The transposable element Tn3 promotes general recombination at the neighboring regions

Transposon Tn3 was inserted into a tRNA operon of the amber suppressor Su+2 on a transducing phage (lambda hcI857nin5pSu+2) by selecting phages with ampicillin resistance and Su- phenotypes. In a strain thus obtained, Tn3 was inserted between the promoter and the first tRNA gene of the operon, which was determined by DNA sequencing. The Su+2 tRNA operon on the transducing phage consisted of two tRNA genes for tRNA(Met) and Su+2 tRNA(2Gln), which was a deletion derivative of the supB-E tRNA operon of E. coli containing seven tRNA genes in the order of promoter-Met-Leu-Gln1-Gln1-Met-Gln2-Gln2. Proliferating the lambda hcI857nin5pSu+2::Tn3 in E. coli cells, a number of phages which had lost Tn3 were isolated, and their tRNA gene compositions as well as the DNA structures of the tRNA operon were analyzed. In many cases the tRNA genes which had been deleted from the original transducing phage were regained from the chromosomal supB-E operon. Thus the loss of Tn3 from the phages was not due to excision of the transposon but due to the replacement of a portion of the tRNA operon, including Tn3, with the host homologous region that did not contain Tn3. This type of replacement takes place rather efficiently as a consequence of Tn3 insertion, owing to the general recombination occurring between homologous tRNA genes of phage and host chromosomes in the presence of either host recA or phage red. No such enhanced recombination in a similar cross between phage and host chromosomes was observed with the Tn3 present in the trans position on an independent plasmid. We conclude that inserting Tn3 in cis promotes general recombination in the neighboring regions. Possible mechanisms for this new type of genetic effect of Tn3 are discussed. During the course of this study, a natural defective mutation (T11) was also detected in one of the duplicated tRNA(2Gln) genes in an E. coli K12 strain we used.     

Physical mapping of stable RNA genes in Bacillus subtilis using polymerase chain reaction amplification from a yeast artificial chromosome library.

A new approach for mapping genes which utilizes yeast artificial chromosome clones carrying parts of the Bacillus subtilis genome and the polymerase chain reaction technique is described. This approach was used to physically map stable RNA genes of B. subtilis. Results from over 400 polymerase chain reactions carried out with the yeast artificial chromosome clone library, using primers specific for the genes of interest and designed from published sequences, were collected. The locations of 10 known rRNA gene regions (rrnO, rrnA, rrnE, rrnD, rrnB, rrnJ-rrnW, and rrnI-rrnH-rrnG) have been determined by this method, and these results correlate with those observed by standard genetic mapping. All rRNA operons, except rrnB, are found between 0 and 90 degrees, while rrnB has been placed in the area of 270 degrees on the chromosome map. Also localized were the tRNA gene clusters associated with the following ribosomal operons: rrnB (21 tRNAs), rrnJ (9 tRNAs), rrnD (16 tRNAs), and rrnO and rrnA (2 internal tRNAs). A previously unmapped four-tRNA gene cluster, trnY, a tRNA gene region that is not associated with a ribosomal operon, was found near the origin of replication. The P-RNA gene, important for processing of tRNAs, was found between map locations 197 and 204 degrees.     

The Bacillus subtilis ochre suppressor sup-3 is located in an operon of seven tRNA genes.

Most Bacillus subtilis tRNA genes have been isolated from lambda libraries by use of probes that hybridize to tRNA or rRNA sequences. None of those genes map to the region of the sup-3 mutation. By cloning of the sup-3 allele, a cluster of seven tRNA genes (the trnS operon) that had not been isolated by other methods was identified. In principle, this approach could be used to isolate at least one more predicted tRNA-containing operon in this bacterium. The trnS operon was shown to contain tRNA genes for Asn (GUU), Ser (GCU), Glu (UUC), Gln (UUG), Lys (UUU), Leu (UAG), and Leu (GAG). The sup-3 mutation was found to be a T-to-A transversion that changes the anticodon of the lysine tRNA from 5'-UUU-3' to 5'-UUA-3'. This result agrees with previous work that determined that the sup-3 mutation causes lysine to be inserted at ochre nonsense mutations.     

Comparative sequence analysis of the symbiosis island of Mesorhizobium loti strain R7A

The Mesorhizobium loti strain R7A symbiosis island is a 502-kb chromosomally integrated element which transfers to nonsymbiotic mesorhizobia in the environment, converting them to Lotus symbionts. It integrates into a phenylalanine tRNA gene in a process mediated by a P4-type integrase encoded at the left end of the element. We have determined the nucleotide sequence of the island and compared its deduced genetic complement with that reported for the 611-kb putative symbiosis island of M. loti strain MAFF303099. The two islands share 248 kb of DNA, with multiple deletions and insertions of up to 168 kb interrupting highly conserved colinear DNA regions in the two strains. The shared DNA regions contain all the genes likely to be required for Nod factor synthesis, nitrogen fixation, and island transfer. Transfer genes include a trb operon and a cluster of potential tra genes which are also present on the strain MAFF303099 plasmid pMLb. The island lacks plasmid replication genes, suggesting that it is a site-specific conjugative transposon. The R7A island encodes a type IV secretion system with strong similarity to the vir pilus from Agrobacterium tumefaciens that is deleted from MAFF303099, which in turn encodes a type III secretion system not found on the R7A island. The 414 genes on the R7A island also include putative regulatory genes, transport genes, and an array of metabolic genes. Most of the unique hypothetical genes on the R7A island are strain-specific and clustered, suggesting that they may represent other acquired genetic elements rather than symbiotically relevant DNA.     

Evolution of a tRNA operon in gamma purple bacteria

Genomic DNA from eubacteria belonging to the gamma-3 subdivision of purple bacteria, as classified by Woese (C.R. Woese, Microbiol. Rev. 51:221-271, 1987), were probed with the argT operon of Escherichia coli encoding 5'-tRNA(Arg)-tRNA(His)-tRNA(Leu)-tRNA(Pro)-3'. The homologous operon from Vibrio harveyi was isolated and sequenced. Comparison of the five available sequences of this tRNA cluster from members of the families Enterobacteriaceae, Aeromonadaceae, and Vibrionaceae led to the conclusion that variations in different versions of this operon arose not only by point mutations but also by duplication and addition-deletion of entire tRNA genes. This data base permitted the formulation of a proposal dealing with the evolutionary history of this operon and suggested that DNA regions containing tRNA genes are active centers (hot spots) of recombination. Finally, since the operon from V. harveyi was not highly repetitive and did not contain tRNA pseudogenes, as in the Photobacterium phosphoreum operon, hybridization of genomic DNAs from different photobacterial strains with probes specific for the repeated pseudogene element was performed. We conclude that the phylogenetic distribution of the repetitive DNA is restricted to strains of P. phosphoreum.     

A Brevibacterium lactofermentum 16S rRNA gene used as target site for homologous recombination

Genes for rRNA are highly conserved and present in multiple copies in most prokaryotic organisms increasing the number of theoretical sites for homologous recombination. They might be targets for integration events between unrelated microorganisms providing that an efficient genetic transfer is present. We have used a plasmid containing a portion of the 16S rRNA gene from the rrnD operon of Brevibacterium lactofermentum to transform the same strain resulting in non-essential inactivation of various rrn operons. Integration of the transforming DNA occurs in all cases. The system may be used to test possible gene transfer at least among closely related strains and is of great interest for integration of foreign DNA and for mapping.     

Euglena gracilis chloroplast transfer RNA transcription units. Nucleotide sequence analysis of a tRNAThr-tRNAGly-tRNAMet-tRNASer-tRNAGln gene cluster

The arrangement and the nucleotide sequence of the tRNA genes in the 2.0-kilobase-pair EcoRI restriction fragment EcoQ of Euglena gracilis Klebs, strain Z Pringsheim chloroplast DNA have been determined. This fragment, cloned in pBR325 to form the plasmid pEZC300, contains five tRNA genes. The DNA insert of this plasmid, a known tRNA gene locus (Orozco, E.M., Jr., and Hallick, R.B. (1982) J. Biol. Chem. 257, 3258-3264) has been mapped by Southern gel analysis using a 32P-labeled oligodeoxynucleotide tRNA gene probe. The DNA sequence of 870 base pairs (bp) from EcoQ containing the entire tRNA gene locus was determined. The organization of this tRNA gene cluster on the E. gracilis chloroplast chromosome is tRNAUUGGln-14-BP spacer-RNAGCUSer-175-bp spacer-tRNACAUMet-12-bp spacer-tRNAGCCGly-5-bp spacer-tRNAUGUThr. The tRNAUUGGln and tRNAGCUSer gene sequences are of the opposite polarity as the other three gene sequences, but of the same polarity as the rRNA genes. The tRNAMet gene is a putative initiator tRNA. The five tRNA genes are separated and flanked by A-T-rich spacer sequences. This gene arrangement is consistent with the model that E. gracilis chloroplast tRNA genes are transcribed into multicistronic tRNA precursors. The DNA sequences have been used to deduce the primary and secondary structures of the tRNAs.     

hisT is part of a multigene operon in Escherichia coli K-12.

The Escherichia coli K-12 hisT gene has been cloned, and its organization and expression have been analyzed on multicopy plasmids. The hisT gene, which encodes tRNA pseudouridine synthase I (PSUI), was isolated on a Clarke-Carbon plasmid known to contain the purF gene. The presence of the hisT gene on this plasmid was suggested by its ability to restore both production of PSUI enzymatic activity and suppression of amber mutations in a hisT mutant strain. A 2.3-kilobase HindIII-ClaI restriction fragment containing the hisT gene was subcloned into plasmid pBR322, and the resulting plasmid (designated psi 300) was mapped with restriction enzymes. Complementation analysis with different kinds of hisT mutations and tRNA structural analysis confirmed that plasmid psi 300 contained the hisT structural gene. Enzyme assays showed that plasmid psi 300 overproduced PSUI activity by ca. 20-fold compared with the wild-type level. Subclones containing restriction fragments from plasmid psi 300 inserted downstream from the lac promoter established that the hisT gene is oriented from the HindIII site toward the ClaI site. Other subclones and derivatives of plasmid psi 300 containing insertion or deletion mutations were constructed and assayed for production of PSUI activity and production of proteins in minicells. These experiments showed that: (i) the proximal 1.3-kilobase HindIII-BssHII restriction fragment contains a promoter for the hisT gene and encodes a 45,000-dalton polypeptide that is not PSUI; (ii) the distal 1.0-kilobase BssHII-ClaI restriction fragment encodes the 31,000-dalton PSUI polypeptide; (iii) the 45,000-dalton polypeptide is synthesized in an approximately eightfold excess compared with PSUI; and (iv) synthesis of the two polypeptides is coupled, suggesting that the two genes are part of an operon. Insertion of mini-Mu d1 (lac Km) phage into plasmid psi 300 confirmed that the hisT gene is the downstream gene in the operon.     

Cloning of chicken embryo tRNA genes using single stranded nucleosomal DNA highly enriched for tRNA complementary sequences

DNA from chicken embryo nucleosome tetramers (about 760 base pairs in size) was enriched for tRNA genes by RPC-5 chromatography. The enriched DNA was hybridized with chicken embryo total tRNA and the hybridized DNA isolated utilizing a) avidinbiotin interaction, b) diazobenzyloxymethyl paper, and c) high temperature RPC-5 chromatography. The obtained single stranded DNA highly enriched for tRNA complementary sequences was hybridized with total DNA from nucleosome monomers (140--190 base pairs in size) and the excess of non hybridized monomer nucleosome DNA removed by Sepharose 4B chromatography. The hybrid molecules obtained were made fully double stranded by incubation with E. coli DNA polymerase I, DNA ligase, and exonuclease III. DNA was inserted into plasmid pBR322 by G-C joining procedure and the recombinant DNA used to transform the E. coli strain chi 1776. More than 70% of the transformants obtained hybridize to chicken embryo total tRNA.     

Analysis of the pediocin AcH gene cluster from plasmid pSMB74 and its expression in a pediocin-negative Pediococcus acidilactici strain.

The 3,500-bp pap operon in the 8,877-bp plasmid pSMB74 contains a cluster of four genes, papABCD, of which papA encodes prepediocin (A. M. Motlagh, M. Bukhtiyarova, and B. Ray, Lett. Appl. Microbiol. 18:305-312, 1994). The cluster without the promoter was cloned in the shuttle vector pHPS9. An Escherichia coli strain and a pediocin-sensitive Pediococcus acidilactici strain transformed with the recombinant plasmid, pMBR1.0, produced pediocin AcH. Deletion analysis by introducing mutations in the four genes in pMBR1.0 revealed that only papA and papD were required for pediocin AcH production and that the gene product of papD has both translocation and processing functions. In the transformed minicells of E. coli chi 925 the proteins of the pap cluster were synthesized, indicating no polar effect due to deletion.     

A hybrid plasmid is a stable cloning vector for the cyanobacterium Anacystis nidulans R2.

Anacystis nidulans R2 is a highly transformable strain which is suitable as a recipient for molecular cloning in cyanobacteria. In an effort to produce an appropriate cloning vector, we constructed a hybrid plasmid molecule, pSG111, which contained pBR328 from Escherichia coli and the native pUH24 plasmid of A. nidulans. pSG111 replicated in and conferred ampicillin and chloramphenicol resistance to both hosts. It contained unique sites for the restriction enzymes EcoRI, SalI, SphI, and XhoI, which could be used for the insertion of exogenous DNA. To demonstrate that a molecule like pSG111 could serve as a shuttle vector for the cloning of A. nidulans genes, we constructed a hybrid plasmid, pRNA404, containing an A. nidulans rRNA operon. This recombinant molecule was genetically and structurally stable during passage through A. nidulans and E. coli. The stability of the hybrid plasmid and the inserted rRNA operon demonstrates the feasibility of cloning in A. nidulans with hybrid vectors, with the subsequent retrieval of cloned sequences.     

Characterization of the umu-complementing operon from R391.

In addition to conferring resistances to antibiotics and heavy metals, certain R factors carry genes involved in mutagenic DNA repair. These plasmid-encoded genes are structurally and functionally related to the chromosomally encoded umuDC genes of Escherichia coli and Salmonella typhimurium. Three such plasmid operons, mucAB, impCAB, and samAB, have been characterized at the molecular level. Recently, we have identified three additional umu-complementing operons from IncJ plasmid R391 and IncL/M plasmids R446b and R471a. We report here the molecular characterization of the R391 umu-complementing operon. The nucleotide sequence of the minimal R plasmid umu-complementing (rum) region revealed an operon of two genes, rumA(R391) and rumB(R391), with an upstream regulatory signal strongly resembling LexA-binding sites. Phylogenetic analysis revealed that the RumAB(R391) proteins are approximately equally diverged in sequence from the chromosomal UmuDC proteins and the other plasmid-encoded Umu-like proteins and represent a new subfamily. Genetic characterization of the rumAB(R391) operon revealed that in recA+ and recA1730 backgrounds, the rumAB(R391) operon was phenotypically indistinguishable from mucAB. In contrast, however, the rumAB(R391) operon gave levels of mutagenesis that were intermediate between those given by mucAB and umuDC in a recA430 strain. The latter phenotype was shown to correlate with the reduced posttranslational processing of the RumA(R391) protein to its mutagenically active form, RumA'(R391). Thus, the rumAB(R391) operon appears to possess characteristics that are reminiscent of both chromosome and plasmid-encoded umu-like operons.     

Staphylococcus aureus has clustered tRNA genes.

The polymerase chain reaction (PCR) was used to detect large tRNA gene clusters in Bacillus subtilis, Bacillus badius, Bacillus megaterium, Lactobacillus brevis, Lactobacillus casei, and Staphylococcus aureus. The primers were based on conserved sequences of known gram-positive bacterial tRNA(Arg) and tRNA(Phe) genes. This PCR procedure detected an unusually large tRNA gene cluster in S. aureus. PCR-generated probes were used to identify a 4.5-kb EcoRI fragment that contained 27 tRNA genes immediately 3' to an rRNA operon. Some of these 27 tRNA genes are very similar, but only 1 is exactly repeated in the cluster. The 5' end of this cluster has a gene order similar to that found in the 9- and 21-tRNA gene clusters of B. subtilis. The 3' end of this S. aureus cluster exhibits more similarity to the 16-tRNA gene cluster of B. subtilis. The 24th, 25th, and 26th tRNA genes of this S. aureus tRNA gene cluster code for three similar, unusual Gly-tRNAs that may be used in the synthesis of the peptidoglycan in the cell wall but not in protein synthesis. Southern analysis of restriction digests of S. aureus DNA indicate that there are five to six rRNA operons in this bacterium's genome and that most or all may have large tRNA gene clusters at the 3' end.     

Molecular cloning of the structural genes of the Escherichia coli deo-operon in plasmid RSF2124]

A specialized phage lambda ddeo carrying the deo operon of Escherichia coli is analyzed by exposing the DNA to the specific restriction endonucleases EcoRI and BamHI. Using the lambda ddeo DNA fragment, obtained by digestion with BamHI and plasmid RSF2124 as a vehicle, the hybrid plasmid pAM1 carrying all the genes of the deo operon is constructed and cloned in E. coli cells. It is shown that the activity of thymidine phosphorylase in the strain AM061, which contains hybrid plasmid pAM1 is 30-fold greater than that in strains of E. coli with chromosomal localization of the deo operon.