Selective advantage of deletions enhancing chloramphenicol acetyltransferase gene expression in Streptococcus pneumoniae plasmids

A hybrid plasmid, pJS37, was made by combining pLS1, which confers tetracycline (Tc) resistance, and pC194, which confers chloramphenicol (Cm) resistance. Both pJS37 (7.3 kb) and its derivative pJS140 (6.0 kb), from which pC194 replication genes were removed, were structurally and segregationally stable when introduced into Streptococcus pneumoniae and grown either in the presence of Tc or in the absence of drug. However, both hybrid plasmids underwent systematic deletion when grown in the presence of Cm. One of the deleted forms, pJS4 (3.4 kb), could not be maintained in the absence of a helper plasmid; two others, pJS3 (4.1 kb) and pJS5 (3.8 kb), lost the tet gene but retained the replication functions of pLS1. They both expressed very high levels of Cm acetyltransferase (CAT), which, in the case of pJS5, were constitutive. Nucleotide sequence determination of the deletion junctions in pJS3 and pJS5 indicated that the deletions occurred, presumably by recombination, between short direct repeats of 6 and 9 bp, respectively. In both cases the tet promoter was juxtaposed to the cat gene. In the case of pJS5, the deletion removed a sequence that sequestered the ribosome-binding site (RBS) for cat, thereby rendering constitutive the production of CAT. The increased resistance to Cm afforded by the hyperexpression of the cat gene apparently provided a positive selective advantage for the accumulation of the deleted forms in the plasmid pool.     

Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites

A method is described for the efficient insertion of mutagenic oligodeoxynucleotide cassettes which allow saturation of a target amino acid codon with multiple mutations. Restriction sites are introduced by oligonucleotide-directed mutagenesis procedures to flank closely the target codon in the plasmid containing the gene. The restriction sites to be introduced are chosen based on their uniqueness to the plasmid, proximity to the target codon and conservation of the final amino acid coding sequence. The flanking restriction sites in the plasmid are digested with the cognate restriction enzymes, and short synthetic duplex DNA cassettes (10-25 bp) are inserted. The mutagenic cassette is designed to restore fully the wild-type coding sequence, except over the target codon, and to eliminate one or both restriction sites. Elimination of a restriction site facilitates selection of clones containing the mutagenic oligodeoxynucleotide cassette. To make the cassettes, single-stranded oligodeoxynucleotides and their complements are synthesized in separate pools containing different codons over the target. This method has been successfully applied to generate 19 amino acid substitutions at position 222 in the subtilisin protein sequence.     

Gene cassettes from the insert region of integrons are excised as covalently closed circles

Integrons are DNA elements which generally include one or more discrete gene cassettes inserted at a specific site. We have recently proposed a model for the acquisition and dissemination of genes found in the insert region of integrons, which requires the existence of circularized gene cassettes. Evidence for the existence of covalently closed circular molecules consisting of one or more gene cassettes has now been obtained. Low levels of small molecules which hybridize to probes specific for individual gene cassettes were detected in plasmid DNA isolated from cells containing a plasmid which includes an integron fragment with three gene cassettes aacC1, orfE and aadA2. These molecules were only detected when the gene encoding the integron DNA integrase was also present and are thus products of site-specific cassette excision. The excised cassettes have been shown to be in the form of covalently closed supercoiled circles, by digestion with restriction enzymes exonuclease III and DNase I. The circular excision products detected included either one cassette, aadA2 or orfE, two cassettes, aacC1 and orfE or all three cassettes. The predicted sequence of the recombinant junction in the excised aadA2 cassette confirmed that excision was precise. The predicted unique sequences of the 59-base elements associated with individual genes in the circular cassette form were compiled, and the sequences of the seven-base core sites which flank 59-base elements are now, with few exceptions, exact inverted repeats.     

2型猪链球菌Fbps基因敲除突变体的构建及毒力分析

目的:敲除2型猪链球菌(SS2)强毒株05ZYH33中的Fbps基因,研究该基因敲除对菌株生物活性及毒力的影响,为深入探讨猪链球菌致病机制提供实验基础。方法:从05ZYH33基因组中扩增Fbps基因上、下游同源臂,从pSET1质粒中扩增氯霉素抗性基因Cm,通过重叠PCR的方法将3个片段整合后连接到温敏自杀载体pSET4s上,电转入05ZYH33感受态细胞,通过改变培养温度实现双交换和质粒丢失,最后经抗性筛选获得敲除株05ZYH33ΔFBPS,分析敲除株的生物学性状,以CD1小鼠作为体外感染模型对突变株和野生株进行毒力比较。结果:PCR分析和测序结果均显示Fbps基因敲除成功,动物实验结果显示Fbps基因敲除后05ZYH33的毒力有所下降。结论:与野生株相比,突变株对小鼠的毒力有所降低。     

Single-stranded hexameric linkers: a system for in-phase insertion mutagenesis and protein engineering

An efficient method for introducing two (or four) codons into a cloned gene has been developed. Single-stranded (ss) hexameric linkers are inserted into a plasmid linearized at cohesive-end restriction sites. The resultant 6 (or 12)-bp insertion creates a new 6-bp restriction site. Plasmids containing linker insertions are enriched by using biochemical selection, or selected by using a kanamycin-resistance (KmR) cassette (biological selection). A total of 57 new linkers have been designed, and compatible KmR cassettes flanked by eleven different restriction sites have been constructed. Two-codon insertions into the tetracycline-resistance (TcR) gene of pBR322 yielded a series of new plasmid vectors. Moreover, proteins with internally duplicated domains have been constructed from beta-lactamase (ApR) insertions into the ApR gene of pBR322. Some of the resulting "gemini" proteins retained the beta-lactamase activity.     

Directional ligation of long-flanking homology regions to selection cassettes for efficient targeted gene-disruption in Candida albicans

PCR-product directed gene disruption with a marker cassette having short homology regions is often used in Candida albicans. However, it is quite inefficient due to the high frequency of non-homologous recombination at non-targeted loci, which necessitates extensive screening to identify the correct disruptants. Thus, many PCR-based methods to introduce long flanking homology regions have been developed to increase the frequency of integration at the targeted loci. However, these methods are not that amenable for use with the widely employed C. albicans marker cassettes having direct repeats, as these repeats tend to recombine during PCR, resulting in shorter amplified products without the selection marker. To circumvent this limitation, we have developed a dinucleotide-sticky-end-ligation strategy to add one flanking homology region to one side of the selection cassette, and the other flanking homology region to the other side of the selection cassette. This method involves release of the selection cassette from the plasmid by digestion with two different restriction enzymes, followed by partial fill-in, to provide a unique two base overhang at each end of the cassette. The flanking homology regions, corresponding to the gene to be disrupted, are individually PCR-amplified, and treated with T4-DNA Polymerase in the presence of appropriate dNTPs to yield two base-5' overhangs. The primers used for the PCR have additional bases at the 5' ends such that after T4 DNA Polymerase treatment, the two flanks will have distinct overhangs compatible with the overhangs of the partially filled-in selection cassette. The selection cassette and the flanks are then ligated together and directly used to transform C. albicans. We have successfully used this method for disruption of several C. albicans genes. We have also used this method to recreate insertion mutations obtained with transposons to reconfirm the mutant phenotypes. This approach can be extended to other organisms like Schizosaccharomyces pombe which also require long flanking regions of homology for targeted gene disruption.     

Long regions of homologous DNA are incorporated into the tobacco plastid genome by transformation.

We investigated the size of flanking DNA incorporated into the tobacco plastid genome alongside a selectable antibiotic resistance mutation. The results showed that integration of a long uninterrupted region of homologous DNA, rather than of small fragments as previously thought, is the more likely event in plastid transformation of land plants. Transforming plasmid pJS75 contains a 6.2-kb DNA fragment from the inverted repeat region of the tobacco plastid genome. A spectinomycin resistance mutation is encoded in the gene of the 16S rRNA and, 3.2 kb away, a streptomycin resistance mutation is encoded in exon II of the ribosomal protein gene rps12. Transplastomic lines were obtained after introduction of pJS75 DNA into leaf cells by the biolistic process and selection for the spectinomycin resistance marker. Homologous replacement of resident wild-type sequences resulted in integration of all, or almost all, of the 6.2-kb plastid DNA sequence from pJS75. Plasmid pJS75, which contains engineered cloning sites between two selectable markers, can be used as a plastid insertion vector.     

PCR- and ligation-mediated synthesis of split-marker cassettes with long flanking homology regions for gene disruption in Candida albicans

Because Candida albicans is a diploid organism, two consecutive steps of gene disruption are required to generate a gene knock-out. The same marker (URA3) is often used for disruption of both copies of the gene. This is possible because, after the first round of disruption, homologous recombination between direct repeats flanking the URA3 marker and the subsequent counterselection allow for the efficient recovery of Ura- revertants. Unfortunately, the URA-blaster disruption cassette cannot be used in a PCR-based disruption approach. The hisG repeats flanking the URA3 gene in the disruption cassette anneal to one another during PCR and thereby prevent amplification of the complete cassette. We explored the use of transformation based on split-marker recombination to circumvent this problem. To avoid any cloning steps and to retain the advantage of long flanking regions for disruption, we combined this with a PCR- and ligation-mediated approach for generating marker cassettes. We used this approach to disrupt the C. albicans FAL1 (ATP-dependent RNA helicase) gene. Long 5' and 3' FAL1-specific regions were amplified by PCR and individually ligated to a URA-blaster cassette. The resulting ligation reactions were used separately as templates to generate two FAL1 disruption cassettes with overlapping URA3 marker regions. Simultaneous transformation with both overlapping disruption cassettes yielded efficient disruption of one FAL1 allele.     

Characterization of pER371-based Streptococcus thermophilus-Escherichia coli shuttle vectors

Native plasmid of Streptococcus thermophilus ST137, pER371 (2.7 kb) linearized at various unique restriction sites was individually subcloned into Escherichia coli plasmid pUC19 to generate the pUER-series recombinants. A selection cassette consisting of chloramphenicol- and erythromycin-resistance genes was spliced into each construct to generate the pMEU shuttle vectors. Electrotransformation of Streptococcus thermophilus with these vectors showed that a ca. 1.7 kb BstEII/ BanII fragment is essential for plasmid replication. A shuttle vector, pMEU149-1 (5.3 kb), was constructed using the minimally required fragment for replication. A chloramphenicol acetyltransferase ( cat) gene was successfully expressed in the ultimate S. thermophilus host by using pMEU149-1. Cloning vectors derived from pER371 should provide valuable alternative gene delivery vehicles for the genetic engineering of lactic acid bacteria.     

The use of a selectable FokI cassette in DNA replacement mutagenesis of the R388 dihydrofolate reductase gene

FokI, a class-IIS restriction endonuclease, cleaves double-stranded DNA to produce a protruding 5' end consisting of four nucleotides, 10-13 residues 3' from the nonpalindromic recognition sequence, GGATG. Cassettes which utilize this separation of cleavage and recognition site have been constructed for the purpose of linker mutagenesis and DNA replacement experiments. The cassettes are flanked by FokI recognition sequences oriented such that the FokI cleavage sites are several nucleotides beyond the cassette/vector fusion sites. FokI excises the cassette and several base pairs of the neighboring vector sequence. The ends produced in the vector by FokI cleavage are generally noncomplementary and suitable for the insertion of a segment of synthesized double-stranded replacement DNA. A cassette which contains a tyrosine tRNA suppressor gene (supF) is selectable by the suppression of amber mutations in the recipient host. A vector containing a pBR322-derived origin of replication, the Escherichia coli xanthine-guanine phosphoribosyl transferase gene as a selectable marker, and no FokI sites has been constructed for use with the FokI cassettes. An experiment which utilized the FokI/supF cassette to modify the N-terminal coding region of the R388 dihydrofolate reductase gene is described.     

Plasmid structural instability associated with pC194 replication functions

The hybrid plasmid pJS37 is composed of the streptococcal plasmid pLS1, which confers tetracycline resistance, and the staphylococcal plasmid pC194, which confers chloramphenicol resistance. When gram-positive bacteria containing pJS37 were grown in the presence of chloramphenicol, four different deleted derivatives accumulated. The deletions in the plasmid enhanced resistance to chloramphenicol by placing the cat gene of pC194 near promoters of pLS1. All four deletions shared a common endpoint that corresponded to the putative target site for DNA strand nicking by the pC194 replication protein, RepH. At the other, variable endpoint, the DNA sequence was similar to the putative RepH target sequence. Alteration of the RepH protein, by in vitro modification of the gene encoding it, eliminated this class of deletions. By extending a previously proposed model for the generation of a different but related class of deletions (B. Michel and S.D. Ehrlich, EMBO J. 5:3691-3696, 1986), a comprehensive model that could generate both classes of deletions is suggested. It proposes that a nicking-closing activity of the plasmid replication protein at its normal target site and, aberrantly, at sites with similar sequence can generate deletions either proximal or distal to the aberrant site during rolling-circle replication of the plasmid.     

猪链球菌2型05Z33强毒株转录调控因子Rgg基因敲除突变体的构建及毒力分析

目的:构建猪链球菌2型强毒株05ZYH33转录调控因子Rgg的基因敲除突变体,观察其生物学性状,并在动物感染实验中比较敲除株与野生株的毒力差异,为进一步研究猪链球菌转录调控因子在致病中的作用提供实验基础。方法:分别以猪链球菌2型05ZYH33基因组和pSET1质粒为模板,扩增基因SSU05_1997两侧各约500 bp的片段为上下游同源臂,氯霉素(Cm)抗性基因为中间片段,采用重叠PCR方法连接3个片段;连接产物先克隆到T载体上,再经过酶切克隆到温度敏感自杀载体pSET4S上;将构建的基因敲除载体pSET4S-1997电转化入05ZYH33感受态细胞,通过改变培养温度筛选出基因敲除突变体05Z33△rgg;对敲除株和野生株的生物学性状及小鼠和猪的致病性进行了初步比较。结果:PCR分析和测序结果均显示基因SSU05_1997完全被Cm抗性基因所替代,基因敲除突变体构建成功;05ZYH33△rgg对小鼠和猪的致病性与野生株相比无明显差异。结论:转录调控因子Rgg可能和猪链球菌2型的毒力无关。     

Intramolecular transposition of insertion sequence IS91 results in second-site simple insertions

A series of plasmids carrying an IRL-kan-IRR transposable cassette, in which IRL and IRR are the left- and right-terminal sequences of IS91, have been constructed. These cassettes could be complemented for transposition with similar efficiency when IS91 transposase was provided either in cis or in trans. A total of 87% of IS91 transposition products were simple insertions of the element, while the remaining 13% were plasmid fusions and co-integrates. When transposase expression was induced from an upstream lac promoter, transposition frequency increased approximately 100-fold. An open reading frame (ORF) present upstream of the transposase gene, ORF121, could be involved in target selection, as mutations affecting this ORF were altered in their insertion specificity. Intramolecular rearrangements were analysed by looking at transposition events disrupting a chloramphenicol resistance gene (cat ) located outside the transposable cassette. Plasmid instability resulting from insertion of an extra copy of IRL-kan-IRR within the cat gene was observed; transposition products contained a second copy of the cassette inserted either as a direct or as an inverted repeat. No deletion or inversion of the intervening DNA was observed. These results could be explained as a consequence of intramolecular transposition of IS91 according to a model of rolling-circle transposition.     

Campylobacter fetus Uses Multiple Loci for DNA Inversion within the 5′ Conserved Regions of sap Homologs

Campylobacter fetus cells possess multiple promoterless sap homologs, each capable of expressing a surface layer protein (SLP) by utilizing a unique promoter present on a 6.2-kb invertible element. Each sap homolog includes a 626-bp 5' conserved region (FCR) with 74 bp upstream and 552 bp within the open reading frame. After DNA inversion, the splice is seamless because the FCRs are identical. In mutant strain 23D:ACA2K101, in which sapA and sapA2 flanking the invertible element in opposite orientations were disrupted by promoterless chloramphenicol resistance (Cm(r)) and kanamycin resistance (Km(r)) cassettes, respectively, the frequency of DNA inversion is 100-fold lower than that of wild-type strain 23D. To define the roles of a 15-bp inverted repeat (IR) and a Chi-like site (CLS) in the FCR, we mutagenized each upstream of sapA2 in 23D:ACA2K101 by introducing NotI and KpnI sites to create strains 23D:ACA2K101N and 23D:ACA2K101K, respectively. Alternatively selecting colonies for Cm(r) or Km(r) showed that mutagenizing the IR or CLS had no apparent effect on the frequency of the DNA inversion. However, mapping the unique NotI or KpnI site in relation to the Cm(r) or Km(r) cassette in the cells that changed phenotype showed that splices occurred both upstream and downstream of the mutated sites. PCR and sequence analyses also showed that the splice could occur in the 425-bp portion of the FCR downstream of the cassettes. In total, these data indicate that C. fetus can use multiple sites within the FCR for its sap-related DNA inversion.