Draft Genome Sequence of Cyanobacterium sp. Strain HL-69, Isolated from a Benthic Microbial Mat from a Magnesium Sulfate-Dominated Hypersaline Lake.

The complete genome sequence of Cyanobacterium sp. strain HL-69 consists of 3,155,247 bp and contains 2,897 predicted genes comprising a chromosome and two plasmids. The genome is consistent with a halophilic nondiazotrophic phototrophic lifestyle, and this organism is able to synthesize most B vitamins and produces several secondary metabolites.

Electrokinesis is a microbial behavior that requires extracellular electron transport.

We report a previously undescribed bacterial behavior termed electrokinesis. This behavior was initially observed as a dramatic increase in cell swimming speed during reduction of solid MnO(2) particles by the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1. The same behavioral response was observed when cells were exposed to small positive applied potentials at the working electrode of a microelectrochemical cell and could be tuned by adjusting the potential on the working electrode. Electrokinesis was found to be different from both chemotaxis and galvanotaxis but was absent in mutants defective in electron transport to solid metal oxides. Using in situ video microscopy and cell tracking algorithms, we have quantified the response for different strains of Shewanella and shown that the response correlates with current-generating capacity in microbial fuel cells. The electrokinetic response was only exhibited by a subpopulation of cells closest to the MnO(2) particles or electrodes. In contrast, the addition of 1 mM 9,10-anthraquinone-2,6-disulfonic acid, a soluble electron shuttle, led to increases in motility in the entire population. Electrokinesis is defined as a behavioral response that requires functional extracellular electron transport and that is observed as an increase in cell swimming speeds and lengthened paths of motion that occur in the proximity of a redox active mineral surface or the working electrode of an electrochemical cell.

Oxygen-dependent autoaggregation in Shewanella oneidensis MR-1.

In aerobic chemostat cultures maintained at 50% dissolved O(2) tension (3.5 mg l(-1) dissolved O(2)), Shewanella oneidensis strain MR-1 rapidly aggregated upon addition of 0.68 mM CaCl(2) and retained this multicellular phenotype at high dilution rates. Confocal microscopy analysis of the extracellular matrix material contributing to the stability of the aggregate structures revealed the presence of extracellular DNA, protein and glycoconjugates. Upon onset of O(2)-limited growth (dissolved O(2) below detection) however, the Ca(2+)-supplemented chemostat cultures of strain MR-1 rapidly disaggregated and grew as motile dispersed cells. Global transcriptome analysis comparing aerobic aggregated to O(2)-limited unaggregated cells identified genes encoding cell-to-cell and cell-to-surface adhesion factors whose transcription increased upon exposure to increased O(2) concentrations. The aerobic aggregated cells also revealed increased expression of putative anaerobic electron transfer and homologues of metal reduction genes, including mtrD (SO1782), mtrE (SO1781) and mtrF (SO1780). Our data indicate that mechanisms involved in autoaggregation of MR-1 are dependent on the function of pilD gene which encodes a putative prepilin peptidase. Mutants of S. oneidensis strain MR-1 deficient in PilD and associated pathways, including type IV and Msh pili biogenesis, displayed a moderate increase in sensitivity to H(2)O(2). Taken together, our evidence indicates that aggregate formation in S. oneidensis MR-1 may serve as an alternative or an addition to biochemical detoxification to reduce the oxidative stress associated with production of reactive oxygen species during aerobic metabolism while facilitating the development of hypoxic conditions within the aggregate interior.

Global transcriptome analysis of Shewanella oneidensis MR-1 exposed to different terminal electron acceptors.

To gain insight into the complex structure of the energy-generating networks in the dissimilatory metal reducer Shewanella oneidensis MR-1, global mRNA patterns were examined in cells exposed to a wide range of metal and non-metal electron acceptors. Gene expression patterns were similar irrespective of which metal ion was used as electron acceptor, with 60% of the differentially expressed genes showing similar induction or repression relative to fumarate-respiring conditions. Several groups of genes exhibited elevated expression levels in the presence of metals, including those encoding putative multidrug efflux transporters, detoxification proteins, extracytoplasmic sigma factors and PAS-domain regulators. Only one of the 42 predicted c-type cytochromes in MR-1, SO3300, displayed significantly elevated transcript levels across all metal-reducing conditions. Genes encoding decaheme cytochromes MtrC and MtrA that were previously linked to the reduction of different forms of Fe(III) and Mn(IV), exhibited only slight decreases in relative mRNA abundances under metal-reducing conditions. In contrast, specific transcriptome responses were displayed to individual non-metal electron acceptors resulting in the identification of unique groups of nitrate-, thiosulfate- and TMAO-induced genes including previously uncharacterized multi-cytochrome gene clusters. Collectively, the gene expression results reflect the fundamental differences between metal and non-metal respiratory pathways of S. oneidensis MR-1, where the coordinate induction of detoxification and stress response genes play a key role in adaptation of this organism under metal-reducing conditions. Moreover, the relative paucity and/or the constitutive nature of genes involved in electron transfer to metals is likely due to the low-specificity and the opportunistic nature of the metal-reducing electron transport pathways.

Trichloroethene reductive dehalogenase from Dehalococcoides ethenogenes: sequence of tceA and substrate range characterization.

The anaerobic bacterium Dehalococcoides ethenogenes is the only known organism that can completely dechlorinate tetrachloroethene or trichloroethene (TCE) to ethene via dehalorespiration. One of two corrinoid-containing enzymes responsible for this pathway, TCE reductive dehalogenase (TCE-RDase) catalyzes the dechlorination of TCE to ethene. TCE-RDase dehalogenated 1,2-dichloroethane and 1, 2-dibromoethane to ethene at rates of 7.5 and 30 micromol/min/mg, respectively, similar to the rates for TCE, cis-dichloroethene (DCE), and 1,1-DCE. A variety of other haloalkanes and haloalkenes containing three to five carbon atoms were dehalogenated at lower rates. The gene encoding TCE-RDase, tceA, was cloned and sequenced via an inverse PCR approach. Sequence comparisons of tceA to proteins in the public databases revealed weak sequence similarity confined to the C-terminal region, which contains the eight-iron ferredoxin cluster binding motif, (CXXCXXCXXXCP)(2). Direct N-terminal sequencing of the mature enzyme indicated that the first 42 amino acids constitute a signal sequence containing the twin-arginine motif, RRXFXK, associated with the Sec-independent membrane translocation system. This information coupled with membrane localization studies indicated that TCE-RDase is located on the exterior of the cytoplasmic membrane. Like the case for the two other RDases that have been cloned and sequenced, a small open reading frame, tceB, is proposed to be involved with membrane association of TCE-RDase and is predicted to be cotranscribed with tceA.

SENTRA, a database of signal transduction proteins.

SENTRA, available via URL http://wit.mcs.anl.gov/WIT2/Sentra/, is a database of proteins associated with microbial signal transduction. The database currently includes the classical two-component signal transduction pathway proteins and methyl-accepting chemotaxis proteins, but will be expanded to also include other classes of signal transduction systems that are modulated by phosphorylation or methylation reactions. Although the majority of database entries are from prokaryotic systems, eukaroytic proteins with bacterial-like signal transduction domains are also included. Currently SENTRA contains signal transduction proteins in 34 complete and almost completely sequenced prokaryotic genomes, as well as sequences from 243 organisms available in public databases (SWISS-PROT and EMBL). The analysis was carried out within the framework of the WIT2 system, which is designed and implemented to support genetic sequence analysis and comparative analysis of sequenced genomes.

Complete sequence of a 184-kilobase catabolic plasmid from Sphingomonas aromaticivorans F199.

The complete 184,457-bp sequence of the aromatic catabolic plasmid, pNL1, from Sphingomonas aromaticivorans F199 has been determined. A total of 186 open reading frames (ORFs) are predicted to encode proteins, of which 79 are likely directly associated with catabolism or transport of aromatic compounds. Genes that encode enzymes associated with the degradation of biphenyl, naphthalene, m-xylene, and p-cresol are predicted to be distributed among 15 gene clusters. The unusual coclustering of genes associated with different pathways appears to have evolved in response to similarities in biochemical mechanisms required for the degradation of intermediates in different pathways. A putative efflux pump and several hypothetical membrane-associated proteins were identified and predicted to be involved in the transport of aromatic compounds and/or intermediates in catabolism across the cell wall. Several genes associated with integration and recombination, including two group II intron-associated maturases, were identified in the replication region, suggesting that pNL1 is able to undergo integration and excision events with the chromosome and/or other portions of the plasmid. Conjugative transfer of pNL1 to another Sphingomonas sp. was demonstrated, and genes associated with this function were found in two large clusters. Approximately one-third of the ORFs (59 of them) have no obvious homology to known genes.

Ecology, physiology, and phylogeny of deep subsurface Sphingomonas sp.

Several new species of the genus Sphingomonas including S. aromaticivorans, S. stygia, and S. subterranea that have the capacity for degrading a broad range of aromatic compounds including toluene, naphthalene, xylenes, p-cresol, fluorene, biphenyl, and dibenzothiophene, were isolated from deeply-buried (>200 m) sediments of the US Atlantic coastal plain (ACP). In S. aromaticivorans F199, many of the genes involved in the catabolism of these aromatic compounds are encoded on a 184-kb conjugative plasmid; some of the genes involved in aromatic catabolism are plasmid-encoded in the other strains as well. Members of the genus Sphingomonas were common among aerobic heterotrophic bacteria cultured from ACP sediments and have been detected in deep subsurface environments elsewhere. The major source of organic carbon for heterotrophic metabolism in ACP deep aquifers is lignite that originated from plant material buried with the sediments. We speculate that the ability of the subsurface Sphingomonas strains to degrade a wide array of aromatic compounds represents an adaptation for utilization of sedimentary lignite. These and related subsurface Sphingomonas spp may play an important role in the transformation of sedimentary organic carbon in the aerobic and microaerobic regions of the deep aquifers of the ACP.

Induction of aromatic catabolic activity in Sphingomonas aromaticivorans strain F199.

Enzyme induction studies with Sphingomonas aromaticivorans F199 demonstrated that both toluene and naphthalene induced expression of both naphthalene and toluene catabolic enzymes. However, neither aromatic compound induced expression of all the enzymes required for complete mineralization of either naphthalene or toluene. Activity measurements in combination with gene sequence analyses indicate that growth on either aromatic substrate in the absence of the other is, therefore, sub-optimal and is predicted to lead to the build-up of metabolites due to imbalance in toluene or naphthalene catabolic enzyme activities. Growth on toluene may be further inhibited by the co-expression of two toluene catabolic pathways, as predicted from gene sequence analyses. One of these pathways may potentially result in the formation of a dead-end intermediate, possibly benzaldehyde. In contrast, either p-cresol or benzoate can support high levels of growth. Analyses of promoter region sequences on the F199 aromatic catabolic plasmid, pNL1, suggest that additional regulatory events are modulated through the interaction of BphR with Sigma54 type promoters and through the binding of a regulator upstream of p-cresol catabolic genes and xylM. We hypothesize that the unusual gene clustering in strain F199 is optimized for simultaneous degradation of multiple aromatic compound classes, possibly in response to the heterogeneous composition of aromatic structures in the fossil organic matter present in the deep Atlantic Coastal Plain sediments from which this bacterium was isolated.

Halomonas campisalis sp. nov., a denitrifying, moderately haloalkaliphilic bacterium.

The isolation and characterization of a denitrifying bacterium that is both moderately halophilic and alkaliphilic is described. The organism was isolated for use in the development of a bioprocess that could potentially reduce the costs of ion exchange resin regenerant disposal. The process of ion exchange, after resin regeneration, produces a briny, alkaline waste that is difficult and expensive to dispose. The biological removal of nitrate and subsequent reuse of these brines can potentially provide a cost-saving alternative to disposing of this waste product. To achieve our objective, a moderately halophilic, alkaliphilic bacterium was isolated from sediment samples taken from the salt plain of Alkali Lake in Washington State (USA). The haloalkaliphilic bacterium, designated strain 4A, is motile with rod-shaped cells that are 3 to 5 microm long and 1 microm wide. Electron acceptors used include oxygen, nitrate, and nitrite. In addition, it has similar specific nitrate reduction rates and biomass yields as non-halophilic denitrifying bacteria. It is capable of using a variety of electron donors. This organism can grow at NaCl concentrations ranging from 0.2 to 4.5 M with optimum growth occurring at 1.5 M and pH values ranging from 6 to 12 with 9.5 being the optimum pH. The temperature range for growth of strain 4A is 4-50 degrees C with optimal growth occurring at 30 degrees C. The G + C content is 66 mol%. Phylogenetic analyses based upon 16S rDNA gene sequence placed isolate 4A in the genus Halomonas. In addition, DNA-DNA hybridization experiments clearly indicate that it is a unique species. Phenotypic and phylogenetic studies indicate that isolate 4A represents a new species. We propose the name Halomonas campisalis for this species and strain 4A (ATCC 700597) as the type strain. Due to its denitrification ability, broad carbon utilization range and its high salinity and pH tolerance this organism, and similar ones, hold promise for the treatment of saline, alkaline waste.

Taxonomic study of aromatic-degrading bacteria from deep-terrestrial-subsurface sediments and description of Sphingomonas aromaticivorans sp. nov., Sphingomonas subterranea sp. nov., and Sphingomonas stygia sp. nov.

Phylogenetic analyses of 16S rRNA gene sequences by distance matrix and parsimony methods indicated that six strains of bacteria isolated from deep saturated Atlantic coastal plain sediments were closely related to the genus Sphingomonas. Five of the strains clustered with, but were distinct from, Sphingomonas capsulata, whereas the sixth strain was most closely related to Blastobacter natatorius. The five strains that clustered with S. capsulata, all of which could degrade aromatic compounds, were gram-negative, non-spore-forming, non-motile, rod-shaped organisms that produced small, yellow colonies on complex media. Their G + C contents ranged from 60.0 to 65.4 mol%, and the predominant isoprenoid quinone was ubiquinone Q-10. All of the strains were aerobic and catalase positive. Indole, urease, and arginine dihydrolase were not produced. Gelatin was not liquified, and glucose was not fermented. Sphingolipids were present in all strains; 2OH14:0 was the major hydroxy fatty acid, and 18:1 was a major constituent of cellular lipids. Acid was produced oxidatively from pentoses, hexoses, and disaccharides, but not from polyalcohols and indole. All of these characteristics indicate that the five aromatic-degrading strains should be placed in the genus Sphingomonas as currently defined. Phylogenetic analysis of 16S rRNA gene sequences, DNA-DNA reassociation values, BOX-PCR genomic fingerprinting, differences in cellular lipid composition, and differences in physiological traits all indicated that the five strains represent three previously undescribed Sphingomonas species. Therefore, we propose the following new species: Sphingomonas aromaticivorans (type strain, SMCC F199), Sphingomonas subterranea (type strain, SMCC B0478), and Sphingomonas stygia (type strain, SMCC B0712).

Recruitment and expression of toluene/trichloroethylene biodegradation genes in bacteria native to deep-subsurface sediments.

Four plasmids, each encoding a combination of either an Escherichia coli or Pseudomonas putida promoter and either toluene dioxygenase or toluene monooxygenase, were electroporated into five bacterial strains isolated from sediments found at depths of 91 to 295 m. Four of these engineered bacterial strains demonstrated both toluene and trichloroethylene degradation activities.

Homology between genes for aromatic hydrocarbon degradation in surface and deep-subsurface Sphingomonas strains.

The cloned genes for aromatic hydrocarbon degradation from Sphingomonas yanoikuyae B1 were utilized in Southern hybridization experiments with Sphingomonas strains from the surface and deep-subsurface environments. One hybridization pattern was obtained with BamHI-digested genomic DNAs for two surface strains, while a differing pattern was seen for five deep-subsurface strains. The cross-hybridizing genes were located in the chromosomes of the surface strains and on plasmids in the deep-subsurface strains.

Physical mapping and characterization of a catabolic plasmid from the deep-subsurface bacterium Sphingomonas sp. strain F199.

A supercoiled 180-kb plasmid, pNL1, has been isolated from the deep-subsurface, chemoheterotrophic Sphingomonas sp. strain F199, and a physical map was generated. Analysis of a pNL1-derived cosmid library indicated that catechol 2,3-dioxygenase activity was linked to two distinct regions of the plasmid. Thus, the genes for aromatic catabolism in this Sphingomonas strain are, at least in part, plasmid encoded.

Aromatic-degrading Sphingomonas isolates from the deep subsurface.

An obligately aerobic chemoheterotrophic bacterium (strain F199) previously isolated from Southeast Coastal Plain subsurface sediments and shown to degrade toluene, naphthalene, and other aromatic compounds (J. K. Fredrickson, F. J. Brockman, D. J. Workman, S. W. Li, and T. O. Stevens, Appl. Environ. Microbiol. 57:796-803, 1991) was characterized by analysis of its 16S rRNA nucleotide base sequence and cellular lipid composition. Strain F199 contained 2-OH14:0 and 18:1 omega 7c as the predominant cellular fatty acids and sphingolipids that are characteristic of the genus Sphingomonas. Phylogenetic analysis of its 16S rRNA sequence indicated that F199 was most closely related to Sphingomonas capsulata among the bacteria currently in the Ribosomal Database. Five additional isolates from deep Southeast Coastal Plain sediments were determined by 16S rRNA sequence analysis to be closely related to F199. These strains also contained characteristic sphingolipids. Four of these five strains could also grow on a broad range of aromatic compounds and could mineralize [14C]toluene and [14C]naphthalene. S. capsulata (ATCC 14666), Sphingomonas paucimobilis (ATCC 29837), and one of the subsurface isolates were unable to grow on any of the aromatic compounds or mineralize toluene or naphthalene. These results indicate that bacteria within the genus Sphingomonas are present in Southeast Coastal Plain subsurface sediments and that the capacity for degrading a broad range of substituted aromatic compounds appears to be common among Sphingomonas species from this environment.

A novel interferon-inducible domain: structural and functional analysis of the human interferon regulatory factor 1 gene promoter.

We have cloned and functionally characterized the human interferon regulatory factor 1 (IRF-1) gene promoter. The promoter contains a CpG island, with several GC boxes, a CAAT box, but no TATA box. IRF-1 mRNA is strongly induced by gamma interferon (IFN-gamma) but more weakly and transiently by IFN-alpha. There are several putative kappa B motifs and numerous AA(G/A)G(G/T)A and GAAANN motifs throughout the promoter. The IRF-1 promoter is not autoregulated by the IRF-1 gene product. IFN inducibility of the promoter was studied with 5' deletion mutants linked to a heterologous reporter gene. Gel mobility shift assays were used to show IFN-inducible factor binding to the IRF-1 promoter. These studies showed that IFN inducibility is conferred by a novel imperfect inverted-repeat arrangement of two GAAANN motifs within a domain, 130 nucleotides upstream of transcription initiation. This inverted repeat binds a factor upon induction with IFN and can confer IFN inducibility on a heterologous promoter. Conversely, point mutations of the inverted repeat are not IFN inducible when linked to the same heterologous promoter.

Human interferon regulatory factor 1: intron-exon organization.

Interferon (IFN) regulatory factor 1 (IRF-1) is a transcriptional regulatory protein that mediates the transcriptional activation of the IFN-alpha and IFN-beta genes by viruses and IFNs. To characterize the mechanisms that govern the level of IRF-1 in cells, we isolated the IRF-1 gene and characterized the structure of its intronic and exonic domains and of its regulatory promoter region. A human placental genomic library was screened with an IRF-1 cDNA probe, and two clones that contained the IRF-1 gene and its 5' regulatory region were obtained. We used these clones to determine the complete nucleotide sequence for the IRF-1 gene, finding that the IRF-1 gene spanned 7.72 kb of DNA and included 10 exons and 9 introns. When the deduced amino acid sequences were compared among different species, the most conserved exons were exons 2, 3, and 4, in which the putative DNA binding domain for the IRF-1 protein is located.