The Virulence of a Dickeya dadantii 3937 Mutant Devoid of Osmoregulated Periplasmic Glucans Is Restored by Inactivation of the RcsCD-RcsB Phosphorelay

Dickeya dadantii is a pectinolytic phytopathogen enterobacterium that causes soft rot disease on a wide range of plant species. The virulence of D. dadantii involves several factors, including the osmoregulated periplasmic glucans (OPGs) that are general constituents of the envelope of proteobacteria. In addition to the loss of virulence, opg-negative mutants display a pleiotropic phenotype, including decreased motility and increased exopolysaccharide synthesis. A nitrosoguanidine-induced mutagenesis was performed on the opgG strain, and restoration of motility was used as a screen. The phenotype of the opg mutant echoes that of the Rcs system: high level activation of the RcsCD-RcsB phosphorelay is needed to activate exopolysaccharide synthesis and to repress motility, while low level activation is required for virulence in enterobacteria. Here, we show that mutations in the RcsCDB phosphorelay system restored virulence and motility in a D. dadantii opg-negative strain, indicating a relationship between the Rcs phosphorelay and OPGs.Osmoregulated periplasmic glucans (OPGs) are general periplasmic constituents of the envelope of most proteobacteria. Their common features are that glucose is the sole constituent sugar, and their abundance in the periplasm increases as the osmolarity of the medium decreases. In Enterobacteriaceae and related bacteria, the glucose backbone synthesis is catalyzed by both products of the opgGH operon (5). Studies of several bacterial pathogens, including Dickeya dadantii, showed the importance of OPGs for virulence (4, 5, 18, 25, 26).Dickeya dadantii is a member of the pectinolytic erwiniae causing soft rot disease in a wide range of plant species (33). The virulence of D. dadantii is associated with the synthesis and the secretion of a set of plant cell wall-degrading enzymes (pectinases, cellulases, and proteases) causing maceration of the plant tissues (22). D. dadantii synthesize OPGs containing 5 to 12 glucose units joined by β,1-2 linkages and branched by β,1-6 linkages that are substituted with succinyl and acetyl residues (11). The opgG or opgH mutants unable to synthesize OPGs show a pleiotropic phenotype. They are nonvirulent on chicory leaves and potato tubers, and synthesis and secretion of pectate-lyases, cellulases, and proteases are reduced (32). Motility is severely reduced, while exopolysaccharide secretion is increased (mucoid phenotype) (32). Data suggest that the opg mutants are impaired in perception of the environment, which prevents D. dadantii from recognizing host cells, suggesting a possible dysfunction of phosphorelay signaling pathways, major systems required for environmental perception in bacteria (6). In these systems, upon stimuli, a kinase/phosphatase sensor autophosphorylates and transfers the phosphate group to a cytoplasmic regulator which modulates expression of target genes.Here, we show that mutations in the rcsC and rcsB genes, encoding, respectively, the sensor and the cognate regulator of the RcsCD-RcsB phosphorelay, suppress several phenotypes of an opgG mutant, including the nonvirulent phenotype on potato tubers. This suggests interactions between the RcsCD-RcsB phosphorelay and OPG molecules and constitutes a first hint at the molecular role of these ubiquitous glycans in virulence.     

Osmoregulated periplasmic glucans of Erwinia chrysanthemi

We report the initial characterization of the osmoregulated periplasmic glucans (OPGs) of Erwinia chrysanthemi. OPGs are intrinsic components of the bacterial envelope necessary to the pathogenicity of this phytopathogenic enterobacterium (F. Page, S. Altabe, N. Hugouvieux-Cotte-Pattat, J.-M. Lacroix, J. Robert-Baudouy and J.-P. Bohin, J. Bacteriol. 183:0000-0000, 2001 [companion in this issue]). OPGs were isolated by trichloracetic acid treatment and gel permeation chromatography. The synthesis of these compounds appeared to be osmoregulated, since lower amounts of OPGs were produced when bacteria were grown in media of higher osmolarities. However, a large fraction of these OPGs were recovered in the culture medium. Then, these compounds were characterized by compositional analysis, high-performance anion-exchange chromatography, matrix-assisted laser desorption mass spectrometry, and (1)H and (13)C nuclear magnetic resonance analyses. OPGs produced by E. chrysanthemi are very heterogeneous at the level of both backbone structure and substitution of these structures. The degree of polymerization of the glucose units ranges from 5 to 12. The structures are branched, with a linear backbone consisting of beta-1,2-linked glucose units to which a variable number of branches, composed of one glucose residue, are attached by beta-1,6 linkages in a random way. This glucan backbone may be substituted by O-acetyl and O-succinyl ester-linked residues.     

Screening for mutants temperature sensitive in protein synthesis by using methionine analogues

Summary Two new mutants are described which are temperature sensitive in protein synthesis. The mutants were obtained by a screening procedure using methionine analogues. The method is based on two findings: a) that in E. coli, and in other members of the Enterobacteriaceae, sensitivity to methionine analogues increases with the growth temperature, and b) that cells which are not synthesizing proteins during treatment with methionine analogues have a shorter lag in resuming growth subsequent to removal of the analogue.     

Coordination of ribosomal RNA synthesis in vertebrate cells

Xenopus embryo cells and HeLa cells were investigated under various conditions to test for coordinate synthesis of high molecular weight (28S and 18S) and low molecular weight (5S) rRNA. Xenopus embryos initiate 28S and 18S rRNA synthesis at gastrulation (Brown and Littna, '64); we found that 5S rRNA synthesis is coordinately initiated with the 28S and 18S rRNAs at the same time in development. Dissociated Xenopus blastula cells were cultured in vitro for several hours to condition the medium; post-gastrula cells were then grown in the conditioned medium to test for the existence of an inhibitor of rRNA synthesis. No inhibitor was detected. Low doses of actinomycin D profoundly inhibit the synthesis of 28S and 18S rRNA in HeLa cells, while 5S rRNA synthesis is less affected by this treatment. Therefore, actinomycin D does not produce a coordinate inhibition of all rRNA species. Similar effects of the antibiotic were found in cultured amphibian cells. Synchronized HeLa cells reinitiating RNA synthesis following mitosis also respond to actinomycin D in a non-coordinate manner.     

Regulation of formation of proteases inBacillus megaterium

Protease was formed only at the end of the growth phase and its synthesis continued in the stationary phase during the growth of the sporulatingBacillus megaterium KM Sp⁺ in complex media with amino acids or peptone and glucose. The enzyme was also not formed during the growth phase in the glucose containing mineral medium and was detected only later during the stationary phase, smaller quantities being observed than those formed in the complex medium. The addition of glucose at the beginning of the synthesis of protease inhibited the production of the enzyme. On the other hand, the addition of the mixture of amino acids under the same conditions stimulated its formation several fold. Cysteine blocked the synthesis of the enzyme unlike other amino acids. Within a certain range the stimulatory effect of amino acids was related to their concentration, being manifested only after a lag period of several hours. The ability to form protease dissappeared after the formation of refractile spores in sporangial cells. Preliminary communication published in Biochem. biophys. Res. Commun. 37: 233, 1969.     

Accumulation of periplasmic enterobactin impairs the growth and morphology of Escherichia coli tolC mutants

TolC is the outer membrane component of tripartite efflux pumps, which expel proteins, toxins and antimicrobial agents from Gram‐negative bacteria. Escherichia coli tolC mutants grow well and are slightly elongated in rich media but grow less well than wild‐type cells in minimal media. These phenotypes have no physiological explanation as yet. Here, we find that tolC mutants have highly aberrant shapes when grown in M9‐glucose medium but that adding iron restores wild‐type morphology. When starved for iron, E. coli tolC mutants synthesize but cannot secrete the siderophore enterobactin, which collects in the periplasm. tolC mutants unable to synthesize enterobactin display no growth or morphological defects, and adding exogenous enterobactin recreates these aberrations, implicating this compound as the causative agent. Cells unable to import enterobactin across the outer membrane grow normally, whereas cells that import enterobactin only to the periplasm become morphologically aberrant. Thus, tolC mutants grown in low iron conditions accumulate periplasmic enterobactin, which impairs bacterial morphology, possibly by sequestering iron and inhibiting an iron‐dependent reaction involved in cell division or peptidoglycan synthesis. The results also highlight the need to supply sufficient iron when studying TolC‐directed export or efflux, to eliminate extraneous physiological effects.     

A carbon catabolite repression mutant of Saccharomyces cerevisiae with elevated hexokinase activity: Evidence for regulatory control of hexokinase PII synthesis

Summary Mutants were investigated that had elevated hexokinase activity and had been isolated previously as resistant to carbon catabolite repression (Zimmermann and Scheel 1977). They were allele tested with mutant strains of Lobo and Maitra (1977), which had defects in one or more of the genes coding for glucokinase and unspecific hexokinases. It was shown, that the mutation abolishing carbon catabolite repression had occured in a gene that was not allelic to any of the structural genes coding for hexokinases. This indicated that a regulatory defect was responsible for elevated hexokinase activity. This agreed with observations that hexokinase activities were like wild-type during growth on non-fermentable carbon sources in hex2 mutants. Recombination between the mutant allele hex2 and mutant alleles hxk1 and hxk2, coding for hexokinase PI and PII respectively, clearly demonstrated that only hexokinase PII was elevated in hex2 mutants. When hex2 mutant cells grown on YEP ethanol were shifted to YEP glucose media, hexokinase activity increased after 30min. This increase depended on de novo protein synthesis. hex2 mutants provide evidence, that carbon catabolite repression and synthesis of hexokinase PII are under common regulatory control.     

TCA cycle activity in Staphylococcus aureus is essential for iron‐regulated synthesis of staphyloferrin A,but not staphyloferrin B: the benefit of a second citrate synthase

Staphylococcus aureus elaborates two citrate‐containing siderophores, staphyloferrin A (SA) and staphyloferrin B (SB), that enhance growth under iron‐restriction, yet, paradoxically, expression of the TCA cycle citrate synthase, CitZ, is downregulated during iron starvation. Iron starvation does, however, result in expression of SbnG, recently identified as a novel citrate synthase that is encoded from within the iron‐regulated SB biosynthetic locus, suggesting an important role for SbnG in staphyloferrin production. We demonstrate that during growth of S. aureus in iron‐restricted media containing glucose, SB is produced but, in contrast, SA production is severely repressed; accordingly, SB‐deficient mutants grow poorly in these media. Hypothesizing that reduced TCA cycle activity hinders SA production, we show that a citZ mutant is capable of SB synthesis, but not SA synthesis, providing evidence that SbnG does not generate citrate for incorporation into SA. A citZ sbnG mutant synthesizes neither staphyloferrin, is severely compromised for growth in iron‐restricted media, and is significantly more impaired for virulence than either of the single‐deletion mutants. We propose that SB is the more important of the two siderophores for S. aureus insofar as it is synthesized, and supports iron‐restricted growth, without need of TCA cycle activity.     

Constitutive appearance of peroxisomes in a regulatory mutant of the methylotrophic yeast Hansenula polymorpha

Summary A selection by glucosamine for mutants of Hansenula polymorpha insensitive to glucose repression of methanol assimilation is described. Constitutive synthesis of enzymes is established in standard batch cultures of glucosegrown cells. Upon prolonged glucose metabolism the phenotype is masked by catabolite inactivation and degradation of enzymes. Addition of the substrate methanol remarkably improves constitutive synthesis by preventing catabolite inactivation and delaying degradation. Regular peroxisomes of reduced number are formed in mutant cells under repressed conditions. No constitutive synthesis is detectable using ethanol as a carbon source. In addition, this alcohol is detrimental to growth of the mutants, indicating that H. polymorpha is constrained to repress synthesis of enzymes involved in the C₁-metabolism when ethanol is present as a substrate.     

Characterization of catalase-negative mutants of methylotrophic yeastHansenula polymorpha

Three recently isolated catalase-negative mutants ofHansenula polymorpha lost the ability to grow on methanol but grew in media containing glucose, ethanol or glycerol. Their incubation in a medium with methanol resulted in an accumulation of hydrogen peroxide and cell death. During growth of a catalase-negative mutant in chemostat on a mixture of methanol and glucose, neither H₂O₂ accumulation nor cell death were observed up to the molar ratio of 10:1 of the two substrates. Cytochrome-c peroxidase and NADH-peroxidase activities were detected in the cells. In methylotrophic yeasts, catalase seems to be an enzyme characteristic of the metabolism of methanol but not needed for the metabolism of multicarbon substrates. The hydrogen peroxide produced during growth of the mutants on mixed substrates is detoxified by cytochrome-c peroxidase and other peroxidases. Translated by Č. Novotny     

Selection of glucose-assimilating variants ofAcinetobacter calcoaceticus LMD 79.41 in chemostat culture

Glucose metabolism has been studied in two strains ofAcinetobacter calcoaceticus. Strain LMD 82.3, was able to grow on glucose and possessed glucose dehydrogenase (EC 1.1.99.17). Glucose oxidation by whole cells was stimulated by PQQ, the prosthetic group of glucose dehydrogenase. PQQ not only increased the rate of glucose oxidation and gluconic acid production but also shortened the lag phase for growth on glucose. Strain LMD 79.41 also possessed glucose dehydrogenase but was unable to grow on glucose. Batch cultures and carbon-limited chemostat cultures growing on acetate in the presence of glucose oxidized the sugar to gluconic acid, which was not further metabolized. However, after prolonged cultivation on mixtures of acetate and glucose, carbon-limited chemostat cultures suddenly acquired the capacity to utilize gluconate. This phenomenon was accompanied by the appearance of gluconate kinase and a repression of isocitrate lyase synthesis. In contrast to the starter culture, cells from chemostats which had been fully adapted to gluconate utilization, were able to utilize glucose as a sole carbon and energy source in liquid and solid media.     

Osmoregulated periplasmic glucans are needed for competitive growth and biofilm formation by Salmonella enterica serovar Typhimurium in leafy-green vegetable wash waters and colonization in mice

Osmoregulated periplasmic glucans (OPGs) are major periplasmic constituents of Gram-negative bacteria. The role of OPGs has been postulated in symbiotic as well as pathogenic host–microorganism interactions. Here, we report the role of OPGs from Salmonella enterica serovar Typhimurium during growth and biofilm formation in leafy-green vegetable wash water. The opgGH mutant strain, which was defective in OPG biosynthesis, initiated the growth at a slower rate in wash waters obtained from spinach, lettuce and green collard and severely impaired biofilm formation. The lack of OPG synthesis did not influence biofilm formation by the opgGH mutant in low-nutrient low-osmolarity laboratory media. In coculture experiments initiated with equal proportions of cells, the opgGH mutant was outnumbered by the wild-type strain under the planktonic as well as the biofilm growth conditions. The opgGH mutant strain poorly colonized mouse organs when introduced orally along with the wild-type strain. This is the first report demonstrating the role of OPGs of Salmonella in competitive colonization of biofilms, planktonic cultures and mouse organs.     

Genetics of alcohol dehydrogenase in Saccharomyces cerevisiae: I. Isolation and genetic analysis of adh mutants

On the basis of allyalcohol resistance, Saccharomyces cerevisiae mutanta were isolated that were deficient in alcohol dehydrogenase (ADH). The mutants were divided into three classes by their different ADH isozyme pattern obtained after starch-gel electrophoresis: adc mutants that did not produce the constitutive ADH, adr mutants from which the glucose repressible enzyme (ADHII) was absent, and adm mutants deficient in ADH activity associated with the mitochondria.Genetic analysis showed that two genes control synthesis of the glucose repressible enzyme ADHII, one gene the constitutive ADHI and a fourth nuclear gene the mitochondrial ADH. None of these four genes showed any linkage.The various mutant types did not show drastic effects on yeast growth on media containing glucose or ethanol as sole carbon sources.     

Yeast mutants without phosphofructokinase activity can still perform glycolysis and alcoholic fermentation

Summary Mutants of Saccharomyces cerevisiae without detectable phosphofructokinase activity were isolated. They were partly recessive and belonged to two genes called PFK1 and PFK2. Mutants with a defect in only one of the two genes could not grow when they were transferred from a medium with a nonfermentable carbon source to a medium with glucose and antimycin A, an inhibitor of respiration. However, the same mutants could grow when antimycin A was added to such mutants after they had been adapted to the utilization of glucose. Double mutants with defects in both genes could not grow at all on glucose as the sole carbon source. Mutants with a single defect in gene PFK1 or PFK2 could form ethanol on a glucose medium. However, in contrast to wild-type cells, there was a lag period of about 2 h before ethanol could be formed after transfer from a medium with only nonfermentable carbon sources to a glucose medium. Wild-type cells under the same conditions started to produce ethanol immediately. Mutants with defects in both PFK genes could not form ethanol at all. Mutants without phosphoglucose isomerase or triosephosphate isomerase did not form ethanol either. Double mutants without phosphofructokinase and phosphoglucose isomerase accumulated large amounts of glucose-6-phosphate on a glucose medium. This suggested that the direct oxidation of glucose-6-phosphate could not provide a bypass around the phosphofructokinase reaction. On the other hand, the triosephosphate isomerase reaction was required for ethanol production. Experiments with uniformly labeled glucose and glucose labeled in positions 3 and 4 were used to determine the contribution of the different carbon atoms of glucose to the fermentative production of CO₂. With only fermentation operating, only carbon atoms 3 and 4 should contribute to CO₂ production. However, wild-type cells produced significant amounts of radioactivity from other carbon atoms and pfk mutants generated CO₂ almost equally well from all six carbon atoms of glucose. This suggested that phosphofructokinase is a dispensable enzyme in yeast glycolysis catalyzing only part of the glycolytic flux.     

Metabolism of acetaldehyde and Custers effect in the yeast

Brettanomyces abstinens growing on different initial glucose concentrations showed an anaerobic inhibition of fermentation. This Custers effect decreased as the initial glucose concentration in the medium increased. Two aldehyde dehydrogenases, one NAD⁺-linked and the other NADP⁺-linked were observed. The results suggest that the NAD⁺-linked enzyme is involved in the production of acetic acid and is repressed by glucose. The NADP⁺-linked enzyme seems to be a constitutive enzyme. Acetyl-CoA synthetase activity also was not greatly affected by the growth conditions.The results support the earlier hypothesis that the Custers effect in Brettanomyces is provoked by the reduction of NAD⁺ in the conversion of acetaldehyde to acetic acid.     

Synthesis of Analogs of Pestalotin

Starch-utilizing mutants of Escherichia coli which can grow well on starch or amylose as the sole carbon source were isolated. The maximal viable cell number of the starch-utilizing mutants on the polysaccharide media reached the same level (4 × 10⁹ cells/ml) as that with glucose medium after incubation for 24 hours at 37°C. The isolated mutants could produce more intracellular α-amylase than the wild-type strain, and the enzyme activity was detected in the extracellular fluid. Polyacrylamide gel electrophoresis showed that the intracellular and extracellular enzymes had similar electrophoretic mobilities. These observations suggested that the ability of growth on the polysaccharide media was due to the excreted α-amylase, which appeared to be identical with the intracellular enzyme.     

Some effects of mannitol on the glucose metabolism of two derivatives of a strain ofRhizobium japonicum differing in mannitol dehydrogenase

The effects of mannitol were investigated by comparing some metabolic features in colonial derivatives, I-110 and L1-110, ofRhizobium japonicum strain 3IIb110, grown either on glucose alone (G-cells) or in glucose media supplemented with mannitol (GM-cells). The polyol stimulated the synthesis of not only mannitol dehydrogenase, which is active in derivative L1-110, but also the nicotinamide adenine dinucleotide (NAD)-linked 6-phosphogluconate (6-PG) dehydrogenase (EC 1.1.1.43). As revealed by radiorespirometry, when GM-cells were allowed to metabolize glucose, they produced relatively more CO₂ from the first and sixth carbons of the sugar than G-cells did. This finding is evidence that NAD-linked 6-PG dehydrogenase might initiate an unknown pathway different from the hexose cycle and the pentose phosphate (PP) pathway. Mannitol exerted no allosteric control on the oxygen consumption and the glucose transport systems. Active uptake of the polyol was correlated with the presence of mannitol dehydrogenase (EC 1.1.1.67); it did not hinder the transport of glucose even though both systems derive their energy for active transport from a common source presumptively characterized as the energized membrane state. Mannitol, however, suppressed by two- or threefold the glucose uptake system. Addition of the polyol to the cell suspensions of both colonial types ofR. japonicum metabolizing glucose caused an immediate 40–50% drop of adenosine triphosphate (ATP) concentrations, owing in part to the mannitol kinase reaction. Type I-110 failed to overcome this reduction of ATP levels, and low growth rates could results. In contrast, type L1-110 offsets the reduction of ATP concentration by oxidizing mannitol as an additional source of energy through mannitol dehydrogenase, fructokinase, and a sequence of glycolytic reactions. The polyol also induced type L1-110 to produce extracellular slimy materials that, apparently, harbor amounts of ATP and proteins.     

Osmoregulated periplasmic glucan synthesis is required for Erwinia chrysanthemi pathogenicity

Erwinia chrysanthemi is a phytopathogenic enterobacterium causing soft rot disease in a wide range of plants. Osmoregulated periplasmic glucans (OPGs) are intrinsic components of the gram-negative bacterial envelope. We cloned the opgGH operon of E. chrysanthemi, encoding proteins involved in the glucose backbone synthesis of OPGs, by complementation of the homologous locus mdoGH of Escherichia coli. OpgG and OpgH show a high level of similarity with MdoG and MdoH, respectively, and mutations in the opgG or opgH gene abolish OPG synthesis. The opg mutants exhibit a pleiotropic phenotype, including overproduction of exopolysaccharides, reduced motility, bile salt hypersensitivity, reduced protease, cellulase, and pectate lyase production, and complete loss of virulence. Coinoculation experiments support the conclusion that OPGs present in the periplasmic space of the bacteria are necessary for growth in the plant host.