Penicillin resistance in bovine Staphylococcus aureus: Genomic evaluation of the discrepancy between phenotypic and molecular test methods.

Staphylococcus aureus is a major pathogen in humans and animals. In cattle, it is one of the most important agents of mastitis, causing serious costs in the dairy industry. Early diagnosis and adequate therapy are therefore 2 key factors to deal with the problems caused by this bacterium, and benzylpenicillin (penicillin) is usually the first choice to treat these infections. Unfortunately, penicillin resistance testing in bovine S. aureus strains shows discrepant results depending on the test used; consequently, the best method for assessing penicillin resistance is still unknown. The aim of this study was therefore to find a method that assesses penicillin resistance in S. aureus and to elucidate the mechanisms leading to the observed discrepancies. A total of 146 methicillin-sensitive S. aureus strains isolated from bovine mastitis were tested for penicillin resistance using a broth microdilution [minimum inhibitory concentration (MIC)] and 2 different disk diffusion protocols. Furthermore, the strains were analyzed for the presence of the bla operon genes (blaI, blaR1, blaZ) by PCR, and a subset of 45 strains was also subjected to whole genome sequencing (WGS). Discrepant results were obtained when penicillin resistance of bovine S. aureus was evaluated by disk diffusion, MIC, and PCR methods. The discrepancies, however, could be fully explained by WGS analysis. In fact, it turned out that penicillin resistance is highly dependent on the completeness of the bla operon promotor: when the bla operon was complete based on WGS analysis, all strains showed MIC ≥1 µg/mL, whereas when the bla operon was mutated (31-nucleotide deletion), they were penicillin sensitive except in those strains where an additional, bla operon-independent resistance mechanism was observed. Further, WGS analyses showed that penicillin resistance is truly assessed by the MIC assay. In contrast, caution is required when interpreting disk diffusion and PCR results.

Clinical screening of oropharyngeal dysphagia in patients with ALS.

OBJECTIVE: It is a major issue to diagnose and detect oropharyngeal dysphagia in the early stage of ALS in order to avoid pulmonary and nutritional complications. The aim of this study was to validate a simple clinical test, the Volume-Viscosity Swallow Test (V-VST), to detect oropharyngeal dysphagia in this population. PATIENTS AND METHODS: Twenty patients were included in this study (mean age: 66.1 ± 8.13, six women). All patients had their swallowing function assessed by videofluoroscopy and V-VST. RESULTS: Among these 20 patients, 15 presented oropharyngeal dysphagia, diagnosed by videofluoroscopy, and five had normal swallowing. Norris score was lower in patients with oropharyngeal dysphagia compared to the patients with normal swallowing (27 ± 6 versus 36 ± 2; P=0.003). Among the 15 patients with oropharyngeal dysphagia, 14 had abnormal V-VST, and only one had normal V-VST. The sensibility of V-VST to detect oropharyngeal dysphagia in these patients with ALS was of 93%, and the specificity was of 80% (P=0.007). There was no significant difference between the two populations for ALSFRS score (22 ± 6 versus 20 ± 6) and body mass index (BMI) (26 ± 6 versus 26 ± 6 kg/m(2)). CONCLUSION: The V-VST presented good sensibility and specificity. It may be interesting to use it systematically for the detection of oropharyngeal dysphagia in ALS, after confirming these results on a wider patient sample.

Effect of surfactants, dispersion and temperature on solubility and biodegradation of phenanthrene in aqueous media.

In the present study surfactant addition with the help of either a mechanical dispersion or a thermal treatment was applied in order to increase the solubility and the bioavailability of phenanthrene in aqueous media, and therefore to promote its biodegradation. Among four tested surfactants (Tween 80, Brij 30, sodium dodecyl sulphate and rhamnolipids), Brij 30 (0.5 gL(-1)) showed the best results allowing us to attain about 20 mgL(-1) of soluble phenanthrene. An additional thermal treatment at 60°C for 24h, 200 rpm permitted to increase the solubility of phenanthrene in the presence of Brij 30 (0.5 gL(-1)) to about 30 mgL(-1). Higher dispersions of phenanthrene particles as well as the reduction of their size were obtained using Ultra-Turrax and French press. The biodegradation of phenanthrene by Pseudomonas putida was then investigated. The reduction of size of phenanthrene particles by mechanical dispersion did not influence its biodegradation, suggesting that P. putida consumed only soluble phenanthrene. The addition of Brij 30 (0.5 gL(-1)) permitted to obtain more phenanthrene metabolized. The use of Brij 30 coupled with a transitory heating of phenanthrene-containing medium at 60°C led to an even more complete biodegradation. This might be a promising way to enhance biodegradation of PAHs.

Influence of intracellular nucleotide and nucleotide sugar contents on recombinant interferon-gamma glycosylation during batch and fed-batch cultures of CHO cells.

Both the macroheterogeneity of recombinant human IFN-gamma produced by CHO cells and intracellular levels of nucleotides and sugar nucleotides, have been characterized during batch and fed-batch cultures carried out in different media. Whereas PF-BDM medium was capable to maintain a high percentage of the doubly- glycosylated glycoforms all over the process, mono-glycosylated and non-glycosylated forms increased during the batch culture using SF-RPMI medium. Intracellular level of UTP was higher in PF-BDM all over the batch culture compared to the SF-RPMI process. UDP-Gal accumulated only during the culture performed in PF-BDM medium, probably as a consequence of the reduced UDP-Glc synthesis flux in SF-RPMI medium. When the recombinant CHO cells were cultivated in fed-batch mode, the UTP level remained at a relatively high value in serum-containing RPMI and its titer increased during the fed-phase indicating an excess of biosynthesis. Besides, an accumulation of UDP-Gal occurred as well. Those results all together indicate that UTP and UDP-Glc syntheses in CHO cells cultivated in SF-RPMI medium in batch process, could be limiting during the glycosylation processes of the recombinant IFN-gamma. At last, the determination of the energetic status of the cells over the three studied processes suggested that a relationship between the adenylate energy charge and the glycosylation macroheterogeneity of the recombinant IFN-gamma may exist.

Intracellular nucleotide and nucleotide sugar contents of cultured CHO cells determined by a fast, sensitive, and high-resolution ion-pair RP-HPLC.

Analysis of intracellular nucleotide and nucleotide sugar contents is essential in studying protein glycosylation of mammalian cells. Nucleotides and nucleotide sugars are the donor substrates of glycosyltransferases, and nucleotides are involved in cellular energy metabolism and its regulation. A sensitive and reproducible ion-pair reverse-phase high-performance liquid chromatography (RP-HPLC) method has been developed, allowing the direct and simultaneous detection and quantification of some essential nucleotides and nucleotide sugars. After a perchloric acid extraction, 13 molecules (8 nucleotides and 5 nucleotide sugars) were separated, including activated sugars such as UDP-glucose, UDP-galactose, GDP-mannose, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine. To validate the analytical parameters, the reproducibility, linearity of calibration curves, detection limits, and recovery were evaluated for standard mixtures and cell extracts. The developed method is capable of resolving picomolar quantities of nucleotides and nucleotide sugars in a single chromatographic run. The HPLC method was then applied to quantify intracellular levels of nucleotides and nucleotide sugars of Chinese hamster ovary (CHO) cells cultivated in a bioreactor batch process. Evolutions of the titers of nucleotides and nucleotide sugars during the batch process are discussed.

Both pH and carbon flux influence the level of rubredoxin in Clostridium butyricum.

The rubredoxin expression level in Clostridium butyricum DSM 5431 grown in continuous culture was monitored using primer extension analyses of the rub gene and a specific enzymatic assay of the iron-sulfur protein. In this way, we showed that variations in rubredoxin content and in rub mRNA level were influenced by the pH of the culture and were directly dependent on the carbon flux. The maximum rubredoxin level reached 1227.3 pmol (mg of proteins)(-1) (i.e. 0.7% of the total protein content) under strictly anaerobic conditions when cells grew at pH 6.5 with an excess of glucose. In addition, primer extension analyses established that the control for all the variations observed operates at the level of gene transcription. Altogether, these results suggested a main function of rubredoxin in Clostridium butyricum independent of the protection against oxygen as has already been reported for Desulfovibrio gigas and Pyrococcus furiosus.

Kinetics and metabolism of cellulose degradation at high substrate concentrations in steady-state continuous cultures of Clostridium cellulolyticum on a chemically defined medium.

The hydrolysis and fermentation of insoluble cellulose were investigated using continuous cultures of Clostridium cellulolyticum with increasing amounts of carbon substrate. At a dilution rate (D) of 0.048 h(-1), biomass formation increased proportionately to the cellulose concentration provided by the feed reservoir, but at and above 7.6 g of cellulose x liter(-1) the cell density at steady state leveled off. The percentage of cellulose degradation declined from 32.3 to 8.3 with 1.9 and 27.0 g of cellulose x liter(-1), respectively, while cellodextrin accumulation rose and represented up to 4.0% of the original carbon consumed. The shift from cellulose-limited to cellulose-sufficient conditions was accompanied by an increase of both the acetate/ethanol ratio and lactate biosynthesis. A kinetics study of C. cellulolyticum metabolism in cellulose saturation was performed by varying D with 18.1 g of cellulose x liter(-1). Compared to cellulose limitation (M. Desvaux, E. Guedon, and H. Petitdemange, J. Bacteriol. 183:119-130, 2001), in cellulose-sufficient continuous culture (i) the ATP/ADP, NADH/NAD+, and q(NADH produced)/q(NADH used) ratios were higher and were related to a more active catabolism, (ii) the acetate/ethanol ratio increased while the lactate production decreased as D rose, and (iii) the maximum growth yield (Y(max)X/S) (40.6 g of biomass per mol of hexose equivalent) and the maximum energetic yield (Y(max)ATP) (19.4 g of biomass per mol of ATP) were lowered. C. cellulolyticum was then able to regulate and optimize carbon metabolism under cellulose-saturated conditions. However, the facts that some catabolized hexose and hence ATP were no longer associated with biomass production with a cellulose excess and that concomitantly lactate production and pyruvate leakage rose suggest the accumulation of an intracellular inhibitory compound(s), which could further explain the establishment of steady-state continuous cultures under conditions of excesses of all nutrients. The following differences were found between growth on cellulose in this study and growth under cellobiose-sufficient conditions (E. Guedon, S. Payot, M. Desvaux, and H. Petitdemange, Biotechnol. Bioeng. 67:327-335, 2000): (i) while with cellobiose, a carbon flow into the cell of as high as 5.14 mmol of hexose equivalent g of cells(-1) x h(-1) could be reached, the maximum entering carbon flow obtained here on cellulose was 2.91 mmol of hexose equivalent g of cells(-1) x h(-1); (ii) while the NADH/NAD+ ratio could reach 1.51 on cellobiose, it was always lower than 1 on cellulose; and (iii) while a high proportion of cellobiose was directed towards exopolysaccharide, extracellular protein, and free amino acid excretions, these overflows were more limited under cellulose-excess conditions. Such differences were related to the carbon consumption rate, which was higher on cellobiose than on cellulose.

Identification of the gene encoding NADH-rubredoxin oxidoreductase in Clostridium acetobutylicum.

NADH-rubredoxin oxidoreductase (NROR), a flavoprotein from the obligately anaerobe Clostridium acetobutylicum is encoded by an ORF (nror) of 1140 nucleotides. Whereas primary structure analysis reveals that NROR has amino acid sequence patterns homologous with those involved in FAD and NAD-binding, the enzyme is distantly related to other flavoproteins in the databank. NROR is highly active for reducing clostridial rubredoxin (Rd) especially against C. acetobutylicum Rd with an efficiency (k(cat)/K(m)) of 400,000 mM(-1)s(-1). These results suggest that Rd from C. acetobutylicum, C. pasteurianum, C. butyricum, and C. cellulolyticum can be interchanged with each other. Since C. acetobutylicum is the sole Clostridium strain that possesses such an enzyme, possible functions are discussed with regard to Desulfovibrio gigas and Pyrococcus furiosus, the only two other anaerobic systems for which a similar activity was reported, but no gene isolated.

Pleiotropic transcriptional repressor CodY senses the intracellular pool of branched-chain amino acids in Lactococcus lactis.

Proteolysis is essential for supplying Lactococcus lactis with amino acids during growth in milk. Expression of the major components of the L. lactis proteolytic system, including the cell wall proteinase (PrtP), the oligopeptide transport system (Opp) and at least four intracellular peptidases (PepO1, PepN, PepC, PepDA2), was shown previously to be controlled negatively by a rich nitrogen source. The transcription of prtP, opp-pepO1, pepN and pepC genes is regulated by dipeptides in the medium. Random insertion mutants derepressed for nitrogen control in the expression of the oligopeptide transport system were isolated using an opp-lacZ fusion. A third of the mutants were targeted in the same locus. The product of the inactivated gene shared 48% identity with CodY from Bacillus subtilis, a pleiotropic repressor of the dipeptide permease operon (dpp) and several genes including genes involved in amino acid degradation and competence induction. The signal controlling CodY-dependent repression was searched for by analysing the response of the opp-lux fusion to the addition of 67 dipeptides with different amino acid compositions. Full correlation was found between the dipeptide content in branched-chain amino acids (BCAA; isoleucine, leucine or valine) and their ability to mediate the repression of opp-pepO1 expression. The repressive effect resulting from specific regulatory dipeptides was abolished in L. lactis mutants affected in terms of their transport or degradation into amino acids, showing that the signal was dependent on the BCAA pool in the cell. Lastly, the repression of opp-pepO1 expression was stronger in a mutant unable to degrade BCAAs, underlining the central role of BCAAs as a signal for CodY activity. This pattern of regulation suggests that, in L. lactis and possibly other Gram-positive bacteria, CodY is a pleiotropic repressor sensing nutritional supply as a function of the BCAA pool in the cell.

Transcriptional pattern of genes coding for the proteolytic system of Lactococcus lactis and evidence for coordinated regulation of key enzymes by peptide supply.

The transcription of 16 genes encoding 12 peptidases (pepC, pepN, pepX, pepP, pepA, pepF2, pepDA1, pepDA2, pepQ, pepT, pepM, and pepO1), P(I) and P(III) proteinases (prtP1 and prtP3), and three transport systems (dtpT, dtpP, and opp-pepO1) of Lactococcus lactis MG1363 was analyzed in response to different environmental factors. Promoter fusions with luciferase reporter genes and/or mRNA analysis were used to study the effects of sugar sources, growth at 37 degrees C, and peptide supply on the transcription of these genes. Only transcription of the pepP gene is modulated by the source of sugar. The presence of potential catabolite-responsive element (CRE) boxes in its promoter region suggests that expression of this gene is directly controlled by catabolic repression. Elevated temperature had no significant effect on the level of transcription of these genes. prtP1, prtP3, pepC, pepN, pepX, and the opp-pepO1 operon are the most highly expressed genes in chemically defined medium, and their expression is repressed 5- to 150-fold by addition of peptide sources such as Casitone in the medium. Moreover, the transcription of prtP1, prtP3, pepC, pepN, and the opp-pepO1 operon is repressed two- to eight-fold by the dipeptides leucylproline and prolylleucine. The transcription of pepDA2 might also be repressed by the peptide sources, but this effect is not observed on the regulation of dtpT, pepP, pepA, pepF2, pepDA1, pepQ, pepT, pepM, and the dtpP operon. The significance of these results with respect to the functions of different components of the proteolytic system in L. lactis are discussed.

Carbon flux distribution and kinetics of cellulose fermentation in steady-state continuous cultures of Clostridium cellulolyticum on a chemically defined medium.

The metabolic characteristics of Clostridium cellulolyticum, a mesophilic cellulolytic nonruminal bacterium, were investigated and characterized kinetically for the fermentation of cellulose by using chemostat culture analysis. Since with C. cellulolyticum (i) the ATP/ADP ratio is lower than 1, (ii) the production of lactate at low specific growth rate (mu) is low, and (iii) there is a decrease of the NADH/NAD(+) ratio and q(NADH produced)/ q(NADH used) ratio as the dilution rate (D) increases in carbon-limited conditions, the chemostats used were cellulose-limited continuously fed cultures. Under all conditions, ethanol and acetate were the main end products of catabolism. There was no shift from an acetate-ethanol fermentation to a lactate-ethanol fermentation as previously observed on cellobiose as mu increased (E. Guedon, S. Payot, M. Desvaux, and H. Petitdemange, J. Bacteriol. 181:3262-3269, 1999). The acetate/ethanol ratio was always higher than 1 but decreased with D. On cellulose, glucose 6-phosphate and glucose 1-phosphate are important branch points since the longer the soluble beta-glucan uptake is, the more glucose 1-phosphate will be generated. The proportion of carbon flowing toward phosphoglucomutase remained constant (around 59.0%), while the carbon surplus was dissipated through exopolysaccharide and glycogen synthesis. The percentage of carbon metabolized via pyruvate-ferredoxin oxidoreductase decreased with D. Acetyl coenzyme A was mainly directed toward the acetate formation pathway, which represented a minimum of 27.1% of the carbon substrate. Yet the proportion of carbon directed through biosynthesis (i.e., biomass, extracellular proteins, and free amino acids) and ethanol increased with D, reaching 27.3 and 16.8%, respectively, at 0.083 h(-1). Lactate and extracellular pyruvate remained low, representing up to 1.5 and 0.2%, respectively, of the original carbon uptake. The true growth yield obtained on cellulose was higher, [50.5 g of cells (mol of hexose eq)(-1)] than on cellobiose, a soluble cellodextrin [36.2 g of cells (mol of hexose eq)(-1)]. The rate of cellulose utilization depended on the solid retention time and was first order, with a rate constant of 0.05 h(-1). Compared to cellobiose, substrate hydrolysis by cellulosome when bacteria are grown on cellulose fibers introduces an extra means for regulation of the entering carbon flow. This led to a lower mu, and so metabolism was not as distorted as previously observed with a soluble substrate. From these results, C. cellulolyticum appeared well adapted and even restricted to a cellulolytic lifestyle.

Cellulose catabolism by Clostridium cellulolyticum growing in batch culture on defined medium.

A reinvestigation of cellulose degradation by Clostridium cellulolyticum in a bioreactor with pH control of the batch culture and using a defined medium was performed. Depending on cellulose concentration, the carbon flow distribution was affected, showing the high flexibility of the metabolism. With less than 6.7 g of cellulose liter(-1), acetate, ethanol, H(2), and CO(2) were the main end products of the fermentation and cellulose degradation reached more than 85% in 5 days. The electron flow from the glycolysis was balanced by the production of H(2) and ethanol, the latter increasing with increasing initial cellulose concentration. From 6.7 to 29.1 g of cellulose liter(-1), the percentage of cellulose degradation declined; most of the cellulase activity remained on the cellulose fibers, the maximum cell density leveled off, and the carbon flow was reoriented from ethanol to acetate. In addition to that of previously indicated end products, lactate production rose, and, surprisingly enough, pyruvate overflow occurred. Concomitantly the molar growth yield and the energetic yield of the biomass decreased. Growth arrest may be linked to sufficiently high carbon flow, leading to the accumulation of an intracellular inhibitory compound(s), as observed on cellobiose (E. Guedon, M. Desvaux, S. Payot, and H. Petitdemange, Microbiology 145:1831-1838, 1999). These results indicated that bacterial metabolism exhibited on cellobiose was distorted compared to that exhibited on a substrate more closely related to the natural ecosystem of C. cellulolyticum. To overcome growth arrest and to improve degradation at high cellulose concentrations (29.1 g liter(-1)), a reinoculation mode was evaluated. This procedure resulted in an increase in the maximum dry weight of cells (2,175 mg liter(-1)), cellulose solubilization (95%), and end product concentrations compared to a classical batch fermentation with a final dry weight of cells of 580 mg liter(-1) and 45% cellulose degradation within 18 days.

Kinetic analysis of Clostridium cellulolyticum carbohydrate metabolism: importance of glucose 1-phosphate and glucose 6-phosphate branch points for distribution of carbon fluxes inside and outside cells as revealed by steady-state continuous culture.

During the growth of Clostridium cellulolyticum in chemostat cultures with ammonia as the growth-limiting nutrient, as much as 30% of the original cellobiose consumed by C. cellulolyticum was converted to cellotriose, glycogen, and polysaccharides regardless of the specific growth rates. Whereas the specific consumption rate of cellobiose and of the carbon flux through glycolysis increased, the carbon flux through the phosphoglucomutase slowed. The limitation of the path through the phosphoglucomutase had a great effect on the accumulation of glucose 1-phosphate (G1P), the precursor of cellotriose, exopolysaccharides, and glycogen. The specific rates of biosynthesis of these compounds are important since as much as 16.7, 16.0, and 21.4% of the specific rate of cellobiose consumed by the cells could be converted to cellotriose, exopolysaccharides, and glycogen, respectively. With the increase of the carbon flux through glycolysis, the glucose 6-phosphate (G6P) pool decreased, whereas the G1P pool increased. Continuous culture experiments showed that glycogen biosynthesis was associated with rapid growth. The same result was obtained in batch culture, where glycogen biosynthesis reached a maximum during the exponential growth phase. Glycogen synthesis in C. cellulolyticum was also not subject to stimulation by nutrient limitation. Flux analyses demonstrate that G1P and G6P, connected by the phosphoglucomutase reaction, constitute important branch points for the distribution of carbon fluxes inside and outside cells. From this study it appears that the properties of the G1P-G6P branch points have been selected to control excretion of carbon surplus and to dissipate excess energy, whereas the pyruvate-acetyl coenzyme A branch points chiefly regulate the redox balance of the carbon catabolism as was shown previously (E. Guedon et al., J. Bacteriol. 181:3262-3269, 1999).

Relationships between cellobiose catabolism, enzyme levels, and metabolic intermediates in Clostridium cellulolyticum grown in a synthetic medium.

Continuous cultures, under cellobiose sufficient concentrations (14. 62 mM) using a chemically defined medium, were examined to determine the carbon regulation selected by Clostridium cellulolyticum. Using a synthetic medium, a q(cellobiose) of 2.57 mmol g cells(-1) h(-1) was attained whereas the highest value obtained on complex media was 0.68 mmol g cells(-1) h(-1) (Payot et al. 1998. Microbiology 144:375-384). On a synthetic medium at D = 0.035 h(-1) under cellobiose excess, lactate and ethanol biosynthesis were able to use the reducing equivalents supplied by acetic acid formation and the H(2)/CO(2) ratio was found equal to 1. At a higher dilution rate (D = 0.115 h(-1)), there was no lactate production and the pathways toward ethanol and NADH-ferredoxin-hydrogenase contributed to balance the reducing equivalents; in this case a H(2)/CO(2) ratio of 1.54 was found. With increasing D, there was a progressive increase (i) in the steady-state concentration of NADH and NAD(+) pools from 11.8 to 22.1 micromol (g cells) (-1), (ii) in the intracellular NADH/NAD(+) ratios from 0.43 to 1.51. On synthetic media, under cellobiose excess the carbon flow was also equilibrated by three overflows: exopolysaccharide, extracellular protein, and amino acid excretions. At D = 0.115 h(-1), 34% of the cellobiose consumed was converted into exopolysaccharides; this deviation of the carbon flow and the increase of the phosphoroclastic activity decreased dramatically the pyruvate excretion and explained the break in lactate production. Whatever the dilution rate, C. cellulolyticum, using ammonium and cellobiose excess, always spilled usual amino acids accompanied by other amino compounds. In vitro, GAPDH, phosphoroclastic reaction, alcohol dehydrogenase, and acetate kinase activities were high under conditions giving high in vivo specific production rates. There were also correlations between the in vitro lactate dehydrogenase activity and in vivo lactate production, but in contrast with the preceding activities, these two parameters decreased with D. All the results demonstrate that C. cellulolyticum was able to optimize carbon catabolism from cellulosic substrates in a synthetic medium.

Induction of lactate production associated with a decrease in NADH cell content enables growth resumption of Clostridium cellulolyticum in batch cultures on cellobiose.

When grown in batch cultures in fermentors with 23.4 mM cellobiose, Clostridium cellulolyticum displayed biphasic growth kinetics not associated with sequential substrate consumption and which led to a twofold higher production of biomass than previously reported. In the first growth phase, acetate was the major product of cellobiose metabolism, since lactate and ethanol productions remained low. Furthermore, an accumulation of intracellular NADH was observed. The transition towards the second growth phase was accompanied by an induction of lactate production, in such a way that lactate became the major product of C. cellulolyticum metabolism. In addition, a decrease in NADH concentration was measured, concomitant with this induction of lactate production and with the growth resumption. During both growth phases, the NADH-ferredoxin reductase-hydrogenase system played a major function in NADH regeneration, since H2 production was 1.4- to 1.5-fold higher than that of CO2. Thus, we found that lactate production serves as an additional catabolic pathway enabling C. cellulolyticum to cope with excesses of carbon and NADH produced. Growth experiments on C. cellulolyticum under an atmosphere of carbon monoxide mimicked this phenomenon and confirmed that a high intracellular level of NADH can provide a barrier to bacterial growth.

Effect of dilution rate, cellobiose and ammonium availabilities on Clostridium cellulolyticum sporulation.

The nutritional and physiological factors affecting sporulation of Clostridium cellulolyticum were studied using steady-state continuous cultures grown in both complex and synthetic media. Under cellobiose limitation, the probability that cells will sporulate appears to be directly related to the growth rate. In complex medium, the highest percentage of sporulation was 20% at a dilution rate of 0.015 h-1 whereas in synthetic medium it was 10% at 0.035 h-1. In both media, when the dilution rate was either higher or lower the percentage of sporulation decreased by between 2% and 4%. At low dilution rates, endospore formation was repressed under cellobiose-sufficient concentrations, suggesting catabolite repression by cellobiose. Furthermore, the concentration of ammonium was important in determining the percentage of sporulation, as ammonium limitation induced extensive sporulation at low growth rates even in an excess of cellobiose. The sporulation process is not triggered when cells are cellobiose-exhausted both in complex and synthetic media. These data suggest that, in C. cellulolyticum, an exogenous supply of carbon is required throughout the sporulation process. In the experimental conditions used in this work, no relationship between glycogen accumulation or glycogen mobilization and endospore formation was detected in C. cellulolyticum.

Growth inhibition of Clostridium cellulolyticum by an inefficiently regulated carbon flow.

Carbon flow in Clostridium cellulolyticum was investigated either in batch or continuous culture using a synthetic medium with cellobiose as the sole source of carbon and energy. Previous experiments carried out using a complex growth medium led to the conclusion that the carbon flow was stopped by intracellular NADH. In this study, results showed that cells cultured in a synthetic medium were better able to control electron flow since the NADH/NAD+ ratios were in the range 0.3-0.7, whereas a ratio as high as 57 was previously found in cells cultured on a complex medium. Furthermore, a specific rate of cellobiose consumption of 2.13 mmol (g cells)-1 h-1 was observed on synthetic medium whereas the highest value obtained on complex medium was 0.68 mmol (g cells)-1 h-1. When C. cellulolyticum was grown in continuous culture and cellobiose in the feed medium was increased from 5.84 to 17.57 mM in stepwise fashion, there was an increase in cellobiose utilization without growth inhibition. In contrast, when the reactor was fed directly with 14.62 mM cellobiose, residual cellobiose was observed (4.24 mM) and growth was limited. These data indicate that C. cellulolyticum is not able to optimize its growth and carbon flow in response to a sudden increase in the concentration of growth substrate cellobiose. This interpretation was confirmed (i) by the study of cellobiose batch fermentation where it was demonstrated that growth inhibition was not due to nutritional limitation or inhibition by fermentation products but was associated with carbon excess and (ii) by the growth of C. cellulolyticum in dialysis culture where no growth inhibition was observed due to the limitation of carbon flow by the low rate of cellobiose diffusion through the dialysis tubing.

The extracellular xylan degradative system in Clostridium cellulolyticum cultivated on xylan: evidence for cell-free cellulosome production.

In this study, we demonstrate that the cellulosome of Clostridium cellulolyticum grown on xylan is not associated with the bacterial cell. Indeed, the large majority of the activity (about 90%) is localized in the cell-free fraction when the bacterium is grown on xylan. Furthermore, about 70% of the detected xylanase activity is associated with cell-free high-molecular-weight complexes containing avicelase activity and the cellulosomal scaffolding protein CipC. The same repartition is observed with carboxymethyl cellulase activity. The cellulose adhesion of xylan-grown cells is sharply reduced in comparison with cellulose-grown cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that cellulosomes derived from xylan- and cellulose-grown cells have different compositions. In both cases, the scaffolding protein CipC is present, but the relative proportions of the other components is dramatically changed depending on the growth substrate. We propose that, depending on the growth substrate, C. cellulolyticum is able to regulate the cell association and cellulose adhesion of cellulosomes and regulate cellulosomal composition.

Carbon and electron flow in Clostridium cellulolyticum grown in chemostat culture on synthetic medium.

Previous results indicated poor sugar consumption and early inhibition of metabolism and growth when Clostridium cellulolyticum was cultured on medium containing cellobiose and yeast extract. Changing from complex medium to a synthetic medium had a strong effect on (i) the specific cellobiose consumption, which was increased threefold; and (ii) the electron flow, since the NADH/NAD+ ratios ranged from 0.29 to 2.08 on synthetic medium whereas ratios as high as 42 to 57 on complex medium were observed. These data indicate a better control of the carbon flow on mineral salts medium than on complex medium. By continuous culture, it was shown that the electron flow from glycolysis was balanced by the production of hydrogen gas, ethanol, and lactate. At low levels of carbon flow, pyruvate was preferentially cleaved to acetate and ethanol, enabling the bacteria to maximize ATP formation. A high catabolic rate led to pyruvate overflow and to increased ethanol and lactate production. In vitro, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and ethanol dehydrogenase levels were higher under conditions giving higher in vivo specific production rates. Redox balance is essentially maintained by NADH-ferredoxin reductase-hydrogenase at low levels of carbon flow and by ethanol dehydrogenase and lactate dehydrogenase at high levels of carbon flow. The same maximum growth rate (0.150 h-1) was found in both mineral salts and complex media, proving that the uptake of nutrients or the generation of biosynthetic precursors occurred faster than their utilization. On synthetic medium, cellobiose carbon was converted into cell mass and catabolized to produce ATP, while on complex medium, it served mainly as an energy supply and, if present in excess, led to an accumulation of intracellular metabolites as demonstrated for NADH. Cells grown on synthetic medium and at high levels of carbon flow were able to induce regulatory responses such as the production of ethanol and lactate dehydrogenase.

Metabolism of cellobiose by Clostridium cellulolyticum growing in continuous culture: evidence for decreased NADH reoxidation as a factor limiting growth.

Previous results indicated that molar growth yields are reduced when Clostridium cellulolyticum is cultured in media containing cellobiose concentrations greater than 1 g I-1. Continuous cultures were examined to determine the physiological basis of these poor growth yields. Acetate was the main product of C. cellulolyticum metabolism, whereas the production of reduced compounds such as ethanol or lactate was low. Such patterns of product formation were accompanied by a 12-fold increase in intracellular NADH concentration when the cellobiose flow was increased. Catabolic enzymic activities were measured in vitro. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), acetate kinase and phosphoroclastic activities were found at similar levels as in cells metabolizing higher substrate concentrations. In contrast, lactate dehydrogenase activity was low and correlated with the rate of lactate production. Furthermore, an inhibition of GAPDH activity by high NADH/NAD+ ratios was established. These results suggested that a decreased NADH reoxidation could be responsible for limiting C. cellulolyticum growth. Lactate and ethanol production were not sufficient to balance out the NADH produced in the GAPDH step of glycolysis. One consequence of poor NADH reoxidation would be an increase in intracellular concentration of NADH, which in turn could inhibit GAPDH activity.