d-Xylose Degradation Pathway in the Halophilic Archaeon Haloferax volcanii

The pathway of d-xylose degradation in archaea is unknown. In a previous study we identified in Haloarcula marismortui the first enzyme of xylose degradation, an inducible xylose dehydrogenase (Johnsen, U., and Schönheit, P. (2004) J. Bacteriol. 186, 6198–6207). Here we report a comprehensive study of the complete d-xylose degradation pathway in the halophilic archaeon Haloferax volcanii. The analyses include the following: (i) identification of the degradation pathway in vivo following ¹³C-labeling patterns of proteinogenic amino acids after growth on [¹³C]xylose; (ii) identification of xylose-induced genes by DNA microarray experiments; (iii) characterization of enzymes; and (iv) construction of in-frame deletion mutants and their functional analyses in growth experiments. Together, the data indicate that d-xylose is oxidized exclusively to the tricarboxylic acid cycle intermediate α-ketoglutarate, involving d-xylose dehydrogenase (HVO_B0028), a novel xylonate dehydratase (HVO_B0038A), 2-keto-3-deoxyxylonate dehydratase (HVO_B0027), and α-ketoglutarate semialdehyde dehydrogenase (HVO_B0039). The functional involvement of these enzymes in xylose degradation was proven by growth studies of the corresponding in-frame deletion mutants, which all lost the ability to grow on d-xylose, but growth on glucose was not significantly affected. This is the first report of an archaeal d-xylose degradation pathway that differs from the classical d-xylose pathway in most bacteria involving the formation of xylulose 5-phosphate as an intermediate. However, the pathway shows similarities to proposed oxidative pentose degradation pathways to α-ketoglutarate in few bacteria, e.g. Azospirillum brasilense and Caulobacter crescentus, and in the archaeon Sulfolobus solfataricus.d-Xylose, a constituent of the polymer xylan, is the major component of the hemicellulose plant cell wall material and thus one of the most abundant carbohydrates in nature. The utilization of d-xylose by microorganisms has been described in detail in bacteria and fungi, for which two different catabolic pathways have been reported. In many bacteria, such as Escherichia coli, Bacillus, and Lactobacillus species, xylose is converted by the activities of xylose isomerase and xylulose kinase to xylulose 5-phosphate as an intermediate, which is further degraded mainly by the pentose phosphate cycle or phosphoketolase pathway. Most fungi convert xylose to xylulose 5-phosphate via xylose reductase, xylitol dehydrogenase, and xylulose kinase. Xylulose 5-phosphate is also an intermediate of the most common l-arabinose degradation pathway in bacteria, e.g. of E. coli, via activities of isomerase, kinase, and epimerase (1).Recently, by genetic evidence, a third pathway of xylose degradation was proposed for the bacterium Caulobacter crescentus, in analogy to an alternative catabolic pathway of l-arabinose, reported for some bacteria, including species of Azospirillum, Pseudomonas, Rhizobium, Burkholderia, and Herbasprillum (2, 3). In these organisms l-arabinose is oxidatively degraded to α-ketoglutarate, an intermediate of the tricarboxylic acid cycle, via the activities of l-arabinose dehydrogenase, l-arabinolactonase, and two successive dehydration reactions forming 2-keto-3-deoxy-l-arabinoate and α-ketoglutarate semialdehyde; the latter compound is further oxidized to α-ketoglutarate via NADP⁺-specific α-ketoglutarate semialdehyde dehydrogenase (KGSADH).² In a few Pseudomonas and Rhizobium species, a variant of this l-arabinose pathway was described involving aldolase cleavage of the intermediate 2-keto-3-deoxy-l-arabinoate to pyruvate and glycolaldehyde, rather than its dehydration and oxidation to α-ketoglutarate (4). Because of the presence of some analogous enzyme activities in xylose-grown cells of Azosprillum and Rhizobium, the oxidative pathway and its variant was also proposed as a catabolic pathway for d-xylose. Recent genetic analysis of Caulobacter crecentus indicates the presence of an oxidative pathway for d-xylose degradation to α-ketoglutarate. All genes encoding xylose dehydrogenase and putative lactonase, xylonate dehydratase, 2-keto-3-deoxylonate dehydratase, and KGSADH were found to be located on a xylose-inducible operon (5). With exception of xylose dehydrogenase, which has been partially characterized, the other postulated enzymes of the pathway have not been biochemically analyzed.The pathway of d-xylose degradation in the domain of archaea has not been studied so far. First analyses with the halophilic archaeon Haloarcula marismortui indicate that the initial step of d-xylose degradation involves a xylose-inducible xylose dehydrogenase (6) suggesting an oxidative pathway of xylose degradation to α-ketoglutarate, or to pyruvate and glycolaldehyde, in analogy to the proposed oxidative bacterial pentose degradation pathways. Recently, a detailed study of d-arabinose catabolism in the thermoacidophilic crenarchaeon Sulfolobus solfataricus was reported. d-Arabinose was found to be oxidized to α-ketoglutarate involving d-arabinose dehydrogenase, d-arabinoate dehydratase, 2-keto-3-deoxy-d-arabinoate dehydratase, and α-ketoglutarate semialdehyde dehydrogenase (3).In this study, we present a comprehensive analysis of the complete d-xylose degradation pathway in the halophilic archaeon Haloferax volcanii. This halophilic archaeon was chosen because it exerts several suitable properties for the analyses. For example, it can be cultivated on synthetic media with sugars, e.g. xylose, an advantage for in vivo labeling studies in growing cultures. Furthermore, a shotgun DNA microarray of H. volcanii is available (7) allowing the identification of xylose-inducible genes, and H. volcanii is one of the few archaea for which an efficient protocol was recently described to generate in-frame deletion mutants.Accordingly, the d-xylose degradation pathway was elucidated following in vivo labeling experiments with [¹³C]xylose, DNA microarray analyses, and the characterization of enzymes involved and their encoding genes. The functional involvement of genes and enzymes was proven by constructing corresponding in-frame deletion mutants and their analysis by selective growth experiments on xylose versus glucose. The data show that d-xylose was exclusively degraded to α-ketoglutarate involving xylose dehydrogenase, a novel xylonate dehydratase, 2-keto-3-deoxyxylonate dehydratase, and α-ketoglutarate semialdehyde dehydrogenase.     

Complete Genome Sequence of the Metabolically Versatile Halophilic Archaeon Haloferax mediterranei, a Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Producer

Haloferax mediterranei, an extremely halophilic archaeon, has shown promise for production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from unrelated cheap carbon sources. Here we report the complete genome (3,904,707 bp) of H. mediterranei CGMCC 1.2087, consisting of one chromosome and three megaplasmids.     

Characterization of Inverted Membrane Vesicles from the Halophilic Archaeon Haloferax volcanii

Membrane-related processes in archaea, the third and most-recently described domain of life, are in general only poorly understood. One obstacle to a functional understanding of archaeal membrane-associated activities corresponds to a lack of archaeal model membrane systems. In the following, characterization of inverted archaeal membrane vesicles, prepared from the halophilic archaeon Haloferax volcanii, is presented. The inverted topology of the vesicles was revealed by defining the orientation of membrane-bound enzymes that in intact cells normally face the cytoplasm or of other protein markers, known to face the exterior medium in intact cells. Electron microscopy, protease protection assays and lectin-binding experiments confirmed the sealed nature of the vesicles. Upon alkalinization of the external medium, the vesicles were able to generate ATP, reflecting the functional nature of the membrane preparation. The availability of preparative scale amounts of inverted archaeal membrane vesicles provides a platform for the study of various membrane-related phenomena in archaea. Received: 27 March 2001/Revised: 13 June 2001     

Complete Genome Sequence of Halalkalicoccus jeotgali B3T,an Extremely Halophilic Archaeon

Halalkalicoccus jeotgali B3^T, isolated from salt-fermented seafood from South Korea, is an extremely halophilic archaeon belonging to the family Halobacteriaceae. Here, we present the complete genome sequence of the type strain H. jeotgali B3^T (3,698,650 bp, with a G+C content of 62.5%), which consists of one chromosome and six plasmids. This is the first complete genome sequence of the Halalkalicoccus species.Extremely halophilic archaea (haloarchaea) are adapted to hypersaline environments and grow optimally in NaCl solutions of 2.6 M or higher (12). These haloarchaea are classified within the family Halobacteriaceae in the order Halobacteriales; currently, this family comprises 28 genera (3), and only 11 complete genome sequences in Halobacteriaceae have been reported. In a study of archaeal diversity in salt-fermented small shrimp or shellfish from South Korea, our laboratory isolated and characterized 5 novel, extremely halophilic archaeal strains of Halobacteriaceae. These strains included Natronococcus jeotgali (9), Halalkalicoccus jeotgali (11), Halorubrum cibi (7), Haloterrigena jeotgali (10) and Haladaptatus cibarius (8). We have now sequenced the genome of Halalkalicoccus jeotgali B3^T; genome sequencing had not been completed or initiated for any strain in this genus when our sequencing project was begun. The genus Halalkalicoccus currently contains only two species, Halalkalicoccus tibetensis (13) and H. jeotgali, and these species exhibit 98.6% gene sequence similarity in their 16S rRNA. The genome of H. jeotgali B3^T is the first of this genus to be sequenced.The complete genome sequence of H. jeotgali B3^T was determined by a whole-genome shotgun strategy using Roche 454 GS (FLX Titanium) pyrosequencing (898,168 reads totaling ∼348 Mb; ∼94-fold coverage of the genome) and a fosmid library (514 reads totaling ∼680 kb) at the Genome Resource Center, KRIBB (Korea Research Institute of Bioscience and Biotechnology). Genome sequences from pyrosequencing were processed by Roche''s software according to the manufacturer''s instructions, and sequences from the fosmid library were processed by PESTAS (6). A total of 898,196 reads were assembled using Newbler Assembler 2.3 (454 Life Science), which generated 54 large contigs (>100 bp in size) with bases having quality scores of 40 and above. The gaps between contigs were closed by primer walking and sequencing of PCR products across the gaps. The annotation was done by merging results obtained from the RAST (Rapid Annotation using Subsystem Technology) pipeline (1), Glimmer 3.02 (2), tRNAscan-SE 1.21 (5), and RNAmmer 1.2 (4).The H. jeotgali B3^T genome is 3,698,650 bases long with a 62.5% G+C content. The chromosome consists of a single circular chromosome (2,809,118 bp, with a G+C content of 65.0%) and six plasmids (406,285 bp, 55.3%; 363,534 bp, 54.2%; 44,576 bp, 58.9%; 44,459 bp, 54.9%; 23,727 bp, 47.6%; 6,951 bp, 60.6%). The genome contains 3,860 predicted coding sequences and 52 RNA genes (determined using RAST). The chromosome is predicted to contain 3,101 coding sequences with a coding intensity of 90.0%, including 47 tRNA genes, 1 5S rRNA gene, 1 16S rRNA gene, and 1 23S rRNA gene. The largest plasmid contains 466 coding sequences with a coding intensity of 81.2% and 2 tRNA genes, while the other five plasmids contain 425, 44, 48, 29, and 5 coding sequences with coding intensities of 80.2%, 84.2%, 83.0%, 69.6%, and 22.8%, respectively (determined using Glimmer3). More detailed analysis of this genome and comparative analysis with other haloarchaea will provide further insight into the genomic differences and metabolism of the extremely halophilic archaea.     

Complete Genome Sequence of Methanothermobacter marburgensis,a Methanoarchaeon Model Organism

The circular genome sequence of the chemolithoautotrophic euryarchaeon Methanothermobacter marburgensis, with 1,639,135 bp, was determined and compared with that of Methanothermobacter thermautotrophicus. The genomes of the two model methanogens differ substantially in protein coding sequences, in insertion sequence (IS)-like elements, and in clustered regularly interspaced short palindromic repeats (CRISPR) loci.Methanothermobacter marburgensis (DSM 2133) (formerly Methanobacterium thermoautotrophicum strain Marburg), a member of the Methanobacteriales (2), was isolated in 1978 from anaerobic sewage sludge in Marburg, Germany (5). The hydrogenotrophic methanogen grows even faster (2 h versus 3 h doubling time) and to higher cell concentrations (3 g versus 1.5 g dry mass per liter) than Methanothermobacter thermautotrophicus (DSM 1053) (formerly Methanobacterium thermoautotrophicum strain ΔH) (20) (for other differences, see references 3 and 19). Both methanogens were used in the last 35 years for the elucidation of the enzymes and coenzymes involved in CO₂ reduction to methane with H₂ (4, 16-18). The genome sequence of M. thermautotrophicus was reported in 1997 (15); that of M. marburgensis is announced here.The genome size of M. marburgensis is 1,639,135 bp (that of M. thermautotrophicus is 1,751,377 bp), the genome G+C content is 48.64% (49.54% for M. thermautotrophicus), and the part coding is 90.94% (91.02% for M. thermautotrophicus). Comparison of the sequences (13) revealed that the two genomes have 1,607 protein coding sequences (CDS) in common and 411 CDS not in common (145 CDS are found only in M. marburgensis and 266 CDS only in M. thermautotrophicus) and show a high degree of synteny. The CDS not in common could be traced back to gene splitting (15%), gene deletion (30%), gene duplication (30%), and lateral gene transfer (24%) events (percentages given are for M. marburgensis). Of the 1,607 CDS in common, approximately 40% show BLAST search expectation values of >10⁻¹⁰⁰ at the protein level, reflecting large differences in sequence divergence. Almost 470 CDS encode conserved hypothetical proteins.The genome of M. marburgensis harbors 15 insertion sequence (IS)-like elements, whereas there is no evidence for a classically organized IS-like element in M. thermautotrophicus. Consistently, a CDS for a transposase is found only in M. marburgensis.In the genome of M. marburgensis there is only one clustered regularly interspaced short palindromic repeat (CRISPR) locus with 36 repeats and only one CRISPR-associated (cas) gene (csa3), indicating that the organism is not protected from invasion by phage and plasmid DNA (7, 8, 10, 12). By comparison, in the genome of M. thermautotrophicus there are three CRISPR loci with 124, 4, and 47 repeats and 18 cas genes that encode proteins involved in adaptation and interference (http://genoweb1.irisa.fr/Serveur-GPO/outils/repeatsAnalysis/CRISPR/). The spacer sequences from locus 2 match DNA sequences found in phage ΨM1 of M. marburgensis (6, 11) and ΨM100 of M. wolfei (9), which supports the observation that M. thermautotrophicus is not lysed by those two phages. Unfortunately, there is no DNA sequence available for phage ΦF1, which is able to lyse M. thermautotrophicus (14), to compare it with the spacer sequences of the CRISPR regions. In the plasmid pM2001 (= pMTBMA4) (4,439-bp circular multicopy plasmid found only in M. marburgensis) (1, 19), no sequence identities for CRISPR spacer sequences of M. thermautotrophicus were found (14).Approximately 200 CDS were identified that are required for the synthesis of the enzymes, coenzymes, and prosthetic groups involved in CO₂ reduction to methane and in the coupling of this process with energy conservation. Some of the genes have been found only recently; others, such as those for coenzyme F₄₃₀ biosynthesis, still remain to be discovered.     

Complete Genome Sequence of the Hydrogenotrophic, Methanogenic Archaeon Methanoculleus bourgensis Strain MS2T, Isolated from a Sewage Sludge Digester

Methanoculleus bourgensis, of the order Methanomicrobiales, is a dominant methanogenic archaeon in many biogas-producing reactor systems fed with renewable primary products. It is capable of synthesizing methane via the hydrogenotrophic pathway utilizing hydrogen and carbon dioxide or formate as the substrates. Here we report the complete and finished genome sequence of M. bourgensis strain MS2(T), isolated from a sewage sludge digester.     

Complete Genome Sequence of Aeromonas hydrophila Phage CC2

Aeromonas hydrophila is one of the major pathogenic bacteria for fish and people. To develop an effective antimicrobial agent, we isolated a bacteriophage from sewage, named CC2, and sequenced its genome. Comparative genome analysis of phage CC2 with its relatives revealed that phage CC2 has higher sequence homology to A. salmonicida phage 65 than to A. hydrophila phage Aeh1. Here, we announce the complete genome sequence of CC2 and report major findings from the genomic analysis.     

Comparative genomic analysis of the Haloferax volcanii DS2 and Halobacterium salinarium GRB contig maps reveals extensive rearrangement.

Anonymous probes from the genome of Halobacterium salinarium GRB and 12 gene probes were hybridized to the cosmid clones representing the chromosome and plasmids of Halobacterium salinarium GRB and Haloferax volcanii DS2. The order of and pairwise distances between 35 loci uniquely cross-hybridizing to both chromosomes were analyzed in a search for conservation. No conservation between the genomes could be detected at the 15-kbp resolution used in this study. We found distinct sets of low-copy-number repeated sequences in the chromosome and plasmids of Halobacterium salinarium GRB, indicating some degree of partitioning between these replicons. We propose alternative courses for the evolution of the haloarchaeal genome: (i) that the majority of genomic differences that exist between genera came about at the inception of this group or (ii) that the differences have accumulated over the lifetime of the lineage. The strengths and limitations of investigating these models through comparative genomic studies are discussed.     

Development of Additional Selectable Markers for the Halophilic Archaeon Haloferax volcanii Based on the leuB and trpA Genes

Since most archaea are extremophilic and difficult to cultivate, our current knowledge of their biology is confined largely to comparative genomics and biochemistry. Haloferax volcanii offers great promise as a model organism for archaeal genetics, but until now there has been a lack of a wide variety of selectable markers for this organism. We describe here isolation of H. volcanii leuB and trpA genes encoding 3-isopropylmalate dehydrogenase and tryptophan synthase, respectively, and development of these genes as a positive selection system. ΔleuB and ΔtrpA mutants were constructed in a variety of genetic backgrounds and were shown to be auxotrophic for leucine and tryptophan, respectively. We constructed both integrative and replicative plasmids carrying the leuB or trpA gene under control of a constitutive promoter. The use of these selectable markers in deletion of the lhr gene of H. volcanii is described.     

Complete Genome Sequence of a Novel Human Parechovirus

Human parechoviruses (HPeVs) belonging to the family Picornaviridae are widely spread pathogens among young children. We report the complete genome sequence of a novel HPeV isolated from the stool sample of a hospitalized child with diarrhea in China. The genome consists of 7,305 nucleotides, excluding the 3′ poly(A) tail, and has an open reading frame that maps between nucleotide positions 675 and 7217 and encodes a 2,180-amino-acid polyprotein. The genome sequence of the virus was sufficiently distinct from the 8 known HPeV types. Phylogenetic analysis based on the complete genome indicated that the HPeV strain represents a new genotype.     

Complete Genome Sequence of Mycobacterium massiliense

Mycobacterium massiliense is a rapidly growing bacterium associated with opportunistic infections. The genome of a representative isolate (strain GO 06) recovered from wound samples from patients who underwent arthroscopic or laparoscopic surgery was sequenced. To the best of our knowledge, this is the first announcement of the complete genome sequence of an M. massiliense strain.     

Complete Genome Sequence of Methanomassiliicoccus luminyensis, the Largest Genome of a Human-Associated Archaea Species

The present study describes the complete and annotated genome sequence of Methanomassiliicoccus luminyensis strain B10 (DSM 24529(T), CSUR P135), which was isolated from human feces. The 2.6-Mb genome represents the largest genome of a methanogenic euryarchaeon isolated from humans. The genome data of M. luminyensis reveal unique features and horizontal gene transfer events, which might have occurred during its adaptation and/or evolution in the human ecosystem.     

Shotgun proteomics of the haloarchaeon Haloferax volcanii

Haloferax volcanii, an extreme halophile originally isolated from the Dead Sea, is used worldwide as a model organism for furthering our understanding of archaeal cell physiology. In this study, a combination of approaches was used to identify a total of 1296 proteins, representing 32% of the theoretical proteome of this haloarchaeon. This included separation of (phospho)proteins/peptides by 2-dimensional gel electrophoresis (2-D), immobilized metal affinity chromatography (IMAC), metal oxide affinity chromatography (MOAC), and Multidimensional Protein Identification Technology (MudPIT) including strong cation exchange (SCX) chromatography coupled with reversed phase (RP) HPLC. Proteins were identified by tandem mass spectrometry (MS/MS) using nanoelectrospray ionization hybrid quadrupole time-of-flight (QSTAR XL Hybrid LC/MS/MS System) and quadrupole ion trap (Thermo LCQ Deca). Results indicate that a SCX RP HPLC fractionation coupled with MS/MS provides the best high-throughput workflow for overall protein identification.     

Complete Genome Sequence of Porcine Circovirus 2d Strain GDYX

Porcine circovirus type 2 (PCV2) is the etiologic agent of porcine circovirus-associated disease, and it is mainly divided into five genotypes. Here, we report the complete genome sequence of PCV2 strain GDYX, which belongs to PCV2d and has a unique amino acid variation at position 169 (S to G).     

Complete Genome Sequence of a Mink Calicivirus in China

We report the complete genome sequence of a novel calicivirus isolated from a diseased mink in China. The complete viral genome is approximately 8.4 kb in length and consists of three open reading frames. The availability of the complete genome sequence is helpful for further investigation into the molecular characteristics and epidemiology of calicivirus in mink.     

Complete Genome Sequence of a Novel Pestivirus from Sheep

We report here the complete genome sequence of pestivirus strain Aydin/04-TR, which is the prototype of a group of similar viruses currently present in sheep and goats in Turkey. Sequence data from this virus showed that it clusters separately from the established and previously proposed tentative pestivirus species.