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tv Deadline White House MSNBC November 30, 2018 1:00pm-2:00pm PST
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in a court filing from robert mueller. while trump's turns on the world stage have included notable low points like siding with vladimir putin over his own intel community, shove aside the leader of montenegro, a nato ally, and blowing off a world war i tribute during a trip to france, his appearance today marks the first of his presidency where he steps on stage as the central subject in an investigation into possible collusion between russia and his own presidential campaign. "the washington post" reporting, quote, in two major developments this week, president trump has been labeled in the parlance of criminal investigations as a major subject of interest, complete with an opaque legal code name, individual 1. one day after the surprise guilty plea from the president's former fixer and lawyer, michael cohen. the picture of just how much trouble the president may be in is becoming clearer. our friend emily jane fox reports in "vanity fair," cohen's guilty plea also came
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together only in the last two weeks, according to people familiar with the matter. it was during that time frame that trump submitted his written answers to questions from mueller's team. including, according to a previous "new york times" report, an explicit question about the president's communication with cohen about the russian real estate deal. the president woke up in argentina today clearly aware of this new reality. one in which robert mueller made clear through his filing yesterday that he knows donald trump was at least trying to do business with russia, even after clinching the nomination of his party. with that in mind, perhaps donald trump sought to move the goal post from his oft repeated i had no business with russia to something more along the lines of, well, of course i'm doing business with russia. i'm a multitasker and you're lucky to have me. i get it. i'm a very good developer, happily living my life. when i see our country going bo in the wrong direction, to put it mildly. against all odds, i decide to
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run for president and continue to run my business. here's my favorite part. very legal and very cool. here to take us through the day's developments and tell us what very legal means, joining us from buenos aires, l.a. times white house correspondent eli stokols, frank figliuzzi, former fbi assistant director for counterintelligence is back, and at the table, heilemann is here of showtime's "the circus." >> oh, i'm here. >> and karine jean-pierre, senior adviser to moveon.org. take us through this collision of the president's now very public. there's been a lot of speculation up until today, up until this point of just how interested robert mueller was specifically in the president's conduct around questions of russia, questions of the trump tower meeting, of his business interest. now we know and now we know as the post reported today, he is the central subject in that
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investigation. >> yeah, that's correct. and it's hard to really get a sense of how much that is weighing on the president as we watch him go from meeting to meeting down here at the g20. but he doesn't seem to be, you know, all that motivated, all that excited to be here. tweeting early this morning about the mueller probe, calling it a witch hunt, defending himself. and moving the goal post. saying once again that that thing he denied for a long time, that now he's been proven to have been doing, now he's basically saying, oh, yeah, what's wrong with doing that anyway? so it's a total 180 on that. not the first time we've seen that from the president. he also was planning to meet with russian president vladimir putin. canceled that meeting earlier in the week and continues to insist that is all about the ukraine, russia's naval aggressions in the sea. it has nothing to do with the russia probe. he's volunteered that information enough times via twitter and in person in terms of talking to the reporters who are in the room ahead of some of
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thesis one-on-one meetings with other leaders that you get a sense he's trying too hard to convince people that that's the case. >> it would also be the first time that any of vladimir putin's conduct, brutal conduct interfered with any of donald trump's plans to rub shoulders with him. it would be an interesting departure. i think he had poisoned disdents in russia and pretheresa may wa speaking out on him on a daily basis. we didn't change anything about our conduct there. i want to ask about the colonel. trump's legal team did not learn until thursday that cohen sat for dozens of hours of interviews in mueller's office according to a senior administration official. i know you often see staff on these trips more than the president himself. is there anything you're picking up from the staff, any sort of fatigue or concern or alarm about the cohen developments? >> not directly, but, you know, talking to people, it does seem
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like you can tell this is weighing on the president quite a bit. throughout this entire investigation, we've seen the white house, the legal team, the president himself caught off guard by developments. learning of things when they break in the news media. not getting a whole lot of advance notice. has to be frustrating for him. continue to have frustrations with the justice department. just feeling a lack of control generally with this probe. i think talking to people around the president, there is a sense that he has that this is maybe nearing a different stage, more dangerous stage for him where there could be more indictments, people very close to him. so i think that's weighing on him pretty heavily as we see him really giving cursory comments during the interactions with reporters at the beginning of a lot of these meetings. unlike other summits where i've seen him, he does not appear to be here in buenos aires with a goal of turning all the tables over and making the summit all about him. he seems distracted to put it
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frankly. >> frank, let me put up the two references that i alluded to at the top of the show these are two references to the president in court filings from the mueller probe this week. one is about roger stone. i believe this was in the charging document for mr. corsi. and it said stone was understood to be in regular contact with then candidate donald j. trump. we know from reporting in multiple news organizations that reference to the president, that reference to stone being in contact with the president, i'm told by a close ally of the president's, that that seemed, to the president's legal team, to be implicating donald trump in the central question of collusion. that that bothered him very much. they took those complaints to the justice department where they were heard. but the president's name not removed from that filing. the other one was in the cohen document yesterday. where cohen said i made this misstatements to be consistent with trump's political messaging and out of loyalty to trump. what is the president's legal
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predicament at this hour, frank? >> well, let's add a third example there. n let's talk about manafort and the revelation that he was not only lying to mueller during his so-called cooperation but funneling information back to the white house and trump. those three together, corsi, stone, cohen, manafort. we've got four. it indicates to me there's a reason why we're seeing the president referred to as individual number 1. it's because he's soon to become defendant number 1. i've learned over the course of my fbi career, nicolle, multiple people cannot lie easily, simultaneously, about the same thing without central coordination. it's actually very hard to coordinate lies and have them happen randomly. so my working theory is that there is a ringmaster here. there is alignment, coordination of statements to congress, to mueller, to the public, and that this is not happening
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haphazardly but rather with cooperation. if that's happening and accurate, then individual number 1 is poised to become defendant number 1. >> let me just follow up. we giggle because the notion there was a coordinated effort. even things they try to do in full view, coordination isn't something you'd ever accuse the trump team of. the campaign, the white house or any facet of his identity and brand. they are not coordinated. so if they actually coordinated lies, is that a separate crime, and what are those crimes? witness tampering? conspiracy? what is that? >> so, obviously, we have lying to congress, lying to federal agents, all violations of federal laws. but on an even larger scale, you certainly could argue that this is an attempt at obstruction, at witness tampering or intimidation. and you know, back to your coordination effort. so the one thing that people seem to do around president trump is try to not invoke his wrath and to ingrateiate
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themselves. these are giant wannabes. they want to tell him i'm about to testify, boss, dwhoot you think? i'm going into mueller's office, boss, what should i say? it's an opportunity for him to be that ringmaster and someone had to do it. and i think it is individual number 1 who is calling the shots. >> do you agree? >> yeah. i agree. and i think, look, it's a complicated question for a variety of reasons. i think one of the things that we have all lapsed into because if you covered the presidential campaign in any close way, the notion that this campaign was a keystone cops operation that they were -- didn't know what they were doing. they were comically disorganized, et cetera, was common place. it's thought by the outcome of the election. really, we have had other presidents, including the guy you worked for who won the electoral college without winning the popular vote. it's not the first time that's ever happened.
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to lose the popular vote by 3.5 million votes and win an electoral college majority is really -- it's a tough needle to thread. that doesn't happen by accident. keystone cops don't pull that off as they bumble and fumble their way around. you go talk to -- >> they knew where to land the plane. >> then you talk about the way he was working with facebook and targeting ads to particular places down the home stretch. that the digital operations were actually pretty sophisticated. how? they were sophisticated in coordination, to use your word, with where the russian fake news was happening. in particular, congressional districts and precincts, states. they were not stupid. and i think that whether donald trump himself was down at the detailed level of running the campaign, that's certainly not true. but when it comes to this level of coordinating things at the highest level and on this question which is the key one here, donald trump is promiscuous in his lies.
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promiscuous in a lot of things but many of the promiscuities. he didn't care about that woman or that person or that person he screwed over. he cares a lot about money. what we're getting to right now is the lies and questions have been lies about protecting a career built on fraud and potentially on financial crime and potentially on money laundering, and those lies because they are all about the one thing he cares about, himself and his money, those are things he has a lot of focus on. the things he has focus on are things he's capable of coordinating. >> this is sort of the ultimate contradiction of the trump era. he also -- there's one thing that everyone has lied about. it's not like one person lied about context with russia and one person forgot about meet with the brits and another forgot about a chitchat with spain. they all lied about the same thing, to frank's point. >> again, the famous bill
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clinton thing of how when you see a turtle on a fence post you know it didn't get there by accident. it's the same thing. you don't lose by 3 1/2 million votes and eke out a narrow electoral college victory without some kind of intelligence and a whole bunch of people telling lies that snap together like a jigsaw puzzle without some coordinated intelligence behind it. whether it's donald trump alone or the rest of the trump family or kushners. but collectively, this low-level, low rent, cheesy crime family that i think they are at heart, there is coordination there and an effort that's worked pretty well to get donald trump to a lot place ofs nobody thought he could get. >> might also include an indictment as president or afterward. let me ask you, frank. i want to get to the questions we know trump has answered. button this part up for us. if you take the list of people, and we're talking about manafort this week because the week started, it feels like 11 weeks ago, mueller ripped up his
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cooperation agreement because he was lying to him. on thursday, cohen pleaded guilty to lying. but way before that, michael flynn pleaded guilty to lying. some have observed it may have been to protect his son from some legal jeopardy. michael flynn was the first person, i believe, in this sort of inner circle to the president who have been caught lying about contacts with ambassador kislyak. it's always a lie about russia. what does this look like from where you sit if you go back to the flynn lies, back to sally yates walking in to white house counsel's office and saying your guy could be blackmailed and you go through yesterday to cohen's lies. all about russia. >> right. soio have hit a whole core of the special counsel inquiry and their purpose in life, which is to get to the question of whether or not an adversarial government penetrated our democracy, our elective system and now has a president compromised in the white house.
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and so why are they lying about this? because all of these people are smart enough to know that this is the central issue that could bring their guy down. no one will sit still for this notion. and the question that mueller's trying to tease out -- the answer he's trying to tease out is when did the russia infection occur. was it a pre-existing condition, to use my medical metaphors? was it something that came well before -- yeah, pre-existing condition meaning the president and his staff came into the campaign compromised and in bed with russia, or did it come in the form of manafort being placed as campaign chair? did it come in the form of stone and/or corsi with social media propaganda, hacking and wikileaks? mueller's working on that. but the answer either way is at some point, our president became compromised and in bed with russia and has been lying about it. >> eli, i want to show you some of the answers the president has offered. what we learned he answered in those written responses and see
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if you have any reporting that adds to it. the president answered a question for robert mueller in writing about when he became aware of the trump tower meeting. he answered a question about what he knew about communication between roger stone, his associates, julian assange or wikileaks. we know he answered this one. what communication did you have with michael cohen, felix sater and others, including foreign nationals about russian real estate developments. and we also know he answered this question. what involvement did you have concerning platform changes regarding arming ukraine? eli, you picking up anything else that we now know he has answered? we know those were on the list of questions that his lawyers obtained from mueller at the very beginning of the year. >> i know at least some of his attorneys are not all that confident, even in the answers that the president put down in writing and sent to the special counsel. we've heard from rudy giuliani saying, look, this is the truth as he remembers it, but, you know, trying to create a little
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leeway here. this is a sticky situation for the president. he's never really played by the rules in business, in politics. he endeared himself to a lot of supporters by flouting a lot of the rules. he's personally operated with this cockiness, this belief that whatever happens, he'd get away with it. that may be coming back to haunt him now until a couple weeks ago in the midterms. he'd never paid a political price. thought he could fix anything. it shook his confidence from what i'm told by people close to him. and a similar situation in terms of the mueller probe advancing, getting to an end stage, comparing his answers to the sworn testimony of several people who have cooperated with the special counsel's office. this is a difficult moment for the president. and i think his confidence in being able to survive this legal mess seems to have eroded a bit. >> you agree? >> this is orange jump suit
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time. and it's been really clear we were going to get to it. a lot of people have been saying that was coming. but this is what got papadopoulos and everybody else. we don't know, necessarily, if what the deals he was working on in russia if they were necessarily illegal, although offering a $50 million penthouse to a foreign leader is probably going to be a problem on legal levels. but lying to congress and investigators is the kind of thing that puts you in jail. if other people find out that you're lying and rat you out, that's what gets you locked up. the number that's always got me through all of this is the 70 hours of interviews and testimony that michael cohen gave. i don't know -- i couldn't tell my life story in 70 hours. i'd be done in 20 or so. whatever it is he told, it wasn't just his business but everybody else's. and there's no way at this point that no one is going to get arrested. and it may happen before the end of the year. >> the president tweeting he lightly looked at doing a building somewhere in russia. >> yeah. and he flip flops. he didn't do it.
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now he did do it. and i think it makes you think, right? the president had to know what was happening was not above board. right? he had to know there was something seemingly wrong with what he was doing and his dealings with russia. but i look at the -- there's a narrative that robert mueller's putting together. and it starts with follow the money. and that is what he's kind of stringing along and that we see. and that makes me think about the founding fathers and they're concerned. this is what they were concerned about. a president that will enrich themselves. that would be corruptible. i mean, we have a president right now who was not vetted. we didn't have his tax returns. we didn't -- the emoluments clause doesn't matter to him and look where we are today. >> that's why they're all there. eli, thank you for joining us from points abroad. we appreciate it. when we come back, we still don't know xat lyexactly who st the first match from which the flame of admiration burns in donald trump for former kgb
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officer vladimir putin. but we do know that doing business with russia was a decades-long dream of donald trump. also turning a blind eye to fraud allegations. acting attorney general matt whitaker, the man atop the nation's law enforcement agency, is in the center of a fraud scandal at a company he once advised. we'll bring you that story and the summit under way in argentina has it all. protests, awkward moments among allies and this image of two of donald trump's closest friends on the world stage sharing a high-five. we'll wonder until the end of time what that was all about. stay with us. i just got my cashback match,
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our point was is that logic could tell you that you don't want the national security adviser to be in a position where the russians have leverage over it. >> and that someone has to say that is the trump story. that was sally yates telling the senate last year why she was so alarmed by mike flynn's lies to the white house about his contact with russians. it's not just about the content of those conversations. it's also about foreign powers knowing that top white house officials have lied. considering how dangerous it was for the russians to know the national security adviser lied. imagine if the president were in that position. yesterday's revelation by michael cohen does just that. in a january 11th, 2017 news conference, trump said the closest i came to russia was in selling a palm beach mansion to a russian oligarch in 2008. while we're just learning how dishonest this was, putin has known it all along. that means throughout trump's
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campaign and presidency, putin has had the power to plunge him into political crisis. joining our conversation, "new york times" political reporter nick confessore. everyone else is still here. so desperate to hear your take on all of this. you've done a ton of reporting on this intersection of russian interests. >> this is really important. so who is donald trump, really? who is he really? he's a guy who licensed hotels and developed them for money. that was his business. russia was his white whale. he always wanted to get there. it's where his deal partners were. people funding his expansions and where his customers were. some of them were buying apartments in new york city. he wanted to be in russia. it's amazing to learn and not surprising, but amazing that up until he had the nomination and perhaps even past it, we don't know, he was trying to get that thing built. and before people say, it's just a hotel. who cares? the key thing is, this was his business. and people think he's not going
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to sell out the country or do crazy things for a hotel deal. but that was his business. his core thing to build hotels. >> his whole family's business. and it's not like he's the only one that went into the white house. the whole family has -- >> it sounds parochial to us, but it's what they did. >> and unclear if it's still what they do. >> correct. >> take me through your tour of donald trump's proposed building sites. >> to start with, we went to moscow for -- i was there almost two weeks last spring for the first episode of the third season of "the circus." and our whole team went and some people left and i was left alone there for a while at the end of the trip with a couple crews and i just went out to have some fun and shoot some stuff. most of which never made it on television but there have been a lot of journalists who have done good work on this. i'm not a pioneer in this area. you read michael isikoff's book and the stuff the guys at the guardian have done, the steele dossier, you piece together a timeline of the 30 years in which donald trump was trying to
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break into the russian real estate market, whether it was the trump hotel or other ventures. and we laid it all out and said let's go do a tour of all the building sites, the places he wanted to build, the places of some historical import where he staged the miss universe pageant. the things he now cites as innocent. well, i had the miss universe pageant there. the reason he had it in russia wases b was because he was trying to get them to build. he never thought -- the guy made a lot of money and lost a lot of money over time in this business. and when he ran for president, he told us at the beginning of the campaign, he thought he had about 30% chance of winning the republican nomination. he never thought he'd be the republican nominee and certainly never thought he'd be president. so the whole thing was teed up as part of this longer continuum. going from site to site in moscow and outside moscow you got this sense of it in a
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geographic way. you stood on the banks of the moscow river where now there's like a shanghai level of development with huge gleaming glass towers. you could easily imagine why a real estate developer like trump, why he went there and said this is going to be a place where i can make a lot of money. as he ran for president, he didn't give up that dream. he thought this is a way to further -- this is a business development exercise, not a race to become president of the united states. this is a way to finish at the end of this and my brand will be bigger. this will lead to greater business opportunities. this presidential campaign is a stepping stone to finally getting that deal done. >> let me ask you. why didn't see say as a candidate, you know, yeah, i'm doing business in russia for all the reasons you just listed. why the big lie? why the lie? >> i think because much better to look -- if you're going to run for president in this country, most people think it would be a cynical, craven, disgusting act to use the quest
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for the presidency as a way of just lining your pockets. what you need to do is convince people you want to make america great again. then when you -- >> make moscow great again. >> when you fall short you say, i gave it my best. that horrible hillary clinton is going to ruin the country but let's go talk to the saudis. let's talk to the russians. i'm a bigger figure now in the world than i've bnch and i'm going to be able to cash in. >> who unpacks the selling of the united states by donald trump? >> that's a really good question. but i do want to touch on something that john just said. the irony of all of this. the reason why the trump tower in moscow even became a possibility is because he was running for president. >> the reason vladimir wanted to help him -- >> right. and so there's this irony at the end of the day where i think the worst decision that donald trump probably will ever make in his life is running for president. and so i think it's just -- the whole thing is just pretty
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insane when you think about it in that way. >> i didn't think of it as his white whale. the godfather when they all want to move to vegas. it's like we all want to move -- they are all moving out there, and he's got don junior, his brother. as the end of the day, this is what i want to do. i don't care what corrupt deals i've got to make in order to get there. yes, it's all clear. most of us know inside and outside, there's lots of people in the trump campaign who never thought he was going to win. once he realized, oh, my gosh, i'm going to become president. once the people connected to him realize this is real, they never stopped. they could have stopped. there were so many -- >> then it just became the reality was, once it became, oh, wait. it would have been great if i fell short because i could have cashed in on that. now i can be president. i can cash in even more on that because i can be president for four years or eight years and on the back side of that, all of the opportunities i'm going to have to make even more money. the presidency if you have no
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principles, no scruples and no concern for the emoluments clause, the president looks like a good gig when it comes to the post-presidential money-making schemes. >> frank, take me -- i'm old enough to remember when you couldn't let a reporter buy you a soda because it would look like you were letting a reporter curry favor with the press office. how did we get from there in 2004 to here where the presidency sounds like it is literally for sale to the most thuggish dictator, in this case vladimir putin, or maybe there was a bidding war. maybe that's what the high-five was. we split him, mbs and vlad. take me through how the law either catches someone like this or do you slip through the cracks? did our laws not imagine someone as corrupt as the trump family? >> yeah, i think we were all operating on a general understanding, nicolle, over our american history that politicians would rise up to become president of the united states with a working
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understanding of our constitution, our laws and our government. what happened here was a sizable percentage of our population said, you know what? enough of that. we need an outsider. how about a businessman. the businessman they selected was someone who was applying manhattan rules of real estate development and trying to get to moscow and other places. the problem is, as soon as you start talking about moscow, you are not in kansas anymore. you have got the intelligence services, the oligarchs, organized crime, all wanting to extract their pound of flesh. as soon as they see that desire in your eye, that carrot of building over there and wanting their money, the stick is going to come out. that's how they operate. they're going to compromise you. they're going to leverage and exploit your desire and that's what happened here. the question is, when it happened and to what extent it happened. >> when we come back, in the know. the acting attorney general of the united states is under scrutiny today after newly released documents show he knew about allegations of fraud at a company he once championed. that scandal costing some people
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acting attorney general matt whitaker, the current head of the justice department today finds himself in the center of an explosive fraud scandal. newly released documents reveal whitaker was more closely involved with a company called world patent marketing. that company shut down in 2017 for defrauding its consumers, allegedly scamming some of their life saving ofs. whitaker previously denied knowing anything about fraudulent activities during his time with the company but the documents released today show that he received multiple complaints from alleged victims. from "the wall street journal" -- mr. whitaker received more than a dozen e-mails and calls in 2015 from people complaining about the company. in most of the cases, mr. whitaker forwarded the e-mails
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to scott cooper. world patent marketing ceo. whitaker played down his role when contacted by the ftc in october 2017, according to notes taken by a staffer of a phone call with mr. whitaker included in the documents. joining us now, "wall street journal" senior writer james grimaldi. take us through your great reporting, and the documents seem to s for themselves. what do we know about how mr. whitaker responded to these fraud allegations? >> we know for sure that he got them. he appears to have at least reviewed some of them. he forwarded them to the ceo as you quoted from our story there. and this is all from records by the way that we got through the freedom of information act. i think we're seeing a body of evidence that's building that shows mr. whitaker was complicit, willfully ignorant or, in fact, ignorant of the schemes that were going on at world patent marketing, none of
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which look good for an acting attorney general. especially for a company that's now under active fbi investigation and had the serious allegations filed against them by the federal trade commission. >> that was my next question for you. so you obtained the documents through a freedom of information request. is it your assumption that the investigation is -- includes all the documents you've seen and more? i mean, what are you -- where does this story go next? it seems to be disqualifying, at best, and potentially some criminal exposure for mr. whitaker. >> the ftc handled it as a civil matter. they shut down the company and moved basicallying it looks like they referred it to the fbi. previously in an earlier story we confirmed that the fbi was under active investigation through contacts that they had made with the victims in the matter. so we don't know where that fbi investigation is going to go. sometimes those can take awhile. but it does put him in a
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compromising position because he oversees the fbi as the acting attorney general. >> frank, can you jump in on that question that was just raised that he's under investigation by the fbi, and he oversees the department of justice which houses the fbi. >> so the highest ranking u.s. government official overseeing all fraud operations, anti-fraud investigations in our government may well be wrapped up in a significant fraud himself. as of today's revelation may have given a false statement or inaccurate statement to an investigator at the ftc resulting in an fbi referral. this is wounding to whitaker. i don't see how he survives this or gets nominated to be ag and makes it. i'm not sure he's -- this choice could not have been worse for the president as an acting ag. he's in a trick bag. if he wants to act out and do something with mueller and
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constrain the mueller invest gairxs he's immediately called before congress to answer for why he's obstructing the investigation. any communications with the white house, on this investigation, are subject to discovery. if he starts to get into the fraud area where his own former company is being investigated, he's accused of obstruction. he literally must stay in his office at this point and hope to god that nothing worse comes out. so i -- this turns out to be the nominee or the acting position that we were all very worried about that essentially is wounded deeply by this and probably going to get worse by the day. >> i'm thinking of george conway. very high minded academic articulation of why matt whitaker can't be attorney general. had to do with the constitution. here it is he may be a criminal. >> think of the credentials that past ags have brought to the job. >> i read "the wall street journal" story thinking only in
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this time would i read this story. >> we think of him as a former semibusinessman wrapped up in a classic kind of low-rent company. a promotional company for patents and inventions. if you look at fraud cases of the ftc or other departments you'll often see these kinds of companies. mills of different kinds. they run different kinds of scams. and it's amazing the acting ag was the lawyer for one of these places or had worked at one of them. >> it doesn't surprise me because this is trump, right? this is trump university. it's that same scam. it's hitting those very working class folks of all colors in ohio who are up at 2:00 in the morning saying i've got an invention. i think i can go to trump university. i'm not surprised he would pick someone who targets the least of these in this country and tries to make money. it's like jordan belfour and "wolf of walstreet." it's an embarrassment that anybody would think --
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>> much, much more impressive than -- >> let me get the last word to james. tell our viewers where you turn for -- are you dealing with attorneys for mr. whitaker? dealing with the justice department? >> the justice department. >> what was the response? >> zero response. they asked us to send them the documents. i said, well, it's a fat file, but, you know, more than 500 pages. one thing i'll point out that even though attorneys at the federal trade commission, when they were trying to serve him a subpoena, they tracked him down. you know where? at the justice department. >> wow. >> and he responded with a voice mail that said, well, you can reach me now. i'm now the chief of staff to attorney general sessions. and that prompted a whole round of e-mails within attorneys, the enforcement attorneys in the ftc who were shocked, who were saying you're not going to believe where he is now. and a series of omgs among staff
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attorneys at the federal trade commission. they were just as shocked as i think maybe you are that he was involved with this company. they shared with him also a particular e-mail that was on the website in which he said, as a former u.s. attorney, i would only align myself with the first class organization. so mr. whitaker was putting, really, his -- his esteem, his integrity as a former u.s. attorney for the southern iowa as part of the credibility that he was loaning to this company that was later found to be a scam, that defrauded people out of thousands of dollars and provided them absolutely no service in return, according to the federal trade commission. >> james, it's a great, great piece of reporting. we're grateful to you for joining us. please come back. we'll stay on this. when we come back, is the trump family eligible for a group rate in legal services because they just might need one. managing my type 2 diabetes wasn't my top priority.
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my 6-year-old is counting me down here. the tentacles of -- >> doing a great job. >> the tentacles of michael cohen's guilty plea reach far beyond the doors to the oval office. it's a new world out there not only for donald trump but for his kids. multiple sources have confirmed to yahoo! news the president's elder daughter ivanka who is now a top white house adviser and his eldest son, don junior, were also working to make trump tower moscow a reality. while their level of involvement is an open question, it seems don junior might have some explaining to do. from natasha bertrand in the atlantic, according to the criminal information followed by mueller on thursday, cohen discussed the moscow deal with trump's family members within the trump organization. donald trump jr., an executive vice president of the trump org told the senate judiciary committee last year he was only
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peripherally aware of the moscow deal in 2016. >> liars. all liars. here's the thing. and i cite someone like emily jane fox who talked about this yesterday and she's right about this. if you know anything and spent time talking to donald trump and being in that world in the past, whether in this campaign or previously. it's just how small it is. and the smallness of it. the claustrophobia of it. how the family in some respects is totally dysfunctional and there are many -- all dysfunctional families are dysfunctional in their own way. the one thing they share is the sense of the tightness of this family around the name and in the physical proximity that they all worked for so long. it's not -- there are just not very many secrets around things that involve money. many secrets among the trumps. not things that involve money that would enrich all of them. so they all knew it was going on. i'm certain. >> the other point and it's important to point this out because it was first explained
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businessman. he inherited a bunch of money. the fantastic "new york times" investigation, the whole story about his wealth is a giant fraud. and his friends -- this is something his friends say about him. he ran a family office with only his family and some administration -- administrative folks in it. >> his campaign was too rich to be bribed or corrupt. but he never actually made his own fortune. he had a lot of failures. relied on his dad to bail him out over and over again. one thing you notice when you read about the trump family and their business is all the kids were involved in all the projects all the time. out there promoting, selling apartments to investors. they are making claims about the actual versus the real vacancy rate in buildings in panama. they are going to sell the tower over there. they're in china. and they were making trips all through the first year of his presidency which caused a lot of
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consternation. so the idea that this trump tower russia, which as we've discussed was a big priority, was somehow the one thing not on their radar screen, does not seem likely to me. >> and the other open question seems to be, what has -- we found out ivanka was using a personal e-mail. they got the spin machine in place right away. oh, you know, just details and logistics. if she was involved in trump tower moscow, if michael cohen has admitted to lying, don't we need to know what's on her e-mail server? >> not only that. she's been making money off of getting trademarks for her shoes in china. so there is -- all of them are so corrupt. what we're finding out is it was a family business, yes, but not only a family business but also sort of a criminal enterprise as well. so they brought that into the campaign and now into the presidency which is really scary. and, yes, she should be. we should be doing oversights and finding out. she's a senior adviser to the president of the united states. not just the daughter of the
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president. and that's what's so amazing about the michael cohen bombshell is that it brought us closer to donald trump. and his family. and there's the question and may give us the answer of what did he know and when did he know it? and know and when did he know it? all the family has been pulled into those two questions now. >> go ahead. >> this always jumps me ahead to where we all think it's going to go and it's the coffee clatcth sort of story we always have. it's much more likely that don jr. or ivanka could get into trouble for lying rather than the president. will he make deals on behalf of his children or is trump really about himself? that's what we'll find out fairly soon. >> frank, we have other people in trump's circle to look to. when mike flynn's son was ensnared, mike flynn made a deal. jared kushner is a famous father
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that was entangled with the law. and it's amazing that there are so many families for whom two generations are ensnared in this criminal investigation. >> look, i've seen this work both ways in public corruption cases. i've seen high-ranking officials told that their child is exposed criminally and they need to cut a deal. and i've seen them walk away from it, much to the d disgruntlement of their spouse. i don't know where this will go, but most likely trump will lash out and fire rosenstein before making a deal for one of his kids. >> frank, thank you for being there for us. when we come back, the high five seen around the world. at something old,
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>> there's something about welcoming the most reviled club pub of the day. they're in the club for the most reviled and the most leverage over trump. >> where they make history is they've got the american history in their back pocket. >> when i look at that picture, i see two men who kill journalists. >> amen. >> and a president who loves them both. who believes vladimir putin over his intel community and who believes mbs over his intel. >> they're basically doing donuts on the national stage. if they actually cared about trump, they wouldn be more subtle. they're basically telling the world, not only do we have this guy in our back pocket, we're going to keep doing it. we'll be right back.
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manafort's corruption blowing up, and putin grinning ear to ear with trump's best friend. >> i've heard them talk in the last 24 hours, and they have said this is the first time they believe that collusion is really on the table for donald trump. and we've been -- many of us on both sides have been saying, oh, not possible, definitely obstruction of justice. this week, this is where we are. we are one step -- robert mueller is one step closer to donald trump. >> as far as we know. it may be three steps closer. >> exactly. >> jason johnson and kareem jean pierre. i'm nicolle wallace. chuck todd starts right now. >> nicolle, you will always be my number
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Published at Monday, April 15th, 2019 - 04:14:24 AM. English Worksheets. By Önde Nordquist.
Lost in their favorite gizmos, today's kids are devoid of the fun learning aspect offered by preschool worksheets. For generations, worksheets for kids have been used by educators to develop logical, lingual, analytical, and problem-solving capabilities. It is a proven fact that children learn quickly in their formative years than at any time in their life. As a result, parents and educators give special importance to grooming kid's mind between 3 to 7 years of age who can be easily moulded to confident youngsters.
We cannot deny the fact that learning proper English grammar is important. Do you want to feel embarrassed each time you speak English with a lot of grammar mistakes? Do people have trouble understanding when you speak English or confused with what you have said? Being able to speak and write in proper English is a main criterion to be successful and broaden your options in job and career advancement. Many people who would otherwise be successful are being held back by their bad command of English grammar.
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People Reveal What Skills They Should Have Mastered By Now But Haven't
Believe it or not, I don't know how to whistle, much to the surprise of just about everyone I know. I also can't blow a bubble while chewing gum, so I'm clearly a failure at life, right?
But at least I know how to do laundry and keep a clean home, unlike some of the people who responded to Redditor biomann when they asked "Adults of reddit, what is something you should have mastered by now, but failed to do so?"
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Handwriting. It has gotten worse not better - peaked at 4th grade.
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Small talk is the bane of my existence. It's required in so many social and business settings but I hit so many walls while going through it. I know the basic format, I know what people want to hear/discuss, but that doesn't stop from me being awkward as fuck some times. It's mostly because I hate small talk and it just feels so fake to me.
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My taxes. I'm 32 and my wife still insists on doing them. Even if I did do them, she would redo them to make sure it was perfect.
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Not letting stupid people's opinions bother me. I get so many ignorant comments that I don't engage with because I think they're a waste of time. But I think about them anyway. Forever.
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Making friends. But most importantly maintaining the friendship.
It just fizzles out. I wonder why.
I am an avoidant according to Attachment Theory. Maybe that's why.
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I'm 44. I was taught the wrong way to tie shoes. My shoes ALWAYS become untied unless I deliberately force myself to tie them the proper way.
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Going to bed and getting up at the same time every day in a consistent routine.
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Every time I bite my tongue or the inside of my cheek, I am reminded how I still have not mastered chewing after decades of chewing daily.
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Calling companies that send me my bills (phone, electricity, internet, etc) and investigating why there was an increase in my payments or an additional charge.
Basically that means I suck at confrontation, I'd rather sit back and just pay it than seek conflict and resolution even though I was in the right to do so.
Laundry. I mean, I can put stuff in the machine and add soap and press a button, but there are so many other ways to remove stains and sort delicates and iron shirts and extra buttons that do whatever, and I don't understand it. My in-laws can bleach stuff! I have no idea how to use bleach without destroying everything in sight.
Press start and the machine goes, that's what I know.
People Reveal How 'Fake It 'Til You Make It' Majorly Backfired On Them
We are told that, if you're not confident, you should just "fake it til you make it."
This is great--in theory. In practice, sometimes "faking it" can have extremely real and terrible consequences, which these people found out the hardest of hard ways.


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Mar 23, 2019, 8:33 am57 ptsGlendon Scott Crawford was a mechanic at General Electric in Schenectady, New York. A tall, slender, middle-aged man with rectangular eyeglasses, he was married with three children. By appearances, he was an unremarkable middle-class American. But beneath Crawford's vanilla exterior lurked a…
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Warren has a spot-on lookalike she met face-to-face FOX News 119
Welcome to Politomix -- the political news wire where left, right and center mix.
Politomix aggregates the day's political news on the web and your mobile device.


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Group is dedicated to PSP tagging and snagging. Challenges, Contests, TUTS, Games for tags, Make friends, etc. Join Group for fun!
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The biosynthesis of the lantibiotics subtilin and nisin is regulated by autoinduction via two-component systems. Although subtilin is structurally closely related to nisin and contains the same lanthionine ring structure, both lantibiotics specifically autoinduce their biosynthesis. Subtilin and also the subtilin-like lantibiotics entianin and ericin autoinduce the two-component system SpaRK of Bacillus subtilis, whereas the biosynthesis of nisin is autoinduced via the two-component system NisRK of Lactococcus lactis. Autoinduction is highly specific for the respective lantibiotic and therefore of major importance for the functional expression of genetically engineered subtilin-like lantibiotics. To identify the structural features required for subtilin autoinduction, subtilin-nisin hybrids and specific point mutations of amino acid position 1 were generated. For subtilin autoinduction, the N-terminal tryptophan is the most important for full SpaK activation. The failure of subtilin to autoinduce the histidine kinase NisK mainly depends on the N-terminal tryptophan, as its single exchange to the aliphatic amino acid residues isoleucine, leucine, and valine provided NisK autoinduction. In addition, the production of subtilin variants which did not autoinduce their own biosynthesis could be rescued upon heterologous coexpression in B. subtilis DSM15029 by the autoinducing subtilin-like lantibiotic entianin.
Lantibiotics are ribosomally synthesized and posttranslationally modified peptides with antimicrobial activity (1, 2). Members of the class I linear lantibiotics are the subtilin-like lantibiotics, produced by several Bacillus subtilis strains, and nisin, produced by various Lactococcus lactis strains (3–6). They exhibit strong antimicrobial activity against multiresistant pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) (3, 7). The presence of the modified amino acids meso-lanthionine and 3-methyllanthionine are name giving for lantibiotics (lanthionine-containing antibiotics) (1). The lantibiotic producers B. subtilis and L. lactis share a similar composition of genes within the lantibiotic gene cluster. The genes lanBTC code for a modification and transport machinery forming a multimeric synthetase complex (8, 9). This complex catalyzes the dehydration of serine and threonine residues (lanB) followed by a nucleophilic intramolecular addition of neighboring cysteine residues (lanC) (10, 11). The genes lanIFEG code for a self-immunity system consisting of an immunity protein and an ABC transporter (12–15). The genes lanRK code for a two-component system composed of a histidine kinase and a response regulator (12, 16).
Subtilin and nisin act as autoinducers for their own biosynthesis via a two-component system (TCS) (17–19). Upon sensing the extracellular lantibiotic subtilin or nisin, the respective histidine kinase gets phosphorylated. The phosphate group is transferred to the response regulator, which in turn induces the transcription of the operons within the lantibiotic cluster. In the case of subtilin, the SpaRK TCS induces the operons spaBTC and spaIFEG and the subtilin structural gene spaS itself (18); in the case of nisin, the NisRK TCS induces the operons nisABTC and nisIFEG (20). In L. lactis, the expression of NisRK is constitutive (20), and nisin biosynthesis is induced with increasing extracellular nisin concentrations. In B. subtilis, subtilin biosynthesis is under dual control (18). In addition to the regulation by SpaRK, biosynthesis is growth phase dependent (21), since the expression of SpaRK is under the control of the major transition state regulator AbrB and the alternative sigma factor H (22, 23). Additionally, as described previously, expression can be influenced by the glucose concentration independently of the growth phase (24).
The subtilin-like lantibiotics (subtilin, ericin S, and entianin) as well as nisin A from L. lactis share a highly similar ring structure. Subtilin-like lantibiotics are closely related and can activate SpaK (25) but cannot activate NisK. Conversely, nisin A is the activator of the NisRK TCS but cannot activate the subtilin TCS, SpaRK (19, 26). Recently, we showed that the N-terminal entianin fragment from amino acids 1 to 20 is sufficient to activate SpaK and that nisin quenches subtilin SpaK activation (25).
Due to the increasing number of multiresistant pathogenic bacteria, there is a strong need for the development of new antibiotics. Lantibiotics such as nisin and subtilin provide excellent tools to genetically engineer new lantibiotics, as they are ribosomally synthesized and the structural genes can be easily modified genetically. Whereas many such mutations have been generated for nisin in recent years, there were no attempts to engineer subtilin-like lantibiotics (19, 27–32). Knowing the structural features of the class I lantibiotics subtilin and nisin needed for autoinduction is an important prerequisite for genetic engineering and the improvement of their antimicrobial activities.
Here we describe an in vivo expression system that enables the expression of subtilin variants and subtilin-nisin hybrid peptides which were used for the investigation of SpaK/NisK specificity. The N-terminal tryptophan is decisive for SpaK activation and further prevents NisK activation. Subtilin-nisin hybrids which were able to activate both the SpaK and the NisK histidine kinases could be generated.
Bacterial and yeast strains.The strains and plasmids used in the present study are listed in Table 1. The oligonucleotides used for construction of the plasmids are listed in Table 2.
pTSp10 was constructed for the deletion of the etnS structural gene in B. subtilis B15029. Therefore, pRS416 (33) was linearized with BamHI and transformed into Saccharomyces cerevisiae CEN.PK2 (34) together with the PCR products TSp26/27 (upstream etnS region), TSp28/29 (downstream etnS region), and TSp30/31 (spectinomycin resistance cassette, amplified from ECE74 [35]). Plasmid pBS09 was constructed for the integration of mutated spaS genes into the amyE locus of B. subtilis. Therefore, gusA of plasmid pMLK83 (36) was removed by EcoRI restriction, resulting in plasmid pSD27. Plasmid pRS426 (37) was linearized with EcoRI and transformed into CEN.PK2 together with the PCR product BS53/54 (the amyE locus integration region with the neomycin resistance cassette, amplified from pSD27), resulting in plasmid pBS09. Plasmid pTSp16 was constructed by the transformation of linearized pBS09 (EcoRI) and the PCR product of TSp45/19 (PspaS-spaS fragment) into CEN.PK2. The mutations W1I, W1I/K2T, W1I/K2T/E4I, W1IK2T/E4I/V12K, and W1I/K2T/E4I/V12K/Q17M of spaS were introduced step by step by oligonucleotides via in vitro mutagenesis PCR (Table 2), resulting in plasmids pTSp25, pTSp33, pTSp34, pTSp45, and pTSp49, respectively. For pCG02 construction, pBS09 was linearized with EcoRI and transformed into CEN.PK2 together with the PCR products TSp45/CG1 (PspaS-BamHI) and CG02/TSp19 (the 3′ region downstream spaS). Plasmid pCG02 was henceforth used as the backbone for the integration of various mutated spaS genes. For example, pTSp99 was created by transformation of BamHI-linearized pCG02 together with the universal oligonucleotides CG11 and CG12 and the oligonucleotide TSp195 into CEN.PK2 (Table 1). Plasmids with mutated subtilin structural genes were transformed into B15029.TSp01. Plasmid pAUT3 was constructed after restriction of pNZ8148 (38) and the PCR product NN16/17 (lacZ, amplified from pSB5 [18]) with NcoI/HindIII and subsequent ligation into Escherichia coli. The plasmid pAUT3 was transformed into L. lactis NZ9800 (39), resulting in nisin reporter strain NZ9800.AUT1.
Transformation for gap repair in Saccharomyces cerevisiae strain CEN.PK2 was performed as described by Schiestl and Gietz (40). Isolation of recombinant plasmids from CEN.PK2 was performed as described by Robzyk and Kassir (41). Plasmids isolated from CEN.PK2 were transformed into E. coli DH5α for further amplification. Isolation of recombinant plasmids from E. coli was carried out by an alkaline extraction procedure (42). Transformation of B. subtilis was performed as described previously (25).
Growth conditions for bacterial and yeast strains.B. subtilis strain B15029 derivatives were grown at 37°C in medium A (43, 44) for optimized production of subtilin and subtilin variants. The autoinduction reporter strain was grown in TY medium containing 0.3 M NaCl (26). For selection, the following antibiotics were used at the indicated concentrations: spectinomycin, 100 μg ml−1; neomycin, 15 μg ml−1; and chloramphenicol, 5 μg ml−1. When selecting with two or more antibiotics simultaneously, the concentrations were reduced to half. L. lactis strains were grown in M17 medium (Fluka) supplemented with 0.5% glucose (GM17) at 30°C. E. coli DH5α strains with recombinant plasmids were grown at 37°C in TB medium (25) containing 100 μg ml−1 ampicillin. S. cerevisiae was grown at 30°C in YPD medium (1% yeast extract, 2% peptone, 2% glucose) or in synthetic dropout medium (0.5% ammonium sulfate, 0.17% yeast nitrogen base, 2% glucose) without uracil for plasmid marker selection.
Autoinduction plate assay.For the rapid detection of lantibiotic production and autoinduction capacity, a simple plate assay was established. In that assay, a fresh overnight culture of subtilin reporter B6633.MB1 was inoculated to an optical density at 600 nm (OD600) of 0.1 and incubated at 37°C until an OD600 of 1 was reached. Subsequently, 100 μl culture was used to inoculate 100 ml cooled down LB agar medium supplemented with 4 mg 5-bromo-4-chloro-3-indoxyl-β-d-galactopyranoside (X-Gal). Thereafter, 10 μl of a fresh overnight culture of the strain of interest was spotted onto the plate. The plate was incubated overnight at 37°C.
Purification of lantibiotics.Culture supernatants of subtilin and subtilin variant producers were prepurified according to a modified protocol described previously (25). Subtilin and subtilin variants were separated on a semipreparative reverse-phase (RP) high-performance liquid chromatography (HPLC) column (5 μm, 110 Å, 250 by 10 mm; Gemini-NX C18 liquid chromatography [LC] column [Phenomenex, CA, USA]) using eluents A (20% acetonitrile [HPLC grade], 0.1% 2,2,2-trifluoroethanoic acid [TFA]) and B (99.9% acetonitrile [HPLC grade], 0.1% TFA). Subtilin and subtilin variants could be separated with a linear gradient of 22% to 26% eluent B within 40 min. Nisin purification was performed as described previously (25). The absorbance was monitored at 214 nm. The collected fractions were dried under vacuum and resuspended in 5% acetonitrile for in vivo tests or in 30% acetonitrile, 0.1% TFA for identification by mass spectrometry.
Quantification of subtilin, subtilin variants, and nisin.Subtilin and subtilin variants were quantified as described previously (24).The absorption of various dilutions of a subtilin stock solution (purified and mass spectrophotometrically confirmed) was monitored at 280 nm. Molarities were calculated using the molar extinction coefficient of subtilin (ε280 = 5,750 liter mol−1 cm−1; ProtParam; ExPASy). Different dilutions were prepared from this stock solution and analyzed by use of an analytical RP-HPLC column (5 μm, 250 by 4.6 mm; Gemini-NX). The absorbances of the samples were recorded at 214 nm and 280 nm. The peak areas were recorded using an Agilent 1200 series ChemStation for LC three-dimensional systems offline program and used to form equations (x = peak area; y = micrograms of lantibiotic), resulting in y = 1,195.8x + 370.97 and y = 77.684x + 7.4 for 214 nm and 280 nm, respectively. Solutions of subtilin variants and nisin were analyzed by analytic RP-HPLC at 214 nm, the peak areas were recorded, and concentrations were determined by using the equation y = 1,195.8x + 370.97.
Measurement of induction capacity by β-galactosidase assay.Samples for β-galactosidase assay were harvested as described previously (25). In short, a fresh overnight culture of subtilin reporter strain B6633.MB1 was inoculated to an OD600 of 0.1 in TY medium containing 0.3 M NaCl. The strain was grown to an optical density of approximately 1.0. Thereafter, 2 ml of the culture was transferred into small test tubes containing different concentrations of lantibiotics. After 1 h, samples were taken and cells were harvested by centrifugation and stored at −20°C for β-galactosidase assay. The β-galactosidase activity was measured as described previously and normalized to the cell density (45). The nisin reporter strain NZ9800.AUT1 was grown at 30°C in GM17 medium supplemented with 25 μg ml−1 chloramphenicol and handled as described above.
Antibiotic activities of lantibiotic variants and hybrids.The lowest concentration that prevented the growth of cells of the lantibiotic-sensitive indicator strain Kocuria rhizophila (formerly called Micrococcus luteus) ATCC 9341 (46) was determined to be the MIC. K. rhizophila was cultivated at 37°C in LB medium. A fresh overnight culture was inoculated to an OD600 of 0.001. For MIC determination, 2-fold serial dilutions of the lantibiotics were made and added to 2 ml culture, resulting in end concentrations of between 0.6 nM and 19.2 nM. The results were evaluated after 12 h of incubation.
Subtilin-nisin hybrids show reduced SpaK induction but trigger NisK induction.Class I lantibiotics like subtilin and nisin act as peptide pheromones and induce their biosynthesis in a quorum-sensing manner via the respective two-component system (TCS). Subtilin and nisin share a highly similar lanthionine ring structure, but their sequences differ at 14 amino acids (Fig. 1). The activation of the corresponding histidine kinase resulting in signal transduction is highly specific for its own cluster-derived lantibiotic (19, 25, 26).
At nontoxic concentrations, nisin cannot activate SpaK, whereas at low concentrations, subtilin provides strong SpaK activation (Fig. 2). This is also the case vice versa, where low nisin concentrations provide a strong NisK response, whereas subtilin cannot activate NisK (Fig. 3).
Knowledge of the structural features of the class I lantibiotics subtilin and nisin needed for autoinduction is an important prerequisite for genetic improvement of their antimicrobial activities.
To investigate the specificity of SpaK autoinduction, the N-terminal amino acids of subtilin were gradually adapted to those of nisin (Fig. 1). The resulting hybrid lantibiotics were expressed in B15029, where the etnS structural gene encoding entianin, a subtilin-like lantibiotic, was deleted. Entianin-producing strain B15029 was used instead of the subtilin producer B6633, as B15029 is easier to transform and to handle. The lantibiotic gene clusters in the two strains are nearly identical and behave absolutely similarly with respect to autoinduction, as shown previously (3, 25). After HPLC purification and quantification as previously described (24, 25), the autoinduction capacities of the hybrid lantibiotics were followed using subtilin reporter strain B6633.MB1 (Fig. 2).
Surprisingly, with subtilin W1I, the first amino acid exchange to isoleucine already resulted in a drastic 50% reduction of the induction capacity. Additional amino acid exchanges did not further significantly reduce the induction capacity. However, when the N-terminal tryptophan was recombined with other exchanges, e.g., hybrid peptide K2T/E4I/V12K/Q17M, the induction capacity was completely restored. These results clearly show the importance of the N-terminal tryptophan residue for the activation of SpaK.
In a corresponding experiment, NisK autoinduction was followed by the subtilin-nisin hybrid lantibiotics described above in the nisin reporter strain NZ9800.AUT1 (Fig. 3).
In contrast to the findings for subtilin, which mediated no NisK activation in nisin reporter strain NZ9800.AUT1, all hybrids where the N-terminal tryptophan of subtilin was replaced by isoleucine mediated moderate NisK activation. NisK activation was already detectable at concentrations of 6 nM (data not shown) and further increased at a higher concentration of 24 nM with respect to the level of activation by all other hybrid peptides (Fig. 3). Exchange of the first 4 amino acid residues of subtilin for those of nisin (subtilin W1I/K2T/E4I) slightly improved the level of autoinduction compared to that achieved with subtilin W1I, whereas a further adaptation of the N-terminal amino acids to residue 17 (subtilin W1IK2T/E4I/V12K/Q17M) did not result in an additional increase in β-galactosidase activity. This shows that the first 4 amino residues of nisin, which has 3 differences in amino acid composition compared to that of subtilin, are an important trigger for NisK activation. However, NisK activation by subtilin hybrid peptides W1I/K2T/E4I, W1IK2T/E4I/V12K, and W1IK2T/E4I/V12K/Q17M in NZ9800.AUT1 was still less efficient than that by nisin, and concentrations 4 times higher (24 nM) were needed to reach 70% of the entire level of autoinduction achieved with nisin. This shows that the C-terminal part of nisin with a further seven amino acid substitutions and the two additional amino acids, histidine and valine, compared to the sequence of subtilin also contributes to full nisin autoinduction.
To further prove the importance of isoleucine at position 1, the level of autoinduction achieved with the subtilin-nisin hybrid K2T/E4I/V12K/Q17M with tryptophan at the N terminus was measured. This hybrid peptide also failed to activate NisK in the reporter strain NZ9800.AUT1, which is in accordance with previous findings, in which replacement of the isoleucine at nisin position 1 by tryptophan abolished nisin autoinduction (19).
Importance of N-terminal amino acids for SpaK and NisK activation.Our current results indicate that the tryptophan N terminus seems to be of major importance for SpaK activation. Therefore, the N-terminal tryptophan was replaced by small amino acids, aromatic amino acids, and charged amino acids. The mutated spaS genes were expressed in B15029 with an etnS deletion and tested in the autoinduction plate assay for color formation on X-Gal plates (see Fig. S1 in the supplemental material). Most subtilin peptides with N-terminal mutations showed autoinduction. Very weak color formation was observed when alanine and positively charged amino acids were at the N terminus of subtilin, whereas strains with peptides with glycine and negatively charged amino acids at the N termini completely failed to form color.
Obviously, peptides with glycine and negatively charged amino acids at the N termini either did not autoinduce or were not accessible for the lantibiotic biosynthesis machinery.
To compare the autoinduction capacities of the mutated lantibiotics, the respective peptides were HPLC purified to homogeneity and quantified, and their autoinduction was followed (Fig. 4).
Similar to the activity obtained with hybrid peptide subtilin W1I, the peptides with leucine and valine showed reduced β-galactosidase activity that was approximately 50% of that of native subtilin. Replacement of tryptophan by other aromatic amino acids (F, Y) did not restore the full autoinduction of native subtilin, and histidine further reduced the level of autoinduction to 10% of that of native subtilin. The amount of peptide produced when the positively charged lysine was at the N terminus was too small for HPLC purification. The peptide with arginine at the N terminus could be purified, but nearly no autoinduction was measured. When these results are taken together with the failure to express SpaS mutations consisting of negatively charged amino acids at the N terminus, these data show that charged residues at the N terminus prevent autoinduction. This also coincides with the strong diminished autoinduction achieved with histidine (pKa of the side chain [pKR] = 6.0) in subtilin W1H, which also had a moderate charge under our experimental conditions.
Most remarkable was the very weak autoinduction obtained with subtilin W1A and the failure of subtilin W1G to activate SpaK, whereas the autoinduction capacity of peptides with valine, leucine, and isoleucine at position 1 was 50% of that of native subtilin. Obviously, the larger aliphatic side chain is advantageous for SpaK activation.
In a corresponding experiment, subtilin peptides with the N-terminal mutations were also tested with the nisin reporter NZ9800.AUT1 (Fig. 5), where 24 nM subtilin with the W1I mutation provided NisK activation that was 40% of that achieved with 6 nM nisin.
As was also found for SpaK, the subtilin peptides with changes to leucine and valine activated NisK, whereas the peptide with alanine failed to significantly activate NisK. Interestingly, with a decreasing size of the aliphatic side chain, autoinduction becomes more diminished (the levels of autoinduction achieved with peptides with I, L, or V at the N terminus were 40%, 30%, and 20% of those achieved with the native peptide). As was also observed for SpaK, the positively charged arginine at the N terminus of subtilin did not provide NisK activation, and in contrast to the activation of SpaK by peptides with aromatic amino acids and histidine at the N terminus, all peptides with these amino acids at the N terminus failed to activate NisK.
Our results show that amino acid residues with larger aliphatic side chains can activate SpaK and NisK as well and that any charge at the N terminus of subtilin prevents the activation of both autoinduction systems. For SpaK, aromatic side chains and, preferably, tryptophan at the N terminus provide activation, whereas for NisK, aromatic side chains are not effective.
A functional analysis to support the findings presented above was performed by determination of the antimicrobial activity of some hybrids and variants against the lantibiotic-sensitive indicator strain Kocuria rhizophila ATCC 9341. All hybrids and variants showed MICs similar to those of subtilin and nisin for K. rhizophila (subtilin and nisin MICs, 4.8 nM), with the MICs of subtilin W1L, W1Y, and W1R, K2T/E4I/V12K/Q17M being 4.8 nM and those of subtilin W1I and W1I/K2T/E4I/V12K/Q17M being 9.6 nM.
Correlation of autoinduction and lantibiotic production.Many subtilin variants and hybrid peptides were produced in reduced amounts. This raised the question of whether the biosynthetic machinery or a reduced autoinduction efficiency was responsible for the decreased levels of production. Therefore, we tested the correlation between lantibiotic production and autoinduction capacity. Using analytical HPLC, we determined the amount of lantibiotic in the culture supernatants of variant peptide producers in comparison to that in the culture supernatant of the wild-type subtilin producer (24). Thereafter, we compared the autoinduction capacities of the purified subtilin variants and subtilin. As aliphatic or aromatic amino acid residues at position 1 caused a reduction in autoinduction capacity to about 50% of that of native subtilin, the amounts of the variants with these changes at position 1 produced was reduced to about 70% of that of subtilin (data not shown). This shows that autoinduction and not the biosynthetic machinery is the strongest determinant of the amount of lantibiotic produced.
The amounts of peptides with positively charged amino acid residues and the small amino acid residue alanine at the N terminus produced were drastically reduced, and peptides with negatively charged residues at the N terminus were not produced at all. To test if a failure of autoinduction was responsible for the lack of peptide production, the respective subtilin variants were coexpressed in a wild-type subtilin-like lantibiotic producer. Hence, the genes coding for subtilin W1A, subtilin W1K, and subtilin W1E were introduced under the control of PspaS into the amyE locus of wild-type strain B15029.
For all three strains, expression of the additionally introduced subtilin variant gene could be shown via analysis of the respective culture supernatants by HPLC (Fig. 6). This shows that the production of mutated subtilin can be restored by coexpression of entianin and excludes the possibility that the lanthionine synthetase complex is not able to interact with and modify the respective precursors. Moreover, the additional amount of the subtilin variants produced did not interfere with the total amount of entianin produced. In each case, the amount of entianin produced was not altered from the amount produced in wild-type strain B15029. Surprisingly, the amount of each subtilin variant produced was significantly higher than the amount of entianin produced, suggesting a higher rate of transcription of the mutated gene introduced ex loco. Thus, the turnover limit of the lanthionine synthetase complex was not reached, which is in accordance with previous findings, where an AbrB deletion strain exhibited a 6-fold-increased subtilin production rate (24).The higher ex loco transcription rate is also in accordance with the previously found discrepancy between autoinduction capacity and the amounts of variants with aliphatic or aromatic amino acid residues at position 1 produced.
As reported previously, subtilin-like lantibiotics are succinylated at the N terminus (S-subtilin) to a certain extent. Succinylation depends on the medium composition and is also strain dependent (24). Remarkably, the amount of succinylation is different for the three subtilin variants (Fig. 6).
Gallidermin quenches entianin-mediated induction.In addition to the importance of tryptophan at the N terminus, the N-terminal ring structure of subtilin also seems to be important for SpaK activation. As recently shown, the subtilin-like lantibiotics entianin and nisin and their corresponding N-terminal fragments compete at the sensory binding site of the histidine kinase SpaK, which suggests that the three N-terminal lanthionine rings are also involved in SpaK activation (25). Gallidermin and epidermin, which are the shortest class I lantibiotics (1, 47, 48), share an identical ring structure with respect to the first two N-terminal lanthionine rings. The third ring of gallidermin and epidermin, however, is very different from that of subtilin and nisin (Fig. 1) and also contains the C-terminal 2-aminovinyl ring structure (47, 49). Thus, gallidermin provided an excellent tool to verify the importance of the third ring for the assumed SpaK interaction.
As shown in Fig. 7, similar to the results obtained with nisin, gallidermin quenches entianin autoinduction to 70% of the level of autoinduction mediated by entianin alone when the lantibiotics are used at equimolar concentrations (6 nM). If gallidermin was applied in 5-fold excess (30 nM), the entianin-mediated autoinduction was reduced to 30% of the level of autoinduction mediated by entianin (6 nM) alone; however, a higher concentration of gallidermin (60 nM) did not further reduce β-galactosidase activity. No SpaK activation was observed with the addition of 60 nM gallidermin alone.
Quenching of subtilin autoinduction by gallidermin clearly showed that the first two lanthionine rings of subtilin-like lantibiotics are sufficient for an interaction with the SpaK sensory binding site.
Taken together, our results show that subtilin-mediated autoinduction via SpaRK is significantly different from nisin-mediated autoinduction via NisRK. Surprisingly, a subtilin peptide with an N-terminal alanine failed to activate SpaK and NisK, whereas peptides with aliphatic side chains with increased sizes activated SpaK and NisK to certain extents. Whereas the size of the I, L, and V side chains had no effect on the activation of SpaK, the activation of NisK was more efficient with side chains of increasing sizes. These data show that hydrophobic interactions support SpaK and NisK activation. A major difference from those findings was observed for peptides with N termini with aromatic amino acids. Whereas tryptophan is needed for full SpaK activation, the affinity pocket of NisK is obviously not accessible to any aromatic side chain.
In summary, the structural constraints of tryptophan at the N terminus and the first two lanthionine rings should be considered for future genetic engineering of subtilin-like lantibiotics with respect to their efficient autoinduction and expression in B. subtilis. More than that, the successful coexpression of variants which do not autoinduce provides additional possibilities for the genetic engineering of subtilin-like lantibiotics and opens new possibilities to improve the antimicrobial activity of lantibiotics and their efficient production in B. subtilis.
This work was supported by DFG (En 134/11-1).
We thank Stephanie Düsterhus for excellent technical support and F. Götz for providing the lantibiotic gallidermin. We also acknowledge Goethe University and the state of Hesse for providing laboratory space, basic support, and the necessary equipment, as well as M. Karas for his support during MS analysis.
- Received 27 July 2015.
- Accepted 1 September 2015.
- Accepted manuscript posted online 4 September 2015.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.02392-15.
- Copyright © 2015, American Society for Microbiology. All Rights Reserved.