Combined use of cultivation-dependent and cultivation-independent methods indicates that members of most haloarchaeal groups in an Australian crystallizer pond are cultivable.

Haloarchaea are the dominant microbial flora in hypersaline waters with near-saturating salt levels. The haloarchaeal diversity of an Australian saltern crystallizer pond was examined by use of a library of PCR-amplified 16S rRNA genes and by cultivation. High viable counts (10(6) CFU/ml) were obtained on solid media. Long incubation times (> or =8 weeks) appeared to be more important than the medium composition for maximizing viable counts and diversity. Of 66 isolates examined, all belonged to the family Halobacteriaceae, including members related to species of the genera Haloferax, Halorubrum, and Natronomonas. In addition, isolates belonging to a novel group (the ADL group), previously detected only as 16S rRNA genes in an Antarctic hypersaline lake (Deep Lake), were cultivated for the first time. The 16S rRNA gene library identified the following five main groups: Halorubrum groups 1 and 2 (49%), the SHOW (square haloarchaea of Walsby) group (33%), the ADL group (16%), and the Natronomonas group (2%). There were two significant differences between the organisms detected in cultivation and 16S rRNA sequence results. Firstly, Haloferax spp. were frequently isolated on plates (15% of all isolates) but were not detected in the 16S rRNA sequences. Control experiments indicated that a bias against Haloferax sequences in the generation of the 16S rRNA gene library was unlikely, suggesting that Haloferax spp. readily form colonies, even though they were not a dominant group. Secondly, while the 16S rRNA gene library identified the SHOW group as a major component of the microbial community, no isolates of this group were obtained. This inability to culture members of the SHOW group remains an outstanding problem in studying the ecology of hypersaline environments.

Bacteremia caused by a Helicobacter pullorum-like organism.

We report a case of bacteremia caused by a Helicobacter pullorum-like organism in a 35-year-old man with pyrexia of unknown origin. Culture of blood samples obtained at admission yielded a motile, spiral-shaped gram-negative rod, and 16S rRNA gene sequencing identified this organism as Helicobacter pullorum-like, showing 23 base differences compared with the recently described "Helicobacter canadensis" (a recently proposed group that had previously been classified within H. pullorum). We believe that this is the first report of bacteremia caused by this organism.

"Helicobacter rappini" isolates from 2 homosexual men.

We report 2 cases of bacteremia due to "Helicobacter rappini" in 2 young, homosexual men, including the first report of H. rappini in a human immunodeficiency virus-positive patient. Blood cultures showed a spiral, fusiform, gram-negative bacterium with bipolar sheathed flagella.

A new simvastatin (mevinolin)-resistance marker from Haloarcula hispanica and a new Haloferax volcanii strain cured of plasmid pHV2.

The mevinolin-resistance determinant, hmg, encodes the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and is a commonly used selectable marker in halobacterial genetics. Plasmids bearing this marker suffer from instability in Haloferax volcanii because the resistance gene was derived from the genome of this species and is almost identical in sequence to the chromosomal copy. In order to reduce the level of homologous recombination between introduced plasmid vectors and the chromosome of Haloferax, a homologue of the hmg determinant was obtained from the distantly related organism, Haloarcula hispanica. The nucleotide sequences of the wild-type genes (hmgA) of these two species are only 78% identical, and the predicted protein sequences show 71% identity. In comparison to the wild-type hmgA gene, the resistance gene from a mutant resistant to simvastatin (an analogue of mevinolin) showed a single base substitution in the putative promoter. Plasmids constructed using the new resistance determinant were stably maintained under selection in Hfx. volcanii and possessed very low recombination rates with the chromosome of this species. In addition, an improved strain of Hfx. volcanii was developed to overcome the plasmid instability and growth reduction observed in the commonly used WFD11 strain.

A member of the delta subgroup of proteobacteria from a pyogenic liver abscess is a typical sulfate reducer of the genus Desulfovibrio.

Strain FH26001/95 (ATCC 700045) was previously isolated from a pyogenic liver abscess from a human. Comparative 16S rRNA gene sequence analysis showed that this strain is related to members of the delta subgroup of the proteobacteria, within a cluster of sulfate-reducing bacteria (Desulfovibrio spp.) and non-sulfate-reducing bacteria (Bilophila wadsworthia and Lawsonia spp.). The phenotype of strain FH26001/95 was found to be typical of members of the genus Desulfovibrio. Growth and substrate transformations were possible at oxygen concentrations of 2 to 5% (vol/vol) but not at oxygen concentrations of 21% (vol/vol) in air. Its isolation from an infection in a human suggests that some members of the genus Desulfovibrio can be considered opportunistic pathogens.

Bacteremia due to Leptotrichia trevisanii sp. nov.

A thin, filamentous, non-motile, aerotolerant, anaerobic, gram-negative bacterium was isolated from the blood of a 46-year-old man who was diagnosed as having acute myeloid leukemia. The organism had a positive catalase reaction but was negative in indole and oxidase tests. A commercially available system failed to identify the bacterium, but 16S rRNA gene sequencing showed it to be most closely related (97% similarity) to a recently isolated Leptotrichia sp. The DNA base composition was 29.7% mol G+C, and the organism produced lactate as the sole end-product of glucose fermentation. These data indicate the isolate is a new species of Leptotrichia for which the name Leptotrichia trevisanii sp. nov. is proposed.

A probable new Helicobacter species isolated from a patient with bacteremia.

A probable new Helicobacter species was isolated from the blood of a 14-month-old aboriginal child who presented with vomiting, diarrhea, fever, and dry cough. The most similar 16S rRNA gene sequence was that of Helicobacter fennelliae CCUG 18820(T) but the new sequence differed from it by at least 32 base substitutions and by the presence of a large (353-nucleotide) intervening sequence.

Sequence and expression of a halobacterial beta-galactosidase gene.

Studies of gene expression in haloarchaea have been greatly hindered by the lack of a convenient reporter gene. In a previous study, a beta-galactosidase from Haloferax alicantei was purified and several peptide sequences determined. The peptide sequences have now been used to clone the entire beta-galactosidase gene (designated bgaH) along with some flanking chromosomal DNA. The deduced amino acid sequence of BgaH was 665 amino acids (74 kDa) and showed greatest amino acid similarity to members of glycosyl hydrolase family 42 [classification of Henrissat, B., and Bairoch, A. (1993) New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 293: 781-788]. Within this family, BgaH was most similar (42-43% aa identity) to enzymes from extremely thermophilic bacteria such as Thermotoga and Thermus. Family 42 enzymes are only distantly related to the Sulfolobus LacS and Escherichia coli LacZ enzymes (families one and two respectively). Three open reading frames (ORFs) upstream of bgaH were readily identified by database searches as glucose-fructose oxidoreductase, 2-dehydro-3-deoxyphosphogluconate aldolase and 2-keto-3-deoxygluconate kinase, enzymes that are also involved in carbohydrate metabolism. Downstream of bgaH there was an ORF which contained a putative fibronectin III motif. The bgaH gene was engineered into a halobacterial plasmid vector and introduced into Haloferax volcanii, a widely used strain that lacks detectable beta-galactosidase activity. Transformants were shown to express the enzyme; colonies turned blue when sprayed with Xgal and enzyme activity could be easily quantitated using a standard ONPG assay. In an accompanying publication, Patenge et al. (2000) have demonstrated the utility of bgaH as a promoter reporter in Halobacterium salinarum.

The ShBle resistance determinant from Streptoalloteichus hindustanus is expressed in Haloferax volcanii and confers resistance to bleomycin.

We have designed a gene cassette for expression of the bleomycin-resistance protein from Streptoalloteichus hindustanus (ShBle) in the extremely halophilic archaeon Haloferax volcanii, and shown that transformed haloarchaea are resistant to bleomycin. Recombinant ShBle was purified by a one-step affinity-chromatography procedure as a correctly folded, dimeric protein. ShBle thus provides a useful haloarchaeal selectable marker and represents the first non-halophilic and soluble heterologous protein to be expressed in the Haloarchaea.

Role of flagellins from A and B loci in flagella formation of Halobacterium salinarum.

Haloarchaeal flagella are composed of a number of distinct flagellin proteins, specified by genes in two separate operons (A and B). The roles of these flagellins were assessed by studying mutants of H. salinarum with insertions in either the A or the B operon. Cells of the flgA- mutant produced abnormally short, curved flagella that were distributed all over the cell surface. The flgA2- strain produced straight flagella, mainly found at the poles. The flgB- mutant had flagella of the same size and spiral shape as wild-type cells, but these cells also showed unusual outgrowths, which appeared to be sacs filled with basal body-like structures. In broth cultures of this mutant, the medium accumulated flagella with basal body-like structures at their ends.

An improved transposon for the halophilic archaeon Haloarcula hispanica.

An improved transposon (ThD73) for Haloarcula hispanica is described. Based on the halobacterial insertion sequence ISH28, it showed little target sequence specificity but was biased toward a lower G+C content. Twenty randomly selected ThD73 mutants were analyzed, and the DNA flanking their insertions revealed several recognizable sequences, including two (unrelated) ISH elements.

Proposal to transfer Halococcus turkmenicus, Halobacterium trapanicum JCM 9743 and strain GSL-11 to Haloterrigena turkmenica gen. nov., comb. nov.

The 16S rRNA gene sequences of Halococcus saccharolyticus and Halococcus salifodinae were closely related (94.5-94.7% similarity) to that of Halococcus morrhuae, the type species of the genus Halococcus. However, Halococcus turkmenicus was distinct from the other members of this genus, with low 16S rRNA similarities when compared to Halococcus morrhuae (88.7%). On the basis of phylogenetic tree reconstruction, detection of signature bases and DNA-DNA hybridization data, it is proposed to transfer Halococcus turkmenicus to a novel genus, Haloterrigena, as Haloterrigena turkmenica gen. nov., comb. nov., and to accommodate Halobacterium trapanicum JCM 9743 and strain GSL-11 in the same species. On the basis of morphological, cultural and 16S rRNA sequence data, it is also proposed that the culture collection strains of Halobacterium trapanicum NCIMB 767, ATCC 43102 and JCM 8979 should be renamed as Halococcus sp.

His1, an archaeal virus of the Fuselloviridae family that infects Haloarcula hispanica.

A novel archaeal virus, His1, was isolated from hypersaline waters in southeastern Australia. It was lytic, grew only on Haloarcula hispanica (titers of up to 10(11) PFU/ml), and displayed a lemon-shaped morphology (74 by 44 nm) previously reported only for a virus of the extreme thermophiles (SSV1). The density of His1 was approximately 1.28 g/ml, similar to that of SSV1 (1.24 g/ml). Purified particles were resistant to low salt concentrations. The genome was linear, double-stranded DNA of 14.9 kb, similar to the genome of SSV1 (15.5 kb). Morphologically, this isolate clearly belongs to the recently proposed Fuselloviridae family of archaeal viruses. It is the first member of this family from the extremely halophilic archaea, and its host, H. hispanica, can be readily manipulated genetically.

"Flexispira rappini" bacteremia in a child with pneumonia.

We describe a case of "Flexispira rappini" bacteremia from a 9-year-old girl who presented with a 5-day history of fever, productive cough, and malaise. A chest X-ray result was compatible with right middle lobe pneumonia. Blood culture grew a gram-negative spiral fusiform bacterium 2 days after the inoculation. Biochemical tests showed the organism to be catalase negative, oxidase positive, sodium hippurate hydrolysis negative, and urea hydrolysis negative. 16S rRNA gene sequencing identified this organism as "F. rappini," showing a six-base substitution from the type strain. This is the first report of "F. rappini" bacteremia in a human, suggesting that this organism has the potential of causing invasive infection, but its role in pneumonia is uncertain and could be unrelated to the bacteremia.

Three cases of Anaerobiospirillum succiniciproducens bacteremia confirmed by 16S rRNA gene sequencing.

We describe three cases of Anaerobiospirillum succiniciproducens bacteremia from Australia. We believe one of these cases represents the first report of A. succiniciproducens bacteremia in a human immunodeficiency virus (HIV)-infected individual. The other two patients had an underlying disorder (one patient had bleeding esophageal varices complicating alcohol liver disease and one patient had non-Hodgkin's lymphoma). A motile, gram-negative, spiral anaerobe was isolated by culturing blood from all patients. Electron microscopy showed a curved bacterium with bipolar tufts of flagella resembling Anaerobiospirillum spp. Sequencing of the 16S rRNA genes of the isolates revealed no close relatives (organisms likely to be in the same genus) in the sequence databases, nor were any sequence data available forA. succiniciproducens. This report presents for the first time the 16S rRNA gene sequence of the type strain of A. succiniciproducens, strain ATCC 29305. Two of the three clinical isolates have sequences identical to that of the type strain, while the sequence of the other strain differs from that of the type strain at 4 nucleotides.

Diversity of alkaliphilic halobacteria: proposals for transfer of Natronobacterium vacuolatum, Natronobacterium magadii, and Natronobacterium pharaonis to Halorubrum, Natrialba, and Natronomonas gen. nov., respectively, as Halorubrum vacuolatum comb. nov., Natrialba magadii comb. nov., and Natronomonas pharaonis comb. nov., respectively.

The 16S rRNA genes of three species of the genus Natronobacterium (Natronobacterium gregoryi, Natronobacterium pharaonis, and Natronobacterium vacuolatum) were sequenced and compared to that of the previously sequenced species Natronobacterium magadii. The sequences revealed that Natronobacterium pharaonis was phylogenetically distinct from the other members of the genus and also from other recognized genera of the family Halobacteriaceae. However, Natronobacterium vacuolatum and Natronobacterium magadii were found to be most closely related to the genera Halorubrum and Natrialba, respectively. An unidentified haloalkaliphile, strain SSL1, was also closely related to Natronobacterium magadii and Natrialba asiatica. On the basis of phylogenetic tree reconstructions, signature bases specific for individual genera, and sequences of spacer regions between 16 and 23S rRNA genes, we propose the following changes: Natronobacterium pharaonis to be transferred to Natronomonas gen. nov. as Natronomonas pharaonis gen. nov., comb. nov.; Natronobacterium vacuolatum to be transferred to the genus Halorubrum as Halorubrum vacuolatum comb. nov.; and Natronobacterium magadii to be transferred to the genus Natrialba as Natrialba magadii.

Purification and analysis of an extremely halophilic beta-galactosidase from Haloferax alicantei.

As a first step in the development of a reporter system for gene expression in halophilic archaea, a beta-galactosidase was purified 140-fold from Haloferax alicantei (previously phenon K, strain Aa2.2). An overproducing mutant was first isolated by UV mutagenesis and screening on agar plates containing X-Gal substrate. Cytoplasmic extracts of the mutant contained 25-fold higher enzyme levels than the parent. Purification of the active enzyme was greatly facilitated by the ability of sorbitol to stabilise enzyme activity in the absence of salt, which allowed conventional purification methods (e.g., ion-exchange chromatography) to be utilised. The enzyme was optimally active at 4 M NaCl and was estimated to be 180 +/- 20 kDa in size, consisting of two monomers (each 78 +/- 3 kDa). It cleaves several different beta-galactoside substrates such as ONP-Gal, X-Gal and lactulose, but not lactose, and also has beta-D-fucosidase activity. No beta-glucosidase, beta-arabinosidase or beta-xylosidase activity could be detected. The amino-acid sequence at the N-terminus and of four proteolytic products has been determined.

Construction and analysis of a recombination-deficient (radA) mutant of Haloferax volcanii.

By deleting the radA open reading frame of an extreme halophile, Haloferax volcanii, we created and characterized a recombination-deficient archaeon. This strain, Hf. volcanii DS52, has no detectable DNA recombination, is more sensitive to DNA damage by UV light and ethylmethane sulfonate, and has a slower growth rate than the wild type. These characteristics are similar to those observed in recombination mutants of Eukarya and Bacteria, and show that the radA gene belongs in the recA/RAD51 family by function as well as sequence homology. In addition, strain DS52 was not transformable by plasmids pWL102 or pUBP2 (which contain pHV2 and pHH1 replicons, respectively), although it was readily transformed by plasmids containing a pHK2 replicon, indicating a role for radA in the maintenance or replication of some halobacterial plasmids. Despite its slower growth rate, Hf. volcanii DS52 was still easy to culture and transform, and should be suitable for use in studies where a recombination-deficient background is desired.

Revised nucleotide sequence of an archaeal insertion element (ISH28) reveals a putative transposase gene.

The published sequence of the insertion element ISH28 contained many small ORFs that were difficult to interpret. We resequenced the entire element and found seven nucleotide differences. The corrected sequence of ISH28 is 938 bp long, and now reveals a single open reading frame of 828 bp. The putative protein is highly similar (49% aa identity) to the predicted transposase of ISH1.

Probable new species of Desulfovibrio isolated from a pyogenic liver abscess.

A fastidious, slowly growing, spiral gram-negative bacterium was isolated from the liver abscess of an 82-year-old man with a 3-week history of febrile illness. The organism was an obligate anaerobe that grew at 37 and 42 degrees C but not at 25 degrees C. Its vibrioid or spiral morphology on Gram staining, rapid progressive motility, electron micrograph features, and biochemical tests were all consistent with the organism belonging to the genus Desulfovibrio. 16S rRNA gene sequencing of this organism demonstrated a 97% similarity to Desulfovibrio desulfuricans with 45 nucleotide differences, suggesting that it is a new species of Desulfovibrio.