Williams, Rachel Jayne; (1996) Studies on the 'amiB' and 'amiS' genes of the amidase operon in Pseudomonas aeruginosa. Doctoral thesis (Ph.D), UCL (University College London).

Preview

Text Studies_on_the_'amib'_and_'ami.pdf Download (19MB) | Preview

Abstract

Pseudomonas aeruginosa strain PAC1 is able to grow on a range of short-chain length aliphatic amides by virtue of an inducible aliphatic amidase, encoded by the amiE gene. Expression of amidase is under positive regulation by AmiR, via a transcription antitermination mechanism, and amide-dependent negative regulation by AmiC. Two additional amidase genes have been sequenced, amiB and amiS, giving the gene order for the amidase operon as amiEBCRS. Analysis of the predicted amino acid sequence of AmiB showed the presence of Parts A and B of the Walker Motif, the consensus sequence found in all nucleotide-binding proteins. The amiB gene has been over-expressed in E. coli as a maltose-binding protein (MBP)-AmiB fusion protein and purified by amylose affinity chromatography. Gel filtration analysis showed that the purified MBP-AmiB was in two forms, soluble aggregated and monomeric, both of which exhibited similar levels of ATPase activity. The Km and Vmax values for ATP have been determined together with additional characterisation including substrate specificity, co-factor dependence and the effect of inhibitors. Hydropathy analysis of the predicted amino acid sequence of AmiS showed that this protein consisted of six hydrophobic stretches characteristic of the transmembrane domains of an integral membrane protein. Various attempts to over-express the PCR- amplified amiS and amiS-poly his. genes in E. coli and P. aeruginosa failed as the proteins could not be detected by SDS-PAGE analysis. The orientation of AmiS in the cytoplasmic membrane was investigated using amiS-TnphoA and amiS-TnlacZ gene fusion analysis. A number of TnlacZ and TnphoA insertions into amiS were identified, sequenced and the enzyme activity determined allowing a prediction of the membrane topology of this protein. The analysis of AmiB and AmiS led to the proposal that these proteins are components of a transport system involved in the active uptake of aliphatic amides. However, mutation of the plasmid-encoded amiB gene did not have a detrimental effect on the ability of the cell to utilise amides as a carbon and/or nitrogen source. In addition, the transport of acetamide and the induction of amidase expression by lactamide was similar in wild-type and amiB mutant strains when these amides were present at low extracellular concentrations, and it has been concluded that AmiB is not involved in the active transport of amides into the cell. The mutational analysis of AmiB has also shown that this protein is not involved either in protecting the amidase proteins against heat-shock or in the utilisation of ammonia (a product of the hydrolysis of amides). Finally, the amidase genes are transcribed on a polycistronic mRNA from the pE promoter upstream of amiE but it has not been possible to clearly identify a full length transcript and, thus, the 3' extent of the operon is unknown. To identify any further 'amidase' genes, a 6 kb DNA fragment situated downstream of amiS was subcloned and a 1.4 kb DNA fragment located immediately downstream of the amidase locus sequenced. A putative transcription terminator of the amidase operon was identified downstream of amiS together with a new open reading frame. The role of Orf2-rjw has not been established although sequence analysis has indicated that it is homologous to members of the Membrane Fusion Protein family.

Download activity - last month

Export as JSONCSVXML

Download activity - last 12 months

Export as JSONCSVXML

Downloads by country - last 12 months

Export as XMLJSONCSV

Archive Staff Only

View Item