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Our next Journal club will be on the 28th of October 2014 at 8 pm on this paper here:

Regulation of the AbrA1/A2 Two-Component System in Streptomyces coelicolor and the Potential of Its Deletion Strain as a Heterologous Host for Antibiotic Production by Sergio Rico, Ana Yepes, Héctor Rodríguez, Jorge Santamaría, Sergio Antoraz, Eva M. Krause, Margarita Díaz, Ramón I. Santamaría, published on October 10, 2014 DOI:10.1371/journal.pone.0109844

Link: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0109844

Background
Streptomyces is a genus of actinobacteria, a group of Gram-positive soil bacteria with an unusual life cycle, involving substrate mycelium, formation of aerial hyphae and spore formation. They are prolific producers of bioactive metabolites, such as clavulinic acid or FK-506 and thus are of great interest for industry. The onset for the production of these metabolites usually happens when nutrients are limited and come along with the developmental stages. The regulation of antibiotic production has been extensively studied mainly in S.coelicolor which is one of the model organisms and many genetic tools are available. For more details see this review here: http://mmbr.asm.org/content/77/1/112.short
Regulation of the AbrA1/A2 Two-Component System in Streptomyces coelicolor and the Potential of Its Deletion Strain as a Heterologous Host for Antibiotic Production
By Sergio Rico, Ana Yepes, Héctor Rodríguez, Jorge Santamaría, Sergio Antoraz, Eva M. Krause, Margarita Díaz, Ramón I. Santamaría
Published on October 10, 2014DOI: 10.1371/journal.pone.0109844
Abstract
The Two-Component System (TCS) AbrA1/A2 from Streptomyces coelicolor M145 is a negative regulator of antibiotic production and morphological differentiation. In this work we show that it is able to auto-regulate its expression, exerting a positive induction of its own operon promoter, and that its activation is dependent on the presence of iron. The overexpression of the abrA2 response regulator (RR) gene in the mutant ΔabrA1/A2 results in a toxic phenotype. The reason is an excess of phosphorylated AbrA2, as shown by phosphoablative and phosphomimetic AbrA2 mutants. Therefore, non-cognate histidine kinases (HKs) or small phospho-donors may be responsible for AbrA2 phosphorylation in vivo. The results suggest that in the parent strain S. coelicolor M145 the correct amount of phosphorylated AbrA2 is adjusted through the phosphorylation-dephosphorylation activity rate of the HK AbrA1. Furthermore, the ABC transporter system, which is part of the four-gene operon comprising AbrA1/A2, is necessary to de-repress antibiotic production in the TCS null mutant. Finally, in order to test the possible biotechnological applications of the ΔabrA1/A2strain, we demonstrate that the production of the antitumoral antibiotic oviedomycin is duplicated in this strain as compared with the production obtained in the wild type, showing that this strain is a good host for heterologous antibiotic production. Thus, this genetically modified strain could be interesting for the biotechnology industry.

Discussion points:
1. Is the paper well written?
2. Were the methods appropriate?
3. Do the results reflect the conclusion?
4. What else could be done?

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Hi everyone, in our next session on Tuesday, the 24th of June at 8 pm (BST) we will look at the following paper:

“Circulating Avian Influenza Viruses Closely Related to the 1918 VirusHave Pandemic Potential” by Watanabe et al from Japan & the US published on the 11th of June 2014 in cell Host&Microbe available here:

http://www.cell.com/cell-host-microbe/pdf/S1931-3128(14)00163-2.pdf

SUMMARY:

Wild birds harbor a large gene pool of influenza A viruses that have the potential to cause influenza pandemics. Foreseeing and understanding this potentialis important for effective surveillance. Our phylogenetic and geographic analyses revealed the global prevalence of avian influenza virus genes whose proteins differ only a few amino acids from the 1918 pandemic influenza virus, suggesting that 1918-like pandemic viruses may emerge in the future. To assess this risk, we generated and characterized a virus composed of avian influenza viral segments with high homology to the 1918 virus. This virus exhibited pathogenicity in mice and ferrets higher than that in an authentic avian influenza virus. Further, acquisition of seven amino acid substitutions in the viral polymerases and the hemagglutinin surface glycoprotein conferred respiratory droplet transmission to the 1918-like avian virus in ferrets, demonstrating that contemporary avian influenza viruses with 1918 virus-like proteins may have pandemic potential.

The paper also got some news coverage, some examples can be found here:

http://www.sciencedaily.com/releases/2014/06/140611131551.htm

http://www.independent.co.uk/news/science/american-scientists-controversially-recreate-deadly-spanish-flu-virus-9529707.html

http://www.dailymail.co.uk/sciencetech/article-2655227/Bird-flu-viruses-unleash-pandemic-ferocious-one-killed-50-MILLION-people-1918-study-shows.html

Discussion points:

1. Was the publication written for an easy understanding?

2. Are the methods chosen appropiately?

3. Is the data well presented?

4. Does the paper achieve what the authors set it out?

5. What do you think of this research? Is it ethically to perform this high-risk research? Or would it be better to study it only theoretically? (to include the public discussion aspect)

Hope to see many of you there,

@Janitensen

Hey everyone,

For the upcoming session (21st of January, 8pm), we will be diving into bacterial metabolism and its regulation in Bacillus subtilis. I have chosen this paper because it is looking at the whole system and not only a small detail. The paper can be found here: http://onlinelibrary.wiley.com/doi/10.1038/msb.2013.66/full.

Title: Transcriptional regulation is insufficient to explain substrate-induced flux changes in Bacillus subtilis

Authors: Victor Chubukov,Markus Uhr, Ludovic Le Chat, Roelco J Kleijn, Matthieu Jules, Hannes Link, Stephane Aymerich, Jörg Stelling, Uwe Sauer

 

I have copied the abstract and the synopsis from the link here:

Abstract

One of the key ways in which microbes are thought to regulate their metabolism is by modulating the availability of enzymes through transcriptional regulation. However, the limited success of efforts to manipulate metabolic fluxes by rewiring the transcriptional network has cast doubt on the idea that transcript abundance controls metabolic fluxes. In this study, we investigate control of metabolic flux in the model bacterium Bacillus subtilis by quantifying fluxes, transcripts, and metabolites in eight metabolic states enforced by different environmental conditions. We find that most enzymes whose flux switches between on and off states, such as those involved in substrate uptake, exhibit large corresponding transcriptional changes. However, for the majority of enzymes in central metabolism, enzyme concentrations were insufficient to explain the observed fluxes—only for a number of reactions in the tricarboxylic acid cycle were enzyme changes approximately proportional to flux changes. Surprisingly, substrate changes revealed by metabolomics were also insufficient to explain observed fluxes, leaving a large role for allosteric regulation and enzyme modification in the control of metabolic fluxes.

Synopsis

Regulation of enzyme expression is one key mechanism by which cells control their metabolic programs. In this work, a quantitative analysis of metabolism in a model bacterium under different conditions shows that expression alone cannot explain the majority of the observed metabolic changes.

  • Most enzymes are indeed highly expressed in conditions where they are more active.
  • Quantitatively, however, the observed changes in expression between conditions do not match the changes in activity for most enzymes.
  • A good quantitative match is only observed for enzymes involved in the TCA cycle.
  • Metabolomics reveals that increased substrate availability explains only a few instances of changes in activity.

Discussion points

  • Was the paper clearly written?
  • Were the methods chosen appropriate?
  • Do the experiments show sufficient proof for the claim that transcriptional regulation is not sufficient to explain flux changes?
  • What experiments could be done next?

So, see you all on the 21st of January, 8 pm! Looking forward to it,

Janitensen

Hey everyone,

I am not sure If we have had this area before…Thought it was interesting as it is on bacterial metabolism and how engineer it…

The discussion will be as usual on a Tuesday night at 8 pm BST on twitter. It will be on the 17th of September.

The paper title is:

Electrochemical Gating of Tricarboxylic Acid Cycle in Electricity-Producing Bacterial Cells of Shewanella

and it can be found here:

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0072901

Abstract

Energy-conversion systems mediated by bacterial metabolism have recently attracted much attention, and therefore, demands for tuning of bacterial metabolism are increasing. It is widely recognized that intracellular redox atmosphere which is generally tuned by dissolved oxygen concentration or by appropriate selection of an electron acceptor for respiration is one of the important factors determining the bacterial metabolism. In general, electrochemical approaches are valuable for regulation of redox-active objects. However, the intracellular redox conditions are extremely difficult to control electrochemically because of the presence of insulative phospholipid bilayer membranes. In the present work, the limitation can be overcome by use of the bacterial genus Shewanella, which consists of species that are able to respire via cytochromes abundantly expressed in their outer-membrane with solid-state electron acceptors, including anodes. The electrochemical characterization and the gene expression analysis revealed that the activity of tricarboxylic acid (TCA) cycle in Shewanella cells can be reversibly gated simply by changing the anode potential. Importantly, our present results forShewanella cells cultured in an electrochemical system under poised potential conditions showed the opposite relationship between the current and electron acceptor energy level, and indicate that this unique behavior originates from deactivation of the TCA cycle in the (over-)oxidative region. Our result obtained in this study is the first demonstration of the electrochemical gating of TCA cycle of living cells. And we believe that our findings will contribute to a deeper understanding of redox-dependent regulation systems in living cells, in which the intracellular redox atmosphere is a critical factor determining the regulation of various metabolic and genetic processes.

Discussion points:

  1. What did you think of the paper? Nicely written? Easy to understand? Aims of the study?
  2. Did you miss any experiment/controls etc that would have been nice to see?
  3. How could this research be used in future?
  4. Could the outer-membrane cytochromes (OMC) of Shewanella be expressed in other genus of bacteria and which effect would it have?  

#microtwjc Week 27

This time it will a bit of an excursion into the world of ecology…I had a few paper that seemed interesting, but in the end I thougt, why not this one, I think it is something we have not covered so far and should definitely be of general interest…It is on nitrogen pollution and its impact on the microbial community in vernal pools.

We meet as usual 8 pm BST on the 28th of May! Hope to see many of you there!

Here is the abstract of the paper and the link to the full paper: http://www.biomedcentral.com/1471-2180/13/78

Shotgun metagenomic analysis of metabolic diversity and microbial community structure in experimental vernal pools subjected to nitrate pulse

By Sarah R Carrino-KykerKurt A Smemo and David J Burke

Background

Human activities have greatly increased nitrogen (N) levels in natural habitats through atmospheric N deposition and nutrient leaching, which can have large effects on N cycling and other ecosystem processes. Because of the significant role microorganisms play in N cycling, high inputs of nitrogenous compounds, such as nitrate (NO3-), into natural ecosystems could have cascading effects on microbial community structure and the metabolic processes that microbes perform. To investigate the multiple effects of NO3– pollution on microbial communities, we created two shotgun metagenomes from vernal pool microcosms that were either enriched with a solution of 10 mg NO3–N (+NO3-) or received distilled water as a control (−N).

Results

After only 20 hours of exposure to NO3-, the initial microbial community had shifted toward one containing a higher proportional abundance of stress tolerance and fermentation environmental gene tags (EGTs). Surprisingly, we found no changes to N metabolism EGTs, even though large shifts in denitrification rates were seen between the +NO3– and –N microcosms. Thus, in the absence of NO3– addition, it is plausible that the microbes used other respiratory pathways for energy. Respiratory pathways involving iron may have been particularly important in our –N microcosms, since iron acquisition EGTs were proportionally higher in the –N metagenome. Additionally, we noted a proportional increase in Acidobacteria and Alphaproteobacteria EGTs in response to NO3– addition. These community shifts in were not evident with TRFLP, suggesting that metagenomic analyses may detect fine-scale changes not possible with community profiling techniques.

Conclusions

Our results suggest that the vernal pool microbial communities profiled here may rely on their metabolic plasticity for growth and survival when certain resources are limiting. The creation of these metagenomes also highlights how little is known about the effects of NO3– pollution on microbial communities, and the relationship between community stability and function in response to disturbance.

A few questions which could be discussed:

  1. How was it written? Easy to understand?
  2. What do you think of the experimental setup and the methodology?
  3. Are you missing any further experiments/controls?
  4. Do you think this approach is useful for analysing environmental pollution?