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Pantoea agglomerans is considered one of the most ubiquitous and versatile organisms that include strains that induce diseases in various crops and occasionally cause opportunistic infections in humans. To develop effective strategies to mitigate its impact on plant health and agricultural productivity, a comprehensive investigation is crucial for better understanding its pathogenicity. One proposed eco-friendly approach involves the enzymatic degradation of quorum sensing (QS) signal molecules like N-acylhomoserine lactones (AHLs), known as quorum quenching (QQ), offering potential treatment for such bacterial diseases. In this study the production of C4 and 3-oxo-C6HSL was identified in the plant pathogenic P. agglomerans CFBP 11141 and correlated to enzymatic activities such as amylase and acid phosphatase. Moreover, the heterologous expression of a QQ enzyme in the pathogen resulted in lack of AHLs production and the attenuation of the virulence by mean of drastically reduction of soft rot disease in carrots and cherry tomatoes. Additionally, the interference with the QS systems of P. agglomerans CFBP 11141 by two the plant growth-promoting and AHL-degrading bacteria (PGP-QQ) Pseudomonas segetis P6 and Bacillus toyonensis AA1EC1 was evaluated as a potential biocontrol approach for the first time. P. segetis P6 and B. toyonensis AA1EC1 demonstrated effectiveness in diminishing soft rot symptoms induced by P. agglomerans CFBP 11141 in both carrots and cherry tomatoes. Furthermore, the virulence of pathogen notably decreased when co-cultured with strain AA1EC1 on tomato plants., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Quorum sensing (QS) plays an important role in the regulation of bacteria-host interactions and ecological fitness in many bacteria. In this study, 2 luxI/R homologs, namely eanI/eanR and rhlI/rhlR, were identified in the genome sequence of Pantoea ananatis LMG 2665 T . To determine a role for these luxI/R homologs in pathogenicity and biofilm formation, mutant bacterial strains lacking either eanI/R or rhlI/R and both of these homologs were generated. The results indicated that both the RhlI/R and EanI/R systems are required for pathogenicity and biofilm formation in strain LMG 2665 T . This is the first study to characterize the biological significance of the RhlI/R QS system in P. ananatis.

The Gram-negative proteobacterium Pantoea stewartii subsp. stewartii causes wilt disease in corn plants. Wilting is primarily due to bacterial exopolysaccharide (EPS) production that blocks water transport in the xylem during the late stages of infection. EsaR, the master quorum-sensing (QS) regulator in P. stewartii, modulates EPS levels. At low cell densities EsaR represses or activates expression of a number of genes in the absence of its acyl homoserine lactone (AHL) ligand. At high cell densities, binding of AHL inactivates EsaR leading to derepression or deactivation of its direct targets. Two of these direct targets are the key transcription regulators RcsA and LrhA, which in turn control EPS production and surface motility/adhesion, respectively. In this study, RNA-Seq was used to further examine the physiological impact of deleting the genes encoding these two second-tier regulators. Quantitative reverse transcription PCR (qRT-PCR) was used to validate the regulation observed in the RNA-Seq data. A GFP transcriptional fusion reporter confirmed the existence of a regulatory feedback loop in the system between LrhA and RcsA. Plant virulence assays carried out with rcsA and lrhA deletion and complementation strains demonstrated that both transcription factors play roles during establishment of wilt disease in corn. These efforts further define the hierarchy of the QS-regulated network controlling plant virulence in P. stewartii.

Pantoea agglomerans YS19, an endophytic diazotrophic bacterium isolated from rice, is characterized by the formation of multicellular aggregate structure called symplasmata, which not only bestow the strong stress-resistance of the bacterium, but also contribute to the specific adaptation in the endophyte-host association. Acyl-homoserine lactones (AHLs), as the important signal molecule in the quorum sensing (QS) system of gram-negative bacteria, were demonstrated to regulate motility, cell-aggregation, and other bacterial behaviors. Here, the production of AHL by P. agglomerans YS19 and its regulation on the symplasmata formation were studied. It was revealed that the production of AHL by YS19 was initiated at the exponential growth stage and from then on, reached the peak values at the stationary growth stage in LB medium. The AHL was identified as N-3-oxooctanoyl-L-homoserine lactone (OOHL) by MALDI-TOF-MS analysis. The AHL synthesis gene pagI and receptor gene pagR in YS19 were cloned and phylogenetic analysis showed that they were high conservative among strains in species of P. agglomerans. It was revealed that AHL promoted the bacterial growth and symplasmata formation of YS19. Meanwhile, the colonization ability and growth-promoting effect of YS19 on the host plant were also enhanced by AHL. These results strongly suggest the pleiotropic effects of the AHL-type QS system in endophytic life of the strain., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Marine snow is a continuous shower of organic and inorganic detritus, and plays a crucial role in transporting materials from the sea surface to the deep ocean. The aims of the current study were to identify N-acyl homoserine lactone (AHL)-based quorum sensing (QS) signaling molecules directly from marine snow particles and to investigate the possible regulatory link between QS signals and extracellular hydrolytic enzymes produced by marine snow bacteria. The marine snow samples were collected from the surface water of China marginal seas. Two AHLs, i.e. 3OC6-HSL and C8-HSL, were identified directly from marine snow particles, while six different AHL signals, i.e. C4-HSL, 3OC6-HSL, C6-HSL, C10-HSL, C12-HSL and C14-HSL were produced by Pantoea ananatis B9 inhabiting natural marine snow particles. Of the extracellular hydrolytic enzymes produced by P. ananatis B9, alkaline phosphatase activity was highly enhanced in growth medium supplemented with exogenous AHL (C10-HSL), while quorum quenching enzyme (AiiA) drastically reduced the enzyme activity. To our knowledge, this is the first report revealing six different AHL signals produced by P. ananatis B9 and AHL-based QS system enhanced the extracellular hydrolytic enzyme in P. ananatis B9. Furthermore, this study first time revealing 3OC6-HSL production by Paracoccus carotinifaciens affiliated with Alphaproteobacteria., (© FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

During quorum sensing in the plant pathogen Pantoea stewartii subsp. stewartii, EsaI, an acyl-homoserine lactone (AHL) synthase, and the transcription factor EsaR coordinately control capsular polysaccharide production. The capsule is expressed only at high cell density when AHL levels are high, leading to inactivation of EsaR. In lieu of detailed structural information, the precise mechanism whereby EsaR recognizes AHL and is hindered by it, in a response opposite to that of most other LuxR homologues, remains unresolved. Hence, a random mutagenesis genetic approach was designed to isolate EsaR* variants that are immune to the effects of AHL. Error-prone PCR was used to generate the desired mutants, which were subsequently screened for their ability to repress transcription in the presence of AHL. Following sequencing, site-directed mutagenesis was used to generate all possible mutations of interest as single, rather than multiple amino acid substitutions. Eight individual amino acids playing a critical role in the AHL-insensitive phenotype have been identified. The ability of EsaR* variants to bind AHL and the effect of individual substitutions on the overall conformation of the protein were examined through in vitro assays. Six EsaR* variants had a decreased ability to bind AHL. Fluorescence anisotropy was used to examine the relative DNA binding affinity of the final two EsaR* variants, which retained some AHL binding capability but remained unresponsive to it, perhaps due to an inability of the N-terminal domain to transduce information to the C-terminal domain.

Pantoea stewartii subsp. stewartii is a proteobacterium that causes Stewart's wilt disease in corn plants. The bacteria form a biofilm in the xylem of infected plants and produce capsule that blocks water transport, eventually causing wilt. At low cell densities, the quorum-sensing (QS) regulatory protein EsaR is known to directly repress expression of esaR itself as well as the genes for the capsular synthesis operon transcription regulator, rcsA, and a 2,5-diketogluconate reductase, dkgA. It simultaneously directly activates expression of genes for a putative small RNA, esaS, the glycerol utilization operon, glpFKX, and another transcriptional regulator, lrhA. At high bacterial cell densities, all of this regulation is relieved when EsaR binds an acylated homoserine lactone signal, which is synthesized constitutively over growth. QS-dependent gene expression is critical for the establishment of disease in the plant. However, the identity of the full set of genes controlled by EsaR/QS is unknown. A proteomic approach previously identified around 30 proteins in the QS regulon. In this study, a whole-transcriptome, next-generation sequencing analysis of rRNA-depleted RNA from QS-proficient and -deficient P. stewartii strains was performed to identify additional targets of EsaR. EsaR-dependent transcriptional regulation of a subset of differentially expressed genes was confirmed by quantitative reverse transcription-PCR (qRT-PCR). Electrophoretic mobility shift assays demonstrated that EsaR directly bound 10 newly identified target promoters. Overall, the QS regulon of P. stewartii orchestrates three major physiological responses: capsule and cell envelope biosynthesis, surface motility and adhesion, and stress response., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

N-Acyl homoserine lactone (AHL) serves as signaling molecule for quorum sensing (QS) in Gram-negative bacteria to regulate various physiological activities including pathogenicity. With the aim of isolating freshwater-borne bacteria that can cause outbreak of disease in plants and portrayed QS properties, environmental water sampling was conducted. Here we report the preliminary screening of AHL production using Chromobacterium violaceum CV026 and Escherichia coli [pSB401] as AHL biosensors. The 16S rDNA gene sequence of isolate M009 showed the highest sequence similarity to Pantoea stewartii S9-116, which is a plant pathogen. The isolated Pantoea sp. was confirmed to produce N-3-oxohexanoyl-L-HSL (3-oxo-C6-HSL) through analysis of high resolution mass tandem mass spectrometry.

Gall formation by Pantoea agglomerans pv. gypsophilae is dependent on the hypersensitive response and pathogenicity (hrp) system. Previous studies demonstrated that PagR and PagI, regulators of the quorum-sensing system, induce expression of the hrp regulatory cascade (i.e., hrpXY, hrpS, and hrpL) that activates the HrpL regulon. Here, we isolated the genes of the Gac/Rsm global regulatory pathway (i.e., gacS, gacA, rsmB, and csrD) and of the post-transcriptional regulator rsmA. Our results demonstrate that PagR and PagI also upregulate expression of the Gac/Rsm pathway. PagR acts as a transcriptional activator of each of the hrp regulatory genes and gacA in a N-butanoyl-L-homoserine lactone-dependent manner as shown by gel shift experiments. Mutants of the Gac/Rsm genes or overexpression of rsmA significantly reduced Pantoea agglomerans virulence and colonization of gypsophila. Overexpression of rsmB sRNA abolished gall formation, colonization, and hypersensitive reaction on nonhost plants and prevented transcription of the hrp regulatory cascade, indicating a lack of functional type III secretion system. Expression of rsmB sRNA in the background of the csrD null mutant suggests that CsrD may act as a safeguard for preventing excessive production of rsmB sRNA. Results presented indicate that the hrp regulatory cascade is controlled directly by PagR and indirectly by RsmA, whereas deficiency in RsmA activity is epistatic to PagR induction.

Pathogenic bacteria interact not only with the host organism but most probably also with the resident microbial flora. In the knot disease of the olive tree (Olea europaea), the causative agent is the bacterium Pseudomonas savastanoi pv. savastanoi (Psv). Two bacterial species, namely Pantoea agglomerans and Erwinia toletana, which are not pathogenic and are olive plant epiphytes and endophytes, have been found very often to be associated with the olive knot. We identified the chemical signals that are produced by strains of the three species isolated from olive knot and found that they belong to the N-acyl-homoserine lactone family of QS signals. The luxI/R family genes responsible for the production and response to these signals in all three bacterial species have been identified and characterized. Genomic knockout mutagenesis and in planta experiments showed that virulence of Psv critically depends on QS; however, the lack of signal production can be complemented by wild-type E. toletana or P. agglomerans. It is also apparent that the disease caused by Psv is aggravated by the presence of the two other bacterial species. In this paper we discuss the potential role of QS in establishing a stable consortia leading to a poly-bacterial disease.

Pantoea ananatis SK-1 produces N-acylhomoserine lactones (AHLs) and regulates expression of some virulence factors through AHL-mediated quorum sensing. In this study, we discovered that the strain SK-02I, which has a disrupted AHL-synthetic gene, showed constitutive cell aggregation. SK-1 has the ability to aggregate, and cell aggregation inhibitory factors are expressed under control of AHL-mediated quorum sensing. One of the transposon mutants, SK-33M, constitutively aggregates without defective AHL production. A homology search revealed that the transposon integration site was located in the adhesin-like yeeJ gene. Based on RT-PCR analysis, transcription of yeeJ is regulated by AHL-mediated quorum sensing. However, because both the wild-type and SK-33M strains induced necrotic symptoms in onion leaves, we conclude that the yeeJ gene is not involved in the pathogenicity of SK-1., (Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Gall formation by Pantoea agglomerans pv. gypsophilae is controlled by hrp/hrc genes, phytohormones, and the quorum-sensing (QS) regulatory system. The interactions between these three components were investigated. Disruption of the QS genes pagI and pagR and deletion of both substantially reduced the transcription levels of the hrp regulatory genes hrpXY, hrpS, and hrpL, as determined by quantitative reverse-transcriptase polymerase chain reaction. Expression of hrpL in planta was inhibited by addition of 20 microM or higher concentrations of the QS signal C(4)-HSL. The pagR and hrpL mutants caused an equivalent reduction of 1.3 orders in bacterial multiplication on bean leaves, suggesting possible mediation of the QS effect on epiphytic fitness of P. agglomerans pv. gypsophilae by the hrp regulatory system. indole-3-acetic acid (IAA) and cytokinin significantly affected the expression of the QS and hrp regulatory genes. Transcription of pagI, pagR, hrpL, and hrpS in planta was substantially reduced in iaaH mutant (disrupted in IAA biosynthesis via the indole-3-acetamide pathway) and etz mutant (disrupted in cytokinin biosynthesis). In contrast, the ipdC mutant (disrupted in IAA biosynthesis via the indole-3-pyruvate pathway) substantially increased expression of pagI, pagR, hrpL, and hrpS. Results presented suggest the involvement of IAA and cytokinins in regulation of the QS system and hrp regulatory genes.

The overall goal of this study was to examine the role of quorum-sensing (QS) signals in a multispecies microbial community. Toward this aim, we studied QS signals produced by an indigenous member and an invading pathogen of the microbial community of the cabbage white butterfly (CWB) larval midgut (Pieris rapae). As an initial step, we characterized the QS system in Pantoea CWB304, which was isolated from the larval midgut. A luxI homolog, designated panI, is necessary for the production of N-acyl-L-homoserine lactones (AHLs) by Pantoea CWB304. To determine whether AHL signals are exchanged in the alkaline environment of the midgut, we constructed AHL-sensing bioluminescent reporter strains in Pantoea CWB304 and a panI mutant of this strain. In the gut of the CWB larvae, the reporter in an AHL-deficient Pantoea CWB304 detected AHLs when coinoculated with the wild type. To study the role of AHL signals produced by a community invader, we examined pathogenesis of Pseudomonas aeruginosa PAO1 in CWB larvae. Mortality induced by P. aeruginosa PAO1 was significantly reduced when signaling was interrupted by either a potent chemical inhibitor of QS or mutations in the lasI and rhlI AHL synthases of P. aeruginosa PAO1. These results show that AHLs are exchanged among bacteria in the alkaline gut of CWB larvae and contribute to disease caused by P. aeruginosa PAO1.

The quorum-sensing (QS) regulatory system of the gall-forming Pantoea agglomerans pv. gypsophilae was identified. Mass spectral analysis, together with signal-specific biosensors, demonstrated that P. agglomerans pv. gypsophilae produced N-butanoyl-l-homoserine lactone (C4-HSL) as a major and N-hexanoyl-l-homoserine lactone (C6-HSL) as a minor QS signal. Homologs of luxI and luxR regulatory genes, pagI and pagR, were characterized in strain P. agglomerans pv. gypsophilae Pag824-1 and shown to be convergently transcribed and separated by 14 bp. The deduced PagI (23.8 kDa) and PagR (26.9 kDa) show high similarity with SmaI (41% identity) and SmaR (43% identity), respectively, of Serratia sp. American Type Culture Collection 39006. PagR possesses characteristic autoinducer binding and a helix-turn-helix DNA-binding domain. Gall formation by P. agglomerans pv. gypsophilae depends on a plasmid-borne hrp/hrc gene cluster, type III effectors, and phytohormones. Disruption of pagI, pagR, or both genes simultaneously in Pag824-1 reduced gall size in gypsophila cuttings by 50 to 55% when plants were inoculated with 10(6) CFU/ml. Higher reductions in gall size (70 to 90%) were achieved by overexpression of pagI or addition of exogenous C4-HSL. Expression of the hrp/hrc regulatory gene hrpL and the type III effector pthG in the pagI mutant, as measured with quantitative reverse-transcriptase polymerase chain reaction, was reduced by 5.8 and 6.6, respectively, compared with the wild type, suggesting an effect of the QS system on the Hrp regulon.

A number of gram-negative bacteria have a quorum-sensing system and produce N-acyl-l-homoserine lactone (AHL) that they use them as a quorum-sensing signal molecule. Pantoea ananatis is reported as a common colonist of wheat heads at ripening and causes center rot of onion. In this study, we demonstrated that P. ananatis SK-1 produced two AHLs, N-hexanoyl-l-homoserine lactone (C6-HSL) and N-(3-oxohexanoyl)-l-homoserine lactone (3-oxo-C6-HSL). We cloned the AHL-synthase gene (eanI) and AHL-receptor gene (eanR) and revealed that the deduced amino acid sequence of EanI/EanR showed high identity to those of EsaI/EsaR from P. stewartii. EanR repressed the ean box sequence and the addition of AHLs resulted in derepression of ean box. Inactivation of the chromosomal eanI gene in SK-1 caused disruption of exopolysaccharide (EPS) biosynthesis, biofilm formation, and infection of onion leaves, which were recovered by adding exogenous 3-oxo-C6-HSL. These results demonstrated that the quorum-sensing system involved the biosynthesis of EPS, biofilm formation, and infection of onion leaves in P. ananatis SK-1.