Pertussis surveillance by small serosurveys of blood donors.

Serosurveys have established data about the distribution of immunoglobulin G (IgG)-antibodies to pertussis toxin (PT) in various populations. We tried to detect whether small serosurveys in blood donors could serve as a simple and inexpensive means to collect information about the circulation of Bordetella pertussis. We screened every donation in 307 adult blood donors aged 19-69 years for IgG-anti-PT by standardised enzyme-linked immunosorbent assays (ELISA), and the donors were followed between 2014 and 2016 for a total of 426 person-years. When we used a vertical survey with cut-offs of 100, 62.5 and 40 IU/ml, respectively, as an indicator for recent contacts with B. pertussis, nine (2.9%), 22 (7.2%) and 54 (17.6%) of donors had IgG-anti-PT titres above the respective levels. During the horizontal observation period of 426 person years, six significant increases and two conversions were found, which lead to an estimate of 1878 contacts/100.000 person-years (1.9% per year). Median and mean IgG-anti-PT concentrations remained relatively stable from year to year during the observation period. Our findings show that small serosurveys of blood donors offer a simple and cheap method for the surveillance of B. pertussis.

Parametric hybrid scattering from light-induced ferroelectric and photorefractive structures.

Processes of photo-induced light scattering are studied in single crystals of LiNbO(3):Fe to uncover the origin of a new part of the entire scattering pattern which can be observed on a viewing screen. The new scattering manifests itself as two arcs enclosing the directly transmitting pump beam. It is shown that this type of scattering is due to a parametric wave-mixing process of coherent optical noise and a pump beam on a combination of photorefractively recorded phase-gratings and photo-induced ferroelectric structures. Phase-matching conditions corresponding to the new scattering are introduced. All photo-induced scattering phenomena contributing to the total scattering pattern are discussed, compared and classified.

Isolation and characterization of Bacillus subtilis sigB operon mutations that suppress the loss of the negative regulator RsbX.

sigmaB, a transcription factor that controls the Bacillus subtilis general stress response regulon, is activated by either a drop in intracellular ATP or exposure to environmental stress. RsbX, one of seven sigmaB regulators (Rsb proteins) whose genes are cotranscribed with sigmaB, is a negative regulator in the stress-dependent activation pathway. To better define the interactions that take place among the Rsb proteins, we analyzed sigB operon mutations which suppress the high-level sigmaB activity that normally accompanies the loss of RsbX. Each of these mutations was in one of three genes (rsbT, -U, and -V) which encode positive regulators of sigmaB, and they all defined amino acid changes which either compromised the activities of the mutant Rsbs or affected their ability to accumulate. sigmaB activity remained inducible by ethanol in several of the RsbX- suppressor strains. This finding supports the notion that RsbX is not needed as the target for sigmaB activation by at least some stresses. sigmaB activity in several RsbX- strains with suppressor mutations in rsbT or -U was high during growth and underwent a continued, rather than a transient, increase following stress. Thus, RsbX is likely responsible for maintaining low sigmaB activity during balanced growth and for reestablishing sigmaB activity at prestress levels following induction. Although RsbX likely participates in limiting the sigmaB induction response, a second mechanism for curtailing unrestricted sigmaB activation was suggested by the sigmaB induction profile in two suppressor strains with mutations in rsbV. sigmaB activity in these mutants was stress inducible but transient, even in the absence of RsbX.

Stress activation of Bacillus subtilis sigma B can occur in the absence of the sigma B negative regulator RsbX.

Environmental stress activates sigma B, the general stress response sigma factor of Bacillus subtilis, by a pathway that is negatively controlled by the RsbX protein. To determine whether stress activation of sigma B occurs by a direct effect of stress on RsbX, we constructed B. subtilis strains which synthesized various amounts of RsbX or lacked RsbX entirely and subjected these strains to ethanol stress. Based on the induction of a sigma B-dependent promoter, stress activation of sigma B can occur in the absence of RsbX. Higher levels of RsbX failed to detectably influence stress induction, but reduced levels of RsbX resulted in greater and longer-lived sigma B activation. The data suggest that RsbX is not a direct participant in the sigma B stress induction process but rather serves as a device to limit the magnitude of the stress response.

The yeast two-hybrid system detects interactions between Bacillus subtilis sigmaB regulators.

SigmaB, the general stress response sigma factor of Bacillus subtilis, is regulated by the products of seven genes (rsbR, S, T, U, V, W, and X) with which it is cotranscribed. Biochemical techniques previously revealed physical associations among RsbW, RsbV, and sigmaB but failed to detect interactions of RsbR, S, T, U, or X with each other or RsbV, RsbW, or sigmaB. Using the yeast two-hybrid system, we have now obtained evidence for such interactions. The yeast reporter system was activated when RsbS was paired with either RsbR or RsbT, RsbR was paired with RsbT, and RsbV was paired with either RsbU or RsbW. In addition, RsbW2 and RsbR2 dimer formation was detected. RsbX failed to show interactions with itself or any of the other sigB operon products.

Reactivation of the Bacillus subtilis anti-sigma B antagonist, RsbV, by stress- or starvation-induced phosphatase activities.

sigma B is a secondary sigma factor that controls the general stress regulon in Bacillus subtilis. The regulon is activated when sigma B is released from a complex with an anti-sigma B protein (RsbW) and becomes free to associate with RNA polymerase. Two separate mechanisms cause sigma B release: an ATP-responsive mechanism that correlates with nutritional stress and an ATP-independent mechanism that responds to environmental insult (e.g., heat shock and ethanol treatment). ATP levels are thought to directly affect RsbW's binding preference. Low levels of ATP cause RsbW to release sigma B and bind to an alternative protein (RsbV), while high levels of ATP favor RsbW-sigma B complex formation and inactivation of RsbV by an RsbW-dependent phosphorylation. During growth, most of the RsbV is phosphorylated (RsbV-P) and inactive. Environmental stress induces the release of sigma B and the formation of the RsbW-RsbV complex, regardless of ATP levels. This pathway requires the products of additional genes encoded within the eight-gene operon (sigB) that includes the genes for sigma B, RsbW, and RsbV. By using isoelectric focusing techniques to distinguish RsbV from RsbV-P and chloramphenicol treatment or pulse-chase labeling to identify preexisting RsbV-P, we have now determined that stress induces the dephosphorylation of RsbV-P to reactivate RsbV. RsbV-P was also found to be dephosphorylated upon a drop in intracellular ATP levels. The stress-dependent and ATP-responsive dephosphorylations of RsbV-P differed in their requirements for the products of the first four genes (rsbR, -S, -T, and -U) of the sigB operon. Both dephosphorylation reactions required at least one of the genes included in a deletion that removed rsbR, -S, and -T; however, only an environmental insult required RsbU to reactivate RsbV.

Relative levels and fractionation properties of Bacillus subtilis σ(B) and its regulators during balanced growth and stress.

sigma B is a secondary sigma factor that controls the general stress response in Bacillus subtilis. sigma B-dependent genes are activated when sigma B is released from an inhibitory complex with an anti-sigma B protein (RsbW) and becomes free to associate with RNA polymerase. Two separate pathways, responding either to a drop in intracellular ATP levels or to environmental stress (e.g., heat, ethanol, or salt), cause the release of sigma B from RsbW. rsbR, rsbS, rsbT, and rsbU are four genes now recognized as the upstream half of an operon that includes sigB (sigma B) and its principal regulators. Using reporter gene assays, we find that none of these four genes are essential for stationary-phase (i.e., ATP-dependent) activation of sigma B, but rsbU and one or more of the genes contained within an rsbR,S,T deletion are needed for stress induction of sigma B. In other experiments, Western blot (immunoblot) analyses showed that the levels of RsbR, RsbS, Rsb, and RsbU, unlike those of the sigB operon's four downstream gene products (RsbV, RsbW, RsbX and sigma B), are not elevated during sigma B activation. Gel filtration and immunoprecipitation studies did not reveal the formation of complexes between any of the four upstream sigB operon products and the products of the downstream half of the operon. Much of the detectable RsbR, RsbS, RsbT, and RsbU did, however, fractionate as a large-molecular-mass (approximately 600-kDa) aggregate which was excluded from our gel filtration matrix. The downstream sigB operon products were not present in this excluded material. The unaggregated RsbR, RsbS, and RsbU, which were retarded by the gel matrix, elated from the column earlier than expected from their molecular weights. The RsbR and RsbS fractionation profile was consistent with homodimers (60 and 30 kDa, respectively), while the RsbU appeared larger, suggesting a protein complex of approximately 90 to 100 kDa.

Separate mechanisms activate sigma B of Bacillus subtilis in response to environmental and metabolic stresses.

sigma B is a secondary sigma factor that controls the general stress response of Bacillus subtilis. sigma B-dependent transcription is induced by the activation of sigma B itself, a process that involves release of sigma B from an inhibitory complex with its primary regulator, RsbW. sigma B becomes available to RNA polymerase when RsbW forms a complex with an additional regulatory protein (RsbV) and, because of this, fails to bind sigma B. Using Western blot (immunoblot) analyses, reporter gene fusion assays, and measurements of nucleotide pool sizes, we provide evidence for two independent processes that promote the binding of RsbW to RsbV. The first occurs during carbon limitation or entry into stationary phase. Activation of sigma B under these circumstances correlates with a drop in the intracellular levels of ATP and may be a direct consequence of ATP levels on RsbW's binding preference. The second activation process relies on the product of a third regulatory gene, rsbU. RsbU is dispensable for sigma B activation during carbon limitation or stationary phase but is needed for activation of sigma B in response to any of a number of different environmental insults (ethanol treatment, salt or acid shock, etc.). RsbU, or a process dependent on it, alters RsbW binding without regard for intracellular levels of ATP. In at least some instances, the effects of multiple inducing stimuli are additive. The data are consistent with RsbW being a regulator at which distinct signals from separate effectors can be integrated to modulate sigma B activity.

The Bacillus subtilis rsbU gene product is necessary for RsbX-dependent regulation of sigma B.

sigma B is a secondary sigma factor of Bacillus subtilis. sigma B-dependent transcription is induced when B. subtilis enters the stationary phase of growth or is exposed to any of a number of different environmental stresses. Three genes (rsbV, rsbW, and rsbX), which are cotranscribed with the sigma B structural gene (sigB), encode regulators of sigma B-dependent gene expression. RsbW and RsbV have been shown to control sigma B activity, functioning as an inhibitory sigma B binding protein and its antagonist, respectively. Using the SPAC promoter (PSPAC) to control the expression of the sigB operon, a ctc::lacZ reporter system to monitor sigma B activity, and monoclonal antibodies to determine the levels of sigB operon products, we have now obtained evidence that RsbX is an indirect regulator of sigma B activity. Genetic data and in vivo measurements argue that RsbX negatively regulates an extension of the RsbV-RsbW pathway that requires the product of an additional regulatory gene (rsbU) which lies immediately upstream of the sigB operon. The results are consistent with RsbU, or a process dependent on RsbU, being able to facilitate the RsbV-dependent release of sigma B from RsbW but normally prevented from doing this by RsbX.