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WatchGuard Firebox Peak X5500e firewall
WatchGuard Firebox: Fiery performer at a nice price
- Client/server-based management system allows true offline editing of configuration, high throughput even when handling attacks, can turn on additional in-the-box features through licensing
- Blocked only a third of the attacks in our test, complex user interface, desperately needs wizards for common setup tasks (public server, VPN), must be online for initial setup, to download updates and user interface
Bottom Line
WatchGuard Firebox Peak X5500e isn't easy to set up, but its use of XML configuration files works wonders for managing configuration across any number of devices and locations. Apart from complex initial configuration, this is a highly manageable, enterprise-grade, proxy-based firewall with impressive throughput, granular control, and an excellent price.
Price$ 5,990.00 (AUD)
When we first began working with the Firebox, we got very frustrated with all of the reboots we had to suffer through while making what we considered minor changes (IP, subnet mask, and so on). But that's because we didn't yet understand WatchGuard's client-server attitude toward configuration. Clearly enterprise in nature, the thick configuration utility wants you to check your configuration changes before you commit them. It's not a handy Web utility that could accidentally paint you into a corner. It wants you to make your changes as a single update so that individual changes can be considered before you hit the return key.
With the Firebox, you could easily have an entire lab configuration (sandbox) to do some initial testing, then pre-edit the changes necessary to drop the config into production. By the same token, you could remove a troubled unit from production and flip it into a lab setting to confirm or deny problems. WatchGuard allows you to save configuration files and swap between them really easily, regardless of whether you're touching the original serial number that the configuration was built on.
WatchGuard's client server approach started us thinking about how well the Firebox line fits regardless of your company size. From the SMB-oriented single console to a team approach with undocked windows spread across the front wall of a NOC, you could find a version of WatchGuard's hardware and combination of software that should fit your needs. This is a stratified product line with software upgrades within the hardware platform allowing you to fit the cost of the unit to your immediate needs but still permitting an easy upgrade path. From smaller Edge units to the Core SMB units all the way to the larger Peak units, the Firebox product line has granular layers allowing a much closer fit to individual company needs. The same stratification can work just as well within a highly distributed enterprise; with varying levels of authority, I could easily see firewall management becoming a team sport.
GUI or CLI?
While we were, in general, impressed by the WatchGuard, it wasn't perfect. The most significant hassle, though, came from the manufacturer's packaging rather than the basic system design; there was no software at all on the CD-ROM, nor were you able to download it from the Firebox's console. You must be able to download it from the WatchGuard site, and the first setup must be on an Internet-connected link since the system wants to do "activation." We asked about this and got the impression from WatchGuard that there is a way around this if you're using it on an isolated network, but that way is not covered in the startup guide (nor is it freely offered by the company's technical support).
Once we got past WatchGuard's system maintenance window and were able to download the Firebox Manager, it wasn't too bad to get through the initial setup. We were advised, though, to not use both the GUI and the CLI since the configs are stored differently. We were told to use one or the other -- a shame since, on so many systems, the GUI is perfect for simple configuration touch-ups while the CLI is there for the heavy lifting. For initial setup, we used the front-panel buttons to give the Firebox an IP address, then connected using the Firebox Manager. You can also do it using the included serial cable to avoid the pain of countless arrow pushes to change the IP address.
Even with the extensive testing (accompanied by the necessary extensive configuration and management that goes with spending weeks on a device's console), we weren't able to work with every single feature on each system. The supercool feature that we couldn't try out on the WatchGuard was the drag-and-drop VPN setup. As long as the console is able to get an encrypted link to both firewalls, you can do a drag and drop from the branch office to the home office for VPN setup.
Speed to burn
With a proxy-oriented architecture such as the Firebox's, you expect to take a hit in absolute packet-passing performance. Typically what you lose in throughput you gain in security, thanks to the proxy's ability to obscure the details of the devices inside the network from the outside world, making it nearly impossible for external devices to connect to them directly. So we were surprised to discover that the Firebox was the fastest UTM in our test -- faster even than the SonicWall, which costs three times as much.
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text:
Rating: 5 out of 5 stars
...What a refreshing and splendidly unique series this is!! I am exceedingly enjoying the inventive twists, new characters, and changed circumstances Maria Grace has created through this series. While some Pride and Prejudice variations may throw one or two wrenches into the plot, Ms. Grace has effectively thrown half a dozen, and you know what? They all seem to work. I am all eagerness to see how everything will pan out in Vol. 3.

subset: commoncrawl


text:
January 26, 2014
Listening to it as a child
Both. I like playing and listening to various styles.
Tough question! I'd have to say all of them
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Hebert, Bellon united during nuptial mass
Susan Marie Hebert became the bride of Kristopher John Bellon during a 7 p.m. nuptials mass on Friday, June 27, 2008. Vows were exchanged at Our Lady Help of Christians Catholic Church in Jennings with Father Charles McMillin officiating.
The bride-to-be is the daughter of David and Beth Hebert of Iota. She is the granddaughter of Mr. and Mrs. Clayton Brooks of Conover, North Carolina and Mrs. Catherine Brooks of Calvert City, Kentucky and the late Mr. and Mrs. Elroy Hebert.
Susan is a 2005 graduate of Iota High School and has attended Louisiana State University at Eunice. She is currently attending McNeese State University where she is majoring in early childhood education.
The groom-elect is the grandson of Mr. and Mrs. John Bellon of Tepetate, Louisiana and Mr. and Mrs. Ronald Dommert of Egan.
Kristopher is a 2003 graduate of Iota High School and attended Louisiana State University at Eunice. He is currently employed with Island Operating Company.
Escorted by her father, the bride was radiant in a strapless organza ball gown featuring a sweetheart neckline. Exquisite beads, pearls and crystals accented the corset top and flowed through the skirt asymmetrically. Multiple inverted pleats at the back and the side gave fullness to the a-line skirt which cascaded into a chapel length train. To complete her ensemble, the bride wore a silver tiara accented with Swarovski crystals complemented by a fingertip veil. She carried a hand tied bouquet of fresh, stemmed flowers of creme Vendella and pink tipped roses.
In keeping with tradition the bride wore a sixpence as something old, her dress as something new, her grandmother's rosary as something borrowed and something blue was her garter.
Attending as matron of honor was Sherry H. Ashford, cousin of the bride; and serving as maid of honor was Katie LaHaye, friend of the bride. Bridal attendants were Rachael Murray, sister of the bride; Amanda Darbonne, cousin of the bride; Kim Dommert, aunt of the groom; and Brittney Hebert, niece of the bride; served as junior bridesmaid. Bridal attendants were beautifully attired in floor length fuscia strapless evening gowns adorned with crystal beading. They carried fresh white roses.
Chosen to stand as best man was Browdie Bellon, brother of the groom. Groomsmen were Buddy Murray, brother-in-law of the bride; Rhett Hebert, cousin of the bride; Andrew Bellon, brother of the groom; Brian Hebert, brother of the bride; and Gavin Paul Ashford served as the miniature groomsmen.
Upon entering the church, guests were ushered to their seats by Ethan Pelsia, cousin of the bride. Participants during the ceremony were Vocalist David J. Medus, Organist Shane R. Monds, accompanied by Trumpeter Kip A. Faulk, M.M.
Scriptural readers included Sheila Sattler, and Joe Bellon.
The ring bearer was Kaleb Murray, nephew of the bride. Others participating during the ceremony were MaKayla Pelsia and Gabrielle Ashford, who greeted guests as they arrived at the church.
On her daughter's special day, the mother of the bride wore a lavender three-piece Dupioni silk suit accented with crystal beading, while the mother of the groom chose to wear a floor length a-line burgundy dress with satin accents. Both were presented with a shoulder corsage of miniature porcelana roses.
Following the ceremony, a reception was held at the Jeff Davis Multipurpose Building in Jennings. The building was elegantly decorated with fresh flowers and greenery throughout.
The wedding cake was a three-tiered confection overlaid with ivory tinted fondant. Each tier was encircled with fondant ribbons. The bride's cake was served by Pam Pelsia.
The groom had a chocolate cake featuring a Louisiana State University theme and was served by Phyllis Maybrier.
Out of town guests registered from North Carolina, Kentucky and Missouri.
Upon returning from a wedding trip to Playa del Carmen, Mexico, the couple will reside in Iota.
On the night prior to the wedding, a rehearsal dinner was held at the St. Joseph Catholic Church Hall in Evangeline. The dinner was hosted by John and Karla Bellon, parents of the groom.


text:
Cemetery Girl
by David Bell
- #3421 in Mystery & Suspense, Thrillers
- #2527 in Fiction & Literature, Thrillers
Get eBook
- Your price$11.99
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A missing child is every parent's nightmare. What comes next is even worse in this riveting thriller from the bestselling author of Since She Went Away and Somebody I Used to Know.
Tom and Abby Stuart had everything: a perfect marriage, successful careers, and a beautiful twelve-year-old daughter, Caitlin. Then one day she vanished without a trace. For a while they grasped at every false hope and followed every empty lead, but the tragedy ended up changing their lives, overwhelming them with guilt and dread, and shattering their marriage.
Four years later, Caitlin is found alive—dirty and disheveled, yet preternaturally calm. She won't discuss where she was or what happened. And when the police arrest a suspect connected to the disappearance, Caitlin refuses to testify, leaving the Stuarts with a choice: Let the man who may be responsible for destroying their lives walk away, or take matters into their own hands. And when Tom decides to try to uncover the truth for himself, he finds that nothing that has happened yet can prepare him for what he is about to discover.
From the Trade Paperback edition.
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This book is very good it keeps your attention from the very beginning and you want to know more! This book tugs on your emotion to the very end!by Erica R on January 23, 2014
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by David Bell
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Ronald A. DeReu, 79, of Annawan, formerly of Atkinson, passed away Wednesday, Sept. 12, 2012, at Geneseo Good Samaritan Village.
A funeral Mass will be at 10 a.m. Monday, Sept. 17, at St. Anthony's Catholic Church, Atkinson, the Rev. R. Anthony Lee officiating. Burial will be in St. Anthony's Cemetery, Atkinson, where military honors will be accorded by the Atkinson American Legion post. Visitation will be from 4-7 p.m. Sunday at Vandemore Funeral Home, Atkinson, with a prayer service at 3:30 p.m. Memorials may be directed to the Atkinson Rescue Squad or the Hammond Henry Hospital Foundation.
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47 years of experience
Riviera Beach, FL 33410 Phone number & directions
A medical license is required for a doctor to practice medicine in a particular state. Requirements vary by state but most require, at a minimum, post-graduate training in the doctor's specific specialty. An NPI number is a national identifier unique to an individual health care provider and is managed by the Center for Medicare & Medicaid Services (CMS).
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text:
Strings should be double quoted. Please refer to the line protocol documentation. https://docs.influxdata.com/influxdb/v1.0/write_protocols/line_protocol_tutorial/
On Saturday, September 17, 2016 at 6:24:07 AM UTC-7, bf3...@gmail.com wrote: > Hi, > > I'm new to influx so I might be missing something. I'm using the line > protocol to insert measurements into influx. At times I use the > "value=%d,msg=<string>" mechanism to add string information to the value > field. I've noticed that I get an error when the msg string contains white > space. Is this expected? If I remove the white space from the msg the > insertion process works. > > Is this expected that strings in mgs can't contain white space? Or am I > doing something wrong on my side? > > Thanks -- Remember to include the InfluxDB version number with all issue reports --- You received this message because you are subscribed to the Google Groups "InfluxDB" group. To unsubscribe from this group and stop receiving emails from it, send an email to influxdb+unsubscr...@googlegroups.com. To post to this group, send email to firstname.lastname@example.com. Visit this group at https://groups.google.com/group/influxdb. To view this discussion on the web visit https://groups.google.com/d/msgid/influxdb/39cde925-8ddd-455c-8998-73cbaff15a38%40googlegroups.com. For more options, visit https://groups.google.com/d/optout.


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The following steps explain how to install your QuickBooks Financial Software on a Terminal Server.
Note: While QuickBooks can run in a Terminal Server environment, only QuickBooks Enterprise Solutions is supported in this environment (with limitations). Enterprise Solutions support engineers do not provide support for the following:
- Setting up, configuring, or enabling terminal services.
- Enabling clients to connect to the server.
- User permission issues for server connections.
- Issues with terminal server connections (for example, connecting to the server or dropping connection).
- Latency or slow performance issues.
Note: Terminal Service is a feature of Windows Server Operating Systems. We recommend researching Terminal Services or engaging a Windows Server professional before making changes in such an environment.
Detailed Instructions
To install QuickBooks on a terminal server.
Note: Log on to the terminal server as an administrator in order to perform this task and close all applications.
Open the Command Prompt.
- On your keyboard, press Windows logo+R. The Run dialog box opens.
- Enter CMD then click OK. The Command Prompt window opens.
- Change the user mode by using this command:
- change user /install then press Enter.
- You could go ahead and safely install the QuickBooks application, but once the application is installed, change back to execution mode by using this command:
- In the Command Prompt, enter change user /execute then press Enter.
To grant permission to log on to QuickBooks Enterprise
Note: You may need to log in as the system administrator to use the following steps to grant the needed permissions.
- Right-click the QuickBooks Enterprise Solutions shortcut on your desktop and select Properties.
- Click the Compatibility tab.
- Select the Allow Non-Administrators to Run This Program option.
- Click Apply, and then click Save.


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As we learn more about bacteria, it is becoming ever clearer that they interact with themselves and the environment with more sophistication than we ever dared to imagine. Of course understanding as much as possible about bacteria's day to day life is very important, as it comes with countless implications for how we understand and treat disease.
In spite of the surplus of women on University campuses, a casual sex culture is thriving. Well, hasn't that always been the case, you might say, and why shouldn't students enjoy a bit of fun?
A team of scientists from the University of Oxford's Wildlife Conservation Research Unit (WildCRU) and World Animal Protection are looking to combine both original thinking and citizen science into a single initiative. Specifically, we are calling out to the world's most creative minds - "Can you think for tigers?"
As the world's leaders come together to discuss climate change, cooling must be part of the debate around global warming. Cooling is currently part of the worsening problem, but it could become an important part of the solution.
In the last week alone, more than 10 million people have visited the NHS Choices website. Often viewed as the public go-to place for a wide-range of health information, it is somewhere many of us turn to when feeling under the weather or looking for advice.
Animals are potentially suffering and it is right to acknowledge that in full, but they are also not suffering for no reason. Granted, they are likely suffering less than the general public already believe them to be.
Morally the blank slate is indefensible as well as intellectually. Is it better to say that discrimination is wrong because everyone is indistinguishable, or that we have free will because people's actions are mysterious? Well no Dr. Pinker rightly points out.
Bristol's commitment to living in a responsible, sustainable way should inspire cities across the globe to join us in building a society we shall be happy to pass on to the next generation. I will continue to push for this locally, nationally and internationally.
Put simply, we are the victims of our own ingenuity. Rapid industrialisation, increased birth and survival rates, and the "green revolution" in food production mean that there are more humans than ever before: in turn this drives more consumption, more need, more demand for resources such as coal and oil that are ultimately damaging to the ecosystems we depend on for our own survival.
It can only be good news that the British public are proving not just to be open-minded, sober and relaxed, but positive and ambitious about AI and the possibilities that AI could bring.
Male spinner dolphins also have odd-looking upturned or curled tail flukes. This might be yet another anatomical oddity that is meant to attract the ladies. Or it might help them swim faster. Nobody knows. Bottom line: adult male spinner dolphins look like they've been crafted out of playdough by a toddler who has no idea what a dolphin should look like.
These unconventional dishes may seem completely bizarre and perhaps stomach-churning to us now, but in the future they could help to solve a global food crisis. Over the next 35 years, the world's population is expected to exceed nine billion, meaning there will be an extra two billion hungry mouths to feed.
Last weekend Art Kinetica set up camp in the Hospital Club, London for a three day festival: Gravity. The festival and accompanying exhibition ties in with the 100th year anniversary of Einstein's Theory of Relativity and marks the start of a three month residency which will showcase the work of 17 'transdisciplinary' artists.
The BRICs should set their local industries the challenge of coming up with the drugs and diagnostics that their citizens need. While the original vision for the BRIC countries in 2001 is on its way to becoming a reality, there is still so much potential to tap. The issue of AMR is one area amongst many, where these nations can lead the world. We hope they take this opportunity.
While lambs' hearts, horsehair and blown glass artillery shells may seem an unlikely combination with which to spark debate around the cultural phenomena of remembrance, these are the materials I've used for my delicate Papaver rhoeas poppy sculptures, currently on show in London.
Word is out. Belief in God will make your children less moral people, so say researchers from the University of Chicago. It's hit the news too, and looking at the comments sections, boy, those un-judgmental atheists are really showing how humble they can be.


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MaxBounty Presents the Affiliate Ball
In preparation for the ASW, I have gotten a chance to know Darren Blatt a bit – the mastermind behind the Affiliate Balls and truth be told, have been pleasantly surprised by his welcoming nature and candidness. Till now I was never the one to partake in these events, but seems this has become a major force for sponsorships for the Affiliate Summit.
Wanting to dig a little deeper, I connected with Darren on FB and thought we would do an interview so people can get to know the man behind the funk and the amazing Affiliate Ball – which btw this year is predominantly sponsored by the fine guys over at MaxBounty.
Darren, tell me a little about how you got started with the Affiliate Ball.
Back when I was in college at Ohio State I was working for a concert venue. I helped them book artists as I always had a feel for what artists were hot. Having this knowledge of how the whole process worked was key, it's certainly not an easy thing to do.
I started marketing online back in 1997. As I attending different trade shows back then I noticed the party scene was all the same. Free drinks at some bar with a DJ. I thought to myself, what if I raised money to pay for big name artists and gave exposure to the sponsors in return. Back then it was to promote my own consulting service which specialized in B2B internet marketing. I would give myself a main sponsorship and share the exposure with other paying sponsors. Well, it worked and my parties only got bigger and better. Over the years I've tweaked this model so sponsors can see more of a ROI rather than just branding.
Now I do this full time and have many online marketing trade shows we do parties at. It started off for selfish reasons and a fun hobby. Now it's a business model.
What was/is the purpose of it, more of a social gathering place or a venue to make some serious contacts?
The purpose was always to give sponsors an alternative marketing platform …read more


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Labor stalwart Lonnie Nelson dies at 83
She was 83 years old and was active in the people's movements and in her Communist Party club until stricken by a stroke a few days before her death, Feb. 12.
Dr. King spoke out against the genocide of Native Americans
It is probably little known what the great freedom fighter had to say about the horrific mistreatment of Native Americans by the U.S.
New York's tale of two cities extends to America
New York Mayor Bill de Blasio's "tale of two cities" could be told in cities across the country.
Enough "shock," time to confront prejudice
It's too late to preach tolerance by the time someone is a full-time corporate employee. Sensitivity must begin before children attend schools, and continue through their formative years.
Sept. 10 primaries a turning point in New York
In the primary elections, hundreds of thousands of Democratic voters selected candidates who advocated a different, more progressive direction for New York City.
Detroit: Through the lens of class and race
The success of the struggle in Detroit will hinge especially on the degree of anti-racist understanding achieved by white people and workers in particular.
States should not reward bigotry
A care model for a state-supported Catholic hospital group excludes same-sex couples. Why should states support such discrimination?
March on Washington volunteer recalls Bayard Rustin, tears
Everyone knows about Dr. King's magnificent speech but I feel it is important that Rustin's role and influence has recently been recognized and written about.
Building a racial justice movement
This week, the nation will celebrate the 50th anniversary of the March on Washington for Jobs and Freedom with events in Washington, D.C., and many other cities.
Bar Association condemns "panic" defense in LGBTQ murders
It's a major step forward that the American Bar Association has now officially condemned "panic" defenses used to justify anti-gay hate crimes.


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Top Definition
an obese monster,someone with to many kids that they don't take care of by a man who doesn't love her and never has
door sunshine ;) 29 april 2012
Trending Now
Can you define these popular missing words?
De e-mails zijn afkomstig van email@example.com. We sturen nooit spam.×


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moviecity.com opened on 2.4.2003 and this domain is 13 years, 5 months old. We see that moviecity.com is using Google Adsense to monetize and , 97969 Alexa Rank and Country rank shows us how good and useful this site is.It is well known webmasters care about W3 Validator and fortunately W3 didn't find any error and warning on moviecity.com. It is important for every website to open quick and be smooth while surfing. We see this site opens in 1032 milliseconds and it isn't a good score at all, you can make your site faster by compressing your scripts and stylesheets. moviecity.com registered under NETWORK SOLUTIONS, LLC. and Its nameservers are ns1.moviecity.com and ns2.moviecity.com.On our researches we see moviecity.com gets visitors with these words : movicity, paul encuentros cercanos, laptv, movicity play, loco por ella, citymix programacion, moviciti holliwood si fueras. This website doesn't have any keyword, we think they should put at least one or two keywords. We see moviecity.com doesn't have DMOZ record that is why we don't think this site is secure to surf but since DMOZ wants money to add your site to Dmoz we can't say this site is 100% secure or not.We see that your website gets most of the users with these missed types;joviecity.com, mviecity.com, m0oviecity.com, moiecity.com, mobviecity.com, movecity.com, movjiecity.com, movicity.com, movidecity.com, movieity.com,
Mostly to be successful, and to obtain top rankings or positions in the search engines.


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Delegation to Jordan
18/09/2016 - EU election observers in the field
15/09/2016 - Statement by High Representative Mogherini on International Day of Democracy
Statement by High Representative Federica Mogherini on the occasion of the International Day of Democracy on 15 September.
06/09/2016 - Call for proposals Support to Civil Society in Jordan
Local publication for call for proposals Support to Civil Society in Jordan
Working together in Jordan
Building the Mediterranean Motorways of the Sea (MoS)
The project aims at promoting the creation of a better maritime transport connections in the Mediterranean.
Top News from the European Union
19/08/16 - Statement by the High Representative/Vice-President Federica Mogherini and Commissioner Christos Stylianides on World Humanitarian Day 2016
- 18/08/16 - Declaration by the High Representative on behalf of the European Union on Syria and the situation in Aleppo
- 17/08/16 - Statement by the EU Delegation and the Embassies of EU Member States in Kyiv on the launch of an e-declaration system without certification in Ukraine
- 08/08/16 - Declaration by the HRVP Federica Mogherini, on behalf of the EU on the occasion of the International Day of the World's Indigenous Peoples, 9 August 2016
- 04/08/16 - Kosovo: new EU Special Representative appointed


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First project under our new name www.mottocreative.com
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What are you working on? Dribbble is a community of designers sharing screenshots of their work, process, and projects.
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DENVER — The man accused of killing 12 people in a Colorado movie theater shooting must undergo a second sanity evaluation by the state mental hospital because the first was "incomplete and inadequate," a judge ruled Wednesday.
The ruling means a months-long delay in James Holmes' case, which has already been in the courts for a year and a half.
DENVER — A man suspected of killing three people and setting fire to a home in southern Colorado has been captured in Oklahoma after a nationwide manhunt, authorities said Sunday.
DENVER (AP) - The U.S. and Canadian military will entertain millions of kids again this Christmas Eve with second-by-second updates on Santa's global whereabouts. But there's something new this year: public criticism.
CENTENNIAL, Colo.
CENTENNIAL, Colo. - A teenager who may have had a grudge against a teacher opened fire Friday with a shotgun at a suburban Denver high school, wounding two fellow students before killing himself.
BOULDER, Colo. (AP) - Astronaut Scott Carpenter is being remembered by friends, family members and dignitaries at a funeral service in Colorado on Saturday.
Carpenter, who grew up in Boulder and lived in Vail, Colo., died Oct.
CENTENNIAL, Colo. — The defendant in the Colorado movie theater shooting had the day of the attack marked "with a unique symbol" on a calendar, a Batman mask and a violent drawing in his apartment, according to testimony Wednesday.
AURORA, Colo. — Survivors of mass shootings in Colorado and Connecticut were among those gathered Friday in a suburban Denver park to honor those killed in the massacre at an Aurora movie theater a year after the attack.
DENVER — Colorado theater shooting suspect James Holmes will be restrained during his trial by wearing a harness under his clothes that will be anchored to the floor, the judge said Thursday.
COLORADO SPRINGS, Colo. - Authorities lifted evacuations in a wide swath of terrain outside Colorado Springs on Friday as they said a surprise rain shower helped them expand containment of a wildfire that has destroyed 400 homes.
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Home > Sunglasses > Carrera > CARRERA 6015/S (N62/8U)
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CARRERA 6015/S is a fashion statement, for which sunshine is not necessarily required, because these sunglasses enhance your natural radiance turning night into day. Would another colour not be a better match for your favourite outfit? Check also the other styles of CARRERA 6015/Sin our 2015, and 2016 Carrera range.
With no frills and state-of-the-art materials and craftsmanship, these men's glasses stand for stylish design and self-confidence. Full rim frames completely enclose the lenses within the frame. For a convinced eyeglass wearer, any other frame style is ruled out, with very few exceptions. This epitome of aviator sunglasses from the house Carrera is distinguished by their characteristically large lenses and thin frame. The model a sure hit, not only in the cockpit of an airplane. Eyeglass frames made of optyl distinguish themselves by containing no plasticizers. Thus, unlike other materials, "embrittlement" of the frame is avoided. Black is one of the undisputed classics. It can be combined with any outfit and simultaneously radiates elegance and naturalness. Brown is the dominant colour for sunglass lenses. They radiate warmth and look very soft. Functionally, you are, of course, on the safe side, too. With 100% UV eye protection, the sun can immediately start shining.
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Copper homeostasis in Enterococcus hirae
Copper is an essential component of life because of its convenient redox potential of 200–800 mV when bound to protein. Extensive insight into copper homeostasis has only emerged in the last decade and Enterococcus hirae has served as a paradigm for many aspects of the process. The cop operon of E. hirae regulates copper uptake, availability, and export. It consists of four genes that encode a repressor, CopY, a copper chaperone, CopZ, and two CPx-type copper ATPases, CopA and CopB. Most of these components have been conserved across the three evolutionary kingdoms. The four Cop proteins have been studied in vivo as well as in vitro and their function is understood in some detail.
- Copper
- Silver
- Enterococcus
- Resistance
- ATPase
- Metallochaperone
1 Introduction
Copper has been known since prehistoric times. It was obtained in the Middle East by charcoal reduction of its ores by about 3500 years before Christ. Approximately 500 years later, the harder bronze, generated by the addition of tin, was discovered; this established the Bronze Age. Copper has continued to be one of mankind's most important metals to this day. Copper is widely distributed in nature and has a relative abundance in the Earth's crust of 68 ppm. Copper is found mainly as the sulfide, oxide, or carbonate, its major ores being copper pyrite (chalcopyrite, CuFeS2), copper glance (chalcocite, Cu2S), cuprite (Cu2O), and malachite (Cu2CO3(OH)2). Weathered minerals give rise to soluble cupric and cuprous salts, such as carbonates and chlorides [1].
Copper is an essential micronutrient both for eukaryotes and prokaryotes. The biological functions of copper are closely related to its properties as a transition metal. Copper has partially filled d orbitals and has more than one stable valence state, Cu(I) being a (d10) cation and Cu(II) being a (d9) cation. As the strongest Lewis acid in Group IB, copper has the ability to mediate electron transport, accepting and donating electrons used by metallo-enzymes like cytochrome c oxidase, lysyl oxidase, or superoxide dismutase [2]. Copper can also be a prosthetic group of microbial enzymes involved in the reduction of nitrite and nitrous oxides. Another class of copper proteins, exemplified by plastocyanins and azurins, act as electron carriers. Second only to iron, copper is the biologically most used transition metal. However, its homeostasis has to be tightly regulated, because it is also toxic. Studies of microbial model systems and the investigation of inherited diseases in copper metabolism led to important insight into the mechanism of copper toxicity and the way cells deal with it.
1.1 The chemical state of copper
The two oxidation states of copper, Cu+ (copper(I)) and Cu2+ (copper(II)), are what make copper a valuable cellular constituent. It participates in biological redox reactions by undergoing cycling between the two oxidation states, thereby taking up and releasing electrons. Such redox-active copper ions are deeply buried in specialized cuproenzymes. While free copper(II) ions are stable in neutral, aqueous solutions exposed to the atmosphere, copper(I) ions can only be maintained in solution at very acidic pH or in complexed form. Copper in both oxidation states avidly binds to biomolecules, preferentially involving thiols. Highly efficient chelators of copper(I) in vitro are o-phenanthroline, bathophenanthroline sulfonate or 8-hydroxyquinoline (reported formation constants for copper–phenanthroline complexes are around 21, irrespective of the ligands on the phenanthroline dipyrimidine ring system [3]). Stable copper(I) complexes are also formed by acetonitrile [4], CN−, or even Tris-buffer [5]. More recently, tetrathiomolybdate has been employed as a very potent copper chelator in experimental systems [6]. This compound has also undergone phase II clinical trials for the removal of copper in people suffering from Wilson disease [7], a condition where copper accumulates in the liver due to a genetic fault in the Wilson copper ATPase, ATP7B [8]. Phenanthrolines complex Cu(I) (and Fe(II)) so strongly that they effectively raise the redox potential to a point at which any reducing equivalent can support the reduction of the higher valency metal ion. This reaction can be counteracted by high concentrations (20 mM) of citrate or lactate, which preferentially bind the oxidized form of copper.
It has been shown that copper ATPases transport copper(I) and that copper chaperones shuttle copper(I) (see below). High backgrounds of non-specific copper binding, strictly anaerobic working conditions, and the use of copper chelators and reductants have to be dealt with when working with copper(I). The use of reducing agents, copper chelators, and the binding of copper(I) to biological material, on the other hand, allow only estimates of the available copper concentration in a given system. These are the major hurdles for in vitro experiments with copper homeostatic components.
1.2 Copper toxicity
The mode of toxicity of copper is not fully understood. Copper toxicity could occur via the production of hydroxyl radicals (OH) in a Fenton-type reaction.
The highly reactive hydroxyl radicals could damage phospholipids and enzymes [9]. Hydroxyl radicals could also attack DNA bases, preferentially guanine residues, and break phosphodiester bonds in single-stranded DNA by modifying the deoxyribose sugars. However, the estimated steady-state concentrations of oxygen and hydrogen peroxide under physiological conditions are 10−5 and 10−9 M, respectively [10]. This makes the generation of hydroxyl radicals by reaction (1) unfavorable, except in hypoxia. Under normal conditions, redox cycling by reactions (2) and (3) should be more favorable.
These combined reactions cause redox cycling of copper at the expense of glutathione (GSH) and O2 to produce GSSG, the reduced form of GSH [11]. Other thiols could be depleted by the same reaction. Extensive thiol depletion would of course impair cell function (most Gram-positive organisms apparently do not contain GSH and its depletion would thus not be a toxicological mechanism in such organisms [12]; however, streptococci and enterococci appear to be exceptions which do contain GSH [13]). GSH has also been shown to be a normal copper ligand, participating in copper transport and/or detoxification [14]. GSH depletion would of course adversely affect such functions.
Kocha et al. have reported that mannitol scavenges hydroxyl radicals and in vitro effectively protects albumin from oxidative damage in a Fe2+/EDTA/H2O2 system, but not in a Cu2+/H2O2 system [15]. They propose that copper, but not iron, first binds to the proteins and then generates the reactive hydroxyl radical at the copper binding site. Whatever the primary toxic effects of copper may be, they are utilized in agriculture for the control of bacterial and fungal diseases on a variety of crops, such as potatoes or grapes [16].
1.3 Model of copper homeostasis in Enterococcus hirae
For the safe management of copper, prokaryotic and eukaryotic cells have developed various mechanisms. Copper homeostasis in E. hirae has been extensively studied and reviewed [17–21] and is the best understood prokaryotic copper homeostasis system. It consists of four genes arranged in the cop operon. The gene products of copA and copB are copper transporting ATPases, copY encodes a copper-responsive repressor, and copZ encodes a chaperone which can catalyze intracellular copper routing (Fig. 1). The cop operon allows growth of E. hirae in a wide range of copper concentrations, from copper-limiting conditions to 8 mM copper. According to the model, CopA takes up copper when copper is limiting, CopB extrudes copper when it reaches toxic concentrations, CopY regulates the expression, and CopZ transfers copper to CopY. In the following section, structural and functional aspects of these components and their interaction will be discussed in detail.
2 Copper ATPases
The E. hirae copper ATPases were discovered fortuitously. While trying to clone a potassium ATPase using an antibody to screen an expression library, a clone encoding the two copper ATPases, CopA and CopB, was isolated. CopA and copB were the first genes encoding copper ATPases to be described [22]. In 1993, a homologous human copper ATPase defective in Menkes disease, a severe X-linked condition of copper deficiency [8], was cloned simultaneously by three groups [23–25]. The discovery of this ATPase, ATP7A or MNK, was shortly followed by the cloning of a second human copper ATPase, ATP7B or WND [26, 27]. Mutations in ATP7B lead to Wilson disease, a failure to secrete excess copper from the liver, resulting in severe liver damage [8]. These discoveries were followed by the description of numerous similar copper ATPases from yeast, plants, insects and bacteria [28, 29]. Together with ATPases pumping a range of other heavy metal ions, namely Cu+, Ag+, Co2+, Zn2+, Cd2+, Hg2+, and Pb2+, they form the large new class of heavy metal ion ATPases [30, 31].
2.1 Structural aspects of copper ATPases
The heavy metal ATPases are a sub-class of the P-type ATPases. These get their name from the fact that the aspartic acid of the universally conserved sequence DKTGT in the 'phosphorylation domain' forms a phosphorylated intermediate in the course of the reaction cycle [32]. Other motifs common to P-type ATPases are the 'actuator domain' of consensus sequence TGE, and the 'nucleotide binding domain' of consensus GCGINDAP (Fig. 2). The X-ray structure for the calcium ATPase of the sarcoplasmic reticulum has been solved [33] and revealed that these three domains are structurally well separated. They have been named the A, P, and N domains, respectively. However, the heavy metal ATPases form a distinct subfamily of the P-type ATPases [34]. Because of the conspicuous feature of the intramembranous CPC or CPH motif (sometimes also SPC) of heavy metal ATPases (Table 1), it has also been suggested that they are called CPx-type ATPases [35]. Phylogenetic analysis now places the P-type ATPases in various subfamilies. A website lists all members of these subfamilies of P-type ATPases (http://biobase.dk/∼axe/Patbase.html).
|Transported ions
|E. coli
|E. hirae
|E. hirae
|E. coli
|S. aureus
|N. crassa
|E. coli
aPutative transmembrane helix number.
The distinguishing CPC/CPH/SPC motifs of the CPx-type ATPases are located in the middle of a predicted membrane helix in the most conserved core structure of these ATPases. The first proline of this motif is a conserved feature of all known P-type ATPases. With the structural information available for the calcium ATPase, it has become clear that this proline is part of the calcium channel across the membrane S, while in most copper translocating ATPases this motif is CPC A. CopA and CopB of E. hirae feature the latter motif, in line with their proposed function in copper transport.
2.2 Evolutionary aspects of copper ATPases
Recently, the oldest known microfossils have been discovered in deep-sea volcanic rock, estimated to be 3.2 billion years old [39]. This suggests that hydrothermal vents have hosted the first living systems on Earth. The hot, acidic seawater encountered at these vents releases metals like iron, manganese, zinc and copper from the volcanic rock [40] and resistance to these metal ions might have been an evolutionary priority for the first life forms. High evolutionary conservation of heavy metal ATPases across phyla and the divergences of heavy metal and non-heavy metal ATPases before the division into eukaryotic and prokaryotic cells [31, 35] support an early evolution of heavy metal ATPases. Copper was probably not an essential trace element in early cells. Rather, it might have become a cellular constituent with the advent of a more oxidized biosphere, a process which started less than 3×109 years ago, and could thus be considered a 'modern' bioelement [41]. Cuproenzymes function almost exclusively in the metabolism of O2, N2O or NO2− which only became necessary with the advent of an oxidizing environment. The first heavy metal ATPases may thus have served solely in the detoxification of the cellular cytoplasm.
2.3 Function of CopA
The working model that CopA serves in copper uptake under copper-limiting conditions still rests on indirect evidence. It is based on the following two observations. First, ΔcopA strains grow poorly in media where copper is limited by complexation with copper chelators. Secondly, ΔcopA strains are more resistant to Ag(I) than the wild-type [42], presumably because Ag+ can fortuitously enter wild-type cells via CopA. CopA endowed with a cleavable histidine tag was expressed in Escherichia coli, solubilized from cell membranes with dodecylmaltoside and purified to homogeneity by Ni-affinity chromatography [43]. Purified CopA was shown to catalyze ATP hydrolysis with and without the histidine tag. It had a pH optimum of 6.3 and a Km for ATP of 0.2 mM. The activity increased three- to four-fold when the protein was reconstituted into proteoliposomes and reached 0.15 μmol min−1 mg−1. The enzyme formed an acylphosphate intermediate, with kinetics of formation and the effects of inhibitors and metal ions upon it supporting a function of CopA in copper transport. Vanadate, a characteristic inhibitor of P-type ATPases, exhibited 50% inhibition at 100 μM. These pieces of evidence strongly suggest that the CopA ATPase provides transport for both Cu+ and Ag+; however, direct transport of copper in vitro has not been shown yet.
Like most heavy metal ATPases, CopA possesses an N-terminal metal binding site with the consensus motif CxxC. Whether this motif is required for function or is dispensable, as in the related CopA ATPase of E. coli [44], is not known at present. However, mutation of the two cysteines of the CxxC motif to serine affected the interaction of CopA with the CopZ chaperone (see below), suggesting a function of the N-terminal metal binding motif in copper transfer from the ATPase to other cellular components.
If CopA indeed supplies copper to the cell, the direction of copper transport relative to the ATP binding site would be opposite to that postulated for most copper ATPases, and actually shown for CopB of E. hirae [45] and the human Menkes and Wilson ATPases [46, 47]. However, evidence for copper transport into cells by a heavy metal ATPase has also been obtained for CtpA of Listeria monocytogenes [48, 49] and CtaA of Synechocystis [37]. A screen for genes induced during infection in Staphylococcus aureus revealed the virulence gene ivi44, which encodes a protein with 50% sequence identity to CopA of E. hirae [50]. This suggests that copper is a limiting nutrient in pathogenesis and copper import becomes a cellular priority when bacteria infect a host. It would obviously be very interesting to test a copA knock-out strain of E. hirae for its ability to infect a host. Finally, all P-type ATPases which have been studied in detail simultaneously transport ions in both directions. The NaK-ATPases exchange Na+ for K+, the KH-ATPases exchange K+ for H+ and what are colloquially called Ca-ATPases are in fact CaH-ATPases. Most likely, Cu-ATPases are CuH-ATPases and it would not require major structural reorganization of the enzyme to reverse the direction of transport for these two ions.
2.4 Function of CopB
CopB, like some other CPx-type ATPases, does not possess an N-terminal CxxC metal binding motif. Instead, it features a histidine-rich N-terminus which most likely also constitutes a metal binding domain. Similar histidine repeat structures were found in two Pseudomonas syringae proteins, psCopA and psCopB, that are components of a plasmid-encoded copper resistance system [51]. PsCopA was shown to be a periplasmic protein that can bind 11 atoms of copper/monomer and was shown to serve in copper resistance by sequestering large amounts of copper in the periplasm [52].
For CopB, copper and silver transport was demonstrated directly. CopB had a Km for Cu+ of 1 μM and a Vmax of 0.07 nmol min−1 mg−1 of membrane protein. 110Ag+ was transported with similar affinity and rate [45]. Since Cu+ and Ag+ were complexed to Tris-buffer and dithiothreitol present in the assay, the Km values must be considered as relative only. The results obtained with membrane vesicles were further supported by 110Ag+ extrusion from whole cells, pre-loaded with this isotope [42]. These findings suggest that CopB functions as a Cu+/Ag+-ATPase for the export of Cu+ and Ag+ in vivo, thereby serving in copper resistance [43, 45] (silver resistance of cells through the action of CopB could not be observed; the reason for this is unknown). Maximal transport was observed at pH 6. Biochemical experiments with purified CopB in reconstituted phospholipid vesicles [53] showed that the enzyme is also vanadate sensitive (I50= 100μM), has a Km for ATP of 0.5 mM and a Vmax for ATP hydrolysis of 1.3 μmol min−1 mg−1 [54].
In an ion-motive ATPase, ATP hydrolysis and transport must be coupled, i.e. there should not be ATP hydrolysis in the absence of the ion(s) transported by this ATPase. Purified CopB exhibited maximal ATPase activity in the absence of added copper ions. Presumably, this was due to the presence of sufficient contaminating copper for full ATPase activity. However, attempts to remove residual copper with standard copper chelators such as bicinchoninic acid or bathocuproine and thereby inhibit CopB did not meet with success. Only tetrathiomolybdate, which strongly interacts with copper and has been used to treat patients with Wilson disease [7, 55], could efficiently scavenge residual copper in purified CopB preparations and reversibly inhibit CopB [6]. These experiments suggested that CopB has a very low Km for copper in the range of 30 nM and advocate tetrathiomolybdate as new tool for the study of copper ATPases. Silver(I), which is also transported by CopB, could similarly activate the ATPase activity of CopB [6]. On mechanistic grounds, it appears unlikely that CopB could transport either monovalent or divalent heavy metal ions. So other heavy metals like zinc, lead, cadmium, mercury or lead, which do not form stable monovalent ions, are unlikely to be substrates of CopB.
2.5 CopB as a model for Menkes and Wilson ATPase
CopB is massively overexpressed in an E. hirae mutant strain deficient in the CopY repressor (see below). Following solubilization from membranes with dodecylmaltoside, it can be purified by Ni-affinity chromatography. The histidine-rich N-terminus of CopB thereby acts as an 'endogenous' histidine tag [53]. This convenient purification procedure of the wild-type enzyme makes CopB readily accessible to biochemical characterization. E. hirae CopB is sufficiently related to the human Menkes and Wilson ATPases that the effect of mutations in the human enzymes can be tested with CopB. It was used to test the impact of some of the mutations occurring in the highly conserved motifs. A C396S mutation was introduced into the conserved CPC motif of CopB to mimic the C1000R mutation in ATP7A, identified in a Menkes patient. This mutant enzyme was not able to reverse the copper sensitive phenotype of a CopB knock-out strain. The purified C396S mutant enzyme did not show any ATPase activity [54]. Similarly, the most frequent Wilson disease mutation H1069Q was introduced into CopB as a H480Q mutation. H480 is part of the HP motif, a motif of unknown function. It is universally conserved in heavy metal ATPases and is also present in a group of related copper proteins, including CopA from P. syringae, a similar protein from Xanthomonas campestris, laccase from four different fungi, and ascorbate oxidase from cucumber [56]. The H480Q mutation eliminated copper transport by CopB as tested in vivo, but the purified protein still exhibited residual ATPase activity. The mutation did not affect the Km for ATP, but reduced Vmax more than 50-fold [54]. The result suggested that the HP motif is not involved in ATP binding, even though the site is very near the ATP binding site. Clearly, site-directed mutagenesis of CopB offers an avenue for the analysis of structure–function relationships of broader significance in copper ATPases.
3 Regulation of the cop operon by the CopY repressor and the CopZ chaperone
Various mechanisms of regulation of copper resistance and tolerance have evolved in different bacterial species. Examples are the plasmid-borne cop system of P. syringae [57] and the pco system of E. coli. They are regulated by two-component regulatory systems, which communicate via series of phosphorylation/dephosphorylation reactions [58]. Transcription of the copA gene in E. coli on the other hand is regulated by the copper-responsive, MerR-like activator, CueR [59, 60]. The cop operon of E. hirae is regulated by the copper-responsive repressor CopY, a protein of 145 amino acids [61]. By database searching, repressors similar to CopY can only be identified in Gram-positive bacteria closely related to E. hirae. This contrasts with the very high evolutionary conservation of the copper ATPases across phyla (see above). It could be hypothesized that ancient copper ATPases were expressed constitutively and only served in detoxification of the cytoplasm. With the advent of an oxidizing environment, copper became a component of redox enzymes and therefore had to be carefully regulated. This could have led to the parallel evolution of different regulatory systems in various organisms.
3.1 In vivo regulation of the cop operon
A ΔcopY deletion strain led to constitutive overexpression of CopA and CopB, while a ΔcopZ strain exhibited reduced levels of expression. This led to the initial suggestion that CopY was a repressor and CopZ was an activator [61]. Given the model that CopA functions in copper uptake and CopB in its extrusion, it would seem paradoxical that both genes are under the same control. Though this could be a safety mechanism: if the cells express only the importer, CopA, under copper-limiting conditions, they would become vulnerable to copper toxicity in case of a sudden increase of ambient copper. Addition, as well as complexation, of ambient copper led to the induction of the operon. This bi-phasic regulation would support a function of the cop genes in both, copper uptake and secretion. The lowest induction of the genes was in standard growth medium containing about 1 μM copper and full induction was obtained with 2 mM copper in the medium. The operon was also induced by 5 μM Cd2+ or Ag+ [62]. According to the current concept, CopY is a copper-responsive repressor: in the Zn2+ form, it is bound to the promoter and suppresses transcription. CopZ can donate copper to CopY, thereby releasing it from the DNA and allowing expression of the downstream cop genes.
3.2 Structure of the CopY repressor
The structure of CopY is bi-partite: the N-terminal half of the protein exhibits around 30% sequence identity to bacterial repressors of β-lactamases, such as MecI of S. aureus (accession BAA82218), PenI of Bacillus licheniformis (accession B28183), or BlaI from an S. aureus transposon Tn552 (accession CAA36951; Fig. 3) [63–65]. For PenI, there is evidence that the N-terminal portion of the protein is the DNA binding domain [66]. The CopY N-terminus also features a QQ motif. For the phage λ cro repressor and phage 434 repressor, a QQ motif in similar positions has been shown to tightly interact with an ACA triplet of the DNA binding sites [67]. Support for a similar interaction of the QQ motif of CopY stems from the observation that mutation of ACA to TCA in the CopY DNA binding sites abolishes CopY–DNA interaction in vitro (see below).
In the C-terminal half of CopY, there are multiple cysteine residues, arranged as CxCx4CxC. The consensus motif CxCx4-5CxC is also present in yeast ACE1 and AMT1, which are copper-responsive transcriptional activators for metallothionein [68, 69], in Mac1, a transcription factor for the Ctr1 and Ctr3 copper transporters of S. cerevisiae [70], and in the N-terminal β-domain of human metallothionein-2 [71]. As experimental work has shown, the CxCx4CxC motif is the site where either one Zn2+ or two Cu+ can be bound. By crosslinking experiments and analytical gel filtration, it could be shown that the CopY repressor is, like most repressors, a homodimer [72].
3.3 Function of the CopY repressor
Binding of CopY to an inverted repeat sequence upstream of the copY gene has been demonstrated in vitro [72]. DNase footprints showed that the CopY dimer protects two discrete sites of 27 and 28 bp, identified in the region between −70 and −11. CopY–DNA interaction occurred in two steps with two discrete apparent half-maximal DNA binding constants. Cu+ released CopY from the promoter with an apparent half-binding constant for Cu(I) of 20 μM. Promoter mutations at A-61T and A-30T abolished the binding of CopY to one or the other binding site, in agreement with the postulated strong interaction of a QQ motif in CopY with an ACA motif in the promoter.
To study the effect of these promoter mutations in vivo, a plasmid-borne expression system encoding a truncated CopB ATPase which was non-functional, but detectable with a specific antibody, was employed. This system was developed due to the lack of success in using β-galactosidase or chloramphenicol acetyltransferase as reporter genes in E. hirae. Mutation of both A-61T and A-30T, but not either alone, abolished the binding of CopY and led to approximately 1000-fold hyperinduction of the promoter [73]. Interestingly, induction by chelation of copper was not affected by these promoter mutations, which suggests that the induction of the cop operon by copper starvation does not proceed via release of CopY from the DNA. The mechanism could involve other elements, such as a sigma factor or a stress response regulator.
In vitro studies showed that CopY binds to DNA as a Zn(II)CopY complex. For the release of CopY from the DNA, two Cu(I) can displace the Zn(II) in CopY. Cu(I) can be donated to CopY by Cu(I)CopZ or suitable copper(I) complexes, such as Cu(I)-acetonitrile [74]. The Cu(I)2CopY complex exhibits luminescence, implying that Cu(I) is sequestered in an environment where it is protected from solvent. X-ray absorption studies suggested a mixture of two- and three-coordinate copper in Cu(I)CopZ, but a purely three-coordinate copper coordination with a Cu–Cu interaction for Cu(I)2CopY. The latter coordination is consistent with the formation of a compact binuclear Cu(I)-thiolate core in the CxCx4CxC binding motif of CopY (Fig. 4) [75].
The displacement of zinc by copper was monitored by the spectral shift of the absorbance maximum from 412 to 500 nm of 2,4-pyridylazoresorcinol upon zinc binding [76]. Two copper(I) ions were required to release the single zinc(II) ion bound per CopY monomer. It is plausible that the Cu(I) ions are being sequestered in a Cu(I)-thiolate cluster as found in the Cu(I)-regulated transcription factor ACE1 and the metallothioneins [69]. The inability of the displaced Zn(II) to bind to CopZ suggests that the metal binding site in CopZ is specific for Cu(I). The combined findings of X-ray absorption spectroscopy and luminescence studies show that CopY binds either one zinc ion or two copper ions to the CxCx4CxC motif, located in a protected, solvent-inaccessible core [76, 77].
The specificity of copper transfer between Cu(I)CopZ and CopY was dependent on electrostatic interactions. Relative copper binding affinities of the proteins were investigated using the chelator diethyldithiocarbamic acid. The findings suggested that CopY has a higher affinity for copper than CopZ. However, this affinity difference does not appear to be the sole factor in the copper exchange: a charge-based interaction between the two proteins is required for the transfer reaction to proceed. Cu(I)MNKr2, the second CopZ-like metal binding domain of the human Menkes ATPase, is not able to donate copper to CopY. It could be shown that this was due to the very different electrostatic surface of MNKr2. A gain-of-function mutant of MNKr2 could be created. In this mutant protein, MNKr2K4, four amino acids were mutated to lysines to mimic the electrostatic surface of CopZ (Fig. 5). Cu-MNKr2K4 functioned in Cu+ transfer to CopY as well as Cu-CopZ. This demonstrated the necessity of four lysine residues on the chaperone for the interaction with CopY. Taken together, these results suggest a mechanism for copper exchange between CopZ and CopY which involves specific protein–protein interaction.
Available data suggest that a similar regulatory mechanism controls the expression of the CadA ATPase, which confers cadmium resistance to E. coli. The CadC repressor was shown in vitro to bind to the cad operator/promoter. Cd2+, Bi3+, and Pb2+ caused the release of CadC from DNA, as shown in band shift experiments [78]. However, the CadC repressor does not exhibit sequence similarity to CopY and whether displacement of zinc from CadC is involved in the process was not shown.
4 The family of copper chaperones
Copper chaperones are members of a diverse group of proteins involved in the intracellular delivery of copper. Studies in yeast suggest that chaperones are required in vivo because intracellular copper concentrations are extremely low, requiring direct protein–protein interaction for copper delivery to copper requiring enzymes [79]. Well studied copper chaperones are the yeast and human chaperones for superoxide dismutase (CCS), delivering copper specifically to superoxide dismutase [80]. Other chaperones serve in the delivery of copper to cytochrome c oxidase (Cox17, Cox19, Sco1, Sco2), to the Menkes/Wilson copper ATPase in humans (Atox1), or the corresponding CCC2 copper ATPase in yeast (Atx1; see [81] for review). Atx1 and CopZ belong to the same family of 8-kDa copper chaperones, which are present in cells of all phyla.
Surprisingly, it was found that the N-termini of most heavy metal ATPases also contain domains that are similar to Atx1 and CopZ [82]. These domains occur in one or two copies in microbial enzymes through to six copies in the human Menkes and Wilson copper ATPases. While the role of these modules in these proteins is still poorly defined, we now have a detailed understanding of the structure and function of Atx1- or CopZ-like domains.
4.1 Structure of CopZ
Detailed structural studies have shown that members of the homologous family of CopZ-like chaperones have very similar folds [83]. The structure of apo-CopZ has been determined by nuclear magnetic resonance (NMR) [84, 85]. The CopZ fold consists of four β-strands forming an antiparallel β-sheet, situated below two α-helices (structure 1CPZ in the PDB Database). This arrangement of secondary structure elements is characteristic of the ferredoxin-like proteins and is known as an 'open-faced β-sandwich'. The two copper binding residues, Cys-11 and Cys-14, of the CxxC motif are located in a loop between β1 and α1, binding copper on an exposed position at one end of the molecule. The exposed copper binding site sets copper chaperones apart from the cuproenzymes known hitherto, which have the copper buried deeply inside the protein. The exposed copper on chaperones must be a functional requirement, facilitating reception and donation of copper.
4.2 Interaction of the CopZ chaperone with the CopA ATPase
As discussed above, CopZ delivers copper to the CopY repressor. By using surface plasmon resonance analysis (on a Biacore apparatus) it could be shown that CopZ also interacts with the CopA copper ATPase, which could be the site of copper loading of CopZ [86]. Interaction between CopZ in solution with CopA immobilized on the surface of the sensor chip was studied in detail. The association rate ka was only slightly affected by copper(I) and maximally exhibited a two-fold change compared to copper-unsupplemented conditions. In contrast, the dissociation rate kd was strongly influenced by added copper and was decreased up to 15-fold by copper(I). Copper could thus modulate the interaction of CopZ with CopA.
To test if the CxxC metal binding motif of CopA plays a key role in the CopZ–CopA interaction, such as by copper bridging, a CopA mutant which had the cysteine residues of the CxxC motif mutated to serine residues (SxxS) was constructed. CopZ still strongly bound to the CopA with the SxxS motif, but the interaction was no longer significantly influenced by copper; all association and dissociation rate constants resembled those observed for wild-type CopA in the absence of added copper. Thus, the CxxC motif increases binding of CopZ to CopA in the presence of copper. However, it is not the guiding element for the interaction of CopZ with CopA. Direct protein–protein interaction must occur. The positively charged surface on CopZ could be interacting with a negatively charged surface on CopA to establish a protein contact. Such an interaction has been shown to take place between the homologous yeast proteins, the CCC2 ATPase and the Atx1 chaperone [87].
If CopA is indeed the site of copper loading of CopZ, copper could be transferred from CopA to CopZ by a ligand transfer mechanism (Fig. 6). Such a mechanism had originally been suggested by Brown et al. for mercury transfer between the MerP mercury chaperone and the mercury transporter [88]. The same model was later adopted by Pufahl et al. for copper transfer from the yeast Atx1 chaperone to the copper ATPase [89]. While plausible, such a ligand transfer mechanism remains to be proven in detail.
4.3 Proteolytic regulation of CopZ
It was observed that CopZ of E. hirae is more labile under high copper stress. Levels of copZ mRNA increased with increasing ambient copper levels and reached a 1000-fold induction at 0.25 mM copper [90]. However, CopZ expression increased only up to 0.5 mM copper and declined at higher copper concentrations, as shown by Western blotting of crude cell lysates. It could be shown that CopZ overexpression is toxic to cells. First, growth of a strain overexpressing CopZ was inhibited by ambient copper in excess of 0.1 mM and was abolished by 1.5 mM copper, while growth of wild-type E. hirae was not markedly affected by this copper concentration. Secondly, CopZ overexpression made E. hirae more sensitive to oxidative stress, such as growth in the presence of H2O2 or paraquat.
Proteolysis of CopZ could be demonstrated in vitro with cytosolic extracts and purified CopZ. Interestingly, apo-CopZ was significantly more resistant to in vitro degradation than Cu-CopZ. The more rapid degradation of Cu-CopZ is in line with its proposed toxicity. Copper bound to CopZ is solvent-exposed [74] and this copper can thus participate in Fenton-type reactions and lead to the generation of reactive hydroxyl radicals and cell damage.
The protease responsible for CopZ degradation was apparently constitutive, but stimulated by copper(I) or Ag(I), a Cu(I) mimetic. The serine protease inhibitors p-phenylmethylsulfonyl fluoride (PMSF) and p-aminobenzamidine inhibited the degradation of CopZ, while N-α-p-tosyl-l-lysine chloromethyl ketone and N-tosyl-l-phenylalanine chloromethyl ketone, which are also serine protease inhibitors, were without effect. The metallo-proteinase inhibitor o-phenanthroline also did not inhibit CopZ degradation. Clearly, the protease degrading CopZ is a serine-type protease. On zymograms, the protease was tentatively identified as a protein of 58 kDa [91, 92]. Fig. 7 summarizes the regulation of the E. hirae cop operon at the current level of understanding. See legend for detail.
Proteolysis as a regulatory mechanism of protein expression has received much less attention than transcriptional and translational regulation [93, 94]. There are, however, a few examples of proteolysis linked to metal ion metabolism. In Saccharomyces cerevisiae, copper-induced degradation was described for the copper importer Ctr1 [95], as well as for the transcription factor Mac1 which is also involved in high affinity copper uptake [96]. An interesting proteolytic process is the degradation of apo-plastocyanin under copper starvation in the green algae Chlamydomonas reinhardtii. Presumably, this serves to preserve copper for use by other, indispensable cuproenzymes [97]. None of the proteases involved in these processes has so far been identified. Copper-induced proteolysis adds yet another level of complexity to the copper homeostatic process.
5 Extracellular copper reductase
Current evidence suggests that copper(I) is the species being transported by copper ATPases or Ctr1-like copper transporters. Yet, copper(I) ions are insoluble in free form at physiological pH and in standard bacteriological media. This suggests that there must be copper ligands and copper reductases involved in the copper homeostatic process. In E. coli it was shown that the copper(II) ions which enter the periplasm can be reduced to copper(I) by the reductase NDH-2 [98, 99]. When E. hirae is grown in media supplemented with 6 mM copper(II), the blue color of the media is lost in several hours due to the presence of copper reductase activity of extracellular or membrane bound nature [20, 100]. An energy (glucose) dependent copper-reducing activity could also be measured with washed E. hirae cells. The activity was strongly inhibited by Hg2+, with an I50 of 1 μM, and much less by Ag+ with an I50 of 50 μM. Other metals, such as nickel(II), tin(II), cadmium(II), platinum(I) and platinum(II), had no significant inhibitory effects at concentrations as high as 100 μM. Platinum(II) was shown to be an inhibitor of the yeast copper and iron reductase Fre1p with an I50 of 50 μM [101] which suggests a different biochemical mechanism of copper reduction by the yeast and the enterococcal enzyme. However, cloning of the E. hirae copper reductase has so far not met with success and more work needs to be done in this field.
6 Concluding remarks
The homeostatic control of copper is a formidable problem for cells and its study has met with difficulties. In this context, the E. hirae model system has, through its simplicity and accessibility, proven to be ideally suited to address fundamental questions of copper homeostasis. The striking homology of major components of copper homeostasis, such as the copper ATPases or the CopZ-like copper chaperones, across phyla shows that copper metabolism has evolved early and has been highly conserved through evolution, from bacteria to humans. Insight gained into the mechanism of bacterial copper homeostasis will thus be widely applicable. Understanding how cells deal with copper is fundamental to understanding copper's role in health and disease. For example, knowledge is still lacking on the minimal required copper intake for humans, as well as the maximal level of copper intake that is still safe.
There is increasing concern about copper contamination of the environment through the use of copper in agricultural pest control, the addition of copper to animal feed, or from the wash-out of copper pipes and roofs. Bacteria are the major players in determining the fate of materials in the environment. Acidithiobacillus ferrooxidans, Leptospirillum ferrooxidans and species of the genus Acicdiphillum were found to be involved in biooxidation of copper sulfide under acidic conditions (bioleaching) and these organisms are extensively used in biogeotechnology [102]. One potentially important impact of bacterial action on environmental copper is copper reduction at the cell surface. The biologically relevant form of copper, i.e. the form that is taken up by cells and transported intracellularly, is copper(I). This may be the primary reason why cells are endowed with copper reductase activity. The known extracellular copper reductase activities, such as those observed in E. hirae and yeast, are far in excess of cellular needs and will have a major impact on the speciation of copper in the environment. Understanding bacterial copper homeostasis at the molecular level will aid definition of the impact of bacteria on environmental copper.
We are grateful to the members of the Solioz lab for their contributions and their enthusiasm. Financial support was received from the Swiss National Foundation, Grant 31-68075.02, and from the International Copper Association.
- © 2003 Federation of European Microbiological Societies.
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