Plasmid transformation of Azospirillum brasilense

Abstract Plasmid transformation of the nitrogen-fixing bacterium Azospirillum brasilense is described. A modification of the method of Hanahan [1] was used to transform this bacterium with the 20-kb plasmid pRK290. The efficiency of transformation ranged from 200–1000 transformants per μg of plasmid DNA according to DNA concentration. Ca²⁺, Mn²⁺ and K⁺ were essential for competence, while Rb⁺ and hexamine cobalt(III) chloride did not appear necessary. The length and the temperature of heat-pulse during transformation affected the efficiency of transformation. The response to different numbers of plasmid molecules was linear, in the range of 0.05–1.0 μg of DNA. No transformants were obtained with pRK290 plasmid DNA linearized with Eco RI. The transformability of different strains of Azospirillum has been compared.     

细菌自然转化的分子机制研究进展

自然环境中,具备自然转化能力的细菌可以自发地从外界获取DNA,从而获得新的遗传性状。为能够自然地被转化,细菌需首先建立一个被称作感受态的生理状态并在此状态下表达DNA摄取和加工相关的基因。DNA摄取基因的表达产物可组装一个能将外源DNA摄入细胞质的蛋白复合物。在细胞质中,进入的DNA可同基因组DNA发生同源重组或建立成一个独立的质粒。一般DNA摄入细胞的过程可分为两个阶段,即从外部基质到细胞周质和跨细胞内膜的转运。近年来,包括作者在内的研究人员发现大肠杆菌中存在新的自然质粒转化模式。本文将首先综述近年来细菌自然转化的分子机制,随后简要介绍大肠杆菌中独特的自然质粒转化模式。     

The genetic transformation machinery: composition, localization, and mechanism

Natural genetic transformation is widely distributed in bacteria. It is a genetically programmed process that is inherent to the species. Transformation requires a specialized membrane-associated machinery for uptake of exogenous double-stranded DNA. It also requires dedicated cytosolic proteins, some of which have been characterized only recently, for the processing of internalized single-stranded DNA fragments into recombination products. A series of observations made in Bacillus subtilis and Streptococcus pneumoniae led to the recent emergence of a picture of a unique, highly integrated machine localized at the cell poles. This dynamic machine, which we propose to name the transformasome, involves both membrane and cytosolic proteins, to internalize, protect, and process transforming DNA. This review attempts to summarize these recent observations with special emphasis on the early stages in DNA processing.     

Significant variation in transformation frequency in Streptococcus pneumoniae

The naturally transformable bacterium Streptococcus pneumoniae is able to take up extracellular DNA and incorporate it into its genome. Maintaining natural transformation within a species requires that the benefits of transformation outweigh its costs. Although much is known about the distribution of natural transformation among bacterial species, little is known about the degree to which transformation frequencies vary within species. Here we find that there is significant variation in transformation frequency between strains of Streptococcus pneumoniae isolated from asymptomatic carriage, and that this variation is not concordant with isolate genetic relatedness. Polymorphism in the signalling system regulating competence is also not causally related to differences in transformation frequency, although this polymorphism does influence the degree of genetic admixture experienced by bacterial strains. These data suggest that bacteria can evolve new transformation frequencies over short evolutionary timescales. This facility may permit cells to balance the potential costs and benefits of transformation by regulating transformation frequency in response to environmental conditions.     

细菌自然转化的分子机制及生物学功能研究进展

基因的水平转移在细菌的进化中起着非常重要的作用。自然界中的细菌之间主要通过3种机制进行基因水平转移:由噬菌体介导的转导、接合转移和自然转化。自然转化是指自然感受态的细菌能够自发地从外界环境中摄取DNA分子并整合到自身基因组上的过程。该现象首先发现于肺炎链球菌,目前至少有83种细菌被发现具有发生自然转化的能力,其中革兰氏阳性菌以肺炎链球菌(Streptococcus pneumoniae,S. pneumoniae)为代表,革兰氏阴性菌以奈瑟氏菌(Neisseria)为代表,对其自然转化机制的研究和认识较为清楚,但不同细菌之间自然转化的机制有所差异。自然转化的生物学功能一直以来有以下几种推测:获取营养、修复DNA损伤、生物进化,而近年来对此认识争论不休。本文将详细描述细菌自然转化的分子机制,并对其主要的生物学功能争论焦点进行评述,以期对细菌自然转化有更深入的理解和认识。     

Polarity of DNA entry in transformation of Streptococcus pneumoniae

Summary DNA transport in Streptococcus pneumoniae was studied using donor molecules labelled either at the 3 or at the 5 end, on one strand only. In contrast to 5 end label, 3 end label was not taken up by the cells indicating that entry is a polarized process. Our results together with those of previous studies are consistent with a model for entry in which double-stranded donor DNA is nicked on binding at the cell surface. Entry of a single strand then proceeds linearly from a newly formed 3 end to the extremity of the donor fragment.     

Site-specific recombination events mediated by the DNA invertase Cin of bacteriophage P1 during transformation

Abstract Bacteriophage P1 encodes the site-specific recombinase Cin which promotes inversion of the C segment, thus controlling the P1 host range. Cin can also mediate inefficient inversion between the normal crossover site cixL and a quasi-crossover site cixQ 1 in inverted orientation. Inversion between cixL and cixQ 1 occurs more frequently in a short period of time after transformation with a plasmid carrying the cin gene, cixL and cixQ 1 than in an established transformant of the plasmid. This is also the case for Cin-mediated deletion on a plasmid containing the cin gene and directly repeated cix sites.     

Genome dynamics in major bacterial pathogens

Pathogenic bacteria continuously encounter multiple forms of stress in their hostile environments, which leads to DNA damage. With the new insight into biology offered by genome sequences, the elucidation of the gene content encoding proteins provides clues toward understanding the microbial lifestyle related to habitat and niche. Campylobacter jejuni, Haemophilus influenzae, Helicobacter pylori, Mycobacterium tuberculosis , the pathogenic Neisseria, Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus are major human pathogens causing detrimental morbidity and mortality at a global scale. An algorithm for the clustering of orthologs was established in order to identify whether orthologs of selected genes were present or absent in the genomes of the pathogenic bacteria under study. Based on the known genes for the various functions and their orthologs in selected pathogenic bacteria, an overview of the presence of the different types of genes was created. In this context, we focus on selected processes enabling genome dynamics in these particular pathogens, namely DNA repair, recombination and horizontal gene transfer. An understanding of the precise molecular functions of the enzymes participating in DNA metabolism and their importance in the maintenance of bacterial genome integrity has also, in recent years, indicated a future role for these enzymes as targets for therapeutic intervention.     

Bacillus subtilis RadA/Sms contributes to chromosomal transformation and DNA repair in concert with RecA and circumvents replicative stress in concert with DisA

Bacillus subtilis radA is epistatic to disA and recA genes in response to methyl methane sulfonate- and 4-nitroquinoline-1-oxide-induced DNA damage. We show that ΔradA cells were sensitive to mitomycin C- and H₂O₂-induced damage and impaired in natural chromosomal transformation, whereas cells lacking DisA were not. RadA/Sms mutants in the conserved H1 (K104A and K104R) or KNRFG (K255A and K255R) motifs fail to rescue the sensitivity of ΔradA in response to the four different DNA damaging agents. A RadA/Sms H1 or KNRFG mutation impairs both chromosomal and plasmid transformation, but the latter defect was suppressed by inactivating RecA. RadA/Sms K255A, K255R and wild type RadA/Sms reduced the diadenylate cyclase activity of DisA, whereas RadA/Sms K104A and K104R blocked it. Single-stranded and Holliday junction DNA are preferentially bound over double-stranded DNA by RadA/Sms and its variants. Moreover, RadA/Sms ATPase activity was neither stimulated by a variety of DNA substrates nor by DisA. RadA/Sms possesses a 5´→3´ DNA helicase activity. The RadA/Sms mutants neither hydrolyze ATP nor unwind DNA. Thus, we propose that RadA/Sms has two activities: to modulate DisA and to promote RecA-mediated DNA strand exchange. Both activities are required to coordinate responses to replicative stress and genetic recombination.     

Plasmid transformation of naturally competent Acinetobacter calcoaceticus in non-sterile soil extract and groundwater

The natural transformation of Acinetobacter calcoaceticus BD413 (trp E27) was characterized with respect to features that might be important for a possible gene transfer by extracellular DNA in natural environments. Transformation of competent cells with chromosomal DNA (marker trp ⁺) occurred in aqueous solutions of single divalent cations. Uptake of DNA into the DNase I-resistant state but not the binding of DNA to cells was strongly stimulated by divalent cations. An increase of transformation of nearly 3 orders of magnitude was obtained as a response to the presence of 0.25 mM Ca²⁺. With CaCl₂ solutions the transformation frequencies approached the highest values obtained under standard broth conditions, followed by MnCl₂ and MgCl₂. It is concluded that transformation requires divalent cations. DNA competition experiments showed that A. calcoaceticus does not discriminate between homologous and heterologous DNA. Furthermore, circular plasmid DNA competed with chromosomal DNA fragments and vice versa. The equally efficient transformation with plasmid pKT210 isolated from A. calcoaceticus or Escherichia coli indicated absence of DNA restriction in transformation. High efficiency plasmid transformation was obtained in samples of non-sterile natural groundwater and in non-sterile extracts of fresh and air-dried soil. Heat-treatment (10 min, 80°C) of the non-sterile liquid samples increased transformation only in the dried soil extract, probably by inactivation of DNases. The results presented suggest that competent cells of A. calcoaceticus can take up free high molecular weight DNA including plasmids of any source in natural environments such as soil, sediment or groundwater.     

Insertional mutagenesis in Bacillus subtilis: mechanism and use in gene cloning

The plasmid pHV32, which replicates in Escherichia coli but not in Bacillus subtilis, transformed B. subtilis-competent cells efficiently when linked in vitro to EcoRI B. subtilis DNA segments. The transformed clones carried pHV32 inserted in their chromosomes, and often displayed a mutant phenotype. One of the transformed clones carried pHV32 inserted close to the thyB gene. We cleaved the DNA extracted from this clone with BglII restriction endonuclease, for which no sites exist on pHV32, ligated the released segments and used them to transform E. coli selecting for pHV32-carried genetic markers. The transformants harbored a hybrid plasmid which carried the B. subtilis thyB gene. Circular molecules composed of pHV32 joined to B. subtilis DNA inserted into the chromosome by a Campbell-like recombination event. Linear molecules, in which pHV32 was flanked by two non-adjacent DNA segments, underwent a double cross-over recombination with the chromosome. In this case the chromosomal sequences between the non-adjacent segments were deleted, and replaced by pHV32 sequences.     

The natural transformation of the soil bacteria Pseudomonas stutzeri and Acinetobacter sp. by transgenic plant DNA strictly depends on homologous sequences in the recipient cells

The nptII(+) gene present in the genome of transgenic potato plants transforms naturally competent cells of the soil bacteria Pseudomonas stutzeri and Acinetobacter BD413 (both harboring a plasmid with an nptII gene containing a small deletion) with the same high efficiency as nptII(+) genes on plasmid DNA (3x10(-5)-1x10(-4) transformants per nptII(+)) despite the presence of a more than 10(6)-fold excess of plant DNA. However, in the absence of homologous sequences in the recipient cells the transformation by nptII(+) dropped by at least about 10(8)-fold in P. stutzeri and 10(9)-fold in Acinetobacter resulting in the latter strain in < or =1x10(-13) transformants per nptII(+). This indicated a very low probability of non-homologous DNA fragments to be integrated by illegitimate recombination events during transformation.     

Sequential action of factors involved in natural competence for transformation of Neisseria gonorrhoeae

Abstract We previously identified and genetically characterized several factors essential for the natural competence of transformation in Neisseria gonorrhoeae . Here we analyse the sequential action of these factors and dissect the overall transformation process into three distinct steps, (i) the sequence-specific uptake of transforming DNA into a DNase-resistant state, (ii) the transfer of DNA to the cytosol and (iii) the processing and recombination of the incoming with the resident DNA. While two pilus-associated factors, PilE and PilC, were previously implicated in the early DNA uptake event, we show here that three competence factors unrelated to pilus biogenesis, ComA, ComL and Tpc, are not essential for DNA uptake and rather act in a subsequent step. The respective mutants, however, lack the characteristic nucleolytic processing observed with the incoming DNA in both wild-type and non-transformable RecA-deficient N. gonorrhoeae , indicating that they are blocked in the processing and/or the delivery of DNA to the cytoplasm. A hypothetical model proposing a sequential action of the known gonococcal competence factors is presented.     

Expression of thymidylate synthetase activity in Bacillus subtilis upon integration of a cloned gene from Escherichia coli

The gene from Escherichia coli encoding thymidylate synthetase was cloned in the plasmid pBR322. The resulting chimeric plasmid, pER2, was effective in transforming both E. coli and Bacillus subtilis to thymine prototrophy. Uncloned linear E. coli chromosomal DNA was unable to transform thymine-requiring strains of B. subtilis to thymine independence. Linearization of the chimeric plasmid, pER2, with restriction enzymes markedly diminished its ability to transform B. subtilis auxotrophs. The Thy+ transformants derived from the transformation of B. subtilis with pER2 DNA did not contain detectable extrachromosomal DNA as demonstrated by Southern hybridization patterns and centrifugation in CsCl gradients of DNA isolated from B. subtilis colonies transformed with the chimeric plasmid. We conclude that the DNA from the chimeric plasmid was integrated into the chromosome of B. subtilis, demonstrating that extensive homology is not required for the integration of foreign DNA. This is the first reported case of a gene from a Gram-negative bacterium functioning in a Gram-positive organism.     

Genetic and molecular events in transformation ofHaemophilus influenzae with plasmid RSF 0885 carrying cloned segments of chromosomal DNA

InHaemophilus influenzae genetic transformation for a plasmid marker is significantly increased when recombinant plasmid RSF 0885 DNA carrying chromosomal DNA segments is used instead of the plasmid DNA alone. Chromosomal DNA by itself, added even a few minutes after the addition of plasmid DNA to competent cells, stopped further uptake of the plasmid DNA. These observations are consistent with the idea that plasmid RSF 0885 contains a ‘degenerate’ version of the required eleven base-pair ‘uptake sequence’ inHaemophilus. The transformation activity of the recombinant plasmid DNA is recoverable after its entry into cells, although the specific biological activity of the re-isolated plasmid DNA is less than that of the parental recombinant plasmid DNA. Therec 1 gene function of the host is necessary for obtaining higher transformation frequencies with recombinant DNA from five different clones. The reduced transformation frequencies seen inrec 1 ^- strain is not all due to a permanent damage to the donor DNA since the recovered recombinant plasmid DNA from such cells can increase the transformation efficiency onrec 1 ⁺ strain.     

Plasmid DNA in a groundwater aquifer microcosm -adsorption, DNAase resistance and natural genetic transformation of Bacillus subtilis

Prokaryotes can exchange chromosomal and plasmid genes via extracellular DNA in a process termed genetic transformation. This process has been observed in the test tube for several bacterial species living in the environment but it is not clear whether transformation occurs in natural bacterial habitats. A major constituent of terrestrial environments are solid particles such as quartz, silt and clay, which have considerable surface areas and which make up the solid-liquid interfaces of the habitat. In previous experiments the adsorption of DNA to chemically purified quartz and clay minerals was shown and the partial protection of adsorbed DNA against DNAase I. In a microcosm consisting of natural groundwater aquifer material (GWA) sampled directly from the environment and groundwater (GW) both linear duplex and supercoiled plasmid DNA molecules bound rapidly and quantitatively to the minerals. The divalent cations required to form the association were those present in the GWA/GW microcosm. The association was stable to extended elution over one week at 23°C. Upon adsorption, the DNA became highly resistant against enzymatic degradation. About 1000 times higher DNAase I concentrations were needed to degrade bound DNA to the same extent as DNA dissolved in GW. Furthermore, chromosomal and plasmid DNA bound on GWA transformed competent cells of Bacillus subtilis. However, in contrast to DNA in solution, on GWA the chromosomal DNA was more active in transformation than the plasmid DNA. The studies also revealed that in the transformation of B. subtilis Mg²⁺ can be replaced by Na⁺, K⁺ or NH₄ The observations suggest that in soil and sediment environments, mineral material with inorganic precipitates and organic matter can harbour extracellular DNA leaving it available for genetic transformation.     

Genetic transformation of Brassica campestris var. rapa protoplasts with an engineered cauliflower mosaic virus genome

Summary A hybrid Cauliflower Mosaic Virus (CaMV) genome containing a selectable marker gene was constructed by replacing the gene VI coding region with the aminoglycoside (neomycin) phosphotransferase type II [APH(3)II] gene from Tn5. This modified viral genome was tested for its infectivity both in planta and in a protoplast transformation system of Brassica campestris var. rapa. Stable, genetically transformed cell lines of B. campestris var. rapa were obtained after transformation. DNA of the hybrid CaMV genome was found to be integrated into high molecular weight plant genomic DNA. Transformation was achieved only when the hybrid genome was supplied together with wild type viral DNA. A possible complementation of the modified CaMV genome with the wild type viral DNA as a helper molecule in planta and in the protoplast system is discussed.     

Interacting proteins Rtt109 and Vps75 affect the efficiency of non-homologous end-joining in Saccharomyces cerevisiae

One of the key pathways for DNA double-stranded break (DSB) repair is the non-homologous end-joining (NHEJ) pathway, which directly re-ligates two broken ends of DNA. Using a plasmid repair assay screen, we identified that the deletion strain for RTT109 had a reduced efficiency for NHEJ in yeast. This deletion strain also had a reduced efficiency to repair induced chromosomal DSBs in vivo. Tandem-affinity purification of Rtt109 recovered Vps75 as a physical interacting protein. Deletion of VPS75 was also shown to have an effect on the efficiency of NHEJ in both the plasmid repair and the chromosomal repair assays. In addition, deletion mutants for both RTT109 and VPS75 showed hypersensitivity to different DNA damaging agents. Our genetic interaction analysis supports a role for RTT109 in DNA damage repair. We propose that one function of the Rtt109-Vps75 interacting protein pair is to affect the efficiency of NHEJ in yeast. Vps75 but not Rtt109 also seem to have an effect on the efficiency of DSB repair using homologous recombination.