Kinetic Modeling and Meta-Analysis of the Bacillus subtilis SigB Regulon during Spore Germination and Outgrowth

The exponential increase in the number of conducted studies combined with the development of sequencing methods have led to an enormous accumulation of partially processed experimental data in the past two decades. Here, we present an approach using literature-mined data complemented with gene expression kinetic modeling and promoter sequence analysis. This approach allowed us to identify the regulon of Bacillus subtilis sigma factor SigB of RNA polymerase (RNAP) specifically expressed during germination and outgrowth. SigB is critical for the cell’s response to general stress but is also expressed during spore germination and outgrowth, and this specific regulon is not known. This approach allowed us to (i) define a subset of the known SigB regulon controlled by SigB specifically during spore germination and outgrowth, (ii) identify the influence of the promoter sequence binding motif organization on the expression of the SigB-regulated genes, and (iii) suggest additional sigma factors co-controlling other SigB-dependent genes. Experiments then validated promoter sequence characteristics necessary for direct RNAP–SigB binding. In summary, this work documents the potential of computational approaches to unravel new information even for a well-studied system; moreover, the study specifically identifies the subset of the SigB regulon, which is activated during germination and outgrowth.

Design to Data for mutants of β-glucosidase B from Paenibacillus polymyxa: I45K, A357S, I20A, I20V, and I20E

ABSTRACTThe relatively small size and scope of most current datasets of biophysical mutation effects in enzymes limit the ability to develop data-driven algorithms enabling accurate generative modeling tools for designing novel enzyme function. Here, the Michaelis-Menten constants (kcat, KM, and kcat/KM) and thermal stability (TM) of five new mutations of β-glucosidase B from Paenibacillus polymyxa (BglB) are characterized. Foldit software was used to create molecular models of the mutants, for which synthetic genes were constructed and the corresponding proteins produced and purified from E. coli. It was found that mutations that disrupted pre-existing hydrogen bonds near the active site had reduced expression in contrast to mutations at the same site that did not affect native hydrogen bonding. This is consistent with previous results showing the relationship between hydrogen bonding and enzyme functionality. These mutants contribute to a growing data set of >100 mutants that have been characterized for expression, kinetic, and thermal properties

Microarray-based determination of the lytic cascade of human herpesvirus 6B

The lytic gene expression of several members of the human herpesvirus family has been profiled by using gene-expression microarrays; however, the lytic cascade of roseoloviruses has not been studied in similar depth. Based on the complete DNA genome sequences of human herpesvirus 6 variant A (HHV-6A) and variant B (HHV-6B), we constructed a cDNA microarray containing DNA probes to their predicted open reading frames, plus 914 human genes. Gene-expression profiling of HHV-6B strain Z29 in SupT1 cells over a 60 h time-course post-infection, together with kinetic classification of the HHV-6B genes in the presence of either cycloheximide or phosphonoacetic acid, allowed the placement of HHV-6B genes into defined kinetic classes. Eighty-nine HHV-6B genes were divided into four different expression kinetic classes: eight immediate-early, 44 early, 33 late and four biphasic. Clustering of genes with similar expression profiles implied a shared function, thus revealing possible roles of previously uncharacterized HHV-6B genes.

IFN-γ production by alloantigen-reactive regulatory T cells is important for their regulatory function in vivo

The significance of cytokine production by CD4+ regulatory T (T reg) cells after antigen exposure in vivo and its impact on their regulatory activity remains unclear. Pretreatment with donor alloantigen under the cover of anti-CD4 therapy generates alloantigen reactive T reg cells that can prevent rejection of donor-specific skin grafts that are mediated by naive CD45RBhighCD4+ T cells. To examine the kinetics and importance of cytokine gene transcription by such alloantigen-reactive T reg cells, pretreated mice were rechallenged with donor alloantigen in vivo. CD25+CD4+ T cells, but not CD25−CD4+ T cells, showed a fivefold increase in IFN-γ mRNA expression within 24 h of reencountering alloantigen in vivo. This expression kinetic was highly antigen-specific and was of functional significance. Neutralizing IFN-γ at the time of cotransfer of alloantigen reactive T reg cells, together with CD45RBhighCD4+ effector T cells into Rag−/− skin graft recipients, resulted in skin graft necrosis in all recipients; the generation and function of alloantigen-reactive T reg cells was impaired dramatically in IFN-γ–deficient mice. These data support a unique role for IFN-γ in the functional activity of alloantigen-reactive T reg cells during the development of operational tolerance to donor alloantigens in vivo.