Lysogeny and lytic viral production during a bloom of the cyanobacterium Synechococcus spp

Lytic viral production and lysogeny were investigated in cyanobacteria and heterotrophic bacteria during a bloom of Synechococcus spp. in a pristine fjord in British Columbia, Canada. Triplicate seawater samples were incubated with and without mitomycin C and the abundances of heterotrophic bacteria, cyanobacteria, total viruses and infectious cyanophage were followed over 24 h. Addition of mitomycin C led to increases in total viral abundance as well as the abundance of cyanophages infecting Synechococcus strain DC2. Given typical estimates of burst size, these increases were consistent with 80% of the heterotrophic bacteria and 0.6% of Synechococcus cells being inducible by the addition of mitomycin C. This is the highest percentage of lysogens reported for a natural microbial community and demonstrates induction in a marine Synechococcus population. It is likely that the cyanophage production following the addition of mitomycin C was much higher than that titered against a single strain of Synechococcus; hence this estimate is a minimum. In untreated seawater samples, lytic viral production was estimated to remove ca. 27% of the gross heterotrophic bacterial production, and a minimum of 1.0% of the gross cyanobacterial production. Our results demonstrate very high levels of lysogeny in the heterotrophic bacterial community, outside of an oligotrophic environment, and the presence of inducible lysogens in Synechococcus spp. during a naturally occurring bloom. These data emphasize the need for further examination of the factors influencing lytic and lysogenic viral infection in natural microbial communities.     

A giant cell surface protein in Synechococcus WH8102 inhibits feeding by a dinoflagellate predator

Diverse strains of the marine planktonic cyanobacterium Synechococcus sp. show consistent differences in their susceptibility to predation. We used mutants of Sargasso Sea strain WH8102 (clade III) to test the hypothesis that cell surface proteins play a role in defence against predation by protists. Predation rates by the heterotrophic dinoflagellate Oxyrrhis marina on mutants lacking the giant SwmB protein were always higher (by 1.6 to 3.9×) than those on wild-type WH8102 cells, and equalled predation rates on a clade I strain (CC9311). In contrast, absence of the SwmA protein, which comprises the S-layer (surface layer of the cell envelope that is external to the outer membrane), had no effect on predation by O. marina. Reductions in predation rate were not due to dissolved substances in Synechococcus cultures, and could not be accounted for by variations in cell hydrophobicity. We hypothesize that SwmB defends Synechococcus WH8102 by interfering with attachment of dinoflagellate prey capture organelles or cell surface receptors. Giant proteins are predicted in the genomes of multiple Synechococcus isolates, suggesting that this defence strategy may be more general. Strategies for resisting predation will contribute to the differential competitive success of different Synechococcus groups, and to the diversity of natural picophytoplankton assemblages.     

Soft X-ray spectro-tomography study of cyanobacterial biomineral nucleation

Quantitative three-dimensional (3D) chemical mapping using angle-scan spectro-tomography in a scanning transmission (soft) X-ray microscope (STXM) has been used for the first time to characterize the early stages of CaCO₃ biomineral nucleation on the surface of planktonic freshwater cyanobacterial cells of the strain Synechococcus leopoliensis PCC 7942. The apparatus for STXM angle-scan tomography is described. Aspects of sample preparation, sample mounting and data acquisition and quantitative analysis and interpretation are discussed in detail. Angle-scan tomography and chemically selective 3D imaging at multiple photon energies has been combined with a complete 2D spectromicroscopic characterization of the biochemical and mineralogical composition. This has provided detailed insights into the mechanisms of mineral nucleation, leading to development of a detailed model of CaCO₃ nucleation by the cyanobacterial strain S. leopoliensis PCC 7942. It shows that Ca is absorbed by the extracellular polymeric substances (EPS) of the cyanobacteria and that CaCO₃ with aragonite-like short-range order is precipitated rather homogeneously within the EPS. The precipitation of the thermodynamically more stable calcite polymorph then starts at Ca-rich hot spots within the EPS and close to the cyanobacteria.     

Initial Phases of biofilm formation in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a facultative Fe(III)- and Mn(IV)-reducing microorganism and serves as a model for studying microbially induced dissolution of Fe or Mn oxide minerals as well as biogeochemical cycles. In soil and sediment environments, S. oneidensis biofilms form on mineral surfaces and are critical for mediating the metabolic interaction between this microbe and insoluble metal oxide phases. In order to develop an understanding of the molecular basis of biofilm formation, we investigated S. oneidensis biofilms developing on glass surfaces in a hydrodynamic flow chamber system. After initial attachment, growth of microcolonies and lateral spreading of biofilm cells on the surface occurred simultaneously within the first 24 h. Once surface coverage was almost complete, biofilm development proceeded with extensive vertical growth, resulting in formation of towering structures giving rise to pronounced three-dimensional architecture. Biofilm development was found to be dependent on the nutrient conditions, suggesting a metabolic control. In global transposon mutagenesis, 173 insertion mutants out of 15,000 mutants screened were identified carrying defects in initial attachment and/or early stages in biofilm formation. Seventy-one of those mutants exhibited a nonswimming phenotype, suggesting a role of swimming motility or motility elements in biofilm formation. Disruption mutations in motility genes (flhB, fliK, and pomA), however, did not affect initial attachment but affected progression of biofilm development into pronounced three-dimensional architecture. In contrast, mutants defective in mannose-sensitive hemagglutinin type IV pilus biosynthesis and in pilus retraction (pilT) showed severe defects in adhesion to abiotic surfaces and biofilm formation, respectively. The results provide a basis for understanding microbe-mineral interactions in natural environments.     

Immunological examination of the glycocalyces ofStaphylococcus aureus strains Wiley and Smith

The immunological examination of the glycocalyces ofStaphylococcus aureus has been concerned with capsular elements while essentially neglecting slime layers. We have found bacterial slime layers to be prevalent in many natural bacterial environments and, in particular, in recently isolatedS. aureus strains Wiley and Smith [1]. Growth in modified staphylococcus 110 medium induces slime layer production in these strains, and investigation of this material has revealed the two slime layers to be immunogenically and antigenically identical. The slime layer of the Smith strain is immunologically distinct from the tight, integral capsule that also comprises the glycocalyx of this strain. The Wiley strain glycocalyx is composed of only a slime layer.     

Network Relationships of Bacteria in a Stable Mixed Culture

We investigated the network relationships of bacteria in a structurally stable mixed culture degrading cellulose. The mixed culture consists of four bacterial strains (a cellulose-degrading anaerobe [strain S], a saccharide-utilizing anaerobe [strain F], a peptide- and acetate-utilizing aerobe [strain 3] and a peptide-, glucose-, and ethanol-utilizing aerobe [strain 5]). Interspecies interactions were examined by analyzing the effects of culture filtrates on the growth of the other strains and by comprehensively analyzing population dynamics in the mixed-culture systems with all possible combinations of the four bacterial strains. The persistence of strain S depends on the effects of strain 5. However, strain 5 is a disadvantaged strain because strain 3 has bacteriocidal activity on strain 5. The extinction of strain 5 is indirectly prevented by strain F that suppresses the growth of strain 3. Although strain F directly has suppressive effects on the growth of strain S, strain F is essential for the persistence of strain S, considering the indirect effects (maintaining strain 5, which is essential for the survival of strain S, by inhibiting strain 3). These indirect relationships form a bacterial network in which all the relationships including suppressive effects were well balanced to maintain the structural stability. In addition to direct metabolite interactions, such kind of indirect relationships could have a great impact on microbial community structure in the natural environment.     

Living under an atomic force microscope

An approach for long‐term in vivo investigations on cyanobacterial cell surface changes at high spatial resolution by Atomic Force Microscopy (AFM) was developed in this study. Until recently, changes of bacterial cell surfaces due to changes of the chemical environment could neither be investigated in situ nor in vivo. However, in vivo investigations give insights into kinetics of cell response to environmental changes and mineral nucleation at the cell's surface. Continuously cultured cyanobacteria of the representative freshwater strain Synechococcus leopoliensis (PCC 7942) were washed and artificially immobilized on poly‐l‐lysine‐coated glass slides. Both immobilization and environmental conditions were optimized in order to facilitate long‐term experiments (> 100 h) with living cells. AFM samples were investigated in situ in two different solutions: Culture medium was used for cultivation experiments and nutrient‐free NaHCO₃/CaCl₂ solutions (supersaturated with respect to calcite) for long‐term characterizations of the changes in cell surface topography. Cell viability under these conditions was investigated by AFM, TEM and epifluorescence microscopy, independently. No indications for extended starvation were found within the relevant timescales. Analysing the influence of Ca²⁺ on the surface of S. leopoliensis, we found significant changes compared to a Ca‐free solution. Few hours after CaCl₂ was added to the circumfluent solution, small protuberances were observed on the cell surface. These are promising results to environmental scientists for a wide range of applications, as cell response to environmental changes can now be monitored online and in vivo at timescales, which are relevant for natural processes. Most especially studies of biomineralization and mineral nucleation on bacterial cell surfaces will profit from this new approach.     

Adhesion of Dissimilatory Fe(III)-Reducing Bacteria to Fe(III) Minerals

The metabolism of dissimilatory iron-reducing bacteria (DIRB) may provide a means of remediating contaminated subsurface soils. The factors controlling the rate and extent of bacterial F(III) mineral reduction are poorly understood. Recent research suggests that molecular-scale interactions between DIRB cells and Fe(III) mineral particles play an important role in this process. One of these interactions, cell adhesion to Fe(III) mineral particles, appears to be a complex process that is, at least in part, mediated by a variety of surface proteins. This study examined the hypothesis that the flagellum serves as an adhesin to different Fe(III) minerals that range in their surface area and degree of crystallinity. Deflagellated cells of the DIRB Shewanella algae BrY showed a reduced ability to adhere to hydrous ferric oxide (HFO) relative to flagellated cells. Flagellated cells were also more hydrophobic than deflagellated cells. This was significant because hydrophobic interactions have been previously shown to dominate S. algae cell adhesion to Fe(III) minerals. Pre-incubating HFO, goethite, or hematite with purified flagella inhibited the adhesion of S. algae BrY cells to these minerals. Transposon mutagenesis was used to generate a flagellum-deficient mutant designated S. algae strain NF. There was a significant difference in the rate and extent of S. algae NF adhesion to HFO, goethite, and hematite relative to that of S. algae BrY. Amiloride, a specific inhibitor of Na + -driven flagellar motors, inhibited S. algae BrY motility but did not affect the adhesion of S. algae BrY to HFO. S.algae NF reduced HFO at the same rate as S. algae BrY. Collectively, the results of this study support the hypothesis that the flagellum of S. algae functions as a specific Fe(III) mineral adhesin. However, these results suggest that flagellum-mediated adhesion is not requisite for Fe(III) mineral reduction.     

Perspectives in the coordinate regulation of cell cycle events in Synechococcus

The concepts of cell theory and the notions of coordinate regulation of the cell cycle have been known for centuries but the conundrum of coordinate regulation of the cell cycle remains to be resolved. The unique characteristics of the cell division cycle of Synechococcus, a photosynthetic bacterium, suggest the existence of a complex network of light/dark responsive gene regulatory factors that coordinate its cell cycle events. Evaluation of the highly ordered cell cycle of Synechococcus led to the construction of workable models that coordinate the cell cycle events. A central issue in bacterial cell growth is the elucidation of the genetic regulatory mechanisms that coordinate the cell cycle events. Synechococcus, a unicellular cyanobacterium, displays a peculiar cell growth cycle. In the light growth conditions, a highly ordered and sequentially coordinated appearances of r-protein synthesis, rRNA synthesis, DNA replication, chromosome segregation, and cell septum formation occur (Figs 1, 2A). Cell membrane syntheses occur predominantly during mid-cell cycle and cell division period. Synthesis of thylakoid (=photosynthetic apparatus) is thought to occur during mid-cell cycle and coincides with a period of peak phospholipid synthesis and oxygen production (Csatorday and Horvath, 1977; Asato, 1979). Cell wall syntheses occur in short discontinuous periods throughout the cell cycle and during cell division (Asato, 1984). Distinct D1 (=G1), C (S) and D2 (=G2) periods as defined by Cooper and Helmstetter (1968) are observed in synchronized cultures of Synechococcus (Asato, 1979). When light grown cultures are placed in the dark, the ongoing cell cycles are aborted in the dark (Fig. 3A) and cell divisions do not occur (Asato, 1983; Marino and Asato, 1986). Upon re-exposure of the cell cultures to the light growth conditions, about 14 h later, new cell cycles are re-initiated. These characteristics of cell growth are considered to be expressions of a unique strategy of obligate phototrophic mode of growth to perpetuate their species (Asato, 2003). Nevertheless, the intermediate metabolism, the synthesis of building block molecules, the genetics and molecular biology in the formation of major macromolecules are similarto heterotrophs such as E. coli. In any case, the genes that are involved in the formation of the cellular structures and the genes that control the orderly appearances of the cell cycle events must be coordinated by novel genetic mechanisms. Currently, there are no known physiological/physical mechanisms, growth rate dependent factors or traditional genetic regulatory mechanisms that could explain the coordinate regulation of the cell cycle events in bacteria (Newton and Ohta, 1992; Vinella and D'Ari, 1995; Donachie, 2001; Margolin and Bernander, 2004). Because the genetic mechanisms of coordinate regulation of cell cycle events in bacteria are largely unexplained, the questions on how Synechococcus coordinates the cell cycle events present a difficult problem to resolve. Nevertheless, the problems with regard to the coordinate regulation of the cell cycle events of Synechococcus must be considered. Possible solutions are developed and described in this article. The proposed schemes do not exclude the formation of other genetic mechanisms on the regulation of cell cycle events in Synechococcus. Although the cell cycle of Synechococcus is not widely known, the issues on the coordinate regulation of the cell cycle events are not trivial since similar regulatory mechanisms most likely occur in other prokaryotes.     

Growth strategy of heterotrophic bacterial population along successional sequence on spoil of brown coal colliery substrate

The bacterial population of brown coal colliery spoil (Sokolov coal mining district, Czechia) was characterized by measuring viable bacterial biomass, the culturable to total cell ratio (C:T), colony-forming curve (CFC) analysis and species and/or biotype diversity. Bacterial representatives that differed in colony-forming growth (fast and/or slow growers) were used for growth-strategy investigation of heterotrophic bacteria. Spoil substrates from the surface (0–50 mm) and the mineral (100–150 mm) layers were sampled on 4 sites undergoing spontaneous succession corresponding to 1, 11, 21 and 43 years after deposition (initial, early, mid and late stages). The bacterial biomass of the surface layer increased during the initial and early stages with a maximum at mid stage and stabilized in the late stage while mineral layer biomass increased throughout the succession. The maxima of C:T ratios were at the early stage, minima at the late stage. Depending on the succession stage the C:T ratio was 1.5–2 times higher in the mineral than the surface layer of soil. An increase in the fraction of nonculturable bacteria was associated with the late succession stage. CFC analysis of the surface layer during a 3-d incubation revealed that the early-succession substrate contained more (75 %) rapidly colonizing bacteria (opportunists, r-strategists) than successively older substrates. The culturable bacterial community of the mineral layer maintained a high genera and species richness of fast growers along the succession line in contrast to the surface layer community, where there was a maximum in the abundance of fast growers in the early stage. There was a balanced distribution of Grampositive and Gram-negative representatives of fast growers in both layers. A markedly lower abundance of slow growers was observed in the mineral in contrast to the surface layer. Gram-positive species dominated the slow growers at the surface as well as in the mineral layers. The growth strategy of the heterotrophic bacterial population along four successional stages on spoil of brown coal colliery substrate in the surface layer displayed a trend indicative of a r-K continuum in contrast to the mineral layer, where an r-strategy persisted. This research was funded by theCzech Science Foundation grant no. 526/03/1259 and by theResearch Plan of the Institute of Soil Biology, Academy of Sciences of the Czech Republic, project no. AV 0Z 6066 0521.     

Biofilm: set-up and organization of a bacterial community

Bacterial attachment on various surfaces mostly takes place in the form of specialised bacterial communities, referred to as biofilm. The biofilm is formed through series of interactions between cells and adherence to surface, resulting in an organised structure. In this review we have been using Pseudomonas aeruginosa as a model microorganism to describe the series of events that occurred during this developmental process. P. aeruginosa is an opportunistic pathogen that has a wide variety of hosts and infectious sites. In addition to biofilm formation in certain tissues, inert surfaces, such as catheters, are also target for bacterial biofilm development. The use of convenient genetic screens has made possible the identification of numerous biofilm-defective mutants, which have been characterised further. These studies have allowed the proposal for a global model, in which key events are described for the different stages of biofilm formation. Briefly, flagellar mobility is crucial for approaching the surface, whereas type IV pili motility is preponderant for surface colonisation and microcolonies formation. These microcolonies are finally packed together and buried in an exopolysaccharide matrix to form the differentiated bio-film. It is obvious that the different stages of biofilm formation also involved perception of environmental stimuli. These stimuli, and their associated complex regulatory networks, have still to be fully characterised to understand the bacterial strategy, which initiates biofilm formation. One such regulatory system, called Quorum sensing, is one of the key player in the initial differentiation of biofilm. Finally, a better understanding, at the molecular level, of biofilm establishment and persistence should help for the design of antimicrobials that prevent bacterial infections.     

Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis

Populations of surface-attached microorganisms comprising either single or multiple species are commonly referred to as biofilms. Using a simple assay for the initiation of biofilm formation (e.g. attachment to an abiotic surface) by Pseudomonas fluorescens strain WCS365, we have shown that: (i) P . fluorescens can form biofilms on an abiotic surface when grown on a range of nutrients; (ii) protein synthesis is required for the early events of biofilm formation; (iii) one (or more) extracytoplasmic protein plays a role in interactions with an abiotic surface; (iv) the osmolarity of the medium affects the ability of the cell to form biofilms. We have isolated transposon mutants defective for the initiation of biofilm formation, which we term surface attachment defective ( sad ). Molecular analysis of the sad mutants revealed that the ClpP protein (a component of the cytoplasmic Clp protease) participates in biofilm formation in this organism. Our genetic analyses suggest that biofilm formation can proceed via multiple, convergent signalling pathways, which are regulated by various environmental signals. Finally, of the 24 sad mutants analysed in this study, only three had defects in genes of known function. This result suggests that our screen is uncovering novel aspects of bacterial physiology.     

Effect of paracrystalline protein surface layers on predation by Bdellovibrio bacteriovorus.

We determined that paracrystalline protein surface arrays (S layers) protected gram-negative eubacteria from predation by Bdellovibrio bacteriovorus. Aquaspirillum serpens VHA and MW5 and Aquaspirillum sinuosum were resistant to predation by B. bacteriovorus 6-5-S when fully covered by their S layers. The S layer of Aeromonas salmonicida A449 protected the cells from predication by B. bacteriovorus 109J. A predacious, plaque-forming vibrio that lysed an S-layer- variant of Caulobacter crescentus but was not predacious on the parental strain which possessed an S layer was isolated from raw sewage. Since S layers are stable components of many bacterial surfaces in nature, they can provide this protective function in both aquatic and terrestrial habitats where Bdellovibrio spp. are found.     

Selection of cyanobacteria isolated from mosquito breeding sites as a potential food source for mosquito larvae.

One way to increase the persistence of larvicidal toxins in mosquito breeding sites is to clone the corresponding genes in microorganisms, such as cyanobacteria, which could serve as a source of food for the larvae. We isolated and cultured 10 strains of cyanobacteria from three mosquito breeding sites along the French Mediterranean coast. Most of the strains were tolerant to a relatively wide range of salt concentrations, and all of them were totally or partially resistant to at least four of the five biological or chemical larvicides used in the local mosquito control program. Six unicellular strains from these habitats and Synechococcus strain PCC 7942, a strain maintained for more than 10 years under laboratory conditions, were assessed for ingestion and digestion by larvae Culex pipiens and Anopheles gambiae mosquitoes. The numbers of cells ingested and digested were dependent on the cyanobacterial strain and varied with the mosquito species. Three of the new isolates, Synechococcus strain PCC 8905 and Synechocystis strains PCC 8906 and PCC 8912, were ingested and digested rapidly by larvae of both mosquito species. Since these strains are also tolerant to larvicides and relatively resistant to elevated salt concentrations, they meet the basic requirements for potential recipients of bacterial genes that encode endotoxins.     

Selection of cyanobacteria isolated from mosquito breeding sites as a potential food source for mosquito larvae.

One way to increase the persistence of larvicidal toxins in mosquito breeding sites is to clone the corresponding genes in microorganisms, such as cyanobacteria, which could serve as a source of food for the larvae. We isolated and cultured 10 strains of cyanobacteria from three mosquito breeding sites along the French Mediterranean coast. Most of the strains were tolerant to a relatively wide range of salt concentrations, and all of them were totally or partially resistant to at least four of the five biological or chemical larvicides used in the local mosquito control program. Six unicellular strains from these habitats and Synechococcus strain PCC 7942, a strain maintained for more than 10 years under laboratory conditions, were assessed for ingestion and digestion by larvae Culex pipiens and Anopheles gambiae mosquitoes. The numbers of cells ingested and digested were dependent on the cyanobacterial strain and varied with the mosquito species. Three of the new isolates, Synechococcus strain PCC 8905 and Synechocystis strains PCC 8906 and PCC 8912, were ingested and digested rapidly by larvae of both mosquito species. Since these strains are also tolerant to larvicides and relatively resistant to elevated salt concentrations, they meet the basic requirements for potential recipients of bacterial genes that encode endotoxins.     

Effect of heat shock on protein synthesis in the cyanobacterium Synechococcus sp. strain PCC 6301.

The response to heat shock at 47 degrees C was examined in the cyanobacterium (blue-green alga) Synechococcus sp. strain PCC 6301. On heat shock, the growth of the cells decreased and they preferentially synthesized a limited number of polypeptides. The rate of synthesis of these proteins increased markedly in the early period of temperature shift up and gradually decreased afterwards. Among the proteins greatly affected by temperature shift up were those with apparent molecular weights of 91,000 (91K), 79K, 78K, 74K, 65K, 64K, 61K, 49K, 45K, 24K, 22K, 18K, 16K, 14K, 12K, and 11.4K, based on their mobilities in sodium dodecyl sulfate-polyacrylamide gels. From these initial studies on Synechococcus sp. strain PCC 6301 we conclude that in cyanobacteria a heat shock response similar to that known to occur in other eucaryotes and procaryotes might exist.     

Radioprotective properties of detoxified lipid A from Salmonella minnesota R595

In the past, the toxicity of bacterial lipopolysaccharide (LPS) or its principal bioactive component, lipid A, has detracted from their potential use as radioprotectants. Recently, a relatively nontoxic monophosphoryl Lipid A (LAM) that retains many of the immunobiologic properties of LPS has been isolated from a polysaccharide deficient Re mutant strain of Salmonella minnesota (R595). The ability of the native endotoxic glycolipid (GL) from S. minnesota (R595) as well as diphosphoryl lipid A (LAD) and nontoxic monophosphoryl lipid A (LAM) derived from GL to protect LPS responsive (CD2F1 or C3H/HeN) and nonresponsive (C3H/HeJ) mice from 60Co gamma irradiation has been studied. Administration of GL, LAD, or LAM to CD2F1 or C3H/HeN mice (400 micrograms/kg) 24 h prior to exposure provided significant radioprotection. No protection was afforded to C3H/HeJ mice. Experiments were also conducted to determine the relative abilities of GL, LAD, and LAM to stimulate hematopoiesis as reflected by the endogenous spleen colony (E-CFU) assay. Protection was not correlated with the ability of these substances to increase E-CFUs or to induce colony-stimulating activity (CSA).     

Modelling the vertical distribution of Prochlorococcus and Synechococcus in the North Pacific Subtropical Ocean

A simple model was developed to examine the vertical distribution of Prochlorococcus and Synechococcus ecotypes in the water column, based on their adaptation to light intensity. Model simulations were compared with a 14-year time series of Prochlorococcus and Synechococcus cell abundances at Station ALOHA in the North Pacific Subtropical Gyre. Data were analysed to examine spatial and temporal patterns in abundances and their ranges of variability in the euphotic zone, the surface mixed layer and the layer in the euphotic zone but below the base of the mixed layer. Model simulations show that the apparent occupation of the whole euphotic zone by a genus can be the result of a co-occurrence of different ecotypes that segregate vertically. The segregation of ecotypes can result simply from differences in light response. A sensitivity analysis of the model, performed on the parameter alpha (initial slope of the light-response curve) and the DIN concentration in the upper water column, demonstrates that the model successfully reproduces the observed range of vertical distributions. Results support the idea that intermittent mixing events may have important ecological and geochemical impacts on the phytoplankton community at Station ALOHA.     

Posttranslational regulation of nitrate assimilation in the cyanobacterium Synechocystis sp. strain PCC 6803

Posttranslational regulation of nitrate assimilation was studied in the cyanobacterium Synechocystis sp. strain PCC 6803. The ABC-type nitrate and nitrite bispecific transporter encoded by the nrtABCD genes was completely inhibited by ammonium as in Synechococcus elongatus strain PCC 7942. Nitrate reductase was insensitive to ammonium, while it is inhibited in the Synechococcus strain. Nitrite reductase was also insensitive to ammonium. The inhibition of nitrate and nitrite transport required the PII protein (glnB gene product) and the C-terminal domain of NrtC, one of the two ATP-binding subunits of the transporter, as in the Synechococcus strain. Mutants expressing the PII derivatives in which Ala or Glu is substituted for the conserved Ser49, which has been shown to be the phosphorylation site in the Synechococcus strain, showed ammonium-promoted inhibition of nitrate uptake like that of the wild-type strain. The S49A and S49E substitutions in GlnB did not affect the regulation of the nitrate and nitrite transporter in Synechococcus either. These results indicated that the presence or absence of negative electric charge at the 49th position does not affect the activity of the PII protein to regulate the cyanobacterial ABC-type nitrate and nitrite transporter according to the cellular nitrogen status. This finding suggested that the permanent inhibition of nitrate assimilation by an S49A derivative of PII, as was previously reported for Synechococcus elongatus strain PCC 7942, is likely to have resulted from inhibition of nitrate reductase rather than the nitrate and nitrite transporter.     

Synergistic effects in mixed Escherichia coli biofilms: conjugative plasmid transfer drives biofilm expansion

Bacterial biofilms, often composed of multiple species and genetically distinct strains, develop under complex influences of cell-cell interactions. Although detailed knowledge about the mechanisms underlying formation of single-species laboratory biofilms has emerged, little is known about the pathways governing development of more complex heterogeneous communities. In this study, we established a laboratory model where biofilm-stimulating effects due to interactions between genetically diverse strains of Escherichia coli were monitored. Synergistic induction of biofilm formation resulting from the cocultivation of 403 undomesticated E. coli strains with a characterized E. coli K-12 strain was detected at a significant frequency. The survey suggests that different mechanisms underlie the observed stimulation, yet synergistic development of biofilm within the subset of E. coli isolates (n = 56) exhibiting the strongest effects was most often linked to conjugative transmission of natural plasmids carried by the E. coli isolates (70%). Thus, the capacity of an isolate to promote the biofilm through cocultivation was (i) transferable to the K-12 strain, (ii) was linked with the acquisition of conjugation genes present initially in the isolate, and (iii) was inhibited through the presence in the cocultured K-12 strain of a related conjugative plasmid, presumably due to surface exclusion functions. Synergistic effects of cocultivation of pairs of natural isolates were also observed, demonstrating that biofilm promotion in this system is not dependent on the laboratory strain and that the described model system could provide relevant insights on mechanisms of biofilm development in natural E. coli populations.