Shiladitya DasSarma

Is gene expression in Halobacterium NRC-1 regulated by multiple TBP and TFB transcription factors?

Sir, Gene expression is regulated by different mechanisms in different organisms. The bacterial core RNA polymerase (a2bb 0) discriminates between subsets of promoters by binding different s factors. Eukaryotes have evolved a more complicated system making use of three RNA polymerases to direct synthesis from different promoter families. Archaea possess a simplified version of RNA polymerase II transcription machinery with a single multisubunit RNA polymerase and a subset, TBP and TFIIB, of general transcription factors (Reeve et al., 1997, Cell 89: 999±1002). However, multiple transcription factor homologues have been identified in several archaea including Halobacterium NRC-1 (Ng et al., 1998, Genome Res 8: 1131±1141), Haloferax volcanii (Thompson et al., 1999, Mol Microbiol 33: 1081±1092) and Pyrococcus horikoshii OT3 (Kawarabayasi et al., 1998, DNA Res 5: 147±155). With the impending completion of the Halobacterium NRC-1 genome project, this extreme halophile is turning out to be a champion of multiple transcription factors, with six tbp and seven tfb genes (http://zdna.micro. umass.edu/haloweb).

A plasmid‐encoded gas vesicle protein gene in a halophilic archaebacterium

The halophilic archaebacterium, Halobacterium halobium, displays spontaneous and revertible genetic variability for the gas vesicle phenotype (Vac) at frequencies as high as 0.5 to 5%. To investigate the mechanism of these high‐frequency mutations, we have cloned a gas vesicle protein gene (gvpA) from the Vac+ wild‐type H. halobium strain, NRC‐1, and determined its nucleotide sequence, transcription start site, and genomic location. The gene sequence predicts that the gas vesicle protein has a molecular weight of 9156 and is relatively hydrophobic except for a hydrophilic C‐terminal region. Northern hybridization analysis shows that the gene is transcribed into a 350‐nucleotide mRNA, and primer extension analysis indicates that transcription begins 20 nucleotides upstream of the ATG start codon. Southern hybridization analysis shows that the gene is encoded by a large H. halobium plasmid. We discuss potential mechanisms for genetic variability of the Vac phenotype and identify sequences in the gvpA promoter region which may function as signals for transcription in H. halobium.

Structure and organization of the gas vesicle gene cluster on the Halobacterium halobium plasmid pNRC100

Halobacterium halobium strain NRC-1 contains intracellular gas-filled vesicles (GVs) that confer buoyancy to the cells. Cloning of the major GV protein (GvpA)-encoding gene, gvpA, and analysis of GV-deficient mutants (Vac-) of H. halobium led to the identification of a region of a 200-kb plasmid, pNRC100, important for GV synthesis. We report here the nucleotide sequence of an 8520-bp region which, including gvpA, contains twelve open reading frames (ORFs) that are organized into two divergent transcription units, gvpAC oriented rightward, and gvpD, E, F, G, H, I, J, K, L, and M located upstream from gvpAC and oriented leftward. Insertions into the gvpA promoter and gvpD and E resulted in the Vac- phenotype. The overall gene organization is highly compact with the end of one ORF overlapping with the beginning of the next in most cases. The gene cluster is bracketed by two ISH8 element copies in inverted orientation, an organization suggestive of a composite transposon. Comparison of predicted amino acid sequences showed homology between GvpA, and the gvpJ and gvpM putative gene products. The putative gvpC gene product contains eight copies of an imperfectly repeated sequence with similarity to repeats in a cyanobacterial GvpC plus a highly acidic C-terminal region not found in the cyanobacterial homologue.

Antigen presentation using novel particulate organelles from halophilic archaea

A presentation vehicle was developed based on particulate gas vesicles produced by halophilic archaea. Gas vesicle epitope displays were prepared using standard coupling methods or recombinant DNA technology. When presented in the context of gas vesicle preparations, either the hapten, TNP, or a model six amino acid recombinant insert in the outer gas vesicle protein, GvpC was rendered immunogenic. Assays to quantify humoral responses indicated that each preparation elicited strong antibody responses in the absence of exogenous adjuvant. Thus, each preparation elicited a humoral response when injected into mice and this response was long lived and exhibited immunologic memory. Recombinant gas vesicle preparations therefore constitute a new, self-adjuvanting carrier/display vehicle for presentation of an array of peptidyl epitopes.

Saturation mutagenesis of the haloarchaeal bop gene promoter: identification of DNA supercoiling sensitivity sites and absence of TFB recognition element and UAS enhancer activity

Transcription from the bop promoter in the haloarchaeon Halobacterium NRC‐1, is highly induced under oxygen‐limiting conditions. A DNA gyrase inhibitor, novobiocin, was previously shown to block bop gene induction and suggested that DNA supercoiling mediates transcriptional induction. A region of non‐B structure was found 3′ to the TATA box within an 11 bp alternating purine–pyrimidine sequence (RY box), which correlated to both increased DNA supercoiling and transcriptional induction. Here, saturation mutagenesis of the RY box region has been used to show that single‐base substitutions of A(r)G either 23 or 19 bp 5′ to the transcription start site temper the effect of DNA supercoiling based on novobiocin insensitivity of transcription. Mutagenesis of the region 5′ to the TATA box showed its involvement in DNA supercoiling modulation of transcription, defined the 3′ end of the upstream activator sequence (UAS) regulatory element, and ruled out the requirement for a TFB (TFIIB) Recognition Element. Spacing between the TATA box and UAS was found to be critical for promoter activity because insertion of partial or whole helical turns between the two elements completely inhibited transcription indicating that the UAS element does not function as a transcriptional enhancer. The results are discussed in the context of DNA melting and flexibility around the TATA box region and the involvement of multiple regulatory and transcription factors in bop promoter activity.

Genetic transformation of a halophilic archaebacterium with a gas vesicle gene cluster restores its ability to float

The halophilic archaebacterium, Halobacterium halobium, and many other aquatic bacteria synthesize gas-filled vesicles for flotation. We recently identified a cluster of 13 genes (gvpMLKJIHGFEDACN) on a 200-kb H. halobium plasmid, pNRC100, involved in gas vesicle synthesis. We have cloned and reconstructed the gvp gene cluster on an H. halobium-E. coli shuttle plasmid. Transformation of H. halobium Vac- mutants lacking the entire gas vesicle gene region with the gvp gene cluster results in restoration of their ability to float. These results open the way toward further genetic analysis of gas vesicle gene functions and directed flotation of other microorganisms with potential biotechnological applications.

Large Deletions in Class III Gas Vesicle-Deficient Mutants of Halobacterium halobium

Summary Mutants of Halobacterium halobium deficient in production of gas vesicles (Vac - and Vac δ- ) arise spontaneously at an extremely high frequency, approximately 1%. In this study, we characterized one class of mutants (class III) with a stable and completely Vac - phenotype. Restriction mapping and Southern analysis of four class III mutants, SD109, SD112A, SD116, and SD118A, demonstrated that the mutants were deleted for the gas vesicle gene cluster and flanking sequences in the 200-kb gas vesicle plasmid, pNRC 100. Deletions resulted from recombination between pNRC 100 fragments Hin dIII-C and Hin dIII-D″ (for SD109, SD112A, and SD116) or Hin dIII-C and an unidentified sequence (for SD118A). In at least three cases, the deletion junctions mapped precisely to the termini of IS elements, IS H2 (for SD112A) and IS H8 (for SD109 and SD118A). These results suggest that deletion formation is a consequence of intramolecular transposition of IS elements, although a multistep mechanism involving recombination between two elements is also possible. Interestingly, a Vac + revenant (SD142R) of a class III mutant (SD142) was isolated from a sectored colony. Southern analysis indicated that SD142 had a deletion of the gvp gene cluster and that SD142R had regained the gas vesicle gene cluster by an unknown mechanism.

Analysis of Left-handed Z-DNA Formation in Short d(CG) Sequences in Escherichia coli and Halobacterium halobium Plasmids STABILIZATION BY INCREASING REPEAT LENGTH AND DNA SUPERCOILING BUT NOT SALINITY

To evaluate the relative importance of alternating d(CG) sequence length, DNA supercoiling, and salt in left-handed Z-DNA formation, plasmids containing short d(CG)sequences (n = 3-17) with the capability of replicating in either Escherichia coli or the halophilic archaeum Halobacterium halobium were constructed. Z-DNA conformation in the d(CG) sequences was assayed by (i) a band shift assay using the Z-DNA-specific Z22 monoclonal antibody (ZIBS assay); (ii) an S1 nuclease cleavage-primer extension assay to map B-Z junctions; and (iii) a BssHII restriction inhibition assay. Using the ZIBS assay on plasmids purified from E. coli, the transition from B-DNA to Z-DNA occurred from d(CG) to d(CG), with 20% of d(CG) and 90% of d(CG) in Z-DNA conformation. These findings were consistent with the results of S1 nuclease cleavage observed at B-Z junctions flanking d(CG) and d(CG) sequences. Resistance to BssHII restriction endonuclease digestion was observed only in supercoiled plasmids containing d(CG) or longer sequences, indicating that shorter d(CG) sequences are in dynamic equilibrium between B- and Z-DNA conformations. When a plasmid containing d(CG) was isolated from a topA mutant of E. coli, it contained 25% greater linking deficiency and 40% greater Z-DNA conformation in the alternating d(CG) region. In plasmids purified from H. halobium, which showed 30% greater linking deficiency than from E. coli, 20-40% greater Z-DNA formation was found in d(CG) sequences. Surprisingly, no significant difference in Z-DNA formation could be detected in d(CG) sequences in plasmids from either E. coli or H. halobium in the NaCl concentration range of 0.1-4 M.

ISOLATION AND CHROMOSOMAL DISTRIBUTION OF NATURAL Z-DNA-FORMING SEQUENCES IN HALOBACTERIUM HALOBIUM

Conditions favoring left-handed Z-DNA such as high salinity (> 4 M), high negative DNA supercoiling, and GC-rich DNA [statistically favoring d(CG)n repeat sequences], are all found in the extremely halophilic archaeum (archaebacterium) Halobacterium halobium. In order to identify and study Z-DNA regions of the H. halobium genome, an affinity chromatography method with high Z-DNA selection efficiency was developed. Supercoiled plasmids were incubated with a Z-DNA-specific antibody (Z22) and passed over a protein A-agarose column, and the bound plasmids were eluted using an ethidium bromide gradient. In control experiments using mixtures of pUC12 (Z-negative) and a d(CG)5-containing (Z-positive) pUC12 derivative, up to 4,000-fold enrichment of the Z-DNA-containing plasmid was demonstrated per cycle of the Z-DNA selection procedure. The selection efficiency was determined by transformation of Escherichia coli DH5α with eluted plasmids and blue-white screening on X-gal plates. Twenty recombinant plasmids containing Z-DNA-forming sequences of H. halobium were isolated from a genomic library using affinity chromatography. Z-DNA-forming sequences in selected plasmids were identified by bandshift and antibody footprinting assays using Z22 monoclonal antibody. Alternating purine-pyrimidine sequences ranging from 8 base pairs (bp) to 13 bp with at least a 6-bp alternating d(GC) stretch were found in the Z22 antibody binding regions of isolated plasmids. The distribution of Z-DNA-forming sequences in the Halobacterium salinarum GRB chromosome was analyzed by dot-blot hybridization of an ordered cosmid library using the cloned H. halobium Z-DNA segments as probe. Among the 11 Z-DNA segments tested, five were found to be clustered in a 100-kilobase pair region of the genome, whereas six others were distributed throughout the rest of the genome.

Restriction Mapping the Genome of Halobacterium halobium Strain NRC-1

Summary To study DNA rearrangements in H. halobium , the complete genome of strain NRC-1 is being restriction mapped. As a first step, we have used pulsed-field gel electrophoresis and enzymes that cut infrequently. Pmel cuts the genome into six fragments. Two of these fragments correspond to the plasmid pNRC100 characterized previously. NRC-1 also has a 400 Kbp plasmid, pNRC200, which is closely related to pNRC100 but is uncut by PmeI. This leaves four PmeI fragments of total size 1,800 Kbp which constitute the chromosome. The chromosome is being mapped by three enzymes which cut infrequently, AflII, AseI and DraI. Southern blotting with junction clones and purified restriction fragments was of limited value because the ubiquitous ISH elements resulted in hybridization to many fragments not necessarily related. Blotting with anonymous unique probes was more helpful. But most information has been obtained from 2D gel electrophoresis using pairs of the enzymes. On the basis of these data, four contigs have been assembled which represent most of the chromosome. Their relationship is being determined.