Crystallographic snapshot of cellulose synthesis and membrane translocation.

Cellulose, the most abundant biological macromolecule, is an extracellular, linear polymer of glucose molecules. It represents an essential component of plant cell walls but is also found in algae and bacteria. In bacteria, cellulose production frequently correlates with the formation of biofilms, a sessile, multicellular growth form. Cellulose synthesis and transport across the inner bacterial membrane is mediated by a complex of the membrane-integrated catalytic BcsA subunit and the membrane-anchored, periplasmic BcsB protein. Here we present the crystal structure of a complex of BcsA and BcsB from Rhodobacter sphaeroides containing a translocating polysaccharide. The structure of the BcsA-BcsB translocation intermediate reveals the architecture of the cellulose synthase, demonstrates how BcsA forms a cellulose-conducting channel, and suggests a model for the coupling of cellulose synthesis and translocation in which the nascent polysaccharide is extended by one glucose molecule at a time.

Native architecture of the photosynthetic membrane from Rhodobacter veldkampii.

The photosynthetic membrane in purple bacteria contains several pigment-protein complexes that assure light capture and establishment of the chemiosmotic gradient. The bioenergetic tasks of the photosynthetic membrane require the strong interaction between these various complexes. In the present work, we acquired the first images of the native outer membrane architecture and the supramolecular organization of the photosynthetic apparatus in vesicular chromatophores of Rhodobacter (Rb.) veldkampii. Mixed with LH2 (light-harvesting complex 2) rings, the PufX-containing LH1-RC (light-harvesting complex 1--reaction center) core complexes appear as C-shaped monomers, with random orientations in the photosynthetic membrane. Within the LH1 fence surrounding the RC, a remarkable gap that is probably occupied (or partially occupied) by PufX is visualized. Sequence alignment revealed that one specific region in PufX may be essential for PufX-induced core dimerization. In this region of ten amino acids in length all Rhodobacter species had five conserved amino acids, with the exception of Rb. veldkampii. Our findings provide direct evidence that the presence of PufX in Rb. veldkampii does not directly govern the dimerization of LH1-RC core complexes in the native membrane. It is indicated, furthermore, that the high membrane curvature of Rb. veldkampii chromatophores (Rb. veldkampii features equally small vesicular chromatophores alike Rb. sphaeroides) is not due to membrane bending induced by dimeric RC-LH1-PufX cores, as it has been proposed in Rb. sphaeroides.

Revealing linear aggregates of light harvesting antenna proteins in photosynthetic membranes.

How light energy is harvested in a natural photosynthetic membrane through energy transfer is closely related to the stoichiometry and arrangement of light harvesting antenna proteins in the membrane. The specific photosynthetic architecture facilitates a rapid and efficient energy transfer among the light harvesting proteins (LH2 and LH1) and to the reaction center. Here we report the identification of linear aggregates of light harvesting proteins, LH2, in the photosynthetic membranes under ambient conditions by using atomic force microscopy (AFM) imaging and spectroscopic analysis. Our results suggest that the light harvesting protein, LH2, can exist as linear aggregates of 4 +/- 2 proteins in the photosynthetic membranes and that the protein distributions are highly heterogeneous. In the photosynthetic membranes examined in our measurements, the ratio of the aggregated to the nonaggregated LH2 proteins is about 3:1 to 5:1 depending on the intensity of the illumination used during sample incubation and on the bacterial species. AFM images further identify that the LH2 proteins in the linear aggregates are monotonically tilted at an angle 4 +/- 2 degrees from the plane of the photosynthetic membranes. The aggregates result in red-shifted absorption and emission spectra that are measured using various mutant membranes, including an LH2 knockout, LH1 knockout, and LH2 at different population densities. Measuring the fluorescence lifetimes of purified LH2 and LH2 in membranes, we have observed that the LH2 proteins in membranes exhibit biexponential lifetime decays whereas the purified LH2 proteins gave single exponential lifetime decays. We attribute that the two lifetime components originate from the existence of both aggregated and nonaggregated LH2 proteins in the photosynthetic membranes.

"Sporotan" a new fluorescent stain for identifying cryptic spores of Rhodobacter johrii.

We propose a new fluorescent stain "sporotan" and staining protocol which aid in the identification of cryptic endospores which are otherwise mistaken as poly-ß-hydroxyalkanoate granules.

Solvent-dependent turn-on probe for dual monitoring of Ag(+) and Zn(2+) in living biological samples.

A novel, solvent-dependent "off-on" probe with benzoylthiourea moiety as the functional receptor and fluorescein as the fluorophore was designed for monitoring of Ag(+) in EtOH-H2O (2:8, v/v) solution and Zn(2+) in CH3CN-H2O (2:8, v/v) solution at physiological range with sufficient selectivity and sensitivity. The Ag(+) promoted desulfurization of thiosemicarbazide functionality in formation of the 1,3,4-oxadiazole and the coordination of Zn(2+) to the O atom and N atom of the spoirolactam moiety and the S atom of the benzoylthiourea moiety were investigated to be the power that promoted the fluorescent enhancement. This probe was tested highly suitable for mapping Ag(+) and Zn(2+) in living human osteosarcoma MG-63 cells and microbial cell-EPS-mineral aggregates, thus, providing a wonderful candidate for tracking Ag(+) and Zn(2+) in biological organisms and processes.

Visualizing tributyltin (TBT) in bacterial aggregates by specific rhodamine-based fluorescent probes.

Here we present the first examples of fluorescent and colorimetric probes for microscopic TBT imaging. The fluorescent probes are highly selective and sensitive to TBT and have successfully been applied for imaging of TBT in bacterial Rhodobacter ferrooxidans sp. strain SW2 cell-EPS-mineral aggregates and in cell suspensions of the marine cyanobacterium Synechococcus PCC 7002 by using confocal laser scanning microscopy.

Biodegradation of Various Aromatic Compounds by Enriched Bacterial Cultures: Part A-Monocyclic and Polycyclic Aromatic Hydrocarbons.

Present study focused on the screening of bacterial consortium for biodegradation of monocyclic aromatic hydrocarbon (MAH) and polycyclic aromatic hydrocarbons (PAHs). Target compounds in the present study were naphthalene, acenaphthene, phenanthrene (PAHs), and benzene (MAH). Microbial consortia enriched with the above target compounds were used in screening experiments. Naphthalene-enriched consortium was found to be the most efficient consortium, based on its substrate degradation rate and its ability to degrade other aromatic pollutants with significantly high efficiency. Substrate degradation rate with naphthalene-enriched culture followed the order benzene > naphthalene > acenaphthene > phenanthrene. Chryseobacterium and Rhodobacter were discerned as the predominant species in naphthalene-enriched culture. They are closely associated to the type strain Chryseobacterium arthrosphaerae and Rhodobacter maris, respectively. Single substrate biodegradation studies with naphthalene (PAH) and benzene (MAH) were carried out using naphthalene-enriched microbial consortium (NAPH). Phenol and 2-hydroxybenzaldehyde were identified as the predominant intermediates during benzene and naphthalene degradation, respectively. Biodegradation of toluene, ethyl benzene, xylene, phenol, and indole by NAPH was also investigated. Monod inhibition model was able to simulate biodegradation kinetics for benzene, whereas multiple substrate biodegradation model was able to simulate biodegradation kinetics for naphthalene.

Continuous cultivation of photosynthetic bacteria for fatty acids production.

In the present work, we introduced a novel approach for microbial fatty acids (FA) production. Photosynthetic bacteria, Rhodobacter sphaeroides KD131, were cultivated in a continuous-flow, stirred-tank reactor (CFSTR) at various substrate (lactate) concentrations. At hydraulic retention time (HRT) 4d, cell concentration continuously increased from 0.97 g dcw/L to 2.05 g dcw/L as lactate concentration increased from 30 mM to 60mM. At 70 mM, however, cell concentration fluctuated with incomplete substrate degradation. By installing a membrane unit to CFSTR, a stable performance was observed under much higher substrate loading (lactate 100mM and HRT 1.5d). A maximum cell concentration of 16.2g dcw/L, cell productivity of 1.9 g dcw/L/d, and FA productivity of 665 mg FA/L/d were attained, and these values were comparable with those achieved using microalgae. The FA content of R. sphaeroides was around 35% of dry cell weight, mainly composed of vaccenic acid (C18:1, omega-7).

[Rhodobaca barguzinensis sp. nov., a new alkaliphilic purple nonsulfur bacterium isolated from a soda lake of the Barguzin Valley (Buryat Republic, eastern Siberia)].

A novel strain, alga-05, of alkaliphilic purple nonsulfur bacteria was isolated from sediments of a small saline (60 g/l) soda lake near Lake Algin (Barguzin Valley, Buryat Republic, Russia). These bacteria contain bacteriochlorophyll a and carotenoids of the alternative spirilloxanthin group with predominating demethylspheroidenone. They are facultative anaerobes; their photosynthetic structures are of the vesicular type and arranged along the cell periphery. Growth of this strain is possible in a salinity range of 5-80 g/l NaCl, with an optimum at 20 g/l NaCl. Best growth occurred at 20-35 degrees C. Analysis of the 16S rRNA gene sequences demonstrated that the studied isolate is closely related to the alkaliphilic purple nonsulfur bacterium Rhodobaca bogoriensis (99% similarity) isolated from soda lakes of the African Rift Zone. According to the results of DNA-DNA hybridization, strain alga-05 has a 52% similarity with the type species of the genus Rhodobaca. On the basis of the obtained genotypic data and some phenotypic properties (dwelling in a hypersaline soda lake of Siberia, moderate halophily, ability to grow at relatively low temperatures, etc.), the isolated strain of purple bacteria was described as a new species of the genus Rhodobaca, Rca. barguzinensis sp. nov.

Identification of "Haematobacter," a new genus of aerobic Gram-negative rods isolated from clinical specimens, and reclassification of Rhodobacter massiliensis as "Haematobacter massiliensis comb. nov.".

Twelve strains of gram-negative, nonfermenting rods recovered mainly from septicemic patients were studied using conventional and molecular methods. The phenotypic profiles of these strains most closely resembled Psychrobacter phenylpyruvicus. They produced catalase, oxidase, urease, and H(2)S (lead acetate paper) but did not produce indole, reduce nitrate or nitrite, or hydrolyze gelatin or esculin. No acid production was observed in a King's oxidation-fermentation base containing d-glucose, d-xylose, d-mannitol, sucrose, lactose, or maltose. All strains were nonmotile and nonpigmented. Most strains produced green discoloration on blood agar. All strains grew at 25 degrees C and 35 degrees C and most grew on MacConkey agar. They shared a common cellular fatty acid (CFA) profile characterized by large amounts (56% to 90%) of 18:1omega7c and the presence of 3-OH-10:0, 16:1omega7c, 16:0, and 19:0cycomega8c that overall was most similar to that of Rhodobacter species but was quite distinct from that of P. phenylpyruvicus. The MICs for most beta-lactams, fluoroquinolones, aminoglycosides, and carbapenems were low. MICs for aztreonam and piperacillin were higher, with MICs for some strains of > 64 mg/liter and > 128 mg/liter, respectively. Polyphasic analysis of these strains, including morphological, biochemical, CFA composition, DNA-DNA hybridization, 16S rRNA gene sequencing, and percent guanine-plus-cytosine (G+C) content analysis, demonstrated that these strains and Rhodobacter massiliensis represent a new genus, "Haematobacter" (proposed name), with the species H. missouriensis (type strain H1892(T) = CCUG 52307(T) = CIP 109176(T)) and H. massiliensis comb. nov. (type strain Framboise(T) = CCUG 47968(T) = CIP 107725(T)) and an unnamed genomospecies.

Structure of the dimeric PufX-containing core complex of Rhodobacter blasticus by in situ atomic force microscopy.

We have studied photosynthetic membranes of wild type Rhodobacter blasticus, a closely related strain to the well studied Rhodobacter sphaeroides, using atomic force microscopy. High-resolution atomic force microscopy topographs of both cytoplasmic and periplasmic surfaces of LH2 and RC-LH1-PufX (RC, reaction center) complexes were acquired in situ. The LH2 is a nonameric ring inserted into the membrane with the 9-fold axis perpendicular to the plane. The core complex is an S-shaped dimer composed of two RCs, each encircled by 13 LH1 alpha/beta-heterodimers, and two PufXs. The LH1 assembly is an open ellipse with a topography-free gap of approximately 25 A. The two PufXs, one of each core, are located at the dimer center. Based on our data, we propose a model of the core complex, which provides explanation for the PufX-induced dimerization of the Rhodobacter core complex. The QB site is located facing a approximately 25-A wide gap within LH1, explaining the PufX-favored quinone passage in and out of the core complex.

Occurrence, overexpression and partial purification of the protein (majastridin) corresponding to the URF6 gene of the Rhodobacter blasticus atp operon.

Antibodies were produced against two antigenic peptides of a protein, which was named majastridin, corresponding to the URF6 gene of the Rhodobacter blasticus atp operon [Tybulewicz, V. L. J., Falk, G. & Walker, J. E. (1984) J. Mol. Biol. 179, 185-214]. A protein band of the expected size is labelled by immunoblotting in Western blots containing the cytosolic fractions from Rb. blasticus and Paracoccus denitrificans but not from Escherichia coli or Rhodospirillum rubrum. Although the protein is present during the entire life cycle of a Rb. blasticus culture, it is most abundant early during the stationary phase. Plasmid constructs of the URF6 gene for overexpression in E. coli were made. These constructs were designed to obtain proteins both with and without His-tagging. In both cases, a protein product was visible in induced cells. The His-tagged protein was purified to 85% on a Ni column and, further, to at least 95% by anion-exchange chromatography. By N-terminal sequencing of the His-tagged protein, its identity was confirmed.