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Hiring a Lab Technician

7 Feb

I’m a Fatboy Slim fan, and when I decided to write today’s post, “Praise You” started playing in my head.  Besides the fact that I need to update my music taste, it reminded me that I want to thank all my faithful followers out there for waiting around for me to post again.

It’s been ages (2 years to the day) since I’ve posted and I salute you both.

Many things have changed since I was blogging last… I’m now the proud SciMom to two little rugrats, and a tenure-track faculty member at Wazzu.  [I feel somehow obligated to assert the obvious fact here that all opinions given in this blog are my own and have absolutely nothing to do with my university.]

That’s right folks, 2 virtual petri dishes that crawl and drool and snot all over the place, AND all the responsibilities of a brand-new, wet-behind-the-ears ass prof (assistant professor, for those of you confused about the abbreviation); I’m just a glutton for punishment.

This has given me a new-found sense of juxtaposition: freedom, weighed down by grant-writing; imagination, mired in the realities of funding priorities; and an awesome new job, potentially debunked at tenure-review time.

I, therefore, chose to attempt to squeeze into my painstakingly time-managed work-day schedule some time for fun-writing (aka, blogging).  The shape and form and overall thrust of this blog will largely remain the same – Microbes do, in fact, still rule the world, and I will continue to extoll their virtues.  However, I hope to also cover science and science events more broadly and may even share the odd job posting. (a clear transition into…)

With my very first blog as a new tenure-track faculty, I will shamelessly let you all know that I need to hire a lab technician.  I need someone with experience in soil microbiology and molecular biology, cultivation-dependent and independent techniques.  The pay is not particularly great to start and I can’t cover relocation costs, but the boss is usually pretty cool and she’s super enthusiastic about what she does.   And you should know that Eastern Washington/northern Idaho is not the big city, but rather the outdoor-enthusiast’s playland.  So, for the full job description and details, contact her directly.

The Trouble With Triclosan

1 Dec

Just a few products you might find Triclosan in.

An article was released online Monday in Environmental Health Perspectives which surprised me. For the last five or six years I’ve been enthusiastically extolling the evils of triclosan in the environment, but the connection with human immune dysfunction really caught me by surprise, most likely because I’m a microbiologist (this is probably not news to all you toxicologists out there). As someone who suffers both indoor and outdoor allergies (including hayfever) as well as allergy-induced asthma, it’s no small thing for the authors to be able to say with such a level of confidence…

“… higher levels of triclosan were associated with greater odds of having been diagnosed with allergies or hayfever (p<0.01).”   Clayton, et al., 2010.

The mechanism of impact on the human immune system isn’t clear yet, but in reading the article I discovered that triclosan has been associated with not only endocrine disruption, but also central nervous system effects and thyroid problems for several years now.

This knowledge becomes particularly disturbing when you consider the fact that since it’s creation in the 1960’s, triclosan (also known by its more descriptive chemical name: 2,4,4’-trichloro-2’-hydroxydiphenyl ether) has been added to countless consumer products as an antimicrobial and preservative, including but certainly not limited to: hand soaps, laundry detergents, toothpastes, wound disinfection solutions, deodorants, facial tissues, plastic kitchen utensils, medical devices, and toys.

If a product’s label says “Antimicrobial” you can bet it contains triclosan or a triclosan derivative. Do a quick literature search and you’ll find we’re discovering new uses for it every month, incorporating it into plastics and personal care products of all kinds. Consequently, triclosan and its derivatives are now found in the urine and tissues of over 75% of all Americans, and in soils, sediments, and waterways all over the world.

Enter, environmental microbial populations.

What you might be thinking is that since triclosan is added to things for its antimicrobial properties, it must be wiping out hordes of bacteria and fungi, reducing their numbers worldwide… well, not exactly.

Some bacteria are naturally resistant to triclosan, like certain Pseudomonas species, while in others it actually causes mutagenesis (directed mutations) of their already existing resistance factors. What that means, is that bacteria have already developed a way to resist or survive a number of toxins, like heavy metals or salts, and when they are exposed to triclosan, their resistance factors actually adjust to treat triclosan in the same manner.

A great example would be common bacterial efflux pumps; these are mechanisms within the cellular membrane designed to “pump” a specific toxin right back out of the cell, where it can’t hurt the bacteria. When a bacterium is exposed to triclosan, these pumps go into over-drive and begin to pump out anything that remotely resembles an antimicrobial or antibiotic.

The tripartite efflux pump of Salmonella, which pumps out antibacterial drugs to foster multidrug resistance. Image: Cambridge Dept of Pathology.

There could hardly be a better use for the old adage, “What doesn’t kill us, make us stronger.”

 This type of triclosan-induced antimicrobial resistance has been proven in model species like E. coli, Salmonella enterica, Staphylococcus aureus, and Mycobacterium tuberculosis, but to date it’s been uncertain if these same effects are true of environmental species (common soil and water bacteria). Pycke, et al.(2010) set out to answer that question and their work was published in a recent article in Applied and Environmental Microbiology.

I guess the good news from their work would be that the species of environmental bacteria the authors tested (Rhodospirillum rubrum) was fairly sensitive to triclosan, and the concentrations needed to inhibit its growth and even kill this bacteria were similar to some of the most sensitive species that have been tested to date.

What this means to you: when you use an antibacterial soap to wash your hands, you’re probably killing off 90% of these types of bacteria on in your hands, i.e. they are very susceptible to high concentrations.

The not so good news is that under low-levels of triclosan exposure, the very same efflux mechanisms that I described above were able to confer resistance to these bacteria, and not just to triclosan, but also to a wide range of antibiotics including tetracycline, chloramphenicol, fluroquinolones, and others.

What this means to you: after you wash your hands with antibacterial soap, and that triclosan is diluted in water and washed out into your septic or local waste-water treatment plant, the environmental bacteria exposed to lower levels of triclosan are not killed, but essentially “turn on” their efflux pumps and become resistant to some of the most common antibiotics in use.

We used to think that a bacterium had to first be exposed to a specific antibiotic in order to become resistant to it (or at least one of his pals was exposed and passed on the genetic resistance); now we know that our own waste-streams, laden with chronic levels of the very antimicrobial products our culture clings to, are fostering even greater levels of antibiotic resistant bacteria in the environment.

To sum up

Two reasons not to buy or use products with antimicrobial chemicals like triclosan or triclosan derivatives:
1.) Increased incidence of hayfever, allergies, and immune dysfunction;
2.) Decimation of the human race by antibiotic resistant diseases. (ok, maybe that’s a little overblown, but you get the idea)

If the one doesn’t make you think twice, surely the other will.

Clayton, E., Todd, M., Dowd, J., & Aiello, A. (2010). The Impact of Bisphenol A and Triclosan on Immune Parameters in the US Population, NHANES 2003-2006 Environmental Health Perspectives DOI: 10.1289/ehp.1002883

Pycke, B., Crabbe, A., Verstraete, W., & Leys, N. (2010). Characterization of Triclosan-Resistant Mutants Reveals Multiple Antimicrobial Resistance Mechanisms in Rhodospirillum rubrum S1H Applied and Environmental Microbiology, 76 (10), 3116-3123 DOI: 10.1128/AEM.02757-09

How does your garden grow?

19 Oct

For a leaf-cutter ant, the answer to that question is not so much “silver bells and cockle-shells” as it is “one nutritious fungus and a variety of beneficial bacteria.”  It may not exactly be the stuff of nursery rhymes, but it’s certainly a fascinating three-way mutualism that makes for a great lesson in microbial ecology, and ecology in general, for that matter.

Leafcutter ant fungus-garden in a lab nest. Source: © Alex Wild 2010

Fungiculture, as it’s called, involves production of food or medicine by cultivating mushrooms or other fungi .  In the insect world, fungiculture is practiced by ambrosia beetles, termites, and gall midges, in addition to leaf-cutter (or attine) ants.  That’s right, millions of years before humans even thought about any type of agriculture, these insects evolved the ability to grow fungi for food (Mueller and Gerardo, 2002). 

Attine ants grow their fungal gardens in underground caverns or chambers and supply the fungus with leaf fragments from live plants collected aboveground.   They literally “feed the fungus” and in return, their larvae are reared exclusively on the fungus itself.  So, the ants provide the fungus with food, protection, and dispersal (fungal spores remain viable within the ant digestive tract and can be deposited via ant dung during the daily travels) while the fungus provides a high-quality food source with increased nutrition and survival for the ants. 

Much like humans, these insect farmers have become dependent on cultivated crops and have even developed task-partitioned societies.  And, just like our crops, theirs can be wiped out by parasites and pests (nematodes and mites), their harvest ruined by weeds and disease (unwanted/harmful fungal species), which can lead to collapse of the colony.  

To protect their fungal gardens, the ants have developed specialized behaviors like grooming and weeding the fungus, as well as producing their own antimicrobials through gland secretions, as well as large-scale application of weed killers.  Only, in this case, when I say “weed killers” I’m not talking about synthetic chemicals like RoundUp, I’m talking about specialized soil bacteria which produce potent antimicrobial compounds with specificity towards the unwanted fungal species.  (Barke, et a.l 2010). 

It’s long been known that the reproductive females in each colony pass on the fungal cultivar, but only recently have we begun to understand that the ant’s gardening toolbox is constantly evolving.  Along with the fungus, the queen also passes down at least one species of bacteria (a Pseudonocardia).  This particular bacterium produces an antifungal compound, specifically aimed at reducing the most common pest of the fungal gardens but which leaves the crop itself untouched.  But beyond that, the gardening ants also pick up a variety of soil bacteria along the way including Streptomyces.   In other words, each of the bacteria makes a unique antimicrobial compound that the ants use to their advantage in their agriculture depending on the particular pest the ants are challenged with.

To recap… 

We have leaf-cutter ants, going out into the world each day, chopping down entire forests so they can bring the good stuff home to feed their fungus.  The ants survive off this fungus, so it’s carefully cultivated and passed on from generation to generation, and they have specialized farmer-ants who will each spend their whole life tending to the fungal garden.  Some of the most important tools these farmers use to keep the fungus healthy and productive include natural soil bacteria.  For the worst pests they use a special bacteria, which was also passed on through the generations, but for more common pests, they can use a variety of bacteria collected from their surroundings: different tools for the different problems. 

So, when I hear  “Mary, Mary quite contrary, how does your garden grow?” I, for one, imagine colorful underground mushroom gardens with their little ant-farmers in their little ant-overalls walking the rows, killing weeds with soil bacteria (kinda like the doozers on Fraggle Rock, but with mushrooms). 

Is that weird?

ResearchBlogging.orgBarke, J., Seipke, R., Grüschow, S., Heavens, D., Drou, N., Bibb, M., Goss, R., Yu, D., & Hutchings, M. (2010). A mixed community of actinomycetes produce multiple antibiotics for the fungus farming ant Acromyrmex octospinosus BMC Biology, 8 (1) DOI: 10.1186/1741-7007-8-109

Mueller, U. (2002). Fungus-farming insects: Multiple origins and diverse evolutionary histories Proceedings of the National Academy of Sciences, 99 (24), 15247-15249 DOI: 10.1073/pnas.242594799

Shall we dance?…

29 Sep

This post was chosen as an Editor's Selection for… says the rhizobial bacteria to the leguminous plant root.  In response, the plant warmly embraces her partner, literally wrapping herself fully around the bacteria, and so the intricate and highly choreographed ballet begins.

Image of a root hair embracing it's bacterial consort, by Euan James, via Life Sciences at the University of Dundee.

But how can a minuscule little bacterium communicate to a “higher plant” in a way to elicit such an overwhelming positive response?  A response which catalyzes this fundamental and truly amazing partnership, on which the world as we know it has depended for millenia?

The answer is simple, these bacteria developed special signaling molecules called nodulation (Nod) factors to communicate with certain plants.  These nodulation factors are diffusible signals, which mimic plant hormones, and stimulate very specific developmental processes within the plant during the initiation of the symbiotic relationship (Oldroyd & Downie, 2008).  They provide the choreography of the dance.

By simply altering the length and degree of saturation (think, saturated vs. unsaturated fats) of the nodulation factors, bacteria can target very specific plants.  This species-specific tuning of the nodulation signals allows an individual bacterium to “speak to” the host it prefers and form a relationship with, for example, a soybean plant rather than an alfalfa plant.

Once the plant recognizes the nodulation factor of the rhizobial bacteria, infection occurs through root hair cells which curl around and entrap the attached bacteria.  The bacteria, in turn, begin to break down the plant cell walls which entrap them, and subsequently initiate the formation of a tube-like structure called the intracellular infection thread or IIT (Ivanov, et al. 2010).

Intracellular infection threads containing fluroescently labeled rhizobial bacteria.  (By Dan Gage)

Intracellular infection threads (IIT) containing fluroescently labeled rhizobial bacteria. (By Dan Gage)

The IIT serves as a biological inoculation needle, so to speak, penetrating the root primordial cells and releasing bacteria into the cytoplasm of the plant.  At this point, the plant cells form a protective membrane around the mass of bacterial cells, where the bacteria differentiate into their nitrogen-fixing stage.  This special intercellular plant membrane, also called the peribacteroid membrane (PBM), allows nutrient exchange and helps protect the bacteria from oxygen, which would destroy the nitrogenase enzyme crucial to the process of gaseous N2 fixation (the process which is central to the nitrogen cycle, controls nitrogen availability, and consequently supports life on this earth).

At every point in this intricate dance, the bacteria and plant are engaged in a complex dialogue; this constant communication prevents activation of the plant defensive systems and the final formation of a functional symbiotic relationship (i.e. an active root nodule).  It’s a beautiful and fascinating process on which we rely, and should duly appreciate.

So, when, in my previous post, I mentioned the fact that it perturbs me when people dismiss the role bacteria play in symbiotic nitrogen fixation, I hope you can see that saying “plants fix nitrogen” for me, is comparable to saying Da Vinci’s paintbrush was responsible for the Mona Lisa, or that Einstein’s pen introduced the Theory of Relativity.  It just doesn’t give credit where credit is due.

ResearchBlogging.orgIvanov, S., Fedorova, E., & Bisseling, T. (2010). Intracellular plant microbe associations: secretory pathways and the formation of perimicrobial compartments. Current Opinion in Plant Biology, 13 (4), 372-377 DOI: 10.1016/j.pbi.2010.04.005

Oldroyd, G., & Downie, J. (2008). Coordinating Nodule Morphogenesis with Rhizobial Infection in Legumes. Annual Review of Plant Biology, 59 (1), 519-546 DOI: 10.1146/annurev.arplant.59.032607.092839

Does Mesquite encroachment alter soil microbiota in the Great Plains of Texas?

17 Sep

Spines and leaflets of the Mesquite.

I grew up in East Texas, where armadillos, cowboy boots, cattle ranching, state pride and Stetsons are just a natural part of daily life. But, I’ll never forget the year my older brother went away to a “summer camp” somewhere in West Texas, where instead of swimming and skiing all summer, the kids were involved in Mesquite removal.  His letters home complained of unendingly deep tap-roots and 6-inch long spines along the branches which would occasionally penetrate the bus tires and leave them stranded in the desert for hours.  This tree, although native to the plains of South and West Texas, is actually considered one of the world’s most problematic invasive species due to its hardy nature and ability to proliferate under adverse conditions.  It is the cattle-rancher’s foe since cattle typically won’t eat it because of the spines, the seeds can be toxic, the taproots deplete the water table, and they often crowd out the grasses and forbs the cattle need to survive.  

So, when I stumbled across this article in the most recent Soil Biology & Biochemistry on bacterial and fungal communities of soils underlying the Mesquite and native grasses of the southern Great Plains in Vernon, TX, I decided to check it out and share it here.

Let’s get my little pet peeve out of the way…

First, I would like to establish the fact that Mesquite (all the various species of Mesquite) are leguminous plants; this means they belong to a family of plants typically referred to as legumes, just like soybeans, peas, alfalfa, and many others.  This is where I want to get an issue of semantics (although I would argue it’s much more than simple semantics) out of the way.  Most people would say that these plants fix nitrogen, or add nitrogen to the soil.  I’m sure you’ve heard this before, and even the Wikipedia article on Mesquite says “Being a legume, it fixes nitrogen in the soil where it grows.”  This is where my head explodes.

PLANTS DO NOT FIX NITROGEN!  In fact there are no known eukaryotic organisms (plants, animals, fungi, etc.) that posses the genes encoding nitrogenase, which is the enzyme required for this process.  That means, no multicellular life-form has the capacity to fix nitrogen at all!  Only bacteria and archaea are capable of fixing atmospheric nitrogen gas into biologically available forms of nitrogen.  These very special bacteria establish symbiotic (a.k.a. mutually beneficial) relationships with leguminous plants by forming nodules on the roots.  The bacteria live in these tiny protected environments, where the plant feeds them carbon (food) and they, in return, feed the plant fixed nitrogen.  There are also free-living nitrogen fixing bacteria in soil and marine environments (we call them diazotrophs), but when you hear someone refer to “nitrogen fixing plants” they are actually referring to the work of bacteria attached within the roots of a specific group of plants.  It kills me every time I hear it.

Moving on… Mesquite encroachment.

Several facts regarding woody plant invasion into grasslands, like the Great Plains, have been established for some time now:

  1. Above-ground productivity is permanently altered – typically reflected as increased biomass production and altered rates of net primary productivity;
  2. Carbon and nitrogen are enriched in the soil directly under the trees; and
  3. Complex and biochemically recalcitrant (resistant to break-down) compounds accumulate in the soil directly under the trees.

So that all seems to make sense if you really think about it… from the macroscopic point of view:  We go from an ecosystem dominated by grasses like Buffalo grass and blue grama, which are very short (typically no more than 6 inches in height), sod-forming grasses with fairly shallow rooting systems, to an ecosystem dominated by large (up to 30 feet or more) trees.  We also know that these trees:   1.)  Form symbiotic associations with not only nitrogen fixing bacteria, but also mycorrhizal fungi which enhance their ability to scavenge phosphorus and other nutrients from a huge volume of soil; and 2.)  Shed their leaves each year to deposit a significant proportion of these accumulated nutrients onto the soil below.  There have been a number of studies establishing these facts, but none to date examine how this all impacts the diversity of soil microbial communities – until now.

What happened in Vernon?

Hollister, et al. (2010) compared soil carbon and nitrogen content, as well as soil fungal and bacterial community genetic diversity amongst the various vegetation types (Mesquite, perennial grasses, midgrass, and shortgrass) of the southern Great Plains near Vernon, TX, by means of cloning and sequencing as well as functional gene microarray technology.

When I say they examined “genetic diversity” in this case, they looked specifically at two different types of genes.  The first set of genes were those we typically used to identify organisms, conserved regions of the microbe’s DNA that allow us to differentiate genus and species, but tell us little to nothing about what the organism actually does.  For these identification genes, they employed standard cloning-and-sequencing protocols and ended up with a clone library for each of the vegetation-types.  In this way they could compare the species diversity of bacteria and fungi between soils.

They found differences in overall community structure, as well as a greater level of diversity (number of species) and richness (evenness of the distribution of these species) in the bacterial and fungal communities under the Mesquite trees than under the short grasses.  This is not terribly surprising if we consider that under the Mesquite trees we already understand that there is greater nutrient availability and substrate diversity.

However, the second type of genes the authors were interested in were those conferring functional ability to the soil organisms, such as genes that encode various enzymes like nitrogenase for N2 fixation, or efflux pumps for contaminant elimination from the cell, etc.  These types of genes may be shared across several genera and tell us very little about the identity of the organism, but give us some insight into what function the organism may be capable of performing in a given ecosystem.  To approach this question, the authors utilized the oh-so-sexy GeoChip Microarray: a commercially available array designed to probe for around 10,000 genes involved in nitrogen, carbon, sulfur and phosphorus cycling, metal reduction and resistance, and organic contaminant degradation.

Using this approach, they found no significant differences between the communities of the different vegetation types.  The authors concluded (albeit “with caution”) a great deal of functional redundancy exists across all of the communities they characterized.

This is where I have some questions…  ok, a lot of questions.

I tend to have a strong opposition reflex against the idea of high levels of functional redundancy because it’s been used so often as an argument against the need for biodiversity.  Perhaps functional redundancy is the answer, but before I can possibly yield to that idea, I need to know more.

In order to draw my own conclusions from the data presented, for one, I’d like to know more about the site history.  How long had each of the veg types been established where sampling took place? What was the grazing and management history at the site? And what was the understory below the Mesquite trees?  Perhaps these particular trees hadn’t been established for a significant period of time to afford appreciable changes in the soil functional diversity, or some of the understory was particularly similar to the plants interspersed with the grasses at the grass sampling locations.  The site was described as “ungrazed” but was wildlife also excluded from the site?

Also, why sample in August?  Most likely for simple logistical reasons, but it means that soil moisture (or rather the lack thereof) and heat were very likely limiting soil microbial activity, both directly and indirectly by limiting plant respiratory activity and therfore root exudation rates.  This brings me to the obvious issue, also cited by the authors as a potential problem for interpreting their results, of the fact that they used DNA rather than RNA for this analysis.  Sure, the organisms under each type of vegetation may contain the genetic information for the same functions (DNA) but the functions they are actually performing at any given time could be drastically different, and only revealed through RNA analyses (or by using more classical techniques).

The authors also make reference to potential design flaws in the GeoChip probes specifically for this study (i.e. 98% of the probes were bacterial in origin, excluding fungal functional genes).  So, then, why not couple the molecular techniques with old-fashioned laboratory functional assays on carbon and nitrogen mineralization or enzyme assays?

What does it all mean?

The authors made a pretty clear case for altered soil microbial community structure and phylogenetic (species) diversity under Mesquite encroachment, but not for soil microbial community function.  This is most likely due to the method applied to the question, but either way, it doesn’t align with virtually everything else we know about the macroecology of these systems.  It does, however, give us valuable insight into the limitations of the GeoChip in assessing soil microbial community function (at least, this is my assertion, which of course can’t be validated without further research).

Overall, a nice application of advanced molecular techniques to the issue of microbial response to Mesquite in a native grassland.  But I have to wonder if there will be more to come on this subject, particularly to double-check the cool and scientifically-sexy technology of functional gene microarrays against the more classical techniques for measuring soil microbial community function.

________________________________________________________________________________________________________ Hollister, E.B., Schadt, C.W., Palumbo, A.V., Ansley, R.J., & Boutton, T.W. (2010). Structural and functional diversity of soil bacterial and fungal communities following woody plant encroachment in the southern Great Plains. Soil Biology & Biochemistry, 42, 1816-1824 : 10.1016/j.soilbio.2010.06.022