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F Pfeifer

Publications and source records attributed to F Pfeifer.

At least 19 recordsLinked to original sources
PubMed1992

Three different but related gene clusters encoding gas vesicles in halophilic archaea.

We present an analysis of the chromosomal region comprising the gene cluster involved in gas vesicle (Vac) synthesis in Haloferax mediterranei (mc-vac-region) and Halobacterium salinarium (c-vac-region) and compare both of them to the plasmid located p-vac-region of H. salinarium. The p-vac-region of 9000 base-pairs (9 kb) is more related to mc-vac (9.4 kb) of Hf. mediterranei than it is to the c-vac-region (8.3 kb) present in the same cell. The Vac- species Hf. volcanii becomes Vac+ following transformation with a fragment containing the entire mc-vac-region. Also the p-vac-region transforms Hf. volcanii to a Vac+ phenotype, indicating that this gene cluster is sufficient for gas vesicle synthesis and does not depend on products of the c-vac-region. Each of these vac-regions contains, in addition to gvpA encoding the major gas vesicle protein, 13 open reading frames named gvpC through gvpO. Ten of these, gvpD through gvpM, are located upstream from gvpA in opposite orientation, while gvpC, gvpN and gvpO are found 3' to gvpA. The absolute requirement of gvpO for gas vesicle synthesis was demonstrated by transformation experiments. Northern analyses with RNA samples isolated during the growth cycle of Hf. mediterranei or of H. salinarium PHH4 revealed that the mc-gvpD or c-gvpD mRNAs occur similar to the respective gvpA mRNA in stationary growth phase, while gvpF-gvpM are transcribed mainly during logarithmic growth. S1-nuclease mapping was performed to determine the transcriptional start site of the gvpD mRNA. The distance between the two divergent start sites of gvpA and gvpD mRNA is 109 base-pairs in mc-vac and p-vac, while in the case of c-vac this distance is 22 base-pairs larger. The conservation of the various gvp products, characteristic features and their possible functions in gas vesicle synthesis are discussed.

Archaeal Proteins↗
PubMed1992

Function and biosynthesis of gas vesicles in halophilic Archaea.

The proteinaceous gas vesicles produced by various microorganisms including halophilic Archaea are hollow, gas-filled structures with a hydrophobic inner and a hydrophilic outer surface. The structural components of gas vesicles and their biosynthesis are still under investigation; an 8-kDa polypeptide appears to be the major constituent of the gas-vesicle envelope. Genetic analysis of the halobacterial gas-vesicle synthesis revealed an unexpected complexity: about 14 genes organized in three transcription units are involved in gas-vesicle structure, assembly, and gene regulation. Here we describe the comparison of three different genomic regions encoding gas vesicles in Halobacterium salinarium (p-vac and c-vac regions) and Haloferax mediterranei (mc-vac region) and speculate on the function of the gene products involved in gas-vesicle synthesis.

Amino Acid Sequence↗
PubMed1992

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 mediterranei, 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 vesicles (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.

Bacterial Proteins↗
PubMed1991

A DNA region of 9 kbp 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 9 kbp. 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.

Amino Acid Sequence↗
PubMed1990

Transposition burst of the ISH27 insertion element family in Halobacterium halobium.

Investigation of the plasmid pHH4 in single colonies of Halobacterium halobium PHH4 indicated transposition of insertion elements in 20% of the colonies. Seven ISH27 insertions were observed as well as one ISH23 insertion. The various copies of ISH27 were compared to the two ISH27 elements already present in pHH4, and to the ISH27 element that was identified in the bacteriopsin (bop) gene of a Bop mutant. These ten copies of ISH27 constitute three types on the basis of DNA sequence identity: ISH27-1 (1398 bp), ISH27-2, and ISH27-3 (1389 bp each). The DNA sequence comparison between the three types indicates a region of 1200 bp where the identity between ISH27-1 and ISH27-2 or ISH27-3 is 82-83%. ISH27-2 and ISH27-3 are 95% identical in this region. The remaining region exhibits a lower DNA similarity (64-74% identity) between the different copies. An open reading frame of 1167 nucleotides spans the more conserved region, and a corresponding transcript could be detected in H. halobium PHH4, but not in H. halobium wild-type. ISH27-1 is 91% identical to members of the insertion sequence-like elements ISH51 of Haloferax volcanii, whereas the other two ISH27 element types are 82-83% identical to ISH51. The transposition 'burst' of ISH27 was only seen after storage of the cells for more than two years at 4 degrees C. Upon continuous cultivation at 37 degrees C no transposition event could be observed, suggesting that stress factor(s) might have caused the high transposition rate.

Amino Acid Sequence↗
PubMed1990

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

In the moderately to extremely halophilic archaebacterium Haloferax 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 microns; this morphology is different from the spindle-shaped gas vesicles found in the Halobacterium halobium wild type which expresses the plasmid-borne p-vac gene, but resembles that of gas vesicles isolated from H. halobium strains expressing the chromosomal c-vac gene. Both the p-vac and the c-vac genes encode very similar structural proteins accounting for the major part of the "membrane" of the respective gas vesicles. The homologous mc-vac gene was isolated from Hf. mediterranei using the p-vac gene as probe. The mc-vac coding region indicates numerous nucleotide differences compared to the p-vac anc c-vac genes; the encoded protein is, however, almost identical to the c-vac gene product. The start point of the 310 nucleotide mc-vac transcript determined by primer extension analysis and S1 mapping was located 20 bp upstream of the ATG start codon, which is at the same relative position as found for the other two vac mRNAs. During the growth cycle, mc-vac mRNA was detectable in Hf. 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 of mc-vac mRNA in cells grown at 25% salt was sevenfold higher than in cells grown in 15% total salt.

Amino Acid Sequence↗
PubMed1989

Expression of two gas vacuole protein genes in Halobacterium halobium and other related species.

The archaebacterium Halobacterium halobium contains two genes encoding gas vacuole proteins (vac). One resides on a large naturally occurring plasmid and encodes a protein of 76 amino acids (p-vac), while the other is a chromosomal gene that encodes a highly similar protein of 79 amino acids (c-vac). Northern analysis determined the c-vac and p-vac mRNA to be approximately 340 nucleotides in length, and S1 mapping of both transcripts indicated that the 5' terminus for each starts at the same relative nucleotide. Three other Halobacterium species producing gas vacuoles were investigated, H. spec. GN101, YC819-9, and SB3. All three contain only a chromosomal c-vac gene, and the 5' terminus of the 340 nucleotide mRNA starts at the same nucleotide as found for H. halobium. The c-vac gene region of H. spec. GN101 contains nine nucleotide exchanges, three of which occur in the coding region with no effect on the amino acid sequence. In contrast, the c-vac gene of H. spec. SB3 has an identical nucleotide sequence to the H. halobium c-vac gene. Gas vacuole production in each of these species was monitored during culture growth by phase contrast microscopy, and the vac mRNA level was determined for each time point. H. halobium p-vac deletion mutants, as well as the halobacterial species GN101 and YC819-9, start to synthesize gas vacuoles in early stationary growth phase with a maximal mRNA content in stationary phase. In contrast, H. halobium wild-type synthesizes gas vacuoles exclusively due to p-vac gene expression with a maximal mRNA level during logarithmic growth, and transcripts of the c-vac gene were not detectable.(ABSTRACT TRUNCATED AT 250 WORDS)

Archaeal Proteins↗
PubMed1989

Insertion elements and deletion formation in a halophilic archaebacterium.

Deletion events that occur spontaneously in 36-kilobase-pair (kbp) plasmid pHH4 from the archaebacterium Halobacterium halobium were investigated. Four different deletion derivatives with sizes ranging from 5.7 to 17 kbp were isolated. Three of these deletion variants derived from pHH4 (pHH6 [17 kbp], pHH7 [16 kbp], and pHH8 [6.3 kbp]), whereas the 5.7-kbp plasmid pHH9 derived from pHH6. Strains containing pHH6, pHH7, or pHH9 each lacked the parental plasmid pHH4, while pHH8 occurred at a 1:1 ratio together with pHH4. Common to all of these plasmids was the 5.7-kbp region of pHH9 DNA. The regions containing the fusion site in the deletion derivatives were investigated and compared with the corresponding area of the parental plasmid. Each deletion occurred exactly at the terminus of an insertion element. In pHH6 and pHH7, a halobacterial insertion element (ISH2) was located at the deletion site. The DNA fused to ISH2 displayed a 7-base-pair (bp) (pHH7) or 10-bp (pHH6) sequence homology to the inverted repeat of ISH2. In the two smaller plasmids, pHH8 and pHH9, an ISH27 element was located at the deletion site. Most likely, all of these smaller plasmids resulted from an intramolecular transposition event. The ISH27 insertion sequence contains a 16-bp terminal inverted repeat and duplicates 5 bp of target DNA during the transposition with the specificity 5'ANNNT3'. Four ISH27 copies were analyzed, and two ISH27 element types were identified that have approximately 85% sequence similarity. The ISH27 insertion elements constitute a family which is related to the ISH51 family characterized for H. volcanii, another halophilic archaebacterium.

Base Composition↗
PubMed1989

Genome structure of Halobacterium halobium: plasmid dynamics in gas vacuole deficient mutants.

Halobacterium halobium contains two gas vacuole protein genes that are located in plasmid pHH1 (p-vac) and in the chromosomal DNA (c-vac). The mutation frequency for these genes is different: the constitutively expressed p-vac gene is mutated with a frequency of 10(-2), while the chromosomal gene expressed in the stationary phase of growth is mutated with a frequency of 10(-5). The difference in the mutation susceptibility is due to the dynamics of plasmid pHH1. p-vac gene mutations are caused (i) by the integration of an insertion element or (ii) by a deletion event encompassing the p-vac gene region. In contrast, c-vac mutants analyzed to date incurred neither insertion elements nor deletions. Deletion events within pHH1 occur at high frequencies during the development of a H. halobium culture. The investigation of the fusion regions resulting from deletion events indicates that insertion elements are involved. The analysis of pHH1 deletion variants led to a 4 kilobase pair DNA region containing the origin of replication of the pHH1 plasmid.

Archaeal Proteins↗
PubMed1988

Two genes encoding gas vacuole proteins in Halobacterium halobium.

The archaebacterium Halobacterium halobium contains two related gas vacuole protein-encoding genes (vac). One of these genes encodes a protein of 76 amino acids and resides on the major plasmid. The second gene is located on the chromosome in a (G + C)-rich DNA fraction and encodes a slightly larger but highly homologous protein consisting of 79 amino acids. The plasmid encoded vac gene is transcribed constitutively throughout the growth cycle while the chromosomal vac gene is expressed during the stationary phase of growth. Comparison of the nucleotide sequences of the two genes indicates differences in the putative promoter regions as well as 35 single base-pair exchanges within the coding regions of the two genes. The majority of the nucleotide exchanges in the coding region occur in the third position of a codon triplet generating the codon synonym. The only differences between the two encoded proteins are the exchange of 2 amino acids (positions 8 and 29) and a deletion of 3 amino acids near the carboxy-terminus of the plasmid encoded vac protein. The genomic DNAs from other halobacterial isolates (Halobacterium sp. SB3, GN101 and YC819-9) were found to contain only a chromosomal vac gene copy. There is a high conservation of the chromosomal vac gene and the genomic region surrounding it among the halobacterial strains investigated.

Amino Acid Sequence↗
PubMed1988

Characterization of a second gene involved in bacterio-opsin gene expression in a halophilic archaebacterium.

Southern blot analysis and nucleotide sequencing of DNA from three bacterio-opsin-deficient mutants of the archaebacterium Halobacterium halobium (M86, W105, and W109) revealed that they each contain an alteration in a region 2,000 to 3,800 base pairs (bp) upstream of the bacterio-opsin gene (bop). Nucleotide sequence analysis of this region, which is also located downstream of the previously characterized brp gene, revealed that it contains an open reading frame (ORF) of 2,022 bp. This 2,022-bp ORF has a start codon which overlaps the stop codon of the brp gene and is read in the same direction. The ORF could encode an acidic protein of 73,334 daltons (674 amino acids) with a predicted secondary structure typical of a soluble protein. Bop mutant M86 contains a 1,883-bp deletion extending from bp 351 of the ORF, to 197 bp beyond the stop codon. Mutant W105 has an ISH2 element integrated at bp 1239 of the ORF, and mutant W109 has an ISH26 element integrated at bp 1889. Our results suggest that the ORF is a gene (designated bat for bacterio-opsin activator gene) involved in bop gene expression.

Amino Acid Sequence↗
PubMed1988

Dynamic plasmid populations in Halobacterium halobium.

Deletion events occurring in the major 150-kilobase-pair (kb) plasmid pHH1 of the archaebacterium Halobacterium halobium were investigated. We found four deletion derivatives of pHH1 in gas-vacuole-negative mutants, two of which (pHH23) [65 kb] and pHH4 [36 kb]) we analyzed. Both plasmids incurred more than one deletion, leading to the fusion of noncontiguous pHH1 sequences. pHH23 and pHH4 overlapped by only 4 kb of DNA sequence. A DNA fragment derived from this region was used to monitor the production of further deletion variants of pHH4. A total of 25 single colonies were characterized, 23 of which contained various smaller pHH4 derivatives. Of the 25 colonies investigated, 2 had lost pHH4 entirely and contained only large (greater than or equal to 100-kb) minor covalently closed circular DNAs. One colony contained the 17-kb deletion derivative pHH6 without any residual pHH4. The sizes of the pHH4 deletion derivatives, produced during the development of a single colony, ranged from 5 to 20 kb. In five colonies, pHH4 was altered by the integration of an additional insertion element. These insertions, as well as copies of the various insertion elements already present in pHH4, presumably serve as hot spots for recombination events which result in deletions. A second enrichment procedure led to the identification of colonies containing either a 16-kb (pHH7) or a 5-kb (pHH8) deletion derivative of pHH4 as the major plasmid. pHH8, the smallest plasmid found, contained the 4 kb of unique DNA sequence shared by pHH23 and pHH4, as well as some flanking pHH4 sequences. This result indicates that the 4-kb region contains the necessary sequences for plasmid maintenance and replication.

Base Sequence↗
PubMed1985

Genome organization in Halobacterium halobium: a 70 kb island of more (AT) rich DNA in the chromosome.

The more A + T rich fractionated component (FII DNA) of the Halobacterium halobium genome constitutes one third of the total DNA and upon isolation consists of covalently closed circular DNA (pHH1 and minor cccDNA) and nonsupercoiled sequences. We have investigated the physical organization of the non cccDNA in FII by a chromosome walk using one copy of the halobacterial insertion element ISH1 as a start point. This chromosome walk led to the isolation of 160 kb of chromosomal DNA containing 70 kb of FII DNA covalently linked to more G + C rich sequences (FI DNA). Copies of three previously characterized insertion elements (ISH1, ISH2, and ISH26) as well as at least 10 other repeated sequences are clustered within this chromosomal FII DNA "island". Unique sequences are found in the FI DNA flanking the FII DNA island as well as in 40 kb of FI DNA surrounding the bacterio-opsin gene. The presence of pHH1 in H. halobium and closely related species correlates with the occurrence of the characterized chromosomal FII DNA island. Halophilic purple membrane producing isolates YC81819-9, GN101, SB3 and GRA lack pHH1 and the 70 kb FII DNA, but contain all of the FI DNA sequences tested. We propose that pHH1 and this chromosomal FII DNA are characteristic genomic components of H. halobium and closely related species, and, that the 70 kb FII DNA might represent a large insertion in the chromosome of H. halobium and closely related species. The conservation of both FI and FII DNA sequences can be used for strain classification and determination of evolutionary relationships among halo-bacteria.

Base Sequence↗
PubMed1984

Characterization of a halobacterial gene affecting bacterio-opsin gene expression.

A substantial number of spontaneous bacterio-opsin mutants of Halobacterium halobium are the result of insertion elements up to 1400 bp upstream of the bacterio-opsin (bop) gene. The nucleotide sequence of 1800 bp upstream of the bop gene has been determined. There is a 1118 bp open reading frame (ORF) located within this region which is transcribed and which coincides with the distribution of insertion elements upstream of the bop gene in Bop mutants. Therefore, we propose that there is a gene (brp gene) 526 bp upstream of the bop gene. This putative gene is transcribed in the opposite direction as the bop gene and could encode a protein of 37,500 D (359 amino acids) with a codon usage similar to bacterio-opsin. The 5' terminus of the brp transcript has been determined. The brp transcript and the bop mRNA are complementary for 13 residues near their 5' termini and both transcripts start at or near the initiating codon of the gene. Both transcripts could form similar hairpin loop structures at their 5' termini which contain possible ribosomal binding sites. The DNA sequences immediately upstream of the bop and the brp genes have significant homologies and there is a short complementary sequence. The role of the brp gene in bacterio-opsin gene expression is unclear.

Amino Acid Sequence↗
PubMed1984

Characterization of insertions affecting the expression of the bacterio-opsin gene in Halobacterium halobium.

We have determined the sequence of the inverted repeats and duplicated target DNA of the halobacterial insertion elements ISH2 (520 bp), ISH23 (900 bp) and ISH24 (3000 bp) associated with bacterio-opsin (bop) mutants. ISH2 has a perfect 19 bp inverted repeat (3,5), while both ISH23 and ISH24 have imperfect inverted repeats of 29 bp and 14 bp respectively. ISH23 was shown to be highly homologous to ISH50 (6). Variable lengths of duplicated target DNA are found when ISH2 and ISH23 (ISH50) transpose into different sites. A 550 bp DNA insert ("ISH25") reverts the Bop mutation caused by ISH24. "ISH25" lacks typical structural features of a transposable element. "ISH25" and ISH24 are found adjacent to each other upstream of the bop gene. An identical arrangement of "ISH25" and ISH24 is found in the cccDNA of H. halobium NRC817. Comparative sequence analysis of both areas suggests that the translocation of "ISH25" to the bop gene region occurred by a recombination event.

Bacteriorhodopsins↗
PubMed1981

Genetic variability in Halobacterium halobium.

Halobacterium halobium exhibits an extraordinary degree of spontaneous variability. Mutants which are defective in the formation of gas vacuoles (vac) arise at a frequency of 10(-2). Other easily detectable phenotypes, like the synthesis of bacterioruberin (Rub) or the synthesis of retinal (Ret) and bacterio-opsin (Ops), the two components which form the purple membrane (Pum) of H. halobium, are lost at a frequency of about 10(-4). With the same frequency a mutant type appears which exhibits an extremely high variability in these phenotypes. With the exception of the ret mutants, all spontaneously arising mutants show alterations, i.e., insertions, rearrangements, or deletions, in the plasmid pHH1. It appears that the introduction of one insertion into pHH1 triggers further insertions, which makes the identification of relationships between phenotypic and genotypic alterations rather difficult. From the analysis of a large number of spontaneous vac mutants and their vac+ revertants it can be concluded that the formation of the gas vacuoles is determined or controlled by plasmid genes. No such conclusion is yet possible for the rub mutants, although all mutants of this type so far analyzed exhibit a defined insertion. pum mutants which have lost the capability of forming bacterio-opsin carry insertions in the plasmid which are distributed over a rather large region of the plasmid. No strains of H. halobium could be obtained which had lost plasmid pHH1 completely.

DNA Transposable Elements↗