The cyclic AMP receptor protein (CRP) is a bacterial regulator that controls more than 100 promoters, including those involved in catabolite repression. In the present study, a null deletion of the crp gene was constructed for Yersinia pestis bv. microtus strain 201. Microarray expression analysis disclosed that at least 6% of Y. pestis genes were affected by this mutation. Further reverse transcription-PCR and electrophoretic mobility shift assay analyses disclosed a set of 37 genes or putative operons to be the direct targets of CRP, and thus they constitute the minimal CRP regulon in Y. pestis. Subsequent primer extension and DNase I footprinting assays mapped transcriptional start sites, core promoter elements, and CRP binding sites within the DNA regions upstream of pla and pst, revealing positive and direct control of these two laterally acquired plasmid genes by CRP. The crp disruption affected both in vitro and in vivo growth of the mutant and led to a >15,000-fold loss of virulence after subcutaneous infection but a <40-fold increase in the 50% lethal dose by intravenous inoculation. Therefore, CRP is required for the virulence of Y. pestis and, particularly, is more important for infection by subcutaneous inoculation. It can further be concluded that the reduced in vivo growth phenotype of the crp mutant should contribute, at least partially, to its attenuation of virulence by both routes of infection. Consistent with a previous study of Y. pestis bv. medievalis, lacZ reporter fusion analysis indicated that the crp deletion resulted in the almost absolute loss of pla promoter activity. The plasminogen activator encoded by pla was previously shown to specifically promote Y. pestis dissemination from peripheral infection routes (subcutaneous infection [flea bite] or inhalation). The above evidence supports the notion that in addition to the reduced in vivo growth phenotype, the defect of pla expression in the crp mutant will greatly contribute to the huge loss of virulence of this mutant strain in subcutaneous infection.

Antimicrobial peptides (AMPs), which present in the non-specific immune system of organism, are amongst the most promising candidates for the development of novel antimicrobials. The modification of naturally occurring AMPs based on their residue composition and distribution is a simple and effective strategy for optimization of known AMPs. In this study, a series of truncated and residue-substituted derivatives of antimicrobial peptide PMAP-36 were designed and synthesized. The 24-residue truncated peptide, GI24, displayed antimicrobial activity comparable to the mother peptide PMAP-36 with MICs ranging from 1 to 4 µM, which is lower than the MICs of bee venom melittin. Although GI24 displayed high antimicrobial activity, its hemolytic activity was much lower than melittin, suggesting that GI24 have optimal cell selectivity. In addition, the crucial site of GI24 was identified through single site-mutation. An amino acid with high hydrophobicity at position 23 played an important role in guaranteeing the high antimicrobial activity of GI24. Then, lipid vesicles and whole bacteria were employed to investigate the membrane-active mechanisms. Membrane-simulating experiments showed that GI24 interacted strongly with negatively charged phospholipids and weakly with zwitterionic phospholipids, which corresponded well with the data of its biological activities. Membrane permeabilization and flow cytometry provide the evidence that GI24 killed microbial cells by permeabilizing the cell membrane and damaging membrane integrity. GI24 resulted in greater cell morphological changes and visible pores on cell membrane as determined using scanning electron microscopy (SEM) and transmission electron microscope (TEM). Taken together, the peptide GI24 may provide a promising antimicrobial agent for therapeutic applications against the frequently-encountered bacteria.

The ferric uptake regulator (Fur) is a predominant bacterial regulator controlling the iron assimilation functions in response to iron availability. Our previous microarray analysis on Yersinia pestis defined the iron-Fur modulon. In the present work, we reannotated the iron assimilation genes in Y. pestis, and the resulting genes in complementation with those disclosed by microarray constituted a total of 34 genome loci (putative operons) that represent the potential iron-responsive targets of Fur. The subsequent real-time reverse transcription-PCR (RT-PCR) in conjunction with the primer extension analysis showed that 32 of them were regulated by Fur in response to iron starvation. A previously predicted Fur box sequence was then used to search against the promoter regions of the 34 operons; the homologue of the above box could be predicted in each promoter tested. The subsequent electrophoretic mobility shift assay (EMSA) demonstrated that a purified His 6 tag-fused Fur protein was able to bind in vitro to each of these promoter regions. Therefore, Fur is a global regulator, both an activator and a repressor, and directly controls not only almost all of the iron assimilation functions but also a variety of genes involved in various non-iron functions for governing a complex regulatory cascade in Y. pestis. In addition, real-time RT-PCR, primer extension, EMSA, and DNase I footprinting assay were used to elucidate the Fur regulation of the ybt locus encoding a virulence-required iron uptake system. By combining the published data on the YbtA regulation of ybt, we constructed a concise Fur/YbtA regulatory network with a map of the Fur-promoter DNA interactions within the ybt locus. The data presented here give us an overview of the iron-responsive Fur regulon in Y. pestis.

BackgroundThe zinc uptake regulator Zur is a Zn2+-sensing metalloregulatory protein involved in the maintenance of bacterial zinc homeostasis. Up to now, regulation of zinc homeostasis by Zur is poorly understood in Y. pestis.ResultsWe constructed a zur null mutant of Y. pestis biovar microtus strain 201. Microarray expression analysis disclosed a set of 154 Zur-dependent genes of Y. pestis upon exposure to zinc rich condition. Real-time reverse transcription (RT)-PCR was subsequently used to validate the microarray data. Based on the 154 Zur-dependent genes, predicted regulatory Zur motifs were used to screen for potential direct Zur targets including three putative operons znuA, znuCB and ykgM-RpmJ2. The LacZ reporter fusion analysis verified that Zur greatly repressed the promoter activity of the above three operons. The subsequent electrophoretic mobility shift assay (EMSA) demonstrated that a purified Zur protein was able to bind to the promoter regions of the above three operons. The DNase I footprinting was used to identify the Zur binding sites for the above three operons, verifying the Zur box sequence as predicted previously in γ-Proteobacteria. The primer extension assay was further used to determine the transcription start sites for the above three operons and to localize the -10 and -35 elements. Zur binding sites overlapped the -10 sequence of its target promoters, which was consistent with the previous observation that Zur binding would block the entry of the RNA polymerase to repress the transcription of its target genes.ConclusionZur as a repressor directly controls the transcription of znuA, znuCB and ykgM-RpmJ2 in Y. pestis by employing a conserved mechanism of Zur-promoter DNA association as observed in γ-Proteobacteria. Zur contributes to zinc homeostasis in Y. pestis likely through transcriptional repression of the high-affinity zinc uptake system ZnuACB and two alternative ribosomal proteins YkgM and RpmJ2.

Background: The transcription regulator PhoP has been shown to be important for Y. pestis survival in macrophages and under various in vitro stresses. However, the mechanism by which PhoP promotes bacterial intracellular survival is not fully understood. Our previous microarray analysis suggested that PhoP governed a wide set of cellular pathways in Y. pestis. A series of biochemical experiments were done herein to study members of the PhoP regulon of Y. pestis biovar Microtus.

BackgroundThe osmotic regulator OmpR in Escherichia coli regulates differentially the expression of major porin proteins OmpF and OmpC. In Yersinia enterocolitica and Y. pseudotuberculosis, OmpR is required for both virulence and survival within macrophages. However, the phenotypic and regulatory roles of OmpR in Y. pestis are not yet fully understood.ResultsY. pestis OmpR is involved in building resistance against phagocytosis and controls the adaptation to various stressful conditions met in macrophages. The ompR mutation likely did not affect the virulence of Y. pestis strain 201 that was a human-avirulent enzootic strain. The microarray-based comparative transcriptome analysis disclosed a set of 224 genes whose expressions were affected by the ompR mutation, indicating the global regulatory role of OmpR in Y. pestis. Real-time RT-PCR or lacZ fusion reporter assay further validated 16 OmpR-dependent genes, for which OmpR consensus-like sequences were found within their upstream DNA regions. ompC, F, X, and R were up-regulated dramatically with the increase of medium osmolarity, which was mediated by OmpR occupying the target promoter regions in a tandem manner.ConclusionOmpR contributes to the resistance against phagocytosis or survival within macrophages, which is conserved in the pathogenic yersiniae. Y. pestis OmpR regulates ompC, F, X, and R directly through OmpR-promoter DNA association. There is an inducible expressions of the pore-forming proteins OmpF, C, and × at high osmolarity in Y. pestis, in contrast to the reciprocal regulation of them in E. coli. The main difference is that ompF expression is not repressed at high osmolarity in Y. pestis, which is likely due to the absence of a promoter-distal OmpR-binding site for ompF.

Background Yersinia pestis is the causative agent of plague. The two transcriptional regulators, PhoP and RovA, are required for the virulence of Y. pestis through the regulation of various virulence-associated loci. They are the global regulators controlling two distinct large complexes of cellular pathways.Methodology/Principal FindingsBased on the LacZ fusion, primer extension, gel mobility shift, and DNase I footprinting assays, RovA is shown to recognize both of the two promoters of its gene in Y. pestis. The autoregulation of RovA appears to be a conserved mechanism shared by Y. pestis and its closely related progenitor, Y. pseudotuberculosis. In Y. pestis, the PhoP regulator responds to low magnesium signals and then negatively controls only one of the two promoters of rovA through PhoP-promoter DNA association.Conclusions/SignificanceRovA is a direct transcriptional activator for its own gene in Y. pestis, while PhoP recognizes the promoter region of rovA to repress its transcription. The direct regulatory association between PhoP and RovA bridges the PhoP and RovA regulons in Y. pestis.

Integration of environmental and endogenous cues at plant shoot meristems determines the timing of flowering and reproductive development. The MADS box transcription factor FLOWERING LOCUS C (FLC) of Arabidopsis thaliana is an important repressor of floral transition, which blocks flowering until plants are exposed to winter cold. However, the target genes of FLC have not been thoroughly described, and our understanding of the mechanisms by which FLC represses transcription of these targets and how this repression is overcome during floral transition is still fragmentary. Here, we identify and characterize TARGET OF FLC AND SVP1 (TFS1) , a novel target gene of FLC and its interacting protein SHORT VEGETATIVE PHASE (SVP). TFS1 encodes a B3-type transcription factor, and we show that tfs1 mutants are later flowering than wild-type, particularly under short days. FLC and SVP repress TFS1 transcription leading to deposition of trimethylation of Iysine 27 of histone 3 (H3K27me3) by the Polycomb Repressive Complex 2 at the TFS1 locus. During floral transition, after downregulation of FLC by cold, TFS1 transcription is promoted by SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), a MADS box protein encoded by another target of FLC/SVP. SOC1 opposes PRC function at TFS1 through recruitment of the histone demethylase RELATIVE OF EARLY FLOWERING 6 (REF6) and the SWI/SNF chromatin remodeler ATPase BRAHMA (BRM). This recruitment of BRM is also strictly required for SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9) binding at TFS1 to coordinate RNAPII recruitment through the Mediator complex. Thus, we show that antagonistic chromatin modifications mediated by different MADS box transcription factor complexes play a crucial role in defining the temporal and spatial patterns of transcription of genes within a network of interactions downstream of FLC/SVP during floral transition.