Role of lon and ClpX in the post-translational regulation of a sigma subunit of RNA polymerase required for cellular differentiation in Bacillus subtilis.

The RNA polymerase sigma subunit, sigmaH (Spo0H) of Bacillus subtilis, is essential for the transcription of genes that function in sporulation and genetic competence. Although spo0H is transcriptionally regulated by the key regulatory device that controls sporulation initiation, the Spo0 phosphorelay, there is considerable evidence implicating a mechanism of post-translational control that governs the activity and concentration of sigmaH. Post-translational control of spo0H is responsible for the reduced expression of genes requiring sigmaH under conditions of low environmental pH. It is also responsible for heightened sigmaH activity upon relief of acid stress and during nutritional depletion. In this study, the ATP-dependent proteases LonA and B and the regulatory ATPase ClpX were found to function in the post-translational control of sigmaH. Mutations in lonA and lonB result in elevated sigmaH protein concentrations in low-pH cultures. However, this is not sufficient to increase sigmaH-dependent transcription. Activation of sigmaH-dependent transcription upon raising medium pH and in cells undergoing sporulation requires clpX, as shown by measuring the expression of lacZ fusions that require sigmaH for transcription and by complementation of a clpX null mutation. A hypothesis is presented that low environmental pH results in the Lon-dependent degradation of sigmaH, but the activity of sigmaH in sporulating cells and in cultures at neutral pH is stimulated by a ClpX-dependent mechanism in response to nutritional stress.

Altered srf expression in Bacillus subtilis resulting from changes in culture pH is dependent on the Spo0K oligopeptide permease and the ComQX system of extracellular control.

The expression of the srf operon of Bacillus subtilis, encoding surfactin synthetase and the competence regulatory protein ComS, was observed to be reduced when cells were grown in a rich glucose- and glutamine-containing medium in which late-growth culture pH was 5.0 or lower. The production of the surfactin synthetase subunits and of surfactin itself was also reduced. Raising the pH to near neutrality resulted in dramatic increases in srf expression and surfactin production. This apparent pH-dependent induction of srf expression required spo0K, which encodes the oligopeptide permease that functions in cell-density-dependent control of sporulation and competence, but not CSF, the competence-inducing pheromone that regulates srf expression in a Spo0K-dependent manner. Both ComP and ComA, the two-component regulatory pair that stimulates cell-density-dependent srf transcription, were required for optimal expression of srf at low and high pHs, but ComP was not required for pH-dependent srf induction. The known negative regulators of srf, RapC and CodY, were found not to function significantly in pH-dependent srf expression. Late-growth culture supernatants at low pH were not active in inducing srf expression in cells of low-density cultures but were rendered active when their pH was raised to near neutrality. ComQ (and very likely the srf-inducing pheromone ComX) and Spo0K were found to be required for the extracellular induction of srf-lacZ at neutral pH. The results suggest that srf expression, in response to changes in culture pH, requires Spo0K and another, as yet unidentified, extracellular factor. The study also provides evidence consistent with the hypothesis that ComP acts both positively and negatively in the regulation of ComA and that both activities are controlled by the ComX pheromone.

Regulation of Bacillus subtilis sigmaH (spo0H) and AbrB in response to changes in external pH.

The RNA polymerase sigma subunit, sigmaH, of Bacillus subtilis is required for the transcription of genes that are induced in late-growth cultures at high cell density, including genes that function in sporulation. The expression of sigmaH-controlled genes is repressed when nutrient broth sporulation medium (Difco sporulation medium [DSM]) is supplemented with high concentrations of glucose and glutamine (DSM-GG), preferred carbon and nitrogen sources of B. subtilis. Under these conditions, the pH of the DSM-GG medium decreases to approximately 5. Raising the pH by the addition of morpholinepropanesulfonic acid (MOPS) or Tris-HCl (pH 7.5) results in a dramatic increase in the expression of lacZ fusions to sigmaH-dependent promoters. Correspondingly, the level of sigmaH protein was higher in cells of late-growth DSM-GG cultures treated with a pH stabilizer. When sigmaH-dependent gene expression was examined in cells bearing a mutation in abrB, encoding the transition state regulator that negatively controls genes transcribed by the sigmaH form of RNA polymerase, derepression was observed as well as an increase in medium pH. Reducing the pH with acetic acid resulted in repression, suggesting that AbrB was not functioning directly in pH-dependent repression but was required to maintain the low medium pH in DSM-GG. AbrB protein levels were high in late-growth, DSM-GG cultures but significantly lower when the pH was raised by Tris-HCl addition. An active tricarboxylic acid (TCA) cycle was required to obtain maximum derepression of sigmaH-dependent transcription, and transcription of the TCA cycle enzyme gene citB was repressed in DSM-GG but derepressed when the pH was artificially raised. The negative effect of low pH on sigmaH-dependent lacZ expression was also observed in unbuffered minimal medium and appeared to be exerted posttranslationally with respect to spo0H expression. However, the addition of amino acids to the medium caused pH-independent repression of both sigmaH-dependent transcription and spo0H-lacZ expression. These results suggest that spo0H transcription or translation is repressed by a mechanism responding to the availability of amino acids whereas spo0H is posttranslationally regulated in response to external pH.

Tn917 transposition in Lactobacillus plantarum using the highly temperature-sensitive plasmid pTV1Ts as a vector.

pTV1Ts, a temperature-sensitive plasmid coding for chloramphenicol (Cm) resistance and carrying the macrolide-lincosamide-steptogramin B (MLS) resistance transposon Tn917, was introduced into strains of Lactobacillus plantarum by electroporation. After two passages in broth medium selecting for MLS resistance at 40 degrees C and subsequent plating on solid medium, two strains, L. plantarum NC4Ts1 and L. plantarum NC7Ts5, lost chloramphenicol resistance but retained MLS resistance, indicative of Tn917 transposition into host DNA. Analysis of DNA from MLSrCms isolates from both strains revealed Tn917 insertions into resident plasmids. Restriction analysis of plasmid DNA from four MLSrCms isolates from NC7Ts5 indicated four different insertion sites.

Formation, regeneration, and transfection of Lactobacillus plantarum protoplasts.

Protoplasts of L. plantarum were produced by mutanolysin-lysozyme digestion at 50 degrees C and regenerated at a frequency of 1.6 to 3.8%. The addition of Tween 80 to the growth medium increased the length of time required for protoplast formation. When transfected with bacteriophage B2 DNA, transfection efficiencies ranged from 25 to 230 PFU/microgram of DNA and from 2.2 X 10(-5) to 4.7 X 10(-4) PFU per recovered protoplast. Total transfectant yield was 3.7 X 10(2) to 3.4 X 10(3) per treatment. Transformations with plasmid DNA were unsuccessful.