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Potential complications when developing gene deletion clones in Xylella fastidiosa.

Johnson KL, Cursino L, Athinuwat D, Burr TJ, Mowery P - BMC Res Notes (2015)

Bottom Line: A combined population was confirmed by PCR, which showed that targeted mutant clones were mixed with non-transformed cells.After repeated transfer and storage the non-transformed cells became the dominant clone present.As a result, careful monitoring of targeted mutant strains must be performed to avoid mixed populations and confounding results.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology and Plant-Microbe Biology, Cornell University New York State Agricultural Experiment Station, Geneva, NY, 14456, USA. johnson3@yahoo.com.

ABSTRACT

Background: The Gram-negative xylem-limited bacterium, Xylella fastidiosa, is an important plant pathogen that infects a number of high value crops. The Temecula 1 strain infects grapevines and induces Pierce's disease, which causes symptoms such as scorching on leaves, cluster collapse, and eventual plant death. In order to understand the pathogenesis of X. fastidiosa, researchers routinely perform gene deletion studies and select mutants via antibiotic markers.

Methods: Site-directed pilJ mutant of X. fastidiosa were generated and selected on antibiotic media. Mutant cultures were assessed by PCR to determine if they were composed of purely transformant cells or included mixtures of non-transformants cells. Then pure pilJ mutant and wildtype cells were mixed in PD2 medium and following incubation and exposure to kanamycin were assessed by PCR for presence of mutant and wildtype populations.

Results: We have discovered that when creating clones of targeted mutants of X. fastidiosa Temecula 1 with selection on antibiotic plates, X. fastidiosa lacking the gene deletion often persist in association with targeted mutant cells. We believe this phenomenon is due to spontaneous antibiotic resistance and/or X. fastidiosa characteristically forming aggregates that can be comprised of transformed and non-transformed cells. A combined population was confirmed by PCR, which showed that targeted mutant clones were mixed with non-transformed cells. After repeated transfer and storage the non-transformed cells became the dominant clone present.

Conclusions: We have discovered that special precautions are warranted when developing a targeted gene mutation in X. fastidiosa because colonies that arise following transformation and selection are often comprised of transformed and non-transformed cells. Following transfer and storage the cells can consist primarily of the non-transformed strain. As a result, careful monitoring of targeted mutant strains must be performed to avoid mixed populations and confounding results.

No MeSH data available.


Related in: MedlinePlus

Aggregation of Xylella fastidiosa and Escherichia coli. A suspension of X. fastidiosa (left) or E. coli (right) in SCP buffer five minutes after vigorous resuspension by vortexing and pipetting. Twenty microliters was pipetted onto slides and viewed by microscopy at 40X. While E. coli cells dispersed, X. fastidiosa present as aggregates, and could not be evenly dispersed.
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Fig6: Aggregation of Xylella fastidiosa and Escherichia coli. A suspension of X. fastidiosa (left) or E. coli (right) in SCP buffer five minutes after vigorous resuspension by vortexing and pipetting. Twenty microliters was pipetted onto slides and viewed by microscopy at 40X. While E. coli cells dispersed, X. fastidiosa present as aggregates, and could not be evenly dispersed.

Mentions: Mixtures of constructed kanamycin-resistant Xf∆pilJ mutant and non-transformed strains may have occurred due to spontaneous antibiotic resistant mutation in wild-type cells, high aggregation rate of X. fastidiosa, or a combination of both events. The ability of bacteria to develop spontaneous resistance to antibiotics is a well known phenomenon [18]. Sub-optimal antibiotic conditions provide particularly favorable conditions for these mutant strains to emerge. Of note, X. fastidiosa mutants were grown at standard, not sub-optimal, kanamycin concentrations [11]. Mixed aggregates of susceptible wild-type with kanamycin-resistant constructed Xf∆pilJ mutant strains may also explain the findings. X. fastidiosa characteristically and spontaneously forms aggregates that are not easily dispersed, compared to bacteria such as Escherichia coli, even after vigorous resuspension by mixing and pipetting (Figure 6). Presumably both aggregated and planktonic cells were transformed during mutant generation, and mixed aggregates of transformed and non-transformed cells formed subsequently. The aggregate formation may result in decreased susceptibility to antibiotics by the non-transformed bacteria [19,20]. After transformation when plating onto selective media (kanamycin) non-transformed bacteria may be “protected” from antibiotics by antibiotic resistant bacteria present in the aggregate, as is found in biofilms [21].Figure 6


Potential complications when developing gene deletion clones in Xylella fastidiosa.

Johnson KL, Cursino L, Athinuwat D, Burr TJ, Mowery P - BMC Res Notes (2015)

Aggregation of Xylella fastidiosa and Escherichia coli. A suspension of X. fastidiosa (left) or E. coli (right) in SCP buffer five minutes after vigorous resuspension by vortexing and pipetting. Twenty microliters was pipetted onto slides and viewed by microscopy at 40X. While E. coli cells dispersed, X. fastidiosa present as aggregates, and could not be evenly dispersed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4403849&req=5

Fig6: Aggregation of Xylella fastidiosa and Escherichia coli. A suspension of X. fastidiosa (left) or E. coli (right) in SCP buffer five minutes after vigorous resuspension by vortexing and pipetting. Twenty microliters was pipetted onto slides and viewed by microscopy at 40X. While E. coli cells dispersed, X. fastidiosa present as aggregates, and could not be evenly dispersed.
Mentions: Mixtures of constructed kanamycin-resistant Xf∆pilJ mutant and non-transformed strains may have occurred due to spontaneous antibiotic resistant mutation in wild-type cells, high aggregation rate of X. fastidiosa, or a combination of both events. The ability of bacteria to develop spontaneous resistance to antibiotics is a well known phenomenon [18]. Sub-optimal antibiotic conditions provide particularly favorable conditions for these mutant strains to emerge. Of note, X. fastidiosa mutants were grown at standard, not sub-optimal, kanamycin concentrations [11]. Mixed aggregates of susceptible wild-type with kanamycin-resistant constructed Xf∆pilJ mutant strains may also explain the findings. X. fastidiosa characteristically and spontaneously forms aggregates that are not easily dispersed, compared to bacteria such as Escherichia coli, even after vigorous resuspension by mixing and pipetting (Figure 6). Presumably both aggregated and planktonic cells were transformed during mutant generation, and mixed aggregates of transformed and non-transformed cells formed subsequently. The aggregate formation may result in decreased susceptibility to antibiotics by the non-transformed bacteria [19,20]. After transformation when plating onto selective media (kanamycin) non-transformed bacteria may be “protected” from antibiotics by antibiotic resistant bacteria present in the aggregate, as is found in biofilms [21].Figure 6

Bottom Line: A combined population was confirmed by PCR, which showed that targeted mutant clones were mixed with non-transformed cells.After repeated transfer and storage the non-transformed cells became the dominant clone present.As a result, careful monitoring of targeted mutant strains must be performed to avoid mixed populations and confounding results.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology and Plant-Microbe Biology, Cornell University New York State Agricultural Experiment Station, Geneva, NY, 14456, USA. johnson3@yahoo.com.

ABSTRACT

Background: The Gram-negative xylem-limited bacterium, Xylella fastidiosa, is an important plant pathogen that infects a number of high value crops. The Temecula 1 strain infects grapevines and induces Pierce's disease, which causes symptoms such as scorching on leaves, cluster collapse, and eventual plant death. In order to understand the pathogenesis of X. fastidiosa, researchers routinely perform gene deletion studies and select mutants via antibiotic markers.

Methods: Site-directed pilJ mutant of X. fastidiosa were generated and selected on antibiotic media. Mutant cultures were assessed by PCR to determine if they were composed of purely transformant cells or included mixtures of non-transformants cells. Then pure pilJ mutant and wildtype cells were mixed in PD2 medium and following incubation and exposure to kanamycin were assessed by PCR for presence of mutant and wildtype populations.

Results: We have discovered that when creating clones of targeted mutants of X. fastidiosa Temecula 1 with selection on antibiotic plates, X. fastidiosa lacking the gene deletion often persist in association with targeted mutant cells. We believe this phenomenon is due to spontaneous antibiotic resistance and/or X. fastidiosa characteristically forming aggregates that can be comprised of transformed and non-transformed cells. A combined population was confirmed by PCR, which showed that targeted mutant clones were mixed with non-transformed cells. After repeated transfer and storage the non-transformed cells became the dominant clone present.

Conclusions: We have discovered that special precautions are warranted when developing a targeted gene mutation in X. fastidiosa because colonies that arise following transformation and selection are often comprised of transformed and non-transformed cells. Following transfer and storage the cells can consist primarily of the non-transformed strain. As a result, careful monitoring of targeted mutant strains must be performed to avoid mixed populations and confounding results.

No MeSH data available.


Related in: MedlinePlus