Specificities and redundancies in the NEL family of bacterial E3 ubiquitin ligases of Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium expresses two type III secretion systems, T3SS1 and T3SS2, which are encoded in Salmonella pathogenicity island 1 (SPI1) and SPI2, respectively. These are essential virulent factors that secrete more than 40 effectors that are translocated into host animal cells. This study focuses on three of these effectors, SlrP, SspH1, and SspH2, which are members of the NEL family of E3 ubiquitin ligases. We compared their expression, regulation, and translocation patterns, their role in cell invasion and intracellular proliferation, their ability to interact and ubiquitinate specific host partners, and their effect on cytokine secretion. We found that transcription of the three genes encoding these effectors depends on the virulence regulator PhoP. Although the three effectors have the potential to be secreted through T3SS1 and T3SS2, the secretion of SspH1 and SspH2 is largely restricted to T3SS2 due to their expression pattern. We detected a role for these effectors in proliferation inside fibroblasts that is masked by redundancy. The generation of chimeric proteins allowed us to demonstrate that the N-terminal part of these proteins, containing the leucine-rich repeat motifs, confers specificity towards ubiquitination targets. Furthermore, the polyubiquitination patterns generated were different for each effector, with Lys48 linkages being predominant for SspH1 and SspH2. Finally, our experiments support an anti-inflammatory role for SspH1 and SspH2.

Comparative secretome analysis reveals cross-talk between type III secretion system and flagella assembly in Pseudomonas plecoglossicida.

The Gram-negative bacterium Pseudomonas plecoglossicida has caused visceral granulomas disease in several farmed fish species, including large yellow croaker (Larimichthys crocea), which results in severe economic losses. Type III secretion systems (T3SS) are protein secretion and translocation nanomachines widely employed by many Gram-negative bacterial pathogens for infection and pathogenicity. However, the exact role of T3SS in the pathogenesis of P. plecoglossicida infection is still unclear. In this study, a T3SS translocators deletion strain (△popBD) of P. plecoglossicida was constructed to investigate the function of T3SS. Then comparative secretome analysis of the P. plecoglossicida wild-type (WT) and △popBD mutant strains was conducted by label-free quantitation (LFQ) mass spectrometry. The results show that knockout of T3SS translocators popB and popD has an adverse effect on the effector protein ExoU secretion, flagella assembly, and biofilm formation. Further experimental validations also confirmed that popB-popD deletion could affect the P. plecoglossicida flagella morphology/formation, adherence, mobility, and biofilm formation. These data indicate that a cross-talk exists between the P. plecoglossicida T3SS and the flagella system. Our results, therefore, will facilitate the further under-standing of the pathogenic mechanisms leading to visceral granulomas disease caused by P. plecoglossicida.

Pangenome insights into the diversification and disease specificity of worldwide Xanthomonas outbreaks.

The bacterial genus Xanthomonas is responsible for disease outbreaks in several hundred plant species, many of them economically important crops. In the era of next-generation sequencing, thousands of strains from this genus have now been sequenced as part of isolated studies that focus on outbreak characterization, host range, diversity, and virulence factor identification. However, these data have not been synthesized and we lack a comprehensive phylogeny for the genus, with some species designations in public databases still relying on phenotypic similarities and representative sequence typing. The extent of genetic cohesiveness among Xanthomonas strains, the distribution of virulence factors across strains, and the impact of evolutionary history on host range across the genus are also poorly understood. In this study, we present a pangenome analysis of 1,910 diverse Xanthomonas genomes, highlighting their evolutionary relationships, the distribution of virulence-associated genes across strains, and rates of horizontal gene transfer. We find a number of broadly conserved classes of virulence factors and considerable diversity in the Type 3 Secretion Systems (T3SSs) and Type 3 Secreted Effector (T3SE) repertoires of different Xanthomonas species. We also use these data to re-assign incorrectly classified strains to phylogenetically informed species designations and find evidence of both monophyletic host specificity and convergent evolution of phylogenetically distant strains to the same host. Finally, we explore the role of recombination in maintaining genetic cohesion within the Xanthomonas genus as a result of both ancestral and recent recombination events. Understanding the evolutionary history of Xanthomonas species and the relationship of key virulence factors with host-specificity provides valuable insight into the mechanisms through which Xanthomonas species shift between hosts and will enable us to develop more robust resistance strategies against these highly virulent pathogens.

Virulence-associated type III secretion systems in Gram-negative bacteria.

Virulence-associated bacterial type III secretion systems are multiprotein molecular machines that promote the pathogenicity of bacteria towards eukaryotic host cells. These machines form needle-like structures, named injectisomes, that span both bacterial and host membranes, forming a direct conduit for the delivery of bacterial proteins into host cells. Once within the host, these bacterial effector proteins are capable of manipulating a multitude of host cell functions. In recent years, the knowledge of assembly, structure and function of these machines has grown substantially and is presented and discussed in this review.

Salmonella Type III Secretion Effector SrfJ: A Glucosylceramidase Affecting the Lipidome and the Transcriptome of Mammalian Host Cells.

Type III secretion systems are found in many Gram-negative pathogens and symbionts of animals and plants. Salmonella enterica has two type III secretion systems associated with virulence, one involved in the invasion of host cells and another involved in maintaining an appropriate intracellular niche. SrfJ is an effector of the second type III secretion system. In this study, we explored the biochemical function of SrfJ and the consequences for mammalian host cells of the expression of this S. enterica effector. Our experiments suggest that SrfJ is a glucosylceramidase that alters the lipidome and the transcriptome of host cells, both when expressed alone in epithelial cells and when translocated into macrophages in the context of Salmonella infection. We were able to identify seventeen lipids with higher levels and six lipids with lower levels in the presence of SrfJ. Analysis of the forty-five genes, the expression of which is significantly altered by SrfJ with a fold-change threshold of two, suggests that this effector may be involved in protecting Salmonella from host immune defenses.

The type III secretion chaperone SctY may shield the hydrophobic export gate-binding C-terminus of its substrate SctX.

Gram-negative bacteria such as Aeromonas and Yersinia spp. have developed mechanisms to inhibit the immune defense of their host. Effector proteins are directly injected into the host cytoplasm from the bacterial cytosol via type III secretion systems (T3SSs), where they modulate the cytoskeleton and signaling of the cell. Assembly of, and secretion via, T3SSs is tightly regulated by a number of bacterial proteins, including SctX (AscX in Aeromonas), the secretion of which is essential for T3SS function. Here, crystal structures of AscX in complex with SctY chaperones from Yersinia or Photorhabdus spp. carrying homologous T3SSs are described. There are crystal pathologies in all cases, with one crystal form diffracting anisotropically and the other two exhibiting strong pseudotranslation. The new structures reveal that the positioning of the substrate is very similar on different chaperones. However, the two C-terminal SctX helices that cap the N-terminal tetratricopeptide repeat of SctY shift and tilt depending on the identity of the chaperone. Moreover, the C-terminus of the α3 helix of AscX exhibits an unprecedented kink in two of the structures. In previous structures, the C-terminus of SctX protrudes beyond the chaperone as a straight helix: a conformation that is required for binding to the nonameric export gate SctV but that is unfavorable for binary SctX-SctY complexes due to the hydrophobicity of helix α3 of SctX. A kink in helix α3 may allow the chaperone to shield the hydrophobic C-terminus of SctX in solution.

Recruitment of heterologous substrates by bacterial secretion systems for transkingdom translocation.

Bacterial secretion systems mediate the selective exchange of macromolecules between bacteria and their environment, playing a pivotal role in processes such as horizontal gene transfer or virulence. Among the different families of secretion systems, Type III, IV and VI (T3SS, T4SS and T6SS) share the ability to inject their substrates into human cells, opening up the possibility of using them as customized injectors. For this to happen, it is necessary to understand how substrates are recruited and to be able to engineer secretion signals, so that the transmembrane machineries can recognize and translocate the desired substrates in place of their own. Other factors, such as recruiting proteins, chaperones, and the degree of unfolding required to cross through the secretion channel, may also affect transport. Advances in the knowledge of the secretion mechanism have allowed heterologous substrate engineering to accomplish translocation by T3SS, and to a lesser extent, T4SS and T6SS into human cells. In the case of T4SS, transport of nucleoprotein complexes adds a bonus to its biotechnological potential. Here, we review the current knowledge on substrate recognition by these secretion systems, the many examples of heterologous substrate translocation by engineering of secretion signals, and the current and future biotechnological and biomedical applications derived from this approach.

The bacterial virulence factor NleA undergoes host-mediated O-linked glycosylation.

Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) are gastrointestinal pathogens responsible for severe diarrheal illness. EHEC and EPEC form "attaching and effacing" lesions during colonization and, upon adherence, inject proteins directly into host intestinal cells via the type III secretion system (T3SS). Injected bacterial proteins have a variety of functions but generally alter host cell biology to favor survival and/or replication of the pathogen. Non-LEE-encoded effector A (NleA) is a T3SS-injected effector of EHEC, EPEC, and the related mouse pathogen Citrobacter rodentium. Studies in mouse models indicate that NleA has an important role in bacterial virulence. However, the mechanism by which NleA contributes to disease remains unknown. We have determined that the following translocation into host cells, a serine and threonine-rich region of NleA is modified by host-mediated mucin-type O-linked glycosylation. Surprisingly, this region was not present in several clinical EHEC isolates. When expressed in C. rodentium, a non-modifiable variant of NleA was indistinguishable from wildtype NleA in an acute mortality model but conferred a modest increase in persistence over the course of infection in mixed infections in C57BL/6J mice. This is the first known example of a bacterial effector being modified by host-mediated O-linked glycosylation. Our data also suggests that this modification may confer a selective disadvantage to the bacteria during in vivo infection.

Crystals of SctV from different species reveal variable symmetry for the cytosolic domain of the type III secretion system export gate.

Type III secretion systems (T3SSs) are proteinaceous devices employed by Gram-negative bacteria to directly transport proteins into a host cell. Substrate recognition and secretion are strictly regulated by the export apparatus of the so-called injectisome. The export gate SctV engages chaperone-bound substrates of the T3SS in its nonameric cytoplasmic domain. Here, the purification and crystallization of the cytoplasmic domains of SctV from Photorhabdus luminescens (LscVC) and Aeromonas hydrophila (AscVC) are reported. Self-rotation functions revealed that LscVC forms oligomers with either eightfold or ninefold symmetry in two different crystal forms. Similarly, AscVC was found to exhibit tenfold rotational symmetry. These are the first instances of SctV proteins forming non-nonameric oligomers.

Enhanced protein translocation to mammalian cells by expression of EtgA transglycosylase in a synthetic injector E. coli strain.

BACKGROUND: Bacterial type III secretion systems (T3SSs) assemble a multiprotein complex termed the injectisome, which acts as a molecular syringe for translocation of specific effector proteins into the cytoplasm of host cells. The use of injectisomes for delivery of therapeutic proteins into mammalian cells is attractive for biomedical applications. With that aim, we previously generated a non-pathogenic Escherichia coli strain, called Synthetic Injector E. coli (SIEC), which assembles functional injectisomes from enteropathogenic E. coli (EPEC). The assembly of injectisomes in EPEC is assisted by the lytic transglycosylase EtgA, which degrades the peptidoglycan layer. As SIEC lacks EtgA, we investigated whether expression of this transglycosylase enhances the protein translocation capacity of the engineered bacterium. RESULTS: The etgA gene from EPEC was integrated into the SIEC chromosome under the control of the inducible tac promoter, generating the strain SIEC-eEtgA. The controlled expression of EtgA had no effect on the growth or viability of bacteria. Upon induction, injectisome assembly was ~ 30% greater in SIEC-eEtgA than in the parental strain, as determined by the level of T3SS translocon proteins, the hemolytic activity of the bacterial strain, and the impairment in flagellar motility. The functionality of SIEC-eEtgA injectisomes was evaluated in a derivative strain carrying a synthetic operon (eLEE5), which was capable of delivering Tir effector protein into the cytoplasm of HeLa cells triggering F-actin polymerization beneath the attached bacterium. Lastly, using ß-lactamase as a reporter of T3SS-protein injection, we determined that the protein translocation capacity was ~ 65% higher in the SIEC-EtgA strain than in the parental SIEC strain. CONCLUSIONS: We demonstrate that EtgA enhances the assembly of functional injectisomes in a synthetic injector E. coli strain, enabling the translocation of greater amounts of proteins into the cytoplasm of mammalian cells. Accordingly, EtgA expression may boost the protein translocation of SIEC strains programmed as living biotherapeutics.

Recent structural advances towards understanding of the bacterial type III secretion injectisome.

The bacterial injectisome is a structurally conserved, syringe-shaped nanomachine that spans the Gram-negative envelope and forms a continuous channel for type III secretion of protein effectors. The injectisome, and the host-modulating effectors it secretes, are essential for the pathogenesis of several Gram-negative bacterial species, and it is a key virulence factor associated with the progression of many clinical and community-based infectious diseases. The molecular structure of the injectisome has been the focus of intense research efforts over the past 30 years, and during this time significant progress has been made in determining the molecular structures of many components. In this review we present major advances in our structural and mechanistic understanding of the injectisome, as facilitated by cryoelectron microscopy approaches.

Analysis of SPI-1 Dependent Type III Secretion and Injection Using a NanoLuc Luciferase-Based Assay.

Studies of bacterial protein secretion have relied on a variety of reporters that allow the tracking of secreted proteins. However, the lack of truly quantitative and highly sensitive reporters has hindered, in particular, the investigation of the kinetics of protein secretion. In this chapter, we describe a luminescence-based assay using NanoLuc luciferase to analyse secretion and injection into host cells of type III secretion (T3S) substrates encoded on Salmonella pathogenicity island-1 (SPI-1). This method has a very high sensitivity and high signal-to-noise ratio. Moreover, the simplicity of the protocol and the rapid determination and quantification of the luminescence makes it ideal for the monitoring of the kinetics of secretion but also convenient for high-throughput screenings. The protocols presented here include (1) Salmonella SPI-1 secretion assay, where the T3S substrates-NanoLuc fusions are detected by luminometry in the bacterial supernatant, and (2) Salmonella injection assays, using the split-Nanoluc (HiBiT/LgBiT) to monitor the injection of T3S substrates-HiBiT fusions into the host cells stably expressing LgBiT.

A truncated mutation of MucA in Pseudomonas aeruginosa from a bronchiectasis patient affects T3SS expression and inflammasome activation.

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic airway infection in bronchiectasis patients and is closely associated with poor prognosis. Strains isolated from chronically infected patients typically have a mucoid phenotype due to the overproduction of alginate. In this study, we isolate a P. aeruginosa strain from the sputum of a patient with bronchiectasis and find that a truncated mutation occurred in mucA, which is named mucA117. mucA117 causes the strain to transform into a mucoid phenotype, downregulates the expression of T3SS and inflammasome ligands such as fliC and allows it to avoid inflammasome activation. The truncated mutation of the MucA protein may help P. aeruginosa escape clearance by the immune system, enabling long-term colonization.

EPEC-induced activation of the Ca2+ transporter TRPV2 leads to pyroptotic cell death.

The enteropathogenic Escherichia coli (EPEC) type III secretion system effector Tir, which mediates intimate bacterial attachment to epithelial cells, also triggers Ca2+ influx followed by LPS entry and caspase-4-dependent pyroptosis, which could be antagonized by the effector NleF. Here we reveal the mechanism by which EPEC induces Ca2+ influx. We show that in the intestinal epithelial cell line SNU-C5, Tir activates the mechano/osmosensitive cation channel TRPV2 which triggers extracellular Ca2+ influx. Tir-induced Ca2+ influx could be blocked by siRNA silencing of TRPV2, pre-treatment with the TRPV2 inhibitor SET2 or by growing cells in low osmolality medium. Pharmacological activation of TRPV2 in the absence of Tir failed to initiate caspase-4-dependent cell death, confirming the necessity of Tir. Consistent with the model implicating activation on translocation of TRPV2 from the ER to plasma membrane, inhibition of protein trafficking by either brefeldin A or the effector NleA prevented TRPV2 activation and cell death. While infection with EPECΔnleA triggered pyroptotic cell death, this could be prevented by NleF. Taken together this study shows that while integration of Tir into the plasma membrane activates TRPV2, EPEC uses NleA to inhibit TRPV2 trafficking and NleF to inhibit caspase-4 and pyroptosis.

Novel Disk Diffusion Assay on Magnesium Oxalate Agar To Evaluate the Susceptibility of Yersinia pestis to Type III Secretion System Inhibitors.

Current methods for screening small molecules that inhibit the plasmid pCD1-encoded Yersinia pestis type III secretion system (T3SS) include lengthy growth curves followed by multistep luminescence assays or Western blot assays to detect secretion, or lack thereof, of effector proteins. The goal of this research was to develop a novel disk diffusion assay on magnesium oxalate (MOX) agar as a simple way to evaluate the susceptibility of Y. pestis to type III secretion system inhibitors. MOX agar produces distinct Y. pestis growth characteristics based on the bacteria's ability or inability to secrete effector proteins; small, barely visible colonies are observed when secretion is activated versus larger, readily visible colonies when secretion is inhibited. Wild-type Y. pestis was diluted and spread onto a MOX agar plate. Disks containing 20 µl of various concentrations of imidocarb dipropionate, a known Y. pestis T3SS inhibitor, or distilled water (dH2O) were placed on the plate. After incubation at 37°C for 48 h, visible colonies of Y. pestis were observed surrounding the disks with imidocarb dipropionate, suggesting that T3S was inhibited. The diameter of the growth of colonies surrounding the disks increased as the concentration of the T3SS inhibitor increased. Imidocarb dipropionate was also able to inhibit Y. pestis strains lacking effector Yops and Yop chaperones, suggesting that they are not necessary for T3S inhibition. This disk diffusion assay is a feasible and useful method for testing the susceptibility of Y. pestis to type III secretion system inhibitors and has the potential to be used in a clinical setting. IMPORTANCE Disk diffusion assays have traditionally been used as a simple and effective way to screen compounds for antibacterial activity and to determine the susceptibility of pathogens to antibiotics; however, they are limited to detecting growth inhibition only. Consequently, antimicrobial agents that inhibit virulence factors, but not growth, would not be detected. Therefore, we developed a disk diffusion assay that could detect inhibition of bacterial virulence factors, specifically, type III secretion systems (T3SSs), needle-like structures used by several pathogenic bacteria to inject host cells with effector proteins and cause disease. We demonstrate that magnesium oxalate (MOX) agar can be used in a disk diffusion assay to detect inhibition of the T3SS of Yersinia pestis, the causative agent of bubonic plague, by small-molecule inhibitors. This assay may be useful for screening additional small molecules that target bacterial T3SSs or testing the susceptibility of patient-derived samples to drugs that target T3SSs.

Complete genome sequence of Salmonella enterica strain K_SA184, multidrug resistance bacterium isolated from lamb (Ovis aries).

Salmonella enterica is a representative foodborne pathogen in the world. The S. enterica strain K_SA184 was isolated from the lamb (Ovis aries), which was collected from a local traditional market in South Korea. In this study, the S. enterica strain K_SA184 was sequenced using PacBio RS II and Illumina NextSeq 500 platforms. The final complete genome of the S. enterica strain K_SA184 consist of one circular chromosome (4,725,087 bp) with 52.3% of guanine + cytosine (G + C) content, 4,363 of coding sequence (CDS), 85 of tRNA, and 22 of rRNA genes. The S. enterica strain K_SA184 genome includes encoding virulence genes, such as Type III secretion systems and multidrug resistance related genes.

VscF in T3SS1 Helps to Translocate VPA0226 in Vibrio parahaemolyticus.

In Vibrio parahaemolyticus, type III secretion system 1 (T3SS1) is a major virulence factor that delivers effectors into the host eukaryotic cytoplasm; however, studies on its infection mechanism are currently limited. To determine the function of the vscF gene, we constructed the vscF deletion mutant ΔvscF and complementation strain CΔvscF. Compared with those of wild-type POR-1 and CΔvscF, the cytotoxic, adherent, and apoptotic abilities of ΔvscF in HeLa cells were significantly reduced (P < 0.01). Furthermore, in infected HeLa cells, the mutant strain reduced the translocation rates of VP1683 and VP1686 effectors compared to the wild-type and complementation strains. A BLAST search showed that vscF is homologous to the MixH needle protein of Shigella flexneri, indicating that the vscF gene encodes the needle protein of T3SS1 in V. parahaemolyticus. Additional translocation assays showed that VPA0226 translocated into the HeLa eukaryotic cytoplasm via T3SS1, secretion assays showed that VPA0226 can be secreted to supernatant by T3SS1, indicating that VPA0226 belongs to the unpublished class of T3SS1 effectors. In conclusion, our data indicate an essential role of vscF in V. parahaemolyticus T3SS1 and revealed that VPA0226 can be secreted into the host cell cytoplasm via T3SS1. This study provides insights into a previously unexplored aspect of T3SS1, which is expected to contribute to the understanding of its infection mechanism.

Stress response, amino acid biosynthesis and pathogenesis genes expressed in Salmonella enterica colonizing tomato shoot and root surfaces.

Salmonella enterica can colonize all parts of the tomato plant. Tomatoes have been frequently implicated in salmonellosis outbreaks. In agricultural settings, Salmonella must overcome stress, nutritional and competition barriers to become established on plant surfaces. Knowledge of the genetic mechanisms underlying Salmonella-plant associations is limited, especially when growing epiphytically. A genome-wide transcriptomic analysis of Salmonella Typhimurium (SeT) was conducted with RNA-Seq to elucidate strategies for epiphytic growth on live, intact tomato shoot and root surfaces. Six plasmid-encoded and 123 chromosomal genes were significantly (using Benjamini-Hochberg adjusted p-values) up-regulated; 54 and 110 detected in SeT on shoots and roots, respectively, with 35 common to both. Key signals included NsrR regulon genes needed to mitigate nitrosative stress, oxidative stress genes and host adaptation genes, including environmental stress, heat shock and acid-inducible genes. Several amino acid biosynthesis genes and genes indicative of sulphur metabolism and anaerobic respiration were up-regulated. Some Type III secretion system (T3SS) effector protein genes and their chaperones from pathogenicity island-2 were expressed mostly in SeT on roots. Gene expression in SeT was validated against SeT and also the tomato outbreak strain Salmonella Newport with a high correlation (R 2 = 0.813 and 0.874, respectively; both p < 0.001). Oxidative and nitrosative stress response genes, T3SS2 genes and amino acid biosynthesis may be needed for Salmonella to successfully colonize tomato shoot and root surfaces.

Design, synthesis, and antimicrobial behavior of novel oxadiazoles containing various N-containing heterocyclic pendants.

BACKGROUND: The gradually elevated outbreak of plant bacterial diseases severely limits agricultural products and small amounts of pesticides can manage them. Our group has previously synthesized and screened the antimicrobial activity of diverse 1,3,4-oxadiazole thioether/sulfone compounds bridged by a sulfur atom at the 2-position of 1,3,4-oxadiazole. However, few studies have evaluated the effect of eliminating the sulfur atom on bioactivity. Herein, a novel type of N-containing heterocyclic pendants-tagged 1,3,4-oxadiazoles bridged by alkyl chains only was systematically synthesized and evaluated for their antimicrobial activities. RESULTS: Bioassay results revealed that antibacterial efficacy increased by 551- and 314-fold against the corresponding phytopathogens Xanthomonas oryzae pv. oryzae and X. axonopodis pv. citri compared to commercial agents bismerthiazol and thiodiazole copper. In vivo trials showed that C 1 exerted remarkable curative activity against rice bacterial blight with a control effectiveness of 52.9% at 200 µg mL-1 . Antibacterial mechanism research found that C 1 could reduce the hypersensitive response behavior and pathogenicity of Xoo through targeting the type III secretion system (T3SS) at a lower drug dose. This outcome was verified by observing the significantly down-regulated proteins and representative genes from the related quantitative proteomics and qRT-PCR assays. CONCLUSION: This study can inspire the design of innovative molecular frameworks targeting the T3SS of phytopathogens for controlling bacterial infections. © 2020 Society of Chemical Industry.

Horizontally Acquired Quorum-Sensing Regulators Recruited by the PhoP Regulatory Network Expand the Host Adaptation Repertoire in the Phytopathogen Pectobacterium brasiliense.

In this study, we examine the impact of transcriptional network rearrangements driven by horizontal gene acquisition in PhoP and SlyA regulons using as a case study a phytopathosystem comprised of potato tubers and the soft-rot pathogen Pectobacterium brasiliense 1692 (Pb1692). Genome simulations and statistical analyses uncovered the tendency of PhoP and SlyA networks to mobilize lineage-specific traits predicted as horizontal gene transfer at late infection, highlighting the prominence of regulatory network rearrangements in this stage of infection. The evidence further supports the circumscription of two horizontally acquired quorum-sensing regulators (carR and expR1) by the PhoP network. By recruiting carR and expR1, the PhoP network also impacts certain host adaptation- and bacterial competition-related systems, seemingly in a quorum sensing-dependent manner, such as the type VI secretion system, carbapenem biosynthesis, and plant cell wall-degrading enzymes (PCWDE) like cellulases and pectate lyases. Conversely, polygalacturonases and the type III secretion system (T3SS) exhibit a transcriptional pattern that suggests quorum-sensing-independent regulation by the PhoP network. This includes an uncharacterized novel phage-related gene family within the T3SS gene cluster that has been recently acquired by two Pectobacterium species. The evidence further suggests a PhoP-dependent regulation of carbapenem- and PCWDE-encoding genes based on the synthesized products' optimum pH. The PhoP network also controls slyA expression in planta, which seems to impact carbohydrate metabolism regulation, especially at early infection, when 76.2% of the SlyA-regulated genes from that category also require PhoP to achieve normal expression levels.IMPORTANCE Exchanging genetic material through horizontal transfer is a critical mechanism that drives bacteria to efficiently adapt to host defenses. In this report, we demonstrate that a specific plant-pathogenic species (from the Pectobacterium genus) successfully integrated a population density-based behavior system (quorum sensing) acquired through horizontal transfer into a resident stress-response gene regulatory network controlled by the PhoP protein. Evidence found here underscores that subsets of bacterial weaponry critical for colonization, typically known to respond to quorum sensing, are also controlled by PhoP. Some of these traits include different types of enzymes that can efficiently break down plant cell walls depending on the environmental acidity level. Thus, we hypothesize that PhoP's ability to elicit regulatory responses based on acidity and nutrient availability fluctuations has strongly impacted the fixation of its regulatory connection with quorum sensing. In addition, another global gene regulator, known as SlyA, was found under the PhoP regulatory network. The SlyA regulator controls a series of carbohydrate metabolism-related traits, which also seem to be regulated by PhoP. By centralizing quorum sensing and slyA under PhoP scrutiny, Pectobacterium cells added an advantageous layer of control over those two networks that potentially enhances colonization efficiency.