Study of Histidine Kinases Involved in Bacillus substilis Differentiation

In response to nutrient deprivation, Bacillus subtilis cells undergo differentiation to form biofilm and spore. This process requires the involvement of a multiple phosphorelay component, wherein phosphate is transferred to a master transcription regulator Spo0A from five histidine kinases (KinA-KinE) via two intermediate phosphotransferases, Spo0F and Spo0B. It has been proposed that the autokinase activity of one or a combination of five kinases is stimulated when the amino (N) terminal ‘sensor’ domain of kinase receives an yet unidentified starvation signal(s), leading to the increase of the phosphorylated (active) form of Spo0A (Spo0A~P) via phosphorelay and resulting in differentiation into biofilm formation and sporulation. However, the underlying mechanisms by which the putative starvation signal(s) stimulate the kinase and the subsequent control of Spo0A activity remain elusive. In order to test, we conducted, domain-swap experiments were performed by fusing a tetramer-forming protein derived from Escherichia coli to the KinA C-terminal domain. Despite the introduction of a foreign domain, the resulting chimeric protein, in a concentration-dependent manner, triggered sporulation by activating Spo0A through phosphorelay, irrespective of nutrient availability. A simultaneous induction system, in which KinC, a kinase that can directly phosphorylate Spo0A, and Spo0A itself are separately controlled by inducible promoters, were constructed. This artificial two-component system can efficiently trigger sporulation even under nutrient rich conditions. However, the sporulation efficiency was significantly impaired when KinC and/or Spo0A induction was too high. Lastly, the role of KinC during the course of starvation was studied. Conventional genetic approaches showed that KinC preferentially and positively controls the expression of the cannibalism (a mechanism to delay sporulation) genes during early stage of starvation in a manner dependent on phosphorelay, resulting in the delay of sporulation. Evidence suggests that the N-terminal domain is essential for forming a stable tetramer as a functional kinase, but possibly not for sensing an as-yet unknown starvation signal. Furthermore, the data suggest that, upon starvation, two different levels (low and high) of Spo0A activity are achieved through two distinct pathways regulated by KinC and KinA, which are essential for the proper cell fate decision to differentiate into biofilm or sporulation.Biology and Biochemistry, Department o

The Threshold Level of the Sensor Histidine Kinase KinA Governs Entry into Sporulation in Bacillus subtilis▿ †

Sporulation in Bacillus subtilis is controlled by a complex gene regulatory circuit that is activated upon nutrient deprivation. The initial process is directed by the phosphorelay, involving the major sporulation histidine kinase (KinA) and two additional phosphotransferases (Spo0F and Spo0B), that activates the master transcription factor Spo0A. Little is known about the initial event and mechanisms that trigger sporulation. Using a strain in which the synthesis of KinA is under the control of an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible promoter, here we demonstrate that inducing the synthesis of the KinA beyond a certain level leads to the entry of the irreversible process of sporulation irrespective of nutrient availability. Moreover, the engineered cells expressing KinA under a σH-dependent promoter that is similar to but stronger than the endogenous kinA promoter induce sporulation during growth. These cells, which we designated COS (constitutive sporulation) cells, exhibit the morphology and properties of sporulating cells and express sporulation marker genes under nutrient-rich conditions. Thus, we created an engineered strain displaying two cell cycles (growth and sporulation) integrated into one cycle irrespective of culture conditions, while in the wild type, the appropriate cell fate decision is made depending on nutrient availability. These results suggest that the threshold level of the major sporulation kinase acts as a molecular switch to determine cell fate and may rule out the possibility that the activity of KinA is regulated in response to the unknown signal(s)

Glucocorticoid-induced leucine zipper (GILZ) regulates testicular FOXO1 activity and spermatogonial stem cell (SSC) function

Spermatogonia stem cell (SSC) self-renewal and differentiation are tightly regulated processes that ensure a continued production of mature sperm throughout male adulthood. In the present study, we investigated the role of glucocorticoid-induced leucine zipper (GILZ) in maintenance of the male germline and spermatogenesis. GILZ was detectable in germ cells of wild type mice on the day of birth, suggesting a role for GILZ in prospermatogonia and SSC pool formation. Gilz KO mice were generated and adult males were azoospermic and sterile. During the first wave of spermatogenesis in Gilz KO mice, spermatogenesis arrested part way through pachytene of meiosis I. Subsequent waves resulted in a progressive depletion of germ cells through apoptosis to ultimately produce a Sertoli cell-only phenotype. Further, in contrast to wild type littermates, PLZF(+) cells were detected in the peri-luminal region of Gilz KO mice at day 6 post-natal, suggesting a defect in prospermatogonia migration in the absence of GILZ. At age 30 days, transient accumulation of PLZF(+) cells in a subset of tubules and severely compromised spermatogenesis were observed in Gilz KO mice, consistent with defective SSC differentiation. GILZ deficiency was associated with an increase in FOXO1 transcriptional activity, which leads to activation of a selective set of FOXO1 target genes, including a pro-apoptotic protein, BIM. On the other hand, no evidence of a heightened immune response was observed. Together, these results suggest that GILZ suppresses FOXO1 nuclear translocation, promotes SSC differentiation over self-renewal, and favours germ cell survival through inhibition of BIM-dependent pro-apoptotic signals. These findings provide a mechanism for the effects of GILZ on spermatogenesis and strengthen the case for GILZ being a critical molecule in the regulation of male fertility

Analysis of the SSC population in Gilz KO testis.

<p>A: Immunohistochemistry for PLZF on testis sections from mice of the indicated postnatal ages (days). Higher magnification insets show the presence of PLZF⁺ cells in the periluminal region of day 6 tubules. B: Immunohistochemistry for SALL4 on testis sections from post-natal day 30 mice. Asterisks in A and B indicate day 14 and 30 tubules containing PLZF and SALL4-positive cells respectively. Scale bars represent 50 µM.</p

Primer sequences for qPCR.

<p>Primer sequences for qPCR.</p

Effect of GILZ deficiency on FOXO1 activity during spermatogenesis.

<p>A: Day 6 old WT and Gilz KO mouse testes were used in immunofluorescence analysis to assess the localization of FOXO1 (green) and GILZ (red) using confocal microscopy (DAPI-stain nuclei shown in blue). High magnification inserts showing localization of FOXO1 and GILZ were also included. Scale bars represent 100 µM and insert scale bars represent 25 µM. B: Quantitative analysis of nuclear FOXO1 positive cells in day 6 old WT and Gilz KO testes. Data represents the mean ± SEM of 6 testes. **, p&lt;0.01. C: Quantitative PCR analysis of <i>Ret</i>, <i>Lhx</i>, <i>Egr4</i>, Sall4, Dppa4 and Bim mRNA expression in day 6, 10 and 14 old WT and Gilz KO testes. Results are expressed as the number of mRNA copies per 10⁶ 18 s rRNA copies. Data represents the mean ± SEM of 4–8 mice per group. **, p&lt;0.01 related to WT controls. D: Protein expression of BIM-EL (extra long) and GILZ was detected in day 20 old WT and Gilz KO testes using Western blotting. Representative images of two individual experiments.</p

Effects of GILZ deficiency on blood-testis barrier and testicular leukocyte.

<p>A: Immunostaining for the blood-testis barrier marker espin was performed on day 20 WT and Gilz KO testes using anti-espin antibody. Scale bars represent 100 µM. Single insert represents the no espin staining negative control. B: Blood-testis-barrier (indicated by arrows) in day 20 old WT and Gilz KO testes samples were examined using transmission electron microscopy. Scale bars represent 5 µM. C: The presence of leukocytes in adult (day 70) WT and Gilz KO testes was examined by immunohistochemistry using CD45 as a marker. Scale bars represent 100 µM.</p

Effect of GILZ deficiency on testicular size.

<p>A: Representative image of WT and Gilz KO testes. B: Testes weights of WT and Gilz KO testes. Data represents the mean ± SEM of 5 mice per group. **P&lt;0.001.</p

GILZ deficiency leads to spermatogenic failure.

<p>A: Testes were collected from 6, 10, 14, 20 and 30 day old and adult WT and Gilz KO mice, processed and stained with PAS. Scale bars represent 100 µM. B: The presence of mature sperm was detected in adult WT but not Gilz KO epididymides. Scale bars represent 100 µM. C: Representative images of TUNEL positive cells in 20 day old WT and Gilz KO testes. Scale bars represent 50 µM. D: Chromatin morphology of pachytene cells was examined using PAS staining (scale bars represent 50 µM) and SCP3 immunofluorescence in day 20 WT and Gilz KO testes.</p