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Suberin has been shown to play a role in protection against and response to stress, including defense against soil-borne pathogens. In soybean, oxidized fatty acids are the most predominant monomers contributing to root suberin, with 18-hydroxy-oleic acid being the most abundant. 18-Hydroxy-oleic acid is predicted to be synthesized by members of the CYP86A family of cytochrome P450 enzyme. Six putative CYP86A genes were identified through phylogenetic analysis of the soybean genome. Two of these, CYP86A37 and CYP86A38 show a root-specific gene expression pattern, and were further analyzed to assess their physiological role in suberin deposition using RNAi knockdown in a hairy root system. Soybean hairy roots were found to be nearly identical to soil-grown roots in terms of anatomy, suberin deposition patterns, and suberin chemistry. The RNAi knockdown of CYP86A37 and CYP86A38 yielded hairy root lines with reduced gene expression and a reduction in the oxidized monomers of suberin, most notably 18-hydroxy-oleic acid. Based on this evidence, CYP86A37 and CYP86A38 are predicted to function as fatty acid [omega]-hydroxylases in vivo. Keywords: Glycine max, soybean, hairy root, suberin, cytochrome P450, CYP86A37, CYP86A38. La subérine s'avère jouer un rôle dans la protection et la réponse au stress, notamment dans la défense contre des agents pathogènes du sol. Chez le soja, les acides gras oxydés sont les principaux monomères qui contribuent à la subérine racinaire, l'acide 18-hydroxy-oléique étant le plus abondant. On prédit que l'acide 18-hydroxyoléique sera synthétisé par les membres de la famille CYP86Adu cytochrome P450. Six gènes présumés du CYP86A ont été identifiés au moyen de l'analyse phylogénétique du génome du soja. Deux d'entre eux, CYP86A37 et CYP86A38 présentent un profil d'expression génique spécifique aux racines et ont été analysés de manière plus approfondie afin d'évaluer leur rôle physiologique dans le dépôt de la subérine, à l'aide d'un knockdown par ARNi dans un système de culture aérienne de racines. Les racines de soja en culture aérienne se sont avérées presque identiques aux racines cultivées en terre en matière d'anatomie, de profils de dépôt de subérine et de chimie de la subérine. Le knockdown des CYP86A37 et CYP86A38 par ARNi produisait des lignées de racines aériennes avec une expression génique réduite et une diminution des monomères oxydés de subérine, plus particulièrement de l'acide 18-hydroxy-oléique. À partir de ces données, on prévoit que les CYP86A37 et CYP86A38 agissent comme [omega]-hydroxylases d'acides gras in vivo. [Traduit par la Rédaction] Mots-clés : Glycine max, soja, racine en culture aérienne, subérine, cytochrome P450, CYP86A37, CYP86A38., Introduction Soybean [Glycine max (L.) Merr.] is an oilseed crop widely cultivated for food, feed, and renewable energy production. However, like all crop plants, soybean production is impacted by both [...]

We show that phage lysogenization, lysogens, and prophage induction are all targeted by CRISPR. The results demonstrate that genomic DNA is not immune to the CRISPR system, that the CRISPR system does not require noncytoplasmic elements, and that the system protects from phages entering and exiting the lysogenic cycle. doi: 10.1128/JB.00644-10

The human microbiome refers to the community of microorganisms, including prokaryotes, viruses, and microbial eukaryotes, that populate the human body. The National Institutes of Health launched an initiative that focuses on describing the diversity of microbial species that are associated with health and disease. The first phase of this initiative includes the sequencing of hundreds of microbial reference genomes, coupled to metagenomic sequencing from multiple body sites. Here we present results from an initial reference genome sequencing of 178 microbial genomes. From 547,968 predicted polypeptides that correspond to the gene complement of these strains, previously unidentified ('novel') polypeptides that had both unmasked sequence length greater than 100 amino acids and no BLASTP match to any nonreference entry in the nonredundant subset were defined. This analysis resulted in a set of 30,867 polypeptides, of which 29,987 (-97%) were unique. In addition, this set of microbial genomes allows for -40% of random sequences from the microbiome of the gastrointestinal tract to be associated with organisms based on the match criteria used. Insights into pan-genome analysis suggest that we are still far from saturating microbial species genetic data sets. In addition, the associated metrics and standards used by our group for quality assurance are presented. doi: 10.1126/science.1183605

The size, shape, and behavior of the modern domesticated dog has been sculpted by artificial selection for at least 14,000 years. The genetic substrates of selective breeding, however, remain largely unknown. Here, we describe a genome-wide scan for selection in 275 dogs from 10 phenotypically diverse breeds that were genotyped for over 21,000 autosomal SNPs. We identified 155 genomic regions that possess strong signatures of recent selection and contain candidate genes for phenotypes that vary most conspicuously among breeds, including size, coat color and texture, behavior, skeletal morphology, and physiology. In addition, we demonstrate a significant association between HAS2 and skin wrinkling in the Shar-Pei, and provide evidence that regulatory evolution has played a prominent role in the phenotypic diversification of modern dog breeds. Our results provide a first-generation map of selection in the dog, illustrate how such maps can rapidly inform the genetic basis of canine phenotypic variation, and provide a framework for delineating the mechanistic basis of how artificial selection promotes rapid and pronounced phenotypic evolution. Canis lupis | evolution doi/10.1073/pnas.0909918107

Bacterial single-stranded DNA-binding proteins (SSBs) help to recruit a diverse array of genome maintenance enzymes to their sites of action through direct protein interactions. For all cases examined to date, these interactions are mediated by the evolutionarily conserved C terminus of SSB (SSB-Ct). The essential nature of SSB protein interactions makes inhibitors that block SSB complex formation valuable biochemical tools and attractive potential antibacterial agents. Here, we identify four small molecules that disrupt complexes formed between Escherichia coli SSB and Exonuclease I (Exol), a well-studied SSB-interacting enzyme. Each compound disrupts Exol/SSB-Ct peptide complexes and abrogates SSB stimulation of Exol nuclease activity. Structural and biochemical studies support a model for three of the compounds in which they compete with SSB for binding to Exol. The fourth appears to rely on an allosteric mechanism to disrupt Exol/SSB complexes. Subsets of the inhibitors block SSB-Ct complex formation with two other SSB-interaction partners as well, which highlights their utility as reagents for investigating the roles of SSB/protein interactions in diverse DNA replication, recombination, and repair reactions. Exonuclease I | inhibitor | PriA | protein complex | RecQ www.pnas.org/cgi/doi/10.1073/pnas.0909191107

Horizontal gene transfer (HGT) is one of the most dominant forces molding prokaryotic gene repertoires. These repertoires can be as small as [approximately equal to]200 genes in intracellular organisms or as large as [approximately equal to]9,000 genes in large, free-living bacteria. In this article we ask what is the impact of HGT from phylogenetically distant sources, relative to the size of the gene repertoire. Using different approaches for HGT detection and focusing on both cumulative and recent evolutionary histories, we find a surprising pattern of nonlinear enrichment of long-distance transfers in large genomes. Moreover, we find a strong positive correlation between the sizes of the donor and recipient genomes. Our results also show that distant horizontal transfers are biased toward those functional groups that are enriched in large genomes, showing that the trends in functional gene content and the impact of distant transfers are interdependent. These results highlight the intimate relationship between environmental and genomic complexity in microbes and suggest that an ecological, as opposed to phylogenetic, signal in gene content gains relative importance in large-genomed bacteria. functional gene content | microbial genomes | scaling | lateral gene transfer doi/10.1073/pnas.0907584106

To understand basic principles of bacterial metabolism organization and regulation, but also the impact of genome size, we systematically studied one of the smallest bacteria, Mycoplosma pneumonioe. A manually curated metabolic network of 189 reactions catalyzed by 129 enzymes allowed the design of a defined, minimal medium with 19 essential nutrients. More than 1300 growth curves were recorded in the presence of various nutrient concentrations. Measurements of biomass indicators, metabolites, and [sup.133]C-glucose experiments provided information on directionality, fluxes, and energetics; integration with transcription profiling enabled the global analysis of metabolic regulation. Compared with more complex bacteria, the M. pneumoniae metabolic network has a more linear topology and contains a higher fraction of multifunctional enzymes; general features such as metabolite concentrations, cellular energetics, adaptability, and global gene expression responses are similar, however. 10.1126/science.1177263

Analysis of marine cyanobacteria and proteobacteria genomes has provided a profound understanding of the life strategies of these organisms and their ecotype differentiation and metabolisms. However, a comparable analysis of the Bacteroidetes, the third major bacterioplankton group, is still lacking. In the present paper, we report on the genome of Polaribacter sp. strain MED152. On the one hand, MED152 contains a substantial number of genes for attachment to surfaces or particles, gliding motility, and polymer degradation. This agrees with the currently assumed life strategy of marine Bacteroidetes. On the other hand, it contains the proteorhodopsin gene, together with a remarkable suite of genes to sense and respond to light, which may provide a survival advantage in the nutrient-poor sun-lit ocean surface when in search of fresh particles to colonize. Furthermore, an increase in [CO.sub.2] fixation in the light suggests that the limited central metabolism is complemented by anaplerotic inorganic carbon fixation. This is mediated by a unique combination of membrane transporters and carboxylases. This suggests a dual life strategy that, if confirmed experimentally, would be notably different from what is known of the two other main bacterial groups (the autotrophic cyanobacteria and the heterotrophic proteobacteria) in the surface oceans. The Polaribacter genome provides insights into the physiological capabilities of proteorhodopsin-containing bacteria. The genome will serve as a model to study the cellular and molecular processes in bacteria that express proteorhodopsin, their adaptation to the oceanic environment, and their role in carbon-cycling. Bacteroidetes | marine bacteria | whole-genome analysis | heterotrophic C[O.sub.2] fixation

The soil actinomycete Kocuria rhizophila belongs to the suborder Micrococcineae, a divergent bacterial group for which only a limited amount of genomic information is currently available. K. rhizophila is also important in industrial applications; e.g., it is commonly used as a standard quality control strain for antimicrobial susceptibility testing. Sequencing and annotation of the genome of K. rhizophila DC2201 (NBRC 103217) revealed a single circular chromosome (2,697,540 bp; G+C content of 71.16%) containing 2,357 predicted protein-coding genes. Most of the predicted proteins (87.7%) were orthologous to actinobacterial proteins, and the genome showed fairly good conservation of synteny with taxonomically related actinobacterial genomes. On the other hand, the genome seems to encode much smaller numbers of proteins necessary for secondary metabolism (one each of nonribosomal peptide synthetase and type III polyketide synthase), transcriptional regulation, and lateral gene transfer, reflecting the small genome size. The presence of probable metabolic pathways for the transformation of phenolic compounds generated from the decomposition of plant materials, and the presence of a large number of genes associated with membrane transport, particularly amino acid transporters and drug efflux pumps, may contribute to the organism's utilization of root exudates, as well as the tolerance to various organic compounds.

Abstract: Recent genome size estimates for Arctic amphipods have revealed the largest genomes known in the Crustacea. Here we provide additional data for 7 species of caridean shrimp collected from [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.08.030 Byline: Manisha Goel (a), Arcady Mushegian (a)(b) Keywords: S. purpuratus; Sea urchin; Comparative genomics; Metabolic pathways; Thiaminase; Urea cycle; Cholesterol; Phosphoglyerate mutase Abstract: The genome sequence of the purple sea urchin Strongylocentrotus purpuratus recently became available. We report the results of functional annotation and initial analysis of more than 2300 proteins predicted to be involved in metabolite transport and enzymatic conversion in sea urchin. The comparison of various reconstructed biosynthetic and catabolic pathways in sea urchin to those known in other genomes suggests the overall similarity of the sea urchin metabolism to that of the vertebrates, with relatively small but non-trivial differences from both vertebrates and protostomes. There are several examples of two parallel, non-orthologous solutions for the same molecular function in sea urchin, in contrast with the other completely sequenced metazoans that tend to contain just one version of the same function. There are also genes that appear to be close phylogenetic neighbors of plant or bacterial homologs, as opposed to homologs in other Metazoa. The evolutionary and functional significance of these variations is discussed. Author Affiliation: (a) Stowers Institute for Medical Research, 1000 E. 50th St., Kansas City, MO 64110, USA (b) Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, Kansas City, KS 66160, USA Article History: Received 8 May 2006; Revised 10 August 2006; Accepted 16 August 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.09.012 Byline: Antonio Fernandez-Guerra (a), Antoine Aze (a), Julia Morales (b), Odile Mulner-Lorillon (b), Bertrand Cosson (b), Patrick Cormier (b), Cynthia Bradham (c), Nikki Adams (d), Anthony J. Robertson (e), William F. Marzluff (f), James A. Coffman (e), Anne-Marie Geneviere (a) Keywords: Sea urchin; Cell cycle; Replication; Mitosis; Checkpoint; Kinases Abstract: A search of the Strongylocentrotus purpuratus genome for genes associated with cell cycle control and DNA metabolism shows that the known repertoire of these genes is conserved in the sea urchin, although with fewer family members represented than in vertebrates, and with some cases of echinoderm-specific gene diversifications. For example, while homologues of the known cyclins are mostly encoded by single genes in S. purpuratus (unlike vertebrates, which have multiple isoforms), there are additional genes encoding novel cyclins of the B and K/L types. Almost all known cyclin-dependent kinases (CDKs) or CDK-like proteins have an orthologue in S. purpuratus; CDK3 is one exception, whereas CDK4 and 6 are represented by a single homologue, referred to as CDK4. While the complexity of the two families of mitotic kinases, Polo and Aurora, is close to that found in the nematode, the diversity of the NIMA-related kinases (NEK proteins) approaches that of vertebrates. Among the nine NEK proteins found in S. purpuratus, eight could be assigned orthologues in vertebrates, whereas the ninth is unique to sea urchins. Most known DNA replication, DNA repair and mitotic checkpoint genes are also present, as are homologues of the pRB (two) and p53 (one) tumor suppressors. Interestingly, the p21/p27 family of CDK inhibitors is represented by one homologue, whereas the INK4 and ARF families of tumor suppressors appear to be absent, suggesting that these evolved only in vertebrates. Our results suggest that, while the cell cycle control mechanisms known from other animals are generally conserved in sea urchin, parts of the machinery have diversified within the echinoderm lineage. The set of genes uncovered in this analysis of the S. purpuratus genome should enhance future research on cell cycle control and developmental regulation in this model. Author Affiliation: (a) Observatoire Oceanologique de Banyuls-Laboratoire Arago, CNRS-UMR7628/UPMC, 66650 Banyuls-sur-Mer, France (b) Station Biologique de Roscoff, CNRS-UMR7150/UPMC, 29680 Roscoff, France (c) Developmental, Cellular, and Molecular Biology Group, Duke University, Durham, NC, USA (d) Department of Biological Sciences, California Polytechnic State University, San Luis Obispo, CA, USA (e) Mount Desert Island Biological Laboratory, Salisbury Cove, ME, USA (f) Program in Molecular Biology, University of North Carolina, Chapel Hill, NC, USA Article History: Received 30 May 2006; Revised 6 September 2006; Accepted 7 September 2006

The main virulence factor of Vibrio cholerae, the cholera toxin, is encoded by the ctxAB operon, which is contained in the genome of the lysogenic filamentous phage CTX[phi]. This phage transmits ctxAB genes between V. cholerae bacterial populations that express toxin-coregulated pilus (TCP), the CTX[phi] receptor. In investigating new forms of ctxAB transmission, we found that V. cholerae filamentous phage VGJ[phi], which uses the mannose-sensitive hemagglutinin (MSHA) pilus as a receptor, transmits CTX[phi] or its satellite phage RS1 by an efficient and highly specific TCP-independent mechanism. This is a novel type of specialized transduction consisting in the site-specific cointegration of VGJ[phi] and CTX[phi] (or RS1) replicative forms to produce a single hybrid molecule, which generates a single-stranded DNA hybrid genome that is packaged into hybrid viral particles designated HybP[phi] (for the VGJ[phi]/CTX[phi] hybrid) and HybRS[phi] (for the VGJ[phi]/RS1 hybrid). The hybrid phages replicate by using the VGJ[phi] replicating functions and use the VGJ[phi] capsid, retaining the ability to infect via MSHA. The hybrid phages infect most tested strains more efficiently than CTX[phi], even under in vitro optimal conditions for TCP expression. Infection and lysogenization with HybP[phi] revert the V. cholerae live attenuated vaccine strain 1333 to virulence. Our results reinforce that TCP is not indispensable for the acquisition of CTX[phi]. Thus, we discuss an alternative to the current accepted evolutionary model for the emergence of new toxigenic strains of V. cholerae and the importance of our findings for the development of an environmentally safer live attenuated cholera vaccine.

Several streptococcal species are able to take up naked DNA from the environment and integrate it into their genomes by homologous recombination. This process is called natural transformation. In Streptococcus pneumoniae and related streptococcal species, competence for natural transformation is induced by a peptide pheromone through a quorum-sensing mechanism. Recently we showed that induction of the competent state initiates lysis and release of DNA from a subfraction of the bacterial population and that the efficiency of this process is influenced by cell density. Here we have further investigated the nature of this cell density-dependent release mechanism. Interestingly, we found that competence-induced pneumococci lysed competence-deficient cells of the same strain during cocultivation and that the efficiency of this heterolysis increased as the ratio of competent to noncompetent cells increased. Furthermore, our results indicate that the lysins made by competent pneumococci are not released into the growth medium. More likely, they are anchored to the surface of the competent cells by choline-binding domains and cause lysis of noncompetent pneumococci through cell-to-cell contact.

Pathogenicity of the gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria depends on a type III secretion (TTS) system which translocates bacterial elector proteins into the plant cell. Previous transcriptome analysis identified a genome-wide regulon of putative virulence genes that are coexpressed with the TTS system. In this study, we characterized two of these genes, xopC and xopJ. Both genes encode Xanthomonas outer proteins (Xops) that were shown to be secreted by the TTS system. In addition, type III-dependent translocation of both proteins into the plant cell was demonstrated using the AvrBs3 effector domain as a reporter. XopJ belongs to the AvrRxv/YopJ family of effector proteins from plant and animal pathogenic bacteria. By contrast, XopC does not share significant homology to proteins in the database. Sequence analysis revealed that the xopC locus contains several features that are reminiscent of pathogenicity islands. Interestingly, the xopC region is flanked by 62-bp inverted repeats that are also associated with members of the Xanthomonas avrBs3 effector family. Besides xopC, a second gene of the locus, designated hpaJ, was shown to be coexpressed with the TTS system. hpaJ encodes a protein with similarity to transglycosylases and to the Pseudomonas syringae pv. maculicola protein HopPmaG. HpaJ secretion and translocation by the X. campestris pv. vesicatoria TTS system was not detectable, which is consistent with its predicted Sec signal and a putative function as transglycosylase in the bacterial periplasm.

We report here the construction of a physical and genetic map of the virulent Wolbachia strain, wMelPop. This map was determined by ordering 28 chromosome fragments that resulted from digestion with the restriction endonucleases FseI, ApaI, SmaI, and AscI and were resolved by pulsed-field gel electrophoresis. Southern hybridization was done with 53 Wolbachia-specific genes as probes in order to determine the relative positions of these restriction fragments and use them to serve as markers. Comparison of the resulting map with the whole genome sequence of the closely related benign Wolbachia strain, wMel, shows that the two genomes are largely conserved in gene organization with the exception of a single inversion in the chromosome.

The bacterial genome projects have suggested a central role for horizontal transfer in bacterial adaptation, but it is difficult to rule out an adaptive role for ordinary genetic change in existing genes. The bacterial systematics literature can readily address the importance of gene acquisition in adaptive evolution, since phenotypic characterization typically assesses presence versus absence of metabolic capabilities, and metabolic gains and losses are most likely due to horizontal transfer and/or gene loss. Bacterial systematists have not geared their studies toward quantitative differences in metabolic capabilities, which are more likely to involve adjustments of existing genes. Here, quantitative variation in metabolism within and between three closely related Bacillus taxa has been assayed. While these taxa show no qualitative (i.e. presence versus absence) differences in resource utilization, they are quantitatively different in utilization of 8% of 95 resources tested. Moreover, 93% of the resources tested showed significant quantitative variation among strains within a single taxon. These results suggest that ordinary genetic changes in existing genes may play an important role in adaptation. If these results are typical, future genomically based assays of quantitative variation in phenotype (e.g. microarray analysis of mRNA concentrations) may identify hundreds of genes whose expression has been modified. A protocol is presented for identifying those modifications of gene expression and those gene acquisitions that are most likely to have played a role in adaptive evolution.

The availability of an ever increasing number of complete genome sequences of diverse prokaryotic taxa has led to the introduction of novel approaches to infer phylogenetic relationships among bacteria. In the present study the sequences of the 16S rRNA gene and nine housekeeping genes were compared with the fraction of shared putative orthologous protein-encoding genes, conservation of gene order, dinucteotide relative abundance and codon usage among 11 genomes of species belonging to the lactic acid bacteria. In general there is a good correlation between the results obtained with various approaches, although it is clear that there is a stronger phylogenetic signal in some datasets than in others, and that different parameters have different taxonomic resolutions. It appears that trees based on different kinds of information derived from whole-genome sequencing projects do not provide much additional information about the phylogenetic relationships among bacterial taxa compared to more traditional alignment-based methods. Nevertheless, it is expected that the study of these novel forms of information will have its value in taxonomy, to determine which genes are shared, when genes or sets of genes were lost in evolutionary history, to detect the presence of horizontally transferred genes and/or confirm or enhance the phylogenetic signal derived from traditional methods. Although these conclusions are based on a relatively small dataset, they are largely in agreement with other studies and it is anticipated that similar trends will be observed when comparing other genomes.

The direct binding of Streptococcus mitis to human platelets is mediated in part by two proteins (PblA and PblB) encoded by a lysogenic bacteriophage (SM1). Since SM1 is the first prophage of S. mitis that has been identified and because of the possible role of these phage-encoded proteins in virulence, we sought to characterize SMI in greater detail. Sequencing of the SM1 genome revealed that it consisted of 34,692 bp, with an overall G+C content of 39 mol%. Fifty-six genes encoding proteins of 40 or more amino acids were identified. The genes of SMI appear to be arranged in a modular, life cycle-specific organization. BLAST analysis also revealed that the proteins of SM1 have homologies to proteins from a wide variety of lambdoid phages. Bioinformatic analyses, in addition to N-terminal sequencing of the proteins, led to the assignment of possible functions to a number of proteins, including the integrase, the terminase, and two major structural proteins. Examination of the phage structural components indicates that the phage head may assemble using stable multimers of the major capsid protein, in a process similar to that of phage r1t. These findings indicate that SMI may be part of a discrete subfamily of the Siphoviridae that includes at least phages r1t of Lactococcus lactis and SF370.3 of Streptococcus pyogenes.

Mycobacterium ulcerans causes Buruli ulcer, the third most prevalent mycobacterial infection of immunocompetent humans after tuberculosis and leprosy. Recent work has shown that the production by M. ulcerans of mycolactone, a novel polyketide, may partly explain the pathogenesis of Buruli ulcer. To search for the genetic basis of virulence in M. ulcerans, we took advantage of the close genetic relationship between M. ulcerans and Mycobacterium marinum by performing genomic suppressive subtractive hybridization of M. ulcerans with M. marinum. We identified several DNA fragments specific to M. ulcerans, in particular, a type I polyketide synthase locus with a highly repetitive modular arrangement. We postulate that this locus is responsible for the synthesis of mycolactone in M. ulcerans.

The nitrifying bacterium Nitrosomonas europaea can obtain all its carbon for growth from C[O.sub.2] and all its energy and reductant for growth from the oxidation of N[H.sub.3] and is considered an obligate chemolithoautotroph. Previous studies have shown that N. europaea can utilize limited amounts of certain organic compounds, including amino acids, pyruvate, and acetate, although no organic compound has been reported to support the growth of N. europaea. The recently completed genomic sequence of N. europaea revealed a potential permease for fructose. With this in mind, we tested if N. europaea could utilize fructose and other compounds as carbon sources to support growth. Cultures were incubated in the presence of fructose or other organic compounds in sealed bottles purged of C[O.sub.2]. In these cultures, addition of either fructose or pyruvate as the sole carbon source resulted in a two-to threefold increase in optical density and protein content in 3 to 4 days. Studies with [[sub.14]C] fructose showed that >90% of the carbon incorporated by the cells during growth was derived from fructose. Cultures containing mannose, glucose, glycerol, mannitoi, citrate, or acetate showed little or no growth. N. europaea was not able to grow with fructose as an energy source, although the presence of fructose did provide an energy benefit to the cells. These results show that N. europaea can be grown in C[O.sub.2]-free medium by using fructose and pyruvate as carbon sources and may now be considered a facultative chemolithoorganotroph.