Transformation and transfection in lysogenic strains of Bacillus subtilis: evidence for selective induction of prophage in competent cells.

Lysogenic strains of Bacillus subtilis 168 were reduced in their level of transformation as compared to non-lysogenic strains. The level of transformation decreased even further if the competent lysogenic cells were allowed to incubate in growth media prior to selection on minimal agar. This reduction in the frequency of transformation was attributable to the selective elimination of transformed lysogenic cells from the competent population. Concurrent with the decrease in the number of transformants from a lysogenic competent population was the release of bacteriophage by these cells. The lysogenic bacteria demonstrated this dramatic release of bacteriophage only if the cells were grown to competence. Both the selective elimination of transformed lysogens and the induction of prophage was prevented by the inhibition of protein synthesis. Additionally, competent lysogenic cells released significantly higher amounts of exogenous donor transforming deoxyribonucleic acid than did competent non-lysogenic cells or competent lysogenic cells incubated with erythromycin. These data establish that the induction of the prophage from the competent lysogenic cells was responsible for the selective elmination of the lysogenic transformants. A model is presented that accounts for the induction of the prophage from competent lysogenic bacteria via the induction of a repair system. It is postulated that a repair system is induced or derepressed by the accumulation of gaps in the chromosomes of competent bacteria. This hypothetical enzyme(s) is ultimately responsible for the induction of the prophage and the selective elimination of transformants.     

Population replacement strategies for controlling vector populations and the use of Wolbachia pipientis for genetic drive

In this video, Jason Rasgon discusses population replacement strategies to control vector-borne diseases such as malaria and dengue. "Population replacement" is the replacement of wild vector populations (that are competent to transmit pathogens) with those that are not competent to transmit pathogens. There are several theoretical strategies to accomplish this. One is to exploit the maternally-inherited symbiotic bacteria Wolbachia pipientis. Wolbachia is a widespread reproductive parasite that spreads in a selfish manner at the extent of its host's fitness. Jason Rasgon discusses, in detail, the basic biology of this bacterial symbiont and various ways to use it for control of vector-borne diseases.     

Competence-related increased enzyme release from Streptococcus sanguis (Wicky) cells.

The ablity of competent and noncompetent Streptococcus sanguis (strain Wicky) cells to release enzymes to the environment was studied. Both competent and noncompetent cells leaked the enzymes tested (aldolase, phosphatase and deoxyribonuclease), but the activities liberated from the competent cells were always roughly 2-fold higher than those released from noncompetent cells. This increased enzyme leakage from competent cells occured in all kinds of media and procedures employed. The leakage of enzymes followed a time-dependent kinetics (different for aldolase and phosphatase), was temperature sensitive and had a pH optimum. The increased enzyme release was most likely not due to cell disruption, but seemed to be rather a consequence of alteration in cell barrier permeability. These results strongly support the "unmasking" model proposed for explanation of competence development in bacteria.     

Repair of U.V. damages in Bacillus subtilis cultures competent for transformation: difference between competent and non-competent fractions.

The repair of U.V. damages to DNA in B. subtilis cultures competent for genetic transformation has been studied. The comparison of survival curves for competent and non competent fractions shows that: i) excision repair is more effective in competent than in non competent bacteria; ii) recombination repair is more effective in non competent than in competent bacteria. These facts support the hypothesis that metabolic conditions and, very likely, DNA replication play a role in the regulation of the efficiency of the two different mechanisms of repair.     

Antigen-initiated B lymphocyte differentiation: non-specific stimulation changes the physical properties of virgin AFC-progenitors in neonatal mouse spleen.

While the virgin AFC-progenitors for an adoptive immune response in neonatal germ-free CBA mouse spleen are small, dense cells, the equivalent cells in the adult are a larger, lighter density population. The effects of injections of unrelated antigens on the physical properties of the AFC-progenitors in neonatal spleen were investigated to test the postulate that the physically distinct "virgin" AFC-progenitors in the adult arose by a process of non-specific activation. Spleen cells from 7-day-old germ-free CBA mice were separated by sedimentation at unit gravity or by density on continuous albumin gradients, and the fractions were tested for NIP-specific AFC-progenitor activity using an adoptive immune assay which gave a direct linear measure of B cell activity. If the donor neonatal animals were injected one day previously with POL or PPD, the NIP-specific AFC-progenitor activity shifted from the typical small, dense lymphocytes to larger, lighter cells. The physical properties of these stimulated AFC-progenitors resembled those of IgM AFC-progenitors in normal adult mice. These results experimentally confirm the theory that environmental stimuli induce a non-specific "activation" of a particular subset of "virgin" B cells.     

Transformation of plasmid DNA into E. coli using the heat shock method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42 degrees C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37 degrees C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.     

Sequence identity of the n-1 product of a synthetic oligonucleotide.

After synthesis and purification of an oligonucleotide, the final product usually contains a low level of n-1 congeneric species. We have sequenced the n-1 population of a 25mer phosphodiester oligonucleotide. The n-1 band was cut from the gel and eluted. Oligonucleotides were tailed with dA and annealed to a dT-tailed plasmid. The recombinant plasmid was ligated and used to transform competent bacteria. Our results show that the n-1 population was heterogeneous. The frequency of truncated nucleotides at the 3'-end was much higher than at the 5'-end of the oligomer. No truncated nucleotides were found in the last four nucleotides at the 5'-end. Our results also show that the chain of oligonucleotides can grow on unreacted sites of a controlled-pore glass support.     

Monitoring temporal and spatial variation in rhizosphere bacterial population diversity: A community approach for the improved selection of rhizosphere competent bacteria

The potential for developing a reliable strategy for selecting rhizosphere competent bacteria, based on an improved understanding of the community diversity and population dynamics of fluorescent pseudomonads, was investigated. Isolates from a collection of over 690 fluorescent pseudomonads, obtained from sugar beet and wheat plants grown in field soils in laboratory microcosms, were genotypically and phenotypically characterised. RFLP rRNA analysis (ribotyping) revealed that the sampled population was composed of 385 related but distinct ribotypes. Most ribotypes were isolated only once and represented a transient colonising population. However, representatives of 26 ribotypes were detected more often, of which five were isolated from rhizosphere soils sampled 7 months after the first sampling. Comparative phenotypic analysis of isolates (motility, antibiotic resistance and production, adherence, fatty acid composition, substrate utilisation patterns) demonstrated that the ability to utilise organic acids as carbon sources correlated with rhizosphere competence. Single inoculum and competitive colonisation studies in planted microcosms confirmed rhizosphere competence, but also demonstrated synergistic interactions. The colonisation ability and population densities of transient strains were significantly increased when co-inoculated with rhizosphere competent isolates. These data demonstrate potential cross-feeding and combined niche exploitation, rather than direct competition, confirming the multi-factorial nature of rhizosphere competence in diverse fluorescent pseudomonad communities. They also highlight the need to consider the use of mixed inocula for plant growth promotion and the systematic selection of strains for effective biotechnological exploitation.     

DNA repair in Bacillus subtilis: excision repair capacity of competent cells.

Competent Bacillus subtilis were investigated for their ability to support the repair of UV-irradiated bacteriophage and bacteriophage DNA. UV-irradiated bacteriophage DNA cannot be repaired to the same level as UV-irradiated bacteriophage, suggesting a deficiency in the ability of competent cells to repair UV damage. However, competent cells were as repair proficient as noncompetent cells in their ability to repair irradiated bacteriophage in marker rescue experiments. The increased sensitivity of irradiated DNA is shown to be due to the inability of excision repair to function on transfecting DNA in competent bacteria. Furthermore, competent cells show no evidence of possessing an inducible BsuR restriction system to complement their inducible BsuR modification enzyme.     

Selection of Fimbriate Transductants of Salmonella typhimurium Dependent on Motility

The ability to form type 1 fimbriae (Fim(+)) was readily transduced to 159 out of 161 wild-type motile Fim(-) FIRN strains of Salmonella typhimurium with phage P22 propagated on a Fim(+) donor strain. Fim(+) clones were isolated from about 35% of tests after the fimbriate bacteria in the transduction mixture had been enriched by culture in aerobic static broth for 48 to 96 hr. A Fim(+) transductant was isolated from only 1 out of 280 tests made with 10 nonmotile recipient FIRN strains that were nonflagellate (Fla(-))- or possessed "paralyzed" flagella (Fla(+) Mot(-)), though motile variants from these strains were fully competent in yielding Fim(+) transductants. The property of motility was thought to facilitate the selective outgrowth of Fim(+) transductant bacteria by enabling them to migrate aerotactically to the surface of the broth where their fimbriae permitted them to float and grow in a pellicle stimulated by the free supply of atmospheric oxygen.     

Adaptive significance of the limited gene expression of the immune system

Evolutionary development of immunity multigene systems seems to involve host-parasite molecular coevolution, as evidenced by comparison of metasoans and unicellular organisms in fixation rates of neutral and adaptive point mutations per one pair of corresponding genes. The estimations we attempted revealed that organisms with maximal restriction of Ig-like gene repertoire expression by an individual immune cell are more adaptive in formation of competent antigen-induced immune response, provided that the magnitude of potential repertoire of functional Ig-like receptors exceeds lymphoid cell population size. In contrast, when phage and bacteria populations are in coevolution, there is no need to limit the repertoire of expressive genes encoding bacteria surface receptor proteins which are recognized by a phage adsorption system. Other hypotheses of evolutionary premises and stimulus of immune genes expression limitation (allelic, isotypic etc.) are critically discussed.     

Effect of lysogeny on transfection and transfection enhancement in Bacillus subtilis.

Strains of Bacillus subtilis 168 lysogenic for bacteriophage phi105 transfer with deoxyribonucleic acid (DNA) isolated from bacteriophage SPO2 at a higher efficiency than non-lysogenic strains. This enhancement of transfection was not the result of recombination between bacteriophages SPO2 and phi105. Superinfection marker rescue increased transfection with DNA from bacteriophage phi105 occurred simultaneously with the addition of the transfecting DNA. Again, this enhancement of transfection was not the result of recombination but rather a protection of the transfecting DNA by the superinfecting bacteriophage. The ability of the superinfecting bacteriophage to protect the transfecting DNA from inactivation was maximal when the bacteria were just becoming competent. Bacteriophage phi1 cannot replicate after the transfection of competent bacteria lacking a functional DNA replication system, whereas bacteriophage phi1 was able to replicate after infection of competent bacteria grown under comparable conditions. These observations support the hypothesis that GAPase and an inducible repair system play an important role in the development of competence.     

Thyroid hormone directly induces hepatocyte competence for estrogen-dependent vitellogenin synthesis during the metamorphosis of Xenopus laevis

Hepatocytes competent for estrogen-dependent vitellogenin synthesis appeared and increased in number in the liver at the metamorphic climax of Xenopus laevis (A. Kawahara, S. Kohara, Y. Sugimoto, and M. Amano, 1987, Dev. Biol. 122, 139-145). The present study was conducted to determine whether cells competent for vitellogenin synthesis could be induced by thyroid hormone in a primary culture of larval hepatocytes. The thyroid hormone, triiodothyronine (T3), directly induced the competent cells in a primary culture of premetamorphic larval hepatocytes in a dose- and duration-dependent manner. The competency acquired in response to T3 persisted after removal of the hormone. Aphidicholin, an inhibitor of DNA synthesis, failed to block this induction, suggesting the presence of a "precursor cell fraction." This cell fraction in the hepatocyte population increased with the progress of metamorphosis. The thyroid hormone is thus considered the cause of competent cell formation at metamorphic climax.     

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。     

A predatory mechanism dramatically increases the efficiency of lateral gene transfer in Streptococcus pneumoniae and related commensal species

Bacteria that are competent for natural genetic transformation, such as pneumococci and their commensal relatives Streptococcus mitis and Streptococcus oralis , take up exogenous DNA and incorporate it into their genomes by homologous recombination. Traditionally, it has been assumed that genetic material leaking from dead bacteria constitutes the sole source of external DNA for competent streptococci. Here we describe a mechanism for active acquisition of homologous DNA that dramatically increases the efficiency of gene exchange between and within the streptococcal species mentioned above. This mechanism gives competent streptococci access to a common gene pool that is significantly larger than their own genomes, a property representing a considerable advantage when these bacteria are subjected to external selection pressures, such as vaccination and treatment with antibiotics.     

Bovine follicular development and its effect on the in vitro competence of oocytes

The current knowledge is reviewed concerning correlations between follicular development in the cow and the competence of matured oocytes to develop into an embryo following IVF and IVC. At the follicular size of 3 mm, some oocytes become competent and the proportion of competent oocytes does not increase during development up to 7 mm. The proportion of competent oocytes increases greatly in follicles > 8 mm in both untreated and gonadotropin-stimulated cows. The competence of in vitro-matured oocytes from these large follicles is lower than the competence of in vivo-matured oocytes. These observations lead to the following concept. Oocytes have acquired an intrinsic capacity to develop into an embryo after IVM-IVF-IVC at the follicular stage of 3 mm, but require an additional "prematuration" to express this competence. In vivo, this prematuration occurs during preovulatory development before the occurrence of the LH surge. In follicles of 3-7 mm, a low level of atresia appears to improve the in vitro competence of oocytes which may act via a prematuration-like effect. A thorough understanding, however, of the effect of atresia and other factors on the competence of this highly heterogeneous oocyte population is still missing. Two routes to improve the embryo yield in ovum pick-up (OPU) practice are discussed.     

Cell competence forAgrobacterium-mediated DNA transfer inPisum sativum L.

Distribution and properties of pea (Pisum sativum L.) cells, competent forAgrobacterium-mediated transformation were analysed byin situ histochemical detection of GUS (-glucuronidase) activity, 4 d after inoculation with engineeredAgrobacterium tumefaciens. The vector system consisted of the hypervirulent disarmed strain EHA101 and the binary plasmid pIBGUS, carrying an intron-containing, 35S-promotor drivengusA (oruidA) gene and two selectable marker genes. Cells competent for transformation were mainly restricted to the dedifferentiating cells neighbouring the vascular system of cotyledon and epicotyl explants. A standardized assay was developed, allowing determination and quantification of factors influencing number and distribution of competent cells. In etiolated seedlings, competence for transformation decreased with the distance of the epicotyl explant from the shoot apex and was specifically induced by the exogenous application of auxins. Transient expression ofgusA afterAgrobacterium-mediated DNA transfer was dramatically reduced upon application of cell-cycle and DNA replication inhibitors aphidicolin, colchicine and nalidixic acid. GUS expression after direct DNA transfer of double-stranded plasmid DNA (via PEG into protoplasts or via particle bombardment of epicotyl segments) was independent of cell-division/DNA replication.A GUS-positive mutant of EHA101 was constructed to allowin situ analysis of attaching bacteria within the plant tissue. Attachment and invasion was inhibited by well-developed cuticula but was restored after chloroform treatment of the tissue surface. Moreover, no correlation was found between distribution of attaching bacteria and the pattern of transformation-competent cells.     

Formation of an animal virus within a bacterium

Conclusions Incubation of competent cells ofB. subtilis with a DNA-like subviral entity from vaccinia virus leads to the formation of complete vaccinia virus in the bacteria. The significance of this finding in regard to the concept of a universal genetic code is discussed.With 2 Figures in the TextA preliminary report of these results was presented at the XI. International Congress of Genetics in The Hague, September, 1963.Habilitandenstipendiat 1962/63 of Deutsche Forschungsgemeinschaft.     

Interactions among cellulolytic bacteria from an anaerobic digester

High cellulolytic activity of particular strains did not cause dominance of one, or a few, species of fiber-digesting bacteria in a cattlewaste anaerobic digester. The population contained a large number of species and varieties with different cellulolytic and fiber-digesting activities. Although mixed cultures of some of these bacteria showed no intereffects, with others, cellulolysis was less or in some cases greater than that shown by individual components of the cultures. The interactions were probably related to effects on growth of the bacteria rather than on activities of components of the cellulase enzyme complex, and culture filtrates of two of the more numerous cellulolytic species ofClostridium affected growth of other cellulolytic bacteria. The inhibitory factor(s) appeared to be of bacteriocin type, but the stimulatory factor(s) was unknown. It was suggested that these interactions are localized or short-lived in the digester, and so the population remains in a dynamic steady state.Some inhibitions of growth of rumen cellulolytic bacteria were caused by the digester bacteria, but it was suggested that factors other than these inhibitions are responsible for the absence of rumen bacteria from anaerobic digesters.     

ComEA Is Essential for the Transfer of External DNA into the Periplasm in Naturally Transformable Vibrio cholerae Cells

The DNA uptake of naturally competent bacteria has been attributed to the action of DNA uptake machineries resembling type IV pilus complexes. However, the protein(s) for pulling the DNA across the outer membrane of Gram-negative bacteria remain speculative. Here we show that the competence protein ComEA binds incoming DNA in the periplasm of naturally competent Vibrio cholerae cells thereby promoting DNA uptake, possibly through ratcheting and entropic forces associated with ComEA binding. Using comparative modeling and molecular simulations, we projected the 3D structure and DNA-binding site of ComEA. These in silico predictions, combined with in vivo and in vitro validations of wild-type and site-directed modified variants of ComEA, suggested that ComEA is not solely a DNA receptor protein but plays a direct role in the DNA uptake process. Furthermore, we uncovered that ComEA homologs of other bacteria (both Gram-positive and Gram-negative) efficiently compensated for the absence of ComEA in V. cholerae, suggesting that the contribution of ComEA in the DNA uptake process might be conserved among naturally competent bacteria.