Classification of organic and biological materials with deep ultraviolet excitation.

We show that native fluorescence can be used to differentiate classes or groups of organic molecules and biological materials when excitation occurs at specific excitation wavelengths in the deep ultraviolet (UV) region. Native fluorescence excitation-emission maps (EEMs) of pure organic materials, microbiological samples, and environmental background materials were compared using excitation wavelengths between 200-400 nm with emission wavelengths from 270 to 500 nm. These samples included polycyclic aromatic hydrocarbons (PAHs), nitrogen- and sulfur-bearing organic heterocycles, bacterial spores, and bacterial vegetative whole cells (both Gram positive and Gram negative). Each sample was categorized into ten distinct groups based on fluorescence properties. Emission spectra at each of 40 excitation wavelengths were analyzed using principal component analysis (PCA). Optimum excitation wavelengths for differentiating groups were determined using two metrics. We show that deep UV excitation at 235 (+/-2) nm optimally separates all organic and biological groups within our dataset with >90% confidence. For the specific case of separation of bacterial spores from all other samples in the database, excitation at wavelengths less than 250 nm provides maximum separation with >6sigma confidence.

Genome sequence of the dissimilatory metal ion-reducing bacterium Shewanella oneidensis.

Shewanella oneidensis is an important model organism for bioremediation studies because of its diverse respiratory capabilities, conferred in part by multicomponent, branched electron transport systems. Here we report the sequencing of the S. oneidensis genome, which consists of a 4,969,803-base pair circular chromosome with 4,758 predicted protein-encoding open reading frames (CDS) and a 161,613-base pair plasmid with 173 CDSs. We identified the first Shewanella lambda-like phage, providing a potential tool for further genome engineering. Genome analysis revealed 39 c-type cytochromes, including 32 previously unidentified in S. oneidensis, and a novel periplasmic [Fe] hydrogenase, which are integral members of the electron transport system. This genome sequence represents a critical step in the elucidation of the pathways for reduction (and bioremediation) of pollutants such as uranium (U) and chromium (Cr), and offers a starting point for defining this organism's complex electron transport systems and metal ion-reducing capabilities.

Microbial survival: the paleome: a sedimentary genetic record of past microbial communities.

Molecular genetic methods were used to analyze the remnants of microbial ecosystems contained within an ancient oceanic microbial habitat that was recovered from a continental drilled core of black shale approximately 100 million years in age. Bacterial ribosomal RNA genes were vertically amplified from the six different depths of a black shale core associated with a phosphate-rich stratum, defined as one of the mid-Cretaceous oceanic anoxic events (OAEs). Although the black shale core was recovered from a terrestrial coring effort, the recovered 16S rRNA gene sequences showed affinity to microbial communities previously seen in deep-sea sedimentary environments (i.e., the microbial assemblage was easily recognizable as a marine community). In particular, a number of 16S rRNA gene clones of oceanic sulfate-reducing bacteria within the delta-Proteobacteria predominated at the OAE layer. The recovered bacterial DNA signatures are consistent with the interpretation that the sequences are derived from the past microbial communities buried in either sea-bottom or subseafloor environments during the sedimentation process and, after ceasing growth, preserved until the present.

A directional electron transfer regulator based on heme-chain architecture in the small tetraheme cytochrome c from Shewanella oneidensis.

The macroscopic and microscopic redox potentials of the four hemes of the small tetraheme cytochrome c from Shewanella oneidensis were determined. The microscopic redox potentials show that the order of reduction is from hemes in the C-terminal domain (hemes 3 and 4) to the N-terminal domain (heme 1), demonstrating the polarization of the tetraheme chain during reduction. This makes heme 4 the most efficient electron delivery site. Furthermore, multi-step reduction of other redox centers through either heme 4 or heme 3 is shown to be possible. This has provided new insights into the two-electron reduction of the flavin in the homologous flavocytochrome c-fumarate reductase.

Identification of 42 possible cytochrome C genes in the Shewanella oneidensis genome and characterization of six soluble cytochromes.

Through pattern matching of the cytochrome c heme-binding site (CXXCH) against the genome sequence of Shewanella oneidensis MR-1, we identified 42 possible cytochrome c genes (27 of which should be soluble) out of a total of 4758. However, we found only six soluble cytochromes c in extracts of S. oneidensis grown under several different conditions: (1) a small tetraheme cytochrome c, (2) a tetraheme flavocytochrome c-fumarate reductase, (3) a diheme cytochrome c4, (4) a monoheme cytochrome c5, (5) a monoheme cytochrome c', and (6) a diheme bacterial cytochrome c peroxidase. These cytochromes were identified either through N-terminal or complete amino acid sequence determination combined with mass spectroscopy. All six cytochromes were about 10-fold more abundant when cells were grown at low than at high aeration, whereas the flavocytochrome c-fumarate reductase was specifically induced by anaerobic growth on fumarate. When adjusted for the different heme content, the monoheme cytochrome c5 is as abundant as are the small tetraheme cytochrome and the tetraheme fumarate reductase. Published results on regulation of cytochromes from DNA microarrays and 2D-PAGE differ somewhat from our results, emphasizing the importance of multifaceted analyses in proteomics.

Aspartic acid racemization and age-depth relationships for organic carbon in Siberian permafrost.

We have analyzed the degree of racemization of aspartic acid in permafrost samples from Northern Siberia, an area from which microorganisms of apparent ages up to a few million years have previously been isolated and cultured. We find that the extent of aspartic acid racemization in permafrost cores increases very slowly up to an age of approximately 25,000 years (around 5 m in depth). The apparent temperature of racemization over the age range of 0-25,000 years, determined using measured aspartic acid racemization rate constants, is -19 degrees C. This apparent racemization temperature is significantly lower than the measured environmental temperature (-11 to -13 degrees C) and suggests active recycling of D-aspartic acid in Siberian permafrost up to an age of around 25,000 years. This indicates that permafrost organisms are capable of repairing some molecular damage incurred while in a "dormant" state over geologic time.

Marinospirillum celere sp. nov., a novel haloalkaliphilic, helical bacterium isolated from Mono Lake.

Two strains of a Gram-negative, helical, haloalkaliphilic bacterium were isolated from Mono Lake (USA). Both strains were mesophilic and grew between 13 and 55 degrees C, with optimum growth at 35-45 degrees C. The optimum pH for growth was 9.5. Growth was observed at NaCl concentrations of 0.5-12% (w/v), with optimum growth at 2% NaCl. Both isolates were motile by means of bipolar tuft flagella, coccoid body-forming and strictly aerobic. It was concluded that they belong to the same species, based on DNA-DNA hybridization values (95% DNA relatedness). DNA G+C contents of the novel strains were 52.1 and 52.3 mol%. On the basis of 16S rRNA gene sequence similarity, both strains were shown to be related closely to the members of the genus Marinospirillum (family Oceanospirillaceae, class Gammaproteobacteria). Sequence similarity of strain v1c_Sn-redT to the type strains of Marinospirillum alkaliphilum, Marinospirillum minutulum, Marinospirillum megaterium and Marinospirillum insulare was 95.0, 92.7, 91.8 and 91.8%, respectively. Chemotaxonomic data [major ubiquinone, Q8; major fatty acids, C18:1(n-7) and C16:0] and physiological and biochemical tests supported the affiliation of the novel strains to the genus Marinospirillum as members of a novel species, for which the name Marinospirillum celere sp. nov. is proposed, with the type strain v1c_Sn-redT (=LMG 24610T=VKM 2416T).

Reproduction and metabolism at - 10 degrees C of bacteria isolated from Siberian permafrost.

We report the isolation and properties of several species of bacteria from Siberian permafrost. Half of the isolates were spore-forming bacteria unable to grow or metabolize at subzero temperatures. Other Gram-positive isolates metabolized, but never exhibited any growth at - 10 degrees C. One Gram-negative isolate metabolized and grew at - 10 degrees C, with a measured doubling time of 39 days. Metabolic studies of several isolates suggested that as temperature decreased below + 4 degrees C, the partitioning of energy changes with much more energy being used for cell maintenance as the temperature decreases. In addition, cells grown at - 10 degrees C exhibited major morphological changes at the ultrastructural level.

Crystal structures at atomic resolution reveal the novel concept of "electron-harvesting" as a role for the small tetraheme cytochrome c.

The genus Shewanella produces a unique small tetraheme cytochrome c that is implicated in the iron oxide respiration pathway. It is similar in heme content and redox potential to the well known cytochromes c(3) but related in structure to the cytochrome c domain of soluble fumarate reductases from Shewanella sp. We report the crystal structure of the small tetraheme cytochrome c from Shewanella oneidensis MR-1 in two crystal forms and two redox states. The overall fold and heme core are surprisingly different from the soluble fumarate reductase structures. The high resolution obtained for an oxidized orthorhombic crystal (0.97 A) revealed several flexible regions. Comparison of the six monomers in the oxidized monoclinic space group (1.55 A) indicates flexibility in the C-terminal region containing heme IV. The reduced orthorhombic crystal structure (1.02 A) revealed subtle differences in the position of several residues, resulting in decreased solvent accessibility of hemes and the withdrawal of a positive charge from the molecular surface. The packing between monomers indicates that intermolecular electron transfer between any heme pair is possible. This suggests there is no unique site of electron transfer on the surface of the protein and that electron transfer partners may interact with any of the hemes, a process termed "electron-harvesting." This optimizes the efficiency of intermolecular electron transfer by maximizing chances of productive collision with redox partners.

Anaerobranca californiensis sp. nov., an anaerobic, alkalithermophilic, fermentative bacterium isolated from a hot spring on Mono Lake.

A novel, obligately anaerobic, alkalithermophilic, chemo-organotrophic bacterium was isolated from the sediment of an alkaline hot spring located on Paoha Island in Mono Lake, California, USA. This rod-shaped bacterium was motile via peritrichous flagella. Isolated strains grew optimally in 5-25 g NaCl l(-1), at pH 9.0-9.5 and at a temperature of 58 degrees C and were fermentative and mainly proteolytic, utilizing peptone, Casamino acids and yeast extract. Optimal growth was seen in the presence of elemental sulfur, polysulfide or thiosulfate with concomitant reduction to hydrogen sulfide. Sulfite was also formed in an equal ratio to sulfide during reduction of thiosulfate. The novel isolate could also reduce Fe(III) and Se(IV) in the presence of organic matter. On the basis of physiological properties, 16S rRNA gene sequence and DNA-DNA hybridization data, strain PAOHA-1(T) (=DSM 14826(T)=UNIQEM 227(T)) belongs to the genus Anaerobranca and represents a novel species, Anaerobranca californiensis sp. nov.

Analysis of the Shewanella oneidensis proteome by two-dimensional gel electrophoresis under nondenaturing conditions.

Proteomes are dynamic, i.e., the protein components of living cells change in response to various stimuli. Protein changes can involve shifts in the abundance of protein components, in the interactions of protein components, and in the activity of protein components. Two-dimensional gel electrophoresis (2-DE) coupled with peptide mass spectrometry is useful for the analysis of relative protein abundance, but the denaturing conditions of classical 2-DE do not allow analysis of protein interactions or protein function. We have developed a nondenaturing 2-DE method that allows analysis of protein interactions and protein functions, as demonstrated in our analysis of the cytosol and crude membrane fractions of the facultative anaerobe Shewanella oneidensis MR-1. Our experiments demonstrate that enzymatic activity is retained under the sample and protein separation methods described, as shown by positive malate dehydrogenase activity results. We have also found protein interactions within both the soluble and membrane fractions. The method described will be useful for the characterization of the functional proteomes of microbial systems.

Thioalkalimicrobium cyclicum sp. nov. and Thioalkalivibrio jannaschii sp. nov., novel species of haloalkaliphilic, obligately chemolithoautotrophic sulfur-oxidizing bacteria from hypersaline alkaline Mono Lake (California).

Two strains of haloalkaliphilic, obligately autotrophic, sulfur-oxidizing bacteria were isolated from the oxygen-sulfide interface water layer of stratified alkaline and saline Mono Lake, California, USA. Strain ALM 1T was a dominant species in enrichment on moderate-saline, carbonate-buffered medium (0.6 M total Na+, pH 10) with thiosulfate as an energy source and nitrate as a nitrogen source. Cells of ALM 1T are open ring-shaped and are non-motile. It has a high growth rate and activity of thiosulfate and sulfide oxidation and very low sulfur-oxidizing activity. Genetic comparison and phylogenetic analysis suggested that ALM 1T (= DSM 14477T = JCM 11371T) represents a new species of the genus Thioalkalimicrobium in the gamma-Proteobacteria, for which the name Thioalkalimicrobium cyclicum sp. nov. is proposed. Another Mono Lake isolate, strain ALM 2T, dominated in enrichment on a medium containing 2 M total Na+ (pH 10). It is a motile vibrio which tolerates up to 4 M Na+ and produces a membrane-bound yellow pigment. Phylogenetic analysis placed ALM 2T as a member of genus Thioalkalivibrio in the gamma-Proteobacteria, although its DNA hybridization with the representative strains of this genus was only about 30%. On the basis of genetic and phenotypic properties, strain ALM 2T (= DSM 14478T = JCM 11372T) is proposed as Thioalkalivibrio jannaschii sp. nov..