Isolation of Thermovenabulum gondwanense from a French hot spring and emended description of the species.

An anaerobic thermophilic bacterium designated CA9F1 was isolated from a thermal spring in France. Strain CA9F1 was observed to grow at temperatures between 55 and 70 °C (optimum 65 °C) and at pH between 6.8 and 9.5 (optimum pH 7.4). Strain CA9F1 does not require salt for growth (0-10 g l(-1) NaCl), with an optimum at 1 g l(-1). The DNA G+C content was determined to be 38.5 mol% (Tm). The major cellular fatty acids identified were C15:0, C16:0, C17:0 iso. Based on phenotypic, chemotaxonomic and genotypic properties, strain CA9F1 was identified as Thermovenabulum gondwanense and this species was studied in more detail. Strain CA9F1 is a Gram-positive bacterium which forms a complex and regular multilayered cell wall structure, here characterised as being due to the presence of an S-layer. The network covers the entire cell surface and forms a hexagonal structure resembling that observed for Deinococcus radiodurans. The main protein component of the S-layer possesses domains comparable to that of the S-layer protein of Halothermothrix orenii. The characteristics of the strain were compared to that of T. gondwanese R270(T) isolated from microbial mats thriving in the thermal waters of a Great Artesian Basin bore runoff channel at 66 °C, in Australia. Significant differences were observed between CA9F1 and the type strain. One of the major physiological differences is the inability of CA9F1 to reduce Fe(III). An emended description of T. gondwanense is given.

Non classical secretion systems.

Bacteria use molecular machines or weapons to colonize, invade or fight other bacteria and eukaryotic cells. In addition to these various secretion systems, two different systems that release bacterial compounds have also been described. The first one corresponds to membrane vesicle formation and to long distance delivery of membrane or soluble components. The second system is dependent of the expression of the colicin lysis genes known for releasing cytoplasmic colicins as well as other soluble proteins. Both systems will be described thereafter.

Architecture of a flagellar apparatus in the fast-swimming magnetotactic bacterium MO-1.

The bacterial flagellum is a motility organelle that consists of a rotary motor and a helical propeller. The flagella usually work individually or by forming a loose bundle to produce thrust. However, the flagellar apparatus of marine bacterium MO-1 is a tight bundle of seven flagellar filaments enveloped in a sheath, and it has been a mystery as to how the flagella rotate smoothly in coordination. Here we have used electron cryotomography to visualize the 3D architecture of the sheathed flagella. The seven filaments are enveloped with 24 fibrils in the sheath, and their basal bodies are arranged in an intertwined hexagonal array similar to the thick and thin filaments of vertebrate skeletal muscles. This complex and exquisite architecture strongly suggests that the fibrils counter-rotate between flagella in direct contact to minimize the friction of high-speed rotation of individual flagella in the tight bundle within the sheath to enable MO-1 cells to swim at about 300 µm/s.

Complex spatial organization and flagellin composition of flagellar propeller from marine magnetotactic ovoid strain MO-1.

Marine magnetotactic ovoid bacterium MO-1 is capable of swimming along the geomagnetic field lines by means of its two sheathed flagellar bundles at a speed up to 300 µm/s. In this study, by using electron microscopy, we showed that, in each bundle, six individual flagella were organized in hexagon with a seventh in the middle. We identified 12 flagellin paralogs and 2 putative flagellins in the genome of MO-1. Among them, 13 were tandemly located on an ~ 17-kb segment while the 14th was on a separated locus. Using reverse transcription PCR and quantitative PCR, we found that all the 14 flagellin or putative flagellin genes were transcribed and that 2 of them were more abundantly expressed than others. A nLC (nanoliquid chromatography)-ESI (electrospray ionization)-MS/MS (mass spectrometry/mass spectrometry) mass spectrometry analysis identified all the 12 flagellin proteins in three glycosylated polypeptide bands resolved by one-dimensional denaturing polyacrylamide gel electrophoresis and 10 of them in 21 spots obtained by means of two-dimensional electrophoresis of flagellar extracts. Most spots contained more than one flagellin, and eight of the ten identified flagellins existed in multiple isoforms. Taken together, these results show unprecedented complexity in the spatial organization and flagellin composition of the flagellar propeller. Such architecture is observed only for ovoid-coccoid, bilophotrichously flagellated magnetotactic bacteria living in marine sediments, suggesting a species and environmental specificity.

Cdk and the anillin homolog Bud4 define a new pathway regulating septin organization in yeast.

In budding yeast, the cortical structure formed by the septins is remodeled at the onset of mitotic exit and delineates a specialized compartment dedicated to cytokinesis. How this septin function is spatially and timely regulated remains poorly understood. In this study, we report a role of the anillin-like protein Bud4 in the formation and the disassembly of the double ring structure formed by the septins at the time of cytokinesis. Bud4 acts with Bud3 in this pathway and in parallel with septin phosphorylation by the p21-activated kinase Cla4 and the septin-dependent kinase Gin4. In addition, we show that the function of Bud4 is regulated by the cyclin-dependent protein kinase Cdk1, the master regulator of cell cycle progression. This result suggests that the Cdks, or a locally specific pool of the kinase, may have a role past mitotic exit.

Magnetotactic bacteria in microcosms originating from the French Mediterranean Coast subjected to oil industry activities.

Magnetotactic bacteria (MTB) mineralize nanosized magnetite or greigite crystals within cells and thus play an important role in the biogeochemical process. Despite decades of research, knowledge of MTB distribution and ecology, notably in areas subjected to oil industry activities, is still limited. In the present study, we investigated the presence of MTB in the Gulf of Fos, French Mediterranean coast, which is subjected to intensive oil industry activities. Microcosms containing sediments/water (1:2, v/v) from several sampling sites were monitored over several weeks. The presence of MTB was revealed in five of eight sites. Diverse and numerous MTB were revealed particularly from one site (named CAR), whilst temporal variations of a homogenous magnetotactic cocci population was shown within the LAV site microcosm over a 4-month period. Phylogenetic analysis revealed that they belonged to Alphaproteobacteria, and a novel genus from the LAV site was evidenced. Among the physicochemical parameters measured, a correlation was shown between the variation of MTB abundance in microcosms and the redox state of sulphur compounds.

The YvcK protein is required for morphogenesis via localization of PBP1 under gluconeogenic growth conditions in Bacillus subtilis.

The YvcK protein was previously shown to be dispensable when B. subtilis cells are grown on glycolytic carbon sources but essential for growth and normal shape on gluconeogenic carbon sources. Here, we report that YvcK is localized as a helical-like pattern in the cell. This localization seems independent of the actin-like protein, MreB. A YvcK overproduction restores a normal morphology in an mreB mutant strain when bacteria are grown on PAB medium. Reciprocally, an additional copy of mreB restores a normal growth and morphology in a yvcK mutant strain when bacteria are grown on a gluconeogenic carbon source like gluconate. Furthermore, as already observed for the mreB mutant, the deletion of the gene encoding the penicillin-binding protein PBP1 restores growth and normal shape of a yvcK mutant on gluconeogenic carbon sources. The PBP1 is delocalized in an mreB mutant grown in the absence of magnesium and in a yvcK mutant grown on gluconate medium. Interestingly, its proper localization can be rescued by YvcK overproduction. Therefore, in gluconeogenic growth conditions, YvcK is required for the correct localization of PBP1 and hence for displaying a normal rod shape.

Calcium ion-mediated assembly and function of glycosylated flagellar sheath of marine magnetotactic bacterium.

Flagella of some pathogens or marine microbes are sheathed by an apparent extension of the outer cell membrane. Although flagellar sheath has been reported for almost 60 years, little is known about its function and the mechanism of its assembly. Recently, we have observed a novel type of sheath that encloses a flagellar bundle, instead of a single flagellum, in a marine magnetotactic bacterium MO-1. Here, we reported isolation and characterization of the sheath which can be described as a six-start, right-handed helical tubular structure with a diameter of about 100 nm, and a pitch of helix of about 260 nm. By proteomic, microscopic and immunolabelling analyses, we showed that the sheath of MO-1 consists of glycoprotein with an apparent molecular mass > 350 kDa. This protein, named sheath-associated protein (Sap), shows homology with bacterial adhesins and eukaryotic calcium-dependent adherent proteins (cadherin). Most importantly, we showed that calcium ions mediate the assembly of the tubular-shaped sheath and disintegration of the sheath was deleterious for smooth swimming of MO-1 cells. The disintegrated sheath was efficiently reconstituted in vitro by adding calcium ions. Altogether, these results demonstrate a novel bacterial Ca(2+) -dependent surface architecture, which is essential for bacterial swimming.

mRNA deep sequencing reveals 75 new genes and a complex transcriptional landscape in Mimivirus.

Mimivirus, a virus infecting Acanthamoeba, is the prototype of the Mimiviridae, the latest addition to the nucleocytoplasmic large DNA viruses. The Mimivirus genome encodes close to 1000 proteins, many of them never before encountered in a virus, such as four amino-acyl tRNA synthetases. To explore the physiology of this exceptional virus and identify the genes involved in the building of its characteristic intracytoplasmic "virion factory," we coupled electron microscopy observations with the massively parallel pyrosequencing of the polyadenylated RNA fractions of Acanthamoeba castellanii cells at various time post-infection. We generated 633,346 reads, of which 322,904 correspond to Mimivirus transcripts. This first application of deep mRNA sequencing (454 Life Sciences [Roche] FLX) to a large DNA virus allowed the precise delineation of the 5' and 3' extremities of Mimivirus mRNAs and revealed 75 new transcripts including several noncoding RNAs. Mimivirus genes are expressed across a wide dynamic range, in a finely regulated manner broadly described by three main temporal classes: early, intermediate, and late. This RNA-seq study confirmed the AAAATTGA sequence as an early promoter element, as well as the presence of palindromes at most of the polyadenylation sites. It also revealed a new promoter element correlating with late gene expression, which is also prominent in Sputnik, the recently described Mimivirus "virophage." These results-validated genome-wide by the hybridization of total RNA extracted from infected Acanthamoeba cells on a tiling array (Agilent)--will constitute the foundation on which to build subsequent functional studies of the Mimivirus/Acanthamoeba system.

Mimivirus and Mimiviridae: giant viruses with an increasing number of potential hosts, including corals and sponges.

Mimivirus, a giant DNA virus (i.e. "girus") infecting species of the genus Acanthamoeba, was first identified in 2003. With a particle size of 0.7microm in diameter, and a genome size of 1.2Mb encoding more than 900 proteins, it is the most complex virus described to date. Beyond its unusual size, the Mimivirus genome was found to contain the first viral homologues of many genes thought to be the trademark of cellular organisms, such as central components of the translation apparatus. These findings revived the debate on the origin of DNA viruses, and the role they might have played in the emergence of eukaryotes. Published and ongoing studies on Mimivirus continue to lead to unexpected findings concerning a variety of aspects, such as the structure of its particle, unique features of its replication cycle, or the distribution and abundance of Mimivirus relatives in the oceans. Following a summary of these recent findings, we present preliminary results suggesting that octocorals might have come in close contact with an ancestor of Mimivirus, and that modern sponges might be host to a yet unidentified, even larger, member of the Mimiviridae.

Isolation and characterization of a magnetotactic bacterial culture from the Mediterranean Sea.

The widespread magnetotactic bacteria have the peculiar capacity of navigation along the geomagnetic field. Despite their ubiquitous distribution, only few axenic cultures have been obtained worldwide. In this study, we reported the first axenic culture of magnetotactic bacteria isolated from the Mediterranean Sea. This magneto-ovoid strain MO-1 grew in chemically defined O(2) gradient minimal media at the oxic-anoxic transition zone. It is phylogenetically related to Magnetococcus sp. MC-1 but might represent a novel genus of Proteobacteria. Pulsed-field gel electrophoresis analysis indicated that the genome size of the MO-1 strain is 5 ± 0.5 Mb, with four rRNA operons. Each cell synthesizes about 17 magnetosomes within a single chain, two phosphorous-oxygen-rich globules and one to seven lipid storage granules. The magnetosomes chain seems to divide in the centre during cell division giving rise to two daughter cells with an approximately equal number of magnetosomes. The MO-1 cell possesses two bundles of seven individual flagella that were enveloped in a unique sheath. They swam towards the north pole with a velocity up to 300 µm per second with frequent change from right-hand to left-hand helical trajectory. Using a magneto-spectrophotometry assay we showed that MO-1 flagella were powered by both proton-motive force and sodium ion gradient, which is a rare feature among bacteria.

Organisation and function of the Phaeospirillum molischianum photosynthetic apparatus.

We have investigated the organisation of the photosynthetic apparatus in Phaeospirillum molischianum, using biochemical fractionation and functional kinetic measurements. We show that only a fraction of the ATP-synthase is present in the membrane regions which contain most of the photosynthetic apparatus and that, despite its complicated stacked structure, the intracytoplasmic membrane delimits a single connected space. We find that the diffusion time required for a quinol released by the reaction centre to reach a cytochrome bc1 complex is about 260 ms. On the other hand, the reduction of the cytochrome c chain by the cytochrome bc1 complex in the presence of a reduced quinone pool occurs with a time constant of about 5 ms. The overall turnover time of the cyclic electron transfer is about 25 ms in vivo under steady-state illumination. The sluggishness of the quinone shuttle appears to be compensated, at least in part, by the size of the quinone pool. Together, our results show that P. molischianum contains a photosynthetic system, with a very different organisation from that found in Rhodobacter sphaeroides, in which quinone/quinol diffusion between the RC and the cytochrome bc1 is likely to be the rate-limiting factor for cyclic electron transfer.

Cross talk between type III secretion and flagellar assembly systems in Pseudomonas aeruginosa.

Pseudomonas aeruginosa cytotoxicity is linked to a type III secretion system (T3SS) that delivers effectors into the host cell. We show here that a negative cross-control exists between T3SS and flagellar assembly. We observed that, in a strain lacking flagella, T3SS gene expression, effector secretion, and cytotoxicity were increased. Conversely, we revealed that flagellar-gene expression and motility were decreased in a strain overproducing ExsA, the T3SS master regulator. Interestingly, a nonmotile strain lacking the flagellar filament (DeltafliC) presented a hyperefficient T3SS and a nonmotile strain assembling flagella (DeltamotAB) did not. More intriguingly, a strain lacking motCD genes is a flagellated strain with a slight defect in swimming. However, in this strain, T3SS gene expression was up-regulated. These results suggest that flagellar assembly and/or mobility antagonizes the T3SS and that a negative cross talk exists between these two systems. An illustration of this is the visualization by electron microscopy of T3SS needles in a nonmotile P. aeruginosa strain, needles which otherwise are not detected. The molecular basis of the cross talk is complex and remains to be elucidated, but proteins like MotCD might have a crucial role in signaling between the two processes. In addition, we found that the GacA response regulator negatively affects the T3SS. In a gacA mutant, the T3SS effector ExoS is hypersecreted. Strikingly, GacA was previously reported as a positive regulator for motility. Globally, our data document the idea that some virulence factors are coordinately but inversely regulated, depending on the bacterial colonization phase and infection types.

Polar positional information in Escherichia coli spherical cells.

Shigella surface protein IcsA and its cytoplasmic derivatives are localized to the old pole of rod-shaped cells when expressed in Escherichia coli. In spherical mreB cells, IcsA is targeted to ectopic sites and close to one extremity of actin-like MamK filament. To gain insight into the properties of the sites containing polar material, we studied the IcsA localization in spherical cells. GFP was exported into the periplasm via the Tat pathway and used as a periplasmic space marker. GFP displayed zonal fluorescence in both mreB and rodA-pbpA spherical E. coli cells, indicating an uneven periplasmic space. Deconvolution images revealed that the cytoplasmic IcsA fused to mCherry was localized outside or at the edge of the GFP zones. These observations strongly suggest that polar material is restricted to the positions where the periplasm possesses particular structural or biochemical properties.

Biogenesis of actin-like bacterial cytoskeletal filaments destined for positioning prokaryotic magnetic organelles.

Magnetosomes comprise a magnetic nanocrystal surrounded by a lipid bilayer membrane. These unique prokaryotic organelles align inside magnetotactic bacterial cells and serve as an intracellular compass allowing the bacteria to navigate along the geomagnetic field in aquatic environments. Cryoelectron tomography of Magnetospirillum strains has revealed that the magnetosome chain is surrounded by a network of filaments that may be composed of MamK given that the filaments are absent in the mamK mutant cells. The process of the MamK filament assembly is unknown. Here we prove the authenticity of the MamK filaments and show that MamK exhibits linear distribution inside Magnetospirillum sp. cells even in the area without magnetosomes. The mamK gene alone is sufficient to direct the synthesis of straight filaments in Escherichia coli, and one extremity of the MamK filaments is located at the cellular pole. By using dual fluorescent labeling of MamK, we found that MamK nucleates at multiple sites and assembles into mosaic filaments. Time-lapse experiments reveal that the assembly of the MamK filaments is a highly dynamic and kinetically asymmetrical process. MamK bundles might initiate the formation of a new filament or associate to one preexistent filament. Our results demonstrate the mechanism of biogenesis of prokaryotic cytoskeletal filaments that are structurally and functionally distinct from the known MreB and ParM filaments. In addition to positioning magnetosomes, other hypothetical functions of the MamK filaments in magnetotaxis might include anchoring magnetosomes and being involved in magnetic reception.

Isolation and characterization of novel marine Roseobacter clade members producing unique intracellular chromium-rich aggregates.

The marine Roseobacter clade comprises one of the largest fractions of heterotrophic marine bacteria and accounts for about 16% of 16S rRNA gene clones retrieved from marine bacterioplankton. Their global distribution seems to be related to oceanic water masses and their environmental and biogeochemical properties. In this study, we report isolation and characterization of novel Roseobacter clade members from the Yellow Sea, China. Phylogenetic analysis of 16S rRNA gene sequences reveals that the new isolates (YSCB1, YSCB2, YSCB3 and YSCB4) are closely related to uncultured Arctic seawater bacterium R7967 (99.57-100% sequence identity) and to the cultured Roseobacter sp. DSS-1 (99.27-99.76% sequence identity) isolated from the southeastern coastal water of the USA. Interestingly, YSCB strains possess unique intracellular chromium-containing aggregates. Therefore, these novel Roseobacter clade members exhibit a peculiar property in mineral biogeneration.

Immunocytochemical localization of scorpion digestive lipase.

The scorpion hepatopancreas consists of digestive diverticula and interstitial tissue. A digestive diverticulum is composed of two differentiated cell types: the secretory zymogene-like cells and the digestive cells which are the most abundant. The scorpion digestive lipase (SDL) has been previously purified from scorpion hepatopancreas, but its cellular localization has not yet been established. Polyclonal antibodies specific to SDL were prepared and used in immunofluorescence and immunogold techniques to determine the cellular location of SDL. Our results clearly established that SDL was detected intracellularly in specific vesicles tentatively named (SDL+) granules of the digestive cells. No immunolabelling was observed in secretory zymogene-like cells. This immunocytolocalization indicates that lipid digestion might occur in specific granules inside the digestive cells, as suggested by previous studies on the scorpion digestive process.

Architecture of the native photosynthetic apparatus of Phaeospirillum molischianum.

The ubiquity and importance of photosynthetic organisms in nature has made the molecular mechanisms of photosynthesis a widely studied subject at both structural and functional levels. A current challenge is to understand the supramolecular assembly of the proteins involved in photosynthesis in native membranes. We have used atomic force microscopy to study the architecture of the photosynthetic apparatus and analyze the structure of single molecules in chromatophores of Phaeospirillum molischianum. Core complexes are formed by the reaction center enclosed by an elliptical light harvesting complex 1. LH2 are octameric rings, assembled either with cores or in hexagonally packed LH2 antenna domains. The symmetry mismatch caused by octameric LH2 packing in a hexagonal lattice, that could be avoided in a square lattice, suggests lipophobic effects rather than specific inter-molecular interactions drive protein organization. The core and LH2 complexes are organized to form a supramolecular assembly reminiscent to that found in Rhodospirillum photometricum, and very different from that observed in Rhodobacter sphaeroides, Rb. blasticus, and Blastochloris viridis.

XcpX controls biogenesis of the Pseudomonas aeruginosa XcpT-containing pseudopilus.

Pseudomonas aeruginosa is an opportunistic gram-negative pathogen equipped with multiple secretion systems. The type II secretion machinery (Xcp secreton) is involved in the release of toxins and enzymes. The Xcp secreton is a multiprotein complex, and most of its components share homology with proteins involved in type IV pili biogenesis. Among them, the XcpT-X pseudopilins possess characteristics of the major constituent of the type IV pili, the pilin PilA. We have shown previously that XcpT can be assembled in a multifibrillar structure that was called the pseudopilus. By using two different microscopic approaches, we show here that the pseudopili are preferentially isolated fibers rather than tight bundles. Moreover, none of the other four pseudopilins are able to form a pseudopilus, suggesting that the assembly of such a structure is a unique property of XcpT. Moreover, we show that 5 of the 12 Xcp proteins are not required for pseudopilus biogenesis, whereas they are for type II secretion. Most interestingly, we showed that one pseudopilin, XcpX, controls the assembly of XcpT into a pseudopilus. Indeed, when the number of XcpX subunits increases, the length of the pseudopilus decreases. Conversely, in the absence of XcpX, the pseudopilus length is abnormally long. Our results indicate that XcpT and XcpX directly interact with each other. Furthermore, this interaction induces a clear destabilization of XcpT. The interaction between XcpT and XcpX could be part of the molecular mechanism underlying the dynamic control of pseudopilus elongation, which could be crucial for type II-dependent protein secretion.

Watching the photosynthetic apparatus in native membranes.

Over the last 9 years, the structures of the various components of the bacterial photosynthetic apparatus or their homologues have been determined by x-ray crystallography to at least 4.8-A resolution. Despite this wealth of structural information on the individual proteins, there remains an urgent need to examine the architecture of the photosynthetic apparatus in intact photosynthetic membranes. Information on the arrangement of the different complexes in a native system will help us to understand the processes that ensure the remarkably high quantum efficiency of the system. In this work we report images obtained with an atomic force microscope of native photosynthetic membranes from the bacterium Rhodospirillum photometricum. Several proteins can be seen and identified at molecular resolution, allowing the analysis and modeling of the lateral organization of multiple components of the photosynthetic apparatus within a native membrane. Analysis of the distribution of the complexes shows that their arrangement is far from random, with significant clustering both of antenna complexes and core complexes. The functional significance of the observed distribution is discussed.