Organization and transcription of the glsA-glnT operon. (A) The glsA, glnT, and ybgG genes are oriented divergently from the glnKL genes. Northern and primer extension analyses in this work revealed that the glsA and glnT genes constituting an operon are transcribed from the promoter of the glsA-glnT (PglsA-glnT) promoter to produce a 2.5-kb mRNA, whereas the ybgG gene is likely transcribed monocistronically to form a 1.1-kb transcript. The two hairpin structures considered ρ-independent transcription terminators were found immediately downstream of glnT and ybgG, respectively. (B) Northern analysis of transcription of the glsA and glnT genes. The RNA samples from strains 168 (lanes 1, 2, 4, 5, 7, and 8) and YCBBd (glnL::pMUTIN) (lanes 3, 6, and 9) grown on glutamine (lanes 1, 3, 4, 6, 7, and 9) and glutamate plus ammonium (lanes 2, 5, and 8) were subjected to Northern analysis using glsA, glnT, and ybgG probes. The arrows indicate a 2.5-kb transcript detected with the glsA and glnT probes and a 1.1-kb transcript detected with the ybgG probe; the former transcript (2.5 kb) was induced with glutamine. The two faint bands (2.8 and 1.7 kb), indicated by arrowheads, resulted from unspecific hybridization to 23 and 16 rRNAs, respectively.

Gel retardation analysis of GlnL interaction with the glsA-glnT promoter region. (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of pure His-tagged GlnL. The purified His-tagged GlnL protein (lane 1) and a crude protein extract of cells of E. coli BL21(DE3) bearing plasmid pGLNL exposed to IPTG (lane 2) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 12.5% gel. The preparation of the crude protein extract and purification of the His-tagged GlnL are described in the text. The arrow indicates His-tagged GlnL. (B) Gel retardation analysis of the binding of His-tagged GlnL to the 230-bp probe containing its recognition site. The analysis was performed as described in the text. The probe (0.02 pmol) was incubated with 0, 0.9, 1.7, 3.4, 6.9, and 13.7 pmol of His-tagged GlnL (lanes 1, 2, 3, 4, 5, and 6, respectively) in a 25-μl reaction mixture. The addition of more His-tagged GlnL to the reaction mixture was not performed due to the low concentration of the protein preparation. The His-tagged GlnL/probe complexes (bound) and free probe (free) are indicated on the right. (C) The increased binding affinity of autophosphorylated His-tagged GlnL to the probe. His-tagged GlnL was treated in the absence and presence of 50 mM acetylphosphate, respectively (GlnL treated and GlnL∼P treated), as described in the text. The probe (0.02 pmol) was incubated with 0, 3.3, 6.6, 9.9, 13.2, and 16.5 pmol of His-tagged GlnL treated without and with acetylphosphate (lanes 1 to 6) in a 25-μl reaction mixture. Lane 7 contains the probe incubated with 33.0 pmol of His-tagged GlnL treated without acetylphosphate.

Catalyzation of deamination of glutamine to glutamate by the GlsA protein. After incubation of the reaction mixture containing glutamine with the crude protein extract prepared from E. coli BL21(DE3) cells carrying either plasmid pET-22b (lane 3) or pGLSA (lane 4) exposed to IPTG, an aliquot of the mixture was subjected to paper chromatography as described in the text. Lanes 1 and 2 contained authentic Na glutamate and glutamine, respectively. The spots were visualized by spraying a ninhydrin solution. Arrows indicate the positions of the glutamine and glutamate spots.

Glutamine transport into cells of the pMUTIN integrant strains YBGJd (glsA::pMUTIN) and YBGHd (glnT::pMUTIN). Cells of strains YBGJd (bars 1 and 2) and YBGHd (bars 3 and 4) were grown with (bars 1 and 3) or without (bars 2 and 4) IPTG, and the glutamine transport into cells was measured as described in the text. The values are averages with standard deviations and were obtained in duplicate experiments.

Mapping of the 5′ end of the glsA-glnT transcript by means of primer extension. Total RNAs from strain 168 grown in glutamate and ammonium (lane 1) and glutamine (lane 2) were annealed with the Ex-glsA primer, and then primer extension was performed as described in the text. Lanes G, A, T, and C contained the products of the respective dideoxy sequencing reactions, as described in the text. The arrow indicates the runoff cDNA resulting from primer extension. The part of the nucleotide sequence of the coding strand corresponding to the ladder is shown with the transcription initiation base (+1) determined in this analysis, and the corresponding −10 and −35 regions are underlined.

DNase I footprinting analysis of the interaction of GlnL with the glsA-glnT region. (A) The analysis was performed as described in the text. The left and right panels are DNase I footprints of the 5′-end-labeled coding and noncoding strands of the DNA probe, respectively. Lanes 1 to 4 contained 0.04 pmol of the ³²P-labeled probe DNA in the reaction mixture (50 μl). Lane 1 contained no His-tagged GlnL. Lanes 2, 3, and 4 contained 27.4, 20.5, and 13.7 pmol of His-tagged GlnL. Lanes G, A, T, and C contained the products of the respective sequencing reactions performed with the same primers as those used for the probe preparation. The protected areas of the coding and noncoding strands are boxed. (B) The nucleotide sequences of the coding and noncoding strands of the region upstream of the glsA-glnT promoter, with the protected sequences (in boldface type), and the −35 and −10 regions of the glsA-glnT promoter (+1 is the transcription initiation base) indicated. The boxed T and ATG are the transcription initiation base and translation initiation codon, respectively. Arrows indicate directly repeated sequences (TTTTGT).

Some defect in utilization of glutamine as the nitrogen source in B. subtilis strain mutated in the gltA and glsA genes, which encoded glutamate synthase and glutaminase, respectively. Cells of strains 168 (open circles), YBGJd (glsA::pMUTIN) (open squares), 1A71 (gltA1) (open triangles), and FU801 (glsA::pMUTIN gltA1) (closed circles) were grown on TBABG plates at 30°C for 14 h. Then the cells were inoculated, to an OD₆₀₀ of 0.05, into MM medium (38) containing 13.6 mM glutamine as the sole nitrogen source and incubated at 37°C with shaking.