text:
An unidentified mother is facing a lot of heat after posting a photo of her 5-year-old son, Sam, wearing pink animal print shoes on his first day of preschool.
According to reports, the mother explained the shoes were for girls but the boy decided he wanted to wear the shoes anyway, telling her, "ninjas can wear pink shoes too."
However, in what was meant to be a momentous moment for a young child (his first day of school) people have began using blogs to verbally attack the mom for letting him wear the shoes.
People are arguing that the pink shoes will turn the young boy gay or will lead to bullying. While others, are on the side of the mom arguing that he is only a kid and it is not a big deal.
The mother of the child has not yet commented on the controversy.
What do you think of the shoes? Was the mother wrong for letting him wear them?
(Photo Credit: Facebook)
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7 thoughts on "Photo of Boy in Pink Shoes Starts On-line Controversy"
"Why change? Everyone has his own style. When you have found it, you should stick to it."
With any research, one will find that pink was the traditional male color until the 1950s. Why would ANY color be considered gender specific? Some men look VERY sexy in a pink shirt that flatters their complexion. It has taken years for "REAL" men to wear pink shirts (my husband still will not), so why are the ADULTS making this a controversy? I'm sure it wasn't the other kindergarten children. What was once a non-gender specific COLOR choice (he LIKES the shoes) has been misconstrued into a problem of monumental importance. Spend your time on volunteer projects people NOT on being the kindergarten fashion police!
What type of job interviews have you been on that asks you to justify/explain what was going on in your life when you were in preschool? The kid is five, not twenty-five.
Wondering how you can make someone gay? Wearing pink shoes?
If you think Keith`s story is incredible,, in the last-month my friend's dad basically brought home $8670 workin a sixteen hour week at home and there best friend's ex-wife`s neighbour has done this for 10-months and actually earnt more than $8670 part-time from there computer. the advice at this website….. Bit40.ℂOℳ
When the boy becomes an adult and starts seeking employment, will he have to face the fact that one day the burden is going to be on him to explain the photo. The smart thing would be for mother to have a written letter saying it was her idea and not his. She could leave the letter with a family attorney or in a bank vault so 20 or 30 something Sam can carry it around to show prospective employers.
What type of job interviews have you been on that asks you to justify/explain what was going on in your life when you were in preschool? The kid is five, not twenty-five.


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Hotels in Worthing, Barbados
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- Most bookedCoral Sands Beach Resort
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"Our stay at the coral beach hotel was a lovely experience the staff were very helpful and friendly.we were close to st Lawrence which made it easy for evening meals. The only minus about the hotel was the bar and restaurant was never open. Apart from that we would definitely stay There again "
A Traveller, Aug 2016, GB
- Worthing Court Apartment HotelNo Guest Reviews yet
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"enjoyable - able to relax - quiet location. I only spent one night there. There was a strong smell of gas in the room from the cooker which was checked and I was reassured all was OK."
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"Located a bit outside Bridgetown but on the main road. You can get a shared mini-bus into town for around 2BBD. The hotel itself is fairly basic but clean. Nothing super fancy but there is free wifi and it is walking distance to many restaurants, bars and beaches."
A Traveller, Sep 2016, IL
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"Came to Barbados for the first time. Stayed in a nice room with a ocean view on the beach."
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"Loved the location of the hotel. Best beach ever. I got to swim with sea turtles every day."
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"Wonderful vacation, close to all amenities - 2 grocery stores to get any staples. Walking distance to a ton of excellent restaurants and truck foods. Beach clean, soft sand & swimmable. We were able to walk to St. Lawrence Gap via beach for many food outlets in one direction and walk to other ..."
A Traveller, May 2016, CA
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"i visited Barbados for Crop over Festival with my friends, my stay at Melrose Beach apt. was great. the apartment was very clean and comfortable. great atmosphere and location it was easy to move around from the apartment.staff were very friendly and welcoming."
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"We loved it.. just returned from a weekend trip. Clean rooms and small hotel. Food is good and priced right. Every room does face the beach. We were on the second floor and had amazing views. Hotel is right at the beginning of "the Gap". walked home after dancing, A 3 minute walk from the cove da..."
A Traveller, Aug 2016, US
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The location of your Worthing hotel is important. If you are traveling to Worthing, &StateorCountry for business, you may want to consider getting a hotel close to the airport or near your meeting venue. By doing so, you can really cut down on transportation costs and time. You may also want to book a hotel that offers free wifi just in case you need to get some work done.
If you are traveling for vacation, figure out which attractions or landmarks you want to see and book a hotel that is within close proximity. Although hotels near attractions might cost a little more, you may find that spending those extra few dollars is worth the convenience. You can even find hotels that align with your interests. For example, if you are looking for a relaxing vacation, a spa hotel may be the perfect choice for you.
You'll also want to determine which type of accommodations will suit your needs before booking a hotel in Worthing. If you're traveling with the kiddos, a family friendly hotel will offer larger rooms and more sleeping arrangements. For those traveling for an extended period of time, booking a hotel suite with a kitchenette is a smart move and will feel a little more like home. If you are budget conscience and just need a place to rest your head, a hostel or motel may be all you need.
If you are looking to score the best discounts and deals, use the filter to sort by price from low to high. You can book a hotel in Worthing for as low as $86 per night. If you are looking to cut down on expenses, look for hotels that include free breakfast and parking. Some hotels even offer free shuttle service to and from nearby places. You can also sort by star rating, proximity, and guest rating.
Before you book, check out user reviews. Hotels.com has 200 reviews in Worthing. We show reviews from Expedia and TripAdvisor so you know that you are reading genuine, reliable reviews. Be sure to download the Hotels.com app so you can book your reservations on the go, view amenities, and save the hotels you want to view and compare later.

subset: commoncrawl


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Hunter Hayes Teaming Up With Stevie Wonder For Special ACM Awards Performance
Hunter Hayes and Stevie Wonder will be performing live together at the ACM Awards this Sunday!
Hunter Hayes and Stevie Wonder will be performing live together at the ACM Awards this Sunday!
If you've been hankering for some new tunes from Hunter Hayes, the wait is almost over. During the 2013 ACM Awards on April 7, the rising country music star has promised to premiere a brand new single for viewers.
Acadiana's own Hunter Hayes has been added to the lineup of the ACM Fan Jam and Post Awards Show at the Orleans Hotel and Casino in Las Vegas on April 7th.
Acadiana's own Hunter Hayes has been announced as one of the performers for the 48th annual ACM Awards on April 7. He's also nominated for 6 awards!
Hunter Hayes kicked off 2013 with a performance at the Grammys and an eye-popping six ACM nominations. The 'Somebody's Heartbreak' singer also expects to have new music for fans this year, and he'll play the first-ever Taste of Country Music Festival this June. As for true romance? Well, he's still searching.
Is Hunter Hayes, one of country music's cutest, most talented and most eligible bachelors, about to come off the market? Could be, as it's being said that he and singer/actress Selena Gomez were seen flirting and even exchanged digits.
While country phenomenon Hunter Hayes may only be 21 years old, he's making a huge splash on the country music scene. The three-time Grammy nominated musician has just garnered six Academy of Country Music Award nominations, becoming the second-most nominated artist this year, right behind Eric Church.
Acadiana's own Hunter Hayes received 6 nominations this morning for the 48th annual Academy of Country Music Awards. Congrats from 97.3 the Dawg!
Hunter Hayes didn't take home his first Grammy award in 2013, but the 21-year-old country newcomer still had a great night at the show. In addition to introducing Carrie Underwood's powerhouse performance, Hayes appeared during the commercial breaks in his new spot for Pepsi.
Carrie Underwood sang snippets of both 'Blown Away' and 'Two Black Cadillacs' at the 2013 Grammy Awards -- ultimately delivering one of the most impressive vocal performances of the night.


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Well, the outspoken B-town beauty will participate in debate which will be held on February 27 at the world's most famous debating society. Read on to know more Mallika Sherawat is back in the news but sadly not coz of her filmi career. The Hisss actor has been invited to be part of a debate […]
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हिन्दी खबरें
ऐश्वर्या ने भुला दिए सारे गिले-शिक़वे, फिर से बड़े पर्दे सलमान के साथ इश्क फरमाते पर दिखेंगी !!
टाइगर श्रॉफ की नई फिल्म 'मुन्ना माइकल' की शूटिंग हुई शुरु
इस साल की दूसरी सबसे बड़ी ओपनर फिल्म बनी एम.एस.धोनी: द अनटोल्ड स्टोरी
जानिए क्यों हैं शाहरुख खान बॉलीवुड के रोमांस किंग ?
ये हैं हमारी बॉलीवुड इंडस्ट्री के बड़बोले स्टार्स


text:
News and analysis from The Center for Michigan • http://thecenterformichigan.net
©2016 Bridge Michigan. All Rights Reserved. • Join us online at http://bridgemi.com
Original article URL: http://bridgemi.com/2013/01/guest-column-states-broken-tax-system-favors-corporations-over-families/
11 January 2013
By Gilda Z. Jacobs/Michigan League for Public Policy
As we enter the budget-setting season in Lansing, we need to step back and ask ourselves some important questions.
Does our revenue structure keep up with the times? Does it improve our education levels and at least keep our quality of life from eroding? Does it 'pay it forward' by offering the next generation the educational opportunities, protection of natural resources and other advantages that were there for us?
Year after year at the annual Revenue Estimating Conference (scheduled for today), we discover that our revenue is falling short of what's needed to pay for education, health care, child protection and other existing public structures. This is due not only to a slow economy, but to an outdated revenue system that acts like it's 1950 by taxing goods but not many services, and missing out on e-trade.
Recent corporate tax breaks mean that businesses will contribute less than 2 percent to the General Fund, the state's main source of funding. But the working poor – who spend long hours in nursing homes, restaurants, child care and other important but low-paid positions — will get a $262 million tax increase this year. This is because our state Earned Income Tax Credit was slashed by 70 percent to help pay for corporate tax breaks.
These changes worsen Michigan's undesirable attributes of rising poverty and growing income inequality. In the face of this, how do we attract families to our state, keep families from leaving and bring in new employers?
Modernizing Michigan's outdated revenue structure will pay off with adequate revenue to pay for a good quality of life — access to quality education, good roads, safe communities and protection of the Great Lakes and other natural resources.
Adjusted for inflation, our General Fund is down more than 15 percent compared with 1968.
Modernizing would pay huge dividends. We would have healthier babies, a more educated and ready workforce, a better quality of life and long-term economic stability and growth. Aren't these the goals we all strive to achieve?
The League has outlined an agenda to move Michigan forward, including a reduction of the income tax on low-income workers and families by restoring the state Earned Income Tax Credit, which was cut from 20 percent of the federal credit to 6 percent.
Other recommendations:
* Ensure that business tax changes are at least revenue neutral by increasing the Corporate Income Tax to replace lost revenues with $550 million lost in fiscal year 2012 alone.
* Modernize the sales and use tax by including more services and capturing Internet sales.
* Scrutinize all forms of spending, not just annual appropriations, including tax credits, deductions, deferrals and exclusions that are forms of "silent spending.''
* Invest in state priorities that are fundamental to job growth and economic development, including education, health, high quality early education and basic income supports.
Let's face it. Our revenue structure is broken. We must pursue tax reforms that modernize our outdated revenue system. It needs to be more dependable. It needs to be fair. Let's start asking the right questions and, hopefully, offering the right answers.
Bridge welcomes guest columns from a diverse range of people on issues relating to Michigan and its future. The views and assertions of these writers do not necessarily reflect those of Bridge or The Center for Michigan.
2 comments from Bridge readers.Add mine!
Ms. Jacobs seems to have one interest, gather more tax dollars. She seems to have no interest on how the means and methods and the impact of gathering those dollars for she is not talking about looking at what the purpose of government and how taxing impacts the State, community, or even the the taxpayers. She shows no interest in providing value for those dollars to the tax payers. She siimply talks about being sure that the government spending is given more and more dollars from those who have been paying irregradless if taxing methods create competitive barriers to those paying the taxes.
Ms. Jacobs shows no interest in working with the tax payers to see what their issues are with the current taxing scheme and working with those tax payer to address the issues to even lower the burden of how those taxes are reported and paid.
Ms. Jacobs claims want balance between those who carry the tax burden and yet she shows little concern of how those who pay the taxes earn the money to pay the taxes she so wants the State to spend.
I have been a tax payer in Michigan and elsewhere, and it happens that my employer in each setting paid taxes and allowed me to pay me so I could pay taxes. In each setting I gain more benefit from the taxes I paid then my employer gain from the taxes they paid. For if it was the schools, I learn not my employer, if it was police or fire my neighborhood was always at greater risk than my place of employement. In my case if it was regulation my employer provide more help (more information, more training, more understanding of what was being regualted) for the State then it ever recieved from the State.
By all appearances from this article it would seem that Ms. Jacob is not concern about the balance between tax payers and taxing, it is simply about taxing business more irregardless of the value they provide to the State, to their communities, to other tax payers. If I were to guess Ms. Jacobs sees the businesses and non-voting payers that she need have little care or consideration for. It is disappointing that she can only see taxpayers as individuals payers or voters and not in the context of the communities they create and support. We have towns that grew around the willingness of business to risk and invest in people and provide them with livelihoods. Ms. Jacobs only sees businesses as a checkbook and she cannot see what those businesses have provided to the people, even those will lower wages.
Sometimes there is merit in stepping back and taking a moment to gain a broader view of the community and how each member impacts the community rather then simply looking at how to spend their money for them.
I agree with everything that Ms. Jacobs writes. Our state's priorities are seriously out of whack. The poor are getting robbed. The schools go without. The police and fire services are cut to the bone.
Our legislature has spent more time regulating the sex lives of the citizens than it has for creating jobs.
What obligations does the community have for its citizens in exchange for taking money in taxes ?
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Free 2004 DNC speeches on iTunes
The iTunes Music Store has recently added speeches from day one and day two of the Democratic National Convention: America 2004 held in Boston, Massachusettes. The free download includes speeches by former Vice President Al Gore; former President Bill Clinton; Senator Hillary Rodham Clinton; former President Jimmy Carter; Senator Ted Kennedy; and Ron Reagan, son of former President Ronald Reagan and many more.
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This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Shewanella oneidensis MR-1 exhibits diverse metal ion-reducing capabilities and thus is of potential utility as a bioremediation agent. Knowledge of the molecular components and regulatory mechanisms dictating cellular responses to heavy metal stress, however, remains incomplete. In a previous work, the S. oneidensis so2426 gene, annotated as a DNA-binding response regulator, was demonstrated to be specifically responsive at both the transcript and protein levels to acute chromate [Cr(VI)] challenge. To delineate the cellular function of SO2426 and its contribution to metal stress response, we integrated genetic and physiological approaches with a genome-wide screen for target gene candidates comprising the SO2426 regulon.
Inactivation of so2426 by an in-frame deletion resulted in enhanced chromate sensitivity and a reduced capacity to remove extracellular Cr(VI) relative to the parental strain. Time-resolved microarray analysis was used to compare transcriptomic profiles of wild-type and SO2426-deficient mutant S. oneidensis under conditions of chromate exposure. In total, 841 genes (18% of the arrayed genome) were up- or downregulated at least twofold in the Δso2426 mutant for at least one of six time-point conditions. Hierarchical cluster analysis of temporal transcriptional profiles identified a distinct cluster (n = 46) comprised of co-ordinately regulated genes exhibiting significant downregulated expression (p < 0.05) over time. Thirteen of these genes encoded proteins associated with transport and binding functions, particularly those involved in Fe transport and homeostasis (e.g., siderophore biosynthetic enzymes, TonB-dependent receptors, and the iron-storage protein ferritin). A conserved hypothetical operon (so1188-so1189-so1190), previously identified as a potential target of Fur-mediated repression, as well as a putative bicyclomycin resistance gene (so2280) and cation efflux family protein gene (so2045) also were repressed in the so2426 deletion mutant. Furthermore, the temporal expression profiles of four regulatory genes including a cpxR homolog were perturbed in the chromate-challenged mutant.
Our findings suggest a previously unrecognized functional role for the response regulator SO2426 in the activation of genes required for siderophore-mediated Fe acquisition, Fe storage, and other cation transport mechanisms. SO2426 regulatory function is involved at a fundamental molecular level in the linkage between Fe homeostasis and the cellular response to chromate-induced stress in S. oneidensis.
Shewanella oneidensis MR-1, a facultatively anaerobic member of the γ-class of Proteobacteria, can respire anaerobically by reducing a wide variety of alternative electron acceptors including fumarate, nitrate, elemental sulfur, and such oxidized metals as Fe(III), Mn(IV), and U(VI) [1-5]. In addition, MR-1 is capable of reducing hexavalent chromium, or chromate [Cr(VI)], to less toxic and sparingly soluble trivalent chromium [Cr(III)] under both aerobic and anaerobic conditions [6-8]; however, there have been no reports to date of the ability of MR-1 to generate energy for growth using Cr(VI) as the sole terminal electron acceptor for anaerobic respiration [9]. This plasticity in the use of alternative electron acceptors for anaerobic respiration has engendered interest in S. oneidensis MR-1 as a model environmental organism with potential utility in the bioremediation of dissolved metal ions, and as a consequence, the complete MR-1 genome was sequenced to advance understanding of Shewanella biology [10].
To expand knowledge of metal stress responses in particular, we previously initiated genome-based studies focused on identifying the molecular components involved in the S. oneidensis response to chromate [11-13], an anthropogenic pollutant widely distributed in the environment due to its prevalent use in manufacturing and military industries [14,15]. Chromate toxicity is associated with the generation of reactive oxygen species during the intracellular partial reduction of Cr(VI) to the highly reactive radical Cr(V) by various in vivo nonspecific reductants or cellular one-electron reductases [16]. Our previous work employing DNA microarrays and multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) demonstrated that a functionally unknown DNA-binding response regulator (designated SO2426) in MR-1 was reproducibly and significantly upregulated at both the transcript and protein levels in response to acute chromate challenge [11,13].
In accordance with its ability to adapt to various environmental conditions, the S. oneidensis MR-1 genome encodes a relatively large repertoire of transcriptional regulators, including 88 predicted two-component regulatory system proteins consisting of 23 histidine protein kinase (HK) genes, 57 response regulators (RR), and 8 HK-RR hybrids [10]. Prototypical two-component systems, which constitute the predominant mechanism used by bacteria for coupling environmental signals to specific adaptive responses, comprise a sensor histidine kinase and a cognate response regulator [17-19]. Fundamental to signal transduction is a phosphotransfer mechanism that forms the core of both mechanistically simple as well as more complex signaling pathways. In this scheme, a membrane-bound sensor kinase catalyzes ATP-dependent autophosphorylation at a conserved histidine residue in the HK cytoplasmic autokinase domain in response to a specific environmental cue. The signal is transduced through transfer of the phosphoryl group from the phosphoHis of the HK to a conserved aspartate residue within the receiver module of a cytosolic RR. Phosphorylation of the RR alters its affinity for DNA, leading to the transcriptional activation or repression of specific genes. An essential feature of two-component systems is that specific histidine kinases and response regulators function as cognate pairs and are often arranged within the same operon. The S. oneidensis SO2426 protein appears to be an orphan RR as defined by the fact that there is no obvious cognate histidine kinase flanking the so2426 gene or proximally located based on the genome annotation [10].
The primary aim of the present study was to investigate the function of the uncharacterized SO2426 regulator within the context of chromate stress by performing a genome-wide screen for potential target genes of this RR in S. oneidensis MR-1. Toward this end, we created an so2426 in-frame deletion mutant and compared its global transcriptional profiles with that of wild-type MR-1 in response to chromate challenge using time-resolved microarray experiments. Comparative transcriptome analysis suggested that SO2426 was associated with the activation of genes involved in siderophore-mediated Fe uptake, Fe storage, and possibly other cation transport processes.
Based on its nucleotide sequence, the so2426 gene of S. oneidensis MR-1 is predicted to encode a 237-amino-acid protein with a calculated molecular mass of 27.4 kDa [10]. Comparative sequence analysis using ClustalW [20] revealed that SO2426 shares sequence similarity to CpxR from Vibrio cholerae (36% identity) and E. coli (34%), and to OmpR from V. cholerae (29%) and E. coli (27%) (Figure (Figure1).1). In addition, SO2426 orthologs are present and conserved among many of the Shewanella species sequenced to date, including Shewanella sp. MR-7 (88% sequence identity), Shewanella sp. MR-4 and sp. ANA-3 (87%), S. baltica OS195 (74%), S. putrefaciens 200 and CN-32 (73%), S. woodyi ATCC 51908 (60%), S. amazonensis SB2B (61%), and S. frigidimarina NCIMB 400 (49%).
The deduced SO2426 protein consists of an N-terminal CheY-like receiver domain and a C-terminal winged-helix DNA-binding domain. The N-terminal portion of SO2426 (amino acids 13–124; see Figure Figure1)1) shares homology with receiver domains and contains the highly conserved signature residues that are thought to constitute the phosphorylated acid-pocket active site [19,21]. In CheY, these residues correspond to Asp12, Asp13, and Asp57, with Thr87 and Lys109 completing the cluster of conserved residues surrounding the phosphorylation site [19]. Thr87 and Lys109, although not absolutely required for phosphorylation, have been implicated in contributing to the phosphorylation-induced conformational change [22-24]. S. oneidensis SO2426 contains three contiguous aspartate residues near its N-terminus at positions 18, 19 and 20, and there is a conserved substitution to serine at the equivalent position of 87. The phosphate-accepting aspartic residue Asp57 and Lys109 (relative to CheY residue numbering) are retained at equivalent positions in the SO2426 primary sequence. It is presently not known whether a cognate histidine kinase interacts with SO2426 or whether phosphorylation plays a regulatory role in the cellular activity of SO2426. The fact that SO2426 contains the highly conserved residues constituting the active phosphorylation pocket (D18, D19, D62, and K109) suggests that the protein might be differentially controlled by phosphorylation. Furthermore, it remains to be determined whether the regulatory activity of SO2426 is modulated in response to environmental or intracellular stimuli.
The C-terminal portion of SO2426 (amino acids 158–235; see Figure Figure1)1) contains a predicted winged helix-turn-helix (wHTH) motif indicative of the DNA-binding domains of response regulators in the OmpR/PhoB subfamily [25-27]. The basic structure of the carboxy-terminal domains of OmpR/PhoB subfamily response regulators is characterized by an amino-terminal four-stranded β sheet, a central three-helical bundle, and a C-terminal β-strand hairpin (the wing), which provides an additional interface for DNA contact [26].
Based on the MR-1 genome annotation, SO2426 is an apparent orphan response regulator given that the so2426 locus is not linked with a gene that encodes a potential cognate histidine kinase. As schematically represented in Figure Figure2A,2A, so2426 is tightly clustered with four functionally unknown downstream genes, all possessing the same transcriptional polarity. Located immediately downstream of so2426 with a short intergenic spacer of 10 bp is a small ORF (132 bp) predicted to encode a soluble hypothetical protein (SO2425), which was shown previously to be induced at the transcriptional level in response to acute chromate challenge [11]. The gene cluster is also characterized by ORFs encoding a zinc carboxypeptidase domain protein (SO2424), a hypothetical protein (SO2423), and a conserved hypothetical protein (SO2422) (Figure (Figure2A).2A). The so2423 ORF overlaps so2422 by 3 bp based on the MR-1 genome annotation (http://www.tigr.org). The so2426 region also includes a gene encoding a putative TonB-dependent receptor (SO2427) positioned approximately 170 bp upstream of so2426. Putative ρ-independent transcription terminators were identified downstream of the so2427 and so2422 ORFs (http://cmr.tigr.org; [28]).
Reverse transcription-PCR (RT-PCR) experiments were performed to determine whether the so2427-so2422 gene cluster is transcribed as a polycistronic mRNA. Primers P1–P9 (Figure (Figure2A)2A) used in the PCR reactions following reverse transcription with random hexamers were designed to amplify regions spanning two or more ORFs in order to establish the transcriptional organization. To assess the reproducibility of the results, multiple independent RT-PCR experiments were carried out using different preparations of total RNA isolated from untreated or chromate-treated MR-1 cells. Figure Figure2B2B shows a representative gel of the RT-PCR results using the entire suite of primers. Each PCR product was confirmed by DNA sequencing. No products were amplified when using either total RNA as the template for PCR amplification (Figure (Figure2B)2B) or primers complementary to an intergenic region located outside of the so2427-so2422 cluster (results not shown), thus indicating the absence of contaminating genomic DNA in the total RNA preparations. S. oneidensis MR-1 genomic DNA was used as a positive control for the PCR conditions (results not shown). Amplicons of the expected sizes, i.e., 0.85 kb for primers P1/P5, 1.9 kb for primers P1/P6, 2.3 kb for primers P1/P7, 2.5 kb for primers P2/P8, and 3.3 kb for primers P9/P3, were obtained (Figure (Figure2B),2B), indicating that the so2427-so2422 region is co-transcribed as a polycistronic mRNA under no-metal and chromate-amended conditions. However, given the presence of a putative ρ-independent terminator immediately downstream of so2427, we cannot rule out the possibility that the product generated from the so2427-so2426 transcript might be due to read-through transcription.
The transcription start site of the so2426 gene was localized using 5' RACE analysis of mRNA transcripts isolated from wild-type cells grown in either the absence or presence of chromate. Two clear 5' termini, both mapping to A residues and corresponding to positions 25 and 27 bp upstream of a second in-frame ATG (amino acid residue M11) (Figure (Figure3),3), were obtained by DNA sequencing of the 5' RACE amplicons. These sites are located at positions 4 and 6 bp downstream of the initial ATG (M1) start codon, indicating that the original sequence annotation for this gene is incorrect and that the second ATG (M11) most likely constitutes the translation start codon for SO2426. The so2426 transcription start point indicated potential -10 and -35 basal promoter elements, 5'-TAatAT and 5'-gTGACA, respectively, with a 15-bp spacing between them. The putative -10 promoter sequence (5'-TAatAT) has a 4/6 match to the consensus σ70-dependent -10 hexamer element (5'-TATAAT) from E. coli. Interestingly, located one base upstream of the -10 hexamer is the extended -10 element 5'-TaTG-3' (Figure (Figure3),3), identified by Mitchell et al. [29] and shown to be an important determinant for promoter activity. Finally, the identified so2426 promoter region contains the putative -35 element 5'-gTGACA, which is a strong match (5/6) to the E. coli consensus -35 hexamer (5'-TTGACA).
Further analysis of the so2426 upstream regulatory region revealed the presence of a putative recognition site for the ferric uptake regulator (Fur), the iron-binding global transcriptional regulator that serves as the dominant sensor of Fe availability in both gram-positive and gram-negative bacteria (for a review, see [30]). A putative palindromic Fur box, 5'-AAATGAtATTgATTcTCgTTT-3', which closely matched our previously derived predicted Fur-binding motif for S. oneidensis MR-1 [31], was identified at positions -5 to +16 immediately downstream of the -10 promoter element and overlapped the mapped transcription start sites for so2426 (Figure (Figure3).3). Identification of a Fur box is consistent with our previous microarray studies demonstrating increased so2426 mRNA levels in a S. oneidensis MR-1 fur knockout strain [31] and suggests that expression of the SO2426 response regulator might be subject to Fur-mediated repression.
A cre-lox recombination method [32,33] was used to create a nonpolar in-frame deletion of the so2426 locus in MR-1 for the purpose of analyzing in depth the functional role of this response regulator in metal stress responses (see Methods for a detailed description). The deleted segment was 721 bp in length and corresponded to positions -77 to +645 (S. oneidensis MR-1 genome coordinates 2534690 and 2533969) from the annotated translational start site, leaving only 69 nucleotides of the so2426 gene intact at the C-terminal end. The growth behavior of the Δso2426 mutant in response to Cr(VI) was examined using a Bioscreen C reader to monitor culture turbidity (OD600) at 30-min intervals under aerobic conditions over 72 h. As shown in Figure Figure4A,4A, growth of the S. oneidensis Δso2426 strain was comparable to the wild type in LB broth without added chromate. By contrast, growth of the Δso2426 mutant was inhibited to a greater extent than the wild type in the presence of 0.3 mM chromate as evident from the longer time interval required before the commencement of exponential growth.
To determine whether deletion of so2426 affected the ability of MR-1 to remove extracellular Cr(VI) from the medium, the wild-type and mutant strains were challenged with 0.3 mM chromate (the same final concentration used for the transcriptomic studies) when cells reached mid-log phase (OD600, 0.5). Residual hexavalent chromium was measured spectrophotometrically at 3-h intervals using the chromogenic 1,5-diphenylcarbazide (DPC) method [34]. The chromate disappearance profiles in Figure Figure4B4B show that the parental MR-1 strain removed 100% of the external Cr(VI) within 9 h, while the Δso2426 mutant transformed approximately 50% within the same time period. No abiotic conversion of chromate was observed for the cell-free LB control (data not shown).
Complementation experiments were performed to ascertain whether the enhanced chromate sensitivity and slower chromate transformation rate for the so2426 null mutant were due to the deletion of the response regulator gene so2426 and not to secondary-site mutations introduced during the mutagenesis procedure. A complemented Δso2426 strain was constructed by cloning the full-length so2426 wild-type ORF plus 100 bp of contiguous upstream sequence into the broad-host-range vector pBBR2426 as described in the Methods. Transformation of pBBR2426 into the Δso2426 mutant resulted in a complemented strain, designated Δso2426(so2426+). Genetic complementation of the so2426 gene in the mutant strain restored growth (data not shown) and Cr(VI) reduction capacity to near wild-type levels (Figure (Figure4B),4B), thus reaffirming the integral role of SO2426 in the cellular response to chromate.
Prior to this study, nothing was known about the regulatory targets of the OmpR family response regulator SO2426. To investigate the effect of the so2426 deletion on global gene expression and to identify candidate target genes under SO2426 control, the chromate-perturbed transcriptomes of the Δso2426 mutant and the S. oneidensis MR-1 wild type were compared using whole-genome microarray analysis. Time-series microarray experiments were performed in which global transcriptional profiles of the wild type and the Δso2426 mutant strains were compared at 5, 30, 60, 90, 180 min, and 24 hours following the addition of K2CrO4 (0.3 mM final concentration) to mid-exponentially growing cells (see Additional file 1 for a complete list of the processed microarray data). Genes with altered expression ratios of ≥ 2-fold for at least one of the time points and having a p value of < 0.05 were considered to be significantly changed and were further analyzed using clustering methods. Overall, microarray expression profiling revealed that 82 (5 min), 90 (30 min), 80 (60 min), 109, (90 min), 443 (180 min), and 465 (24 h) genes were induced in the Δso2426 mutant under chromate exposure, whereas 125 (5 min), 81 (30 min), 56 (60 min), 105 (90 min), 314 (180 min), and 364 (24 h) genes were repressed. The total number of differentially expressed genes was substantially less at the shorter, acute exposure times (i.e., 5, 30, 60, and 90 min) compared to the longer exposure periods (i.e., 180 min and 24 h), at which point other effects such as accumulation of intracellular chromium (hexavalent and/or reduced forms) and stationary phase growth are likely impacting global expression profiles.
Quantitative real-time RT-PCR (qRT-PCR) analysis was employed to assess the general quality of the microarray data by providing an independent measurement of gene expression for a small subset of genes. The five genes (so0404, so2426, so3670, so1826, and so3585) selected for comparative qRT-PCR displayed downregulated or unaltered expression patterns at the 180-min time point of chromate exposure based on microarray hybridization. Comparison of gene expression levels as determined by microarray and qRT-PCR analyses indicated a high degree of concordance, with a Pearson's correlation coefficient (r) value of 0.99 (data not shown). For the mutant strain, both microarray hybridization and qRT-PCR indicated that so2426 expression levels were not detectable above background, thus providing additional confirmation of deletion of the so2426 gene.
Pairwise complete-linkage clustering was used to identify groups of potentially co-ordinately regulated genes among the 841 ORFs (18% of the arrayed genome) showing a twofold or greater statistically significant (p < 0.05) change in expression for at least one time point during Cr(VI) exposure. One particularly noteworthy cluster of similarly expressed genes (Cluster A in Figure Figure5)5) comprises 46 genes with functions distributed across the following role categories: cellular processes (2 genes), energy metabolism (9 genes), hypothetical proteins (17 genes), protein synthesis (1 gene), regulatory functions/signal transduction (4 genes), and transport and binding proteins (13 genes). These clustered genes displayed a distinct pattern of downregulated expression in the Δso2426 mutant relative to the wild-type MR-1 strain, particularly at the earlier time points (5, 30, 60, and 90 min) of chromate exposure (see Table Table1).1). This is consistent with our previous studies demonstrating that the wild-type so2426 gene was induced at the transcriptional level early in Cr(VI) exposure, i.e. 90 min or less, but not at 24 h post-treatment [11,12]. Closer inspection of the 46 potentially co-regulated genes (Table (Table1)1) revealed that many of the transport/binding, hypothetical, and regulatory genes were shown previously to be upregulated in wild-type MR-1 cells in response to chromate treatment [11,13], thus suggesting that these ORFs are candidate gene targets of positive control by SO2426. The genes constituting Cluster A are discussed in greater detail in the sections below.
Cluster B (Figure (Figure5)5) includes 20 genes, three of which encode AcrB/AcrD/AcrF family proteins (SO1882, SO3484, SO4692) presumably involved in drug efflux and resistance. All of these 20 genes showed decreased expression in the Δso2426 mutant except at the 24-h time point, where mRNA levels increased (Figure (Figure55 and Additional file 1). Notably, Cluster B also contains genes so0392 (putative lipoprotein) and so4688 (glycosyl transferase, group 2 family protein), both with annotated functions associated with the cell envelope, as well as cpxA (so4478). In E. coli, CpxA is the sensory inner membrane kinase that functions with its cognate partner, a cytoplasmic response regulator (CpxR), in a prototypical two-component system to sense and respond to perturbations in the bacterial cell envelope (reviewed in [35]). S. oneidensis cpxR also displayed downregulated temporal expression profiles in the Δso2426 mutant under chromate challenge but grouped with genes in Cluster A.
Thirteen of the genes in Cluster A have annotated functions in metal transport and binding, in particular Fe acquisition and homeostasis: ftn (ferritin), so1580 (TonB-dependent heme receptor), so1771 (GntP family permease), so2045 (cation efflux family protein), alcA (siderophore biosynthesis protein), so3031 and so3032 (both putative siderophore biosynthesis proteins encoded immediately downstream of alcA), so3033 (ferric alcaligin siderophore receptor), so3063 (sodium:alanine symporter family protein), so4150 (putative transporter), viuA (ferric vibriobactin receptor), so4712 (putative ABC transporter, ATP-binding protein), and so4743 (putative TonB-dependent receptor). In contrast to the TonB-dependent receptor genes so1580 and so4743, either no significant change or a maximal twofold decrease at the 180-min time point was observed for so2427 (putative TonB-dependent receptor), located directly upstream of the so2426 response regulator gene. Of the 13 genes with transport and binding functions in Cluster A, ten ORFs (ftn, so1580, so2045, alcA-so3031-so3032, so3033, so3063, viuA, so4743) were shown in an earlier study to be induced in chromate-challenged wild-type MR-1 cells compared to untreated cells [11,13]. Temporal expression patterns for these genes demonstrated that they were downregulated 2- to 20-fold in the Δso2426 strain over the 180-min time course (Table (Table1),1), suggesting that SO2426 acts as a direct or indirect positive regulator of a subset of Fe uptake and storage genes in S. oneidensis.
The alcA-so3031-so3032 operon encodes proteins required for siderophore biosynthesis in MR-1 [36], and the product of gene so3033 is predicted to allow for the cellular utilization of the structurally undetermined MR-1 siderophore. The differential profiles for these four genes were characterized by a peak in down-regulated expression (ranging from ~7- to 17-fold) at the 180-min time interval post chromate addition, followed by no significant change in expression at 24 h (Table (Table1).1). The first gene in the siderophore biosynthetic operon shows 48% sequence identity to Bordetella pertussis alcA, which is required for alcaligin production [37], while the two downstream genes (so3031 and so3032, respectively) diverge from alcB and alcC and are annotated as putative siderophore biosynthesis genes. While the structural identity of the MR-1 siderophore has not been elucidated, we predict that MR-1 likely produces a siderophore similar to putrebactin [38], a novel cyclic dihydroxamate siderophore characterized from Shewanella putrefaciens strain 200 and structurally similar to alcaligin.
Consistent with the transcriptomic data, further physiological evidence for the involvement of the SO2426 response regulator in controlling siderophore-dependent iron acquisition was obtained by performing semiquantitative liquid CAS assays in which relative siderophore production levels in supernatants of the Δso2426 mutant were compared to those in wild-type MR-1 cultures. Following 24-h growth in LB medium in the absence of added FeCl3 or chromate, the Δso2426 mutant (A630, 0.739 ± 0.02) exhibited essentially no detectable siderophore production over that of the cell-free control (A630, 0.726 ± 0.005), whereas wild-type MR-1 cultures (A630, 0.108 ± 0.06) produced approximately 7-fold more siderophore than the Δso2426 mutant. As expected, addition of 50 μM FeCl3 to the culture medium reduced siderophore production by the wild-type to near background levels (an approximately 6-fold reduction) but did not affect siderophore accumulation in the Δso2426 mutant. In the presence of 0.3 mM chromate, replicate cultures of wild-type MR-1 exhibited increasing levels in relative siderophore production over time (Figure (Figure6),6), which was consistent with previous microarray expression data showing induction of siderophore biosynthesis genes in response to chromate exposure [11]. By contrast, the temporal profile for the Δso2426 mutant showed an initial moderate increase and then a dramatic reduction in siderophore excretion compared to the wild type (Figure (Figure6).6). Collectively, these data support the hypothesis that SO2426 is a positive regulator of siderophore-mediated Fe uptake.
Cluster A also includes an S. oneidensis gene that is involved in Fe storage, i.e., the ftn gene (so0139) encoding ferritin. The ftn mRNA expression levels were down-regulated 4- to 20-fold in the SO2426 null mutant across the entire temporal range, with the peak in repression occurring at the 180-min time point (Table (Table1).1). Although iron is an essential micronutrient for most organisms, free excess Fe in the presence of oxygen is potentially toxic because of its tendency to participate in Fenton chemistry reactions, which generate cell-damaging reactive oxygen intermediates. By scavenging and storing intracellular free Fe in a remobilizable form, Ftn protects against Fe(II)-mediated formation of hydroxyl radicals [39,40]. Studies have indicated that ferritins make important contributions to both Fe storage as well as cellular protection against oxidative stress [41,42] and that ftn transcription is co-ordinately regulated by iron and redox stress in some bacteria [43]. S. oneidensis ftn was shown previously to be induced at a low level (~2-fold) in chromate-exposed wild-type MR-1 cells in contrast to untreated cells [11]. The induction of ftn in S. oneidensis might act, at least partially, to mitigate chromate-generated oxidative stress by sequestering excess free Fe available for the catalysis of Fenton reactions and the formation of additional reactive hydroxyl radicals.
The majority of coordinately repressed genes in Cluster A belong to the functional category of hypothetical and conserved hypothetical proteins (Table (Table1).1). Most notably, genes comprising the conserved hypothetical so1188-so1189-so1190 operon exhibited the highest degree of down-regulated expression in the chromate-exposed Δso2426 mutant relative to the wild type, with decreases in mRNA ratios ranging from 45- to > 300-fold over the 180-min period. No significant changes in expression, however, were observed for these genes at the 24-h time point. In addition, the hypothetical gene so2425, which is co-transcribed with so2426 in a polycistronic transcript (shown in this study), was repressed as much as 11-fold over the 180-min period, pointing to the possibility that the so2426 operon may be under positive autoregulatory control. Surprisingly, the functionally unknown genes so2424, so2423, and so2422, which also form part of the so2426 operon, showed either a less than 2-fold change or no significant differential expression over the time course.
In addition to metal transport and hypothetical genes described above, the temporal expression profiles for four transcriptional regulatory genes (so0916, so0544, cpxR, so4567) were altered in the Δso2426 mutant. The function of the regulators encoded by so0916, so0544, and so4567 in S. oneidensis as well as their target genes are not known. However, gene so4477 encodes a homolog of CpxR, the response regulator component of the CpxAR envelope stress response system, which has been well characterized in E. coli (reviewed in [35,44]). The annotated cpxR gene in S. oneidensis MR-1 was downregulated as much as threefold in response to the so2426 deletion under chromate stress conditions (Table (Table1).1). This is in contrast to the two- to three-fold induction measured for cpxR in chromate-treated wild-type MR-1 cells [11]. Located immediately downstream of cpxR in an apparent operon is cpxA, which was downregulated as much as 3-fold in the mutant but grouped in a separate cluster (Cluster B in Figure Figure55).
In E. coli, the CpxAR two-component pair constitutes a stress response system the main function of which is to sense and respond to cell envelope distress, principally misfolded periplasmic proteins, by activating genes encoding proteases and folding catalysts [35,45,46]. However, the complexity, extent, and response overlap of the Cpx system have become progressively apparent, as evident from its involvement in outer membrane porin expression [47], stationary-phase survival [48], and the response to high pH stress [49] to name a few. Although the specific cellular role of the CpxR homolog in S. oneidensis has not been established, it is intriguing to consider the possibility that interplay between CpxR and SO2426 may occur in response to metal stress. It is conceivable that the CpxA-CpxR two-component system may respond to oxidative damage imposed on biomolecules as a consequence of reactive oxygen species generated during intracellular partial Cr(VI) reduction.
While hypothetical and metal transport/binding genes are dominant in Cluster A (representing 65%), other notable genes with ≥ 2-fold-repressed mRNA levels in the Δso2426 mutant include ORFs codifying a putative bicyclomycin resistance (Bcr) protein (SO2280) and a ferric iron reductase protein (SO3034). Bcr proteins are members of the major facilitator superfamily class of membrane efflux pumps and play important roles in drug resistance [50]. In a previous work, the so2280 gene in a wild-type MR-1 genetic background exhibited 2- to 4-fold upregulated expression at the transcript level in response to short-term chromate exposure, i.e., up to 90 min [11], but was shown in this study to be repressed as much as 4-fold in Cr(VI)-challenged Δso2426 cells (Table (Table1),1), suggesting that the so2280 gene is a direct or indirect target of SO2426-mediated activation under chromate conditions. Although the cellular function of SO2280 is presently unknown, its co-ordinately regulated expression with the siderophore biosynthesis operon (alcA-so3031-so3032) and gene so3033, encoding a ferric alcaligin siderophore receptor, is intriguing. Studies in other bacterial systems have shown that certain proteins resembling Bcr at the amino acid level function in siderophore export from the cell [51].
Downregulated expression of gene so3034 (a putative ferric iron reductase) peaked at the 90-min time point with a 4-fold decrease in mRNA levels (Table (Table1).1). Ferric iron reductases are thought to be involved in reducing siderophore-complexed Fe(III) to Fe(II), thus enabling dissociation of the high-affinity Fe3+-chelating siderophore from iron [30]. The entire so3030-so3034 gene cluster, which is predicted to be involved in siderophore production and utilization, exhibits similar temporal expression profiles in the mutant, suggesting a role for the SO2426 response regulator in coordinating these functions. It is not clear whether the other differentially expressed energy metabolism genes (fdhG, fdnH, fdhE, so4503, so4506, so4509) contribute directly to chromate stress response or show altered transcription as a result of secondary effects associated with the so2426 deletion. These genes were not observed to be induced in response to chromate exposure in our previous transcriptomic studies using wild-type S. oneidensis MR-1 [11].
S. oneidensis SO2426 is an apparent orphan response regulator with an N-terminal domain that contains an aspartate residue at the canonical receiver phosphorylation site and a predicted C-terminal wHTH motif indicative of response regulators in the OmpR subfamily. Although previous work in this laboratory demonstrated enhanced mRNA levels for the so2426 gene in a S. oneidensis Fur-deficient strain [31] and in chromate-challenged wild-type MR-1 cells [11], the basic molecular function of this predicted transcriptional regulator remained undefined. In this study, by integrating functional genomics and genetic approaches, we have shown that SO2426 is a direct or indirect positive regulator of siderophore-mediated Fe transport systems, the Fe storage gene ftn, and other as-yet uncharacterized cation transport genes. At this time, however, we cannot discount the possibility of negative regulation by SO2426 as well. Clustering analysis of temporal transcriptome data revealed a distinctive subset of 46 coordinately expressed genes that were consistently downregulated in the so2426 deletion mutant during the initial 180-min period of chromate exposure. Many of these genes – namely, ftn, the alcA-so3031-so3032 siderophore biosynthesis operon, so1580 (TonB-dependent heme receptor), so4743 (TonB-dependent receptor), viuA, so2280 (bcr), as well as a number of hypothetical genes – constituted the predominant molecular response to acute chromate stress in wild-type MR-1 [11]. The transcriptomic analyses described here identified a number of possible directly SO2426-regulated genes, including those of unknown function, that may serve as targets for future studies.
One possible hypothesis that can be generated from the collective data described here is that S. oneidensis MR-1 employs at least two control mechanisms for regulating Fe acquisition via siderophores under conditions of external Fe insufficiency: derepression as a result of decreased Fur activity and activation by SO2426 to enhance needed expression of Fe(III) transport systems. CAS siderophore assays clearly demonstrated that the Δso2426 mutant was deficient in siderophore production compared to wild-type MR-1 cultures in the presence and absence of chromate, indicating that SO2426 plays a positive role in coordinating the regulation of siderophore-dependent Fe uptake mechanisms in S. oneidensis. Furthermore, the impaired chromate reduction rate of the so2426 deletion strain points to a connection between Fe transport and chromate stress response that requires further delineation at the molecular level. Our previous global studies characterizing the transcriptome and proteome of wild-type MR-1 in response to sub-lethal acute doses of chromate demonstrated a dramatic and unexpected induction of genes involved in Fe sequestration and uptake in contrast to unchallenged cells [11,13]. Two possible explanations for this observation are (i) Fe limitation under external chromate conditions, and (ii) direct or indirect displacement of the Fe(II) cofactor from Fur-binding sites under elevated Cr(VI), resulting in decreased DNA-binding activity of the Fur repressor and subsequent derepression of target genes. Exposure to other metals has been shown to perturb intracellular Fe pools and hence influence Fur-mediated repression [52]. Future studies will focus on experimentally confirming direct gene targets of SO2426 control and further elucidating the linkage between regulation of Fe homeostasis and chromate stress response at molecular and physiological levels.
All bacterial strains and plasmids used in this study are listed in Table Table2.2. S. oneidensis strain MR-1 [4,53] was used as the wild type. The Δso2426 strain, a derivative of MR-1, contains an in-frame deletion of the so2426 locus on the chromosome (see below for a detailed description of mutant construction). For growth studies, wild-type and mutant strains were cultivated aerobically with shaking (250 rpm) at 30°C in Luria-Bertani (LB) medium (pH 7.2) alone or in media amended with 0.3 mM (final concentration) of K2CrO4. Optical density was monitored at a wavelength of 600 nm (OD600) in triplicate using either a Spectronic 20D+ spectrophotometer (Thermo Electron Cooperation, Waltham, MA) or the Bioscreen C microbiological culture system (Growth Curves USA, Piscataway, NJ) as described elsewhere [36].
For transcriptome analyses, three separate batch cultures each of wild-type MR-1 and the Δso2426 mutant were grown to mid-exponential phase (OD600, 0.5) in 100 ml of LB broth, followed by addition of 2 M K2CrO4 to a final concentration of 0.3 mM. The two different sets of chromate-challenged cultures (wild type and mutant), each consisting of three biological replicates, were grown aerobically in parallel at 30°C. Cells were harvested for RNA extraction at 5, 30, 60, 90, 180 min, and 24 h post-chromate exposure. For RT-PCR and 5' RACE experiments, wild-type S. oneidensis MR-1 cells were grown to mid-log phase (OD600, 0.5) in LB broth at 30°C and then exposed to chromate (final concentration, 1 mM) for 30 min before harvesting cells for RNA extraction. Untreated MR-1 cells were grown in parallel and used as the control.
Disappearance of extracellular Cr(VI) was quantified spectrophotometrically using the 1,5-diphenylcarbazide (DPC) method as described elsewhere [34]. Cultures were assayed at different time points (5, 30, 60, 90, 180 min, and 24 h) post chromate-challenge to determine the amount of residual Cr(VI) remaining in the medium by measuring absorbance at 540 nm using a Varian (Cary-1E) UV-visible spectrophotometer (Hewlett-Packard, Wilmington, DE). Cell-free LB medium containing 0.3 mM K2CrO4 served as the abiotic negative control and was monitored in parallel with the experimental samples.
The chrome azurol S (CAS) assay for detection of siderophore production was performed essentially as described by Schwyn and Neilands [54]. Siderophore biosynthesis and excretion by the Δso2426 mutant was compared with those by the wild-type MR-1 strain in LB broth with or without the addition of 50 μM FeCl3 or 0.3 mM K2CrO4. Cultures of these strains were grown aerobically to stationary phase (OD600 > 1.0) at 30°C for 24 h. Cell-free supernatants of S. oneidensis cultures were mixed 1:1 with the CAS assay solution and equilibrated at room temperature for 2 h before the absorbance at 630 nm was measured. The relative siderophore production was calculated as the ratio of the control (uninoculated LB medium) A630 to the wild-type or mutant strain supernatant A630. All CAS measurements were performed in triplicate, and at least two independent determinations were conducted.
An S. oneidensis MR-1 in-frame deletion mutant lacking the so2426 locus was generated using the cre-lox recombination system as described elsewhere [32,33]. The application of this mutagenesis strategy to targeted S. oneidensis MR-1 genes has been described previously [55]. The primer sequences used in the generation and verification of the Δso2426 mutant are listed in Table Table3.3. PCR was used to amplify a 900-bp region upstream and a 840-bp region downstream of the so2426 gene using primer pairs Del900-F/Del900-R and Del840-F/Del840-R, respectively. The amplified regions were cloned into the kanamycin-resistant (Kmr) plasmid pJK100 [32]. The resulting plasmid, pJK102, carrying a correct construct was introduced into the E. coli strain WM3064 [32], a diaminopimelic acid (DAP) auxotroph, and then subsequently moved into MR-1 via conjugation with WM3064. The resultant MR-1 strain was Kmr/Tets and contained the loxP-Kmr-loxP cassette. Removal of the kanamycin cassette and the helper plasmid was performed as described elsewhere [33]. A cre-recombinase enzyme-producing plasmid, pCM157/Tetr, was introduced into MR-1 to resolve the loxP-Kmr-loxP cassette through conjugation. The Tetr plasmid pCM157 was cured from MR-1 by continuous culturing in non-selective LB medium. The in-frame deletion of so2426 was confirmed by PCR amplification using several sets of primers (ISU2426/ISD2426, OP840, OP900, kanF/kanR in Table Table3)3) and DNA sequencing.
The Δso2426 mutant was complemented by reintroduction of the wild-type so2426 allele on a low-copy-number plasmid. For this, the so2426 open reading frame with 100 bp of upstream sequence was amplified from S. oneidensis MR-1 genomic DNA using primers 2426com-F and 2426com-R (Table (Table3).3). The PCR product was gel purified, digested, and ligated into the BamH1 and HindIII sites of the plasmid pBBR1MCS-5 [56], which contains a gentamicin resistance (Gmr) cassette. The resultant construct harboring the complete S. oneidensis so2426 gene with its endogenous promoter was designated pBB2426. The pBB2426 plasmid was electroporated into E. coli strain WM3064 and then introduced into the S. oneidensis Δso2426 mutant and wild-type MR-1 strains via conjugation. In addition, plasmid pBBR1MCS5 without the insert (empty vector) was also transferred into the mutant and wild-type strains via conjugation with WM3064. Gmr Shewanella colonies were verified for the presence of the so2426 gene by PCR analysis and DNA sequencing.
For microarray profiling and real-time RT-PCR experiments, total cellular RNA was isolated from S. oneidensis cultures using the TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RNA preparations were treated with RNase-free DNase I (Ambion Applied Biosystems, Foster City, CA) to remove residual genomic DNA and subsequently further purified using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's RNA cleanup protocol. Total RNA was quantitated as described previously [36]. For RT-PCR and 5'-RACE analyses, total RNA was extracted from S. oneidensis cultures using the RNeasy Mini Kit (Qiagen). Chromosomal DNA contamination was removed by incubating total purified RNA with 3 U of RNase-free DNase (Ambion Applied Biosystems) for 30 min at 37°C. The DNA digestion reaction was performed twice for each RNA sample. The quantity and purity of the RNA was assessed as described previously [36]. The integrity of all RNA samples was assessed visually using 1% agarose gel electrophoresis and ethidium bromide staining.
Transcriptome analyses were performed with a microarray containing 4,197 PCR amplicons and 451 specific 50-mer oligonucleotides, covering approximately 94% of the total predicted gene content of S. oneidensis MR-1. Fabrication of the MR-1 arrays has been described in detail elsewhere [57]. Synthesis of the two differentially labeled cDNA pools (wild-type MR-1 and the Δso2426 mutant) to be compared, microarray prehybridization and hybridization, and post-hybridization washings were performed as described previously [36]. Temporal gene expression analysis was performed using six independent microarray hybridizations (three biological replicates × two dye-swap reactions) for each of six time points, with each slide containing two spots representing each gene at different array locations for a total of 12 signal intensity measurements per gene per time point. Image quantification, data normalization, and analysis of gene expression data for statistical significance were conducted as described by Brown et al. [36]. The time-series microarray expression profiles of the Δso2426 mutant relative to the parental strain were clustered using Hierarchical Clustering Explorer (HCE) [58]. During the clustering process, only genes with an expression value of at least ≥ 2-fold or ≤ 0.5-fold in one or more of 6 expression profiling time points were included in the analyses. As a result, a dataset of 841 genes was clustered based on average linkage using Euclidean distance.
The microarray data reported in this study have been deposited in MIAME-compliant format at Gene Expression Omnibus on the NCBI website [59] under series accession number GSE12129. The statistically analyzed microarray output is provided in Additional file 1.
Reverse transcriptase, quantitative real-time PCR (qRT-PCR) was used to provide an independent assessment of gene expression for five selected genes (so0404, so2426, so3670, so1826, and so3585), which exhibited different expression patterns (i.e., down-regulated or no change). These genes are predicted to encode a hypothetical protein (SO0404), a DNA-binding response regulator (SO2426), proteins involved in transport and binding of cations (SO3670 and SO1826), and a putative azoreductase (SO3585). Relative expression patterns for each selected gene were independently confirmed using the following primer pairs, which were designed using the program Primer3 http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi: SO0404, 5'-AGTATAACCAAGCGCCAGTA and 3'-GCATCGGTATTAACTTGCTC; SO2426, 5'-GCAGAAGGATTTAGGTCGAT and 3'-CGGTGTTGATTAAAGTACGC; SO3670, 5'-TCTAAACAGTCGCAGGAGCA and 3'-GCGCCATATTGCTATCCATT; SO1826, 5'-GGGTGTCCCAAGCTAGT CAA and 3'-GAGCATTACTCGTCCCCTGA; SO3585, 5'-CGAGGCTATCCATC ACTTAG and 3'-TGGAAAACACGATAAAGACC. Reverse transcription was performed on the 180-min time point samples with 3 μg of total cellular RNA and 2.5 mM random hexamers using Superscript™ II RNase H- Reverse Transcriptase (Invitrogen) as described previously [31]. Quantitative PCR was carried out in an iCycler iQ® real-time PCR system (BioRad, Hercules, CA) in 50-μl reaction mixtures containing 1 μl cDNA, 600 nM forward and reverse primers, and iQ SYBR green supermix (BioRad) according to the manufacturer's instructions and conducted under the following conditions: 30 sec at 95°C, followed by 40 cycles of 15 sec at 95°C, 30 sec at the specific annealing temperature, and 30 sec at 72°C. The qRT-PCR reactions were performed in triplicate for each of the three biological replicates tested. Standards for each gene of interest were included in the analysis as described previously [31]. The final Δso2426/WT ratio for each target gene and the standard error were calculated as described elsewhere [31], and the linear correlation between the qRT-PCR and microarray data was determined based on the log mean values using Sigma-Plot version 9.0 (SPSS Inc., Chicago, IL).
Reverse transcription-PCR (RT-PCR) was performed to investigate the transcription of the so2427-so2422 gene cluster on the MR-1 chromosome. First-strand cDNA was synthesized from 1 μg of DNase-treated total RNA isolated from MR-1 cultures grown in LB broth under either non-stress (no chromate added) or chromate stress (1 mM, 30-min exposure) conditions. Random hexamers (250 ng), dNTPs (10 mM), 5× First-Strand buffer (4 μl), 0.1 M DTT (1 μl), RNaseOUT (1 μl), recombinant RNase inhibitor (40 U), and Superscript™ II Reverse Transcriptase (200 U; Invitrogen) were added to the reaction mixture, which was incubated at 25°C for 10 min, 42°C for 50 min, and then 70°C for 15 min in accordance with the recommendations of the supplier. Synthesized cDNA was used as the template in subsequent PCR amplification reactions with gene-specific oligonucleotide primers P1–P9 (Table (Table3)3) spanning the so2427-so2422 gene region. The TripleMaster PCR System (Eppendorf, Westbury, NY) was used for amplification, with primers added to each reaction mixture at a final concentration of 250 nmol. The reaction mixtures were subjected to 30 cycles of denaturation at 94°C for 60 s, annealing at 44–52°C (depending on the primer Tm) for 60 s, and an extension at 72°C for 1–2 min in an Eppendorf Mastercycler ep gradient thermocycler. Each amplified product was analyzed by 1% agarose gel electrophoresis with ethidium bromide staining. In addition, each PCR product obtained was cloned into the pGEM-T vector (Promega, Madison, WI) and sequenced at Purdue University's Low-Throughput DNA Sequencing Laboratory to confirm that they were the expected loci of each respective gene region.
The transcriptional start site of the so2426 gene was localized using the 5' RACE System for Rapid Amplification of cDNA Ends version 2.0 (Invitrogen) according to the manufacturer's instructions. Briefly, cDNA was generated using Superscript™ II reverse transcriptase (42°C, 1 h; Invitrogen) in a reaction containing 2 μg of total cellular RNA (from non-stressed or chromate-stressed MR-1 cells) and a so2426-specific primer (2426-GSP1: 5'-TAACCGATTGAATTGTT-3'). Nested PCR was performed using two so2426-specific primers (2426-GSP2: 5'-TGCACTAACCGTCGCTCTATGGCCTGCAAA-3' and 2426-GSP3: 5'-GCACCAAGTTCATAGCTTCGTATCCTGTCT-3') and the manufacturer-supplied abridged anchor and abridged universal anchor primers. The 5'-RACE products were analyzed by agarose gel electrophoresis and cloned into the pGEM-T vector (Promega) prior to DNA sequencing.
KC created the deletion mutant analyzed in this work; performed the microarray experiments, qRT-PCR verifications, and the initial data analysis; and carried out the growth studies and Cr(VI) reduction assays. WW performed the operon structure analysis, transcription start site determination, and CAS siderophore assays. X–FW carried out statistical and clustering analyses of the microarray data. KC and X–FW both contributed to the manuscript writing. DKT conceived and coordinated the study and performed the majority of the manuscript writing. All authors read and approved the final manuscript.
Complete processed microarray dataset for the S. oneidensis Δso2426 mutant compared to the wild type under chromate conditions. This file provides the complete statistically analyzed microarray output for all six time points (5, 30, 60, 90, 180 min, and 24 h) during chromate (0.3 mM) exposure starting with the Gene ID, mean Δso2426 mutant/WT expression ratio, n (number of signal values out of 12 total per gene included in the statistical analysis), statistical significance, gene name, gene product, main role category, and subrole category. ArrayStat™ (Imaging Research, Inc., Ontario, Canada) was used to determine the common error of normalized expression values, remove outliers, and determine statistical significance via a z test for two independent conditions and the false discovery rate method (nominal α, confidence cutoff of p < 0.05).
We thank Dr. Joel Klappenbach for plasmids and E. coli strain WM3064 used in site-directed mutagenesis and Dr. Steven Brown for assistance with the construction of the so2426 deletion mutant. We also thank Drs. Jizhong Zhou and Liyou Wu for S. oneidensis MR-1 microarrays, and Dr. Gene Wickham for helpful comments concerning the manuscript. This research was supported in part by the Office of Science (BER), U. S. Department of Energy, Grant No. DE-FG02-06ER64163, to DKT.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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What is the difference berween DOM and TRU. Why is DOM higher but this pays a higher dividend? Also how can you get info when they decide in Jan. if they will continue the section 29?
WHY DO US RTs exist at all? I cannot see the point in them , except maybe to take some of the risk?
Your voice reflects from that brick wall with the uttermost of sincerity. I always say "help those who help themselves", but I do agree that it is important to present the facts as you see them, so that others don't get led astray with mis-information.
Thanks for your posts.
That same person posted very similar crap not too long ago on another RT board and was corrected by several of us there.
Now, to post the same nonsense here or on any other RT board again must mean he just has his mind made up and is not going to let the facts change his views one iota.
Maybe he is older and going senile and I shouldn't have been so hard on him. Oh well... if he is coherent enough to post on Yahoo boards, his mind should not be so far gone as to not be able to read and comprehend the simple fact that US RTs have no managements and are not allowed, by law, to make any of the decisions that he is bitching about them doing poorly.
Hay, Need High Yield, don't hold back! Let it all hang out. Tell it how it is, don't sugar coat it!!!!! Don't let idiots like this bother you.
Suggest you do some due diligence on US RTs before you buy/sell/short or do anything here!
You are completely in the dark and clueless as to what these US RTs are all about!
THERE IS NO MANAGEMANT OF US RTs!!!
THERE IS NO MANAGEMANT OF US RTs!!!
Say that to yourself until you understand what that means. There is NO ONE to fire, NOBODY is playing golf, NOBODY buying cars, etc.
Just a single bank EMPLOYEE that deposits the trust's income, cuts the distribution checks and makes the required SEC filings, period!
"...tell em to get their asses in gear. "
Seems the ONLY one needing to get their ass in gear is YOU! You need to do an inklinking of homework to learn even the basic fundamentals of US RTs before spewing out such utter nonsense about them on these boards!
Yes, see May 14,2002 from tirtz 10-q report. It seems the Can. folks got the right idea. Us trust management got to get out from behind their Houston , Dallas ivory tower desks and find out why costs have ski rocked? Same crap as wcom, dyn, mir, management rather play golf buy new cars, buying 2nd homes instead of doing business. Fire these duds and get someone that STICKS to business not playtime for bonzo. Usually their email or phone numbers are at the bottom of PRs give em a call and tell em to get their asses in gear.
US RTs MAY have new additional wells drilled on the properties in which they have an interest. This must be under the control and the decision of the operator, not the trust. Also, there may be secondary recovery methods employed on their existing wells or rework them for added recovery and finally, new wells can be drilled to different pay zones on thier properties.
However, US RTs CAN NOT purchase or lease additional properties, buy additional reserves from someone else or merge with another O&G company, whereas Canadian RTs can and frequently do so.
Hope that helps.
SJT is the champion of reserve replenishers (they have been paying for almost 20 years without much overall difference in production); and, to my knowledge, also HGT and TELOZ increase their reserves by drilling and well improvement. Definitely TRU and CRT do NOT replenish their reserves. Others (including DOM) I am not sure about. Does this information conflict with the analyses of you other readers of RT threads?
"I'm now beginning to think more decidedly in terms favoring Canadian R/T's!"
I hope you didn't take that from my post.
Canadian Rts open up another whole can of worms.
The possibility of adding to reserves sounds good but in reality, they must pay for those new reserves with debt or more units. So management and timing and prices paid are critical to Canadian RTs.
Most people who say that Canadian RTs can replace their reserves haven't taken the time to do the simple math but are bowing to that slogan without realling considering the ramifications.
Few of the Canadian RTs have been around long enough to see if that theory even works.
As a clue to what I'm talking about, suppose US RTs could expand. If US RT "A" that is priced at $10.00 and pays $1.00 per year in distributions on its reserves with a 10 year remaining life buys out US RT "B", also priced at $10.00 and paying out $1.00 yearly from their reserves with a 10 year life, what happens at the end of those 10 years?
It's not nearly as simple as some believe. The Canadians can buy reserves but the VAST BULK of the income of those reserves are used to pay the distributions on those units paid for those reserves with only a tiny portion that will go to the prior unitholders.
Debt adds another risk that US RTs don't have. So, managements ability to find UNDERPRICED reserves during low commodity periods will be critical for the long term success of the Canadian RTs.
Most Canadian RTs have reserve lives of 7-12 years whereas many of the US RTs have been producing for many years and will likely continue to produce years after many of us are dead.
Finally, Canadian RTs do not share the same tax benefits as their US counterparts.
Do some serious due diligence before buying any RT, US or Canadian. They pay these high yields for a reason!
graham99: Re: your #3660, and
NHY: Re: your #3661:
Many sincere thanks to you both for these clarifications of the differences between Stateside and Canadian R/T's. What you both say, makes much sense! Based on this information, I'm now beginning to think more decidedly in terms favoring Canadian R/T's!
Good luck!
t now makes


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Illustrated interview : FML's bone
Oskar's website if you feel like you wanna laugh: http://www.mojocomic.com/
If you want to be the next published artist, send an email to alice [at] fmylife.com including a link to your website/blog/DA. But DON'T send your illustration right away! You need first to get in touch with me, and I'll tell you what you have to do!
If you want to be the next published artist, send an email to alice [at] fmylife.com including a link to your website/blog/DA. But DON'T send your illustration right away! You need first to get in touch with me, and I'll tell you what you have to do!
FML video: Fake Busy
This new video was put together by Ben Giroux and Ray Chase, Co-Creators of AwkwardUniverse.com
The actors in this one are great, don't you just love them? We do. And the way the video is produced is not too shabby either. By the way, you can follow Ben and Ray on Twitter if you want to (say hi to them from us) @BenGiroux and @Raychases.
Welcome to the new FML!
Our website is now 2 and a half years-old, and we thought that this would be the ideal arbitrary time to unveil what the whole team has been working on for the past few weeks. Here's thus the new Fmylife. To go on a magical mystery tour, step right this way…
"This is an evolution."
And not a revolution, to paraphrase the guy with the black turtleneck sweater. Ever since our website was launched, it has tried to follow the same policy: to have a clear and simple appearance, and to keep the spotlight on the content, i.e. the FMLs. You reading this right now proves that you like our concept and ethos, and we kept these notions in mind when we started thinking about how to jazz things up a bit.
As you can see, we finally plumped for giving you more or less the same as before, but improved and updated. The website has been streamlined and given a bit of an overhaul, some stuff has been moved around, and pages have undergone some alterations. Nothing too radical, but some parts of it have been given a lick of paint, panels have been beaten into smoother shapes here and there, all with the sole purpose of making your time spent with us more pleasant, and your interactions with the website easier. And of course, we've also added some funky new functions.
The categories are obviously still present, and do the same things as before. Underneath them, three new buttons will give you a quick and obvious access to random FMLs, the Top FMLs as well as the page containing all of the illustrated FMLs.
Everything has been redesigned to make things clearer. One thing hasn't changed though: your account settings are still available via the "Edit" link. However, two new links have materialized, notably "My Comments" which will take you straight to the comments you are following, and a new function that will display all the stories that you have commented on. Everything concerning moderating has been moved to "My Profile".
The website is now tailored for anyone with a visual handicap. To access the adapted version, just click on the link at the bottom right-hand side of the website.
Previously, the only option you had in the comments section was to be able to follow certain comments. That's all in the past. Today, a powerful organisation system has made its appearance. It makes it possible to display comments in a page format (dividing up the list of comments into pages of 50 items) but also sort them according to the amount of votes (the comments are reorganised to display the more popular ones first). You'll also be able to choose to display your own personal comments only. And the possibility to flag comments is back. If a comment seems to be out of line, you can click on the little signpost, give the reason why you're flagging it and sent it to us. This function should however only be used in extreme cases, just to help our moderating team weed out offensive stuff.
And are there any really amazing new additions? Yes! The little progress bar which has appeared in your member's area when you recently logged on to the website has maybe given the game away, but that's too bad. A new function is now available which should be a great addition for all the loyal FML users, who have lost count of the amount of time they devote to the website. The badges have arrived!
These little guys will congratulate your activity and loyalty, and will be credited to you when you perform certain things on the website. Some are publicly visible, some are secret, and once you've got some, you'll be able to display them with honour on your comments as well as on your profile. There's a whole bunch of them to be collected, and you already know that you want them all.
Click here to discover the badge page
This article doesn't cover each new addition to the website, but you'll soon be able to work out what's different, and we have made quite a few changes. Don't hesitate to look around the website to check everything out (and maybe earn some secret badges on the way). If you come across a technical glitch, if something doesn't happen the way it should or even if you come across a small spelling mistake, please feel free to send us an email to email@example.com and we will get back to you as soon as we can.
Illustrated interview : FML's euthanasia
It's summer! And don't think that because of the sun, the sand, the sexy-almost-naked-people-damn-you-swimsuits that we're surrounded by, that we'll not provide you a new comic today. As Flufflybum told you last week, we're welcoming today Jeffrey Jang for a new illustration AND an illustrated interview. Let's do this :
-Hello Jeff, it's been a while, how are you ?
-I am good! I'm excited to be able to share my art on FMyLife again.
-Speaking of that, why did you decided to do a second round?
-I wanted to do a second round because when I first did it, I got a lot of great feedback and found a lot of people who liked Boximals. I am hoping to do the same again to both new and old faces.
-And why did you choose this particular FML story?
-I chose my FML story because I thought it was pretty terrible and funny at thesame time of how the mother took the $100 to euthanize the dog. I have a dog myself, and I think it'd be great if he could take the situation into his own hands (paws?). I chose to show that in my comic.
-Are you suggesting the dog should committ suicide (consternation, horror, etc. ...)?
-Hahaha no! The dog should punish his owner for making such a silly decision with the money.
-Okay... it's better... What are you working on right now?
-I am very busy with my new job as an illustrator right now, but still have a few projects going on. I am working on developing another group of characters, as well as still looking for freelance design work to do on the side. Maybe I'll get a chance to show those characters on FMyLife some day!
-We'd love to. But tell us more about this new job!
-I'm an illustrator for a company in Vancouver that focuses on developing games for big brands to promote things like movies or tv series. I work on things like icons, interface designs and characters!
-It's pretty great. What are your next projects?
-Most of my time in the future will be on Boximals and hopefully my Cloud Six characters, but like I said the new job is my priority currently so it might take some time. I wish there were more hours in the day.
-We all do. *sigh*. As you're featured on FML, you can say anything you want to the FML audience.. go ahead!
-Thanks to everyone who went and let me know how much they liked Boximals on the Facebook page when the first comic went up. It really encouraged me to keep sticking with Boximals, and to work on other ideas I have. I hope everyone likes the second Boximals comic!
-*hug* We love you too. Thank you very much Jeffrey. Now dear readers don't waste anymore time and go read today's comic :
Jeff's website full of boxes, love and laughs: http://www.jjang.ca/
If you want to be the next published artist, send an email to alice [at] fmylife.com including a link to your website/blog/DA. But DON'T send your illustration right away! You need first to get in touch with me, and I'll tell you what you have to do!
If you want to be the next published artist, send an email to alice [at] fmylife.com including a link to your website/blog/DA. But DON'T send your illustration right away! You need first to get in touch with me, and I'll tell you what you have to do!


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Get cagey with the Savoy House Connell collection, designed by Brian Thomas! Great industrial style comes courtesy of the cages protecting each shade of clear seeded glass and the sleek arms are finished in bold English bronze.
Savoy House's Elizabeth 2-light sconce is a beautiful Versailles-inspired crystal fixture with sparkling oversized crystal pendalogues, cream beeswax candle covers and a rich New Tortoise Shell finish with Silver accents. ADA compliant.
The Savoy House Orsay collection, designed by Brian Thomas , combines vintage lighting inspirations with modern steel finishes and clear glass globes for an appealing industrial look.
With it's abundant curls and smooth, clean classic lines the Spirit collection is definitely and eye pleaser. The Pewter finish and White Marble glass finish off the collection with a classic yet stylish look.


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A number of pop heavyweights have come together to feature in Global's latest ad campaignMedia
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Look who it is!
Jennifer Aniston walked the red carpet at the premiere of fiance Justin Theroux's new HBO series, "The Leftovers," at NYU Skirball Center on Monday, June 23, in New York City. The couple looked stunning as usual as they cuddled up and posed for photographers.
Aniston wore a black blouse and miniskirt and rocked tousled hair as her husband-to-be looked dapper in a Dior Homme suit.
The 45-year-old actress couldn't keep her hands off of her 42-year-old beau, who plays suburban police chief Kevin Garvey on "The Leftovers." According to E! News, the show focuses on a group of people who are left behind following the sudden unexplained disappearance of a percentage of the world's population.
"It feels great [to support him]," Aniston told Us Weekly on the red carpet.
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York, ONCanada
June 17, 2013
Do you love the arts? Do you like meeting up? Do you like making friends?
Well, you've come to the right place.
Members of this group are creative and intellectual with a desire to expand our circles of friends to enjoy life and explore the arts in its myriad forms. The group is all about getting together to enjoy creative discussions with friends, to view art, the making of art and how art influences our lives.
And the best thing is, it's run by all of us, collectively. Each of us can decide where to go, what to do, and invite others to join in. We are friends creating events focused on enjoying creativity, together.
Join us!
See our Group Guidelines on the next page...
3,016 International Cinema Fans
1,513 DBNers
2,806 Vancouver Lifestylins
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