Flagella-driven Motility Research Articles

AcsS Negatively Regulates the Transcription of type VI Secretion System 2 Genes in Vibrio parahaemolyticus.

The type VI secretion system 2 (T6SS2) gene cluster of Vibrio parahaemolyticus comprises three operons: VPA1027-1024, VPA1043-1028, and VPA1044-1046. AcsS is a LysR-like transcriptional regulator that play a role in activating flagella-driven motility in V. parahaemolyticus. However, its potential roles in other cellular pathways remain poorly understood. In this study, we conducted a series of experiments to investigate the regulatory effects of AcsS on the transcription of VPA1027 (hcp2), VPA1043, and VPA1044. The findings revealed that AcsS indirectly inhibits the transcription of these genes. Additionally, deletion of acsS resulted in enhanced adhesion of V. parahaemolyticus to HeLa cells. However, disruption of T6SS2 alone or in conjunction with AcsS significantly diminished the adhesion capacity of V. parahaemolyticus to HeLa cells. Therefore, it is suggested that AcsS suppresses cell adhesion in V. parahaemolyticus by downregulating the transcription of T6SS2 genes.

Involvement of ArlI, ArlJ, and CirA in archaeal type IV pilin-mediated motility regulation.

Many prokaryotes use swimming motility to move toward favorable conditions and escape adverse surroundings. Regulatory mechanisms governing bacterial flagella-driven motility are well-established; however, little is yet known about the regulation underlying swimming motility propelled by the archaeal cell surface structure, the archaella. Previous research showed that the deletion of the adhesion pilins (PilA1-6), subunits of the type IV pili cell surface structure, renders the model archaeon Haloferax volcanii non-motile. In this study, we used ethyl methanesulfonate mutagenesis and a motility assay to identify motile suppressors of the ∆pilA[1-6] strain. Of the eight suppressors identified, six contain missense mutations in archaella biosynthesis genes, arlI and arlJ. In trans expression of arlI and arlJ mutant constructs in the respective multi-deletion strains ∆pilA[1-6]∆arlI and ∆pilA[1-6]∆arlJ confirmed their role in suppressing the ∆pilA[1-6] motility defect. Additionally, three suppressors harbor co-occurring disruptive missense and nonsense mutations in cirA, a gene encoding a proposed regulatory protein. A deletion of cirA resulted in hypermotility, while cirA expression in trans in wild-type cells led to decreased motility. Moreover, quantitative real-time PCR analysis revealed that in wild-type cells, higher expression levels of arlI, arlJ, and the archaellin gene arlA1 were observed in motile early-log phase rod-shaped cells compared to non-motile mid-log phase disk-shaped cells. Conversely, ∆cirA cells, which form rods during both early- and mid-log phases, exhibited similar expression levels of arl genes in both growth phases. Our findings contribute to a deeper understanding of the mechanisms governing archaeal motility, highlighting the involvement of ArlI, ArlJ, and CirA in pilin-mediated motility regulation.IMPORTANCEArchaea are close relatives of eukaryotes and play crucial ecological roles. Certain behaviors, such as swimming motility, are thought to be important for archaeal environmental adaptation. Archaella, the archaeal motility appendages, are evolutionarily distinct from bacterial flagella, and the regulatory mechanisms driving archaeal motility are largely unknown. Previous research has linked the loss of type IV pili subunits to archaeal motility suppression. This study reveals three Haloferax volcanii proteins involved in pilin-mediated motility regulation, offering a deeper understanding of motility regulation in this understudied domain while also paving the way for uncovering novel mechanisms that govern archaeal motility. Understanding archaeal cellular processes will help elucidate the ecological roles of archaea as well as the evolution of these processes across domains.

FlgV forms a flagellar motor ring that is required for optimal motility of Helicobacter pylori.

Flagella-driven motility is essential for Helicobacter pylori to colonize the human stomach, where it causes a variety of diseases, including chronic gastritis, peptic ulcer disease, and gastric cancer. H. pylori has evolved a high-torque-generating flagellar motor that possesses several accessories not found in the archetypical Escherichia coli motor. FlgV was one of the first flagellar accessory proteins identified in Campylobacter jejuni, but its structure and function remain poorly understood. Here, we confirm that deletion of flgV in H. pylori B128 and a highly motile variant of H. pylori G27 (G27M) results in reduced motility in soft agar medium. Comparative analyses of in-situ flagellar motor structures of wild-type, ΔflgV, and a strain expressing FlgV-YFP showed that FlgV forms a ring-like structure closely associated with the junction of two highly conserved flagellar components: the MS and C rings. The results of our studies suggest that the FlgV ring has adapted specifically in Campylobacterota to support the assembly and efficient function of the high-torque-generating motors.

Flagella-Driven Motility Is Critical to the Virulence of Xanthomonas fragariae in Strawberry.

Xanthomonas fragariae (X. fragariae) is the causal agent of angular leaf spots (ALS) in strawberry plants. Recently, a study in China isolated X. fragariae strain YL19, which was observed to cause both typical ALS symptoms and dry cavity rot in strawberry crown tissue; this was the first X. fragariae strain to have both these effects in strawberry. In this study, from 2020 to 2022, we isolated 39 X. fragariae strains from diseased strawberries in different production areas in China. Multilocus sequence typing (MLST) and phylogenetic analysis showed that X. fragariae strain YLX21 was genetically different from YL19 and other strains. Tests indicated that YLX21 and YL19 had different pathogenicities toward strawberry leaves and stem crowns. YLX21 did not cause ALS symptoms, rarely caused dry cavity rot in strawberry crown after wound inoculation, and never caused dry cavity rot after spray inoculation, but it did cause severe ALS symptoms after spray inoculation. However, YL19 caused more severe symptoms in strawberry crowns under both conditions. Moreover, YL19 had a single polar flagellum, while YLX21 had no flagellum. Motility and chemotaxis assays showed that YLX21 had weaker motility than YL19, which may explain why YLX21 tended to multiply in situ within the strawberry leaf rather than migrate to other tissues, causing more severe ALS symptoms and mild crown rot symptoms. Taken together, the new strain YLX21 helped us reveal critical factors underlying the pathogenicity of X. fragariae and the mechanism by which dry cavity rot in strawberry crowns forms.

Flagella-driven motility is a target of human Paneth cell defensin activity.

In the mammalian intestine, flagellar motility can provide microbes competitive advantage, but also threatens the spatial segregation established by the host at the epithelial surface. Unlike microbicidal defensins, previous studies indicated that the protective activities of human α-defensin 6 (HD6), a peptide secreted by Paneth cells of the small intestine, resides in its remarkable ability to bind microbial surface proteins and self-assemble into protective fibers and nets. Given its ability to bind flagellin, we proposed that HD6 might be an effective inhibitor of bacterial motility. Here, we utilized advanced automated live cell fluorescence imaging to assess the effects of HD6 on actively swimming Salmonella enterica in real time. We found that HD6 was able to effectively restrict flagellar motility of individual bacteria. Flagellin-specific antibody, a classic inhibitor of flagellar motility that utilizes a mechanism of agglutination, lost its activity at low bacterial densities, whereas HD6 activity was not diminished. A single amino acid variant of HD6 that was able to bind flagellin, but not self-assemble, lost ability to inhibit flagellar motility. Together, these results suggest a specialized role of HD6 self-assembly into polymers in targeting and restricting flagellar motility.

Loss of Flagella-Related Genes Enables a Nonflagellated, Fungal-Predating Bacterium To Strengthen the Synthesis of an Antifungal Weapon.

Loss of flagellar genes causes a nonmotile phenotype. The genus Lysobacter consists of numerous environmentally ubiquitous, nonflagellated bacteria, including Lysobacter enzymogenes, an antifungal bacterium that is beneficial to plants. L. enzymogenes still has many flagellar genes on its genome, although this bacterium does not engage in flagella-driven motility. Here, we report that loss of certain flagellar genes allows L. enzymogenes to strengthen its evolutionarily gained capacity in fungal killing. To clarify why this bacterium loses flagellar genes during the evolutionary process, we cloned several representative flagellar genes from Xanthomonas oryzae, a flagellated, phylogenetically related species of Lysobacter, and introduced them individually into L. enzymogenes to mimic genomic reacquisition of lost flagellar genes. Heterogeneous expression of the three X. oryzae flagellar structural genes (Xo-motA, Xo-motB, Xo-fliE) and one flagellar regulatory gene (Xo-fleQ) remarkably weakened the bacterial capacity to kill fungal pathogens by impairing the synthesis of an antifungal weapon, known as the heat-stable antifungal factor (HSAF). We further investigated the underlying mechanism by selecting Xo-FleQ as the representative because it is a master transcription factor responsible for flagellar gene expression. Xo-FleQ inhibited the transcription of operon genes responsible for HSAF synthesis via direct binding of Xo-FleQ to the promoter region, thereby decreasing HSAF biosynthesis by L. enzymogenes. These observations suggest a possible genome and function coevolution event, in which an antifungal bacterium deletes certain flagellar genes in order to enhance its ability to kill fungi. IMPORTANCE It is generally recognized that flagellar genes are commonly responsible for the flagella-driven bacterial motility. Thus, finding nonflagellated bacteria partially or fully lost flagellar genes is not a surprise. However, the present study provides new insights into this common idea. We found that loss of either certain flagellar structural or regulatory genes (such as motA, motB, fliE, and fleQ) allows a nonflagellated, antifungal bacterium (L. enzymogenes) to stimulate its fungal-killing capacity, outlining a genome-function coevolution event, where an antifungal bacterium "smartly" designed its genome to "delete" crucial flagellar genes to coordinate flagellar loss and fungal predation. This unusual finding might trigger bacteriologists to reconsider previously ignored functions of the lost flagellar genes in any nonflagellated, pathogenic, or beneficial bacteria.

Swarming Motility Assays in Salmonella.

Salmonella enterica has six subspecies, of which thesubspecies enterica is the most important for human health. The dispersal and infectivity of this species are dependent upon flagella-driven motility. Two kinds of flagella-mediated movements have been described-swimming individually in bulk liquid and swarming collectively over a surface substrate. During swarming, the bacteria acquire a distinct physiology, the most significant consequence of which is acquisition of adaptive resistance to antibiotics. Described here are protocols to cultivate, verify, and study swimming and swarming motility in S. enterica, and an additional "border-crossing" assay, where cells "primed" to swarm are presented with an environmental challenge such as antibiotics to assess their propensity to handle the challenge.

Site-Specific Isotope Labeling of FliG for Studying Structural Dynamics Using Nuclear Magnetic Resonance Spectroscopy.

To understand flagella-driven motility of bacteria, it is important to understand the structure and dynamics of the flagellar motor machinery. We have conducted structural dynamics analyses using solution nuclear magnetic resonance (NMR) to elucidate the detailed functions of flagellar motor proteins. Here, we introduce the analysis of the FliG protein, which is a flagellar motor protein, focusing on the preparation method of the original stable isotope-labeled protein.

Altering Microbiomes with Hydroxyapatite Nanoparticles: A Metagenomic Analysis.

Hydroxyapatite (HAp), the most abundant biological material among mammals, has been recently demonstrated to possess moderate antibacterial properties. Metagenomics provides a series of tools for analyzing the simultaneous interaction of materials with larger communities of microbes, which may aid in optimizing the antibacterial activity of a material such as HAp. Here, a microbiome intrinsic to the sample of sandy soil collected from the base of an African Natal plum (Carissa macrocarpa) shrub surrounding the children’s sandbox at the Arrowhead Park in Irvine, California was challenged with HAp nanoparticles and analyzed with next-generation sequencing for hypervariable 16S ribosomal DNA base pair homologies. HAp nanoparticles overwhelmingly reduced the presence of Gram-negative phyla, classes, orders, families, genera and species, and consequently elevated the relative presence of their Gram-positive counterparts. Thermodynamic, electrostatic and chemical bonding arguments were combined in a model proposed to explain this selective affinity. The ability of amphiphilic surface protrusions of lipoteichoic acid in Gram-positive bacteria and mycolic acid in mycobacteria to increase the dispersibility of the bacterial cells and assist in their resistance to capture by the solid phase is highlighted. Within the Gram-negative group, the variability of the distal, O-antigen portion of the membrane lipopolysaccharide was shown to be excessive and the variability of its proximal, lipid A portion insufficient to explain the selectivity based on chemical sequence arguments. Instead, flagella-driven motility proves to be a factor favoring the evasion of binding to HAp. HAp displayed a preference toward binding to less pathogenic bacteria than those causative of disease in humans, while taxa having a positive agricultural effect were largely captured by HAp, indicating an evolutionary advantage this may have given it as a biological material. The capacity to selectively sequester Gram-negative microorganisms and correspondingly alter the composition of the microbiome may open up a new avenue in environmental and biomedical applications of HAp.

Adaptation of the periplasm to maintain spatial constraints essential for cell envelope processes and cell viability.

The cell envelope of Gram-negative bacteria consists of two membranes surrounding a periplasm and peptidoglycan layer. Molecular machines spanning the cell envelope depend on spatial constraints and load-bearing forces across the cell envelope and surface. The mechanisms dictating spatial constraints across the cell envelope remain incompletely defined. In Escherichia coli, the coiled-coil lipoprotein Lpp contributes the only covalent linkage between the outer membrane and the underlying peptidoglycan layer. Using proteomics, molecular dynamics, and a synthetic lethal screen, we show that lengthening Lpp to the upper limit does not change the spatial constraint but is accommodated by other factors which thereby become essential for viability. Our findings demonstrate E. coli expressing elongated Lpp does not simply enlarge the periplasm in response, but the bacteria accommodate by a combination of tilting Lpp and reducing the amount of the covalent bridge. By genetic screening, we identified all of the genes in E. coli that become essential in order to enact this adaptation, and by quantitative proteomics discovered that very few proteins need to be up- or down-regulated in steady-state levels in order to accommodate the longer Lpp. We observed increased levels of factors determining cell stiffness, a decrease in membrane integrity, an increased membrane vesiculation and a dependance on otherwise non-essential tethers to maintain lipid transport and peptidoglycan biosynthesis. Further this has implications for understanding how spatial constraint across the envelope controls processes such as flagellum-driven motility, cellular signaling, and protein translocation.

Role of DegQ in differential stability of flagellin subunits in Vibrio vulnificus

Biofilm formation of Vibrio vulnificus is initiated by adherence of flagellated cells to surfaces, and then flagellum-driven motility is not necessary during biofilm maturation. Once matured biofilms are constructed, cells become flagellated and swim to disperse from biofilms. As a consequence, timely regulations of the flagellar components’ expression are crucial to complete a biofilm life-cycle. In this study, we demonstrated that flagellins’ production is regulated in a biofilm stage-specific manner, via activities of a protease DegQ and a chaperone FlaJ. Among four flagellin subunits for V. vulnificus filament, FlaC had the highest affinities to hook-associated proteins, and is critical for maturating flagellum, showed the least susceptibility to DegQ due to the presence of methionine residues in its DegQ-sensitive domains, ND1 and CD0. Therefore, differential regulation by DegQ and FlaJ controls the cytoplasmic stability of flagellins, which further determines the motility-dependent, stage-specific development of biofilms.

Pseudomonas Flagella: Generalities and Specificities.

Flagella-driven motility is an important trait for bacterial colonization and virulence. Flagella rotate and propel bacteria in liquid or semi-liquid media to ensure such bacterial fitness. Bacterial flagella are composed of three parts: a membrane complex, a flexible-hook, and a flagellin filament. The most widely studied models in terms of the flagellar apparatus are E. coli and Salmonella. However, there are many differences between these enteric bacteria and the bacteria of the Pseudomonas genus. Enteric bacteria possess peritrichous flagella, in contrast to Pseudomonads, which possess polar flagella. In addition, flagellar gene expression in Pseudomonas is under a four-tiered regulatory circuit, whereas enteric bacteria express flagellar genes in a three-step manner. Here, we use knowledge of E. coli and Salmonella flagella to describe the general properties of flagella and then focus on the specificities of Pseudomonas flagella. After a description of flagellar structure, which is highly conserved among Gram-negative bacteria, we focus on the steps of flagellar assembly that differ between enteric and polar-flagellated bacteria. In addition, we summarize generalities concerning the fuel used for the production and rotation of the flagellar macromolecular complex. The last part summarizes known regulatory pathways and potential links with the type-six secretion system (T6SS).

Architecture and Assembly of the Bacterial Flagellar Motor Complex.

One of the central systems responsible for bacterial motility is the flagellum. The bacterial flagellum is a macromolecular protein complex that is more than five times the cell length. Flagella-driven motility is coordinated via a chemosensory signal transduction pathway, and so bacterial cells sense changes in the environment and migrate towards more desirable locations. The flagellum of Salmonella enterica serovar Typhimurium is composed of a bi-directional rotary motor, a universal joint and a helical propeller. The flagellar motor, which structurally resembles an artificial motor, is embedded within the cell envelop and spins at several hundred revolutions per second. In contrast to an artificial motor, the energy utilized for high-speed flagellar motor rotation is the inward-directed proton flow through a transmembrane proton channel of the stator unit of the flagellar motor. The flagellar motor realizes efficient chemotaxis while performing high-speed movement by an ingenious directional switching mechanism of the motor rotation. To build the universal joint and helical propeller structures outside the cell body, the flagellar motor contains its own protein transporter called a type III protein export apparatus. In this chapter we summarize the structure and assembly of the Salmonella flagellar motor complex.

Under Elevated c-di-GMP in Escherichia coli, YcgR Alters Flagellar Motor Bias and Speed Sequentially, with Additional Negative Control of the Flagellar Regulon via the Adaptor Protein RssB.

In Escherichia coli and Salmonella, the c-di-GMP effector YcgR inhibits flagellar motility by interacting directly with the motor to alter both its bias and speed. Here, we demonstrate that in both of these bacteria, YcgR acts sequentially, altering motor bias first and then decreasing motor speed. We show that when c-di-GMP levels are high, deletion of ycgR restores wild-type motor behavior in E. coli, indicating that YcgR is the only motor effector in this bacterium. Yet, motility and chemotaxis in soft agar do not return to normal, suggesting that there is a second mechanism that inhibits motility under these conditions. In Salmonella, c-di-GMP-induced synthesis of extracellular cellulose has been reported to entrap flagella and to be responsible for the YcgR-independent motility defect. We found that this is not the case in E. coli Instead, we found through reversion analysis that deletion of rssB, which codes for a response regulator/adaptor protein that normally directs ClpXP protease to target σS for degradation, restored wild-type motility in the ycgR mutant. Our data suggest that high c-di-GMP levels may promote altered interactions between these proteins to downregulate flagellar gene expression.IMPORTANCE Flagellum-driven motility has been studied in E. coli and Salmonella for nearly half a century. Over 60 genes control flagellar assembly and function. The expression of these genes is regulated at multiple levels in response to a variety of environmental signals. Cues that elevate c-di-GMP levels, however, inhibit motility by direct binding of the effector YcgR to the flagellar motor. In this study conducted mainly in E. coli, we show that YcgR is the only effector of motor control and tease out the order of YcgR-mediated events. In addition, we find that the σS regulator protein RssB contributes to negative regulation of flagellar gene expression when c-di-GMP levels are elevated.

Flagella-Driven Motility of Bacteria.

The bacterial flagellum is a helical filamentous organelle responsible for motility. In bacterial species possessing flagella at the cell exterior, the long helical flagellar filament acts as a molecular screw to generate thrust. Meanwhile, the flagella of spirochetes reside within the periplasmic space and not only act as a cytoskeleton to determine the helicity of the cell body, but also rotate or undulate the helical cell body for propulsion. Despite structural diversity of the flagella among bacterial species, flagellated bacteria share a common rotary nanomachine, namely the flagellar motor, which is located at the base of the filament. The flagellar motor is composed of a rotor ring complex and multiple transmembrane stator units and converts the ion flux through an ion channel of each stator unit into the mechanical work required for motor rotation. Intracellular chemotactic signaling pathways regulate the direction of flagella-driven motility in response to changes in the environments, allowing bacteria to migrate towards more desirable environments for their survival. Recent experimental and theoretical studies have been deepening our understanding of the molecular mechanisms of the flagellar motor. In this review article, we describe the current understanding of the structure and dynamics of the bacterial flagellum.

Rhizospheric life of Salmonella requires flagella-driven motility and EPS-mediated attachment to organic matter and enables cross-kingdom invasion

Salmonella is an established pathogen of the members of the kingdom Animalia. Reports indicate that the association of Salmonella with fresh, edible plant products occurs at the pre-harvest state, i.e. in the field. In this study, we follow the interaction of Salmonella Typhimurium with the model plant Arabidopsis thaliana to understand the process of migration in soil. Plant factors like root exudates serve as chemo-attractants. Our ex situ experiments allowed us to track Salmonella from its free-living state to the endophytic state. We found that genes encoding two-component systems and proteins producing extracellular polymeric substances are essential for Salmonella to adhere to the soil and roots. To understand the trans-kingdom flow of Salmonella, we fed the contaminated plants to mice and observed that it invades and colonizes liver and spleen. To complete the disease cycle, we re-established the infection in plant by mixing the potting mixture with the fecal matter collected from the diseased animals. Our experiments revealed a cross-kingdom invasion by the pathogen via passage through a murine intermediate, a mechanism for its persistence in the soil and invasion in a non-canonical host. These results form a basis to break the life-cycle of Salmonella before it reaches its animal host and thus reduce Salmonella contamination of food products.

Essential Role of σ Factor RpoF in Flagellar Biosynthesis and Flagella-Mediated Motility of Acidithiobacillus caldus.

Acidithiobacillaceae, an important family of acidophilic and chemoautotrophic sulfur or iron oxidizers, participate in geobiochemical circulation of the elements and drive the release of heavy metals in mining associated habitats. Because of their environmental adaptability and energy metabolic systems, Acidithiobacillus spp. have become the dominant bacteria used in bioleaching for heavy metal recovery. Flagella-driven motility is associated with bacterial chemotaxis and bacterial responses to environmental stimuli. However, little is known about how the flagellum of Acidithiobacillus spp. is regulated and how the flagellum affects the growth of these chemoautotrophic bacteria. In this study, we analyzed the flagellar gene clusters in Acidithiobacillus strains and uncovered the close relationship between flagella and the sulfur-oxidizing systems (Sox system). The σ28 gene (rpoF) knockout and overexpression strains of Acidithiobacillus caldus were constructed. Scanning electron microscopy shows that A. caldus ΔrpoF cells lacked flagella, indicating the essential role of RpoF in regulating flagella synthesis in these chemoautotrophic bacteria. Motility analysis suggests that the deletion of rpoF resulted in the reduction of swarming capability, while this capability was enhanced in the rpoF overexpression strain. Both static cultivation and low concentration of energy substrates (elemental sulfur or tetrathionate) led to weak growth of A. caldus ΔrpoF cells. The deletion of rpoF promoted bacterial attachment to the surface of elemental sulfur in static cultivation. The absence of RpoF caused an obvious change in transcription profile, including genes in flagellar cluster and those involved in biofilm formation. These results provide an understanding on the regulation of flagellar hierarchy and the flagellar function in these sulfur or iron oxidizers.

Polar landmark protein HubP recruits flagella assembly protein FapA under glucose limitation in Vibrio vulnificus.

How motile bacteria recognize their environment and decide whether to stay or navigate toward more favorable location is a fundamental issue in survival. The flagellum is an elaborate molecular device responsible for bacterial locomotion, and the flagellum-driven motility allows bacteria to move themselves to the appropriate location at the right time. Here, we identify the polar landmark protein HubP as a modulator of polar flagellation that recruits the flagellar assembly protein FapA to the old cell pole, thereby controlling its activity for the early events of flagellar assembly in Vibrio vulnificus. We show that dephosphorylated EIIAGlc of the PEP-dependent sugar transporting phosphotransferase system sequesters FapA from HubP in response to glucose and hence inhibits FapA-mediated flagellation. Thus, flagellar assembly and motility is governed by spatiotemporal control of FapA, which is orchestrated by the competition between dephosphorylated EIIAGlc and HubP, in the human pathogen V. vulnificus.

Induction by Bradyrhizobium diazoefficiens of Different Pathways for Growth in D-mannitol or L-arabinose Leading to Pronounced Differences in CO2 Fixation, O2 Consumption, and Lateral-Flagellum Production.

Bradyrhizobium diazoefficiens, a soybean N2-fixing symbiont, constitutes the basic input in one of the most prominent inoculant industries worldwide. This bacterium may be cultured with D-mannitol or L-arabinose as carbon-plus-energy source (C-source) with similar specific growth rates, but with higher biomass production with D-mannitol. To better understand the bacterium’s carbon metabolism, we analyzed, by liquid chromatography and tandem mass spectrometry (MS), the whole set of proteins obtained from cells grown on each C-source. Among 3,334 proteins identified, 266 were overproduced in D-mannitol and 237 in L-arabinose, but among these, only 22% from D-mannitol cultures and 35% from L-arabinose cultures were annotated with well defined functions. In the D-mannitol-differential pool we found 19 enzymes of the pentose-phosphate and Calvin–Benson–Bassham pathways and accordingly observed increased extracellular-polysaccharide production by D-mannitol grown bacteria in a CO2-enriched atmosphere. Moreover, poly-3-hydroxybutyrate biosynthesis was increased, suggesting a surplus of reducing power. In contrast, the L-arabinose-differential pool contained 11 enzymes of the L-2-keto-3-deoxyarabonate pathway, 4 enzymes for the synthesis of nicotinamide-adenine dinucleotide from aspartate, with those cultures having a threefold higher O2-consumption rate than the D-mannitol cultures. The stoichiometric balances deduced from the modeled pathways, however, resulted in similar O2 consumptions and ATP productions per C-mole of substrate. These results suggested higher maintenance-energy demands in L-arabinose, which energy may be used partly for flagella-driven motility. Since B. diazoefficiens produces the lateral-flagella system in only L-arabinose, we calculated the O2-consumption rates of a lafR::Km mutant devoid of lateral flagella cultured in L-arabinose or D-mannitol. Contrary to that of the wild-type, the O2-consumption rate of this mutant was similar on both C-sources, and accordingly outcompeted the wild-type in coculture, suggesting that the lateral flagella behaved as parasitic structures under these conditions. Proteomic data are available via ProteomeXchange with identifier PXD008263.

A Chemotaxis-Like Pathway of Azorhizobium caulinodans Controls Flagella-Driven Motility, Which Regulates Biofilm Formation, Exopolysaccharide Biosynthesis, and Competitive Nodulation.

The genome of the Azorhizobium caulinodans ORS571 contains a unique chemotaxis gene cluster (che) including five chemotaxis genes: cheA, cheW, cheY1, cheB, and cheR. Analysis of the role of the chemotaxis cluster of A. caulinodans using deletion mutant strains revealed that CheA or the Che signaling pathway controls chemotaxis behavior and flagella-driven motility and plays important roles in formation of biofilms and production of extracellular polysaccharides (EPS). Furthermore, the deletion mutants (ΔcheA and ΔcheA-R) were defective in competitive adsorption and colonization on the root surface of host plants. In addition, a functional CheA or Che pathway promoted competitive nodulation on roots and stems. Interestingly, a nonflagellated mutant, ΔfliM, displayed a phenotype highly similar to that of the ΔcheA or ΔcheA-R mutant strains. These findings suggest that through controlling flagella-driven motility behavior, the chemotaxis signaling pathway in A. caulinodans coordinates biofilm formation, EPS, and competitive colonization and nodulation.