Impact of Self-Association on the Architectural Properties of Bacterial Nucleoid Proteins

The chromosomal DNA of bacteria is folded into a compact body called the nucleoid, which is composed essentially of DNA (～80%), RNA (～10%), and a number of different proteins (～10%). These nucleoid proteins act as regulators of gene expression and influence the organization of the nucleoid by bridging, bending, or wrapping the DNA. These so-called architectural properties of nucleoid proteins are still poorly understood. For example, the reason why certain proteins compact the DNA coil in certain environments but make the DNA more rigid instead in other environments is the subject of ongoing debates. Here, we address the question of the impact of the self-association of nucleoid proteins on their architectural properties and try to determine whether differences in self-association are sufficient to induce large changes in the organization of the DNA coil. More specifically, we developed two coarse-grained models of proteins, which interact identically with the DNA but self-associate differently by forming either clusters or filaments in the absence of the DNA. We showed through Brownian dynamics simulations that self-association of the proteins dramatically increases their ability to shape the DNA coil. Moreover, we observed that cluster-forming proteins significantly compact the DNA coil (similar to the DNA-bridging mode of H-NS proteins), whereas filament-forming proteins significantly increase the stiffness of the DNA chain instead (similar to the DNA-stiffening mode of H-NS proteins). This work consequently suggests that the knowledge of the DNA-binding properties of the proteins is in itself not sufficient to understand their architectural properties. Rather, their self-association properties must also be investigated in detail because they might actually drive the formation of different DNA-protein complexes.     

The Bacterial Chromatin Protein HupA Can Remodel DNA and Associates with the Nucleoid in Clostridium difficile

The maintenance and organization of the chromosome plays an important role in the development and survival of bacteria. Bacterial chromatin proteins are architectural proteins that bind DNA and modulate its conformation, and by doing so affect a variety of cellular processes. No bacterial chromatin proteins of Clostridium difficile have been characterized to date.Here, we investigate aspects of the C. difficile HupA protein, a homologue of the histone-like HU proteins of Escherichia coli. HupA is a 10-kDa protein that is present as a homodimer in vitro and self-interacts in vivo. HupA co-localizes with the nucleoid of C. difficile. It binds to the DNA without a preference for the DNA G + C content. Upon DNA binding, HupA induces a conformational change in the substrate DNA in vitro and leads to compaction of the chromosome in vivo.The present study is the first to characterize a bacterial chromatin protein in C. difficile and opens the way to study the role of chromosomal organization in DNA metabolism and on other cellular processes in this organism.     

Defining the Role of ATP Hydrolysis in Mitotic Segregation of Bacterial Plasmids

Hydrolysis of ATP by partition ATPases, although considered a key step in the segregation mechanism that assures stable inheritance of plasmids, is intrinsically very weak. The cognate centromere-binding protein (CBP), together with DNA, stimulates the ATPase to hydrolyse ATP and to undertake the relocation that incites plasmid movement, apparently confirming the need for hydrolysis in partition. However, ATP-binding alone changes ATPase conformation and properties, making it difficult to rigorously distinguish the substrate and cofactor roles of ATP in vivo. We had shown that mutation of arginines R36 and R42 in the F plasmid CBP, SopB, reduces stimulation of SopA-catalyzed ATP hydrolysis without changing SopA-SopB affinity, suggesting the role of hydrolysis could be analyzed using SopA with normal conformational responses to ATP. Here, we report that strongly reducing SopB-mediated stimulation of ATP hydrolysis results in only slight destabilization of mini-F, although the instability, as well as an increase in mini-F clustering, is proportional to the ATPase deficit. Unexpectedly, the reduced stimulation also increased the frequency of SopA relocation over the nucleoid. The increase was due to drastic shortening of the period spent by SopA at nucleoid ends; average speed of migration per se was unchanged. Reduced ATP hydrolysis was also associated with pronounced deviations in positioning of mini-F, though time-averaged positions changed only modestly. Thus, by specifically targeting SopB-stimulated ATP hydrolysis our study reveals that even at levels of ATPase which reduce the efficiency of splitting clusters and the constancy of plasmid positioning, SopB still activates SopA mobility and plasmid positioning, and sustains near wild type levels of plasmid stability.     

Chromosome Transfer by Autonomous Transmissible Plasmids: the Role of the Bacterial Recombination (rec) System

The ability of autonomous transmissible plasmids or sex factors to transfer chromosomal genes to F⁻ recipient bacteria has been investigated by using a series of rec⁺ and recA⁻ donor strains. It is concluded that chromosome transfer by most sex factors is virtually dependent upon the functional integrity of the bacterial recombination system. However, evidence is presented that suggests the existence of plasmid-specified mechanisms of interaction with the chromosome which are independent of the bacterial recombination system.     

Polymeric Structures and Dynamic Properties of the Bacterial Actin AlfA

AlfA is a recently discovered DNA segregation protein from Bacillus subtilis that is distantly related to actin and the bacterial actin homologues ParM and MreB. Here we show that AlfA mostly forms helical 7/3 filaments, with a repeat of about 180 Å, that are arranged in three-dimensional bundles. Other polymorphic structures in the form of two-dimensional rafts or paracrystalline nets were also observed. Here AlfA adopted a 16/7 helical symmetry, with a repeat of about 387 Å. Thin polymers consisting of several intertwining filaments also formed. Observed helical symmetries of AlfA filaments differed from those of other members of the actin family: F-actin, ParM, or MreB. Both ATP and guanosine 5′-triphosphate are able to promote rapid AlfA filament formation with almost equal efficiencies. The helical structure is only preserved under physiological salt concentrations and at a pH between 6.4 and 7.4, the physiological range of the cytoplasm of B. subtilis. Polymerization kinetics are extremely rapid and compatible with a cooperative assembly mechanism requiring only two steps: monomer activation followed by elongation, making AlfA one of the most efficient polymerizing motors within the actin family. Phosphate release lags behind polymerization, and time-lapse total internal reflection fluorescence images of AlfA bundles are consistent with treadmilling rather than dynamic microtubule-like instability. High-pressure small angle X-ray scattering experiments reveal that the stability of AlfA filaments is intermediate between the stability of ParM and the stability of F-actin. These results emphasize that actin-like polymerizing machineries have diverged to produce a variety of filament geometries with diverse properties that are tailored for specific biological processes.     

The Population Biology of Bacterial Plasmids: A PRIORI Conditions for the Existence of Conjugationally Transmitted Factors

A mathematical model for the population dynamics of conjugationally transmitted plasmids in bacterial populations is presented and its properties analyzed. Consideration is given to nonbacteriocinogenic factors that are incapable of incorporation into the chromosome of their host cells, and to bacterial populations maintained in either continuous (chemostat) or discrete (serial transfer) culture. The conditions for the establishment and maintenance of these infectious extrachromosomal elements and equilibrium frequencies of cells carrying them are presented for different values of the biological parameters: population growth functions, conjugational transfer and segregation rate constants. With these parameters in a biologically realistic range, the theory predicts a broad set of physical conditions, resource concentrations and dilution rates, where conjugationally transmitted plasmids can become established and where cells carrying them will maintain high frequencies in bacterial populations. This can occur even when plasmid-bearing cells are much less fit (i.e., have substantially lower growth rates) than cells free of these factors. The implications of these results and the reality and limitations of the model are discussed and the values of its parameters in natural populations speculated upon.     

The Population Biology of Bacterial Plasmids: A PRIORI Conditions for the Existence of Mobilizable Nonconjugative Factors

A mathematical model for the population dynamics of nonconjugative plasmids that can be mobilized by conjugative factors is presented. In the analysis of the properties of this model, primary consideration is given to the conditions under which these nonself-transmissible extrachromosomal elements could become established and would be maintained in bacterial populations. The results of this analysis demonstrate the existence of conditions where, as a consequence of infectious transmission via mobilization, nonconjugative plasmids could become established and be maintained even when the bacteria carrying them have lower reproductive fitnesses than plasmid-free members of the population. However, these existence conditions are stringent and suggest therefore, that it is highly unlikely that plasmids of this type would become established and maintained without some direct selection favoring their carriage. The general implications of these results and limitations of the model are discussed. Brief consideration is also given to the implications of these theoretical findings to the problems of the spread of multiple antibiotic resistance plasmids (R-factors) and the risk of contaminating natural populations of bacteria with chimeric plasmids produced by work with recombinant DNA.     

Bacterial diversity in the snow over Tibetan Plateau Glaciers

Bacterial diversity and cell abundance in the snow of the four glaciers (Guoqu, Zadang, East Rongbuk and Palong No. 4) located in different climatic zones of the Tibetan Plateau were investigated through culture-independent molecular analysis of 16S rRNA gene clone library and flow cytometry approaches. Cell abundance ranged from 0.68 × 10³ to 720 × 10³ cells mL⁻¹, with higher values in the northern glaciers than in the southern ones. Bacterial diversity was unexpectedly high in the snow habitats of the world’s highest plateau, with 15 common genera distributed widely among the glaciers. The bacterial diversity in the snow at different glaciers was related to the surrounding environments. The Guoqu Glacier, to the north near the desert zone and with the lowest temperature, preserved more bacteria closely related to a cold environment and soil than the other glaciers. However, in the Palong No. 4 Glacier located in the south warm region around vegetation, most bacteria were phylogenetically related to plant-associated bacteria.     

Contribution of the Earthworm Lumbricus rubellus (Annelida,Oligochaeta) to the Establishment of Plasmids in Soil Bacterial Communities

The contribution of the carthworm Lumbricus rubellus in spreading plasmids from a nonindigenous bacterial species to the soil microbial community was studied with Escherichia coli strains as donor organisms. The selected donor strains harbored marker-gene tagged plasmids with different transfer properties and host ranges. Prototrophic benzoate degrading indigenous bacteria were analyzed as potential recipients. In filter-mating experiments, donor strains were mixed with bacterial cell consortia extracted from earthworm casts (feces) and incubated on nutrient agar at 28°C. Transfer was detected with the broad host range IncP plasmid pRP4luc; with the IncQ plasmid, pSUP104luc, but only when it was present in a mobilizing donor strain; and with the transposon delivery vector pUTlux. No transfer was detected with the nonmobilizable pUCluc and the mobilizable pSUP202luc, both of narrow host range. In microcosm studies with E. coli inoculated soil incubated at 12°C, transconjugants were only detected in casts of L. rubellus but not in bulk soil, indicating that the gut passage was a precondition for plasmid transfer. Plasmid pRP4luc was transferred at higher frequencies than detected in filter mating. Results of the filter matings were confirmed except that transfer of pUTlux could not be detected. The majority of transconjugants isolated in this study lost their acquired plasmid upon further cultivation. Stable transconjugants, however, were obtained and identified at the 16S rRNA gene level as members of the β- and γ-subgroups of Proteobacteria. Incubation of E. coli and selected transconjugants in soil microcosms with L. rubellus demonstrated that the gut passage resulted in a slight but significant reduction of ingested cells. In contrast to the donor strains, however, the population sizes of transconjugants in bulk soil and in casts did not decrease over time. This demonstrated that the transferred plasmids had established themselves in the soil microbial community.     

Light Structures Phototroph,Bacterial and Fungal Communities at the Soil Surface

The upper few millimeters of soil harbour photosynthetic microbial communities that are structurally distinct from those of underlying bulk soil due to the presence of light. Previous studies in arid zones have demonstrated functional importance of these communities in reducing soil erosion, and enhancing carbon and nitrogen fixation. Despite being widely distributed, comparative understanding of the biodiversity of the soil surface and underlying soil is lacking, particularly in temperate zones. We investigated the establishment of soil surface communities on pasture soil in microcosms exposed to light or dark conditions, focusing on changes in phototroph, bacterial and fungal communities at the soil surface (0–3 mm) and bulk soil (3–12 mm) using ribosomal marker gene analyses. Microbial community structure changed with time and structurally similar phototrophic communities were found at the soil surface and in bulk soil in the light exposed microcosms suggesting that light can influence phototroph community structure even in the underlying bulk soil. 454 pyrosequencing showed a significant selection for diazotrophic cyanobacteria such as Nostoc punctiforme and Anabaena spp., in addition to the green alga Scenedesmus obliquus. The soil surface also harboured distinct heterotrophic bacterial and fungal communities in the presence of light, in particular, the selection for the phylum Firmicutes. However, these light driven changes in bacterial community structure did not extend to the underlying soil suggesting a discrete zone of influence, analogous to the rhizosphere.     

High-resolution Structures of the Ligand Binding Domain of the Wild-type Bacterial Aspartate Receptor

The high-resolution structures of the wild-type periplasmic domain of the bacterial aspartate receptor have been determined in the absence and presence of bound aspartate to 1.85 and 2.2 Å resolution, respectively. As we reported earlier, in the refined structure of the complexed form of the crosslinked cysteine mutant receptor, the binding of the aspartate at the first site was mediated through four bridging water molecules while the second site showed an occupant electron density that best fit a sulfate group, which was present in the crystallization solution at high concentration. In the wild-type periplasmic domain structure two aspartate residues are bound per dimer, but with different occupancies. There exists a “strong” aspartate-binding site whose binding is again mediated by four water molecules while the second site contains aspartate whoseB-factor is about 10% higher, signifying weaker binding. The interaction between the second, “weaker” aspartate with the three ligand-binding arginine side-chains is slightly different from the first site. The major difference is that there are three water molecules mediating the binding of aspartate at the second site, whereas in the first site there are four bridging water molecules. The fact that aspartate-complexed crystals of the wild-type were grown with a large excess aspartate while the cross-linked crystals were grown with equal molar aspartate may explain the difference in the stoichiometry observed. The conservation of the four bridging water molecules in the strong aspartate site of both the cross-linked and wild-type periplasmic domain may reflect an important binding motif.The periplasmic domain in the apo form is a symmetrical dimer, in which each of the subunits is equivalent, and the two aspartate binding sites are identical. Upon the binding of aspartate, the subunits are no longer symmetrical. The main difference between the aspartate-bound and unbound forms is in a small, rigid-body rotation between the subunits within a dimer. The rotation is similar in both direction and magnitude in the crosslinked and wild-type periplasmic domains. The presence of the second aspartate in the wild-type structure does not make any additional rotation compared to the single-site binding. The conservation of the small angular changein vitrosuggests that the inter-subunit rotation may have relevance to the understanding of the mechanism of transmembrane signal transductionin vivo.     

Self-Assembly of the Bacterial Cytoskeleton-Associated RNA Helicase B Protein into Polymeric Filamentous Structures

The Escherichia coli RNA degradosome proteins are organized into a helical cytoskeletal-like structure within the cell. Here we describe the ATP-dependent assembly of the RhlB component of the degradosome into polymeric filamentous structures in vitro, which suggests that extended polymers of RhlB are likely to comprise a basic core element of the degradosome cytoskeletal structures.The RNA degradosome plays an essential role in normal RNA processing and degradation. Within the cell, the degradosome proteins (RNA helicase B [RhlB], RNase E, polynucleotide phosphorylase [PNPase], and enolase) (4, 13, 15, 16) are organized into coiled structures that resemble the pole-to-pole helical structures of the MreB and MinCDE bacterial cytoskeletal systems (4, 12, 13). However, the degradosomal structures are also present in cells that lack the MreB and MinCDE cytoskeletal elements, suggesting that the degradosomal structures may be part of an independent class of prokaryotic cytoskeletal elements (19-21).One of the degradosomal proteins, RhlB, is organized into similar helical cellular structures in cells that lack the other degradosome proteins (Fig. ​(Fig.11 A). In addition, RhlB recruits PNPase to the helical framework in the absence of other degradosome proteins, suggesting that the RhlB structures are core elements of the degradosomal cytoskeletal-like elements of the cell (Fig. ​(Fig.1B)1B) (20). The cellular RhlB structures could be generated in two ways: (i) individual RhlB molecules may bind to an as-yet-undefined underlying track, or (ii) RhlB may polymerize to form the filamentous helical structures independent of any underlying template.Open in a separate windowFIG. 1.The RhlB filamentous cytoskeletal-like structures. (A) Cellular organization of RhlB based on immunofluorescence microscopy using purified anti-RhlB antibody in the absence of RNase E filamentous elements in AT8 cells (rne^1-417), which fail to generate RNase E coiled structures because of the absence of the RNase E cytoskeletal localization domain (20). (B) Proposed model for the cytoskeletal-like organization of the RNA degradosome (modified from reference 20). Arcs depict the RNase E (blue) and RhlB (red) helical strands. It is not known whether the RNase E helical strand is formed by RNase E polymerization or by the association of RNase E with an unknown underlying cytoskeletal structure. Enolase (Eno) and PNPase are shown in gray. Molecular dimensions and stoichiometry of the proteins were arbitrarily chosen to simplify the figure. (C to H) Electron micrographs of uranyl acetate-stained RhlB filaments (C, E, and H) and RhlB sheets (D). Unless otherwise indicated, the sample contained 9 μM RhlB, 2 mM ATP, 5 mM MgCl₂, and 5 mM CaCl₂. (E) Calcium was omitted. (F) ATP was omitted. (G and H) ATP was replaced by ATPγS (G) or AMP-PNP (H). Samples were loaded on glow-discharged 300-mesh carbon-coated copper grids and then stained. Images were taken with a JEOL 100CX transmission electron microscope. Magnification, ×10,000 to 50,000.Here we report that RhlB can self-assemble into extended polymeric structures in vitro in a process that requires ATP binding but not ATP hydrolysis. It is likely that extended RhlB polymers such as those described here are the basic components of the RhlB filamentous helical elements that comprise the core of the degradosomal cytoskeletal structures of the Escherichia coli cell.Evidence that RhlB can self-assemble into filamentous polymeric structures came from electron microscopic studies of purified His-tagged RhlB negatively stained with 2% uranyl acetate. This staining showed large numbers of long uniform filamentous structures when the purified protein was incubated in the presence of ATP and Ca²⁺ (Fig. ​(Fig.1C).1C). The filaments were 25 ± 1.8 nm wide (n = 91; mean ± standard deviation) and were generally more than 10 μm long. Some wider sheets were also observed (Fig. ​(Fig.1D).1D). Optimal assembly of the RhlB filamentous structures required ATP and Ca²⁺, as shown by the observation that only occasional single structures were present when the polymerization reaction was carried out in the absence of Ca²⁺ (Fig. ​(Fig.1E)1E) or ATP (Fig. ​(Fig.1F).1F). The polymeric RhlB-His structures were observed with approximately similar frequencies when ATP was replaced by the nonhydrolyzable ATP analog adenosine 5′-(γ-thiotriphosphate) (ATPγS) or AMP-PNP (Fig. 1G and H).Cellular localization studies showed that the presence of the His tag did not interfere with the ability of RhlB to form the helical cellular structures. Thus, RhlB-His was present in extended helical filamentous structures that were indistinguishable from those formed by untagged RhlB (20, 21). Similarly, the RhlB-His structures recruited PNPase to the helical framework in a manner similar to untagged RhlB (20) (see Fig. S1 in the supplemental material).Immunogold staining showed that the filaments and sheets were decorated with gold particles when stained with mouse anti-His tag antibody and gold-labeled secondary antibody (Fig. ​(Fig.22 A to C), confirming that the structures were composed of RhlB. In contrast, the structures were not decorated with gold particles in the absence of the primary antibody or when mouse anti-His tag antibody was replaced by nonimmune mouse IgG (Fig. ​(Fig.2D2D and E). The polymeric structures were observed with C-terminally His-tagged RhlB, which is functional in terms of helicase activity (8), but not when the tag was present at the amino terminus of the protein, where the His tag may interfere with RhlB self-assembly.Open in a separate windowFIG. 2.Immunogold electron microscopy of RhlB structures. Samples were prepared as describe for Fig. ​Fig.1C,1C, except that the grids were stained with 2% uranyl acetate after exposure to primary and/or secondary antibodies as indicated. (A to C) RhlB structures decorated with 10-nm gold particles in samples stained with mouse anti-His tag monoclonal antibody and gold-labeled secondary antibody. (A) single filaments; (B) clustered filaments; (C) a single RhlB filament and RhlB sheet. Arrows indicate gold particles. (D) The primary mouse anti-His tag antibody was replaced by mouse IgG. (E) The primary antibody was omitted.Changes in light scattering were used to follow the course of polymerization and to compare polymerization conditions in a more quantitative way than is possible by electron microscopy. The initial rate of increase in scattering was used to estimate polymerization rate (see Table S1 in the supplemental material). Significant rates of polymerization were observed in the presence of ATP and Ca²⁺, whereas there was very little increase in light scattering in the absence of nucleotide and/or Ca²⁺ (Fig. ​(Fig.33 A). ADP was less effective than ATP, whereas AMP and cyclic AMP (cAMP) were inactive (Fig. ​(Fig.3B).3B). In the presence of Ca²⁺, the extent and rate of RhlB polymerization varied as a function of ATP concentration (Fig. ​(Fig.3C).3C). Millimolar concentrations of Ca²⁺ were required to produce a measurable rate of polymerization in the light scattering assay (Fig. ​(Fig.3A).3A). It is not known how these relatively high concentrations of Ca²⁺ promote the in vitro polymerization of RhlB and other cytoskeletal proteins, such as MreB and FtsZ (1, 11, 12, 14, 23).Open in a separate windowFIG. 3.RhlB polymerization. (A and B) RhlB polymerization as shown by 90° light scattering is indicated in arbitrary units (a.u.). RhlB polymerization was followed at room temperature in a 1-cm light path quartz cuvette using a Hitachi fluorometer (FL-2500) set to 400 V with excitation and emission wavelengths set at 455 nm and a slit width of 10 nm. The reaction (100 μl volume) was performed in a polymerization buffer (50 mM Tris, 50 mM KCl, 5 mM MgCl₂; pH 8) as indicated. (A) The sample contained 9 μM RhlB-His, 1 mM ATP, and either 10 mM CaCl₂, 7.5 mM CaCl₂, or no CaCl₂ (squares). In the lower three curves the samples lacked ATP or CaCl₂, as indicated. (B) The sample contained 9 μM RhlB-His, 7.5 mM CaCl₂, and 1 mM adenosine nucleotides: ATP, ADP, AMP, cAMP, AMP-PNP, and ATPγS. (C) The sample contained 9 μM RhlB-His, 7.5 mM CaCl₂, and ATP as indicated (1 mM, 0.75 mM, 0.5 mM, 0.25 mM, or 0.1 mM ATP). (D) Effects of Ca²⁺ concentration on RhlB sedimentation in the presence of 2 mM ATP. RhlB in the pellet, expressed as a percentage of total RhlB present in the polymerization reaction mixture, was plotted against calcium concentration. The insert shows an example of a Coomassie blue-stained gel of supernatant (S) and pellet (P) fractions from the sedimentation assay in the presence of ATP and Ca²⁺ (see results for ATP in Table ​Table11).The nonhydrolyzable ATP analogs ATPγS and AMP-PNP were approximately equivalent to ATP in promoting polymerization as monitored by the light scattering assay (Fig. ​(Fig.3B)3B) as well as in the electron microscopic studies. This suggests that nucleotide binding, but not hydrolysis, is required to promote RhlB polymerization. In this regard, RhlB resembles a number of other proteins, including F-actin, MreB, and MinD, where polymerization is induced by nucleotide binding (2, 5, 6, 18, 22). In these systems, subsequent ATP hydrolysis induces depolymerization, providing the basis for the dynamic behavior of the polymers within the cell. RhlB is an RNA-dependent ATPase (7), but it is not yet known whether ATP hydrolysis is associated with depolymerization in the RhlB system.Similar results were obtained when the extent of polymerization was monitored by a sedimentation assay, measuring the proportion of RhlB in the pellet fraction after centrifugation at 278,000 × g for 10 min (Table ​(Table1;1; Fig. ​Fig.3D).3D). Essentially all of the protein was sedimentable at pH 8 in the presence of Ca²⁺ and ATP. RhlB sedimentation returned to background levels when EGTA or EDTA was added to the reaction mixture (Table ​(Table1),1), confirming the Ca²⁺ requirement for RhlB polymerization in the electron microscopic and light scattering analyses. ATPγS was equivalent to ATP in the sedimentation assay, confirming the results described above. The relatively high background of RhlB sedimentation was not affected by prespinning the samples prior to addition of nucleotides and/or Ca²⁺.

TABLE 1.

Sedimentation assay for RhlB polymerization

Role of Bacterial Surface Structures on the Interaction of Klebsiella pneumoniae with Phagocytes

Phagocytosis is a key process of the immune system. The human pathogen Klebsiella pneumoniae is a well known example of a pathogen highly resistant to phagocytosis. A wealth of evidence demonstrates that the capsule polysaccharide (CPS) plays a crucial role in resistance to phagocytosis. The amoeba Dictyostelium discoideum shares with mammalian macrophages the ability to phagocytose and kill bacteria. The fact that K. pneumoniae is ubiquitous in nature and, therefore, should avoid predation by amoebae, poses the question whether K. pneumoniae employs similar means to counteract amoebae and mammalian phagocytes. Here we developed an assay to evaluate K. pneumoniae-D. discoideum interaction. The richness of the growth medium affected the threshold at which the cps mutant was permissive for Dictyostelium and only at lower nutrient concentrations the cps mutant was susceptible to predation by amoebae. Given the critical role of bacterial surface elements on host-pathogen interactions, we explored the possible contribution of the lipopolysaccharide (LPS) and outer membrane proteins (OMPs) to combat phagoyctosis by D. discoideum. We uncover that, in addition to the CPS, the LPS O-polysaccharide and the first core sugar participate in Klebsiella resistance to predation by D. discoideum. K. pneumoniae LPS lipid A decorations are also necessary to avoid predation by amoebae although PagP-dependent palmitoylation plays a more important role than the lipid A modification with aminoarabinose. Mutants lacking OMPs OmpA or OmpK36 were also permissive for D. discoideium growth. Except the LPS O-polysaccharide mutants, all mutants were more susceptible to phagocytosis by mouse alveolar macrophages. Finally, we found a correlation between virulence, using the pneumonia mouse model, and resistance to phagocytosis. Altogether, this work reveals novel K. pneumoniae determinants involved in resistance to phagocytosis and supports the notion that Dictyostelium amoebae might be useful as host model to measure K. pneumoniae virulence and not only phagocytosis.     

Investigation of the Cell Surface Structures of Agrobacteria Involved in Bacterial and Plant Interactions

Agrobacterial cells produced straight microfibrils not only when in contact with wheat seedling roots, but also when in contact with each other. After 2 h of incubation, agrobacterial cells were found to form aggregates, in which the cells were in contact either directly or through thick straight microfibrils (bridges) of an unknown composition. The majority of the microfibrils were susceptible to attack by cellulase, although some of them showed resistance to this enzyme. Like the wild-type flagellated agrobacteria, their bald mutants produced long straight microfibrils. The cell surface structures of agrobacteria were examined by labeling them immunocytochemically with colloidal gold–conjugated antibodies against O-specific lipopolysaccharides, Vir proteins, and cellulase. Agrobacterial cells treated with acetosyringone and brought into contact were found to contain subpolar and polar cell surface structures. Antibodies against the VirB2 protein were able to interact with a tuft of thin microfibrils located on one pole of the agrobacterial cell whose virgenes were induced by acetosyringone but were unable to interact with the surface structures of the agrobacterial cells aggregated in liquid medium in the absence of wheat seedlings.     

Protection of Bacterial Spores in Space, a Contribution to the Discussion on Panspermia

Spores of Bacillus subtilis were exposed to space in theBIOPAN facility of the European Space Agency onboard of the Russian Earth-orbiting FOTON satellite. The spores were exposed either in dry layers without any protecting agent, or mixed withclay, red sandstone, Martian analogue soil or meteorite powder,in dry layers as well as in so-called `artificial meteorites', i.e. cubes filled with clay and spores in naturally occurring concentrations. After about 2 weeks in space, their survival was tested from the number of colony formers. Unprotected spores in layers open to space or behind a quartz window were completely or nearly completely inactivated (survival rates in most cases10^-6). The same low survival was obtained behind a thin layer of clay acting as an optical filter. The survival rate was increased by 5 orders of magnitude and more, if the spores in the dry layer were directly mixed with powder of clay,rock or meteorites, and up to 100% survival was reached in soilmixtures with spores comparable to the natural soil to spore ratio. These data confirm the deleterious effects of extraterrestrial solar UV radiation. Thin layers of clay, rock or meteorite are only successful in UV-shielding, if they are indirect contact with the spores. The data suggest that in a scenario of interplanetary transfer of life, small rock ejecta ofa few cm in diameter could be sufficiently large to protectbacterial spores against the intense insolation; however, micron-sized grains, as originally requested by Panspermia, may notprovide sufficient protection for spores to survive. The data arealso pertinent to search for life on Mars and planetaryprotection considerations for future missions to Mars.     

Bacterial diversity in the mountains of South-West China: Climate dominates over soil parameters

Certain patterns in soil bacterial diversity and community composition have become evident from metagenomics studies on a range of scales, from various parts of the world. For example, soil pH has generally been seen as dominating variation in bacterial diversity, above all other soil and climate parameters. It is important however to test the generality of these relationships by studying previously un-sampled areas. We compared soil bacterial diversity and community composition under a wide range of climatic and edaphic conditions in mountainous Yunnan Province, SW China. Soil samples were taken from a range of primary forest types and altitudes, reflecting the great variation of forest environments in this region. From each soil sample, DNA was extracted and pyrosequenced for bacterial 16S rRNA gene identification. In contrast to other recent studies from other parts of the world, pH was a weaker predictor of bacterial community composition and diversity than exchangeable Ca²⁺ concentration, and also the more poorly defined environmental parameter of elevation. Samples from within each forest type clustered strongly, showing the distinctive pattern of their microbial communities on a regional scale. It is clear that on a regional scale in a very heterogeneous environment, additional factors beyond pH can emerge as more important in determining bacterial diversity.     

Anaerobic Methane Oxidation and Sulfate Reduction in Bacterial Mats on Coral-Like Carbonate Structures in the Black Sea

A detailed study of the processes of anaerobic methane oxidation and sulfate reduction in the bacterial mats occurring on coral-like carbonate structures in the region of methane seeps in the Black Sea, as well as of the phenotypic diversity of sulfate-reducing bacteria developing in this zone, has been performed. The use of the radioisotopic method shows the microbial mat structure to be heterogeneous. The peak activity of the two processes was revealed when a mixture of the upper (dark) and underlying (intensely pink) layers was introduced into an incubation flask, which confirms the suggestion that methanotrophic archaea and sulfate-reducing bacteria closely interact in the process of anaerobic methane oxidation. Direct correlation between the rate of anaerobic methane oxidation and the methane and electron acceptor concentrations in the medium has been experimentally demonstrated. Several enrichment and two pure cultures of sulfate-reducing bacteria have been obtained from the near-bottom water and bacterial mats. Both strains were found to completely oxidize the substrates to CO₂ and H₂S. The bacteria grow at temperatures ranging from −1 to 18 (24)°C, with an optimum in the 10–18°C range, and require the presence of 1.5–2.5% NaCl and 0.07–0.2% MgCl 2⋅6H₂O. Regarding the aggregate of their phenotypic characteristics (cell morphology, spectrum of growth substrates, the capacity for complete oxidation), the microorganisms isolated have no analogues among the psychrophilic sulfate-reducing bacteria already described. The results obtained demonstrate the wide distribution of psychrophilic sulfate-reducing bacteria in the near-bottom water and bacterial mats covering the coral-like carbonate structures occurring in the region of methane seeps in the Black Sea, as well as the considerable catabolic potential of this physiological group of psychrophilic anaerobes in deep-sea habitats__________Translated from Mikrobiologiya, Vol. 74, No. 3, 2005, pp. 420–429.Original Russian Text Copyright © 2005 by Pimenov, Ivanova.