Characterization of bromoethanesulfonate-resistant mutants of Methanococcus voltae: evidence of a coenzyme M transport system.

Mutants of Methanococcus voltae were isolated that were resistant to the coenzyme M (CoM; 2-mercaptoethanesulfonic acid) analog 2-bromoethanesulfonic acid (BES). The mutants displayed a reduced ability to accumulate [35S]BES relative to the sensitive parental strain. BES inhibited methane production from CH3-S-CoM in cell extracts prepared from wild-type sensitive or resistant strains. BES uptake required the presence of both CO2 and H2 and was inhibited by N-ethylmaleimide and several reagents that are known to disrupt energy metabolism. The mutants showed normal uptake of isoleucine and were not cross-resistant to either azaserine or 5-methyltryptophan and, thus, were neither defective in general energy-dependent substrate transport nor envelope permeability. Both HS-CoM and CH3-S-CoM prevented the uptake of BES and protected cells from inhibition by it. We propose that M. voltae has an energy-dependent, carrier-mediated uptake system for HS-CoM and CH3-S-CoM which can also mediate uptake of BES.     

Isolation of a coenzyme M-auxotrophic mutant and transformation by electroporation in Methanococcus voltae.

An auxotrophic mutant of Methanococcus voltae was isolated that required coenzyme M (CoM) for growth. With the mutant as a recipient, conditions were developed that allowed the introduction of wild-type CoM+ DNA into the mutant methanogen via electroporation. This method also allowed the rescue of both a histidine and purine auxotroph as well as the introduction of DNA determining resistance to the CoM analog 2-bromoethanesulfonic acid. Electroporation of the CoM(+)-determining DNA was 50- to 80-fold more efficient than natural transformation.     

Isolation and characterization of an archaebacterial viruslike particle from Methanococcus voltae A3.

Small amounts of a 23-kilobase covalently closed circular DNA molecule were isolated from unwashed cells of Methanococcus voltae A3. Further investigation indicated the presence of greater quantities of the circular DNA in the culture supernatant, complexed with protein in a manner rendering the DNA resistant to DNase. Electron-microscopic examination of supernatant material revealed the presence of particles which morphologically resemble virus. Phenol extraction of viruslike particle preparations resulted in the recovery of DNase-sensitive open-circular DNA molecules. As many as 30 viruslike particles per cell were recovered from some cultures. Hybridization data clearly indicated the presence of a chromosomally integrated copy of the viruslike particle DNA. Although M. voltae PS was not observed to produce viruslike particles, DNA homologous to the viruslike particle DNA was detected in its chromosome. A mutant of M. voltae A3 was isolated which produced no particles; its DNA was deleted for 80% of the integrated viruslike particle DNA. Despite any similarities to lysogenic bacteriophages of eubacteria, neither infectivity nor inducibility of the viruslike particles could be demonstrated.     

Fine mapping and molecular cloning of mutations in the herpes simplex virus DNA polymerase locus.

Mutations in five phenotypically distinct mutants derived from herpes simplex virus type 1 strain KOS which lie in or near the herpes simplex virus DNA polymerase (pol) locus have been fine mapped with the aid of cloned fragments of mutant and wild-type viral DNAs to distinct restriction fragments of 1.1 kilobase pairs (kbp) or less. DNA sequences containing a mutation or mutations conferring resistance to the antiviral drugs phosphonoacetic acid, acyclovir, and arabinosyladenine of pol mutant PAAr5 have been cloned as a 27-kbp Bg+II fragment in Escherichia coli. These drug resistance markers have been mapped more finely in marker transfer experiments to a 1.1-kbp fragment (coordinates 0.427 to 0.434). In intratypic marker rescue experiments, temperature-sensitive (ts), phosphonoacetic acid resistance, and acyclovir resistance markers of pol mutant tsD9 were mapped to a 0.8-kbp fragment at the left end of the EcoRI M fragment (coordinates 0.422 to 0.427). The ts mutation of pol mutant tsC4 maps within a 0.3-kbp sequence (coordinates 0.420 to 0.422), whereas that of tsC7 lies within the 1.1-kbp fragment immediately to the left (coordinates 0.413 to 0.420). tsC4 displays the novel phenotype of hypersensitivity to phosphonoacetic acid; however, the phosphonoacetic acid hypersensitivity phenotype is almost certainly not due to the mutation(s) conferring temperature sensitivity. The ts mutation of mutant tsN20--which does not affect DNA polymerase activity--maps to a 0.5-kbp fragment at the right-hand end of the EcoRI M fragment (coordinates 0.445 to 0.448). The mapping of the mutations in these five mutants further defines the limits of the pol locus and separates mutations differentially affecting catalytic functions of the polymerase.     

Transport of coenzyme M (2-mercaptoethanesulfonic acid) and methylcoenzyme M [(2-methylthio)ethanesulfonic acid] in Methanococcus voltae: identification of specific and general uptake systems.

A transport system for coenzyme M (2-mercaptoethanesulfonic acid [HS-CoM]) and methylcoenzyme M [(2-(methylthio)ethanesulfonic acid (CH3-S-CoM)] in Methanococcus voltae required energy, showed saturation kinetics, and concentrated both forms of coenzyme M against a concentration gradient. Transport required hydrogen and carbon dioxide for maximal uptake. CH3-S-CoM uptake was inhibited by N-ethylmaleimide and monensin. Both HS-CoM and CH3-S-CoM uptake showed sodium dependence. In wild-type M. voltae, HS-CoM uptake was concentration dependent, with a Vmax of 960 pmol/min per mg of protein and an apparent Km of 61 microM. Uptake of CH3-S-CoM showed a Vmax of 88 pmol/min per mg of protein and a Km of 53 microM. A mutant of M. voltae resistant to the coenzyme M analog 2-bromoethanesulfonic acid (BES) showed no uptake of CH3-S-CoM but accumulated HS-CoM at the wild-type rate. While the higher-affinity uptake system was specific for HS-CoM, the lower-affinity system mediated uptake of HS-CoM, CH3-S-CoM, and BES. Analysis of the intracellular coenzyme M pools in metabolizing cells showed an intracellular HS-CoM concentration of 14.8 mM and CH3-S-CoM concentration of 0.21 mM.     

Characterization of the histidine decarboxylase gene of Staphylococcus epidermidis TYH1 coded on the staphylococcal cassette chromosome

Histamine production from histidine in fermented food results in food spoilage, and is harmful to consumers. From fish-miso, we have isolated a new bacterial strain Staphylococcus epidermidis TYH1, which produced histamine under acidic condition in the medium supplemented with glucose. Using oligonucleotides deduced from the histidine decarboxylase gene (hdcA) of Lactobacillus hilgardii, about 14-kbp DNA region of the TYH1 genome was cloned and sequenced. This region contained two putative genes hdcA(TYH1) and hdcP(TYH1) encoding proteins HdcA(TYH1) (310 amino acid residues) and HdcP(TYH1) (495 residues), respectively. Nucleotide sequence around this hdc cluster showed similarity to SCCpbp4 region of S. epidermidis ATCC 12228. Downstream of the cluster, ccrA, ccrB (Type II, respectively) and pbp4 were located. The CcrA and CcrB proteins catalyzed excision of the hdc cluster from the TYH1 chromosome, upon introduction into the TYH1 strain via multicopy plasmid. When hdcA(TYH1) was expressed in Staphylococcus warneri M, histamine was extracellularly accumulated in dependence on exogenous histidine. These results indicate that the gene encoding a histidine decarboxylase resides in a movable genetic element, SCC. This new element is designated as SCChdc.     

Palindromic structure and polypeptide expression of 36 kilobase pairs of heterogeneous Epstein-Barr virus (P3HR-1) DNA.

Among the Epstein-Barr virions (EBV) produced by the P3HR-1 (HR-1) cell line are a defective subpopulation with rearranged viral DNA designated heterogeneous DNA (het DNA). These defective virions are responsible for the capacity of HR-1 virus to induce early antigen in Raji c cells and for trans activation of latent EBV in X50-7 cells. Virions with het DNA are independent replicons which pass horizontally from cell to cell rather than being partitioned vertically. We analyzed the structure and defined several polypeptide products of het DNA to understand these remarkable biologic properties. A 36-kilobase-pair (kbp) stretch of het DNA was cloned (as two EcoRI fragments of 20 and 16 kbp) from virions released from a cellular subclone of HR-1 cells. The unusual aspect of the 20-kbp fragment was the linkage of sequences of BamHI-M and BamHI-B', which are not adjacent on the standard EBV genome. The 16-kbp fragment was a palindrome in which at least two additional recombinations on each side of the palindrome had linked regions of the standard EBV genome which are not normally contiguous. The 20-kbp het DNA fragment was attached to at least one and possibly both ends of the 16-kbp het DNA fragment. We identified antigenic polypeptides produced in COS-1 cells after gene transfer of various cloned het DNA fragments. The 20-kbp fragment encoded a cytoplasmic antigen of about 95 kilodaltons (kDa). The 16-kbp fragment encoded antigens located in the nucleus, nuclear membrane, and cytoplasm. These were represented by several polypeptides, the most prominent of which were about 55, 52, and 36 kDa. The 36-kDa polypeptide was localized to a 2.7-kbp BamHI fragment which had homology to standard BamHI-W and BamHI-Z. Another polypeptide of 50 kDa found in the nucleus was mapped to the 7.1-kbp BamHI het DNA fragment which spans the EcoRI site linking the 20- and 16-kbp fragments of het DNA. Thus, HR-1 het DNA encodes several discrete polypeptide products, one or more of which could be responsible for the unusual biologic properties of the virus. The composition, regulation, and ultimately the expression of some of these products relative to standard EBV is probably altered by the genomic rearrangements of het DNA.     

Excretion of amino acids by 1,2,4-triazole-3-alanine-resistant mutants of Methanococcus voltae.

In contrast to wild-type cells, it was found that triazole-alanine-resistant mutants of Methanococcus voltae excreted histidine, proline, phenylalanine, and tyrosine in various combinations. These results suggest that a form of general amino acid biosynthetic control may operate in this methanogen. We also show that wild-type M. voltae excretes methionine.     

Analysis of drug resistance in the archaebacterium Methanococcus voltae with respect to potential use in genetic engineering

The sensitivity of the methanogenic archaebacterium Methanococcus voltae to 12 inhibitors was tested in liquid medium. Four compounds appeared to be inhibitors of growth. Their MICs were as follows: pseudomonic acid, 0.1 micrograms/ml (0.19 microM); puromycin, 2 micrograms/ml (3.6 microM); methionine sulfoximine, 30 micrograms/ml (170 microM); and fusidic acid, 100 micrograms/ml (170 microM). On solid medium, the MICs were similar and the frequency of spontaneous resistance was found to be 5 X 10(-5) (methionine sulfoximine), 10(-7) (pseudomonic acid), and less than 10(-7) (puromycin and fusidic acid). Pseudomonic acid was found to inhibit isoleucyl-tRNA synthetase activity as measured by the in vitro aminoacylation of M. voltae tRNA with L-[U-14C]isoleucine. Fusidic acid and puromycin were shown to inhibit poly(U)-dependent polyphenylalanine synthesis in S30 extracts. Acetylpuromycin was inhibitory at much higher concentrations both in vivo and in vitro for M. voltae. Thus, the pac gene of Streptomyces alboniger, which is responsible for acetylation of puromycin and which conferred resistance to puromycin when introduced in eubacteria and eucaryotes, is a potential selective marker in gene transfer experiments with M. voltae. The latter was recently shown to be transformable. The same would be true for the cat gene of Tn9, which encodes resistance to fusidic acid in eubacteria in addition to resistance to chloramphenicol.     

Excretion of amino acids by 1,2,4-triazole-3-alanine-resistant mutants of Methanococcus voltae

In contrast to wild-type cells, it was found that triazole-alanine-resistant mutants of Methanococcus voltae excreted histidine, proline, phenylalanine, and tyrosine in various combinations. These results suggest that a form of general amino acid biosynthetic control may operate in this methanogen. We also show that wild-type M. voltae excretes methionine.     

Analysis of drug resistance in the archaebacterium Methanococcus voltae with respect to potential use in genetic engineering.

The sensitivity of the methanogenic archaebacterium Methanococcus voltae to 12 inhibitors was tested in liquid medium. Four compounds appeared to be inhibitors of growth. Their MICs were as follows: pseudomonic acid, 0.1 micrograms/ml (0.19 microM); puromycin, 2 micrograms/ml (3.6 microM); methionine sulfoximine, 30 micrograms/ml (170 microM); and fusidic acid, 100 micrograms/ml (170 microM). On solid medium, the MICs were similar and the frequency of spontaneous resistance was found to be 5 X 10(-5) (methionine sulfoximine), 10(-7) (pseudomonic acid), and less than 10(-7) (puromycin and fusidic acid). Pseudomonic acid was found to inhibit isoleucyl-tRNA synthetase activity as measured by the in vitro aminoacylation of M. voltae tRNA with L-[U-14C]isoleucine. Fusidic acid and puromycin were shown to inhibit poly(U)-dependent polyphenylalanine synthesis in S30 extracts. Acetylpuromycin was inhibitory at much higher concentrations both in vivo and in vitro for M. voltae. Thus, the pac gene of Streptomyces alboniger, which is responsible for acetylation of puromycin and which conferred resistance to puromycin when introduced in eubacteria and eucaryotes, is a potential selective marker in gene transfer experiments with M. voltae. The latter was recently shown to be transformable. The same would be true for the cat gene of Tn9, which encodes resistance to fusidic acid in eubacteria in addition to resistance to chloramphenicol.     

Genetic transformation in the methanogen Methanococcus voltae PS.

Mutations causing requirements for histidine, purine, and vitamin B12 were obtained in strain PS of Methanococcus voltae (archaebacteria) upon irradiation with UV or gamma rays. The first two mutations were shown to revert at low frequencies and were used to demonstrate the occurrence of transformation with homologous, wild-type DNA. The transformation rates obtained for these presumably chromosomal markers were in the range of 2 to 100 transformants per microgram of DNA. Mutants resistant to 2-bromoethanesulfonate and to 5-methyl-DL-tryptophan were also isolated.     

Single-Molecule and FRET Fluorescence Correlation Spectroscopy Analyses of Phage DNA Packaging: Colocalization of Packaged Phage T4 DNA Ends within the Capsid

Linear DNAs of any sequence can be packaged into empty viral procapsids by the phage T4 terminase with high efficiency in vitro. Packaging substrates of 5 kbp and 50 kbp, terminated by energy transfer dye pairs, were constructed from plasmid and λ phage DNAs. Nuclease and fluorescence correlation spectroscopy (FCS) assays showed that ∼ 20% of the substrate DNA was packaged and that the DNA dye ends of the packaged DNA were protected from nuclease digestion. Upon packaging, both 5-kbp and  50-kbp DNAs produced comparable fluorescence resonance energy transfer (FRET) between Cy5 and Cy5.5 double-dye terminated DNAs. Single-molecule FRET (sm-FRET) and photobleaching analysis shows that FRET is intramolecular rather than intermolecular upon packaging of most procapsids and demonstrates that single-molecule detection allows mechanistic analysis of packaging in vitro. FRET-FCS and sm-FRET measurements are comparable and show that both the 5-kbp and the  50-kbp packaged DNA ends are held within 8-9 nm of each other, within the dimensions of the long axis of the procapsid portal. The calculated distribution of FRET distances is relatively narrow for both FRET-FCS and sm-FRET, suggesting that the two packaged DNA ends are held at the same fixed distance relative to each other in most capsids. Because one DNA end is known to be positioned for ejection through the portal, it can be inferred that both DNAs ends are held in proximity to the portal entrance and ejection channel. The analysis suggests that a DNA loop, rather than a DNA end, is translocated by the packaging motor to fill the procapsid.     

Neomycin resistance as a selectable marker in Methanococcus maripaludis.

We cloned the aminoglycoside phosphotransferase genes APH3'I and APH3'II between the Methanococcus voltae methyl reductase promoter and terminator in a plasmid containing a fragment of Methanococcus maripaludis chromosomal DNA. The resulting plasmids encoding neomycin resistance transformed M. maripaludis at frequencies similar to those observed for pKAS102 encoding puromycin resistance. The antibiotic geneticin was not inhibitory to M. maripaludis.     

Genetic requirements for the function of the archaeal virus SSV1 in Sulfolobus solfataricus: construction and testing of viral shuttle vectors.

Directed open reading frame (ORF) disruption and a serial selection technique in Escherichia coli and the extremely thermophilic archaeon Sulfolobus solfataricus allowed the identification of otherwise cryptic crucial and noncrucial viral open reading frames in the genome of the archaeal virus SSV1. It showed that the 15. 5-kbp viral genome can incorporate a 2.96-kbp insertion without loss of viral function and package this DNA properly into infectious virus particles. The selection technique, based on the preferential binding of ethidium bromide to relaxed DNA and the resulting inhibition of endonuclease cleavage to generate a pool of mostly singly cut molecules, should be generally applicable. A fully functional viral shuttle vector for S. solfataricus and E. coli was made. This vector spreads efficiently through infected cultures of S. solfataricus, its replication is induced by UV irradiation, it forms infectious virus particles, and it is stable at high copy number in both S. solfataricus and E. coli. The classification of otherwise unidentifiable ORFs in SSV1 facilitates genetic analysis of this virus, and the shuttle vector should be useful for the development of genetic systems for Crenarchaeota.     

Expression of Mycoplasma pulmonis antigens in Escherichia coli

The expression of Mycoplasma pulmonis antigen in Escherichia coli was investigated by cloning genomic DNA derived from M. pulmonis m 53, and the DNA fragment participating in antigen expression was identified. When the DNA library of M. pulmonis was screened by colony immunoassay using anti-M. pulmonis serum, 10 recombinant clones expressing seroreactive antigens were obtained. The recombinant plasmids isolated from these clones included 3.7-6.5 kilobase pair (kbp) DNA inserts, while all clones contained a common 2.3-kbp DNA fragment. Subcloning of initial DNA inserts showed that the common 2.3-kbp fragment is essential for antigen expression. Moreover, antiserum against the recombinant antigen generated from the 2.3-kbp DNA fragment recognized a native M. pulmonis antigen. The reactivity of this antiserum was absorbed specifically with M. pulmonis. These results suggest that the cloned 2.3-kbp DNA fragment codes an antigen specific to M. pulmonis.     

Complementation of argG and hisA mutations of Escherichia coli by DNA cloned from the archaebacterium Methanococcus voltae

DNA derived from the methanogenic archaebacterium Methanococcus voltae was digested with PstI restriction endonuclease and cloned into the PstI site of pBR322. The recombinant plasmids generated were used to transform a multiply auxotrophic strain of Escherichia coli with selection for tetracycline resistance. Plasmids complementing the argG(pAW1) or hisA(pAW2) mutations were isolated and characterized. Nick-translated pAW1 and pAW2 hybridized to the predicted M. voltae PstI fragments but not to digested E. coli DNA. A novel 55,000-dalton protein was synthesized in UV-irradiated cells by pAW1, whereas pAW2 synthesized a novel 26,000-dalton protein. Derivatives of pAW1 carrying insertion elements no longer complemented the argG mutation and failed to produce the 55,000-dalton protein. When an AccI fragment was deleted from pAW2, complementation of hisA did not occur and no 26,000-dalton protein was synthesized. The effect of orientation of the cloned DNA within the vector on complementation and polypeptide synthesis was examined.     

In vitro model for the nuclear transport of the hepadnavirus genome.

Hepadnaviruses contain a DNA genome, but they replicate via an RNA intermediate, synthesized by the cellular RNA polymerase II in the nucleus of the infected cell. Thus, nuclear transport of the viral DNA is required in the viral life cycle. Protein-free DNA is only poorly imported into the nucleus, so one or more of the viral proteins must be involved in the transport of the viral genome. In order to identify these viral proteins, we purified woodchuck hepadnavirus (WHV) core particles from infected woodchuck liver, isolated WHV DNA, and extracted the covalent complex of viral polymerase from the particles using urea. Intact core particles, the polymerase-DNA complex, or protein-free WHV DNA from core particles was added to digitonin-permeabilized HuH-7 cells, in which the cytosol was substituted by rabbit reticulocyte lysate (RRL) and an ATP-generating system. The distribution of the viral genome was analyzed by semiquantitative PCR or by hybridization in total nuclei, RRL, nuclear membranes, and nucleoplasm. The polymerase-DNA complex was efficiently transported into the nucleus, as indicated by the resistance of the nucleus-associated DNA to a short-term treatment with DNase I of the intact nuclei. The DNA within core particles stayed mainly in the cytosol and remained protected against DNase I. A minor part of the encapsidated DNA was bound to nuclei. It was protected against DNase I but became accessible after disruption of the nuclei. Deproteinized viral DNA completely remained in the cytosol. These data show that the viral polymerase is probably sufficient for mediating the transport of a hepadnavirus genome into the nucleus and that the viral core particles may release the genome at the nuclear membrane.     

Prevalence of Lysogeny among Soil Bacteria and Presence of 16S rRNA and trzN Genes in Viral-Community DNA

Bacteriophages are very abundant in the biosphere, and viral infection is believed to affect the activity and genetic diversity of bacterial communities in aquatic environments. Lysogenic conversion, for example, can improve host fitness and lead to phage-mediated horizontal gene transfer. However, little is known about lysogeny and transduction in the soil environment. In this study we employed atrazine-impregnated Bio-Sep beads (a cell immobilization matrix) to sample active microbiota from soils with prior pesticide exposure history. Once recovered from soil, the bead communities were induced with mitomycin C (MC), and viral and bacterial abundances were determined to evaluate the incidence of inducible prophage in soil bacteria. The inducible fraction calculated within bead communities was high (ca. 85%) relative to other studies in aquatic and sedimentary environments. Moreover, the bacterial genes encoding 16S rRNA and trzN, a chlorohydrolase gene responsible for dehalogenation of atrazine, were detected by PCR in the viral DNA fraction purified from MC-induced bead communities. A diverse collection of actinobacterial 16S rRNA gene sequences occurred within the viral DNA fraction of induced, water-equilibrated beads. Similar results were observed in induced atrazine-equilibrated beads, where 77% of the cloned sequences were derived from actinobacterial lineages. Heterogeneous 16S rRNA gene sequences consisting of fragments from two different taxa were detected in the clone libraries. The results suggest that lysogeny is a prevalent reproductive strategy among soil bacteriophages and that the potential for horizontal gene transfer via transduction is significant in soil microbial communities.