text:
- 4 beds
- 4.5 baths
- $2,595,000 price
- 5897391 listing id
- Half Baths: 1
- Square Feet: 5,015 sqft
- Year Built: 2019
- Neighborhood: Desert Mountain
- Status: Active
CONSTRUCTION HAS BEGUN on this wonderful designed home by award winning Architector, PC, (Tor Barstad) The home features what every buyer desires in today's luxury home, an open GREAT ROOM floor plan with NO steps in the entire home, four bedroom en suites, study, expansive outdoor patio spaces with an outdoor family room. Great location with quick access to the main gate and to the redesigned Renegade golf course that features one of the best practice facilities in Arizona. Great views of the Continental Mountains, Black Mountain, Continental Peak, Lone Mountain and sunsets. The home is being built by Mike Reuter Construction Inc. who has built approximately 28 homes at Desert Mountain. See document section for floor plan. Estimated completion March 2020.
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74 Pictures
Luxury Mediterranean-styled estate with three bedrooms and an office in a Main House, plus a two-room Guest Casita, with golf and mountain views. With all-day solar exposure on the back patio, you can also directly see sunsets every night of the year, as well as the colors of the sunsets...
Listing ID 5843634
9966 E CHIRICAHUA Pass Scottsdale, AZ
4 beds, 5 baths, 5,562 sqft, $2,450,000
Listing Agent: Davis Driver
Courtesy of: RE/MAX Fine Properties
28 Pictures
Overlooking the 15th green of the Chiricahua golf course within Desert Mountain's premier Village of Saguaro Forest this exquisite home offers breathtaking golf course, lush Sonoran Desert and mountain views. The 100 year old reclaimed Oak plank floors, Venetian plaster walls, extensive use of...
Listing ID 5686341
41445 N 95TH Street Scottsdale, AZ
4 beds, 5 baths, 6,200 sqft, $2,495,000
Listing Agent: Daniel Wolski
Courtesy of: Russ Lyon Sotheby's International Realty
Courtesy of: Russ Lyon Sotheby's International Realty
Office Phone: (480) 488-2998
Copyright Arizona Regional Multiple Listing Service, Inc. All rights reserved. Information is deemed reliable but not guaranteed.
Updated: 19th June, 2019 6:30 PM.
9974 E HIDDEN VALLEY Road Scottsdale, AZ


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SET of 4 (Four) 2011 Buick Regal 19" OEM 245/40/19 245/40R19 Rims Wheels & Tires
Descriptiondescription ITEM # 00000000 Hello and thank you for clicking on our listing! Up for sale is a set of... read more
Out of stock
- The bolt pattern for this rim is 5X114.3
- Hollander Interchange Number: 4101
- Center caps ARE included.
- Wheels do contain a few scratches. Refer to pictures!
- Wheels spin perfectly on the balance machine.
- Wheels are not bent nor warped!
- Please refer to pictures for precise details
- Please check fitment with your local dealer before purchasing. Thank you!
- Valve stems ARE included
We try to respond to all e-mails within 15 minutes of being received during normal business hours.
frequently asked questions
- Can we pick up these items locally?
Sure, no problem! Please give us a call (954-228-3597) at least 24 hours in advance so we can have your item ready for you to pick up!
- What is OEM?
OEM stands for Original Equipment Manufacturer. This means that these wheels are the same ones that come stock straight from the dealership!
- Will I receive the exact item pictured?
Yes! We take pictures of every item we sell at multiple angles. We want to make sure you know exactly what you're getting before it arrives.
payment & shipping options
PayPal is the preferred payment method online. All of our items are shipped via UPS ground or USPS. After the item has been purchased, UPS or USPS will pick it up within 1 business day. All tracking numbers will be uploaded to eBay for easier tracking. Customers in Hawaii, Alaska and Puerto Rico are welcome, however, please message us before purchasing. We also welcome customers from Canada, however please note you will be responsible for all applicable duties and taxes, and incur a $30 s/h charge on top of the shipping (usually free) for your order.
All shipments over $100 will be sent with delivery confirmation, meaning a signature will be required on delivery. All shipments over $200 will be sent with adult delivery confirmation, meaning an adult over 21 years of age must sign for the package to be delivered. You may opt out of this requirement by sending us a message with your order, stating you'd prefer to 'opt out of delivery confirmation'.
Additional information
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In a possible lead-up to its plans on becoming an ATS, on March 26, 2018, San Francisco-based Coinbase announced it was adding support for ERC20 into all its trading platforms. ERC20 is the Ethereum technical standard that the majority of ICO tokens are based on. "This paves the way for supporting ERC20 assets across Coinbase products in the future, though we aren't announcing support for any specific assets or features at this time," Coinbase said in a blog post.
But… it's also possible that the institutional investors that are negative on bitcoin's prospects (and there's no shortage of those) may use the futures markets to put money behind their conviction. It's much easier to sell a futures contract with a lower-than-market price than it is to actually short bitcoin. These investors may well send signals to the actual bitcoin market that sends prices tumbling.
People are getting excited about Hempcoin (THC) because it's slowly but surely starting to re-surface again and receive some of the media's attention that it deserves. Even though a couple of competitors recently showed up (PotCoin and CannabisCoin) – Hempcoin is actually the oldest technologies and coins – not just in the industry – but in the crypto world altogether. Hempcoin was founded back in 2014 and its sole purpose is to act as a digital currency for the Agriculture/Farming industry and naturally – the Hemp/Marijuana field.
Always pay attention to Bitcoin. Most altcoins (every cryptocurrency except Bitcoin) are pegged more closely to Bitcoin than Asian currencies were to the USD during the Asian Financial Crisis. If Bitcoin price pump drastically, altcoins price can go down as people try to exit altcoins to ride the BTC profits; inversely, if Bitcoin prices dump drastically, altcoin prices can go down, too, as people exit altcoins to exchange back into fiat. The best times for altcoin growth appear when Bitcoin shows organic growth or decline, or remains stagnant in price.
BitMEX also has a weekly rebalancing for all their contracts, but currently their most popular product is the Daily 100x maximum leverage contract. This settles daily, but otherwise the rest of the contracts are handled on Friday, a little bit after OKCoin's 8:00AM UTC. See this website to get a countdown to the settlements on the different exchanges. The policies of exchanges are changing often so this information may be outdated by the time you read it (though we will try to keep it as up to date as possible).
If an investor gets the timing of the oscillations right, they can make money at every point along the way, going long when the market goes up and short when it drops. However, it is also difficult to come across any reliable strategy that has thus far been able to predict which events influence the price of bitcoin to which extent. The initial calling off of the Segwit2x fork is a good example of that. Shortly after the news broke, the market appeared to be divided into two camps – those who saw less value because they would not receive the equivalent amount of their holdings in the new currency ("dividends"), and those who saw the news as a consolidation of bitcoin's strength. The two camps pushed the price in opposite directions in a way that made it hard to predict which side would have the upper hand at which point in time.
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The truth is that bitcoin is the hottest trading market right now, hotter than stock trading, oil trading, gold trading and any other market at this point. The reason people believe this is going to continue to be a hot market is because blockchain technology which is what allows transactions to happen without a central exchange. Here is another strategy on how to draw trend lines with fractals.
The Funds may be eligible to elect alternative tax treatment with respect to PFIC shares. Under an election that currently is available in some circumstances, a Fund generally would be required to include in its gross income its share of the earnings of a PFIC on a current basis, regardless of whether distributions were received from the PFIC in a given year. If this election were made, the special rules, discussed above, relating to the taxation of excess distributions, would not apply. Another election would involve marking to market a Fund's PFIC shares at the end of each taxable year, with the result that unrealized gains would be treated and reported as though they were realized as ordinary income on the last day of the taxable year. Any mark-to-market losses and any loss from an actual disposition of PFIC shares would be deductible by the Fund as ordinary losses to the extent of any net mark-to-market gains included in income in prior years. Making either of these two elections may require a Fund to liquidate other investments (including when it is not advantageous to do so) to meet its distribution requirements, which also may accelerate the recognition of gain and affect the Fund's total return. Dividends paid by PFICs will not be eligible to be treated as "qualified dividend income." Because it is not always possible to identify a foreign corporation as a PFIC, the Fund may incur the tax and interest charges described above in some instances.
I under-performed the market in 2017. A crypto market that went up several hundred percent and having read 24 books on finance and trading throughout the process, these were my biggest takeaways. Remember, these are notes I wrote to myself, so they may not work for your trading style. This version was summarized exclusively for CryptoMarket360 – a full version is hyperlinked at the bottom.
• The bitcoin exchanges on which bitcoin trades are relatively new and, in most cases, largely unregulated and, therefore, may be more exposed to fraud and security breaches than established, regulated exchanges for other products. Over the past several years, a number of Bitcoin Exchanges have been closed due to fraud, failure, security breaches or governmental regulations. The nature of the assets held at Bitcoin Exchanges make them appealing targets for hackers and a number of Bitcoin Exchanges have been victims of cybercrimes. No Bitcoin Exchange is immune from these risks. Fraudulent activity can increase volatility and have an adverse effect on the price of bitcoin, the general acceptance of bitcoin as an investment or means of currency and could have a negative impact on the bitcoin futures contracts in which the Fund invests and the value of the Fund.
The Funds are not required to enter into forward currency contracts for hedging purposes. It is possible, under certain circumstances, that the Fund may have to limit its currency transactions to qualify as a "regulated investment company" ("RIC") under the Internal Revenue Code. The Funds do not intend to enter into a forward currency contract with a term of more than one year, or to engage in position hedging with respect to the currency of a particular country to more than the aggregate market value (at the time the hedging transaction is entered into) of their portfolio securities denominated in (or quoted in or currently convertible into or directly related through the use of forward currency contracts in conjunction with money market instruments to) that particular currency.
In the event an order is cancelled, the Authorized Participant will be responsible for reimbursing the Fund for all costs associated with cancelling the order, including costs for repositioning the portfolio, provided the Authorized Participant shall not be responsible for such costs if the order was cancelled for reasons outside the Authorized Participant's control or the Authorized Participant was not otherwise responsible or at fault for such cancellation. Upon written notice to the Distributor, such cancelled order may be resubmitted the following Business Day, with a newly constituted Portfolio Deposit or Fund Securities to reflect the next calculated NAV.
When you hear "margin", you may be thinking that you are borrowing money to trade bitcoin futures. This is not quite true. A key feature of these futures contracts is that the leverage comes from counterparties providing it to one another, not from the exchange lending funds and not from any bitcoin being lent from third parties. The contracts are simply like stocks with a market price, which represents the agreements between traders to take the opposing sides of where price of bitcoin will go, so no actual bitcoins are being exchanged per se, however the profit and loss between counterparties is very real!
Each of the Funds may enter into repurchase agreements with financial institutions in pursuit of its investment objectives, as "cover" for the investment techniques it employs, or for liquidity purposes. Under a repurchase agreement, a Fund purchases a debt security and simultaneously agrees to sell the security back to the seller at a mutually agreed-upon future price and date, normally one day or a few days later. The resale price is greater than the purchase price, reflecting an agreed-upon market interest rate during the purchaser's holding period. While the maturities of the underlying securities in repurchase transactions may be more than one year, the term of each repurchase agreement will always be less than one year. The Funds follow certain procedures designed to minimize the risks inherent in such agreements. These procedures include effecting repurchase transactions generally with major global financial institutions. The creditworthiness of each of the firms that is a party to a repurchase agreement with the Funds will be monitored by the Advisor. In addition, the value of the collateral underlying the repurchase agreement will always be at least equal to the repurchase price, including any accrued interest earned on the repurchase agreement. In the event of a default or bankruptcy by a selling financial institution, a Fund will seek to liquidate such collateral which could involve certain costs or delays and, to the extent that proceeds from any sale upon a default of the obligation to repurchase were less than the repurchase price, the Fund could suffer a loss. A Fund also may experience difficulties and incur certain costs in exercising its rights to the collateral and may lose the interest the Fund expected to receive under the repurchase agreement. Repurchase agreements usually are for short periods, such as one week or less, but may be longer. It is the current policy of the Funds not to invest in repurchase agreements that do not mature within seven days if any such investment, together with any other illiquid assets held by the Fund, amounts to more than 15% of the Fund's total net assets. The investments of each of the Funds in repurchase agreements at times may be substantial when, in the view of the Advisor, liquidity, investment, regulatory, or other considerations so warrant.
• Rolling Futures Contract Risk — The Fund will invest in and have exposure to bitcoin futures contracts and is subject to risks related to "rolling" such contracts. Rolling occurs when the Fund closes out of a futures contract as it nears its expiration and replaces it with a contract that has a later expiration. The Fund does not intend to hold futures contracts through expiration, but instead intends to "roll" its futures positions. When the market for these futures contracts is such that the prices are higher in the more distant delivery months than in the nearer delivery months, the sale during the course of the rolling process of the more nearby contract would take place at a price that is lower than the price of the more distant contract. This pattern of higher futures contract prices for longer expiration contracts is often referred to as "contango." Alternatively, when the market for futures contracts is such that the prices are higher in the nearer months than in the more distant months, the sale during the course of the rolling process of the more nearby contract would take place at a price that is higher than the price of the more distant contract. This pattern of higher futures prices for shorter expiration futures contracts is referred to as "backwardation." Extended periods of contango could cause significant losses for the Fund. The Advisor will utilize active management techniques to seek to mitigate the negative impact or, in certain cases, benefit from the contango or backwardation present in the various futures contract markets, but there can be no guarantee that it will be successful in doing so.
8. Except for the Managed Futures Strategy ETF, the Crude Oil Strategy ETF and the CDS Short North American HY Credit ETF, concentrate (i.e., hold more than 25% of its assets in the stocks of a single industry or group of industries) its investments in issuers of one or more particular industries, except that a Fund will concentrate to approximately the same extent that its index concentrates in the stocks of such particular industry or industries. For purposes of this limitation, securities of the U.S. government (including its agencies and instrumentalities) and tax-free securities of state or municipal governments and their political subdivisions (and repurchase agreements collateralized by government securities) are not considered to be issued by members of any industry. For purposes of determining whether the Funds are concentrated in an industry or group of industries, each Fund may concentrate its investment in the securities of companies engaged in a single industry or group of industries to approximately the same extent as its benchmark and in accordance with its investment objective and policies as disclosed in the Prospectus and SAI.
The CFE has determined that if the Bitcoin Network is forked and a new blockchain is created, the form of bitcoin on which CFE bitcoin futures contracts and their final settlement values will be based is the form of bitcoin in U.S. dollars traded on the Gemini Exchange. The Gemini Exchange has indicated that it will support the network that has the greatest cumulative computational difficulty for the forty-eight hour period following a given fork. If the Gemini Exchange is unable to make a conclusive determination about which network has the greatest cumulative computational difficulty after forty-eight hours, or Gemini determines in good faith that this is not a reasonable criterion upon which to make a determination, the Gemini Exchange will support the network which it deems in good faith is most likely to be supported by a greater number of users and miners. The Gemini Exchange has indicated it will consult with CFE in the event of a fork. If the Gemini Exchange were to offer trading in multiple forms of bitcoin in U.S. dollars, the CFE would designate the form of bitcoin traded on the Gemini Exchange that would serve as the basis for CFE bitcoin futures contracts and their final settlement values.
It's important to realise that you need to do your own research and come up with your own strategy for cryptocurrency trading. If you are short on time and want to play it safe; the easiest cause of action is to simply diversify into several different coins and then wait a year or more. However, if you want to maximise profits you should learn how to swing trade cryptocurrency.
The Advisor and its affiliated persons may come into possession from time to time of material nonpublic and other confidential information about companies which, if disclosed, might affect an investor's decision to buy, sell, or hold a security. Under applicable law, the Advisor and its affiliated persons would be prohibited from improperly disclosing or using this information for their personal benefit or for the benefit of any person, regardless of whether the person is a client of the Advisor. Accordingly, should the Advisor or any affiliated person come into possession of material nonpublic or other confidential information with respect to any company, the Advisor and its affiliated persons will have no responsibility or liability for failing to disclose the information to clients as a result of following its policies and procedures designed to comply with applicable law. However, each Matching Fund is managed using what is commonly referred to as an index strategy in an attempt to simulate either the daily movement or a multiple, the inverse or an inverse multiple of the daily movement of its index, and the use of such index strategies may reduce conflicts of interest compared to funds using non-index investment strategies.
S&P 500 Dividend Aristocrats ETF; S&P MidCap 400 Dividend Aristocrats ETF; Russell 2000 Dividend Growers ETF; Equities for Rising Rates ETF; Morningstar Alternatives Solution ETF; S&P 500 Ex-Energy ETF, S&P 500 Ex-Financials ETF, S&P 500 Ex-Health Care ETF, S&P 500 Ex-Technology ETF 4:00 p.m. (3:30 p.m. if in cash) in order to receive that day's closing NAV per Share
Bitcoin is maintained on the decentralized, open source protocol of the peer-to-peer bitcoin computer network (the "Bitcoin Network"). No single entity owns or operates the Bitcoin Network. The infrastructure of the Bitcoin Network is collectively maintained by a decentralized user base. The Bitcoin Network is accessed through software, and software governs bitcoin's creation, movement, and ownership. The value of bitcoin is determined in great part by the supply of (which is limited), and demand for, bitcoin in the global exchange markets for the trading of bitcoin (individually, "Bitcoin Exchanges" and collectively, the "Bitcoin Exchange Market"), market expectations for the adoption of bitcoin and the volume of private user-to-user transactions.
Some Centra investors have their doubts, and a plaintiffs' law firm has brought a class action complaint against Centra demanding the investors' money back. The complaint is fun -- Centra had a "Blog/Media Bounty" program to "Reward Experienced Writers who write quality Reviews, Articles About the Centra Project and the ICO crowdsale" -- but not that fun, because the plaintiffs' lawyers don't actually need to prove that Centra was a scam. Their job is much easier: All they need to do is prove that the tokens Centra sold in its initial coin offering were securities. If they were securities, they were sold illegally: They were offered publicly without being registered with the Securities and Exchange Commission, or being exempt from registration. And one remedy for the illegal sale of securities is that the buyers can demand their money back -- whether or not Centra is legitimate, whether or not it is actually using the money to build a cryptocurrency debit card, whether or not it made any misleading statements in the ICO.
Altcoins is the general term associated with the cryptocurrencies launched after Bitcoin's success. At first, these were mere copies mimicking the original Bitcoin. Today, there are over 1,000 of these, and the list just keeps growing. Most crypto coins are launched following an ICO (Initial Coin Offering – a form of crowdfunding) in which the developers raise cash by offering a limited number of initial coins to finance technological development. So far, besides the list below, we can find names, such as Namecoin, Peercoin, Bytecoin, Deutsche eMark, Novacoin, Cryptogenic Bullion, Quark, DarkCoin and Mangocoinz (for smartphones).
In addition, the Advisor, any of its affiliates or employees and the Funds have a policy not to enter into any agreement or other understanding—whether written or oral—under which brokerage transactions or remuneration are directed to a broker to pay for distribution of a Fund's shares. The table below sets forth the brokerage commissions paid by each Fund for the period noted for each Fund. Because each of the New Funds was not operational at the end of the Trust's last fiscal year, information on brokerage commissions paid by the Fund is not included in this SAI.
Always learn from your mistakes. Never accept a total loss. Always evaluate the situation and try to figure out why it happened. Take that experience as an asset for your next move, which will be better because you are know more now than you knew before. We all start off as amateurs, and we have all lost money throughout out trading experience. In his first month of trading, Miles went from $1,000 to $300. I've lost a lot by selling at losses inspired by fear. No one is perfect, no one wins every single trade. Don't let the losses discourage you, because the reality is they're making you better trader if you choose to learn from them.
Certain of the Funds are likely to obtain substantial exposure to the price movements of bitcoin by holding bitcoin linked exchange-traded notes ("ETNs") that provide exposure to the price of bitcoin. ETNs are unsecured, unsubordinated debt securities of an issuer that are listed and traded on a U.S. stock exchange. An ETN's returns are generally linked to the performance of a particular market benchmark or strategy minus applicable fees. ETNs do not provide principal protection and may or may not make periodic coupon payments. ETNs are subject to credit risk, which is the risk that the issuer cannot pay interest or repay principal when it is due. Additionally, the value of an ETN may be influenced by time to maturity, level of supply and demand, volatility and lack of liquidity in the underlying market (e.g., the commodities market), changes in interest rates or the issuer's credit rating, and other economic, legal, political or geographic events. The value of an investment in an ETN may be impacted by fees associated with the ETN. Structural aspects of the ETNs may impact their market value. Trading by affiliates of an ETN sponsor may create conflicts of interest. The issuer of an ETN may be unable to meet its obligations. The potential impact of Bitcoin Network forks on the value of a bitcoin ETN is unclear. ETNs issued by special purpose vehicles may include greater risk. ETNs are subject to risks associated with the underlying asset.
Bitcoin isn't just an unknown commodity: it will always be an unknown commodity. Bitcoin doesn't have the fundamentals that investors typically use to analyze an asset. Most stocks or bonds can be analyzed based on some trait of the instrument. Stocks have P/E ratios and dividends, for example, while bonds have return percentages. Bitcoin has no fundamentals that can be easily measured.
• The bitcoin exchanges on which bitcoin trades are relatively new and, in most cases, largely unregulated and, therefore, may be more exposed to volatility, fraud and security breaches than established, regulated exchanges for other products. Over the past several years, a number of Bitcoin Exchanges have been closed due to fraud, failure, security breaches or governmental regulations. The nature of the assets held at Bitcoin Exchanges make them appealing targets for hackers and a number of Bitcoin Exchanges have been victims of cybercrimes. No Bitcoin Exchange is immune from these risks. Fraudulent activity can increase volatility and have an adverse effect on the price of bitcoin, the general acceptance of bitcoin as an investment or means of currency and could have a negative impact on the bitcoin futures contracts in which the Fund invests and the value of the Fund.
Each Fund may buy and write (sell) options for the purpose of realizing its investment objective. By buying a call option, a Fund has the right, in return for a premium paid during the term of the option, to buy the asset underlying the option at the exercise price. By writing a call option a Fund becomes obligated during the term of the option to sell the asset underlying the option at the exercise price if the option is exercised. By buying a put option, a Fund has the right, in return for a premium paid during the term of the option, to sell the asset underlying the option at the exercise price. By writing a put option, a Fund becomes obligated during the term of
The first decentralized cryptocurrency, bitcoin, was created in 2009 by pseudonymous developer Satoshi Nakamoto. It used SHA-256, a cryptographic hash function, as its proof-of-work scheme.[16][17] In April 2011, Namecoin was created as an attempt at forming a decentralized DNS, which would make internet censorship very difficult. Soon after, in October 2011, Litecoin was released. It was the first successful cryptocurrency to use scrypt as its hash function instead of SHA-256. Another notable cryptocurrency, Peercoin was the first to use a proof-of-work/proof-of-stake hybrid.[18] IOTA was the first cryptocurrency not based on a blockchain, and instead uses the Tangle.[19][20] Many other cryptocurrencies have been created though few have been successful, as they have brought little in the way of technical innovation.[21] On 6 August 2014, the UK announced its Treasury had been commissioned to do a study of cryptocurrencies, and what role, if any, they can play in the UK economy. The study was also to report on whether regulation should be considered.[22]
Two members of the Silk Road Task Force—a multi-agency federal task force that carried out the U.S. investigation of Silk Road—seized bitcoins for their own use in the course of the investigation.[86] DEA agent Carl Mark Force IV, who attempted to extort Silk Road founder Ross Ulbricht ("Dread Pirate Roberts"), pleaded guilty to money laundering, obstruction of justice, and extortion under color of official right, and was sentenced to 6.5 years in federal prison.[86] U.S. Secret Service agent Shaun Bridges pleaded guilty to crimes relating to his diversion of $800,000 worth of bitcoins to his personal account during the investigation, and also separately pleaded guilty to money laundering in connection with another cryptocurrency theft; he was sentenced to nearly eight years in federal prison.[87]
* The "Fund Complex" consists of all operational registered investment companies under the 1940 Act that are advised by ProShare Advisors LLC and any registered investment companies that have an investment adviser that is an affiliated person of ProShare Advisors LLC. Investment companies that are non-operational (and therefore, not publicly offered) as of the date of this SAI are excluded from these figures.
Bitcoin isn't just an unknown commodity: it will always be an unknown commodity. Bitcoin doesn't have the fundamentals that investors typically use to analyze an asset. Most stocks or bonds can be analyzed based on some trait of the instrument. Stocks have P/E ratios and dividends, for example, while bonds have return percentages. Bitcoin has no fundamentals that can be easily measured.
Swap agreements are generally traded in OTC markets and have only recently become subject to regulation by the CFTC. CFTC rules, however, do not cover all types of swap agreements. Investors, therefore, may not receive the protection of CFTC regulation or the statutory scheme of the Commodity Exchange Act in connection with a Fund's swap agreements. The lack of regulation in these markets could expose investors to significant losses under certain circumstances, including in the event of trading abuses or financial failure by participants. Unlike in futures contracts, the counterparty to uncleared OTC swap agreements is generally a single bank or other financial institution, rather than a clearing organization backed by a group of financial institutions. As a result, the Fund is subject to increased counterparty risk with respect to the amount it expects to receive from counterparties to
Coinbase is not alone in making moves toward becoming an ATS. Recently, mobile payment app company Circle acquired Poloniex, another U.S.-based exchange, with plans to "clean up" the exchange. A slide that was initially leaked from a Circle presentation stated, "Circle has briefed the SEC on the transaction and indicated that upon closing that we will begin the process of registering the new entity with the SEC and FINRA as a Broker/Dealer and in turn as a licensed ATS…"
In this guide we don't want to deal with social loss risk, so use the FCA-regulated, London-based bitcoin derivatives exchange CryptoFacilities. They offer contracts with 2% margin requirement (50x leverage) as well as a 15% margin requirement (6.5x leverage). They are 100% bitcoin based, but they don't accept US customers. Sign up here to get started:


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joaoluisbrito - in reply to Jurikles
Prusas don't have high precision, but an E3D steel nozzle might. And if you're talking about the MK3 even if it's brand new it might not be adjusted correctly, and even then I don't know what your settings are for perimeters, infill, etc.
It would have been highly irresponsible of me not to test that, don't you think? I use it every other day and have shipped half a hundred kits with it included.
If you need further reference there should be some build pictures in the make section, good luck!


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Structural changes of bacterial nanocellulose pellicles induced by genetic modification of Komagataeibacter hansenii ATCC 23769
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Bacterial nanocellulose (BNC) synthesized by Komagataeibacter hansenii is a polymer that recently gained an attention of tissue engineers, since its features make it a suitable material for scaffolds production. Nevertheless, it is still necessary to modify BNC to improve its properties in order to make it more suitable for biomedical use. One approach to address this issue is to genetically engineer K. hansenii cells towards synthesis of BNC with modified features. One of possible ways to achieve that is to influence the bacterial movement or cell morphology. In this paper, we described for the first time, K. hansenii ATCC 23769 motA+ and motB+ overexpression mutants, which displayed elongated cell phenotype, increased motility, and productivity. Moreover, the mutant cells produced thicker ribbons of cellulose arranged in looser network when compared to the wild-type strain. In this paper, we present a novel development in obtaining BNC membranes with improved properties using genetic engineering tools.
KeywordsKomagataeibacter Motility Cell division Bacterial nanocellulose
Bacterial nanocellulose (BNC) synthesized by bacteria from Komagataeibacter genus is one of the most intensively studied biopolymers due to its unique properties such as high mechanical strength, chemical stability, hydrophilicity, crystallinity, and biocompatibility. BNC is the most abundant renewable organic material produced in the biosphere (Gallegos et al. 2016; Ryngajłło et al. 2018). Because of these properties, BNC has a wide range of potential biomedical applications, including wound dressings, medical implants, drug delivery, vascular grafts, and scaffolds for tissue engineering (Svensson et al. 2005; de Oliveira Barud et al. 2016; Picheth et al. 2017). In recent years, many studies have been carried to develop BNC scaffolds for biomedical use (Wu et al. 2016; Cielecka et al. 2018; Jacek et al. 2018). Nevertheless, the main problem for tissue engineering is the limited porosity of the BNC that makes the ingrowth of the eukaryotic cells is impossible.
The properties of a BNC membrane closely depend on its morphology. One of the recent approaches aiming to change the morphology of BNC involves the regulation of bacterial cell movement, by using different types of electromagnetic fields (Sano et al. 2010; Baah-Dwomoh et al. 2015; Fijałkowski et al. 2015). Use of electrokinetic force to control bacterial motion of Komagataeibacter xylinus allowed for the differentiation of orientation and organization of bacterial nanocellulose fibers (Baah-Dwomoh et al. 2015). Furthermore, the intrinsic cell motility was shown to be important for pellicle formation in many species (Hölscher et al. 2015). Modification of bacterial nanocellulose structure has mainly been achieved by chemical or mechanical modifications of the cellulose matrix or via changing culturing conditions, while there are no successful results in changing the BNC architecture using genetic engineering tools (Chanliaud and Gidley 1999; Luo et al. 2008; Lee et al. 2014). Nevertheless, genetic engineering remains a promising approach to enable a fine control over nanocellulose synthesis towards production of diverse BNC-based biomaterials.
The studies, which involved genetic modifications of the Komagataeibacter strains, focused mainly on the genes affecting efficiency of cellulose biosynthesis (Jacek et al. 2019). These genetic modifications included the expression of a foreign gene, the gene disruption, or gene overexpression. One of the most impressive examples of these genetic modifications is the K. xylinus mutant which uses lactose from whey as a carbon source, producing 28-fold more cellulose from lactose than the wild-type strain (Battad-Bernardo et al. 2004). Another successful application of recombinant strains for bacterial nanocellulose production from wastes is a mutant of K. xylinus with enhanced ability to produce BNC from a hydrolysate of a potato pulp (Zhong et al. 2013).
Bacterial motility is a complex phenomenon playing diverse roles in biofilm formation. Most of the motile bacteria move with flagella, which is an essential multifunctional organellum involved in the initial stages of biofilm formation (Bogino et al. 2013). Bacterial cells can move through the liquid environment or translocate over the surface of solids, using various mechanisms. Bacterial chemotaxis is a kind of movement by which microorganisms efficiently and rapidly react to the changes in the chemical composition of their surroundings, approach chemically beneficial environments, and avoiding unfavorable ones (Tamar et al. 2016). The core components of the chemotaxis mechanism are universal for all motility systems and highly conserved among bacteria; however, some, often complex, variations have been identified (Szurmant and Ordal 2004). According to literature data, strains of the Komagataeibacter genus exhibit chemotactic movement (Basu et al. 2018).
In Gram-negative bacteria, cellular motility and cell division are described as one of the essential factors for biofilm formation (Pratt and Kolter 1998; Merritt et al. 2007; Niba et al. 2007). Many reports suggest that proteins MotA/MotB, ExbB/ExbD, and TolQ/TolR which form transmembrane proton channels are important in biofilm formation (Abbas et al. 2007; Søgaard-Andersen 2011; Guttenplan and Kearns 2013; Islam and Mignot 2015; Santos et al. 2015). MotA and MotB are membrane-bound proteins, which form a heterohexamer MotA4/MotB2 and are indispensable for bacterial motility. MotA/MotB works as the stator of the bacterial flagellar motor to couple proton influx with torque generation (Morimoto et al. 2010). It has been reported that Gram-negative bacterium Myxococcus xanthus that glides over surfaces without the aid of flagella has AglS, AglR, and AglQ, which are MotA/MotB homologs (Søgaard-Andersen 2011). Flagellar motor proteins MotA/MotB and gliding proteins AglQ-AglL/AglS share homologies with the components of the Ton and Tol systems (ExbB/ExbD and TolQ/TolR)—systems that also form proton channels and utilize a proton-motive force (PMF). However, these proteins are not involved in motility but in the active transport of substrates across the outer membrane (Cascales et al. 2001; Sun et al. 2011; Gray et al. 2015). ExbB and ExbD proteins form a complex, which is a component of the energy transducing Ton system, and which functions to harness the energy of the PMF at the cytoplasmic membrane to support active transport of iron-siderophore complexes and vitamin B12 across the outer membrane in Gram-negative bacteria (Andrews et al. 2003; Postle and Kadner 2003; Noinaj et al. 2010). TolQ and TolR together with TolA form a complex in the cytoplasmic membrane that plays an essential role in maintaining outer membrane integrity and cell division (Yeh et al. 2010; Teleha et al. 2013).
Currently, there is no information explaining potential role of MotA/ExbB/TolQ and MotB/ExbD/TolR in Komagataeibacter genus. Moreover, little is known about the role of motility and cell division on bacterial nanocellulose biosynthesis and properties. In 1976, Brown et al. first characterized the movement of Komagataeibacter hansenii cells in relation to the BNC biosynthesis (Brown et al. 1976). They observed that cell motility rate correlates with the actual biosynthesis and extrusion of bacterial nanocellulose microfibrils. Consequently, they hypothesized that cell movement is caused by the propulsion force of BNC microfibrils secretion by the bacterium (Brown et al. 1976).
Herein, for the first time, K. hansenii ATCC 23769 was modified by using genetic engineering tools, in order to obtain the loosened structure of BNC membranes. In this study, we created and investigated two overexpression mutants; motA+ and motB+. Moreover, to get more information about the functions of the MotA and MotB proteins, we investigated their subcellular location. Our results expanded the understanding the role of K. hansenii motility and cell morphology in BNC structure architecture.
Materials and methods
Bacterial cell culture conditions and growth media
Komagataeibacter hansenii ATCC 23769 was cultivated in SH medium (Schramm and Hestrin medium) at 30 °C. The SH medium for K. hansenii cultivation was used in two forms: liquid—for agitated and stationary cultures and with 2% (w/v) agar (Difco, USA)—for plate cultures. One-liter culture medium contained 20.0 g glucose (POCH, Poland), 5.0 g yeast extract (BTL, Poland), 5.0 g bacterial peptone (BTL, Poland), 2.7 g sodium phosphate dibasic (Chempur, Poland), 1.15 g citric acid (Chempur, Poland), and 0.5 g magnesium sulfate (Chempur, Poland). The pH was adjusted to 5.7 with 80% acetic acid (Chempur, Poland) before sterilization.
Plasmid construction and transformation
For motA+ and motB+ mutants, pTI99 plasmid (Hokkaido University) was used (Table 2). Initially, motA and motB were amplified from the genomic DNA of K. hansenii ATCC 23769 with primers pTI-motA_for, pTI-motA_rev primers and pTI-motB_for, pTI-motB_rev primers, respectively (Table S1). The amplified DNA fragments and pTI99 plasmid were treated BamHI and HindIII and ligated. The resulting constructs were transformed using the heat shock method into E. coli TOP10F' cells (Froger and Hall 2008). Then, K. hansenii ATCC 23769 was transformed with control pTI99, pTI99-motA, pTI99-motB by electroporation, in accordance with the method used in previous studies (Jacek et al. 2018). The cells were incubated at 30 °C for 3 h, diluted, and transferred to SH/agar plates containing ampicillin (200 μg/mL) for screening of recombinants (Fig. S4).
MotA-GFP and MotB-GFP fusion protein generation and expression
To construct plasmids carrying the motA-gfp and motB-gfp fusion genes, the motA and motB genes were amplified by PCR using the K. hansenii ATCC 23769 genomic DNA as a template and the motA_gfp_F, motA_gfp_R primers and motB_gfp_F, motB_gfp_R primers, respectively. The amplified DNA fragments were treated with EcoRI and BamHI (for motA), KpnI, and BamHI (for motB), and then ligated with a pTIE plasmid digested with the same restriction enzymes. All plasmids, including the control vector pTIE-GFP, were introduced into K. hansenii ATCC 23769 by electroporation (Hall et al. 1992).
Bacterial growth curves
To monitor cell growth and survival, viable cell counts were determined by serial dilution. A single colony was inoculated into a test tube containing 5 mL of liquid SH medium with/without ampicillin and grown at 30 °C in static conditions. Every 24 h, 1.5% of cellulase (Sigma-Aldrich, Denmark) was added and incubated for 3 h and periodically stirred. Then, serial dilutions were made in 0.9% NaCl and spread on SH with agar plates with/without ampicillin. The experiments were carried out in triplicate.
K. hansenii ATCC 23769 cell length determination
Liquid cultures in SH medium with/without 200 μg/mL ampicillin of four tested K. hansenii ATCC 23769 strain variants (wild-type, control, motA+, and motB+ mutants) were used for cell length determination. During 6 days of incubation, 10 μL portions of cells suspension were fixed to the microscope slide and stained with crystal violet, every 24 h. From each slide, 20 pictures were taken, with use of light microscope Olympus BX 51 (Olympus, Japan) under 400 times magnification. Cells lengths were measured with Makroaufmassprogram software of at least 10 cells randomly chosen from each picture. Distribution of obtained values comprises at least 300 measurements of individual cells for each strain.
E. coli TOP10F' cell length determination
Liquid cultures in LB medium supplemented with/without 100 μg/mL ampicillin of four tested E. coli TOP10F' strain variants (wild-type, control, motA+, and motB+ mutants) were used for cell morphology determination. After 12 h of incubation, 10 μL portions of cells suspension were fixed to the microscope slide and stained with crystal violet and observed in light microscope Olympus BX 51 (Olympus, Japan) under 1000 times magnification. Representative pictures were taken for each strain.
Swarming motility assay
Pre-cultures of three tested K. hansenii ATCC 23769 strain variants (wild-type, control and mutant) were diluted to reach optical density of 0.1 at 600 nm. Next, 2 μL portions from each equilibrated culture were inoculated onto five 0.3% agar SH plates containing 2% (v/v) cellulase (from Trichoderma reesei ATCC 26921, Sigma-Aldrich, Germany). Spots of mutant culture were accompanied by spots of wild-type and control strains on the same plates. After 3 days of incubation the diameters of colonies were measured. Measurement was repeated at 24 h interval two more times. Experiments were performed in five replicates and the results are given as means.
Bacterial nanocellulose biosynthesis
Liquid culture for inoculum was prepared by transferring a single bacterial colony grown on SH agar seed medium into 5 mL of a liquid SH medium, both with or without antibiotics, which was then incubated at 30 °C for 4 days. Five percent of the inoculum culture was introduced into a 250-mL Erlenmeyer flask containing 50 mL of SH medium. The culture was then incubated statically at 30 °C for 7 days. After incubation, membranes were picked and soaked in 2% NaOH solution for one night, and, next in 1.5% acetic acid solution for 4 h. Further on, the membranes were washed with distilled water until neutral pH was reached. After purification, the cellulose membranes were dried at 55 °C in an oven to constant weight. The productivity of BNC was quantified, based on the dry weight of the BNC pellicles, collected at the end of the 7-day cultivation and expressed in grams per liter. Experiments were performed in quadruplicate and the results are given as means.
Scanning electron microscopy
Morphology and microstructural features of the membranes were investigated, using the scanning electron microscope (SEM) FEI QUANTA 250 FEG, operating at 2 kV, magnification ×5000, ×20,000, and ×40,000. Prior to the analysis, BNC membranes were purified according to the methodology described by Fang et al. (Fang et al. 2015). The pellicles were freeze-dried in Christ Alpha model 1-4 LSC plus. Before the SEM analysis, the samples were coated with gold/palladium. After the observation of three biological replicates, representative micrographs were taken in triplicates for each magnification. The diameters of fibers and pores were determined with the Makroaufmassprogram, with 50 fibers and pores from the SEM micrographs.
Sample preparation and fluorescence microscopy
The cells were grown at 30 °C for 48 h in SH medium with the appropriate antibiotics. A small portion of a culture was dropped on the slide glass and cover glass was placed. Cells were observed under a Nikon Eclipse TS100 microscope (Nikon Instruments, Tokyo, Japan). The microscope settings were as follows: excitation at 340–380 nm, emission at 435–485 nm.
RNA isolation and quantitative reverse transcription PCR analysis
Statistical analysis
All data were expressed as mean ± standard deviation (SD). The statistical analysis was calculated using Student's t test, and statistical significance was set at *p < 0.05 and **p < 0.001 and extreme significance was set at ***p < 0.0001.
Bioinformatical analysis of the target genes
BLASTp search with the K. hansenii ATCC 23769 MotA and MotB sequences against the genomes from Alphaproteobacteria class
Accession no.
Genome annotation
Coverage (%)
Identity (%)
MotA/TolQ/ExbB proton channel family protein
Gluconacetobacter diazotrophicus PA1 5
Flagellar motor protein MotA
Gluconobacter japonicus NBRC 103476
Flagellar motor protein MotA
Acetobacter tropicalis NBRC 101654
OmpA/MotB domain protein
Rhizobium gallicum IE4872
Chemotaxis MotB protein
Rhodospirillum centenum SW
Flagellar motor rotation protein MotB
Magnetospirillum magnetotacticum MS-1
Knowing different roles of the paralogous proteins, we searched for sequence similarity and predicted transmembrane topology. Results of transmembrane domains prediction suggested 4 TM domains with large cytoplasmic domain between 2nd and 3rd TMs in MotA candidate (Fig. S2 a), which would make it more similar to MotA not ExbB/TolQ proteins (they possess 3 TM domains). Overall architecture of MotB (one TM domain, OmpA-like domain at C terminus, mostly periplasmic) again shows closer similarity to the stator protein then to the ExbD/TolR paralogs (Fig. S2 b).
Although, bacteria of the Komagataeibacter genus do not have flagellum, sequence-based predictions show that the investigated proteins probably form a proton pump. However, the protein partner of these proteins, to which energy is transformed, is to be elucidated experimentally and, moreover, it cannot be a FliH-like flagellum protein as it is absent in Komagataeibacter.
K. hansenii ATCC 23769 motA+ and motB+ mutants' phenotype—cell size and swarming motility
Based on the putative roles of MotA/ ExbB/TolQ and MotB/ExbD/TolR homologs (Hossain et al. 2005; Belas 2013; Teleha et al. 2013), we decided to limit K. hansenii ATCC 23769 mutant strains phenotype studies to cell lengths, motility, cellulose productivity, and pellicle microarchitecture.
Bacterial strains and plasmids used in this study
Relevant characteristics
Eschericha coli
F'lacIqTn10(TetR) mcrAΔ(mrr-hsdRMS-mcrBC) ϕ80lacZΔM15 ΔlacX74 deoR recA1 araD139 Δ(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG
Invitrogen, USA
Komagataeibacter hansenii
Wild-type strain
Hokkaido University, Japan
Creation pTI99-motA and pTI99-motB vectors
Hokkaido University, Japan
Creation MotA-GFP, MotB-GFP vectors
Hokkaido University, Japan
K. hansenii motA+ overexpression mutant
This study
K. hansenii motB+ overexpression mutant
This study
K. hansenii MotA-GFP overexpression mutant
This study
K. hansenii MotB-GFP overexpression mutant
This study
We observed that overexpression of motA and motB genes caused an elongate cell phenotype under normal growth conditions. In some microorganisms, swarming motility is associated with cell elongation (Fraser and Hughes 1999). Furthermore, it has been shown that some bacteria become elongated during swarming (Little et al. 2018). In Gram-negative bacteria, the Tol-Pal complex is widely conserved and may have various physiological functions such as maintenance of cell envelope integrity, interaction with outer membrane porins, expression of lipopolysaccharide surface antigens and virulence factors, facilitation of infection by filamentous DNA phage, and improvement resistance to detergents (Cascales et al. 2000; Gaspar et al. 2000; Cascales et al. 2001). Filamentous phenotype implies a defect in cell division such that cells continue to grow in the absence of septation. Cells that seem to be elongated can also form by the failure of cell separation following successful division, consequently resulting in cells that are connected end-to-end in long chains (Kearns and Losick 2005; Patrick and Kearns 2008). Teleha et al. reported that tolQ and tolR E. coli overexpression mutants' cells display an elongated phenotype, suggesting role of these genes in cell division (Teleha et al. 2013). Therefore, we decided to investigate the effect of motA and motB overexpression on E. coli TOP10F' cell morphology. No differences were observed in cell morphology between the motA+, motB+ mutants and the control with pTI99 and the wild-type strain (Fig. S3).
In order to test if cells with increased motility could form cellulose pellicles with loosened network, we performed motility assay for K. hansenii ATCC 23769 WT, control, and motA+ motB+ mutants. Motility assay showed that K. hansenii ATCC 23769 is able to spread over moist surfaces in a process called "swarming." Observation of the bacterial colonies spreading on the soft agar is commonly interpreted as presence of swarming motility in the tested microorganisms.
Swarming typically involves collective movement of a dense population of bacteria cells. It is a common manner of motility that allows bacteria to rapidly colonize a surface, leading to the biofilm formation (Donlan 2002). In some microorganisms, swarming motility is associated with cell elongation (Fraser and Hughes 1999). Furthermore, it has been shown that some bacteria become elongated during swarming (Little et al. 2018).
Characterization of BNC membranes produced by mutant strains
High porosity is significant feature of 3-D biopolymer scaffolds used in tissue engineering because of its essential effects on cell growth. It is generally assumed that pore size of a scaffold may affect the cell adhesion and proliferation (Bäckdahl et al. 2006). Material obtained from mutants has a great application potential as a scaffold in tissue engineering due to the fact that it has significantly enlarged pores when compared to wild-type membranes and there are possibilities to control the pores sizes.
Impact of bacterial motility and cell morphology on bacterial nanocellulose production
Before comparing the yields of bacterial nanocellulose, we examined the growth rates of motA+ and motB+ mutants over a period of 6 days to investigate the effect of motA and motB genes overexpression on the cell growth. We decided to monitor the growth rate of each culture by its colony-forming unit (CFU) since, under the static growth condition, cells producing cellulose are fixed in the cellulose membrane formed at the surface of the medium, which makes monitoring cell growth by optical density imprecise.
In order to investigate the efficiency in the production of BNC by mutants, wild type, pTI99, and motA+, motB+ were grown for 3 days in 5 mL SH medium under static conditions; for overexpression mutants, ampicillin (200 μg/mL) was added, and then 5% of inoculum was transferred into 50 mL SH medium. After growth under static conditions for 7 days, BNC membranes were harvested and purified (Fig. 5b) and their dry weights were determined. The results shown in Fig. 5c, d revealed that the mutants produced more BNC, when compared with the wild-type. MotA+ and motB+ mutants produced 6% and 20% more cellulose respectively, in contrast to WT and pTI99. These results suggest that overexpression of motA and motB genes not only affected the morphology, but also the yield, of bacterial nanocellulose production.
There is no information explaining the impact of motility and cell length on BNC biosynthesis by K. hansenii ATCC 23769. Our results indicate that motA, motB genes may have an impact not only on the BNC structure but also on the cellulose yield. In the latest work, Basu et al. also noticed that there is a correlation between cell motility and cellulose yields (Basu et al. 2018). Several studies suggest that motility and cell size are involved in biofilm formation in many different microbes (O'Toole and Kolter 1998; Pratt and Kolter 1998; Choy et al. 2004; Merritt et al. 2007; Niba et al. 2007). It has been demonstrated that deletion mutant of motA was non-motile and displayed reduced ability to form biofilm (Hossain and Tsuyumu 2006). Further, it was reported that the tonB1 gene of Pseudomonas aeruginosa is not only required for transport of molecules involved in biofilm formation, but also in quorum sensing (Abbas et al. 2007).
Subcellular localization of MotA and MotB proteins
Relative gene expression
Herein, for the first time, a new approach in modifying the structure of bacterial nanocellulose using genetic engineering tools is presented. We chose two genes encoding proteins which are homologs of MotA/ExbB/TolQ and MotB/ExbD/TolR proteins. Homologs of selected proteins are specific for Gram-negative bacteria and are involved in diverse activities. Two motility proteins MotA and MotB are essential for rotating the proton-driven flagellar motor (Kojima and Blair 2004). Furthermore, ExbB and ExbD participates in active transport mechanisms in TonB system, responsible mainly for macromolecules and complexes with ferric ions uptake but as well saccharides and DNA may be transported by these systems (Braun and Braun 2002; Maki-Yonekura et al. 2018). Third role of such systems was most frequently observed for TolA/TolQ homologs with its involvement in cell division regulation but by yet-unknown mechanism (Germon et al. 2001; Gerding et al. 2007).
Very high level of motA and motB sequence conservation between Komagataeibacter strains suggests involvement of the tested genes in yet-unknown but important mechanism, presumably connected with single cellulose chain secretion and further assembly in bacterial nanocellulose ribbon. Although bioinformatic studies have proven that the tested genes are more closely related to motA and motB homologs, we found them to be involved not only with bacterial movement but also in diverse activities, such as cell division, productivity of cellulose, and creation of 3-D cellulose network with larger pores and thicker fibers.
Our results suggested that cell size elongation influenced cellulose ribbons diameter and spatial arrangement as well. MotA+ and motB+ mutants produced membranes with loosened structure and thicker fibers compared to the wild-type strain. Therefore, this result gives a new line of evidence that cellulose fiber assembly occurring outside the K. hansenii ATCC 23769 cells is correlated with cell size. Cell elongation lead to fiber thickening. A possible explanation of this phenomenon could be in this manner. Bacteria grow by cell division. Cellulose networks are supposed to form together with cell division (Watanabett and Yamanaka 1995). It is considered that, in every cell division, a ribbon is divided into two in the horizontal direction; thus, this action is reiterated with every cell division and with every separation of cells, and these reiterations result in networks of cellulose ribbons (Yamanaka et al. 1989). The same effect with cell elongation induced by, e.g., chloramphenicol addition to the medium was previously described to induce loosening of cellulose network (Yamanaka et al. 2000).
Interestingly, motA+ and motB+ overexpression mutants showed significantly increased cellulose productivity as well. Several studies suggest that motility and cell size are involved in biofilm formation in many different microbes (O'Toole and Kolter 1998; Pratt and Kolter 1998; Choy et al. 2004; Merritt et al. 2007; Niba et al. 2007). It has been demonstrated that deletion mutant of motA was non-motile and displayed reduced ability to form biofilm (Hossain and Tsuyumu 2006). Despite the fact that both motA and motB play an essential role in the biofilm formation in Gram-negative bacteria, there is no evidence of the role of these genes in Komagataeibacter genus.
Localization of MotA-GFP and MotB-GFP occurred at the cell pole. Furthermore, we have observed that both MotA and MotB have a polar location when cells are short, while bipolar when cells are longer. Similar results were obtained in bacteria with polar flagella (Koerdt et al. 2009; Wang et al. 2013). In Vibrio alginolyticus, PomA and PomB proteins, which are MotA and MotB homologs, are localized to a cell pole dependent on the presence of the polar flagellum (Fukuoka et al. 2005; Kojima 2015). The polar localization of proteins is a crucial functional feature, since it is critical for correct performance of several essential cellular processes including growth and cell division as well as cell motility (Shapiro et al. 2002; Ryan and Shapiro 2003; Treuner-Lange and Søgaard-Andersen 2014). Similar localization of Mot proteins was observed for in bacteria with polar flagella (Koerdt et al. 2009). Since, bacteria from the Komagataeibacter genus do not have flagella, we suppose that this feature is not essential for BNC production and maybe was reduced during evolution.
The RT-qPCR results indicated that the overexpression of motA and motB genes in K. hansenii ATCC 23769 strain resulted in a higher expression level of motA, motB, groEs, groEL, dnaJ, if2, tu, ugp, and bscA genes. GroES/EL and DnaJ contributes to diverse cellular functions, including stress responses, motility, and pathogenesis (Shi et al. 1992; Susin et al. 2006). Translational factors are involved in many mechanisms, including replication, transcription, RNA processing, DNA repair, regulation of translation, malignant transformation, and regulation of cell growth and development (Caldas et al. 2000; Madison et al. 2012). Translation initiation factor (if2) and translation elongation factors (tu), in addition to their role in translation, might be implicated in protein folding and protection from stress. Several studies have reported upregulation of molecular chaperons and stress-responsive genes, e.g., by ethanol (Okamoto-Kainuma et al. 2004). Furthermore, the expression level of ugp and bcsA genes was upregulated in the motA+ and the motB+ mutants, which might explain the observed increase in the efficiency of BNC production. These observations are in agreement with the previous reports suggesting that MotA and MotB proteins are important in biofilm formation (Hossain and Tsuyumu 2006).
In this study, we show for the first time the functional role of MotA and MotB in the regulation of bacterial cellulose production and creation of porous BNC network. By control of K. hansenii motility and cell size, it is possible to direct cells to produce well-defined three-dimensional scaffold for tissue regeneration and repair. Therefore, we believe that it is necessary to discover and examine other genes related to cell division or motility, as this may in the future allow obtaining cellulose with desirable properties depending on the application.
We would like to thank Prof. K. Tajima for kindly providing the pTI99, pTIE plasmids, and K. hansenii ATCC 23769 strain. We also thank Dr Katarzyna Kubiak for valuable discussion at the initial steps of the studies. Furthermore, we would like to thank Jolanta Płoszyńska for her help with the media preparation.
Compliance with ethical standards
Conflict of interest
The authors declare they have no conflict of interest.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Supplementary material
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text: I have just started playing a little while ago and just purchased my 3rd uke yesterday. I am replacing this concert uke with the Koaloha sceptre. Can't wait! Anyway this has to go. I bought it from a music store on line at a discounted price a month ago. It has blemishes. I am posting pics so you know what you are getting. The only thing that "concerns" me is the bridge. So you know though, I have replaced strings and play this at least an hour a day and there has been no change in it but I want full disclosure here. That is also why I am only asking $130 and that INCLUDES shipping (CONUS of course). All knicks/scratches are cosmetic only (came that way). This plays very nice with aquila strings I have on it. It retails at $270 at our local music store. Any questions please feel free to PM. I can accept Paypal or money order if you want to mail it to me. Thanks for looking, Dean


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