On the track of natural transformation in soil

Abstract The understanding of microbial gene transfer including how bacteria acquire and disseminate genes in natural environments will provide data on the role of horizontal transfer in evolution. This understanding has been stimulated in recent years by concern about the impact of genetically engineered microorganisms on natural environments. This prospect has increased interest in determining the regulatory mechanisms of indigenous microbial populations as well as detecting genetic interactions between bacteria introduced into soil and the indigenous microflora. This paper will review the strategies developed to demonstrate whether the different steps required by natural bacterial transformation (the uptake of naked DNA by competent bacteria) could actually occur in soil. This will include a review on the release of DNA from microbial cells by passive or active mechanisms, its persistence by adsorption of extracellular DNA onto major soil components such as sand or clay minerals and the uptake of DNA by competent bacteria.     

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。     

Evidence for the active role of a novel nuclease from Helicobacter pylori in the horizontal transfer of genetic information

Helicobacter pylori is a gram-negative bacterium that colonizes the human stomach, causes gastritis, and is associated with ulcers and gastric cancer. H. pylori is naturally competent for transformation. Natural genetic transformation is believed to be essential for the genetic plasticity observed in this species. While the relevance of horizontal gene transfer in H. pylori adaptiveness and antibiotic resistance is well documented, the DNA transformation machinery components are barely known. No enzymatic activity associated with the transformation process has been determined experimentally and described. We isolated, microsequenced, and cloned a major DNA nuclease from H. pylori. This protein, encoded by the open reading frame hp0323, was expressed in Escherichia coli. The purified protein, NucT, has a cation-independent thermostable nuclease activity that preferentially cleaves single-stranded DNA. NucT is associated with the membrane. NucT-deficient H. pylori strains are one or more orders of magnitude less efficient than the parental strain for transformation with either chromosomal or self-replicating plasmid DNA. To the best of our knowledge, NucT is the first nuclease identified in a gram-negative natural transformation system, and its existence suggests that there is a mechanism of DNA processing and uptake similar to the mechanisms in well-studied gram-positive systems.     

Plasmid transfer to indigenous marine bacterial populations by natural transformation

Abstract Horizontal gene transfer among microbial populations has been assumed to occur in the environment, yet direct observations of this phenomenon are rare or limited to observations where the mechanism(s) could not be explicitly determined. Here we demonstrate the transfer of exogenous plasmid DNA to members of indigenous marine bacterial populations by natural transformation, the first report of this process for any natural microbial community. Ten percent of marine bacterial isolates examined were transformed by plasmid DNA while 14% were transformed by chromosomal DNA. Transformation of mixed marine microbial assemblages was observed in 5 of 14 experiments. In every case, acquisition of the plasmid by members of the indigenous flora was accompanied by modification (probably from genetic rearrangement or methylation) that altered its restriction enzyme digestion pattern. Estimation of transformation rates in estuarine environments based upon the distribution of competency and transformation frequencies in isolates and mixed populations ranged from 5 × 10⁻⁴ to 1.5 transformants/1 day. Extrapolation of these rates to ecosystem scales suggests that natural transformation may be an important mechanism for plasmid transfer among marine bacterial communities.     

Shuffling genes around in hot environments: the unique DNA transporter of Thermus thermophilus

Natural transformation permits the transport of DNA through bacterial membranes and represents a dominant mode for the transfer of genetic information between bacteria and between microorganisms of distant evolutionary lineages and even between members of different domains. This phenomenon, known as horizontal, or lateral, gene transfer, has been a major force for genome plasticity over evolutionary history, and is largely responsible for the spread of fitness-enhancing traits, including antibiotic resistance and virulence factors. In particular, for adaptation of prokaryotes to extreme environments, lateral gene transfer seems to have played a crucial role. Here, we present a survey of the natural transformation machinery of the thermophile Thermus thermophilus HB27. A tentative model of the transformation machinery comprising of components similar to proteins of type IV pili and type II secretion systems is presented. A comparative discussion of the subunits and the structure of the DNA translocator and the underlying mechanism of transfer of free DNA in T. thermophilus highlights conserved and unique features of the DNA translocator in T. thermophilus . We hypothesize that the extraordinary broad substrate specificity and the high efficiency of the T. thermophilus DNA uptake system is of major importance for thermoadaptation and interdomain DNA transfer in hot environments.     

Type IV pili-related natural transformation systems: DNA transport in mesophilic and thermophilic bacteria

Horizontal gene flow is a driving force for bacterial adaptation. Among the three distinct mechanisms of gene transfer in bacteria, conjugation, transduction, and transformation, the latter, which includes competence induction, DNA binding, and DNA uptake, is perhaps the most versatile mechanism and allows the incorporation of free DNA from diverse bacterial species. Here we review DNA transport machineries mediating uptake of naked DNA in gram-positive and gram-negative bacteria. Different putative models of transformation machineries comprising components similar to proteins of type IV pili are presented. Emphasis is placed on a comparative discussion of the underlying mechanisms of DNA transfer in mesophilic and extremely thermophilic bacteria, highlighting conserved and distinctive features of these transformation machineries.     

Intergeneric natural plasmid transformation between E. coli and a marine Vibrio species

Natural transformation is the mechanism of procaryotic gene transfer that involves the uptake and expression of genetic information encoded in extracellular DNA. This process has been regarded as a mechanism to transfer genes (primarily chromosomal markers) between closely related strains or species. Here we demonstrate the cell-contact-dependent transfer of a non-conjugative plasmid from a laboratory E. coli strain to a marine Vibrio species, the first report of intergeneric natural plasmid transformation involving a marine bacterium. The nucleic acid synthesis inhibitors nalidixic acid and rifampicin inhibited the ability of the E. coli to function as a donor. However, dead cells also served as efficient donors. There was an obligate requirement for cell contact. No transfer occurred in the presence of DNase I, when donors and recipients were separated by a 0.2-micron filter, or when spent medium alone was used as a source of transforming DNA. These results indicate that contact-mediated intergeneric plasmid exchange can occur in the absence of detectable viable donor cells and that small non-conjugative plasmids can be spread through heterogeneous microbial communities by a process previously not recognized, natural plasmid transformation. These findings are important in the assessment of genetic risk to the environment, particularly from wastewater treatment systems and the use of genetically engineered organisms in the environment.     

Functional characterization of the competence protein DprA/Smf in Escherichia coli

In several bacterial species that show natural transformation, dprA has been described as a competence gene. The DprA protein has been suggested to be involved in the protection of incoming DNA. However, members of the dprA gene family (also called smf) can be detected in virtually all bacterial species, which suggests that their gene products have a more general function. We examined the function of the DprA/Smf homologue of Escherichia coli. Escherichia coli dprA/smf is able to partially restore transformation in a Haemophilus influenzae dprA mutant, which shows that dprA/smf genes from competent and noncompetent species are interchangeable with respect to their involvement in natural transformation. From this, we conclude that natural transformation is probably an additional function of these genes. Subsequently, the dprA/smf gene was deleted in various recombination mutants of E. coli, and the resultant phenotype was tested. All the resultant E. coli dprA/smf mutants did not differ from their parent strains with respect to transformation, Hfr-conjugation, recombination and DNA repair. Therefore, a role of DprA/Smf in DNA recombination could not be established and the basic function of dprA/smf remains unclear.     

Characterization of Helicobacter pylori factors that control transformation frequency and integration length during inter-strain DNA recombination

Helicobacter pylori is a genetically diverse bacterial species, owing in part to its natural competence for DNA uptake that facilitates recombination between strains. Inter-strain DNA recombination occurs during human infection and the H. pylori genome is in linkage equilibrium worldwide. Despite this high propensity for DNA exchange, little is known about the factors that limit the extent of recombination during natural transformation. Here, we identify restriction-modification (R-M) systems as a barrier to transformation with homeologous DNA and find that R-M systems and several components of the recombination machinery control integration length. Type II R-M systems, the nuclease nucT and resolvase ruvC reduced integration length whereas the helicase recG increased it. In addition, we characterized a new factor that promotes natural transformation in H. pylori, dprB. Although free recombination has been widely observed in H. pylori, our study suggests that this bacterium uses multiple systems to limit inter-strain recombination.     

Regulation of natural genetic transformation and acquisition of transforming DNA in Streptococcus pneumoniae

The ability of pneumococci to take up naked DNA from the environment and permanently incorporate the DNA into their genome by recombination has been exploited as a valuable research tool for 80 years. From being viewed as a marginal phenomenon, it has become increasingly clear that horizontal gene transfer by natural transformation is a powerful mechanism for generating genetic diversity, and that it has the potential to cause severe problems for future treatment of pneumococcal disease. This process constitutes a highly efficient mechanism for spreading β-lactam resistance determinants between streptococcal strains and species, and also threatens to undermine the effect of pneumococcal vaccines. Fortunately, great progress has been made during recent decades to elucidate the mechanism behind natural transformation at a molecular level. Increased insight into these matters will be important for future development of therapeutic strategies and countermeasures aimed at reducing the spread of hazardous traits. In this review, we focus on recent developments in our understanding of competence regulation, DNA acquisition and the role of natural transformation in the dissemination of virulence and β-lactam resistance determinants.     

Bacterial conjugation: everybody's doin' it.

Recent evidence suggests that bacterial conjugation is ubiquitous in the bacterial world and that DNA transfer between different genera-kingdoms is possible. It has also been demonstrated that many bacterial gene transfer systems resemble each other at the molecular level and that others are a blend of two or more of these systems. Thus, in the absence of a sexual cycle, bacterial conjugation, along with bacteriophages, transposons, and natural transformation systems, forms a potent force for gene dissemination and repair in the eubacteria and simple eukaryotes.     

DNA Transport and Natural Transformation in Mesophilic and Thermophilic Bacteria

Comparative genome analyses revealed a massive DNA exchange between microbes of distant evolutionary lineages. This phenomenon known as horizontal, or lateral, gene transfer has a tremendous impact in the evolution of prokaryotes. Here, the process of DNA transport via genetic transformation is discussed. This review will focus on the process of DNA uptake mediated by type IV pilin-like proteins in Gram-positive and Gram-negative bacteria. Three tentative models of transformation machineries comprising components similar to proteins of type IV pili and type II secretion are presented. A comparative discussion of the structure of DNA translocators and the underlying mechanism of transfer of free DNA in mesophilic and extremely thermophilic bacteria highlights conserved and distinctive features of the DNA translocators in mesophilic and thermophilic bacteria.     

Plasmid and chromosomal gene transfer in Bacillus subtilis and Escherichia coli in natural transformation

The transfer of hDHFR gene by natural transformation between Escherichia coli and Bacillus subtilis cells has been studied, as well as intrageneric gene transfer in Bacillus subtilis. The gene was transferred by natural transformation in bacterial cells and included into the chromosome or the plasmid.     

Adaptation through genetic time travel? Fluctuating selection can drive the evolution of bacterial transformation

Natural transformation is a process whereby bacteria actively take up DNA from the surrounding environment and incorporate it into their genome. Natural transformation is widespread in bacteria, but its evolutionary significance is still debated. Here, we hypothesize that transformation may confer a fitness advantage in changing environments through a process we term ‘genetic time travel’: by taking up old genes that were retained in the environment, the bacteria may revert to a past genotypic state that proves advantageous in the present or a future environment. We scrutinize our hypothesis by means of a mathematical model involving two bacterial types (transforming and non-transforming), a single locus under natural selection and a free DNA pool. The two bacterial types were competed in environments with changing selection regimes. We demonstrate that for a wide range of parameter values for the DNA turnover rate, the transformation rate and the frequency of environmental change, the transforming type outcompetes the non-transforming type. We discuss the empirical plausibility of our hypothesis, as well as its relationship to other hypotheses for the evolution of transformation in bacteria and sex more generally, speculating that ‘genetic time travel’ may also be relevant in eukaryotes that undergo horizontal gene transfer.     

Growth phase-specific evolutionary benefits of natural transformation in Acinetobacter baylyi

Natural transformation in bacteria facilitates the uptake and genomic integration of exogenous DNA. This allows horizontal exchange of adaptive traits not easily achieved by point mutations, and has a major role in the acquisition of adaptive traits exemplified by antibiotic resistance determinants and vaccination escape. Mechanisms of DNA uptake and genomic integration are well described for several naturally transformable bacterial species; however, the selective forces responsible for its evolution and maintenance are still controversial. In this study we evolved transformation-proficient and -deficient Acinetobacter baylyi for 175 days in serial transfer cultures where stress was included. We found that natural transformation-proficient populations adapted better to active growth and early stationary phase. This advantage was offset by the reduced performance in the late stationary/death phase. We demonstrate fitness trade-offs between adaptation to active growth and survival in stationary/death phase caused by antagonistic pleiotropy. The presented data suggest that the widely held assumption that recombination speeds up adaptation by rapid accumulation of multiple adaptive mutations in the same genetic background is not sufficient to fully account for the maintenance of natural transformation in bacteria.     

Cj0011c, a periplasmic single- and double-stranded DNA-binding protein, contributes to natural transformation in Campylobacter jejuni

Campylobacter jejuni is an important bacterial pathogen causing gastroenteritis in humans. C. jejuni is capable of natural transformation, which is considered a major mechanism mediating horizontal gene transfer and generating genetic diversity. Despite recent efforts to elucidate the transformation mechanisms of C. jejuni, the process of DNA binding and uptake in this organism is still not well understood. In this study, we report a previously unrecognized DNA-binding protein (Cj0011c) in C. jejuni that contributes to natural transformation. Cj0011c is a small protein (79 amino acids) with a partial sequence homology to the C-terminal region of ComEA in Bacillus subtilis. Cj0011c bound to both single- and double-stranded DNA. The DNA-binding activity of Cj0011c was demonstrated with a variety of DNAs prepared from C. jejuni or Escherichia coli, suggesting that the DNA binding of Cj0011c is not sequence dependent. Deletion of the cj0011c gene from C. jejuni resulted in 10- to 50-fold reductions in the natural transformation frequency. Different from the B. subtilis ComEA, which is an integral membrane protein, Cj0011c is localized in the periplasmic space of C. jejuni. These results indicate that Cj0011c functions as a periplasmic DNA receptor contributing to the natural transformation of C. jejuni.     

Genetics of ectomycorrhizal fungi: progress and prospects

The variability within and among ectomycorrhizal species provides a substantial genetic resource and the potential to increase forest productivity and environmental sustainability. Two parallel and interacting approaches, classical and molecular genetics, are being developed to acquire the genetic information underpinning selection of improved ectomycorrhizal strains. Determining the genetic traits of the fungi which contribute to symbiosis and plant function are being followed using natural variability combined with classical and molecular genetic manipulations. Classical and molecular manipulations for breeding rely on key information including sexual and parasexual reproduction, postmeiotic nuclear behaviour, mating-types and vegetative incompatibility mechanisms. Progress in the manipulation of genomes of ectomycorrhizal fungi will depend on efficient methods for gene cloning and DNA transformation. Gene transfer into fungal cells have been shown to be successful and include treatment of protoplasts and intact mycelium with naked DNA in the presence of polyvalent cations, electroporation, and microbombardment. The merits and limitations of these methods are discussed. Using this technology the expression of foreign DNA, the functional analysis of fungal DNA sequences, as well as molecular exploitation for commercial purposes can be carried out. This review concentrates on these aspects of fungal molecular biology and discusses the applications of the experimental systems that are currently available to ectomycorrhizal fungi. As it is essential to be able to define the traits which a breeder is seeking to improve, availability of genetically defined strains that are isogenic for a character or differ only in one character and a thorough knowledge of the biochemistry of the symbiosis will be necessary before any genetic manipulation be carried out. Genetic variability of ectomycorrhizal strains has been assessed by DNA fingerprinting. This approach allows the evaluation of DNA variability and the exchange of genetic information in natural populations, the identification of species and isolates by DNA polymorphisms, and tracking the environmental fate of the introduced fungi to determine their survival, growth, and dissemination within the soil.     

Agrobacterium-Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool

Agrobacterium tumefaciens and related Agrobacterium species have been known as plant pathogens since the beginning of the 20th century. However, only in the past two decades has the ability of Agrobacterium to transfer DNA to plant cells been harnessed for the purposes of plant genetic engineering. Since the initial reports in the early 1980s using Agrobacterium to generate transgenic plants, scientists have attempted to improve this “natural genetic engineer” for biotechnology purposes. Some of these modifications have resulted in extending the host range of the bacterium to economically important crop species. However, in most instances, major improvements involved alterations in plant tissue culture transformation and regeneration conditions rather than manipulation of bacterial or host genes. Agrobacterium-mediated plant transformation is a highly complex and evolved process involving genetic determinants of both the bacterium and the host plant cell. In this article, I review some of the basic biology concerned with Agrobacterium-mediated genetic transformation. Knowledge of fundamental biological principles embracing both the host and the pathogen have been and will continue to be key to extending the utility of Agrobacterium for genetic engineering purposes.