The amino acid sequence of a Bacillus subtilis phosphoprotein that matches an orfY-tsr coding sequence

The soil bacterium Bacillus subtilis can utilize exogenous proline as a sole nitrogen or carbon source. The proline-inducible putBCP (formerly ycgMNO) operon encodes proteins responsible for proline uptake and two-step oxidation of proline to glutamate. We now report that a gene (formerly ycgP, now designated prcR) located downstream of the putBCP operon is essential for B. subtilis cells to utilize proline as a sole nitrogen or carbon source. Disruption of the prcR gene also abolished proline induction of putB transcription. prcR expression is not subject to autoregulation and proline induction. The PrcR protein shows no significant amino acid sequence similarity to the known transcriptional activators for proline utilization genes of other bacteria, but it does show partial amino acid sequence similarity to the transcriptional regulator PucR for the purine degradation genes of B. subtilis. PrcR orthologues of unknown function are present in some other Bacillus species. Primer-extension analysis suggests that both putB and prcR are transcribed by a σA-dependent promoter. Deletion and mutation analysis revealed that an inverted repeat (5′-TTGTGG-N5-CCACAA-3′) centred at position −76 relative to the transcriptional initiation site of putB is essential for putB expression. Electrophoretic mobility shift assays showed that the purified His-tagged PrcR was capable of binding specifically to this inverted repeat. Altogether, these results suggest that PrcR is a PucR-type transcriptional activator that mediates expression of the B. subtilis putBCP operon in response to proline availability.

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Two Regions of GerE Required for Promoter Activation in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.184.1.241-249.2002 ◽

2002 ◽

Vol 184 (1) ◽

pp. 241-249 ◽

Cited By ~ 14

Author(s):

Dinene L. Crater ◽

Charles P. Moran

Keyword(s):

Crystal Structure ◽

Bacillus Subtilis ◽

Amino Acid ◽

Amino Acid Sequence ◽

In Vitro Transcription ◽

Dna Binding Domain ◽

Promoter Activation ◽

Transcription Activators

ABSTRACT GerE from Bacillus subtilis is the smallest member of the LuxR-FixJ family of transcription activators. Its 74-amino-acid sequence is similar over its entire length to the DNA binding domain of this protein family, including a putative helix-turn-helix (HTH) motif. In this report, we sought to define regions of GerE involved in promoter activation. We examined the effects of single alanine substitutions at 19 positions that were predicted by the crystal structure of GerE to be located on its surface. A single substitution of alanine for the phenylalanine at position 6 of GerE (F6A) resulted in decreased transcription in vivo and in vitro from the GerE-dependent cotC promoter. However, the F6A substitution had little effect on transcription from the GerE-dependent cotX promoter. In contrast, a single alanine substitution for the leucine at position 67 (L67A) reduced transcription from the cotX promoter, but not from the cotC promoter. The results of DNase I protection assays and in vitro transcription reactions lead us to suggest that the F6A and L67A substitutions define two regions of GerE, activation region 1 (AR1) and AR2, that are required for activation of the cotC and cotX promoters, respectively. A comparison of our results with those from studies of MalT and BvgA indicated that other members of the LuxR-FixJ family may use more than one surface to interact with RNA polymerase during promoter activation.

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