Physical and genetic characterization of the genome of Magnetospirillum magnetotacticum, strain MS-1.

Pulsed-field gel analysis of Magnetospirillum magnetotacticum, strain MS-1, indicates that the genome is a single, circular structure of about 4.3 mb. A few genes, identified by sequence similarity, have been localized and arranged in a map with dnaA, indicating the presumed origin of replication. There are at least two rRNA operons. In addition, rRNA genes are found on a 40 kb, possibly extrachromosomal, structure. The genes thought to be involved in magnetite synthesis, bfr and magA, are located in the same 17% of the genome. A one base pair-overlap seen in the bfr genes of MS-1 is found also in the closely related magnetic strain AMB-1, but not in the non-magnetic relative A. itersonii.

Paleoproterozoic snowball earth: extreme climatic and geochemical global change and its biological consequences.

Geological, geophysical, and geochemical data support a theory that Earth experienced several intervals of intense, global glaciation ("snowball Earth" conditions) during Precambrian time. This snowball model predicts that postglacial, greenhouse-induced warming would lead to the deposition of banded iron formations and cap carbonates. Although global glaciation would have drastically curtailed biological productivity, melting of the oceanic ice would also have induced a cyanobacterial bloom, leading to an oxygen spike in the euphotic zone and to the oxidative precipitation of iron and manganese. A Paleoproterozoic snowball Earth at 2.4 Giga-annum before present (Ga) immediately precedes the Kalahari Manganese Field in southern Africa, suggesting that this rapid and massive change in global climate was responsible for its deposition. As large quantities of O(2) are needed to precipitate this Mn, photosystem II and oxygen radical protection mechanisms must have evolved before 2.4 Ga. This geochemical event may have triggered a compensatory evolutionary branching in the Fe/Mn superoxide dismutase enzyme, providing a Paleoproterozoic calibration point for studies of molecular evolution.

Evidence for two types of subunits in the bacterioferritin of Magnetospirillum magnetotacticum.

In order to investigate the role of bacterioferritin (Bfr) in the biomineralization of magnetite by microorganisms, we have cloned and sequenced the bfr genes from M. magnetotacticum. The organism has two bfr genes that overlap by one nucleotide. Both encode putative protein products of 18 kDa, the expected size for Bfr subunits, and show a strong similarity to other Bfr subunit proteins. By scanning the DNA sequence databases, we found that a limited number of other organisms, including N. gonorrhea, P. aeruginosa, and Synechocystis PCC6803, also have two bfr genes. When the sequences of a number of microbial Bfrs are compared with each other, they fall into two distinct types with the organisms mentioned above having one of each type. Differences in heme- and metal-binding sites and ferroxidase activities of the two types of subunits are discussed.

The isolation and characterization of the gene (dfr1) encoding dihydrofolate reductase (DHFR) in Schizosaccharomyces pombe.

A sequence encoding dihydrofolate reductase (DHFR) was isolated from a Schizosaccharomyces pombe cDNA library by selecting for trimethoprim resistance in Escherichia coli. The sequence was found to be functional in both Saccharomyces cerevisiae and Sz. pombe. When present on a multicopy plasmid, it confers increased resistance to concentrations of the drug methotrexate that are otherwise inhibitory for the standard yeast strains. The sequence was mapped by DNA hybridizations between genes adh1 and ade5 on chromosome III of Sz. pombe. The 1.6-kb insert contains a 1.5-kb open reading frame (ORF) with strong sequence similarity to other described DHFR-encoding genes. The similarity, however, is limited to a 678-bp sequence, occupying the 3'-half of the ORF. No similarity to other described DNA sequences or proteins could be found for the 5'-half. Southern and Northern blots indicate that the entire insert is present intact in the Sz. pombe genome and produces a 1.7-kb RNA transcript.

Control of prophage integration and excision in bacteriophage P2: nucleotide sequences of the int gene and att sites.

Integration of bacteriophage P2 into the Escherichia coli host genome involves recombination between two specific attachment sites, attP and attB, one on the phage and the other on the host genome, respectively. The reaction is controlled by the product of the phage int gene, a basic polypeptide of about 37 kDa [Ljungquist and Bertani, Mol. Gen. Genet. 192 (1983) 87-94]. The int gene appears to be expressed differently by an infecting phage, as opposed to a prophage [Bertani, Proc. Natl. Acad. Sci. USA 65 (1970) 331-336]. A 1200-bp region of P2 DNA containing the int gene and attP, the prophage hybrid ends attL and attR, and one bacterial attachment site, the preferred site locI from E. coli strain C, have all been sequenced. An open reading frame coding for a polypeptide of 337 amino acids corresponds to the int gene. The gene has no obvious promoter sequence preceding it. The int gene transcript seems to continue past the attP site downstream from it, suggesting a possible explanation for the previously observed difference in integration and excision. A comparison of the four attachment sites reveals a common 'core' sequence of 27 bp: 5'-AAAAAATAAGCCCGTGTAAGGGAGATT-3'. The P2 nip1 mutation, which increases prophage excision [Calendar et al., Virology 47 (1972) 68-75], was found to lie within the int gene itself. The P2 saf variant, which has altered site preference [Six, Virology 29 (1966) 106-125], has a bp substitution within the core sequence. Three deletion/substitution mutants, vir22, vir94 and del3, also have altered core sequences.

DNA sequences of bacteriophage P2 early genes cox and B and their regulatory sites.

Part of the early operon of the temperate phage P2 of Escherichia coli, including genes cox (involved in prophage excision) and B (required for phage specific DNA synthesis), was sequenced. The results are consistent with an early promoter spanning the repressor binding sites, a leader sequence of about 80 bases which overlaps the leader sequence of the repressor gene for about 30 bases, and coordinate transcription of genes cox and B with a termination signal after the B gene. In addition, the data provide amino acid sequences for the Cox and B proteins of 91 and 166 residues, respectively and reveal a hitherto undetected coding sequence between genes cox and B that has the potential to produce a very basic polypeptide of 56 residues. Slight structural similarities between the P2 Cox protein and the analogous Xis protein of phage lambda were noted and the P2 B gene product was compared with proteins that interact with the DnaB protein of E. coli.

DNA sequences of the repressor gene and operator region of bacteriophage P2.

The nucleotide sequence of the repressor gene C of the temperate phage P2 has been determined. It codes for a nonbasic polypeptide, 99 amino acids long. Twelve repressor-defective mutants have been mapped. All but one are located within the presumed coding part of the gene. There is a strong promoter sequence and an 8-base-pair inverted repeat preceding the gene. The P2 repressor protein shows structural similarity to other DNA-binding proteins. The operator region for the early replication functions was located by sequencing the DNA of three virulent mutants. The sequence indicates that there are two repressor-binding sites. In addition, one of the sites shows sequence homology with part of the operator region of the biotin operon of Escherichia coli.

Properties and products of the cloned int gene of bacteriophage P2.

Fragments of DNA of the temperate phage P2, generated by treatment with the restriction enzyme PstI, have been cloned into the plasmid pBR322. One such fragment, which has its endpoints within phage genes T and C, carries the structural P2 int gene as well as its promoter and the phage att site. When introduced into a suitable bacterial host, the cloned fragment mediates the integration and excision of int- mutants of P2 and recombination within the phage att site in mixed infection. All these activities are independent of the orientation of the fragment within the plasmid. When introduced into minicells, the fragment produces, in addition to the products of genes D and U, a protein of 35-37,000 daltons identified as the int protein. A study of the map location of two amber int mutants, together with the sizes of the polypeptides they produce, indicates that the P2 int gene is transcribed from right to left on the P2 map, i.e. starting near gene C and proceeding toward att.

Constitutive expression of bacteriophage P2 early genes resulting from a tandem duplication.

A tandem-duplication mutant of bacteriophage P2 was isolated. Physically, its particles are characterized by a higher buoyant density and lower heat stability than the wild type, both consequences of increased DNA content. Genetically, the mutant is easily recognized by its insensitivity to control by the immunity-specific repressor. The duplication covers part of gene B, necessary for phage DNA replication. To explain the immunity-insensitivity of the duplication it is proposed that the promoter, but not the operator site, in the early gene operon is duplicated in this mutant. By crosses with a gene-B mutant, a recombinant carrying a heterozygous duplication was isolated.

Split-operon control of a prophage gene.

Both prophage integration in bacteriophage P2 and the reverse event, prophage excision, are known to require a specific phage gene product, the so-called int function. We find that P2 can integrate efficiently at a free attachment site also in an immune host (i.e., in the presence of phage specific repressor) provided the superinfecting phage is not deficient in int function. Prophage P2, on the other hand, is not excised from the host chromosome even in a derepressed lysogen unless int function is supplied by a superinfecting phage. Thus, the int function of P2 is expressed constitutively by the superinfecting phage, but is not expressed by the prophage even in the absence of phage repressor. It is proposed that the int function of P2 is not controlled by phage repressor, but belongs instead to a constitutive operon that is physically disrupted by prophage integration.