The act operon controls the level and time of C-signal production for Myxococcus xanthus development

The C-signal is a morphogen that controls the assembly of fruiting bodies and the differentiation of myxospores. Production of this signal, which is encoded by the csgA gene, is regulated by the act operon of four genes that are co-transcribed from the same start site. The act A and act B genes regulate the maximum level of the C-signal, which never rises above one-quarter of the maximum wild-type level of CsgA protein in null mutants of either gene. The act A and act B mutants have the same developmental phenotype: both aggregate, neither sporulates, both prolong rippling. By sequence homology, act A encodes a response regulator, and act B encodes a sigma-54 activator protein of the NTRC class. The similar phenotypes of act A and act B deletion mutants suggest that the two gene products are part of the same signal transduction pathway. That pathway responds to C-signal and also regulates the production of CsgA protein, thus creating a positive feedback loop. The act C and act D genes regulate the time pattern of CsgA production, while achieving the same maximum level. An act C null mutant raises CsgA production 15 h earlier than the wild type, whereas an act D null mutant does so 6 h later than wild type. The loop explains how the C-signal rises continuously from early development to a peak at the time of sporulation, and the act genes govern the time course of that rise.     

Robust and sensitive control of a quorum‐sensing circuit by two interlocked feedback loops

The quorum‐sensing (QS) response of Vibrio fischeri involves a rapid switch between low and high induction states of the lux operon over a narrow concentration range of the autoinducer (AI) 3‐oxo‐hexanoyl‐L ‐homoserine lactone. In this system, LuxR is an AI‐dependent positive regulator of the lux operon, which encodes the AI synthase. This creates a positive feedback loop common in many bacterial species that exhibit QS‐controlled gene expression. Applying a combination of modeling and experimental analyses, we provide evidence for a LuxR autoregulatory feedback loop that allows LuxR to increase its concentration in the cell during the switch to full lux activation. Using synthetic lux gene fragments, with or without the AI synthase gene, we show that the buildup of LuxR provides more sensitivity to increasing AI, and promotes the induction process. Elevated LuxR levels buffer against spurious variations in AI levels ensuring a robust response that endows the system with enhanced hysteresis. LuxR autoregulation also allows for two distinct responses within the same cell population.     

Relative roles of the fla/che P(A), P(D-3), and P(sigD) promoters in regulating motility and sigD expression in Bacillus subtilis

Three promoters have been identified as having potentially important regulatory roles in governing expression of the fla/che operon and of sigD, a gene that lies near the 3' end of the operon. Two of these promoters, fla/che P(A) and P(D-3), lie upstream of the >26-kb fla/che operon. The third promoter, P(sigD), lies within the operon, immediately upstream of sigD. fla/che P(A), transcribed by E sigma(A), lies >/=24 kb upstream of sigD and appears to be largely responsible for sigD expression. P(D-3), transcribed by E sigma(D), has been proposed to participate in an autoregulatory positive feedback loop. P(sigD), a minor sigma(A)-dependent promoter, has been implicated as essential for normal expression of the fla/che operon. We tested the proposed functions of these promoters in experiments that utilized strains that bear chromosomal deletions of fla/che P(A), P(D-3), or P(sigD). Our analysis of these strains indicates that fla/che P(A) is absolutely essential for motility, that P(D-3) does not function in positive feedback regulation of sigD expression, and that P(sigD) is not essential for normal fla/che expression. Further, our results suggest that an additional promoter(s) contributes to sigD expression.     

L. monocytogenes-induced actin assembly requires the actA gene product, a surface protein.

The intracellular pathogenic bacterium L. monocytogenes can spread directly from cell to cell without leaving the cytoplasm. The mechanism of this movement, generated through bacterially induced actin polymerization, is not understood. By analyzing an avirulent Tn917-lac mutant defective for actin polymerization, we have identified a bacterial component involved in this process. The transposon had inserted in actA, the second gene of an operon. Gene disruption of downstream genes and transformation of the mutant strain with actA showed that the actA gene encodes a surface protein necessary for bacterially induced actin assembly. Our results indicate that it is a 610 amino acid protein with an apparent molecular weight of 90 kd.     

Positive feedback and noise activate the stringent response regulator rel in mycobacteria

Phenotypic heterogeneity in an isogenic, microbial population enables a subset of the population to persist under stress. In mycobacteria, stresses like nutrient and oxygen deprivation activate the stress response pathway involving the two-component system MprAB and the sigma factor, SigE. SigE in turn activates the expression of the stringent response regulator, rel. The enzyme polyphosphate kinase 1 (PPK1) regulates this pathway by synthesizing polyphosphate required for the activation of MprB. The precise manner in which only a subpopulation of bacterial cells develops persistence, remains unknown. Rel is required for mycobacterial persistence. Here we show that the distribution of rel expression levels in a growing population of mycobacteria is bimodal with two distinct peaks corresponding to low (L) and high (H) expression states, and further establish that a positive feedback loop involving the mprAB operon along with stochastic gene expression are responsible for the phenotypic heterogeneity. Combining single cell analysis by flow cytometry with theoretical modeling, we observe that during growth, noise-driven transitions take a subpopulation of cells from the L to the H state within a "window of opportunity" in time preceding the stationary phase. It is these cells which adapt to nutrient depletion in the stationary phase via the stringent response. We find evidence of hysteresis in the expression of rel in response to changing concentrations of PPK1. Hysteresis promotes robustness in the maintenance of the induced state. Our results provide, for the first time, evidence that bistability and stochastic gene expression could be important for the development of "heterogeneity with an advantage" in mycobacteria and suggest strategies for tackling tuberculosis like targeting transitions from the low to the high rel expression state.     

Androgen regulates follicle-stimulating hormone beta gene expression in an activin-dependent manner in immortalized gonadotropes

Little is known about the molecular mechanisms of androgen regulation of the FSHbeta gene; however, studies suggest that it consists of a complex feedback loop that involves multiple mechanisms acting at both the level of the hypothalamus and the pituitary. In the present study, we address androgen regulation of the FSHbeta gene in immortalized gonadotrope cells and investigate the roles of activin and GnRH in androgen action. Using transient transfection assays in the FSHbeta-expressing mouse gonadotrope cell line, LbetaT2, we demonstrate that androgens stimulate expression of an ovine FSHbeta reporter gene in a dose-dependent manner. Mutation of either of two conserved androgen response elements at -245/-231 and -153/-139 within the proximal region of the ovine FSHbeta gene promoter abolishes this stimulation, and androgen receptor binds directly to the -244 ARE in vitro. Androgen induction of the FSHbeta reporter gene is also dependent upon the activin autocrine loop present in the LbetaT2 cells, as well as an activin-response element at -138/-124 of the FSHbeta gene. However, activin regulation of other genes remains unaffected by androgens. In addition, androgens stimulate expression of a mouse GnRH receptor reporter gene, and thus may indirectly augment the response of the FSHbeta gene to GnRH. Taken together, these data demonstrate that, in mouse gonadotropes, androgens act directly on the ovine FSHbeta gene to stimulate expression by a mechanism that is dependent upon activin, as well as acting indirectly, potentially through a second mechanism that may be dependent upon induction of GnRH receptor.     

Isolation of Listeria monocytogenes mutants with high-level in vitro expression of host cytosol-induced gene products

The facultative intracellular bacterial pathogen Listeria monocytogenes dramatically increases the expression of several key virulence factors upon entry into the host cell cytosol. actA, the protein product of which is required for cell-to-cell spread of the bacterium, is expressed at low to undetectable levels in vitro and increases in expression more than 200-fold after L. monocytogenes escape from the phagosome. To identify bacterial factors that participate in the intracellular induction of actA expression, L. monocytogenes mutants expressing high levels of actA during in vitro growth were selected after chemical mutagenesis. The resulting mutant isolates displayed a wide range of actA expression levels, and many were less sensitive to environmental signals that normally mediate repression of virulence gene expression. Several isolates contained mutations affecting actA gene expression that mapped at least 40 kb outside the PrfA regulon, supporting the existence of additional regulatory factors that contribute to virulence gene expression. Two actA in vitro expression mutants contained novel mutations within PrfA, a key regulator of L. monocytogenes virulence gene expression. PrfA E77K and PrfA G155S mutations resulted in high-level expression of PrfA-dependent genes, increased bacterial invasion of epithelial cells and increased virulence in mice. Both prfA mutant strains were significantly less motile than wild-type L. monocytogenes. These results suggest that, although constitutive activation of PrfA and PrfA-dependent gene expression may enhance L. monocytogenes virulence, it may conversely hamper the bacterium's ability to compete in environments outside host cells.