Elongation factor P modulates Acinetobacter baumannii physiology and virulence as a cyclic dimeric guanosine monophosphate effector.

Cyclic diguanosine monophosphate (c-di-GMP) is widely used by bacteria to control biological functions in response to diverse signals or cues. A previous study showed that potential c-di-GMP metabolic enzymes play a role in the regulation of biofilm formation and motility in Acinetobacter baumannii. However, it was unclear whether and how A. baumannii cells use c-di-GMP signaling to modulate biological functions. Here, we report that c-di-GMP is an important intracellular signal in the modulation of biofilm formation, motility, and virulence in A. baumannii. The intracellular level of c-di-GMP is principally controlled by the diguanylate cyclases (DGCs) A1S_1695, A1S_2506, and A1S_3296 and the phosphodiesterase (PDE) A1S_1254. Intriguingly, we revealed that A1S_2419 (an elongation factor P [EF-P]), is a novel c-di-GMP effector in A. baumannii. Response to a c-di-GMP signal boosted A1S_2419 activity to rescue ribosomes from stalling during synthesis of proteins containing consecutive prolines and thus regulate A. baumannii physiology and pathogenesis. Our study presents a unique and widely conserved effector that controls bacterial physiology and virulence by sensing the second messenger c-di-GMP.

An anthranilic acid-responsive transcriptional regulator controls the physiology and pathogenicity of Ralstonia solanacearum.

Quorum sensing (QS) is widely employed by bacterial cells to control gene expression in a cell density-dependent manner. A previous study revealed that anthranilic acid from Ralstonia solanacearum plays a vital role in regulating the physiology and pathogenicity of R. solanacearum. We reported here that anthranilic acid controls the important biological functions and virulence of R. solanacearum through the receptor protein RaaR, which contains helix-turn-helix (HTH) and LysR substrate binding (LysR_substrate) domains. RaaR regulates the same processes as anthranilic acid, and both are present in various bacterial species. In addition, anthranilic acid-deficient mutant phenotypes were rescued by in trans expression of RaaR. Intriguingly, we found that anthranilic acid binds to the LysR_substrate domain of RaaR with high affinity, induces allosteric conformational changes, and then enhances the binding of RaaR to the promoter DNA regions of target genes. These findings indicate that the components of the anthranilic acid signaling system are distinguished from those of the typical QS systems. Together, our work presents a unique and widely conserved signaling system that might be an important new type of cell-to-cell communication system in bacteria.

tRNA modification enzyme MiaB connects environmental cues to activation of Pseudomonas aeruginosa type III secretion system.

Pseudomonas aeruginosa, a major inhabitant of numerous environmental reservoirs, is a momentous opportunistic human pathogen associated with severe infections even death in the patients suffering from immune deficiencies or metabolic diseases. Type III secretion system (T3SS) employed by P. aeruginosa to inject effector proteins into host cells is one of the pivotal virulence factors pertaining to acute infections caused by this pathogen. Previous studies showed that P. aeruginosa T3SS is regulated by various environmental cues such as calcium concentration and the host signal spermidine. However, how T3SS is regulated and expressed particularly under the ever-changing environmental conditions remains largely elusive. In this study, we reported that a tRNA modification enzyme PA3980, designated as MiaB, positively regulated T3SS gene expression in P. aeruginosa and was essential for the induced cytotoxicity of human lung epithelial cells. Further genetic assays revealed that MiaB promoted T3SS gene expression by repressing the LadS-Gac/Rsm signaling pathway and through the T3SS master regulator ExsA. Interestingly, ladS, gacA, rsmY and rsmZ in the LadS-Gac/Rsm signaling pathway seemed potential targets under the independent regulation of MiaB. Moreover, expression of MiaB was found to be induced by the cAMP-dependent global regulator Vfr as well as the spermidine transporter-dependent signaling pathway and thereafter functioned to mediate their regulation on the T3SS gene expression. Together, these results revealed a novel regulatory mechanism for MiaB, with which it integrates different environmental cues to modulate T3SS gene expression in this important bacterial pathogen.

Gram-negative bacteria resist antimicrobial agents by a DzrR-mediated envelope stress response.

BACKGROUND: Envelope stress responses (ESRs) are critical for adaptive resistance of Gram-negative bacteria to envelope-targeting antimicrobial agents. However, ESRs are poorly defined in a large number of well-known plant and human pathogens. Dickeya oryzae can withstand a high level of self-produced envelope-targeting antimicrobial agents zeamines through a zeamine-stimulated RND efflux pump DesABC. Here, we unraveled the mechanism of D. oryzae response to zeamines and determined the distribution and function of this novel ESR in a variety of important plant and human pathogens. RESULTS: In this study, we documented that a two-component system regulator DzrR of D. oryzae EC1 mediates ESR in the presence of envelope-targeting antimicrobial agents. DzrR was found modulating bacterial response and resistance to zeamines through inducing the expression of RND efflux pump DesABC, which is likely independent on DzrR phosphorylation. In addition, DzrR could also mediate bacterial responses to structurally divergent envelope-targeting antimicrobial agents, including chlorhexidine and chlorpromazine. Significantly, the DzrR-mediated response was independent on the five canonical ESRs. We further presented evidence that the DzrR-mediated response is conserved in the bacterial species of Dickeya, Ralstonia, and Burkholderia, showing that a distantly located DzrR homolog is the previously undetermined regulator of RND-8 efflux pump for chlorhexidine resistance in B. cenocepacia. CONCLUSIONS: Taken together, the findings from this study depict a new widely distributed Gram-negative ESR mechanism and present a valid target and useful clues to combat antimicrobial resistance.

Engineered Sucrose Metabolism Improves the Smut Disease Suppression Potency of Pseudomonas sp. ST4.

Sporisorium scitamineum and Ustilago maydis are two fungal pathogens causing severe sugarcane and maize diseases, respectively. Sexual mating of compatible sporidia is essential for these pathogens to form infections dikaryotic mycelia and cause smut diseases. We showed recently that in the presence of exogenous glucose, the Pseudomonas sp. strain ST4 could block the fungal mating and display a strong disease suppression potency on S. scitamineum. With the aim of conferring strain ST4 the ability to metabolize sucrose in plants for glucose production, we identified a strong native promoter pSsrA in strain ST4 and additional promoter elements to facilitate translation and peptide translocation for the construction of a fusion gene encoding sucrose metabolism. The cscA gene encoding sucrose hydrolase from Pseudomonas protegens Pf-5 was fused to the promoter pSsrA, a translational coupler bicistronic design and a Tat signal peptide, which was then cloned into mini-Tn7 transposon. This synthetic gene cassette was integrated into the chromosome of strain ST4, and the resultant engineered strain ST4E was able to hydrolyze sucrose with high efficiency and displayed elevated inhibitory activity on the mating and virulence of S. scitamineum and U. maydis. The findings from this study provide a valuable device and useful clues for the engineering of sucrose metabolism in non- or weak-sucrose-utilizing bacterial strains and present an improved biocontrol agent against plant smut pathogens. IMPORTANCE Sporisorium scitamineum and Ustilago maydis are typical dimorphic fungi causing severe sugarcane and maize smut diseases, respectively. Sexual mating of compatible sporidia is essential for these pathogens to form infections dikaryotic mycelia and cause smut diseases. We previously demonstrated that the biocontrol strain Pseudomonas sp. ST4 could block the fungal mating and displays a strong suppression potency on smut diseases, while it was unable to utilize the host-sourced sucrose for glucose production critical for antifungus efficiency. In this study, we constructed a high-expression gene cassette for minitransposon-mediated genome integration and sucrose hydrolysis in the bacterial periplasmic space. The resultant engineered strain ST4E was able to hydrolyze sucrose and inhibit the mating and hyphal growth of S. scitamineum and U. maydis. These findings provide a valuable tool and useful clues for the engineering of sucrose metabolism in non- or weak-sucrose-utilizing bacterial strains and present an improved biocontrol agent against plant smut pathogens.

The GacA-GacS Type Two-Component System Modulates the Pathogenicity of Dickeya oryzae EC1 Mainly by Regulating the Production of Zeamines.

The GacS-GacA type two-component system (TCS) positively regulates pathogenicity-related phenotypes in many plant pathogens. In addition, Dickeya oryzae EC1, the causative agent of soft rot disease, produces antibiotic-like toxins called zeamines as one of the major virulence factors that inhibit the germination of rice seeds. The present study identified a GacS-GacA type TCS, named TzpS-TzpA, that positively controls the virulence of EC1, mainly by regulating production of the toxin zeamines. RNA-seq analysis of strain EC1 and its tzpA mutant showed that the TCS regulated a wide range of virulence genes, especially those encoding zeamines. Protein-protein interaction was detected between TzpS and TzpA through the bacterial two-hybrid system and pull-down assay. In trans expression of tzpA failed to rescue the defective phenotypes in both the ΔtzpS and ΔtzpSΔtzpA mutants. Furthermore, TzpA controls target gene expression by direct binding to DNA promoters that contain a Gac-box motif, including a regulatory RNA rsmB and the vfm quorum-sensing system regulator vfmE. These findings therefore suggested that the EC1 TzpS-TzpA TCS system mediates the pathogenicity of Dickeya oryzae EC1 mainly by regulating the production of zeamines.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The cis-2-Dodecenoic Acid (BDSF) Quorum Sensing System in Burkholderia cenocepacia.

It has been demonstrated that quorum sensing (QS) is widely employed by bacterial cells to coordinately regulate various group behaviors. Diffusible signal factor (DSF)-type signals have emerged as a growing family of conserved cell-cell communication signals. In addition to the DSF signal initially identified in Xanthomonas campestris pv. campestris, Burkholderiadiffusible signal factor (BDSF) (cis-2-dodecenoic acid) has been recognized as a conserved DSF-type signal with specific characteristics in both signal perception and transduction from DSF signals. Here, we review the history and current progress of the research on this type of signal, especially focusing on its biosynthesis, signaling pathways, and biological functions. We also discuss and explore the huge potential of targeting this kind of QS system as a new therapeutic strategy to control bacterial infections and diseases.

The Two-Component System FleS/FleR Represses H1-T6SS via Cyclic di-GMP Signaling in Pseudomonas aeruginosa.

The type VI secretion system (T6SS) is an important translocation apparatus that is widely employed by Gram-negative bacteria to deliver toxic effectors into eukaryotic and prokaryotic target cells, causing host damage and providing competitive advantages in polymicrobial environments. The genome of Pseudomonas aeruginosa harbors three T6SS clusters (H1-T6SS, H2-T6SS, H3-T6SS). Activities of these systems are tightly regulated by a complicated signaling network which remains largely elusive. In this study, we focused on a previously characterized two-component system FleS/FleR, and performed comparative transcriptome analysis between the PAO1 wild-type strain and its isogenic ΔfleR mutant, which revealed the important role of FleS/FleR in regulating multiple physiological pathways including T6SS. Gene expression and bacterial killing assays showed that the expression and activity of H1-T6SS are repressed in the wild-type strain owing to the high intracellular c-di-GMP content. Further explorations demonstrated that c-di-GMP relies on the transcription factor FleQ to repress H1-T6SS and its synthesis is controlled by a global regulator AmrZ which is induced by the active FleS/FleR. Interestingly, repression of H1-T6SS by FleS/FleR in PAO1 is independent of RetS which is known to regulate H1-T6SS by controlling the central post-transcriptional factor RsmA. Together, our results identified a novel regulator of H1-T6SS and provided detailed mechanisms of this signaling pathway in PAO1. IMPORTANCE Pseudomonas aeruginosa is an opportunistic human pathogen distributed widely in the environment. The genome of this pathogen contains three T6SS clusters which contribute significantly to its virulence. Understanding the complex regulatory network that controls the activity of T6SS is essential for the development of effective therapeutic treatments for P. aeruginosa infections. In this study, transcriptome analysis led to the identification of a novel regulator FleS/FleR which inversely regulates H1-T6SS and H2-T6SS in P. aeruginosa PAO1. We further revealed a detailed FleS/FleR-mediated regulatory pathway of H1-T6SS in PAO1 which involves two additional transcriptional regulators AmrZ and FleQ and the second messenger c-di-GMP, providing important implications to develop novel anti-infective strategies and antimicrobial drugs.

Agrobacteria reprogram virulence gene expression by controlled release of host-conjugated signals.

It is highly intriguing how bacterial pathogens can quickly shut down energy-costly infection machinery once successful infection is established. This study depicts that mutation of repressor SghR increases the expression of hydrolase SghA in Agrobacterium tumefaciens, which releases plant defense signal salicylic acid (SA) from its storage form SA ß-glucoside (SAG). Addition of SA substantially reduces gene expression of bacterial virulence. Bacterial vir genes and sghA are differentially transcribed at early and later infection stages, respectively. Plant metabolite sucrose is a signal ligand that inactivates SghR and consequently induces sghA expression. Disruption of sghA leads to increased vir expression in planta and enhances tumor formation whereas mutation of sghR decreases vir expression and tumor formation. These results depict a remarkable mechanism by which A. tumefaciens taps on the reserved pool of plant signal SA to reprogram its virulence upon establishment of infection.

Structural basis of a novel repressor, SghR, controlling Agrobacterium infection by cross-talking to plants.

Agrobacterium tumefaciens infects various plants and causes crown gall diseases involving temporal expression of virulence factors. SghA is a newly identified virulence factor enzymatically releasing salicylic acid from its glucoside conjugate and controlling plant tumor development. Here, we report the structural basis of SghR, a LacI-type transcription factor highly conserved in Rhizobiaceae family, regulating the expression of SghA and involved in tumorigenesis. We identified and characterized the binding site of SghR on the promoter region of sghA and then determined the crystal structures of apo-SghR, SghR complexed with its operator DNA, and ligand sucrose, respectively. These results provide detailed insights into how SghR recognizes its cognate DNA and shed a mechanistic light on how sucrose attenuates the affinity of SghR with DNA to modulate the expression of SghA. Given the important role of SghR in mediating the signaling cross-talk during Agrobacterium infection, our results pave the way for structure-based inducer analog design, which has potential applications for agricultural industry.