An endolithic microbial community in dolomite rock in central Switzerland: characterization by reflection spectroscopy, pigment analyses, scanning electron microscopy, and laser scanning microscopy.

A community of endolithic microorganisms dominated by phototrophs was found as a distinct band a few millimeters below the surface of bare exposed dolomite rocks in the Piora Valley in the Alps. Using in situ reflectance spectroscopy, we detected chlorophyll a (Chl a), phycobilins, carotenoids, and an unknown type of bacteriochlorophyll-like pigment absorbing in vivo at about 720 nm. In cross sections, the data indicated a defined distribution of different groups of organisms perpendicular to the rock surface. High-performance liquid chromatography analyses of pigments extracted with organic solvents confirmed the presence of two types of bacteriochlorophylls besides chlorophylls and various carotenoids. Spherical organisms of varying sizes and small filaments were observed in situ with scanning electron microscopy and confocal laser scanning microscopy (one- and two-photon technique). The latter allowed visualization of the distribution of phototrophic microorganisms by the autofluorescence of their pigments within the rock. Coccoid cyanobacteria of various sizes predominated over filamentous ones. Application of fluorescence-labeled lectins demonstrated that most cyanobacteria were embedded in an exopolymeric matrix. Nucleic acid stains revealed a wide distribution of small heterotrophs. Some biological structures emitting a green autofluorescence remain to be identified.

Generation of bioaerosols during manual mail unpacking and sorting.

AIMS: The dynamics of bioaerosol generation in specific occupational environments where mail is manually unpacked and sorted was investigated. METHODS AND RESULTS: Total number of airborne particles was determined in four different size classes (0.3-0.5, 0.5-1, 1-5 and >5 microm) by laser particle counting. Time dependent formation of bioaerosols was monitored by culturing methods and by specific staining followed by flow cytometry. Besides handling of regular mail, specially prepared letters ('spiked letters') were added to the mailbags to deliberately release powdered materials from letters and to simulate high impact loads. These letters contained various dry powdered biological and nonbiological materials such as milk powder, mushrooms, herbs and cat litter. Regarding the four size classes, particulate aerosol composition before mail handling was determined as 83.2 +/- 1.0, 15.2 +/- 0.7, 1.7 +/- 0.4 and 0.04 +/- 0.02%, respectively, whereas the composition changed during sorting to 66.8 +/- 7.9, 22.3 +/- 3.6, 10.4 +/- 4.0 and 0.57 +/- 0.27%, respectively. Mail processing resulted in an increase in culturable airborne bacteria and fungi. Maximum concentrations of bacteria reached 450 CFU m(-3), whereas 270 CFU of fungi were detected. CONCLUSIONS: Indoor particle concentrations steadily increased during mail handling mostly associated with particles of diameters >1 microm. However, it was not possible to distinguish spiked letters from nonspiked by simple particle counting and CFU determinations. SIGNIFICANCE AND IMPACT OF STUDY: The dynamics of bioaerosol generation have to be addressed when monitoring specific occupational environments (such as mail sorting facilities) regarding the occurrence of biological particles.

Effect of medium composition, flow rate, and signaling compounds on the formation of soluble extracellular materials by biofilms of Chromobacterium violaceum.

Biofilms of the homoserine-lactone-defective strain Chromobacterium violaceum CV026 were grown on silicone surfaces in a flow chamber. The effect of medium flow rate, different levels of N-butanoyl-homoserine lactone, and nutrients on biofilm activity was studied by quantifying the proteins and exopolysaccharides attached to the cells. To compare the effect of each of the three variables within a wide range, the experiments were designed as a full factorial search with three levels for each variable. Calculated contour plots demonstrated that N-acyl homoserine lactone is an important determinant of both the amount and the composition of the compounds excreted into the medium.

Seasonal and spatial community dynamics in the meromictic Lake Cadagno.

The seasonal and spatial variations in the community structure of bacterioplankton in the meromictic alpine Lake Cadagno were examined by temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rDNA fragments. Two different amplifications were performed, one specific for the domain Bacteria (Escherichia coli positions 8-536) and another specific for the family Chromatiaceae (E. coli positions 8-1005). The latter was followed by semi-nested reamplification with the bacterial primer set, allowing comparison of the two PCR approaches by TTGE. The TTGE patterns of samples from the chemocline and the anoxic monimolimnion were essentially identical, whereas the oxic mixolimnion displayed distinctively different banding patterns. For samples from the chemocline and the monimolimnion, dominant bands in the Bacteria-specific TTGE profiles comigrated with bands obtained by the semi-nested PCR approach specific for Chromatiaceae. This observation suggested that Chromatiaceae are in high abundance in the anoxic water layer. All dominant bands were excised and sequenced. Changes in the community structure, as indicated by changes in the TTGE profiles, were observed in samples taken at different times of the year. In the chemocline, Chomatium okenii was dominant in the summer months, whereas Amoebobacter purpureus populations dominated in autumn and winter. This change was confirmed by fluorescent in situ hybridization.

Rhythmic activity of uptake hydrogenase in the prokaryote Rhodospirillum rubrum.

Growth of Rhodospirillum rubrum was followed in cultures kept under anoxic conditions at constant temperature in either continuous light (LL, 32 degrees C) or continuous darkness (DD, 32 degrees C and 16 degrees C). In DD, only small modifications of the turbidity were detected; linear regression analysis nevertheless gives a very significant slope (t(34) = 13.07, p < 10(-14), with R2 of 0.834). Mean generation times reflected these differences of growth with 11.9+/-0.5 h in LL and 43.2+/-1.1 h in DD at 32 degrees C and 37.4+/-1.0 h at 16 degrees C cultures. The uptake hydrogenase (Hup) activity has been followed in situ in whole cells of R. rubrum grown in the same conditions, and a clear ultradian rhythm of activity has been observed. Indeed, after about 12 h in the new media, a rapid rise of hydrogenase activity was observed in both LL and DD cultures after which it decreased again to very low values. The activity of Hup continued to show such fluctuations during the rest of the experiment, both in DD and in LL, during the growth and stationary phases. The Lomb-Scargle power periodogram method demonstrates the presence of a clear rhythmic Hup activity both in LL and DD. In the LL-grown cultures, the oscillating activity is faster and continues throughout the growth and the stationary phases, with an ultradian period of 12.1+/-0.5 h. In DD, the slow-growing bacteria showed an ultradian oscillatory pattern of Hup activity with periods of 15.2+/-0.5 h at 32 degrees C and 23.4+/-2.0 h at 16 degrees C. The different periods obtained for LL- and DD-grown bacteria are significantly different.

In situ determination of sulfide turnover rates in a meromictic alpine lake.

A push-pull method, previously used in groundwater analyses, was successfully adapted for measuring sulfide turnover rates in situ at different depths in the meromictic Lake Cadagno. In the layer of phototrophic bacteria at about 12 m in depth net sulfide consumption was observed during the day, indicating active bacterial photosynthesis. During the night the sulfide turnover rates were positive, indicating a net sulfide production from the reduction of more-oxidized sulfur compounds. Because of lack of light, no photosynthesis takes place in the monimolimnion; thus, only sulfide formation is observed both during the day and the night. Sulfide turnover rates in the oxic mixolimnion were always positive as sulfide is spontaneously oxidized by oxygen and as the rates of sulfide oxidation depend on the oxygen concentrations present. Sulfide oxidation by chemolithotrophic bacteria may occur at the oxicline, but this cannot be distinguished from spontaneous chemical oxidation.

Bacterial diversity and community composition in the chemocline of the meromictic alpine Lake Cadagno as revealed by 16S rDNA analysis.

Using different techniques of molecular biology we investigated the bacterial diversity of the chemocline of the meromictic Lake Cadagno. Cloning of a total community 16S rDNA PCR product and subsequent screening with a combination of amplified ribosomal DNA restriction analysis and temporal temperature gradient gel electrophoresis (TTGE) analysis revealed that 30 of 47 randomly selected clones were unique. Partial sequencing and comparative analysis indicated a high bacterial diversity dominated by the gamma-Proteobacteria (33.3%). Most of these rDNA clone sequences were not closely related to any 16S rDNA sequence in the database. In a second approach, the TTGE pattern from an environmental sample was compared with the migration of the cloned 16S rDNA fragments. Four clone types were identified on the environmental pattern by excising and sequencing comigrating bands, three of which were well represented in the library: two Chromatiaceae species and one sequence affiliated with the Desulfobulbus assemblage. Using the fluorescent in situ hybridization technique we essentially confirmed the results of the cloning experiments and the TTGE analysis.

Reduction of selenite and detoxification of elemental selenium by the phototrophic bacterium Rhodospirillum rubrum.

The effect of selenite on growth kinetics, the ability of cultures to reduce selenite, and the mechanism of detoxification of selenium were investigated by using Rhodospirillum rubrum. Anoxic photosynthetic cultures were able to completely reduce as much as 1. 5 mM selenite, whereas in aerobic cultures a 0.5 mM selenite concentration was only reduced to about 0.375 mM. The presence of selenite in the culture medium strongly affected cell division. In the presence of a selenite concentration of 1.5 mM cultures reached final cell densities that were only about 15% of the control final cell density. The cell density remained nearly constant during the stationary phase for all of the selenite concentrations tested, showing that the cells were not severely damaged by the presence of selenite or elemental selenium. Particles containing elemental selenium were observed in the cytoplasm, which led to an increase in the buoyant density of the cells. Interestingly, the change in the buoyant density was reversed after selenite reduction was complete; the buoyant density of the cells returned to the buoyant density of the control cells. This demonstrated that R. rubrum expels elemental selenium across the plasma membrane and the cell wall. Accordingly, electron-dense particles were more numerous in the cells during the reduction phase than after the reduction phase.

Development of a laboratory-scale leaching plant for metal extraction from fly ash by thiobacillus strains.

Semicontinuous biohydrometallurgical processing of fly ash from municipal waste incineration was performed in a laboratory-scale leaching plant (LSLP) by using a mixed culture of Thiobacillus thiooxidans and Thiobacillus ferrooxidans. The LSLP consisted of three serially connected reaction vessels, reservoirs for a fly ash suspension and a bacterial stock culture, and a vacuum filter unit. The LSLP was operated with an ash concentration of 50 g liter, and the mean residence time was 6 days (2 days in each reaction vessel). The leaching efficiencies (expressed as percentages of the amounts applied) obtained for the economically most interesting metal, Zn, were up to 81%, and the leaching efficiencies for Al were up to 52%. Highly toxic Cd was completely solubilized (100%), and the leaching efficiencies for Cu, Ni, and Cr were 89, 64, and 12%, respectively. The role of T. ferrooxidans in metal mobilization was examined in a series of shake flask experiments. The release of copper present in the fly ash as chalcocite (Cu(2)S) or cuprite (Cu(2)O) was dependent on the metabolic activity of T. ferrooxidans, whereas other metals, such as Al, Cd, Cr, Ni, and Zn, were solubilized by biotically formed sulfuric acid. Chemical leaching with 5 N H(2)SO(4) resulted in significantly increased solubilization only for Zn. The LSLP developed in this study is a promising first step toward a pilot plant with a high capacity to detoxify fly ash for reuse for construction purposes and economical recovery of valuable metals.

Optical fiber-based in situ spectroscopy of pigmented single colonies.

We have adapted a commercially available fiber-optic spectroradiometer with diode array detection to record reflection and absorption spectra from single, 1-mm-diameter bacterial colonies. A careful assessment of the performance of the spectroradiometer for this application is reported. In a model study employing colonies from various phototrophic bacteria, we show that the reflectance spectra are reliable within the range of 450 to 820 nm, whereas the transmission spectra yield accurate peak intensities and absorption maxima from 400 to 900 nm. For screening of populations of about 10(sup4) colonies, fiber-optic transmission spectroscopy provides an attractive and inexpensive alternative to present techniques based on charge-coupled device imaging technology.

Biodegradation of cyclic and substituted linear oligomers of poly(3-hydroxybutyrate).

Cyclic oligo(3-hydroxybutyrate), oligo(3-HB), was synthesized and purified, resulting in oligolides that contained three to seven (R)-3-hydroxybutyrate units (triolides up to heptolides). In addition, linear 3-HB octamers obtained as either tert-butyl or methyl esters were substituted with different end groups at the hydroxy end. The hydroxy terminus was replaced by either a benzyloxy, trifluoroacetoxy, crotonyloxy (S)-3-hydroxybutyryloxy, or fluorenylmethylcarbonyloxy (FMOC) group. P(3-HB) hairpin loops occurred on the surface of certain regions of the polymer, especially of lamellar crystallites. Cyclic 3-HB oligomers provide a model system for these loops. It is assumed that they provide attachment points for the depolymerizing enzymes. All of the (R)-oligolides tested were degraded except the (R)-triolide. Triolides were not degraded, suggesting that enzymatic attack was prevented presumably by steric hindrance on the rigid ring system. Unsubstituted linear octamers were degraded. Biodegradation was prevented when the hydroxy terminus was protected by the FMOC group, but was not dependent on a free hydroxy terminal group; all other protecting groups did not prevent degradation. Substitution of the carboxy end of a methyl or tert-butyl ester group did not influence biodegradation.

Purification of an LHI-RC-complex of Rhodospirillum rubrum by solubilization of chromatophores with a short-chain lecithin.

Chromatophores from Rhodospirillum rubrum were solubilized using the detergent 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC). The solubilization curves are sigmoidal reaching a plateau at a detergent/protein ratio of 2-3 µmol/mg corresponding to 75-90% solubilized protein. The BChl-binding proteins are stable over a large range of DHPC/protein ratios. A complex of BChl-binding proteins containing both LHI- and RC-polypeptides (LHI-RC-complex) was purified using a two step procedure. RC photochemical activity as well as absorption and near-IR CD spectra showed the complex to be active and stable after purification in presence of DHPC.

An improved procedure and new vectors for transposon Tn5 mutagenesis of the phototrophic bacterium Rhodospirillum rubrum.

A detailed examination of vectors and procedures used for Tn5 mutagenesis of the phototrophic purple non-sulfur bacterium Rhodospirillum rubrum has been performed. The mobilizable Tn5 suicide vectors currently available show a frequency of Tn5 mutagenesis for R. rubrum of approx. 10(-7)-10(-8), approx. 100-1000-fold lower than observed for the related bacteria Rhodobacter capsulatus and Rhodobacter sphaeroides. Using the blue-to-red reversion of a blue-green mutant, R. rubrum ST6, containing a single Tn5 lesion in one of the early genes for carotenoid biosynthesis, we have shown that the frequency of precise excision of a chromosomally inserted Tn5 element, to restore the wild-type phenotype in the absence of selection, is 10(-6). We have constructed three new suicide vectors for Tn5 mutagenesis, where the transposase encoded by the IS50R element was placed in the same (pSUPEG11, pSUPEG21) or in the opposing (pSUPEG22) orientation from the weak promoter of the RK2-derived tetR gene. With the vector pSUPEG11, the frequency of Tn5 mutagenesis was increased to 10(-5), approx. 100-fold higher than observed previously.

Characterization and partial purification of an ATPase and inorganic pyrophosphatase of the archaebacterium Methanobacterium thermoautotrophicum.

ATPase and inorganic pyrophosphatase (PPase) activities have been detected in several methanogenic bacteria. These activities are believed to play a crucial role in energy metabolism. In the present study we have investigated some characteristics of the ATPase and the inorganic PPase activities of Methanobacterium thermoautotrophicum. Although these proteins migrate identically on non-dissociating gels, they are catalyzed by distinct enzymes which are separable by biochemical purification methods. The partially purified enzymes are composed of at least two subunits. The ATPase subunits have molecular masses of about 43 and 33 kDa and the inorganic PPase such of about 31 and 25 kDa. After purification, the PPase and the ATPase did not hydrolyze ATP and PP(i), respectively. The membrane-bound ATPase and PPase activities are distinguished in response to sodium fluoride, by the effects of divalent cations, by the temperature ranges for activities and the solubilization behaviour by different extractants. Most investigated catalytic and structural properties of the ATPase do not suit the current criteria for classifying this enzyme under either the F-, V- or P-ATPases.

Short-chain phosphatidylcholines as superior detergents in solubilizing membrane proteins and preserving biological activity.

The solubilization of plasma and organelle membranes by diheptanoylphosphatidylcholine (DHPC) has been studied. This short-chain phosphatidylcholine is shown to act as a mild detergent, solubilizing effectively both kinds of membranes at DHPC concentrations of 10-20 mM (0.5-1%). The size of the resulting mixed protein-lipid-DHPC micelles ranges between 5 and 8 nm. The protein conformation and hence the enzymatic activity are well preserved over a rather large DHPC concentration range (up to 4-5 times the DHPC concentration required for solubilizing the membranes). Evidence is presented that short-chain phosphatidylcholines are superior to most detergents commonly used by biochemists. This is true not only regarding its excellent dispersing power on both phospholipid bilayers (Gabriel & Roberts, 1986) and biological membranes but also as to its capacity to preserve the native protein structure and hence enzymatic activity in the solubilized state. Due to its special properties DHPC lends itself very well not only to membrane solubilization but also to the purification of the solubilized membrane proteins and reconstitution of the proteins into simple lipid bilayers. Concerning the mechanism of membrane solubilization, evidence indicates that DHPC interacts primarily with the lipid bilayer of the membrane and not with the membrane proteins. DHPC solubilizes membranes by being distributed into the lipid bilayer and breaking it up. In the resulting small mixed micelles, the protein remains associated with its preferred intrinsic membrane lipids and is thus stabilized. The protein-intrinsic lipid complex is successfully shielded from unfavorable contacts with H2O by DHPC-intrinsic lipid interactions.

Optimization of the Sistrom Culture Medium for Large-Scale Batch Cultivation of Rhodospirillum rubrum under Semiaerobic Conditions with Maximal Yield of Photosynthetic Membranes.

The defined medium A of W. R. Sistrom (W. R. Sistrom, J. Gen. Microbiol. 22:77-85, 1960) has been modified to allow the growth of Rhodospirillum rubrum in large-scale batch cultures under dark, semiaerobic conditions. The simultaneous use of two substrates, NH(4)-succinate (46 mM) and fructose (0.3%), which are utilized in aerobic and fermentative metabolism, respectively, leads to very high cell densities with a maximal yield of photosynthetic membranes.

Two-dimensional crystallization of the light-harvesting complex from Rhodospirillum rubrum.

Homogeneous detergent-solubilized B873 light-harvesting complexes from a carotenoid-less mutant of the purple non-sulfur bacterium, Rhodospirillum rubrum G9, were reassembled spontaneously into two-dimensional (2D) hexagonal arrays during extensive and controlled dialysis. As the complexes contain only 1 to 2 mol phospholipid per mol alpha beta dimer, the arrays formed by a self assembly process are primary due to protein-protein interactions. The hexagonal lattices were analyzed by negative stain electron microscopy and digital image processing. They exhibited a unit cell size of 12.3 nm, in close agreement with the particle diameter of the active photo-unit in native chromatophore membranes. The unit cell contains a central 5 nm stain-filled depression, embraced by a ring with an outer diameter of 10 nm.

Gene expression of the B875 light-harvesting prepolypeptides from Rhodospirillum rubrum in Escherichia coli.

The gene coding for the prepolypeptides of alpha and beta, obtained as a 429 bp fragment from chromosomal DNA of Rhodospirillum rubrum S1 by polymerase chain reaction amplification, were cloned in tandem into the high-level expression vector pOTSNco 12 for expression in Escherichia coli. The vector pOTSNco12 is a derivative of the pAS vector system, which contains the strong lambda PL promotor and is under tight control by the cI857 repressor encoded by the expression strain AR58. Induction of transcription from the lambda PL promotor is achieved by shifting the growth temperature from 32 to 42 degrees C. Expression of the gene products was monitored by sodium dodecylsulfate polyacrylamide gel electrophoresis and western blotting. The expressed B875 light-harvesting prepolypeptides were located in the E. coli inner membrane and could not be removed by washing with high salt. The amount of expressed B875 light-harvesting prepolypeptides was estimated to be about 0.1% of the total soluble protein.

Incorporation of reaction centers into submitochondrial particles resulting in light induced electron transfer.

Conditions for the incorporation of reaction centers, isolated from Rhodospirillum rubrum, into submitochondrial particles have been studied. Incorporation of the reaction centers into the lipid bilayer occurs in both orientations. Electron flow from the light activated reaction center to the b-c1 complex is demonstrated. Preliminary data on the reaction kinetics of the b cytochromes are given.

Interaction of horse cytochrome c with the photosynthetic reaction center of Rhodospirillum rubrum.

Mitochondrial cytochrome c (horse), which is a very efficient electron donor to bacterial photosynthetic reaction centers in vitro, binds to the reaction center of Rhodospirillum rubrum with an approximate dissociation constant of 0.3-0.5 microM at pH 8.2 and low ionic strength. The binding site for the reaction center is on the frontside of cytochrome c which is the side with the exposed heme edge, as revealed by differential chemical acetylation of lysines of free and reaction-center-bound cytochrome c. In contrast, bacterial cytochrome c2 was found previously to bind to the detergent-solubilized reaction center through its backside, i.e., the side opposite to the heme cleft [Rieder, R., Wiemken, V., Bachofen, R., and Bosshard, H. R. (1985). Biochem. Biophys. Res. Commun. 128, 120-126]. Binding of mitochondrial cytochrome c but not of mitochondrial cytochrome c2 is strongly inhibited by low concentrations of poly-L-lysine. The results are difficult to reconcile with the existence of an electron transfer site on the backside of cytochrome c2.