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It is going to happen to you at one point or another. You are driving down the road and all of a sudden your tire goes flat. Speaking from personal experience this is not a good feeling. You will need to get to the side of the road, out of the line of traffic and hopefully have a spare. The spare can be done ahead of time with tire mounting port jefferson ny. This will ensure that if you have a problem it can be resolved.
Looking for ware
When looking at your tires make sure that you don't have any ware. A ware pattern is where one side of a tire looks lower or worn down than another side. This can be caused by an imbalance where one side rides higher or lower. You can get this checked out at any repair shop.
Remove rocks and other debris
When riding in your car you will enter a lot of different terrain. Some of these may be construction sites, off road areas and even hazardous situations to do with the weather. All of these conditions can expose your tire from outside influences such as nails, glass and miscellaneous sharp object. These objects can puncture your tire or create cuts and gashes. Unattended to these can eventually cause your tire to fail.
Rotate your tires
Every six months or so it is a good idea to have your tires rotated. This means moving the front tires to the back of the vehicle and the back tires to the front. This will allow your tires to build equal ware.
When it comes to taking care of your vehicle the tires are just as important as anything else. When you have tire issues they can leave you high and dry.


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How To: Restrict the ArcIMS Tasker service on Windows
Instructions provided describe a restrictive environment for the ArcIMS Tasker on Windows. This article may be useful when installing ArcIMS in high-security environments.
Warning: This article is intended as a suggestion when installing ArcIMS in a secure environment. This configuration has not been tested and is not considered certifiedby ESRI. Support is still available when using ArcIMS in this configuration, but these settings may need to be removed in order to continue any troubleshooting. To install any Service Pack or updates switching of the ArcIMS services to run as the user that originally installed ArcIMS is required.
The ArcIMS Tasker cleans up old image files created by the ArcIMS spatial server. It is installed on the same machine as the ArcIMS Application Server.
· The machine on which the Tasker is running is referred to as the TaskerMachine in this article.
· The machine on which the spatial server outputs images, files, and zip files, is referred to as the OutputMachine in this article. This is not necessarily the same as the TaskerMachine.
· The Windows account under which the Tasker runs is referred to as the 'aimsTasker' account for this article.
The aimsTasker account should be a local Windows account if the TaskerMachine and the OutputMachine are the same. Otherwise this must be a Windows-domain account.
The aimsTasker account does not need to be part of any Windows groups.
The aimsTasker account needs the following user rights on the TaskerMachine:
· Log on as a service
· Bypass Traverse Checking
The aimsTasker account needs the following user rights on the OutputMachine if different from the TaskerMachine:
· Access the computer from the network
· Bypass Traverse Checking
Related Information
- Restrict the ArcIMS Application Server service on Windows
- Restrict the ArcIMS Monitor service and Spatial Server on Windows

subset: commoncrawl


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Introduction: Knex Sword
This is my first knex weapon but not my last so thanks.Don't forget to rate or subscribe.
Step 1: Handle.
This is the handle make the part you hold long enough for you to put both hand,s on the handle.
Step 2: Blade
This should be aatleast a few inches longer than the blade, it only breaks on the weakest part of the blade.
Be the First to Share
15 Discussions
but it doesnt cut; kinda defeats the purpose of having a sword...
did you expect a k'nex sword to actually be able to cut anything in the first place?
ironically I made my own sword with my own design and is almost just like this one. the main difference is that I connected yellow connectors to the white ones in the middle of the blade using green and white rods. that way it has more of an edge.
this a cool sword but i followed the pics and it was a bit small so i made it longer but still prity cool
well i actually didt have enought peices to make it longer but still wat evs
is this the better version of mine that you mentioned in november
heck yeah
300th commenter.
lol its pretty cool
nice man
cool i subed & rated it 5.0 good job can u sub me?
no soz 4 late reply
kk, lol =D


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Jan 23, 2017 - 06:07 pm .- Ahead of the 44th annual March for Life in Washington, D.C. a survey shows a "consensus" favoring a 20-week abortion ban, which President Trump has pledged to sign into law if passed by Congress.
Dec 14, 2016 - 10:01 am .- On Tuesday, Ohio Gov. John Kasich (R) signed a bill banning abortions performed after five months of pregnancy in most cases, citing evidence that unborn babies can feel pain at this point.
Mar 30, 2016 - 03:02 am .- A new Utah law requiring anesthesia for unborn babies during late-term abortions shows the contradiction between society's view of life and legal abortion, a leading pro-life doctor has said.
Mar 16, 2016 - 04:27 pm .- As the Senate considers a bill protecting unborn human life past 20 weeks of pregnancy, medical experts insist these children do feel pain and must be protected by law.
May 13, 2015 - 06:35 pm .- Two years after abortionist Kermit Gosnell was convicted of committing three first-degree murders at his abortion clinic, the House passed a bill Wednesday that effectively bans abortions after 20 weeks of pregnancy.
Mar 22, 2014 - 06:03 am .- Pro-life advocates praised the South Carolina House's passage of a bill to ban abortions after 19 weeks into pregnancy – the point at which science has indicated unborn children can feel pain.
Feb 17, 2014 - 04:25 am .- A new chapter published by Dr. Carlo Bellieni in a prestigious German encyclopedia explores the topic of fetal pain, stating that it is a proven fact that the unborn are able to feel pain, even as early as 20 weeks.


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Aggregators are parasites, only slightly more benign than plagiarists – and sooner or later, parasites kill the host. Someone has to actually create words for other people to steal. It's just that actually paying for people to be creative is expensive.
In the yard on a sunny afternoon tending my kid goats, I get a telephone call from freelance journalist Alexander Zaitchik. He said his editors were asking him about W. Cleon Skousen and his relationship to the public lands grab, and that he called me because I was the "expert on these things." Although I've written almost a dozen pieces on the subject, I demurred, as he's a noted professional who in 2009 wrote Common Nonsense, a book about Glenn Beck and Skousen's influence. Alex and I became acquainted after I wrote Constitutional Crisis in the Heart of Dixie that was published in CounterPunch, the piece a description of the LDS Church/Koch business and ideological connections to the lands transfer / Sagebrush Rebellion movement.
Dixie and other pieces that were published in the Southern Utah Independent would later gain some traction after a contingent of Mormons led by the Bundys and LaVoy Finicum took over the Malheur National Wildlife Refuge in Oregon. The day after it began, I declined several interviews, including Oregon Public Broadcasting, and later accepted one, Real News, to speak of the Mormon contingent and their influence. In CounterPunch, I later chronicled meeting Finicum when he was supposed to be at the standoff, breaking the story that the principles were traveling freely out of Oregon, questioning that the FBI had no perimeter to prevent recruitment, and that this allowance was white privilege. The piece and themes were later 'aggregated' by Raw Story, Alternet, The Guardian and Salon – sometimes without attribution.
My investigative journalism, amateur and without the benefit of a line editor, is steeped in political science and public administration combined with an inside knowledge of the public lands players, their organizations, and bureaucracies. I am humbled by the number of professional journalists with whom I have since become acquainted, and I continue in a sense to be a student in the field. I call my work "compelled folk art."
I told Zaitchik that I had no Skousen anti-public lands quotes and reminded him of my SUIndy two-part 50 Year Leap, whereby I describe Skousen Theo-constitutionalism and the indoctrination network the LDS Church directed him to develop. I also told him that two weeks prior I wrote Donors Trump Administration, again published in CounterPunch, detailing the political motivations and players who are acting on ready made Koch Donors Trust / American Legislative Exchange legislative policy during the first 100 days, beginning, "What seems to be the best kept secret is the importance of the Koch Donors Trust network and political minions from both Utah and Wisconsin to a Trump Administration."
Later the same day, Amanda Marcotte follows me on Twitter. Three days later Salon publishes her piece Behind the Western land war: How the fringe ideology of anti-government cranks is becoming the GOP mainstream: Nutty right-wing theories meet the fossil-fuel industries, and the result is an all-out war on public land.
Aggregate journalism in the internet age is somewhere between ambulance chasing and field reporting. It's certainly not investigative, no original. It's capitalizing on the work of others. It's retail, inherently akin to filling the shelves with plastic products. Often lacking substance, more often clickbait, it becomes throw-away, the idea to create a constant stream of content on the backs of hacks like me.
Marcotte is an Aggregate Journalist, a blogger doing it for a living primarily covering feminism politics. She describes herself as a Politics Writer at Salon and has contributed to other media aggregators including Raw Story and Slate.
Salon's business model and quality moved from the investigative journalism of Glenn Greenwald to a low grade, high traffic, aggregate model on purpose, chronicled in Kelsey Sutton and Peter Sterne's Politico piece The fall of Salon.com: How a digital trailblazer and progressive powerhouse lost its way:
"Twelve current and former employees said they were discouraged from doing original journalism out of a concern that time spent reporting could be better spent writing commentary and aggregate stories. "
Marcotte is referenced in Sutton and Sterne's piece, noting allegations of Salon's bias against "Bernie Bros." for a commentary she wrote during the 2016 Democratic primary. No doubt trafficking well, the stories an indication that as Salon strives for success in the aggregate form Marcotte is a good fit.
Behind the Western land war grabs from two sources. First, the Center for Biological Diversity recently published their "15 Top Public Lands Enemies in Congress", the medal sweep being Skousen theo-constitutional Mormons. Marcotte explains the report then centers on several Utah Congresspeople, sliding to the Bundy's use of Skousen ideology. Her second grab was from Zaitchik who forwarded my work to Marcotte and demurred to me the Skousen / Koch connections with the anti-public lands radicals. This is likely the reason for Marcotte's follow via Twitter.
Marcotte had three primary sources she utilized while aggregating her story – and appears to have knowingly only attributed two, "clipping" my blood, sweat, and tears. I maintain Marcotte and her CounterPunch reading editors at Salon listened to the secrets I told in Donors Trump. Aggregating Behind the Western land war, they knowingly stole my thesis, neither crediting me nor Counterpunch.
Beyond thesis and attribution, I largely write about public lands and my partner works in public land management. Our vocational endeavors crossed professionally mid-life, our ideas crossing like Mary Matalin and James Carville but not quite. So, not only have I never asked for compensation for my journalism but my subject matter creates possible retribution and tests our thankfully strong relationship. I – we – have skin in the game.
Consider my SUIndy piece Mike Noel owes an apology for his 'there will be bloodshed' statement made during Malheur at a McCarthy-esque Congressional Field Hearing jointly hosted in St. George, Utah by CBD Enemies list entries Representatives #2 Rob Bishop, #6 Chris Stewart, and #10 Jason Chaffetz. All of these politicians, including Senators #3 Oral Hatch and #1 Mike Lee, are lobbying President Trump to make Noel Director of the Bureau of Land Management, our nation's largest public lands agency. Asshat Noel's comment was directed at civil servants like my partner. He could be her boss.
In the aggregate, although I give of myself freely, Marcotte and Salon, through no fault of Zaitchik who attributed, got my goat.
And, after all of this, Marcotte and Salon, like Sam Levin and the Guardian, still get it wrong: They do not Bite the Bullet and name the Mormon problem concerning public lands policy in the west. All continue to fail to address the role of theology and Prophet directed theo-constitutional indoctrination programs Skousen was to implement, and all continue to fail to address the LDS Church corporate, for profit motivation that is based in both real estate and one of the nation's largest agricultural holdings.
Never plagiarize. Always attribute.
– Society of Professional Journalists, Code of Ethics
Of course this piece is directed at Marcotte, Salon and professional journalism as a whole. To give you the perspective of a folk artist.
I called up a journalist I've gotten to know, Christopher Ketcham, to ask his opinion. Ketcham has lived in Mormon country, writes about public lands politics, and has used my work as a guide to understanding the religious threat to the public domain. "What you are experiencing happens all the time to writers," Ketcham told me. "Not enough of us speak out to tell smooth operators like Salon to fuck off."
Consider the Columbia Journalism Review piece Why Plagiarize when you can rip off a writers thoughts? Washington Post Senior Editor Marc Fisher concludes, "In the cultural battles over aggregation, the word "plagiarism" gets bandied about all too loosely. Often, the grumbling about aggregation is really more about basic courtesy."
Fisher quotes fellow editor Steve Buttry, "There was always a genre of story we called the 'clip job,' where a reporter parachuted in, did some original reporting to advance the story, but mainly relied on a whole lot of stuff from other sources that wasn't credited. What's new is that there are people out there being plagiarism cops."
This is what Marcotte and Salon did, and I have become a cop – as the SPJ Code of Ethics suggests I should. While I don't mean to be too sensitive and I continue to altruistically believe in building upon one another, this story is telling, contributing to my education in the world and state of journalism.
The internet allows for a new form of journalism and journalist, and I must thank editors like Jeffrey St. Clair and Joshua Frank of Counterpunch, along with Josh Warburton and Darren Edwards of the Southern Utah Independent. They all gave me a chance, will likely give YOU a chance, even if your grammar sucks, to break news, investigate and report without the undue influence of retail aggregate journalism. Attribution matters not only to the author but also the rags that publish them. I'm certain CP and the SUIndy could have used the extra foot traffic to help pay the bills. Subscribe and donate, and give them some clicks.


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This product is only available in our physical, brick and mortar stores only, and is not eligible for shipping. Inventory varies by store, and is not accurate online.
Please call your local store for inventory listings!Locate a store near you!
A perfect transition from classic Greece to America's Garden Scene. The Grecian series combines the natural look of stone with the durability and economy of polypropylene. Measures 18 in. Diameter x 14.5 in. high.
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Back somewhere between the invention of the wheel and the Dark Ages a group called "Air Supply" had a hit song called "Lost In Love". Actually, it was in the 80's, but that was a while ago. But that idea fits the two sons in Jesus' third parable. They were surrounded by the lavish love of a gracious father and neither of them were able to experience it. I have been examining why that was true.
The younger son got caught in the pleasure trap. He thought that fulfillment and happiness were "out there" somewhere to be captured. So he took his fortune and began a wanton search only to come up bankrupt and in bondage. He had to abuse grace to learn how to use it.
The older son was missing out because he was caught in the performance trap. He thought he could earn the father's love by his faithful service. He didn't know he could get it by grace. Unfortunately, he refused grace.
He apparently believed that since he had served the father faithfully he deserved a better deal than what he had. Since he felt he was getting cheated, he refused to celebrate with his father over the return of his little brother. It seems that people who have trouble receiving grace also have trouble giving it.
So, surrounded by lavish love, he lived feeling unloved. God's love is real. His love is free. It is unconditional. But we need to know how to receive it or we will never experience it. Feeling the need to earn love, he spurned love.
If you are like this older son and have trouble accepting the Father's love because you feel the need to earn it, learn from the younger brother. When you come to the Father confessing with a contrite heart, not expecting anything, the Father gives you everything! If you come seeking His face you receive His grace. It's not about Him accepting what you've done, it's about you receiving what He's done.
The younger son partied and the older one pouted.


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I just love this colour.. have purchased everything u have done in this fuscia colour.. the embroidery detail is beautiful.. fabric and style perfect... just love the sleeves... i will be purchasing additional colours today


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Thur May XVII
Nice easy fitness session with Wong. Played badly at soccer. Had a letter from Gran saying Grandad had offered her a trip to Auck. for her Birthday.
As I have been writing this I have done the maths and realised that this would have been Gran's 60th birthday. In the 1980s a trip to the other side of the world was still a pretty generous thing to be giving someone as a gift, even for a special birthday such as that and even if your youngest daughter and her family were temporarily residing there.
I am not sure how often my grandparents had been on a plane, but I am sure it can't have been that often. In fact, I can only recall them making two trips abroad apart from this one, both in the 1970s. One was to the Canary Islands and one to Holland and I am pretty sure those were both cruises.
Quite what my parents thought of all of this I don't know. I only found out much later in life that having Gran and Grandad to stay was pretty hard work for them. At this stage all that the four of us children knew was that two of our favourite people in the world would be coming to visit and we couldn't wait for them to arrive.


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2019 Exhibitors
ORR Protection Systems - Booth #713
Since 1971, ORR Protection Systems has grown to be the nation's largest privately owned safety and fire protection company in the country. Some of the largest and most successful companies in the world trust ORR to protect their mission critical business processes from failure due to fire. Orr designs, installs and services fire alarm, detection and suppression systems. Building on our skills and reputation, ORR has emerged as an industry expert in special-hazards, mission-critical fire protection. We custom design every system, ensuring maximum protection of people, property and business processes. Visit us at www.orrprotection.com to learn more about ORR.


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Education must not, and WILL NOT STOP.
MBA ESG has taken extensive measures to ensure the safety of our students and staff during these challenging times.
Learn More.
Date of Visit: August 21, 2019
Project: Vineyard visit
Visit Guide: Sabine Temin
KĀDU (pronounced kaa-du) is the first premium wine offering from the Karnataka winery of Sula Vineyards, India's leading wine producer. The Kādu wines are the most significant launch for Sula in the past 10 years. Kādu, meaning "wild" in Kannada, is also India's first 'Wine for a Cause' as well as India's first 'Wildlife Wine' devoted to the cause of tiger conservation.
The visit helped students in understanding the process of wine-making and its supply chain. It also enlightened the students about the corporate culture of the wine industry. But overall, students will agree that the best part of the entire tour was the wine tasting.
Our MBA in Luxury Management program includes a number of such visits to industries that are associated with luxury. These visits are crucial to understanding how India stands in the luxury segment and what kind of careers students can expect in it.


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Lots of customers have reached out to us regarding the changes to Coke Zero's ingredients and the impact that this recipe change may have on maintaining ketosis. With so much conflicting information out there, we asked our Nutritional Expert, Ashley English, to clarify exactly what this means for particularly our Total Solution customers.
First, what is ketosis?
Ketosis is a state of metabolism whereby your body uses fat rather than carbohydrates as its main fuel source. Our Total Solution Plan can stimulate fasted ketosis because you're eating fewer calories than normal.
Remember, however, that you do not need to enter into a state of ketosis in order to lose weight on our diet.
- Typically has a positive appetite-suppressing effect
- Can (after the first few days) make you feel more energised!
And, what are ketones?
Coke Zero and Ketosis
What has changed?
Coca-Cola has announced it is to change its Coke Zero recipe and rename it Coca-Cola Zero Sugar in the UK to make it "taste more and look more" like the original Coke.
Ahead of the now-implemented Sugar Tax in the UK, Coca-Cola wanted to encourage people to reduce their sugar intake: did you know, for instance, that the original 'Coca-Cola' recipe contained nine teaspoons of sugar in a 330ml can.
The refreshed Coca-Cola Zero Sugar drink includes sodium citrate, which can refer to any of the three sodium salts of citric acid, an ingredient of the product used as a preservative.
What is citric acid?
Citric acid is a weak organic acid found in citrus fruits. It is a natural preservative and is also used to add an acidic (sour) taste to foods and soft drinks.
How does citric acid affect nutritional ketosis?
The theory that citric acid consumption inhibits ketosis is not clear-cut.
It is thought citric acid may affect ketosis by impacting metabolism within the liver, primarily during the Krebs Cycle (Citric Acid Cycle) which citric acid is an intermediate of.
Some people have reported that while they have seen citric acid consumption prevent them entering ketosis and increase appetite, they have not seen it impact ketosis once it has been established.
What does this mean for me?
Everybody is different, in size, shape and sensitivity to certain foods.
Even if avoiding citric acid, establishing and maintaining ketosis varies from person to person. It is important for the individual to determine their threshold and establish whether citric acid has any effect on ketosis, appetite or fat loss on a ketogenic diet.
(However, this does not mean that coca-cola zero will stop you from losing weight – as long as you are in a significant calorie deficit, you will lose weight!)
Is there anything I can use instead?
While water is always the best way to stay hydrated, we know that occasionally you might need a sweet fix. Many Total Solution customers also use Dr. Pepper Zero as a sweet fix to help them on their way. Other brands such as Tesco and Asda's own brand Diet Coke and Sainsburys Sparkling Water, along with green tea are also suitable (but always check the label).
Try our Exante Water Enhancer Multipack for a convenient pick me up.


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Age: 22
Hometown: New York, New York
Current town: Verona, New Jersey
Job description: Artist, Photograper, Assistant to Deborah Kass, Cat Lover
Bio: Swooning over lush National Geographic landscapes for years inspired me at an early age, alerting my subconscious that I was to be a photographer. Upon my entrance into the BFA program for Photography at Montclair State University, my intent was to hone my skills and focus successfully on travel imagery, however this training ground sparked my conscious with new ideas, themes, and image making techniques, I was inundated with a zest to create. Now, women, water, and special processes are my main dig. It is my desire as a young artist to enhance and support feminist and bodily dialogue with images that interact with people and settings that may at times appear unnerving, but lovely.
What are you working on/ describe your work:
In one word my focus is: feminism, as a person and artist.
My senior thesis is a portrait series I created called The 21st Century Feminist, which features contrived yet serious images of women literally 'taking back' the rolls and ideas that are ingrained in society.
My current work, Memories Lost, is a series that combines full rolls of 120 film shot through a Diana F+ plastic camera, and later exposed onto platinum-palladium coated vellum. The goal is to make film-like progressions of haphazard, melancholic, dreamy settings that make one feel as if they are recalling a distant memory.
Select links:
Upcoming projects:
Memories Lost will be followed by a series called Le peuple de le mer, or the people of the sea. This features bodies submerged under water, taking the images from on top of water plane. Body functions and gender roles will be the creative focus. By taking away the concrete shape of a persons image and gender, and instead water-painting them in a more ethereal quality, it is my hope for the viewer to see the form, and not the gender or shape.
What's your art background / when did you begin really focusing on art?
Combine a 6th grade art show, a spiritual experience at the Grand Canyon, and my first on site photo class in the field at National Parks, and there you have the special combo that encouraged my eye for imagery.
What's been happening in your life/ what's next for you?
My feminist BFA senior thesis was a year long battle of love and hate, it's completion brought much celebration, along with health trials. My craving to eat McDonald's every day, and spending way too much time in bed watching Downton Abbey ended quickly, as I am now graduated, and continue my marriage to my work and career.
Too bad post grad life doesn't bring on as much cash as it does cheer, but it did bring focus. In almost a comic 360 turn of events I am now all about vegan cooking, kundalini yoga, and getting back into my faith at church!
I'd like to tell you about my super graceful fall off a ladder in the studio, but that might mess up my chi.
Describe your current state of mind / what's inspiring you?
Working on Memories Lost has been such a gift, it has pushed me to shoot multiple rolls every day. With my camera attached like an appendage my settings vary from the subway, to an art gallery, to the woods. Even a simple trek from my house to a leafy setting has been turned into a still series. This project has enabled me to look at every space life brings me and turn it into a continuation, a memory. I started off creating memories I couldn't remember, and now I am creating a visual diary to leave to my future self.
Any exhibits coming up/ past exhibits you'd like to mention?
I am so excited to announce the following upcoming exhibitions:
-It's an honor to be a featured artist in this years RAW Natural Born Artist Show: Marvel, which will take place at the Public Assembly in Brookyln, NY at the end of August. Get a ticket, and come out for a great experience!
I am so excited to say that I will be a part of Ground Art's upcoming HOME exhibition on March 7th!
Following that, I will be in the DWELL show on March 10th at Hamilton Street Gallery in Boundbrook, New Jersey.
Finally, I will be in the Double Exposure show at the Blue Door Gallery, New York, on March 22nd.
I have recently been a part of:
-Work from The 21st Century Feminist showed at Sylvia White Gallery in Ventura, CA. Be A Saint Show in February 2013.
-I gifted a few images to the Index Art Center, Newark, NJ, for their fundraiser/auction February 2013.
-February 2013: images from Memories Lost were in the 50/50 show at the Covington Artist's Enterprise Center in Covington, KY.
Is any of your work political?
I'm not voice of a generation, rather I like to do my small part. Gender and sexual equality is top of mind for me, and I feel it is my duty not only as an artist, but as a woman, to advocate for equality across the board. I go back and forth between loud and subtle ways of showing this in my work.
What is one of the biggest challenges you face as an artist?
Funding and studio space seems to be the challenge for most budding artists, I am not unique in this. Finding shows and residencies is top of mind.
Has the meaning of your work changed over time?
Art can be therapeutic; as a young artist I needed to get the art therapy out of my system and then focus on artistic issues. My time-line was a gradual stumble to this realization. My earliest work dealt with the disintegration of my family and life as I knew it. I was cutting my chops at this time, and learning. Instead of looking at myself, I looked at the world around me and felt challenged by aesthetically pleasing work, that had no 'meat' to it. This frustration was my segue into being more active in the current feminist movement. Again, I was frustrated with the world around me, but also how feminism was perceived. This became The 21st Century Feminist. The series, thankfully, has been successful, and my most memorable work to date. It began my marriage to my work; I learned how to roar.
While working on this, I conjured more ideas on bodies, and how they are treated, and how they treat us. I have thought abut how bodies are perceived, and how they are manipulated through photography. I am very dissatisfied with the straight portrait photography that dominates the photo world right now. Certainly, it should be celebrated, but I wish to see more. So, I have another developing series of body image, as seen through water. Water is healing, yet finite. It covers the earth, yet we do not cherish it. The same can be said for our bodies, despite the handicapped turmoil that we might endure. Through this, I learned how to whisper.
What are you listening to these days?
Flying Lotus, Fiona Apple, Thurston Moore, Albatros L79 and Delicate Steve, tons of Black Keys, and even more Michael Jackson. Along with phone messages from my mother who can't stand texting.
What are a few of your favorite spots in your area?
I like to spend my Sunday mornings at Mitsuwa, a Japanese supermarket, in Edgewater, NJ. Mondays I like all you can eat sushi at Sushi X in Ridgewood. Tuesdays is Bottino in Chelsea when I am working at Paul Kasmin Gallery. You can find me at Aquarian Yoga Center in Montclair almost any night of the week. Wednesday is my Verona Park crawl time. Thursday it's Gowanus, Brooklyn. Friday has me de-stressing at Tierney's pub for a cider. Saturday, my bed is THE perfect spot for me.
Favorite artists and why/ people in your field whom you most admire:
Cindy Sherman will always be my portrait goddess and my first, and biggest influence. Old favorites include Marina Abramović, Robert Mapplethorpe and Sophie Calle.
I am completely involved with Michelangelo Caravaggio, Georges de La Tour and Artemisia Gentileschi. I've also come to really admire Robyn Cumming, Marilyn Minter, Wangechi Mutu, Deborah Kass, and Katy Grannan.
Rising stars I highly suggest are: Maritza de la Vega, Jillian Keats, Dustin Gramando, Maria Sturm, and anything you see on the New York Times Lens Blog.
What are you reading?
Subway signs for acne doctors and a 40 day journey through the entire New Testament. Also Bossypants, by Tina Fey.
Favorite authors, nonfiction:
I am only going to list one because I find it THAT important: I can not get enough of Just Kids by Patti Smith. Any art student that is struggling or looking for motivation, or is setting out into the world should read this book.
Your website(s):


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Claudius: The Man Pulling the Strings
When seeing a series of events, the purchase or result of each celebration is dependent within the actions of people people involved. From exactly what a university person says or will and every small thing in between, an entire group of events can completely alter. In Hamlet, by William Shakespeare, the activities of the primary characters enjoy a huge part in the book. Anyone can just read the book uncomplicated; it is much more interesting, nevertheless , to look at the order of events in the book and pull alternative results. When observing the chain of occasions in Hamlet, one figure in particular stands out. King Claudius plays a huge role in the play. Claudius' actions, big or small, all significantly impact the way the story unfolds.
If it was not for Claudius killing California king Hamlet in the first place, there would be simply no play. Early on in the enjoy, it is obvious that Hamlet feels psychologically dead by the death of his dad; his mother's quick matrimony with his granddad did not support, either. In agony, Hamlet says " Within a month, \ ere yet the salt of most unrighteous tears \ had still left the flushing in her galled eyes, \ the girl married" (1. 2 . 158-161). It could be declared that Hamlet now has some anger against Claudius and believes he was the murderer of King Hamlet. Even though this individual feels suspect, Hamlet are not able to act on a gut feeling alone. Hamlet had to be sure of who was accountable for killing his father. Hamlet cannot seek revenge against a think. However , Hamlet catches a rest. Luckily to get Hamlet, he gets all the info he requires from a ghost that the night guards have seen. Hamlet is informed the ghosting looks like his dead father. Hamlet fulfills this ghosting in person and he certainly reveals him self as the ghost of Hamlet's daddy. The ghost of Ruler Hamlet explains to Hamlet the way the murderer gets rid of him.
Sleeping in my orchard,
A snake stung me. So the complete ear of Denmark
Through a cast process of my personal death
Rankly abused. Although know, hard noble youngsters,
The snake that performed sting they will father's existence
Now wears his top. (1. your five. 42-47)
The ghost of King Hamlet gives Hamlet crucial details regarding his death. Due to important conference, Hamlet has enough purpose to believe Claudius. Now, Hamlet begins his lengthy path that could lead to his destruction also to the destruction of others. In the event that Claudius hardly ever kills Hamlet's dad, then your whole perform changes. After murdering the King, Claudius userps the crown and marries wrongfully to Gertrude, which causes Hamlet to begin to obtain second thoughts about his uncle, Claudius. It was not time but, however , to kill Claudius; Hamlet should find a way to prove the ghost advised the truth initial.
Claudius continues to be paranoid of Hamlet, which usually further effects the buy of situations. Hamlet changes drastically if he and the ghosting speak also because of this, Claudius sets two people up to spy on him. Both Gertrude and Claudius be anxious of Hamlet's new attitude and are frightened for his sanity. That they both choose to arrange for Rosencrantz and Guildenstern to meet with Hamlet so that they can spy on him. Hamlet satisfies Rosencrantz and Guildenstern, his childhood close friends, whom he hasn't seen in many years. During his 1st conversation with them, Hamlet asks the 2: But i want to conjure
You by the legal rights of our fellowship, by the consonancy
Of our junior, by the accountability of our at any time
Preserved appreciate and by also dear an improved
Proposer can transform you withal: be possibly and direct
With me whether you had been sent pertaining to or no. (1. 2 . 309-311)
It happens to Hamlet that they have absolutely no reason to get in Denmark and they should have been delivered for simply by his parents. Soon after, Hamlet is minted with a cerebrovascular accident of wizard. Thanks to his meeting with Rosencrantz and Guildenstern, Hamlet confronts a way to expose his uncle. Hamlet's great plan only came to him because of his location. In the event that Hamlet hardly ever saw the touring actors, this idea wouldn't happen. As three speak, a grouping of touring actors...


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Francis Hills
I'm an Englishman in New York, sound familiar?
I became a photographer back in 2002 and was a digital evangelist for Hasselblad and traveled the world promoting professional digital photography before we had iPhones. I've been on TV for shows like Sweden's Next Top Model and have photographed unbelievably cool people - both well-known and not so well-known. I live vicariously through performers.
I've worked across the world for lots of publications and have had what you'd call a "varied" career. From bouncing around in helicopters with the French Foreign Legion in Africa, to photographing 300 naked people for charity, I haven't exactly seen it all, but I've seen a lot.
studio: +1.646.257.4076. email: email@example.com
If you'd like to view my corporate work please visit FIGJAMPHOTO.com
Francis Hills Editorial Fashion Photoshoot
West Edmonton Mall | Back To School
Francis Hills on Top Model & Covershot
Notable client list:
Apple / Barclays Capital / Biohaven Pharmaceutical / Blue Mercury / Breast Cancer Research Foundation / Brown Harris Stevens / Burson Cohn & Wolfe / Cheesecake Factory / Cole Haan / Crate & Barrel / Douglas Elliman / Elle Magazine / Emmy Magazine / Entertainment Weekly / Esquire Magazine / The FT / Gotham Magazine / GQ / Halstead Property / Hamptons Magazine / Hasselblad / Hewlett-Packard / The Josephine Herrick Project / Matador Content / Macerich Malls/ Ocean Drive / Oprah / Parade / PBS / The Radio Times / Red Book / St. Andrews University / Sandisk / The Scotsman Magazine / Terra Holdings / Time / TV Guide / UBS Group AG / Vegas Magazine / Williams Sonoma / Zink Magazine


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Idols, idols everywhere! We make so many things into idols…sex, drugs, and rock and roll are only the tip of the iceberg! God is a jealous God and He detests idols of every kind. Learn how to recognize when you have made something into an idol and what to do to get that image burned out of your soul! This teaching contains an amazing soaking portion where Katie speaks over you for more than hour with scriptures and repentance decrees. This teaching will jump-start your healing journey!
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FtsI (also called PBP3) of Escherichia coli is a transpeptidase required for synthesis of peptidoglycan in the division septum and is one of several proteins that localize to the septal ring. FtsI comprises a small cytoplasmic domain, a transmembrane helix, a noncatalytic domain of unknown function, and a catalytic (transpeptidase) domain. The last two domains reside in the periplasm. We used PCR to randomly mutagenize ftsI, ligated the products into a green fluorescent protein fusion vector, and screened ∼7,500 transformants for gfp-ftsI alleles that failed to complement an ftsI null mutant. Western blotting and penicillin-binding assays were then used to weed out proteins that were unstable, failed to insert into the cytoplasmic membrane, or were defective in catalysis. The remaining candidates were tested for septal localization and ability to recruit another division protein, FtsN, to the septal ring. Mutant proteins severely defective in localization to the septal ring all had lesions in one of three amino acids—R23, L39, or Q46—that are in or near the transmembrane helix and implicate this region of FtsI in septal localization. Mutant FtsI proteins defective in recruitment of FtsN all had lesions in one of eight residues in the noncatalytic domain. The most interesting of these mutants had lesions at G57, S61, L62, or R210. Although separated by ∼150 residues in the primary sequence, these amino acids are close together in the folded protein and might constitute a site of FtsI-FtsN interaction.
Cell division in Escherichia coli requires approximately a dozen proteins, all of which localize to a ring structure at the division site (Fig. 1A) (for a recent review, see reference 15). The division proteins localize in a defined order, which starts with assembly of the tubulin homolog FtsZ into a contractile ring at the midcell. Subsequent recruitment of the other division proteins is thought to result in the assembly of a complex that mediates inward growth of the cell envelope. According to the current model, the last four proteins recruited to the septal ring are FtsW, FtsI, FtsN, and AmiC (in that order) (4). The precise roles of FtsW and FtsN in septum assembly are not yet known. FtsI is a transpeptidase that introduces peptide cross-linking into the peptidoglycan cell wall in the division septum (6, 37). AmiC is a periplasmic amidase that hydrolyzes peptide cross-links and contributes to the separation of daughter cells after division (25).
The ftsI gene encodes a protein of 588 amino acids, but a proteolytic processing event removes 11 residues from the C terminus (32). Processing is not required for FtsI function (22). The membrane topology of FtsI has been studied with protein fusions, which revealed that the protein can be divided into three domains: an amino-terminal cytoplasmic domain (23 amino acids), a single transmembrane helix (17 residues), and a large periplasmic domain (537 residues) (7). The periplasmic domain appears to comprise two parts, a noncatalytic domain of unknown function and a catalytic domain that is directly responsible for introducing cross-links into septal peptidoglycan (reviewed in reference 33). A cartoon of the domain structure of FtsI is shown in Fig. 1B.
Several reports have implicated the cytoplasmic domain and membrane-spanning segment in targeting of FtsI to the septal ring (12, 17, 20, 40). The most direct evidence comes from analysis of hybrid proteins in which these domains have been replaced with the corresponding parts of other membrane proteins. Such hybrid proteins fail to complement temperature-sensitive and null alleles of ftsI and also fail to localize to the septal ring (20, 40).
The catalytic domain extends from residues 237 to 577 and is the site of the transpeptidase activity responsible for cross-linking the peptidoglycan cell wall (1). The catalytic domain exhibits homology to other transpeptidases involved in peptidoglycan metabolism (18). In particular, the active site contains a universally conserved serine residue (S307) that forms a covalent bond with the peptide substrate during transpeptidation, a reaction that proceeds via an acyl enzyme intermediate. The catalytic domain also binds β-lactam antibiotics, which mimic a transpeptidase substrate and serve as suicide inhibitors by forming a long-lived covalent adduct with the catalytic serine (28, 34). Thus, FtsI is also known as penicillin-binding protein 3 (PBP3).
The noncatalytic domain extends from residues 41 to 236. This domain was first recognized as a distinct structural element when sequence comparisons revealed that it was conserved among a subset of transpeptidases called the class B high-molecular-mass penicillin-binding proteins (16). It has been proposed that the noncatalytic domain interacts with other proteins of the septal ring and that these interactions are important for septal localization of FtsI, recruitment of downstream proteins such as FtsN, and regulation of the transpeptidase activity of FtsI (33). Marrec-Fairley and coworkers have characterized several mutants with properties consistent with these roles. Most notably, they have suggested that changing D58 to V prevents localization of FtsI to the septal ring and changing both R210 and R213 to Q (a double mutation) interferes with a protein-protein interaction but not septal localization per se (29). These interpretations were based on the recessive nature of the D58V lesion and the dominant nature of the R210Q-R213Q lesions. The abilities of the mutant proteins to localize and/or interact with other division proteins were not tested directly.
The three-dimensional structure of an FtsI homolog, PBP2x from Streptococcus pneumoniae, has been solved by X-ray crystallography (Fig. 1C) (13, 35). The catalytic domain looks like a typical penicillin-binding domain, except that the active site is at the center of a long groove that is not seen in other penicillin-binding enzymes. This groove presumably accommodates the two peptides to be joined in the cross-linking reaction. The noncatalytic domain has a strikingly elongated shape. One end fits into a pocket in the catalytic domain, and two long arms extend into solution, creating a large central cavity that could accommodate another protein in vivo. This arrangement suggests that the noncatalytic domain regulates the catalytic domain and that the arms might engage in protein-protein interactions (35). This arrangement also suggests why the noncatalytic domain appears to be required for folding of the catalytic domain (17); these domains interact so extensively that neither is likely to be stable alone.
To identify sequences in FtsI that are needed for localization to the septal ring and for interaction with other division proteins, we have isolated and characterized mutant forms of the protein that fail to support cell division but appear to be normal for insertion into the membrane and catalytic activity (penicillin binding). These mutants are described in this paper. Single-amino-acid substitutions that severely impaired localization invariably mapped to the membrane-spanning segment rather than the noncatalytic domain. We also found several mutant proteins that were defective in recruitment of FtsN to the septal ring. These proteins had single-amino-acid substitutions in the noncatalytic domain and might define a site of FtsI-FtsN interaction. Finally, we found one lesion in the noncatalytic domain that renders FtsI incapable of supporting cell division, but the protein appears essentially normal in all of our assays.
Bacterial strains and plasmids.Strain construction was done by generalized transduction with P1 (31), specialized transduction with lambda InCh (9), or transformation with integration vectors (21).
The E. coli strains used were MG1655, DHB4 [F′ lacIq pro/λ− ΔlacX74 galE galK thi rpsL phoR ΔphoA(PvuII) ΔmalF3], EC295 [MG1655 ftsI23(Ts) leu::Tn10], EC812 [MG1655 ftsI::cat Δ(λattL-lom)::bla lacIq PBAD-ftsI], and EC1137 [MG1655 attL::pDSW539 (KanrlacIq P209-gfp-ftsI)]. MG1655 and DHB4 have been described elsewhere (5, 8). EC295 was constructed by transducing MG1655 to Tetr with P1 grown on LMG64 (20) and screening for temperature sensitivity. EC812 was constructed in several steps. First, an arabinose-controlled ftsI gene was introduced into the chromosome of MC4100 from plasmid pDSW172 (pBAD24-ftsI) using λ InCh1 (19). The PBAD-ftsI was then moved into MG1655 by transduction with P1 to make EC811. Finally, an ftsI::cat allele from JE7947 (23) was transduced into EC811 to make EC812. Strain EC1137 was constructed by integrating pDSW539 into the λ att site (21). Derivatives that express mutant ftsI alleles were constructed similarly from plasmids pDSW546 to -559. These strains were EC1138 (R23H), EC1139 (L39P), EC1140 (Q46H), EC1141 (G57D), EC1142 (S61P), EC1143 (L62P), EC1144 (V86E), EC1145 (R141C), EC1146 (H177R), EC1147 (G188E), EC1148 (R210C), EC1149 (R210H), EC1150 (S61F), and EC1155 (R23C).
The plasmid pDSW287 was constructed by replacing the 1.3-kb promoter-bearing EcoRV-EcoRI fragment in pDSW254 with the corresponding fragment carrying a weaker promoter, P206, from pDSW209 (40). The construction of pDSW478 (pBAD18-Kan-ftsI) has been described elsewhere (14). Derivatives that express mutant alleles of ftsI were constructed by replacing a 1.5-kb EcoRI-KpnI fragment that carries most of ftsI. The resulting plasmids were pDSW479 (R23H), pDSW480 (L39P), pDSW481 (G57D), pDSW484 (Q46H), pDSW486 (S61P), pDSW487 (L62P), pDSW488 (V86E), pDSW489 (R141C), pDSW490 (H177R), pDSW492 (G188E), pDSW494 (R210C), pDSW495 (R210H), pDSW496 (R23C), and pDSW498 (S61F).
The plasmid pDSW521 (P206-gfp-ftsI) was constructed by PCR amplifying the gfp-ftsI fusion gene from pDSW287 using primers P324 (5′-CACGATATCAGCCAACAGACCATGAGTAAAGGAG-3′) and P304 (5′-GTGAAGCTTACGATCTGCCACCTGTCCC-3′). The 2.5-kb product was digested with EcoRV and HindIII (the sites are underlined) and ligated into pTH18-kr (24) that had been cut with EcoRI, made blunt ended by filling it with T4 DNA polymerase, and then cut with HindIII. Derivatives that carry mutant alleles of ftsI were obtained directly during the screen or constructed by subcloning from pDSW287 derivatives by replacing a 560-bp EcoRI-XmnI fragment in pDSW521 with the corresponding fragment from the pDSW287 derivatives. The resulting plasmids were pDSW562 (R23C), pDSW563 (R23H), pDSW566 (L39P), pDSW567 (Q46H), pDSW568 (G57D), pDSW569 (S61F), pDSW570 (S61P), pDSW571 (L62P), pDSW572 (V86E), pDSW573 (R141C), pDSW574 (H177R), pDSW575 (G188E), pDSW576 (R210C), and pDSW577 (R210H).
The vectors used to integrate gfp-ftsI fusion genes into the chromosome were constructed in several steps. The gene for gfp was amplified from pDSW207 (40) using the primers P459 (CAAGGATCCAGGAGGATGAATATGAAAGGAGAAGAACTTTTC) and P460 (CGTGAGCTCTCAGAATTCTTTGTATAGTTCATCCATGCC). The 0.8-kb product was cut with BamHI and SacI (the sites are underlined) and ligated into the same sites of pAH144 (21) to create pDSW499. Then, a 1.7-kb SphI-NdeI restriction fragment that carries lacIq and P206-gfp′ from pDSW287 was ligated into pDSW499 that had been cut with SphI and NdeI. The resulting construct was designated pDSW535. This plasmid was digested with SphI and NheI, and the 2.5-kb fragment carrying lacIq, P209-gfp, and tL3 was ligated into the same sites of pAH120 (21) to create pDSW537. Finally, ftsI was cloned into pDSW537 to create pDSW539. This was accomplished by PCR amplifying ftsI from pDSW287 with the primers GFP666-F (GAGACCACATGGTCCTTCTTGAG) and P500 (ACGGACAATTGAAGCTTCTAGAATTACGATCTGCCACCTGTCC). The 1.8-kb product was cut with EcoRI (a site in the pDSW287 template plasmid) and MfeI (the site is underlined) and ligated into the EcoRI site of pDSW537 to yield pDSW539. Derivatives that express mutant alleles of ftsI were constructed by replacing a 1.5-kb EcoRI-KpnI fragment that carries most of ftsI. The resulting plasmids were pDSW546 (R23C), pDSW547 (R23H), pDSW548 (L39P), pDSW549 (Q46H), pDSW550 (G57D), pDSW551 (S61F), pDSW552 (S61P), pDSW553 (L62P), pDSW554 (V86E), pDSW555 (R141C), pDSW556 (H177R), pDSW557 (G188E), pDSW558 (R210C), and pDSW559 (R210H).
Media and reagents.The medium was Luria broth (LB) (10 g of tryptone, 5 g of yeast extract, 10 g of NaCl per liter, with 15 g of agar/liter for plates). l-Arabinose and d-glucose were used to modulate the expression of genes under the control of the PBAD promoter (19), and IPTG (isopropyl-β-d-thiogalactoside) was used to induce expression from lac promoters. Antibiotics were used at various concentrations as indicated in the description of each experiment.
Molecular biological procedures.Standard protocols for cloning and analysis of DNA, PCR, and electroporation were used (3). Restriction enzymes were from New England Biolabs (Beverly, Mass.). PCR was performed with SuperMix from Life Technologies (Rockville, Md.). Oligonucleotides were from Integrated DNA Technologies (Coralville, Iowa). DNA was sequenced at the DNA Core Facility of the University of Iowa using dye-terminator cycle-sequencing chemistry.
Isolation of mutant ftsI alleles.Random mutagenesis of the ftsI gene was performed by PCR with SuperMix, which contains Taq DNA polymerase. Amplification reaction mixtures had a volume of 100 μl and included 10 ng of pDSW235 plasmid template, which carries a wild-type gfp-ftsI fusion gene. The primers used were 5′-GAGACCACATGGTCCTTCTTGAG-3′, which anneals in gfp, and 5′-TGCTCTAGATTACGATCTGCCACCTGT-3′, which anneals to the 3′ end of ftsI. Amplification was done for 25 cycles with denaturation at 94°C, annealing at 52°C, and extension at 72°C. The 1.8-kb amplification product was digested with EcoRI and XbaI (sites at the gfp-ftsI junction and in the primer [underlined], respectively). The digested fragment was ligated into the same sites of pDSW287 or pDSW521. The resulting plasmid pools were introduced into EC812 by electroporation, selecting for kanamycin resistance on LB plates containing 40 μg of kanamycin/ml, 25 μg of ampicillin/ml, and 0.2% arabinose at 30°C. The colonies were then patched in a grid pattern onto similar plates that contained 0.0002% arabinose to predeplete the cells of wild-type ftsI expressed from the chromosome. Colonies that grew on the low-arabinose plates were tested for complementation by replica plating them onto the same medium with either 0.2% arabinose or 0.2% glucose. EC812 derivatives carrying an empty vector (pDSW286 or pTK18kr) and gfp-ftsI (pDSW286 or pDSW521) served as negative and positive controls, respectively.
Assays for expression, membrane insertion, catalytic activity, and stability of mutant FtsI proteins by Western blotting and penicillin binding.Western blotting was done as described previously (40). Briefly, proteins were separated on sodium dodecyl sulfate (SDS)-10% polyacrylamide gels and transferred to nitrocellulose. Then, FtsI and green fluorescent protein (GFP)-FtsI were probed with a polyclonal anti-FtsI serum, followed by a secondary antibody conjugated to horseradish peroxidase, which in turn was detected with a chemiluminescent substrate. The blots were visualized with an LAS-1000 luminescent imager from Fuji (Stamford, Conn.). Penicillin binding was assayed with Bocillin-FL from Molecular Probes (Eugene, Oreg.) essentially as described previously (14, 20). For initial screening of expression, catalytic activity, and membrane insertion, we assayed penicillin binding in spheroplasts using a procedure similar to one previously used to demonstrate catalytic activity and membrane insertion for FtsI "swap" proteins (20). Cells harboring plasmids that expressed gfp-ftsI fusions were induced with 100 μM IPTG for 5 h and then converted to spheroplasts. The spheroplasts were incubated with 20 μM Bocillin FL for 20 min at 30°C. Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE), and the fluorescent label incorporated into GFP-FtsI was visualized and quantified with a Typhoon 8600 imager from Amersham (Piscataway, N.J.). A penicillin-binding assay was also used to determine thermostability (14). Cells harboring pBAD18-Kan derivatives that expressed ftsI without the gfp moiety were induced for 2 h with 0.2% arabinose. Then, the membranes were isolated by extraction of soluble proteins with a nonionic detergent (14). The isolated membranes were incubated at 47 or 33°C for different lengths of time, and residual penicillin-binding activity was assayed with Bocillin FL at 30°C.
Localization of GFP-FtsI in merodiploids.To visualize GFP-FtsI in merodiploids, strains were grown overnight at 37°C in LB containing 10 μg of kanamycin/ml. The next morning, the cultures were diluted 1:2,000 in LB containing 5 mM IPTG and were grown at 30°C to an optical density at 600 nm of ∼0.3, at which time they were fixed and analyzed by fluorescence microscopy with a GFP filter set as described previously (30). The typical exposure time was 12 s. Digital images were captured as 12-bit images, converted to 8-bit TIFF format, and imported to Adobe Photoshop and Canvas for the preparation of figures. At the time the cells were fixed for microscopy, the cells from 1 ml of culture were collected by centrifugation and resuspended in 100 μl of loading buffer for Western blotting.
Localization of GFP-FtsI and FtsN in filaments depleted of FtsI.Strains were grown overnight at 37°C in LB containing 40 μg of kanamycin/ml, 10 μg of chloramphenicol/ml, 25 μg of ampicillin/ml, and 0.2% arabinose. The next morning, the cultures were diluted 1:50 in LB containing 40 μg of kanamycin/ml, 0.02% arabinose, and 100 μM IPTG. These cultures were grown at 30°C to an optical density at 600 nm of ∼0.3, and then the arabinose was removed by pelleting 1 ml of culture in a microcentrifuge tube and resuspending it in 1 ml of LB. The cells were washed in this manner three times before being diluted 1:250 in LB containing 40 μg of kanamycin/ml, 100 μM IPTG, and 0.2% arabinose or 0.2% glucose. Growth was continued for ∼4.5 to 5 h, at which time the glucose-grown cells had become filaments ∼20 to 30 μm long, except in the case of strains that expressed GFP-FtsI fusions that complemented the ftsI null mutation. The cells were fixed with cross-linking agents directly in growth medium and permeabilized by treatment with lysozyme as described previously (36). Anti-FtsN serum was used at a dilution of 1:200 overnight at 4°C. The secondary antibody was goat anti-rabbit conjugated to Texas Red (Molecular Probes) at 1:400 for 2 h at room temperature. For photography of Texas Red, the filter set was from Chroma Technology Corp. (Brattleburo, Vt.) and comprised a 555- to 560-nm-wavelength excitation filter, a 595-nm dichroic mirror (long pass), and a 645- to 675-nm emission filter. The typical exposure time was 2 s. Images were captured as 8-bit images, converted to TIFF format, and exported to Adobe Photoshop and Canvas for the preparation of figures. GFP-FtsI was also visualized in these filaments as described above for merodiploids. GFP-FtsI was photographed first to minimize bleaching. Staining with DAPI (4′,6′-diamidino-2-phenylindole) was done to visualize nucleoids and to verify proper segregation (not shown). At the time the cells were fixed for microscopy, the cells from 1 ml of culture were collected by centrifugation and resuspended in 100 μl of loading buffer for Western blotting.
Quantitation of fluorescence intensity.Localization of GFP-FtsI and FtsN was quantified using the line profile tool in Image-Pro Plus version 4.1 from Media Cybernetics (Silver Spring, Md.). Transects ≥3 μm long were centered on the brightest GFP-FtsI bands (green channel). An average transect was then calculated after centering ≥24 transects on their brightest pixel. The average transects for wild-type and the various mutant proteins could not be compared directly owing to substantial day-to-day variation in the absolute fluorescence intensities in our images. However, relative fluorescence intensities were consistent between experiments, so to compare mutants to the wild type, we scaled the average transects as follows. First, the background fluorescence intensity, meaning the intensity away from the septal band, was determined by averaging the intensity values in 1-μm regions to the left (−1.5 to −0.5 μm) and right (+0.5 to +1.5 μm) of the peak. Values along each transect were then divided by the background intensity to produce a normalized transect. Finally, the baseline was set to zero by subtracting 1 from each pixel. This procedure was then repeated for FtsN after the transects were automatically replicated from the green channel to the corresponding position in the red channel.
Antiserum for detection of FtsN by immunofluorescence microscopy.Polyclonal antibodies against the periplasmic domain of FtsN were raised in New Zealand White rabbits (Covance, Denver, Pa). The protein used as the antigen was obtained as follows. The periplasmic domain of FtsN (residues 56 to 319) was fused to the maltose-binding protein (MBP) in pMAL-c2 and purified by affinity chromatography on amylose-agarose as described previously (3). The final preparation was ∼95% pure, and the yield was ∼3 mg/liter of culture. Characterization of the antiserum by Western blotting revealed two bands corresponding to FtsN and MBP. Antibodies against MBP were depleted by incubating the serum with MBP-agarose that was made by incubating purified MBP2* from New England Biolabs with AminoLink coupling gel from Pierce Biotechnology, Inc. (Rockford, Ill.). One milliliter of antiserum was diluted with 1 ml of phosphate-buffered saline and passed over a column containing 200 μl of immobilized MBP slurry. Subsequent Western blotting revealed only a single protein. This band was present in a malE mutant but disappeared upon depletion of FtsN. The specificity of the antibody in immunofluorescence microscopy was verified by detection of FtsN as a band of fluorescence at the midcell in about half of the cells from a wild-type population and by showing that this signal was lost when the cells were depleted of FtsN (data not shown).
Model of FtsI.The program O (27) was used to model the three-dimensional structure of FtsI based on the Cα coordinates for PBP2x deposited in the Brookhaven Protein Data Bank (PDB) under the PDB identification codes 1PMD and 1K25. First, the B subunit of 1K25 was superimposed on 1PMD. Then, the alpha carbon backbone for amino acids 76 to 633 of 1PMD and amino acids 66 to 77 of the B subunit of 1K25 were combined into a single PDB file.
Isolation of mutants.We screened for ftsI mutants that failed to support division and were either recessive to wild type or exhibited dominant-negative phenotypes only at high levels of expression. To isolate such mutants, we constructed strain EC812, in which ftsI was expressed from an arabinose-inducible promoter integrated into the chromosome at the λ attachment site. This strain grew normally on media containing arabinose but failed to form colonies on media containing glucose. The growth of EC812 on media containing glucose was rescued by a plasmid that expresses a bright variant of gfp (gfpmut2 [11]) fused to the wild-type ftsI gene. We used two plasmids in this study, both of which direct expression of gfp-ftsI fusions under the control of IPTG-regulated promoters, although IPTG was not present in the plates during the initial screen to ensure low levels of expression. Plasmid pDSW287 has a copy number of ∼20 and overproduces GFP-FtsI ∼5-fold in the absence of induction. The other plasmid, pDSW521 has a copy number of ∼5 and produces GFP-FtsI at roughly the normal chromosomal level in the absence of induction.
To randomly mutagenize ftsI, we amplified the gene by PCR with Taq DNA polymerase, which resulted in ∼1 mutation per kb (data not shown). Since the ftsI gene is ∼1.8 kb long, most of the amplified copies had an amino acid substitution, even though some mutations are silent. The PCR products were ligated into a GFP fusion vector. The resulting plasmids were transformed into EC812 on LB medium containing arabinose. The transformants were then tested for failure to grow on LB medium containing glucose, when division depends on the GFP-FtsI protein produced from the plasmid. Noncomplementing plasmids were recovered from DNA minipreps, analyzed by restriction digestion to verify the presence of a gfp-ftsI fusion gene, and transformed back into EC812 to confirm the mutant phenotype. The candidates were then screened with a combination of Western blotting and penicillin-binding assays to weed out those that expressed proteins which were truncated, unstable, or catalytically inactive or failed to insert into the membrane. In particular, it should be noted that the penicillin-binding assay requires that FtsI perform catalysis on a β-lactam substrate and that this substrate is not able to diffuse across the cytoplasmic membrane. Thus, a positive result in this assay, which was done with spheroplasts, implies that the mutant protein in question retained catalytic activity and inserted into the membrane. Candidates that passed these tests were examined by fluorescence microscopy to determine whether the respective GFP-FtsI fusion protein localized to the septal ring, as indicated by a fluorescent band at the midcell. In parallel, DNA sequencing was used to identify the amino acid substitution(s) in the products of these alleles. If multiple substitutions were found, subcloning was used to identify the relevant lesion. Proteins whose defects could not be attributed to a single lesion were not studied further.
We started with ∼7,500 transformants generated from 15 independent PCRs. About 5% of the transformants recovered on arabinose exhibited poor complementation on glucose. Most of these produced proteins that were markedly unstable or that lacked penicillin-binding activity. Ultimately, we found only 23 plasmids that passed all of the tests. Of these, eight had striking defects in septal localization, i.e., ≤5% of the cells had a fluorescent band at the midcell compared to ∼50% for wild-type GFP-FtsI. The remaining 15 fusion proteins exhibited near-normal levels of septal localization.
Sequence changes.Although mature FtsI is 577 amino acids long, the 23 mutant proteins were found to have lesions in only 11 amino acids (Table 1). The fact that most of the targeted amino acids were altered multiple times in independent mutants indicates that our analysis was reasonably thorough. Lesions in the eight localization-defective proteins mapped to three residues clustered in a 23-amino-acid region from R23 to Q46. These amino acids are in or near the membrane-spanning segment, implying that the membrane-spanning segment is important for septal localization. In the 15 mutant proteins that appeared to be proficient in localization, the lesions mapped to 8 amino acids in a 153-amino-acid region extending from Gly 57 to Arg 210. All of these amino acid substitutions are in the noncatalytic domain. Apparently this domain is important for something other than septal localization.
Localization of the mutant FtsI proteins.To study localization more carefully, each of the mutant gfp-ftsI alleles was integrated into the chromosome in single copy at the λ attachment site (21). The resulting strains were merodiploids, because a wild-type copy of ftsI was present at the normal chromosomal locus. Expression of gfp-ftsI was driven from a weak IPTG-inducible promoter, P206 (40). Cells were grown to mid-exponential phase in LB containing 5 mM IPTG, fixed with cross-linking agents, and examined by fluorescence microscopy. About 40% of the cells exhibited localization of wild-type GFP-FtsI to the septal ring (Fig. 2 and Table 1). The frequencies of localization for various mutant proteins ranged from 0 to 40%. Proteins with lesions in or near the membrane-spanning segment had localization frequencies ranging from 0% for L39P to 5% for Q46H. In contrast, proteins with lesions in the noncatalytic domain had localization frequencies ranging from 17 to 40%, close to wild type. (Formally, Q46 is considered to be in the noncatalytic domain, but just barely—the membrane-spanning segment probably ends at R40.) Western blotting indicated that the various mutant gfp-ftsI fusions were expressed at levels similar to those of the wild-type gfp-ftsI fusion and that this level was about half that of authentic ftsI expressed from its normal chromosomal locus (Fig. 3A). Thus, there was no reason to suspect that high expression levels were masking a mild localization defect in the cases of the mutant FtsI proteins with lesions in the noncatalytic domain.
We also assayed localization in cells (filaments) depleted of wild-type FtsI. These studies were done with EC812 derivatives transformed with low-copy-number plasmids that express gfp-ftsI fusions under the control of a lac promoter. Note that this is the genetic configuration used to isolate the mutants. Characterization of this system by Western blotting demonstrated that (i) EC812 produced normal amounts of FtsI when the inducer arabinose was present; (ii) FtsI levels were reduced ∼10-fold after several hours of growth in the presence of glucose, at which time the cells had become filaments ∼20 μm long; (iii) in the absence of IPTG, the level of GFP-FtsI fusion protein produced from the plasmid was similar to what is normal for FtsI from the chromosome; and (iv) induction with 100 μM IPTG results in severalfold overproduction of the GFP-FtsI fusion protein and the appearance of significant amounts of proteolytic breakdown products (Fig. 3B).
Results of experiments to test the effect of depleting FtsI on localization of the "localization-defective" mutants are shown in Fig. 4 and Table 2. Depletion of FtsI had no effect on the morphology of cells that express a wild-type gfp-ftsI fusion from the plasmid, because the protein localizes well and supports cell division. Interestingly, localization of the R23C, R23H, and Q46H proteins improved greatly upon depletion of the wild-type protein. Only the L39P protein appeared to have a severe localization defect after depletion of the wild-type protein, although even in that case, localization improved somewhat. Figure 4 also shows that the R23C and Q46H proteins support division reasonably well in broth when produced at this level (100 μM IPTG). We later found that all of the localization-defective proteins could rescue EC812 on plates when induced with enough IPTG (see below). One interpretation of these findings is that FtsI localization involves recognition of a limiting factor at the septal ring, and the localization-defective mutants compete poorly for this factor, which might be FtsW (30). Chen et al. made a similar observation in their study of localization-defective mutants of ftsQ and also suggested that this phenomenon reflects competition for a limiting component of the septal ring (10). An alternative interpretation is that the lesions impair dimerization of FtsI. The oligomeric state of FtsI in vivo is not yet known.
All of the mutant GFP-FtsI proteins that localized well in merodiploids also localized well when wild-type FtsI was depleted (Fig. 5, left, and Table 2). This result appears to exclude the formal possibility that localization defects were masked in the merodiploids because the wild-type FtsI recruited the mutant proteins to the septal ring, perhaps by forming a dimer or some other oligomer. Rather, the mutant FtsI proteins with lesions in the domain of unknown function really are proficient in septal localization, even though they fail to support cell division. In the experiment shown, 100 μM IPTG was used to induce gfp-ftsI alleles from the plasmid. Similar results were obtained with depletion filaments grown in the presence of 10 μM IPTG, which resulted in GFP-FtsI levels essentially the same as those of FtsI (not shown).
Localization of FtsN.Because localization of FtsN to the septal ring depends upon FtsI (2), we determined the abilities of the mutant forms of FtsI to recruit FtsN. To do this, we used indirect immunofluorescence microscopy to visualize FtsN in filamentous cells depleted of wild-type FtsI but containing GFP-FtsI fusion proteins that were produced from a plasmid. FtsN was detected with a polyclonal primary antibody followed by a secondary antibody conjugated to Texas Red. Filaments were photographed using filters appropriate for green (GFP-FtsI) and red (FtsN) light.
At the resolution afforded by light microscopy, FtsN colocalized with wild-type GFP-FtsI to the septal ring (Fig. 5, middle). In the filamentous cells that expressed GFP fusions to mutant forms of FtsI, the results were more complex. In most cases, FtsN appeared to localize poorly in the filaments, but some of the mutant proteins, such as the H177R protein, appeared to recruit FtsN rather well. Nevertheless, we found it difficult to make simple yes-no calls as to whether FtsN localized to a particular site, largely because many of these sites had weak accumulations of fluorescence that were too bright to ignore. To score FtsN localization more objectively, we used computer software to draw transects across the strongest GFP-FtsI bands and let the computer replicate these transects in the same position in the red (FtsN) image. The fluorescence intensities in these transects were then measured, normalized, and averaged over at least 24 GFP-FtsI bands as described in Materials and Methods. The results are shown in Fig. 5 (right) and Table 2 and agree with more subjective impressions of localization. With wild-type GFP-FtsI, which recruited FtsN very well, the red fluorescence was about twice as bright as the green. The H177R mutant protein also recruited FtsN well—the ratio of red to green was 87% of that for the wild type. At the other extreme, the S61F and L62P mutant proteins appeared to have only 17% of the wild-type recruitment activity.
Western blot analysis showed that the different GFP-FtsI proteins were produced at similar levels (Fig. 3B) and that these levels were ∼4-fold above what is normal for FtsI expressed from the chromosome. This level of expression should be adequate for FtsN recruitment, if a particular mutant protein were proficient in this function. The same blots also revealed that the filaments contained residual FtsI. While some of this FtsI could be mutant protein derived from the fusions by proteolysis, residual FtsI was also seen in an EC812 derivative carrying a plasmid that did not express a gfp-ftsI fusion. These observations raise the possibility that residual wild-type FtsI, rather than mutant GFP-FtsI, is responsible for the weak localization of FtsN observed in some cases. If so, then mutant proteins such as S61F and L62P are probably more defective for recruitment of FtsN than they appear.
Complementation and dominance.Mutant FtsI proteins that localize efficiently to the septal ring but fail to recruit FtsN ought to have dominant-negative effects. Indeed, merodiploids expressing gfp fusions to localization-proficient mutant proteins were slightly elongated (Fig. 2). Still, the fact that we recovered such mutants implies that any dominant-negative effects are mild. Conversely, elevated production of some localization-defective proteins appeared to rescue cell division (Fig. 4). These observations were confirmed by testing for complementation and dominant-negative effects in various ftsI mutant backgrounds (Table 3). With no IPTG, the gfp-ftsI fusions are expressed at normal levels, and the localization-defective proteins failed to complement either a depletion strain or a temperature-sensitive mutant. The presence of 100 μM IPTG resulted in ∼4-fold overproduction and some complementation by each mutant allele. In contrast, the set of proteins that exhibited fairly normal localization did not complement at either level of production. These proteins were not very toxic in the depletion strain, even when 100 μM IPTG was used for induction. However, in the temperature-sensitive background, which has ∼10% as much FtsI protein as the wild type even at the permissive temperature (data not shown), there was considerable toxicity. In fact, most of the localization-proficient alleles could not be transformed into the temperature-sensitive background, and those that we did recover were not genetically stable upon restreaking. Fusion genes that express localization-defective proteins, however, were not toxic in this background and even complemented slightly when mildly overproduced.
Penicillin-binding activity and thermostability.Our initial screen included a penicillin-binding assay done with spheroplasts isolated from cells that overproduced GFP-FtsI fusion proteins. We therefore anticipated that the mutant proteins described here would fractionate with the membranes and exhibit essentially normal reactivity toward a β-lactam. To verify this expectation, cell envelopes were isolated from strains that overproduced wild-type and mutant forms of FtsI (without the GFP). Cell envelopes were incubated at 30°C with a fluorescent β-lactam, and then the proteins were separated by SDS-PAGE and the gel was visualized with a fluorescence imager. As expected, wild-type and mutant proteins bound similar amounts of the β-lactam (Fig. 3C).
We also used a penicillin-binding assay to determine the half-lives of wild-type and mutant forms of FtsI at 47°C as described previously (14). Briefly, cell envelopes were incubated at 47°C for various lengths of time and then transferred to 30°C and assayed for binding of a fluorescent β-lactam. The stability assay was done at 47°C because at that temperature wild-type FtsI has a half-life of ∼5 min, so we anticipated that even mild stability problems would be reflected in very short half-lives for the mutant proteins. As summarized in Table 4, 10 of the mutant proteins (R23C, R23H, L39P, Q46H, G57D, S61F, S61P, L62P, R210C, and R210H) had half-lives similar to that of the wild type, indicating that the lesions do not cause a folding defect. The remaining mutants (V86E, R141C, H177R, and G188E) had distinctly shorter half-lives. For these mutants, we also determined the half-life at 33°C. At the lower temperature, the wild-type, V86E, and R141C proteins had half-lives of ∼33 h, while the H177R and G188E proteins had half-lives of ∼5 h.
Amino acid substitutions that impair septal localization fall in or near the membrane-spanning segment.Our results indicate that amino acids 23, 39, and 46 of E. coli FtsI are important for localization to the septal ring. The amino acid substitutions recovered at these sites reduced localization by a factor of 8 or more but did not prevent insertion of the protein into the membrane, impair catalytic activity as judged by a penicillin-binding assay, or reduce the stability of the protein. Interestingly, changes at R23 (to C and H) were recovered four times, and changes at Q46 (all to H) were recovered three times. Given that mature FtsI is 577 amino acids long and that the mutants were obtained after random mutagenesis, these statistics imply that if other parts of FtsI are important for localization, a different approach will be needed to find them.
Residues R23, L39, and Q46 fall within or near the transmembrane helix, defined operationally as extending from R23 to R40 (7). Several other observations implicate the membrane-spanning segment in septal localization. An FtsI protein, constructed by site-directed mutagenesis, that has lesions at both ends of the membrane-spanning segment was previously described (20). Although this form of FtsI supports division, it localizes poorly (41). It was also shown that an FtsI protein with an extra leucine in the transmembrane helix does not support cell division (20) and does not localize to the septal ring (M. Wissel and D. Weiss, unpublished data).
Our results provide little support for previous speculation that the noncatalytic domain, which resides in the periplasm, engages in protein-protein interactions involved in recruiting FtsI to the septal ring. Otherwise, we should have found localization-defective mutants with lesions in this domain. We did, however, find several amino acid substitutions in the domain that had modest effects on septal localization and that may hint at a role for the domain in targeting FtsI to the septal ring. A previous report suggested that the D58V amino acid substitution prevented FtsI from localizing to the septal ring (29). This conclusion was based on the recessive nature of the mutation rather than on a direct assay of the ability of the mutant protein to localize. We have constructed the D58V protein by site-directed mutagenesis and have confirmed that it does not support cell division, but in our hands it localizes almost as well as the wild type (J. Wendt and D. Weiss, unpublished data). In these respects, the D58V protein is similar to the G57D protein that we found by random mutagenesis.
How the membrane-spanning segment of FtsI targets the protein to the septal ring is a matter of speculation. It might serve as a binding site for another protein, such as FtsI itself (if the protein is a dimer) or FtsW, which localizes immediately ahead of FtsI and is needed to recruit FtsI to the septal ring (30). Alternatively, changes in the membrane-spanning segment might subtly perturb the positioning of the periplasmic domain and thus impair dimerization or interaction with another protein. However, this hypothesis suggests that the real localization determinants might be in the noncatalytic domain, despite our inability to find lesions in that domain that prevent septal localization (other than Q46H at the beginning of the domain). Perhaps no single amino acid substitution in the noncatalytic domain impairs the putative interaction sufficiently to prevent complementation without causing additional defects.
Amino acid substitutions that impair recruitment of FtsN affect a domain of unknown function.Our results indicate that amino acids 57, 61, 62, and 210 of E. coli FtsI are important for recruitment of FtsN to the septal ring but are not critical for septal localization of FtsI, insertion of the protein into the membrane, catalytic activity as judged by a penicillin-binding assay, or stability. Substitutions at these positions were isolated repeatedly: changes at G57 (all to D) were recovered three times, changes at S61 (to F and P) were recovered twice, changes at L62 (all to P) were recovered three times, and changes at R210 (to C and H) were recovered twice. Since these mutants were obtained after random mutagenesis, it is remarkable that lesions conferring a relatively specific defect in FtsN recruitment repeatedly mapped to the same four residues. The four amino acids targeted by our mutant hunt are in the noncatalytic domain, which we now conclude is needed for recruitment of FtsN to the septal ring. This inference is consistent with previous evidence that the periplasmic domain of FtsN directs that protein to the septum (2, 12). Our finding that R210 is important for recruitment of FtsN confirms and extends findings of Marrec-Fairley and coworkers, who reported that an R210Q-R213Q double-mutant protein was toxic and inferred that it probably localizes to the septal ring but fails to interact properly with another protein (29).
The three-dimensional structure of FtsI is not known, but the structure of a homolog, PBP2x from S. pneumoniae, has been determined by X-ray diffraction (13, 35). We used an alignment of the two proteins to identify the amino acids in PBP2x that correspond to G57, S61, L62, and R210 of FtsI. Despite being separated by 150 residues in the primary sequence, these amino acids appear close together in the folded protein (Fig. 1C). They cluster near the end of one of the arms that were suggested on the basis of the structure to be involved in protein-protein interactions (35). Our results suggest that one of these interactions is with FtsN, although biochemical evidence will be needed to prove this point. Because the resolution in the PBP2x structure is low, it is not possible to say whether any of these amino acids have solvent-exposed side chains that could interact with FtsN directly.
Whether the defects in FtsN recruitment account for the failure of these mutant proteins to support division is not yet known. There is enough residual localization of FtsN (17 to 30% of wild-type activity) that, if this were the only defect, we would expect the mutant ftsI alleles to complement to some extent. But the mutant proteins might be more defective than they appear, because Western blotting revealed some residual wild-type FtsI protein in the filaments used to assay FtsN recruitment.
Other lesions in the domain of unknown function.Our screen identified several other critical amino acids in the noncatalytic domain: V86, R141, H177, and G188. Although lesions at these sites resulted in a failure to complement, further characterization indicated that the defects in these proteins are complex. Each mutant protein was somewhat less stable than the wild type. This instability does not by itself account for the failure to complement, because the steady-state level of each of the mutant proteins is similar to that of the wild type. Still, the instability suggests that parts of FtsI distant from the lesions might have been disrupted. One of the lesions, G188E, is at the start of a block of sequence, G188IEGVEKSFD, that is highly conserved among class B penicillin-binding proteins (18) and that has previously been shown to be important for the folding and stability of FtsI (17). The V86E and H177R proteins localize about half as well as the wild type, suggesting that the domain of unknown function has a role in septal localization, even though our best localization-defective mutants mapped to the transmembrane helix. The V86E, R141C, and G188E proteins did not recruit FtsN very well and lend further support to the notion that the noncatalytic domain is needed for recruitment of FtsN. The H177R protein is in some ways the most intriguing one to come out of the study. Although it does not support cell division, it is essentially normal in everything we can measure—septal localization, recruitment of FtsN, and penicillin-binding activity. Perhaps FtsI has functions that we do not know of. Alternatively, the H177R protein might point to the limitations of our assays. For example, the H177R lesion might alter the orientation of FtsI with respect to the peptidoglycan wall so that transpeptidation is not possible, but because the active site is not disrupted, penicillin binding is not impaired. Finally, Höltje and coworkers have used affinity chromatography to recover complexes that contain a host of murein enzymes, including FtsI, Slt70, MltA, MltB, PBP1b, PBP1c, PBP2, PBP4, PBP7, and AmiC (26, 38, 39). Thus, the H177R mutant (and possibly others too) might be defective in formation of a proposed "holoenzyme" for peptidoglycan synthesis.
The lesions in these mutant proteins are scattered throughout the domain of unknown function. In PBP2x, the amino acids that correspond to V86, H177, and G188 are near the interface between the noncatalytic and catalytic domains (Fig. 1C). This may explain why lesions at these sites reduce stability. R141 of FtsI is difficult to map onto the structure of PBP2x because it falls in a region where PBP2x has an ∼30-amino-acid insertion that is not present in the E. coli protein. Nevertheless, it appears safe to infer that R141 is in one of the arms of the noncatalytic domain, opposite the arm that contains residues implicated in FtsN recruitment.
We thank Ryan Arends, Jennie Blair, Calista Mitchell, Kari Schmidt, Ryan Seipke, Victor Torres, James Urton, Jennifer Wendt, and Brian Wulff for help with experiments and Subramanian Ramiswamy for help producing Fig. 1C.
This work was supported by a grant from the National Institutes of Health (GM59893) to D.S.W. M.C.W. was supported in part by a National Science Foundation Research Training Grant (DBI9602247). The DNA facility is supported by the Diabetes and Endocrinology Research Center with National Institutes of Health grant DK25295 and by the School of Medicine.
- Received 3 September 2003.
- Accepted 17 October 2003.
- Copyright © 2004 American Society for Microbiology


text:
I want to focus on one word, and one word only. That word is fairness. Not gender. Not transgender. Not activism. Not rights. Not perception. Simply fairness. That's the issue here, and that's where we need to keep our focus.
The question is not why a man identifies as a woman (or vice versa).
The question is this: Is it fair for a biological male to compete against biological females?
According to a story posted on Trib Live, a "A transgender woman [CeCe, formerly Craig, Telfer] who competed as a man as recently as last year won an NCAA women's track national championship on Saturday."
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Yet, less than 18 months ago, in January, 2018, "Telfer finished eighth in a field of nine in the Men's 400 meters at the Middlebury Winter Classic in Vermont."
So, competing against fellow males, Telfer was second to last in a field of 9. Now, competing against females, Telfer came in first in a field of 8. That's quite a turnaround.
And Telfer did not simply win. He won decisively, finishing with a time of 57.53, more than one full second ahead of second-place finisher Minna Sveard, who ran a time of 59.21, edging out the third place finisher Sidney Trinidad, who finished in 59.29. (This picture says it all.)
So, instead of Sveard taking a first place, she ends up with a second place, despite training hard and running her best. The deck was simply stacked against her. Is that fair?
Like it or not, there's a massive difference between first and second, between gold and silver.
And what if you were competing in the Olympics and were bumped from third (a bronze medal) to fourth (no medal at all)? How big a difference is that?
Interestingly, the story on Trib Live is positive from beginning to end.
It uses standard, LGBT terminology, identifying Telfer as a "transgender woman" and referring to her as "she."
It quotes Telfer's coach, Zach Emerson, who said, "It was tough conditions out here with the wind and the heat over the last three days but, as she has over the last six months, CeCe proved herself to be tough enough to handle it. Today was a microcosm of her entire season; she was not going to let anything slow her down. I've never met anybody as strong as her mentally in my entire life."
And it notes that, "The NCAA allows male athletes to compete as women if they suppress their testosterone levels for a full calendar year. Before that, they compete on mixed teams—with men and women—in the men's division but not the women's."
Yet, despite this glowing, LGBT-affirming coverage, readers of the article felt that the competition was not fair, as indicated by their overwhelmingly negative response to a poll contained in the article.
The poll asked, "Do you believe that a person born as a male but identifying as a female should be allowed to compete in women's competitive sports events?"
As of this writing, 16,982 readers responded, with 4% saying, "Yes," 4% saying, "I'm not sure," and a staggering 92% saying, "No."
They were shouting as one, "This is not fair!"
It doesn't mean Telfer is an evil person.
It doesn't mean Telfer is not a devoted athlete.
It doesn't mean Telfer is not deeply sincere about identifying as female.
It simply means that, when it comes to athletic competition, there's a reason men do not compete against women.
We have the NBA and the WNBA for a reason.
We have the PGA and the LGPA for a reason.
We have men's world records and women's world records for a reason.
This is biology, not bigotry; common sense, not bias.
I'm aware of the statement in the NCAA's Transgender Handbook which reads: "According to medical experts on this issue, the assumption that a transgender woman competing on a women's team would have a competitive advantage outside the range of performance and competitive advantage or disadvantage that already exists among female athletes is not supported by evidence."
To that I say, bah, humbug.
No amount of medical experts will convince me that Telfer did not have an unfair advantage competing against women. No amount of experts will convince me that Craig Telfer, who finished second to last among male competitors, did not have a biological advantage as CeCe Telfer when he finished first among females.
Writing in World magazine, Ray Hacke notes that, "According to the website LetsRun.com ... 'Prior to joining [Franklin Pierce University's] women's team this season, Telfer was a mediocre D-II athlete who never came close to making it to nationals in the men's category. In 2016 and 2017, Telfer ranked 200th and 390th, respectively, among D-II men' before sitting out the 2018 season."
Hacke continues, "If this narrative sounds eerily familiar, it should: Biologically male sprinter Terry Miller of Bloomfield High set a state record of 6.95 seconds in the girls' 55 meters at Connecticut's indoor track and field championships back in February. Miller also set meet records in the 100 and 200 at Connecticut's outdoor championships last spring."
But, "Like Telfer, Miller was nowhere near elite when competing against boys. After declaring himself a girl and competing as one, Miller became a champion."
Accordingly, Hacke quotes Robert Johnson, who previously wrote on LetsRun.com, "The fact that Telfer can change [his] gender and immediately become a national champion is proof positive as to why women's sports needs protection."
These women work hard and make great sacrifices to reach their athletic and personal goals. They are competitors, and they deserve an even playing field.
Something needs to be done to protect them. Fairness is the operative word.
Dr. Michael Brown (www.askdrbrown.org) is the host of the nationally syndicated Line of Fire radio program. His latest book is Evangelicals at the Crossroads: Will We Pass the Trump Test? Connect with him on Facebook or Twitter, or YouTube.
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