Influence of Carbohydrate Starvation and Arginine on Culturability and Amino Acid Utilization of Lactococcus lactis subsp. lactis

Two strains of Lactococcus lactis subsp. lactis were used to determine the influence of lactose and arginine on viability and amino acid use during carbohydrate starvation. Lactose provided energy for logarithmic-phase growth, and amino acids such as arginine provided energy after carbohydrate exhaustion. Survival time, cell numbers, and ATP concentrations increased with the addition of arginine to the basal medium. By the onset of lactose exhaustion, the concentrations of glycine-valine and glutamate had decreased by as much as 67% in L. lactis ML3, whereas the serine concentration increased by 97% during the same period. When no lactose was added, the concentrations of these amino acids remained constant. Similar trends were observed for L. lactis 11454. Without lactose or arginine, L. lactis ML3 was nonculturable on agar but was viable after 2 days, as measured by fluorescent viability stains and intracellular ATP levels. However, L. lactis 11454 without lactose or arginine remained culturable for at least 14 days. These data suggest that lactococci become viable but nonculturable in response to carbohydrate depletion. Additionally, these data indicate that amino acids other than arginine facilitate the survival of L. lactis during carbohydrate starvation.     

Fatty acid production from amino acids and alpha-keto acids by Brevibacterium linens BL2

Low concentrations of branched-chain fatty acids, such as isobutyric and isovaleric acids, develop during the ripening of hard cheeses and contribute to the beneficial flavor profile. Catabolism of amino acids, such as branched-chain amino acids, by bacteria via aminotransferase reactions and alpha-keto acids is one mechanism to generate these flavorful compounds; however, metabolism of alpha-keto acids to flavor-associated compounds is controversial. The objective of this study was to determine the ability of Brevibacterium linens BL2 to produce fatty acids from amino acids and alpha-keto acids and determine the occurrence of the likely genes in the draft genome sequence. BL2 catabolized amino acids to fatty acids only under carbohydrate starvation conditions. The primary fatty acid end products from leucine were isovaleric acid, acetic acid, and propionic acid. In contrast, logarithmic-phase cells of BL2 produced fatty acids from alpha-keto acids only. BL2 also converted alpha-keto acids to branched-chain fatty acids after carbohydrate starvation was achieved. At least 100 genes are potentially involved in five different metabolic pathways. The genome of B. linens ATCC 9174 contained these genes for production and degradation of fatty acids. These data indicate that brevibacteria have the ability to produce fatty acids from amino and alpha-keto acids and that carbon metabolism is important in regulating this event.     

Catabolite inhibition and sequential metabolism of sugars by Streptococcus lactis.

Growth of galactose-adapted cells of Streptococcus lactis ML(3) in a medium containing a mixture of glucose, galactose, and lactose was characterized initially by the simultaneous metabolism of glucose and lactose. Galactose was not significantly utilized until the latter sugars had been exhausted from the medium. The addition of glucose or lactose to a culture of S. lactis ML(3) growing exponentially on galactose caused immediate inhibition of galactose utilization and an increase in growth rate, concomitant with the preferential metabolism of the added sugar. Under nongrowing conditions, cells of S. lactis ML(3) grown previously on galactose metabolized the three separate sugars equally rapidly. However, cells suspended in buffer containing a mixture of glucose plus galactose or lactose plus galactose again consumed glucose or lactose preferentially. The rate of galactose metabolism was reduced by approximately 95% in the presence of the inhibitory sugar, but the maximum rate of metabolism was resumed upon exhaustion of glucose or lactose from the system. When presented with a mixture of glucose and lactose, the resting cells metabolized both sugars simultaneously. Lactose, glucose, and a non-metabolizable glucose analog (2-deoxy-d-glucose) prevented the phosphoenolpyruvate-dependent uptake of thiomethyl-beta-d-galactopyranoside (TMG), but the accumulation of TMG, like galactose metabolism, commenced immediately upon exhaustion of the metabolizable sugars from the medium. Growth of galactose-adapted cells of the lactose-defective variant S. lactis 7962 in the triple-sugar medium was characterized by the sequential metabolism of glucose, galactose, and lactose. Growth of S. lactis ML(3) and 7962 in the triple-sugar medium occurred without apparent diauxie, and for each strain the patterns of sequential sugar metabolism under growing and nongrowing conditions were identical. Fine control of the activities of preexisting enzyme systems by catabolite inhibition may afford a satisfactory explanation for the observed sequential utilization of sugars by these two organisms.     

Nonculturable bacteria: programmed survival forms or cells at death's door?

Upon starvation and growth arrest, Escherichia coli cells gradually lose their ability to reproduce. These apparently sterile/nonculturable cells initially remain intact and metabolically active and the underlying molecular mechanism behind this sterility is something of an enigma in bacteriology. Three different models have been proposed to explain this phenomenon. The first theory suggests that starving cells become nonculturable due to cellular deterioration, are moribund, and show some of the same signs of senescence as aging organisms. The two other theories suggest that genetically programmed pathways, rather than stochastic deterioration, trigger nonculturability. One "program" theory suggests that nonculturability is the culmination of an adaptive pathway generating dormant survival forms, similar to spore formation in differentiating bacteria. The other "program" theory states that starved cells lose viability due to activation of genetic modules mediating programmed cell death. The different models will be reviewed and evaluated in light of recent data on the physiology and molecular biology of growth-arrested E. coli cells.     

Membrane fatty acid and virulence changes in the viable but nonculturable state of Vibrio vulnificus

The nonculturable state of Vibrio vulnificus and, for comparison, that of Escherichia coli were studied in artificial-seawater microcosms at 5 degrees C. Total cell counts were monitored by acridine orange epifluorescence, metabolic activity by direct viable counts, and culturability by plate counts on selective and nonselective media. Whereas total counts remained constant, plate counts of V. vulnificus suggested nonculturability by day 24. In contrast, direct viable counts indicated significant cell viability throughout 32 days of incubation. As an indication of the metabolic changes that occurred as cells entered the state of nonrecoverability, membrane fatty acid analyses were performed. At the point of nonculturability of V. vulnificus, the major fatty acid species (C16 and C16:1) had decreased 57% from the T0 level, concomitant with the appearance of several short-chain acids. Although the bacteria were still recoverable, a similar trend was observed with E. coli. Electron microscopy of nonculturable V. vulnificus showed that the cells were rounded and reduced in size and contained fewer ribosomes. Mouse infectivity studies conducted with these cells suggested loss of virulence.     

Membrane fatty acid and virulence changes in the viable but nonculturable state of Vibrio vulnificus.

The nonculturable state of Vibrio vulnificus and, for comparison, that of Escherichia coli were studied in artificial-seawater microcosms at 5 degrees C. Total cell counts were monitored by acridine orange epifluorescence, metabolic activity by direct viable counts, and culturability by plate counts on selective and nonselective media. Whereas total counts remained constant, plate counts of V. vulnificus suggested nonculturability by day 24. In contrast, direct viable counts indicated significant cell viability throughout 32 days of incubation. As an indication of the metabolic changes that occurred as cells entered the state of nonrecoverability, membrane fatty acid analyses were performed. At the point of nonculturability of V. vulnificus, the major fatty acid species (C16 and C16:1) had decreased 57% from the T0 level, concomitant with the appearance of several short-chain acids. Although the bacteria were still recoverable, a similar trend was observed with E. coli. Electron microscopy of nonculturable V. vulnificus showed that the cells were rounded and reduced in size and contained fewer ribosomes. Mouse infectivity studies conducted with these cells suggested loss of virulence.     

Fatty Acid Production from Amino Acids and α-Keto Acids by Brevibacterium linens BL2

Low concentrations of branched-chain fatty acids, such as isobutyric and isovaleric acids, develop during the ripening of hard cheeses and contribute to the beneficial flavor profile. Catabolism of amino acids, such as branched-chain amino acids, by bacteria via aminotransferase reactions and α-keto acids is one mechanism to generate these flavorful compounds; however, metabolism of α-keto acids to flavor-associated compounds is controversial. The objective of this study was to determine the ability of Brevibacterium linens BL2 to produce fatty acids from amino acids and α-keto acids and determine the occurrence of the likely genes in the draft genome sequence. BL2 catabolized amino acids to fatty acids only under carbohydrate starvation conditions. The primary fatty acid end products from leucine were isovaleric acid, acetic acid, and propionic acid. In contrast, logarithmic-phase cells of BL2 produced fatty acids from α-keto acids only. BL2 also converted α-keto acids to branched-chain fatty acids after carbohydrate starvation was achieved. At least 100 genes are potentially involved in five different metabolic pathways. The genome of B. linens ATCC 9174 contained these genes for production and degradation of fatty acids. These data indicate that brevibacteria have the ability to produce fatty acids from amino and α-keto acids and that carbon metabolism is important in regulating this event.     

Glycosyl hydrolases of cell wall are induced by sugar starvation in Arabidopsis

Three Arabidopsis genes encoding a putative beta-galactosidase (At5g56870), beta-xylosidase (At5g49360) and beta-glucosidase (At3g60140) are induced by sugar starvation. The deduced proteins belong to the glycosyl hydrolase families 35, 3 and 1, respectively. They are predicted to be secretory proteins that play roles in modification of cell wall polysaccharides based on amino acid similarity. The beta-galactosidase encoded by At5g56870 was identified as a secretory protein in culture medium of suspension cells by mass spectrometry analysis. This protein was specifically detected under sugar-starved conditions with a specific antibody. Induction of these genes was repressed in suspension cells grown with galactose, xylose and glucose, as well as with sucrose. In planta, expression of the genes and protein accumulation were detected when photosynthesis was inhibited. Glycosyl hydrolase activity against galactan also increased during sugar starvation. The amount of monosaccharide in pectin and hemicellulose in detached leaves decreased in response to sugar starvation. These findings suggest that the cell wall may function as a storage reserve of carbon in addition to providing physical support for the plant body.     

Physiological,biochemical and molecular analysis of sugar-starvation responses in tomato roots

Two-month-old tomato plants were submitted to day/night cycles and to prolonged darkness in order to investigate the physiological and biochemical response to sugar starvation in sink organs. Roots appeared particularly sensitive to the cessation of photosynthesis, as revealed by the reduction of the growth rate and the decline of the carbohydrate and protein content. Therefore, excised tomato roots were used as a model to deepen the characterization of sugar starvation symptoms. In excised roots, the endogenous sugars were rapidly exhausted and significant degradation of protein was observed. Glutamine and asparagine accounted for most of the nitrogen released by protein breakdown. Respiration declined and proliferation- and growth-associated genes were repressed soon after the beginning of the sugar depletion. Among the genes studied, only the gene encoding asparagine synthetase was strongly induced. All the starvation symptoms were reversible when the roots were resupplied with sugar. When the culture conditions deteriorated, the metabolic and molecular changes led to the triggering of apoptosis of the root cells.     

Sugar Repression of Mannitol Dehydrogenase Activity in Celery Cells

We present evidence that the activity of the mannitol-catabolizing enzyme mannitol dehydrogenase (MTD) is repressed by sugars in cultured celery (Apium graveolens L.) cells. Furthermore, this sugar repression appears to be mediated by hexokinases (HKs) in a manner comparable to the reported sugar repression of photosynthetic genes. Glucose (Glc)-grown cell cultures expressed little MTD activity during active growth, but underwent a marked increase in MTD activity, protein, and RNA upon Glc starvation. Replenishment of Glc in the medium resulted in decreased MTD activity, protein, and RNA within 12 h. Addition of mannoheptulose, a competitive inhibitor of HK, derepressed MTD activity in Glc-grown cultures. In contrast, the addition of the sugar analog 2-deoxyglucose, which is phosphorylated by HK but not further metabolized, repressed MTD activity in mannitol-grown cultures. Collectively, these data suggest that HK and sugar phosphorylation are involved in signaling MTD repression. In vivo repression of MTD activity by galactose (Gal), which is not a substrate of HK, appeared to be an exception to this hypothesis. Further analyses, however, showed that the products of Gal catabolism, Glc and fructose, rather than Gal itself, were correlated with MTD repression.     

Bioenergetic consequences of lactose starvation for continuously cultured Streptococcus cremoris.

Streptococcus cremoris cells that had been grown in a chemostat were starved for lactose. The viability of the culture remained essentially constant in the first hours of starvation and subsequently declined logarithmically. The viability pattern during starvation varied with the previously imposed growth rates. The death rates were 0.029, 0.076, and 0.298 h-1 for cells grown at dilution rates of 0.07, 0.11 and 0.38 h-1, respectively. The proton motive force and the pools of energy-rich phosphorylated intermediates in cells grown at a dilution rate of 0.10 h-1 fell to zero within 2 h of starvation. The culture, however, remained fully viable for at least 20 h, indicating that these energy-rich intermediates are not crucial for survival during long-term lactose starvation. Upon starvation, the intracellular pools of several amino acids depleted with the proton motive force, while large concentration gradients of the amino acids alanine, glycine, aspartate, and glutamate were retained for several hours. A quantitative analysis of the amino acids released indicated that nonspecific protein degradation was not a major cause of the loss in viability. The response of the energy metabolism of starved S. cremoris cells upon refeeding with lactose was monitored. Upon lactose starvation, the glycolytic activity and the rate of proton motive force generation decreased rapidly but the steady-state level of the proton motive force decreased significantly only after several hours. The decreasing steady-state level of the proton motive force and consequently the capacity to accumulate amino acids after the addition of lactose correlated well with the loss of viability. The response of the energy metabolism of starved S. cremoris cells upon refeeding with lactose was monitored. Upon lactose starvation, the glycolytic activity and the rate of proton motive force generation decreased rapidly but the steady-state level of the proton motive force decreased significantly only after several hours. The decreasing steady-state level of the proton motive force and consequently the capacity to accumulate amino acids after the addition of lactose correlated well with the loss of viability. It is concluded that a regulatory loss of glycolytic capacity has pivotal role in the survival of S. cremoris under the conditions used.     

Growth and β-galactosidase synthesis in aerobic chemostat cultures of Kluyveromyces lactis

Growth and β-galactosidase (β-gal) expression were characterized in the yeast Kluyveromyces lactis strain NRRL Y-1118 growing in aerobic chemostat cultures under carbon, nitrogen or phosphate limitation. In lactose or galactose-limited cultures, β-gal accumulated in amounts equivalent to 10–12% of the total cell protein. The induced β-gal expression was repressed when cells were grown under N- or P-limitation. In lactose medium, enzyme levels were 4–8 times lower than those expressed in C-limited cultures. A similar response was observed when galactose was the carbon source. These results suggest that a galactose-dependent signal (in addition to glucose) may have limited induction when cells were grown in carbon-sufficient cultures. Constitutive β-gal expression was highest in lactate-limited and lowest in glucose-limited media and was also repressed in glucose-sufficient cultures. Other K. lactis strains (NRRL Y-1140 and CBS 2360) also showed glucose repression (although with different sensitivity) under non-inducing conditions. We infer that these strains share a common mechanism of glucose repression independent of the induction pathway. The kinetics of β-gal induction observed in C-limited cultures confirms that β-gal induction is a short-term enzyme adaptation process. Applying a lactose pulse to a lactose-limited chemostat culture resulted in ‘substrate-accelerated death’. Immediately after the pulse, growth was arrested and β-gal was progressively inactivated. Yeast metabolism in C-limited cultures was typically oxidative with the substrate being metabolized solely to biomass and CO₂. Cells grown under P- or N-limitation, either with glucose or lactose, exhibited higher rates of sugar consumption than C-limited cells, accumulated intracellular reserve carbohydrates and secreted metabolic products derived from the glycolytic pathway, mainly glycerol and ethanol. Received 16 October 1997/ Accepted in revised form 17 April 1998     

Branched-chain amino acid transport in Streptococcus agalactiae

The transport of the branched-chain amino acids in Streptococcus agalactiae was characterized. Glucose-grown cells were able to utilize only glucose as an energy source for transport of L-leucine, whereas lactose-grown cells could utilize both glucose and lactose. It was determined from metabolic inhibitor studies that energy from glycolysis and substrate level phosphorylation was required for active transport. Energy was found to be coupled to transport by the action of adenosine triphosphatase and the generation of a proton motive force. The branched-chain amino acids were found to share a common transport system that may consist of multiple components.