Population Heterogeneity in Corynebacterium glutamicum ATCC 13032 caused by prophage CGP3

The genome of Corynebacterium glutamicum type strain ATCC 13032 (accession number BX927147) contains three prophages, CGP1, CGP2, and CGP3. We recently observed that many genes within the CGP3 prophage region have increased mRNA levels in a dtxR deletion mutant that lacks the master regulator of iron homeostasis (J. Wennerhold and M. Bott, J. Bacteriol. 188:2907-2918, 2006). Here, we provide evidence that this effect is due to the increased induction of the prophage CGP3 in the dtxR mutant, possibly triggered by DNA damage caused by elevated intracellular iron concentrations. Upon induction, the CGP3 prophage region is excised from the genome and forms a circular double-stranded DNA molecule. Using quantitative real-time PCR, an average copy number of about 0.1 per chromosome was determined for circular CGP3 DNA in wild-type C. glutamicum. This copy number increased about 15-fold in the dtxR mutant. In order to visualize the CGP3 DNA within single cells, a derivative of the wild type was constructed that contained an array of tet operators integrated within the CGP3 region and a plasmid-encoded YFP-TetR fusion protein. As expected, one to two fluorescent foci that represented the chromosomally integrated CGP3 prophage were detected in the majority of cells. However, in a small fraction (2 to 4%) of the population, 4 to 10 CGP3 DNA molecules could be observed in a single cell. Interestingly, the presence of many CGP3 copies in a cell often was accompanied by an efflux of chromosomal DNA, indicating the lysis of the corresponding cell. However, evidence for the formation of functional infective CGP3 phage particles could not be obtained.     

ORF90, a Gene Required for Photoreactivation in Rhodobacter capsulatus SB1003 Encodes a Cyclobutane Pyrimidine Dimer Photolyase

Based on deduced amino-acid sequence similarities to class-I photolyases, the open reading frame ORF90 was identified from the genome sequence of Rhodobacter capsulatus SB1003. Photoreactivation activity is not detectable in an ORF90 deletion mutant of R. capsulatus SB1003. The phenotype of R. capsulatus wild-type cells was restored by plasmid borne ORF90 of R. capsulatus DeltaORF90. Furthermore, we detected an ORF90-related CPD-specific photoreactivation activity in R. capsulatus cell extracts. The results show that the gene product of ORF90 is involved in photoreactivation and encodes a class-I cyclobutane pyrimidine dimer photolyase.     

Isolation and genetic analysis of an Agrobacterium tumefaciens avirulent mutant with a chromosomal mutation produced by transposon mutagenesis.

A transposon 5 (Tn5) insertion was introduced into the genome of A. tumefaciens (A-208 strain harboring a nopaline type Ti-plasmid) using a conjugative pJB4JI plasmid containing Tn5. Five thousand transconjugants were assayed for virulence on carrot (Daucus carota L.) disks; 54 isolates were avirulent or very attenuated. The cellular localization (plasmid or chromosome) of the Tn5 insertion in those isolates were identified by Southern hybridization analysis. An avirulent mutant (B-90 strain) with the Tn5 insertion in the chromosome was selected and characterized. The mutant had the same growth rate as that of the parent strain in L-broth. The mutant and the parent strain had similar attachment ability to carrot root cells. Tn5 was inserted into one site of the chromosome. The wild-type target chromosomal region (1281 base pairs) was cloned and sequenced. An open reading frame (ORF) consisting of 395 base pairs was identified. The wild-type DNA fragment (1.6 kb) containing the ORF introduced into B-90 strain complemented the avirulent phenotype of the strain. A soluble protein was predicted from the ORF. The Tn5 was inserted near the 3'-terminal of the ORF. Homology search of this ORF found no significant homology to known genes and proteins. Thus, the ORF identified in this paper seems to be a new chromosomal virulence gene of A. tumefaciens.     

Chromosomal replication incompatibility in Dam methyltransferase deficient Escherichia coli cells.

Dam methyltransferase deficient Escherichia coli cells containing minichromosomes were constructed. Free plasmid DNA could not be detected in these cells and the minichromosomes were found to be integrated in multiple copies in the origin of replication (oriC) region of the host chromosome. The absence of the initiation cascade in Dam- cells is proposed to account for this observation of apparent incompatibility between plasmid and chromosomal copies of oriC. Studies using oriC-pBR322 chimeric plasmids and their deletion derivatives indicated that the incompatibility determinant is an intact and functional oriC sequence. The seqA2 mutation was found to overcome the incompatability phenotype by increasing the cellular oriC copy number 3-fold thereby allowing minichromosomes to coexist with the chromosome. The replication pattern of a wild-type strain with multiple integrated minichromosomes in the oriC region of the chromosome, led to the conclusion that initiation of DNA replication commences at a fixed cell mass, irrespective of the number of origins contained on the chromosome.     

Identification, cloning, and characterization of the Escherichia coli sohA gene, a suppressor of the htrA (degP) null phenotype.

Two extragenic suppressors which allow temperature-sensitive htrA mutant Escherichia coli bacteria to grow at 42 degrees C and simultaneously acquire a cold-sensitive phenotype at 30 degrees C were isolated. The cold-sensitive phenotype exhibited by one of the mutants was used to clone the corresponding wild-type copy of the suppressor gene. This was done through complementation with a mini-mu plasmid E. coli DNA library, by selection for colonies which were no longer cold sensitive, at 30 degrees C. The cloned suppressor gene was shown to complement the cold-sensitive phenotype of both suppressor mutations. It was mapped to 68 min on the E. coli chromosome through hybridization to the Kohara library of overlapping lambda transducing bacteriophages, which covers the entire E. coli chromosome. The complementing gene was further subcloned on an 830-base-pair (bp) DNA fragment. DNA sequencing revealed the presence of an open reading frame (ORF) of 333 bp which could encode a protein of 12,359 Mr. Subcloning of various DNA fragments from within this 830-bp DNA fragment suggests that this ORF is most likely responsible for suppression of the cold-sensitive phenotype of the htrA suppressor bacteria. By using a T7 polymerase system to overproduce plasmid-encoded proteins, a protein of approximately 12,000 Mr was produced by this cloned DNA fragment. This ORF defines a previously undiscovered gene in E. coli, called sohA (suppressor of htrA).     

The asgE locus is required for cell-cell signalling during Myxococcus xanthus development

In response to starvation, Myxococcus xanthus undergoes a multicellular developmental process that produces a dome-shaped fruiting body structure filled with differentiated cells called myxospores. Two insertion mutants that block the final stages of fruiting body morphogenesis and reduce sporulation efficiency were isolated and characterized. DNA sequence analysis revealed that the chromosomal insertions are located in open reading frames ORF2 and asgE, which are separated by 68 bp. The sporulation defect of cells carrying the asgE insertion can be rescued phenotypically when co-developed with wild-type cells, whereas the sporulation efficiency of cells carrying the ORF2 insertion was not improved when mixed with wild-type cells. Thus, the asgE insertion mutant appears to belong to a class of developmental mutants that are unable to produce cell-cell signals required for M. xanthus development, but they retain the ability to respond to them when they are provided by wild-type cells. Several lines of evidence indicate that asgE cells fail to produce normal levels of A-factor, a cell density signal. A-factor consists of a mixture of heat-stable amino acids and peptides, and at least two heat-labile extracellular proteases. The asgE mutant yielded about 10-fold less heat-labile A-factor and about twofold less heat-stable A-factor than wild-type cells, suggesting that the primary defect of asgE cells is in the production or release of heat-labile A-factor.     

Isolation and Genetic Analysis of an Agrobacterium tumefaciens Avirulent Mutant with a Chromosomal Mutation Produced by Transposon Mutagenesis

A transposon 5 (Tn5) insertion was introduced into the genome of A. tumefaciens (A-208 strain harbouring a nopaline type Ti-plasmid) using a conjugative pJB4JI plasmid containing Tn5. Five thousand transconjugants were assayed for virulence on carrot (Daucus carota L.) disks; 54 isolates were avirulent or very attenuated. The cellular localization (plasmid or chromosome) of the Tn5 insertion in those isolates were identified by Southern hybridization analysis. An avirulent- mutant (B-90 strain) with the Tn5 insertion in the chromosome was selected and characterized. The mutant had the same growth rate as that of the parent strain in L-broth. The mutant and the parent strain had similar attachment ability to carrot root cells. Tn5 was inserted into one site of the chromosome. The wild-type target chromosomal region (1281 base pairs) was cloned and sequenced. An open reading frame (ORF) consisting of 395 base pairs was identified. The wild-type DNA fragment (1.6 kb) containing the ORF introduced into B-90 strain complemented the avirulent phenotype of the strain. A soluble protein was predicted from the ORF. The Tn5 was inserted near the 3′-terminal of the ORF. Homology search of this ORF found no significant homology to known genes and proteins. Thus, the ORF identified in this paper seems to be a new chromosomal virulence gene of A. tu?efaciens.     

Cytoplasmic filament-deficient mutant of Treponema denticola has pleiotropic defects

In Treponema denticola, a ribbon-like structure of cytoplasmic filaments spans the cytoplasm at all stages of the cell division process. Insertional inactivation was used as a first step to determine the function of the cytoplasmic filaments. A suicide plasmid was constructed that contained part of cfpA and a nonpolar erythromycin resistance cassette (ermF and ermAM) inserted near the beginning of the gene. The plasmid was electroporated into T. denticola, and double-crossover recombinants which had the chromosomal copy of cfpA insertionally inactivated were selected. Immunoblotting and electron microscopy confirmed the lack of cytoplasmic filaments. The mutant was further analyzed by dark-field microscopy to determine cell morphology and by the binding of two fluorescent dyes to DNA to assess the distribution of cellular nucleic acids. The cytoplasmic filament protein-deficient mutant exhibited pleiotropic defects, including highly condensed chromosomal DNA, compared to the homogeneous distribution of the DNA throughout the cytoplasm in a wild-type cell. Moreover, chains of cells are formed by the cytoplasmic filament-deficient mutant, and those cells show reduced spreading in agarose, which may be due to the abnormal cell length. The chains of cells and the highly condensed chromosomal DNA suggest that the cytoplasmic filaments may be involved in chromosome structure, segregation, or the cell division process in Treponema.     

The histidine permease gene (HIP1) of Saccharomyces cerevisiae

The histidine-specific permease gene (HIP1) of Saccharomyces cerevisiae has been mapped, cloned, and sequenced. The HIP1 gene maps to the right arm of chromosome VII, approx. 11 cM distal to the ADE3 gene. The gene was isolated as an 8.6-kb BamHI-Sau3A fragment by complementation of the histidine-specific permease deficiency in recipient yeast cells. We sequenced a 2.4-kb subfragment of this BamHI-Sau3A fragment containing the HIP1 gene and identified a 1596-bp open reading frame (ORF). We confirmed the assignment of the 1596-bp ORF as the HIP1 coding sequence by sequencing a hip1 nonsense mutation. Analysis of the amino acid (aa) sequence of the HIP1 gene reveals several hydrophobic stretches, but shows no obvious N-terminal signal peptide. We have constructed a deletion of the HIP1 gene in vitro and replaced the wild-type copy of the gene with this deletion. The hip1 deletion mutant can grow when it is supplemented with 30 mM histidine, 50 times the amount required for the growth of HIP1 cells. Revertants of this deletion mutant able to grow on a normal level of histidine arise by mutation in unlinked genes. Both these observations suggest that there are additional, low-affinity pathways for histidine uptake.     

Deoxyribonucleic acid-binding properties and membrane protein composition of a competence-deficient mutant of Haemophilus influenzae.

A mutant of Haemophilus influenzae was isolated which was completely unable to take up double-stranded homologous deoxyribonucleic acid (DNA) at normal physiological conditions but which took up DNA equally as well as the wild type at low pH (pH 4.4). The properties of the mutant provide evidence for the existence of two different mechanisms for DNA entry in the H. influenzae transformation system. With the aid of the mutant the optimal conditions for entry of DNA by these two mechanisms were determined, and the dependence of entry and the specific transforming activity of the entered DNA on competence was examined. The mechanism of entry of DNA at neutral pH, which is not functioning in the mutant, effected entry of homologous DNA only, whereas the mechanism involved in entry of DNA at low pH also effected entry of heterologous DNA. This suggests that the mutant is lacking a protein which recognizes the specific base sequence(s) required for entry. Comparison of the protein composition of the membranes of mutant cells subjected to a growth regimen provoking competence in wild-type cells with that of competent wild-type cells revealed that the mutant is impaired in the synthesis of a protein with a molecular weight of 22,500.     

Bovine papillomavirus mutant temperature sensitive for transformation, replication and transactivation.

The genetic analysis of the papillomaviruses has been hampered by the lack of mutants conditionally defective for viral biological activities. We report here the construction and characterization of a temperature-sensitive papillomavirus mutant. The mutation is predicted to insert the sequence Pro-Arg-Ser-Arg into the N-terminal half of the bovine papillomavirus type 1 (BPV1) ORF E2 protein, the major viral regulatory protein. The cloned mutant viral DNA displays temperature-sensitive defects in the induction of focus formation in mouse C127 cells, in its establishment as an extrachromosomal plasmid and in transactivation of a BPV1 enhancer. Genetic experiments confirm that this pleiotropic phenotype is caused by the insertion mutation in ORF E2 and that the transformation and replication defects of the mutant at 37 degrees C are corrected in trans by wild-type E2 gene activity. Most cell lines stably transformed by the mutant at 32.5 degrees C display a reduced ability to overgrow a monolayer of normal cells following temperature shift to 37 degrees C and the mutant viral DNA after temperature shift is present in decreased copy number and/or in an integrated state. These results provide strong genetic evidence that continued ORF E2 activity is required for maintenance of BPV1-induced transformation and for normal viral DNA replication.     

A yeast nuclear gene, MRS1, involved in mitochondrial RNA splicing: nucleotide sequence and mutational analysis of two overlapping open reading frames on opposite strands.

We have cloned a 1.6-kb fragment of yeast nuclear DNA, which complements pet- mutant MK3 (mrs1). This mutant was shown to be defective in mitochondrial RNA splicing: the excision of intron 3 from the mitochondrial COB pre-RNA is blocked. The DNA sequence of the nuclear DNA fragment revealed two open reading frames (ORF1 with 1092 bp; ORF2 with 735 bp) on opposite strands, which overlap by 656 bp. As shown by in vitro mutagenesis, ORF1, but not ORF2, is responsible for complementation of the splice defect. Hence, ORF1 represents the nuclear MRS1 gene. Disruption of the gene (both ORFs) in the chromosomal DNA of the respiratory competent yeast strain DBY747 (long form COB gene) leads to a stable pet- phenotype and to the accumulation of the same mitochondrial RNA precursors as in strain MK3. The amino acid sequence of the putative ORF1 product does not exhibit any homology with other known proteins, except for a small region of homology with the gene product of another nuclear yeast gene involved in mitochondrial RNA splicing, CBP2. The function of the MRS1 (ORF1) gene in mitochondrial RNA splicing and the significance of the overlapping ORFs in this gene are discussed.     

Genetic analysis of bovine papillomavirus type 1 trans-acting replication factors.

The establishment of bovine papillomavirus type 1 in somatic mammalian cells is mediated by extrachromosomal replication and stable maintenance of the viral genome as a multicopy nuclear plasmid. Previous studies indicated the requirement of viral gene expression for bovine papillomavirus type 1 replication and plasmid maintenance (M. Lusky and M. R. Botchan, Cell 36:391-401, 1984; Turek et al., Proc. Natl. Acad. Sci. U.S.A. 79:7914-7918, 1982). To define the viral genes which are necessary for this process, we constructed a series of specific mutations within the viral genome and assayed the resulting mutants for their ability to replicate extrachromosomally in mouse C127 cells. We report here that the bovine papillomavirus type 1 trans-acting replication factors were encoded by at least two distinct viral genes since the mutants fell into two complementation groups, rep and cop. Mutants (rep-) affecting the E1 open reading frame (ORF) failed to replicate bovine papillomavirus type 1 DNA extrachromosomally and would integrate into chromosomal DNA. We suggest that this gene product is one of the factors required to specifically preclude the integration event. Mutants (cop-) affecting the E7 ORF were maintained in the extrachromosomal state; however, the copy number of the mutant genomes was reduced 100-fold compared with that of wild-type DNA. Analysis of single-cell subclones showed that each cell contained the mutant genomes at a copy number of one to two, indicating that the cop- phenotype did not reflect a simple segregation defect. We propose that the gene defined by mutations in the E7 ORF played a crucial role in stably maintaining the copy number of the viral plasmid at high levels. Genomes with mutations in the cop and rep complementation groups, when cotransfected, rescued the wild-type phenotype, extrachromosomal replication with a high, stable copy number for both types of plasmids. Therefore, the gene products acted in trans, and the mutations were recessive to the wild-type functions. One specific rep- mutant showed a 30-fold-increased transformation efficiency when compared with that of the wild-type genome. In addition, morphological transformation mediated by the cop- mutants appeared to be unstable. These results imply that either or both of the replication functions played some role in regulating the expression of the viral transforming functions.     

Amplified DNA in Streptomyces fradiae.

A spontaneous mutant of Streptomyces fradiae contained an amplifiable unit of DNA with a sequence length of approximately 10.5 kilobases that was amplified to approximately 500 copies per chromosome. The amplified DNA appears to be cryptic. SalI fragments of the amplified DNA were cloned into Escherichia coli to construct a restriction map and characterize the amplified DNA. The amplified DNA contained tandem repeats of the amplifiable unit of DNA. The unit had an average base composition of 71% guanine plus cytosine, similar to the chromosomal DNA of Streptomyces species. At least a portion of the amplifiable unit of DNA was present at a low copy number in the wild-type strain. The phenotype of amplified DNA was designated Ads1SF for amplified DNA sequence 1 in S. fradiae.