinone; Sigma). E. coli was cultivated in LB medium (Luria-Bertani, Difco Laboratories) at 37°C with aeration by shaking at 180 rpm. Antibiotics were used as needed at the following concentrations: for S. agalactiae, 5 µg/ml erythromycin, 1000 µg/ml kanamycin, 4 µg/ml chloramphenicol, 5 µg/ml tetracycline; for E. coli, 100 µg/ml erythromycin, 20 µg/ml chloramphenicol.
RNA extraction, Northern blot and RACE PCR
RNA was extracted from S. agalactiae cultures as described [66]. Cells from 20 ml culture with an OD600 of 0.5∼0.8 (exponential growth phase) or OD600∼1.5 were harvested by centrifugation at 6000×g during 10 min. Total RNA was extracted with a guanidine isothycionate and phenol-chloroform step [67], using the TRIzol Reagent (Invitrogen).
For Northern blot analysis, RNA samples (30 µg) were mixed with an equal volume of glyoxal load dye (Ambion) and were electrophoretically separated on a 0.9% agarose glyoxal gel [68]. RNA samples were transferred to a Biodyne B membrane (Pall) according to manufacturer's instructions. Hybridization and detection were performed using ECL direct nucleic acid labeling and detection systems (Amersham). A ∼500 base pair (bp) DNA labeled fragment of each ORF tested was used as probe. Primers used for probe generation are shown in Table S3.
The pefA transcriptional start was mapped using a 5′/3′ rapid amplification cDNA ends (RACE) kit, second generation (Roche Applied Science) according to supplier's instructions. Mapping was realized with RNA extracted from respiring cells, using primers: 5′TAGATGTAGGTGCTAACGTCG3′ and 5′CTTGTTGAGCCGTTGACAACG3′.
β-galactosidase assays
Plasmid pTCV-lac is a low copy number plasmid used to evaluate promoter activities in S. agalactiae [32]. A DNA fragment containing gbs1753 promoter was PCR-amplified with primers 5′GCGTAGAATTCATTAAATGGAG3′, containing an EcoRI site (underlined) and 5′TATCTCGGATCCCTATTTCTGAT3′, containing a BamHI site (underlined). After digestion of the amplified fragment by EcoRI and BamHI, the gbs1753 promoter was cloned into plasmid pTCV-lac, resulting in plasmid pTCV-J11 carrying the Pgbs1753-lacZ (or PpefA-lacZ) fusion. The plasmids pTCV-lac and pTCV-J11 were subsequently transformed into S. agalactiae by electroporation and recombinant clones were obtained by erythromycin selection. The same strategy was performed to generate a Pgbs0119-lacZ fusion using the primers 5′ATACGCCAGAATTCTCGGCGAC3′ and 5′CCTTGGATCCTTTGATGTGAAC3′, giving rise to plasmid pTCV-J21.
Mutagenesis and screening conditions
Random insertional mutagenesis was performed on S. agalactiae with pG+host8::ISS1 basically as described [69]. Both Pgbs1753-lacZ (reporter for gbs1753 transcription) and pG+host8::ISS1 plasmids were established in S. agalactiae. The strain was grown at 30°C with kanamycin for 2.5 h. The temperature was then shifted to 37°C for 2.5 h and plated on M17G with erythromycin, kanamycin and 80 µg/ml X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside, BioMerieux), to obtain ∼100 CFU per plate. After 24 h incubation at 37°C, deep blue colonies were selected. Stable ISS1 mutants were isolated by growth at 30°C without erythromycin as described [70]. The flanking chromosomal DNA was sequenced using primer 5′AGGGCATGGAAACAATTCGAGG3′. From the 5000 mutants we screened, 7 were in the pefR locus. Stable ISS1 mutants were then retested for gbs1753 up-regulation.
Gbs1402 (PefR) overexpression and purification
The gbs1402 ORF was amplified by PCR from S. agalactiae NEM316 using primers 5′CACCATGGAGAATCCTCTTCAAAA3′ and 5′TAATCAAAATCTTCCATCGCC3′. This amplified fragment was cloned into the E. coli expression plasmid Champion pET100 directional TOPO vector (Invitrogen) and transformed into E. coli TOP10. After plasmid verification by sequencing, the plasmid pET100-J14 was electroporated in E. coli BL21 (DE3). Protein expression was induced at an OD600 = 0.6 by addition of 1 mM IPTG for 2 h. Cells were harvested by centrifugation at 6000 rpm for 10 min and resuspended in 10 ml binding buffer (Tris HCl 50 mM pH 8, NaCl 300 mM, Imidazole 50 mM). Cells were broken by shaking with glass beads in a Fast Prep apparatus (MP Biomedicals). The lysate was centrifuged at 14000 rpm for 15 min at 4°C. The recombinant His-tagged fusion protein was purified by nickel affinity chromatography using His-Select affinity (Sigma) equilibrated with binding buffer according to supplier's instructions. The elution was carried out with binding buffer containing 300 mM imidazole. All fractions were collected and analyzed by SDS-PAGE. Purified proteins were dialyzed against 50 mM Tris HCl pH 8, 80 mM NaCl and 6.25% glycerol. Protein concentrations were determined by the Lowry assay (Bio-Rad).
Immunoblotting analysis
Strains harboring PaphA-3-katA-His6 plasmid were incubated overnight under static conditions with or without 1 µM heme or PPIX. Cell growth was harvested by centrifugation at 6000×g during 10 min. Cells were broken by shaking with glass beads in a Fast Prep apparatus (MP Biomedicals). Immunoblotting analysis was done with rabbit anti-KatA antiserum as described, and kindly provided by Dr. L. Hederstedt [19]. Detection was performed using ECL Plus Western blotting detection reagents (GE Healthcare).
Electrophoretic mobility shift assay (EMSA)
A 281 bp DNA fragment containing the gbs1753 promoter region and a 248 bp DNA fragment containing the gbs1402 promoter region were generated by PCR from genomic DNA of S. agalactiae and purified using the PureLink PCR Purification Kit (Invitrogen). Binding assays with PefR were carried out using a binding buffer (5% glycerol, 20 mM Tris HCl pH 8, 50 mM KCl, 0.2 mM MgCl2, 1 mM EDTA, 0.2 mM DTT, 0.3 µM BSA) in a final volume of 15 µl at 37°C. Reaction mixtures were incubated at 37°C for 20 min, after which they were analyzed by gel electrophoresis on an 8% polyacrylamide gel in TBE 1× buffer, stained with ethidium bromide. Reactions were carried out in the presence of a 116 bp non-specific competitor DNA (internal coding gbs1753 sequence) where specified. Where tested, heme or PPIX were added 20 min after protein and DNA components. Gel shift experiments were performed with two independent batches of purified PefR.
Construction of Δgbs1753 (ΔpefA), Δgbs1752 (ΔpefB), Δgbs1401 Δgbs1400 (ΔpefCD) and Δgbs1402 (ΔpefR) mutants and ppefR plasmid construction
In-frame pefA, pefB, pefCD, and pefR deletion mutants were constructed using the strategy described below. Briefly, the two regions flanking the locus to be deleted were independently amplified by PCR (see Table S3 for primers). Amplified fragments were digested by HindIII or EcoRV enzyme and ligated to each other. The resulting fragments were amplified by PCR, digested by EcoRI enzyme and cloned into the thermosensitive shuttle plasmid pG+host5. Electroporation of S. agalactiae strains and allelic exchange were done as described [70]. In-frame deletions were confirmed by PCR and sequence analysis. The pefR gene was amplified (Table S3 for primers), and cloned using EcoRI and BamHI on fragment ends, onto plasmid pRC2 cut with the same enzymes; the resulting plasmid, pRC2-J14, is referred to as ppefR in Results.
PPIX and metalloporphyrin sensitivity tests
Stock solutions (10 mM) of PPIX, GaPPIX, ZnPPIX, and ZnMPIX (Frontier Scientific) were prepared in DMSO. PPIX sensitivity of S. agalactiae NEM316 and its derivatives was examined on early stationary growing cells (OD600 = 1.6). For PPIX light sensitivity tests, serial dilutions were spotted on plates and exposed to visible light for 0, 10 or 50 minutes. A 500 W halogen lamp with a glass lid placed at a distance of 55 cm was used as light source. Metalloporphyrin sensitivity tests on plates were performed as described [12]: Briefly, 1∶100 dilutions of stationary-phase cultures were prepared in M17G soft (0.7%) agar, and poured over M17G plates. Metalloporphyrin (heme, 5 µl of a 0.2 mM solution; GaPPIX, 5 µl of a 10 mM solution) was spotted onto the plates. All plates were incubated for 20 h at 37°C and then photographed.
Oxygen consumption.measurements
Oxygen consumption was determined as described [17]. Briefly, S. agalactiae WT and ΔpefR late exponential phase cultures, prepared in aerobic fermentation or respiration conditions, were washed twice with PBS at 4°C and resuspended in the same buffer to obtain a 1 ml bacterial suspension at OD600 = 1.0. Oxygen consumption was followed with a Clark-type oxygen electrode (Liquid-Phase Oxygen electrode unit DW1, Hansatech instruments). The maximum oxygen consumption rate was measured following the addition of 10 mM glucose.
Mouse virulence assay
Pathogen-free 6 week-old Swiss CD1 mice (Charles River laboratories) were infected intravenously with S. agalactiae NEM316 or derivatives. Groups of 7–8 mice were anesthetized with ketamine (100 µg/g, Merial) and xylazine (12 µg/g, Bayer) and were inoculated in the eye vein with 108 CFU. Bacterial counts were determined in parallel by plating serial dilutions in saline buffer on M17G. Cells were harvested from mid-log phase and washed in a solution of NaCl 0.9%. Mortality was observed over a 10-day period. Animal experiments were performed in triplicate.
To find the conserved sequence between pef promoter regions, sequence were aligned with ClustalW and manually refined. A search for the 23 bp consensus in S. agalactiae NEM316 genome was done by visual inspection, and using BlastN program with EXPECT threshold set at 100. Pef protein homologs were identified using Genome Region Comparison, http://cmr.jcvi.org/cgi-bin/CMR/CmrHomePage.cgi, and/or were retrieved from genomic Blast databases, http://blast.ncbi.nlm.nih.gov/Blast.cgi/, using BlastP program with an E-value threshold set at 0.01.
Supporting Information
Figure S1.
A. PPIX displaces PefR from its DNA target. Gel mobility shift analysis of the effect of PPIX on PefR binding to the pefAB (right) and pefRCD (left) promoter regions. Samples contained 2 pmoles of PpefAB or PpefRCD fragment and 30 pmoles of PefR, to which were added either sample buffer, 120 pmoles (left) or 240 pmoles (right) of PPIX. On the two top panels, lanes 1–2 were juxtaposed to lane 3, which were separated on the initial same gel. B. Heme, but not iron, displaces PefR from its DNA target. Samples contained 2 pmoles of PpefAB fragment and 32 pmoles of PefR, to which were added either sample buffer, 160 pmoles heme, or 160 pmoles iron. Lanes 1–2 were juxtaposed to lane 3–4, which were separated on the initial same gel.
Figure S2.
Differential heme (FePPIX) and GaPPIX sensitivity of the pef mutants. Stationary phase cultures of WT, ΔpefR, ΔpefA ΔpefCD or ΔpefB ΔpefCD strains were spread on M17G plates and 1 nmole of heme (A) or 50 nmoles of GaPPIX (B) were pipetted directed onto plates. Plates were incubated 24 hours, and then photographed. Representative results of at least 3 experiments are shown.
Table S1.
Transcriptome analysis in respiration versus aerobic fermentation conditions. Cells were grown in M17 medium with 1% glucose, supplemented or not with a mixture of 10 µM vitamin K2 (a menaquinone) and 48 µg/ml hemoglobin as heme source. Cells were harvested at OD600 = 0.3 for RNA extraction. Total RNA was extracted and analyzed by hybridization on NEM316 derived whole genome DNA macroarray as described [66]. Fold change in expression is the mean of two independent macroarray experiments. Shown are results of S. agalactiae genes whose expression was induced or repressed at least 2-fold in respiration compared to aeration conditions. Genes further studied in this work are in bold. Gene assignments are according to the Sagalist web site (http://genolist.pasteur.fr/SagaList/) and BLAST searches (http://blast.ncbi.nlm.nih.gov/Blast.cgi/).
Table S2.
Distribution of pef regulon and cytochrome genes among Lactobacillales. * Reorganization of gene or domain order; # frameshift mutation in pefA homologous gene. In bold, species containing the complete pef regulon; ** Imperfect repeats, characteristic of MarR DNA binding sites, are present in respective pefAB and pefRCD promoter regions of this strain; *** gbs1401 and gbs1400 ORFs are missing in S. agalactiae 2603V/R [43].
Table S3.
Primers used in this study.
We are grateful to IERP for technical assistance in animal studies. We thank Lars Hederstedt (Lund University, Sweden) for the generous gift of plasmid and anti-KatA antibody. We are grateful to Marie-Agnès Petit for application of ANI program and for critical and thoughtful reading of the manuscript, Gilles Lamberet for precious help in oxygen uptake determinations and Olivier Son and Isabelle Poquet for advice in gel shift assays (Micalis). We thank Cécile Wandersman, Patrick Trieu-Cuot, and Philippe Glaser (Institut Pasteur), Claire Poyart (Hôpital Cochin), and Philippe Bouloc (Institut de Génétique et Microbiologie) for stimulating discussions, and the members of the Micalis 'Batiment 222' lab for their support in the course of this study.
Author Contributions
Conceived and designed the experiments: AF DL PG AG. Performed the experiments: AF DL ADB EC. Analyzed the data: AF DL PG AG. Wrote the paper: AF AG.
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text:
Russia's Drive for Global Economic Power: A Challenge for the Obama Administration - Россия наращивает мировую экономическую мощь, что вызывает беспокойство у администрации Обамы.
|Russia's Drive for Global Economic Power: A Challenge for the Obama Administration
| Russia's Drive for Global Economic Power:
A Challenge for the Obama Administration
By Ariel Cohen and Lajos Szaszdi.
January 30, 2009
Until the recent global financial crisis, Russia's economic revival during the presidency of Vladimir Putin had helped to restore the country's standing as a major player in the world arena. Yet, prosperity has come with some unintended consequences. Russia's invasion of Georgia was fueled by Russia's economic growth and newfound wealth.
This economic comeback is largely the result of Russia's oil and natural gas exports, coupled with the high prices that other Russian commodities have enjoyed in world markets. With the seventh-largest oil reserves and the largest gas reserves in the world, and as the leading exporter of oil and gas, the Kremlin is using its energy exports, revenue from arms and metals sales, and investments abroad in the mining and energy sectors to extend Russia's influence worldwide.
The interruption of gas supply to Ukraine and the rest of Europe in January 2009 resulted in the worst energy crisis in Europe since the Arab Oil Embargo of 1973, and once again raised questions about Russia's reliability as an energy supplier. In the recent past, Russia has already prevented Caspian oil and gas supplies from flowing freely to the European markets; has threatened to disrupt oil exports that pass through Georgian territory when it invaded Georgia last August; has acquired, and is in the process of acquiring, major European energy companies, as well as pipelines, refineries, and other assets in more than a dozen countries. Moscow is also targeting the strategic Middle Eastern oil sector and is displacing Western energy companies operating in OPEC founding member Venezuela.
Beyond that, Russia has dominant global positions in the strategic and precious metals sectors including titanium, platinum, and other precious metals used in aerospace industries, electronics, and military and automotive production. A major Kremlin-connected oligarch owns the world's largest aluminum company and has been accused of corrupt practices in the U.S., Germany, Nigeria, and Guinea, while the Russian banking sector is tied in with organized crime.
Moscow's expanding business interests have made Europe highly – and dangerously – dependent on Russian oil, gas, and raw materials. Russia currently supplies two-thirds of Europe's imported natural gas – 42 percent of total European consumption; Central and Eastern European countries depend on Russian gas for more than 90 percent of their needs. By 2030, Europe will import 84 percent of its gas needs. Europe has not developed alternative sources of gas, and has rejected nuclear power and coal. Since natural gas is supplied by pipelines controlled by Gazprom, the Russian state gas monopoly, these countries cannot easily turn to other suppliers. Thus, Europe has tied itself to dependence on a commodity supplier with a track record of geopolitical intimidation as opposed to a free-market relationship.
Severe repercussions for Europe's national security dependence on Russian energy are widely recognized by the European Union and individual countries. Europe has now "stepped up its attempts to reduce its exposure to potential Russian blackmail over energy supplies," reports Ian Traynor in The Guardian. The European Commission unveiled "an ambitious strategy aimed at weakening Russian giant Gazprom's domination of Europe's gas imports." "We must not sleepwalk into Europe's energy dependence crisis," said Jose Manuel Barroso, EU Commission President. Russia is trying to replicate this model in other areas as well, such as electricity and raw-materials exports by state-owned corporations, as demonstrated below.
Russia also aims to become a major energy supplier and provider of raw materials to countries of the Asia-Pacific region, including China, Japan, South Korea, and the United States. Such a goal, if accomplished, will greatly enhance Russian leverage in the Pacific Rim.
The Tools for Global Cooperation
The U.S. should cooperate with its friends and allies on combating excessive dependency (beyond 25-30 percent) on Russian strategic raw materials and energy exports, such as oil, gas, coal, and electricity. What is needed is a global security system for tracking investment activities by Russia and other anti-Western governments in industries and sectors with defense and security implications.
One of those tools is the Committee on Foreign Investment in the United States (CFIUS). CFIUS is an inter-agency committee of the United States government that reviews the national security implications of foreign investments in U.S. companies or operations. Chaired by the Secretary of the Treasury, CFIUS coordinates representatives from nine U.S. agencies including the Departments of Defense, State, Commerce, and Homeland Security.
The U.S. Treasury recently published final rules to strengthen security reviews of foreign investments in U.S. businesses. As the former Treasury Secretary Henry Paulson put it, the final regulations are intended to "strengthen the CFIUS process in a manner that reaffirms America's longstanding policy of openness to investment, consistent with the protection of our national security." The regulations clarify that transactions in which a foreign entity acquires less than a 10 percent stake in a U.S. business are not automatically exempt from a CFIUS review. Under the new procedures, a foreign investor in a U.S. business considered "critical infrastructure" is encouraged to consult with the CFIUS panel before filing a formal notice. This is a wise step in improving oversight of investments in critical infrastructure, resources, and financial systems on which our nation and our alliances depend.
The U.S. should also increase cooperative effortsamong the international intelligence and law enforcement agencies and independent experts to keep track of how the Russian state and oligarchs may be laundering money and engaging in corruption and unfair competition. The Obama Administration should encourage, without dictating investment decisions,U.S. and other multinational companies to compete with Russian companies like Gazprom for pipeline and energy projects, as well as promote alternative market-based sources of energy and unconventional sources of fuels worldwide to counter any over-dependency on energy from countries such as Russia, Iran and Venezuela, which overtly seek to counter the West's economic and military strength.
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Russia and Greece call for a Cyprus settlement based on UN resolutions, Lavrov says after his talks with Kotzias
The Cyprus problem was one of the issues discussed during the meeting which the Foreign Ministers of Russia and Greece, Sergey Lavrov and Nikos Kotzias, had on Wednesday in Moscow.
In his statements at a joint press conference, the Russian Foreign Minister said, as reported by Tass news agency, that Moscow and Athens have similar or the same stance on resolving regional conflicts. "We exchanged views on the Cyprus issue," he said, adding that "Russia and Greece both call for resolving this long-standing issue based on United Nations Security Council resolutions and agreements reached by the two communities."
On his part, Kotzias expressed hope that "we will reopen the Cyprus issue � in the right way � and resolve that as well." He noted that this is a more difficult issue than the dispute over North Macedonia's name and the issues that have been pending with Albania "because in those two we were the larger party and we were determined to resolve the issues."
Moreover, Kotzias said that he thanked Lavrov, and he wants to do so publicly, for Russia's stance on the Cyprus issue,
Cyprus has been divided since 1974, when Turkish troops invaded and occupied 37% of its territory.
Source: Cyprus News Agency


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Our resident Pugs at the time where George & his little brother Archie. Two very naughty Pugs, perhaps among the naughtiest Pugs in the world! But then i guess we all think that of our Pugs! Full of energy and mischief. George was a Majestically Handsome Black Boy. Archie is Cute, Cheeky Chops, Fawn Boy. He's
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Archie was very lonely and our 2 young children missed George's massive character so much we decided to look for another friend for Archie and to are suprise shortly after loosing George Miss Rosie joined our family.
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It took a couple of weeks but Archie and Rosie are now best of pals
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