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David Dundee and Gillian Oliver.
The Spousal Support Advisory Guidelines is an academic paper released by the federal Department of Justice in July 2008. It is not a law and is not expected to become a law.
When someone is entitled to receive spousal support, the Advisory Guidelines describes several different formulas that can be used to calculate how much support should be paid and the length of time it should be paid for.
While no one is required to use the Advisory Guidelines, lawyers and the courts routinely use them in making decisions about spousal support.
This section provides an introduction to the Spousal Support Advisory Guidelines. It discusses what the courts have had to say about the Advisory Guidelines and describes how its formulas work, how they can be restructured for fairness, and what exceptions exist to the formulas.
In 2001, the federal Department of Justice struck an advisory working group to look into the feasibility of uniform guidelines for the calculation of spousal support. The group was composed of judges, family law lawyers, law school faculty members, and social workers. The group was led by Professors Carol Rogerson and Rollie Thompson, both gifted and highly qualified academics with strong backgrounds in family law.
In January 2005, Professors Rogerson and Thompson released their first paper, Spousal Support Advisory Guidelines: A Draft Proposal, for public comment and feedback. After touring the country speaking to judges, lawyers and academics, and monitoring the case law on the draft Advisory Guidelines as it developed over several years, Rogerson and Thompson released their final paper, Spousal Support Advisory Guidelines, in July 2008.
The Advisory Guidelines is not a law, and people involved in family law disputes are not bound by it. As of July 2008, the Department of Justice said that it had no plans to turn the Advisory Guidelines into a law and didn't intend to do so. I've heard nothing to suggest a contrary intention since.
The Spousal Support Advisory Guidelines is an attempt to capture how the majority of Canadian court decisions on spousal support have determined how much support should be paid, and how long support should be paid for, in mathematical formulas. It is not intended to change the law on spousal support, rather it is intended to normalize future decisions about spousal support based upon how the general majority of past court decisions have dealt with the issue.
The Advisory Guidelines attempts to reflect current practice under the existing law. The Advisory Guidelines is not based on any particular theory of spousal support; it is an independent attempt to reflect the results currently found in the case law on the subject.
The essential concept underlying the Advisory Guidelines is the calculation of support based on the total disposable income available to both parties, rather than looking at each party's needs and means separately. The formulas work with the total amount of money collectively available to a couple.
Income sharing does not mean an equal division of income, however, and the ranges the Advisory Guidelines proposes when child support is paid is between 40% and 46% of the total disposable income available to both parties. When no child support is paid, the Advisory Guidelines gives the recipient a share of the difference between the recipient's income and the payor's income, which increases with the length of the relationship.
The Advisory Guidelines does not deal with whether a spouse is entitled to receive support. Entitlement is, of course, the first question to be decided when dealing with an application for spousal support. The Advisory Guidelines will only be used when a spouse is found to be entitled to spousal support.
Where child support is paid, the amount of support payable will be calculated taking into consideration the payment of child support and the different tax rules relating to spousal support and child support. The parties' net incomes are used to determine spousal support.
In many cases, the Advisory Guidelines sets out a range of years that support will be paid for. In certain cases, such as long marriages, where the dependant spouse is older or when child support is paid, support will be paid for an indefinite period of time.
The key factors in determining the length of time for which support will paid are the length of the marriage plus any period of time the parties lived together before marriage, the age of the recipient of support and the age of the youngest child.
The Advisory Guidelines has both floors and ceilings: where a payor's income is below $20,000, no spousal support will be payable; and, where a payor's income exceeds $350,000, the payor should pay at the amount for incomes of $350,000. The payor's income above that ceiling will be taken into account at the discretion of the court. Although, in a recent case our Court of Appeal warned that the court would still have to be given reasons to depart from the Guidelines ranges, even when the payor’s income is substantially above the $350,000 ceiling: Hathaway v Hathaway 2014 BCCA 310.
To every rule there is an exception, and the Advisory Guidelines is no different. The ranges the Advisory Guidelines formulas produce aren't carved in stone. Factors such as advanced age, illness, debt load and so forth may suggest that the results for amount, duration, or both should be ignored.
The Advisory Guidelines also allows for the restructuring of a support award to pay more for a shorter time, to pay less for a longer time, or to pay it all up front in one lump sum. Restructuring keeps the total amount paid within the results generated by the Advisory Guidelines formulas.
The Advisory Guidelines is written for married or formerly married spouses, because the federal government only has the authority to make rules about spousal support for married couples. The Advisory Guidelines is therefore based on the factors and tests set out in the Divorce Act rather than those set out in the different provincial laws about family breakdown and spousal support.
That said, the Advisory Guidelines works just as well to decide spousal support for married spouses and unmarried spouses since the law that applies to determine spousal support for unmarried spouses in British Columbia, the Family Law Act, works in exactly the same way as the Divorce Act.
The Advisory Guidelines has melded seamlessly into family law practice in this province and is routinely used to determine spousal support, whether in the course of litigation, arbitration, mediation, or negotiation. Professor Thompson was exactly correct when he predicted that lawyers and the courts would come to rely on the Advisory Guidelines by custom. Frankly, the Advisory Guidelines offers a seductively easy solution to a difficult problem.
My views on the Advisory Guidelines have mellowed with time, and while many of my basic concerns remain, they have assumed a lesser significance. Arbitrariness is good, I have concluded. After all, the Child Support Guidelines are essentially arbitrary and so are many other principles of family law. Take the shared custody exception for the calculation of child support. Why 40%? Why not 38.5 or 41.3%? The answer to that question is, I think, "why not?" There has to be a tipping point somewhere, and exactly where on the scale that tipping point lies is immaterial as long as it has is some rational, defensible foundation.
The point of the arbitrariness of the Child Support Guidelines is to reduce conflict by setting out a fixed sum that must be paid depending on nothing more than the payor's income and the number of children. The same principle applies to the Spousal Support Advisory Guidelines, although the formulas are a lot more complicated. If an arbitrary set of numbers reduces conflict and saves money, I'm all for it, as long as those arbitrary numbers have a certain fundamental reasonableness to them.
Until fairly recently, my enthusiasm for the Advisory Guidelines was primarily tempered by the absence of public, free spousal support calculators. This seemed to me to be an appropriate function of government, particularly as it was government that funded the Advisory Guidelines project, however the Department of Justice had no enthusiasm for the project. Making things worse, the two main manufacturers of spousal support software, DivorceMate and ChildView, would not sell their product to people who weren't employed in the justice system in some capacity.
This all changed in April 2011 when DivorceMate stepped up to the plate with a free public website, mysupportcalculator.ca. The website performs child support calculations under the Child Support Guidelines and spousal support calculations under the Advisory Guidelines. The results of the spousal support calculations do not precisely match the results produced by their bells-and-whistles product for professionals and do not account for all of the factors that can impact on the results (such as source of income, tax benefits, deductions and credits, payments to special expenses, and so forth). However, the results will be fine for most people most of the time. DivorceMate deserves much credit for making this resource available, and I thank them for it, whether the resource is a revenue-generating advertising platform or not.
On 4 July 2005, the British Columbia Supreme Court released its first judgment considering the Advisory Guidelines, in the case of W. v. W, 2005 BCSC 1010. The judge said that the Advisory Guidelines "provide a cross check against the assessment made under existing law", and that the formulas provided in the Advisory Guidelines are "consistent with the law in British Columbia". She then made an order for spousal support using the Advisory Guidelines as a check, without expressly applying the Advisory Guidelines to determine the issue. The Advisory Guidelines, she held, is not law, and is not intended to become law.
On 19 July 2005, the court released its second judgment on the Advisory Guidelines, M.S. v. W.S., 2005 BCSC 939. In this case, the judge held, rather emphatically, that the court is not bound by the Advisory Guidelines in determining spousal support and that "equitable distribution can be achieved in a variety of ways and need not be calculated according to a strict formula."
This view was softened later in 2005 by the Court of Appeal in the case of Yemchuk v. Yemchuk, 2005 BCCA 406. In this case, the court held that the Advisory Guidelines reflects the general results seen in the case law on spousal support. While the court stopped well short of saying that the Advisory Guidelines must be used to determine spousal support, it did consider the Advisory Guidelines a useful tool and a factor to be considered in making an order for spousal support, and made an order for support that was within a hair's breadth of the numbers the Advisory Guidelines formulas produced.
As a result of Yemchuk, the law in British Columbia was that the Advisory Guidelines is a factor to be taken into account in fixing the amount and duration of an order for spousal support, but that it is no more than a factor. This changed with Redpath v. Redpath, 2006 BCCA 338, a 2006 decision of the Court of Appeal, in which the court held that it is an appealable error for a judge to fail to consider the results produced by the Advisory Guidelines. This moves things well beyond Yemchuk, as now a trial judge must consider the Advisory Guidelines formula results in making a decision on spousal support. In 2010 the Court of Appeal went even further in Domirti v. Domirti, 2010 BCCA 472, ruling that an award of spousal support that falls substantially outside the Advisory Guidelines ranges may be an appealable error.
The law in British Columbia, then, is that the results of the Advisory Guidelines calculations must be considered when making a decision on spousal support. The Advisory Guidelines is, in other words, all but mandatory in this province.
Of course, this also means that lawyers must become proficient in using the Advisory Guidelines formulas or they risk giving their clients and the courts incorrect information. The Without Child Support calculations, discussed later, are not terribly complex, but the With Child Support calculations demand a basic knowledge of the federal and provincial benefits and credits relating to children, the different deductions that apply to employment and self-employment income, received versus taxable dividends, and a few other wrinkles relating to the calculation of after-tax income. Owning the software required to do the Advisory Guidelines calculations isn't enough. Lawyers need to know how to expertly use that software, with tax and income issues in mind, and the software has to do the math and actually perform those calculations correctly.
Many courts in Canada's other provinces and territories have talked about the Advisory Guidelines in much the same way as our courts have, although with varying degrees of enthusiasm. Alberta has led the charge against the Advisory Guidelines and is the province in which it is least likely to be used to determine a spousal support obligation.
Here is a sampling of judicial comment on the Advisory Guidelines.
"Although the Spousal Support Advisory Guidelines may be of some assistance to the court on an initial application for spousal support, it must be noted that they are advisory only and are not binding on the court. More importantly, the authors of the guidelines make it very clear in the Executive Summary ... that the advisory guidelines do not deal with the effect of a prior agreement on spousal support and that this issue, like entitlement, is outside the scope of the advisory guidelines and would continue to be dealt with under the evolving law guided by the Supreme Court of Canada's recent decision in Miglin v. Miglin."
"Counsel urged the court to consider the Spousal Support Advisory Guidelines. These are not law."
"These Guidelines are not mandatory and are only a suggestion. There has been very little judicial analysis of the Guidelines as they are new."
"I suggest that courts will likely use these Guidelines as a bench mark to see what the support amount would be if the Guidelines were applies. Thus it will serve as another method of calculation, which when coincidentally echoing judicial discretion, will become referenced with approval. In other cases where the courts are either astonished by the payment proposition, that is that the math creates a number that is perceived to be too high or inadequate, the courts will shy away from its application."
"The provisions of the Divorce Act as interpreted by the Supreme Court of Canada are the law in this country with respect to spousal support. The Spousal Support Advisory Guidelines are the work of two university professors, Carol Rogerson and Rollie Thompson, assisted by a small committee. Those with strong views to the contrary were not involved, nor was there widespread discussion of the guidelines prior to their publication. They have not been enacted by the Parliament of Canada or any Provincial Legislature nor are they the subject of any governmental regulation.
"The Guidelines are a cause for concern. There is no doubt that they are useful for a judge who does not wish to make a thorough and careful analysis of each case and wants a quick answer. However, it is not the role of judges to opt out for an easy answer. Rather judges are bound by the Divorce Act and the case law which require judges to do individual justice in each case and not look for a 'cookie cutter' answer.
"As well, the Guidelines are stated to be experimental. It is not the function of courts to experiment on the citizens of this country.
"The authors of the Guidelines state that the Guidelines do not change the law. However, in my view, they attempt to do so. For example, they advocate income sharing which has rarely been accepted in this country except for exceptionally long term marriages. It will not be long before someone will argue that disparity in income equals entitlement. As indicated by Chouinard, J. in Messier v. Delage, a person does not acquire a lifetime pension as a result of marriage. Likewise, marriage is not an insurance policy. The Guidelines also ignore the distribution of matrimonial property which the Supreme Court of Canada in Boston v. Boston, said was relevant. They ignore the goal of self-sufficiency as set out in the Divorce Act. Also they fail to take into account the growing trend, at least in Alberta, towards shared parenting.
"The Guidelines purport not to deal with entitlement. Under the Divorce Act there are degrees of entitlement based on a multitude of factors. However, the ranges set out in the Guidelines are not sufficient to cover the many possible degrees of entitlement.
"The Guidelines set an arbitrary and presumptive range for both amount and duration. The onus is reversed requiring the payor to justify an exception to the Guidelines. The presumptive nature of the Guidelines does away with a claim based on evidence.
"The stated purpose of the Guidelines is to bring more certainty and predictability to the determination of spousal support. As a result individual justice is sacrificed for consistency. Every case is different on its facts. There are often many variables. Flexibility and discretion are needed for individual circumstances.
"Judges should exercise extreme caution in using the Guidelines. The Divorce Act and the decisions of the Supreme Court of Canada call on judges to undertake a thorough analysis in each case. Not to do so is to disregard the views of the highest court in this country and to make a mockery of stare decisis."
However, despite these differences in judicial attitude elsewhere, until or unless the Supreme Court of Canada rules otherwise, BC remains a pro-Guidelines jurisdiction.
One formula for when child support is not being paid (the Without Child Support Formula), as might be the case if the couple have no children or if all of the children are adults and financially independent at the time of separation.
Another formula for when there is a legal obligation to pay child support (the With Child Support Formula), whether child support is actually being paid or not.
when all of the children for whom support is being paid are older than the age of majority.
The amount of support is 1.5 to 2 percent of the difference between the parties' gross incomes for each year of marriage.
For example, say a relationship is 10 years long, and Party A has a gross income of $50,000 and Party B has an income of $20,000. The difference between the parties' incomes is $30,000. Party B would have share in the difference of 15 to 20 percent (1.5 times 10 and 2 times 10), or between $4,500 and $6,000 per year. On a monthly basis, support would be paid at $375 to $500.
The length of time support will be paid is 0.5 to 1 year for each year of the relationship. If a couple have been together for more than 20 years, or if the age of the dependant party plus the number of years of the relationship equals 65, support will be paid indefinitely.
Using the same example, support would be payable for 5 to 10 years (0.5 times 10 and 1 times 10). If, however, the dependent party was 55 at the time of separation, support would be paid indefinitely (55 years of age plus 10 years equals 65).
The maximum amount payable under this formula ranges from 37.5% of the difference between the parties' gross incomes to 50% of the difference between the parties' incomes, net of taxes and benefits.
The maximum time spousal support can be payable under this formula ranges from an amount of time equal to the duration of the parties' cohabiting relationship to an indefinite amount of time.
This formula is a lot more complex. In this formula, child support is taken out of the payor's gross income and the recipient's income, taxes are taken into account, and government benefits are added to the recipient's income. The reason why child support is deducted from the recipient's income is to reflect the costs that parents bears in raising the children.
The amount of support is 40 to 46% of the payor's individual net disposable income plus the recipient's net disposable income.
The payor's net disposable income is their gross income, minus taxes and minus their child support obligation, including the tax credits they receive as a result of paying spousal support.
The recipient's net disposable income is their gross income, minus taxes and minus their notional child support obligation, plus any government benefits they receive, less the taxes payable as a result of receiving spousal support.
Say the parties have an 8 year old child, the payor has a gross income of $50,000 and the recipient has an income of $20,000. The payor's net disposable income is $26,710 ($50,000 minus taxes of $15,570, minus annual child support of $5,112, minus EI deductions of $772, minus CPP deductions of $1,831). The recipient's net disposable income is $15,045 ($20,000 minus taxes of $4,410, minus notional child support of $2,052, minus EI deductions of $396, minus CPP deductions of $816, plus child tax benefit of $1,208, plus national child benefit of $1,511).
The family's net disposable income is $41,755 ($26,710 plus $15,045). 40 percent of the net disposable income is $16,702; 46 percent of the income is $19,207. After deducting the recipient's net disposable income, the difference between the recipient's income and 40 percent of the family's disposable income is $1,657 per year, and $4,162 for 46 percent.
On a monthly basis, spousal support would be between $138 and $346. The total the payor would pay each month would be spousal support plus $426 in child support.
The length of time for which support will be paid ranges from the longest of two formulas used to determine the low end of the range to the longest of two formulas used to determine the high end of the range. If a couple have been together for more than 20 years, or if the age of the dependant party plus the number of years of the relationship equals 65, support will be paid indefinitely.
I told you it was complex. To quote Professor Thompson, "this is not a calculation you can do on the back of an envelope, you will need a computer program." This formula requires a detailed understanding of how income is determined under the Advisory Guidelines and of the various government benefits, tax deductions and tax credits that can apply to adjust net income.
I've written a paper on the subject for the Department of Justice, "Obtaining Reliable and Repeatable SSAG Calculations," which is available to the public. Be warned: it's a bit dry.
The maximum amount payable under this formula is the range the formula sets out: 40% to 46% of the difference between the payor's net disposable income and the recipient's net disposable income.
The maximum time spousal support can be payable under this formula is an indefinite amount of time.
the number of years until the youngest child starts and leaves full-time school.
the children are step-children to the payor.
These formulas use modified or hybrid versions of the Without Child Support and main With Child Support formulas.
The Advisory Guidelines includes a few ways to accommodate circumstances that might make the formulas' results unfair to the people involved. The Advisory Guidelines requires that the parties first attempt to use the ranges to solve the problem, but allows for the results to be restructured if adjustments within the ranges fail to solve the problem.
pay the total amount payable over the period of the award in a single lump sum.
The point of each option is that the total amount payable under the formula results stays the same. The total amount is just paid sooner or later than the results for duration would normally require.
Restructuring will not work if the length of time support is to be paid for is indefinite. In cases like this, the best option is probably to build in a review date, a date on which the recipient's entitlement to receive spousal support will be checked.
If adjusting support within the ranges doesn't work and if restructuring support doesn't work, the Advisory Guidelines allows parties to depart from the numbers produced by the formulas to accommodate their special circumstances. The authors of the Advisory Guidelines are clear that these exceptions are to be treated as a last resort.
In shorter marriages, the results produced by the formulas might not reflect a recipient's right to be compensated for a sacrifice made in the course of the marriage. Circumstances that might fall into this exception would include: giving up a job to be with the payor; or, moving across the country to be with the payor and losing a job or a business.
Under this exception, the results produced by the formulas for amount and duration will not apply.
The length of time the formulas require support to be paid may not be adequate for a recipient who is ill or disabled, or otherwise unable to become self-sufficient.
Under this exception, the results produced by the formula for duration will not apply.
A payor who winds up being responsible to pay for debts incurred during the marriage may not be able to meet the payments required by the formula for amount. This will particularly be the case for families whose debts exceed their assets.
Under this exception, the results produced by the formula for amount will not apply.
In cases where a payor has an obligation to provide support to other people, such as prior spouses or children from previous relationships, the payor may not be able to pay spousal support at the amount required by the formula.
Under this exception, the payor's income is adjusted to deduct the amount of support paid as a result of the previous relationship before calculating the amount spousal support to be paid for the present relationship.
The Advisory Guidelines allows still other exceptions from the formulas to address situations where a child has special needs that result in the parents having greater expenses than other parents; where a relationship was very short but resulted in significant economic loss to the recipient ― like the loss of a career opportunity or an expensive move; where payment of support within the ranges for amount is not sufficient to meet the recipient's needs; and, where the payor's income is not taxed.
If you think your situation falls into one of these exceptions, or one of the exceptions discussed above, you should seriously consider hiring a lawyer for advice about your situation and how the Advisory Guidelines may apply.
This information applies to British Columbia, Canada. Last reviewed for legal accuracy by David Dundee and Gillian Oliver, June 9, 2017.
The obligation of a party to prove their case; the burden of proof. The onus usually lies on the party who makes a claim, although in certain circumstances this burden is reversed, usually by operation of statute.
The remainder of a person’s annual income after the mandatory deductions have been paid, which may include CPP, EI, income taxes and union or professional dues. For self-employed persons, necessary and reasonable business and operating expenses may also be deducted to determine net income.

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After a revision of the Diagnostic and Statistical Manual (DSM), and thousands of public comments later, the Social Security Administration (SSA) has published significant revisions to its mental impairment listings. SSA had issued a Notice of Proposed Rule Making in August 2010, proposing what at the time appeared to be major changes to the listings. As with most SSA revisions, there is good with the bad. SSA has added a new listing pertaining to Post Traumatic Stress Disorders (PTSD), but has eliminated Listing 12.05C, which encompasses low IQ scores combined with another “significant” impairment. SSA has recognized that Licensed Clinical Social Workers frequently serve as therapists, renaming both as “clinical mental health counselors,” but are still not acceptable medical sources. It also added social workers, shelter staff, and other community support and outreach workers to the list of examples of non-medical sources of evidence.
It has acknowledged some of the unique circumstances of claimants facing homelessness, including an example of a situation that makes it difficult to provide longitudinal medical evidence. It included a recognition that periods of lack of treatment or noncompliance may result from a claimant’s mental disorder. And it rejected suggestions it adopt the use of symptoms validity testing to identify malingering.
But SSA removed from the final regulations proposed language about the effects of work-related stress and the questionable validity of mental status exams. Instead, mental status exams are among the list of evidence from medical sources that SSA will consider, along with psychiatric or psychological rating scales. On the other hand, SSA removed all references to standardized tests to inform assessments, except in relation to Listing 12.05.
Also included in the introductory language at 12.00 is an acknowledgement that evidence of functioning in an unfamiliar setting does not necessarily show how a claimant would function on a sustained basis in a work setting. See §12.00C6a. It also specifies how different levels of support and structure should be evaluated. A “complete picture” of daily activities should be considered, with a recognition that the ability to perform “some routine activities without help and support does not mean that you do not have a mental disorder or that you are not disabled.” By way of example, §12.00D3a cites routine activities such as taking care of personal needs, cooking, shopping, paying bill, living alone, or driving.
SSA did not, as previously proposed, eliminate the special technique, known as the psychiatric review technique. It agreed with commenters who believed it is a useful tool for adjudicators and helps increase consistency in decision making.
SSA backed away from broad changes to the “A” – or diagnostic – criteria of the listings. Each category still contains “A” criteria. Introductory section 12.00A describes how the listings are arranged. Section 12.00B gives examples of the mental disorders evaluated under each category, or listing.
SSA defends the removal of “activities of daily living” (ADL) as a criterion by claiming it will continue to evaluate how a person performs ADL; it will use that evaluation as a principal source of information rather than a criterion of disability. The focus of the “B” criteria is instead on the mental abilities a person uses to perform work activities. Examples of the ability to understand, remember or apply information include following one-or-two step oral instructions. Interacting with others includes the ability to handle conflicts with others, responding to social cues, and keeping interactions free from excessive irritability. Adapting or managing oneself encompasses the ability to regulate emotions and control behavior, including but not limited to responding to demands, and maintaining personal hygiene. “Repeated episodes of decompensation” has been eliminated. See §12.00E for the complete listing of examples, which, according to SSA, are non-exhaustive.
In response to comments, SSA did agree to change “and” in the previous version of the criteria to “or,” acknowledging that a claimant need not demonstrate a limitation in each of the three parts of B1 and B3. It also acknowledges in Section 12.00F3f that if a claimant has a “marked” or “extreme” limitation in any single part of B1 or B3, s/he has that degree of limitations for that particular B criterion.
The evaluation of the new “B” criteria will be more comparable to that used to evaluate functional equivalency in children, requiring two marked limitations or one extreme. SSA has created a five-point rating scale (none, mild, moderate, marked, and extreme) to evaluate limitations under the B criteria. As with childhood functional equivalency, SSA’s definitions in 12.00F2 for these terms are less than crystal clear. For example, “mild” means functioning is “slightly limited,” while moderate represents fair, “marked” is seriously limited, and “extreme” means an inability to function on a sustained basis. SSA acknowledges, however, in response to comments, that “extreme” does not mean a total lack or loss of ability to function. In response to comments about the potentially confusing use of these terms by clinicians, SSA added language to §12.00F3a acknowledging the use of these descriptors by clinicians will not always be the same as the degree of limitation specified by the “B” criteria.
SSA retains the “C” criteria as an alternative severity criterion for those situations where a claimant has achieved marginal adjustment, but whose symptoms are diminished because of psychosocial supports or treatment. SSA retained the two year documentation requirement and the requirement that the disorders are “serious and persistent” from the current listings. In 12.00G2c, SSA has substituted the term “deterioration” for “decompensation” in the evaluation of “marginal adjustment.” According to SSA, “decompensation” refers to an extreme state of deterioration, often leading to hospitalization, that exceeds the degree of impairment intended in the “C” criteria. Examples of deterioration, however, include becoming unable to function outside of more restrictive setting without additional support.
Note that Listings 12.07 (Somatic symptom disorder), 12.08 (Personality and impulse control disorders). 12.10 (Autism Spectrum Disorder), 12.11 (Neurodevelopmental disorders), and 12.13 (eating disorders) not include the “C” criteria. According to SSA, experts and program experience indicate the unique situations described in the “C” criteria typically do not apply to these disorders.
No longer known as “Organic Mental Disorders,” the new listing requires a “significant cognitive decline in one or more cognitive areas, and the usual B criteria.” Examples include dementia related to various medical conditions, including Alzheimer’s. It also covers traumatic brain injuries (TBI) and substance induced cognitive disorders.
Examples in this category include schizophrenia, schizoaffective disorder, delusional disorder, and psychotic disorders due to another medical condition.
The listing has been reorganized to reflect the diagnostic criteria from the DSM-5 and the American Association on Intellectual and Developmental Disabilities. It now has only two paragraphs: 12.05A for those claimants whose cognitive limitations prevent them from taking a standardized intelligence test, and 12.05B, for those who are able to be tested. Neither section contains the current “capsule definition,” but each contains three subparagraphs, the last of which requires evidence that demonstrates or supports the conclusion that the disorder began prior to age 22. Per §12.00H4, if evidence recorded before age 22 is not available, SSA will require evidence about current intellectual and adaptive functioning and the history of the disorder to support the conclusion the disorder began before age 22. Examples include school records indicating a history of special education, statements from employers or supervisors and from people who may be able to describe the claimant’s functioning in the past and currently.
The first subparagraph of each section requires “significantly sub average general intellectual functioning,” which for 12.05A is measured by the inability to function at a cognitive level necessary to participate in standardized intellectual testing. For 12.05B, it is measured by a full scale IQ score of 70 or below OR a full scale score of 71-75 accompanied by a verbal or performance IQ score (or comparable part score) of 70 or below. This is a significant change from the standard in the current listing, which relies on the lowest score, which might not necessarily be the full scale. SSA claims the full scale scores are the most reliable evidence that a person has an intellectual disability, and not another impairment that affects cognition.
SSA’s prefatory comments contain fairly detailed discussion about its decision to rely on the full scales scores. It has also made clear that only “qualified specialists, Federal and State agency medical and psychological consultants, and other contracted medical and psychological experts, may conclude that an obtained IQ scores(s) is not an accurate reflection of a claimant’s general intellectual functioning.” See §12.00H2d. Will this mean the ALJ cannot decide test scores are not valid?
The second of the three required subparagraphs require significant deficits in adaptive functioning. In terms of 12.05A, that will be demonstrated by dependence on others for personal needs. For 12.05B, it will be manifested by meeting the B criteria. Per §12.00H3c, standardized tests of adaptive functioning will not be required, but will be considered if they already exist. According to §12.00H3d, the fact that the claimant can engage in everyday activities such as caring for personal needs, preparing simple meals, or driving a car, will not always disprove deficits in adaptive functioning. Nor will lack of deficits in one area negate deficits in another. And pursuant to §12.00H3e, past work activity will not necessarily disprove deficits. SSA will consider, for example, whether the job required extra time or supervision, or involved more limited duties. Helpful nuggets that will require lots of extra digging and preparation by advocates!
Of concern is SSA’s cross-reference to new listing 12.11 – Neurodevelopmental disorders, discussed infra, which includes specific learning disability and borderline intellectual functioning (BIF). According to SSA, other mental impairments such as specific learning disability and BIF do not involve the same nature or degree of sub average intellectual functioning and deficits of adaptive function as intellectual disabilities. Query regarding how consultative examination reports diagnosing BIF even when IQ scores are below 70 will be treated?
Examples include symptom disorder, illness anxiety disorder, and conversion disorder – disorders characterized by physical symptoms that are not feigned but cannot be fully explained by a general medical condition, mental disorder, substance use, or culturally sanctioned behavior or experience.
In addition to personality disorders, examples of disorders evaluated under this listing include intermittent explosive disorder, which was added to both the adult and childhood version in response to comments.
The current reference listing for Substance Abuse Disorders was eliminated.
SSA’s preface contains extensive discussion of its decision making involving this category. In response to comments, it removed references to Asperger’s disorder. But it declined to specify that the core nature of Autism Spectrum Disorder (ASD), as suggested by commenters, is not an intellectual impairment but a social and behavioral disability. According to SSA, some people with ASD do have cognitive disorders, so all four of the B criteria are used to evaluate individual cases. In the examples of impairments evaluated under this listing, SSA acknowledges ASD may or may not be accompanied by an intellectual impairment, and may or may not be accompanied by a language impairment.
These include disorders characterized by onset during the developmental period, and include learning disorder, borderline intellectual functioning, and tic disorders, such as Tourette syndrome. Section 12.00B9a sets forth possible signs and symptoms, including but not limited to abnormalities in cognitive processing, deficits in attention or impulse control, low frustration tolerance, or deficits in social skills.
The new category of eating disorders, which previously only existed in the childhood listings, include, by way of example, anorexia nervosa, bulimia nervosa, binge-eating disorders, and avoidant/restrictive food disorder.
This category includes posttraumatic stress disorder and other specified related disorders such as adjustment-like disorders with prolonged duration. The disorders are characterized by “experiencing or witnessing a traumatic or stressful event, or learning of a traumatic event occurring to a close family member or friend, and the psychological aftermath of clinically significant effects on functioning.” Section 12.00 B11a lists examples of relevant symptoms and signs.
These disorders are currently considered under listing 12.06 for anxiety disorders. The new listing reflects the DSM-5, which created a new category for trauma and stress related disorders.
The changes to the children’s listings mirror to a large extent those in the adult listings. Of note, Listing 112.12 Developmental and Emotional Disorders of Newborns and Younger Infants, is now Listing 112.14 – Developmental disorders in infants and toddlers. The B criteria are unique to that listing. And Section 112.00I2 provides “additional guidance” for calculating corrected chronological age.
Listing 112.15 – Trauma-and-stress-related disorders – also has slightly different criteria than the adult version, including an alternative diagnosis of Reactive attachment disorder.
Of concern is SSA’s refusal to include Oppositional Defiant Disorder and Conduct Disorder as examples of impairments under Listing 112.08 – Personality and impulse-control disorders. According to SSA, “these impairments do not typically result in marked limitation in one of the ‘paragraph B’ criteria or extreme limitation in one of the criteria.” Advocates will need to make sure that adjudicators do not interpret this to preclude consideration of these impairments under the alternative functional equivalency evaluation in childhood claims.
New regulations will become effective at all levels of adjudication on January 17, 2017, including in pending cases. Federal courts will be expected to review appeals under the rules in effect when the decisions were rendered. A court remand, however, will be governed by the new rules.


ZHRO SOLUTIONS, LLC, a Nevada limited liability company, and ADVANCED GREEN INNOVATIONS, LLC, a Nevada limited liability company, Defendants.
This matter is before the Court on Plaintiff Woodward, Inc.'s Response to the Court's Order to Show Cause [Docket No. 12]. Plaintiff asserts that this Court has subject matter jurisdiction pursuant to 28 U.S.C. § 1332(a). Docket No. 11 at 4-5, ¶ 17; Docket No. 12 at 3.
On June 13, 2018, the Court issued an order to show cause identifying several deficiencies in plaintiff's jurisdictional allegations. Docket No. 7. Specifically, the Court found that: (1) plaintiff's allegation, “upon information and belief, ” that defendant ZHRO Solutions' sole member is Advanced Green Innovations, LLC was not sufficient to establish subject matter jurisdiction; (2) plaintiff had failed to identify the citizenship of Roy McLester,  one of two members of Advanced Green Technologies, LLC; and (3) plaintiff had failed to identify the citizenship of the KL-998 Trust UTD December 28, 1999 (“KL-998 Trust”), the sole member of Cochise Investments, LLC. See Id. at 2-4.
On June 21, 2018, plaintiff filed an amended complaint along with a response to the Court's show cause order. See Docket Nos. 11, 12. As stated in plaintiff's response, the amended complaint affirmatively alleges that Advanced Green Innovations, LLC is ZHRO's sole member without resorting to plaintiff's “information and belief.” Docket No. 11 at 3, ¶ 9; Docket No. 12 at 7. Additionally, the complaint asserts that both Roy McAlister and Kenneth Losch, the trustee of KL-998 Trust, are citizens of Arizona. Docket No. 11 at 4, ¶¶ 12-14; Docket No. 12 at 8, 11. Plaintif f contends that this latter allegation is sufficient to establish the citizenship of the KL-998 Trust because, consistent with the Supreme Court's decision in Americold Realty Trust v. Conagra Foods, Inc., 136 S.Ct. 1012 (2016), “the inquiry for purposes of diversity jurisdiction should focus on the citizenship of the trustee.” Docket No. 12 at 11. The Court disagrees.
Americold's confusion regarding the citizenship of a trust is understandable and widely shared. The confusion can be explained perhaps, by tradition. Traditionally, a trust was not considered a distinct legal entity, but a “fiduciary relationship” between multiple people. Such a relationship was not a thing that could be haled into court; legal proceedings involving a trust were brought by or against the trustees in their own name. And when a trustee files a lawsuit or is sued in her own name, her citizenship is all that matters for diversity purposes. For a traditional trust, therefore, there is no need to determine its membership, as would be true if the trust, as an entity, were sued. Many States, however, have applied the ‘trust' label to a variety of unincorporated entities that have little in common with this traditional template. . . . So long as such an entity is unincorporated, we apply our “oft-repeated rule” that it possesses the citizenship of all of its members. But neither this rule nor Navarro limits an entity's membership to its trustees just because the entity happens to call itself a trust.
Id. at 1016. As courts have recognized, this language is susceptible to several interpretations. For example, it could mean that “there is no need to determine entity membership for diversity purposes when a ‘traditional trust' is sued as an entity.” See Zoroastrian Ctr. & Darb-E-Mehr of Metro. Washington, D.C. v. Rustam Guiv Found. of New York, 822 F.3d 739, 749 (4th Cir. 2016). Alternatively, the passage from Americold could reflect the principle that “a trust sued as an entity must prove entity membership because it is a separate legal person from the individual trustees.” Id. Three circuits have directly confronted this ambiguity. All three have interpreted Americold as establishing that, in contrast to a business trust, the citizenship of a traditional trust is determined solely by the citizenship of its trustees. See GBForefront, L.P. v. Forefront Mgmt. Grp., LLC, 888 F.3d 29, 34 (3d Cir. 2018); Raymond Loubier Irrevocable Trust v. Loubier, 858 F.3d 719, 729 (2d Cir. 2017); Wang ex rel. Wong v. New Mighty U.S. Trust, 843 F.3d 487, 494 (D.C. Cir. 2016).
Although the Tenth Circuit has not yet addressed the issue, the Court finds the reasoning of the Second, Third, and D.C. Circuits persuasive. However, the fact that “traditional trusts retain the citizenship of their trustees” for purposes of diversity jurisdiction, Docket No. 12 at 9, does not resolve the jurisdictional issue here. Plaintiff has not alleged that the KL-998 Trust is a traditional trust or presented any facts from which the Court could reach that conclusion. See, e.g., GBForefront, L.P., 888 F.3d at 40-41 (explaining two inquiries a court should undertake in deciding whether a trust is a “traditional” or “business” trust for purposes of diversity jurisdiction). The Court finds that plaintiff's response to the order to show cause, which fails to analyze whether the KL-998 Trust is a traditional trust or a business trust, is insufficient to enable the Court to determine the citizenship of defendants or whether the Court has jurisdiction.
Plaintiff argues that, “[i]n the event that the Court believes the citizenship of the KL-998 Trust's beneficiaries are relevant to the jurisdictional inquiry, ” plaintiff should be permitted “limited discovery of the KL-998 Trust to identify its current beneficiaries.” Docket No. 12 at 11. The Court disagrees. Permitting such discovery at the outset of a case would undermine the well-established rule that “[t]he party invoking federal jurisdiction bears the burden of establishing such jurisdiction as a threshold matter.” Radil v. Sanborn W. Camps, Inc., 384 F.3d 1220, 1224 (10th Cir. 2004); see also Lowery v. Ala. Power Co., 483 F.3d 1184, 1216 (11th Cir. 2007) (“[S]hould the plaintiff request leave to conduct discovery to support its assertion that the case is properly before the court, the court would deny such a request. In such a situation, the court would not reserve ruling on the motion to dismiss in order to allow the plaintiff to look for what the plaintiff should have had - but did not - before coming through the courthouse doors, even though the court would have the inherent power to do so.”); Celli v. Shoell, 40 F.3d 324, 327 (10th Cir. 1994) (“[T]he presumption is that [federal courts] lack jurisdiction unless and until a plaintiff pleads sufficient facts to establish it.”). The Court sees no reason to depart from its prior rulings denying requests for jurisdictional discovery where parties have failed to plead a sufficient basis for federal jurisdiction at the outset of a case. See, e.g., Nancy P. Assad Trust v. Berry Petroleum Co., No. 13-cv-00544-PAB, 2013 WL 1151912, at *2-3 (D. Colo. Mar. 20, 2013) (denying jurisdictional discovery where plaintiff had failed to identify the members of the defendant LLCs).
Plaintiff argues that “[r]equiring a plaintiff . . . to conclusively establish the citizenship of a private trust's beneficiaries at the pleading stage for purposes of subject matter jurisdiction without limited discovery would be prejudicial” because such a plaintiff would be forced to pursue its lawsuit in state court. Docket No. 12 at 12 n.6. But a state forum is not presumptively prejudicial. See, e.g., Assad Trust, 2013 WL 1151912, at *3 (finding that there had been no showing of prejudice where “there [was] no indication that dismissing [the] case [would] prejudice plaintiff by depriving it of a forum in which to assert its claims”). And, as plaintiff itself suggests, plaintiff would have the option of conducting jurisdictional discovery in state court and then moving to dismiss the case without prejudice to its re-filing in a federal forum. Docket No. 12 at 12 n.6. For these reasons, the Court finds that there is no basis to permit jurisdictional discovery at this time. Cf. Abrego Abrego v. The Dow Chem. Co., 443 F.3d 676, 691 (9th Cir. 2006) (where party invoking federal jurisdiction has “failed to present to the district court any pleading, evidence, or admission that establishes that it is more likely than not that jurisdiction lies, ” it is “well within the court's discretion to remand to state court rather than ordering jurisdictional discovery, with the knowledge that later-discovered facts may prompt a second attempt at removal”).
ORDERED that, on or before 5:00 p.m. on July 6, 2018, plaintiff shall show cause why this case should not be dismissed due to the Court's lack of subject matter jurisdiction.


text:
The SOS response is a well-known regulatory network present in most bacteria and aimed at addressing DNA damage. It has also been linked extensively to stress-induced mutagenesis, virulence and the emergence and dissemination of antibiotic resistance determinants. Recently, the SOS response has been shown to regulate the activity of integrases in the chromosomal superintegrons of the Vibrionaceae, which encompasses a wide range of pathogenic species harboring multiple chromosomes. Here we combine in silico and in vitro techniques to perform a comparative genomics analysis of the SOS regulon in the Vibrionaceae, and we extend the methodology to map this transcriptional network in other bacterial species harboring multiple chromosomes.
Our analysis provides the first comprehensive description of the SOS response in a family (Vibrionaceae) that includes major human pathogens. It also identifies several previously unreported members of the SOS transcriptional network, including two proteins of unknown function. The analysis of the SOS response in other bacterial species with multiple chromosomes uncovers additional regulon members and reveals that there is a conserved core of SOS genes, and that specialized additions to this basic network take place in different phylogenetic groups. Our results also indicate that across all groups the main elements of the SOS response are always found in the large chromosome, whereas specialized additions are found in the smaller chromosomes and plasmids.
Our findings confirm that the SOS response of the Vibrionaceae is strongly linked with pathogenicity and dissemination of antibiotic resistance, and suggest that the characterization of the newly identified members of this regulon could provide key insights into the pathogenesis of Vibrio. The persistent location of key SOS genes in the large chromosome across several bacterial groups confirms that the SOS response plays an essential role in these organisms and sheds light into the mechanisms of evolution of global transcriptional networks involved in adaptability and rapid response to environmental changes, suggesting that small chromosomes may act as evolutionary test beds for the rewiring of transcriptional networks.
Bacteria are frequently exposed to a broad range of stressors that may cause, directly or indirectly, DNA damage. First described in Escherichia coli, the SOS response comprises the rapid and concerted activation of a specific set of genes aimed at addressing DNA damage [1, 2]. The synchronized activation of SOS genes is mediated by the transcriptional repressor LexA . The E. coli LexA protein forms dimers that are able to recognize a palindromic binding motif, commonly referred to as the LexA or SOS box, which has been shown to follow a CTGT-N8-ACAG consensus [3, 4]. Binding sites conforming tightly to the LexA-binding motif are typically located near or overlapping the RNA-polymerase binding-site on the promoter region of SOS genes . When bound to their target sites, LexA dimers physically hinder RNA-polymerase binding and thereby effectively repress gene expression. The SOS response is induced through the activation of the RecA protein [6, 7]. Single-stranded DNA fragments, generated by replication fork stall or enzymatic processing of broken DNA ends , bind and activate RecA . In its active form, RecA will induce the auto-catalytic cleavage of the LexA dimer and several other transcriptional repressors, such as the λ phage CI repressor. Cleaved LexA is unable to bind its target sites, thus activating the expression of SOS genes . The E. coli SOS transcriptional network comprises more than 40 genes involved in diverse forms of DNA repair, error-prone DNA replication and cell division arrest, and it includes also both the recA and lexA genes [5, 10, 11]. Once DNA damage has been addressed, activated RecA levels fall rapidly and newly synthesized LexA returns the system to its repressed state.
The SOS response was first identified in response to UV treatment, but it is also intrinsically linked to the natural response against antibiotics that damage DNA . Furthermore, the need to bypass lesions that cannot be readily repaired using translesion synthesis polymerases (TLS) establishes the SOS response as a de facto mechanism for stress-induced mutagenesis [13–15]. In addition, the SOS response has long been known to promote prophage induction  and the activation of virulence factors, like Shiga-like toxins in enterohemorrhagic E. coli. Independent studies have shown that activation of the SOS response also triggers the dissemination of other mobile elements, such as pathogenicity islands . Finally, it has been reported that several antibiotics that do not cause direct damage to bacterial DNA (e.g. β-lactams) are capable of inducing the SOS response through indirect routes , and that such induction is capable of triggering dissemination of mobile elements carrying antibiotic resistance determinants . These findings have irrevocably transformed the image of the SOS response from a DNA-repair textbook paradigm into a key component in the fight against virulence and antibiotic resistance [21–23].
The recent evidence for a tight link between dissemination of antibiotic resistance, pathogenicity and the SOS response is perhaps most clearly outlined in the genus Vibrio. This group of heterotrophic and aerobic γ-Proteobacteria encompasses at least 12 species known for being pathogenic in human hosts . Vibrio species are usually linked to intestinal infections (e.g. Vibrio cholerae or Vibrio parahaemolyticus) acquired by drinking of contaminated water or eating of raw sea animals [25–27], but they are also well documented as opportunistic pathogens associated with high morbidity rates (e.g. Vibrio alginolyticus or Vibrio vulnificus) [28–30]. The genome of Vibrio species is composed of 2 circular chromosomes (known as large/1 and small/2) and in most cases it is known to harbor several virulence determinants, including toxins and colonization factors, that are strongly linked to the well-known severity and harshness of classical Vibrio infections, such as cholera [31–33]. In 2004, Beaber et al. showed that transfer of the V. cholerae SXT integrative-conjugative element (ICE) was controlled by the SOS response through RecA-dependent cleavage of the SXT SetR repressor . The SXT is a 100 kbp element encoding genes that confer resistance to several antibiotics, such as chloramphenicol, sulphamethoxazole or trimethoprim, and is now found in almost all clinical isolates of V. cholerae from Asia and Africa . The fact that SOS-mediated activation of SXT conjugation could be induced by antibiotics, such as ciprofloxacin, put forward for the first time a mechanism by which the use of antibiotic agents might promote the dissemination of antibiotic resistance determinants . Soon thereafter, Quinones et al. showed that induction of the CTX prophage, which encodes the cholera toxin, is regulated by the SOS repressor LexA in conjunction with the phage repressor RstR, and the same group recently proved that transcription of the rstR gene is also regulated by LexA [36, 37]. Most recently, the SOS response has been shown to regulate the activity of the integrase gene of the V. cholerae superintegron, leading to heightened rates of recombination excision of integron cassettes upon induction of the SOS response by different antibiotics . The V. cholerae superintegron encompasses more than 150 gene cassettes and is known to be a source of genetic diversity and antibiotic resistance genes [39, 40]. SOS regulation of the integrase gene has later been confirmed in chromosomal superintegrons of the Vibrio genus and in most mobile integron classes, establishing a mechanism for the long-term safe storage of otherwise harmful adaptive traits, such as antibiotic resistance determinants .
Since its initial description in E. coli, the SOS response has been described in many other bacteria and, with some notable exceptions (e.g. the ε-Proteobacteria), it is considered a universal stress response in the Bacteria domain . In contrast to other transcriptional networks, like the arg regulon , the SOS response has undergone extensive evolutionary changes in the binding motif recognized by the repressor protein LexA. As a consequence, the original description of a CTGT-N8-ACAG binding motif in E. coli has been extended to most β- and γ-Proteobacteria species [43, 44], but has been replaced by alternative descriptions in many different classes and phyla. The universality of the E. coli LexA-binding motif was first challenged by the description of a GAAC-N4-GTTC LexA-binding motif in the Firmicutes Bacillus subtilis. This new motif was later confirmed to be the LexA-binding motif in the Actinobacterium Mycobacterium tuberculosis and, by extension, in all Gram-positive bacteria . Following this initial discovery, research has unearthed a plethora of LexA-binding motifs in different bacterial groups, ranging from a GTTC-N7-GTTC direct repeat in the α-Proteobacteria to a TTAC-N3-GTAA palindrome in Bdellovibrio bacteriovorus[47, 48]. The identification of the LexA-binding motif in several bacterial groups has led to the outlining of a loosely conserved core of genes that seem to constitute the core or ancestral LexA regulon. These include the two main SOS genes (lexA and recA), members of the nucleotide excision repair (NER) system (most notably the excinuclease subunit A, uvrA) and several TLS polymerases . Recently, the canonical view of the SOS response as a DNA repair system has shifted considerably towards a mutagenesis-centered perspective following the identification of a mutagenesis cassette (imuA-imuB-dnaE2) that is widely distributed in the Bacteria domain. Whenever present, this cassette is persistently regulated by LexA, being the only transcriptional unit regulated by the SOS repressor in several organisms [15, 48–50].
The rapid increase in the number of completely sequenced bacterial genome sequences in the early 2000 s led to the development of comparative genomics approaches for the analysis of transcriptional networks [42, 51–53]. The main premise in these approaches is that binding sites for a transcription factor, if functional, will tend to be preserved by evolution. Hence, the analysis of multiple genomes will identify functional binding sites as conserved regions across several genomes, in spite of significant changes to their immediate neighborhood. Functional conservation of binding sites can be exploited in two different ways. In motif discovery applications, the upstream regions of orthologous genes can be scanned to identify consistent instances of a candidate binding motif [54–56]. In contrast, transcriptional regulatory network analyses use a known binding motif to search multiple genomes and elucidate the composition of the regulatory network at different levels [42, 57, 58]. In this approach, the identification of a candidate binding site upstream of an ortholog in different species bolsters the a priori low confidence of the in silico prediction made on each individual genome [59, 60].
A basic tenet of comparative genomic approaches for regulon analysis is the assumption that the transcription factor-binding motif is conserved across the species being studied. In spite of the apparent diversity in LexA-binding motifs across the Bacteria domain, research on different phyla has also shown that changes in LexA-binding motif tend to be monophyletic for the clades in which they take place [61–63]. This has enabled research into the composition and evolution of the SOS response in several bacterial groups using comparative genomics approaches. These studies have revealed that, beyond the conserved core outlined above, the genetic composition of the SOS response can vary substantially between and within Proteobacteria classes [23, 43, 64]. Because the original collection of binding sites may not belong to any of the species under analysis, a good practice in comparative genomics involves the experimental validation of in silico predictions in at least one of the organisms under study, in order to validate the computational method and the parameters used for the analysis .
In this work, we take advantage of previously developed methodology to map for the first time the SOS regulatory network in the Vibrionaceae family. We apply computational analyses to the 14 complete genome sequences available for this family, which encompass nine different Vibrionaceae species, and we validate our findings in vitro using V. parahaemolyticus as an experimental model. Our analysis thus provides a first glimpse at the genetic organization of the SOS response in a complex and heterogeneous family of bacteria comprising more than a hundred species. In order to gain insight into the adaptation of the SOS response to genomes containing multiple chromosomes, which are a defining feature of the Vibrionaceae, we extend the comparative genomics analysis to two additional phylogenetic groups (the β- and α-Proteobacteria) that contain many species with multiple chromosomes. Our results provide comprehensive support to the notion that the SOS response is strongly associated with dissemination of antibiotic resistance and pathogenicity in the Vibrio genus, and identify new genes that might play a role in this association. We also show that the core constituents of the SOS response in all the groups analyzed are located systematically in the large chromosome, indicating that the SOS response is a fundamental component of these genomes. The observed distribution of non-core genes among the smaller chromosomes and plasmids puts forward a mechanism for the dynamic evolution of a critical response network involved in the regulation of many essential genes, and supports the hypothesis of plasmid origin for the smaller chromosomes of organisms with multiple chromosome genomes.
To analyze the SOS regulon of the Vibrionaceae, we downloaded all the available complete genome sequences from the NCBI RefSeq database  and we performed independent searches of both chromosomes and sequenced plasmids using a collection of known binding sites from E. coli. As in previous work, to define the family-wide SOS regulon we included only putative sites that were observed consistently upstream of orthologous genes in at least three separate species. The results of the comparative genomics analysis shown in Figure 1 suggest that, in accordance with other LexA regulons, the SOS response of the Vibrionaceae comprises more than 20 genes [5, 66]. Given the relative phylogenetic proximity between the Vibrionaceae and E. coli, it is not surprising to find that many of the genes described as SOS regulated in E. coli are also members of the Vibrionaceae SOS regulon. These include the two main regulators of the SOS network (lexA and recA), as well as canonical SOS genes involved in DNA repair (recN, yebG, yigN, uvrA, uvrD and the ruvAB operon) and translesion synthesis (dinB and umuDC). Nonetheless, differences in the mode of regulation of certain genes can be observed in the Vibrionaceae. The uvrA gene forms a divergent gene pair with ssb in E. coli and other γ-Proteobacteria, sharing a single LexA-binding site . Expression of both genes has been shown to be SOS regulated, even though SOS induction of ssb is known to be moderate [67, 68]. This arrangement (Figure 2A) is preserved in many Vibrionaceae (e.g. V. parahaemolyticus), but has been disrupted by a bacteriophage insertion in other species (e.g. Vibrio fischeri). The fact that independent LexA-binding sites for ssb and uvrA have been preserved in these species points to a selective advantage in the regulation of both genes by the SOS response. In contrast, the ruvCAB operon yields a different story (Figure 2B). In E. coli and close relatives, this operon is split into the ruvC gene and the ruvAB operon, and only the latter is regulated by the SOS response [5, 43]. In Aliivibrio salmonicida and other species, the operon retains its ancestral ruvCAB configuration but displays independent LexA-binding sites upstream of both ruvC and the ruvAB tandem, which presumably incorporates a secondary promoter. The operon has later been disrupted by the insertion of one (V. vulnificus) or two (V. parahaemolyticus) genes and the regulation of ruvC has apparently been lost in the process. This suggests that the selective pressure for SOS regulation of ruvC is not as strong as the one operating on ruvAB, in agreement with recent results that emphasize the independent and vital role of the ruvAB complex in rescuing stalled replication forks .
Tabulated description of the LexA regulon of the Vibrionaceae. Colors indicate the presence and location of the gene and patterns denote presence (plain) or absence (patterned) of one or more LexA-binding sites in its promoter region. E. coli genes and their corresponding regulation are shown for comparative purposes. Abbreviations are as follows: Eco, E. coli; Vch, V. cholerae; Vha, V. harveyi; Vpa, V. parahaemolyticus; Vsp, Vibrio splendidus; Vvu, V. vulnificus; Asa, A. salmonicida; Vfi, V. fischeri; Ppr, Photobacterium profundum.
Schematic representation of the uvrA - ssb divergent pair and the ruvCAB operon in different Vibrionaceae species. The figure illustrates the persistence of ssb regulation despite gene insertions and the disruption of the ruvCAB operon by gene insertions in some Vibrio species. Genes are represented by filled arrows. Grey-filled arrows correspond to genes that do not constitute part of the canonical uvrA-ssb and ruvCAB elements. Black triangles indicate predicted LexA-binding sites. GenBank accession numbers and absolute genome coordinates for each bacterial species are provided for reference.
The comparative genomics analysis also identifies putative binding sites upstream of several genes that are not members of the E. coli LexA regulon but that have been shown experimentally to be SOS regulated in at least one of the species under analysis. The promoter of the luxR gene of V. fischeri, for instance, has been shown to bind LexA and its own product (LuxR), which share the same binding site [70, 71], and our analysis indicates that this cross-regulation is maintained in the closely related A. salmonicida. Similarly, we identify LexA-binding sites in the promoter of the aforementioned rstR gene in all the V. cholerae strains harboring the CTX prophage . Close examination of these promoter regions in classical and El Tor biovars reveals that the LexA binding site mediating the regulation of rstR has been selectively maintained in spite of substantial changes to its promoter region . In CTX phages of the classical biotype, the region encompassing the LexA-binding site contains several insertions with respect to El Tor biotypes, in which the combined RstR-LexA regulation has been analyzed in detail  (Additional file 1). These insertions, together with the specific sequence of the LexA-binding site, can in fact be used reliably to determine the CTX biotype of Vibrio sequences. The variability of the rstR promoter is in contrast with that observed in other SOS regulated elements of V. cholerae. For instance, the promoter of the setR gene in the integrating-conjugative element SXT shows remarkable conservation of promoter elements and SetR-binding sites among strains (Additional file 2). Significant conservation is also observed within each biotype for the rstR promoter, suggesting that both variants of CTX and the SXT element have spread recently among V. cholerae strains. Our analysis also uncovers the recently reported regulation of Vibrio superintegron integrase genes [38, 41] and the systematic regulation of the imuA-imuB-dnaE2 mutagenesis cassette in all the species in which it is present . Interestingly, the presence of this mutagenesis cassette correlates neatly with the location of the superintegron (found in the large chromosome in all the species harboring the cassette and in the small chromosome otherwise). This suggests that both traits are the product of a single reorganization event that took place before the split of V. cholerae from other Vibrionaceae, (such as Vibrio harveyi), a fact that is in consonance with the existing 16 S rRNA phylogeny for this family .
To validate the results of the in silico comparative genomics analysis, we performed electro-mobility shift assays (EMSA) of putatively regulated promoters of V. parahaemolyticus using the purified LexA protein of this organism. EMSA were performed on promoters of three canonical SOS genes (lexA, recA and imuA) for positive control, and on the promoters of genes flagged as candidate new members of the LexA regulon in the comparative genomics analysis: topB, recG, mutH and the two genes coding for proteins of unknown function (unfA [VP1264] and unfB [VP1428]). The results of the EMSA shown in Figure 3 confirm that the putative LexA-binding sites identified in the comparative genomics analysis are bound by LexA in V. parahaemolyticus, thus providing experimental validation for the in silico approach. Furthermore, competition assays establish that LexA-binding to these sites is specific (Additional file 3). Hence, even though they do not constitute conclusive proof of SOS regulation, these results provide direct evidence of LexA-binding to the promoter region of five genes that have never been described previously as members of the SOS response. The apparent LexA regulation of recG makes intuitive sense. Even though it is not LexA regulated in E. coli, the product of recG is involved in the generation of Holliday junctions after replication fork stall and is known to interact with other members of the DNA recombination repair system . The same logic applies to topB, which encodes a DNA topoisomerase III that has been shown in vitro to interact in E. coli with the products of ssb and recQ in the resolution of converging replication forks .
EMSA of the promoter region for several V. parahaemolyticus genes in the presence (+) or absence (-) of purified LexA protein from this organism. Competition assays (Additional file 3) were conducted to validate the specificity of LexA binding. The absence of purified LexA protein is used as a negative control for each EMSA. The lexA, recA and imuA promoter regions are used as positive controls.
The putative SOS regulation of mutH, which encodes the DNA mismatch repair (MMR) endonuclease MutH, is more unexpected than that of recG or topB because no members of the MMR system have been reported to be SOS regulated previously. However, increased concentrations of MutH have been shown to lead to a predominance of MMR over very short patch repair (VSP), mediated by the Vsr endonuclease , and prevalence of MMR over VSP may be desirable to minimize the mutagenic effects of VSP. Finally, the V. parahaemolyticus EMSA confirm binding of LexA to the promoters of two genes coding for proteins of unknown function (VP1264 and VP1428). Homologs of the VP1264 gene, here termed unfA, have been annotated as members of the superfamily II of DNA and RNA helicases, for which recQ is the most well-known representative in bacteria. Hence, binding of LexA to the unfA promoter in the Vibrionaceae might be linked to the regulation of topB reported above and is likely to be involved in DNA repair processes. The gene VP1428 (unfB), however, has no known homologs outside the Vibrionaceae and its product is consistently annotated as hypothetical in all the Vibrio species analyzed here. Importantly, unfB appears to be present and associated with a putative LexA-binding site only in those Vibrio species that are known to be human pathogens. Given the well-founded relationship of the SOS response with dissemination of antibiotic resistance and pathogenicity in the Vibrio genus, it seems reasonable to postulate that the unfB gene product may be involved in such processes in Vibrio species.
Multiple chromosome genomes have been described and appear to have evolved independently in at least five different bacterial clades [31, 76–79]. Besides the γ-Proteobacteria, to which the Vibrionaceae belong, complete genome sequences composed of multiple chromosomes are available for the α-Proteobacteria (Rhizobiaceae, Brucellaceae and Rhodobacteraceae families), the β-Proteobacteria (Burkholderiaceae and Comamonadaceae families), the Chloroflexi (Sphaerobacteraceae), the Deinococci (Deinococcaceae) and the Spirochaetes (Leptospiraceae). Having validated the comparative genomics approach in the Vibrionaceae, we decided to extend the analysis to other phylogenetic groups that present genomes with multiple chromosomes in order to analyze the adaptation of a complex genetic network, like the SOS response, to such genomic environments. The lack of a well defined LexA-binding motif and/or more than one complete genome sequence within a given phylogenetic group restricted our analysis to the β-Proteobacteria, to which the here-validated LexA-binding motif can be applied  and to the α-Proteobacteria, in which a suitable LexA-binding motif has already been experimentally validated for comparative genomic approaches .
The results of the comparative genomics analysis for α- and β-Proteobacteria are presented, respectively, in Figure 4 and Figure 5. These support previous results reporting significant variation in the composition of the SOS system across bacterial groups [64, 66]. In particular, the results on α- and β-Proteobacteria reveal a conserved core for the SOS regulon that comprises only the lexA and recA genes, an inducible TLS polymerase (dinB and/or polB), the NER excinuclease subunit A (uvrA) and the mutagenesis cassette imuA-imuB-dnaE2. Beyond this small core, the three phylogenetic groups analyzed here present numerous differences and some relevant similarities. A feature common to α- and β-Proteobacteria is the lack of LexA regulation of the recN gene, which is heavily regulated (up to three LexA-binding sites) in E. coli and in the Vibrionaceae, and which had been formerly identified as a key component of the SOS response . On the other hand, the recG gene of some α-Proteobacteria appears to be regulated by LexA, suggesting that the presence of LexA-binding sites upstream of recG reported in the Vibrionaceae might be due to an ancestral regulation of this gene. The same reasoning can be applied in the case of the ruvCAB operon, the promoter of which harbors putative LexA-binding sites in the α-Proteobacteria in spite of substantial genomic rearrangements. In a similar vein, the identification of putative LexA-binding sites in the promoter of β-Proteobacteria Helicase c2 coding genes is congruent with the apparent regulation of unfA and recG in the Vibrionaceae.
Tabulated description of the predicted LexA regulon of α-Proteobacteria species with multiple chromosome genomes. Colors indicate the presence and location of the gene and patterns denote presence (plain) or absence (patterned) of one or more LexA-binding sites in its promoter region. E. coli genes and their corresponding regulation are shown for comparative purposes. Eco, E. coli; Arad, Agrobacterium radiobacter; Atu, A. tumefaciens; Avi, Agrobacterium vitis; Bab, Brucella abortus; Bca, Brucella canis; Bme, Brucella melitensis; Bov, Brucella ovis; Bsu, Brucella suis; Oant, Ochrobactrum anthropi; Pden, Paracoccus denitrificans; Rsph, Rhodobacter sphaeroides; Sme, S. meliloti.
Tabulated description of the predicted LexA regulon of β-Proteobacteria species with multiple chromosome genomes. Colors indicate the presence and location of the gene and patterns denote presence (plain) or absence (patterned) of one or more LexA-binding sites in its promoter region. E. coli genes and their corresponding regulation are shown for comparative purposes. Eco, E. coli; B. 383, Burkholderia sp.; Bamb, Burkholderia ambifaria; Bcen, Burkholderia cenocepacia; Bglu, Burkholderia glumae; Bmal, Burkholderia mallei; Bmul, Burkholderia multivorans; Bphy, Burkholderia phymatum; Bphyt, Burkholderia phytofirmans; Bpse, Burkholderia pseudomallei; Btha, Burkholderia thailandensis; Bvie, Burkholderia vietnamiensis; Bxen, Burkholderia xenovorans; Reut, Ralstonia eutropha; Rmet, Ralstonia metallidurans; Rpic, Ralstonia pickettii; Ctai, Cupriavidus taiwanensis; Vpar, Variovorax paradoxus.
In the α-Proteobacteria, the comparative genomics analysis identifies several genes not belonging to the shared SOS core that have already been reported as SOS regulated in some species with single chromosomes (parE in Sinorhizobium meliloti and tag, comM and dnaE in Caulobacter crescentus) [64, 80]. Conversely, the analysis of the β-Proteobacteria suggests that two genes coding for hypothetical proteins might be LexA regulated in this class. One of them (hypA) has no homologs outside β-Proteobacteria species with multiple chromosomes, whereas the other (hypB) appears to be exclusive to the Burkholderia genus. Beyond the consistent lack of recN regulation, however, the predicted α- and β-Proteobacteria SOS networks share an additional trait that is not observed in the γ-Proteobacteria: the apparent regulation of a gene coding for a predicted photolyase (splB). The splB gene identified here codes for a protein homologous to the spore photoproduct lyase of B. subtilis, which is known to address DNA lesions generated by UV radiation in B. subtilis spores . The fact that the homologs of this gene present LexA-binding sites in two groups where LexA recognizes markedly divergent LexA-binding motifs suggests that the splB gene is a functional member of the SOS response in both groups. This finding is remarkable because it is the first evidence of a photoreactivation enzyme regulated by the SOS response. Together with the predicted regulation of mutH in V. parahaemolyticus, it extends the conventional reach of this transcriptional response to include the direct regulation of MMR and photoreactivation components.
The apparent regulation of splB by the SOS response also points to an adaptive component in the specialization of this transcriptional network in different bacterial groups. As opposed to the Vibrionaceae, α- and β-Proteobacteria are mainly soil bacteria . Soil habitats can lead to bacteria being exposed to high levels of UV light in combination with dehydration. This condition is encountered often by B. subtilis spores, and the ensuing DNA lesions are repaired by SlpB . Hence, it seems plausible that α- and β-Proteobacteria might benefit from regulation of photoreactivation processes by the SOS response. The case of the splB gene thus provides a window into the process of adaptation of this transcriptional network to different ecological niches. More generally, the three LexA regulons analyzed in this work show evidence of radial divergence from a small shared core, and this divergent process has endowed each group with a different set of specialized SOS genes. The cooption of the SOS response to regulate dissemination of antibiotic resistance, pathogenicity and virulence determinants and its enhanced repertoire of repair genes in the Vibrionaceae has already been discussed. Presumably, the loss of regulation for some repair genes (e.g. ruvCAB) in the β-Proteobacteria and the regulation of DNA glycosilases (tag) and competence genes (comM) follows a similar, if not so obvious, adaptive rationale.
A main result of the comparative genomics analysis over several phylogenetic groups containing species with multiple chromosome genomes is the observation that the core genes of the SOS response are found predominantly in the large chromosome. Genomes with multiple chromosomes may originate through different mechanisms (e.g. chromosome duplication or partition), but the prevailing view on the evolution of multiple chromosomes in bacteria points to a plasmid origin of the smaller chromosomes . This is supported by the presence of plasmid-like origins of replication in several small chromosomes , and by the greater proportion of housekeeping genes, synteny and gene conservation in the large chromosomes [84, 85]. Few comparative genomics studies have analyzed regulatory networks in species with multiple chromosomes and none of them has done so in a systematic way. Nonetheless, the scant data available suggest that genes belonging to primary regulatory networks, like those involved in anaerobic respiration, tend to reside in the large chromosome , whereas the genes composing more specialized regulons (like the KdgR regulon involved in pectin catabolism) are located mainly in the small chromosomes .
The comparative genomics analysis conducted here is thus the first systematic study of the distribution of a regulatory network in genomes with multiple chromosomes. The near-universality of the SOS response in the Bacteria domain and the well-documented housekeeping role of many of its members (e.g. recA) [23, 87] establish it as an essential component of the genetic makeup of most bacteria. Hence, our analysis provides sound support to the hypothesis that the genomic distribution of primary regulons in species with multiple chromosomes is predominantly skewed towards the large chromosome. On the other hand, our study reveals also that family-specific additions to the SOS network, as well as duplications of core SOS genes, are most frequently located in small chromosomes and plasmids. The genetic content of small chromosomes has been shown recently to evolve faster, putting forward the notion that these secondary chromosomes act as evolutionary test beds subject to reduced purifying selection . Here we build up on this idea to suggest that small chromosomes and plasmids might provide ideal grounds for the rewiring of transcriptional networks and, specifically, for systems like the SOS response that must strike a delicate balance between regulation of essential genes and rapid adaptability to ecological changes.
This work provides for the first time a detailed description of the SOS regulatory network in a bacterial family of clinical importance. This analysis identifies and experimentally validates new genes bound by LexA in the Vibrionaceae and supports the previously reported link between this stress response, the dissemination of antibiotic resistance and the adaptation of Vibrio species to human pathogenicity. The mapping of the SOS response in other bacterial groups with multiple chromosome genomes reveals a pattern of recent adaptation to specific environmental niches. Furthermore, it shows that the key elements of this response are consistently located in the large chromosome and it suggests smaller chromosomes and plasmids may serve as test beds for the rewiring of transcriptional networks.
Bacterial species with completely sequenced genomes composed of multiple chromosomes were identified through filtered queries on the NCBI Entrez system . Complete chromosome and plasmid sequences for species with multiple chromosome genomes were downloaded from the RefSeq database in GenBank (.gbk) format . Collections of binding sites for the γ/β- and the α-Proteobacteria were derived from experimental data from E. coli and several α-Proteobacteria species as described previously [43, 64].
Comparative genomics analyses were carried out using xFITOM, a program for binding site search in genomic sequences [59, 90]. All chromosome sequences were searched for putative LexA-binding sites using the R i index  and a motif-normalized threshold as reported previously . Putative LexA-sites identified in a single species were considered candidate sites if they were located within -300 to +50 bp of a gene translation start point and their score was larger than the average score of the original collection minus one standard deviation, as described previously . Gene homology was established on the basis of best reciprocal BLAST hits using BLASTP . A gene was considered to possess a reliable LexA-binding site within a given family if candidate sites were located upstream of its homologs in at least three different species. The detailed results for the searches in the three phylogenetic groups analyzed in this work can be found in Additional file 4.
The V. parahaemolyticus lexA gene was amplified using suitable primers (Additional file 5) and cloned into a pET15b vector (Additional file 6). The V. parahaemolyticus LexA protein was over-expressed and purified as described previously . DNA probes were constructed using two complementary 61 bp synthetic oligonucleotides (Additional file 6). EMSA experiments were performed as reported previously , using 40 nM of V. parahaemolyticus LexA and 20 ng of each DIG-marked DNA probe in the binding mixture. For EMSA competitive assays (Additional file 1), 200 fold of either specific or non-specific non-labeled DNA was added to the binding mixture. In all cases samples were loaded in 6% non-denaturing Tris-glycine poly-acrylamide gel. Digoxigenin-labeled DNA-protein complexes were detected following the manufacturer's protocol (Roche).
This work was supported by the UMBC Office of Research through a Special Research Assistantship/Initiative Support (SRAIS) award, and by grants BFU2008-01078/BMC from the Ministerio de Ciencia e Innovación de España and 2009SGR-1106 from the Generalitat de Catalunya. NSA was supported by a fellowship from the Fundació Cellex. Writing of this paper was supported by a UMBC SFF award.
The authors are indebted to Dr. Richard E. Wolf for kindly allowing NSA to perform part of the experimental work reported here in his lab, and to Dr. Lanyn P. Taliaferro for her assistance in setting up EMSA. The authors wish to thank the two anonymous reviewers for their insightful comments and Dr. Michael C. O'Neill for proofreading the final version of this manuscript.
NSA performed the protein purification and mobility-shift assays, carried out searches for LexA-binding sites and integrated the data. JB, SC and IE conceived the study and participated in its design and coordination. SC designed and directed the in vitro studies. IE coordinated the study, designed and supervised the in silico analyses, supervised the in vitro studies, curated the data and drafted the manuscript. All authors read and approved the final manuscript.

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