Glycosylate and move! The glycosyltransferase Maf is involved in bacterial flagella formation.

The flagella of various Gram-negative bacteria are decorated with diverse glycan structures, amongst them nonulosonic acids related to the sialic acid family. Although nonulosonic sugar biosynthesis pathways have been dissected in various pathogens, the enzymes transferring the sugars onto flagellin are still poorly characterized. The deletion of genes coding for motility associated factors (Mafs) found in many pathogenic strains systematically gives rise to nonflagellated bacteria lacking specific nonulosonic sugars on the flagellins, therefore, relating Maf function to flagellin glycosylation and bacterial motility. We investigated the role of Maf from our model organism, Magnetospirillum magneticum AMB-1, in the glycosylation and formation of the flagellum. Deletion of the gene amb0685 coding for Maf produced a nonflagellated bacterium where the flagellin was still produced but no longer glycosylated. Our X-ray structure analysis revealed that the central domain of Maf exhibits similarity to sialyltransferases from Campylobacter jejuni. Glycan analysis suggested that the nonulosonic carbohydrate structure transferred is pseudaminic acid or a very close derivative. This work describes the importance of glycosylation in the formation of the bacterial flagellum and provides the first structural model for a member of a new bacterial glycosyltransferase family involved in nonulosonic acids transfer onto flagellins.

Opposite and Coordinated Rotation of Amphitrichous Flagella Governs Oriented Swimming and Reversals in a Magnetotactic Spirillum.

UNLABELLED: Current knowledge regarding the mechanism that governs flagellar motor rotation in response to environmental stimuli stems mainly from the study of monotrichous and peritrichous bacteria. Little is known about how two polar flagella, one at each cell pole of the so-called amphitrichous bacterium, are coordinated to steer the swimming. Here we fluorescently labeled the flagella of Magnetospirillum magneticum AMB-1 cells and took advantage of the magnetically controllable swimming of this bacterium to investigate flagellar rotation in moving cells. We identified three motility behaviors (runs, tumbles, and reversals) and two characteristic fluorescence patterns likely corresponding to flagella rotating in opposite directions. Each AMB-1 locomotion mode was systematically associated with particular flagellar patterns at the poles which led us to conclude that, while cell runs are allowed by the asymmetrical rotation of flagellar motors, their symmetrical rotation triggers cell tumbling. Our observations point toward a precise coordination of the two flagellar motors which can be temporarily unsynchronized during tumbling. IMPORTANCE: Motility is essential for bacteria to search for optimal niches and survive. Many bacteria use one or several flagella to explore their environment. The mechanism by which bipolarly flagellated cells coordinate flagellar rotation is poorly understood. We took advantage of the genetic amenability and magnetically controlled swimming of the spirillum-shaped magnetotactic bacterium Magnetospirillum magneticum AMB-1 to correlate cell motion with flagellar rotation. We found that asymmetric rotation of the flagella (counterclockwise at the lagging pole and clockwise at the leading pole) enables cell runs whereas symmetric rotation triggers cell tumbling. Taking into consideration similar observations in spirochetes, bacteria possessing bipolar ribbons of periplasmic flagella, we propose a conserved motility paradigm for spirillum-shaped bipolarly flagellated bacteria.

Comparative genomic analysis provides insights into the evolution and niche adaptation of marine Magnetospira sp. QH-2 strain.

Magnetotactic bacteria (MTB) are capable of synthesizing intracellular organelles, the magnetosomes, that are membrane-bounded magnetite or greigite crystals arranged in chains. Although MTB are widely spread in various ecosystems, few axenic cultures are available, and only freshwater Magnetospirillum spp. have been genetically analysed. Here, we present the complete genome sequence of a marine magnetotactic spirillum, Magnetospira sp. QH-2. The high number of repeats and transposable elements account for the differences in QH-2 genome structure compared with other relatives. Gene cluster synteny and gene correlation analyses indicate that the insertion of the magnetosome island in the QH-2 genome occurred after divergence between freshwater and marine magnetospirilla. The presence of a sodium-quinone reductase, sodium transporters and other functional genes are evidence of the adaptive evolution of Magnetospira sp. QH-2 to the marine ecosystem. Genes well conserved among freshwater magnetospirilla for nitrogen fixation and assimilatory nitrate respiration are absent from the QH-2 genome. Unlike freshwater Magnetospirillum spp., marine Magnetospira sp. QH-2 neither has TonB and TonB-dependent receptors nor does it grow on trace amounts of iron. Taken together, our results show a distinct, adaptive evolution of Magnetospira sp. QH-2 to marine sediments in comparison with its closely related freshwater counterparts.

Gear up! An overview of the molecular equipment used by Myxococcus to move, kill, and divide in prey colonies.

Myxococcus relies on motility to efficiently invade and predate a prey colony. Upon contact with prey, Myxococcus temporarily halts its motility and initiates prey cell lysis, which involves two contact-dependent predatory machineries, the Kil system and the needleless T3SS*. Predatory cells grow as they invade and feed on prey cells. When dividing, Myxococcus cells systematically pause their movements before division. This highlights a high level of co-ordination between motility and contact-dependent killing but also with cell division. In this review, we give an overview of the different nanomachines used by Myxococcus to move on surfaces, kill by contact, and divide, and we discuss the potential regulatory mechanisms at play during these different processes.

The magnetosome membrane protein, MmsF, is a major regulator of magnetite biomineralization in Magnetospirillum magneticum AMB-1.

Magnetotactic bacteria (MTB) use magnetosomes, membrane-bound crystals of magnetite or greigite, for navigation along geomagnetic fields. In Magnetospirillum magneticum sp. AMB-1, and other MTB, a magnetosome gene island (MAI) is essential for every step of magnetosome formation. An 8-gene region of the MAI encodes several factors implicated in control of crystal size and morphology in previous genetic and proteomic studies. We show that these factors play a minor role in magnetite biomineralization in vivo. In contrast, MmsF, a previously uncharacterized magnetosome membrane protein encoded within the same region plays a dominant role in defining crystal size and morphology and is sufficient for restoring magnetite synthesis in the absence of the other major biomineralization candidates. In addition, we show that the 18 genes of the mamAB gene cluster of the MAI are sufficient for the formation of an immature magnetosome organelle. Addition of MmsF to these 18 genes leads to a significant enhancement of magnetite biomineralization and an increase in the cellular magnetic response. These results define a new biomineralization protein and lay down the foundation for the design of autonomous gene cassettes for the transfer of the magnetic phenotype in other bacteria.

Comprehensive genetic dissection of the magnetosome gene island reveals the step-wise assembly of a prokaryotic organelle.

Although membrane-bounded compartments are commonly considered a unique eukaryotic characteristic, many species of bacteria have organelles. Compartmentalization is well studied in eukaryotes; however, the molecular factors and processes leading to organelle formation in bacteria are poorly understood. We use the magnetosome compartments of magnetotactic bacteria as a model system to investigate organelle biogenesis in a prokaryotic system. The magnetosome is an invagination of the cell membrane that contains a specific set of proteins able to direct the synthesis of a nanometer-sized magnetite crystal. A well-conserved region called the magnetosome island (MAI) is known to be essential for magnetosome formation and contains most of the genes previously implicated in magnetosome formation. Here, we present a comprehensive functional analysis of the MAI genes in a magnetotactic bacterium, Magnetospirillum magneticum AMB-1. By characterizing MAI deletion mutants, we show that parts of its conserved core are not essential for magnetosome biogenesis and that nonconserved genes are important for crystal formation. Most importantly, we show that the mamAB gene cluster encodes for factors important for magnetosome membrane biogenesis, for targeting of proteins to this compartment and for several steps during magnetite production. Altogether, this genetic analysis defines the function of more than a dozen factors participating in magnetosome formation and shows that magnetosomes are assembled in a step-wise manner in which membrane biogenesis, magnetosome protein localization, and biomineralization are placed under discrete genetic control.

The HtrA/DegP family protease MamE is a bifunctional protein with roles in magnetosome protein localization and magnetite biomineralization.

Magnetotactic bacteria contain nanometre-sized, membrane-bound organelles, called magnetosomes, which are tasked with the biomineralization of small crystals of the iron oxide magnetite allowing the organism to use geomagnetic field lines for navigation. A key player in this process is the HtrA/DegP family protease MamE. In its absence, Magnetospirillum magneticum str AMB-1 is able to form magnetosome membranes but not magnetite crystals, a defect previously linked to the mislocalization of magnetosome proteins. In this work we use a directed genetic approach to find that MamE, and another predicted magnetosome-associated protease, MamO, likely function as proteases in vivo. However, as opposed to the complete loss of mamE where no biomineralization is observed, the protease-deficient variant of this protein still supports the initiation and formation of small, 20 nm-sized crystals of magnetite, too small to hold a permanent magnetic dipole moment. This analysis also reveals that MamE is a bifunctional protein with a protease-independent role in magnetosome protein localization and a protease-dependent role in maturation of small magnetite crystals. Together, these results imply the existence of a previously unrecognized 'checkpoint' in biomineralization where MamE moderates the completion of magnetite formation and thus committal to magneto-aerotaxis as the organism's dominant mode of navigating the environment.

C-terminal domain of the Uup ATP-binding cassette ATPase is an essential folding domain that binds to DNA.

The Uup protein belongs to a subfamily of soluble ATP-binding cassette (ABC) ATPases that have been implicated in several processes different from transmembrane transport of molecules, such as transposon precise excision. We have demonstrated previously that Escherichia coli Uup is able to bind DNA. DNA binding capacity is lowered in a truncated Uup protein lacking its C-terminal domain (CTD), suggesting a contribution of CTD to DNA binding. In the present study, we characterize the role of CTD in the function of Uup, on its overall stability and in DNA binding. To this end, we expressed and purified isolated CTD and we investigated the structural and functional role of this domain. The results underline that CTD is essential for the function of Uup, is stable and able to fold up autonomously. We compared the DNA binding activities of three versions of the protein (Uup, UupDeltaCTD and CTD) by an electrophoretic mobility shift assay. CTD is able to bind DNA although less efficiently than intact Uup and UupDeltaCTD. These observations suggest that CTD is an essential domain that contributes directly to the DNA binding ability of Uup.

Misic, a general deep learning-based method for the high-throughput cell segmentation of complex bacterial communities.

Studies of bacterial communities, biofilms and microbiomes, are multiplying due to their impact on health and ecology. Live imaging of microbial communities requires new tools for the robust identification of bacterial cells in dense and often inter-species populations, sometimes over very large scales. Here, we developed MiSiC, a general deep-learning-based 2D segmentation method that automatically segments single bacteria in complex images of interacting bacterial communities with very little parameter adjustment, independent of the microscopy settings and imaging modality. Using a bacterial predator-prey interaction model, we demonstrate that MiSiC enables the analysis of interspecies interactions, resolving processes at subcellular scales and discriminating between species in millimeter size datasets. The simple implementation of MiSiC and the relatively low need in computing power make its use broadly accessible to fields interested in bacterial interactions and cell biology.

Synthetic riboswitches that induce gene expression in diverse bacterial species.

We developed a series of ligand-inducible riboswitches that control gene expression in diverse species of Gram-negative and Gram-positive bacteria, including human pathogens that have few or no previously reported inducible expression systems. We anticipate that these riboswitches will be useful tools for genetic studies in a wide range of bacteria.

Deletion of the Escherichia coli uup gene encoding a protein of the ATP binding cassette superfamily affects bacterial competitiveness.

Bacteria use a variety of mechanisms for intercellular communication. Here we show that deletion of the uup gene, which encodes a soluble ATP binding cassette (ABC) ATPase, renders the mutant strain sensitive to its parent when they are grown together in the same medium. Our data suggest that the decrease in viability of the mutant is dependent on direct cell-to-cell contact with the parent strain. Furthermore, we show that the presence of intact Walker B motifs in Uup is required for immunity or resistance to the parental strain, suggesting that ATP hydrolysis is an important determinant of this phenotype.

Genome Sequence of Luminous Piezophile Photobacterium phosphoreum ANT-2200.

Bacteria of the genus Photobacterium thrive worldwide in oceans and show substantially varied lifestyles, including free-living, commensal, pathogenic, symbiotic, and piezophilic. Here, we present the genome sequence of a luminous, piezophilic Photobacterium phosphoreum strain, ANT-2200, isolated from a water column at 2,200 m depth in the Mediterranean Sea. It is the first genomic sequence of the P. phosphoreum group. An analysis of the sequence provides insight into the adaptation of bacteria to the deep-sea habitat.

Swimming behaviour and magnetotaxis function of the marine bacterium strain MO-1.

Magnetotactic bacteria (MTB) have the unique capacity to align and swim along the geomagnetic field lines downward to the oxic-anoxic interface in chemically stratified water columns and sediments. They are most abundant within the first few centimetres of sediments below the water-sediment interface. It is unknown how MTB penetrate into the sediment layer and swim in the pocket water, while their movements are restricted by the alignment along the magnetic field lines. Here we characterized the swimming behaviour of the marine fast-swimming magnetotactic ovoid bacterium MO-1.We found that it rotates around and translates along its short body axis to the magnetic north (northward). MO-1 cells swim forward constantly for a minimum of 1770 µm without apparent stopping. When encountering obstacles, MO-1 cells squeeze through or swim southward to circumvent the obstacles. The distance of southward swimming is short and inversely proportional to the magnetic field strength. Using a magnetic shielding device, we provide direct evidence that magnetotaxis is beneficial to MO-1 growth and becomes essential at low cell density. Environmental implications of the fast-swimming magnetotactic behaviour of magnetococci are discussed.

Magnetosomes: how do they stay in shape?

Biology textbooks taught us that eukaryotes could be easily distinguished from the far less complex bacteria. One criterion is that eukaryotes can segregate their DNA into a lipid-bounded compartment called a nucleus which isolates DNA replication and transcription from the rest of the cytoplasmic content. The second criterion is that eukaryotes can compartmentalize their cytoplasm so as to isolate specific pathways, enzymes and chemical reactions in membrane-bounded subcellular compartments called organelles. Time and high resolution imaging taught us that the story is a little more complicated. In fact, bacteria too can isolate cell components in subcellular compartments, including, in rare cases, their DNA. Clearly, some bacteria also have the capacity to isolate reactions that require a specific chemistry or that generate toxic byproducts within specialized organelles. Despite the significant advances made in the field of bacterial cell biology in the past 15 years, little is known about the mechanisms employed by bacteria to shape, position and segregate organelles, or how the cells can discriminate and address specific proteins to these compartments. Then, if eukaryotes did not invent organelles or the nucleus, who did? Are bacteria with a complex cell plan providing us with an unexpected opportunity to investigate how organelles came to exist? Is it possible that the mechanisms leading to cell compartmentalization in eukaryotes were invented by bacteria? Or, by studying how bacterial organelles are formed, will we discover new ways to control membrane curvature, target proteins, organize and segregate organelles?

Cell biology of prokaryotic organelles.

Mounting evidence in recent years has challenged the dogma that prokaryotes are simple and undefined cells devoid of an organized subcellular architecture. In fact, proteins once thought to be the purely eukaryotic inventions, including relatives of actin and tubulin control prokaryotic cell shape, DNA segregation, and cytokinesis. Similarly, compartmentalization, commonly noted as a distinguishing feature of eukaryotic cells, is also prevalent in the prokaryotic world in the form of protein-bounded and lipid-bounded organelles. In this article we highlight some of these prokaryotic organelles and discuss the current knowledge on their ultrastructure and the molecular mechanisms of their biogenesis and maintenance.

ATP hydrolysis is essential for the function of the Uup ATP-binding cassette ATPase in precise excision of transposons.

In Escherichia coli K-12, the RecA- and transposase-independent precise excision of transposons is thought to be mediated by the slippage of the DNA polymerase between the two short direct repeats that flank the transposon. Inactivation of the uup gene, encoding an ATP-binding cassette (ABC) ATPase, led to an important increase in the frequency of precise excision of transposons Tn10 and Tn5 and a defective growth of bacteriophage Mu. To provide insight into the mechanism of Uup in transposon excision, we purified this protein, and we demonstrated that it is a cytosolic ABC protein. Purified recombinant Uup binds and hydrolyzes ATP and undergoes a large conformational change in the presence of this nucleotide. This change affects a carboxyl-terminal domain of the protein that displays predicted structural homology with the socalled little finger domain of Y family DNA polymerases. In these enzymes, this domain is involved in DNA binding and in the processivity of replication. We show that Uup binds to DNA and that this binding is in part dependent on its carboxyl-terminal domain. Analysis of Walker motif B mutants suggests that ATP hydrolysis at the two ABC domains is strictly coordinated and is essential for the function of Uup in vivo.