Multikinase Networks: Two-Component Signaling Networks Integrating Multiple Stimuli.

Bacteria depend on two-component systems to detect and respond to threats. Simple pathways comprise a single sensor kinase (SK) that detects a signal and activates a response regulator protein to mediate an appropriate output. These simple pathways with only a single SK are not well suited to making complex decisions where multiple different stimuli need to be evaluated. A recently emerging theme is the existence of multikinase networks (MKNs) where multiple SKs collaborate to detect and integrate numerous different signals to regulate a major lifestyle switch, e.g., between virulence, sporulation, biofilm formation, and cell division. In this review, the role of MKNs and the phosphosignaling mechanisms underpinning their signal integration and decision making are explored.

Transcriptional organization and regulation of the Pseudomonas putida K1 type VI secretion system gene cluster.

The type VI secretion system (T6SS) is an antimicrobial molecular weapon that is widespread in Proteobacteria and offers competitive advantages to T6SS-positive micro-organisms. Three T6SSs have recently been described in Pseudomonas putida KT2440 and it has been shown that one, K1-T6SS, is used to outcompete a wide range of phytopathogens, protecting plants from pathogen infections. Given the relevance of this system as a powerful and innovative mechanism of biological control, it is critical to understand the processes that govern its expression. Here, we experimentally defined two transcriptional units in the K1-T6SS cluster. One encodes the structural components of the system and is transcribed from two adjacent promoters. The other encodes two hypothetical proteins, the tip of the system and the associated adapters, and effectors and cognate immunity proteins, and it is also transcribed from two adjacent promoters. The four identified promoters contain the typical features of σ70-dependent promoters. We have studied the expression of the system under different conditions and in a number of mutants lacking global regulators. P. putida K1-T6SS expression is induced in the stationary phase, but its transcription does not depend on the stationary σ factor RpoS. In fact, the expression of the system is indirectly repressed by RpoS. Furthermore, it is also repressed by RpoN and the transcriptional regulator FleQ, an enhancer-binding protein typically acting in conjunction with RpoN. Importantly, expression of the K1-T6SS gene cluster is positively regulated by the GacS-GacA two-component regulatory system (TCS) and repressed by the RetS sensor kinase, which inhibits this TCS. Our findings identified a complex regulatory network that governs T6SS expression in general and P. putida K1-T6SS in particular, with implications for controlling and manipulating a bacterial agent that is highly relevant in biological control.

RetS Regulates Phage Infection in Pseudomonas aeruginosa via Modulating the GacS/GacA Two-Component System.

In Pseudomonas aeruginosa, the complex multisensing regulatory networks RetS-GacS/GacA have been demonstrated to play key roles in controlling the switch between planktonic and sessile lifestyles. However, whether this multisensing system is involved in the regulation of phage infection has not been investigated. Here, we provide a link between the sensors RetS/GacS and infection of phages vB_Pae_QDWS and vB_Pae_W3. Our data suggest that the sensors kinases RetS and GacS in Pseudomonas aeruginosa play opposite regulatory functions on phage infection. Mutation in retS increased phage resistance. Cellular levels of RsmY and RsmZ increased in PaΔretS and were positively correlated with phage resistance. Further analysis demonstrated that RetS regulated phage infection by affecting the type IV pilus (T4P)-mediated adsorption. The regulation of RetS on phage infection depends on the GacS/GacA two-component system and is likely a dynamic process in response to environmental signals. The findings offer additional support for the rapid emergence of phage resistance. IMPORTANCE Our knowledge on the molecular mechanisms behind bacterium-phage interactions remains limited. Our study reported that the complex multisensing regulatory networks RetS-GacS/GacA of Pseudomonas aeruginosa PAO1 play key roles in controlling phage infection. The main observation was that the mutation in RetS could result in increased phage resistance by reducing the type IV pilus-mediated phage adsorption. The bacterial defense strategy is generally applicable to various phages since many P. aeruginosa phages can use type IV pilus as their receptors. The results also suggest that the phage infection is likely to be regulated dynamically, which depends on the environmental stimuli. Reduction of the signals that RetS favors would increase phage resistance. Our study is particularly remarkable for uncovering a signal transduction system that was involved in phage infection, which may help in filling some knowledge gaps in this field.

Direct Inhibition of RetS Synthesis by RsmA Contributes to Homeostasis of the Pseudomonas aeruginosa Gac/Rsm Signaling System.

The Gac/Rsm system is a global regulator of Pseudomonas aeruginosa gene expression. The primary effectors are RsmA and RsmF. Both are RNA-binding proteins that interact with target mRNAs to modulate protein synthesis. RsmA/RsmF recognize GGA sequences presented in the loop portion of stem-loop structures. For repressed targets, the GGA sites usually overlap the ribosome binding site (RBS) and RsmA/RsmF binding inhibits translation initiation. RsmA/RsmF activity is controlled by several small non-coding RNAs (sRNA) that sequester RsmA/RsmF from target mRNAs. The most important sequestering sRNAs are RsmY and RsmZ. Transcription of rsmY/rsmZ is directly controlled by the GacSA two-component regulatory system. GacSA activity is antagonized by RetS, a hybrid sensor kinase. In the absence of retS, rsmY/rsmZ transcription is derepressed and RsmA/RsmF are sequestered by RsmY/RsmZ. Gac/Rsm system homeostasis is tightly controlled by at least two mechanisms. First, direct binding of RsmA to the rsmA and rsmF mRNAs inhibits further synthesis of both proteins. Second, RsmA stimulates rsmY/rsmZ transcription through an undefined mechanism. In this study we demonstrate that RsmA stimulates rsmY/rsmZ transcription by directly inhibiting RetS synthesis. RetS protein levels are elevated 2.5-fold in an rsmA mutant. Epistasis experiments demonstrate that the rsmA requirement for rsmY/rsmZ transcription is entirely suppressed in an rsmA, retS double mutant. RsmA directly interacts with the retS mRNA and requires two distinct GGA sites, one of which overlaps the RBS. We propose a model wherein RsmA inhibits RetS synthesis to promote rsmY/rsmZ transcription and that this acts as a checkpoint to limit RsmA/RsmF availability. IMPORTANCE The Pseudomonas aeruginosa Gac/Rsm system controls ∼500 genes and governs a critical lifestyle switch by inversely regulating factors that favor acute or chronic colonization. Control of gene expression by the Gac/Rsm system is mediated through RsmA and RsmF, small RNA-binding proteins that interact with target mRNAs to inhibit or promote protein synthesis and/or mRNA stability. RsmA/RsmF activity is governed by two small non-coding RNAs (RsmY and RsmZ) that sequester RsmA/RsmF from target mRNAs. The GacSA two-component regulatory system plays a pivotal role in the Gac/Rsm system by controlling rsmYZ transcription. This study provides insight into the control of homeostasis by demonstrating that RsmA directly targets and inhibits expression of RetS, an orphan sensor kinase critical for rsmYZ transcription.

RetS inhibits Pseudomonas aeruginosa biofilm formation by disrupting the canonical histidine kinase dimerization interface of GacS.

Bacterial signaling histidine kinases (HKs) have long been postulated to function exclusively through linear signal transduction chains. However, several HKs have recently been shown to form complex multikinase networks (MKNs). The most prominent MKN, involving the enzymes RetS and GacS, controls the switch between the motile and biofilm lifestyles in the pathogenic bacterium Pseudomonas aeruginosa. While GacS promotes biofilm formation, RetS counteracts GacS using three distinct mechanisms. Two are dephosphorylating mechanisms. The third, a direct binding between the RetS and GacS HK regions, blocks GacS autophosphorylation. Focusing on the third mechanism, we determined the crystal structure of a cocomplex between the HK region of RetS and the dimerization and histidine phosphotransfer (DHp) domain of GacS. This is the first reported structure of a complex between two distinct bacterial signaling HKs. In the complex, the canonical HK homodimerization interface is replaced by a strikingly similar heterodimeric interface between RetS and GacS. We further demonstrate that GacS autophosphorylates in trans, thus explaining why the formation of a RetS-GacS complex inhibits GacS autophosphorylation. Using mutational analysis in conjunction with bacterial two-hybrid and biofilm assays, we not only corroborate the biological role of the observed RetS-GacS interactions, but also identify a residue critical for the equilibrium between the RetS-GacS complex and the respective RetS and GacS homodimers. Collectively, our findings suggest that RetS and GacS form a domain-swapped hetero-oligomer during the planktonic growth phase of P. aeruginosa before unknown signals cause its dissociation and a relief of GacS inhibition to promote biofilm formation.

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.

Engineering glucose metabolism for enhanced muconic acid production in Pseudomonas putida KT2440.

Pseudomonas putida KT2440 has received increasing attention as an important biocatalyst for the conversion of diverse carbon sources to multiple products, including the olefinic diacid, cis,cis-muconic acid (muconate). P. putida has been previously engineered to produce muconate from glucose; however, periplasmic oxidation of glucose causes substantial 2-ketogluconate accumulation, reducing product yield and selectivity. Deletion of the glucose dehydrogenase gene (gcd) prevents 2-ketogluconate accumulation, but dramatically slows growth and muconate production. In this work, we employed adaptive laboratory evolution to improve muconate production in strains incapable of producing 2-ketogluconate. Growth-based selection improved growth, but reduced muconate titer. A new muconate-responsive biosensor was therefore developed to enable muconate-based screening using fluorescence activated cell sorting. Sorted clones demonstrated both improved growth and muconate production. Mutations identified by whole genome resequencing of these isolates indicated that glucose metabolism may be dysregulated in strains lacking gcd. Using this information, we used targeted engineering to recapitulate improvements achieved by evolution. Deletion of the transcriptional repressor gene hexR improved strain growth and increased the muconate production rate, and the impact of this deletion was investigated using transcriptomics. The genes gntZ and gacS were also disrupted in several evolved clones, and deletion of these genes further improved strain growth and muconate production. Together, these targets provide a suite of modifications that improve glucose conversion to muconate by P. putida in the context of gcd deletion. Prior to this work, our engineered strain lacking gcd generated 7.0 g/L muconate at a productivity of 0.07 g/L/h and a 38% yield (mol/mol) in a fed-batch bioreactor. Here, the resulting strain with the deletion of hexR, gntZ, and gacS achieved 22.0 g/L at 0.21 g/L/h and a 35.6% yield (mol/mol) from glucose in similar conditions. These strategies enabled enhanced muconic acid production and may also improve production of other target molecules from glucose in P. putida.

Study of the sRNA RsmY involved in the genetic regulation of the synthesis of alginate and alkyl resorcinols in Azotobacter vinelandii.

Azotobacter vineladii is a Gram-negative bacterium that produces alginate and poly-hydroxybutyrate (PHB), two polymers of biotechnological interest. This bacterium has the ability to form desiccation-resistant cysts. In the cyst the membrane phospholipids are replaced with a family of phenolic lipids called alkylresorcinols (ARs). The alginate, PHB, and ARs are controlled by the GacS/A two-component system and the small regulatory RNA (sRNA) RsmZ1, belonging to the Rsm (Csr) regulatory system. The Rsm (Csr) systems usually possess two or more sRNAs, in this regard A. vinelandii is the bacterium with the highest number of rsm-sRNAs. Originally, the presence of two sRNAs of the RsmY family (RsmY1 and RsmY2) was reported, but in a subsequent work it was suggested that they conformed to a single sRNA. In this work we provide genetic evidence confirming that rsmY1 and rsmY2 constitute a single gene. Also, it was established that rsmY mutation decreased alginate and ARs production, but did not affect the PHB synthesis. Transcriptional studies showed that rsmY has its higher expression during the stationary growth phase, and in the absence of RsmZ1, rsmY increases its transcription. Interestingly, rsmY expression was influenced by the carbon source, but its expression did not correlate with alginate production.

Hydrogen cyanide produced by Pseudomonas chlororaphis O6 is a key aphicidal metabolite.

A biocontrol bacterium, Pseudomonas chlororaphis O6 promotes plant health through multifaceted mechanisms. In this study, we used P. chlororaphis O6 mutants to examine metabolites with aphicidal activity. Direct application of intact P. chlororaphis cells to the surface of second-instar nymphs of the green peach aphid resulted in no mortality. However, nymphs died when exposed only to the volatiles produced by the P. chlororaphis O6 wild-type strain grown on rich media. Mutants lacking the production of two antibiotics, phenazine and pyrrolnitrin, or the insect toxin FitD retained the aphicidal potential of the wild-type strain. However, the volatiles produced by mutants deficient in the production of hydrogen cyanide (HCN) or defective in the synthesis of the global regulator GacS, which regulates HCN synthesis, showed no aphicidal activity. Direct application of potassium cyanide caused mortality of green peach aphid nymphs. These results indicate that HCN production by a plant probiotic is involved in preventing insect growth.

Fluorescent pseudomonads pursue media-dependent strategies to inhibit growth of pathogenic Verticillium fungi.

Verticillium species represent economically important phytopathogenic fungi with bacteria as natural rhizosphere antagonists. Growth inhibition patterns of Verticillium in different media were compared to saprophytic Aspergillus strains and were significantly more pronounced in various co-cultivations with different Pseudomonas strains. The Brassica napus rhizosphere bacterium Pseudomonas fluorescens DSM8569 is able to inhibit growth of rapeseed (Verticillium longisporum) or tomato (Verticillium dahliae) pathogens without the potential for phenazine or 2,4-diacetylphloroglucinol (DAPG) mycotoxin biosynthesis. Bacterial inhibition of Verticillium growth remained even after the removal of pseudomonads from co-cultures. Fungal growth response in the presence of the bacterium is independent of the fungal control genes of secondary metabolism LAE1 and CSN5. The phenazine producer P. fluorescens 2-79 (P_phen) inhibits Verticillium growth especially on high glucose solid agar surfaces. Additional phenazine-independent mechanisms in the same strain are able to reduce fungal surface growth in the presence of pectin and amino acids. The DAPG-producing Pseudomonas protegens CHA0 (P_DAPG), which can also produce hydrogen cyanide or pyoluteorin, has an additional inhibitory potential on fungal growth, which is independent of these antifungal compounds, but which requires the bacterial GacA/GacS control system. This translational two-component system is present in many Gram-negative bacteria and coordinates the production of multiple secondary metabolites. Our data suggest that pseudomonads pursue different media-dependent strategies that inhibit fungal growth. Metabolites such as phenazines are able to completely inhibit fungal surface growth in the presence of glucose, whereas GacA/GacS controlled inhibitors provide the same fungal growth effect on pectin/amino acid agar.

A bacterial symbiont is converted from an inedible producer of beneficial molecules into food by a single mutation in the gacA gene.

Stable multipartite mutualistic associations require that all partners benefit. We show that a single mutational step is sufficient to turn a symbiotic bacterium from an inedible but host-beneficial secondary metabolite producer into a host food source. The bacteria's host is a "farmer" clone of the social amoeba Dictyostelium discoideum that carries and disperses bacteria during its spore stage. Associated with the farmer are two strains of Pseudomonas fluorescens, only one of which serves as a food source. The other strain produces diffusible small molecules: pyrrolnitrin, a known antifungal agent, and a chromene that potently enhances the farmer's spore production and depresses a nonfarmer's spore production. Genome sequence and phylogenetic analyses identify a derived point mutation in the food strain that generates a premature stop codon in a global activator (gacA), encoding the response regulator of a two-component regulatory system. Generation of a knockout mutant of this regulatory gene in the nonfood bacterial strain altered its secondary metabolite profile to match that of the food strain, and also, independently, converted it into a food source. These results suggest that a single mutation in an inedible ancestral strain that served a protective role converted it to a "domesticated" food source.

HemN2 Regulates the Virulence of Pseudomonas donghuensis HYS through 7-Hydroxytropolone Synthesis and Oxidative Stress.

Compared to pathogens Pseudomonas aeruginosa and P. putida, P. donghuensis HYS has stronger virulence towards Caenorhabditis elegans. However, the underlying mechanisms haven't been fully understood. The heme synthesis system is essential for Pseudomonas virulence, and former studies of HemN have focused on the synthesis of heme, while the relationship between HemN and Pseudomonas virulence were barely pursued. In this study, we hypothesized that hemN2 deficiency affected 7-hydroxytropolone (7-HT) biosynthesis and redox levels, thereby reducing bacterial virulence. There are four hemN genes in P. donghuensis HYS, and we reported for the first time that deletion of hemN2 significantly reduced the virulence of HYS towards C. elegans, whereas the reduction in virulence by the other three genes was not significant. Interestingly, hemN2 deletion significantly reduced colonization of P. donghuensis HYS in the gut of C. elegans. Further studies showed that HemN2 was regulated by GacS and participated in the virulence of P. donghuensis HYS towards C. elegans by mediating the synthesis of the virulence factor 7-HT. In addition, HemN2 and GacS regulated the virulence of P. donghuensis HYS by affecting antioxidant capacity and nitrative stress. In short, the findings that HemN2 was regulated by the Gac system and that it was involved in bacterial virulence via regulating 7-HT synthesis and redox levels were reported for the first time. These insights may enlighten further understanding of HemN-based virulence in the genus Pseudomonas.

GacA reduces virulence and increases competitiveness in planta in the tumorigenic olive pathogen Pseudomonas savastanoi pv. savastanoi.

GacS/GacA is a widely distributed two-component system playing an essential role as a key global regulator, although its characterization in phytopathogenic bacteria has been deeply biased, being intensively studied in pathogens of herbaceous plants but barely investigated in pathogens of woody hosts. P. savastanoi pv. savastanoi (Psv) is characterized by inducing tumours in the stem and branches of olive trees. In this work, the model strain Psv NCPPB 3335 and a mutant derivative with a complete deletion of gene gacA were subjected to RNA-Seq analyses in a minimum medium and a medium mimicking in planta conditions, accompanied by RT-qPCR analyses of selected genes and phenotypic assays. These experiments indicated that GacA participates in the regulation of at least 2152 genes in strain NCPPB 3335, representing 37.9 % of the annotated CDSs. GacA also controls the expression of diverse rsm genes, and modulates diverse phenotypes, including motility and resistance to oxidative stresses. As occurs with other P. syringae pathovars of herbaceous plants, GacA regulates the expression of the type III secretion system and cognate effectors. In addition, GacA also regulates the expression of WHOP genes, specifically encoded in P. syringe strains isolated from woody hosts, and genes for the biosynthesis of phytohormones. A gacA mutant of NCPPB 3335 showed increased virulence, producing large immature tumours with high bacterial populations, but showed a significantly reduced competitiveness in planta. Our results further extend the role of the global regulator GacA in the virulence and fitness of a P. syringae pathogen of woody hosts.

Characterisation of pellicle-forming ability in clinical carbapenem-resistant Acinetobacter baumannii.

Background: Acinetobacter baumannii was reported to have resistance towards carbapenems and the ability to form an air-liquid biofilm (pellicle) which contributes to their virulence. The GacSA two-component system has been previously shown to play a role in pellicle formation. Therefore, this study aims to detect the presence of gacA and gacS genes in carbapenem-resistant Acinetobacter baumannii (CRAB) isolates recovered from patients in intensive care units and to investigate their pellicle forming ability. Methods: The gacS and gacA genes were screened in 96 clinical CRAB isolates using PCR assay. Pellicle formation assay was performed in Mueller Hinton medium and Luria Bertani medium using borosilicate glass tubes and polypropylene plastic tubes. The biomass of the pellicle was quantitated using the crystal violet staining assay. The selected isolates were further assessed for their motility using semi-solid agar and monitored in real-time using real-time cell analyser (RTCA). Results: All 96 clinical CRAB isolates carried the gacS and gacA genes, however, only four isolates (AB21, AB34, AB69 and AB97) displayed the ability of pellicle-formation phenotypically. These four pellicle-forming isolates produced robust pellicles in Mueller Hinton medium with better performance in borosilicate glass tubes in which biomass with OD570 ranging from 1.984 ± 0.383 to 2.272 ± 0.376 was recorded. The decrease in cell index starting from 13 hours obtained from the impedance-based RTCA showed that pellicle-forming isolates had entered the growth stage of pellicle development. Conclusion: These four pellicle-forming clinical CRAB isolates could be potentially more virulent, therefore further investigation is warranted to provide insights into their pathogenic mechanisms.

Acinetobacter baumannii coordinates central metabolism, plasmid dissemination, and virulence by sensing nutrient availability.

Plasmid conjugation plays an important role in the dissemination of antibiotic-resistance genes. The emergence of multidrug-resistant isolates of Acinetobacter baumannii poses grave challenges in treating infections caused by this notorious nosocomial pathogen. Yet, the composition, functionality, and regulation of conjugative machinery utilized by A. baumannii remain poorly understood. Here, we found that conjugation of the major plasmid pAB3 of A. baumannii is mediated by a type IVB secretion system similar to the Dot/Icm transporter of Legionella pneumophila. Furthermore, the expression of the structural genes of the Dot/Icm-like system is co-regulated with genes involved in central metabolism by the GacS/GacA two-component system in response to various metabolites, including intermediates of the tricarboxylic acid cycle. Loss of GacS/A also severely impaired bacterial virulence. These results establish that A. baumannii coordinates metabolism with plasmid conjugation and virulence by sensing nutrient availability, which may be exploited to develop inhibitory agents for controlling the spread of drug-resistance genes and virulence factors. IMPORTANCE Plasmid conjugation is known to be an energy-expensive process, but our understanding of the molecular linkage between conjugation and metabolism is limited. Our finding reveals that Acinetobacter baumannii utilizes a two-component system to co-regulate metabolism, plasmid transfer, and virulence by sensing reaction intermediates of key metabolic pathways, which suggests that nutrient availability dictates not only bacterial proliferation but also horizontal gene transfer. The identification of Dot/Icm-like proteins as components of a conjugation system involved in the dissemination of antibiotic-resistance genes by A. baumannii has provided important targets for the development of agents capable of inhibiting virulence and the spread of anti-microbial-resistance genes in bacterial communities.

HrgS (Avin_34990), a novel histidine-kinase related to GacS, regulates alginate synthesis in Azotobacter vinelandii.

Azotobacter vinelandii is a soil bacterium that produces alginates, a family of polymers of biotechnological interest. In A. vinelandii, alginate production is controlled by the two-component system GacS/GacA. GacS/GacA, in turn, regulates the Rsm post-transcriptional regulatory system establishing a cascade that regulates alginate biosynthesis by controlling the expression of the algD biosynthetic gene. In Pseudomonas aeruginosa, GacS/GacA is influenced by other histidine-kinases constituting a multicomponent signal transduction system. In this study, we explore the presence of GacS-related histidine-kinases in A. vinelandii and discover a novel histidine-kinase (Avin_34990, renamed HrgS). This histidin-kinase acts as a negative regulator of alginate synthesis by controlling the transcription of the sRNAs belonging to the Rsm post-transcriptional regulatory system, for which a functional GacS is required.

Genetic Regulation of Alginate Production in Azotobacter vinelandii a Bacterium of Biotechnological Interest: A Mini-Review.

Alginates are a family of polymers composed of guluronate and mannuronate monomers joined by ß (1-4) links. The different types of alginates have variations in their monomer content and molecular weight, which determine the rheological properties and their applications. In industry, alginates are commonly used as additives capable of viscosifying, stabilizing, emulsifying, and gelling aqueous solutions. Recently, additional specialized biomedical uses have been reported for this polymer. Currently, the production of alginates is based on the harvesting of seaweeds; however, the composition and structure of the extracts are highly variable. The production of alginates for specialized applications requires a precise composition of monomers and molecular weight, which could be achieved using bacterial production systems such as those based on Azotobacter vinelandii, a free-living, non-pathogenic bacterium. In this mini-review, we analyze the latest advances in the regulation of alginate synthesis in this model.

Corrigendum: Genetic Regulation of Alginate Production in Azotobacter vinelandii a Bacterium of Biotechnological Interest: A Mini-Review.

[This corrects the article DOI: 10.3389/fmicb.2022.845473.].

The Type VI Secretion Systems in Plant-Beneficial Bacteria Modulate Prokaryotic and Eukaryotic Interactions in the Rhizosphere.

Rhizosphere colonizing plant growth promoting bacteria (PGPB) increase their competitiveness by producing diffusible toxic secondary metabolites, which inhibit competitors and deter predators. Many PGPB also have one or more Type VI Secretion System (T6SS), for the delivery of weapons directly into prokaryotic and eukaryotic cells. Studied predominantly in human and plant pathogens as a virulence mechanism for the delivery of effector proteins, the function of T6SS for PGPB in the rhizosphere niche is poorly understood. We utilized a collection of Pseudomonas chlororaphis 30-84 mutants deficient in one or both of its two T6SS and/or secondary metabolite production to examine the relative importance of each T6SS in rhizosphere competence, bacterial competition, and protection from bacterivores. A mutant deficient in both T6SS was less persistent than wild type in the rhizosphere. Both T6SS contributed to competitiveness against other PGPB or plant pathogenic strains not affected by secondary metabolite production, but only T6SS-2 was effective against strains lacking their own T6SS. Having at least one T6SS was also essential for protection from predation by several eukaryotic bacterivores. In contrast to diffusible weapons that may not be produced at low cell density, T6SS afford rhizobacteria an additional, more immediate line of defense against competitors and predators.