Novel streptococcal-integration shuttle vectors for gene cloning and inactivation.

Seven new streptococcal integration shuttle vectors have been constructed which contain different antibiotic-resistance-encoding genes capable of expression in both Streptococcus sp. and Escherichia coli. These plasmids can replicate in E. coli, but not in streptococci because of the absence of a streptococcal origin of replication. The size, antibiotic resistance, and number of unique restriction sites available for cloning for each plasmid are as follows: pSF141 (7.6 kb, CmR and KmR, 7 sites), pSF143 (5.7 kb, TcR, 6 sites), pSF148 (7.3 kb, CmR and SpR, 7 sites), pDL285 (3.4 kb, KmR, 3 sites), pDL286 (3.1 kb, SpR, 4 sites), pSF151 (3.5 kb, KmR, 10 sites), pSF152 (3.2 kb, SpR, 9 sites). If these plasmids carry a fragment of streptococcal DNA they can specifically integrate into the chromosome via Campbell-like, homologous recombination. Therefore, they should be useful for gene inactivation, cloning, chromosomal walking, or linkage analysis in streptococci. The availability of these integration plasmids resistant to different antibiotics, along with the previously described plasmid, pVA891 (ErR), should also allow the construction of mutants possessing multiple insertionally inactivated genes useful for a variety of genetic studies.     

Construction of a novel gene bank of Bacillus subtilis using a low copy number vector in Escherichia coli

Low copy number vector plasmid pCT571 was constructed to clone Bacillus subtilis genomic fragments in Escherichia coli. pCT571 confers KmR, TcR and CmR in E. coli and CmR in B. subtilis. It has unique restriction sites within the KmR and TcR markers to allow screening for recombinant plasmids by insertional inactivation of these genes. It contains the pSC101 replicon and replicates normally at six to eight copies per chromosome equivalent in E. coli. It also contains oriVRK2, which when supplied with the product of the trfA gene of RK2 in trans, allows pCT571 to replicate at 35-40 copies per chromosome equivalent. A B. subtilis gene bank was created by cloning partially Sau3A-digested and size-fractionated fragments of B. subtilis chromosomal DNA into the BamHI site of pCT571. DNA from 1097 KmR TcS transformants was extracted and analysed electrophoretically as supercoiled DNA and after digesting with EcoRI or EcoRI and SalI. Approximately 1000 hybrid plasmids were found with reasonably sized B. subtilis fragments. The mean size of the inserts in pCT571 is 8 kb, ranging from 4 to 20 kb in different plasmids. The gene bank covers most of the B. subtilis chromosome, as demonstrated by the results of screening the gene bank for selectable nutritional markers in E. coli and B. subtilis. Hybrid plasmids which complement E. coli mutants for arg, his, lys, met, pdx, pyr and thr markers were identified from the gene bank. In B. subtilis the presence of argC, cysA, dal, hisA, ilvA, leuA, lys, metB, metC, phe, purA, purB, thr and trpC was established by transformation experiments. The effects of copy number on cloning and long-term maintenance in the bacterial strains were also investigated. At high copy number some hybrid plasmids cannot be maintained at all, while others show an increased rate of structural deletions and rearrangements.     

Genetic transfer systems in Bacteroides: cloning and mapping of the transferable tetracycline-resistance locus

Conjugation systems that transfer antibiotic resistance in the absence of detectable plasmids are common in Bacteroides, but the mechanism of transfer is poorly understood. We found that linked transfer of tetracycline (TcR) and clindamycin (ClR) resistance by Bacteroides fragilis strain 1126 is induced by growth in either Tc or Cl. We cloned the transferable TcR locus as a 13 kb fragment on the shuttle vector pPH6 in Escherichia coli and showed that this region expresses TcR in Bacteroides but not E. coli. The TcR gene was mapped to a 3 kb region and the ClR gene was shown not to be present in the 13 kb insert. Homologous TcR genes are found in B. fragilis V479 and 1792. Using pulsed-field electrophoresis, the transferable TcR gene was shown to be physically associated with high molecular-weight DNA, suggesting that it is located on the chromosome. A new TcR shuttle vector, pPH7 delta 1.1, was constructed to facilitate use of this selective marker in Bacteroides genetics.     

Cultivar-specific avirulence and virulence functions assigned to avrPphF in Pseudomonas syringae pv. phaseolicola, the cause of bean halo-blight disease

The avrPphF gene was cloned from Pseudomonas syringae pathovar phaseolicola (PPH:) races 5 and 7, based on its ability to confer avirulence towards bean cultivars carrying the R1 gene for halo-blight resistance, such as Red Mexican. avrPphF comprised two open reading frames, which were both required for function, and was located on a 154 kb plasmid (pAV511) in PPH: Strain RW60 of PPH:, lacking pAV511, displayed a loss in virulence to a range of previously susceptible cultivars such as Tendergreen and Canadian Wonder. In Tendergreen virulence was restored to RW60 by avrPphF alone, whereas subcloned avrPphF in the absence of pAV511 greatly accelerated the hypersensitive resistance reaction caused by RW60 in Canadian Wonder. A second gene from pAV511, avrPphC, which controls avirulence to soybean, was found to block the activity of avrPphF in Canadian Wonder, but not in Red Mexican. avrPphF also conferred virulence in soybean. The multiple functions of avrPphF illustrate how effector proteins from plant pathogens have evolved to be recognized by R gene products and, therefore, be classified as encoded by avirulence genes.     

Comparative analysis of the P-1-group plasmids of incompatibility

Wide host range plasmids (IncP-1) R906, R751 and R702 have several cleavage sites for BamHI, HindIII and EcoRI enzymes, in contrast to RP4 plasmid. Using these enzymes, deletion mutants of R906 plasmid have been obtained in vitro which only lost short DNA fragments (1 to 14 kb). A narrow host range pAV1 plasmid of the same incompatibility group has been transformed into the cells of Escherichia coli. pAV1 is stably maintained in the new host and retains its narrow host range in the course of conjugation. Different restriction fragments of R702, R751, R906 and R906-derived deletion mutants hybridize with the nick-translated probe of RP4 DNA. It is suggested that the wide host range plasmids have a similarity in structural and functional organization.     

Developmental regulation of the gene for formate dehydrogenase in Neurospora crassa.

We have isolated and characterized a gene, fdh, from Neurospora crassa which is developmentally regulated and which produces formate dehydrogenase activity when expressed in Escherichia coli. The gene is closely linked (less than 0.6 kb apart) to the leu-5 gene encoding mitochondrial leucyl-tRNA synthetase; the two genes are transcribed convergently from opposite strands. The expression patterns of these genes differ: fdh mRNA is found only during conidiation and early germination and is not detectable during mycelial growth, while leu-5 mRNA appears during germination and mycelial growth. The structure of the fdh gene was determined from the sequence of cDNA and genomic DNA clones and from mRNA mapping studies. The gene encodes a 375-amino-acid-long protein with sequence similarity to NAD-dependent dehydrogenases of the E. coli 3-phosphoglycerate dehydrogenase (serA gene product) subfamily. In particular, there is striking sequence similarity (52% identity) to formate dehydrogenase from Pseudomonas sp. strain 101. All of the residues thought to interact with NAD in the crystal structure of the Pseudomonas enzyme are conserved in the N. crassa enzyme. We have further shown that expression of the N. crassa gene in E. coli leads to the production of formate dehydrogenase activity, indicating that the N. crassa gene specifies a functional polypeptide.     

BamHI-SacI RFLP and Gm analysis of the immunoglobulin IGHG genes in the Northern Selkups (west Siberia): new haplotypes with deletion, duplication and triplication

Gm immunoglobulin allotypes have been studied in 1157 individuals of seven Northern Selkup populations, which account for 80% of the entire population of this west Siberian tribe. This study confirms that the northern Selkup populations are a Caucasoid-Mongoloid hybrid. Restriction fragment length polymorphism (RFLP) analysis of the IGHG genes using double BamHI-SacI digests, performed on 475 DNA samples, allowed us to describe nine new BamHI-SacI haplotypes (BS47 to BS55), eight of them being characterized by IGHG gene deletion or duplication: G1 (BS49) or G4 (BS55) deletion, G4 duplication (BS51), GP-G2-G4 multigene deletion (BS50), duplication (BS48, BS53 and BS54) or triplication (BS52). A new rare Gm haplotype 15,16*;1,17;23 has been found associated with BS52. The BS51 haplotype characterized by a duplicated G4 gene (additional 7.85 kb G4 band identifying a new G4*C5 allele) was always found associated with the Gm 5*;3;23 haplotype. A high RFLP diversity has been observed for the Northern-Mongoloid haplotype Gm 15,16*;1,17;.. which was found (1) with the BS27 haplotype characterized by a 3-exon hinge G3 gene, (2) with two different GP-G2-G4 multigene duplications, BS53 and BS54 haplotypes, which differ by the size of the duplicated G4 genes, and (3) with the BS55 haplotype characterized by a G4 deletion. In the Northern Selkups, haplotypes with duplicated genes were observed at a higher frequency (24%) than haplotypes with deleted genes (6%).     

Spontaneous deletions of the chromosome-mobilizing plasmid R68.45 in Pseudomonas aeruginosa PAO

In Pseudomonas aeruginosa strain PAO the chromosome-mobilizing IncP-1 plasmid R68.45 was unstable whereas the parent plasmid R68 was stable. The instability of R68.45 was observed in rec+ and rec strains within about 100 generations after conjugal transfer of the plasmid and, to a lesser extent, in established R68.45 donor strains. Two phenotypically distinct classes of R68.45 derivatives were obtained: (i) CbR (carbenicillin-resistant), TcR (tetracycline-resistant), KmR (kanamycin-resistant), Tra+ (transfer proficient), Cma- (chromosome-mobilizing ability), lacking the duplicated IS21 copy typical of R68.45 and indistinguishable from R68 by restriction enzyme analysis; (ii) CbR TcR KmS Tra- Cma-, due to deletion of one IS21 copy, the adjacent KmR gene, and a variable part of the Tra-1 region including, in most cases, the origin of transfer (oriT). Both types of deletion derivatives were stable. R68.45 derivatives lacking the Tra-2 region were not recovered spontaneously, but could be constructed in vitro and were stable in strain PAO. Deletion formation of type ii as well as Cma did not depend on homologous recombination and can be ascribed to functions of the duplicated IS21. Chromosome mobilization does not appear to require obligatory transfer of the entire R68.45 plasmid. Four ClaI restriction sites were mapped on R68 extracted from P. aeruginosa. One of these sites was cryptic, presumably because of methylation, when the plasmid was prepared from Escherichia coli (dam+).     

Cloning, expression in Escherichia coli and nucleotide sequence of a tetracycline-resistance gene from Streptomyces rimosus

Determinants of tetracycline resistance have been cloned from two different tetracycline-producing industrial strains of Streptomyces into Streptomyces lividans using the plasmid vector pUT206. Three plasmids, pUT250 and pUT260 with a 9.5 and a 7.5 kb insert respectively of Streptomyces rimosus DNA, and pUT270 with a 14.0 kb insert of Streptomyces aureofaciens DNA, conferring resistance to tetracycline, have been isolated. By in vitro sub-cloning, a similar fragment of 2.45 kb containing the tetracycline resistance gene (tet347) was further localized on these plasmids. The S. rimosus gene has been cloned into Escherichia coli and expressed under the control of lambda pL or Lpp promoters. Differential protein extraction of E. coli cells revealed the presence of an additional membrane-embedded protein in tetracycline-resistant cells. On the basis of available restriction endonuclease maps, the tet347 gene is probably identical to the tetB gene from S. rimosus recently identified by T. Ohnuki and co-workers as responsible for the reduced accumulation of tetracycline. The nucleotide sequence of a 2052 bp DNA fragment containing the TcR structural gene from S. rimosus has been determined. The amino acid sequence of the tet347 protein (Mr35818) deduced from the nucleotide sequence shows a limited but significant homology to other characterized tetracycline transport acting determinants from pathogenic bacteria.     

Heteroduplex formation by human Rad51 protein: effects of DNA end-structure, hRP-A and hRad52.

Purified human Rad51 protein (hRad51) catalyses ATP-dependent homologous pairing and strand transfer reactions, characteristic of a central role in homologous recombination and double-strand break repair. Using single-stranded circular and partially homologous linear duplex DNA, we found that the length of heteroduplex DNA formed by hRad51 was limited to approximately 1.3 kb, significantly less than that observed with Escherichia coli RecA and Saccharomyces cerevisiae Rad51 protein. Joint molecule formation required the presence of a 3' or 5'-overhang on the duplex DNA substrate and initiated preferentially at the 5'-end of the complementaryx strand. These results are consistent with a preference for strand transfer in the 3'-5' direction relative to the single-stranded DNA. The human single-strand DNA-binding protein, hRP-A, stimulated hRad51-mediated joint molecule formation by removing secondary structures from single-stranded DNA, a role similar to that played by E. coli single-strand DNA-binding protein in RecA-mediated strand exchange reactions. Indeed, E. coli single-strand DNA-binding protein could substitute for hRP-A in hRad51-mediated reactions. Joint molecule formation by hRad51 was stimulated or inhibited by hRad52, dependent upon the reaction conditions. The inhibitory effect could be overcome by the presence of hRP-A or excess heterologous DNA.     

Molecular genetics of Pseudomonas syringae pathovar pisi: plasmid involvement in cultivar-specific incompatibility.

A mutant (PF24) of the race 1 strain, 299A, of Pseudomonas syringae pv. pisi has been characterized in terms of its interactions with pea (Pisum sativum) cultivars. The mutant showed a changed reaction (avirulence to virulence) with a group of pea cultivars, including cvs. Belinda and Puget, previously thought to contain resistance genes R1 and R3. Avirulence towards cv. Puget was restored by transfer of any one of five cosmid clones from a race 3 (strain 870A) gene library to a rifampicin-resistant derivative of PF24. These observations were in agreement with a revised race-specific resistance genotype for Belinda and similar cultivars comprising a single resistance gene, R3. An incompatible interaction was observed between strain PF24 and cvs. Vinco (postulated to harbour race-specific resistance genes R1, R2, R3 and R5) and Hurst's Greenshaft (R4 and possibly R1), indicating that the mutant retains at least one avirulence gene (A1 or A1 and A4). Mutant PF24 showed loss of a cryptic plasmid (pAV212) compared with its progenitor, strain 299A. A subclone (pAV233) of one of the race 3 restoration clones showed strong hybridization with similar-sized digestion fragments in race 3 plasmid DNA, confirming the A3 gene to be plasmid-borne. Strong cross-hybridization was also observed with a single 3.27 kb EcoRI fragment of plasmid DNA present in strain 299A but absent from strain PF24. This is consistent with the corresponding A3 determinant being located on pAV212 in the race 1 strain 299A. The novel avirulence gene corresponding to A3 in strain 870A is provisionally designated avrPpi3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intergeneric conjugation in Thiobacillus versutus.

In plate matings with Escherichia coli HB101/pUW965::Tn5 (KmR) Thiobacillus versutus reacted as an efficient recipient, producing 10(-2) to 10(-3) kanamycin resistant (KmR) T. versutus exconjugants per donor cell. Analysis of agarose gels of plasmid DNA extracted from the exconjugants confirmed that the suicide vector pUW964 did not persist in the recipient, implying that the kanamycin resistance of the exconjugants is based on effective transposition of Tn5 in T. versutus as well as function of the E. coli kanamycin gene. Transfer was equally efficient when a nalidixate-resistant T. versutus mutant was used as recipient. Hybridization evidence for the presence of Tn5 was consistently negative. The significance of this anomalous result is discussed.     

Cloning and nucleotide sequences of the mdh and sucD genes from Thermus aquaticus B

A 3 kb DNA fragment containing the gene (mdh) encoding malate dehydrogenase (MDH) from the thermophile Thermus aquaticus B was cloned in Escherichia coli and its nucleotide sequence determined. Comparative analysis showed the nucleotide sequence to be very closely related to that determined for the Thermus flavus mdh gene and flanking regions, with no differences between the predicted amino acid sequences of the MDHs. A proximal open reading frame, identified as the sucD gene, and the mdh gene may be parts of the same operon in T. aquaticus B. Expression of the T. aquaticus B mdh gene in E. coli was found to be at a relatively low level. A simple method for purification of thermostable MDH from the E. coli clone containing the T. aquaticus B mdh gene is presented.     

Cloning of the Streptococcus thermophilus beta-galactosidase gene and its expression in Escherichia coli and Bacillus subtilis cells

The beta-galactosidase gene from the chromosome of Streptococcus thermophilus, strain 6 kb, has been cloned on a vector plasmid pBR322. The corresponding gene has been found to be located on the Pst1 DNA fragment. The restriction map of this 6 kb fragment has been constructed. The shortening of the DNA fragment carrying the beta-galactosidase gene has been achieved by digestion of the recombinant derivative of pBR322 by the restriction endonuclease Sau3A under the conditions of incomplete hydrolysis. The obtained fragments have been cloned into the BamHI site in the berepliconed shuttle vector pCB20 for grampositive and gramnegative bacteria. The obtained recombinant plasmids contained the beta-galactosidase gene in the inserted fragments of different length. Expression of the cloned beta-galactosidase gene in Escherichia coli and Bacillus subtilis cells has been studied.     

Transposon insertion mutagenesis of Pseudomonas aeruginosa with a Tn5 derivative: application to physical mapping of the arc gene cluster

For insertional mutagenesis of Pseudomonas aeruginosa, a derivative of the kanamycin-resistance (KmR) transposon Tn5 was constructed (Tn5-751) that carried the trimethoprim-resistance (TpR) determinant from plasmid R751 as an additional marker. Double selection for KmR and TpR avoided the isolation of spontaneous aminoglycoside-resistant mutants which occur at high frequencies in P. aeruginosa. As a delivery system for the recombinant transposon, plasmid pME305, a derivative of the broad-host-range plasma RP1, proved effective; pME305 is temperature-sensitive at 43 degrees C for maintenance in Escherichia coli and P. aeruginosa and deleted for IS21 and the KmR and primase genes. In matings with an E. coli donor carrying pME9(= pME305::Tn5-751), transposon insertion mutants of P. aeruginosa PAO were recovered at approx. 5 X 10(-7)/donor at 43 degrees C. Among Tn5-751 insertional mutants 0.9% were auxotrophs. A thr::Tn5-751 mutation near the recA-like locus rec-102 is useful for the construction of recombination-deficient strains. Several arc::Tn5-751 mutants could be isolated that were defective in anaerobic utilization of arginine as an energy source. From three of these mutants the arc gene region was cloned into an E. coli vector plasmid. Since Tn5-751 has a single EcoRI site between the TpR and KmR genes, EcoRI-generated fragments carrying either resistance determinant plus adjacent chromosomal DNA could be selected separately in E. coli. Thus, a restriction map of the arc region was constructed and verified by hybridization experiments. The arc genes were tightly clustered, confirming earlier genetic evidence.     

Molecular cloning, partial purification, and characterization of a haemin-binding lipoprotein from Haemophilus influenzae type b

A library of genomic DNA fragments from Haemophilus influenzae type b (Hib) DL42 was constructed in plasmid pBR322, transformed into Escherichia coli strain RR1, and screened for recombinant clones with haemin-binding activity by plating onto haemin-containing agar. Expression of haemin-binding activity by clones correlated with the expression of a protein with an apparent molecular weight of 51,000 (51K) that was also recognized by anti-Hib strain DL42 serum in immunoblots. One recombinant clone, designated pHM2, with the smallest DNA insert (3.62 kb) was characterized further. Ethanol inhibition of expression of pHM2 in minicells revealed that the 51K protein was the result of a processing event involving a larger precursor. E. coli RR1(pHM2) adsorbed haemin in liquid suspensions as well as from solid media. Subcloning of a 2.6 kb fragment of pHM2 into a shuttle vector permitted the construction of a recombinant Hib clone, DL42(pHM1002), which overexpressed the 51K haemin-binding protein. This 51K protein appears to be peripherally associated with the inner, and possibly outer, membranes of Hib. Affinity chromatography on haemin-agarose was utilized to purify the haemin-binding protein from both E. coli RR1(pHM2) and Hib DL42(pHM1002) to near homogeneity. The use of the antibiotic globomycin in a minicell expression system and radioimmunoprecipitation analysis of Hib proteins intrinsically radiolabelled with [3H]-palmitate indicated that the 51K haemin-binding protein is a lipoprotein.