E. coli happy Excess of amino acids

E. coli moderately happy Interconversion of amino acids Too much alanine Strategy glucose Too little phenylalanine

E. coli moderately happy Interconversion of amino acids Too much alanine Transaminase Oxaloacetic acid (OAA) Aspartate Transaminase Enough phenylalanine Aspartate

E. coli concerned Formation of amino acids de novo Alpha-keto acids but no amino acids Oxaloacetic acid (OAA) nitrate

E. coli concerned Formation of amino acids de novo Nitrogen Starvation nitrate Nitrate reductase α-ketoglutarate ammonia Glutamine synthetase glutamine glutamate Glutamine synthetase glutamate

E. coli concerned Formation of amino acids de novo Nitrogen Starvation Nitrate reductase Glutamine synthetase Presence of amino acid

Nitrogen Starvation E. coli concerned Formation of amino acids de novo Environmental signal Intracellular signal Sensation of nitrogen starvation Presence of nitrate Presence of histidine Response Nitrate reductase Glutamine synthetase Histidine degradation

Control of glutamine synthetase P Active glutamine synthetase transcription gln translation Posttranscriptional control ATP Adenylylation Transcriptional control Inactive glutamine synthetase

Control of glutamine synthetase Nitrogen Starvation pppGpp! ??? Translation of signal Stringent response evoked by N starvation …but wrong response! If not pppGpp then what?

Control of glutamine synthetase Autogenous regulation? Effect of Gln mutants on nitrogen control (Ntr) Mutations mapping to gln affect global N control GS need not be active to exert control

Control of glutamine synthetase Mutations outside of glnA X Y Maybe all those glnA mutations were really in X or Y??? Yes and no So GS STILL regulates itself? Maybe not.

Control of glutamine synthetase Mutations outside of glnA P glnA X Y transcription

Control of glutamine synthetase Mutations outside of glnA P glnA X Y stop transcription Polar effect of nonsense mutations Plausible,… ...but is it true?

Control of glutamine synthetase Are “regulatory” glnA merely polar Control of glutamine synthetase Are “regulatory” glnA merely polar? How to tell? P NtrC glnA X Y P NtrC glnA X Y Complementation: If not polar, then yes If polar, then no

Control of glutamine synthetase Current understanding of glnA operon ntrB ntrC N-regulated gene expression P Phosp’n of NtrC NtrB NtrC UMP Control of NtrB Uridyl’n of PII PII (GlnB) Uridylyltransferase (UT) (GlnD)

Nitrogen Starvation E. coli concerned Formation of amino acids de novo Environmental signal ??? Intracellular signal Sensation of nitrogen starvation Presence of nitrate Presence of histidine Response Nitrate reductase Glutamine synthetase Histidine degradation

Control of glutamine synthetase How is nitrogen status sensed? Uridylyltransferase (UT) UMP - + Active PII Inactive PII Uridylyltransferase (UT) glutamine α-ketoglutarate

E. coli concerned Formation of amino acids de novo Nitrogen Starvation nitrate Nitrate reductase α-ketoglutarate ammonia Glutamine synthetase glutamine glutamate Glutamine synthetase glutamate