text: NEW YORK ( TheStreet) -- Shares of Cresud (Nasdaq: CRESY) were gapping up Monday morning with an open price 10.9% higher than Friday's closing price. The stock closed at $11.72 Friday and opened today's trading at $13. The average volume for Cresud has been 168,000 shares per day over the past 30 days. Cresud has a market cap of $639.6 million and is part of the consumer goods sector and food & beverage industry. Shares are up 4.4% year to date as of the close of trading on Friday. Cresud Sociedad Anonima Comercial Inmobiliaria Financiera Y Agropecuaria engages in the production of basic agricultural commodities in Brazil and other Latin American countries. The company has a P/E ratio of 13.2, below the average food & beverage industry P/E ratio of 41.1 and below the S&P 500 P/E ratio of 17.7. TheStreet Ratings rates Cresud as a hold. Among the primary strengths of the company is its robust revenue growth -- not just in the most recent periods but in previous quarters as well. At the same time, however, we also find weaknesses including disappointing return on equity, poor profit margins and a generally disappointing performance in the stock itself. You can view the full Cresud Ratings Report. Get more investment ideas from our investment research center. Interested in other stocks that are gapping up? Get free SMS text alerts sent to you when the action happens by texting UP to 95370 or select from multiple alert options.


text:
How to place an order in OVH?
Ordering an OVH Service
To make an order you must complete 6 steps.
Step 1: Choose a domain name
On our homepage type in the domain name you would like to have associated with an offer and then click on Search. You will then see the domain or domain names which you have selected and these following options:
- Domain registered at OVH
- Domain Available
- Domain Transferable, after owner's consent.
You can then add and choose other domains while you are still on Step 1.
Once you have finished selecting your domain, move on to Step 2 by clicking on the Step 2 button.
Step 2: Service Selection
This step allows you to choose the services you desire for your domain.
3 Possible Services for your Domain
1. The domain is available
Choose between :
- Create the domain
- Do not create domain
2. The domain is registered with OVH
Choose between :
- No renewal available
- Renew the domain
3. The domain is transferable with consent of owner
Choose between :
- Transfer the domain
- Do not transfer domain
2 Possible Services for Hosting
1.The domain already has a host
Choose between:
- Do not renew service of host
- Renew service of this host
- Change to a host proposed by OVH
2. The domain does not have a host
Choose between:
- Do not host this domain
- Host this domain on a different GP, plan
After the selection of domain services, you can move on to Step 3 by clicking Continue
Step 3: Duration choices
According to the chosen host and your domain extension, you will be able to modify the duration of your service.
According to the type of service and your domain extension you may choose the duration of services.
- for a .com, .org, .net, you have the option of keeping the domain for up to 10 years!
- for a GP, only a 36 month renewal is available.
- for a plan, choose between 3 and 12 months.
After choosing the duration, you can move on to Step 4 by clicking on Continue
Step 4: Identification
This step asks you for your identification information. There are two ways of doing this step:
1.You already possess a NIC Handle
In this case, you only need to enter your NicHandle and password to be identified.
If you already have a NIC Handle, but do not remember your password, or the NIC handle itself, please follow the instructions for LostNicHandle or LostNicHandlePass
2.You do not have a NIC Handle
In this case, please follow the procedure to create a NicHandle. (see NicHandle manual)
Once that is finished, you can then move on to Step 5 and click on Continue
Step 5: Registration of the owner, contacts and DNS
For this step, you will be asked to supply the owner's personal information. By default this information carries onto your NIC Handle.
Next, create the NIC Handles corresponding to others contacts whom you desire to give access.
Finally, the DNS information is ours by default (dns15.ovh.net and ns15.ovh.net).
Click on Continue to proceed to the final step.
Step 6: Payment
Here your order is finished, and we supply you with a purchase order. Follow the instructions below on payment procedures.
We will then send an e-mail confirmation and a link that permits access to the purchase order.
Means of Payment
- Debit/Credit Card
Click on the credit card icons and you will be forwarded to our banking interface to finish the payment.
- Cheque
Please write your purchase order number on the back of the cheque and then mail it to the following address:
OVH Hosting LTD
5 Fitzwilliam Place
Dublin 2
Attention: we do not accept foreign checks (only UK banks).
- Money Orders or International Business
You must go to the post office with the amount to be paid in cash (be careful for extra fees!).
Be sure to write the Order Number in the "correspondence" section and then send the money order to the following address:
OVH Hosting LTD
5 Fitzwilliam Place
Dublin 2
- Administrative Money Order
Attention: We only accept bank/credit transfers from civil services (school, town hall, ...) !
You must contact us and give us your Purchase Order number.
The billing services will create the bill from your Purchase Order Number.
We will send this bill to you by mail, accompanied with a bank identification slip and a letter with procedure instructions to follow.
You must contact, by fax, 01-6394091 in order to verify and validate the bank/credit transfer payment process.
The rest of the procedure is the same as the other types of payments.
- The creation of a Bank Card Payment Account
Only the payment (not just creation of account) can start your domain or website hosting services.
- Validate the E-mail address for your NIC Handle e-mail
Be careful to verify your e-mail address of your NIC Handle.
Or, if you wish to change your contact information, go to Manager v3.

subset: commoncrawl


text:
|By NightBird
|Part of the Avatar: Birth of a Nation continuity.
|"I wonder who decided to build Ba Sing Se's walls with earth... Can't a master bend the earth and make a hole on them?"
|— Zorin in Rising
Zorin Tsao is the teenage female protagonist on Avatar: Birth of a Nation.
Ever serene and rebellious, Zorin is almost the typical black sheep of the family. Cunning like a fox, and strong as a lion, she made her name among the Noble families with her intellect, sarcasm, and beauty. As a kid, Zorin was praised for her intellectuality but deemed a rebel after disobeying her parents' orders repeatedly. She dislikes following the way of a stereotypical noble woman, although disliking more to perform any physical task. She enjoys her privileges as nobility member at its fullest. Her rebellious nature leaded to her father sending her to the Earth Kingdom after embarrassing her mother at a Tea Ceremony. Zorin as most teenagers posses the desire to see the world with her own eyes, but her love for commodity and the noble's privileges had kept her from travelling.
Early lifeZorin was born in the Fire Nation to the Tsao family. The Tsao were a noble clan during the Fire Sages' regime, the Tsao were subordinates to the sages, as there were local administrators of small region of the Fire Empire. Zorin's parents were both Firebenders, revered for their dedication to their people, which gained them the favor of the theocracy. Zorin was the first daughter the couple had, before her two males were born. Zorin proved her prowess for Firebending early on life, which prompted her parents into teaching her the bending art.
When she reached 10, she had not only surpassed her older brothers in Firebending, but also her intelligence was regarded highly by the Sages. One sage declared that, "She her only sin was to be born a noble, she would be a perfect sage."
Life of a prodigy
Charismatic and regarded as the Tsao's finest daughter, marriage arrangement proposals arrived to her parents as soon as Zorin celebrated her thirteen birthday. Zorin's mother disliked the idea of giving her daughter to any suitor at a young age. She replied that anyone what wished to marry her should wait until she became a woman.
When Zorin reached fourteen years old, she transformed into a beautiful woman, whom most noble heirs coveted. As most teenagers, she began to question the authority of her parents, and began to seek for independence. Zorin became more rebellious to the point, of disregarding any order from her family. Her serene personality persisted, but with a sharp tongue and a bright mind, aside her beauty, she became a rebel without a cause
See more
For the collective works of the author, go here.


text:
Custom fields are LOST on every UPDATE
Five MONTHS ago I posted a note about this problem. At that time I thought I found a bug #10511 which is still not solved in the code, although, there seems to be a patch. WHICH MUST BE APPLIED AFTER EVERY UPDATE….
But I'm not sure that this bug addresses the problem.
My problem is that if you put in a CUSTOM field, like
SLIPPERY 1, or SERIES or some other field, which some plugins require, then when you UPDATE the field is deleted.
This is a ROYAL PAIN IN THE KIESTER… CAN I BE MORE CLEAR? DO I NEED TO??? DO I SEEM TO BE SHOUTING???
Well, nobody is fixing bug #10511, and based on it's description, I'm not even sure that it applies. I'm not using ENCLOSURES of any type. I am not sure that we are EVEN TALKING ABOUT THE SAME THING.
Just to be crystal clear. When adding or editing a post, scroll down to CUSTOM FIELDS and enter SLIPPERY and 1 in the CUSTOM FIELDS section and click ADD CUSTOM FIELD. You will see the field being added in the list above. Then if you click UPDATE at the top right, you find the CUSTOM FIELD has DISAPPEARED!!!!!!!!!!!!!!!!!!!!!
AM I SHOUTING>… After 5 months and several updates, I am entitled to SHOUT. This is annoying at Heck. Excuse my French.
I am using FIREFOX 3.5.7 with wordpress running on PHP 5.x.
Thank you so much for listening…… Or for clarifying my misunderstanding about how to use this OBVIOUS INTERFACE…..
Thank you so much,
- The topic 'Custom fields are LOST on every UPDATE' is closed to new replies.


text:
Ryan O. White
RYAN O. WHITE is a partner in the Indianapolis office of Taft Stettinius & Hollister LLP and a member of the firm's Intellectual Property Group. Ryan counsels individual and corporate clients in all areas of intellectual property law, including the preparation, procurement, licensing and enforcement of patents, as well as the drafting and review of licensing and supplier agreements, IP protection clauses and confidentiality and nondisclosure agreements.
Ryan's practice experience also includes preparing and rendering oral and written patentability, freedom to practice, non-infringement and validity opinions, as well as conducting intellectual property due diligence investigations and audits of usage and ownership. In addition to preparing and facilitating the ongoing prosecution of U.S. patent applications, Ryan has experience coordinating the filing and prosecution of PCT and national stage foreign patent applications. Ryan has experience working with a diverse range of clients, from small start-up businesses, to Fortune 100 companies.
Ryan has experience spanning many technical disciplines, including working with businesses in the biotech, biochemistry, biomedical, chemical, Internet and mechanical engineering industries. He has also worked extensively with technologies within the medical device and surgical sectors, including orthopedic implants, minimally invasive and computer assisted surgical procedures, bioabsorbable stents, catheter devices, general infusion and pain management pumps, wound closure devices, medical imaging systems, immunoassay platforms, as well as diagnostic workstations and drug delivery devices. In addition to his practice experience within the intellectual property field, Ryan also has worked with clients on various FDA issues, including advising clients on governmental regulatory and compliance related matters.
Ryan is admitted to practice law in both Indiana and Ohio and is registered before the United States Patent and Trademark Office. He is also admitted to practice law before the United States District Court for the Southern District of Ohio, the Court of Appeals for Sixth Circuit and the Court of Appeals for the Federal Circuit.
Ryan serves on the board of directors for the Sociedad Amigos de Colombia (SADCO), a non-profit organization that focuses on creating a caring community by drawing together Latin American cultures in central Indiana, as well as by offering financial and educational support to those in need.
|Law Updates | In the News | News Releases | Upcoming Events | Subscribe


text:
I realize that there is a great benefit to getting licensed in VA and that by far, if possible, is the better choice. However, I currently have two dogs and a toddler and although able to close off the kitchen to them while I work, would be unable to do so constantly as that is where the doggie door and only exit to the yard. I know that if I do not get licensed, I cannot sell items that need refrigeration, must clearly label my food as coming from a non-inspected kitchen and may not be able to deliver to certain venues. However, since this simply may be the only avenue open to me, I was wondering if there were any other bakers from VA or other states operating soley under cottage law without licensing? How have you done? Where have you sold? Advertising? Any help would be greatly appreciated.
i'm from va and i do cakes for friends and stuff i would love to do my own at home business but i am lost...what is cottage law exactly?
Mudderteresa, the cottage food law in VA permits use of a home based kitchen to bake cakes, etc. and sell them without being inspected. Different counties may or may not allow this, as well as may or may not require business licenses depending on profit etc. You are limited to creating items that are not perishable and must be labeled that they are are from a kitchen that isn't inspected by the state. There are couple more things, but can't remember them now. If you look up on the forums Cottage food laws in VA, there is a lot of info on them. You can also still use your home based kitchen and also get licensed. It apparently isn't super strenuous and allows you to do a lot more things. I can't really do it because I rent and have pets that I won't be able to keep out of the kitchen all the time, but if you are interested, you should contact the VA Department of Agriculture and they will send you a packet that explains everything.


text:
Expand Messages
- Shlomo,Only those who have priesthood can give the sacrament of baptism. One cannot give what one does not have authority to. Further, it is not enough to be baptized in the name of Jesus. We see this clearly in the Acts: "But when they believed Philip (who was ordained as deacon by Apostles) as he preached the things concerning the kingdom of God and the name of Jesus Christ, both men and women were baptized. Then Simon himself also believed; and when he was baptized he continued with Philip, and was amazed, seeing the miracles and signs which were done. Now when the apostles who were at Jerusalem heard that Samaria had received the word of God, they sent Peter and John to them, who, when they had come down, prayed for them that they might receive the Holy Spirit. For as yet He had fallen upon none of them. They had only been baptized in the name of the Lord Jesus." (Acts 8:12-16).
If the baptism in the name of Jesus Christ (which the people of Samaria had accepted already) is enough, why did the apostles have to send them Peter and John in order that they may receive the Holy Spirit? What we understand here is that because Philip was a deacon (and had no priesthood) his baptism was not acceptable by the apostles. Philip baptized, as John used to, by water only but without the Holy Spirit.
The Syrian Orthodox Church gives the Chrism (Confirmation) to those baptized by non-apostolic Churches, because she believes that they have not received the Holy Spirit yet.
C. Eugene Kaplan
--- In SOR-Forum@y..., mor_efraim@y... wrote:
>I would like to know on Protestant Baptism, one say have to rebaptism again, why? Is there any books/diciplines from the Apostles on it?
>Thank you.
Get more from the Web. FREE MSN Explorer download : http://explorer.msn.com
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text:
Are you looking for a partner to work with on a large scale or technically complex project? We have a depth of experience with national and international brands and organisations, helping them deliver bespoke projects on time and on brief.
Scroll down to take a look at our case studies and a taste of what we do.
4 million loyalty cards. 2 months. Distributed to 900 national locationsRead the case study
It doesn't end there. Here are a few more happy clients...
Feel free to get in touch to discuss your project.
Call us today on +44 1428 729306 or contact us online


text:
Confidential Client
I worked with a digital agency who sought design research and strategy consulting for one of their pharmaceutical clients. This client was in the process of launching a new prescription drug for heart failure patients. In addition to the drug, they were interested in designing a 12 week aftercare program for patients and caregivers, along with a mobile application to support patient care.
I worked directly with the agency and their client to design the functionality for the mobile application within the context of the aftercare program. We began with qualitative and quantitative research methods—including surveys, interviews, and ethnographic studies—to better understand the unique, nuanced situation of heart failure patients. We discovered that the biggest problems revolved around medication adherence and caregiver communication (in many cases, there were primary caregivers with a network of secondary caregivers).
I facilitated multiple collaborative design workshops with clients and a partner agency to better understand patient and caregiver contexts, frame the problems we intended on solving, and rapidly ideate solutions. I also managed an interaction designer who designed the first prototypes of the app. Early prototypes focused on health tracking (exercise, medication, diet) and communication facilitation between multiple caregivers. Some of the documentation we presented included user personas, experience maps, flow diagrams, strategic launch plan, and wireframes.
As with many pharmaceutical projects, the iron fist of the FDA determined that another round of trials was needed before the drug can be approved. The project is on hold until completion of those approvals.


text:
Spam link insertion hack
I run a few blogs and some of them have been compromised by hackers. I discovered foreign links within my articles as well as the sidebar, typically cialis and pill links.
I already have removed those foreign links and changed passwords (mysql, cpanel etc.), however I cannot help but wonder how did they get through. From what I've observed, they haven't actually gained access to WordPress; they altered the database directly. Template and WordPress files were left unchanged. In some cases, links were inserted into <img> tags, thus breaking them, which leads me to believe that links were inserted into post_content data randomly.
I have searched, however I am not sure what search terms to use; most results are ambigious.
I would appreciate advice on how to protect my blogs from these attacks; or at least proper search terms that I can use.
Thank you,
- The topic 'Spam link insertion hack' is closed to new replies.


text: This directional racerback bikini top is part of the exclusive London Edit collection and the perfect partner to the matching Beach Volleyball Bikini Bottoms. Inspired by British sporting style, feel supported due to a completely opaque powermesh lining. Instil confidence into every workout in the water with technical all-Sensitive fabric, while enjoying unsurpassed feminininity with contrasting feminine straps.


text: This newly built home embodies style, sophistication, and exquisite design. Located between Las Lomitas and La Entrada schools. This home comes with many top-of-the-line amenities, including a gourmet kitchen with island counter-top, Thermador appliances, spacious rooms, formal living room and dining room. Downstairs, a bedroom with en suite bathroom; upstairs, two bedrooms and master suite. Beautiful and tranquil back yard with fire pit, perfect for hosting or relaxation. Available for rent starting beginning of August for $10,750 per month. * Renter to verify enrollment and availability of schools. Information deemed reliable but not guaranteed. ** house is available for rent unfurnished and staging is no longer present.


text:
Aug. 25, 2012
LARAMIE, Wyo. -
The University of Wyoming Cowgirl volleyball team won the 2012 UniWyo Wyoming Invitational after posting a perfect 4-0 record. On Saturday, in Laramie at the UniWyo Sports Complex, Wyoming defeated Eastern Washington (0-4) in its first match moving their record to 3-0. In their second match, the Cowgirls pulled away from the Seattle Redhawks (2-2) winning in four sets to capture the tournament title. The Cowgirls opened their season 4-0 for the second time in three seasons, the third time since 2002 and the second time under head coach Carrie Yerty. Adding to the Cowgirls excellent weekend, junior libero Becky Stewart was named the tournament's Most Valuable Player, while senior Jodi Purdy and junior Reese Plante earned all-tournament team honors.
"I think any time you start 4-0 it is a great confidence booster for your team," Yerty said. "Through these first four games our team has played with a great level of intensity, but we have been very focused and relaxed on the floor. As for Becky earning the tournament MVP she has really linked our team so far. She has great reading skills and can move all over the floor."
Wyoming vs. Eastern Washington
The Cowgirls opened the first set against Eastern Washington with a 5-0 run on kills by Jodi Purdy, Erin Kirby and a combined block by Purdy and Kirby. EWU came back and scored four of the next seven points to make the score 8-4. Led by a Plante kill and a Purdy service ace the Cowgirls mounted a 7-2 run making the score 15-6. Wyoming did not let up advancing the lead to 22-11 before Eastern Washington managed a 6-0 run on three UW attack errors. With the score 22-17, junior Calli Miller came up with kill that shut the door on the Eagles. Following the Miller kill, UW scored two straight points including the set-winning block by Kirby and Miller. Miller led UW in points in the first set scoring five on four kills and two block assists. Kirby added 4.5 points on three kills and three block assists. Junior Rachel Keyes assisted on six kills for UW.
After trading the first eight points of the second set, the Cowgirls went on a 4-0 run on Purdy kills, a Whitney Smith ace and a combined block by Katharine Feldman and Plante to give UW a 8-4 advantage. After the Eagles ended the Cowgirl run with a kill, UW went on another run scoring six of the next eight points making its lead 14-8. The teams traded the next 15 points but the Cowgirl lead was insurmountable and the Cowgirls closed the set 25-14. Senior Purdy and junior Plante led in points with five and 4.5, respectively. Purdy scored her five points on five kills, while Plante added three kills and three block assists. On the defensive end, libero Stewart recorded seven digs. Sophomore setter Smith added five assists in the second set.
The final set began with the Cowgirls and the Eagles trading the first 12 points. With the game tied at six apiece Eastern Washington scored three straight points making the score 9-6. The Cowgirls answered that with a 10-1 run on back-to-back Miller kills, followed by combined blocks from Plante, Miller and Feldman to take the lead, 16-10. Wyoming rattled off nine of the next 12 points on Miller, Plante and Purdy kills as well as a service ace from freshman Kaitlyn Johnson to win the third set and the match 25-15. Miller led the Cowgirl scoring with 4.5 points on four kills and one block assist. Purdy, Plante and Camille Coffman each added three points apiece in the third set. Purdy and Coffman each had three kills, while Plante had one kill, one ace and two block assists. Setters Keyes and Smith added six assists, while libero Stewart added five digs and one assist.
For the match, Purdy recorded her second double-double of the season with 10.5 points and 10 digs. Also in double-figure points were Plante (10) and Miller (10). Keyes added 15 assists, while Smith added 14. Stewart hit double figures in digs with 15.
Wyoming vs. Seattle
The first set, the Cowgirls and the Redhawks battled keeping the set close all the way to 14-13 UW. The Cowgirls scored eight of the next 10 points taking a 22-15 lead. Seattle stormed back to pull within five at 24-19, but a Plante kill closed the set for UW at 25-19. Miller led the Cowgirl scoring in the first set with five points on four kills and two block assists. Plante hit a perfect 3-3 on attacks in the set and added two block assists for four total points. Purdy added three points on three kills. Keyes and Smith added five and six assists, respectively. Defensively, Stewart recorded five digs.
Set No. 2 began the same way as the first with the teams trading the first 16 points. With the score 9-7, Wyoming made its first run. Plante started the 7-3 run with a kill, followed by back-to-back blocks from Shanahan and Kirby, as well as kills from Shanahan, Miller and two from Kirby. Following the run the score went Wyoming 16, Seattle 10. The Cowgirls reeled off nine of the next 13 points including, blocks by Plante and Coffman and the set-winning kill by Shanahan. Kirby led the scoring in the second set with 4.5 points on three kills and three assisted blocks. Shanahan added four points for UW on two kills and four block assists. Purdy also added two kills and one block assist for 3.5 points. Keyes led the way with seven total assists.
In the third set, Wyoming and Seattle stayed close throughout with neither team gaining more than a two point advantage throughout. With the set tied at 22 the teams traded points to 25 before Seattle scored two-consecutive points to take the set. As with their only set loss on Friday evening, the Cowgirls recorded the most errors in the third set with eight. Miller led the scoring with 4.5 points on four kills and one block assist. Purdy and Kirby each added four points each.
The fourth and final set played much the same as the third with neither team able to gain a decisive advantage. UW and SU traded points all the way to a 20-20 tie. Seattle scored two straight points on Wyoming errors to take a 22-20 lead. Back-to-back kills from Kirby and Coffman tied the match back at 22. The Redhawks scored on a kill to take a 23-22 lead, but a well-timed Purdy kill and an attack error by SU gave the Cowgirls a 24-23 advantage. On final play of the match, Cowgirls Plante and Shanahan combined to block a Seattle attack giving the Cowgirls the 25-23 set win and the match victory. Kirby led UW in scoring in the fourth set with six kills. Plante added three kills and a block assist for 3.5 points, while Purdy added three points on three kills. Keyes proved to be an offensive weapon in the set adding nine assists for UW.
For the match four Cowgirls scored in double-figures led by, Kirby with 15 total points on 12 kills and six block assists. Purdy added 12 kills, one ace and a block assist for 13.5 points, while Miller added 11.5 points on 10 kills and three block assists. Also in double figures was Plante who scored 10 points on seven kills and six block assists. Keyes added 28 assists for the match and fellow setter Smith added 16. Libero Stewart added 13 digs to the Cowgirl total.
Other action in the UniWyo Wyoming Invitational:
9 a.m. Eastern Washington (0-3) vs. Presbyterian (1-2), 3-0
11 a.m. San Francisco (2-1) vs. Seattle (2-1), 3-0
5 p.m. Presbyterian (1-3) vs. San Francisco (3-1), 3-0
2012 UniWyo Wyoming Invitational Team Places, MVP and All-Tournament Team:
Tournament MVP and All-Tournament team honors are as follows. The team was chosen as follows, the first-place team received the MVP and two individuals, the second-place team also received two individuals, while third and fourth-place teams each earned one selection.
First place team - Wyoming (4-0)
MVP - Becky Stewart, L, Jr.
All-Tournament Team members -
Jodi Purdy, OH, Sr.
Reese Plante, OH, Jr.
Second place team - San Francisco (3-1)
All-Tournament team members -
Valentina Zaloznik, OH, So.
Jocelyn Levig, OH/MB, Sr.
Third place team - Seattle (2-2)
All-Tournament team member -
Kaytlyn Dill, OH, Sr.
Fourth place team - Presbyterian (1-3)
All-Tournament team member -
Mary Trimpe, L, RsSo.
Fifth place team - Eastern Washington (0-4)


text:
The 2016 Toyota Camry comes in four main trim levels, plus a new model. The core trim levels are LE, SE, XLE, and XSE, and for 2016 Toyota adds a sport-oriented Special Edition model that fits between the SE and XSE. These trims essentially consolidate the Camry's equipment to a conventional lineup (LE and XLE) and a sporty lineup (SE, Special Edition, and XSE). The XE, SE, and Special Edition are offered only with the 2.5-liter inline-4, while the XSE and XLE come with the 2.5 or the 3.5-liter V-6.
The Camry's feature set is in step with that of its rivals.
Standard equipment in the 2016 Camry LE includes 16-inch steel wheels, power heated side mirrors, intermittent wipers, a power driver's seat, air conditioning, cruise control, a tilt/telescopic steering wheel, power windows and locks, keyless entry, and a rearview camera. Buyers also get the Entune 6.1-inch touchscreen audio and infotainment system with a CD player, auxiliary audio input, USB port, voice recognition, Bluetooth hands-free calling and audio streaming, and six speakers.
SE models add to the LE equipment with 17-inch graphite-finish alloys, a sport suspension, projector-beam headlamps, black trim bezels, a color-keyed rear spoiler, chrome-tipped exhaust, smoked-chrome upper-grille and rear garnish trim, and a sport mesh front grille. Inside, they get SofTex faux leather upholstery, a leather-trimmed steering wheel, and brighter Optitron gauges.
The Special Edition adds to the SE equipment a sunroof, keyless ignition, Qi wireless cellphone charging, Entune Audio Plus with Connected Navigation Scout GPS Link app, and blue interior accents including contrast stitching, gauge cluster and trim, and exclusive floor mats.
The XSE model adds luxury equipment to the SE with sport Ultrasuede seats, dual-zone climate control, LED low and high-beam headlights, LED running lamps, and 18-inch machined alloy wheels. When equipped with the V-6, the XSE also gets a universal garage-door opener, an auto-dimming rearview mirror, vanity mirrors, reading lights, a sunroof, an acoustic windshield, and dual chrome-tipped exhaust outlets.
Top XLE models retain a feature list that some might say belongs to a luxury car, as they add keyless ignition, leather seats with power lumbar support, a power front passenger seat, and 17-inch Super Chrome alloys. XSE and XLE models with the 4-cylinder engine get the Entune Audio Plus system that brings Sirius satellite radio, HD radio, HD traffic and weather services, and the new smartphone navigation integration.
XSE V-6 and XLE models also add Qi wireless charging. XSE and XLE models with the V-6 get an Entune Premium Audio system with audio playback capability, HD predictive traffic, doppler map overlay, and the Entune App Suite, which lets you run Bing search, Pandora, Yelp, and others on the touchscreen using your smartphone's connection.
Equipment for 2016 Toyota Camry Hybrid models roughly parallels that of 4-cylinder Camrys of the same trim level, though the Hybrid is only offered in LE, SE, and XLE trim levels.


text:
Climate Effects on Rainfall Index Insurance Purchase Decisions
Rainfall Index insurance is a pilot insurance product offered to producers of hay and pasture in 9 states. This analysis examines the expected payoff of the RI insurance for bi-monthly periods based on rainfall shortage probabilities in alternative climate phases. Differences in expected returns indicate that selection of ENSO-specific optimal intervals may result in higher returns than those based on pooled rainfall series.
If you experience problems downloading a file, check if you have the proper application to view it first. In case of further problems read the IDEAS help page. Note that these files are not on the IDEAS site. Please be patient as the files may be large.
|Date of creation:
|Contact details of provider:
| Web page: http://www.saea.org/
More information through EDIRC
References listed on IDEAS
Please report citation or reference errors to , or , if you are the registered author of the cited work, log in to your RePEc Author Service profile, click on "citations" and make appropriate adjustments.:
- Miguel Carriquiry & Daniel E. Osgood, 2008. "Index Insurance, Probabilistic Climate Forecasts, and Production," Center for Agricultural and Rural Development (CARD) Publications 08-wp465, Center for Agricultural and Rural Development (CARD) at Iowa State University.
- Martin, Steven W. & Barnett, Barry J. & Coble, Keith H., 2001. "Developing And Pricing Precipitation Insurance," Journal of Agricultural and Resource Economics, Western Agricultural Economics Association, vol. 26(01), July.
- Denis Nadolnyak & Dmitry Vedenov & James Novak, 2008. "Information Value of Climate-Based Yield Forecasts in Selecting Optimal Crop Insurance Coverage," American Journal of Agricultural Economics, Agricultural and Applied Economics Association, vol. 90(5), pages 1248-1255.
- Podesta, Guillermo & Letson, David & Messina, Carlos & Royce, Fred & Ferreyra, R. Andres & Jones, James & Hansen, James & Llovet, Ignacio & Grondona, Martin & O'Brien, James J., 2002. "Use of ENSO-related climate information in agricultural decision making in Argentina: a pilot experience," Agricultural Systems, Elsevier, vol. 74(3), pages 371-392, December.
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text:
Blackburn closing in on keeper
By Neil Foster
Last Updated: 01/01/70 1:00am
The 21-year-old spent time on loan at Rovers towards the end of the season and, according to the club's official website, is close to signing a deal in the next few days.
The youngster has dual citizenship - German and American - and so is keen to work with Rovers' number one keeper, US international Brad Friedel (pictured).
The Ewood Park outfit released third-choice shot-stopper Ryan Robinson earlier in the year and so they require back up for the giant American and for number two Alan Kelly.
Yelldell is highly rated by Blackburn's management and should sign in time to meet up with his new team-mates for pre-season training.


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Child Psychiatry Job – Long Beach, CA #3010
Child Psychiatry Job – Long Beach, CA #3010
Our client based out of Long Beach, CA is growing and is seeking a Child & Adolescent Psychiatrist to join their team:
• Prominent California health group is expanding!
• Board Certified Psychiatrist or Board Eligible with plans to take boards
• Experience working with children and adults preferred
• Flexible models - EMPLOYED OR CONTRACTED POSITION!
• Practice at their new urgent care clinic to open in the fall of 2017
• Serving a lifespan population with 18 chairs
• Daytime live coverage or evening Telepsychiatry coverage needed!
• Benefits may include comprehensive health, 401K, CME, Vacation, ESOP
• Competitive salary!
• Not near Long Beach, but enjoy telepsych??– this STILL may be the position for you!
• Collaborative environment looking for a team player
Long Beach is a coastal city and port in Southern California. Long Beach is the 36th most populous city in the United States and the 7th most populous in California.
Moored in its Queensway Bay, RMS Queen Mary is a retired ocean liner and museum ship. The waterfront Aquarium of the Pacific features touch tanks and a shark lagoon. Modern and contemporary works are on display at the Museum of Latin American Art. Rancho Los Cerritos is a 19th-century adobe home and museum set in expansive gardens.
Long Beach, depending on the reporting location, has a Mediterranean climate, with strong semi-arid climate characteristics. Days in Long Beach are mostly sunny, as in Southern California in general.
The Long Beach Museum of Art, sited in the historic Elizabeth Milbank Anderson residence, is owned by the City of Long Beach, and operated by the Long Beach Museum of Art Foundation. Long Beach also features the Museum of Latin American Art, founded in 1996 by Dr. Robert Gumbiner. It is the only museum in the western United States that exclusively features modern and contemporary Latin American art.
For more information on this and other GREAT psychiatry jobs, contact Molly Brown at SELECT inc, (877) 499-9889 or (register or log to show contact information).
This position is specific for a Board Prepared or Board Certified Child & Adolescent Psychiatrist. Requirements then are that the successful candidate would have a degree as an M.D. or D.O. with proved residency training in Psychiatry and a Fellowship in C&A Psychiatry.
Job Posted: 08/18/17
Select, Inc. is hiring! Interested? Submit your info below.
Contact Information
P.O. Box 706
Bowling Green KY 42102-0706


text:
- Hydroscope
- Hydroscope Hy"dro*scope, n. [Hydro-, 1 + -scope.]
1. An instrument designed to mark the presence of water,
especially in air. --Weale.
[1913 Webster]
2. A kind of water clock, used anciently for measuring time, the water tricking from an orifice at the end of a graduated tube.
The Collaborative International Dictionary of English. 2000.


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|51 m²
|Property type
|Minimum Stay
|3 - 5 nights
'The Pool House Suite'
Come and relax at one of St. John's newest Luxury Boutique Inn. Located high up in North Chocolate Hole/ Gifft Hill, on St John's dramatic south shore. Enjoy amazing views of the St. John south shore bays and beaches with vista over the Caribbean Sea out to St. Croix.
The owners live in a separate villa on the property with their little dogs Millie and Lucy.and they are available for any questions or anything you may need to make your stay complete! St John Guest Suites Villa boasts two private and luxurious rental suites and two luxury guest bedrooms. Has a larger party? Rent all four units!
Sugar Cane Suite - VRBO Listing #499095
Sea View Guest Room - VRBO Listing #555929
The Lilly Guest Room - VRBO Listing #513590
We will provide you a welcome basket of coffee, soft drinks, fruit, cheese & crackers so you can rest and recharge after your travels arriving on our beautiful island. We will pick you up at the ferry station and take you to your rental car. We will have you follow us up to your villa, so your arrival is painless!
The Pool House Suite is on the main floor of the complex (no steps) and is next to the spectacular infinity edge pool. The suite has stunning panoramic views of the Caribbean Sea from its private deck.
Take full advantage of taking a dip with sea turtles and starfish inlaid in the stunning, shared, infinity pool overlooking the blue waters of the Caribbean Sea just steps from your suite. Pool floats for the pool or beach are perfect to let you completely relax.
To spoil yourself, we have a friend who does Massage in your suite or the porch by the pool! We can also arrange for poolside yoga. The Westin Spa and Gym are 3 minutes away; you can join for your stay, to keep up your exercises routine. Reservations during the high season and holidays should be booked a month ahead!
The suite features a brand new king sized pillow top bed, luxury 100% cotton linens, down pillows, and a lovely sitting area with two club chairs and an ottoman. Beautiful Travertine stone floors throughout the suite.
We have wireless Vi Broadband Internet and Dish TV on your wall mounted Flat screen television
The new bathroom has a private toilet closet, double sinks, and a beautiful stone and shell shower. Luxury towels and linens are provided.
The kitchenette is fully equipped for easy in suite dining. It includes a microwave/convection oven, electric burner, electric frypan, refrigerator/freezer, sink, toaster, coffee maker (and filters), hot water kettle, dishes, silverware and barware. Assorted spices are also provided.
Also, by the pool is an outdoor kitchen with a built-in grill, sink, ice maker, gas burners and a refrigerator.
The suite is also supplied with beach towels, a cooler, beach bag cooler, ice packs, beach chairs, and beach floats.
The onsite owners will make sure your stay is as enjoyable as possible.
The villa is only 5 - 8 minutes away from all the dining and shopping options of Cruz Bay. Ready for the beach? Hop in your jeep and after a stunning 15 minute drive through the national park land you will have a plethora of white sandy beaches to choose from, some rated the best in the world. The small town of Coral Bay, on the other side of the island, is a 20-minute drive from the villa.
Christmas/New Years 2015 Check-ins and Check outs Saturday and Sundays only.
Our Property Extends From The End Of Rock Ridge Road (No Access) to Gift Hill Road (Where We Have Access), Fabulous Panoramic Ocean Views, 15 minutes to North Shore Beaches, 1/2 mile to South Shore Beaches. One mile into Cruz Bay. Cab access easy if you do not feel like driving to town or back. We have a list of cabs that will pick you up and bring you back home.
|Cleaning Fee


text:
Circuit Breaker Enclosure, Surface, 3R
GENERAL ELECTRIC| Zoro #: G0932373| Mfr #: TK4V1200R
Zoro #: G0932373Mfr. Part #: TK4V1200R
$2157.15 /ea
This item ships free. DetailsStandard ground shipping
Technical Specifications
|Zoro #: G0932373 Mfr #: TK4V1200R
|Mounting Style: Surface
|Item: Circuit Breaker Enclosure
|NEMA Rating: 3R
|Color: Gray
|Finish: Painted
|Material: steel
|Length: 47.75"
|Depth: 8.94"
|Amps: 1200
|Width: 23.38"
|Country of Origin (subject to change): Mexico
Product Description
Circuit Breaker Enclosure, Mounting Style Surface, NEMA Rating 3R, 1200 Amps, Gray, Painted Finish, Material Steel, Length 47.75 In., Width 23.38 In., Depth 8.94 In., For Use With GE SK, TKMA, THKMA Circuit Breakers


text:
Lanvin Happy Python Metallic Shoulder Bag
Lanvin Happy Python Metallic Shoulder Bag £1,799
This incredibly super stunning Lanvin Happy Python Metallic Shoulder bag is without a doubt, one of the most beautiful party bags I have seen in my life. This Lanvin Happy metallic Python bag is ideal if you're going to a hot party, a night out on the town, or a very upmarket restaurant. This is a bag that should be preserved only for special occasions, ones that are worthy of its spectacular beauty. Where do I start to dissect the gorgeousness of this bag? OK, lets start with the metallic python leather. As you can see, this Lanvin shoulder bag is made with metallic python leather, and has a dual toned gradient of gold and pink (great combination of color). The front of the Happy Python Metallic shoulder bag is an incredibly detailed beautiful clasp, which reeks high quality. The gold plate with the what looks like a wooden clasp gives the bag an interesting edge.
The chain strap of this Lanvin Happy Python bag has a leather handle at the top with interwoven material on the one side and features a stunning piece of black material at the top of the strap and what looks like a silver coin charm. The shoulder strap also doubles up so you can carry it in your hands. This sexy, stunning, Lanvin Happy Python Metallic Shoulder bag really pushes boundaries. It's not safe, but hey, safe is boring. Why not dazzle up your wardrobe with this stunner. I know I'm going to be saving up for it! One thing is for sure is that this Happy Python Metallic shoulder bag will definitely make you happy! Buy through Luisa Via Roma


text:
Round Metal-Trim Sunglasses
Standard Price $6.99 Sales Price $2.99
Select Color
Select size
Details content below
- California Proposition 65 WARNING: This product contains a chemical known to the State of California to cause birth defects or other reproductive harm. California Health and Safety Code Section 25249.6
Product Fit:5.5" wide by 2.5" tall
Product Care:wipe clean / imported
These round, retro sunglasses are extra trendy, with wire metal frames that outline the fun reflective lenses!
Returns content below
Return or exchange in any Charlotte Russe store within 30 days of your ship date, or use the mailing label included in your package to send it back right away.
We do not accept returns on jewelry, swim, intimates, nail polish, fragrance, gift cards, opened beauty products or anything marked as final sale.
You Might Like These Too
Style: 302247436


text:
Phone: 609-841-9740
E-mail: firstname.lastname@example.com
Office Hours: Monday thru Friday 9:00am to 5:00pm
If you are only available evenings or weekends a meeting time can be made upon your request.
LME Designs, LLC is located in Haddon Heights, NJ. Conveniently located ten minutes from Cherry Hill, NJ and Center City Philadelphia.
Call today for a Free quote, 609-841-9740. Or complete the Request a Quote form and click the "Click Here To Send" button.
Request A Free Quote!
Talk to you soon! 🙂


text:
Dog Training
Our Goal is to provide training programs which result in a confident, obedient, and happy dog. We understand every pet is unique and we strive to bring out the best in each. It doesn't matter if this is a puppy getting their first training, an older dog with long term bad habits or a dog with aggressive tendencies and behaviors.
Our Industry-leading results and customer service will allow each owner to achieve the maximum control of their dog on and off leash while still allowing your pet to "act like a dog".
Our Dog training programs address such things as:
Basic Puppy Manners
Come When Called
Leash PullingHouse Breaking
Control Aggression
Seperation Anxiety
Nipping And Chewing
Basic And Advanced Obedience
Nuisance Barking
K-9 Work Dogs
Tricks And Agility


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- Tie My ShoesVote A
- Drive a CarVote B
- Cook on the StoveVote C
- Roll my TongueVote D
- Not flexibleVote E
- WhistleVote F
- Other (You Name it!!) 😉Vote G
Most Helpful Guy
Pronounce the letter "L" at the end of a word. Seriously, I can't do it.
For example wool, steel, foul, etc. Like if I said "steel wool" I would pronounce it "steo woe". It's similar to how people from Boston say "caw" instead of "car".33|00|0
Hahahaha sorry I had to laugh lol
My friends used to laugh too. This is an American Midwest thing. It's not all of the Midwest, I think it's probably highly localized. When I moved out west my friends used to get me to say steel wool. I'd try and try and couldn't do it, and they'd all get a kick out of it. My girlfriend moved out there and she couldn't do it either.
Most Helpful Girl
I can't drive a car (where I'm from we can only drive at 18), can't whistle, but I used to be able to, I can't tell the time with analog clocks, I can't roll my Rs, I can't throw or catch for shit, I suck at tying shoe laces, I only learnt how to ride a bike this year (my best friend spent 3 days teaching me 😂) anddd there's more but we would be here all day 😂11|00|0
My twin hahaha


text:
A Mississippi hunter last seen Thursday is the subject of a search in the Pearl River Wildlife Management Area, according to the St. Tammany Parish Sheriff's Office.
Robert Varnado, 42, was hunting with a friend when the two got separated. After the friend didn't hear from Varnado by Saturday, he reported Varnado missing, according to a post on the sheriff's Facebook page.
Deputies searched Saturday and Sunday but did not find the 5-foot-6, 157-pound Varnado.
According to the Facebook post, Varnado has numerous tattoos, including a Star of David under his right eye and tear drops under his left eye.
Anyone who sees or hears from Varnado is asked to call Detective Justin Parker at (985) 726-7852.


text:
The murein peptide amidase MpaA is a cytoplasmic enzyme that processes peptides derived from the turnover of murein. We have purified the enzyme from Escherichia coli and demonstrated that it efficiently hydrolyses the γ-D-glutamyl-diaminopimelic acid bond in the murein tripeptide (L-Ala-γ-D-Glu-meso-Dap), with Km and kcat values of 0.41±0.05 mM and 38.3±10 s−1. However, it is unable to act on the murein tetrapeptide (L-Ala-γ-D-Glu-meso-Dap-D-Ala). E. coli MpaA is a homodimer containing one bound zinc ion per chain, as judged by mass spectrometric analysis and size-exclusion chromatography. To investigate the structure of MpaA we solved the crystal structure of the orthologous protein from Vibrio harveyi to 2.17 Å (1Å=0.1 nm). Vh_MpaA, which has identical enzymatic and biophysical properties to the E. coli enzyme, has high structural similarity to eukaryotic zinc carboxypeptidases. The structure confirms that MpaA is a dimeric zinc metalloprotein. Comparison of the structure of MpaA with those of other carboxypeptidases reveals additional structure that partially occludes the substrate-binding groove, perhaps explaining the narrower substrate specificity of the enzyme compared with other zinc carboxypeptidases. In γ-proteobacteria mpaA is often located adjacent to mppA which encodes a periplasmic transporter protein previously shown to bind murein tripeptide. We demonstrate that MppA can also bind murein tetrapeptide with high affinity. The genetic coupling of these genes and their related biochemical functions suggest that MpaA amidase and MppA transporter form part of a catabolic pathway for utilization of murein-derived peptides that operates in γ-proteobacteria in addition to the established murein recycling pathways.
- Escherichia coli
- murein tripeptide
- protein structure
- transporter
- Vibrio harveyi
- zinc carboxypeptidase
Peptidoglycan (murein) is the major structural component of bacterial cell walls that forms a mesh-like layer outside the cytoplasmic membrane and is essential for maintaining cell growth with high turgor pressure and for conserving cell shape [1,2]. It consists of glycan strands composed of alternating units of GlcNAc (N-acetylglucosamine) and MurNAc (N-acetylmuramic acid), linked together by β-1,4 glycosidic bonds [3]. In Escherichia coli the glycans are decorated with pentapeptides (L-Ala-γ-D-Glu-meso-Dap-D-Ala-D-Ala) that are attached to the carboxy groups of MurNAc in nascent peptidoglycan. The strength of the wall is a result of approximately half of these peptides forming covalent bonds with peptides on adjacent glycan strands [4]. They are linked by bonds between the amino group of m-Dap (meso-diaminopimelic acid) at position 3 of one peptide and the carboxy group of the D-Ala residue at position 4 of adjacent peptide [2]. In mature peptidoglycan there is subsequent cleavage of one or both of the D-Ala residues, leaving the tetrapeptide, or the tripeptide as the common form present in the mature polymer, with only traces of unprocessed pentapeptide [4,5].
The biosynthesis of peptidoglycan is a complex process that takes place in three subcellular compartments: the cytoplasm, the cytoplasmic membrane and the periplasm (Figure 1). The pentapeptide is assembled on a UDP-MurNAc molecule through the action of a series of Mur enzymes to create the UDP-MurNAc-pentapeptide [6]. The phospho-MurNAc-pentapeptide motif of the latter precursor is then transferred on to the undecaprenyl-phosphate carrier lipid by the MraY membrane translocase, generating lipid I. Subsequent addition of a GlcNAc motif by the MurG transferase yields the lipid II intermediate which is flipped to the periplasmic face of the inner membrane where disaccharide-pentapeptide units are finally inserted into peptidoglycan by murein synthases [5].
The cell wall of bacteria is a dynamic structure which is continually remodelled during cell growth and division. In E. coli 40–50% of the peptidoglycan is broken down and reused each generation for the synthesis of new murein [7]. In this process, murein is degraded by the action of murein-hydrolysing enzymes which include lytic transglycosylases, amidases and endopeptidases. The resulting anhydro-muropeptides (GlcNAc-anhMurNAc-L-Ala-γ-D-Glu-meso-Dap-D-Ala) are imported into the cytoplasm by a specific permease (AmpG), where they are efficiently reused in a well-studied murein recycling pathway [7,8] (Figure 1). In the cytoplasm, the muropeptides are broken down by an amidase AmpD to yield GlcNAc-anhMurNAc and the tetrapeptide (L-Ala-γ-D-Glu-meso-Dap-D-Ala) [9]. The tetrapeptide is subsequently hyrolysed by LD-carboxypeptidase LdcA [10] to produce D-Ala and Mtp (murein tripeptide, L-Ala-γ-D-Glu-meso-Dap). Under normal growth conditions these tripeptides are ligated to UDP-MurNAc by Mpl (murein peptide ligase) and thus re-enter the pathway for de novo peptidoglycan synthesis [11] (Figure 1).
A second pathway of peptidoglycan turnover uses the outer membrane protein AmiD, which is homologous with the cytoplasmic AmpD protein [12]. AmiD activity releases tri- and tetra-peptides in the periplasm from both the intact murein, but also from released muropeptides, and indeed there is strong evidence that E. coli actually secrete both peptides into the growth medium [13]. The released tri- and tetra-peptides are poor substrates for AmpG [8] and the tripeptide is recycled via the MppA/OppBCDF ABC transporter (ATP-binding cassette transporter) [14,15]. A route for uptake of the tetrapeptide has yet to be identified.
It was proposed that tri- and tetra-peptides were recycled via another enzyme, MpaA, which was identified on the basis of its homology with a sporulation-related enzyme ENP1 from Bacillus sphaericus that cleaves the γ-D-Glu-meso-Dap amide bond from both tri- and tetra-peptide [16,17] and which is a member of the peptidase family M14 [CPA (carboxypeptidase A) family] [18]. Although in vitro biochemical characterization of this enzyme has not been reported, there is genetic evidence which suggests a function for this enzyme in E. coli [17]. In the normal recycling pathway described by Park and Uehara [7], cytoplasmically released Mtp is ligated to UDP-MurNAc by Mpl. Strains lacking mpl accumulate Mtp in the cytoplasm and a strain lacking both mpl and mpaA accumulated significantly higher levels of Mtp, suggesting that its normal function is to breakdown Mtp in the cell [17]. This enzyme clearly competes with Mpl for substrate and so possibly has a catabolic function that is regulated to ensure that it is active only when peptidoglycan biosynthesis is not a limiting factor for growth. The product of the MpaA-catalysed reaction on the Mtp substrate, L-Ala-D-Glu, is then converted into L-Ala-L-Glu and subsequently into L-Ala and L-Glu by YcjG epimerase and PepD peptidase respectively [20]. The amino acids produced can be used as carbon and nitrogen sources by E. coli.
To determine the precise function of MpaA in E. coli, we have characterized the protein in vitro to study its kinetic parameters and substrate specificity, determined its oligomeric state and solved the structure of the Vibrio harveyi MpaA (Vh_MpaA) protein by X-ray crystallography, revealing the structural basis of the action of a zinc carboxypeptidase with a very narrow substrate specificity.
Restriction endonucleases were purchased from New England Biolabs, oligonucleotides were produced by MWG-Biotech, and DNA purification kits were obtained from Macherey-Nagel. DNA sequencing was performed at the technology facility in the University of York. Tripeptides L-Ala-γ-D-Glu-L-Lys, L-Pro-L-Phe-L-Lys and L-Lys-L-Glu-L-Lys were purchased from Alta Bioscience. L-Ala-D-Glu-meso-Dap was purchased from Anaspec (Cambridge Bioscience). γ-D-Glu-meso-Dap was purchased from Invitrogen. MurNAc-L-Ala-γ-D-Glu-meso-Dap, UDP-MurNAc-L-Ala-γ-D-Glu-meso-Dap, UDP-MurNAc-L-Ala-γ-D-Glu-meso-Dap-D-Ala, L-Ala-γ-D-Glu-meso-Dap-D-Ala and L-[14C]Ala-γ-D-Glu-meso-Dap were prepared as described previously [11]. GlcNAc-MurNAc (anhydro)-Mtp and GlcNAc-MurNAc (anhydro)-Mtp-D-Ala were prepared as described previously [8]. Genomic DNA of V. harveyi ATCC BAA-1116 was purchased from LGC standards.
Bacterial strains, plasmids and growth conditions
E. coli strain DH5α (Invitrogen) was used for cloning and maintenance of plasmid constructs, whereas E. coli strain BL21 (DE3) (Novagen) was used for expression of recombinant protein. Both strains were grown routinely in LB (Lennox broth) with antibiotic (kanamycin at a concentration of 30 μg/ml) and growth was monitored at A650 using a Jenway 6305 UV–Vis spectrophotometer. Selenomethionine-labelled Vh_MpaA was produced in a methionine auxotroph strain, B834 (DE3), of E. coli. Strains, plasmids and primers used in the present study are listed in Table 1.
Cloning, expression and purification of MpaA and MppA
The LIC (ligation-independent cloning) method was used for cloning of mpaA into pETYSBLIC3C vector. This vector is based on the pET-28a vector (kanamycin-resistant, 30 μg/ml) and allows expression of recombinant proteins with a His6 tag on N-terminus of the protein [21]. The mpaA gene was amplified from genomic DNA of E. coli K-12 MG1665 using PCR primers Ec_MpaAF and Ec_MpaAR and from genomic DNA of V. harveyi ATCC BAA-116 (gene VIBHAR_07057) using the PCR primers Vh_MpaAF and Vh_MpaAR (Table 1). Primers were designed such that a LIC tail was added to the PCR product, which was designed to be complementary to the LIC cassette in pETYSBLIC3C. PCR-amplified fragments were cloned into pETYSBLIC3C. The resultant expression vectors were named as pAM6092 and pAM6093. The nucleotide sequence was verified by DNA sequencing.
For synthesis of recombinant proteins, each construct (pAM6092 and pAM6093) was introduced into BL21 (DE3) cells. For enzymology, the transformed cells were grown aerobically in 5 ml of LB for 8 h, and used to inoculate 50 ml of LB for overnight aerobic growth at 37°C. This was then used to inoculate 625 ml of LB at 37°C. Cells were allowed to grow aerobically to an A650 of 0.4–0.6 before production of recombinant protein was induced with 0.4 mM IPTG (isopropyl β-D-thiogalactopyranoside), followed by further overnight aerobic incubation at 37°C. Cells were harvested by centrifugation at 4430 g for 15 min at 4°C and resuspended in buffer A [50 mM Tris/HCl (pH 8), 500 mM NaCl and 10 mM imidazole] supplemented with a cocktail of protease inhibitors (Complete™ EDTA-free tablets, Roche). The cells were lysed by sonication and cell debris was removed by centrifugation at 38000 g for 30 min at 4°C and the supernatant containing overexpressed protein was collected. Ni2+-affinity chromatography was used for the purification of the His6-tagged protein using a 5 ml His-trap column (GE Healthcare), connected to an ÄKTA purifier P-900. The soluble fraction containing the protein of interest was passed over a column pre-equilibrated with buffer A. Weakly bound contaminants were removed by washing the column with 20 CV (column volumes) of buffer A and the recombinant protein was eluted by applying a linear gradient of imidazole (0–500 mM). To remove the His6 tag, purified protein was incubated with HRV3C protease in a ratio of 1:100 at 4°C overnight and then separated from the protease by a second Ni2+-affinity chromatography step [a 5 ml His-trap column (GE Healthcare)] where the protease is retained on the column through an N-terminal His6 tag. Four residues from the affinity tag (Gly-Pro-Ala-Met) remain attached to protein following HRV3C cleavage. Fractions containing cleaved protein were pooled, dialysed and concentrated using Vivaspin 5 kDa MWCO (molecular-mass cut-off) concentrators. Protein concentrations were determined from the absorbance at 280 nm (A280) using calculated molar absorption coefficients of 45790 cm−1·M−1 and 28360 cm−1·M−1 for Ec_MpaA and Vh_MpaA respectively. E. coli MppA was purified as described previously [15].
For crystallization, pAM6093 was transformed into a methionine auxotroph strain, B834 (DE3) of E. coli. The transformed cells were grown aerobically in 5 ml of LB for 8 h, and used to inoculate 50 ml of LB for overnight aerobic growth at 37°C. This was then used to inoculate 625 ml of M9 minimal medium supplemented with 2.5 g of glucose, 2 mM MgSO4, 25 μg/ml FeSO4·7H20, 25 mg of each amino acid except methionine, 25 mg of L-selenomethionine, a cocktail of vitamins (riboflavin, niacinamide, pyridoxine monohydrochloride and thiamine, each at 1 μg/ml) and the antibiotic kanamycin at 30 μg/ml. Cells were allowed to grow aerobically to an A650 of 0.4–0.6 before synthesis of recombinant protein was induced with 0.4 mM IPTG, followed by a further 4 h aerobic incubation at 37°C. A purification protocol similar to that for the native enzyme/protein was used for purification of selenomethionine-labelled protein. Crystallization experiments used protein with a His6 tag.
Protein analysis by MS
A Micromass LCT Premier XE mass spectrometer using a nanospray source was used for ES (electrospray)-MS. For analysis of MpaA under denaturing conditions, purified MpaA (after cleavage of the His6 tag) was dialysed into 10 mM Tris buffer (pH 8) and concentrated to 100 μM. Concentrated protein was diluted to 1 μM in 1:1 acetonitrile/water containing 0.1% formic acid. The sample was passed into the ES-MS and the data were collected over 3 min within an m/z range of 100–2000. For analysis under native conditions, purified MpaA was dialysed into 200 mM ammonium acetate and then concentrated to 100 μM. Data were collected over 30 min within an m/z range of 100–8000. The raw m/z data were deconvoluted to mass spectra using the MaxEnt1 routine in the MassLynx software provided by the manufacturer (Waters).
SEC (size-exclusion chromatography)-MALLS (multi-angle laser light scattering)
For SEC-MALLS, samples of MpaA (100 μl of 1 mg/ml in 20 mM Tris buffer, pH 8) were applied to a Superdex 200 10/300 GL SEC column (GE Healthcare) at 0.5 ml/min with an HPLC system (Shimadzu), linked to a Wyatt Dawn Heleos MALLS detector and a Wyatt Optilab rEX unit, to determine the refractive index. The buffer was 150 mM NaCl and 20 mM Tris (pH 8). Data were analysed with the program Wyatt ASTRA version 18.104.22.168.
Enzymatic assays for MpaA amidase activity
The standard assay mixture (25 μl) contained 100 mM Tris/HCl (pH 8), 0.2 mM tripeptide L-[14C]Ala-γ-D-Glu-meso-Dap (40 Bq) and purified MpaA [5 ng of protein in 5 μl of 50 mM Tris buffer (pH 8), containing 0.5 mg/ml BSA]. Mixtures were incubated at 37°C for 30 min, and the reactions were stopped by addition of 10 μl of acetic acid. Radiolabelled substrate [14C]L-Ala-γ-D-Glu-meso-Dap and reaction product [14C]L-Ala-D-Glu were separated by TLC on silica gel plates LK6D (Whatman), using ethanol/1 M ammonium acetate (pH 3.8) (5:2) as a mobile phase. The radioactive spots were located and quantified with a radioactivity scanner (Multi Tracemaster LB285; Berthold). When other compounds were tested as substrates, assay conditions were modified: the amount of MpaA enzyme was appropriately adjusted (from 5 ng to 10 μg), as well as the time of incubation (from 30 min to 16 h). Some enzymatic assays used unlabelled compounds as substrates; in that case, reaction mixtures were analysed by HPLC, using a C18 ODS Hypersil 3 μm column (250 mm×4.6 mm; Grace). Elution was performed either using isocratic conditions with 0.05% TFA (trifluoroacetic acid) or by applying a gradient of acetonitrile (0–20% acetonitrile in 0.05% TFA) at a flow rate of 0.6 ml/min. Detection was at 214 nm. In some cases, the enzymatic digestion of tripeptide-derivative compounds was also analysed by measuring the release of the C-terminal amino acid residue (Dap or lysine) using a Hitachi L8800 amino acid analyser (ScienceTec).
For determination of the kinetic constants, the same assay was used with various concentrations of Mtp. In all cases, the substrate consumption was <20% and linearity was observed within the interval even at the lowest substrate concentration. The data were fitted to the equation v=VmaxS/(Km+S), using the Simplex algorithm (MDFitt software developed by M. Desmadril, IBBMC, Orsay, France).
Crystallization of Vh_MpaA
For crystallization, selenomethionine-labelled Vh_MpaA was concentrated to 20 mg/ml in 50 mM Tris/HCl (pH 8). Crystallization screening experiments were performed using several commercial kits (Index, Hampton I and II, Newcastle, PACT and Morpheus) by hanging-drop vapour diffusion procedures in 96-well plates set up with a Mosquito nanolitre pipetting robot (TTP Labtech). The plates were sealed to avoid evaporation and incubated at 20°C. The initial crystallization droplet contained 150 nl of a 20 mg/ml protein solution in 50 mM Tris/HCl (pH 8.0) mixed with 150 nl of well solution containing 0.1 M Mes/imidazole (pH 6.5), a 0.02 M mix of monosaccharides (Morpheus condition F2) and a mix of PEG [poly(ethylene glycol)] 8000 and ethylene glycol. Optimization experiments led to crystal growth over 7–10 days in 0.12 M Mes/imidazole (pH 6.5), 0.01 M D-glucose, 8% PEG 8000 and 20% ethylene glycol. Prior to flash-freezing in liquid nitrogen, crystals were soaked for 30 s in a cryoprotectant solution of 0.12 M Mes/imidazole (pH 6.5), 8% PEG 8000 and 40% ethylene glycol. SAD (single-wavelength anomalous dispersion) data were collected from a cryo-cooled crystal (100 K) on beamline I02 at the Diamond Light Source, UK, using a Mar CCD (charge-coupled device) detector. The SAD data were processed using the programme Xia2 [22]. The diffraction data statistics are summarized in Table 2.
X-ray data collection, structure solution and refinement
ShelxCDE [23] was used to solve the structure of Vh_MpaA using SAD data collected from a selenomethionine crystal. ShelxD was used to locate the two selenium atoms present in the two molecules of the Vh_MpaA asymmetric unit. The location of the two selenium atoms allowed phasing of the Vh_MpaA structure and the space group was unambiguously determined to be P6522 following the density modification step performed by ShelxE. The resultant electron-density map was of sufficient quality to allow 244 of the 258 residues to be built using the programme Buccaneer [24]. The Vh_MpaA model produced by Buccaneer was refined iteratively by maximum likelihood methods in REFMAC5 [25] and model building using COOT [26] to give Rfactor and Rfree values of 19.6% and 23.2% respectively. The refinement statistics are given in Table 2. The structure was validated using COOT and MOLPROBITY [27]. The co-ordinates and experimental structure factors have been deposited in the RCSB PDB under the accession code 4AXV. The dimer interface was analysed using PISA [28].
Fluorescence spectroscopy
Protein fluorescence experiments used a FluoroMax 4 fluorescence spectrometer (Horiba Jobin-Yvon) with connecting water bath at 25°C. Ligand free protein (MppA*) was purified as described previously [15] and was used at a concentration of 0.05 μM in 50 mM potassium phosphate (pH 7.8). Protein was excited at 297 nm with slit-widths of 3.5 nm and emission was monitored at 330 nm with slit-widths of 3.5 nm. To determine the Kd for tetrapeptide binding, the protein was titrated with increasing concentrations of the tetrapeptide and the corresponding fluorescence change was monitored in time-acquisition mode. The cumulative fluorescence change was plotted in SigmaPlot and the Kd was calculated from the hyperbolic fit of the binding curve.
ITC (isothermal titration calorimetry)
Calorimetry experiments were performed using a VP-ITC instrument (MicroCal) by taking 1.4 ml of MppA* in the calorimeter cell and 400 μl of tetrapeptide ligand in the syringe. The concentration of ligand in the syringe was typically 10-fold that in the cell, whereas the cell concentration was chosen according to a c value of 50, where c=[macromolecule]/(predicted) Kd.
Experiments were carried out in 50 mM potassium phosphate buffer (pH 7.8), at 25°C. Both cell and syringe solutions were degassed at 20°C for 10 min before use. The titrations were performed as follows. A single preliminary injection of 5 μl of ligand solution was followed by 28 injections (10 μl), delivered at an injection speed of 10 μl/s. Injections were made at 3 min intervals with a stirring speed of 307 rev./min. Raw titration data were integrated and fit to a one-site model of binding using MicroCal Origin version 7.0.
Overexpression and purification of Ec_MpaA
To investigate the biochemical properties of Ec_MpaA, a recombinant mpaA gene was expressed in E. coli to produce an N-terminally His6-tagged protein. SDS/PAGE analysis of cell extracts showed that induced cells accumulated a recombinant protein with a molecular mass of approximately 28 kDa with 30–40% of the overproduced protein present in the soluble fraction (Supplementary Figure S1 at http://www.BiochemJ.org/bj/448/bj4480329add.htm). Soluble protein was purified using Ni2+-affinity chromatography and the His6 tag was removed using HRV3C protease to produce native MpaA with four additional amino acid residues (Gly-Pro-Ala-Met) at the N-terminus. After purification, the protein was ~99% pure as judged by Coomassie Blue staining of SDS/PAGE gels (Figure 2A, inset), with a final yield of 12 mg/l. ES-MS under denaturing conditions was used to determine the molecular mass of the recombinant MpaA protein to be 26783±1 Da (Figure 2A), which is consistent with the predicted mass of the protein after removal of the His6 tag (26782 Da).
MpaA is a homodimer in solution containing two bound zinc ions
The purified Ec_MpaA was further examined using ES-MS, this time under native conditions that often preserves non-covalent interactions between proteins and their ligands [29,30]. We observed a major species with mass of 26846 Da and a minor species with a mass of 53692 Da (Figure 2B). The difference in mass of the species observed under denaturing conditions (26783±1 Da) and that observed under native conditions (26846±1) is 63 Da. These data indicate that Ec_MpaA contains a zinc ion (65.3 Da) consistent with its assignment based on structure to the family of Zn2+-dependent amidases. Also, the second peak of 53692±2 Da is exactly twice that of the native zinc-bound form, suggesting that MpaA forms a homodimer which bind two molecules of zinc. To investigate the native oligomeric state of Ec_MpaA further we analysed the protein using SEC-MALLS (Figure 2C), which revealed a single symmetric peak for MpaA with a calculated mass of 54±1kDa. This is a close match to that observed by native ES-MS for a dimer species and so confirms that Ec_MpaA is a dimer in solution.
The enzymatic activity of MpaA is highly specific to L-Ala-γ-D-Glu-meso-Dap (Mtp)
The MpaA enzyme has been demonstrated to cleave L-Ala-γ-D-Glu-meso-Dap (Mtp) to L-Ala-γ-D-Glu and Dap in a qualitative assay [17] and we used the purified enzyme to characterize this reaction further in vitro. MpaA was able to hydrolyse Mtp at a rate of 200 nmol·s−1·mg−1 (Table 3). The kinetic parameters of MpaA towards Mtp were determined using substrate concentrations ranging from 0.07 to 0.7 mM. The calculated Km and kcat values were 0.41±0.05 mM and 38.3±10 s−1 respectively (Table 3). The pH-dependence of the MpaA-catalysed reaction was investigated revealing a rather flat curve between pH 7.2 and pH 9, with an optimum value at approximately 8.0 (Supplementary Figure S2 at http://www.BiochemJ.org/bj/448/bj4480329add.htm). The activity of MpaA was totally inhibited by incubation with 10 mM EDTA (results not shown) suggesting that the enzyme requires the presence of metal ions.
It has been suggested that Mtp is also a substrate for MpaA [17], but surprisingly we saw no activity with this substrate. Although the enzyme cannot tolerate a C-terminal extension in the peptide substrate, we investigated the ability of the related tripeptide L-Ala-γ-D-Glu-L-Lys to be used as a substrate and this did give a low rate of cleavage, approximately 1.5% of that seen with Mtp. The enzyme also had extremely low, but detectable, activities with the MurNAc tripeptide and the dipeptide γ-D-Glu-meso-Dap, and did not act on a number of other peptidoglycan precursors and their analogues (Table 3).
These data suggest that Mtp is the physiological substrate of MpaA and that the enzyme cannot tolerate many changes in this structure, including the length of the peptide and the presence of a γ-D-Glu-meso-Dap bond. The lack of activity on the tetrapeptide demonstrates that MpaA does not exhibit any endopeptidase activity.
The structure of Vh_MpaA resembles eukaryotic carboxypeptidases
In order to probe further into the mechanism of MpaA action we sought to determine the structure of E. coli MpaA, but were unable to grow crystals. However, we were able to grow crystals and determine the crystal structure to 2.17 Å (1 Å=0.1 nm) resolution of MpaA from V. harveyi (Vh_MpaA, VIBHAR_07057) (Table 2). This orthologue was chosen because V. harveyi is a γ-proteobacterium that contains a divergently transcribed mpaA/mppA gene pair described later in the present paper. We achieved a high level of production of Vh_MpaA and confirmed its mass using ES-MS (Supplementary Figure S3A at http://www.BiochemJ.org/bj/448/bj4480329add.htm). Incorporation of L-selenomethionine to Vh_MpaA was confirmed using ES-MS (Supplementary Figure S4 at http://www.BiochemJ.org/bj/448/bj4480329add.htm). Vh_MpaA was also found to be a dimer in solution by SEC-MALLS and ES-MS suggested that, as for Ec_MpaA, the V. harveyi MpaA dimer also binds two zinc ions (Supplementary Figures S3B and S3C). Vh_MpaA is able to hydrolyse Mtp (specific activity of 190 nmol·s−1·mg−1) (Supplementary Table S1 at http://www.BiochemJ.org/bj/448/bj4480329add.htm), but not murein tetrapeptide, confirming that it is a functional orthologue (the two proteins are 56% identical and 67% similar over 230 amino acids) (Supplementary Figure S5 at http://www.BiochemJ.org/bj/448/bj4480329add.htm) of the E. coli MpaA protein. We crystallized a 258 residue polypeptide consisting of Vh_MpaA fused at its N-terminus to a 22 residue sequence containing a His6 tag. The first 15 residues of this sequence are not defined in the electron-density maps and they are assumed to be disordered. The remaining seven residues of the tag are observed as they make crystal packing interactions (results not shown). In the refined model of Vh_MpaA, residue numbering starts at the first methionine residue of the native protein (1–235). There is one Zn2+ ion (at the active site) and 46 water molecules associated with each molecule of Vh_MpaA.
The overall fold is one seen in zinc metallocarboxypeptidases [31] of both prokaryotes and eukaryotes. It comprises a central eight-stranded mixed β-sheet with surrounding α-helices (Figure 3A). A DALI search of the PDB revealed that Vh_MpaA has the highest structural similarity to a number of eukaryotic general carboxypeptidases giving Z-scores of 20±0.5 for ~200 equivalent residues with an rmsd (root mean square deviation) of 2.7±0.1 Å. The three best matches were CPD (carboxypeptidase D) from duck (PDB code 1H8L) [32], the catalytic domain of human CPN (carboxypeptidase N) (PDB code 2NSM) [31] and the Drosophila melanogaster CPD (PDB code 3MN8) [33], with the closest bacterial match being the carboxypeptidase T from Thermoactinomyces vulgaris (PDB code 1OBR; Z-score of 19.2 over 205 residues with an rmsd of 2.8 Å) [34] (Supplementary Figure S6 at http://www.BiochemJ.org/bj/448/bj4480329add.htm). A sequence alignment of Vh_MpaA with duck CPD, human CPN and D. melanogaster CPD is shown in Supplementary Figure S7 (at http://www.BiochemJ.org/bj/448/bj4480329add.htm), which reveals that all three eukaryotic carboxypeptidases have an additional C-terminal TT (transthyretin) domain, characteristic of the CPN/E subfamily of carboxypeptidases, that is missing in Vh_MpaA [31,32], but that the catalytic domains of all four proteins align well at the sequence and structural level (Supplementary Figures S6 and S7).
Active site of Vh_MpaA and a possible structural basis for substrate specificity
Comparison of the active site of MpaA with that of the well-studied human CPA reveals that there is conservation of both the zinc-co-ordinating residues His48, Glu51 and His156 and other residues integral to catalysis in this enzyme, Glu209, Arg98, Arg88 and Asn97 (Figure 3B). Tyr248 in human CPA, whose contribution to catalysis has long been controversial [35], corresponds to Tyr188 in Vh_MpaA. This residue is in an 'up' conformation in Vh_MpaA away from the active site. This conservation of catalytic residues, together with the close structural superimposition, suggests a common catalytic mechanism for CPA and Vh_MpaA [31]. According to this mechanism, upon binding of substrate, the α-carboxylate forms a bidentate salt bridge to the side chain of Arg145 (corresponding to Arg98 in MpaA) and a polar interaction with the side-chain amide of Asn144 (Asn97 in Vh_MpaA). Glu270 (Glu209 in Vh_MpaA) acts as a base to activate a nucleophilic water molecule which attacks the carbonyl carbon (the carbonyl bond being polarized by the zinc) of the scissile peptide bond. This generates a tetrahedral anionic intermediate which is stabilized by interactions with the side chain of Arg127 (Arg88 in Vh_MpaA) and the zinc in a quasi-oxyanion hole. Following collapse of this intermediate and peptide bond cleavage, Arg127 (Arg88 in Vh_MpaA) and Arg145 (Arg98 in Vh_MpaA) stabilize the carboxylates of the peptide and amino acid products respectively, with the newly formed amino group interacting with Glu270 (Glu209 in Vh_MpaA) [36].
Unlike the carboxypeptidases, MpaA exhibits narrow substrate specificity. In the absence of structural data on the MpaA–Mtp complex, we displayed the amino acid and pentapeptide ligands from the human CPA-cleaved hexapeptide product complex [36] in the active site of Vh_MpaA following least squares superimposition of the secondary structure elements in the two proteins. As can be seen in Figure 3(C), the Arg113–Ser122 loop serves as a flap which closes around the peptide-binding groove and clashes with the N-terminus of the N-terminal pentapeptide cleavage product. This argues that this loop restricts the length of substrates in Vh_MpaA.
Next we attempted to introduce the Mtp ligand from the MppA–Mtp crystal structure into Vh_MpaA. This is not straightforward as the D-stereochemistry at Glu2 of the Mtp and its γ-peptide linkage differ from the structures of conventional peptides [15]. A pose derived by fitting Mtp into the electron density for the pentapeptide ligand in the carboxypeptidase-cleaved hexapeptide complex, placed the L-Ala-D-Glu residues over the P2–P1 residues of the pentapeptide with the oxygen of the scissile peptide bond in co-ordination distance of the zinc ion (Figure 3C) and with the α-carboxylate of Dap in salt-bridging distance of Arg98. As shown in Figure 3(C), the rest of the Dap molecule extends into the protein beyond the volume occupied by the P1′ amino acid product in the human carboxypeptidase structure. This highly speculative modelling shows that Mtp can be accommodated in the pocket, but it is not clear how the protein interacts with the various functional groups to determine specificity for Mtp. We note, however, that the S1′ pocket in Vh_MpaA, where Dap would sit, is more accessible from the solvent than in the 'classical' human carboxypeptidase which has an enclosed 'dead-end' pocket [36], suggesting that there may not be multiple contacts between Dap and MpaA.
Analysis of the dimer interface in Vh_MpaA
Analysis of the intermolecular interactions in the crystal using the programme PISA [28] reveals a prominent protein–protein interface in which ~2050 Å2 or 17% of the surface area on each MpaA subunit is buried, indicating that Vh_MpaA forms a homodimer (Figure 4). This observation is consistent with both the MS and SEC-MALLS data for the protein (Supplementary Figure S3). The residues involved in the dimer interface span the full length of the protein and the dimer interface is stabilized by a network of intermolecular hydrogen bonds (Supplementary Figure S7). At the heart of the interface, residues at the C-terminus of helix α2 are enclosed in a surface cavity in the partner subunit. Core residues in this interface are Ala10, Ala26, Ile55, Ser77, Asn83, Gln84, Gly86 and Val123 with Arg6, Thr7, Phe12, Pro27, Leu29, Thr52, Gln93, Trp114, Ser214, Asp216 and Glu220 prominent contributors (>50 Å2 of surface area buried in the interface) to the rim [37]. As far as we are aware, this is the first observation of a dimeric bacterial zinc carboxypeptidase.
As noted above, access to the active site is restricted by an extended hairpin element with flanking β-strands that resembles a flap (Figure 3C). In the dimer, the flap residues of each chain participate in three interchain ion pairs (His117–Glu220, Asp122–Arg6 and Lys124–Glu8) and a further three interchain hydrogen bonds. Furthermore, the side chain of Trp114 is packed into a cavity in the partner subunit formed by the N-terminal 310 helix and helices α4 and α6 (Supplementary Figure S7). The movement of the flap to allow entry of substrate into the active site would be expected to be accompanied by disruption of these predominantly polar interactions. Thus the opposing subunit in this unusual homodimeric carboxypeptidase has the potential to influence the structure and accessibility of the active site in the partner subunit through interactions that determine the conformation and dynamics of the flap. Whether, and to what extent, this determines the kinetics or the specificity of the MpaA-catalysed reaction is not yet established, but this question could be addressed through detailed comparative studies of MpaA mutants in which the dimer interface is disrupted. Site-directed mutagenesis of residues 83–86 may provide a route to monomeric MpaA derivatives.
Analysis of the genome context of mpaA in γ-proteobacteria reveals a potential scavenging pathway for peptidoglycan-derived peptides
In E. coli K-12 the mpaA amidase gene is likely to be monocistronic and is convergently transcribed from the ycjG gene which encodes an L-Ala-D-Glu epimerase that is probably needed for the cell to utilize the product of the MpaA reaction [20,38]. Examination of the genome context of mpaA across a range of γ-proteobacteria (Figure 5) reveals that the genetic association of mpaA and ycjG is conserved in Salmonella enterica, Klebsiella pneumoniae and Yersinia pestis. Furthermore, in these three genomes the divergently oriented mppA transporter gene is adjacent to the mpaA amidase gene, an arrangement that is not seen in E. coli due to the interposition of three additional coding sequences. The adjacent mppA/mpaA arrangement is also seen in other γ-proteobacteria such as Erwinia carotovora. In the species V. harveyi, V. parahaemolyticus and Vibrio sp. Ex25 we observed a similar arrangement of the mpaA amidase gene linked to a gene encoding an oligopeptide-binding protein, which we have called mppA* and which has the same amino acid sequence changes that we identified for the MppA transporter protein that distinguish it from its ancestor the general oligopeptide-binding protein OppA [15]. Examining the phylogeny of the OppA and MppA/MppA* proteins from these bacteria suggests that the Mtp-binding function has evolved twice from the oppA genes in these lineages (Supplementary Figure S8 at http://www.BiochemJ.org/bj/448/bj4480329add.htm).
The conserved gene organization of the mppA transporter genes with the mpaA amidase gene and ycjG seen in S. enterica, K. pneumoniae and Y. pestis, suggests that the three gene products have a functional and likely physiological connection. Given that MppA is the Mtp-binding protein component of the MppAOppBCDF transporter [14,15], the uptake of Mtp may be coupled to its catabolism via the MpaA amidase and YcjG epimerase (Figure 1). Although Mtp makes up approximately 7% of the total peptide content of the cell wall, the most abundant peptide is the murein tetrapeptide (L-Ala-γ-D-Glu-meso-Dap-L-Ala), making up approximately 36% [4]. To investigate whether the tetrapeptide could be taken up by E. coli we examined whether it would bind to E. coli MppA. Using tryptophan fluorescence spectroscopy we observed a ~5.5% quench in the fluorescence upon addition of excess ligand, which could be titrated to yield a Kd of 0.140±0.013 μM (Figure 6A). We also measured binding of tetrapeptide to MppA via ITC (Figure 6B) which yielded an equilibrium dissociation constant of 0.111±0.003 μM, in close agreement with the data derived from fluorescence. Although binding tetrapeptide with high affinity, the protein is unable to bind murein pentapeptide (L-Ala-γ-D-Glu-meso-Dap-L-Ala-L-Ala) or muropeptides (results not shown).
These data demonstrate that MppA transporter protein can recognize both the murein tri- and tetra-peptides and so we propose that both free peptides are taken up by the MppAOppBCDF transporter into the cell. The known cytoplasmic LD-carboxypeptidase LdcA enzyme is able to cleave the tetrapeptide to tripeptide [10], which is then the substrate for the MpaA amidase and subsequently YcjG (Figure 1).
Cell-wall turnover is an enzymatic process that results in the loss of peptidoglycan components and has been reported in many bacteria [7]. The turnover products are generally re-utilized through a process known as cell-wall recycling [7]. In E. coli a major turn over product (muropeptide, GlcNAc-anhMurNAc-L-Ala-γ-D-Glu-meso-Dap-D-Ala) is known to be taken up by AmpG permease and further processed by lytic enzymes to release free cytoplasmic Mtp, which then re-enters the peptidoglycan biosynthetic pathway [7]. There is now clear evidence of an alternative route for the utilization of cell-wall-derived peptides by E. coli, which has been postulated for many years since E. coli was shown to secrete Mtp and murein tetrapeptide into the growth medium [13]. Release of these peptides follows their cleavage from the murein by AmiD, an outer membrane amidase [12]. The presence of a transporter MppAOppBCDF which recognises the murein tri- and tetra-peptide suggests that E. coli takes up these peptides for their re-utilization [14,15]. The genetic linkage of the mpaA amidase gene, the mppA transporter gene and the ycjG epimerase gene (Figure 5) suggests that E. coli has a route to facilitate uptake and catabolism of murein tri- and tetra-peptides. As the MpaA amidase does not recognize the tetrapeptide, the LdcA enzyme must be used to convert tetrapeptide transported by MppAOppBCDF into tripeptide that can be broken down by MpaA (Figure 1). LdcA is an important enzyme as, in its absence, free tetrapeptide is incorporated into new peptidoglycan via Mpl, which results in a less cross-linked and hence weaker cell wall [10,11]. This observation is also consistent with our data that the MpaA amidase does not recognize the tetrapeptide or it would have not accumulated in the ldcA background. In addition, in the present study we demonstrate that the transporter MppA recognizes both tri- and tetra-peptide with high affinity, yet it was notable that cytoplasmically expressed recombinant MppA purified with only tripeptide bound [15], suggesting that even though tetrapeptide is being taken up by these cells, the action of LdcA means that tripeptide is the only form that is present in significant concentrations in the cytoplasm.
The likely function of this catabolic pathway is to allow E. coli to utilize exogenous murein-derived peptides present in the growth medium from other Gram-negative bacteria as well as recycling its own murein peptides released in the periplasm. The relative affinity of the MpaA amidase for Mtp is approximately 4-fold lower than Mpl (Km values of ~0.4 compared with ~0.1 mM) for Mtp and so, given a limiting amount (low micromolar) of substrate, it will be preferentially recycled into peptidoglycan rather than being catabolized. Only when higher concentrations are present is there likely to be significant catabolism via MpaA. It is interesting that the MpaA amidase can also recognize, albeit poorly, the murein tripeptide that would be derived from some Gram-positive bacteria where the Dap is replaced by lysine, although whether this is physiologically important needs to be determined. Little is known about the regulation either mpaA, mppA, ycjG or ldcA, but they do not appear to be within well-known catabolic regulons for carbon (CRP) or nitrogen (Ntr), suggesting that perhaps they are constitutively expressed.
The MpaA amidases from both E. coli and V. harveyi were easily overexpressed and purified in active forms. Previously efforts to overproduce E. coli MpaA in E. coli were unsuccessful [17], probably due to misidentification of the start codon, which resulted in the expression of a recombinant protein with an additional 20 amino acid residues at the N-terminus. We used the start codon as determined from the EcoGene database of E. coli [39] resulting in successful overproduction of MpaA. The structure of the Vh_MpaA protein revealed a classical zinc carboxypeptidase with complete conservation of key active-site residues. Clearly the MpaA enzyme is a special carboxypeptidase as it has high substrate specificity compared with more general carboxypeptidases such as the human CPA and CPB. The capping region that we propose constrains the size of the peptide substrate that can be recognized by MpaA (Figure 3C) is analogous to similar regions seen in the L-Ala-D-Glu epimerase enzyme that uses the products from the MpaA reaction and where a capping domain restricts the active site and plays a direct role in ligand recognition [40]. Similarly in the YkfC protein from Bacillus subtilis, which is functionally orthologous to MpaA, but is instead an NIpC/P60 cysteine peptidase, there is capping of the entry to the active site by an SH3 (Src homology 3) domain in the proteins which have been proposed to function in restricting the range of substrates that the enzymes can use [41,42]. The other unusual feature of MpaA as a member of the zinc carboxypeptidase family is that it forms a homodimer, and we have described how this dimerization might make a contribution to the ability of the enzyme to have a narrow substrate specificity for a zinc carboxypeptide.
Using purified E. coli MpaA we were able to confirm much of the data of Uehara and Park [17], who demonstrated that MpaA specifically cleaves the γ-D-Glu-meso-Dap amide bond of Mtp and that replacement of the Dap by lysine resulted in a 98.5% drop in activity (Table 3). Uehara and Park [17] tested the activity of cell extracts prepared from E. coli mpaA-overproducing cells on several substrates and observed that MpaA was not active on UDP-MurNAc pentapeptide. We also confirmed that the purified MpaA does not act on the tri-and penta-peptide-containing nucleotide precursor, confirming that MpaA does not interfere with the peptidoglycan biosynthetic pathway. Also, we show that MpaA is very poorly active with MurNAc-tripeptide (specific activity being 0.005 compared with 200 nmol·s−1·mg−1) and is totally inactive towards a disaccharide(anhydro)-tripeptide, demonstrating that it is a zinc carboxypeptidase with a rather narrow substrate range.
As the dipeptide meso-Dap-D-Ala has been detected in cell extracts of E. coli [13] this is presumably being produced in the periplasm and it was suggested that MpaA amidase would also be able to hydrolyse murein tetrapeptide [17], hence acting as an endopeptidase as well. This was unlikely as the MpaA protein lacks a signal peptide and is probably a cytoplasmic protein. Regardless, the results of the present study demonstrate that MpaA cannot hydrolyse murein tetrapeptide. This suggests that there must be an alternative route in E. coli to produce the meso-Dap-D-Ala product in the periplasm. The YkfC protein from B. subtilis is able to cleave tri-, tetra- and penta-peptides with L-lysine at the third position [20] and there are four members of this family in E. coli, two of which, YdhO and YafL, are predicted to be periplasmically localized [43], whereas two others, NlpC and Spr, are predicted to be outer membrane lipoproteins. One or more of these may encode the tetrapeptide endopeptidase activity responsible for the release of meso-Dap-D-Ala.
Our analysis of the genetic linkage between mpaA and mppA also suggests that the Mtp-binding function has evolved twice within the γ-proteobacteria from the ancestral oligopeptide binding protein (Supplementary Figure S8). The fact that the MppA transporter binds tripeptide and tetrapeptide, but not pentapeptide or muropeptides, reflects its ancestry as OppA has a preference for tri- and tetra-peptides [44]. We expressed the V. harveyi MppA* protein in E. coli, but unfortunately it was totally insoluble and so verification of the hypothesis of dual evolution of MppA-like protein requires further experimental confirmation. It is also of interest that the orthologue of E. coli ycjG in B. cereus, ykfB, is adjacent to ykfC and also to genes for uncharacterized oppA-like binding proteins (Figure 5), suggesting perhaps that a murein peptide binding function has evolved independently for a third time in this bacterial lineage.
In conclusion, we have in the present study determined the biochemical and structural properties of the zinc metallocarboxypeptidase MpaA and confirmed its likely role in utilization of Mtp as a nitrogen and carbon source. Our discovery that MppA is able to bind murein tetrapeptide as well as tripeptide, and the genetic conservation of mppA, mpaA and the L-Ala-D-Glu epimerase encoded by ycjG together suggest that these genes function in a catabolic manner to allow E. coli to utilize exogenous murein-derived peptides as nutrients.
Abbas Maqbool and Gavin Thomas designed the research. Abbas Maqbool and Mireille Hervé performed the experiments. All authors analysed the results of the experiments. Abbas Maqbool, Anthony Wilkinson and Gavin Thomas wrote the paper.
This work was funded through a scholarship from the HEC (Higher Education Commission) Pakistan to A.M.
We thank Johan Turkenburg and Sam Hart for help with data collection, staff at the DIAMOND light Source at Harwell for excellent X-ray facilities, Eleanor Dodson for help with structure solution and deposition and Dr Andrew Leech in the Technology Facility, Department of Biology, University of York for help with interpretation of data from ITC, SEC-MALLS and ES-MS.
The co-ordinates and experimental structure factors have been deposited in the RCSB PDB under accession code 4AXV.
Abbreviations: CPA, carboxypeptidase A; CPD, carboxypeptidase D; CPN, carboxypeptidase N; ES, electrospray; GlcNAc, N-acetylglucosamine; IPTG, isopropyl β-D-thiogalactopyranoside; ITC, isothermal titration calorimetry; LB, Lennox broth; LIC, ligation-independent cloning; MALLS, multi-angle laser light scattering; m-Dap, meso-diaminopimelic acid; Mpl, murein peptide ligase; Mtp, murein tripeptide; MurNAc, N-acetylmuramic acid; PEG, poly(ethylene glycol); rmsd, root mean square deviation; SAD, single-wavelength anomalous dispersion; SEC, size-exclusion chromatography; TFA, trifluoroacetic acid
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