An Osmoregulatory Mechanism Operating through OmpR and LrhA Controls the Motile-Sessile Switch in the Plant Growth-Promoting Bacterium Pantoea alhagi

Abstract

Adaptation to osmotic stress is crucial for bacterial growth and survival in changing environments. Although a large number of osmotic stress response genes have been identified in various bacterial species, how osmotic changes affect bacterial motility, biofilm formation, and colonization of host niches remains largely unknown. In this study, we report that the LrhA regulator is an osmoregulated transcription factor that directly binds to the promoters of the flhDC, eps, and opgGH operons and differentially regulates their expression, thus inhibiting motility and promoting exopolysaccharide (EPS) production, synthesis of osmoregulated periplasmic glucans (OPGs), biofilm formation, and root colonization of the plant growth-promoting bacterium Pantoea alhagi LTYR-11Z. Further, we observed that the LrhA-regulated OPGs control RcsCD-RcsB activation in a concentration-dependent manner, and a high concentration of OPGs induced by increased medium osmolarity is maintained to achieve the high level of activation of the Rcs phosphorelay, which results in enhanced EPS synthesis and decreased motility in P. alhagi Moreover, we showed that the osmosensing regulator OmpR directly binds to the promoter of lrhA and promotes its expression, while lrhA expression is feedback inhibited by the activated Rcs phosphorelay system. Overall, our data support a model whereby P. alhagi senses environmental osmolarity changes through the EnvZ-OmpR two-component system and LrhA to regulate the synthesis of OPGs, EPS production, and flagellum-dependent motility, thereby employing a hierarchical signaling cascade to control the transition between a motile lifestyle and a biofilm lifestyle.IMPORTANCE Many motile bacterial populations form surface-attached biofilms in response to specific environmental cues, including osmotic stress in a range of natural and host-related systems. However, cross talk between bacterial osmosensing, swimming, and biofilm formation regulatory networks is not fully understood. Here, we report that the pleiotropic regulator LrhA in Pantoea alhagi is involved in the regulation of flagellar motility, biofilm formation, and host colonization and responds to osmotic upshift. We further show that this sensing relies on the EnvZ-OmpR two-component system that was known to detect changes in external osmotic stress. The EnvZ-OmpR-LrhA osmosensing signal transduction cascade is proposed to increase bacterial fitness under hyperosmotic conditions inside the host. Our work proposes a novel regulatory mechanism that links osmosensing and motile-sessile lifestyle transitions, which may provide new approaches to prevent or promote the formation of biofilms and host colonization in P. alhagi and other bacteria possessing a similar osmoregulatory mechanism.

Keywords: LrhA; OPGs; OmpR; Rcs phosphorelay; biofilm; exopolysaccharides; motility; osmosensing.

FIG 1

LrhA promotes biofilm formation and inhibits swimming motility in P. alhagi. (A) Biofilm formation by the WT, mutant, and complemented strains was displayed with crystal violet staining (top) and quantified with optical density measurement. (B) The swimming motility of the indicated strains was tested in 0.3% agar tryptone plates, and the diameters of swimming zones were measured (top). (C) Growth curves of the indicated strains in LB and M9 media at 30°C. Data are shown as means ± standard deviations (SD) from three experiments. ***, P ≤ 0.001 (Student’s t test). lg N (CFU ml⁻¹) refers to the log₁₀ number of CFU per ml.

FIG 2

The P. alhagi lrhA gene is required for attachment and endophytic colonization of wheat roots. (A) Attachment and endophytic colonization of wheat roots by WT, ΔlrhA mutant, and complemented strains. The indicated strains were inoculated separately into wheat roots. (B) Competition between the WT strain containing pBBR1MCS-1 and the ΔlrhA mutant containing pKT100 for attachment and endophytic colonization of wheat roots. The two strains were coinoculated at a 1:1 ratio in wheat roots. Data are shown as means ± SD from three independent experiments. ***, P ≤ 0.001 (Student’s t test). (C) Confocal microscopy analysis of the competition between the GFP-labeled WT and the mCherry-labeled ΔlrhA mutant for wheat root attachment after 15 h of incubation. A 1:1 mixture of the two strains was used for inoculation. The GFP-labeled WT cells and mCherry-labeled ΔlrhA mutant cells are visible in green and red, respectively, and the wheat root cells stained with 4′,6-diamidino-2-phenylindole (DAPI) are visible in blue. Scale bar = 50 μm.

FIG 3

LrhA promotes EPS production by positively regulating the expression of the eps operon. (A) β-Galactosidase activity of the eps promoter in the indicated strains. Strains were grown in LB medium. (B) EMSA was performed to analyze interactions between His₆-LrhA and the eps promoter. Increasing amounts of LrhA (0.2, 0.4, 0.8, and 1.2 μg) and 30 ng DNA probe were used. As negative controls, a 450-bp fragment from the wza (B1H58_14485) coding region instead of the eps promoter (control A) and bovine serum albumin (BSA) instead of His₆-LrhA (control B) were included in the binding assays. A total of 1.2 μg of LrhA protein was added to the control A, and the same amount of BSA protein was added to the control B. (C) CR-binding assays of the WT, ΔlrhA mutant, and complemented strains. (D) EPS quantification of the indicated strains. **, P ≤ 0.01; ***, P ≤ 0.001 (Student’s t test).

FIG 4

LrhA negatively regulates the expression of flhDC. (A) β-Galactosidase activity of the flhDC promoter in the indicated strains that were grown to stationary phase in LB medium. ***, P ≤ 0.001. (B) LrhA (0.5, 1, 2, and 4 μg) EMSA of the flhDC promoter probe (−61 to −507) (30 ng). A 447-bp fragment from the opgH coding region instead of the flhDC promoter and 4 μg of LrhA protein were used in control A, while BSA (4 μg) instead of LrhA was used in control B.

FIG 5

LrhA positively regulates the expression of opgGH, which is required for the synthesis of OPGs involved in the regulation of motility and biofilm formation. (A) β-Galactosidase activity of the opgGH promoter in the indicated strains grown to stationary phase in LB medium. (B) LrhA (0.1, 0.2, 0.4, and 1.0 μg) EMSA of the opgGH promoter probe (−1 to −346) (30 ng). A 346-bp fragment from the flhC coding region instead of the opgGH promoter and 1.0 μg of LrhA protein was added to control A. The same amount of BSA was used in control B. (C) Quantification of OPGs in the indicated strains. (D) The swimming motility of WT, ΔopgH mutant, the ΔopgH/opgH complemented, and the WT/opgGH opgGH-overexpressing strains was tested, and the diameters of swimming zones were measured (top). (E) Biofilm formation by the WT, ΔopgH mutant, ΔopgH/opgH, and WT/opgGH strains displayed with crystal violet staining (top) and quantified with optical density measurement. **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 6

OPGs regulate biofilm formation and motility by controlling the phosphorylation level of RcsB. (A) Separation of RcsB-P from unphosphorylated RcsB in the indicated strains in vivo by Phos-tag SDS-PAGE and Western blot analysis. The results presented are representative of three independent experiments performed. (B) Quantification of the ratio of RcsB-P or unphosphorylated RcsB to the total RcsB in the indicated strains. The data reported are the averages of the results from three independent experiments. (C) The swimming motility of the WT, ΔrcsB mutant, ΔrcsB/rcsB and ΔrcsB/rcsB^D56N strains was tested, and the diameters of swimming zones were measured (top). (D) Biofilm formation by the WT, ΔrcsB mutant, ΔrcsB/rcsB, and ΔrcsB/rcsB^D56N strains displayed with crystal violet staining (top) and quantified with optical density measurement. **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 7

LrhA regulation is modulated by osmotic stress. (A) β-Galactosidase activities of the opgGH promoter in the WT strain and the ΔlrhA mutant grown in the indicated media. (B) Quantification of OPGs in the WT strain grown in LOS medium and LOS medium supplemented with NaCl (450 mM) or sorbitol (600 mM). (C) β-Galactosidase activities of the lrhA promoter in the WT strain and the ΔompR mutant grown in the indicated media. (D) β-Galactosidase activities of the eps and flhDC promoters in the WT strain and the ΔlrhA mutant grown in the indicated media. **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 8

Osmoregulation of biofilm formation, motility, and root attachment by P. alhagi. (A) Biofilm formation by the WT strain and the ΔlrhA mutant grown in the indicated media. (B) Swimming motility of the WT strain and the ΔlrhA mutant grown in the indicated media with 0.3% agar. (C) Attachment of wheat roots by the WT strain and the ΔlrhA mutant after 1 and 15 h of incubation in 1/2 MS medium supplemented with 450 mM NaCl or 600 mM sorbitol. Data are shown as means ± SD of the results from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 9

OmpR positively regulates the expression of lrhA by directly binding to the lrhA promoter. (A) β-Galactosidase activities of the lrhA promoter (WT motif versus mutant motif) in the WT, ΔompR mutant, and complemented strains. Strains were grown in LB medium. ***, P ≤ 0.001; ns, not significant (Student’s t test). (B) OmpR (0.25, 0.5, 1, and 2 μg) EMSA of the lrhA promoter region (+10 to −451) (50 ng). A 447-bp fragment from the opgH coding region instead of the lrhA promoter and 2 μg of OmpR protein were added to control A. The same amount of BSA was added to control B. (C) OmpR (0.5, 1, and 2 μg) EMSA of the lrhA promoter region (WT motif) versus the lrhA promoter region with mutations in the putative OmpR binding site (mutant motif).

FIG 10

Model for the osmoregulatory mechanism operating through OmpR and LrhA that links osmotic changes to host colonization in the plant drought resistance-promoting bacterium P. alhagi. At low osmolarity (left), the OmpR-P levels decrease, and the level of LrhA remains low. The result is that the expression of the eps and opgGH operons is not activated, and flhDC would be derepressed. At high osmolarity (right), the concentration of OmpR-P increases, and it activates the expression of LrhA. Sequentially, LrhA activates transcription of eps and opgGH and inhibits transcription of flhDC, which results in enhanced EPS production and biofilm formation and reduction in motility. The blue gradient at the bottom represents the switch between planktonic and biofilm lifestyles in response to osmotic stress. OM, outer membrane; IM, inner membrane.