Natural Competence of Xylella fastidiosa Occurs at a High Frequency Inside Microfluidic Chambers Mimicking the Bacterium's Natural Habitats

Abstract

Xylella fastidiosa is a xylem-limited bacterium that is the causal agent of emerging diseases in a number of economically important crops. Genetic diversity studies have demonstrated homologous recombination occurring among X. fastidiosa strains, which has been proposed to contribute to host plant shifts. Moreover, experimental evidence confirmed that X. fastidiosa is naturally competent for recombination in vitro. Here, as an approximation of natural habitats (plant xylem vessels and insect mouthparts), recombination was studied in microfluidic chambers (MCs) filled with media amended with grapevine xylem sap. First, different media were screened for recombination in solid agar plates using a pair of X. fastidiosa strains that were previously reported to recombine in coculture. The highest frequency of recombination was obtained with PD3 medium, compared to those with the other two media (X. fastidiosa medium [XFM] and periwinkle wilt [PW] medium) used in previous studies. Dissection of the media components led to the identification of bovine serum albumin as an inhibitor of recombination that was correlated to its previously known effect on inhibition of twitching motility. When recombination was performed in liquid culture, the frequencies were significantly higher under flow conditions (MCs) than under batch conditions (test tubes). The recombination frequencies in MCs and agar plates were not significantly different from each other. Grapevine xylem sap from both susceptible and tolerant varieties allowed high recombination frequency in MCs when mixed with PD3. These results suggest that X. fastidiosa has the ability to be naturally competent in the natural growth environment of liquid flow, and this phenomenon could have implications in X. fastidiosa environmental adaptation. IMPORTANCEXylella fastidiosa is a plant pathogen that lives inside xylem vessels (where water and nutrients are transported inside the plant) and the mouthparts of insect vectors. This bacterium causes emerging diseases in various crops worldwide, including recent outbreaks in Europe. The mechanisms by which this bacterium adapts to new hosts is not understood, but it was previously shown that it is naturally competent, meaning that it can take up DNA from the environment and incorporate it into its genome (recombination). In this study, we show that the frequency of recombination is highest when the bacterium is grown under flow conditions in microfluidic chambers modeled after its natural habitats, and recombination was still high when the medium was amended with grapevine sap. Our results suggest that this bacterium is able to recombine when growing inside plants or insects, and this can be a mechanism of adaptation of this pathogen that causes incurable diseases. N atural competence is a phenomenon that allows bacteria to take up DNA segments from the environment and incorporate them into the genome via homologous recombination (1). Natural competence was first demonstrated in Streptococcus pneumoniae in 1928 by Frederick G...

Natural Competence Rates Are Variable Among Xylella fastidiosa Strains and Homologous Recombination Occurs In Vitro Between Subspecies fastidiosa and multiplex

“…This suggested that the insertion at pilQ rendered BBI64 noncompetent and nonmotile. Another strain (pglA _ -KmR) that lacked twitching and competence in a previous study (Kandel et al 2016) showed an insertion in the pilM (PD1695) coding region, which was also confirmed by further sequencing (data not shown). No frame-shift mutation was observed in the competence-related genes in another noncompetent and nonmotile strain, Georgia Plum (data not shown).…”

“…Strain WM1-1 had the highest recombination frequency and showed highest motility among strains, while the two noncompetent strains were nonmotile. Positive correlation between recombination frequency and twitching motility was also suggested in our previous study using different media components (Kandel et al 2016). Since components of type IV pili are involved in both natural competence and twitching motility in several naturally competent gram-negative bacteria (Seitz and Blokesch 2013a), including X. fastidiosa (Kung and Almeida 2014), the activity of type IV pili could govern both of these phenomena.…”

“…Cells developed competence when entering into the exponential growth phase Almeida 2011, 2014), appeared to prefer DNA from self sources over foreign sources (Kung and Almeida 2011), and required flanking region homology for recombination (Kung et al 2013). One key knowledge gap is related to the variability of natural competence among strains, as previous studies were based on a few closely related strains belonging to the same subsp., namely, fastidiosa (Kandel et al 2016;Almeida 2011, 2014;Kung et al 2013). Since both genotypic and phenotypic differences have been described in X. fastidiosa strains (Antonova and Hammer 2015;Coletta-Filho et al 2017;Oliver et al 2014Oliver et al , 2015Parker et al 2012;Scally et al 2005), their natural competence abilities could differ, as is the case reported in other naturally competent bacteria (Bossé et al 2009;Coupat et al 2008;Evans and Rozen 2013;Fujise et al 2004;Gromkova et al 1998;Maughan and Redfield 2009;Sikorski et al 2002).…”

“…However, very limited information exists regarding natural competence in X. fastidiosa, as only a few studies have been performed (Kandel et al 2016;Almeida 2011, 2014;Kung et al 2013). Studies so far have demonstrated natural competence of X. fastidiosa in vitro in batch cultures (Kandel et al 2016;Kung and Almeida 2011), solid media (Kandel et al 2016;Kung and Almeida 2014), and in microfluidic chambers (MC), a system designed to closely resemble the bacterium's natural habitat (Kandel et al 2016).…”

“…Studies so far have demonstrated natural competence of X. fastidiosa in vitro in batch cultures (Kandel et al 2016;Kung and Almeida 2011), solid media (Kandel et al 2016;Kung and Almeida 2014), and in microfluidic chambers (MC), a system designed to closely resemble the bacterium's natural habitat (Kandel et al 2016). Natural competence occurred at a high frequency under flow conditions in MC, including conditions with grapevine sap as growth medium (Kandel et al 2016).…”