Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Apr 17:10:746.
doi: 10.3389/fmicb.2019.00746. eCollection 2019.

AmrZ and FleQ Co-regulate Cellulose Production in Pseudomonas syringae pv. Tomato DC3000

Daniel Pérez-Mendoza  1 , Antonia Felipe  1 , María Dolores Ferreiro  1 , Juan Sanjuán  1 , María Trinidad Gallegos  1
Affiliations

AmrZ and FleQ Co-regulate Cellulose Production in Pseudomonas syringae pv. Tomato DC3000

Daniel Pérez-Mendoza et al. Front Microbiol. .

Abstract

Pseudomonas syringae pv. tomato DC3000 carries the wssABCDEFGHI operon for the synthesis of acetylated cellulose, whose production is stimulated by increasing the intracellular levels of the second messenger c-di-GMP. This enhances air-liquid biofilm formation and generates a wrinkly colony morphotype in solid media. In the present study we show that cellulose production is a complex process regulated at multiple levels and involving different players in this bacterium. Using different in vitro approaches, including Electrophoretic Mobility Shift Assay (EMSA) and footprint analysis, we demonstrated the interrelated role of two transcriptional regulators, AmrZ and FleQ, over cellulose production in Pto DC3000 and the influence of c-di-GMP in this process. Under physiological c-di-GMP levels, both regulators bind directly to adjacent regions at the wss promoter inhibiting its expression. However, just FleQ responds to c-di-GMP releasing from its wss operator site and converting from a repressor to an activator of cellulose production. The additive effect of the double amrZ/fleQ mutation on the expression of wss, together with the fact that they are not cross-regulated at the transcriptional level, suggest that FleQ and AmrZ behave as independent regulators, unlike what has been described in other Pseudomonas species. Furthermore, this dual co-regulation exerted by AmrZ and FleQ is not limited to cellulose production, but also affects other important phenotypes in Pto DC3000, such as motility and virulence.

Keywords: AmrZ; FleQ; Pseudomonas syringae; c-di-GMP; cellulose; transcriptional regulation.

PubMed Disclaimer

Figures

FIGURE 1
FIGURE 1
Cellulose production in Pto DC3000. (A) Effect of c-di-GMP on the expression of the wss operon in different genetic backgrounds. Total RNAs were obtained from bacteria grown in MMR at 20°C for 24 h. Results show qRT-PCR of wssB in the wild type strain (Pto) and mutants amrZ, amrZ/fleQ and fleQ, the latter also complemented, with pJB3Tc19 (in the absence of pleD∗, white bars) or with pJB3pleD∗ (in the presence of pleD∗, gray bars). Expression values were normalized with the housekeeping gene gyrA and referred to the wild type condition in the absence of pleD∗. The graph shows the average mRNA levels and error bars correspond to the standard deviation of three biological replicates; a–e denote ANOVA categories with significant differences (P < 0.01). (B) Cellulose production and colony morphology. Pto and the amrZ, fleQ and amrZ/fleQ mutants were grown in MMR with CF (100 μg/ml) for 24 h at 20°C, and the fluorescence emission of the cell attached CF in liquid cultures was measured. The graph shows the average amount of cellulose produced by the indicated strains in the absence (white bars) and in the presence of pleD∗ (gray bars) as fluorescence (in arbitrary units) referred to total cell protein. Error bars correspond to the standard deviation of three biological replicates and a-d denote ANOVA categories with significant differences (P < 0.01). Representative colony morphology of the different strains grown in agar plates supplemented with Congo Red and Calcofluor is included below. 5 μl of bacterial suspensions at OD660 = 1.0 were placed on the surface of MMR plates with CR (50 μg/ml, top) or CF (100 μg/ml, bottom) and pictured after incubation at 20°C for 3 days.
FIGURE 2
FIGURE 2
In vitro transcription. Multiple round transcription assays were carried out as described in Materials and Methods. The assays were performed in the absence (-) or in the presence of 0.5 μM FleQ. When indicated, c-di-GMP or c-di-AMP (0.25 mM) or AmrZ (50 nM) were also added to the reaction. C- indicates a reaction without template DNA. The 232 nucleotide mRNA synthesized from PwssA is indicated by arrowheads. The graph shows the average amount of the mRNA produced as percentage of the condition without any protein (-). Error bars correspond to the standard deviation of six different transcription assays and a–c denote ANOVA categories with significant differences (P < 0.01).
FIGURE 3
FIGURE 3
In vitro binding of FleQ to the wss promoter region. (A) EMSA with different fragments of the fleSR and wss operon. Binding reactions were carried out as described in Experimental Procedures. The indicated fragments were incubated without protein and with 1 μM of native FleQ. In presence of FleQ, shifted fleS3, wssA2 and wssH fragments were not observed. (B) Binding of FleQ to the wss promoter in the presence of different nucleotides. Binding reactions were carried out in the absence (-) and in the presence of 1 μM FleQ and 0.5 mM of ATP, GTP, c-di-GMP or c-di-AMP. (C) Binding of FleQ to the wss promoter in the presence of AmrZ. Binding reactions were carried out in the absence (-) and in the presence of 1 μM FleQ, 50 nM AmrZ and 0.5 mM of ATP, GTP, c-di-GMP or c-di-AMP (0.5 mM). Putative shifted FleQ-DNA, AmrZ-DNA and FleQ-AmrZ-DNA complexes are indicated.
FIGURE 4
FIGURE 4
Identification of the FleQ binding site in the wss promoter region by footprinting analysis. (A) DNAse I and DMS footprint. DNA probes corresponding to the wssA upstream region 5′ end-labeled on either the top or the bottom strand were prepared and incubated without (lanes -) and with FleQ (1 μm) and/or AmrZ (50 nM) and c-di-GMP (0.5 mM). After partial digestion with DNase I or treatment with DMS and partial digestion with piperidine, the DNAs were subjected to urea-PAGE. Nucleotide sequences protected by FleQ and AmrZ are indicated on the left and right, respectively, of each panel; ∗, indicates hyperreactivity. (B) Localisation of the FleQ binding site at the wss promoter. The boxes indicate the regions protected from DNAse I by FleQ (red) and AmrZ (green) in the top and bottom strands. The -10 and -35 regions and the wss transcriptional start site are in bold.
FIGURE 5
FIGURE 5
Expression of amrZ and fleQ. Total RNAs were obtained from bacteria grown in MMR at 20°C for 24 h. Results show qRT-PCR of amrZ in the wild type (Pto) and fleQ mutant and of fleQ in the wild type and amrZ mutant, with pJB3Tc19 (in the absence of pleD∗, white bars) or with pJB3pleD∗ (in the presence of pleD∗, gray bars). Expression values were normalized with the housekeeping gene gyrA and referred to the wild type condition in the absence of pleD∗. The graph shows the average mRNA levels and error bars correspond to the standard deviation of three biological replicates; a-c denote ANOVA categories with significant differences (P < 0.01).
FIGURE 6
FIGURE 6
Motility of Pto DC3000 and its mutants. Observation and quantification of motility and syringafactin production were carried out in the different genetic backgrounds. Mean values are indicated for the different strains with the empty plasmid pBBR1MCS (white), pBBR1MCS::amrZ (dark gray), and pBBR1MCS::fleQ (light gray). Error bars correspond to the standard deviation of at least three biological replicates. a–d denote ANOVA categories with significant differences (P < 0.01). (A) Swimming assays. The indicated strains were punctured in the center of LB (0.3% agar) plates and incubated 48 h at 20°C, when pictures were taken and swimming halos measured. The graphic shows the respective average diameter of the halos. (B) Surface motility assays. Bacterial suspensions of the indicated strains were deposited on the surface of PG-agar (0.5% agar) plates and incubated 24 h at 20°C, when pictures were taken. The respective areas of motility were calculated with ImageJ. (C) Syringafactin production. Comparison of surfactant-induced halos around bacterial colonies grown on LB (1% agar) for 24 h at 20°C and visualized by the atomized oil assay. Bar = 1 cm. The graphic shows the respective biosurfactant areas.
FIGURE 7
FIGURE 7
Bacterial growth and symptom development on tomato leaves. (A) Time course of growth of Pto DC3000 and the amrZ, fleQ, amrZ/fleQ mutants in the primary leaves of tomato plants. Colony forming units (cfu) were quantified at 0, 3, 6, and 10 days post-inoculation (dpi) with approximately 108 cfu/ml by spray. Data represent the averages from three experiments with their standard deviations. (B) Development of symptoms induced on tomato leaves 10 days after inoculation with Pto DC3000 and different mutants.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Arora S. K., Ritchings B. W., Almira E. C., Lory S., Ramphal R. (1997). A transcriptional activator, FleQ, regulates mucin adhesion and flagellar gene expression in Pseudomonas aeruginosa in a cascade manner. J. Bacteriol. 179 5574–5581. 10.1128/jb.179.17.5574-5581.1997 - DOI - PMC - PubMed
    1. Baraquet C., Harwood C. S. (2013). Cyclic diguanosine monophosphate represses bacterial flagella synthesis by interacting with the Walker A motif of the enhancer-binding protein FleQ. Proc. Natl. Acad. Sci. U.S.A. 110 18478–18483. 10.1073/pnas.1318972110 - DOI - PMC - PubMed
    1. Baraquet C., Harwood C. S. (2016). FleQ DNA binding consensus sequence revealed by studies of FleQ-dependent regulation of biofilm gene expression in Pseudomonas aeruginosa. J. Bacteriol. 198 178–186. 10.1128/JB.00539-15 - DOI - PMC - PubMed
    1. Baraquet C., Murakami K., Parsek M. R., Harwood C. S. (2012). The FleQ protein from Pseudomonas aeruginosa functions as both a repressor and an activator to control gene expression from the pel operon promoter in response to c-di-GMP. Nucleic Acids Res. 40 7207–7218. 10.1093/nar/gks384 - DOI - PMC - PubMed
    1. Barnhart D. M., Su S., Baccaro B. E., Banta L. M., Farrand S. K. (2013). CelR, an ortholog of the diguanylate cyclase PleD of Caulobacter, regulates cellulose synthesis in Agrobacterium tumefaciens. Appl. Environ. Microbiol. 79 7188–7202. 10.1128/AEM.02148-13 - DOI - PMC - PubMed