Reconstitution of Spiroplasma swimming by expressing two bacterial actins in synthetic minimal bacterium

Cell motility may originate from the movements of housekeeping proteins after they were amplified and transmitted to cell outside. However, this process is not supported by experimental observations. The swimming motility of Spiroplasma, commensal or parasitic bacteria are caused by the helicity switching of a ribbon-like cytoskeleton, that is composed of six proteins evolved from a nucleosidase and bacterial actin called MreB. Here, we expressed these proteins in a synthetic minimal bacterium, JCVI-syn3B whose genome was computer-designed and chemically synthesized. The synthetic bacterium showed swimming motility with the common features with Spiroplasma swimming. Moreover, combination of two proteins showed helical cell shape and swimming, suggesting that the swimming was originated from differentiation and coupling of MreB proteins

Polyunsaturated fatty acids cause physiological and behavioral changes in Vibrio alginolyticus and Vibrio fischeri

Vibrio alginolyticus and Vibrio (Aliivibrio) fischeri are Gram-negative bacteria found globally in marine environments. During the past decade, studies have shown that certain Gram-negative bacteria, including Vibrio species (cholerae, parahaemolyticus, and vulnificus) are capable of using exogenous polyunsaturated fatty acids (PUFAs) to modify the phospholipids of their membrane. Moreover, exposure to exogenous PUFAs has been shown to affect certain phenotypes that are important factors of virulence. The purpose of this study was to investigate whether V. alginolyticus and V. fischeri are capable of responding to exogenous PUFAs by remodeling their membrane phospholipids and/or altering behaviors associated with virulence. Thin-layer chromatography (TLC) analyses and ultra-performance liquid chromatography-electrospray ionization mass spectrometry (UPLC/ESI-MS) confirmed incorporation of all PUFAs into membrane phosphatidylglycerol and phosphatidylethanolamine. Several growth phenotypes were identified when individual fatty acids were supplied in minimal media and as sole carbon sources. Interestingly, several PUFAs acids inhibited growth of V. fischeri. Significant alterations to membrane permeability were observed depending on fatty acid supplemented. Strikingly, arachidonic acid (20:4) reduced membrane permeability by approximately 35% in both V. alginolyticus and V. fischeri. Biofilm assays indicated that fatty acid influence was dependent on media composition and temperature. All fatty acids caused decreased swimming motility in V. alginolyticus, while only linoleic acid (18:2) significantly increased swimming motility in V. fischeri. In summary, exogenous fatty acids cause a variety of changes in V. alginolyticus and V. fischeri, thus adding these bacteria to a growing list of Gram-negatives that exhibit versatility in fatty acid utilization and highlighting the potential for environmental PUFAs to influence phenotypes associated with planktonic, beneficial, and pathogenic associations.

Swimming of the Trophont Zooid of Vorticella Convallaria

Vorticella convallaria is a ciliated protozoan found in freshwater habitats. In the sessile or stalked trophont form, V. convallaria is shaped somewhat like a balloon as it has a body or zooid (the head of the balloon) that is about 40 μm large with cilia around its oral part, and a stalk (the string of a balloon) anchoring the zooid to a solid surface. When a trophont zooid of V. convallaria detached from the stalk, the zooid swims around in water by creating water flow using its oral cilia. In contrast to the stalk contraction of V. convallaria that has been well studied, the swimming motility of V. convallaria is little known. In this study, we measured the swimming trajectories of the stalkless trophont zooid of V. convallaria using video microscopy and Hele-Shaw cells with a gap height of 25 μm, traced the swimming zooid using image processing, and analyzed the swimming motion in terms of swimming velocity and mean square displacement. The stalkless trophont zooid of V . convallaria was found to swim in circular patterns with intermittent ballistic motions in the confinement, and the average swimming speed ranged from 20 μm/s to 110 μm/s. Since the swimming pattern of V. convallaria appeared to be affected by the level of confinement, we will continue characterizing the ciliate’s swimming in the Hele-Shaw cell with different gap heights. Our study is expected to reveal the swimming motility of V. convallaria and to advance general understanding of swimming of microorganisms.

Molecular Mechanisms Underlying the Regulation of Biofilm Formation and Swimming Motility by FleS/FleR in Pseudomonas aeruginosa

Pseudomonas aeruginosa, a major cause of nosocomial infection, can survive under diverse environmental conditions. Its great adaptive ability is dependent on its multiple signaling systems such as the two-component system (TCS). A TCS FleS/FleR has been previously identified to positively regulate a variety of virulence-related traits in P. aeruginosa PAO1 including motility and biofilm formation which are involved in the acute and chronic infections, respectively. However, the molecular mechanisms underlying these regulations are still unclear. In this study, we first analyzed the regulatory roles of each domains in FleS/FleR and characterized key residues in the FleS-HisKA, FleR-REC and FleR-AAA domains that are essential for the signaling. Next, we revealed that FleS/FleR regulates biofilm formation in a c-di-GMP and FleQ dependent manner. Lastly, we demonstrated that FleR can regulate flagellum biosynthesis independently without FleS, which explains the discrepant regulation of swimming motility by FleS and FleR.

Involvement of RpoN in Regulating Motility, Biofilm, Resistance, and Spoilage Potential of Pseudomonas fluorescens

Pseudomonas fluorescens is a typical spoiler of proteinaceous foods, and it is characterized by high spoilage activity. The sigma factor RpoN is a well-known regulator controlling nitrogen assimilation and virulence in many pathogens. However, its exact role in regulating the spoilage caused by P. fluorescens is unknown. Here, an in-frame deletion mutation of rpoN was constructed to investigate its global regulatory function through phenotypic and RNA-seq analysis. The results of phenotypic assays showed that the rpoN mutant was deficient in swimming motility, biofilm formation, and resistance to heat and nine antibiotics, while the mutant increased the resistance to H2O2. Moreover, the rpoN mutant markedly reduced extracellular protease and total volatile basic nitrogen (TVB-N) production in sterilized fish juice at 4°C; meanwhile, the juice with the rpoN mutant showed significantly higher sensory scores than that with the wild-type strain. To identify RpoN-controlled genes, RNA-seq-dependent transcriptomics analysis of the wild-type strain and the rpoN mutant was performed. A total of 1224 genes were significantly downregulated, and 474 genes were significantly upregulated by at least two folds at the RNA level in the rpoN mutant compared with the wild-type strain, revealing the involvement of RpoN in several cellular processes, mainly flagellar mobility, adhesion, polysaccharide metabolism, resistance, and amino acid transport and metabolism; this may contribute to the swimming motility, biofilm formation, stress and antibiotic resistance, and spoilage activities of P. fluorescens. Our results provide insights into the regulatory role of RpoN of P. fluorescens in food spoilage, which can be valuable to ensure food quality and safety.

Pectobacterium brasiliense 1692 Chemotactic Responses and the Role of Methyl-Accepting Chemotactic Proteins in Ecological Fitness

To adapt to changing environmental niches, bacteria require taxis, a movement toward or away from a stimulus (ligand). Chemotaxis has been studied in some members of the Soft Rot Pectobacteriaceae (SRP), particularly members of the genus Dickeya. On the contrary, there are fewer studies on this topic for the other genus in the SRP group, namely Pectobacterium. This study evaluated chemotactic responses in Pectobacterium brasiliense (Pb 1692) to various ligands. A total of 34 methyl-accepting chemotactic proteins (MCPs) were identified in the Pb 1692 genome and the domain architectures of these MCPs were determined. Four Pb 1692 MCPs previously shown to be differentially expressed during potato tuber infection were selected for further functional characterization. Toward this end, Pb 1692 mutant strains each lacking either AED-0001492, AED-0003671, AED-0000304, or AED-0000744 were generated. Two of these mutants (AED-0001492 and AED-0003671), were attenuated in their ability to grow and respond to citrate and are thus referred to as MCPcit2 and MCPcit1, respectively, while the other two, AED-0000304 (MCPxyl) and AED-0000744 (MCPasp), were affected in their ability to respond to xylose and aspartate, respectively. Trans-complementation of the mutant strains restored swimming motility in the presence of respective ligands. The four MCP mutants were not affected in virulence but were significantly attenuated in their ability to attach to potato leaves suggesting that ecological fitness is an important contribution of these MCPs toward Pb 1692 biology.

Structure and polymerization dynamics of bacterial actin MreB3 and MreB5 involved in Spiroplasma swimming.

MreB is a bacterial protein belonging to the actin superfamily. It polymerizes into an antiparallel double-stranded filament that generally functions for cell shape determinations by maintaining the cell wall synthesis. Spiroplasma eriocheiris, a helical wall-less bacterium, has five classes of MreB homologs (SpeMreB1-5) that are responsible for its swimming motility. SpeMreB5 is likely responsible for generating the driving force for the swimming motility. However, molecular profiles involved in the swimming motility are poorly understood. Additionally, SpeMreB3 has distinct sequence features from the other SpeMreBs. Here, we have revealed the structures and polymerization dynamics of SpeMreB3 and SpeMreB5. Both SpeMreBs formed antiparallel double-stranded filaments with different characters; SpeMreB3 formed short filaments with slow polymerization, and SpeMreB5 filaments further assembled into bundle structures such as raft and paracrystal. SpeMreB5 filaments hydrolyzed ATP at a constant rate and were depolymerized immediately after ATP depletion. The Pi release rate of SpeMreB3 was much slower than that of SpeMreB5. Our crystal structure of SpeMreB3 and Pi release measurements of SpeMreB3 and SpeMreB5 mutant variants explain that the cause of the slow Pi release is the lack of the amino acid motif "E ... T - X - [DE]", found in almost all MreBs, which probably takes roles to adjust the position and eliminate a proton of the putative nucleophilic water for γ-Pi of AMPPNP. These results show that SpeMreB3 has unique polymerization dynamics without bundle formations, whereas SpeMreB5 shows bundle formations, and its polymerization dynamics occur in the same manner as other actin superfamily members.