Complete Genome Sequence of the Marine, Chemolithoautotrophic, Ammonia-Oxidizing Bacterium Nitrosococcus oceani ATCC 19707

The gammaproteobacterium Nitrosococcus oceani (ATCC 19707) is a gram-negative obligate chemolithoautotroph capable of extracting energy and reducing power from the oxidation of ammonia to nitrite. Sequencing and annotation of the genome revealed a single circular chromosome (3,481,691 bp; G+C content of 50.4%) and a plasmid (40,420 bp) that contain 3,052 and 41 candidate protein-encoding genes, respectively. The genes encoding proteins necessary for the function of known modes of lithotrophy and autotrophy were identified. Contrary to betaproteobacterial nitrifier genomes, the N. oceani genome contained two complete rrn operons. In contrast, only one copy of the genes needed to synthesize functional ammonia monooxygenase and hydroxylamine oxidoreductase, as well as the proteins that relay the extracted electrons to a terminal electron acceptor, were identified. The N. oceani genome contained genes for 13 complete two-component systems. The genome also contained all the genes needed to reconstruct complete central pathways, the tricarboxylic acid cycle, and the Embden-Meyerhof-Parnass and pentose phosphate pathways. The N. oceani genome contains the genes required to store and utilize energy from glycogen inclusion bodies and sucrose. Polyphosphate and pyrophosphate appear to be integrated in this bacterium's energy metabolism, stress tolerance, and ability to assimilate carbon via gluconeogenesis. One set of genes for type I ribulose-1,5-bisphosphate carboxylase/oxygenase was identified, while genes necessary for methanotrophy and for carboxysome formation were not identified. The N. oceani genome contains two copies each of the genes or operons necessary to assemble functional complexes I and IV as well as ATP synthase (one H⁺-dependent F₀F₁ type, one Na⁺-dependent V type).     

Complete genome sequence of the marine, chemolithoautotrophic, ammonia-oxidizing bacterium Nitrosococcus oceani ATCC 19707

The gammaproteobacterium Nitrosococcus oceani (ATCC 19707) is a gram-negative obligate chemolithoautotroph capable of extracting energy and reducing power from the oxidation of ammonia to nitrite. Sequencing and annotation of the genome revealed a single circular chromosome (3,481,691 bp; G+C content of 50.4%) and a plasmid (40,420 bp) that contain 3,052 and 41 candidate protein-encoding genes, respectively. The genes encoding proteins necessary for the function of known modes of lithotrophy and autotrophy were identified. Contrary to betaproteobacterial nitrifier genomes, the N. oceani genome contained two complete rrn operons. In contrast, only one copy of the genes needed to synthesize functional ammonia monooxygenase and hydroxylamine oxidoreductase, as well as the proteins that relay the extracted electrons to a terminal electron acceptor, were identified. The N. oceani genome contained genes for 13 complete two-component systems. The genome also contained all the genes needed to reconstruct complete central pathways, the tricarboxylic acid cycle, and the Embden-Meyerhof-Parnass and pentose phosphate pathways. The N. oceani genome contains the genes required to store and utilize energy from glycogen inclusion bodies and sucrose. Polyphosphate and pyrophosphate appear to be integrated in this bacterium's energy metabolism, stress tolerance, and ability to assimilate carbon via gluconeogenesis. One set of genes for type I ribulose-1,5-bisphosphate carboxylase/oxygenase was identified, while genes necessary for methanotrophy and for carboxysome formation were not identified. The N. oceani genome contains two copies each of the genes or operons necessary to assemble functional complexes I and IV as well as ATP synthase (one H(+)-dependent F(0)F(1) type, one Na(+)-dependent V type).     

Worldwide Distribution of Nitrosococcus oceani, a Marine Ammonia-Oxidizing γ-Proteobacterium, Detected by PCR and Sequencing of 16S rRNA and amoA Genes

Diversity of cultured ammonia-oxidizing bacteria in the γ-subdivision of the Proteobacteria was investigated by using strains isolated from various parts of the world ocean. All the strains were very similar to each other on the basis of the sequences of both the 16S rRNA and ammonia monooxygenase genes and could be characterized as a single species. Sequences were also cloned directly from environmental DNA from coastal Pacific and Atlantic sites, and these sequences represented the first Nitrosococcus oceani-like sequences obtained directly from the ocean. Most of the environmental sequences clustered tightly with those of the cultivated strains, but some sequences could represent new species of Nitrosococcus. These findings imply that organisms similar to the cultivated N. oceani strains have a worldwide distribution.     

Characterization of a New Bacteriocin Operon in Sakacin P-Producing Lactobacillus sakei, Showing Strong Translational Coupling between the Bacteriocin and Immunity Genes

Previous studies of genes involved in the production of sakacin P by Lactobacillus sakei Lb674 revealed the presence of an inducible promoter downstream of the known spp gene clusters. We show here that this promoter drives the expression of an operon consisting of a bacteriocin gene (sppQ), a cognate immunity gene (spiQ), another gene with an unknown function (orf4), and a pseudoimmunity gene containing a frameshift mutation (orf5). The leader peptide of the new one-peptide bacteriocin sakacin Q contains consensus elements that are typical for so-called “double-glycine” leader peptides. The mature bacteriocin shows weak similarity to the BrcA peptide of the two-peptide bacteriocin brochocin C. Sakacin Q has an antimicrobial spectrum that differs from that of sakacin P, thus expanding the antimicrobial properties of the producer strain. The genes encoding sakacin Q and its cognate immunity protein showed strong translational coupling, which was investigated in detail by analyzing the properties of a series of β-glucuronidase fusions. Our results provide experimental evidence that production of the bacteriocin and production of the cognate immunity protein are tightly coregulated at the translational level.     

Worldwide distribution of Nitrosococcus oceani,a marine ammonia-oxidizing gamma-proteobacterium,detected by PCR and sequencing of 16S rRNA and amoA genes

Diversity of cultured ammonia-oxidizing bacteria in the gamma-subdivision of the Proteobacteria was investigated by using strains isolated from various parts of the world ocean. All the strains were very similar to each other on the basis of the sequences of both the 16S rRNA and ammonia monooxygenase genes and could be characterized as a single species. Sequences were also cloned directly from environmental DNA from coastal Pacific and Atlantic sites, and these sequences represented the first Nitrosococcus oceani-like sequences obtained directly from the ocean. Most of the environmental sequences clustered tightly with those of the cultivated strains, but some sequences could represent new species of NITROSOCOCCUS: These findings imply that organisms similar to the cultivated N. oceani strains have a worldwide distribution.     

Amphitrite ornata, a Marine Worm, Contains Two Dehaloperoxidase Genes

Amphitrite ornata, a terebellid polychaete, inhabits marine environments that are contaminated by biogenically produced halometabolites. These halogenated organic compounds are toxic and quite diverse. To survive in this environment, A. ornata produces a novel dehaloperoxidase (DHP I) that detoxifies haloaromatic compounds. In this study we identified and characterized two dehaloperoxidase genes, designated dhpA and dhpB, from an A. ornata complementary DNA library. The deduced amino acid sequences (DHP A and DHP B) of the two dhp genes both contain 137 amino acid residues, but they differ at 5 amino acid positions. Allelic variation was observed for both genes as well. Polymerase chain reaction–restriction fragment length polymorphism assays of genomic DNA from 19 in individuals showed that each individual contains both the dhpA and the dhpB genes. Therefore, the two types of DHP are encoded by separate genes and are not alleles of a single gene. Furthermore, DHP A and DHP B may have different substrate specificities since they have amino acid differences in the active site. Accepted January 16, 2001.     

Cloning and Characterization of the Ribosomal Protein Genes in the spc Operon of a Prokaryotic Endosymbiont of the Pea Aphid, Acyrthosiphon kondoi

To correlate a prokaryotic endosymbiont in the pea aphid, Acyrthosiphonkondoi, with the endosymbionts in related aphid species as wellas with free-living bacteria and subcellular organelles, andto study the mode of its gene expression within aphid cells,we have cloned and characterized the genes encoding ribosomalproteins S3, L16, L29, S17, L14, L24, L5, S14, S8, L6, L18,S5, L30, L15 and secretion protein Y (Sec Y) from the S10 andspc ribosomal protein gene operons of this endosymbiont. Theorganization of these genes is identical to that in Escherichiacoli, and their nucleotide sequences are highly similar (87%identity) to the corresponding E. coli genes. They are muchless similar to the corresponding chloroplast and mitochondrialgenes. The guanine plus cytosine G+C content of the genes ofthe A. kondoi endosymbiont is much higher than those of theendosymbionts in related aphid species reported so far. It appearseither that the A. kondoi endosymbiont is derived from an ancestralbacterium different from those in other aphids or that its G+Ccontent increased in a relatively short time after the evolutionarydivergence of its host.     

Purification and Characterization of Two Endoxylanases from Clostridium acetobutylicum ATCC 824

Two endoxylanases produced by C. acetobutylicum ATCC 824 were purified to homogeneity by column chromatography. Xylanase A, which has a molecular weight of 65,000, hydrolyzed larchwood xylan randomly, yielding xylohexaose, xylopentaose, xylotetraose, xylotriose, and xylobiose as end products. Xylanase B, which has a molecular weight of 29,000, also hydrolyzed xylan randomly, giving xylotriose and xylobiose as end products. Xylanase A hydrolyzed carboxymethyl cellulose with a higher specific activity than xylan. It also exhibited high activity on acid-swollen cellulose. Xylanase B showed practically no activity against either cellulose or carboxymethyl cellulose but was able to hydrolyze lichenan with a specific activity similar to that for xylan. Both xylanases had no aryl-β-xylosidase activity. The smallest oligosaccharides degraded by xylanases A and B were xylohexaose and xylotetraose, respectively. The two xylanases demonstrated similar K_m and V_max values but had different pH optima and isoelectric points. Ouchterlony immunodiffusion tests showed that xylanases A and B lacked antigenic similarity.     

Comparative Diversity of Ammonia Oxidizer 16S rRNA Gene Sequences in Native, Tilled, and Successional Soils

Autotrophic ammonia oxidizer (AAO) populations in soils from native, tilled, and successional treatments at the Kellogg Biological Station Long-Term Ecological Research site in southwestern Michigan were compared to assess effects of disturbance on these bacteria. N fertilization effects on AAO populations were also evaluated with soils from fertilized microplots within the successional treatments. Population structures were characterized by PCR amplification of microbial community DNA with group-specific 16S rRNA gene (rDNA) primers, cloning of PCR products and clone hybridizations with group-specific probes, phylogenetic analysis of partial 16S rDNA sequences, and denaturing gradient gel electrophoresis (DGGE) analysis. Population sizes were estimated by using most-probable-number (MPN) media containing varied concentrations of ammonium sulfate. Tilled soils contained higher numbers than did native soils of culturable AAOs that were less sensitive to different ammonium concentrations in MPN media. Compared to sequences from native soils, partial 16S rDNA sequences from tilled soils were less diverse and grouped exclusively within Nitrosospira cluster 3. Native soils yielded sequences representing three different AAO clusters. Probes for Nitrosospira cluster 3 hybridized with DGGE blots from tilled and fertilized successional soils but not with blots from native or unfertilized successional soils. Hybridization results thus suggested a positive association between the Nitrosospira cluster 3 subgroup and soils amended with inorganic N. DGGE patterns for soils sampled from replicated plots of each treatment were nearly identical for tilled and native soils in both sampling years, indicating spatial and temporal reproducibility based on treatment.     

Regulation and Characterization of Two Nitroreductase Genes, nprA and nprB, of Rhodobacter capsulatus

Among photosynthetic bacteria, strains B10 and E1F1 of Rhodobacter capsulatus photoreduce 2,4-dinitrophenol (DNP), which is stoichiometrically converted into 2-amino-4-nitrophenol by a nitroreductase activity. The reduction of DNP is inhibited in vivo by ammonium, which probably acts at the level of the DNP transport system and/or physiological electron transport to the nitroreductase, since this enzyme is not inhibited by ammonium in vitro. Using the complete genome sequence data for strain SB1003 of R. capsulatus, two putative genes coding for possible nitroreductases were isolated from R. capsulatus B10 and disrupted. The phenotypes of these mutant strains revealed that both genes are involved in the reduction of DNP and code for two major nitroreductases, NprA and NprB. Both enzymes use NAD(P)H as the main physiological electron donor. The nitroreductase NprA is under ammonium control, whereas the nitroreductase NprB is not. In addition, the expression of the nprB gene seems to be constitutive, whereas nprA gene expression is inducible by a wide range of nitroaromatic and heterocyclic compounds, including several dinitroaromatics, nitrofuran derivatives, CB1954, 2-aminofluorene, benzo[a]pyrene, salicylic acid, and paraquat. The identification of two putative mar/sox boxes in the possible promoter region of the nprA gene and the induction of nprA gene expression by salicylic acid and 2,4-dinitrophenol suggest a role in the control of the nprA gene for the two-component MarRA regulatory system, which in Escherichia coli controls the response to some antibiotics and environmental contaminants. In addition, upregulation of the nprA gene by paraquat indicates that this gene is probably a member of the SoxRS regulon, which is involved in the response to stress conditions in other bacteria.     

Characterization of Two Extracellular Polysaccharides from Marine Bacteria

Two bacterial isolates from the intertidal zone produced significant quantities of extracellular polysaccharide with interesting properties. One polysaccharide was named PS 3a24; the other was named PS 3a35. The relative proportion of sugars in PS 3a35 was 51.6% glucose, 39.0% galactose, 3.1% mannose, and 6.3% rhamnose, with a trace of an unidentified sugar. PS 3a24 was composed of 40.2% glucose, 57.2% galactose, and 2.6% mannose. PS 3a35 contained 6% pyruvate, whereas PS 3a24 contained no pyruvate. Both exhibited high specific viscosity, pseudoplasticity, and stability over a wide range of pH in the presence of a variety of salts. The viscosity of PS 3a35 was relatively insensitive to increasing temperature, whereas that of PS 3a24 showed an irreversible drop on heating.     

Characterization of the rrnB Operon of the Plant Pathogen Rhodococcus fascians and Targeted Integrations of Exogenous Genes at rrn Loci

A 6.0-kb SalI DNA fragment containing an entire rRNA operon (rrnB) was cloned from a cosmid gene bank of the phytopathogenic strain Rhodococcus fascians D188. The nucleotide sequence of the 6-kb fragment was determined and had the organization 16S rRNA-spacer-23S rRNA-spacer-5S rRNA without tRNA-encoding genes in the spacer regions. The 5′ and 3′ ends of the mature 16S, 23S, and 5S rRNAs were determined by alignment with the rrn operons of Bacillus subtilis and other gram-positive bacteria. Four copies of the rrn operons were identified by hybridization with an rrnB probe in R. fascians type strain ATCC 12974 and in the virulent strain R. fascians D188. However, another isolate, CECT 3001 (= NRRL {"type":"entrez-nucleotide","attrs":{"text":"B15096","term_id":"2122845","term_text":"B15096"}}B15096), also classified as R. fascians, produced five rrn-hybridizing bands. An integrative vector containing a 2.5-kb DNA fragment internal to rrnB was constructed for targeted integration of exogenous genes at the rrn loci. Transformants carrying the exogenous chloramphenicol resistance gene (cmr) integrated in different rrn operons were obtained. These transformants had normal growth rates in complex medium and minimal medium and were fully stable for the integrated marker.

Rhodococcus fascians is a gram-positive bacterium with a high G+C content belonging to the group of lower actinomycetes (14) closely related to corynebacteria. Strains of this species are of interest because they are phytopathogenic (32), causing the formation of galls on dicotyledonous plants (30) and malformations of bulbs of monocotyledonous plants (24).The molecular genetics of nonpathogenic corynebacteria have received considerable attention (for reviews see references 23 and 29), but there are no advanced recombinant DNA tools for studying molecular genetics of plant-pathogenic bacteria such as R. fascians. Several plasmids, including circular and high-molecular-weight linear plasmids, are present in strains of R. fascians (7, 11). Conjugative plasmids carrying genes determining resistance to cadmium salts (10) or chloramphenicol (12) have been characterized. One of these plasmids, pRF2, was used to develop bifunctional vectors that also replicate in Escherichia coli (12). By using these vectors, transformation of R. fascians strains has been obtained by electroporation (9, 11).Some genes associated with phytopathogenicity were found in a 200-kb linear plasmid in R. fascians D188 (7, 8). Chromosomal genes also appear to be required to produce plant disease (7). In order to clone and study additional genetic determinants involved in plant pathogenicity, there is a need to develop a system for chromosomal integration and expression of homologous or heterologous DNA in well-characterized dispensable sites of the R. fascians chromosome. As part of an effort in this direction, we characterized the nucleotide sequence and organization of an rRNA operon (rrnB) of R. fascians D188. Although the 5S rRNA gene of this species (21) and the 16S rRNA gene were amplified by PCR and used in phylogenetic analyses of gram-positive bacteria previously (26), the complete organization of the rrn operons and the number of rrn loci were not established.Gene targeting is a useful strategy for introducing exogenous genes into specific chromosomal regions. Due to their repetitive nature, rRNA operons are very suitable targets for chromosomal integration of foreign DNA fragments without modification of growth rates and viability characteristics (5). In this paper we describe the characterization of the rrnB locus and the use of rRNA-encoding regions of R. fascians D188 as target sites for integration and expression of the exogenous gene cmr, a gene conferring chloramphenicol resistance (12).     

Cloning and Characterization of Three Fatty Alcohol Oxidase Genes from Candida tropicalis Strain ATCC 20336

Candida tropicalis (ATCC 20336) converts fatty acids to long-chain dicarboxylic acids via a pathway that includes among other reactions the oxidation of ω-hydroxy fatty acids to ω-aldehydes by a fatty alcohol oxidase (FAO). Three FAO genes (one gene designated FAO1 and two putative allelic genes designated FAO2a and FAO2b), have been cloned and sequenced from this strain. A comparison of the DNA sequence homology and derived amino acid sequence homology between these three genes and previously published Candida FAO genes indicates that FAO1 and FAO2 are distinct genes. Both genes were individually cloned and expressed in Escherichia coli. The substrate specificity and K_m values for the recombinant FAO1 and FAO2 were significantly different. Particularly striking is the fact that FAO1 oxidizes ω-hydroxy fatty acids but not 2-alkanols, whereas FAO2 oxidizes 2-alkanols but not ω-hydroxy fatty acids. Analysis of extracts of strain H5343 during growth on fatty acids indicated that only FAO1 was highly induced under these conditions. FAO2 contains one CTG codon, which codes for serine (amino acid 177) in C. tropicalis but codes for leucine in E. coli. An FAO2a construct, with a TCG codon (codes for serine in E. coli) substituted for the CTG codon, was prepared and expressed in E. coli. Neither the substrate specificity nor the K_m values for the FAO2a variant with a serine at position 177 were radically different from those of the variant with a leucine at that position.     

Identification and Characterization of Two Quiescent Porin Genes, nmpC and ompN, in Escherichia coli BE

The genomic DNA of the B^E strain of Escherichia coli has been scrutinized to detect porin genes that have not been identified so far. Southern blot analysis yielded two DNA segments which proved highly homologous to, yet distinct from, the ompC, ompF, and phoE porin genes. The two genes were cloned and sequenced. One of them, designated ompN, encodes a porin which, due to low levels of expression, has eluded prior identification. The functional properties (single-channel conductance) of the OmpN porin, purified to homogeneity, closely resemble those of the OmpC porin from E. coli K-12. The second DNA fragment detected corresponds to the nmpC gene, which, due to an insertion of an IS1 element in its coding region, is not expressed in E. coli B^E.     

Abundance,Composition and Activity of Ammonia Oxidizer and Denitrifier Communities in Metal Polluted Rice Paddies from South China

While microbial nitrogen transformations in soils had been known to be affected by heavy metal pollution, changes in abundance and community structure of the mediating microbial populations had been not yet well characterized in polluted rice soils. Here, by using the prevailing molecular fingerprinting and enzyme activity assays and comparisons to adjacent non-polluted soils, we examined changes in the abundance and activity of ammonia oxidizing and denitrifying communities of rice paddies in two sites with different metal accumulation situation under long-term pollution from metal mining and smelter activities. Potential nitrifying activity was significantly reduced in polluted paddies in both sites while potential denitrifying activity reduced only in the soils with high Cu accumulation up to 1300 mg kg⁻¹. Copy numbers of amoA (AOA and AOB genes) were lower in both polluted paddies, following the trend with the enzyme assays, whereas that of nirK was not significantly affected. Analysis of the DGGE profiles revealed a shift in the community structure of AOA, and to a lesser extent, differences in the community structure of AOB and denitrifier between soils from the two sites with different pollution intensity and metal composition. All of the retrieved AOB sequences belonged to the genus Nitrosospira, among which species Cluster 4 appeared more sensitive to metal pollution. In contrast, nirK genes were widely distributed among different bacterial genera that were represented differentially between the polluted and unpolluted paddies. This could suggest either a possible non-specific target of the primers conventionally used in soil study or complex interactions between soil properties and metal contents on the observed community and activity changes, and thus on the N transformation in the polluted rice soils.