Hello:dish

Next Tuesday’s #microtwjc paper will be “Total synthesis of a functional designer eukaryotic chromosome“, published last week or so in Science (you may have also seen the accompanying media attention). Apologies for it not being open access but the importance of this paper overshadowed that need. I think. If anyone has any problems accessing it just let me know via this or on twitter.

This paper documents the design, building and biological characterisation of a synthetic yeast chromosome, specifically chromosome III. For a primer on yeast genetics have a look at this. This is the first chromosome to be generated in what has been called the ‘yeast 2.0′ project - an international effort to generate a yeast with a completely synthetic genome (and actually carried out mainly by undergraduates). The paper is important for a number of reasons: 1) yeast is a model organism in its own right, engineering of a complete chromosome (or genome) will aid our understanding of not just yeast biology but of biology in general. 2) yeast are useful in their own right (see this: and this) - S. cerevisiae is my 4th favourite organism, I think. Synthesising the yeast genome will aid our exploitation of this organism, and 3) This is a stepping stone to synthetic ‘higher eukaryotic’ genomes (like us or our domestic animals) – the generation of synthetic higher animals may aid the development of new medical treatments and economic benefits. Remember where we were only a couple of years ago with mycoplasma. Whatever you want to call it. 

The paper is pretty straightforward but has a lot of supplementary data (no surprise there for a Science paper), which actually covers the bulk of this work (biological characterisation) – worth a read to see if there are any downsides to synthetic genomes(!). So have fun reading. Here are a couple of discussion points for you to think about (the usually ‘is this paper written well’ also applies).

1) what do you make of the authors design principles? They screwed around a great deal with this chromosome.

2) what do you think of the biological effect of the synthetic chromosome? how much change should be tolerated?

3) what would you do with this system? They mentioned ‘scrambling’ the genome to uncover hidden biology of yeast but what else could you do?

4) how hard would a human chromosome be to generate?

Thanks,

Connor

Hello #MicroTwJc Fans!

Next Tuesday’s #microtwjc paper (which has just been published this month) is one for all bacteriologists (and virologists that like bacteria!) as it has implications for all bacteria due to the conservation of the bacteria cell cycle.

Title“FtsZ Placement in Nucleoid-Free Bacteria”

Authors: Manuel Pazos, Mercedes Casanova, Pilar Palacios, William Margolin, Paolo Natale, and Miguel Vicente

Abstract: We describe the placement of the cytoplasmic FtsZ protein, an essential component of the division septum, in nucleoid-free Escherichia coli maxicells. The absence of the nucleoid is accompanied in maxicells by degradation of the SlmA protein. This protein, together with the nucleoid, prevents the placement of the septum in the regions occupied by the chromosome by a mechanism called nucleoid occlusion (NO). A second septum placement mechanism, the MinCDE system (Min) involving a pole-to-pole oscillation of three proteins, nonetheless remains active in maxicells. Both Min and NO act on the polymerization of FtsZ, preventing its assembly into an FtsZ-ring except at midcell. Our results show that even in the total absence of NO, Min oscillations can direct placement of FtsZ in maxicells. Deletion of the FtsZ carboxyl terminal domain (FtsZ*), a central hub that receives signals from a variety of proteins including MinC, FtsA and ZipA, produces a Min-insensitive form of FtsZ unable to interact with the membrane-anchoring FtsA and ZipA proteins. This protein produces a totally disorganized pattern of FtsZ localization inside the maxicell cytoplasm. In contrast, FtsZ*-VM, an artificially cytoplasmic membrane-anchored variant of FtsZ*, forms helical or repetitive ring structures distributed along the entire length of maxicells even in the absence of NO. These results show that membrane anchoring is needed to organize FtsZ into rings and underscore the role of the C-terminal hub of FtsZ for their correct placement.

Discussion Points

  • Was the paper clearly written, presented etc. Did it all make sense?
  • Were the methods sound? Was there anything extra that you would have done? How were the stats?
  • Were the conclusions supported by the results?
  • How much of this is new? What are the practical implications?
  • What experiments would you do next?
  • Hope to see you on Tuesday 1st April, 8pm BST :)  (thats right! our clocks will have changed and we’ll be on summer time!)

Hi all.

Tuesday’s #microtwjc paper is this one:

Clonal Expansion during Staphylococcus aureus Infection Dynamics Reveals the Effect of Antibiotic Intervention

Gareth McVicker, Tomasz K. Prajsnar, Alexander Williams, Nelly L. Wagner, Michael Boots, Stephen A. Renshaw, Simon J. Foster

Abstract:

To slow the inexorable rise of antibiotic resistance we must understand how drugs impact on pathogenesis and influence the selection of resistant clones. Staphylococcus aureus is an important human pathogen with populations of antibiotic-resistant bacteria in hospitals and the community. Host phagocytes play a crucial role in controlling S. aureus infection, which can lead to a population “bottleneck” whereby clonal expansion of a small fraction of the initial inoculum founds a systemic infection. Such population dynamics may have important consequences on the effect of antibiotic intervention. Low doses of antibiotics have been shown to affect in vitro growth and the generation of resistant mutants over the long term, however whether this has any in vivo relevance is unknown. In this work, the population dynamics of S. aureus pathogenesis were studied in vivo using antibiotic-resistant strains constructed in an isogenic background, coupled with systemic models of infection in both the mouse and zebrafish embryo. Murine experiments revealed unexpected and complex bacterial population kinetics arising from clonal expansion during infection in particular organs. We subsequently elucidated the effect of antibiotic intervention within the host using mixed inocula of resistant and sensitive bacteria. Sub-curative tetracycline doses support the preferential expansion of resistant microorganisms, importantly unrelated to effects on growth rate or de novo resistance acquisition. This novel phenomenon is generic, occurring with methicillin-resistant S. aureus(MRSA) in the presence of β-lactams and with the unrelated human pathogen Pseudomonas aeruginosa. The selection of resistant clones at low antibiotic levels can result in a rapid increase in their prevalence under conditions that would previously not be thought to favor them. Our results have key implications for the design of effective treatment regimes to limit the spread of antimicrobial resistance, where inappropriate usage leading to resistance may reduce the efficacy of life-saving drugs.

Author Summary:

Staphylococcus aureus is a major cause of human disease, made even more notable due to the spread of antibiotic resistance. We used a combination of animal models to study the spread of bacteria between organs during an infection and the resulting effect of antibiotic intervention. We found that S. aureus infection is highly clonal, following a “bottleneck” in which very few bacterial cells found each abscess. Despite previous in vitro research, the effect of antibiotics on S. aureus infection was poorly understood. We utilized our systemic infection models to study intervention with sub-curative antibiotic doses, such as one might encounter upon failing to complete an antibiotic course. We have shown that such doses are able to support the preferential expansion of antibiotic-resistant organisms during a mixed infection. This selection is due to the clonal pattern of infection, occurring despite a lack of effect on growth rate or on the spontaneous generation of resistance. Furthermore, it is generic to multiple pathogen species, including Pseudomonas aeruginosa, and antibiotic classes, such as with methicillin-resistant S. aureus (MRSA) in the presence of oxacillin. Given the current debate in the field, our results have important implications for the design of properly-controlled treatment regimes.

Discussion Points

  1. Was the paper clearly written, presented etc. Did it all make sense?
  2. Were the methods sound? Was there anything extra that you would have done? How were the stats?
  3. Were the conclusions supported by the results?
  4. How much of this is new? What are the practical implications?
  5. What experiments would you do next?

Hope to see you on Tuesday 18th March, 8pm GMT :)

For the next session (Tuesday 4th March, 8pm GMT) we will be discussing the following paper: Effect of spaceflight on Pseudomonas aeruginosa final cell density is modulated by nutrient and oxygen availability available from the following link http://www.biomedcentral.com/1471-2180/13/241

Abstract

Background: Abundant populations of bacteria have been observed on Mir and the International Space Station.
While some experiments have shown that bacteria cultured during spaceflight exhibit a range of potentially
troublesome characteristics, including increases in growth, antibiotic resistance and virulence, other studies have
shown minimal differences when cells were cultured during spaceflight or on Earth. Although the final cell density
of bacteria grown during spaceflight has been reported for several species, we are not yet able to predict how
different microorganisms will respond to the microgravity environment. In order to build our understanding of how
spaceflight affects bacterial final cell densities, additional studies are needed to determine whether the observed
differences are due to varied methods, experimental conditions, or organism specific responses.

Results: Here, we have explored how phosphate concentration, carbon source, oxygen availability, and motility
affect the growth of Pseudomonas aeruginosa in modified artificial urine media during spaceflight. We observed
that P. aeruginosa grown during spaceflight exhibited increased final cell density relative to normal gravity controls
when low concentrations of phosphate in the media were combined with decreased oxygen availability. In
contrast, when the availability of either phosphate or oxygen was increased, no difference in final cell density was
observed between spaceflight and normal gravity. Because motility has been suggested to affect how microbes
respond to microgravity, we compared the growth of wild-type P. aeruginosa to a ΔmotABCD mutant deficient in
swimming motility. However, the final cell densities observed with the motility mutant were consistent with those
observed with wild type for all conditions tested.

Conclusions: These results indicate that differences in bacterial final cell densities observed between spaceflight and
normal gravity are due to an interplay between microgravity conditions and the availability of substrates essential for
growth. Further, our results suggest that microbes grown under nutrient-limiting conditions are likely to reach higher cell
densities under microgravity conditions than they would on Earth. Considering that the majority of bacteria inhabiting
spacecrafts and space stations are likely to live under nutrient limitations, our findings highlight the need to explore the
impact microgravity and other aspects of the spaceflight environment have on microbial growth and physiology

Discussion points?

Was the paper well written?
Were the methods used appropiate?
Were the experiments carried out adequately?
What else can be done?

In Tuesday’s #microtwjc we will be discussing viral miRNAs:

Zhang S, Sroller V, Zanwar P, Chen CJ, Halvorson SJ, et al. (2014) Viral MicroRNA Effects on Pathogenesis of Polyomavirus SV40 Infections in Syrian Golden Hamsters. PLoS Pathog 10(2): e1003912. doi:10.1371/journal.ppat.1003912

Abstract:

Effects of polyomavirus SV40 microRNA on pathogenesis of viral infections in vivo are not known. Syrian golden hamsters are the small animal model for studies of SV40. We report here effects of SV40 microRNA and influence of the structure of the regulatory region on dynamics of SV40 DNA levels in vivo. Outbred young adult hamsters were inoculated by the intracardiac route with 1×107 plaque-forming units of four different variants of SV40. Infected animals were sacrificed from 3 to 270 days postinfection and viral DNA loads in different tissues determined by quantitative real-time polymerase chain reaction assays. All SV40 strains displayed frequent establishment of persistent infections and slow viral clearance. SV40 had a broad tissue tropism, with infected tissues including liver, kidney, spleen, lung, and brain. Liver and kidney contained higher viral DNA loads than other tissues; kidneys were the preferred site for long-term persistent infection although detectable virus was also retained in livers. Expression of SV40 microRNA was demonstrated in wild-type SV40-infected tissues. MicroRNA-negative mutant viruses consistently produced higher viral DNA loads than wild-type SV40 in both liver and kidney. Viruses with complex regulatory regions displayed modestly higher viral DNA loads in the kidney than those with simple regulatory regions. Early viral transcripts were detected at higher levels than late transcripts in liver and kidney. Infectious virus was detected infrequently. There was limited evidence of increased clearance of microRNA-deficient viruses. Wild-type and microRNA-negative mutants of SV40 showed similar rates of transformation of mouse cells in vitro and tumor induction in weanling hamsters in vivo. This report identified broad tissue tropism for SV40 in vivo in hamsters and provides the first evidence of expression and function of SV40 microRNA in vivo. Viral microRNA dampened viral DNA levels in tissues infected by SV40 strains with simple or complex regulatory regions.

Author’s Summary:

The recent discovery of virally encoded microRNAs (miRNAs) raises the possibility of additional regulatory processes being involved in viral replication, immune recognition, and host cell survival. In this study, we sought to characterize the effect of SV40-encoded miRNAs and the structure of the viral regulatory region on infections in outbred Syrian golden hamsters. Results revealed that SV40 has a wide tissue tropism, including liver, kidney, spleen, lung, and brain, with kidney the preferred site for long-term persistent infection. Significant increases in tissue-associated viral DNA loads were observed with miRNA-negative mutant strains, whereas the presence of SV40 miRNAs had no effect on tumor induction and little effect on viral clearance. Our results provide the first evidence for SV40 miRNA expression and function in an in vivoanimal model and highlight the complexity of regulation of SV40 viral replication and persistent infections.

Discussion points

  1. Was the paper well written etc?
  2. Were all of the methods appropriate?  Were there any experiments missing that you think needed to be there?
  3. What future work would you like to see done?

Hope to see you there, Tues 18th Feb at 8pm GMT

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