Staphylococcus aureus is a Gram-positive spherical bacterium which grows as a series of clusters. This type of bacteria is usually found in the nose, oral cavity, gastrointestinal tract and skin of humans. During 1884, Rosenbach described two pigmented colonies of staphylococci and gave them two types the nomenclature; Staphylococcus aureus which forms a yellow colony and Staphylococcus albus which forms a white colony. Staphylococcus aureus tends to disrupt red blood cells on blood agar. The bacterium understudy can grow under aerobic respiration as well as by fermentation (Todar Kenneth, PhD). Glycolysis or Embden-Meyerhof pathway is a ten step aerobic process which converts one molecule of glucose to two molecules of pyruvate. During the course of this process a net of 2 ATP and 2 NADH is produced. In gluconeogeneis, glucose is produced from non-carbohydrate sources such as pyruvate and glycerol. Gluconeogenesis is the reverse pathway of glycolysis but in some steps different enzymes are used. However, steps 1, 3 and 10 in glycolysis are irreversible (Mount St.Mary College).
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Glyceraldehyde-3-phosphate dehydrogenase (GADPH) is an enzyme in glycolysis which converts to 1, 3-diphosphoglycerate from Glyceraldehyde-3-phosphate (oxidative phosphorylative step). GADPH roles are important in protein binding and cell signaling of organisms, immune illusion of bacteria and also cell maintenance. Most bacteria contain one GAPDH gene but there are a few exceptions to this general perception. The Gram-positive bacterium Bacillus subtilis possesses two gaps that code for GAPDH proteins with opposing functions in glucose metabolism which differs from the more likely case of both GADPH performing both roles. Bacillus subtilis gapA GAPDH functions in glycolysis which converts G3P to 1,3dPG and the cofactor is NAD+. GapB GAPDH is gluconeogenic and the cofactor is NADH when converting 1,3dPG to G3P. Bacillus subtilis gapA expression is repressed by CcgR when 1, 6-bisphosphate (FBP) is absent since there would be no conversion of FBP to G3P and DHAP and thus the second half of the cycle would not continue. CcGR binds to the operator sequence blocking transcript elongation and thus repressing the glycotytic operon. However, no evidence suggests CcGR participates in regulating GapB.
Staphylococcus aureus is a very efficient pathogen that causes many infections in humans and animals. S.aureus has two GAPDH homologues which are known as gapA and gapB. The gapA gene is situated in the glycolytic operon which encodes a glycolytic GAPDH. The glycolytic regulator CcgR which regulates gapA in B.subtilis is also found in the S. aureus glycolytic operon and is termed gapR. The gapB gene is found within a single open reading frame along with other genes which are involved in DNA replication and repair. The actual role of GapB and GapR inside of S. aureus has not been been discovered but GapB is presumed to have gluconeogenic GAPDH. However, this statement may not be accurate. GapB in B.subtilis had a much higher catalytic efficiency using NADP+ than NAD+ but the GapB protein from S. aureus which had a small NAD+ dependent GAPDH activity and no NADP+ dependent GAPDH activity. In this regard the authors' objective was to investigate the function of GapB in S. aureus and to identifyif it is important in virulence.
The results showed that GAPDH activity is not expressed in the GapB protein. This was demonstrated by cloning gapA and gapB reading frames in S. aureus strain 8325.4 into E.coli protein expression vector pLEICS03. The gapR glycolytic operon was also into included into the vector as a measure of negative due it being a regulatory protein. The recombinants and 200 ng purified protein was assayed for GAPDH activity of NAD+ or NADP+ as a cofactor. The result of this procedure resulted in gapA showing a high affinity towards NAD+ but a less with NADP+ which can be assumed that gapA has glycolytic GAPDH activity and not gluconeogenic. However, gapB and the regulatory protein gapR showed no enzyme activity for NAD+ or NADP+. There was a chance that the enzyme activity in gapB was affected by the His tag but this was proved to be inaccurate when the TEV protease cleavage removed the tag and the gapB activity was still nonexistent. This proved that gapB does not have GAPDH activity or the recombinant was nonfunctional. Even though GapB lacked NADP+ dependent GAPDH activity, the essential amino acid residues and G3P binding which determines GAPDH activity was present which suggested that gapB had a gluconeogenic GAPDH role.
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To investigate further mutations were incorporated into gapA and gapB loci. GapA function was impaired thus allowing the expression of the glycolytic operon which Northern blotting established using a phosphoglycerate kinase probe. GAPDH activity was tested in the 8325-4 âˆ†gapA and the âˆ†gapB; no significant difference between the activity of the wild type and âˆ†gapB mutant or the âˆ†gapA mutant and the double mutant which is observed by the graph because the mutant activity was much lower than the wild type. This shows that in vivo GapB does not show NAD+ GADPH activity but it is constant that GapA in S. aureus does show NAD+ activity because since its' expression was impaired for this experiment and thus the expression should be lowered. NADP+ which is gluconeogenic was lower in âˆ†gapA mutant and âˆ†gapB mutant than the wild type. Both of these mutants showed NADP+ activity in vivo but this activity was not present in the purified protein. The NADP+ GADPH activity in both of the double mutants was not significantly lower than the single mutants and therefore this suggests that there may be another enzyme responsible for this in vivo activity.
S. aureus growth was investigated with respect to the role of each GAPDH homologue. A twenty-four hour growth curve was carried out in a TSB medium which contains Casamino acids and nitrogenous substances with glucose or pyruvate as an alternative carbon source. Glucose and pyruvate increases the carbon flow into the TCA cycle which increases the growth rate but pyruvate should bypass the glycolytic pathway. The âˆ†gapB mutant showed no significant growth in comparison to the wild type while in under primary carbon metabolism. âˆ†GapA and the double mutant âˆ†gapA âˆ†gapB did show a decrease in growth TSB and TSB in glucose when compared to the wild type. âˆ†GapA grew in the presence of pyruvate and thus glycolysis did not occur in that particular strain but downstream processes such as the TCA cycle were still able to function. A TM medium contains no glucose but contained Casamino acids which contain amino acids that can be converted into an intermediate in the TCA cycle and thus can be used to produce pyruvate and hence activate gluconeogenesis. In this absence of glucose âˆ†GapA the growth rate was the same as that as the wild type which suggests that it plays a vital role in gluconeogenesis. âˆ†GapB and the double mutant âˆ†GapA âˆ†GapB growth rate were minute in the absence of glucose. However, the wild type and âˆ†GapA growth rate was significant when pyruvate was added but this was not the case for âˆ†GapB mutants which an inference can be made that a loss in gapB also loses the ability to synthesize glucose.
When glucose was added the growth rate of âˆ†GapB mutant was significantly enhanced with the wild type strain but âˆ†GapA growth rate was significantly lowered. There was also an increase in growth in the âˆ†GapA âˆ†GapB compare to that of the âˆ†GapA. Since it was proved that gapA grows significantly in the presence of pyruvate and amino acids but minutely in glucose. It can be deduced that gapA is gluconeogenic and gapB (âˆ†GapB mutants) which grows more efficiently in the presence of glucose but not in the presence of pyruvate and amino acids is glycolytic. GapA does not control glycolytic and glucogenic GADPH activity in S. aureus because GapB is proven to be active in the gluconeogenic pathway.
The âˆ†gapB does not use secondary carbon sources so the function of this mutant was tested using secondary carbon sources of succinate, glutamate and glycerol which can enter both glycolysis and gluconeogenesis. The âˆ†gapA mutant grew in all mediums except in the presence of glucose and glycerol while âˆ†gapB only grew in the presence of glucose and glycerol when compared to the wild type strain. Glycerol goes into the gluconeogenic pathway following the conversion to G3P and hence GapB should be active in S.aureus prior to this point and hence has gluconeogenic GAPDH. GADPH proteins are active in both pathways but not at the same time. Northern blot analysis determines the gapA and gapB expression after five hours in a TM medium in the presence and absence of 1% glucose. This increased growth rate can affect cell density and pH which can alter gene expression. In order to make sure any changes were only with respect to the presence of absence of glucose, glucose was added at zero minutes and at one hour before being harvested. The gapA probe had many transcripts from other genes transcribed with it while gapB only had three from the gapB reading frame. The northern blot figure does show that gapA is expressed while gapB is repressed because if you look at the blots, gapB is only observed when glucose is not present. While in the presence of casamino acids, gapA is inactive and gapB isactive. The similarity in the role of gapR in S. aureus to CcgR in B. subtilis was accessed by inserting a mutation into gapR. The gapR probe was used in place of the gapA and it showed that it expressed low levels of gapA in the wild type. GapR expression is increased under glycolysis and gluconeogenesis compared to the wild type. When in gluconeogenic conditions, glycolysis is repressed and glucose is limited.
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The expression of âˆ†gapR increases under glycolysis and gluconeogenesis compared to the wild type which is supported by the densitometry analysis table and thus the author's observation is supported. GapB limits expression in gluconeogenic conditions and also control of the glycolytic operon. In order to determine how much glucose is needed to alternate from gapA to gapB, the GADPH mutants were placed in a TM medium of varying glucose concentration. Above 0.05% glucose concentration, âˆ†gapB grows equivalent to the wild type while âˆ†gapA and âˆ†gapA âˆ†gapB double mutant growth is inhibited. The switch from gluconeogenesis to glycolysis is at 0.01% glucose and this switch is called carbon catabolite repression (CCR). When glucose is present, secondary carbon sources are repressed. At 0.1% glucose âˆ†gapA starts to decrease and âˆ†gapB increases; glucose metabolism is induced while secondary carbon metabolism genes are repressed. Both pathways should be disrupted in the double mutant but the growth rate of the double mutant increased up until 0.01% of glucose and as the concentration of glucose further increased, the growth rate of the double mutant decreased.
The reason this does not occur in âˆ†gapB is because the glycolytic pathway is tightly regulated, the mutant must use its environmental carbon sources first. Determining the role of GapA and GapB in host infection, Galleria mellonella larvae was infected with the wild type and each of the GAPDH mutant strains. Pathogenesis is measured by the percent viability of the infected larvae at 24, 48, and 72 hour post infection. Mutants gapA and gapB proved essential in the full pathogenic phenotype of S. aureus. The survival of the âˆ†gapA, âˆ†gapB and the âˆ†gapA âˆ†gapB double mutant was smaller than the wild type. For 72 hours post infection viability in the Galleria, the wild type had the lowest survival followed by the âˆ†gapA then the âˆ†gapB and finally the âˆ†gapA âˆ†gapB double mutant which had a 100 % survival rate. This showed that gapA and gapB is necessary for host infection.
GapB is essential in anabolic carbon metabolism in S. aureus. GapR regulated the expression of GapA in glycolysis and the GapB level of expression in gluconeogenesis. Gap A and GapB is important in the virulence of S. aureus along with glycolysis and secondary carbon metabolism. Recombinant His-tagged GapB protein had some or no GAPDH activity with NAD+ or NADPH as a cofactor. Even though recombinant Gap B shows no GAPDH activity in vitro it is a gluconeogenic enzyme. GapA does not function only in gluconeogenic GAPDH activity, âˆ†gapA activity increases when glucose is lacking indicating the disruption of glycolysis at this point but secondary carbon sources are being utilized indicating secondary carbon metabolism is still functioning. On page 5226 of the paper gave reference to a figure 1D but there is no evidence of this figure. GapA expression is large when glucose is present but is repressed gluconeogenic conditions and vice versa for GapB. Data suggests that gapA is glycolytic and gapB functions in gluconeogenic. In my opinion, this method proved to be effective in determining glycolytic and gluconeogenic ability of GapA and GapB. However, another experiment that could have been carried out was to test the amount of glucose produced from the gapA mutant in a TSM medium. This would determine if gluconeogenesis is actually being carried out (Purves et al. 2010).