Constitutive Synthesis Research Articles

Investigating Autocatalytic Gene Expression Systems through Mechanistic Modeling

A structured model of gene expression, which incorporates the stochastic behavior of cellular processes, was developed to examine the “all-or-none” phenomenon observed in autocatalytic systems (e.g. the lac operon). Autocatalytic expression systems typically have the genes encoding the inducer transport proteins controlled by internal inducer levels, so that transport of the inducer increases production of the transport protein. The model was able to predict the unique behaviors of autocatalytic expression systems that have been experimentally observed and provided valuable insight into the role of population heterogeneity in these systems. The simulations substantiate the importance of stochastic processes on induction of gene expression in autocatalytic systems. The simulation results show that the all-or-none phenomenon is governed largely by random cellular events, and that population-averaged variations in gene expression are due to changes in the frequency of full gene induction in individual cells rather than to uniform variations in gene expression across the entire population. In addition, the model shows how concentrations of inducer too low to induce expression in uninduced cells can maintain induction in pre-induced cultures. A comparison of induction behaviors from an autocatalytic system and a system having constitutive synthesis of the transport protein showed that transport protein levels must be decoupled from inducer control to achieve homogeneous expression of a gene of interest in all cells of a culture.

Proteolytic cleavage of the repressor (BlaI) of β-lactamase synthesis in Staphylococcus aureus

The proteolytic cleavage of BlaI was shown to correlate with β-lactamase synthesis in Staphylococcus aureus. BlaI was found to be autoregulatory when expressed from the blaZ promoter. Insertion of a 10-bp linker into the SnaBI site of blaRI resulted in constitutive synthesis of β-lactamase.

Proteolytic cleavage of the repressor (BlaI) of beta-lactamase synthesis in Staphylococcus aureus.

The proteolytic cleavage of BlaI was shown to correlate with beta-lactamase synthesis in Staphylococcus aureus. BlaI was found to be autoregulatory when expressed from the blaZ promoter. Insertion of a 10-bp linker into the SnaBI site of blaRI resulted in constitutive synthesis of beta-lactamase.

Dietary Protein or Arginine Deficiency Impairs Constitutive and Inducible Nitric Oxide Synthesis by Young Rats

Effects of dietary protein or arginine deficiency on constitutive and lipopolysaccharide (LPS)-induced nitric oxide (NO) synthesis were determined in young rats by quantifying urinary nitrate excretion. In Experiment 1, 30-d-old rats (n = 16) were divided randomly into two groups (n = 8/group) and pair-fed on the basis of body weight semipurified isocaloric diets containing 20 or 5% casein. In Experiment 2, 30-d-old rats (n = 24) were divided randomly into three groups (n = 8) and pair-fed on the basis of body weight purified isonitrogenous and isocaloric diets (composed of amino acids) containing 0.0, 0.3 or 1.0% L-arginine. In both experiments, daily collection of urine was initiated 10 d after the start of pair-feeding. On d 17 after the pair-feeding was initiated, LPS (1 mg/kg body wt) was injected intraperitoneally into rats, and urine was collected daily for an additional 7 d. In Experiments 3 and 4, activities of constitutive and inducible NO synthases were measured in macrophages and various tissues from protein- or arginine-deficient rats (n = 6). Body weight was lower in rats fed the 5% casein diet or the 0.0 and 0.3% arginine diets than in those fed 20% casein or 1% arginine, respectively. Dietary protein or arginine deficiency decreased serum concentrations of arginine and urinary nitrate excretion before and after LPS treatment, indicating impaired constitutive and inducible NO synthesis. Protein malnutrition reduced constitutive and inducible NO synthase activities in brain, heart, jejunum, lung, skeletal muscle and spleen, and inducible NO synthase activity in macrophages. Because NO is a mediator of the immune response and is the endothelium-dependent relaxing factor, impaired NO synthesis may help explain immunodeficiency and cardiovascular dysfunction in protein- or arginine-deficient subjects.

MICROBIAL GENES EXPRESSED IN TRANSGENIC PLANTS TO IMPROVE DISEASE RESISTANCE

The development of plant transformation and gene cloning techniques has opened new avenues for augmenting disease and insect resistance in crops. The concept of ‘Genetically Acquired Resistance’ now encompasses a variety of strategies based on the transgenic expression in plant of genes from many different origins. Some of the most potentially useful transgenes capable of enhancing resistance to attack by microbes, viruses and insects have been obtained from bacteria and fungi, which are regarded as rich sources of desirable traits for plant genetic improvement. The main research directions have included: (i) the enhancement of the plant anti-microbial and insecticidal arsenal by the constitutive or inducible synthesis of various pathogen inhibiting compounds; (ii) the alteration of the plant response to pathogens by the appropriate induction of the local or systemic defence mechanisms, or by the interference with plant-pathogen signalling and (iii) the inactivation of pathogen toxins or the improvement of plant resistance to them. Although transgenic plants that are insect- resistant or bear other useful traits obtained from microbes, such as herbicide-resistance or enhanced food quality, are being commercially marketed, genetically- engineered crops exhibiting resistance to fungal or bacterial diseases have yet to reach the marketplace. The main focus of this paper addresses the application of bacterial or fungal genes to improve plant resistance to insect, fungi, bacteria and viruses, and the usefulness of these microbial genomes in the development of new transgenic technologies. In addition, perspectives dealing with environmental concerns and important biosafety questions resulting from these technologies are briefly discussed.

Characterization of the vanD glycopeptide resistance gene cluster from Enterococcus faecium BM4339.

VanD-type resistance to glycopeptides in Enterococcus faecium BM4339 is due to constitutive synthesis of D-alanyl-D-lactate-terminating peptidoglycan precursors (B. Périchon, P. Reynolds, and P. Courvalin, Antimicrob. Agents Chemother. 41:2016-2018, 1997). The sequence of a 5,780-bp fragment was determined and revealed six open reading frames. The 3' distal part encoded the VanHD dehydrogenase, the VanD ligase, and the VanXD DD-dipeptidase, which were highly similar to the corresponding proteins in VanA and VanB types of resistance. The deduced VanYD protein was homologous to penicillin-binding proteins that display DD-carboxypeptidase activity. The 5' end coded for the putative VanRD-VanSD two-component regulatory system. Due to a frameshift mutation in the chromosomal ddl gene, BM4339 produced an impaired D-alanine:D-alanine ligase. However, since expression of the resistance genes is constitutive, growth of E. faecium BM4339 was not dependent on the presence of glycopeptides in the culture medium.

Heterologous expression of soluble methane monooxygenase genes in methanotrophs containing only particulate methane monooxygenase.

The methanotrophs Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b contain particulate methane monooxygenase (pMMO) and soluble methane monooxygenase (sMMO) genes. Other methanotrophs such as Methylomicrobium album BG8 and Methylocystis parvus OBBP contain only pMMO genes. Although molecular genetic techniques are poorly developed in methanotrophs, sMMO genes were expressed in methanotrophs normally containing only pMMO genes. This was achieved by conjugation using broad-host-range plasmids containing the native promoter and sMMO genes from Mc. capsulatus (Bath) and Ms. trichosporium OB3b. sMMO genes derived from Ms. trichosporium OB3b were expressed in an active form in Mcy. parvus OBBP and in Mm. album BG8. Therefore, all of the genes required for active sMMO synthesis were contained on the broad-host-range plasmids and were expressed in the heterologous hosts. Constitutive synthesis of pMMO was observed in Mm. album BG8 when grown at high and low copper-to-biomass ratios, while transcription of the recombinant sMMO genes was only observed under growth conditions of low copper-to-biomass ratios. Therefore, the regulatory protein(s) for sMMO synthesis was also present on the plasmid used, or the heterologous host contained a regulatory system for sMMO. Expression of sMMO genes in methanotrophs containing only pMMO will assist further investigations on the expression and regulation of MMO genes in methanotrophs.

Functional analysis of the two zinc fingers of SRE, a GATA-type factor that negatively regulates siderophore synthesis in Neurospora crassa.

Multiple GATA factors, zinc finger DNA binding proteins that recognize consensus GATA elements, exist in Neurospora crassa. One of them, SRE, is involved in controlling the iron metabolic pathway of N. crassa. In N. crassa, iron transport is mediated by a number of small cyclic peptides, known as siderophores. The siderophore synthesis pathway is negatively regulated by SRE; a loss-of-function sre mutant strain showed partial constitutive synthesis of siderophore. In the research presented here, the negative function of SRE was further confirmed by a heterokaryon test and by gene complementation. SRE was expressed as a GST fusion protein. In vitro EMSA revealed that SRE binds specifically to DNA molecules containing GATA sequence elements. Autoregulation of sre gene expression appears possible because the sre gene promoter itself contains GATA sequences. Mutations were introduced into sre that lead to amino acid substitutions in each of the zinc fingers that will disrupt their function. In vitro EMSA revealed that both N-terminal and C-terminal zinc fingers of SRE are involved in DNA binding. This feature is different from that found with the vertebrate two zinc finger GATA factors. Invivo tests, accomplished by transforming the mutant sre genes into sre rip mutant, showed that SRE with mutations in either or both zinc fingers still maintained its function under low-iron conditions. In contrast, these mutant SRE proteins fail to function under high-iron conditions. Our results predict the presence of other positive or negative regulators of the siderophore synthetic pathway.

Gene expression, synthesis, and secretion of interleukin 18 and interleukin 1beta are differentially regulated in human blood mononuclear cells and mouse spleen cells.

Interleukin (IL)-18, formerly called interferon gamma (IFN-gamma)-inducing factor, is biologically and structurally related to IL-1beta. A comparison of gene expression, synthesis, and processing of IL-18 with that of IL-1beta was made in human peripheral blood mononuclear cells (PBMCs) and in human whole blood. Similar to IL-1beta, the precursor for IL-18 requires processing by caspase 1. In PBMCs, mature but not precursor IL-18 induces IFN-gamma; in whole human blood stimulated with endotoxin, inhibition of caspase 1 reduces IFN-gamma production by an IL-1beta-independent mechanism. Unlike the precursor for IL-1beta, precursor for IL-18 was expressed constitutively in PBMCs and in fresh whole blood from healthy human donors. Western blotting of endotoxin-stimulated PBMCs revealed processed IL-1beta in the supernatants via an caspase 1-dependent pathway. However, in the same supernatants, only unprocessed precursor IL-18 was found. Unexpectedly, precursor IL-18 was found in freshly obtained PBMCs and constitutive IL-18 gene expression was present in whole blood of healthy donors, whereas constitutive IL-1beta gene expression is absent. Similar to human PBMCs, mouse spleen cells also constitutively contained the preformed precursor for IL-18 and expressed steady-state IL-18 mRNA, but there was no IL-1beta protein and no spontaneous gene expression for IL-1beta in these same preparations. We conclude that although IL-18 and IL-1beta are likely members of the same family, constitutive gene expression, synthesis, and processing are different for the two cytokines.

Targeted Reduction of Nucleoside Triphosphate Hydrolase by Antisense RNA Inhibits Toxoplasma gondii Proliferation

Nucleoside triphosphate hydrolase (NTPase) is a very abundant protein secreted by the obligate intracellular parasite Toxoplasma gondii shortly after invasion of the host cell. When activated by dithiols, NTPase is one of the most potent apyrases known to date, but its physiological function remains unknown. The genes encoding NTPase have been cloned (Bermudes, D., Peck, K. R., Afifi-Afifi, M., Beckers, C. J. M., and Joiner, K. A. (1994) J. Biol. Chem. 269, 29252-29260). We have recently shown that the enzyme is tightly controlled within the vacuolar space and may influence parasite exit from the host cell (Silverman, J. A., Qi, H., Riehl, A., Beckers, C., Nakaar, V., and Joiner, K. A (1998) J. Biol. Chem. 273, 12352-12359). In the present study, we have generated an antisense NTP RNA construct in which the 3'-untranslated region is replaced by a hammerhead ribozyme. The constitutive synthesis of the chimeric antisense RNA-ribozyme construct in parasites that were stably transfected with this construct resulted in a dramatic reduction in the steady-state levels of NTPase. This inhibition was accompanied by a decrease in the capacity of the parasites to replicate. The reduction in parasite proliferation was due to a specific effect of antisense NTP RNA, since a drastic inhibition of hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) expression by a chimeric antisense HXGPRT RNA-ribozyme construct did not alter NTPase expression nor compromise parasite replication. These data implicate NTPase in an essential parasite function and suggest that NTPase may have more than one function in vivo. These results also establish that it is possible to study gene function in apicomplexan parasites using antisense RNA coupled to ribozymes.

Pathogen-induced elicitin production in transgenic tobacco generates a hypersensitive response and nonspecific disease resistance.

The rapid and effective activation of disease resistance responses is essential for plant defense against pathogen attack. These responses are initiated when pathogen-derived molecules (elicitors) are recognized by the host. We have developed a strategy for creating novel disease resistance traits whereby transgenic plants respond to infection by a virulent pathogen with the production of an elicitor. To this end, we generated transgenic tobacco plants harboring a fusion between the pathogen-inducible tobacco hsr 203J gene promoter and a Phytophthora cryptogea gene encoding the highly active elicitor cryptogein. Under noninduced conditions, the transgene was silent, and no cryptogein could be detected in the transgenic plants. In contrast, infection by the virulent fungus P. parasitica var nicotianae stimulated cryptogein production that coincided with the fast induction of several defense genes at and around the infection sites. Induced elicitor production resulted in a localized necrosis that resembled a P. cryptogea-induced hypersensitive response and that restricted further growth of the pathogen. The transgenic plants displayed enhanced resistance to fungal pathogens that were unrelated to Phytophthora species, such as Thielaviopsis basicola, Erysiphe cichoracearum, and Botrytis cinerea. Thus, broad-spectrum disease resistance of a plant can be generated without the constitutive synthesis of a transgene product.

Dietary and systemic phenylalanine utilization for mucosal and hepatic constitutive protein synthesis in pigs.

The objective of this study was to quantify the utilization of dietary and systemic phenylalanine for mucosal and hepatic constitutive protein synthesis in piglets. Seven female piglets (7.6 kg) bearing arterial, portal, peripheral venous, and gastric catheters were fed a high-protein diet and infused intragastrically with U-13C-labeled protein and intravenously with [2H(phenyl)5]phenylalanine ([2H5]phenylalanine) for 6 h. The isotopic enrichment of the two phenylalanine tracers was measured in arterial and portal blood, in mucosal and hepatic-free and protein-bound phenylalanine, and in very low-density apolipoprotein B-100, albumin, and fibrinogen. The relative isotopic enrichments of the tracers in mucosal-free (ratio of 2H5- to U-13C-labeled = 0.20 +/- 0.05) and protein-bound (0.32 +/- 0.08) phenylalanine differed significantly (P < 0.01). Although this suggests preferential use of arterial phenylalanine for mucosal protein synthesis, on a molar basis, 59 +/- 6% of the mucosal protein was derived from dietary phenylalanine. There were significant differences (P < 0.025) between the relative labeling of the two tracers in arterial (ratio of 2H5- to U-13C-labeled = 1.25 +/- 0.48) and portal (ratio of 2H5- to U-13C-labeled = 0.72 +/- 0.18) phenylalanine. The mean ratio of the two tracers in all proteins of hepatic origin that were analyzed (0.69 +/- 0.18) was similar to that of portal phenylalanine. We conclude that in the fed state portal phenylalanine is preferentially used for constitutive as well as secreted hepatic protein synthesis.

Natural body temperature and kinetics of heat-shock protein synthesis in the toad-headed agamid lizard Phrynocephalus interscapularis

Abstract We demonstrated that in the desert lizard Phrynocephalus interscapularis constitutive synthesis of both members of the hsp70 family of heat shock proteins takes place in summer. Elevation of environmental and body temperatures during the day was accompanied by a pronounced (2.0-2.5 times) increase in hsp70 synthesis. Synthesis of hsps continues during the day and drops to a basal level at night. Northern hybridization analysis demonstrated a higher content of hsp-coding RNA in the cells of P. interscapularis under normal physiological conditions in comparison with the lizard Lacerta vivipara, which inhabits regions with moderate climates.

OncogenicrasFails to Restore anin VivoTumorigenic Phenotype in Embryonic Stem Cells Lacking Vascular Endothelial Growth Factor (VEGF)

Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis. Previous studies have shown that the ability of murine embryonic stem (ES) cells to form teratocarcinomas in nude mice is substantially reduced following targeted inactivation of the VEGF gene. We sought to determine whether VEGF−/− ES cells' tumorigenic phenotype can be rescued by transfection with a mutant H-ras.VEGF−/− ES cells were transfected with expression vector which directs the constitutive synthesis of oncogenic Val-12ras.Expression ofrasprotein was documented by Western blot analysis. We injected several clones with different levels of Val-12rasexpression in nude mice. In agreement with our earlier report, VEGF−/− ES cells formed much smaller tumors than control ES cells. However, none of theras-expressing clones tested formed tumors larger than those derived from parental VEGF−/− cells. Thus, pluripotent cells such as ES cells are unable to compensate for the loss of VEGF even in the presence of a potent oncogenic stimulus such as mutantras.These findings strengthen the hypothesis that VEGF-mediated angiogenesis is crucial for effectivein vivotumor growth.

Nitric oxide inhibits lipopolysaccharide-induced apoptosis in pulmonary artery endothelial cells.

Our group recently reported that cultured sheep pulmonary artery endothelial cells (SPAECs) became resistant to lipopolysaccharide (LPS)-induced apoptosis several days after constitutive synthesis of nitric oxide (NO) after adenoviral (Ad) transfer of inducible NO synthase (iNOS) or exposure to the NO donor S-nitroso-N-acetylpenicillamine (SNAP) (E. Tzeng, Y.-M. Kim, B. R. Pitt, A. Lizonova, I. Kovesdi, and T. R. Billiar. Surgery 122: 255-263, 1997). In the present study, we confirmed this observation by establishing stable transfectants after retroviral gene transfer [replication-deficient retrovirus (DFG)] of human iNOS (DFG-iNOS) SPAECs and then used all three approaches (Ad, DFG, and SNAP) to determine underlying mechanisms of this phenomenon. Continuous endogenous production of NO in itself did not cause apoptosis as assessed by phase-contrast microscopy, nuclear morphology, and internucleosomal DNA fragmentation. Prolonged (72-96 h) synthesis of NO, however, after DFG- or replication-deficient adenovirus (Ad. CMV)-iNOS or SNAP (100 microM, 96 h) inhibited LPS-induced apoptosis. The kinetics of such protection suggested that NO may be inducing other gene products. Ad-mediated transfer of manganese superoxide dismutase (MnSOD) decreased the sensitivity of wild-type SPAECs to LPS-induced apoptosis. MnSOD, however, was not induced in an NG-monomethyl-L-arginine (L-NMMA)-sensitive time-dependent fashion after Ad.CMV-iNOS. Other inducible genes that may be affected by NO and that may protect against potential oxidant-mediated LPS-induced apoptosis including 70-kDa heat shock protein, heme oxygenase-1, metallothionein, and Bcl-2 also were not elevated in an L-NMMA-sensitive, time-dependent fashion. Although the candidate gene product underlying NO-induced protection remains unclear, we did note that prolonged synthesis of NO inhibited LPS-induced activation of an interleukin-1beta-converting enzyme-like cysteine protease (cysteine protease protein-32-like) in a dithiothreitol-sensitive fashion, suggesting that S-nitrosylation of an important downstream target of convergence of apoptotic signals may contribute to the sensitivity of SPAECs to LPS.

Relationship between IkappaBalpha constitutive expression, TNFalpha synthesis, and apoptosis in EBV-infected lymphoblastoid cells.

In order to understand the role of NF-kappaB in EBV transformation we have established stably transfected IkappaBalpha into lymphoblastoid cells. Two clones were obtained in which the loss of NF-kappaB binding activity correlated with the constitutive expression of the transgenic IkappaBalpha. Protein latency expression was determined by immunocytochemistry. Expression of surface markers, intracytoplasmic content of cytokines cell cycle analysis after BrdU incorporation and DNA staining with propidium iodide were studied by flow cytometry. Percentage of apoptotic cells was determined by in-situ labelling of DNA strand breaks. No significative changes in EBV latency nor in cell surface marker expression was found. In contrast, intracytoplasmic TNFalpha levels were strongly reduced in transfected clones. Furthermore, 30% of IkappaBalpha transfected cells were apoptotic after 8 h of TNFalpha treatment. This correlated with a strong reduction of BrdU incorporation after 24 h of TNFalpha treatment. No effect was seen with non transfected cells or with cells transfected with a control plasmid. Our results suggest that the TNFalpha gene could be one of the targets of NF-kappaB in EBV infected cells and that NF-kappaB protects EBV-infected cells from apoptosis induced by TNFalpha, which may favour the proliferative effect of this cytokine.

Human mast cells express stem cell factor.

Stem cell factor (SCF) is a major cytokine regulator of mast cell growth and function. The present study demonstrates that human mast cells are able to produce SCF. Constitutive synthesis of SCF mRNA was seen in the mast cells isolated from human lung and skin by RT-PCR. This was confirmed by in situ hybridization in conjunctival mast cells of both tryptase-only (MCT) and tryptase/chymase (MCTC) subsets. SCF protein product was found in conjunctival MCT and MCTC mast cells by immunohistochemistry. Soluble SCF protein was detected in the culture supernatant of isolated lung mast cells by ELISA, and cross-linkage of IgE receptor (Fc epsilon-RI) on the lung mast cells in culture did not alter SCF mRNA expression, or the secreted soluble SCF protein. This was consistent with the finding that levels of SCF mRNA expression in conjunctival mast cells were similar between normal subjects and patients with seasonal allergic conjunctivitis (SAC). This study shows that human mast cells themselves are a cellular source of SCF, as well as being target cells for this growth factor. SCF may regulate mast cell growth and function via both paracrine and autocrine mechanisms. The production of SCF by mast cells may be regulated via mechanisms other than IgE receptor-mediated pathways.

Acquisition of cellular resistance to 9-nitro-camptothecin correlates with suppression of transcription factor NF-kappa B activation and potentiation of cytotoxicity by tumor necrosis factor in human histiocytic lymphoma U-937 cells.

Resistance of tumor cells to chemotherapeutic agents is a major problem in cancer therapy. Continuous exposure of human histiocytic lymphoma U-937 cells to 9-nitro-camptothecin (9NC), an inhibitor of the nuclear DNA topoisomerase I, induces resistance to this drug. Because of the involvement of the nuclear factor NF-kappa B in the expression of several growth regulatory genes, we examined the activation of this transcription factor in 9NC-resistant U-937 cells. We found that resistance to increasing concentrations of 9NC correlated with resistance to tumor necrosis factor (TNF)-dependent activation of NF-kappa B. However, the constitutive synthesis of NF-kappa B proteins remained unaffected. Cellular resistance was not unique to TNF, as other activators of NF-kappa B, including interleukin-1, phorbol ester and hydrogen peroxide, also had no effect. There was no difference between 9NC-sensitive and -resistant cells in the activation of NF-kappa B by okadaic acid. Other transcription factors, including AP-1 and Oct-1, were not affected in the resistant cells. When examined for the inhibitory subunit of NF-kappa B (I kappa B alpha), resistant cells showed a faster rate of resynthesis than the control. Interestingly, although 9NC resistance correlated with resistance to TNF-dependent NF-kappa B activation, TNF-dependent cytotoxicity in these cells was enhanced by several hundred fold despite a significant decrease in the number of TNF receptors. In conclusion, our results suggest that NF-kappa B activation may play a role in tumor cell killing by 9NC but not by TNF.

Construction of versatile high-level expression vectors for Bartonella henselae and the use of green fluorescent protein as a new expression marker.

Expression vectors suitable for directing high levels of protein synthesis in Bartonella henselae have been constructed based on the mobilisable broad-host-range (IncQ) plasmidpMMB206. They confer kanamycin resistance and feature the taclac (tac-lacUV5 in tandem) promoters in front of a polylinker followed by the rrnB transcriptional stop point. While expression of genes fused to the taclac promoter is constitutive in one vector, the lacIq gene carried by the other vector allows a controlled, IPTG-inducible gene expression. These vectors were tested by subcloning a mutated gfp gene coding for the green fluorescent protein (GFP) from Aequorea victoria into the multiple cloning site and introducing the resulting plasmids into Escherichia coli and B. henselae. GFP expression was determined by measuring fluorescence via flow cytometry or directly by immunoblotting. Compared to E. coli, expression of GFP in B. henselae was more tightly controlled by lacIq and resulted in much higher levels of both IPTG-induced and constitutive gene expression. In vitro infection of endothelial cells indicated that GFP expression does not adversely affect the interaction of B. henselae with host cells. These data demonstrate that (i) the established expression vectors are useful for directing controlled or constitutive high-level protein synthesis in B. henselae and (ii) that GFP is a valuable expression marker which may has important applications in studying the bacterial genetics and cellular interactions of this emerging human pathogen.

Melanin Synthesis Is Associated with Changes in Hyphopodial Turgor, Permeability, and Wall Rigidity inGaeumannomyces graminisvar.graminis

Mycelia ofGaeumannomyces graminisvar.graminisform large cells called hyphopodia with deeply lobed, melanized walls. Like appressoria produced by other pathogens, hyphopodia develop on hydrophobic surfaces, but it is not clear that hyphopodia function as platforms for host penetration. In appressoria, melanin synthesis is linked to the generation of enormous turgor pressures that provide the necessary force for plant penetration. In the present study, hyphopodial turgor was measured in a wild-type strain ofG. graminisvar.graminis,a mutant exhibiting constitutive synthesis of melanin (referred to as the dark mutant), and a melanin-deficient strain (thr). These experiments demonstrate that hyphopodia of the wild-type strain generate higher pressures than the dark mutant and that nonmelanizedthrhyphopodia generate minuscule internal pressures. Melanization of the wall is also associated with an increase in its rigidity. These data correlate with differences in wall permeability consistent with a recent model for turgor generation by appressoria.