Structure of the gas vesicle plasmid in Halobacterium halobium: inversion isomers, inverted repeats, and insertion sequences

Ng, Wai Lap; Kothakota, Srinivas; DasSarma, Shiladitya

doi:10.1128/jb.173.6.1958-1964.1991

Cited by 54 publications

(37 citation statements)

References 31 publications

(41 reference statements)

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“…The minichromosomes share a 145-kb region of identity within which exist 2 copies of 33-37-kb inverted repeats. The majority (69 of 91) of IS elements were also localized to the minichromosomes, which, along with the other genomic repeats, accounts for the dynamic nature of the genome (Charlebois and Doolittle 1989;Ng et al 1991;Hackett et al 1994). Altogether, 14% of the genome, including the 91 IS elements, 33-37-kb inverted repeats, and 145-kb duplication in pNRC100 and pNRC200, is repeated DNA.…”

mentioning

confidence: 99%

Understanding the Adaptation of Halobacterium Species NRC-1 to Its Extreme Environment through Computational Analysis of Its Genome Sequence

Kennedy¹,

Ng²,

Salzberg³

et al. 2001

Genome Res.

303

267

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The genome of the halophilic archaeon Halobacterium sp. NRC-1 and predicted proteome have been analyzed by computational methods and reveal characteristics relevant to life in an extreme environment distinguished by hypersalinity and high solar radiation: (1) The proteome is highly acidic, with a median pI of 4.9 and mostly lacking basic proteins. This characteristic correlates with high surface negative charge, determined through homology modeling, as the major adaptive mechanism of halophilic proteins to function in nearly saturating salinity. (2) Codon usage displays the expected GC bias in the wobble position and is consistent with a highly acidic proteome. (3) Distinct genomic domains of NRC-1 with bacterial character are apparent by whole proteome BLAST analysis, including two gene clusters coding for a bacterial-type aerobic respiratory chain. This result indicates that the capacity of halophiles for aerobic respiration may have been acquired through lateral gene transfer. (4) Two regions of the large chromosome were found with relatively lower GC composition and overrepresentation of IS elements, similar to the minichromosomes. These IS-element-rich regions of the genome may serve to exchange DNA between the three replicons and promote genome evolution. (5) GC-skew analysis showed evidence for the existence of two replication origins in the large chromosome. This finding and the occurrence of multiple chromosomes indicate a dynamic genome organization with eukaryotic character.

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mentioning

confidence: 99%

Understanding the Adaptation of Halobacterium Species NRC-1 to Its Extreme Environment through Computational Analysis of Its Genome Sequence

Kennedy¹,

Ng²,

Salzberg³

et al. 2001

Genome Res.

303

267

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“…The H. halobium gvpA gene was found to map to a 200-kb plasmid, pNRC100, which suffers rearrangements, such as insertions, deletions, and inversions, at a high frequency. The instability of pNRC100 results from the recombinational activity promoted by many repeated elements, including at least 17 copies of well-characterized insertion sequence (IS) elements and a pair of very large (-35-kb) inverted repeat sequences that mediate the inversion of the intervening single-copy regions (21). About 12 Vac-mutants were characterized in detail and shown to have either insertions or deletions of the gvpA gene region ( Fig.…”

mentioning

confidence: 99%

The rightward gas vesicle operon in Halobacterium plasmid pNRC100: identification of the gvpA and gvpC gene products by use of antibody probes and genetic analysis of the region downstream of gvpC

et al. 1993

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The extreme halophile Halobacterium halobium synthesizes intracellular gas-filled vesicles that confer buoyancy. A cluster of 13 genes on the 200-kb endogenous plasmid pNRC100 has been implicated in the biosynthesis of gas vesicles. Here, we show that two gas vesicle proteins are encoded by genes in the rightward operon, gvpA and gvpC, by Western blotting (immunoblotting) analysis with antibodies directed against LacZ-GvpA and LacZ-GvpC fusion proteins. Our results are consistent with previous data showing that the gvpA gene product is the major gas vesicle protein and demonstrate for the first time that thegvpC gene product is also present in H. halobium gas vesicles. Northern (RNA) blotting analysis showed two RNA species, an abundant 0.35-kb transcript of gvpA and a minor 2.5-kb transcript of gvpAC, and a third gene 3' to gvpAC, named gvpN. The gvpN gene encodes a hypothetical acidic protein with a molecular weight of 39,000 and a nucleotide binding motif. We used a site-directed mutagenesis method involving double recombination in Escherichia coli to insert a kanamycin resistance cassette just beyond the stop codon of gvpN. Introduction of the mutated gene cluster into an H. halobium mutant with a deletion of the entire gas vesicle gene cluster resulted in gas vesicle-positive transformants; this result suggests that gvpN is the last gene of the rightward gas vesicle transcription unit. We discuss the design and utility of the kanamycin resistance cassette for the mutagenesis of other genes in large operons.Many aquatic bacteria, such as the extreme halophile Halobacterium halobium, produce intracellular gas-filled vesicles that provide buoyancy and allow cells to float at the surface of liquid cultures (3, 27). The vesicles contain ambient gas and are surrounded by a rigid membrane composed of protein with little or no lipids. The vesicle shape is generally cylindrical in the midsection and conical at the ends. There are two types of gas vesicles in wild-type H. halobium NRC-1; the vast majority (99%) are lemon or spindle shaped, and a few (1%) are more elongated or cylindrical. Studies on cyanobacterial vesicles have suggested that vesicle biosynthesis is initiated with the cones and proceeds by the addition of protein subunits to the central cylindrical region (18,31). The accumulation of gases within vesicles is thought to result from the differential permeability of the membrane to gases and water (34).Our initial interest in gas vesicles resulted from the observation that H. halobium gas vesicle-deficient (Vac-) mutants arise at an extremely high frequency, about 1%. Cloning of the first gas vesicle protein gene, gvpA, opened the way to an investigation of the genetic basis of the mutation (6, 29). The H. halobium gvpA gene was found to map to a 200-kb plasmid, pNRC100, which suffers rearrangements, such as insertions, deletions, and inversions, at a high frequency.

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“…Furthermore, a gas-vesicle protein (ORF H0698) that was detected in both large inverted repeats identified on the Halobacterium sp. plasmid pNRC 100 (Ng et al, 1991) revealed identity to the putative ISBst12 transposase. These two large inverted repeats were found to mediate inversion of the intervening single-copy region (Ng et al, 1991).…”

Section: Discussionmentioning

confidence: 99%

“…plasmid pNRC 100 (Ng et al, 1991) revealed identity to the putative ISBst12 transposase. These two large inverted repeats were found to mediate inversion of the intervening single-copy region (Ng et al, 1991). The transposase encoded by ISBst12 showed identity to a putative transposase encoded by IS22-1 of V. cholerae as well as to two members of the IS66 family, namely to a protein encoded by IS66 of A. tumefaciens and to a protein encoded by ISRm14 of S. meliloti.…”

Section: Discussionmentioning

confidence: 99%

“…However, by using the  program (Altschul et al, 1997), the predicted amino acid sequence of the ORF encoded by ISBst12 revealed sequence identities to a putative transposase of Deinococcus radiodurans , a gasvesicle protein (ORF H0698) encoded by the Halobacterium sp. plasmid pNRC 100 (Ng et al, 1991), a putative transposase encoded by IS22-1 of Vibrio cholerae (Yamasaki et al, 1999), a protein encoded by IS66 found in the T-DNA region of the mutant Tiplasmid pTiA66 of Agrobacterium tumefaciens (IS66 family) (Machida et al, 1984) and a protein encoded by ISRm14 of Sinorhizobium meliloti (Schneiker et al, 1999) (see Table 2). The results obtained by sequence comparison supported that the ORF of ISBst12 encodes a putative bacterial transposase.…”

Section: Characterization Of the Putative Transposase Encoded By Isbst12mentioning

confidence: 99%

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ISBst12, a novel type of insertion-sequence element causing loss of S-layer-gene expression in Bacillus stearothermophilus ATCC 12980 The GenBank accession number for the sequence reported in this paper is AF162268.

et al. 2000

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The cell surface of the surface layer (S-layer)-carrying strain of Bacillus stearothermophilus ATCC 12980 is completely covered with an oblique lattice composed of the S-layer protein SbsC. In the S-layer-deficient strain, the S-layer gene sbsC was still present but was interrupted by a novel type of insertion sequence (IS) element designated ISBst12. motifs were identified at the N-terminal part of the putative transposase. As revealed by Southern blotting, ISBst12 was present in multiple copies in the Slayer-deficient strain as well as in the S-layer-carrying strain. Northern blotting indicated that S-layer gene expression is already inhibited at the transcriptional level, since no sbsC-specific transcript could be identified in the S-layer-deficient strain. By using PCR, ISBst12 was also detected in B. stearothermophilus PV72/p6, in its oxygen-induced strain variant PV72/p2 and in the S-layer-deficient strain PV72/T5.

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Structure of the gas vesicle plasmid in Halobacterium halobium: inversion isomers, inverted repeats, and insertion sequences

Cited by 54 publications

References 31 publications

Understanding the Adaptation of Halobacterium Species NRC-1 to Its Extreme Environment through Computational Analysis of Its Genome Sequence

Understanding the Adaptation of Halobacterium Species NRC-1 to Its Extreme Environment through Computational Analysis of Its Genome Sequence

The rightward gas vesicle operon in Halobacterium plasmid pNRC100: identification of the gvpA and gvpC gene products by use of antibody probes and genetic analysis of the region downstream of gvpC

ISBst12, a novel type of insertion-sequence element causing loss of S-layer-gene expression in Bacillus stearothermophilus ATCC 12980 The GenBank accession number for the sequence reported in this paper is AF162268.

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