Physical map of the genome of Acholeplasma oculi ISM1499 and construction of a Tn4001 derivative for macrorestriction chromosomal mapping.

A physical chromosomal map of Acholeplasma oculi ISM1499 was constructed by using field inversion gel electrophoresis. To assist in the ordering of the chromosomal fragments, a modified transposon, Tn4001.1064, was constructed. It was also used to rescue mycoplasmal chromosomal sequences adjacent to transposon insertion sites in a one-step cloning procedure. The total size of the A. oculi ISM1499 genome was estimated to be 1,633 kb. The restriction enzyme sites for ApaI, BssHII, EagI, and SmaI were positioned on the map along with several transposon insertion sites.     

Comparison of two Xanthomonas campestris pathovar campestris genomes revealed differences in their gene composition

For the Xanthomonas campestris pathovar campestris wild-type strain B100 a plasmid-based clone library was constructed. The plasmids carried chromosomal fragments of 3-4 kb in size that were tagged in vitro with the artificial transposon KAN-2. More than 3000 of the transposon target sites were characterized by DNA sequencing. The sequences obtained were compared to the recently published genome of Xanthomonas campestris pathovar campestris strain ATCC 33913. Most of the sequenced clones derived from strain B100 matched the chromosomal sequence of strain ATCC 33913. An alignment to the circular map of this chromosome revealed that the similarities were statistically distributed over the entire genome of strain ATCC 33913. The similarity was obvious for protein coding sequences, as well as for mobile genetic elements. However, four regions in the genome of Xanthomonas campestris pathovar campestris strain ATCC 33913, ranging in size from 11 to 37 kb, were not represented in the sequenced clone library of Xanthomonas campestris pathovar campestris strain B100. On the other hand, 1.2% of the sequenced clones originating from Xanthomonas campestris pathovar campestris strain B100 showed no or insignificant similarities to the genome of strain ATCC 33913.     

Strain-specific retrotransposon-mediated recombination in commercially used <Emphasis Type="Italic">Aspergillus niger</Emphasis> strain

Transposons are usually present in multiple copies in their hosts' genomes. Recombination between two transposon copies can result in chromosomal rearrangements. Here, we describe a recombination event between two copies of the retrotransposon ANiTa1 within the genome of the fungus Aspergillus niger (strain CBS513.88). The observed chromosomal rearrangement appears to be strain-specific, as the corresponding genomic region in another strain, ATCC1015, shows a different organization. Strain ATCC1015 actually seems to lack full-length ANiTa1 copies and possesses only solo LTR sequences. Presumably strain ATCC1015 was once colonized by ANiTa1, but then the genome subsequently lost the ANiTa1 copies. The striking genomic differences in ANiTa1 copy distribution leading to differences in the chromosomal structure between the two strains, ATTC1015 and CBS513.88, suggest that the activity of transposons may profoundly affect the evolution of different fungal strains.     

Transposon mutagenesis of Mycoplasma gallisepticum by conjugation with enterococcus faecalis and determination of insertion site by direct genomic sequencing

Few genetic systems for studying mycoplasmas exist, but transposon Tn916 has been shown to transpose into the genomes of some species and can be used as an insertional mutagen. In the current study, the ability of Enterococcus faecalis to serve as a donor for the conjugative transfer of transposon Tn916 into the genome of the avian pathogen Mycoplasma gallisepticum strain PG31 was examined. Transconjugants were obtained at a frequency of > or =6 x 10(-8) per recipient CFU. To determine the transposon insertion site, an oligonucleotide primer corresponding to the 3' end of Tn916 was designed for the purpose of directly sequencing genomic DNA without PCR amplification. Using the direct sequencing approach, Tn916 was shown to insert into any of numerous sites in the M. gallisepticum genome. This is the first report of conjugal transposition of Tn916 into the M. gallisepticum genome. The ability to determine transposon insertion sites in mycoplasmas by genomic sequencing has not been previously described and allows rapid sequence analysis of transposon-generated mutants.     

A physical map of the sulfur-dependent archaebacterium Sulfolobus acidocaldarius 7 chromosome.

A chromosomal map of the sulfur-dependent thermoacidophilic archaebacterium Sulfolobus acidocaldarius 7 was constructed with four restriction enzymes: NotI, BssHII, RsrII, and EagI. The map indicated that the chromosome is a single circular DNA of 2,760 +/- 20 kb (mean +/- standard error of the mean). rRNA genes were also mapped. They were located at one site in the genome.     

Specificity of Tn9 insertion into the genome of bacteriophage lambda att80 depending on its preceding location

It was shown that the site of previous integration (the donor site) of Tn9 affects the specificity of its next integration into the target molecule--phage lambda att80 DNA. The transposon integration sites were mapped by restriction and heteroduplex analysis following Tn9 transposition from chromosomal sites of Escherichia coli K-12 differing in location and Tn9 stability. When transposed from chromosomal galT::IS1 gene, Tn9 inserted into the site with coordinates 44,5 +/- 2 kb of lambda att80; when transposed from chromosomal attTn9A site, the transposon inserted into the sites with coordinates 31 +/- 0,7 kb or 33,3 +/- 0,5 kb. In the course of transposition of Tn9 from chromosomal attTn9N site the transposon inserted into the lambda att80 site with coordinates 26,5 +/- 5 kb. In the latter case, the increase of Tn9 single-stranded loop and the appearance of two new HindIII cleavage sites were observed in heteroduplex experiments. The data were interpreted as indicating structural rearrangements of Tn9 or linked sequences in the course of transposition.     

Physical maps of the genomes of three Bacillus cereus strains.

NotI restriction maps of the chromosomes from Bacillus cereus ATCC 10876, ATCC 11778, and the B. cereus type strain ATCC 14579 have been established and compared with the previously established map of B. cereus ATCC 10987. Between 10 and 14 NotI fragments were observed, ranging from 15 to 1,300 kb, in digests of DNA from the various strains. The sizes of the genomes varied between 5.4 and 6.3 Mb. The maps were constructed by hybridization of 42 random probes, prepared from B. cereus ATCC 10987 libraries, to fragments from partial and complete NotI digests, separated by pulsed-field gel electrophoresis. Nine probes were specific for ATCC 10987 only. Probes for five B. subtilis and five B. cereus genes were also used. The NotI restriction fragment patterns of the four strains were strikingly different.     

Analysis of Tn916-induced mutants ofClostridium acetobutylicum altered in solventogenesis and sporulation

Summary The conjugative transposon Tn916 was used for mutagenesis ofClostridium acetobutylicum ATCC 824. Tetracycline-resistant mutants were screened for loss of granulose synthesis and five classes of granulose mutants, that contained single transposon insertions, were identified on the basis of altered solvent production. Class 1 mutants did not make acetone or butanol, lacked activity of enzymes induced during solventogenesis, and did not sporulate, indicating that they are regulatory mutants. The class 2 mutant strains also did not produce acetone but did form small amounts of butanol and ethanol while the class 3 mutants produced low amounts of all solvents. Class 4 and 5 mutants produced essentially the same or higher amounts of solvents than the parent strain. Transposon insertions in the class 1 mutants were used as markers for in vitro synthesis of flanking chromosomal DNA using Tn916-specific primers. The DNA fragments were labeled to produce specific probes. Transposon insertion sites in the chromosomes of 13 different class 1 regulatory mutants were compared by hybridization of the specific probes to Southern blots of restriction endonuclease-digested parental chromosomal DNA. Insertions in two mutants appeared to be, in the same region of the chromosome. These results predict, that multiple regulatory elements are required to induce solvent production and sporulation.     

Intramolecular transposition and inversion in plasmid R6K

Selection was made in Escherichia coli K-12 recA hosts carrying plasmid R6K for ampicillin hyperresistance. Twenty-two selected strains were found to carry mutant plasmids, which, from electron microscopy and restriction enzyme analysis, were concluded to arise by a duplication of transposon Tn2660, which confers ampicillin resistance, in all cases the duplicate transposon being in an inverted orientation with respect to the resident Tn2660. A mutant of R6K, pSJC301, which was temperature sensitive for ampicillin resistance was produced by in vitro hydroxylamine treatment of R6K deoxyribonucleic acid. A plasmid hybrid, pSJC102, was constructed by cloning the EcoRI R6K fragment carrying the wild-type beta-lactamase gene into the EcoRI site of ColE1. pSJC301 and pSJC102 were transformed into the same recA host strain to form a stable biplasmid strain. Ampicillin-hyperresistant mutants were selected from this strain and screened for plasmids with a duplication of transposon Tn2660, which occurred with equal frequency in either pSJC301 or pSJC102; of 12 characterized, all were inverse repeats of the resident transposon. All six Tn2660 inserts into pSJC301 determined temperature-sensitive ampicillin resistance, and all six inserts into pSJC102 determined wild-type ampicillin resistance, from which it was inferred that transposition of a duplicate Tn2660 occurs predominantly as an intramolecular event, at least in the multicopy R6K plasmid. In all 28 insertion mutants of R6K, there was an inversion of the deoxyribonucleic acid between the two transposons, whereas in only one of six insertion mutants of pSJC102, inversion had occurred. These results are discussed in terms of current models of transposition.     

Characterization of a Chlamydomonas Transposon, Gulliver, Resembling Those in Higher Plants

While pursuing a chromosomal walk through the mt+ locus of linkage group VI of Chlamydomonas reinhardtii, I encountered a 12-kb sequence that was found to be present in approximately 12 copies in the nuclear genome. Comparison of various C. reinhardtii laboratory strains provided evidence that the sequence was mobile and therefore a transposon. One of two separate natural isolates interfertile with C. reinhardtii, C. smithii (CC-1373), contained the transposon, but at completely different locations in its nuclear genome than C. reinhardtii; and a second, CC-1952 (S1-C5), lacked the transposon altogether. Genetic analysis indicated that the transposon was found at dispersed sites throughout the genome, but had a conserved structure at each location. Sequence homology between the termini was limited to an imperfect 15-bp inverted repeat. An 8-bp target site duplication was created by insertion; transposon sequences were completely removed upon excision leaving behind both copies of the target site duplication, with minor base changes. The transposon contained an internal region of unique repetitive sequence responsible for restriction fragment length heterogeneity among the various copies of the transposon. In several cases it was possible to identify which of the dozen transposons in a given strain served as the donor when a transposition event occurred. The transposon often moved into a site genetically linked to the donor, and transposition appeared to be nonreplicative. Thus the mechanism of transposition and excision of the transposon, which I have named Gulliver, resembles that of certain higher plant transposons, like the Ac transposon of maize.     

First Chromosomal Restriction Map of Actinobacillus pleuropneumoniae and Localization of Putative Virulence-Associated Genes

Combined physical and genetic maps of the genomes of Actinobacillus pleuropneumoniae AP76 (serotype 7 clinical isolate) and of A. pleuropneumoniae ATCC 27088 (serotype 1 reference strain) were constructed by using the restriction endonucleases ApaI, AscI, NotI, and SalI. The chromosome sizes as determined by the addition of estimated fragment sizes were 2.4 Mbp, and both maps had a resolution of approximately 100 kbp. The linkages between the ApaI, AscI, NotI, and SalI fragments and their relative positions were determined by (i) fragment excision and redigestion and (ii) partial digests of defined fragments and Southern blot using end-standing probes. The single SalI site within the chromosome of strain A. pleuropneumoniae AP76 was defined as position 1 of the map; for the map of A. pleuropneumoniae ATCC 27088, the corresponding SalI site was chosen. Putative virulence-associated genes (apx, omlA, sodA, tbpBA, ureC, and a repeat element) and housekeeping genes (glyA, metJ, recA, and rhoAP) were positioned on the physical maps and located on the ApaI and NotI fragments of A. pleuropneumoniae serotype reference strains.     

The genome of Haemophilus influenzae Rd has a unique NotI site

Previous analysis of physical maps of Haemophilus influenzae, which is circular and 1.9 Mb in length [Lee and Smith, J. Bacteriol. 170 (1988) 4402-4405; Kauc et al., J. Bacteriol. 171 (1989) 2474-2479], did not detect any NotI (GCGGCCGC) restriction sites. A transposon, Tn916, was constructed to contain a NotI linker cloned into its NciI site and introduced into the H. influenzae chromosome. NotI digestion of chromosomes containing a Tn916-associated NotI site followed by separation of fragments by field-inversion gel electrophoresis revealed the presence of two fragments obtained by two NotI cuts, one in Tn916 and the other, a unique, 'natural' NotI site in the original chromosomal DNA. The examination of other Haemophilus strains demonstrated the presence of one or more NotI sites in all of those tested.     

Construction of a physical map of a kanamycin (Km) transposon, Tn5, and a comparison to another km transposon, Tn903

Summary A cleavage map of Tn5, a kanamycin (Km) transposon from plasmid JR67, was constructed from pMKI, a composite plasmid of ColE1 and Tn5, and compared to that of Tn903, a Km transposon from plasmid R6-5. The two transposons showed marked heterogeneity in both the structural gene for Km resistance and the inverted repeat regions as evidenced by their distinctly different restriction maps. This result suggests separate paths of evolution for the two Km transposons.     

Isolation of a repeated DNA sequence from Bordetella pertussis

A repeated DNA sequence in the genome of Bordetella pertussis has been demonstrated. At least 20 copies of this sequence could be observed in either BamHI or EcoRI restriction enzyme digests of chromosomal DNA; fragments carrying the repeated DNA sequence ranged in size from about 1.5 to 20 kbp. The repeated DNA sequence was cloned from two separate regions of the B. pertussis genome, as shown by restriction enzyme site maps of the two clones and by hybridization studies. A small number of differences in the pattern of hybridization of the repeated DNA sequence to chromosomal DNA from several strains of B. pertussis was observed. No repeated DNA sequences were observed in one strain each of B. parapertussis and B. bronchiseptica, and there was no hybridization of B. pertussis DNA to Escherichia coli chromosomal DNA. The repeated DNA sequence was subcloned on a 2.54 kbp BamHI fragment from one of the two original clones. Restriction enzyme digests and hybridization studies showed that the repeated DNA sequence was about 1 kbp in size and had a single, internal ClaI site.     

Transposon mutagenesis of Mycoplasma gallisepticum

There are few systems available for studying the genetics of the important avian respiratory pathogen, Mycoplasma gallisepticum. These techniques are needed to develop a mechanism to study the molecular pathogenesis of M. gallisepticum. Tn916 has the ability to transpose into the M. gallisepticum genome by both transformation and conjugation. In this study, PEG-mediated transformation was employed for the transfer of Tn916 into M. gallisepticum and create a transposon mutant library. Transformants were obtained at a frequency of approximately 5 x 10(-8) per recipient CFU. A total of 424 MG/Tn916 mutants were constructed and sequence data from the transposon junctions of 71 mutants was obtained and used to identify transposon insertion sites. Insertions were found throughout the genome in nearly all of the major gene categories, making this the first extensive characterization of a transposon mutant library of M. gallisepticum. Transposon stability was also examined, and it was determined that for two mutants the element was stably maintained in vivo in the absence of selective pressure.     

Identification of a plasmid-borne parathion hydrolase gene from Flavobacterium sp. by southern hybridization with opd from Pseudomonas diminuta

Parathion hydrolases have been previously described for an American isolate of Pseudomonas diminuta and a Philippine isolate of Flavobacterium sp. (ATCC 27551). The gene which encodes the broad-spectrum organophosphate phosphotriesterase in P. diminuta has been shown by other investigators to be located on a 66-kilobase (kb) plasmid. The intact gene (opd, organophosphate-degrading gene) from this degradative plasmid was cloned into M13mp10 and found to express parathion hydrolase under control of the lac promoter in Escherichia coli. In Flavobacterium sp. strain ATCC 27551, a 43-kb plasmid was associated with the production of parathion hydrolase by curing experiments. The M13mp10-cloned fragment of the opd gene from P. diminuta was used to identify a homologous genetic region from Flavobacterium sp. strain ATCC 27551. Southern hybridization experiments demonstrated that a genetic region from the 43-kb Flavobacterium sp. plasmid possessed significant homology to the opd sequence. Similar hybridization did not occur with three other native Flavobacterium sp. plasmids (approximately 23, 27, and 51 kb) present within this strain or with genomic DNA from cured strains. Restriction mapping of various recombinant DNA molecules containing subcloned fragments of both opd plasmids revealed that the restriction maps of the two opd regions were similar, if not identical, for all restriction endonucleases tested thus far. In contrast, the restriction maps of the cloned plasmid sequences outside the opd regions were not similar. Thus, it appears that the two discrete bacterial plasmids from parathion-hydrolyzing soil bacteria possess a common but limited region of sequence homology within potentially nonhomologous plasmid structures.     

Identification of a plasmid-borne parathion hydrolase gene from Flavobacterium sp. by southern hybridization with opd from Pseudomonas diminuta.

Parathion hydrolases have been previously described for an American isolate of Pseudomonas diminuta and a Philippine isolate of Flavobacterium sp. (ATCC 27551). The gene which encodes the broad-spectrum organophosphate phosphotriesterase in P. diminuta has been shown by other investigators to be located on a 66-kilobase (kb) plasmid. The intact gene (opd, organophosphate-degrading gene) from this degradative plasmid was cloned into M13mp10 and found to express parathion hydrolase under control of the lac promoter in Escherichia coli. In Flavobacterium sp. strain ATCC 27551, a 43-kb plasmid was associated with the production of parathion hydrolase by curing experiments. The M13mp10-cloned fragment of the opd gene from P. diminuta was used to identify a homologous genetic region from Flavobacterium sp. strain ATCC 27551. Southern hybridization experiments demonstrated that a genetic region from the 43-kb Flavobacterium sp. plasmid possessed significant homology to the opd sequence. Similar hybridization did not occur with three other native Flavobacterium sp. plasmids (approximately 23, 27, and 51 kb) present within this strain or with genomic DNA from cured strains. Restriction mapping of various recombinant DNA molecules containing subcloned fragments of both opd plasmids revealed that the restriction maps of the two opd regions were similar, if not identical, for all restriction endonucleases tested thus far. In contrast, the restriction maps of the cloned plasmid sequences outside the opd regions were not similar. Thus, it appears that the two discrete bacterial plasmids from parathion-hydrolyzing soil bacteria possess a common but limited region of sequence homology within potentially nonhomologous plasmid structures.     

Isolation, characterization, and complementation of a paralyzed flagellar mutant of Rhodobacter sphaeroides WS8.

A paralyzed Rhodobacter sphaeroides mutant strain (PARA1) was isolated by a motility screening procedure following mutagenesis of wild-type R. sphaeroides WS8-N with the transposable element TnphoA (Tn5 IS50L::phoA). PARA1 synthesized a wild-type level of flagellin, as detected by Western immunoblotting with antiflagellar antiserum. Flagellar staining showed that flagellin was assembled into apparently normal external flagellar filaments. Electron micrographs of basal body structures from PARA1 showed that some ring structures that were present were similar to those in wild-type R. sphaeroides WS8-N. PARA1 cells were nonmotile under all growth conditions. No pseudorevertants to motility were seen when PARA1 was grown in the presence of kanamycin to select for the presence of the transposon. The presence of the single copy of TnphoA in the PARA1 chromosome was demonstrated by Southern blotting. Western blotting of cytoplasmic, periplasmic, and membrane fractions of PARA1 with anti-alkaline phosphatase antiserum showed that the transposon had been inserted in-frame into a gene encoding a membrane protein. A SalI restriction endonuclease fragment was cloned from the chromosome of PARA1; this fragment contained a portion of the transposon and R. sphaeroides DNA sequence 5' of the site of insertion. This flanking R. sphaeroides DNA sequence was used to probe an R. sphaeroides WS8 cosmid library. A cosmid designated c19 hybridized to the probe, and a SalI restriction endonuclease fragment derived from this cosmid restored wild-type motility to PARA1 when introduced into this mutant strain by conjugation. The significance of this finding in a bacterium with unidirectionally rotating flagella is discussed.     

Mycoplasma gallisepticum: Influence of cell invasiveness on the outcome of experimental infection in chickens

Recently we have shown that a low (R(low)) and a high laboratory passage (R(high)) of the poultry pathogen Mycoplasma gallisepticum prototype strain R differ markedly in their capability to invade non-phagocytic eukaryotic cells. In the present study the infection traits of these two mycoplasma passages were compared in an in vivo setting. After aerosol inoculation of chickens, M. gallisepticum was re-isolated from the inner organs of birds infected with R(low), whereas no mycoplasma was recovered from the inner organs of birds infected with R(high). These results indicate that the two mycoplasma populations derived from strain R differ in their capacity to cross the mucosal barrier and suggest that cell invasion may play a major role in the observed systemic spreading of M. gallisepticum in its chicken host.