Development of competence of Haemophilus influenzae

Spencer, Hugh T. (The Johns Hopkins University School of Hygiene and Public Health, Baltimore, Md.), and Roger M. Herriott. Development of competence of Haemophilus influenzae. J. Bacteriol. 90:911-920. 1965.-A chemically defined nongrowth medium was developed for the induction of competence of Haemophilus influenzae by a stepdown procedure. Cells grown logarithmically in Heart Infusion Broth became competent after being transferred to a medium which consisted of amino acids, sodium fumarate, and inorganic salts. Chloramphenicol (2 mug/ml) or l-valine (1 mug/ml) in the nongrowth medium inhibited development of competence. The inhibitory action of l-valine was reversed by comparable concentrations of l-isoleucine. Kinetic studies of the development of competence showed a variable capacity of competent cells to take up deoxyribonucleic acid and reaffirmed earlier findings that competence was not transmissible in H. influenzae. Addition of nicotinamide adenine dinucleotide, thiamine, calcium pantothenate, uracil, and hypoxanthine to the medium for competence resulted in a minimal growth medium in which reduced levels of competence were developed.     

Competence Factor Production in Chemically Defined Media by Noncompetent Cells of Group H Streptococcus Strain Challis

Chemically defined media for competence factor (CF) production by group H Streptococcus strain Challis-6 are described. CF is produced by noncompetent cells in a glutamate-free medium in which the cells cannot attain competence and by cells prior to their competence development in a glutamate-containing medium. Glutamate was required for competence development, but was not necessary for growth or CF production. Exacting cultural conditions required for the consistent production of relatively high amounts of CF in defined medium and for its recovery are detailed. The most important requirements include the selection of isolates (like Challis-6) which grew well in another defined medium, early harvest of CF because of its demonstrated instability on continued incubation in defined medium, incubation at 37 C, and the addition of glucose. The CF production was more rapid with increasing inocula and with reduced aeration. Aspartate, cystine, and NaCl were not required. Under the conditions described, large amounts of CF were consistently obtained in the culture filtrates of Challis-6 as measured by the induction of competence in strain Wicky cells and their subsequent transformation at frequencies of 6% or greater.     

Potentially lethal damage repair is due to the difference of DNA double-strand break repair under immediate and delayed plating conditions

Cells plated immediately after irradiation on nutrient agar (immediate plating) exhibit a lower survival than cells which are kept under nongrowth conditions before plating (delayed plating). The difference between the survival curves obtained after immediate plating and delayed plating is considered to exhibit the cell's capacity to repair potentially lethal damage. In yeast evidence has been presented previously for the DNA double-strand break (DSB) as the molecular lesion involved in the repair of potentially lethal damage observed at the cellular level. Radiation-induced DSB are repaired in cells plated on nutrient agar, i.e., under growth conditions, as well as in cells kept under nongrowth conditions. In this paper DSB repair under growth and nongrowth conditions is studied with the help of the yeast mutant rad54-3 which is temperature conditional for DSB repair. It is shown that the extent of repair of potentially lethal damage can be varied by shifting the relative fractions of repair of DSB under growth conditions versus nongrowth conditions. Repair of DSB in cells plated on nutrient agar is promoted when glucose is substituted by Na-succinate as an energy source. As a result the immediate plating survival curve approaches the delayed plating survival curve, thus reducing the operationally defined repair of potentially lethal damage. We show that this reduced potentially lethal damage repair is caused, however, by a higher amount of DSB repair in cells immediately plated on succinate agar as compared to glucose agar.     

Episodic Selection and the Maintenance of Competence and Natural Transformation in Bacillus subtilis

We present a new hypothesis for the selective pressures responsible for maintaining natural competence and transformation. Our hypothesis is based in part on the observation that in Bacillus subtilis, where transformation is widespread, competence is associated with periods of nongrowth in otherwise growing populations. As postulated for the phenomenon of persistence, the short-term fitness cost associated with the production of transiently nongrowing bacteria can be compensated for and the capacity to produce these competent cells can be favored due to episodes where the population encounters conditions that kill dividing bacteria. With the aid of a mathematical model, we demonstrate that under realistic conditions this “episodic selection” for transiently nongrowing (persisting) bacteria can maintain competence for the uptake and expression of exogenous DNA transformation. We also show that these conditions for maintaining competence are dramatically augmented even by rare episodes where selection favors transformants. Using experimental populations of B. subtilis and antibiotic-mediated episodic selection, we test and provide support for the validity of the assumptions behind this model and the predictions generated from our analysis of its properties. We discuss the potential generality of episodic selection for the maintenance of competence in other naturally transforming species of bacteria and critically evaluate other hypotheses for the maintenance (and evolution) of competence and their relationship to this hypothesis.     

Reduction of porous media permeability from in situ Leuconostoc mesenteroides growth and dextran production

In situ growth of bacteria in a porous medium can alter the permeability of that media. This article reveals that the rate of permeability alteration can be controlled by the inoculation strategy, nutrient concentrations, and injection rates. Based on experimental observations a phenomenological model has been developed to describe the inoculation of the porous medium, the in situ growth of bacteria, and the permeability decline of the porous medium. This model consists of two phases that describe the bacteria in the porous medium: (1) the nongrowth phase in which cell transport and retention are occurring; and (2) the growth phase in which the retained cells grow and plug the porous media. Transition from the transport phase to the growth phase is governed by the growth lag time of the cells within the porous medium. The importance of the inoculum injection strategy and the nutrient injection strategy is illustrated by the model. (c) 1996 John Wiley & Sons, Inc.     

Growth medium-independent genetic competence mutants of Bacillus subtilis.

The development of competence in Bacillus subtilis is normally dependent on the growth medium. Expression of late competence genes occurs in glucose-minimal salts-based media but not in complex media. Expression is also inhibited when glutamine is added to competence medium and when glycerol is substituted for glucose. Mutations have been identified in two regulatory loci, mecA and mecB, which render competence development independent of these variables. Although in mec mutants the expression of late competence genes, as well as of competence itself, occurred in all media tested, this expression was still growth stage regulated. Thus at least some forms of medium-dependent and growth stage-specific regulation are genetically separable. One of the mecB mutations (mecB31) conferred oligosporogenicity. The mecB mutations were tightly linked by transformation to rif, lpm, and std markers and were located between rif-2103 and cysA14. The mecA42 mutant was linked by transduction to argC4.     

Microbial Maintenance: A Critical Review on Its Quantification

Microbial maintenance is an important concept in microbiology. Its quantification, however, is a subject of continuous debate, which seems to be caused by (1) its definition, which includes nongrowth components other than maintenance; (2) the existence of partly overlapping concepts; (3) the evolution of variables as constants; and (4) the neglect of cell death in microbial dynamics. The two historically most important parameters describing maintenance, the specific maintenance rate and the maintenance coefficient, are based on partly different nongrowth components. There is thus no constant relation between these parameters and previous equations on this subject are wrong. In addition, the partial overlap between these parameters does not allow the use of a simple combination of these parameters. This also applies for combinations of a threshold concentration with one of the other estimates of maintenance. Maintenance estimates should ideally explicitly describe each nongrowth component. A conceptual model is introduced that describes their relative importance and reconciles the various concepts and definitions. The sensitivity of maintenance on underlying components was analyzed and indicated that overall maintenance depends nonlinearly on relative death rates, relative growth rates, growth yield, and endogenous metabolism. This quantitative sensitivity analysis explains the felt need to develop growth-dependent adaptations of existing maintenance parameters, and indicates the importance of distinguishing the various nongrowth components. Future experiments should verify the sensitivity of maintenance components under cellular and environmental conditions.     

Suppression of early competence mutations in Bacillus subtilis by mec mutations.

Although competence normally develops only in glucose-minimal salts media, mecA and mecB mutations permit the expression of competence and of late competence genes in complex media as well (D. Dubnau and M. Roggiani, J. Bacteriol. 172:4048-4055, 1990). The expression of late competence genes is dependent on the products of the regulatory genes comA, comB, comP, sin, abrB, spo0H, and spo0A. We show here that this list must be extended to include degU, csh-293, and spo0K. mecA and -B mutations bypass most of these requirements, making the expression of late competence genes and of competence itself independent of all of these regulatory genes, with the exceptions of spo0A and spo0K (in the case of mecB). The expression of late competence genes in mec mutants that are deficient for each of the bypassed regulatory functions is still under growth stage-specific regulation. The implications of these findings are discussed, and a provisional scheme for the flow of information during the development of competence is proposed.     

Boron controls suspensor development in embryogenic cultures of Larix decidua

Suspensor formation in somatic embryos of Larix decidua Mill. was. studied in relation to its dependence on nutritional factors of the culture medium. Protoplasts exhibiting embryogenic competence were prepared from an embryogenic suspension culture, immobilized in Ca-alginate and cultured in liquid media. Developmental patterns showed qualitative differences in the two basic media used. In one medium, containing 100 μ M boric and, compact colonies developed, most of which displayed formation of polar suspensors after 3 to 4 weeks. In contrast, suspensor development never occurred in the other medium, containing 10 μ M borate, yet colonies of loosely connected cytoplasmically dense cells were regenerated without loss of embryogenic competence. Detailed examination of the constituents of the two media led to the conclusion that suspensor development is dependent on boron concentration: below 35 μ M boron, suspensor formation is blocked completely, whereas with an increase up to 1 μ M boron there is a progressively faster development of suspensor-bearing embryos.     

Antibiotics and UV radiation induce competence for natural transformation in Legionella pneumophila

Natural transformation by competence is a major mechanism of horizontal gene transfer in bacteria. Competence is defined as the genetically programmed physiological state that enables bacteria to actively take up DNA from the environment. The conditions that signal competence development are multiple and elusive, complicating the understanding of its evolutionary significance. We used expression of the competence gene comEA as a reporter of competence development and screened several hundred molecules for their ability to induce competence in the freshwater living pathogen Legionella pneumophila. We found that comEA expression is induced by chronic exposure to genotoxic molecules such as mitomycin C and antibiotics of the fluoroquinolone family. These results indicated that, in L. pneumophila, competence may be a response to genotoxic stress. Sunlight-emitted UV light represents a major source of genotoxic stress in the environment and we found that exposure to UV radiation effectively induces competence development. For the first time, we show that genetic exchanges by natural transformation occur within an UV-stressed population. Genotoxic stress induces the RecA-dependent SOS response in many bacteria. However, genetic and phenotypic evidence suggest that L. pneumophila lacks a prototypic SOS response and competence development in response to genotoxic stress is RecA independent. Our results strengthen the hypothesis that competence may have evolved as a DNA damage response in SOS-deficient bacteria. This parasexual response to DNA damage may have enabled L. pneumophila to acquire and propagate foreign genes, contributing to the emergence of this human pathogen.     

Exponential or shouldered survival curves result from repair of DNA double-strand breaks depending on postirradiation conditions

The yeast mutant rad54-3 is temperature conditional for the rejoining of DNA double-strand breaks, but cells do proliferate at both the restrictive and permissive temperatures. Thus, after irradiation with 30 MeV electrons, survival curves can be obtained which may or may not involve double-strand break rejoining under certain experimental conditions. Because of this special property of rad54-3 cells, it was possible to demonstrate that rejoining of radiation-induced double-strand breaks under nongrowth conditions yields exponential survival curves the slopes of which decrease as a function of the rejoining time. These survival data suggest that, under nongrowth conditions, the rejoining of double-strand breaks is an unsaturated process and lacks binary misrepair. In contrast, whenever rejoining of double-strand breaks occurs under growth conditions, shouldered survival curves are observed. This is true for immediate plating as well as for delayed plating survival curves. It is proposed that it is the unsaturated rejoining of double-strand breaks under nongrowth conditions, lacking binary misrepair, which is responsible for potentially lethal damage repair.     

Carbohydrate utilization by Trichomonas gallinae: effects on growth and metabolic activity under nongrowth conditions

Trichomonas gallinae used 13 of 29 carbohydrates for growth. Quantitative relationships between final populations, acid production, and cellular glycogen contents varied depending on the substrate. The effect of growth on different carbohydrates on the subsequent utilization of carbohydrates by cells under nongrowth conditions was studied by measuring carbohydrate uptake, changes in cellular glycogen content, and gas production. Two major utilization patterns were found. Cells grown on maltose or starch used these substrates well, but cells grown on other sugars did not. All cells used glucose, fructose, galactose, and mannose, but cells grown on maltose or starch did not use them as well as cells grown on other sugars. All cells used ribose slightly but not xylose or arabinose. Turanose, a disaccharide yielding high populations in growth medium, was not used under nongrowth conditions.     

Single cell analysis of gene expression patterns of competence development and initiation of sporulation in Bacillus subtilis grown on chemically defined media

AIM: Understanding the basis for the heterogeneous (or bistable) expression patterns of competence development and sporulation in Bacillus subtilis. METHODS AND RESULTS: Using flow cytometric analyses of various promoter-GFP fusions, we have determined the single-cell gene expression patterns of competence development and initiation of sporulation in a chemically defined medium (CDM) and in biofilms. CONCLUSIONS: We show that competence development and initiation of sporulation in a CDM are still initiated in a bistable manner, as is the case in complex media, but are sequential in their timing. Furthermore, we provide experimental proof that competence and sporulation can develop under conditions that normally do not trigger these processes. SIGNIFICANCE AND IMPACT OF THE STUDY: Some pathogens are able to develop natural competence, which is a serious medical problem with the increased acquired multi-drug resistance of these organisms. Another adaptive microbial response is spore formation. Because of their heat resistance and hydrophobicity, spores of a variety of species are of major concern for the food industry. Using the model organism B. subtilis, we show that competence development and sporulation are initiated in a bistable and sequential manner. We furthermore show that both processes may be noise-based, which has major implications for the control of unwanted differentiation processes in pathogenic and food-spoilage micro-organisms.     

Formation of competent Bacillus subtilis cells.

The process of competent cell formation for transformation has been studied with early-stationary-phase (T1) cells of Bacillus subtilis which had been grown in an enriched Spizizen minimal medium and transferred to a second synthetic medium. Rifampin, chloramphenicol, and tunicamycin were strong inhibitors of competent cell formation, as well as vegetative growth. After formation, competent cells were no longer sensitive to the above agents. Methicillin and an inhibitor of chromosomal replication, hydroxyphenylazouracil, did not inhibit the development of competence. A D-alanine-requiring mutant strain developed competence even in the absence of D-alanine in the second medium. A T1-stage culture showed the activity of extracellular serine protease which is necessary for sporulation. Competent cell formation was completely blocked by 0.7 M ethanol, which is a specific inhibitor of early events during sporulation, including forespore septum formation. Competent cells were formed even in media which supported sporulation. The development of competence was also studied with spo0 mutants at 10 different loci. Most spo0 mutations repressed the development of competence except for spo0C, spo0G, and spo0J. These results suggest that competent cells are formed from early sporulating cells with the synthesis of cell wall materials and by factors whose genes are activated by the supply of nutrients. It is suggested that common steps are involved both in forespore septation and in competent cell formation.     

A primary culture technique of adult retina for regeneration studies on adult CNS neurons

This protocol details a tissue culture technique that allows for quantified regeneration studies on adult retinal ganglion cells (RGCs), that is, CNS neurons. The method may also allow for elucidation of molecular cues, for example of signals relevant in neuronal survival and axon regeneration. The procedure relies on fractioned stripe culture of previously injured retina in defined culture media. Naive dendritic cell contacts of RGCs are preserved, and the system is independent of growth factors. In contrast to other techniques, the protocol is based on tissue grown from adult animals; it dispenses immature co-cultures and evaluates the outgrowth of unmyelinated neurites in a milieu lacking CNS myelin. The technique is suitable for rodent retina from mouse or rat. A growth-conditioning injury of the optic nerve is set 10 days before retinal explantation. Explants are cultured for 5-7 days. Mere preparation of a single retina should be completed within 20 min.     

The kinetics of cometabolism

Experimental observations indicate that the rates of cometabolic transformation are linked to the consumption of growth substrate during growth and to the consumption of cell mass and/or energy substrate in the absence of growth substrate. Three previously proposed models (models 1 through 3) describing the kinetics of cometabolism by resting cells are compared, and the interrelationships and underlying assumptions for these models are explored. Models 1 to 3 are shown to converge at high concentrations of the nongrowth substrate. An expression describing nongrowth substrate transformation in the presence of growth substrate is proposed, and this expression is integrated with an expression for cell growth to give a single unstructured model (model 4) that encompasses models 1 to 3 and describes cometabolism by both resting and growing cells. Model 4 couples transformation of nongrowth substrate to consumption of growth substrate and biomass, and predicts that cometabolism will result, and decreased specific growth rates for a cometabolizing population. Competitive inhibition can also be incorporated in the model. Experimental aspects of model calibration and verification are discussed. The need for models that distinguish between the exhaustion of cell activity and cell death is emphasized. (c) 1993 Wiley & Sons, Inc.     

The primary role of comA in establishment of the competent state in Bacillus subtilis is to activate expression of srfA.

The establishment of genetic competence in Bacillus subtilis requires the genes of the competence regulon which function in the binding, processing, and transport of DNA. Their expression is governed by multiple regulatory pathways that are composed of the comA, comP, sin, abrB, spo0H, spo0K, spo0A, degU, and srfA gene products. Among these, srfA is thought to occupy an intermediate position in one of the pathways that controls late competence gene expression. The full expression of srfA requires the gene products of comP, comA, and spo0K. To determine the role of these genes in the regulation of competence development, the expression of the srfA operon was placed under control of the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter Pspac and the expression of the Pspac-srfA construct was examined in mutants blocked in early competence. By monitoring the IPTG-induced expression of Pspac-srfA with a srfA-lacZ operon fusion, it was observed that srfA expression was no longer dependent on the products of comP, comA, and spo0K. Production of the lipopeptide antibiotic surfactin in Pspac-srfA-bearing cells was induced in the presence of IPTG and was independent of ComP and ComA. Competence development was induced by IPTG and was independent of comP, comA, and spo0K in cells carrying Pspac-srfA. These results suggest that the ComP-ComA signal transduction pathway as well as Spo0K is required for the expression of srfA in the regulatory cascade of competence development. Studies of Pspac-srfA also examined the involvement of srfA in the growth stage-specific and nutritional regulation of a late competence gene.