Proline Utilization by Bacillus subtilis: Uptake and Catabolism

Abstract

L-Proline can be used by Bacillus subtilis as a sole source of carbon or nitrogen. We traced L-proline utilization genetically to the putBCP (ycgMNO) locus. The putBCP gene cluster encodes a high-affinity proline transporter (PutP) and two enzymes, the proline dehydrogenase PutB and the ⌬ 1 -pyrroline-5-carboxylate dehydrogenase PutC, which jointly catabolize L-proline to L-glutamate. Northern blotting, primer extension, and putB-treA reporter gene fusion analysis showed that the putBCP locus is transcribed as an L-proline-inducible operon. Its expression was mediated by a SigA-type promoter and was dependent on the proline-responsive PutR activator protein. Induction of putBCP expression was triggered by the presence of submillimolar concentrations of L-proline in the growth medium. However, the very large quantities of L-proline (up to several hundred millimolar) synthesized by B. subtilis as a stress protectant against high osmolarity did not induce putBCP transcription. Induction of putBCP transcription by external L-proline was not dependent on L-proline uptake via the substrate-inducible PutP or the osmotically inducible OpuE transporter. It was also not dependent on the chemoreceptor protein McpC required for chemotaxis toward L-proline. Our findings imply that B. subtilis can distinguish externally supplied L-proline from internal L-proline pools generated through de novo synthesis. The molecular basis of this regulatory phenomenon is not understood. However, it provides the B. subtilis cell with a means to avoid a futile cycle of de novo L-proline synthesis and consumption by not triggering the expression of the putBCP L-proline catabolic genes in response to the osmoadaptive production of the compatible solute L-proline.

Virus Evolution toward Limited Dependence on Nonessential Functions of the Host: the Case of Bacteriophage SPP1

“…YcgM is a proline dehydrogenase, the first enzyme of the pathway for oxidation of proline to glutamate (42,43). Its function has been demonstrated in B. subtilis, and the name of the gene has been changed to putB (43).…”

“…YcgM is a proline dehydrogenase, the first enzyme of the pathway for oxidation of proline to glutamate (42,43). Its function has been demonstrated in B. subtilis, and the name of the gene has been changed to putB (43). The gene efp encodes the universally conserved protein elongation factor P, which binds to the ribosome and stimulates the synthesis of peptide bonds (41).…”

“…SPP1 plating in knockout strains carrying a complementing allele of the relevant gene that is expressed ectopically under an inducible P xyl promoter at the amyE locus are shown labeled as complement. The SPP1 plaque phenotype on the putB found to be necessary for efficient phage infection fall into three classes: (i) genes from the yuk operon (yukE, yukD, yukC, yukB, and yueB) coding for components of the Esat-6-like secretion system (40); (ii) protein synthesis (efp [41]); (iii) intermediary metabolism (ycgM [42,43]). As it was elegantly demonstrated by the groups of M. A. Santos and C. São-José, YueB and other proteins encoded by the yuk operon are required for phage SPP1 irreversible binding to the B. subtilis cell surface and are possibly involved in postadsorption events, such as phage genome transfer to the host cell cytoplasm (13,32,40).…”

“…It has been proposed that the catabolism of L-proline maintains an active level of cellular metabolism (43). SPP1 was titrated on the putB-deficient strain in LB semisolid medium supplemented with glutamate to verify if an exogenous supply of this amino acid could compensate for the absence of PutB and prolong the period of host cell competence for SPP1 infection.…”

Phylogenetic analysis of substrate-binding subunits in an osmoprotectant system

“…These three transporters use energy released by ATP hydrolysis to import solutes (Du et al, 2011). The other transporters are OpuD, a transporter of the BCCT family (betaine-choline-carnitine transporter) that uses the energy of the driving force of sodium symport transport for importing solutes (Ziegler et al, 2010), and OpuE, a transporter from the sodium/solute symport family that is specific for importing proline (Moses et al, 2012).…”