The main aim of this set of experiments was to determine if AmiB was involved in the accumulation of ammonia. Figure 3.14(c) shows the concentration of total ammonia present in the assay media during the growth of the wild-type and mutant cells. The concentration of ammonia in BM-N+lOmM acetamide medium subject to identical conditions as the assays but without cells was also measured (Figure 3.14(c)). This control indicated that during the assay period the non-enzymatic hydrolysis of acetamide was minimal.
Similar rapid increases in the concentration of extracellular ammonia were observed in the acetamide and acetamide/succinate media, which peaked at
around 9 mM at 180 min. As the complete hydrolysis of 10 mM acetamide
yields 10 mM ammonia, these results indicate that in these two assay media all available acetamide had been hydrolysed at around 180 min. With reference to the amidase activities measured in these media (Figure 3.14(b)) these observations reveal that although greater than two-fold more amidase activity was measured in the cells grown in the acetamide media, the lower activity in the cells grown in the presence of succinate/acetamide was enough to hydrolyse all available acetamide. Thus, the induced levels of amidase found in acetamide- grown cells are far in excess of that required.
Surprisingly, little ammonia was present in the acetamide/pyruvate media after 60 min. although both wild-type and mutant cells grown in this media exhibited the highest levels of amidase activity (Figure 3.14(b)). These results suggest that either pyruvate prevents the hydrolysis of acetamide by amidase or that pyruvate enhances the incorporation of ammonia into glutamate/glutamine. The latter situation seems the most probable although the mechanism by which this occurs is unknown.
After 180 min. the concentration of ammonia in the acetamide/succinate and acetamide/pyruvate media decreased to a final concentration of between 3 and 4 mM at 600 min. This is in contrast to the ammonia utilisation of the cells grown in the acetamide media which utilised approximately 1 to 2 mM of the available ammonia. This difference is due to the greater growth of the cultures in the media containing two carbon sources.
In all of these investigations the ammonia present in the media did not decrease
to growth-limiting concentrations and it is possible that diffusion of NH3 into
the cells was sufficient for growth. The differential rates of ammonia excretion for the wild-type and mutant cells grown in the different media were calculated ( 2 .2 .2 .4 .3 ) and plotted (Figure 3 .1 5 ). These plots correlate the ammonia excretion with cell growth between two time points such that a zero differential rate indicates no overall increase or decrease in the medium ammonia concentration with respect to growth. A high positive differential rate indicates the export of ammonia and vice versa. In all cases, there is essentially no difference between the wild-type and mutant cells. The amidase present in the
24 - 16 _ 0 100 200 300 400 500 600 24 - 20 _ 16 - o - C 1 2- 0 100 200 300 400 500 600
Time (min.) Time (min.)
24 - 20 - 16 - 12 - 0 100 200 300 400 500 600 Time (min.)
Figure 3.15 Differential rate of ammonia excretion by PAC452pSW101 and PAC452pMW22.
(a) Differential rate of ammonia excretion when grown in BM-N+lOmM
Acetamide. (b) Differential rate of ammonia excretion when grown in
BM-N+lOmM Acetamide+1% Succinate, (c) Differential rate of ammonia excretion when grown in BM-N+lOmM Acetamide+1% Pyruvate.
cells appears to hydrolyse the acetamide very rapidly leading to an initial net efflux of ammonia. After approximately 100 min. an overall decrease in the
differential rate occurs until 2 0 0 min., when the differential rate stabilises at
zero. These plots support the prediction that the diffusion of NH3 into the cells
gives the necessary nitrogen requirement for growth in that the decrease in the medium ammonia concentration is equal to the increase in the growth of the culture.
The growth, amidase activity and ammonia utilisation of the mutant cells was almost identical to that of the wild-type cells indicating quite emphatically that AmiB is not involved in any of these processes when the cells are grown in the presence of acetamide, acetamide/succinate or acetamide/pyruvate.
3 . 6 S u m m a ry o f C h a p ter
The attempts to find a role for the AmiB protein described in this Chapter have been unsuccessful. The initial analysis investigated the effect of insertion and deletion
mutations of either the chromosomal or plasmid-encoded amiB gene upon cell growth
in the presence of acetamide as the carbon and/or nitrogen source. If the AmiB protein was one component, together with AmiS, of an energy-dependent amide uptake system then the lack of AmiB would be expected to be disadvantageous to growth which required the products of acetamide hydrolysis. The analysis of the effects of the
chromosomal amiB insertion mutation were eventually discontinued as the mutation
resulted in the inhibition of transcription of the amiCRS genes, probably due to the
convergent transcription of the inserted aacCl gene, and the apparent metabolic load
placed on the strain by the constitutive expression of the aacCl gene.
Assays were thereafter carried out comparing the growth of PAC452 carrying the
plasmid-encoded wild-type amidase genes (pSW lOl) and an amiB deletion derivative
(pMW22) in the presence of lOmM acetamide as the carbon and/or nitrogen source. The lack of AmiB was not detrimental to the growth of the culture indicating that this protein is not involved in the transport of acetamide when it is present at lOmM concentrations.
Farin (1976) argued that the entry of amides into P. aeruginosa was by passive
diffusion and that this could be at a sufficient rate in bacteria possessing active amidase to be of no hindrance to amide utilisation. However, this may not be the case when
very low extracellular concentrations of amides are present and one possibility was that the AmiB and AmiS proteins could be primarily involved in the accumulation of amides when they are present at low concentrations (ie. a ‘scavenging’ mechanism), Wachira (1994) concluded that AmiB was not involved in the induction of amidase expression by 0.2% lactamide. To test the ‘scavenging’ mechanism hypothesis the induction of
amidase expression in the wild-type and a m iB mutant cells using reduced
concentrations of lactamide was determined. It was thought that if the lack of AmiB hindered the uptake of the low concentrations of lactamide then a decrease in the induced levels of amidase activity in the mutant cells would be observed. However, the
induction of amidase activity was not adversely affected by the amiB mutation giving an
initial indication that AmiB is not a part of a ‘scavenging’ mechanism.
To confirm these observations, the ability of the wild-type and mutant cells to accum ulate low extracellular concentrations (100|iM ) of t^C-acetamide was determined. Two plasmids were constructed (pRW35 and pRW36) similar to pSWlOl and pMW22 except that they did not produce amidase due to the introduction of a
frameshift mutation in the amiE gene. When PAC452pRW35 and PAC452pRW36
were grown under inducing conditions there was no significant difference in the accumulation of i^C-acetamide showing that AmiB is not involved in the transport of acetamide irrespective of the extracellular concentrations of this substrate. However, the results showed that PAC452pRW35 grown under inducing conditions accumulated more acetamide than either the same strain grown under non-inducing conditions or a control strain which was deleted for the amidase operon (PAC452pKT231). Thus, the transport of acetamide could be aided by the presence of an amide-specific inner
membrane pore, for example that encoded by the amiS gene, particularly when low
concentrations of acetamide are available. Unfortunately, due to a lack of time the
construction of amiS gene mutations to check this hypothesis were not carried out.
The fact that the lack of AmiB did not have a detrimental affect upon the growth of the cells on acetamide, upon the induction of amidase expression by reduced concentrations of inducer and, finally, upon the accumulation of acetamide when present at low
extracellular concentrations clearly shows that the product of the amiB gene is not an
essential component of an energy-dependent transport system for the uptake of acetamide. Furthermore, it is highly unlikely that such a transport system is encoded
by the amidase operon as the induced expression of the amiBCRS genes (pRW35) in
the labelled uptake studies. Unfortunately, the accumulation of other amides could not be tested due to the lack of radio-labelled derivatives. The hypothesis of passive diffusion of amides (Farin, 1976) was based, in part, on the findings of Cohen and Bangham (1967) who examined the permeation of amides into artificial membrane vesicles and found that the aliphatic amides tested penetrated these structures. It is likely therefore that the accumulation of other amides would be similar to that observed with acetamide. Thus the search for a possible role for AmiB was widened.
The first idea for another role for AmiB was based on the known functions of homologous proteins. The AmiB homolog HS104 had been shown to mediate the resolubilisation of heat-inactivated proteins and it seemed apt to determine if AmiB was involved in the protection of any of the amidase operon proteins at higher temperatures. An approximate 50% reduction in the levels of amidase activity were observed in both the wild-type (PAC452pSW10I) and mutant (PAC452pMW22) cells when placed at 4 IOC compared to cultures grown at 370C. As similar reductions in activity and increases in activity upon return to 370C were measured in both the wild-type and mutant cultures it is unlikely that AmiB protects either amidase or the AmiR protein from heat shock.
Finally, the extracellular concentration of ammonia was measured during the growth of wild-type and mutant cultures. The idea was to investigate if AmiB was involved in ammonia transport. The majority of excess ammonia is excreted from the cell by passive diffusion (NHg) whilst the difference in pH across the membrane prevents the intracellular accumulation of the non-permeating ammonium ions. It was hoped that during the final stages of growth, the ammonia concentration would become limiting and that the affect, if any, of the lack of AmiB on the energy-dependent transport of
NH4+ would be observed. Unfortunately, this situation did not arise as the minimal
extracellular concentration of ammonia was 3 mM. The results of these experiments did, however, raise some interesting questions. It appeared that in the presence of pyruvate the amidase activity increased whilst the concentration o f extracellular ammonia remained low for the initial 60 min. The reasons for this are not clear although it seems that pyruvate enhances amidase expression, probably via catabolite derepression, and the incorporation of ammonia into glutamate/glutamine. The extent of repression of amidase expression was measured when two catabolite repressors were present in the same medium. The repression of amidase synthesis in a medium containing acetate and succinate was severe indicating that succinate acts as the
dominant repressor. In contrast, no repression was evident when the cells were grown in the presence of pyruvate and acetamide indicating that pyruvate is the dominant carbon source and alleviates the moderate repression of amidase synthesis by acetate.
Although the role of the AmiB protein was not identified, it became clear that purification of the protein for characterisation of its enzymatic activity would be a useful exercise. The amino acid sequence of AmiB contains the conserved nucleotide-binding motif (Walker, 1982) and is highly homologous to the C-terminal part of ClpA, the
regulatory subunit of an energy-dependent protease in E. coliy which has been shown
to bind and hydrolyse ATP. The following Chapter describes the attempts to over- express and purify AmiB and the subsequent enzymatic analysis.