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Loot tables
Mobs and Bosses will drop Gold, Items and crafting materials based on the type of mob or boss. For example, a wolf is not going to be carrying a sack of gold. It may instead drop "pelts" that can be traded for gold.[2]
- Loot tables will favor dropping crafting materials as opposed to trash items.[2]
- Loot tables are disabled for player controlled monsters.[3]
- The developers intend to test dynamic loot tables during the test phases.[4]
Crafted items
Crafted items will be on par with best in slot items.[5]
- Highest tier items will be a combo of both crafted and boss dropped.[6]
- The rarity of the items will be commensurate with the effort required to gather, process and craft the items.[5]
- It may be possible for crafters to determine what their crafted items will look like.[7]
- At the moment, harnessing corruption to warp items into demonic or decayed things is not on the cards.[8]
- A crafter's name is embedded in the items they craft.[9]
We believe that every item that exists in the world should in some way reflect its creator. As such, there will be extreme versatility in our crafting system – giving crafters the ability to create unique items that represent their strengths and weaknesses.[10]
Crafting and World Bosses will be the main way to obtain high-level gear. There are some exceptions to this in the form of Legendary items (single server drops or discoveries), and other equipment that may drop wholesale, but we want Artisans and top tier raiding to produce the lion's share of high level, high effectiveness equipment.[4] – Sarah Flanagan
Ashes of Creation intends to use traditional loot rules, selected by the party leader.[11]
- Lootmaster
- A master looter (or lootmaster) is a player designated by the party leader to decide how loot is distributed in a dungeon or raid party.
- Round-robin
- With round-robin looting, party members take turns looting.
- Need before greed
- This is a traditional need before greed system based on dice rolls.
- Bidding system
- Intrepid are investigating a potential bidding system, which allows players to bid on items instead of rolling for them.[11]
- The highest bidder wins the item.
- The gold then goes into a pool that is split among the rest of the party members.
Loot tagging
Gatherers may have the ability to "spoil" a boss' loot.[12]
If you want to be able to get those mats from the boss, they're just not going to drop for anybody. They will only drop for a master gatherer who is capable of extracting those resources from the creature itself... The reason why we say there's a lot of interdependencies between the crafters of the world and the raiders of the world and our PvPers of the world is because we don't want to house the capability to attain these things all within the adventuring class. We want there to be an influence necessary from the craftsmen's guild to come in to either come and participate and spoil the boss and gather the goods or something along those lines.[12] – Steven Sharif
The dynamics of who has the authority to gather the loot will be decided based on testing.[12]
- Party leader.
- First tagging raid.
- Most damage done.
With regards to the dynamics of authority, you know, who gathers those things, exactly what is that: The party leader of the first tagging raid. Is that the most damage dealing. Is that the you know whatever: Those are things we've got a test in testing and see how they turn out.[12] – Steven Sharif
The loot tagging system is still being finalized.[12]
AoE looting
AoE (Area of effect) looting is not included in the looting mechanics at the time of writing. It may be added based on feedback.[13]
Caravan looting
Caravans create an open PvP zone that flags players for combat (purple).[15]
- Players will be able to state their intentions to attack, defend or ignore via a user interface.[16][17]
- A group will be required to successfully attack a caravan.[18][19]
Caravan destruction
If a caravan is destroyed (becomes a wreckage) it will drop a portion of the goods it was transporting.[18][20][21]
- Caravan components may also drop when a caravan is destroyed. These components may be salvageable by the caravan owner or by other players, in the case of high grade components.[22]
- Caravans drop certificates for heavy goods that are redeemable at the origin node for a portion of the goods.[23][21]
The caravan becomes a wreckage upon destruction and that wreckage is an interfaceable item that players can come up to and they can receive certificates for a portion of the goods inside the caravan. Now the idea with that certificate is that it must be taken back to the point of origin, or at least a region within that point of origin. We'll see about that last part because there's a few things I want to test in the Alpha from a gameability standpoint. The reason why for this is because what might happen is you may have some type of collaboration within a guild to kind of game that system. Hey I'm gonna reach this caravan just to the border of the region and then we're all destroy it, collect the goods and take it to you know that region's warehouse; and have to skip out on the last half of the way. So it must successfully reach its destination before the goods can be considered a part of that region.[21] – Steven Sharif
In-game currency is referred to as "gold".[2] This may be a placeholder name.
Mount and pet drops
Mounts and pets are dropped by world bosses on a very rare basis.[26]
Temporary mounts
Temporary mounts that drop from bosses will age and eventually die.[27]
Player death
We don't have deleveling, instead what we have is experience debt. Now the more experience debt you accrue, the greater the detriment to your character; not to the point where you can not get out of the debt. There will always be a way forward to remove your debt.[29] – Steven Sharif
- A non-combatant (green player) who dies suffers normal penalties, which include:[30]
- A combatant (purple player) who dies suffers these same penalties, but at half the rate of a non-combatant.[30]
- A corrupt (red player) suffers penalties at three or four times the rate of a non-combatant, and has a chance to drop any carried/equipped items based on their current corruption score. This includes:[30]
- Death penalties do not differ between PvP and PvE, but this is subject to change.[37]
- Death by drowning is possible.[38]
Item rendering
In-game rendering of dropped items (such as Gear and Currency) is not a planned feature.[39]
See also
- ↑
- ↑ 2.0 2.1 2.2 Livestream, 24 May 2017 (44:14).
- ↑ Livestream, 3 May 2017 (35:25).
- ↑ 4.0 4.1 February 8, 2019 - Questions and Answers.
- ↑ 5.0 5.1 Livestream, 5 May 2017 (14:45).
- ↑
- ↑ Livestream, 24 May 2017 (24:19).
- ↑
- ↑
- ↑ About Ashes of Creation.
- ↑ 11.0 11.1 Group dynamics blog.
- ↑ 12.0 12.1 12.2 12.3 12.4 Podcast, 4 August 2018 (1:44:54).
- ↑ Livestream, 15 May 2017 (48:30).
- ↑ Livestream, 28 March 2020 (1:27:28).
- ↑
- ↑
- ↑ Livestream, 22 May 2017 (40:40).
- ↑ 18.0 18.1 Livestream, 15 May 2017 (45:20).
- ↑ Livestream, 22 May 2017 (40:41).
- ↑ Livestream, 15 December 2017 (1:04:25).
- ↑ 21.0 21.1 21.2 Interview, 15 April 2019 (26:59).
- ↑ Interview, 15 April 2019 (28:28).
- ↑
- ↑ Ashes of Creation Press Kit.
- ↑ Livestream, 26 May 2017 (54:00).
- ↑
- ↑
- ↑ Livestream, 28 March 2020 (1:58:24).
- ↑ Podcast, 23 April 2018 (49:21).
- ↑ 30.0 30.1 30.2 30.3 30.4 30.5 30.6 30.7 Podcast, 5 May 2017 (43:05).
- ↑ Livestream, 19 May 2017 (13:37).
- ↑ Livestream, 27 September 2018 (47:46).
- ↑ Livestream, 17 November 2017 (35:20).
- ↑ Interview, 11 May 2018 (15:41).
- ↑ Interview, 27 April 2017 (9:28).
- ↑ Interview, 11 May 2018 (3:43).
- ↑ Livestream, 15 May 2017 (36:23).
- ↑ Livestream, 28 July 2017 (50:22).
- ↑ Livestream, 24 May 2017 (13:19).


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Restaurant Recipes
Takeout Fakeout: 10 Restaurant-Inspired Dishes to Make Tonight
Takeout Fakeout: 10 Restaurant-Inspired Dishes to Make Tonight
We don't like to put words into others' mouths, but we have a sneaking suspicion that "it's not delivery, it's
DiGiorno homemade" will quickly become your motto after a brief perusal of our homemade versions of takeout superstars. Whether your go-to order is a fully loaded burrito, soothing miso soup, California rolls, or something in between, chances are we've got a recipe for your favorite fix, so click on through and get inspired.
0previous images
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subset: commoncrawl


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Healthy Origins, Sunflower E, 400 IU, 120 Softgels (Discontinued Item)
Flash Deals are limited time, limited quantity promotions, so act fast! Limited to: 1 unit of 1 flash deal product per order. If you order more than one unit or re-order or order other flash deals, you will pay our regular low price. Flash Deals are ineligible from all other promotions.
Healthy Origins, Sunflower E, 400 IU, 120 Softgels (Discontinued Item)
Shipping Weight:?0.14 kgSwitch to Imperial unitsShipping WeightThe Shipping Weight includes the product, protective packaging material and the actual shipping box. In addition, the Shipping Weight may be adjusted for the Dimensional Weight (e.g. length, width & height) of a package. It is important to note that certain types of products (e.g. glass containers, liquids, fragile, refrigerated or ice packed) will often require protective packaging material. As such, these products will reflect a higher Shipping Weight compared to the unprotected product.
- Product Code: HOG-15158
- UPC Code: 603573151584
- Package Quantity: 120 Count
6.1 x 6.1 x 10.9 cm , 0.11 kgSwitch to Imperial units
- Soy Free
- Natural
- Non-GMO
- Supports Healthy Hair, Skin and Nails
- SunE900
- Dietary Supplement
Healthy Origins Sunflower E-400 IU is a great alternative to traditional Vitamin E supplements which are commonly derived from GMO Soy. Vitamin E from sunflower oil does not contain any major food allergens and is guaranteed to be free of any GMOs (genetically modified organisms).
Healthy Origins Sunflower E-400 IU may help support cardiovascular health as well as hair, skin and nail health.
Suggested Use
Directions: As a dietary supplement for adults; take one (1) softgel daily, preferably with a meal, or as directed by a physician.
Other Ingredients
Gelatin, glycerin, water, rosemary extract.
Does not contain sugar, yeast, wheat, gluten, soy, barley, fish, shellfish, peanuts, egg or milk. No preservatives, artificial colors or artificial flavors.
Keep out of reach of children
While iHerb strives to ensure the accuracy of its product images and information, some manufacturing changes to packaging and/or ingredients may be pending update on our site. Although items may occasionally ship with alternate packaging, freshness is always guaranteed. We recommend that you read labels, warnings and directions of all products before use and not rely solely on the information provided by iHerb.
|Supplement Facts
|Serving Size: 1 Softgel
|Amount Per Serving
|Vitamin E (as natural d-alpha tocopherol) (Sun E 900)
|Percent Daily Values (DV) are based on a 2,000 calorie diet.


text:
Are You Interested in Finding an Assisted Living Facility in Stuart, FL?
We must accommodate our living needs at each stage in our lives. When we are seniors, we need to consider communities that anticipate our requirements with respect to medical care and aid. As you get older, too, you need to ensure both your privacy and independence. By choosing assisted living, you can remain independent but have access to medical attention 24/7.
A Better Quality of Life
An assisted living facility in Stuart, FL also allows you to obtain help with daily living activities such as preparing meals, taking medications, and grooming. If you are a senior who is anticipating your future care needs, assisted living communities enable you to lead a better-quality life.
Every phase of a person's life is important. Therefore, you need to know your options in an assisted living facility if you are a senior with certain health conditions. By taking this step, you can live more securely and happily.
Besides following a maintenance-free lifestyle, seniors at an assisted living facility can take part in social activities and dine in a separate dining area. This type of community also may feature areas that take care of seniors with memory care needs for conditions such as Alzheimer's or dementia.
Who to Contact for Assistance
When making a selection for assisted living facilities, turn to a senior housing agency such as Oasis Senior Advisors – Treasure Coast. You cannot put off making a decision, especially if you have age-related conditions. The sooner you take this step, the better it will be for you and your family.
Assisted living allows seniors to live in a studio or one-bedroom apartment in a secure and safe building. Seniors can set up their living arrangements so they can schedule companionship and care during certain times of the day. They typically have access to a dining room where they can enjoy delicious and well-planned meals. Activities such as bingo are scheduled during the day to keep seniors occupied in fun and motivating pursuits. You can connect with them on Facebook for more updates!


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Hello and welcome as I join with 'Five on Friday'. As the weather seems to be cooling and the evenings drawing in, I find myself not really wanting to let summer go just yet. My five random summer photos reflect my mood perfectly, summer days with winter preparation.
Summer holidays and carrying on with routine jobs and shopping, are not so easy with teens, that don't really want to be taken out for some 'fresh air and a change of scenery'. These bold, bright and beautiful flowers, complete with a sunflower, were purchased on one such occasion. Chosen with a chorus of 'can we go home now'.


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Fire shot out of the clothes dryer in Zinc Spa and Nail Bar on Saturday afternoon. Flames had reached the ceiling and, if Wrentham Fire Dept arrived a few minutes later, the situation could have turned drastically worse. But as fortune would have it, the fire station is located just across the way from the 60 South Street business. "They called us right away," said Wrentham Fire Chief Jay McMorrow. "There was significant fire coming out of the dryer."
He added, "You never want to have a fire in the center of your town."
The Wrentham firefighters were able to extinguish the flames quickly and remained on the scene to make sure it hadn't spread up into the ceiling and beyond. The chief said Wrentham's building inspector was being called into to evaluate the exhaust system in the building.
Norfolk Fire responded to the situation in Wrentham Center.
In a demonstration of neighbors caring for neighbors, the owners of Town Pizza invited the Zinc Spa workers over for pizza and a chance to get out of the cold November air.


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Source: Wikipedia
Kathryn Elizabeth Smith (May 1, 1907 – June 17, 1986), known professionally as Kate Smith and The First Lady of Radio, was an American singer, a contralto, best known for her rendition of Irving Berlin's "God Bless America".
Read more about Kate Smith at Wikipedia or at the Internet Movie Database
The Rich and Complicated History of 'God Bless America'
NY Times Music, 2019-07-03 19:25:19
Kate Smith's rendition is now tainted by her own legacy, but Irving Berlin's love letter to his adopted country still has the power to stir.
Who was Kate Smith and why is she suddenly a pariah for recording racist songs decades ago?
USA Today Music, 2019-04-23 21:33:14
Why is Kate Smith in the news – and not in a good way? Everything we know about the flap around the late singer's history of racist recordings.
Kate Smith Statue Removed by NHL's Philadelphia Flyers Amid Racism Allegations
Billboard, 2019-04-22 09:36:10
The Philadelphia Flyers removed a statue of late singer Kate Smith outside NHL team's arena Sunday, two days after covering it amid allegations...
Nearby you will also find Cliff Arquette, Gloria Stuart, Sam Wood, and many others.


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The drum shop is a special place. When I started on my way to becoming a professional drummer, I studied at Jack and Johnie's Drumshop in Kansas City. Jack Miller and John Terry were two of the best drummers in Kansas City during the 1950s. John Terry still teaches and plays in L.A.
Not only did I take lessons at Jack and Johnie's, but I would also hang around for the entire day, talking—and sometimes practicing—with other students. We would exchange information, discuss equipment, and occasionally ask Jack or John to settle a difference of opinion.
The traditional drum shop is a friendly place. At Henry Adler's shop in New York City (no longer there), I met and talked with many famous drummers. I once watched Buddy Rich pick out a pair of hi-hats in the back room of Henry's store. After he found a pair he liked, he played a five-minute solo, just on the hi-hats. Henry and I were the only ones there, and it was a thrilling experience.
Louie Bellson used to drop in at Henry's whenever he was in town. He would talk to everyone, give advice, and sometimes grab a pair of sticks and demonstrate his point on the ever-present practice pad on the counter.
In many ways, Bob Yeager's Pro Drum Shop in L.A. is typical of the traditional drum shop. Unlike many large music stores, this shop has a complete repair department. The Pro Drum Shop also stocks obsolete parts, just in case your set isn't new and the manufacturer is no longer in business. In addition, this store offers a large selection of books, music, and learning materials.
No one is ever in a hurry in a drum shop. If you want to take extra time to select sticks or pick out a cymbal, that's okay. There are no high-pressure or heavy sales tactics. The owner will have time to give advice and help whenever it is needed.
Much can be learned in a drum shop. In many cases, the owner is also a drum teacher. He or she is as interested in drumming as you are. Group discussions about technique, tuning, playing styles, and music in general are commonplace. Where else can you hang out with a group of drummers in a friendly atmosphere? You may also learn something in an easy and non-stressful way.
Drum shops come in all sizes. Ed Hameric started Atlanta Pro Percussion in a very small store. Today, it is one of the largest in the country. His stock of drumsets and cymbals is as complete as any I have seen. Ed still plays every week, promotes clinics constantly, and is as interested in drumming as he ever was.
Drum shops often offer very specialized equipment not found in the average full-line music store. The Modern Drum Shop in New York City is a favorite hangout for pros. Owner Joe Cusatis (an old friend) makes his own drums, sticks, and other items found nowhere else. Joe, like most drum shop owners, is happy to provide personalized service for any drummer with special needs.
Drum shops also promote clinics. It takes time, effort, and money to have clinics on a regular basis. Often, these clinics will feature top drummers and percussionists. But many shops also promote clinics by great young players who may not yet have established a big name. These clinics tend to be less of a show, more down to earth, and often more educational. Clinics of this type are most often held in the store. Drum Headquarters, in St. Louis, Missouri, is a good example. The people there ask clinicians to do two clinics, -because they can only accommodate so many people at one time. This allows everyone to be close to the artists, and makes for an intimate and friendly atmosphere.
Once in a great while, a person who is not a drummer will run a drum shop. Bill Crowden's Drums Ltd. in Chicago is a good example. Bill doesn't claim to be a player, but he loves the drum business and drummers. He sells drums, cymbals, and books, and he regularly promotes clinics. He also has a great collection of rare old pedals, snare drums, and unusual instruments from all over the world. I sometimes feel that somehow Bill "thinks" like a drummer.
Most drum shops have a pot of coffee going or available all the time. Sometimes it is not the greatest coffee—especially late in the day—but it's the thought that counts.
I should add that there are a great many full-line music stores that have excellent drum departments. Usually, it is a separate area in the store devoted just to percussion. In a great many cases, these departments are carrying on the tradition of the drum shop. They have their own little world amidst the guitar and keyboard areas. The people who run these departments are just as dedicated as those in the established traditional shops.
One of the great advantages to traveling with a group or band is the opportunity to visit drum shops in various cities and parts of the country. The people at drum shops are always happy to see you, whether you're a "big name" or not. There are so many drum shops across the country that I can't possibly mention them all. As my examples in this article, I've only referred to a few that are run by friends of mine. However, as far as I'm concerned, anyone who runs a drum shop is a friend of mine.
Every drum shop is a labor of love. Drum shop owners are not in it just for the money. Their hearts are in it. The people who start drum shops are drummers, not people with business degrees. They enjoy being around drums and drummers. All drum shops contribute something special to that unusual fraternity that is like no other: the special fraternity of drummers. Drum shops help to bring drummers together. Visit your local drum shop or favorite drum department soon. The people there will be glad to see you.


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Unique opportunity to lease this entire building comprising A1 Retail Unit and two 3 bedroom flats. Located at the southern end of Kentish Town a short walk from Camden Town.
Location: Located just north of Camden Town along the Kentish Town Road just past the junction with St Pancras Way before the Prince of Wales Road. The shop is with in walking distance of Kentish Town West, Camden Road, Kentish Town and Camden Town Stations. Local occupiers include Lidl, Tesco Metro, Nando's, Franchi and The Abbey Tavern to name a few. Description: The property is comprised of a self contained an A1 super market/off licence on the ground floor, three bedroom flat on the first floor and a three bedroom flat on the second. The residential aspect of the building is currently generating £49,200 per annum. The market has been trading as a successful supermarket for many years in the same ownership. Comprised of a serving counter at the front, retail area, butchers at the rear, cold room, office, rear yard and WC. Accommodation: Ground floor Commercial Unit 157 sq m / 1,689 sq ft First floor 3 bed flat Second floor 3 bed flat Amenities CCTV Tiled Floor Display Fridges/freezers Cold Room Rear Yard Terms: Available on assignment of an existing 15 year Full Repairing and Insuring lease with approximately 8 years remaining, 3 yearly rent reviews. Premium sought for the benefit of the lease, fixtures & fittings and goodwill. Rates: Parties are advised to make their own enquiries with the local authority. Currents rates payable are approximately £15,000 per annum. Viewing: Strictly via the Landlord's sole agent Drivers and Norris. Parking: No
For further details on this property please call us on:
020 7607 5001


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NATIONWIDE MUTUAL FIRE INSURANCE COMPANY v. ACE AMERICAN INSURANCE COMPANY et al
ATTENTION: It is possible that this information may no longer be current and therefore may be inaccurate. The index contains both open and closed cases and is not a complete list of cases in which an ACE Insurance Group company is involved. This information is provided to give interested persons an idea of the issues disputed in the indexed cases. For a full understanding of a case, one should read the rest of the court file, including the response. For the most up-to-date and complete information on a case, visit www.pacer.gov or contact the clerk of the relevant court.
"Nationwide Fire seeks recovery under equitable subrogation, equitable contribution and equitable indemnity for sums that it paid to settle an action originaly filed against its insured that Nationwide Fire contends should have been paid in full or in part by other insurers. Nationwide Fire also seeks declaratory judgment pursuant to 28 U.S.C. Section 2201 and Federal Rules of Civil Procedure Rule 57, finding that the other insurers are obligated to reimburse Nationwide Fire for the amounts that Nationwide Fire paid in settlement."
The provided text is an excerpt from a document filed in this case. For a full understanding of the case, one should read the complete court file, including the response.


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On the Supreme Court's decision to allow All-India Permit holding diesel cabs to continue ferrying BPM employees in Delhi-NCR
NASSCOM is thankful to the Supreme Court as well as the Government of India for understanding the genuine concerns of the BPM industry on the ban put on the plying of diesel based cabs in the Delhi-NCR region. We also thank Amicus Curiae Harish Salve and applaud the decision towards formulating a plan to phase out diesel cabs.
This is welcome news for the BPM industry, which has thrived in the region helping create job opportunities and supporting economic growth. The ban on cabs had put immense pressure on the sector and business continuity; potentially denting its image at a global level. Due to the nature of its work, the BPM industry functions 24×7 and its employees work across shifts day & night. The industry is cognizant of the safety of its employees, especially women hence we are heavily dependent of cab services to ferry its employees to and from their door-step.
NASSCOM pledges to continue its focus on promoting public transport and the use of non-polluting vehicles by the industry, and will continue to work towards creating more initiatives around sustainable transportation.


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Taking Up the Recorder as an Adult
The Recorder Is Very Suitable for an Adult to Take Up
The recorder has the advantage of needing little financial outlay. It is relatively easy to start playing but requires much practice to master. It can be enjoyed on its own or in an ensemble. the latter being most enjoyable and also providing social life. The stimulus of playing with others spurs one on to put more time to practice and so great progress is made.
Making a Start
Rosario Montenegro mentioned the recorder as a good instrument to take up as an adult and she is quite right. It is also an instrument where you can join in with a group of recorder players and have a really good time together. One big advantage is that the recorder is much cheaper to buy than other instruments unless you go in for a really plush one. The recorder has a thumbhole at the back which is covered by the left thumb, then on the front, there are three holes at the top to be covered by your first,second and third fingers of the left hand. Below that are three holes to be covered by your first, second and third fingers of your right hand. It is important to use the hands correctly as explained. Then there is a double hole at the bottom of the instrument to be covered by the right-hand little finger. You have to make sure that you are covering the holes properly. Having got this sorted you need to abandon the right hand for the time being and use the top three holes and thumb hole only to start with. This gives you the chance to get used to covering those holes properly.
Now you need to blow very gently into the mouthpiece producing a steady stream of air only slightly more than a gentle breathing. With thumbhole and three holes covered you should produce the note G. Above this is A (just two fingers down), above that B (one finger down, all with the thumbhole covered) so if you play BAGA BBB AAA BBB BAGA BBB GAABAG you should eventually recognize it as Mary had a little lamb. If you can do that you are on your way, so get a book and get cracking. If anybody can do it(and they can) then so can you. I wish you every success.
Fingering for a C on descant. Flatten your fingers so that the holes are completely closed by the pads of your fingers. Any escaping air will alter the tuning or make your recorder squeak. All the fingers are covering the holes for the note C and the thumb is covering the back hole too.
The Back Hole Covered by the Left Thumb
The Back Hole Uncovered
A Good Basic Start
The Note "G" on the Descant Recorder
When I Heard Piers Adams
I had quite forgotten till now that I had personally heard a very talented recorder player perform at my daughter's school about sixteen years ago. It was none other than Piers Adams. You can hear him in the Youtube video below and judge for yourself. He is at present working in America so if you get the chance to hear him, take it, it will be well worthwhile. He plays with his ensemble, Red Priest. I guess this name comes from Vivaldi who was known by this name because he was a priest and had red hair. This man's playing is a delight and very far removed from the awful wailings of a school child just making their first attempt at the instrument. Piers is often called a modern day Pied Piper and he could certainly charm the birds out of the trees. I remember the performance I heard was absolutely captivating. We were all spellbound. He also has a very charming stage presence and sent many of us home with weak knees that night. Take a look on his website to read more.
The Extraordinary Talent of Piers Adams and Friends
The Note "A" on Descant
Talent, Is It Necessary?
Don't worry about talent. The ability to stick with something, to keep going against the odds is far more important than talent. There are plenty of people out there with talent, but what have they done with it. Nothing. If you are prepared to work hard, and practise, practise, practise you will get huge enjoyment from your improvement. But increase the practise slowly or you will knot your fingers up. Start with ten minutes a day, or three sessions of ten minutes a day and gradually increase to half an hour a day. Remember there is no question of your getting to the Juilliard this is strictly for pleasure. No one may ever want to listen to you play except you but that is what it is all about, your own satisfaction in progress. Then if you have some talent too you will do even better.
All These Four Notes Have the Back Hole Closed
The thumb of the right hand sits at the back of the recorder at the level of the hole below G. It is there just to steady the instrument and be in readiness for when these holes are needed. Later you will find that the left-hand back hole is pinched by pressing halfway across with the nail. This is used to give higher notes. A good tutor book will help you with these things.
The Note "B" on the Descant
The Note "C" on Descant
© 2009 Liz Mackay
Do you find this lens helpful?
Yes! Very helpful Thank You.
Hi Liz,
At what age did you start learning the recorder?
Thank you for your suggestion regarding the book/tutor. I have ordered the book via Amazon and will let you know how I get on.
Incidentally, I do not know where everyone is that sends in their comments. I am in England.
I am also interested in your comments about 'Red Priest' who I can vouch for as worth seeing. They played a concert at my local music society and entranced everyone.
I admire you enthusiasm for learning an instrument as an adult. In my case my boyfriend has just taken up the saxaphone so I did not want to be left out and have decided to take up the descant recorder. I learned it as a child at school so am full of enthusiasm. To see if I am any good at it and can make a tolerable noise I thought I would undertake a bit of self tuition. If the sound is not too painful I will look for a proper teacher. I, therefore, need to identify an initial book/tutor suitable for an adult rather than a child. Any suggestions?
Wonderful lens! Makes me want to take out my recorder and start learning again!
Your thoughts on talent being put to use and to practice, practice, practice reminded my of something my son told me once. "Some people say that practice makes perfect but really its perfect practice that makes perfect." What an encouragement you are to others and the low cost of the recorder as compared to other instruments is an excellent point. So well done!
Very good info about the recorder. I had an eighth grade music teacher that thought all students should learn how to play an instrument. The recorder is what he chose, since it was the most affordable for everyone. I still have mine and occasionally play it.
Great lens Liz. I have lenrolled it to a couple of mine and added it to my Chopin lens featured lenses. 5*
My son and I play the recorder with a small group of other homeschoolers and really enjoy it! It's a great instrument for kids and adults alike!
Well done Liz! :) Good tutorial! BTW, I used to play this as a kid! ;)
Very good step by step pictures - very clear on what to do. 5*s
Nice work on this lens, Liz...well done - I do like the step-by-step pictures too!
This is wonderful! Nice work!
What a good looking lens! I particularly enjoyed the photos ;)


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This year is the first year that the Princess will be choosing what she wants to be for Halloween. Her first year she was the most adorable Lady Bug and last year she was Madonna. My friend has two boys and they went as Elvis and Tommy Lee and our musical menagerie were just too cute! My friend made the costume with a nothing more than a body suit, fabric glitter, lace and sticky jewels. I added the hair piece and the tell-tale beauty mark and she captured the hearts of all the people whose door she knocked on. Frankly, I think she was more of a treat than a trick!
Now this year, the Princess has been flip-flopping between Pirates and Vampires, Zombies and Princess' (go figure!) Now Hubby wants her to go as a zombie, while I think she would make an adorable vampire, but the Princess can't seem to make up her mind. We were given a princess costume that would be very nice, however, for those of you aware of Canadian Halloween's, the costume doesn't quite look the same with a snow suit underneath. What was once a cute princess costume quickly turns into the Pilsbury Dough Boy. I remember my mother always picking out our costumes and we always looked like pudgy versions of what we were going as. One year I went as Xena the Warrior Princess and with my snow suit underneath, I was Xena the buffet Princess. Just not a good look. Oh the dilemma!


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Type IV pili (T4P) are surface-exposed fibers that mediate many functions in bacteria, including locomotion, adherence to host cells, DNA uptake (competence), and protein secretion and that can act as nanowires carrying electric current. T4P are composed of a polymerized protein, pilin, and their assembly apparatuses share protein homologs with type II secretion systems in eubacteria and the flagella of archaea. T4P are found throughout Gram-negative bacterial families and have been studied most extensively in certain model Gram-negative species. Recently, it was discovered that T4P systems are also widespread among Gram-positive species, in particular the clostridia. Since Gram-positive and Gram-negative bacteria have many differences in cell wall architecture and other features, it is remarkable how similar the T4P core proteins are between these organisms, yet there are many key and interesting differences to be found as well. In this review, we compare the two T4P systems and identify and discuss the features they have in common and where they differ to provide a very broad-based view of T4P systems across all eubacterial species.
Type IV pili (T4P) are polymers of the major pilin protein that are displayed on the surfaces of many Gram-negative bacteria and were recently identified in a select number of Gram-positive species (1–3). In Gram-negative bacteria, T4P mediate specialized forms of locomotion, called gliding motility and twitching motility, which are independent of the flagella (3–5). In addition to motility, T4P carry out a multitude of functions, including adherence to eukaryotic cells, microcolony formation, DNA uptake, and protein secretion, and some even function as nanowires carrying electric current (6–9). T4P are polymerized from an inner membrane-bound pool of pilin monomers and are held together in the core of the pilus filament via hydrophobic interactions among the conserved N-terminal segments of the proteins (10–13). Beyond this common N-terminal segment, pilins can vary substantially in size and sequence, producing pili with unique surface features, interaction partners, and functions (1, 3).
The canonical T4P system is closely related to several other systems in bacteria and archaea. The type II secretion (T2S) system in Gram-negative bacteria, the competence systems (Com) for DNA uptake in Gram-positive bacteria (14), and archaeal flagella (recently renamed archaella [15]) all have structural homologs of proteins found in T4P systems, including the major building blocks of the filament (16, 17) (Fig. 1). The central proteins in all these systems in Gram-negative bacteria are (i) the major pilin subunit (including the archaeal flagellin), (ii) a prepilin peptidase that processes pilins to a mature form, (iii) an assembly ATPase, (iv) an inner membrane core protein, and (v) an outer membrane secretin channel (Fig. 1). Conserved sequences within these proteins can be used to identify T4P and related systems in any organism for which the genome sequence has been determined.
T4P were first described for Pseudomonas aeruginosa, in the 1970s (18, 19), and subsequently were identified on a vast number of Gram-negative bacteria, including many human pathogens. They were initially considered to be exclusive to Gram-negative bacteria, in part based on the architecture of the assembly apparatus, which spans both bacterial membranes and the intervening periplasm. Filaments resembling T4P were reported for Streptococcus sanguinis and Clostridium difficile (20, 21), two Gram-positive pathogenic bacteria, but T4P-dependent twitching or gliding motility was not described. The availability of multiple-strain genome sequences for Clostridium perfringens led to the identification of T4P gene products in that species and a realization that all of the clostridial species sequenced up to that time (2006) contained likely T4P-associated gene products (22). Since then, hundreds of additional Gram-positive bacterial genomes have been sequenced and it is clear T4P genes are nearly universal in clostridia and also occur sporadically in other families.
The goal of this review is to assimilate available data for Gram-positive T4P and compare them with Gram-negative T4P to define commonalities and differences in order to understand the evolution of these important molecular machines, particularly as they relate to pathogenesis. We also note many similarities between Gram-positive T4P and Gram-negative T2S systems. This is of profound interest because virtually every Gram-positive human pathogen secretes toxins as part of the infection process, yet the secretion mechanisms are not well defined. Determining if a T2S-like mechanism is utilized for toxin secretion in pathogens that have T4P will be valuable for understanding virulence mechanisms. Although the Com systems are related to T4P and are widespread in Gram-positive bacteria, they are not covered in this review; instead, the reader is directed to other reviews of the Com systems (14, 23). Tad operons (for tight adherence), believed to encode a subset of T4P, are also present in many Gram-positive species (24) but have not been studied to any extent and are not discussed extensively here. We focus on the T4P systems in two Clostridium species, C. perfringens and C. difficile, chosen because they are the best-characterized examples so far of T4P in Gram-positive bacteria. These important human and animal pathogens are only very distantly related within the extremely diverse Clostridiales group (25); thus, the T4P features they have in common may be shared by many clostridia. Many of the observations and comparisons presented here are based on genomic sequences; new genome sequences coming into the public domain on nearly a daily basis are likely to reveal new information and, possibly, exceptions to the general trends we discuss in this review. We show here that the clostridial pilus systems share features with the type IVa and IVb pilus systems and the T2S systems, consistent with them being the precursors from which these various systems evolved. Because there is a vast array of research related to T4P and related systems, we apologize to investigators whose work is not cited in this review.
The major components that make up the Gram-negative T4P and T2S systems and the Gram-positive T4P systems are illustrated in Fig. 1 and listed in Table 1. Unfortunately, the nomenclature for homologous proteins varies from species to species. To minimize complexity, we designated the homologs of T4P proteins found in the clostridia in the same fashion as that for P. aeruginosa, i.e., PilA for the major pilin, PilB for the assembly ATPase, PilC for the inner membrane core protein, PilD for the prepilin peptidase, and PilT for the retraction ATPase (22). We encourage others who identify and characterize these proteins in other Gram-positive species to follow this practice. For this review, proteins are referred to by their descriptive generic names wherever possible, based on their sequence homology to proteins whose functions are well established.
The T4P filament is composed of thousands of copies of the major pilin polymerized from a pool of subunits in the cytoplasmic membranes of Gram-negative bacteria (Fig. 1). Pilins are synthesized in a prepilin form with a signal peptide that is rich in basic residues and typically has a glycine at the −1 position preceding the cleavage site. The type IV pilins are divided into two subclasses: the type IVa pilins have a 5- to 7-amino-acid signal peptide and a phenylalanine as the first residue of the mature protein, whereas type IVb pilins have longer signal peptides (up to 26 residues) and have a hydrophobic residue other than phenylalanine at the mature N terminus. Upon translocation of the prepilin across the inner membrane via the Sec secretion machinery (26), the signal sequence on the cytoplasmic side of the membrane is removed by a dedicated membrane-bound prepilin peptidase, called PilD in many species (27–29). Only the processed pilin is competent for polymerization (30). The N-terminal ∼50 residues of the mature pilin form an extended α-helix (31–33). The N-terminal hydrophobic half of this α-helix anchors the C-terminal globular domain in the inner membrane prior to pilus assembly and forms a helical array in the filament core in the assembled pilus filament, with the globular domain forming the outer shell of the filament (11, 12). Minor pilins share N-terminal sequence homology with the major pilin and act to optimize filament assembly or retraction; some have been found to be incorporated into the pilus itself and to influence pilus functions (34–44).
The mechanism by which pilin polymerization occurs is not well understood but involves, at a minimum, an assembly ATPase and an inner membrane core protein (45–51). One model that has been proposed is that upon binding ATP, the assembly ATPase undergoes a conformational change that is transmitted via the core protein to the base of the pilus, wherein the core protein functions as a lever that pushes the pilus out of the membrane a sufficient distance to allow a new pilin monomer to diffuse from the inner membrane into a gap at the base of the pilus filament (11) (Fig. 2). After hydrolysis of the ATP to ADP and inorganic phosphate (Pi), the complex returns to its resting state and the cycle is repeated, iteratively adding one subunit at a time to the base of the filament. Pilus assembly requires three or more additional inner membrane accessory proteins that recruit the amphitropic ATPase, stabilize the inner membrane assembly platform, and interact with T4P components in the outer membrane (45, 46, 52–59). For pilus retraction, the assembly process is presumably reversed and involves a retraction ATPase, i.e., PilT or PilU (Fig. 1) (60–63), although this motor enzyme is not present in all T4P systems (2). The mechanism by which the retraction ATPase induces pilus disassembly is not understood, but the force generated by this process has been measured as >100 pN for a single PilT hexamer, making it the strongest biological motor that has been examined thus far (64).
In Gram-negative bacteria, the pilus must pass through the outer membrane barrier. This is accomplished via the secretin protein, which forms an oligomeric gated channel in the outer membrane (Fig. 1) (55, 65–69). The periplasmic domains of the inner membrane accessory proteins PilN and PilO contact the secretin channel via PilP, forming a periplasmic conduit through which the pilus grows (not shown in Fig. 1) (52, 55). Gram-positive bacteria have different requirements because they lack an outer membrane and have a thick peptidoglycan layer. The pili (sometimes called fimbriae [70]) that are most commonly associated with Gram-positive bacteria are assembled by sortases, which polymerize pilin subunits at the base of the filament and then covalently attach the filament to either the peptidoglycan or the cytoplasmic membrane (reviewed in reference 71).
A few T4P- and T2S-related genes were found by chance in Gram-positive species, mostly clostridia, due to proximity to an unrelated gene of interest. The first full genome sequence obtained for a Clostridium species was that of Clostridium acetobutylicum ATCC 824 (72), a nonpathogenic bacterium used in solvent production. The T4P-related genes found in that strain were initially annotated as encoding components of the general secretory pathway (72), of which T2S represents the terminal branch, but were later described as a fimbrilin protein exporter system (73). Once sequences became available for 3 strains of C. perfringens (74, 75), a Gram-positive pathogen, T4P-like genes were identified that were highly conserved among the strains (22). Immunofluorescence studies demonstrated the presence of two putative pilin proteins on the surfaces of the bacteria, and short (200 to 300 nm) pili were visible on the surface by use of field-emission scanning electron microscopy (22). Intriguingly, these bacteria exhibit a distinctive type of gliding motility on agar surfaces, characterized by the alignment of individual bacteria in an end-to-end conformation to form chains that moved away from the central colony (22) (see Video S1 in the supplemental material). Because C. perfringens lacks flagellar and chemotaxis genes, it seemed likely that the T4P-like gene products were responsible for this motility. Mutations in the pilC1 (previously named pilC; see T4P Operons in Clostridia) and pilT genes, encoding a putative inner membrane core protein and a retraction ATPase, respectively, inhibited gliding motility and reduced expression of the surface pilins (22). These results suggested that pilC1 and pilT are necessary for pilin polymerization and gliding motility, marking the first demonstration of T4P-dependent motility in a Gram-positive bacterium.
Analysis of the 7 other clostridial genome sequences available at that time, including that of the opportunistic enteric pathogen C. difficile, indicated that each carried T4P-associated genes (22). Clostridium beijerinckii, a species closely related to C. perfringens, has a similar arrangement of T4P-associated genes but also has flagella. In the liquid film on the surface of 0.5% agar plates, C. beijerinckii is highly motile via flagellum-mediated swimming, but on 4% agar plates, which have less water on the surface, the bacterium demonstrates gliding motility similar to that seen with the nonflagellated C. perfringens, suggesting that an increased viscosity of the surrounding medium forces a switch from swimming to gliding motility (22).
Most, if Not All, Species of Clostridia Sequenced to Date Have T4P-Associated Gene ProductsThe gene sequences for the key assembly proteins found in C. perfringens, PilA, PilB, PilC, and PilD (Table 1), were used to identify homologs present in other clostridia. The genome sequences of ∼40 randomly chosen species, spread over the >800 species in the Clostridia class, were analyzed using the BLAST program (http://www.ncbi.nlm.nih.gov/), and homologs of pilB, pilC, and pilD were identified in each of these (data not shown). Among these were two clostridial species in genera of particular interest: Epulopiscium, comprising multiple-spore-forming bacteria large enough to be seen with the naked eye (76); and Heliobacterium, comprising the only photosynthetic Gram-positive bacteria (77).
A useful program, PilFind, was developed by M. Pohlschröder and colleagues to detect candidate pilins in many Gram-positive species (24). PilFind takes advantage of conserved features of the type IV pilin amino acid sequence: a signal peptide that typically ends in Gly(−1), followed by a hydrophobic stretch of ∼25 amino acids with a glutamate at position 5 of the mature protein. By searching for the canonical peptide-terminal motif (A/G)X4(D/E), PilFind identified candidate pilin genes in Gram-positive genomes, including several in C. perfringens and C. difficile (Table 2). Some of these gene assignments are questionable, as they are not associated with other pilus assembly genes or their protein sequences bear little resemblance to those of type IV pilins. Others, such as CPE1839 and CPE1841, appear to be part of an uncharacterized C. perfringens T4P operon.
T4P in Other Gram-Positive GeneraStreptococcus sanguinis has both a T4P operon and a com operon, making it the only known species in the genus Streptococcus with both systems (78). Xu et al. suggested the T4P genes were acquired by horizontal gene transfer, perhaps from a clostridial species (78). Imam et al. (24) used the PilFind program to identify a T4P operon in a Bacillus species, NRRL_B-14911, that also possesses com and tad operons. To our knowledge, this is the only Gram-positive organism reported to have all three types of pilin-related complexes. It seems that T4P operons are ubiquitous in clostridia but rare in bacilli, whereas com operons are quite common in the bacilli but have not been found in any clostridial species thus far.
Most of the clostridial genomes we have analyzed thus far possess multiple gene clusters encoding T4P components (22). Both C. perfringens and C. difficile have a T4P operon, referred to here as the primary operon, that encodes all the proteins required to produce a pilus, plus additional gene clusters that encode single pilin proteins and/or pilus assembly components (Fig. 3). Because some of the T4P genes on the primary operons of C. perfringens and C. difficile were discovered after their homologs on the secondary operons, they are given a designation of "2," whereas those genes that were identified initially are given a designation of "1." The primary T4P operon encodes one or more major pilins (PilA; CD3513 in C. difficile), a prepilin peptidase (PilD), an assembly ATPase (PilB), an inner membrane core protein (PilC), inner membrane accessory proteins (PilM and PilO; and PilN in C. perfringens), three putative minor pilins, and, in the case of C. difficile, a retraction ATPase. These primary operons resemble those encoding type IVb pili in Vibrio cholerae, enterotoxigenic Escherichia coli (ETEC), enteropathogenic E. coli (EPEC), and Citrobacter rodentium, where all pilus assembly proteins are encoded on a single operon (79–84). Such an arrangement is also seen for the genes encoding the T2S system (85). In contrast, genes encoding type IVa pilus assembly proteins are more numerous and are distributed in small clusters throughout the genome (2).
In addition to the apparently complete primary T4P operon, both C. perfringens and C. difficile possess two or three secondary gene clusters that encode a single pilin and/or one or more pilus assembly components (Fig. 3). Both organisms have a cluster of three syntenic genes, encoding a pilin, an assembly ATPase (PilB1), and an inner membrane core protein (PilC1). C. perfringens has a single T4P gene, pilT, immediately upstream of ftsA and ftsZ, which are essential for development of the division septum (86) (Fig. 3A).
Both C. perfringens and C. difficile encode multiple type IV pilins, identified by their small size (less than 200 amino acids), the presence of a signal peptide containing basic residues and ending in glycine, and an ∼25-amino-acid hydrophobic N-terminal stretch with a glutamate at position 5 of the mature protein. Several pilin or putative pilin genes are located on the primary T4P operon, and others are present in secondary T4P operons or are on their own (Fig. 3). In addition, the pilA2 locus on the primary T4P operon in many C. perfringens strains contains multiple pilin genes, called pilA2-1, pilA2-2, etc. The primary T4P operons also encode proteins that are not immediately recognizable as type IV pilins. Although they possess a prepilin signal sequence and a hydrophobic mature N terminus, they are substantially larger than a typical pilin protein and may not have a Glu5 residue: C. perfringens CPE2280 has 322 amino acids, including Glu5, whereas CPE2279 and C. difficile CD3506 have 693 and 499 amino acids, respectively, and a hydrophobic residue at position 5. These adjacent genes may encode minor pilins. A similar gene arrangement is seen in the EPEC type IVb bundle-forming pilus (bfp) operon and the T2S operons, whereby the major pilin/pseudopilin gene is located at the 5′ end and the minor pilin/pseudopilin genes are clustered near the 3′ end. In contrast, minor pilin genes in the type IVa pilus systems of Gram-negative bacteria are located on their own operon (2). Most type IVa pilus systems and T2S systems, as well as the EPEC type IVb pili, possess several minor pilins/pseudopilins, one of which is uncharacteristically large for a type IV pilin and has a hydrophobic residue at position 5. These proteins belong to the GspK family of minor pilins/pseudopilins, which are critical to T2S dynamics and functions (87–91) and also play a role in type IVa pilus assembly (39, 92). The X-ray crystal structure of the minor pseudopilin GspK from the T2S system of ETEC was solved as a ternary complex with the minor pseudopilins GspI and GspJ (93). The bulky GspK protein sits atop this complex, which is predicted to form a cap at the tip of the pseudopilus. Such a bulky structure may prevent the pseudopilus from growing out through the secretin channel. Similarly, the clostridial GspK minor pilins may affect the passage of T4P through the peptidoglycan cell wall, thereby influencing dynamic assembly and disassembly.
C. perfringensThe four designated pilins in C. perfringens strain 13, PilA1, PilA2, PilA3, and PilA4, are small proteins (117 to 176 amino acids) that share a conserved hydrophobic N terminus with a Glu5 residue (Fig. 4). Apart from this N-terminal segment, these pilins differ substantially in (i) the lengths of their signal peptides, from 7 amino acids in PilA4 to 23 amino acids in PilA1 (Fig. 4A); (ii) the lengths of their mature proteins, from 117 amino acids for PilA1 to 176 amino acids for PilA3; and (iii) their amino acid sequences beyond residue 25 (Fig. 4B). The mature PilA1, PilA2, and PilA4 pilins from C. perfringens strain 13 begin with Phe1-Thr2, typical of the type IVa pilins and T2S pseudopilins of Gram-negative bacteria, whereas PilA3 begins with Leu1-Ser2, which matches the N-terminal sequence of bundlin, the major type IVb pilin of the EPEC bundle-forming pilus. The PilA3 gene lies between CPE2280 and CPE2279, and these three genes likely encode minor pilins. The large size and lack of Glu5 for CPE2279 are characteristic of the GspK superfamily.
The C. perfringens pilin sequences were compared among various strains (Fig. 4C; see Fig. S1 in the supplemental material). PilA1, PilA3, and PilA4 sequences are highly conserved, but PilA3 pilins fall into two discrete groups, A and B, based on length, the presence of short amino acid insertions, and whether they have a serine or glycine at position 21. The PilA2 proteins are much more variable than the PilA1, PilA3, and PilA4 proteins, particularly beyond their N-terminal ∼50 residues (Fig. 4C; see Fig. S1B), yet they also fall into two distinct groups, based on signal peptide length and the sequence of the N-terminal ∼55 amino acids, which correspond to the N-terminal α-helix, α1, in the Gram-negative type IV pilins. The PilA2 pilins in group 2 have a 4-residue G/SPFF insert within α1, at position 21, which shifts the register of the N-terminal α-helix relative to that in the group 1 PilA2 pilins. As α1 is the polymerization domain, the presence of this insertion may affect the ability of these pilins to assemble into pilus filaments. Furthermore, the N-terminal residues of the group 2 PilA2 pilins are Leu1-Ser2, as seen for the PilA3 minor pilins (Fig. 4A; see Fig. S1C) and for EPEC bundlin. In fact, the group 2 PilA2 pilins are very similar in sequence to the PilA3 pilins: type D PilA2-1 is identical to PilA3 from the same strain and differs by only a few residues from group A PilA3 pilins in other strains, and strain F4969 and NCTC 8239 PilA2 pilins are identical to the PilA3 pilins in the respective strains and highly similar to SM101 PilA3, all from group B (see Fig. S1E). Thus, PilA2 and PilA3 are identical in some C. perfringens strains, suggesting that the minor pilin genes arose from duplication of the major pilin genes.
The sequence heterogeneity of the pilins at the PilA2 locus in C. perfringens (Fig. 4C; see Fig. S1B in the supplemental material) implies evolutionary selection, the exact nature of which is unknown. In some obligate human pathogens, such as the pathogenic Neisseria species, pilin sequence heterogeneity occurs via a dedicated homologous recombination system that helps these organisms to resist antibody-mediated host immune responses (94). However, rather than being an obligate pathogen, C. perfringens is one of the most ubiquitous bacteria found in nature (95), and tissue infections of living animals likely represent a minor niche, suggesting that immune evasion does not exert a strong selective pressure. What seems more likely is that the PilA2 pilus, which has been shown to be important for attachment to host cells (see Functions of T4P Systems in Gram-positive Bacteria), might be highly variable to facilitate attachment to different surfaces, including but not exclusive to animal cells.
C. difficileThe seven putative pilins of C. difficile have fairly short signal peptides (5 to 12 residues for strain 630) (Fig. 5A). The mature proteins vary in length (from 108 to 262 residues) and are dissimilar in sequence beyond their N-terminal 25 residues, with the pilins encoded at loci CD3513 and CD2305, carried on separate operons, being related most closely overall (Fig. 5B). The N-terminal sequence is hydrophobic, with the exception of Glu5, but is not as well conserved among the seven pilins within a single C. difficile strain as it is in the C. perfringens pilins. The arrangement of the pilin genes on the C. difficile primary T4P operon suggests that CD3513 encodes the major pilin and CD3508, CD3507, and CD3506 encode minor pilins. Similar to the C. perfringens putative minor pilin CPE2279, CD3506 is large for a pilin (499 amino acids) and lacks a Glu5 residue, suggestive of a GspK family protein. CD3506 also has an alanine, rather than phenylalanine, at position 1. Individual pilins are highly conserved among the three C. difficile strains (see Fig. S2 in the supplemental material) but do not resemble the C. perfringens pilins beyond their hydrophobic N termini (not shown).
Predicting Type IV Pilin Structures in ClostridiaThe Gram-negative type IV pilins have a conserved architecture of an ∼53-amino-acid N-terminal α-helical stalk, α1, and a globular C-terminal domain with a central 4- or 5-stranded antiparallel β-sheet (1). α1 anchors the pilin subunit in the inner membrane prior to pilus assembly, and it holds together the subunits in the pilus filament via hydrophobic interactions (11, 12). The regions flanking the β-sheet vary substantially in sequence, length, and structure among the pilins. These regions, i.e., the αβ-loop between α1 and the β-sheet and the D-region, delineated by the disulfide bond formed by two conserved cysteines, in large part define the pilus surface and at least some of its functions. Though similar in overall architecture, the type IVa and IVb pilins have distinct topologies: the D-region and C-terminal loop of the type IVa pilins are continuous and lie at the periphery of the globular domain, whereas the C-terminal segment of the type IVb pilins forms the central strand of the β-sheet (1). The clostridial type IV pilins lack the conserved cysteines but share the hydrophobic N-terminal segment, and their assembly proteins are homologous to those found in Gram-negative T4P systems. Thus, they are likely to share the canonical type IV pilin architecture. Structure predictions using the online servers Phyre2 (96) and Fugue (97) yielded high-confidence models for C. perfringens PilA1 and PilA2 and C. difficile pilins CD3513, CD3294, CD2305, and CD0755, based on sequences and predicted secondary structure homologies with full-length type IVa pilin structures from Neisseria gonorrhoeae, Pseudomonas aeruginosa, and Dichelobacter nodosus (Protein Data Bank [PDB] ID 2PIL, 1OQW, and 3SOK, respectively), as well as the major T2S pseudopilin PulG structure from Klebsiella pneumoniae (PDB ID 1T92). We were able to model only fragments of the remaining pilins, primarily for the N-terminal α-helices. Despite the noted similarities between clostridial type IV pilins and the Gram-negative type IVb pilins, no models were obtained using type IVb pilins as templates, even though there are several such structures available in the Protein Data Bank.
Predictive models are shown for PilA1 and PilA2 from C. perfringens and the CD3513 pilin from C. difficile (Fig. 6), all of which are predicted to be major pilins based on their gene positions within the primary T4P operons and, in some cases, based on functional data. The αβ-loop and D regions are colored green and magenta, respectively, based on their respective positions in the template structures. C. perfringens PilA1 is a 117-amino-acid protein and is thus too short to possess a D region (Fig. 6A). High-quality models were obtained for C. perfringens PilA2 by using both N. gonorrhoeae GC pilin and D. nodosus FimA as templates. The FimA-based model is shown in Fig. 6B, because FimA, like the clostridial pilins, lacks the conserved cysteine pair that delineates the D-region in most Gram-negative type IV pilins yet retains the conserved D-region loop found in the type IVa pilins (33). The D-region loop in FimA is stabilized by hydrophobic interactions, and this may also be the case for some of the clostridial type IV pilins. The cysteines are not conserved in D. nodosus, C. perfringens, and C. difficile, most likely because these anaerobic bacteria live in a highly reducing environment that does not support disulfide bond formation. However, the cysteines are also lacking in the type IV pilins of two aerobic Gram-positive bacteria, S. sanguinis and Bacillus sp. NRRL_B-14911, suggesting that the absence of cysteines may be a characteristic of T4P in Gram-positive bacteria.
While the clostridial pilin models shown in Fig. 6 were derived from type IVa pilins, high-confidence models were also produced based on the T2S pseudopilin PulG from K. pneumoniae (98). This is despite the fact that the PulG crystal structure is for a truncated protein lacking the N-terminal 28-residue segment that bears the highest degree of sequence similarity to the Gram-positive pilins. Pseudopilins generally have smaller globular domains than type IV pilins. Most, like PulG, have a 3-stranded β-sheet and no disulfide-bonded cysteines. A characteristic feature of the major pseudopilins is a conserved loop–α-helix–loop structure in the αβ-loop region, between the N-terminal α-helix and the β-sheet, which contains a signature motif (99). In contrast, the type IV pilins display substantial sequence and structural variability in the αβ-loop. Although clostridial pilins can be modeled as pseudopilins, the pseudopilin αβ-loop signature motif is not present in the pilins we examined. Atomic structures of the clostridial pilins, once available, will likely aid in their classification and provide insights into their filament architecture and functions.
Prepilin Peptidases (PilD)C. perfringens possesses a single prepilin peptidase gene, pilD, encoding a homolog of the PilD prepilin peptidase (22), but C. difficile has two putative prepilin peptidase genes, pilD1 (CD3503) and pilD2 (CD3504), that are adjacent in the primary T4P cluster in strain 630 (Fig. 3). The three clostridial PilD proteins were aligned with each other and with PilD proteins from T4P systems and a T2S system from Gram-negative species (Fig. 7; see Fig. S3 in the supplemental material). Interestingly, C. difficile PilD2 is more similar in length and sequence to the single C. perfringens PilD homolog (41% identity for 254 residues) than to C. difficile PilD1 (25% for 207 residues), suggesting a common origin for their corresponding genes, with C. difficile pilD1 perhaps arising from duplication of pilD2 followed by genetic drift to its present form. Furthermore, C. perfringens PilD and C. difficile PilD2 align more closely with the prepilin peptidases from the type IVa pilus and T2S systems than with those of the type IVb pili. C. difficile PilD1 appears to have an N-terminal truncation that is not seen in the other prepilin peptidases analyzed. The prepilin peptidases are polytopic membrane proteins belonging to the GXGD-type aspartyl protease family, which includes preflagellin peptidase and presenilin, a human enzyme that plays an important role in Alzheimer's disease (27, 100–102). All three PilD proteins possess two conserved aspartates that are critical for peptidase activity, the second of which lies in the GXGD consensus sequence. In terms of substrate specificity, the clostridial prepilin peptidases likely do not discriminate between the lengths of the pilin signal peptides, as these vary substantially within C. perfringens (Fig. 4A), which appears to have only a single PilD protein. However, whereas most of the C. perfringens and C. difficile pilins have a highly positively charged sequence immediately preceding the target glycine at the −1 position of the signal peptide, the putative minor C. difficile pilins, CD3508, CD3507, and CD3506, which are encoded in the same operon as the two PilD proteins, have a glutamate at the −3 position (Fig. 5A). Thus, C. difficile PilD1 may recognize this negative charge near the cleavage site, with the more closely related C. difficile PilD2 and C. perfringens PilD having specificity for the more positively charged signal peptides found on the majority of the clostridial prepilins. Interestingly, glutamates are also present in type IVb pilin signal peptides: at the −3 position in EPEC bundlin and at the −2, −11, and −12 positions in V. cholerae TcpA, along with an aspartate at the −9 position. There are also two and three glutamates in the ETEC CofA and LngA signal peptides, respectively. Importantly, V. cholerae employs two signal peptidases: one that is encoded by tcp on the T4P operon and processes the major pilin (TcpA) and a second prepilin peptidase, PilD, that acts on the minor pilin (TcpB) and components of the T2S system (103). Separate prepilin peptidases may help to maintain a correct stoichiometry of major versus minor pilins.
Assembly ATPases (PilB)There are two assembly ATPases, PilB1 and PilB2, in both C. perfringens (22) and C. difficile. These proteins belong to the GspE superfamily of secretion nucleoside triphosphatases (NTPases) (104). In each species, PilB2 is encoded on the primary T4P operon, along with all key pilus assembly components, whereas PilB1 is encoded on a secondary operon that also encodes a pilin and an inner membrane core protein (Fig. 3). Alignment of the four clostridial PilB proteins with T4P and T2S assembly ATPases shows significant sequence homology (40 to 50% identity), including the signature Walker A and Walker B boxes, the Asp and His boxes, and the tetracysteine motif found in other secretion ATPases (105, 106) (Fig. 8; see Fig. S4 in the supplemental material). All of the clostridial PilB sequences are more similar to those of the type IVa pilus and T2S assembly ATPases (40 to 50% identity) than to those of the type IVb enzymes. Secretion ATPases have a bilobed structure that binds ATP in its cleft via the Walker, Asp, and His boxes in the C-terminal lobe (107–110). The C. perfringens and C. difficile PilB2 proteins both possess an N-terminal ∼60-residue extension present in the type IVa assembly ATPases from N. gonorrhoeae and P. aeruginosa, whereas the PilB1 proteins lack this segment, as do the type IVb pilus assembly ATPases and the T2S ATPases (Fig. 8). The N-terminal regions of assembly ATPases from V. cholerae T2S systems and type IVb pili interact with cytoplasmic domains of inner membrane accessory proteins (46, 111). Thus, differences in the N-terminal domains suggest that there are unique interaction partners for the two ATPases within each clostridial system.
Inner Membrane Core ProteinsGenes encoding the PilB secretion NTPases are typically paired with those encoding PilC, the inner membrane core protein, also referred to as the platform protein or the polytopic inner membrane protein. PilC proteins belong to the GspF superfamily of secretion proteins (112). Since the clostridia lack an outer membrane, we refer to their PilC homologs as "membrane core proteins" or just "core proteins." The C. perfringens and C. difficile assembly ATPase genes, pilB1 and pilB2, are adjacent to genes encoding the T4P-associated PilC1 and PilC2 proteins, respectively, in C. perfringens and C. difficile (Fig. 3). Alignment of the four PilC proteins from C. perfringens and C. difficile with PilC proteins from Gram-negative T4P and T2S systems shows that the PilC2 proteins encoded on the primary operons are more similar to each other than they are to the PilC1 proteins (see Fig. S5 in the supplemental material), and they possess an extended N-terminal region found in the Gram-negative type IVa pilus and T2S system core proteins but absent in clostridial PilC1 and the type IVb pilus PilC proteins (Fig. 9). Most PilC homologs are predicted to have three transmembrane α-helices, TM1 to TM3, with an N-terminal cytoplasmic domain (cyto1), a central cytoplasmic domain (cyto2), a small periplasmic loop between TM1 and TM2, and a short periplasmic C-terminal segment, as shown schematically for V. cholerae TcpE (Fig. 9). Only a few residues are highly conserved among PilC homologs. These include the EXXG sequence in cyto1. X-ray crystal structures for the cyto1 domains of each of the type IVa, type IVb, and T2S PilC homologs show a conserved 6-helix bundle predicted to interact with the cytoplasmic face of the inner membrane, exposing the conserved Glu and Gly residues in an otherwise hydrophobic groove on the face of the bundle that is directed away from the membrane (113–115). This face may interact with partner proteins in filament assembly, including the assembly ATPase and cytoplasmic domains of the accessory proteins. While most of cyto1 forms the 6-helix bundle in V. cholerae type IVb TcpE, Thermus thermophilus PilC (type IVa) and V. cholerae EpsF (T2S) have an additional ∼60 residues at the N terminus that were not resolved in their crystal structures. This segment appears to also be present in the clostridial PilC2 protein and may represent a specialized cytoplasmic binding site in these proteins.
It is not clear whether the ATPase and inner membrane core protein encoded on the secondary T4P gene cluster work with assembly components encoded on the primary T4P operon or use as yet unidentified components to make pili. Presumably, each ATPase-core protein pair operates within its own inner membrane assembly platform. In P. aeruginosa, a recombinant PilB protein fused to yellow fluorescent protein (PilB-YFP) was found to localize to the poles of the cells in a PilC-dependent manner, as this PilB-YFP fusion protein was diffusely distributed throughout the cytoplasm in a pilC mutant background (116). A similar situation was seen in C. perfringens, where a PilB1-YFP fusion protein was found to localize to the poles of the cell but polar localization was lost in a strain lacking PilC1 (117). This suggests that PilB and PilC proteins interact, either directly or indirectly, in both Gram-negative and Gram-positive bacteria.
Inner Membrane Accessory ProteinsThe clostridial proteins PilM, PilN, and PilO are not closely related in sequence to their homologs in Gram-negative bacteria, but they share key features with each system. These putative membrane accessory proteins are encoded on the primary operons of C. perfringens and C. difficile (Fig. 3). C. perfringens PilM belongs to the HSP70 actin superfamily and is predicted by the membrane topology server TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) to be a cytoplasmic protein, while PilN and PilO each have a single N-terminal transmembrane segment and a periplasmic C-terminal domain (Fig. 10). The predicted membrane topologies of the C. perfringens PilM, PilN, and PilO proteins resemble those of the Gram-negative T4P. The crystal structure of the cytoplasmic PilM protein from Thermus thermophilus was solved in complex with a peptide corresponding to the N-terminal cytoplasmic segment of PilN, implying that PilN anchors PilM to the inner membrane (56). PilM is an ATP binding protein that resembles the actin-like protein FtsA. The periplasmic domain of P. aeruginosa PilO has a ferredoxin-like fold, as shown by its crystal structure, and the corresponding region of PilN is thought to have a homologous structure, based on predicted secondary structure similarity with PilO (53). These periplasmic PilN/PilO domains interact to form a stable heterodimer. Furthermore, the PilM, PilN, and PilO proteins, which are encoded on a dedicated pilMNOPQ operon in Gram-negative bacteria, form a complex that connects the inner membrane pilus assembly platform to the outer membrane secretin complex PilQ via the periplasmic lipoprotein PilP (52, 54, 55, 118–120). While C. perfringens lacks PilP and PilQ homologs, the presence of an apparent cytoplasmic membrane complex that extends into the region corresponding to the periplasm of Gram-negative bacteria implies that the pilus assembly apparatus extends into the peptidoglycan layer of C. perfringens.
C. difficile PilM2, encoded on the C. difficile primary T4P operon, appears to be the product of a gene fusion between pilM and pilN: it is predicted to have a single transmembrane segment with an N-terminal cytoplasmic domain that is homologous to PilM from Gram-negative bacteria and a C-terminal periplasmic domain that is homologous to PilN (Fig. 10). A discrete pilN gene is not present in C. difficile. The type IVb pili and the T2S systems also have only two transmembrane accessory proteins, one with both a cytoplasmic domain and a periplasmic domain, analogous to C. difficile PilM2, and the other with only a periplasmic domain, analogous to C. difficile PilO (Fig. 10). The cytoplasmic domains of the EPEC type IVb protein BfpC and the V. cholerae T2S protein EpsL are homologous in structure to T. thermophilus PilM, despite very limited sequence homology (56, 59, 121). Similarly, the periplasmic domains of V. cholerae EpsL and EpsM have ferredoxin folds (111, 121) like that shown for P. aeruginosa PilO (53). Thus, each filament system has a cluster of membrane accessory proteins with a single cytoplasmic domain and two periplasmic domains, but this is accomplished using three proteins for C. perfringens and type IVa pili and only two proteins for C. difficile, EPEC, and the T2S systems.
Genes encoding the inner membrane accessory proteins of the type IVb and T2S systems are clustered in the same operon as other pilus assembly genes, as they are in both C. perfringens and C. difficile (Fig. 3). Interestingly, the secondary operon of C. difficile, which encodes a single type IV pilin (CD3294), a membrane core protein (PilC1), and an assembly ATPase (PilB1), also has a pilM gene, CD3293 (Fig. 3B). Its gene product, which we call PilM1, lacks a transmembrane segment and is predicted to be cytoplasmic, similar to PilM in C. perfringens and the type IVa pili. The two genes downstream of pilM1, CD3292 and CD3291, encode gene products with predicted topologies matching those of C. perfringens PilN and PilO but without recognizable sequence similarity to these proteins. Thus, this secondary operon of C. difficile appears to encode a minimal but complete set of T4P assembly proteins.
The role of the clostridial PilM protein on the cytoplasmic side of the cell membrane likely mimics its role in Gram-negative bacteria, which is to interact with the inner membrane core protein and the assembly ATPase to form a functional pilus assembly platform (45, 46, 59, 111, 122–125). The roles of the PilM, PilN, and PilO proteins on the outer face of the cell membrane are less obvious in the absence of a secretin channel and outer membrane. These proteins likely interact with proteins that are unique to Gram-positive bacteria to form a channel through the thick peptidoglycan cell wall that allows pilus extension and surface display, and may also allow passage of exoproteins for release into the extracellular milieu.
Retraction ATPases (PilT)In Gram-negative T4P systems, pili that have been shown to be retractile possess a second ATPase, usually called PilT, that somehow facilitates active disassembly of the pili. Some species, including P. aeruginosa, possess two retraction ATPases: PilT and PilU (105, 126). PilT homologs have some amino acid sequence similarity to assembly ATPases, including the Walker A and B boxes and Asp and His boxes, but have unique sequence motifs (AIRNLIRE and GMQTXXXXLXXLXXXXXI) in their C termini (127, 128). These signature PilT motifs are also present in the putative PilT proteins of C. perfringens and C. difficile (Fig. 11; see Fig. S6 in the supplemental material). C. perfringens and C. difficile PilT proteins are 56% identical in sequence (http://www.cbs.dtu.dk/services/TMHMM/). This similarity is much higher than that of other proteins in the pilus assembly machinery. They also share ∼50% identity with P. aeruginosa PilT, suggesting that conservation of this protein sequence is somehow beneficial for bacterial survival. Retraction ATPases have not been identified for the T2S systems or for V. cholerae or ETEC type IVb pili, but the EPEC type IVb pili possess a putative retraction ATPase, BfpF, that is required for EPEC dissemination in cultured epithelial cells and for full virulence (129, 130). However, the amino acid sequence of BfpF is much less conserved, with a poorly matched AIRNLIRE sequence (SISSKIRS) and no GMQT sequence (see Fig. S6).
In Gram-negative bacteria, pilT mutants tend to be hyperpiliated, because the pili can assemble but not disassemble (4). However, in at least one Gram-negative bacterium, Francisella tularensis, disruption of the pilT gene results in loss of pilus assembly (51). Similarly, a C. perfringens pilT mutant is nonpiliated (22). While the mechanism is not understood at this point, it may rely on a regulatory pathway that terminates pilus assembly when genes in the assembly/retraction machinery are disrupted.
An unusual feature of the pilT gene in C. perfringens is its location in an apparent operon with the ftsA and ftsZ genes (Fig. 3A). In most clostridia, the pilT gene is located within the primary T4P operon, as is the case with C. difficile (Fig. 3B), or it is nearby. Reverse transcription-PCR (RT-PCR) experiments with C. perfringens indicated that there is a transcript that runs from the pilT gene into the ftsA gene (131). The synteny of the pilT/ftsA/ftsZ genes is maintained in the relatives of C. perfringens that fall into cluster I (132), such as Clostridium botulinum and Clostridium novyi (unpublished data). Why pilT expression is linked to genes involved in creating the division septum is unknown, but an intriguing observation is that T4P in C. perfringens tend to be located mostly at the poles (117; unpublished data). Since the division septum represents a new pole after division and separation occur, it may be necessary for the pilus assembly/retraction proteins to be located in this area prior to cell division. Coordination of pole formation with pilus assembly is well established for Gram-negative bacteria (133, 134). Cowles and Gitai showed that the MreB cytoskeleton is required for PilT localization to the poles in P. aeruginosa (135). This may be true in Gram-positive bacteria as well.
SecretinsSecretins are proteins that form a ring-shaped oligomeric gated channel in the outer membranes of Gram-negative bacteria through which the pilus grows (55, 136–141). Because the large majority of Gram-positive bacteria lack an outer membrane, it is not surprising that no secretin homologs have been found, except in species in the genera Desulfotomaculum and Pelomaculum (24), which are closely related to each other. The secretin homologs found in these bacteria are smaller (375 amino acids) than those seen in Gram-negative bacteria, such as the P. aeruginosa PilQ protein (714 amino acids), and they appear to comprise only the C-terminal half of the Gram-negative secretins that oligomerize to form a transmembrane β-barrel. Whether or not the Desulfotomaculum homolog actually functions as a secretin remains to be determined, but it is located in the main T4P operon (24), and thus it is likely to play a role in T4P functionality. In addition to lacking secretins, Gram-positive bacteria also lack homologs of PilF and PilP, which are lipoproteins found in the type IVa systems that are involved in assembly of the secretin in the Gram-negative outer membrane and in anchoring the secretin complex to the T4P inner membrane complex, respectively (52, 55, 119, 120, 142).
Sporomusa and Selenomonas organisms are members of the Firmicutes that have an outer membrane (143) and have characteristics of both Gram-negative and Gram-positive bacteria. For example, Sporomusa organisms are able to produce heat-stable endospores, a feature usually restricted to Gram-positive bacteria (144). Selenomonas sputigena has a T4P system with a 447-amino-acid secretin homolog that is comparable in size to the Desulfotomaculum secretin, with 17.8% identity, but may be related more closely in evolutionary history to P. aeruginosa PilQ, with 26.9% sequence identity.
In Gram-negative bacteria, T4P have been linked to many cellular functions, including twitching motility, gliding motility, DNA uptake, protein secretion, adherence to eukaryotic cells, biofilm formation, and acting as nanowires carrying electric current (1, 2, 145–148). Since the functions associated with T4P in Gram-positive bacteria are just beginning to be investigated, only a few examples are discussed here.
Gliding MotilityC. perfringens lacks flagella but can glide on the surfaces of agar plates by lining up in an end-to-end orientation to form long filaments that can be seen moving away from a colony (22) (see Video S1 in the supplemental material). Cells in the filaments also appear to align laterally, with filaments sliding along one another. Mutations in the retraction ATPase pilT gene and the core protein pilC gene result in nonmotile C. perfringens strains that have no regular end-to-end connectivity (22), but the exact nature of the cell-cell interactions is still not known.
Unlike C. perfringens, most clostridia possess flagella and can swim via flagellum-mediated motility. As noted above, C. beijerinckii swims in the liquid film on the surface on 0.5% agar plates, but on 4% agar, it moves via gliding motility in a manner similar to that seen with C. perfringens. For C. difficile, another flagellated species, bacteria can be seen swimming on 0.5% agar media (see Video S2 in the supplemental material) and gliding when the agar concentration is increased to 2% (see Video S3). Thus, this type of motility switch may be common among the clostridia. It is not clear whether the increase in viscosity induces a switch in protein expression or simply allows the pili greater access to the agar surface and to each other. Mutagenesis of T4P-associated genes in these species will determine if the gliding motility is in fact dependent on T4P.
Adherence to Host CellsGiven that many Gram-negative pathogens utilize T4P as adherence organelles, it would not be surprising if Gram-positive pathogens that have T4P do the same. Evidence in support of this comes from experiments in which the C. perfringens PilA2 gene from strain 13 was heterologously expressed in N. gonorrhoeae lacking its native pilin, PilE (149). While N. gonorrhoeae normally adheres to genitourinary tract epithelial cells, the strain expressing C. perfringens PilA2 was unable to adhere to such cells but instead bound to myoblasts (muscle cells) and fibroblasts (149), which are host cell types that C. perfringens would encounter in a gas gangrene infection (150). In addition, C. perfringens itself adheres to myoblasts in a PilT-dependent manner (149). Since C. perfringens pilT mutants are nonpiliated, these results suggest that the T4P are important for adherence.
In hamsters infected orally with C. difficile strain 630, bacteria were associated with crypt cells, and immunogold labeling of these bacteria with antibodies to the CD3507 pilin revealed a pattern of gold particles bound to distinct pilus-like filaments projecting from the cells (151). Whether or not CD3507 or these filaments are involved in adherence remains to be determined. We predict, based on sequence and gene location, that CD3507 is a minor pilin (Fig. 3) that is incorporated at low levels into pilus filaments, which are composed primarily of the major pilin, CD3513. Finally, the T4P of Ruminococcus albus play a role in adherence of this bacterium to cellulose fibers, an important initial step in cellulose degradation by this bacterium (152).
Other T4P FunctionsC. perfringens T4P are important for maximum biofilm formation, as mutants lacking the PilT and PilC1 proteins show significantly reduced numbers of bacteria in biofilms (153). Interestingly, immunofluorescence labeling of PilA1 and PilA2 in mature C. perfringens biofilms suggested that the pilin proteins are part of the extracellular matrix (153), a pattern seen in some Gram-negative bacterial biofilms (154).
The nature of a last universal common ancestor preceding eubacterial and archaeal evolution is not without controversy (155), but it seems clear that T4P-like structures must have been acquired early in organismal evolution, since they are widespread in eubacteria and archaea. Because many trees of life depict thermophilic clostridia as one of the most ancient types of eubacteria (e.g., see reference 156), and because T4P are ubiquitous in clostridia, it seems likely that a clostridium-like eubacterial organism is the source of T4P in all eubacteria. Our analysis shows that the clostridial T4P share commonalities with both the type IVa and IVb pilus systems and the T2S system but are not easily classified into a single system. These observations are consistent with clostridial T4P being precursors to all three systems.
How, then, did Gram-negative bacteria inherit T4P? One hypothesis regarding the origin of Gram-negative bacteria is that they are the result of an endosymbiotic event between an ancient clostridium and an ancient actinobacterium that produced a Gram-negative organism (157). If the ancient clostridium carried T4P genes as part of its genome, then this system would have been retained and evolved further in Gram-negative bacteria. The endosymbiotic origin of Gram-negative bacteria is certainly a controversial hypothesis (e.g., see reference 158), but it does explain the similarity of T4P systems throughout the Gram-positive and Gram-negative clades. Since the EPEC and ETEC type IVb pilus operons are located on virulence plasmids (84), it is also possible that the type IVb pilus components in enteric bacteria such as EPEC were acquired from a clostridial species via horizontal transfer of an intact T4P operon similar to the primary operons found in C. perfringens and C. difficile.
Are Some of the T4P Systems in Gram-Positive Bacteria Actually T2S Systems?Defining features that distinguish type IV pilins from T2S pseudopilins are that they form filaments on the bacterial surface under normal expression conditions and that these filaments mediate characteristic T4P functions, such as motility and adhesion. Empirical data support the classification of the C. perfringens primary operon as encoding a T4P rather than a T2S system: PilA1 and PilA2 are present on the surfaces of C. perfringens cells; their expression correlates with the presence of pilus-like filaments on the bacterial surface and with gliding motility (22); disruption of either the core membrane protein, PilC1, or the putative retraction ATPase, PilT, results in a loss of pili and gliding motility (22); and expression of PilA2 in N. gonorrhoeae correlates with binding to muscle cells (149). Furthermore, this system possesses a retraction ATPase, an enzyme that is not associated with T2S systems. Examination of Gram-positive bacterial genomes with predicted T4P systems shows PilT homologs are present in most or all of these species, suggesting that active pilus retraction occurs.
The distinction between T4P and T2S systems is not an absolute one even among Gram-negative bacteria. Some T4P systems, like those of V. cholerae and D. nodosus, secrete protein substrates (7, 8), and some T2S systems have been shown to display pseudopilus filaments on the bacterial surface when the major pseudopilin is overexpressed (88, 160, 161). The distinction between T4P and T2S systems may not be meaningful for Gram-positive bacteria. The difference between displaying a long pilus on the bacterial surface versus a short pseudopilus in the periplasm is likely determined in part by interactions between the growing filament and the outer membrane secretin, which allows passage of T4P but not T2S pseudopili. Given that most Gram-positive bacteria lack an outer membrane, all pilus filaments should, in theory, be displayed on the cell surface, provided that they can traverse the peptidoglycan layer. Some of these pili could function as pistons to extrude proteins that have been transported from the cytoplasm via the Sec secretion machinery in the cell membrane. Such a mechanism may explain how some of the many toxins and degradative enzymes are exported into the extracellular space by Gram-positive bacteria. A T4P/T2S system could provide a conduit in the peptidoglycan through which a pseudopilus could actively extrude larger folded proteins (Fig. 1). We predict that upon further investigation, a secretion function will be revealed for at least some of the Gram-positive T4P systems.
How Do Pili Pass through the Thick Peptidoglycan Layer of Gram-Positive Bacteria?Regardless of the ultimate length and surface display of the pilus in Gram-positive organisms and whether or not they export proteins into the extracellular space, they must pass through the physical barrier of the peptidoglycan layer (Fig. 1). In Bacillus subtilis, the peptidoglycan layer is permeable only to globular proteins smaller than 25 kDa (162). Thus, it is unlikely that a solid pilus cylinder of 6 to 9 nm could pass through the cell wall without requiring significant local restructuring of the cell wall. Little is known about this aspect of the pilus assembly process for Gram-negative bacteria, which have a thinner but nonetheless substantial peptidoglycan barrier. FimV, a peptidoglycan binding protein in P. aeruginosa, has been shown to promote secretin complex formation and surface piliation (163). These functions are dependent on a peptidoglycan binding domain in FimV (163), suggesting that this protein provides a link between the peptidoglycan layer and the secretin to help guide the pilus through the periplasm and across the outer membrane. FimV homologs have not been identified in the clostridia; thus, they may use another system to allow passage of the pilus through the peptidoglycan layer. Peptidoglycan binding proteins have also been identified that participate in T2S system formation (for example, see reference 164). Most likely the PilM-PilN-PilO protein complex, which is conserved among the Gram-negative and Gram-positive T4P systems, plays a key role in ushering the pilus filament through the peptidoglycan cell wall, perhaps by linking the inner membrane assembly platform with a protein channel through this semipermeable barrier.
It is clear from the evidence presented in this review that T4P are widespread among the clostridia and are also present in other Gram-positive species. We are only beginning to explore their structure, assembly mechanisms, and functions in this major class of bacteria and to understand the extent of their involvement in bacterial ecology and pathogenesis. The Gram-positive T4P represent an entirely new opportunity to study the mechanism and functionality of T4P and related filament systems. Since the clostridial T4P systems are among the simplest ones identified in bacteria thus far (2), as they require no outer membrane machinery, they should be excellent model systems for analysis of T4P assembly and retraction. In the past, genetic systems were limited for studying the clostridia, but recently, the ability to construct in-frame deletions (165, 166) and the development of transposon mutagenesis systems (167, 168) have greatly accelerated progress in using genetic approaches to study macromolecular processes in a few of these species, in particular C. perfringens. The development and use of fluorescent protein fusion tags (117) based on low-GC optimized green fluorescent protein genes (169) will allow tracking of the constituent proteins in the T4P complex in real time. We envision an exciting and productive future for researchers in this field.
We thank John Varga for helpful bioinformatic analyses.
This work was supported by National Institutes of Health grant R21 AI088298 and NSF grant 1057871 to S.M. and by a Discovery Grant from the Natural Sciences and Engineering Research Council to L.C.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/MMBR.00063-12.
- Copyright © 2013, American Society for Microbiology. All Rights Reserved.
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Author Bios
Stephen Melville received his Ph.D. from the University of California, Davis. He did postdoctoral training at the University of California, Los Angeles, in the laboratory of Robert Gunsalus, studying the global transcriptional regulatory protein FNR. He then did postdoctoral work in the laboratory of Abraham L. Sonenshein at Tufts University School of Medicine, where he began working with the Gram-positive pathogen Clostridium perfringens, which has been the focus of his research career ever since. After joining the University of Tennessee, Memphis (now The University of Tennessee Health Sciences Center), as an Assistant Professor in 1995, he moved to Virginia Tech as an Associate Professor in 2001, where he has remained. In recent years, his work has focused on the role of type IV pili (T4P) in Clostridium perfringens gliding motility and pathogenesis. He is using the relatively simple T4P system in C. perfringens as a model to understand the basic mechanisms of T4P assembly in both Gram-positive and Gram-negative bacteria.
Lisa Craig received her Ph.D. from Simon Fraser University (SFU) in Burnaby, British Columbia, Canada, in 1998. She began her work on type IV pili as a postdoctoral fellow in the laboratory of John Tainer at the Scripps Research Institute in La Jolla, CA. In 2005, she returned to SFU as a faculty member, where she is currently an Associate Professor in the Molecular Biology and Biochemistry Department. Dr. Craig has performed extensive structure-function studies on type IV pili from a number of Gram-negative bacterial pathogens, including Vibrio cholerae, Neisseria gonorrhoeae, and enterotoxigenic Escherichia coli. Her current research interests are focused on type IV pilus assembly and retraction and secretion mechanisms. Dr. Craig is funded by the Natural Sciences and Engineering Research Council and the Canadian Institutes of Health Research (CIHR). She is the recipient of a CIHR New Investigator Award and a Michael Smith Foundation for Health Research Scholar Award.