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Will payment for past wrongs end future wrongs?
Dayvid Figler
Nevada Legislature. June 3, 2019. Photo by David Calvert.
There have been quite a few solemn celebrations over the passage of AB267 in the 2019 Nevada legislative session. The law which provides compensation for individuals wrongfully convicted and incarcerated sailed to the governor's desk on the bipartisan wings of very emotional testimony, including that from DeMarlo Berry. Mr. Berry's true story stemmed from a case in which he served more than 20 years in a Nevada prison for a murder he absolutely did not commit. The measure, sponsored by Assembly Representatives Yeager, Fumo, McCurdy and Flores and in the Senate by Chris Brooks, caught the twittery feels of many.
Governor Sisolak:
Signing #AB267 alongside DeMarlo Berry on Friday was one of the most meaningful actions I've taken as #nvgov. While we can't give someone back lost time, we must do all we can to make it right. This law is a good start toward righting wrongs of the past.
Assemblyman Yeager:
It makes me so proud that Nevada has the strongest compensation law in the country. Nice to be at the top of a good list! #NVLeg #AB267
The Culinary Union:
Proud to stand with DeMarlo Berry in the fight for criminal justice reform. Thank you @GovSisolak for signing #AB267 today. The criminal justice system has a real impact on the lives of working families and wrongful convictions disproportionately impact communities of color.
In fact, this wrongful compensation bill has rightfully been touted nationally as a huge victory for the unfortunate victims of "wrongs of the past" and a measure (only tangentially) supporting Nevada's commitment to criminal justice reform. As the injustice DeMarlo Berry was forced to endure begins its journey towards recompense in the form of slightly more than $2 million dollars in formulaic compensation from taxpayer money, the question unasked despite all the hearings and accolades comes to the fore:
Will this legislation stop or even slow down wrongful convictions?
The answer, of course, is as complicated as any individual case. But by focusing solely on the high-price-tagged "I'm sorry" decades after the devastating impact was suffered, this important action is really only a half measure — especially when it comes unadorned with sustained talk about how we got (and get) to that wrongfulness. Thus, this new law is only a "tangential" criminal justice reform. A justifiably expensive remedy that requires a commensurately pricey deep-dive into the flaws of our system. And maybe the taxpayers will revolt for what potentially can be millions and millions more for the wrongful conviction of the innocent. But maybe that day will never come because we aren't looking at more safeguards on the front-end or creating disincentives for the bad behavior of those who investigate and prosecute cases. We aren't saying much about how the existence of wrongful convictions almost necessarily means that some people at the levers of power aren't paying much heed to carefully protecting the presumption, let alone the reality, of innocence.
SHOULDN'T WE ALSO PUNISH THE PEOPLE WHO CAUSE WRONGFUL CONVICTIONS?
Most wrongful convictions start their journey with a failure to fully and timely investigate claims of innocence or with intentionally or recklessly withholding evidence of innocence. Of course, the over-the-cliff drop of injustice occurs when a wily investigator or prosecutor compounds the likelihood of the ultimate uh-oh by proceeding in a single-minded effort to convince a judge or jury to convict by also utilizing all sorts of legal (and marginally ethical) tricks to accomplish the task. These type of things include: pushing hard to insert hearsay, prior criminal acts and bad character of the defendant; bolstering favorable witnesses; excluding unfavorable witnesses; shifting burdens; making misleading arguments, and all the other strategies that regularly occur in an adversarial criminal justice system. Of course, sometimes it's just a simple matter of a very good prosecutor against an overmatched defense lawyer – and of course that equation usually bodes poorly for justice.
Yet, rare is the desire to beat up on the police or prosecutors and the people who work with them. Say what you will of bad vibes towards law enforcement and prosecutors, most of the populace holds them in a place of general admiration and voraciously defends the law and order approach to society. As a result, there is rarely a direct impact on the wrongdoer even after post-conviction, actual innocence is discovered.
Let's look at an oft-discussed case in Nevada of a wrongful conviction – Fred Steese. There are complex reasons why Fred Steese spent more than two decades in prison for a murder he did not commit. But foremost of the reasons is that prosecutors from the Clark County district attorney's office withheld evidence that Mr. Steese was not in Nevada at the time of the murder from seeing the light of day prior to his trial.
It appears Mr. Steese should also get compensation from AB267, though the District Attorney's office has made his path more convoluted than Mr. Berry's for the simple fact that they — despite evidence, despite a finding from a District Court judge of actual innocence, despite a full pardon from the State of Nevada, despite the seeming regret of one of the two DA's involved in the horrorshow destroying Fred Steese's life — continue to refuse to acknowledge their office's egregious misconduct. But it's really simple. Had the DA turned over evidence that Fred Steese wasn't in Nevada – he would have never been convicted.
And what happened to those DAs? Have they been forced to pay out of their own pockets for their conduct? Have they been disbarred? Is there even a formal blemish on their record? Well, no. Both (now former) DAs retired with benefits from their DA positions and are currently unencumbered as they both sit as District Court judges accruing new benefits and a taxpayer-funded salary until they decide to voluntarily step down or lose an election. District Court judges, mind you, who exercise their discretion in new criminal cases using their own moral compass of what is right and wrong to guide their decisions. Stunning.
So along with all the hallelujahs over AB267, where's the talk of preventing the DAs (or punishing the DAs) for being bad actors, apart from being called out by judges? Is compensation out of taxpayer funds for the misconduct of the DA long after-the-fact really the end of the conversation? When we talk about criminal justice reform, are we just rearranging chairs on the deck of the Titanic if we don't also tackle the potential for abuse of the DAs and the lack of consequence when that abuse is exposed?
Same story with the police and the coroner. Let's look at another high-profile case of innocence: Kristen Blaise Lobato. There, it became pretty clear from the evidence that the police solely focused on third-hand rumors that Ms. Lobato was involved in some wrongdoing – somewhere – and decided to link that to the dead body of a rapist found in Las Vegas. The timing didn't work to show guilt. The science didn't work to show guilt. The logic didn't work to show guilt. And yet, 16 years in prison before a series of court decisions forced the hand of the district attorney to finally give up (but not without a fight) until the very end. Also, no apology or acknowledgement that the police and coroner messed up, or that the prosecutors zealously abdicated their duty to seek justice. Ms. Lobato should be covered by AB267 as well.
We talk about justice in the form of paying people for the flagrant flaws of a system that is far too unencumbered with restraint for us to guarantee that cases like the above won't happen again. This, of course, is just a portion of the discussion about the actually innocent and wrongfully convicted.
But what about the over-charged and the marginally culpable? What about the over-punished? And the less-drastic-but-still-indefensible misconduct of prosecutors who are still stacking counts to force negotiations, unjustifiably removing people of color from juries, making improper arguments in court that sway emotions over evidence, offering evidence in violation of well-established laws, and pursuing convictions they should know will result in acquittals?
AB267 doesn't cover any of these situations, nor do most of the criminal justice "reforms" the 2019 Legislature seems to be a little too self-congratulatory about passing. So what about the back-end of wrongful convictions? Does AB267 at least encourage the discovery of more cases where compensation should kick?
Yeah, about that….
WE NEED MORE AND MORE EFFECTIVE CONVICTION REVIEW UNITS
There is no systemic state process or agency charged with sussing out wrongful convictions, although in what might be argued to be a companion piece to AB267, the Nevada Legislature made it a little easier for a claim of factual innocence to be evaluated by a court with AB356, which creates a new procedure with fewer procedural barriers. Under this new law, a person with new evidence doesn't need to rely on a benevolent district attorney to get a day in court, but can petition the court directly at any time. A favorable finding by the court regarding innocence then starts an adversarial process which the DA can oppose (and appeal if they lose).
Even with the new law, though, it's still a tough road to prove "innocence." More likely, a court will acknowledge that errors or misconduct made in trial (or even plea-bargained) proceedings were not harmless, which only requires the case to be retried – not the exoneration of the individual. Then, it becomes a matter of the district attorney discretion to re-try the case (or offer a too-good-to-turn-down plea negotiation). Statewide, the DA offices don't have a great track record of giving up – even when it seems inevitable that they may lose the trial and be the impetus for the trigger to type of compensation offered by AB267.
Of course, therein lies the rub! It's not the DA's money. It never is. The DA offices around the state are never punished in any form for losing cases, even cases they knew they shouldn't pursue. Rarely do they get fired or sanctioned for misusing their considerable power. At worst, they win the unrighteous case, and then a guy stays in prison for 20 years with the taxpayers being stuck with the bill to pay the apology.
So is any agency trying to help with discovering whether there are wrongful convictions still stinging? Whether there are people in prison who just don't belong in prison?
The short answer is : ummmmmm?
The effort in the Nevada Legislature to create statewide offices of a Public Integrity Unit Commission failed this last session. However, in October 2016, the Clark County DA's office, for whatever motivation, proactive or responsive, announced the formation of its own Conviction Review Unit (CRU) as the first (and still only) of its kind in Nevada. The proclaimed concept? To "review factual claims of innocence, often times when new evidence is brought to the attention of prosecutors." Clark County DA Steve Wolfson even hired a seasoned, well-regarded defense attorney to head up the unit, Dan Silverstein. The office developed a referral form, established criteria and ostensibly set out to right prior wrongs – as the story goes. Indeed, the office takes credit for the exoneration of DeMarlo Berry, albeit with a righteous asterisk.
I asked Mr. Silverstein what the criteria would be for an exoneration like the one offered in DeMarlo Berry's case. He listed it as follows:
(1) the conviction occurred in Clark County, Nevada. (2) The convicted offender is a living person. (3) The claim is based on actual innocence, not diminished or lesser responsibility, ineffective assistance of counsel, legal error, or some other basis. (4) The claim is supported by new evidence capable of being investigated and potentially substantiated. The Conviction Review Unit is not a "thirteenth juror."
This unit takes referrals from anyone, including defense attorneys, and apparently scans appellate documents for claims of actual innocence. One hopeful foray into the CRU was to re-evaluate the convictions of people based on bunk field-tests of drugs, but that went nowhere. To date, there have been 148 petitions for review by this unit, with only one resulting in any exoneration-level action from this unit whatsoever, that being DeMarlo Berry.
And that's where the asterisk comes in. It wasn't an independent investigation, per se, by the CRU that swayed the day, but years of litigation and evidence gathered by the Rocky Mountain Innocence Center, including the corroborated confession of the actual killer. This proved to be the decisive factor getting the blessing of the CRU in June 2017. The announcement, however, perhaps overshadowing the fact that even with this evidence, the same District Attorney's office was fighting Mr. Berry's position (including actual innocence) as late as an oral argument in a Nevada Supreme Court case on November 13, 2015, and then was further fighting to preserve the conviction, to some degree, through the fall of 2016.
So with almost three years of operation, and only one endorsed exoneration, there are only two possible conclusions: (1) Wrongful convictions, despite the anecdotal evidence to the contrary, are no ones fault and are really rare things not deserving of more support for identifying; or (2) We may have some institutional problems that no amount of payback after will ever fix or stop.
Either way, the happy day at the Legislature is not the end, but the beginning of a motive (now financial) to not let wrongful convictions happen at all and figure out with more ardor (perhaps by a neutral body) if they are in our midst. Congratulations on AB267, ya'll; let's start talking about ways to make writing those payment checks as few and far between as court cases rife with error, misconduct and miscarriages of justice.
Dayvid Figler is a private criminal defense attorney based in Las Vegas. He previously served as an associate attorney representing indigent defendants charged with murder for the Clark County Special Public Defender's office. During his legal tenure, he served a brief appointment as a Las Vegas Municipal Court judge. Figler has been cited as a noted legal expert in many places including the New York Times, National Public Radio, Newsweek, USA Today, Court TV and the Los Angeles Times. His award-winning radio essays have appeared on KNPR as well as on NPR's All Things Considered program.
https://thenevadaindependent.com/article/will-payment-for-past-wrongs-end-future-wrongs


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Amalia .
Amalia Appears on the Front Cover of Morro Bay Life Newspaper
Following Amalia's performance at the Morro Bay Open Space Alliance, Amalia reconnected with Ruth Ann Angus who runs a non-violence peace organization and writes for the Morro Bay Life Newspaper. Amalia was asked if she could be featured in the newspaper and she was ecstatic to find that she was on the front cover! Amalia enjoyed speaking with Ruth Ann Angus, the interviewer, and is so honored to have an article in this month's paper. The article discusses Amalia's long term hopes for a career in music, dives into the meaning behind her song "Renegades, and explores her life apart from concerts and protests.
Amalia Plays Protest Song "Renegades" at Climate Change Strike in SLO
On May 3rd, Amalia played at the Climate Strike that was held at the park across from the Fremont theatre in San Luis Obispo. The protest was organized by the SLO County Youth for Environmental Action and Morro Bay High School's Students for Social Justice Club. Amalia was honored when she was asked to play her protest song, "Renegades," that she also performed at the March walkouts for gun control the previous year and at the Women's March in 2017. She feels as though music should be used to make positive change in the world and bring awareness on crucial issues. Climate change is a critical problem that her generation is faced with. In Amalia's song, she sings "No one's ever had to save the world before, they're giving us something we can't ignore." She believes that in order to save the planet we have to act now. Generations before believed they needed to save the world but hers is the first that has to.
Amalia Performs at the Annual Meeting for Morro Bay's Open Space Alliance
The Morro Bay Open Space Alliance(MBOSA) is a non-profit organization that seeks to conserve land in and around Morro Bay. Amalia was asked by a member to perform at the annual meeting on April 25th. Growing up in Morro Bay, Amalia loved to be outdoors and explore in nature, she has lived in Morro Bay her whole life and hopes that the beautiful land is preserved. At the meeting, she performed her empowering song, "Renegades" and Forest Wars, along with her award-winning song, "The Simplicity of Things," which emphasizes appreciating the simple things in life. It was very inspiring for her to watch the local community raise funds to support this cause and she hopes that others take action and save the land that is left.
Rotary Scholarship Fundraiser Concert at Windows on the Water
The Rotary Club of Los Osos hosted its Annual Scholarship Dinner at Windows on the Water on Sunday April 14th from 5-8pm.The event held a silent auction and raffle as well as live entertainment by Amalia. All proceeds benefit scholarships for Los Osos graduating seniors of Morro Bay High School. The Los Osos Rotary Club is a non-profit Service Club, that does Community & International Service. Amalia was delighted to support this cause because she knows many of the seniors this year and will be an incoming senior this fall.
Central Coast Music Convention
Amalia attended the 2019 Central Coast Music Con put on by Big Big SLO this March held at SLO Brew Rock. The event is for Venues, Promoters, Bands, Musicians, Event Planners, Music Businesses, and anyone else looking to connect with the local music industry. Amalia also went last year and really enjoyed networking and meeting other young musicians. This year she was asked to play in the Songwriter Showcase. She performed a 15 minute set following local musicians Dante Marsh and Noach Tangeras. Amalia set up a booth with her CD's, email list, and business cards to network. The convention included a variety of workshops that Amalia attended and performances by other musicians. This was such a wonderful opportunity for Amalia to connect with the local industry and get her name out there!
Amalia Performs with Little Village Foundation at Freight and Salvage Coffeehouse
After performing at Hardly Strictly Bluegrass Festival with the other artists on the National Protest album, "Raise Your Voice," Amalia was asked to come back to San Francisco to perform at the showcase concert for the Little Village Foundation. The Little Village Foundation is a non-profit cultural organization and record label that searches out, discovers, records and produces music that otherwise would not be heard beyond the artist's family and community. The LVF put the student protest album together following the Parkland shooting in February 2018. Amalia and 2 others from the album met at the infamous coffee house, Freight and Salvage, in Berkeley, San Francisco. Amalia enjoyed reuniting with her friends and adventuring backstage before the concert. She played her song "Renegades" from the protest album, which she wrote and performed at the Women's March in 2016. Amalia's song applies to many cultural conflicts her generation faces and represents the voice of the youth. She was able to connect with other talented musicians that collaborated with LVF and perform a wonderful show!
Amalia Wins for "Best Song" at Songwriters at Play Competition
The Morro Bay Wine Seller was one of the first venues that Amalia started playing live at in 2014. She gained performing experience every Thursday night at their open mic night which has given her confidence on stage. Though Amalia has moved on to her own solo concerts, she participated last night in the Songwriters at Play contest at the Morro Bay Wine Seller and played two of her newest songs: ¨Inked Tears¨ and ¨Breathing Fine¨. She competed against other adult songwriters and won the Best Song Award for her song Breathing Fine! She wrote the song about how heartbreak can feel like the end of the world but no matter what we get through it: ¨You said you couldn't live without me but i see that you're still breathing fine.¨ You can find a video of the performance on her Instagram(@amalia.songwriter).
Ruth K
Audience Moved to Tears at Hardly Strictly Bluegrass Festival
Amalia along with 10 talented young musicians from around the country came together last Sunday to Debut their new album in front of Thousands at Hardly Strictly Bluegrass. Here is MIke Kappus review of the concert. "The most moving set I've ever seen over 18 years of San Francisco's wonderful Hardly Strictly Bluegrass Festival was this Sunday by the students who participated in the Little Village Foundation album project, Raise Your Voice. An added plus was Bay Area Grammy winner, Fantastic Negrito acting as Emcee for the festival set. Little Village just released the album last Friday, a compilation of songs by students from across the country who participated in the anti-gun violence protests following the February 14, 2018 mass shooting at Marjory Stoneman Douglas High School in Parkland, FL. The first track on the record is "Shine" by Parkland students, Sawyer Garrity and Andrea Peña and 10 other songs and spoken word pieces share powerful, personal messages by students standing up for change with their passionately delivered songs. All proceeds from the sale of the album benefit Everytown for Gun Safety. Buy the record wherever you get your music, support the cause and bring it the profile it deserves. This is a crucial time to speak up and support others who are speaking up for all of us. In the meantime, make sure now that you are registered to vote and vote for those who prioritize people over party and profit."

redpajama_set_name: RedPajamaCommonCrawl


The federal government has issued guidance to Canada's spy agency on using contentious new anti-terrorism laws.
OTTAWA — The federal government has issued guidance to Canada's spy agency on using contentious new anti-terrorism laws — but most of the instructions won't be made public.
"One of our greatest concerns with C-51 is that CSIS has been given extraordinary new powers, including the power to break the law and violate the Constitution,'' said Josh Paterson, executive director of the British Columbia Civil Liberties Association.
"While some details need to remain secret, it's troubling that so much of the minister's high-level, general direction to CSIS on how the agency should interpret its new powers is blacked out. The government should be more transparent about the direction it is giving our spy agencies as to how they should be accountable."
The direction set out "overarching parameters for CSIS's use of its new powers'' under C-51 and Bill C-44, which addressed matters including source protection and use of judicial warrants, says a July 29 memo to Blaney.
Elements of the direction that were released to The Canadian Press mention fundamental principles CSIS must follow, including respect for the rule of law and the use of operational methods "reasonable and proportional'' to the threat.
The direction also stresses the fair treatment of human sources who help CSIS and says the spy service's activities abroad must respect Canada's foreign policy interests as well as the security of overseas missions and personnel.
"The really interesting stuff, I'm sure, is in the redacted portions,'' said University of Ottawa law professor Craig Forcese, co-author of "False Security,'' a book critiquing C-51.
Forcese said the direction should include things like guidance to CSIS about being careful not to step on the RCMP's toes and potentially derail a criminal prosecution.
"I think we need to know some of these details,'' said Forcese, who questioned classification of parts of the direction as cabinet secrets.
"Avoiding political interference or the appearance of it is crucial for the integrity of the regime."
Scott Bardsley, a spokesman for Goodale, said responsibilities for Access to Information requests are delegated within each department and are processed outside of ministers' offices.
"Avoiding political interference or the appearance of it is crucial for the integrity of the regime. We don't have a comment on particular decisions,'' he said.


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L. Messi
'Messi is human, I can catch the penalties' - Netherlands keeper Noppert 'ready' for potential shootout battle with Argentina star
11:16 SAST 2022/12/08
Netherlands keeper Andries Noppert will face Lionel Messi's Argentina next at the World Cup and is feeling confident if the game goes to penalties.
Netherlands to face Argentina
Noppert ready for Messi
Keeper confident about penalties
WHAT HAPPENED? Dutch coach Louis van Gaal raised a few eyebrows by making Andries Noppert his first-choice goalkeeper for the World Cup, but the 6ft 8in Heerenveen stopper has played a key role in the team's run to the quarter-finals. Two-time winners Argentina are up next, but Noppert, who only made his international debut in the Netherlands' World Cup opener against Senegal, is not fazed about the prospect of taking on a forward considered by many to be the greatest ever to play the game.
WHAT THEY SAID: Quizzed on the prospect of a penalty shootout battle against Messi, Noppert told reporters: "I'm always ready for that, and he can also miss, we saw it at the beginning of this tournament, so he's the same like us – a human. For sure he's good, but for sure I can catch the penalties."
THE BIGGER PICTURE: Messi has been in good form at the World Cup already, scoring three goals so far for Lionel Scaloni's side. He netted a penalty in Argentina's loss to Saudi Arabia but did also miss from 12 yards against Poland in the group stages. Saturday's game will go to penalties if the contest is level after normal and extra time.
DID YOU KNOW? Netherlands have lost just one of their nine meetings with Argentina in all competitions (W4 D4), with that loss coming in the 1978 World Cup final. However, the most recent of those four draws did result in a penalty shootout defeat, in the last meeting between the sides in the 2014 World Cup semi-final.
WHAT NEXT? The Netherlands face Argentina on Saturday in Qatar. The winners of the match will take on either Croatia or Brazil in the World Cup semi-finals.
Transfer news: Latest rumours from Kaizer Chiefs, Orlando Pirates, Mamelodi Sundowns & all PSL teams
'Only Benni can advise Foster, Tau must keep quiet' - Fans urge Burnley's new signing to listen to Bafana legend
'Messi will laugh' but 'it's no longer funny being Ronaldo' - Taunts as CR7 and Al-Nassr knocked out of Saudi Super Cup
Done Deals: Confirmed transfers by PSL clubs in January 2023
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The theme of the conference is "Geometry Assurance in Product Realization". Geometry assurance refers to all activities in the product realization process aiming at minimizing the effects of variation in the final product.
Do you have any wishes about the program? Is there any particular person you would like to hear as key note speaker? Is there any particular company you would like to hear more about? Take this opportunity to influence the content of the conference to include just that you want. We cannot promise that we manage to arrange it, but we promise to try!
Send your ideas to: organizer@cat2016.se.


© Ocean Renewable Power Co.
Ocean Renewable Power Co.'s TidGen® Power System prior to installation at the company's Cobscook Bay Tidal Energy Project site in Maine.
Maine is a pioneer in the development of clean energy, especially wind, biomass, and hydropower. The state has the highest renewable portfolio standard in the country, requiring 40 percent of electricity to come from clean energy sources by 2017—approximately double the goal of most of the other 28 states with similar policies. Maine also has a wind capacity goal of 8 gigawatts by 2030, 5 GW of which must come from offshore. Today, half of the electricity generated in Maine is from renewable power, with 1.8 GW of installed capacity.
Research by The Pew Charitable Trusts, released during a briefing at the University of Maine in Orono, found that the state attracted $900 million in private investment from 2009 to 2013. This amount is projected to grow by an additional $1.9 billion from 2014 to 2023.
Maine has a significant wind resource along its Atlantic shoreline and on the crests of the Appalachian range in its northwestern region. Its on- and offshore potential could supply nearly 47 times the state's current electricity needs. As of December 2014, the state had 13 completed projects totaling 431 megawatts in capacity, and another 1,261 MW were in various stages of development.
In 2013, Maine became the first state to connect an offshore turbine to the power grid when the DeepCwind Consortium, led by the University of Maine, launched the VolturnUS prototype project. The demonstration, a multimillion-dollar private-public partnership, is the culmination of a five-year collaboration to develop and install a floating turbine off Castine, a town about 35 miles south of Bangor. The university is completing final design and engineering work on a full-scale floating platform and projects that it would be deployed farther offshore.
Maine, as the most heavily forested state, generates 2.1 million kilowatt-hours of electrical power from biomass—one-quarter of the total electricity generation in the state and the most per capita of any state. Biomass is energy made from wood and wood waste-derived products such as pellets.
Maine's university system plays an important role in research and development in the biomass sector, just as it does well with wind power. Researchers at the Forest Bioproducts Research Institute at the University of Maine study ways to bolster commercialization of fuels, chemicals, and advanced materials from forest bioproducts. The institute also fosters collaboration among researchers, wood suppliers, and end users. In 2006, it obtained a $6.9 million award from the National Science Foundation and a matching state grant to develop facilities to support Maine's biomass industry. The ultimate goal is to build the research infrastructure that leads to a forest-based biorefinery in Maine using trees instead of oil to make fuel and other products and augment the pulp and paper and building products industries.
Marine and hydrokinetic technologies convert the energy of waves, tides, and river and ocean currents into electricity. These systems represent an emerging industry with hundreds of potentially viable technologies. They also offer operators unique advantages, including close proximity to major coastal areas of high energy demand, capacity predictability, and the ability to leverage solutions and lessons learned from more mature renewable industries such as wind and solar.


From Madysin Taylor: Awareness of gratitude will allow you to savor and, above all, appreciate your life with renewed grace.
Sometimes we forget to take the time to recognize the richness that defines our lives. This may be because many of the messages we encounter as we go about our affairs prompt us to think about what we don't have rather than all the abundance we do enjoy. Consequently, our gratitude exists in perpetual conflict with our desire for more, whether we crave time, convenience, wealth, or enlightenment. Yet understanding and truly appreciating our blessings can be as simple as walking a mile in another's shoes for a short period of time. Because many of us lead comparatively insular lives, we may not comprehend the full scope of our prosperity that is relative to our sisters and brothers in humanity.


The international furore over cats in New Zealand took my attention from Perissodactyla for a week to also post on my other blog but I am back now. While away I have been thinking about how to start the topic of populations and how we conserve or manage them.
Getting populations to grow is a central objective of wildlife conservation – fundamental to the rescue of rare and declining species. Larger populations are less vulnerable to unexpected catastrophes, like extreme weather events, and more robust to environmental changes, especially because they retain a greater diversity of individuals – genetic and cultural variation.
Grevy's zebra. Only around 750 remain in the wild.
Larger populations are also more likely to sustain a harvest by another component of the ecosystem's biodiversity, like a predator or parasite, or by people for food, clothing or recreation. It is more difficult to extinguish or over-harvest a larger population and diverse populations are more adaptable to changing circumstances. Thus, conservation is successful when it makes populations larger.
Reducing or maintaining a population at a particular size is a central objective of wildlife management. Population growth may need to be controlled or reversed to prevent a species from being at densities where it impacts other values of the landscape, like other plant and wildlife diversity, or the services that the ecosystem provides, like clean water. Some populations are pest species because they impose a social or economic cost on communities – impacting the quality of peoples' lives. A pest population's decline is the first step towards its eradication.The Perissodactyla are interesting because species are rare or endangered but also common. They are conserved in some ecosystems, but regarded as a pest in others.
The Javan and Sumatran rhinoceros are amongst the most endangered large mammal species on the planet. Black rhinoceros of southern and eastern Africa are critically endangered and illegal hunting for their horns threatens their extinction.
The world's tapir were recently common but are in rapid decline due to deforestation in equatorial Asia and South America.
Plains zebra is amongst the most common ungulates on the African continent, but Grevy's zebra number only around 750 individuals and they are endangered. Domestic horses are one of our most common large animals and released to re-wild or become feral on the landscape are regarded as pest species by some.
Posting under the category 'Population growth' I will explain how populations change and how that change can be measured and calculated. The uncertainties inherent in estimates of population growth will be discussed and the reliability of estimates for horses and rhinoceros debated. I will use characters of these species lives to calculate the maximum population growth rate that it could achieve under best conditions. I will evaluate some of the extraordinary claims about how fast these populations can grow or how slowly they are growing – hoping to inform the debates around the world about the conservation and management of wild Perissodactyla.


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Topologically-guided continuous protein crystallization controls bacterial surface layer self-assembly
Colin J. Comerci1,2 na1,
Jonathan Herrmann3,4 na1,
Joshua Yoon5,2,
Fatemeh Jabbarpour3,4,
Xiaofeng Zhou6,
John F. Nomellini7,
John Smit ORCID: orcid.org/0000-0002-5204-32787,
Lucy Shapiro6,
Soichi Wakatsuki ORCID: orcid.org/0000-0001-5896-79683,4 &
W. E. Moerner ORCID: orcid.org/0000-0002-2830-209X1,2,5
Nature Communications volume 10, Article number: 2731 (2019) Cite this article
Biomaterials – proteins
Biopolymers in vivo
Cellular microbiology
Nanoscale biophysics
Single-molecule biophysics
Many bacteria and most archaea possess a crystalline protein surface layer (S-layer), which surrounds their growing and topologically complicated outer surface. Constructing a macromolecular structure of this scale generally requires localized enzymatic machinery, but a regulatory framework for S-layer assembly has not been identified. By labeling, superresolution imaging, and tracking the S-layer protein (SLP) from C. crescentus, we show that 2D protein self-assembly is sufficient to build and maintain the S-layer in living cells by efficient protein crystal nucleation and growth. We propose a model supported by single-molecule tracking whereby randomly secreted SLP monomers diffuse on the lipopolysaccharide (LPS) outer membrane until incorporated at the edges of growing 2D S-layer crystals. Surface topology creates crystal defects and boundaries, thereby guiding S-layer assembly. Unsupervised assembly poses challenges for therapeutics targeting S-layers. However, protein crystallization as an evolutionary driver rationalizes S-layer diversity and raises the potential for biologically inspired self-assembling macromolecular nanomaterials.
Assembling a macromolecular structure on the micron scale often requires input energy and spatial coordination by enzymes and other cellular processes1,2,3. S-layers, however, exist outside the cell envelope and lack access to many cytosolic components, including ATP4,5,6. How do microbes continuously assemble a crystalline macromolecular structure on a highly curved cell surface undergoing drastic changes during normal cell growth? To answer this question, we perform time-resolved, super-resolution fluorescence imaging and single-molecule tracking (SMT) of S-layer assembly on living C. crescentus cells. In C. crescentus, the S-layer is made of a single 98 kDa SLP, RsaA, which accounts for around 30% of the cell's total protein synthesis7. RsaA, like other SLPs, self-assembles into crystalline sheets upon the addition of divalent calcium (Ca2+) in vitro8,9,10,11,12. Given the many fitness-related functions ascribed to crystalline bacterial S-layers, we hypothesized that SLP self-assembly may play a role in generating the S-layer coat in vivo4,6.
RsaA covers the cellular surface of C. crescentus by forming a 22 nm-repeat hexameric crystal lattice and is non-covalently anchored to an ∼18 nm thick LPS outer membrane13,14,15,16,17 (Fig. 1a–c). The surface topology of stalked and predivisional C. crescentus includes a cylindrical stalk measuring roughly 100 nm in diameter while the crescentoid cell body approaches 800 nm in width18,19 (Fig. 1d). This large variety of curved topologies guarantees that crystal distortion and defects within the S-layer lattice structure are present and enable complete coverage of the bacterial surface4,13,20,21. We can use Gaussian curvature, the product of the maximum and minimum curvatures at a given point, to quantify the cellular topology22. Crystalline defects cluster at regions with high absolute values of Gaussian curvature such as the cell poles and division plane, while grain boundaries occur where Gaussian curvature approaches zero such as the cell body (Fig. 1d)20,21.
Localized S-layer assembly occurs independently of secretion. a Schematic of the C. crescentus cell envelope with the S-layer crystal lattice (red/orange) anchored to the outer membrane (OM) via an 18 nm thick LPS layer (yellow), peptidoglycan (PG), and inner membrane (IM). b Model of the RsaA S-layer structure (EMD-3604) applied to the surface of a 100 nm diameter cylinder. c STED fluorescence microscopy image of a CysRsaA cell labeled with STAR RED. d 3D mesh representation of predivisional C. crescentus topology with absolute value of Gaussian curvature projected onto the surface (gray shading). e Schematic of 2-color pulse-chase experiment to image the sites of S-layer assembly where fluorophores DY-480XL and STAR RED were added to CysRsaA cells 30 min apart. f Confocal (top) and STED images (bottom) show localized assembly of natively produced S-layer at the cell poles (arrows), division plane (triangles), and crack-like features on the cell body (asterisks). g Schematic of pulse-chase experiment where saturating quantities of purified, fluorescently labeled CysRsaA are added to the media of ΔRsaA cells. h STED images show localization of newly incorporated CysRsaA protein at the cell poles (arrows), division plane (triangle), and other locations. i Overview of analysis method. Top: Cells were horizontally aligned with the stalk on the left side and the cell boundary was identified (non-gray region). Middle: S-layer was identified in both channels via intensity thresholding, and the binary image is projected along the normalized cell axis. Bottom: The projected image created two binary profiles of the S-layer for the upper and lower half of the cell in each color channel (red and green lines). j S-layers produced natively (red, representative cell in red boxed image Fig. 1f) or by exogenous protein addition (blue, representative cell in blue boxed image Fig. 1h) show preferential incorporation at the cell poles and division plane, while control cells (black, representative cell in black boxed image Fig. 1c) show uniform labeling. Shaded regions show 95% confidence intervals for n = 81, 75, and 57 cells for native, exogenous, and control cells, respectively. Scale bars = 1 μm unless noted
Specifically labeling the S-layer in vivo has proven difficult due to the SLP's life cycle and functions, which include secretion, refolding, anchoring, and crystallization11,16,17,23,24. Previously, electron microscopy of the C. crescentus S-layer was performed by inserting cysteine residues into the RsaA sequence and labeling the protein with nanogold via maleimide chemistry13. One such variant, henceforth referred to as CysRsaA, consists of a small tail added to the N-terminus of RsaA, which includes a single cysteine residue13 (Supplementary Fig. 1, Methods). CysRsaA-producing cells divide normally and create a stable S-layer at a rate similar to that of WT cells, and protein can be extracted and purified as a monomeric species (Supplementary Fig. 1). By examining the dynamic behavior of S-layer assembly using specific small-molecule labels and living cells, we find that protein self-assembly alone is sufficient to create and maintain the S-layer. The topology of the cell creates localized defects and boundaries within the S-layer lattice, which guides continuous protein crystallization to specific regions of the cell surface.
Imaging localized S-layer assembly in living cells
Covalently modifying CysRsaA with membrane-impermeable fluorophores via maleimide chemistry is a robust, highly specific labeling scheme for RsaA and enables live-cell STimulated Emission Depletion (STED) fluorescence microscopy showing a complete S-layer (Fig. 1c; Supplementary Fig. 2). Pulse-chase STED imaging of living cells was performed using DY-480XL as the pulse fluorophore and STAR RED as the chase with a 30 min delay (Fig. 1e, f). Using only stalked and predivisional cells oriented in the same direction, we found highly localized S-layer assembly in growing cells characterized largely by new protein enrichment at both cell poles and the division plane (Fig. 1f, i, j; Supplementary Fig. 3), in agreement with observations by electron microscopy25. However, high-efficiency labeling coupled with super-resolution STED imaging revealed nano-scale crack-like features of new RsaA on the cell body, indicating additional sites of localized S-layer assembly (Fig. 1f). We sought to determine the factors that contribute to S-layer assembly by examining how RsaA secretion, cell wall growth, and the presence of an existing S-layer structure affect the location of S-layer assembly in living cells.
Secretion alone does not explain localization
To determine whether RsaA secretion is necessary to localize S-layer assembly, we added purified, fluorescently labeled CysRsaA to cultures of cells with a genomic deletion of the rsaA gene (∆RsaA). Saturating quantities (600 nM) of DY-480XL labeled CysRsaA were introduced as a pulse, followed by a wash, 30 min of cell growth, and another saturating quantity of STAR RED labeled CysRsaA as chase (Fig. 1g). This experiment revealed that exogenously added CysRsaA incorporates in a manner favoring the poles and division plane (Fig. 1h). However, reconstituting S-layer assembly in this way creates more widespread, punctate fluorescence signal on the cell body (Fig. 1h, j, blue) compared to the crack-like features seen in native S-layer assembly (Fig. 1f, j, red). This effect is attributable to structural differences between the native and exogenous S-layers and is discussed below. Previous immuno-gold staining and electron microscopy of RsaF, the outermost component of the RsaA secretion apparatus, indicated its diffuse localization23,26. Therefore, the non-uniform reconstitution of S-layer assembly with purified protein suggests that a factor independent of RsaA secretion contributes to assembly localization.
Cell wall growth alone does not explain localization
To investigate the effect of changing cell wall surface area on S-layer assembly, we manipulated peptidoglycan (PG) insertion using the specific MreB perturbing compound, A2227. Under normal conditions, the cell wall grows mainly at the polar-proximal base of the stalk and around the middle of the cell body, which eventually becomes the division plane2. These regions alone are insufficient to explain localized S-layer assembly due to the additional appearance of signal at the pole opposite the stalk (Fig. 1j). At 2 μg/mL, A22 delocalizes MreB but still allows some peptidoglycan addition along the cell body, leading to lemon-shaped cells that divide slowly27 (Supplementary Fig. 4). At 25 μg/mL, A22 fully inhibits peptidoglycan insertion and surface area addition stops completely except at division planes where constriction began before drug exposure27 (Supplementary Fig. 4). Using a pulse-chase labeling scheme after drug treatment (Fig. 2a), we found that cells treated with 2 μg/mL A22 maintain bipolar localized S-layer assembly, but the amount of new RsaA incorporated along the cell body increased (compare red and green highlighted columns in Fig. 2b, c). At 25 μg/mL A22, bipolar localized S-layer assembly is disrupted as evidenced by a decrease in signal at both poles (Fig. 2b, c, black). Native cell wall growth is asymmetric to maintain the shape of the cell and lengthen the stalk1,2, yet S-layer assembly remains largely symmetrically localized at both poles with or without A22.
Cell wall growth is insufficient to explain polar localized S-layer assembly. a Schematic of pulse-chase labeling of CysRsaA cells treated with A22 (to inhibit MreB) for 1 h or cephalexin (to inhibit cell division) for 3 h. b Upper: Schematic showing effect of A22 treatment on PG insertion in C. crescentus, where untreated cells add PG primarily at the stalk pole and division plane, cells treated with 2 μg/mL A22 exhibit delocalized PG insertion along the entire cell with enrichment at the division plane, and cells treated with 25 μg/mL A22 have PG insertion exclusively at the division plane. Lower: STED images of S-layer assembly for treated cells with signal at the poles, division planes, and crack-like features marked by arrows, triangles, and asterisks, respectively. c Quantitation of S-layer assembly localization on cells treated with 2 μg/mL A22 (green) show similar degrees of polar localization, but enhanced localization along the cell body compared to untreated cells (red curve; reproduced from Fig. 1j). Polar localized S-layer assembly is decreased in cells treated with 25 μg/mL A22 (black), including the pole opposite the stalk (black dashed box). Shaded regions show 95% confidence intervals for n = 81, 54, and 63 cells for 0 μg/mL, 2 μg/mL, and 25 μg/mL A22 treatment, respectively. d Quantitation of S-layer assembly localization on cells treated with 35 μg/mL cephalexin show localization at both poles. Shaded region shows 95% confidence interval for n = 25 cells. e Upper: Schematic showing effect of 35 μg/mL cephalexin, which halts PG insertion during cell division. Bottom: STED image of CysRsaA cell treated with cephalexin shows localized S-layer assembly at both poles (arrows) while also adding S-layer at crack-like features (asterisks) along the cell body. Scale bars = 1 μm
Treating cells with cephalexin prevents cell division by inhibiting peptidoglycan insertion at the division plane28 (Supplementary Fig. 5). In cephalexin-treated cells, S-layer assembly follows untreated localization patterns, but with more crack-like features of new RsaA on the significantly elongated cell body (Fig. 2d, e). Taken together, both drug treatments show that S-layer assembly on the cell body appears to correlate with localized cell wall growth while bipolar localization is not driven by PG insertion alone.
Assembly occurs in patches without a pre-existing S-layer
Physically removing the S-layer from a CysRsaA cell provides a clean surface with which to observe the cell replacing its own S-layer, which we term de novo assembly. Calcium depletion (50 μM CaCl2 instead of 500 μM CaCl2 normally present in minimal growth medium) has been shown to cause shedding of the RsaA S-layer29,30. After culturing CysRsaA cells overnight in media containing 50 μM CaCl2, supplementing the media with 500 μM CaCl2 causes a new S-layer to be produced on the surface of C. crescentus (Fig. 3a). A time course of de novo S-layer assembly using only one fluorophore label at different time points displays the appearance of several S-layer patches per cell within an hour of calcium introduction (Fig. 3b, c). By 2 h, cells have produced a mostly complete S-layer, which agrees with the 2 h doubling time of C. crescentus (Fig. 3c; Supplementary Fig. 6). Localization analysis of de novo S-layer assembly at 30 min reveals its exclusion at the poles in sharp contrast to S-layer assembly in cells with a pre-existing S-layer, which prefers the poles and division plane (red curve, Fig. 3j).
A new S-layer assembles by crystallization and location depends on crystal size. a Coomassie-stained SDS–PAGE of protein samples extracted from the surface of CysRsaA cells grown in low calcium M2G (50 μM CaCl2, t < 0 min) and then switched to 500 μM CaCl2 (0 < t < 180 mins) show de novo accumulation of the S-layer. b Schematic of de novo S-layer assembly experiment where CysRsaA cells are resuspended in M2G with 500 μM CaCl2 and then their S-layer is labeled with STAR RED. c A STED imaging time course of STAR RED labeled CysRsaA cells shows that de novo assembly of a new S-layer occurs at discrete patches that grow larger over time. d Schematic of 2-color pulse-chase labeling between 30 and 60 minutes after calcium addition. e STED imaging reveals growth of S-layer patches from their perimeter (red signal at the edges of green patch) and is confirmed by line profiles of the dashed line (inset). f Schematic of experiment where low concentrations of STAR RED-labeled CysRsaA are incubated with ΔRsaA cells for 15 mins. g STED images show exogenous CysRsaA nucleates small puncta of S-layer. h Schematic of 2-color stepwise addition of 5 nM DY-480XL labeled CysRsaA followed by 10 nM STAR RED labeled CysRsaA. i STED imaging shows that puncta on the cell surface formed by exogenous addition of purified CysRsaA grow from their perimeter, confirmed by line profiles of the dashed line (inset). j Quantitation of S-layer assembly localization for native S-layer assembly (red), de novo native S-layer assembly (black), and S-layer assembly by exogenous addition of labeled CysRsaA (blue). Shaded regions show 95% confidence intervals for n = 100, 50, and 65 cells for native, de novo, and exogenous protein addition, respectively. k Quantitation of the number of S-layer puncta and average number of RsaA molecules per punctum on ΔRsaA cells upon addition of purified CysRsaA protein. Error bars represent SEM. A model of an RsaA crystal of about the measured size is shown (inset). Scale bars = 1 μm (c, e, g, i)
RsaA nucleates and crystallizes on the cell surface
To determine how these patches grew, we performed pulse-chase imaging of de novo S-layer assembly (Fig. 3d). This experiment revealed that initial S-layer patches expand from the perimeter of each patch (Fig. 3e), consistent with nucleation and growth characteristic of in vitro SLP crystallization observed by time-resolved atomic force microscopy10,31. To further evaluate this mode of assembly, very low concentrations (0.5 to 10 nM) of purified, STAR RED labeled CysRsaA were added to cultures of ∆RsaA cells (OD600nm = 0.2) and puncta were observed (Fig. 3f, g). The number of puncta appeared dependent on CysRsaA concentration whereas the number of molecules in each punctum did not strongly correlate with CysRsaA concentration from 0.5 to 10 nM (Fig. 3g, k). These observations are consistent with fast nucleation occurring at the measured CysRsaA concentrations and limited further growth of the observed puncta during the 15 min incubation. Adding 5 nM of exogenously purified and labeled DY-480XL CysRsaA followed by 10 nM of STAR RED CysRsaA revealed expansion of fluorescent puncta from the perimeter, as observed with two-color native de novo S-layer assembly (Fig. 3e, h, i). These observations are consistent with each punctum representing a nucleated RsaA protein crystal of at least 7 RsaA hexamers (Fig. 3k, inset). Large, natively grown S-layer crystals initially exclude the cell poles (Fig. 3c, j); however, introducing exogenously purified RsaA to ∆RsaA cells creates small S-layer crystals that appear more uniformly distributed along the cell axis (Fig. 3j).
Observing RsaA diffuse and crystallize on the cell surface
If de novo S-layer assembly occurs through nucleation and 2D crystallization of RsaA, then protein self-assembly could also be responsible for continuous S-layer growth. Since protein crystallization is concentration-dependent, we predict that once secreted, RsaA monomers should be able to diffuse while non-covalently anchored to the LPS outer membrane until incorporated into a nucleating or growing S-layer crystal. Indeed, at 1 nM exogenous CysRsaA, cells exhibit weak diffuse fluorescence suggestive of diffusing molecules (Supplementary Fig. 7). Therefore, we employed single-molecule tracking (SMT) to dynamically track the location of individual CysRsaA monomers anchored to the LPS outer membrane. ∆RsaA cells were first pre-treated with 2.5 nM Cy3-CysRsaA to form sparse, immobile S-layer crystalline patches or seeds on the LPS outer membrane (Fig. 4a, green). Then, 1 nM AlexaFluor647-CysRsaA (Fig. 4a, red) was added to the growth media, allowing single molecules to flow into the microscope's viewing area. Once attached to the LPS outer membrane, CysRsaA molecules were imaged for several minutes at a time (Fig. 4b). CysRsaA tracks included entirely immobile molecules, entirely mobile molecules, and molecules that appear to sample mobile and immobile states during the experiment (Fig. 4b, c; Supplementary Figs 8, 9). Correlating mobility of the molecule with distance measurements from a nearby S-layer seed (Cy3 signal) reveals behavior consistent with binding at the edge of a growing S-layer crystal (Fig. 4c, d; Supplementary Fig. 9). Tracking CysRsaA anchored to the LPS outer membrane while utilizing a double helix point-spread function for 3D localization18 allowed calculation of an apparent diffusion coefficient, D = 0.077μm2/s (Fig. 4e, Methods). A comparison with the recent SMT of a similarly sized transmembrane signaling protein anchored to the inner membrane of C. crescentus, CckA (D = 0.0082μm2/s)32, reveals that CysRsaA diffuses an order of magnitude faster.
RsaA monomers diffuse on the outer surface and bind to S-layer crystals. a Schematic of 2D, 2-color single-molecule tracking of CysRsaA on a ΔRsaA cell. Cells were incubated with 2.5 nM Cy3-labeled CysRsaA (green) to create small fluorescent seed crystals. Single molecules of AlexaFluor647 labeled CysRsaA diffused through the agarose mounting pad and bound to the cell surface. b Single-molecule tracking (SMT) shows mobile and immobile single-molecule tracks. The cell boundary is sketched in orange. Scale bar = 1 μm. c Upper: The RMSD from a nearby seed of an example track shows binding of the molecule from ∼16 to 43 s. Binding is defined as RMSD < 57.3 nm (red shaded region, see also Supplementary Fig. 8). Lower: Distance from the nearest nucleation seed (dNS) for the same track shows the molecule binds ∼200 nm from the center of a crystal patch. Green shaded region shows the 300 nm threshold used for determining crystal patch proximity. d A quantitative Venn diagram displaying the total time (circle indicated by gray area), and the fractions of time molecules spend near a crystal patch and unbound (green area), near a crystal patch and bound (yellow area), and bound but not near a crystal patch (red area), shows that when molecules cease diffusing, they are almost always in close proximity to an existing crystal patch. e MSD analysis from 3D SMT of AlexaFluor647 labeled CysRsaA molecules revealing a diffusion coefficient of D = 0.077μm2/s. Shaded regions represent SEM. f A generalizable model for S-layer assembly including secretion, diffusion, and incorporation at gaps within the S-layer lattice. g A model for S-layer crystal boundaries/defects (white) mapped onto the C. crescentus surface (red), suggested by Gaussian curvature calculations (gray colorbar, right)
Based on these observations, we propose a model of S-layer assembly in C. crescentus whereby RsaA monomers are secreted randomly and subsequently explore the LPS outer membrane by diffusion until captured and incorporated into the growing crystalline S-layer structure (Fig. 4f). While SMT of RsaA was performed on ∆RsaA cells, it is plausible that RsaA diffuses either on or within the LPS outer membrane, depending on whether an existing S-layer structure is present above the site of RsaA secretion. Protein diffusion within the LPS outer membrane in gram-negative bacteria has been observed by fluorescence recovery after photobleaching, but individual LPS molecules are immobile33. In C. crescentus, the LPS outer membrane is ∼18 nm thick, while the size of a single RsaA monomer can be approximated by its radius of gyration (Rg = 5.8 nm)14,30. Several crystal structures of S-layer anchoring domains from gram-positive bacteria have been elucidated, revealing multiple polysaccharide binding sites per subunit34,35. Assuming that the N-terminal domain of RsaA has similar multiple carbohydrate binding surfaces, a plausible molecular basis for anchored extracellular protein diffusion is that S-layer proteins migrate along the cell surface by alternately binding and releasing the polysaccharides of neighboring LPS molecules.
Our results imply that continuous native S-layer crystallization occurs at gaps, defects, and grain boundaries within the S-layer structure caused by localized cell wall growth or the inherent topology of the cell surface (Fig. 4g). Reconstituting S-layer assembly using the stepwise addition of exogenously purified CysRsaA yielded a non-uniform pattern of S-layer assembly, indicating a secretion-independent process (Fig. 1j). During this experiment, the long crack-like features observed on the cell body during native assembly (Fig. 1f) are replaced by more widespread, punctate fluorescence signal (Fig. 1h). Reconstituting the S-layer with purified CysRsaA creates small, punctate S-layer crystal patches on the cell surface (Fig. 3g, Supplementary Fig. 7), which are significantly smaller than natively produced S-layer crystals (Fig. 3c, g). Therefore, the morphological difference in fluorescence signal on the cell body between native and exogenous cases could reflect the difference in molecular boundaries created by large versus small S-layer crystals. Small crystals present more boundaries for new S-layer incorporation, rationalizing the increased and less localized fluorescence signal along the cell body observed when reconstituting S-layer assembly by exogenous protein addition.
Increasing localized cell wall growth along the cell body (treatment with 2 μg/mL A22) increases S-layer assembly at that location; however, preventing asymmetric cell elongation (treatment with 25 μg/mL A22) disrupts S-layer assembly at both poles, indicating that an additional factor is coordinating this process (Fig. 2c)2. When the cell lacks an existing S-layer and assembles a new one using a few large crystalline patches, S-layer coverage initially excludes the poles rather than preferentially assembling there (Fig. 3j). Decreasing the size of nucleated S-layer crystals by adding exogenous RsaA to ∆RsaA cells disrupts polar exclusion (Fig. 3j), indicating that an underlying tether within the LPS is not responsible for localizing S-layer assembly.
The molecular defects and boundaries created by imposing a large crystalline lattice on a variably curved surface are sufficient to explain these observations20,21. Regions of non-zero Gaussian curvature values on a 3D model of the C. crescentus topology correlate with the polar and divisional regions of S-layer assembly observed in this study (Figs. 1d, j, 4g). At these regions, we expect small crystalline patches with more defects, allowing for more local RsaA incorporation. Along the cell body, changes in curvature are less dramatic, which produce longer grain boundaries between large crystalline patches20,21. The crack-like features of new RsaA we observe on the cell body further support the model that S-layer assembly patterning reflects growth at natural imperfections in the S-layer crystalline lattice (Figs 1f and 2b, e).
In C. crescentus, RsaA deletion and the subsequent loss of an S-layer disrupts normal cell growth, suggesting a connection between the S-layer structure and cellular fitness30. Given the central role of RsaA crystallization in S-layer assembly and this connection to fitness, the protein's ability to self-assemble may drive natural selection of the RsaA amino acid sequence. Remarkably small but stable RsaA protein crystals consist of only ∼50 molecules (Fig. 3g, k), suggesting that fast, efficient nucleation at low concentrations may be another selectable trait supporting protein self-assembly. SLPs are exceptionally diverse in sequence, varying widely in size (40–200 kDa) and fold4. Functional convergence of diverse crystalline structures can be rationalized by selection driven by protein self-assembly, which can occur independently of overall fold and instead requires just a few key surface residues making symmetric, planar crystal contacts36,37. Similarly, diverse SLPs in archaea prefer charged (acidic or basic) amino acids to facilitate nutrient uptake through the nanoporous S-layer—a function that evolves independently of protein fold38.
The mechanisms by which bacteria build, maintain, and evolve their S-layers are important to human health and our ability to treat and respond to bacterial pathogens such as C. difficile, A. salmonicida, and B. anthracis39,40,41. Additionally, S-layers have been exploited as nanomaterials in a variety of applications4,42, including high-density organization and display of organic or inorganic molecules tethered to RsaA in particular43,44. This study proposes a mechanism by which bacteria can control extracellular structures without direct intracellular feedback, exploiting the biophysics of macromolecular 2D crystalline self-assembly on curved 3D surfaces. In particular, defects and natural imperfections within the S-layer lattice serve as sites of new S-layer growth. Further manipulation of this seemingly unsupervised assembly pathway may lead to treatments that target cell surface structures such as the S-layer or allow enhanced utilization of S-layers as self-assembling macromolecular nanomaterials.
Three strains were used in this study and are available from the corresponding author S.W. upon request. C. crescentus NA1000 (ATCC 19089), which is referred to as wild type (WT) throughout the text, was used as a control for fluorescent labeling, S-layer protein production, and drug treatment response. An RsaA-negative strain of NA1000, referred to as ∆RsaA, was generated via clean genomic deletion. We amplified 802 bp upstream and 806 bp downstream of rsaA using primer pairs RsaAUpstreamF/R (5′-CTACGTAATACGACTCAGGCCGCGATCAGTGCCGACGCG-3′ and 5′-ACGTTCGCTTAGGCCATGAGGATTGTCTCCCAAAAAAAATCCCACACCC-3′) and RsaADownstreamF/R (5′-TGGCCTAAGCGAACGTCTGATCCTCGCCTAG-3′ and 5′-CGGCCGAAGCTAGCGGGCCATGGTGGCCATCTGGATC-3′). The two fragments were inserted into SpeI and EcoRI-linearized pNPTS138 by Gibson assembly. The resulting plasmid was electroporated into C. crescentus NA1000. Deletion mutant was isolated using a two-round selection approach45.
For fluorescence microscopy and protein purification, a cysteine mutant of RsaA, CysRsaA, was created previously13 and obtained from the Smit Laboratory (University of British Columbia). Briefly, RsaA with a 7-residue N-terminal tail was cloned into the p4A vector and introduced to background strain JS1023 by electroporation13,17. JS1023 contains the repBAC operon to enable plasmid replication as well as an amber mutation within the native rsaA gene and a disruptive insertion within the gene for an S-layer associated protease, sap17. All liquid and plated cultures involving CysRsaA included 2 μg/mL chloramphenicol.
S-layer protein purification
Purified RsaA in the absence of CaCl2 was previously shown to partially unfold at 28 °C30. Therefore, CysRsaA samples were kept cold (<4 °C) at all times unless otherwise noted. CysRsaA protein was purified similarly to previously reported methods30,46. CysRsaA-producing C. crescentus cells were grown to early stationary phase at 30 °C in PYE medium, shaking at 200 rpm. The culture was then pelleted by centrifugation and stored at −80 °C. Approximately 1 g of cell pellet was thawed on ice, re-suspended with 10 mL of ice cold 10 mM HEPES buffer pH 7.0, and centrifuged for 4 min at 18,000 rcf. This washing step was performed three times. The pellet was then separated into 10 aliquots and 600 μL of 100 mM HEPES buffer pH 2.0 was added to each aliquot. These cell suspensions were incubated on ice for 15 min and then spun for 4 min at 18,000 rcf. The supernatants were then pooled and neutralized (pH = 7) by the addition of 5 N NaOH. To remove free divalent cations and reduce cysteine side chains, 5 mM Ethylenediaminetetraacetic acid (EDTA) and 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were added. The protein solution was then syringe filtered using a 0.22 μm PES syringe filter and 5 mL were injected onto a Highload Superdex200 16/600 size exclusion column (GE Healthcare). During size exclusion chromatography, the running buffer consisted of 50 mM Tris/HCl pH 8.0 and 150 mM NaCl. Monomeric CysRsaA consistently eluted at ∼0.55 column volumes (Supplementary Fig. 1). From 1 g of pelleted cells, we consistently purified at least 1 mg of monomeric CysRsaA protein. Purity was assessed by SDS–PAGE (Supplementary Fig. 1).
Time-resolved Blot
WT or CysRsaA-producing C. crescentus cells were grown in modified M2G medium containing 50 μM CaCl2 (normally 500 μM) to log phase (OD600nm = 0.5). At t = 0 min, CaCl2 was adjusted to 500 μM using a sterile 1 M stock. The culture was then incubated at 30 °C, shaking at 180 rpm with 0.5 mL aliquots removed every 15 minutes. Aliquots were immediately spun down and snap frozen in LN2. Soluble RsaA extraction was performed as above (without chromatography) and analyzed by SDS–PAGE.
Fluorescent labeling of the S-layer protein
CysRsaA protein was fluorescently labeled by covalent maleimide chemistry both in vivo and in vitro. For in vivo experiments, 1 μM STAR RED (Abberior) or DY-480XL (Dyomics) Cys-reactive fluorescent label was introduced to 1 mL of log-phase (0.1 < OD600nm < 1.0) CysRsaA cells in minimal medium (M2G) at 30 °C. After 15 minutes, cells were washed with 1 mL of M2G once if another fluorophore was to be added next or twice if the next step was imaging. Complete labeling was evidenced by highly spatially complementary fluorescent images in pulse-chase labeled cells (Fig. 1b).
For in vitro experiments, purified CysRsaA was buffer exchanged into 50 mM HEPES pH 7.0 and 150 mM NaCl using a 30 kDa MWCO centrifugal concentrator (Sartorius). Overnight labeling of CysRsaA protein ( > 20 μM) was performed on ice with the addition of 1 mM TCEP and at least 5-fold stoichiometric excess of maleimide-derivatized, cell-impermeable fluorescent dyes, STAR RED and DY-480XL, which are well-suited for confocal and STED imaging. Cell-impermeable Sulfo-Cy3 (Lumiprobe) was used as a diffraction-limited indicator for S-layer seed crystals. AlexaFluor647 (ThermoFisher) was used to track single CysRsaA molecules since SMT requires a particularly bright and photostable fluorophore. The next day, three successive 1:30 dilutions were performed to remove unbound dye molecules using a 30 kDa MWCO centrifugal concentrator (Sartorius) and buffer containing 50 mM Tris/HCl pH 8.0 and 150 mM NaCl. Absorbance measurements at 280 nm and the known absorbance peak for each fluorophore determined labeling efficiency, which varied from 50 to 90%.
Growth curves
For growth curve analysis of WT and ΔRsaA C. crescentus strains, 10 μL of mid-log phase cultures (OD600nm = 0.5) were added to 90 μL of M2G containing varying amounts of A22 (Cayman Chemical) or cephalexin (Frontier Scientific) in a sterile, black-walled, 96-well transparent plate (Corning). While incubating the plate at 29 °C and shaking at 600 rpm between readings, OD600nm was measured every 5 min for up to 23 h using an Infinite M1000 microplate reader (Tecan).
Confocal and STED microscopy
Images were acquired on a bespoke 2-color fast scanning STED microscope22. Briefly, the pulsed 750 nm depletion beam is provided by a titanium-sapphire mode-locked oscillator (Mira 900D, Coherent) running at 80 MHz, providing an average power of 120–130 MW/cm2 at the sample plane. A vortex phase plate imparts the STED donut shape (RPC Photonics). 530 nm and 635 nm pulsed diode lasers are used for excitation (LDH-P-FA-530B & LDH-P-C-635B, PicoQuant), providing an average power of 40–60 kW/cm2 and 50–80 kW/cm2, respectively. The laser beams are focused and fluorescence is collected through an oil immersion objective (Plan Fluor 100 × /1.3 NA, Nikon). A 7.5 kHz resonant mirror (Electro-Optical) scans the beams along the fast axis, while the slow axis is scanned using a piezo stage (PD1375, Mad City Labs). Fluorescence is collected through a ∼0.7 Airy unit (AU) and 0.8 AU pinhole (red and green channels, respectively), spectrally filtered from 650 to 710 nm or 550 to 615 nm, and detected on a Si APD detector (SPCM-ARQH-13, Perkin Elmer). Microscope control and image acquisition arose from a custom LabView program running on an FPGA (PCIe-7842R, National Instruments). Confocal images are taken using no depletion laser, a pixel size of 100 nm, and an average pixel dwell time of ∼0.25 ms/pixel. STED images have a pixel size of 20 nm, and an average pixel dwell time of 0.1 ms/pixel for the red channel. Images in the green channel are the sum of two frames, smoothed with a σ = 0.9 pixel Gaussian filter and an average pixel dwell time of 30μs/pixel/frame. STED images have a FWHM resolution of ∼60 nm and ∼80 nm in the red and green channels, respectively22,47.
Binary cell profile analysis
S-layer cell profiles were analyzed using a custom MATLAB algorithm (Supplementary Figure 3). STED images were aligned to the gradient of the transmitted light image via cross-correlation to correct for sample drift during scanning. Well-separated cells were selected by hand and aligned to be horizontal with the stalk on the left side using a radon transform (except Fig. 3j, where stalks cannot easily be identified on cells for de novo assembly and exogenous addition experiments). A cell axis was determined by fitting a second order polynomial to the maximum intensity transmitted light pixels. For the cephalexin treated cells, a smoothing spline was used instead of a second order polynomial due to the longer cell length. Cells were classified as swarmer or stalked, and swarmer cells were omitted from analysis. For each cell, a 200 nm wide cell outline was defined. For conditions with a mostly complete S-layer (Figs. 1d and 2c), first the outer cell boundary was determined by thresholding a normalized sum of the two-color channels, and then this outer boundary was eroded to form the 200 nm cell outline. For analysis including conditions with an incomplete S-layer (Fig. 3e), the center of the cell outline was determined by finding the maximum gradient of the transmitted light image around the cell axis, followed by expansion to a 200 nm wide cell outline. Then, the presence of S-layer was determined by applying a binary intensity threshold to the STED images smoothed using a Gaussian filter with σ = 0.9 pixels (18 nm). An upper and lower binary cell profile were determined by projecting each pixel onto the cell axis and binning into 40 equi-length bins, where a single pixel identified as positive for S-layer in a bin makes the entire bin positive. The fraction of cells with S-layer at each position along the normalized cell axis can then be computed (combining both upper and lower halves), yielding a binomial observation for the probability of finding S-layer at a given position. 95% confidence intervals were determined using the Wilson score interval. All imaging experiments except for Figs 2e and 3e, i were performed on cell populations from at least two independently created samples.
Puncta quantification
Counting the number of crystal puncta per cell upon exogenous addition of RsaA was performed by identifying spots with signal greater than 10 standard deviations above background. Quantitation of the number of RsaA monomers per punctum was performed using a custom MATLAB algorithm. In-focus puncta were fit using non-linear least squares to an asymmetric Gaussian. These fits were used to determine the number of photons per punctum. The number of photons per RsaA monomer was determined by imaging single molecules of RsaA-STAR RED in vitro on a poly-L-lysine coated coverslip. By calculating the photons per molecule of both STED and confocal images of the in vitro sample, a photobleaching correction factor was determined by comparing the number of molecules identified in both confocal and STED images of identical fields-of-view. The final number of RsaA monomers per punctum was corrected for photobleaching, as well as the in vitro labeling efficiency of the exogenously added RsaA, as determined by absorbance at 280 nm and the absorbance of STAR RED at 640 nm.
2D and 3D single-particle tracking microscopy
Images were acquired on a custom-built 2-color inverted microscope (Olympus, IX71) for imaging Cy3 and AlexaFluor647 (Supplementary Figure 10). Labeled biological samples were mounted on a 2D micrometer stage and in contact with an oil-immersion objective (Olympus, ×100, 1.4 NA, UPLANSAPO). Shutters were used in a sequential, interleaved fashion, where the sample was first exposed to the 641 nm laser (Coherent Cube, 100 mW) for 1.8 s at an intensity of 88.5 W/cm2, then the 514 nm laser (Coherent Sapphire, 100 mW) for 0.2 s at an intensity of 1.6 W/cm2. Emission is collected with the objective, passes through a dichroic filter (Semrock, FF425/532/656-Di01), and another dichroic filter (594LP), which allows us to spectrally separate the emission. Alexa647 emission is detected on one EMCCD camera (Andor, DU-897U-CS0-#BV) with two emission filters (Chroma, 680–60; Chroma, 655LP) and Cy3 is detected on a separate EMCCD camera (Andor, DU-897U-CS0-#BV) with an emission filter (Semrock, 578–105), both recorded at a framerate of 20 frames/s and an electron-multiplying gain of 200. During the first ∼10 s of acquisition, the intensity of the 641 nm laser is briefly increased to a higher intensity to allow the fluorescent dyes to bleach down approximately to the single-molecule regime before reducing the intensity for optimal tracking. Spatial correlation between the two cameras is determined by detecting Tetraspeck beads (Invitrogen), which appear in both channels. 3D tracking was performed with a double-helix phase mask placed at the Fourier plane in the detection side of our microscope. Imaging conditions are similar to our 2D single-particle tracking experiments, except the 641 nm laser was used for detecting labeled RsaA molecules with an intensity of 885 W/cm2. All image acquisition was performed through software made by Andor. Pixel size is 163 nm. All tracks shown (Fig. 4b and Supplementary Figure 9) are down-sampled 5X for clarity.
3D Calibration with Fiducials
In 3D single-molecule tracking, the double helix point spread function (DH-PSF) allowed extraction of the xyz position of individual emitters in the field of view48. Spatial calibrations utilized fluorescent beads (FluoSpheres 0.2 µm, crimson fluorescent (625/645)), spin-coated from 1% polyvinyl alcohol onto a glass coverslip. Using a piezo-electric z-motion stage, calibrations were acquired over a 3 µm range along the z axis with a 50 nm step-size with 30 frames measured at each z-height. This calibration step produces template images of the DH-PSF, which are used for the identification of single-molecule signals during post-processing of the raw data. All imaging was performed at 25 °C. Using the easyDHPSF MATLAB program49, a z-axis calibration over a 3 µm range is obtained via a 2D Double-Gaussian fit, which provides us with xy positions, width, amplitudes, and offset levels of each lobe of the fluorescent bead.
Mobility analysis of 2D RsaA tracking data
Images of single molecules and beads were analyzed using custom-built MATLAB code. Within one C. crescentus cell, a 2D symmetric Gaussian fit is applied to a single labeled RsaA molecule of interest, which provides an estimate of its xy position at that point in time. Linking together the trajectory of the same molecule over time generates tracks. In order to determine whether the detected molecules were bound or not, we first calculated the root mean squared deviation (RMSD) from the mean position over a 1 s sliding window (20 frames). Bound molecules will have a relatively low RMSD (near the localization precision) compared to a molecule freely diffusing within the cell. By performing this for every track (9 tracks) within the same cell, we were able to generate a histogram of all RMSD values (6625 RMSD values), which shows a clear peak with a tail (Supplementary Figure 8), where the left-most peak arises from the localization precision error for bound molecules. The Gaussian fit of the lowest RMSD population was utilized to determine a binding threshold (RMSD < 57.3 nm) corresponding to 2σ above the mean. Molecules were classified as bound if RMSD < Threshold or unbound if RMSD ≥ Threshold).
2-Color analysis of 2D RsaA/nucleation site tracking data
The xy location of each nucleation site, labeled with Cy3, was separately determined by fitting a 2D symmetric Gaussian. The locations of these nucleation sites was found to be stationary over the ∼15 min imaging period. Using the 2D RsaA trajectories analyzed earlier, we calculated the dNS, which is defined as the distance between the RsaA molecule and the nearest nucleation site. By calculating both the RMSD and the dNS, we can categorize each frame as the following: bound only, bound and close to a nucleation site, or close to a nucleation site only (Supplementary Figure 9).
3D Mean Square Displacement (MSD) Analysis
Images of RsaA imaged in 3D were analyzed using custom-built MATLAB code for analyzing DH-PSF data. A 2D Double-Gaussian fit was applied to each emitter in the field-of-view, which provides us with x, y, and θ information from the tilt of the two lobes of the double-helix. We use the calibration obtained earlier to convert our estimates to xyz values. For each individual track, the MSD is computed over a series of time lags starting from 50 ms. We then pool the data over all 30 trajectories to obtain a 3D MSD plot. The diffusion coefficient is extracted by fitting the following equation to the first 4 time lags:
$${\mathrm{MSD}}_{3{\mathrm{D}}} = 6{\mathrm{D}}\left( {\tau - \left( {\frac{{{\mathrm{\tau }}_{\mathrm{E}}}}{3}} \right)} \right) + 2(s_1^2 + s_2^2 + s_3^2)$$
where D is the diffusion coefficient, τ is the time lag, τE is the exposure time of the camera (50 ms), and si is the localization error in the ith dimension (sx,y,z = 93 nm, 93 nm, 91 nm).
Gaussian curvature analysis
Point cloud localization data of positions on the C. crescentus surface were obtained previously by a covalently surface-attached fluorophore18. A surface mesh was extracted with the Poisson Surface Reconstruction algorithm, available through MeshLab, an open source software package50. Gaussian curvature information can be extracted from the computed mesh22.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
Data supporting the findings of this manuscript are available from the corresponding authors upon reasonable request. A reporting summary for this Article is available as a Supplementary Information file. The code used in this study is either open access49,50 or is described above and available upon reasonable request. Matlab analysis was performed using version 9.0.0.341360. The source data underlying Figs 1j, 2c, d, 3a, j, k, 4c-e and Supplementary Figs 1b, c, 3c, 4a, b, 5a, b, 6c, 8, 9 are provided as a Source Data file.
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This work was supported in part by the National Institute of General Medical Sciences: Grants No. R01-GM086196 to W.E.M. and L.S., R35-GM118067 to W.E.M., and R35-GM118071 to L.S., and in part by the US Department of Energy, Laboratory Directed Research and Development under contract No. DE-AC02–76SF00515 and Biology and Environmental Research to S.W. L.S. is a Chan Zuckerberg Biohub Investigator. J.S. was supported by the Natural Sciences and Engineering Research Council of Canada Discovery Program (Grant No. RGPIN 36574–11). C.J.C., J.H., and J.Y. were supported in part by the National Science Foundation Graduate Research Fellowship Program (NSF-GRFP). J.H. was supported in part by the US Department of Energy Office of Science Graduate Student Research Program (DOE-SCGSR). The authors thank Greg Stewart/SLAC for graphic design support.
These authors contributed equally: Colin J. Comerci, Jonathan Herrmann.
Biophysics Program, Stanford University, Stanford, 94305-5101, CA, USA
Colin J. Comerci & W. E. Moerner
Department of Chemistry, Stanford University, Stanford, 94305-4401, CA, USA
Colin J. Comerci, Joshua Yoon & W. E. Moerner
Department of Structural Biology, Stanford University, Stanford, 94305-5101, CA, USA
Jonathan Herrmann, Fatemeh Jabbarpour & Soichi Wakatsuki
Bioscience Division, SLAC National Accelerator Laboratory, Menlo Park, 94025, CA, USA
Department of Applied Physics, Stanford University, Stanford, 94305-4090, CA, USA
Joshua Yoon & W. E. Moerner
Department of Developmental Biology, Stanford University, Stanford, 94305-5329, CA, USA
Xiaofeng Zhou & Lucy Shapiro
Department of Microbiology and Immunology, University of British Columbia, Vancouver, V6T 1Z3, BC, Canada
John F. Nomellini & John Smit
Colin J. Comerci
Jonathan Herrmann
Joshua Yoon
Fatemeh Jabbarpour
Xiaofeng Zhou
John F. Nomellini
John Smit
Lucy Shapiro
Soichi Wakatsuki
W. E. Moerner
Conceptualization: C.J.C., J.H., L.S., S.W., and W.E.M. Investigation: C.J.C., J.H., J.Y., and F.J. Formal analysis: C.J.C., J.H., J.Y., X.Z., and W.E.M. Resources: X.Z., J.N., J.S., and L.S. Writing—original draft: C.J.C. and J.H. Writing—review and editing: All authors. Supervision and funding acquisition: J.S., L.S., S.W., and W.E.M.
Correspondence to Soichi Wakatsuki or W. E. Moerner.
The authors declare no competing interests.
Peer review information: Nature Communications thanks Behzad Rad, Han Remaut and other anonymous reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
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Comerci, C.J., Herrmann, J., Yoon, J. et al. Topologically-guided continuous protein crystallization controls bacterial surface layer self-assembly. Nat Commun 10, 2731 (2019). https://doi.org/10.1038/s41467-019-10650-x
DOI: https://doi.org/10.1038/s41467-019-10650-x
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House, however, pointed to the PlayStation 4's market dominance and denied that Pro was in any way a response to Microsoft's Project Scorpio project. "When Playstation 4 Pro was being conceived there wasn't a word about Project Scorpio in the marketplace. I would suggest that 'reactive' isn't our approach this time around," he said.
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