Felicitas Pfeifer

Diversity of Archaea in hypersaline environments characterized by molecular-phylogenetic and cultivation studies

The diversity of Archaea from three different hypersaline environments was analyzed and compared by polymerase chain reaction (PCR)-based molecular phylogenetic techniques and cultivation approaches. The samples originated from a crystallization pond of a solar saltern in Spain (FC); an alkaline lake in Nevada, USA, (EMF); and a small pond from a slag heap of a potassium mine in Germany (DIE). Except for two 16S rDNA sequences that were related to crenarchaeota from soil and did not apparently belong to the indigenous halophilic community, all sequences recovered from environmental DNA or cultivated strains grouped within the Halobacteriaceae. Mostly 16S rDNA sequences related to the genera Halorubrum and Haloarcula were detected in sample FC, and organisms belonging to these genera were also recovered by cultivation. In contrast, sequences related to five different groups of halophilic archaea were amplified from sample DIE (including novel lineages with only uncultivated phylotypes), but the organisms that were cultivated from this sample fell into different groups (i.e., Natronococcus, Halorubrum, or unaffiliated) and did not overlap with those predicted using the culture-independent approach. With respect to the highly alkaline sample, EMF, four groups were predicted from the environmental 16S rDNA sequences, two of which (Natronomonas and Haloarcula) were also recovered through cultivation together with Natronococcus isolates. In summary, we found that halophilic archaea dominate the archaeal populations in these three hypersaline environments and show that culturability of the organisms predicted by molecular surveys might strongly depend on the habitat chosen. While a number of novel halophilic archaea have been isolated, we have not been able to cultivate representatives of the new lineages that were detected in this and several other environmental studies.

Purification and analysis of an extremely halophilic β-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.

Distribution, formation and regulation of gas vesicles

A range of bacteria and archaea produce intracellular gas-filled proteinaceous structures that function as flotation devices in order to maintain a suitable depth in the aqueous environment. The wall of these gas vesicles is freely permeable to gas molecules and is composed of a small hydrophobic protein, GvpA, which forms a single-layer wall. In addition, several minor structural, accessory or regulatory proteins are required for gas vesicle formation. In different organisms, 8–14 genes encoding gas vesicle proteins have been identified, and their expression has been shown to be regulated by environmental factors. In this Review, I describe the basic properties of gas vesicles, the genes that encode them and how their production is regulated. I also discuss the function of these vesicles and the initial attempts to exploit them for biotechnological purposes.

A DNA region of 9kbp contains all genes necessary for gas vesicle synthesis in halophilic archaebacteria

We determined the minimal size of the genomic region necessary for gas vesicle synthesis in halophilic archaebacteria by transformation experiments, comparative DNA sequence analysis and investigation of gas vesicle (Vac) mutants. The comparison of the three genomic regions encoding gas vesicles in Halobacterium halobium (p‐vac‐ and c‐vac‐region) and Haloferax mediterranei (mc‐vac‐region) indicates high DNA sequence similarity throughout a contiguous sequence of 9kbp. In each case, this area encompassed at least 13 open reading frames (ORFs). Ten of these ORFs (gvpD to gvpM) were located 5′ to the vac gene encoding the major gas vesicle protein, but were transcribed from the opposite strand. At least two ORFs (gvpC, and gvpN) were located 3′ to each vac gene and transcribed from the same strand as the respective vac gene. In the p‐vac‐region present on plasmid pHH1 these ORFs were transcribed as at least three units, one transcript encompassing gvpD‐gvpE, the second encompassing ORFs gvpF to gvpM, and the third unit comprising the ORFs located 3′ to the p‐vac gene. In H. halobium Vac mutants copies of the insertion elements ISH2, ISH23, ISH26 or ISH27 were found to be integrated throughout the p‐vac‐region. The de novo synthesis of gas vesicles was tested by transformation of the Vac‐negative species, Haloferax volcanii, with various subfragments of the mc‐vac‐ or p‐vac‐region cloned into vector plasmids. In contrast to a fragment containing the entire 9 kbp region, none of the subfragments tested was sufficient to promote gas vesicle synthesis. However, gas vesicle synthesis could be restored in each Vac mutant containing an ISH element when the entire transcription unit encompassing the mutated gene on pHH1 was present in the wild‐type form on the vector construct.

Plasmid pHH1 of Halobacterium salinarium: characterization of the replicon region, the gas vesicle gene cluster and insertion elements.

The DNA sequence of the 5.7 kb plasmid pHH9 containing the replicon region of the 150 kb plasmid pHH1 from Halobacterium salinarium was determined. The minimal region necessary for stable plasmid maintenance lies within a 2.9 kb fragment, as defined by transformation experiments. The DNA sequence contained two open reading frames arranged in opposite orientations, separated by an unusually high AT-rich (60–70% A + T) sequence of 350 bp. All H. salinarium strains (H. halobium, H. cutirubrum) investigated harbour endogenous plasmids containing the pHH1 replicon; however, these pHH1-type plasmids differ by insertions and deletions. Adjacent to the replicon, and separated by a copy of each of the insertion elements ISH27 and ISH26, is the 9 kb p-vac region required for gas vesicle synthesis. Analysis of these and other ISH element copies in pHH1 revealed that most of them lack the target DNA duplication usually found with recently transposed ISH elements. These results underline the plasticity of plasmid pHH1.

Expression of the major gas vesicle protein gene in the halophilic archaebacterium Haloferax mediterranei is modulated by salt

SummaryIn the moderately to extremely halophilic archaebacteriumHaloferax mediterranei gas vacuoles are not observed before the stationary phase of growth, and only when the cells are grown in media containing more than 17% total salt. Under the electron microscope, isolated gas vesicles appear as cylindrical structures with conical ends that reach a maximal length of 1.5 μm; this morphology is different from the spindle-shaped gas vesicles found in theHalobacterium halobium wild type which expresses the plasmid-borne p-vac gene, but resembles that of gas vesicles isolated fromH. halobium strains expressing the chromosomalc-vac gene. Both the p-vac and thec-vac genes encode very similar structural proteins accounting for the major part of the “membrane” of the respective gas vesicles. The homologousmc-vac gene was isolated fromHf. mediterranei using thep-vac gene as probe. Themc-vac coding region indicates numerous nucleotide differences compared to thep-vac ancc-vac genes; the encoded protein is, however, almost identical to thec-vac gene product. The start point of the 310 nucleotidemc-vac transcript determined by primer extension analysis and S1 mapping was located 20 by upstream of the ATG start codon, which is at the same relative position as found for the other twovac mRNAs. During the growth cycle,mc-vac mRNA was detectable inHf. mediterranei cells grown in 15% as well as 25% total salt, with a maximal level in the early stationary phase of growth. The relative abundance ofmc-vac mRNA in cells grown at 25% salt was sevenfold higher than in cells grown in 15% total salt.

Improved shuttle vectors for Haloferax volcanii including a dual-resistance plasmid.

Two new Haloferax-Escherichia shuttle vectors are described, pMDS20 and pMLH3. These vectors contain the E. coli ColE1 plasmid ori region and ampicillin-resistance(ApR)-conferring bla gene, and the Haloferax pHK2 replicon region and novobiocin-resistance(NbR)-encoding gyrB gene, enabling maintenance and selection in both hosts. Plasmid pMLH3 has, in addition, a H. volcanii mevinolin-resistance (MvR) determinant and restriction sites allowing insertional inactivation of either marker, to facilitate the identification of Haloferax transformants harbouring cloned sequences. Sequencing of gyrA, within the NbR determinant, and the pHK2 ori region has been completed so the complete sequence of both pMDS20 and pMLH3 is now known.

Functional analysis of the gas vesicle gene cluster of the halophilic archaeon Haloferax mediterranei defines the vac-region boundary and suggests a regulatory role for the gvpD gene or its product

A series of deletions introduced into the gvp gene cluster of Haloferax mediterranel, comprising 14 genes involved in gas vesicle synthesis (mc‐vac‐region), was investigated by transformation experiments. Gas vesicle production and the expression of the gvpA gene encoding the major gas vesicle protein, GvpA, was monitored in each Haloferax volcanii transformant. Whereas transformants containing the entire mc‐vac‐region produced gas vesicies (Vac+), various deletions in the region 5′ to gvpA (encompassing gvpD‐gvpM) or 3′ to gvpA (containing gvpC, gvpN and gvpO) revealed Vac− transformants. All these transformants expressed gvpA and contained the 8 kDa GvpA protein as shown by Western analysis. However, transformants containing the gvpA gene by itself indicated a lower level of GvpA than observed with each of the other transformants. None of these transformants containing deletion constructs assembled the GvpA protein into gas vesicles. In contrast, transformants containing a construct carrying a 918 bp deletion internal to gvpD exhibited a tremendous gas vesicle overproduction, suggesting a regulatory role for the gvpD gene or Its product. This is the first assignment of a functional role for one of the 13 halobacterial gvp genes found in addition to gvpA that are involved in the synthesis of this unique structure.

Biofilm formation by haloarchaea

A fluorescence-based live-cell adhesion assay was used to examine biofilm formation by 20 different haloarchaea, including species of Halobacterium, Haloferax and Halorubrum, as well as novel natural isolates from an Antarctic salt lake. Thirteen of the 20 tested strains significantly adhered (P-value  < 0.05) to a plastic surface. Examination of adherent cell layers on glass surfaces by differential interference contrast, fluorescence and confocal microscopy showed two types of biofilm structures. Carpet-like, multi-layered biofilms containing micro- and macrocolonies (up to 50 μm in height) were formed by strains of Halobacterium salinarum and the Antarctic isolate t-ADL strain DL24. The second type of biofilm, characterized by large aggregates of cells adhering to surfaces, was formed by Haloferax volcanii DSM 3757T and Halorubrum lacusprofundi DL28. Staining of the biofilms formed by the strongly adhesive haloarchaeal strains revealed the presence of extracellular polymers, such as eDNA and glycoconjugates, substances previously shown to stabilize bacterial biofilms. For Hbt. salinarum DSM 3754T and Hfx. volcanii DSM 3757T , cells adhered within 1 day of culture and remained viable for at least 2 months in mature biofilms. Adherent cells of Hbt. salinarum DSM 3754T showed several types of cellular appendages that could be involved in the initial attachment. Our results show that biofilm formation occurs in a surprisingly wide variety of haloarchaeal species.

Eight of Fourteen gvp Genes Are Sufficient for Formation of Gas Vesicles in Halophilic Archaea

The minimal number of genes required for the formation of gas vesicles in halophilic archaea has been determined. Single genes of the 14 gvp genes present in the p-vac region on plasmid pHH1 of Halobacterium salinarum (p-gvpACNO and p-gvpDEFGHIJKLM) were deleted, and the remaining genes were tested for the formation of gas vesicles in Haloferax volcanii transformants. The deletion of six gvp genes (p-gvpCN, p-gvpDE, and p-gvpHI) still enabled the production of gas vesicles in H. volcanii. The gas vesicles formed in some of these gvp gene deletion transformants were altered in shape (Delta I, Delta C) or strength (Delta H) but still functioned as flotation devices. A minimal p-vac region (minvac) containing the eight remaining genes (gvpFGJKLM-gvpAO) was constructed and tested for gas vesicle formation in H. volcanii. The minvac transformants did not form gas vesicles; however, minvac/gvpJKLM double transformants contained gas vesicles seen as light refractile bodies by phase-contrast microscopy. Transcript analyses demonstrated that minvac transformants synthesized regular amounts of gvpA mRNA, but the transcripts derived from gvpFGJKLM were mainly short and encompassed only gvpFG(J), suggesting that the gvpJKLM genes were not sufficiently expressed. Since gvpAO and gvpFGJKLM are the only gvp genes present in minvac/JKLM transformants containing gas vesicles, these gvp genes represent the minimal set required for gas vesicle formation in halophilic archaea. Homologs of six of these gvp genes are found in Anabaena flos-aquae, and homologs of all eight minimal halobacterial gvp genes are present in Bacillus megaterium and in the genome of Streptomyces coelicolor.