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.

Leaf Spot Disease of Michelia alba caused by Alternaria alternata in China.

The perennial deciduous tree Michelia alba is a widely cultivated street plant in China. In June 2021 and March 2022, M. alba trees with leaf spots were found in the green belt of the approximately 200,000 m2 community (32.62°N, 116.98°E) of Tianjia'an District, Huainan, Anhui, China, where approximately half of the M. alba trees had brown leaf spots surrounded with irregular yellow halos ranging from 2 to 6 mm in diameter (Fig S1A). The leaves of M. alba trees with multiple lesions became blighted. To isolate the potential pathogens causing leaf spot symptoms in M. alba trees, twenty fragments (2 cm2) were excised from the margin of the necrosis on symptomatic leaves, immersed in 1% sodium hypochlorite for 45 s, and then washed three times in sterile water. The fragments were plated and incubated on potato dextrose agar (PDA) at 25 °C and 15 dark green fungal colonies were obtained 5 days later. Single-spore isolates of the fungal colonies plated on potato carrot agar (PCA, Simmons 2007) produced gray, floccose colonies, which reached 71 mm after 7 days at 25 °C were obtained 5 days later (Fig S1C). Optical microscopy analysis showed that single-spore isolates formed sparsely branched chains with pale brown conidiophores on PCA after incubation at 25 °C in darkness for 7 days. The conidia were ellipsoidal, inverted rod, or ovoid, light brown, and 10.0 to 52.5 × 4.5 to 22.7 µm, with zero to four longitudinal or oblique and zero to eight transverse septa (n = 50). Partial conidia are 2.5 to 27.5 × 0.6 to 3.7 µm with cylindrical light brown beaks (n = 50) (Fig S1D, E). The cultural and morphological characteristics of the isolated fungi were consistent with the description of Alternaria alternata (Woudenberg et al. 2015). To further characterize the isolated fungi, the genomic DNA of three representative strains (BYL-1, BYL-2 and BYL-3) were extracted from their mycelia, respectively. ITS region and housekeeping genes GPD, and TEF, were amplified and sequenced using ITS4/ITS5 (White et al. 1990), Gpd1/Gpd2 (Berbee et al. 1999), and EF1-728F/EF1-986R (Carbone and Kohn 1999), primer pairs, respectively. BLAST analysis showed that the isolates BYL-1 (GenBank accession nos. OP325693, OP405008, and OP405009), BYL-2 (GenBank accession nos. PP057859, PP138442, and PP138444), and BYL-3 (GenBank accession nos. PP057860, PP138443, and PP138445) shared 99 to 100% identity with Alternaria alternata (GenBank accession nos. AF347032.1, AY278809.1, KC584693.1), which suggested that all the three isolates belong to A. alternata. The identifications were further confirmed by phylogenetic analysis based on combined DNA sequences data of ITS, GPD, and TEF. As showed in Fig S2, the strains of BYL-1 , BYL-2 and BYL-3 formed a robust clade with A. alternata CBS918.96. Taken together, the morphology and molecular assays suggest that strain BYL-1 is A. alternata. To test pathogenicity, the isolate BYL-1 was cultured on PCA for 7 days to prepare conidial suspensions, and the spore concentration was adjust to a final concentration of 105 spores/ml. The leaves of 3-5-leaf stage of six 5-years-old natural planting M. alba plants were sprayed with conidial suspensions and sterile distilled water, respectively. The petiole of each inoculated leaves of M. alba were secured with sterile wet cotton, and covered with plastic bags to prevent moisture evaporation after incubation. After a 3- to 5- day of inoculation, necrotic lesions appeared on the leaves inoculated with conidial suspensions, whereas no necrotic lesion was observed in the control leaves inoculated with sterile distilled water (Fig S1B). To fulfill the Koch,s postulates, fungi were re-isolated from the margin of necrotic lesions and identified as A. alternata by DNA sequencing the ITS gene. To our knowledge, this is the first report of A. alternata causing leaf spot on M. alba. Because the disease could cause damage to the foliage influencing the greening and ornamental effects of these trees, control measures may need to be implemented during daily management.

Complete Genome Resource of Bacillus velezensis J17-4, an Endophyte Isolated from Stem Tissues of Rice.

Dickeya zeae is the causative agent of rice root rot disease and causes severe harvest and economic losses. In this study, the Bacillus velezensis strain J17-4 with significant antagonism against D. zeae was used to generate DNA for sequencing. After assembly, a high-quality complete genome comprising only a circular chromosome was available. The genome sequence consists of a total of 3,877 prediction coding sequences and nine types of gene clusters involved in secondary metabolite production. This genome data will provide information for understanding the underlying mechanism of strain J17-4 antagonist against D. zeae and a new useful source for comparative genomics studies between strains isolated from various habitats.

Characterization of the Arn lipopolysaccharide modification system essential for zeamine resistance unveils its new roles in Dickeya oryzae physiology and virulence.

The zeamines produced by Dickeya oryzae are potent polyamine antibiotics and phytotoxins that are essential for bacterial virulence. We recently showed that the RND efflux pump DesABC in D. oryzae confers partial resistance to zeamines. To fully elucidate the bacterial self-protection mechanisms, in this study we used transposon mutagenesis to identify the genes encoding proteins involved in zeamine resistance in D. oryzae EC1. This led to the identification of a seven-gene operon, arnEC1 , that encodes enzyme homologues associated with lipopolysaccharide modification. Deletion of the arnEC1 genes in strain EC1 compromised its zeamine resistance 8- to 16-fold. Further deletion of the des gene in the arnEC1 mutant background reduced zeamine resistance to a level similar to that of the zeamine-sensitive Escherichia coli DH5α. Intriguingly, the arnEC1 mutants showed varied bacterial virulence on rice, potato, and Chinese cabbage. Further analyses demonstrated that ArnBCATEC1 are involved in maintenance of the bacterial nonmucoid morphotype by repressing the expression of capsular polysaccharide genes and that ArnBEC1 is a bacterial virulence determinant, influencing transcriptional expression of over 650 genes and playing a key role in modulating bacterial motility and virulence. Taken together, these findings decipher a novel zeamine resistance mechanism in D. oryzae and document new roles of the Arn enzymes in modulation of bacterial physiology and virulence.

Hfq Is a Critical Modulator of Pathogenicity of Dickeya oryzae in Rice Seeds and Potato Tubers.

The frequent outbreaks of soft-rot diseases caused by Dickeya oryzae have emerged as severe problems in plant production in recent years and urgently require the elucidation of the virulence mechanisms of D. oryzae. Here, we report that Hfq, a conserved RNA chaperone protein in bacteria, is involved in modulating a series of virulence-related traits and bacterial virulence in D. oryzae EC1. The findings show that the null mutation of the hfqEC1 gene totally abolished the production of zeamine phytotoxins and protease, significantly attenuated the production of two other types of cell wall degrading enzymes, i.e., pectate lyase and cellulase, as well as attenuating swarming motility, biofilm formation, the development of hypersensitive response to Nicotiana benthamiana, and bacterial infections in rice seeds and potato tubers. QRT-PCR analysis and promoter reporter assay further indicated that HfqEC1 regulates zeamine production via modulating the expression of the key zeamine biosynthesis (zms) cluster genes. Taken together, these findings highlight that the Hfq of D. oryzae is one of the key regulators in modulating the production of virulence determinants and bacterial virulence in rice seeds and potato tubers.

Putrescine Is an Intraspecies and Interkingdom Cell-Cell Communication Signal Modulating the Virulence of Dickeya zeae.

The infections caused by Dickeya zeae become a severe problem in recent years, but the regulatory mechanisms that govern the bacterial virulence remain to be fragmental. Here we report the investigation of potential involvement of polyamines in regulation of D. zeae virulence. We showed that null mutation of speA encoding arginine decarboxylase dramatically decreased the bacterial swimming motility, swarming motility and biofilm formation, and exogenous addition of putrescine effectively rescues the defective phenotypes of D. zeae. HPLC and mass spectrometry analysis validated that speA was essential for production of putrescine in D. zeae. In addition, we demonstrated that D. zeae EC1 could detect and response to putrescine molecules produced by itself or from host plant through specific transporters. Among the two transporters identified, the one represented by PotF played a dominated role over the other represented by PlaP in modulation of putrescine-dependent biological functions. Furthermore, we provided evidence that putrescine signal is critical for D. zeae EC1 bacterial invasion and virulence against rice seeds. Our data depict a novel function of putrescine signal in pathogen-host communication and in modulation of the virulence of an important plant bacterial pathogen.

A Substrate-Activated Efflux Pump, DesABC, Confers Zeamine Resistance to Dickeya zeae.

Zeamines are a family of polyamino phytotoxins produced by Dickeya zeae EC1. These phytotoxins are also potent antibiotics against a range of microorganisms. To understand how D. zeae EC1 can protect itself from the antimicrobial activity of zeamines, we tested whether the ABC transporter genes within the zms (zeamine synthesis) gene cluster were related to zeamine resistance. Our results ruled out the possible involvement of these ABC transporters in zeamine resistance and instead unveiled an RND (resistance-nodulation-cell division) efflux pump, DesABC, which plays an important role in zeamine resistance in D. zeae EC1. The desAB genes are located next to the zms gene cluster, but desC is at a distant location in the bacterial genome. Null mutation of the desABC genes in a zeamine-minus derivative of strain EC1 led to about an 8- to 32-fold decrease in zeamine tolerance level. This efflux pump was zeamine specific and appeared to be conserved only in Dickeya species, which may explain the high potency of zeamines against a wide range of bacterial pathogens. Significantly, expression of the desAB genes was abolished by deletion of zmsA, which encodes zeamine biosynthesis but could be induced by exogenous addition of zeamines. The results suggest that sophisticated and coordinated regulatory mechanisms have evolved to govern zeamine production and tolerance. Taken together, these findings documented a novel signaling role of zeamines and the first resistance mechanism against zeamines, which is a family of potent and promising antibiotics against both Gram-positive and Gram-negative bacterial pathogens.IMPORTANCE Zeamines are a family of newly identified phytotoxins and potent antibiotics produced by D. zeae EC1. Unlike most bacterial organisms, which are highly sensitive, D. zeae EC1 is tolerant to zeamines, but the mechanisms involved are unknown. Our study showed, for the first time, that a new RND efflux pump, DesABC, is indispensable for D. zeae EC1 against zeamines. We found that the DesABC efflux pump was zeamine specific and appeared to be conserved only in the Dickeya species, which may explain the high potency of zeamines against a wide range of bacterial pathogens. We also showed that expression of DesABC efflux system genes was induced by zeamines. These findings not only provide an answer to why D. zeae EC1 is much more tolerant to zeamines than other bacterial pathogens but also document a signaling role of zeamines in modulation of gene expression.

Fis is a global regulator critical for modulation of virulence factor production and pathogenicity of Dickeya zeae.

Dickeya zeae is the causal agent of rice foot rot disease, which has recently become a great threat to rice planting countries and regions. The pathogen produces a family of phytotoxins named zeamines that is critical for bacterial virulence, but little is known about the signaling pathways and regulatory mechanisms that govern zeamine production. In this study, we showed that a conserved transcriptional regulator Fis is involved in the regulation of zeamine production in D. zeae strain EC1. Deletion mutants were markedly attenuated in the virulence against rice seed germination. Transcriptome and phenotype analyses showed that Fis is a potent global transcriptional regulator modulating various virulence traits, including production of extracellular enzymes and exopolysaccharides, swimming and swarming motility, biofilm formation and cell aggregation. DNA gel retardation analysis showed that Fis directly regulates the transcription of key virulence genes and the genes encoding Vfm quorum sensing system through DNA/protein interaction. Our findings unveil a key regulator associated with the virulence of D. zeae EC1, and present useful clues for further elucidation of the regulatory complex and signaling pathways which govern the virulence of this important pathogen.

Genetic Modulation of c-di-GMP Turnover Affects Multiple Virulence Traits and Bacterial Virulence in Rice Pathogen Dickeya zeae.

The frequent outbreaks of rice foot rot disease caused by Dickeya zeae have become a significant concern in rice planting regions and countries, but the regulatory mechanisms that govern the virulence of this important pathogen remain vague. Given that the second messenger cyclic di-GMP (c-di-GMP) is associated with modulation of various virulence-related traits in various microorganisms, here we set to investigate the role of the genes encoding c-di-GMP metabolism in the regulation of the bacterial physiology and virulence by construction all in-frame deletion mutants targeting the annotated c-di-GMP turnover genes in D. zeae strain EC1. Phenotype analyses identified individual mutants showing altered production of exoenzymes and phytotoxins, biofilm formation and bacterial motilities. The results provide useful clues and a valuable toolkit for further characterization and dissection of the regulatory complex that modulates the pathogenesis and persistence of this important bacterial pathogen.