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Re-solidify disturbed agar plates

Re-solidify disturbed agar plates


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I am stuck in trouble while pouring LB agar onto my petri dishes. I had to add kanamycin to my plates just before it started to solidify, out of forgetfulness, which I tried to mix using the tip. The plate was such a disaster! Is there a way to melt the agar in the plate and re-solidify it evenly.
I use falcon sterile plastic petri dish.


Yes. It is quite simple actually. Do this inside a clean laminar hood:

  • Take a clean sterile scalpel blade or any other sterile long object. 1ml pipette tip would also do.
  • Cut the agar on your plate. Scrape off cleanly.
  • Put it in a sterile glass bottle.
  • Heat in microwave
  • Add kanamycin
  • Pour back

Another solution is to coat the agar surface with kanamycin just like spread plating. Check the appropriate concentrations.


Ask an Expert: Agar Petri dish tests?

Do you plan to do swabs on something else besides leaves? What is your hypothesis? What question are you trying to answer by your experiments?
I'm assuming you are using nutrient agar plates because that is what most students use, so you will get some fungi--molds and yeasts--growing on the agar as well as some bacteria. Try to keep the plates at 78-85F (26-30C). If they are too cold, the microorganisms won’t grow very well.

You could ask the question: "Are there more bacteria or fungi on the upper or lower surface of a leaf? The upper surface may be exposed to sunlight and the UV in sunlight is harmful to many types of bacteria. Thus you might predict that you would find more species of bacteria on the underside of a leaf than on the top. Fungi are more resistant to sunlight and might not show this effect.

You would have to do swabs from several species of plant because leaves vary a lot in structure and microbial colonization. If you have a limited number of plates, you can divide the agar area in half by drawing a line across the center on the bottom of the Petri dish with a Sharpie. Then you can do two swabs on it--one from the top surface of a leaf and one from the bottom and compare them directly by taking a photo.

When you do swabs on nutrient agar there is a chance of growing a dangerous human pathogen, so after you have swabbed them, seal the lid to the bottom with tape and DO NOT open them. You can take pictures through the clear plastic lid.

After your experiment is all done, autoclave the plates if you can, and if not, fill a pail with 10% Clorox and put the plates in the liquid. Leave them there for a couple of days. The Clorox will seep inside and kill the bacteria. Then you can dispose of them in the biohazard waste bin at your school.


Abstract

A new mutant of Arabidopsis named rha1 is characterized and the gene involved cloned. In roots, the mutant shows minimal right-handed slanting, reduced gravitropic response, notable resistance to 2,4-D, but scarce resistance to IAA and NAA. The roots also show a clear resistance to the auxin transport inhibitors TIBA and NPA, and to ethylene. Other characteristics are a reduced number of lateral roots and reduced size of shoot and root in the seedlings. The gene, cloned through TAIL-PCR, was found to be a heat-shock factor that maps on chromosome 5, close to and above the RFLP marker m61. The rha1 structure, mRNA, and translation product are reported. Since, so far, no other gravitropic mutant has been described as mutated in a heat-shock factor, rha1 belongs to a new group of mutants disturbed in slanting, gravitropism, and auxin physiology. As shown through the RT-PCR analyses of its expression, the gene retains the function connected with heat shock. If the characteristics connected with auxin physiology are considered, however, it is also likely that the gene, as a transcription factor, could be involved in root circumnutation, gravitropic response, and hormonal control of differentiation. Since GUS staining under the gene promoter was localized mainly in the mature tissues, rha1 does not seem to be involved in the first steps of gravitropism, but is rather related to the general response to auxin. The alterations in slanting (mainly due to reduced chiral circumnutation) and gravitropism lead to the supposition that the two processes may have, at least in part, common origins.


Abstract

Plant deaths had been observed in the sub-alpine and alpine areas of Australia. Although no detailed aetiology was established, patches of dying vegetation and progressive thinning of canopy suggested the involvement of root pathogens. Baiting of roots and associated rhizosphere soil from surveys conducted in mountainous regions New South Wales and Tasmania resulted in the isolation of eight Phytophthora species Phytophthora cactorum, Phytophthora cryptogea, Phytophthora fallax, Phytophthora gonapodyides, Phytophthora gregata, Phytophthora pseudocryptogea, and two new species, Phytophthora cacuminis sp. nov and Phytophthora oreophila sp. nov, described here. P. cacuminis sp. nov is closely related to P. fallax, and was isolated from asymptomatic Eucalyptus coccifera and species from the family Proteaceae in Mount Field NP in Tasmania. P. oreophila sp. nov, was isolated from a disturbed alpine herbfield in Kosciuzsko National Park. The low cardinal temperature for growth of the new species suggest they are well adapted to survive under these conditions, and should be regarded as potential threats to the diverse flora of sub-alpine/alpine ecosystems. P. gregata and P. cryptogea have already been implicated in poor plant health. Tests on a range of alpine/subalpine plant species are now needed to determine their pathogenicity, host range and invasive potential.


DISCUSSION

Posttranscriptional regulation of gravitropism

A proteomics analysis during early time points after cold gravistimulation of the GPS response was performed to identify additional components of the gravitropic signaling pathway. Rapid changes have previously been observed in gene expression ( Kimbrough et al., 2004 ) and in signaling molecules, but this is the first study to look at changes in protein abundance in response to reorientation to the gravity vector in the inflorescence stems of Arabidopsis.

Classic second messengers such as Ca 2+ and inositol 1,4,5-triphosphate have been shown to flux in as little as 1 min after gravistimulation ( Perera et al., 1999 , 2001 Toyota et al., 2008 ). Other components such as a change in pH and generation of reactive oxygen species have been shown to be associated and necessary for a gravitropic response ( Scott and Allen, 1999 Joo et al., 2001 ). Using a two-dimensional gel electrophoresis-based proteomics approach, Young et al. (2006) identified adenosine kinase 1 as increasing in expression by 1.8-fold 12 min after gravistimulation as compared with the mechanostimulated controls. Adenosine kinase 1 has thus been implicated in the signal transduction events of gravitropism however, it was not identified in the present study, possibly because of the different tissue types used (root vs. inflorescence stems). Phosphorylation of proteins involved in gravitropism has also been observed rapidly after gravistimulation. Chang et al. (2003) identified a 50-kDa soluble protein that was phosphorylated upon gravistimulation in as little as 5 min. Changes in phosphorylation pattern for this protein were also observed between upper and lower halves of oat pulvini ( Chang et al., 2003 ). It is clear that gravitropic events are being regulated post transcriptionally and even post translationally.

Here, we showed that a rapid change in protein abundance occurs after cold gravistimulation during the GPS treatment. Of the 82 proteins that changed in expression after 2 and/or 4 min, none were found in the study by Kamada et al. (2005) , perhaps because of differences in tissues used (root tip vs. inflorescence stem tissue), time points analyzed (0.5 and 3 h after gravistimulation vs. 2 and 4 min after gravistimulation), or proteomics technique (two-dimensional gel electrophoresis vs. iTRAQ analysis). After 0.5 and 3 h of gravistimulation, all the phases of gravitropism have occurred, perception, signal transduction, and a growth response, so the time points used by Kamada et al., (2005) do not target a specific phase, and thus the proteins identified may be involved in the later events of gravitropism. The time points analyzed here were chosen to specifically target the signaling events of gravitropism utilizing the GPS treatment.

The 82 differentially quantified proteins identified in this study, could represent 82 novel proteins involved in a plant's response to gravity. Using a functional analysis as our guide, we chose to analyze two proteins in more depth: HSP81-2 and GSTF9 to validate the proteomics approach. Mutants were obtained for the genes encoding these proteins, and plants subjected to a phenotypic analysis. Although neither mutant showed obvious growth defects, both demonstrated reduced gravitropic curvature in the inflorescence stem during the GPS response (Fig. 8), abnormal root waving and skewing phenotypes (Figs. 5, 6), and reduced root curvature after gravistimulation (Fig. 7). These data suggest that GSTF9 and HSP81-2 represent conserved proteins in the gravitropic response because they are required for a full response in both roots and shoots.

Insights into HSP81-2

HSP81-2 was identified in this study to decrease in abundance after 4 min of gravistimulation (Table 1). The hsp81-2 mutant showed an altered root skewing phenotype compared with the wild type (Figs. 5, 6A), a reduced gravitropic response in the roots (Fig. 7) and reduced curvature in the inflorescence stems in response to the GPS treatment (Fig. 8A). HSP81-2 (also referred to as Hsp90.2) is a member of the heat shock protein 90 (Hsp90) gene family. The Hsp90 gene family consists of seven identified members in Arabidopsis with differing tissue specificities ( Krishna and Gloor, 2001 ). The major function of Hsp90 is to assist in protein folding like other chaperones. Hsp90 gene family plays a key role in signal transduction pathways, cell-to-cell communication, cell-cycle control, differentiation, and apoptosis ( Young et al., 2001 ). HSP81-2 was previously shown to be involved in plant stress responses, specifically salt, and drought stress ( Song et al., 2009 ). Overexpression lines of HSP81-2 in Arabidopsis showed a decreased tolerance to salt and drought stress, but an increased tolerance to high Ca 2+ concentrations ( Song et al., 2009 ). These data implicate HSP81-2 to be involved in abiotic stress signaling, possibly through a Ca 2+ pathway. It has been shown that Ca 2+ fluxes are an integral part of gravitropic signaling, and HPS81-2 could be modulating the flux of Ca 2+ that is responsible for the gravitropic signaling pathway.

HSP81-2 is not the first heat shock related protein to be shown to be involved in gravitropism. Root Handedness 1 (RHA1) was identified by its lack of right-handed slanting on tilted agar plates ( Fortunati et al., 2008 ). RHA1 was also identified as a heat shock factor (HSF) and proposed to be involved in the auxin response phase of gravitropism because of its reduced sensitivity to auxin transport inhibitors ( Fortunati et al., 2008 ). HSPs are thought to interact with the J-domain-containing proteins in the molecular chaperone system to bind specific substrates and modulate their activity ( Bukau and Horwich, 1998 ). ALTERED RESPONSE TO GRAVITY 1 (ARG1) and a paralog ARG1-LIKE2 (ARL2) have been shown to be necessary for a full gravitropic response in roots and hypocotyls and have been implicated in being integral components in the gravitropic signaling pathway ( Boonsirichai et al., 2003 Guan et al., 2003 ). ARG1 and ARL2 both encode J-domains at the N-terminus of their proteins ( Guan et al., 2003 ). These two proteins could represent interacting partners for HSP81-2 (or other HSPs like RHA1, which also displays an altered gravitropic response), but more work needs to be done to determine whether such an interaction between these proteins exist. An interaction between J-domain proteins and HSPs could represent an important process in the signaling events of gravitropism.

Insights into GSTF9

GSTs are a family of 55 enzymes (in Arabidopsis) that typically add glutathione to foreign chemicals to protect the cell from oxidizing agents ( Dixon and Edwards, 2010 ). It is no surprise that most of these enzymes are involved in stress responses however, they also perform various other glutathione-independent roles. Two GSTs were identified in this study: GSTF9 and GSTU20.

Significantly increased levels of GSTU20 were seen after 2 min with significantly lower levels after 4 min (online Appendix S3). GSTU20 has been shown to interact with an auxin-induced protein, far-red insensitive 219, using a yeast two-hybrid assay ( Chen et al., 2007 ). GSTU20 is thought to be involved in jasmonic acid stabilization or transportation ( Dixon and Edwards, 2010 ). Jasmonic acid has been implicated in the gravitropic response pathway as having indirect effects on auxin transport ( Muday and Rahman, 2008 ). A jasmonic-acid-deficient mutant in rice showed a reduced response to gravistimulation compared with wild type ( Gutjahr et al., 2005 ). Hormone synthesis, stabilization, and transport are necessary to allow for a differential growth response upon gravistimulation. It is also clear that hormones coordinate their signaling to elicit responses. GSTU20 could stabilize and allow for transport of hormones after a gravistimulation since GSTU20 levels increase after 2 min of gravistimulation.

GSTF9 was also found to be significantly increased after 2 min and significantly decreased after 4 min, similar to GSTU20 (Table 1 Appendix S3). Mutant gstf9 displayed a significantly different root skewing pattern (Figs. 5, 6A), a reduced gravitropic response in the roots (Fig. 7), and reduced curvature in the inflorescence stems during the GPS response (Fig. 8A) as compared with WT. Little is known about the function of GSTF9, but it is constitutively expressed and highly abundant in Arabidopsis ( Wagner et al., 2002 ). Because of the protein expression pattern and its altered gravitropic phenotype, GSTF9 could play a role in a plant's response to gravity through the synthesis of growth hormones or some other, as yet to be determined mechanism.

In conclusion, although only two of the 82 proteins identified were analyzed for gravitropic phenotype, all could play a role in gravitropic signal transduction. Further work on these proteins may reveal crucial aspects involved in the early events of gravitropic signal transduction.

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Practical Work for Learning

Class practical

Microbes are found everywhere, but they are mostly far too small to be seen by the naked eye. This activity allows students to discover that microbes are found in a range of different habitats, to explore the variety of microorganisms around us and to compare the range of microorganisms that are found in different places. This activity also introduces skills for safe handling of microbial material.

This practical is based on an investigation called Finding and growing microbes (286 KB) published in Practical Microbiology for Secondary Schools © Society for General Microbiology.

Lesson organisation

Give each working group (2 students) one nutrient agar plate and one malt extract agar plate. Nutrient agar supports the growth of a wide range of bacteria and fungi from the soil and air. Malt extract agar supports better growth of fungi, because the low pH and nutrient content reduce the competition from bacteria. Students could discuss which habitat they would like to investigate, and how to make sure their results will be reliable. Students will also need to discuss how they can reliably compare their results with other groups working with samples from the same habitat, and also with groups working with samples from different habitats.

Provide pond water, and soil suspended in sterile water in sterile containers. Discuss how to transfer these to the agar plates without introducing contamination.

Discovering the range of microbes in the environment could lead to a discussion of how hard it is to keep samples, containers and other equipment uncontaminated when carrying out microbial investigations. This observation could link into How Science Works, with a discussion about why it is important to minimize or prevent microbial contamination in commercial, medical or forensic contexts.

Apparatus and Chemicals

Material per group investigating pond water

Sterilised bottle containing 10 cm 3 pond water

Material per group investigating soil
Sterilised bottle containing 1 g of soil suspended in 100 cm 3 of sterile watere

Apparatus and materials (each group)

Nutrient agar plate, 1 and malt extract agar plate, 1

VirKon solution 1% w/v (see manufacturer’s instructions)

Sterile dropping pipettes, 2

Sterile swabs, 2 (Note 1) or 2 sterile glass or plastic spreaders

Health & Safety and Technical notes

For detailed information on health and safety issues with regard to microbiology investigations in schools and colleges, refer to Basic Practical Microbiology – a Manual available free from the Society for General Microbiology (SGM) email This email address is being protected from spambots. You need JavaScript enabled to view it. or go to the safety area on the SGM website – http://www.microbiologyonline.org.uk/teachers/safety-information

  • Do not collect microbes to culture from toilets.
  • Do not culture from human body fluids or skin other than hands and fingers.
  • Do not incubate plates above 25 °C.
  • Invert agar plates before incubating
  • Tape the lids and base of agar plates together with 2-4 short strips of adhesive tape.
  • After incubation, seal plates before inspection.
  • Take steps to kill cultures if there is a risk of the plates being opened (e.g. treatment with methanal – Note 2). Alternatively plates can be sealed in zip -lock bags before inspection.
  • Count the plates out and in again if there is any chance of students taking them away. This is good practice if plates have not been methanal-treated to kill the microbes.
  • Sterilise cultures after use, and dispose of according to guidance in Basic Practical Microbiology – a Manual (p 17).

1 Prepare moistened sterile swabs by sterilising cotton buds in Universal bottles with a little water (see CLEAPSS Laboratory Handbook 15.2).

2 Stop the growth of a culture completely by placing a piece of filter paper the size of the plate inside the lid of the inverted plate. Carefully add 40 % carefully to soak the filter paper, and replace the base. Leave for 24 hours. Remove the filter paper, remove any surplus liquid, and reseal the plate.

Ethical issues

There are no ethical issues with this procedure.

Procedure

A full risk assessment must be carried out before embarking on any practical microbiological investigation. See Basic Practical Microbiology – a Manual (page2).

Preparation

Basic Practical Microbiology – a Manual (BMP) has information on standard techniques relevant to this experiment.

a Refer to Standard techniques for details of handling agar and preparing your own agar plates, or buy ready-prepared plates (Basic Practical Microbiology (BMP) page 12 see Suppliers below).

b Prepare a suitable solution to disinfect the work area both before the investigation and afterwards. Suitable disinfectants include sodium chlorate(I) (hypochlorite) at concentrations greater than 1% (refer to CLEAPSS Hazcard 89), or 1% VirKon used according to manufacturer’s instructions (BPM page 7).

c Sterilise pipettes if needed to inoculate plates with water or soil mixture (BPM p 7).

d Sterilise bottles for collecting samples (BPM p 7).

e Sterilise spreaders if needed (BPM p 7).

f Prepare sterile swabs if needed (Note 1).

g Prepare soil sample suspended in sterile water.

h Collect pond water sample.

Investigation

a Keep one nutrient agar plate and one malt extract plate unopened as controls.

b Divide students into 3 large groups according to the habitat they are investigating – air, pond water or soil. Within these groups students can work in pairs.

c Provide each pair with two agar plates – one nutrient agar and one malt extract agar.

d Students label each plate on the bottom with name, date and habitat.

e Students inoculate or expose their plates according to their chosen habitat. If students are using liquids – but not using sterile swabs or spreaders – allow time for the liquid to be absorbed into the agar before the dish is moved.

Exposure to air

Students chose a place to leave both their agar plates open to the air. They take the lids off each dish and keep them open until the end of the lesson.
Then they replace the lids.

Students shake the bottle of pond water gently to mix the contents. They remove the top, flame the neck of the bottle in the Bunsen burner, and draw up a small amount of water with a sterile dropping pipette. The neck should be flamed again and the top replaced. Students lift the lid from the nutrient agar plate and dispense 2–3 drops on to the surface of the agar. They discard the pipette into the beaker of disinfectant. They then use a sterile glass spreader or swab to spread the drops evenly over the agar. They should discard the swab or spreader into the beaker of disinfectant.

They should follow the same procedure for the malt extract agar plate.

Soil suspended in sterile water

Students should follow the same procedure for pond water, using the bottle of soil suspension.

f Students tape the lids onto the plates before incubation to ensure they cannot be opened accidentally. Do this by fixing with 2 or 4 short strips of adhesive tape at opposite edges of the dish. Do not seal completely – this may promote the growth of anaerobic pathogens and/or prevent the growth of aerobic organisms.

g Invert plates and incubate at temperatures below 25 °C for 2-3 days. This reduces the risk of culturing microbes pathogenic in humans.

h Seal plates and place in zip-lock bags if required.

i Students observe, describe, and draw the colonies on the plate.

The following website may be useful as a starting point to help describe the colonies.

Teaching notes

Culturing microorganisms like this can be fascinating for students and teachers. If you or your technician are unsure about what safety precautions are necessary, seek advice from MiSAC / SGM or CLEAPSS who run regular courses on basic practical microbiology.

Exposure of the agar plates in a variety of places should, after incubation, produce growth of bacteria and fungi. The number of colonies may be a reflection of the disturbance of the air by convection currents or people. Microbes in disturbed air may not be detected, as they are held in suspension. Soil and water will probably yield more microbial colonies than air.

It’s worth making sure students understand that each colony on the final plate is a collection of thousands or millions of microorganisms. The individual organisms are microscopic and mostly too small to see with the naked eye which is why we have to grow colonies on a plate like this. The agar contains suitable nutrients for growth.

You could develop self-assessment or peer-assessment of these investigations, and use it as a starting point for planning more complex investigations.

Health and safety checked, September 2008

Downloads

Download the student sheet Microbes all around us (61 KB) with questions and answers.
Download the original protocol from SGM Finding and growing microbes (286 KB) .

Web links

Society for General Microbiology – Basic Practical Microbiology – A Manual, an excellent manual of laboratory techniques and Practical Microbiology for Secondary Schools, a selection of tried and tested practicals using microorganisms.
www.microbiologyonline.org.uk
email This email address is being protected from spambots. You need JavaScript enabled to view it.

(Websites accessed October 2011)

© 2019, Royal Society of Biology, 1 Naoroji Street, London WC1X 0GB Registered Charity No. 277981, Incorporated by Royal Charter


Isolation of Pythium oligandrum and other necrotrophic mycoparasites from soil

Samples of soil and other material from horticultural, grassland, arable, woodland, moorland, fresh-water and coastal sites were assayed for the presence of mycoparasites by incubation on agar precolonized by Phialophora sp. Of the total 164 samples, 84% contained one or more of the mycoparasites, Pythium oligandrum, Trichoderma viride, Gliocladium roseum and an unidentified Pythium sp. (‘Pythium SWO’).

P. oligandrum was found in 29% of all samples, 45% of samples from ‘disturbed’ sites (gardens, arable lands, managed grasslands), but only 11% of samples from ‘natural’ sites subject to minimal disturbance (woodlands, moorlands, permanent pastures, coastal sites and fresh-water sediments). It was commonest at pH 5.5.6.5. Pythium SWO was found in 17% of all samples but was probably underestimated because of competition from P. oligandrum on isolation plates. Its distribution was similar to that of P. oligandrum, but it was much less common in horticultural and grassland sites. The mycoparasite P. acanthicum was found only twice, and P. periplocum was not found, although both could grow well on Phialophora-precolonized agar. Overall, mycoparasitic pythia were found in 38% of samples — at a similar frequency to T. viride (45%) and G. roseum (40%).

T. viride and G. roseum occurred in similar numbers of ‘naturalrs and ‘disturbed’ sites, and showed different spectra of occurrence from the mycoparasitic pythia, although there was much overlap.

In preliminary experiments, pentachloronitrobenzene, chloramphenicol, gallic acid and aureomycin were moderately or markedly inhibitory to both mycoparasitic and non-mycoparasitic Pythium spp. Although a medium containing benzylpenicillin, streptomycin, nystatin and PCNB, with or without rose bengal and gallic acid, selectively isolated Pythium spp. from soil, it did not detect P. oligandrum in a soil known to contain it. Phialophora-precolonized agar plates did so and could be used in a ‘Most Probable Number’ technique to estimate populations of P. oligandrum in air-dry soil.


Materials and Methods

Plant materials and growth conditions

Arabidopsis thaliana seedlings were used in this study. The pin1-5 (CS69067), eir1-1 (CS8058), eir1-4 (CS859601), pin4-3 (CS9368), pin5-3 (SALK-021738), pin6 (SALK-095142), pin7-3 (CS9367), pin8 (SALK-044651), and DR5rev:GFP (CS9361) seeds were obtained from the Arabidopsis Biological Resource Center (ABRC, Ohio State University, Columbus, OH, USA). The tir1-1 (N3798), pin3-4 (N9363), fer-4 (N69044), aux1-7 (N9583), and DII-VENUS (N799173) seeds were obtained from the Nottingham Arabidopsis Stock Centre (NASC, Nottingham, UK). The tir1-1 afb1-1 afb2-1 afb3-1 quadruple mutant (Dharmasiri et al., 2005b ) and pin3-3 pin4-3 pin7-1 triple mutant (Waldie & Leyser, 2018 ) seeds were described previously.

Seeds surface-disinfected using 70% ethanol were incubated at 4°C for 3 d and then transferred to a growth room set at 24°C and at c. 50% humidity. Seedlings were grown on half-strength Murashige and Skoog agar (½MS agar) plates under long days (LDs 16 h : 8 h, light : dark cycles). White light with an intensity of 100 μmol m −2 s −1 was applied using fluorescent FL40EX-D tubes (Focus, Bucheon, Korea).

Measurement of root bending angles

For obstacle avoidance, seedlings were grown on vertically oriented MS agar plates (1% agar) for 5 d. Corrosion-resistant BA-400 blades (CUTTERMALLKOREA, Seoul, Korea) were used as obstacles. Obstacles were installed vertically on the MS agar plates and 5-d-old seedlings were laid 2 mm above the obstacles. Root growth and movement were photographed every 20 min. To measure rate of first bending, the angle between the roots was measured at 0 min and at annotated time points after contact with the barrier using ImageJ software (https://imagej.nih.gov/ij). For the second bending, the angles between the root tips and the barrier were measured 200 min after the roots touched the obstacle.

Confocal microscopy

Here, 5-d-old DR5rev:GFP or DII-VENUS seedlings were transferred to obstacle-installed MS agar plates. Fluorescence during first and second bending was observed 20−40 min and 150−200 min after the roots touched obstacles, respectively. To visualise root cells, roots were immersed in 10 μM propidium iodide (PI) solution for 30 s before observing fluorescence. Seedlings were placed on slide glasses and were subjected to fluorescence imaging using an LSM 800 confocal microscope (Carl Zeiss, Oberkochen, Germany). Fluorescence images were analysed using Zen 2.5 lite software (https://www.zeiss.com).

For quantification of fluorescence intensities, we used ImageJ software. When using DR5rev:GFP reporter plants, GFP fluorescence intensities at the later root cap cells, which are located at 100−200 μm from the root tip were analysed to quantify lateral auxin distributions. PI fluorescence intensities at the same region were also quantified as controls.

Pharmacological treatment

For N-1-naphthylphthalamic acid (NPA Sigma-Aldrich, St Louis, MO, USA, cat. no. 33371), 5-(4-chlorophenyl)-4H-1,2,4-triazole-3-thiol (yucasin Carbosynth, Berkshire, UK cat. no. FC122238), and l -kynurenine ( l -Kyn Sigma-Aldrich cat. no. K8625) treatments, 5-d-old seedlings grown on MS agar plates were transferred to the obstacle-installed plates containing 25 μM NPA, 50 μM yucasin, or 1 μM l -Kyn. The roots grew for c. 5 h in the medium before making contact with the obstacle. For ethylene glycol tetraacetic acid (EGTA) treatment, 5-d-old seedlings were transferred to 2 mM EGTA-containing plates before measuring obstacle avoidance. As up to 10 mM EGTA is used to block Ca 2+ signalling in Arabidopsis (Zhang & Mou, 2009 Ma et al., 2017 ), we applied 2 mM EGTA in this article.

Statistical analysis

The statistical significance between two means of measurements was analysed using Student's t-test with the P-value < 0.05. Numbers of replications are annotated in the figure legends. Quantitative data are displayed as standard deviations of the mean (SD).


Discussion

The modular features of yeast promoters offered space to design and synthesize artificial promoters that promoted high-efficient gene expression. However, major modules owned highly conservative base motifs and offer little space of redesign [15, 16]. Redden’s work selected several well-performed artificial upstream activating site (UAS) and core promoter elements from respective more than 10 6 options [8]. The process of FACS, colony and sequencing analysis offered a standard procedure for promoter selection. In our work, the strength of redesigned promoter was correlated with the expression of a certain enzyme CrtY (Fig. 1). As a result, the yeast strains with different initial level of lycopene presented the transformation of lycopene into beta-carotene to differentiate extents (Additional file 1: Figure S2). Our work directly combined the demand of metabolic optimization in Y. lipolytica with promoter engineering, offering an approach of rapid screening of artificial promoters. The three partitions of 10-base T-rich or G/C-rich elements were joined together as the artificial 30 bases of core promoters of Y. lipolytica (Table 1).

The options of different promoter upstream sequences and different starting strains affected the variance of colony-color distribution, facilitating the screening of desirable engineered promoters. It was suggested that the higher metabolic flux could be an amplifier of the impacts of engineered promoters (Fig. 2). The HE and HG libraries showed obvious large amounts of orange colonies besides yellow colonies than the LE and LG libraries, implying the different ratios of lycopene and beta-carotene with much higher yields would obviously affect yeast phenotype to different extents. Different promoter’s upstream sequences also introduced different influences. The PEXP1 with 30 artificial bases were more susceptible than PGPD, as the red-color colonies of different HE libraries covered around 5.5–65% but all HG libraries remained 53% red-color colonies. PGPD was more robust than PEXP1.

The different yields of lycopene and beta-carotene produced by the selected strains showed that not all the artificial promoters remained active (Fig. 3). Some red colonies like LE1-Y1, LE6-Y3 and HE6-R did not produce beta-carotene, implying the absence of the activity of CrtY. The selected best-performed artificial core promoters like LE7-Y1, LG7-Y1 and HG3-O1 obtained typical more T-rich elements or higher T-percentage than weak promoters (Additional file 1: Tables S2, S3). Portola’s recent work observed no meaningful correlation between nucleosome occupancy and promoter strength, not in accordance with previous opinion that AT rich sequences were associated with low nucleosome affinity and high promoter activity [27, 28]. Our work here found that the artificial strong promoters and natural strong ones both shared the common feature of T-rich elements and higher T-percentage. The conclusion was also verified when the best-performed artificial core promoters were combined with other upstream promoter sequences (Fig. 4).

The selected champion promoters in HE and HG libraries still did not transformed all the substrate lycopene into beta-carotene (Fig. 3, Additional file 1: Figure S4). This meant the starting high level of lycopene offered enough space for selection of improved promoters. The redesigned artificial promoters share different base orders and lengths from the traditional sequences. The de novo designed characteristic allows unique and orthogonal targeting of the promoter, which can be used as barcode. The unique barcode is beneficial to tuning and analysis of gene expression, targeting and recombination. Due to their diversity and independence of natural sequences, the artificial core promoters are valuable to the research of synthetic biology at pathway and genome level.


Discussion

Individual cell morphology varies widely within and across bacterial species (50, 51), but in most cases it is not clear how cell shape relates to emergent structure and ecology of cell collectives such as biofilms. Here we have found that some isolates of V. cholerae produce long cell filaments under nutrient-limited media, including conditions closely matched to the natural marine environment. This cell morphology generates a pronounced advantage in chitin surface colonization and a matrix-independent biofilm architecture that permits rapid surface occupation and high dispersal rates. However, this advantage comes at a cost: Filamentous biofilms are ultimately displaced by matrix-secreting strains in long-term competition experiments. Filamentation is thus advantageous when patches of chitin turn over quickly, such that faster colonization and reversible attachment are more important. Our results demonstrate that the shape of V. cholerae variants can tune the relative investment into surface colonization, long-term biofilm robustness, and dispersal back into the planktonic phase. These are fundamental elements of fitness for any biofilm-producing microbe (13, 52), and they are especially important for the marine ecology of V. cholerae as it colonizes, consumes, disperses from, and then recolonizes chitin in its marine environment.

Bacterial cell shape can serve a broad range of adaptive functions (49 ⇓ –51). The curved shape of Caulobacter crescentus, for example, promotes the formation of biofilms as hydrodynamic forces reorient single cells to optimize daughter cell attachment (53) this process also nucleates clonal clusters under strong flow (54, 55). Simulations and experiments with engineered variants of Escherichia coli suggest that rod-shaped bacteria can obtain a competitive advantage over spherical cells in colonies on agar plates, because rod-shaped cells burrow underneath spherical cells and spread more effectively to access fresh nutrients on the colony periphery (56). Filamentation has been observed in a wide variety of bacteria and eukaryotic microbes this morphology is implicated in assisting spatial spread through soil or host tissue and defense against phagocytosing amoeboid predators (50, 57, 58). In this work, we have shown that filamentation can alter surface colonization, biofilm architecture, and, as a result, the relative investment into rapid surface occupation versus long-term competitive success in biofilms.

The biophysical bases of our results are a topic of future work, but here we note that single filaments of V. cholerae can rapidly bend in shear flow, despite the stiffness of the bacterial cell wall (59). Our results demonstrate that sufficiently long bacteria can behave as elastic filaments (60). In analogy with the stretching behavior of polymers in flows that approach and split at the interface with an obstacle (i.e., extensional flows), we expect that filamentous bacteria experience shear that stretches them into alignment with stationary surfaces in the flow path (61 ⇓ –63). This phenomenon presumably increases the dwell time and contact area of filaments in proximity to obstacles in flow (SI Appendix, Fig. S12). We speculate that this process, in conjunction with previously known surface-adhesion mechanisms such as MSHA pili (46, 47), promotes rapid attachment and wrapping of filaments around chitin particles (SI Appendix, Fig. S6).

Following surface attachment, filamentation allows the construction of biofilms in which cell–cell contacts generate a mesh network that is not dependent on the major polysaccharide or cell–cell adhesion components of the V. cholerae biofilm matrix. In this respect, filamentous biofilms may be analogous to a polymeric gel in which elongated cell bodies are directly associated through physical entanglement (64). Here, this cell network is more natively inclined to fast surface spreading and subsequent dispersal but also porous and prone to physical invasion by competing strains or species (SI Appendix, Fig. S10). This strategy of rapid colonization and biomass accumulation but high reversibility of surface association is particularly well suited to fluctuating environments in which resource patches are short-lived. This could be the case when particles are small and quickly consumed, or when disturbance events are common and destroy or disrupt particles with high frequency.

Understanding the transitions between individual cell behavior and collective properties that emerge among biofilm-dwelling bacteria is a topic of increasingly intense interest in the microbiology community. Our results here suggest that changes in cell shape are a fundamental element of this individual-to-collective transition. For V. cholerae, and potentially other microbes producing biofilms on and dispersing from particle to particle, filamentation may offer a surface-occupation strategy better suited to transient environments by shifting a biofilm’s ability to spread over surfaces, at the expense of long-term competitive ability against matrix-replete, more highly adhesive cell groups. The ecological scope and impact of this cell morphology for V. cholerae and its relatives in realistic multispecies communities remain open questions for future work.



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