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The Amateur Traveler talks to Edith about the mountainous region of Austria where she grew up, the Tyrol. Edith designs for us both a winter and a summer itinerary, both of which involve getting outside and enjoying the beautiful mountains. In the winter we would ski and in the summer we would hike. When we are not hiking or enjoying one of the mountain alms then we can explore the palace at Innsbruck, a hiking museum and the Höfemuseum which is a farm museum. Edith talks about traditional foods such as Wiener Schnitzel, Kaiserschmarrn (sweet pancake dish), Kaspressknödel (fried cheese dumplings) and Graukäse (literally grey cheese). To work off that rich food (and of course beer) she will recommend we hike between the mountain huts or up her favorite hike Nochspitze.

The Amateur Traveler talks to Lee from Atlanta about side trips that you can take when you travel to Shanghai this year for Expo 2010. Lee is currently living in China and talks about 3 different weekend trips to destinations that are located near Shanghai. Lee starts us the in history-rich city of Nanjing the site of “China’s Holocaust” in WW 2. Nanjing has been the capital for 6 different Chinese governments and still retains its ancient walls in addition to modern shopping and the tomb of Sun Yat-sen. It was also the location of the world’s worst civil war, the Taiping Rebellion. After Nanjing we explore the many gardens and the canals of Suzhou. Finally Lee takes us to Hangzhou with its beautiful West Lake which the Chinese compare to heaven.

News

Spirit Air to experiment with carry-on bag fees
Airlines losing 3000 bags – every hour of every day
Women try to smuggle dead relative onto flight
Paying to use the plane potty?
Ky. Judge Rules in Favor of Online Travel Sites

Show Notes

Expo 2010 Shanghai China
Travel to Shanghai, China – Episode 26

Nanjing
Nanjing, China
Nanking Massacre
John Rabe – a German businessman and Nazi party member who is best known for his efforts to stop the atrocities of the Japanese army during the Nanking Occupation
Sun Yat-sen – “Father of the Chinese Nation”
Ming Dynasty
Cheongsam or Qipao – traditional Chinese dress
Taiping Rebellion – largest civil war in history started by Hong Xiuquan who declared himself the brother of Jesus.

Restaurants
Skyway Bakery and Deli
Les 5 Sens
Tairyo Teppanyaki

Suzhou
Gardens – Master of the Nets, Lion’s Forest, Humble Administrator
Mingtown Suzhou International Youth Hostel

Hangzhou
Tea Museum
A History of Chinese in California
Braised Pork Belly – Hangzhou Dongpo Pork
Sweet and Sour Pork Recipe
Wushanyi International Youth Hostel

Shanghai
Captain Youth Hostel

elong.com, ctrip.com – for accompodations

Community

Egypt Photo Tour
Amateur Traveler Newsletter
Does Amateur Traveler work for you at work?

The Amateur Traveler talks to Italophile Ira Bernstein who returns to the show to talk about day trips in Tuscany that you can take from Florence. We talk about visiting the walled city of Siena and its famous bareback horse race the palio, viewing the many towers of San Gimignano, climbing the leaning tower of Pisa (after you take the obligatory picture of someone holding it up), and walking on the walls of Lucca. Each town has its own character and attractions. Arezzo has a jewelry museum and the home of the “Father of Humanism” Petrarch. Fiesole is a rich enclave, a suburb of Florence with views of the city and a roman theatre. Montepulciano and Montalcino are both known for their wines. Ira talks about some of the events that the locals enjoy like the palios, flea markets and flag throwing competitions. So even if you just get as far out of Florence as Piazzale Michelangelo there are many places to explore in Tuscany.

The Amateur Traveler talks to Zoë Dawes from TheQuirkyTraveler.com about England’s Lake District. This beautiful pastoral area was made famous by the poets and artists in the romantic period, most notably William Wordsworth. Not far from industrial Manchester, the lake district is still a refuge for hikers and and other tourists tucked away in northern England. Zoë describes some of her favorite hikes like the coffin trail along the shores of Lake Windermere and favorite villages like Ambleside.

The Amateur Traveler talks to JD Andrews about his recent trip to Trinidad and Tobago. JD, who used to work for a cruise line, has visited many of the islands of the Caribbean but this was his first visit to Trinidad.

The Amateur Traveler talks to Kirk Horsted in a two part episode on the Twin Cities of Minneapolis and Saint Paul, Minnesota. In this second half Kirk looks at the state capital of Saint Paul, Minnesota.

Hear about travel to Vienna, Austria as the Amateur Traveler talks to Gerhard Strassgschwandtner who is a tour guide and owner of the Third Man Museum in Vienna.

Hear about travel to Salzburg, Austria as the Amateur Traveler talks to Gina Czupka about her time living in Salzburg. Salzburg is a "compact walkable city. Everywhere you go there is incredible architecture. It's got a beautiful mountain setting. It's just a picturesque easy place to experience the old-world feel of Europe."

Hear about a road trip around Ontario as the Amateur Traveler talks to guidebook author Carolyn Heller, author of the Moon guide to Ontario. 

In preparation for the 2017 500th anniversary of the Reformation, Chris (the Amateur Traveler) took a road trip through "Luther Land" and the heart of Germany.

Hear about travel to 4 national parks in British Columbia as the Amateur Traveler talks to Carolyn B. Heller author of "Moon Vancouver & Canadian Rockies Road Trip".

Hear about a road trip in Central Mexico as the Amateur Traveler talks to Amie from thetravelingtogetherjournal.com about a trip they took near Mexico City.

Hear about travel to Southern New Brunswick as the Amateur Traveler talks to Kirsten Maxwell from kidsareatrip.com about her recent travel to the Province with her family.

Hear about a Route 66 road trip as the Amateur Traveler talks to Austin Coop from 2laneamerica.com and guidedroute66tours.com about the Mother Road.

Hear about a road trip in Kansas as the Amateur Traveler talks to John McKenzie of johnthetraveler.com about the state of his birth. 

Hear about an Oregon Coast road trip as the Amateur Traveler talks to Jessica Baker from BoundlessBakers.com about a portion of their year and a half long road trip.

Sen. Ted Cruz was apparently about to get into mullet territory. He hadn’t had a haircut in three months. Things were looking grim for the Texas senator. Thankfully, Shelley Luther is able to do something about that. Luther is both famous and free after a viral clip of her standing up to a judge who…

The post Ted Cruz Stops by Shelly Luther’s Salon for a Trim, ‘True Authenticity’ Leaves Her Sobbing appeared first on The Western Journal.

By easing distancing measures, the Greater Montreal area could experience a substantial increase in the number of deaths per day due to the coronavirus, according to a new document published by the Institut national de santé publique du Québec (INSPQ) in collaboration with experts from Laval University.

After spending two months living in Mérida and years of trips traveling through the Yucatán Peninsula, I’ve finally put together the best itinerary for two weeks in Mexico. Most travelers go to Mexico to lie on the beach for a week, maybe go on an excursion to a ruin or a cenote, and head straight …

Two Weeks in Mexico: The BEST Yucatán Road Trip Itinerary Read More »

The post Two Weeks in Mexico: The BEST Yucatán Road Trip Itinerary appeared first on Adventurous Kate.

With a bright, 8.2-inch HD display and the flexibility to work in almost any vehicle, the Lanmodo Automotive Vast Night Vision System is a near-perfect alternative to factory-installed systems.

The post Brighten Up Your After-Dark Road-Tripping with Lanmodo’s Vast Automotive Night Vision System appeared first on Vagabondish.

If you've never had enough room to take your bike road-tripping, Oyama's origami-inspired folding electric bike is here to change your mind.

The post I Never Thought I’d Want (or Need) an Electric Folding Bike … But This Changed My Mind appeared first on Vagabondish.

We love to cook. But RV kitchens are notoriously — almost impossibly — small. The limited workspace can challenge even the most skilled food truck chefs. The key to actually enjoying cooking in such a confined space is finding the right tools that pack maximum versatility into a compact, lightweight, and durable package. Here are seven of our favorite RV kitchen gadgets. The Best RV Kitchen Gadgets for Every On-the-Go Chef Flexible Wood Cutting Board The easiest way to keep […]

The post 7 Must-Have Gadgets to Trick Out Your RV Kitchen for 2019 appeared first on Vagabondish.

My flight was booked! My dream of hiking the Inca Trail, and walking through the sun gate to Machu Picchu was finally becoming a reality. But then, the reality hit me. I have never done an overnight hike before! What do I pack? How do I get into shape? Is it safe? Do I go alone or with a group? As excited as I was, I realized I had a lot of planning ahead of me, but every moment of preparation (and sometimes extreme anxiety) was all worth it. Here are a few tips and tricks that helped me not only survive the Inca Trail, but have the trip of a lifetime!

1. Find an Incredible Tour Group

Before booking my trip, I was not sure how I felt about going with a tour group. I was worried that traveling with a group of strangers would take away from the trip. However, after plenty of research, I decided to go with Active Adventures on the Ultimate Peru Adventure.

Our trip leader was not only full of helpful and interesting information, but became a friend who gave local tips, and made me feel comfortable and safe. I became close friends with my small tour group of 10 people, and still keep in touch with them. Picking a quality tour group like Active Adventures not only helped take the stress out of travelling logistics, but is truly what made my trip an enjoyable experience. (And for all the foodies out there, our Inca Trail chefs were incredible).

2. Get Fit

On the Inca Trail we had people of all ages, sizes, and fitness levels. However, I highly recommend preparing your body a little, and familiarizing yourself with how your body reacts to high altitude. But, don’t worry, you don’t need to an extreme amount of training. To prepare, I went on one to two hikes a week, and did plenty of dog walking. My hikes on average ranged from 3 to 7 miles.

However, as fit as you are, everybody reacts differently to altitude. Try finding a hike in your area that has somewhat of a higher altitude. I only had the opportunity to do one higher altitude hike. It was not as high as the Inca Trail, but it still gave me an idea of how my body feels in high altitude. But don’t stress out too much! Your tour leader has helped many people hike the Inca Trail, and will be there to help you if you start to feel sick, or simply need a little extra motivation.

3. Technology Tools

I am a firm believer in digital detoxing when traveling, but when hiking in the wilderness, it is comforting to know that I have helpful resources at my fingertips if I ever need it. When hiking or traveling, there are always a few resources and apps I like to have on my phone or iTouch to stay safe.

As a traveler, we are more vulnerable to security threats or identity theft on public computers and Wi-Fi. I choose to use a VPN when traveling for Wi-Fi security in hotels, to avoid computer viruses, and to gain access to content that is geo-blocked. For my Inca Trail trek I downloaded the MotionX GPS app. The app supports GPS navigation without the need for cell service. Thankfully our guides kept us on track, but it was comforting knowing that if anything happened, I could use my phone as a backup for navigation help.

4. Make a Visit to Your Doctor

When traveling anywhere, it is important to check in with your doctor. Different regions of the world may require various vaccinations, or medication. Before visiting your doctor, it is also helpful to do a little bit of your own research as to what medications or vaccinations are needed for travel. Your doctor may be familiar with vaccinations needed for Peru, but you are the only person who knows what your trip will consist of. Be sure to mention to your doctor if you are going to the Peruvian Amazon, or high altitude.

For the Inca Trail I recommend packing the following medications:

-Altitude sickness medication (Acetazolamide)

                       -Imodium

                       -Antibiotics (Ciprofloxacin)

                       -Bug spray (not technically a medication, but important to bring as well)

                       -Ibuprofen (always good to have)

This list is most likely what your doctor will recommend as well, but be sure to do your own research as well. You may need malaria pills or certain vaccinations depending on where else your trip is going, or personal previous medical history.

5. Pack Light

The hardest part of my trip (aside from trekking up the stairs of the Inca Trail) was packing! There are several limitations on how much you pack on the Inca Trail in order to maintain that porters are being treated fairly and respectfully. Each porter is limited to carrying 20kg. This includes all blankets, clothes, and the porter’s personal belongings. Talk to your Active Adventure tour guide before leaving on your trip to find out up to date information on how much you are able to pack, and information on sleeping bags and pads.

Aside from packing light, there are of course certain items you will want to pack, depending on the time of year your trek is. I used Her Packing List, along with the information provided by Active Adventures to help me in packing everything I need.

6. Have the Time of Your Life

Last, but not least, be ready to have incredible experiences and memories that will last a life time. When I first booked my trip to Peru and the Inca Trail I had certain expectations and ideas of what the trip would be like. I was looking forward to seeing Machu Picchu, but what I found was that the most memorable and exciting parts of my trip were not necessarily seeing the big tourist spots like Machu Picchu, but the journey getting there, and the unexpected surprises that came along the way. Peru and its people exceeded all expectations, and opened my mind in ways I did not know were possible.

Hi, my name is Jess Signet. My parents were travelers since before I was born. Even in the womb, I was able to travel all over the place! Boy, did things NOT change as I grew older!
Knowing there’s more to the world than the bubble I live in made me want to travel even further. Traveling is my drug and I’m addicted. (Please, no intervention!)

[jump to video]

Long story (not very) short? One night in 2003--after killing it in front of audience of about 30 lucky people in Oakland--The Long Winters needed a place to crash, and my wife and I were happy to oblige. 

So, they drove their Big Stinky Blue Van over the bridge, slept on our floor, and by breakfast the next morning, it'd become clear to me that I'd provided lodging to a man who was not only very likely a member of my karass--he was also one of the smartest bullshit artists I'd ever met. 

Almost eight years later, although I don't see him nearly as much as I'd like, I still count the guy as one of my best pals ever.

That's John Roderick. And, I think you need to know about him.

John doesn't read this site--he's more of a Twitter person--so I don't risk feeding his astounding excess of dignity by saying he's one of the most gifted writers and bon vivants of our generation. He's just the best. In large part because he's congenitally incapable of suffering bullshit.

This was never more apparent than the Saturday morning in 2007 when we sat in my back yard and talked about a lot of stuff. Playing guitar, advertising on the web, the evil work of promoters, and why everyone is always trying to shortchange everyone on copper pipe. 

That talking became a four-part interview I ran on the late and occasionally lamented The Merlin Show, and, to this day, it's one of my favorite things I've been lucky enough to post to the web.

So, y'know how I'm definitely "not for everyone?" Well, John is really "not for everyone."

He's opinionated and arrogant and undiplomatic and unironically loves Judas Priest--meaning everyone will find at least one thing not to like about him. Despite being hairy and enjoying laying on your bed, John is not exactly a teddy bear. 

But, John's also right a lot. And, he never sands off the edges of his personality or opinions to make you theoretically "like" him. Which, it will come as no surprise to you, is a big reason I love the guy more than a free prime rib dinner. 

So, why the jizzfest about that awful jerk, John Roderick?

Because, as I noted the other day on the Twitter, in our first episode of Back to Work I misattributed a line that should have been credited to John. Which in itself is unimportant, except inasmuch as finding that link to correct the error got me watching our 50-some minutes of chatting again. I also received some at-responses and emails that reminded me how much people enjoyed our chat. 

But, really it made me realize how much that rambling morning in my back yard still resonates so much with stuff I care a lot about. Independence. Agency. Directness. And, never apologizing for wanting to get paid. Also, guitars and talkative hippies.

So, anyway. John. 

I edited all four parts of the video into one big (streamable/downloadable) movie that should make it way easier to watch at a sitting. Should that interest you. Which it may not. Which, as ever, is totally fine, and kind of the point.

But. If you like Dan and my new show (and, seriously—God bless you magnificent bastards who helped briefly make B2W the most popular podcast in the world [gulp]), I think you'll really like this interview a lot too. I hope so, anyway.

Thus, submitted for your disapproval, permit me to present my four-year-old visit with the acerbic, opinionated, and reportedly unlikeable bullshit artist whom I respect and adore more than just about anybody. 

Meet Hotrod.

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The DML News App offers the best in news reporting.

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The DML News App offers the best in news reporting.

The post REPORT: US slams new restrictions on journalists from this country appeared first on Dennis Michael Lynch.

Police officers responding to a call about a man with a machete in Oppenheimer Park Friday afternoon found what they were looking for, and then some.

The following article, Woman Shoots Three Teenage McDonald’s Workers For Telling Her She Couldn’t Eat In Dining Room Over COVID19 Restrictions, was first published on 100PercentFedUp.com.

An angry Oklahoma woman shot at a group of teenagers working at a local McDonald’s after they explained to her that the dining room was closed due to coronavirus restrictions. 32-year-old Gloricia Woody was arrested by the Oklahoma City Police and charged with the shooting. From the Oklahoma City Police -Last night, officers were called […]

Continue reading: Woman Shoots Three Teenage McDonald’s Workers For Telling Her She Couldn’t Eat In Dining Room Over COVID19 Restrictions ...

Heterotrimeric G proteins are the core upstream elements that transduce and amplify the cellular signals from G protein–coupled receptors (GPCRs) to intracellular effectors. GPCRs are the largest family of membrane proteins encoded in the human genome and are the targets of about one-third of prescription medicines. However, to date, no single therapeutic agent exerts its effects via perturbing heterotrimeric G protein function, despite a plethora of evidence linking G protein malfunction to human disease. Several recent studies have brought to light that the Gq family–specific inhibitor FR900359 (FR) is unexpectedly efficacious in silencing the signaling of Gq oncoproteins, mutant Gq variants that mostly exist in the active state. These data not only raise the hope that researchers working in drug discovery may be able to potentially strike Gq oncoproteins from the list of undruggable targets, but also raise questions as to how FR achieves its therapeutic effect. Here, we place emphasis on these recent studies and explain why they expand our pharmacological armamentarium for targeting Gq protein oncogenes as well as broaden our mechanistic understanding of Gq protein oncogene function. We also highlight how this novel insight impacts the significance and utility of using G(q) proteins as targets in drug discovery efforts.

Interferon-regulated myxovirus resistance protein B (MxB) is an interferon-induced GTPase belonging to the dynamin superfamily. It inhibits infection with a wide range of different viruses, including HIV-1, by impairing viral DNA entry into the nucleus. Unlike the related antiviral GTPase MxA, MxB possesses an N-terminal region that contains a nuclear localization signal and is crucial for inhibiting HIV-1. Because MxB previously has been shown to reside in both the nuclear envelope and the cytoplasm, here we used bioinformatics and biochemical approaches to identify a nuclear export signal (NES) responsible for MxB's cytoplasmic location. Using the online computational tool LocNES (Locating Nuclear Export Signals or NESs), we identified five putative NES candidates in MxB and investigated whether their deletion caused nuclear localization of MxB. Our results revealed that none of the five deletion variants relocates to the nucleus, suggesting that these five predicted NES sequences do not confer NES activity. Interestingly, deletion of one sequence, encompassing amino acids 505–527, abrogated the anti-HIV-1 activity of MxB. Further mutation experiments disclosed that amino acids 515–519, and Pro-515 in particular, regulate MxB oligomerization and its binding to HIV-1 capsid, thereby playing an important role in MxB-mediated restriction of HIV-1 infection. In summary, our results indicate that none of the five predicted NES sequences in MxB appears to be required for its nuclear export. Our findings also reveal several residues in MxB, including Pro-515, critical for its oligomerization and anti-HIV-1 function.

Treatment of patients with triple-negative breast cancer (TNBC) is limited by a lack of effective molecular therapies targeting this disease. Recent studies have identified metabolic alterations in cancer cells that can be targeted to improve responses to standard-of-care chemotherapy regimens. Using MDA-MB-468 and SUM-159PT TNBC cells, along with LC-MS/MS and HPLC metabolomics profiling, we found here that exposure of TNBC cells to the cytotoxic chemotherapy drugs cisplatin and doxorubicin alter arginine and polyamine metabolites. This alteration was because of a reduction in the levels and activity of a rate-limiting polyamine biosynthetic enzyme, ornithine decarboxylase (ODC). Using gene silencing and inhibitor treatments, we determined that the reduction in ODC was mediated by its negative regulator antizyme, targeting ODC to the proteasome for degradation. Treatment with the ODC inhibitor difluoromethylornithine (DFMO) sensitized TNBC cells to chemotherapy, but this was not observed in receptor-positive breast cancer cells. Moreover, TNBC cell lines had greater sensitivity to single-agent DFMO, and ODC levels were elevated in TNBC patient samples. The alterations in polyamine metabolism in response to chemotherapy, as well as DFMO-induced preferential sensitization of TNBC cells to chemotherapy, reported here suggest that ODC may be a targetable metabolic vulnerability in TNBC.

Interferon-regulated myxovirus resistance protein B (MxB) is an interferon-induced GTPase belonging to the dynamin superfamily. It inhibits infection with a wide range of different viruses, including HIV-1, by impairing viral DNA entry into the nucleus. Unlike the related antiviral GTPase MxA, MxB possesses an N-terminal region that contains a nuclear localization signal and is crucial for inhibiting HIV-1. Because MxB previously has been shown to reside in both the nuclear envelope and the cytoplasm, here we used bioinformatics and biochemical approaches to identify a nuclear export signal (NES) responsible for MxB's cytoplasmic location. Using the online computational tool LocNES (Locating Nuclear Export Signals or NESs), we identified five putative NES candidates in MxB and investigated whether their deletion caused nuclear localization of MxB. Our results revealed that none of the five deletion variants relocates to the nucleus, suggesting that these five predicted NES sequences do not confer NES activity. Interestingly, deletion of one sequence, encompassing amino acids 505–527, abrogated the anti-HIV-1 activity of MxB. Further mutation experiments disclosed that amino acids 515–519, and Pro-515 in particular, regulate MxB oligomerization and its binding to HIV-1 capsid, thereby playing an important role in MxB-mediated restriction of HIV-1 infection. In summary, our results indicate that none of the five predicted NES sequences in MxB appears to be required for its nuclear export. Our findings also reveal several residues in MxB, including Pro-515, critical for its oligomerization and anti-HIV-1 function.

Invitation Only Research Event

Marilyn Goudreault
Jan 1, 2009; 8:157-171
Research

Leigh Anderson
Apr 1, 2006; 5:573-588
Research

Heterotrimeric G proteins are the core upstream elements that transduce and amplify the cellular signals from G protein–coupled receptors (GPCRs) to intracellular effectors. GPCRs are the largest family of membrane proteins encoded in the human genome and are the targets of about one-third of prescription medicines. However, to date, no single therapeutic agent exerts its effects via perturbing heterotrimeric G protein function, despite a plethora of evidence linking G protein malfunction to human disease. Several recent studies have brought to light that the Gq family–specific inhibitor FR900359 (FR) is unexpectedly efficacious in silencing the signaling of Gq oncoproteins, mutant Gq variants that mostly exist in the active state. These data not only raise the hope that researchers working in drug discovery may be able to potentially strike Gq oncoproteins from the list of undruggable targets, but also raise questions as to how FR achieves its therapeutic effect. Here, we place emphasis on these recent studies and explain why they expand our pharmacological armamentarium for targeting Gq protein oncogenes as well as broaden our mechanistic understanding of Gq protein oncogene function. We also highlight how this novel insight impacts the significance and utility of using G(q) proteins as targets in drug discovery efforts.

2 March 2020

Heme-regulatory motifs (HRMs) are present in many proteins that are involved in diverse biological functions. The C-terminal tail region of human heme oxygenase-2 (HO2) contains two HRMs whose cysteine residues form a disulfide bond; when reduced, these cysteines are available to bind Fe3+-heme. Heme binding to the HRMs occurs independently of the HO2 catalytic active site in the core of the protein, where heme binds with high affinity and is degraded to biliverdin. Here, we describe the reversible, protein-mediated transfer of heme between the HRMs and the HO2 core. Using hydrogen-deuterium exchange (HDX)-MS to monitor the dynamics of HO2 with and without Fe3+-heme bound to the HRMs and to the core, we detected conformational changes in the catalytic core only in one state of the catalytic cycle—when Fe3+-heme is bound to the HRMs and the core is in the apo state. These conformational changes were consistent with transfer of heme between binding sites. Indeed, we observed that HRM-bound Fe3+-heme is transferred to the apo-core either upon independent expression of the core and of a construct spanning the HRM-containing tail or after a single turnover of heme at the core. Moreover, we observed transfer of heme from the core to the HRMs and equilibration of heme between the core and HRMs. We therefore propose an Fe3+-heme transfer model in which HRM-bound heme is readily transferred to the catalytic site for degradation to facilitate turnover but can also equilibrate between the sites to maintain heme homeostasis.

The dextransucrase DSR-OK from the Gram-positive bacterium Oenococcus kitaharae DSM17330 produces a dextran of the highest molar mass reported to date (∼109 g/mol). In this study, we selected a recombinant form, DSR-OKΔ1, to identify molecular determinants involved in the sugar polymerization mechanism and that confer its ability to produce a very-high-molar-mass polymer. In domain V of DSR-OK, we identified seven putative sugar-binding pockets characteristic of glycoside hydrolase 70 (GH70) glucansucrases that are known to be involved in glucan binding. We investigated their role in polymer synthesis through several approaches, including monitoring of dextran synthesis, affinity assays, sugar binding pocket deletions, site-directed mutagenesis, and construction of chimeric enzymes. Substitution of only two stacking aromatic residues in two consecutive sugar-binding pockets (variant DSR-OKΔ1-Y1162A-F1228A) induced quasi-complete loss of very-high-molar-mass dextran synthesis, resulting in production of only 10–13 kg/mol polymers. Moreover, the double mutation completely switched the semiprocessive mode of DSR-OKΔ1 toward a distributive one, highlighting the strong influence of these pockets on enzyme processivity. Finally, the position of each pocket relative to the active site also appeared to be important for polymer elongation. We propose that sugar-binding pockets spatially closer to the catalytic domain play a major role in the control of processivity. A deep structural characterization, if possible with large-molar-mass sugar ligands, would allow confirming this hypothesis.

Bacterial biofilms are cellular communities that produce an adherent matrix. Exopolysaccharides are key structural components of this matrix and are required for the assembly and architecture of biofilms produced by a wide variety of microorganisms. The human bacterial pathogens Escherichia coli and Salmonella enterica produce a biofilm matrix composed primarily of the exopolysaccharide phosphoethanolamine (pEtN) cellulose. Once thought to be composed of only underivatized cellulose, the pEtN modification present in these matrices has been implicated in the overall architecture and integrity of the biofilm. However, an understanding of the mechanism underlying pEtN derivatization of the cellulose exopolysaccharide remains elusive. The bacterial cellulose synthase subunit G (BcsG) is a predicted inner membrane–localized metalloenzyme that has been proposed to catalyze the transfer of the pEtN group from membrane phospholipids to cellulose. Here we present evidence that the C-terminal domain of BcsG from E. coli (EcBcsGΔN) functions as a phosphoethanolamine transferase in vitro with substrate preference for cellulosic materials. Structural characterization of EcBcsGΔN revealed that it belongs to the alkaline phosphatase superfamily, contains a Zn2+ ion at its active center, and is structurally similar to characterized enzymes that confer colistin resistance in Gram-negative bacteria. Informed by our structural studies, we present a functional complementation experiment in E. coli AR3110, indicating that the activity of the BcsG C-terminal domain is essential for integrity of the pellicular biofilm. Furthermore, our results established a similar but distinct active-site architecture and catalytic mechanism shared between BcsG and the colistin resistance enzymes.

4 March 2020 , Volume 96, Number 2

4 March 2020 , Volume 96, Number 2

19 December 2013

Chi-Liang Eric Yen
Nov 1, 2008; 49:2283-2301
Thematic Reviews

M el-Saadani
Apr 1, 1989; 30:627-630
Articles

M. Mahmood Hussain
Jan 1, 2003; 44:22-32
Reviews

AE Christian
Nov 1, 1997; 38:2264-2272
Articles

JE Hixson
Mar 1, 1990; 31:545-548
Articles

4 March 2020 , Volume 96, Number 2