Bacterial gene transfer by natural genetic transformation in the environment.

Natural genetic transformation is the active uptake of free DNA by bacterial cells and the heritable incorporation of its genetic information. Since the famous discovery of transformation in Streptococcus pneumoniae by Griffith in 1928 and the demonstration of DNA as the transforming principle by Avery and coworkers in 1944, cellular processes involved in transformation have been studied extensively by in vitro experimentation with a few transformable species. Only more recently has it been considered that transformation may be a powerful mechanism of horizontal gene transfer in natural bacterial populations. In this review the current understanding of the biology of transformation is summarized to provide the platform on which aspects of bacterial transformation in water, soil, and sediments and the habitat of pathogens are discussed. Direct and indirect evidence for gene transfer routes by transformation within species and between different species will be presented, along with data suggesting that plasmids as well as chromosomal DNA are subject to genetic exchange via transformation. Experiments exploring the prerequisites for transformation in the environment, including the production and persistence of free DNA and factors important for the uptake of DNA by cells, will be compiled, as well as possible natural barriers to transformation. The efficiency of gene transfer by transformation in bacterial habitats is possibly genetically adjusted to submaximal levels. The fact that natural transformation has been detected among bacteria from all trophic and taxonomic groups including archaebacteria suggests that transformability evolved early in phylogeny. Probable functions of DNA uptake other than gene acquisition will be discussed. The body of information presently available suggests that transformation has a great impact on bacterial population dynamics as well as on bacterial evolution and speciation.     

Study of the epidemic significance of noncultivated forms of Vibrio cholerae by the polymerase chain reaction]

A specific method of the isolation of the cholera toxin gene by the directional amplification of DNA in the polymerase chain reaction (PCR) has been developed. The product of this reaction has a molecular weight of 440 sequence pairs and is a DNA fragment located on the A-subunit of V. cholerae gene vct. The sensitivity of the method permits the detection of one bacterial cell in the reaction mixture. The method is effective when V. cholerae purified DNA, cell lysates and the DNA of total microflora isolated from the water of natural springs are used. The study of water samples from natural water bodies by the method of PRC has revealed cholera toxin genes of V. cholerae noncultivated forms ni 5 out of 7 water samples taken from natural water bodies at the regions of Azerbaijan endemic for cholera and made it possible to evaluate the number of V. cholerae. The prospects of using PCR for the control of the epidemiological situation in regions endemic for cholera are discussed.     

DNA enzyme generated by a novel single-stranded DNA expression vector inhibits expression of the essential bacterial cell division gene ftsZ

Rapid emergence of antibiotic-resistant bacterial pathogens has created urgent demand for the discovery and development of new antibacterial agents directed toward novel targets. Antisense oligodeoxynucleotides (AS-ODN) and their modified forms have been utilized to block gene expression in bacterial cells, showing potential for developing highly specific and efficacious antibacterial agents. In this study, a tetracycline-regulated expression vector was developed for generating single-stranded DNA (ssDNA) of a desired target sequence in bacterial cells. This inducible ssDNA expression vector was tested for producing a DNA enzyme designed to specifically cleave ftsZ mRNA. Our results indicate that the expressed DNA enzyme molecules not only repress ftsZ gene expression and but also inhibit bacterial cell proliferation. Although we believe that the cleavage of ftsZ mRNA by the expressed DNA enzyme molecules is responsible for the inhibitory effects on ftsZ gene expression and bacterial cell proliferation, the antisense mechanism could also be responsible for the biological effects. The ability of this ssDNA expression system to selectively modulate gene expression may provide a powerful strategy in determining the contribution of a given gene product to bacterial growth or pathogenesis and opens a new venue for developing antibacterial agents.     

Potential Human Pathogenic Bacteria in a Mixed Urban Watershed as Revealed by Pyrosequencing

Current microbial source tracking (MST) methods for water depend on testing for fecal indicator bacterial counts or specific marker gene sequences to identify fecal contamination where potential human pathogenic bacteria could be present. In this study, we applied 454 high-throughput pyrosequencing to identify bacterial pathogen DNA sequences, including those not traditionally monitored by MST and correlated their abundances to specific sources of contamination such as urban runoff and agricultural runoff from concentrated animal feeding operations (CAFOs), recreation park area, waste-water treatment plants, and natural sites with little or no human activities. Samples for pyrosequencing were surface water, and sediment collected from 19 sites. A total of 12,959 16S rRNA gene sequences with average length of ≤400 bp were obtained, and were assigned to corresponding taxonomic ranks using ribosomal database project (RDP), Classifier and Greengenes databases. The percent of total potential pathogens were highest in urban runoff water (7.94%), agricultural runoff sediment (6.52%), and Prado Park sediment (6.00%), respectively. Although the numbers of DNA sequence tags from pyrosequencing were very high for the natural site, corresponding percent potential pathogens were very low (3.78–4.08%). Most of the potential pathogenic bacterial sequences identified were from three major phyla, namely, Proteobacteria, Bacteroidetes, and Firmicutes. The use of deep sequencing may provide improved and faster methods for the identification of pathogen sources in most watersheds so that better risk assessment methods may be developed to enhance public health.     

Use of suppressor subtraction hybridization for finding strain-specific sequences in bacterial genomes

Comparisons of bacterial genomes demonstrate that even strains of one species may strikingly differ in gene set. Strain-specific genes are of considerable interest, as they may be responsible for distinguishing features, such as virulence or drug resistance, of the strain and may be employed as markers in epidemiological or evolutionary studies. Suppression subtractive hybridization (SSH) was shown to be suitable for generating a set of DNA fragments differing between two closely related bacterial strains. More than 95% DNA fragments selected by SSH proved to be specific for Staphylococcus aureus strains ZW compared with strain 29213.     

Use of DNA probes in the study of silage colonization by Lactobacillus and Pediococcus strains

A technique to monitor lactic acid bacteria inoculants in silage, based on specific DNA probes, was developed and used to evaluate the colonization properties of two strains of Lactobacillus plantarum and one strain of Pediococcus pentosaceus which were used as maize silage inoculants in farm conditions. The results indicated that these three strains were able to dominate the natural microflora of the silage, representing more than the 95% of the bacterial biomass of the maize silage. These studies indicate that the colony hybridization with specific DNA probes may be an effective method for monitoring bacteria and evaluating the colonization properties of inoculants in maize silage.     

The Use of Suppression Subtractive Hybridization to Identify Strain-Specific Sequences in Bacterial Genomes

Comparisons of bacterial genomes demonstrate that even strains of one species may strikingly differ in gene set. Strain-specific genes are of considerable interest, as they may be responsible for distinguishing features, such as virulence or drug resistance, of the strain and may be employed as markers in epidemiological or evolutionary studies. Suppression subtractive hybridization (SSH) was shown to be suitable for generating a set of DNA fragments differing between two closely related bacterial strains. More than 95% DNA fragments selected by SSH proved to be specific for Staphylococcus aureus strains ZW compared with strain 29213.     

Effects of physiological self-crowding of DNA on shape and biological properties of DNA molecules with various levels of supercoiling

DNA in bacterial chromosomes and bacterial plasmids is supercoiled. DNA supercoiling is essential for DNA replication and gene regulation. However, the density of supercoiling in vivo is circa twice smaller than in deproteinized DNA molecules isolated from bacteria. What are then the specific advantages of reduced supercoiling density that is maintained in vivo? Using Brownian dynamics simulations and atomic force microscopy we show here that thanks to physiological DNA–DNA crowding DNA molecules with reduced supercoiling density are still sufficiently supercoiled to stimulate interaction between cis-regulatory elements. On the other hand, weak supercoiling permits DNA molecules to modulate their overall shape in response to physiological changes in DNA crowding. This plasticity of DNA shapes may have regulatory role and be important for the postreplicative spontaneous segregation of bacterial chromosomes.     

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.     

Exploration of horizontal gene transfer between transplastomic tobacco and plant-associated bacteria

The likelihood of gene transfer from transgenic plants to bacteria is dependent on the transgene copy number and on the presence of homologous sequences for recombination. The large number of chloroplast genomes in a plant cell as well as the prokaryotic origin of the transgene may thus significantly increase the likelihood of gene transfer from transplastomic plants to bacteria. In order to assess the probability of such a transfer, bacterial isolates, screened for their ability to colonize decaying tobacco plant tissue and possessing DNA sequence similarity to the chloroplastic genes accD and rbcL flanking the transgene (aadA), were tested for their ability to take up extracellular DNA (broad host-range pBBR1MCS-3-derived plasmid, transplastomic plant DNA and PCR products containing the genes accD-aadA-rbcL) by natural or electrotransformation. The results showed that among the 16 bacterial isolates tested, six were able to accept foreign DNA and acquire the spectinomycin resistance conferred by the aadA gene on plasmid, but none of them managed to integrate transgenic DNA in their chromosome. Our results provide no indication that the theoretical gene transfer-enhancing properties of transplastomic plants cause horizontal gene transfer at rates above those found in other studies with nuclear transgenes.     

Molecular cloning of Renibacterium salmoninarum DNA fragments

A Renibacterium salmoninarum enriched recombinant DNA library was constructed to isolate DNA fragments which could be used as probes to detect gene sequences specific for the causative agent of bacterial kidney disease in salmonid fish. One fragment of 149 base pairs was isolated and its specificity and sequence determined. This probe may prove useful in the design of diagnostic tests for the disease in asymptomatic fish and ova.     

Environmental bacteriophage-host interactions: factors contribution to natural transduction

Over the past two decades the potential for the exchange of bacterial genes in natural environments through transduction (bacteriophage-mediated gene transfer) has been well established. Studies carried out by various laboratories throughout the world have demonstrated that both chromosomal and plasmid DNA can be successfully transduced in natural environments ranging from sewer plants to rivers and lakes. Transduction has been shown to take place in the gills of oysters and the kidneys of mice. Model studies have demonstrated the ability of transduction to maintain genetic material in bacterial gene pools that would otherwise be lost because of negative fitness. Thus, transduction may affect the course of bacterial evolution. Identification of natural transduction has led to the investigation of the dynamics of bacteriophage host interactions in natural aquatic environments and to the exploration of various environmental factors that affect virus-host interactions. Two important environmental factors which affect virus-host interactions are the metabolic state of the host and the exposure of the host to DNA-damaging stresses such as solar UV light. Recent researches on these two areas of virus-host relationships are reviewed.     

Rapid determination of bacterial ribosomal RNA sequences by direct sequencing of enzymatically amplified DNA

Ribosomal RNA sequences are an appealing target for bacterial classification as well as for development of group- or species-specific DNA probes. Using the polymerase chain reaction and synthetic primers, the feasibility of this gene amplification technique for rapid sequence determination of the major 16S ribosomal RNA domains from small amounts of input DNA is demonstrated. Information useful for phylogenetic classification as well as for construction of specific DNA probes may be obtained by comparison with known sequences.     

Phenylethynylpyrene-labeled oligonucleotide probes for excimer fluorescence SNP analysis of 23S rRNA gene in clarithromycin-resistant Helicobacter pylori strains

The use of phenylethynylpyrene excimer forming pair in the design of specific fluorescent probes for determination of A2144G (A2143G and/or A2143C) mutations in 23S rRNA gene of Helicobacter pylori is described. Analysis of fluorescence spectra of model duplexes revealed optimal positions of fluorophore residues in the probe sequences for maximum efficiency of SNP detection. Application of excimer forming probes for analysis of DNA samples isolated from natural bacterial strains of H. pylori was demonstrated.     

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.     

Use of DNA probes in the study of silage colonization by Lactobacillus and Pediococcus strains

P.S. COCCONCELLI, E. TRIBAN, M. BASSO AND V. BOTTAZZI. 1991. A technique to monitor lactic acid bacteria inoculants in silage, based on specific DNA probes, was developed and used to evaluate the colonization properties of two strains of Lactobacillus plantarum and one strain of Pediococcus pentosaceus which were used as maize silage inoculants in farm conditions. The results indicated that these three strains were able to dominate the natural microflora of the silage, representing more than the 95% of the bacterial biomass of the maize silage. These studies indicate that the colony hybridization with specific DNA probes may be an effective method for monitoring bacteria and evaluating the colonization properties of inoculants in maize silage.     

Preparation of a DNA gene probe for detection of mercury resistance genes in gram-negative bacterial communities.

A DNA gene probe was prepared to study genetic change mechanisms responsible for adaptation to mercury in natural bacterial communities. The probe was constructed from a 2.6-kilobase NcoI-EcoRI DNA restriction fragment which spans the majority of the mercury resistance operon (mer) in the R-factor R100. The range of specificity of this gene probe was defined by hybridization to the DNA of a wide variety of mercury-resistant bacteria previously shown to possess the mercuric reductase enzyme. All of the tested gram-negative bacteria had DNA sequences homologous to the mer probe, whereas no such homologies were detected in DNA of the gram-positive strains. Thus, the mer probe can be utilized to study gene flow processes in gram-negative bacterial communities.