Comparative Analysis of anti-Shine- Dalgarno Function in Flavobacterium johnsoniae and Escherichia coli

The anti-Shine-Dalgarno (ASD) sequence of 16S rRNA is highly conserved across Bacteria, and yet usage of Shine-Dalgarno (SD) sequences in mRNA varies dramatically, depending on the lineage. Here, we compared the effects of ASD mutagenesis in Escherichia coli, a Gammaproteobacteria which commonly employs SD sequences, and Flavobacterium johnsoniae, a Bacteroidia which rarely does. In E. coli, 30S subunits carrying any single substitution at positions 1,535–1,539 confer dominant negative phenotypes, whereas subunits with mutations at positions 1,540–1,542 are sufficient to support cell growth. These data suggest that CCUCC (1,535–1,539) represents the functional core of the element in E. coli. In F. johnsoniae, deletion of three ribosomal RNA (rrn) operons slowed growth substantially, a phenotype largely rescued by a plasmid-borne copy of the rrn operon. Using this complementation system, we found that subunits with single mutations at positions 1,535–1,537 are as active as control subunits, in sharp contrast to the E. coli results. Moreover, subunits with quadruple substitution or complete replacement of the ASD retain substantial, albeit reduced, activity. Sedimentation analysis revealed that these mutant subunits are overrepresented in the subunit fractions and underrepresented in polysome fractions, suggesting some defect in 30S biogenesis and/or translation initiation. Nonetheless, our collective data indicate that the ASD plays a much smaller role in F. johnsoniae than in E. coli, consistent with SD usage in the two organisms.

Social motility of biofilm-like microcolonies in a gliding bacterium

Bacterial biofilms are aggregates of surface-associated cells embedded in an extracellular polysaccharide (EPS) matrix, and are typically stationary. Studies of bacterial collective movement have largely focused on swarming motility mediated by flagella or pili, in the absence of a biofilm. Here, we describe a unique mode of collective movement by a self-propelled, surface-associated biofilm-like multicellular structure. Flavobacterium johnsoniae cells, which move by gliding motility, self-assemble into spherical microcolonies with EPS cores when observed by an under-oil open microfluidic system. Small microcolonies merge, creating larger ones. Microscopic analysis and computer simulation indicate that microcolonies move by cells at the base of the structure, attached to the surface by one pole of the cell. Biochemical and mutant analyses show that an active process drives microcolony self-assembly and motility, which depend on the bacterial gliding apparatus. We hypothesize that this mode of collective bacterial movement on solid surfaces may play potential roles in biofilm dynamics, bacterial cargo transport, or microbial adaptation. However, whether this collective motility occurs on plant roots or soil particles, the native environment for F. johnsoniae, is unknown.

The Type 9 Secretion System Is Required for Flavobacterium johnsoniae Biofilm Formation

Flavobacterium johnsoniae forms biofilms in low nutrient conditions. Protein secretion and cell motility may have roles in biofilm formation. The F. johnsoniae type IX secretion system (T9SS) is important for both secretion and motility. To determine the roles of each process in biofilm formation, mutants defective in secretion, in motility, or in both processes were tested for their effects on biofilm production using a crystal violet microplate assay. All mutants that lacked both motility and T9SS-mediated secretion failed to produce biofilms. A porV deletion mutant, which was severely defective for secretion, but was competent for motility, also produced negligible biofilm. In contrast, mutants that retained secretion but had defects in gliding formed biofilms. An sprB mutant that is severely but incompletely defective in gliding motility but retains a fully functional T9SS was similar to the wild type in biofilm formation. Mutants with truncations of the gldJ gene that compromise motility but not secretion showed partial reduction in biofilm formation compared to wild type. Unlike the sprB mutant, these gldJ truncation mutants were essentially nonmotile. The results show that a functional T9SS is required for biofilm formation. Gliding motility, while not required for biofilm formation, also appears to contribute to formation of a robust biofilm.

A highly active phosphate-insensitive phosphatase is widely distributed in nature

The regeneration of bioavailable phosphate from immobilised organophosphorus represents a key process in the global phosphorus cycle and is facilitated by enzymes known as phosphatases. Most bacteria possess at least one of three major phosphatases, known as PhoA, PhoX and PhoD, whose activity is optimal under alkaline conditions. The production and activity of these three phosphatase families is negatively regulated by phosphate availability and thus these enzymes play a major role in scavenging phosphorus only during times of phosphate scarcity. Here, we reveal a previously overlooked phosphate-insensitive phosphatase, PafA, prevalent in Bacteroidetes, which is highly abundant in nature and represents a major route for the remineralisation of phosphate in the environment. Using Flavobacterium johnsoniae as the model, we reveal PafA is highly active towards phosphomonoesters. Unlike other major phosphatases, PafA is fully functional in the presence of its metabolic product, phosphate, and is essential for growth on phosphorylated carbohydrates as a sole carbon source. PafA, which is constitutively produced under all growth conditions tested, rapidly remineralises phosphomonoesters producing significant quantities of bioavailable phosphate that can cross feed into neighbouring cells. pafA is both abundant and highly expressed in the global ocean and abundant in plant rhizospheres, highlighting a new and important enzyme in the global phosphorus cycle with applied implications for agriculture as well as biogeochemical cycling. We speculate PafA expands the metabolic niche of Bacteroidetes by enabling utilisation of abundant organophosphorus substrates in the presence of excess phosphate, when other microbes are rendered incapable.

The Monoheme c Subunit of Respiratory Alternative Complex III Is Not Essential for Electron Transfer to Cytochrome aa 3 in Flavobacterium johnsoniae

Energy conversion is a fundamental process of all organisms, realized by specialized protein complexes, one of which is alternative complex III (ACIII). ACIII is a functional analogue of well-known mitochondrial complex III, but operates according to a different, still unknown mechanism.

Biofilm Spreading by the Adhesin-Dependent Gliding Motility of Flavobacterium johnsoniae: 2. Role of Filamentous Extracellular Network and Cell-to-Cell Connections at the Biofilm Surface

Flavobacterium johnsoniae forms a thin spreading colony on nutrient-poor agar using gliding motility. As reported in the first paper, WT cells in the colony were sparsely embedded in self-produced extracellular polymeric matrix (EPM), while sprB cells were densely packed in immature biofilm with less matrix. The colony surface is critical for antibiotic resistance and cell survival. We have now developed the Grid Stamp-Peel method whereby the colony surface is attached to a TEM grid for negative-staining microscopy. The images showed that the top of the spreading convex WT colonies was covered by EPM with few interspersed cells. Cells exposed near the colony edge made head-to-tail and/or side-to-side contact and sometimes connected via thin filaments. Nonspreading sprB and gldG and gldK colonies had a more uniform upper surface covered by different EPMs including vesicles and filaments. The EPM of sprB, gldG, and WT colonies contained filaments ~2 nm and ~5 nm in diameter; gldK colonies did not include the latter. Every cell near the edge of WT colonies had one or two dark spots, while cells inside WT colonies and cells in SprB-, GldG-, or GldK-deficient colonies did not. Together, our results suggest that the colony surface structure depends on the capability to expand biofilm.

Crystal structures of two camelid nanobodies raised against GldL, a component of the type IX secretion system from Flavobacterium johnsoniae

GldL is an inner-membrane protein that is essential for the function of the type IX secretion system (T9SS) in Flavobacterium johnsoniae. The complex that it forms with GldM is supposed to act as a new rotary motor involved in the gliding motility of the bacterium. In the context of structural studies of GldL to gain information on the assembly and function of the T9SS, two camelid nanobodies were selected, produced and purified. Their interaction with the cytoplasmic domain of GldL was characterized and their crystal structures were solved. These nanobodies will be used as crystallization chaperones to help in the crystallization of the cytoplasmic domain of GldL and could also help to solve the structure of the complex using molecular replacement.

Large-scale vortices with dynamic rotation emerged from monolayer collective motion of gliding Flavobacteria

A collective motion of self-driven particles has been a fascinating subject in physics and biology. Sophisticated macroscopic behavior emerges through a population in thousands or millions of bacterial cells, propelling itself by flagellar rotation and its chemotactic response. Here we found a series of collective motions accompanying successive phase-transitions in a non-flagellated rod-shaped soil bacterium, Flavobacterium johnsoniae, which was driven by a surface cell movement known as gliding motility. When we spot the cells on an agar plate with a low level of nutrients, the bacterial community exhibited vortex patterns that spontaneously appeared as lattice and integrated into a large-scale circular plate. All patterns exhibit with monolayer of bacteria, which enable to visualize an individual cell with a high resolution among a wide-range pattern two-dimensionally. The single cells moved at random orientation, but the cells connected with one another showed left-turn biased trajectories in starved environment. This feature is possibly due to the collision of cells inducing a nematic alignment of dense cells as self-propelled rods. Subsequently, each vortex oscillated independently, and then transformed to the rotating mode as an independent circular plate. Notably, the rotational direction of the circular plate was counterclockwise without exception. The plates developed accompanying rotation with constant angular velocity, suggesting that the mode is an efficient strategy for bacterial survival. Importance Self-propelled bacteria propelled by flagella rotation often display highly organized dynamic patterns at high cell densities. Here we found a new mode of collective motion in non-flagellated bacteria: vortex patterns were spontaneously appeared as lattice and integrated into a large-scale circular plate comprising hundreds of thousands of cells, which exhibited unidirectional rotation in a counterclockwise manner and expanded in size on agar. A series of collective motions was driven by gliding motility of the rod-shaped soil bacterium Flavobacterium johnsoniae. In a low nutrient environment, single cells moved at random orientation while cells at high density moved together as a unitary cluster. This might be an efficient strategy for cells of this species to find nutrients.

Natural Transformation of Riemerella columbina and Its Determinants

In a previous study, it was shown that Riemerella anatipestifer, a member of Flavobacteriaceae, is naturally competent. However, whether natural competence is universal in Flavobacteriaceae remains unknown. In this study, it was shown for the first time that Riemerella columbina was naturally competent in the laboratory condition; however, Flavobacterium johnsoniae was not naturally competent under the same conditions. The competence of R. columbina was maintained throughout the growth phases, and the transformation frequency was highest during the logarithmic phase. A competition assay revealed that R. columbina preferentially took up its own genomic DNA over heterologous DNA. The natural transformation frequency of R. columbina was significantly increased in GCB medium without peptone or phosphate. Furthermore, natural transformation of R. columbina was inhibited by 0.5 mM EDTA, but could be restored by the addition of CaCl2, MgCl2, ZnCl2, and MnCl2, suggesting that these divalent cations promote the natural transformation of R. columbina. Overall, this study revealed that natural competence is not universal in Flavobacteriaceae members and triggering of competence differs from species to species.