Decreased Growth Yield Research Articles

Isocitrate dehydrogenase mutation in Vibrio anguillarum results in virulence attenuation and immunoprotection in rainbow trout (Oncorhynchus mykiss)

BackgroundVibrio anguillarum is an extracellular bacterial pathogen that is a causative agent of vibriosis in finfish and crustaceans with mortality rates ranging from 30% to 100%. Mutations in central metabolism (glycolysis and the TCA cycle) of intracellular pathogens often result in attenuated virulence due to depletion of required metabolic intermediates; however, it was not known whether mutations in central metabolism would affect virulence in an extracellular pathogen such as V. anguillarum.ResultsSeven central metabolism mutants were created and characterized with regard to growth in minimal and complex media, expression of virulence genes, and virulence in juvenile rainbow trout (Oncorhynchus mykiss). Only the isocitrate dehydrogenase (icd) mutant was attenuated in virulence against rainbow trout challenged by either intraperitoneal injection or immersion. Further, the icd mutant was shown to be immunoprotective against wild type V. anguillarum infection. There was no significant decrease in the expression of the three hemolysin genes detected by qRT-PCR. Additionally, only the icd mutant exhibited a significantly decreased growth yield in complex media. Growth yield was directly related to the abundance of glutamate. A strain with a restored wild type icd gene was created and shown to restore growth to a wild type cell density in complex media and pathogenicity in rainbow trout.ConclusionsThe data strongly suggest that a decreased growth yield, resulting from the inability to synthesize α-ketoglutarate, caused the attenuation despite normal levels of expression of virulence genes. Therefore, the ability of an extracellular pathogen to cause disease is dependent upon the availability of host-supplied nutrients for growth. Additionally, a live vaccine strain could be created from an icd deletion strain.

Use of Microcalorimetry To Determine the Costs and Benefits to Pseudomonas putida Strain KT2440 of Harboring Cadmium Efflux Genes

A novel microcalorimetric approach was used to analyze the responses of a metal-tolerant soil bacterium (Pseudomonas putida strain KT2440) to metal resistance gene deletions in cadmium-amended media. As hypothesized, under cadmium stress, the wild-type strain benefited from the resistance genes by entering the exponential growth phase earlier than two knockout strains. In the absence of cadmium, strain KT1, carrying a deletion in the main component (czcA1) of a Cd/Zn chemiosmotic efflux transporter (CzcCBA1), grew more efficiently than the wild type and released ∼700 kJ (per mole of biomass carbon) less heat than the wild-type strain, showing the energetic cost of maintaining CzcCBA1 in the absence of cadmium. A second mutant strain (KT4) carrying a different gene deletion, ΔcadA2, which encodes the main Cd/Pb efflux transporter (a P-type ATPase), did not survive beyond moderate cadmium concentrations and exhibited a decreased growth yield in the absence of cadmium. Therefore, CadA2 plays an essential role in cadmium resistance and perhaps serves an additional function. The results of this study provide direct evidence that heavy metal cation efflux mechanisms facilitate shorter lag phases in the presence of metals and that the maintenance and expression of tolerance genes carry quantifiable energetic costs and benefits.

Alterations of cellular physiology in Escherichia coli in response to oxidative phosphorylation impaired by defective F1-ATPase.

The physiological changes in an F1-ATPase-defective mutant of Escherichia coli W1485 growing in a glucose-limited chemostat included a decreased growth yield (60%) and increased specific rates of both glucose consumption (168%) and respiration (171%). Flux analysis revealed that the mutant showed approximately twice as much flow in glycolysis but only an 18% increase in the tricarboxylic acid (TCA) cycle, owing to the excretion of acetate, where most of the increased glycolytic flux was directed. Genetic and biochemical analyses of the mutant revealed the downregulation of many TCA cycle enzymes, including citrate synthase, and the upregulation of the pyruvate dehydrogenase complex in both transcription and enzyme activities. These changes seemed to contribute to acetate excretion in the mutant. No transcriptional changes were observed in the glycolytic enzymes, despite the enhanced glycolysis. The most significant alterations were found in the respiratory-chain components. The total activity of NADH dehydrogenases (NDHs) and terminal oxidases increased about twofold in the mutant, which accounted for its higher respiration rate. These changes arose primarily from the increased (3.7-fold) enzyme activity of NDH-2 and an increased amount of cytochrome bd in the mutant. Transcriptional upregulation appeared to be involved in these phenomena. As NDH-2 cannot generate an electrochemical gradient of protons and as cytochrome bd is inferior to cytochrome bo3 in this ability, the mutant was able to recycle NADH at a higher rate than the parent and avoid generating an excess proton-motive force. We discuss the physiological benefits of the alterations in the mutant.

Correlation of acetate catabolism and growth yield in Staphylococcus aureus: implications for host-pathogen interactions.

Recently, we reported that the prototypical Staphylococcus aureus strain RN6390 (a derivative of NCTC 8325) had significantly reduced aconitase activity relative to a diverse group of S. aureus isolates, leading to the hypothesis that strain RN6390 has impaired tricarboxylic acid (TCA) cycle-mediated acetate catabolism. Analysis of the culture supernatant from RN6390 confirmed that acetate was incompletely catabolized, suggesting that the ability to catabolize acetate can be lost by S. aureus. To test this hypothesis, we examined the carbon catabolism of the S. aureus strains whose genome sequences are publicly available. All strains catabolized glucose and excreted acetate into the culture medium. However, strains NCTC 8325 and N315 failed to catabolize acetate during the postexponential growth phase, resulting in significantly lower growth yields relative to strains that catabolized acetate. Strains NCTC 8325 and RN6390 contained an 11-bp deletion in rsbU, the gene encoding a positive regulator of the alternative sigma factor sigma(B) encoded by sigB. An isogenic derivative strain of RN6390 containing the wild-type rsbU gene had significantly increased acetate catabolism, demonstrating that sigma(B) is required for acetate catabolism. Taken together, the data suggest that naturally occurring mutations can alter the ability of S. aureus to catabolize acetate, a surprising discovery, as TCA cycle function has been demonstrated to be involved in the virulence, survival, and persistence of several pathogenic organisms. Additionally, these mutations decrease the fitness of S. aureus by reducing the number of progeny placed into subsequent generations, suggesting that in certain situations a decreased growth yield is advantageous.

Antimicrobial activity of sodium lactate

Sodium lactate (5%) used in neutral culture media has an antimicrobial effect which rises above that what would be expected from its water activity lowering effect. This antimicrobial effect was found to exist towards various lactic acid bacteria, Staphylococcus aureus, and Salmonella typhimurium. It is particularly evident from increased lag phase, decreased growth yield, and somewhat less from decreased growth rate. Adverse growing conditions, such as suboptimum temperature, increase its anti-microbial effect. Escherichia coli is an exception, as its growth rate is hardly affected by 5% sodium lactate, although its growth yield is lowered. The antimicrobial activity of sodium lactate evidently reflects how various micro-organisms can cope with this compound.

Isolation and genetic study of Triethyltin-resistant mutants of Saccharomyces cerevisiae

Three mutants of Saccharomyces cerevisiae resistant to triethyltin (an inhibitor of mitochondrial ATPase) on non-fermentative media, and non-resistant to this drug on fermentative media, were isolated and named TTR1, TTR2 and TTR3. Apart from triethyltin resistance, these mutants show the following common characteristics: (1) Increased intracellular cytochrome c concentration. (2) Increased respiration rate. (3) Decreased growth yield. (4) Increased growth sensitivity to several drugs inhibiting oxidative phosphorylation: namely, CCCP (permeabilizing inner mitochondrial membrane to protons), valinomycin (permeabilizing inner mitochondrial membrane to potassium) and oligomycin (inhibitor of mitochondrial ATPase). (5) Increased sensitivity to carbon source starvation. For each mutant, these characteristics appeared to be due to a single pleiotropic nuclear mutation. Mutation TTR1 causes additional phenotypic characteristics which do not appear in mutants TTR2 and TTR3: (1) Pinkish coloration of colonies which is more pronounced after a long growth period. (2) Inability of the cells to store glycogen. (3) Growth defect of the cells on a galactose-containing medium. (4) Inability of a diploid homozygote mutant strain to sporulate. All these phenotypic characteristics have already been described in yeast mutants deregulated in cAMP-dependent protein phosphorylation. Crossing of a strain bearing the TTR1 mutation with a strain mutated in the adenylate cyclase structural gene suggested that the TTR1 phenotype is due to a modification in regulation of cAPK by cAMP, making cell multiplication possible without intracellular cAMP.

Enhanced Growth of Complex Communities of Dental Plaque Bacteria in Mucin-Limited Continuous Culture

The possibility that bacteria in plaque can grow at rates which are unaffected by the availability of dietary foods, because of their ability to metabolise salivary proteins, has been investigated by Keevil et al. using an in vitro continuous culture model. Plaque was collected aseptically from 13 school children, pooled and inoculated into a chemostat containing complex medium plus glucose. The principal genera enriched during growth at dilution rates of 0.05 and 0.2 h -1 (corresponding to mean generation times of 13.9 and 3.5 h, respectively, which might occur in vivo ) were streptococci (including Streptococcus mutans ) peptostreptococci, lactobacilli, fusiforms, veillonellae and Bacteroides spp . Addition of 0.1 per cent (w/v) mucin resulted in an increased growth yield, due to increases in the numbers of anaerobes, but the recovery of peptostreptococci and veillonellae decreased. Importantly, Actinomyces spp . increased and the appearance of spirochaetes was noted in all cultures fed with mucin. Withdrawal of glucose from mucin supplemented complex medium resulted in decreased growth yield, the loss of Neisseria spp . and decreased recovery of streptococci and lactobacilli. Replacement of the medium with artificial saliva containing 0.1 per cent (w/v) mucin enriched for Bacteroides spp ., but lower numbers of the other genera found in plaque were also maintained at both growth rates. This suggests that the chemostat can be used successfully to model the oral ecosystem, with salivary proteins providing the principal source of carbon and nitrogen for growth. Keywords: Mucin enrichment; plaque bacteria. mucin-limited continuous culture; enrichment of complex microbial communities.

Enhanced Growth of Complex Communities of Dental Plaque Bacteria in Mucin-Limited Continuous Culture

The possibility that bacteria in plaque can grow at rates which are unaffected by the availability of dietary foods, because of their ability to metabolise salivary proteins, has been investigated by Keevil et al. using an in vitro continuous culture model. Plaque was collected aseptically from 13 school children, pooled and inoculated into a chemostat containing complex medium plus glucose. The principal genera enriched during growth at dilution rates of 0.05 and 0.2 h -1 (corresponding to mean generation times of 13.9 and 3.5 h, respectively, which might occur in vivo ) were streptococci (including Streptococcus mutans ) peptostreptococci, lactobacilli, fusiforms, veillonellae and Bacteroides spp . Addition of 0.1 per cent (w/v) mucin resulted in an increased growth yield, due to increases in the numbers of anaerobes, but the recovery of peptostreptococci and veillonellae decreased. Importantly, Actinomyces spp . increased and the appearance of spirochaetes was noted in all cultures fed with mucin. Withdrawal of glucose from mucin supplemented complex medium resulted in decreased growth yield, the loss of Neisseria spp . and decreased recovery of streptococci and lactobacilli. Replacement of the medium with artificial saliva containing 0.1 per cent (w/v) mucin enriched for Bacteroides spp ., but lower numbers of the other genera found in plaque were also maintained at both growth rates. This suggests that the chemostat can be used successfully to model the oral ecosystem, with salivary proteins providing the principal source of carbon and nitrogen for growth. Keywords: Mucin enrichment; plaque bacteria. mucin-limited continuous culture; enrichment of complex microbial communities.