Global Transcription Factor Research Articles

Minor Alterations in Core Promoter Element Positioning Reveal Functional Plasticity of a Bacterial Transcription Factor.

ABSTRACTIscR is a global transcription factor that regulates Fe-S cluster homeostasis and other functions in Escherichia coli by either activating or repressing transcription. While the interaction of IscR with its DNA sites has been studied, less is known about the mechanism of IscR regulation of transcription. Here, we show that IscR recruits RNA polymerase to an activated promoter and that IscR binding compensates for the lack of an optimal RNA polymerase σ70 −35 promoter element. We also find that the position of the −35 promoter element within the IscR DNA site impacts whether IscR activates or represses transcription. RNA polymerase binding at a distally positioned −35 element within the IscR site results in IscR activation. Molecular modeling suggests that this position of the −35 element allows IscR and RNA polymerase to bind to the promoter from opposite faces of the helix. Shifting the −35 element 1 nucleotide upstream within the IscR binding site results in IscR repression and a steric clash of IscR and RNA polymerase binding in the models. We propose that the sequence similarity of the IscR binding site with the −35 element is an important feature in allowing plasticity in the mechanism of IscR regulation.

Transcription Factor Signatures May Predict the Prognosis and Status of the Immune Microenvironment of Primary Lower-Grade Gliomas.

AimTranscription factor (TF) in glioma, including proliferation, invasion/migration, and tumor microenvironment, has been receiving increasing attention. However, there are still no systematical analyses based on global TF. Herein, using global TF target gene sets, we comprehensively investigated their relationship with prognosis and potential biological effect in lower-grade glioma (LGG). We aimed to develop a less-biased prognostic model and provide new insight for personalized management of this disease.MethodsTF target gene sets were collected from MSigDB and GRID database followed by ssGSEA calculating normalized enrichment score. Comprehensive survival analysis was combined with Kaplan–Meier and Cox algorithms. Consensus cluster and lasso regression were performed to develop prognostic signatures with validation of ROC and independent external cohort. Approaches of xCell/CIBERSORT/TIMER were involved in analyzing the immune microenvironment. We also correlated identified prognostic signatures with tumor mutational burden (TMB) and m6A genes.ResultsFourteen TFs were significantly screened based on survival. Patients were classified into 2 prognosis-related clusters based on 14-TFs features. The function of differentially expressed TF target genes between Cluster1/2 was enriched mostly on glioma invasion/migration. The prognostic model was trained by 6 out of 14-TFs followed by generating risk-score as an independent prognostic indicator. We found differences between the high/low-risk group in TMB and the immune microenvironment, where the high-risk group represented “hot-tumor”. Besides, 6-TFs were correlated with m6A regulation genes.ConclusionOur findings suggested that the 6-TFs model could be used to predict prognosis and predict the status of the immune microenvironment in LGG.

Temporal evolution of master regulator Crp identifies pyrimidines as catabolite modulator factors

The evolution of microorganisms often involves changes of unclear relevance, such as transient phenotypes and sequential development of multiple adaptive mutations in hotspot genes. Previously, we showed that ageing colonies of an E. coli mutant unable to produce cAMP when grown on maltose, accumulated mutations in the crp gene (encoding a global transcription factor) and in genes involved in pyrimidine metabolism such as cmk; combined mutations in both crp and cmk enabled fermentation of maltose (which usually requires cAMP-mediated Crp activation for catabolic pathway expression). Here, we study the sequential generation of hotspot mutations in those genes, and uncover a regulatory role of pyrimidine nucleosides in carbon catabolism. Cytidine binds to the cytidine regulator CytR, modifies the expression of sigma factor 32 (RpoH), and thereby impacts global gene expression. In addition, cytidine binds and activates a Crp mutant directly, thus modulating catabolic pathway expression, and could be the catabolite modulating factor whose existence was suggested by Jacques Monod and colleagues in 1976. Therefore, transcription factor Crp appears to work in concert with CytR and RpoH, serving a dual role in sensing both carbon availability and metabolic flux towards DNA and RNA. Our findings show how certain alterations in metabolite concentrations (associated with colony ageing and/or due to mutations in metabolic or regulatory genes) can drive the evolution in non-growing cells.

The effect of ArcA on the growth, motility, biofilm formation, and virulence of Plesiomonas shigelloides

BackgroundThe anoxic redox control binary system plays an important role in the response to oxygen as a signal in the environment. In particular, phosphorylated ArcA, as a global transcription factor, binds to the promoter regions of its target genes to regulate the expression of aerobic and anaerobic metabolism genes. However, the function of ArcA in Plesiomonas shigelloides is unknown.ResultsIn the present study, P. shigelloides was used as the research object. The differences in growth, motility, biofilm formation, and virulence between the WT strain and the ΔarcA isogenic deletion mutant strain were compared. The data showed that the absence of arcA not only caused growth retardation of P. shigelloides in the log phase, but also greatly reduced the glucose utilization in M9 medium before the stationary phase. The motility of the ΔarcA mutant strain was either greatly reduced when grown in swim agar, or basically lost when grown in swarm agar. The electrophoretic mobility shift assay results showed that ArcA bound to the promoter regions of the flaK, rpoN, and cheV genes, indicating that ArcA directly regulates the expression of these three motility-related genes in P. shigelloides. Meanwhile, the ability of the ΔarcA strain to infect Caco-2 cells was reduced by 40%; on the contrary, its biofilm formation was enhanced. Furthermore, the complementation of the WT arcA gene from pBAD33-arcA+ was constructed and all of the above features of the pBAD33-arcA+ complemented strain were restored to the WT level.ConclusionsWe showed the effect of ArcA on the growth, motility, biofilm formation, and virulence of Plesiomonas shigelloides, and demonstrated that ArcA functions as a positive regulator controls the motility of P. shigelloides by directly regulating the expression of flaK, rpoN and cheV genes.

Structural basis of transcription activation by the global regulator Spx

Spx is a global transcriptional regulator in Gram-positive bacteria and has been inferred to efficiently activate transcription upon oxidative stress by engaging RNA polymerase (RNAP) and promoter DNA. However, the precise mechanism by which it interacts with RNAP and promoter DNA to initiate transcription remains obscure. Here, we report the cryo-EM structure of an intact Spx-dependent transcription activation complex (Spx–TAC) from Bacillus subtilis at 4.2 Å resolution. The structure traps Spx in an active conformation and defines key interactions accounting for Spx-dependent transcription activation. Strikingly, an oxidized Spx monomer engages RNAP by simultaneously interacting with the C-terminal domain of RNAP alpha subunit (αCTD) and σA. The interface between Spx and αCTD is distinct from those previously reported activators, indicating αCTD as a multiple target for the interaction between RNAP and various transcription activators. Notably, Spx specifically wraps the conserved –44 element of promoter DNA, thereby stabilizing Spx–TAC. Besides, Spx interacts extensively with σA through three different interfaces and promotes Spx-dependent transcription activation. Together, our structural and biochemical results provide a novel mechanistic framework for the regulation of bacterial transcription activation and shed new light on the physiological roles of the global Spx-family transcription factors.

Beyond the Biosynthetic Gene Cluster Paradigm: Genome-Wide Coexpression Networks Connect Clustered and Unclustered Transcription Factors to Secondary Metabolic Pathways.

ABSTRACTFungal secondary metabolites are widely used as therapeutics and are vital components of drug discovery programs. A major challenge hindering discovery of novel secondary metabolites is that the underlying pathways involved in their biosynthesis are transcriptionally silent under typical laboratory growth conditions, making it difficult to identify the transcriptional networks that they are embedded in. Furthermore, while the genes participating in secondary metabolic pathways are typically found in contiguous clusters on the genome, known as biosynthetic gene clusters (BGCs), this is not always the case, especially for global and pathway-specific regulators of pathways’ activities. To address these challenges, we used 283 genome-wide gene expression data sets of the ascomycete cell factory Aspergillus niger generated during growth under 155 different conditions to construct two gene coexpression networks based on Spearman’s correlation coefficients (SCCs) and on mutual rank-transformed Pearson’s correlation coefficients (MR-PCCs). By mining these networks, we predicted six transcription factors, named MjkA to MjkF, to regulate secondary metabolism in A. niger. Overexpression of each transcription factor using the Tet-On cassette modulated the production of multiple secondary metabolites. We found that the SCC and MR-PCC approaches complemented each other, enabling the delineation of putative global (SCC) and pathway-specific (MR-PCC) transcription factors. These results highlight the potential of coexpression network approaches to identify and activate fungal secondary metabolic pathways and their products. More broadly, we argue that drug discovery programs in fungi should move beyond the BGC paradigm and focus on understanding the global regulatory networks in which secondary metabolic pathways are embedded.IMPORTANCE There is an urgent need for novel bioactive molecules in both agriculture and medicine. The genomes of fungi are thought to contain vast numbers of metabolic pathways involved in the biosynthesis of secondary metabolites with diverse bioactivities. Because these metabolites are biosynthesized only under specific conditions, the vast majority of the fungal pharmacopeia awaits discovery. To discover the genetic networks that regulate the activity of secondary metabolites, we examined the genome-wide profiles of gene activity of the cell factory Aspergillus niger across hundreds of conditions. By constructing global networks that link genes with similar activities across conditions, we identified six putative global and pathway-specific regulators of secondary metabolite biosynthesis. Our study shows that elucidating the behavior of the genetic networks of fungi under diverse conditions harbors enormous promise for understanding fungal secondary metabolism, which ultimately may lead to novel drug candidates.

MaAreB, a GATA Transcription Factor, Is Involved in Nitrogen Source Utilization, Stress Tolerances and Virulence in Metarhizium acridum.

The nitrogen catabolite repression (NCR) pathway is involved in nitrogen utilization, in which the global GATA transcription factor AreA plays an indispensable role and has been reported in many fungi. However, relatively few studies are focused on AreB, another GATA transcription factor in the NCR pathway and the functions of AreB are largely unknown in entomopathogenic fungi. Here, we characterized MaAreB in the model entomopathogenic fungus Metarhizium acridum. Sequence arrangement found that MaAreB had a conserved GATA zinc finger DNA binding domain and a leucine zipper domain. Disruption of MaAreB affected the nitrogen utilization and led to decelerated conidial germination and hyphal growth, decreased conidial yield, and lower tolerances to UV-B irradiation and heat-shock. Furthermore, the MaAreB mutant (ΔMaAreB) exhibited increased sensitivity to CFW (Calcofluor white), decreased cell wall contents (chitin and β-1,3-glucan) and reduced expression levels of some genes related to cell wall integrity, indicating that disruption of MaAreB affected the cell wall integrity. Bioassays showed that the virulence of the ΔMaAreB strain was decreased in topical inoculation but not in intra-hemocoel injection. Consistently, deletion of MaAreB severely impaired the appressorium formation and reduced the turgor pressure of appressorium. These results revealed that MaAreB regulated fungal nitrogen utilization, cell wall integrity and biological control potential, which would contribute to the functional characterization of AreB homologous proteins in other insect fungal pathogens, and even filamentous fungi.

Composition of Transcription Machinery and Its Crosstalk with Nucleoid-Associated Proteins and Global Transcription Factors.

The coordination of bacterial genomic transcription involves an intricate network of interdependent genes encoding nucleoid-associated proteins (NAPs), DNA topoisomerases, RNA polymerase subunits and modulators of transcription machinery. The central element of this homeostatic regulatory system, integrating the information on cellular physiological state and producing a corresponding transcriptional response, is the multi-subunit RNA polymerase (RNAP) holoenzyme. In this review article, we argue that recent observations revealing DNA topoisomerases and metabolic enzymes associated with RNAP supramolecular complex support the notion of structural coupling between transcription machinery, DNA topology and cellular metabolism as a fundamental device coordinating the spatiotemporal genomic transcription. We analyse the impacts of various combinations of RNAP holoenzymes and global transcriptional regulators such as abundant NAPs, on genomic transcription from this viewpoint, monitoring the spatiotemporal patterns of couplons—overlapping subsets of the regulons of NAPs and RNAP sigma factors. We show that the temporal expression of regulons is by and large, correlated with that of cognate regulatory genes, whereas both the spatial organization and temporal expression of couplons is distinctly impacted by the regulons of NAPs and sigma factors. We propose that the coordination of the growth phase-dependent concentration gradients of global regulators with chromosome configurational dynamics determines the spatiotemporal patterns of genomic expression.

Single-Target Regulators Constitute the Minority Group of Transcription Factors in Escherichia coli K-12.

The identification of regulatory targets of all transcription factors (TFs) is critical for understanding the entire network of genome regulation. A total of approximately 300 TFs exist in the model prokaryote Escherichia coli K-12, but the identification of whole sets of their direct targets is impossible with use of in vivo approaches. For this end, the most direct and quick approach is to identify the TF-binding sites in vitro on the genome. We then developed and utilized the gSELEX screening system in vitro for identification of more than 150 E. coli TF-binding sites along the E. coli genome. Based on the number of predicted regulatory targets, we classified E. coli K-12 TFs into four groups, altogether forming a hierarchy ranging from a single-target TF (ST-TF) to local TFs, global TFs, and nucleoid-associated TFs controlling as many as 1,000 targets. Using the collection of purified TFs and a library of genome DNA segments from a single and the same E. coli K-12, we identified here a total of 11 novel ST-TFs, CsqR, CusR, HprR, NorR, PepA, PutA, QseA, RspR, UvrY, ZraR, and YqhC. The regulation of single-target promoters was analyzed in details for the hitherto uncharacterized QseA and RspR. In most cases, the ST-TF gene and its regulatory target genes are adjacently located on the E. coli K-12 genome, implying their simultaneous transfer in the course of genome evolution. The newly identified 11 ST-TFs and the total of 13 hitherto identified altogether constitute the minority group of TFs in E. coli K-12.

Global Gene Expression Profiling and Transcription Factor Network Analysis of Cognitive Aging in Monozygotic Twins.

Cognitive aging is one of the major problems worldwide, especially as people get older. This study aimed to perform global gene expression profiling of cognitive function to identify associated genes and pathways and a novel transcriptional regulatory network analysis to identify important regulons. We performed single transcript analysis on 400 monozygotic twins using an assumption-free generalized correlation coefficient (GCC), linear mixed-effect model (LME) and kinship model and identified six probes (one significant at the standard FDR < 0.05 while the other results were suggestive with 0.18 ≤ FDR ≤ 0.28). We combined the GCC and linear model results to cover diverse patterns of relationships, and meaningful and novel genes like APOBEC3G, H6PD, SLC45A1, GRIN3B, and PDE4D were detected. Our exploratory study showed the downregulation of all these genes with increasing cognitive function or vice versa except the SLC45A1 gene, which was upregulated with increasing cognitive function. Linear models found only H6PD and SLC45A1, the other genes were captured by GCC. Significant functional pathways (FDR < 3.95e-10) such as focal adhesion, ribosome, cysteine and methionine metabolism, Huntington's disease, eukaryotic translation elongation, nervous system development, influenza infection, metabolism of RNA, and cell cycle were identified. A total of five regulons (FDR< 1.3e-4) were enriched in a transcriptional regulatory analysis in which CTCF and REST were activated and SP3, SRF, and XBP1 were repressed regulons. The genome-wide transcription analysis using both assumption-free GCC and linear models identified important genes and biological pathways implicated in cognitive performance, cognitive aging, and neurological diseases. Also, the regulatory network analysis revealed significant activated and repressed regulons on cognitive function.

Investigation of flavonoid expression and metabolite content patterns during seed formation of Artemisia sphaerocephala Krasch.

AbstractFlavonoids are a group of phenolic secondary metabolites in plants that have important physiological, ecological and economic value. In this study, using the desert plant Artemisia sphaerocephala Krasch. as the sample material, the content and components of the total flavonoids in its seeds at seven different developmental stages were determined. In addition, the genes involved in flavonoid metabolism were identified by full-length transcriptome sequencing (third-generation sequencing technology based on PacBio RS II). Their expression levels were analysed by RNA-seq short reading sequencing, to reveal the patterns and regulation mechanisms of flavonoid accumulation during seed development. The key results were as follows: the content of total flavonoids in mature seeds was 15.05 mg g−1, including five subclasses: flavonols, chalcones, flavones, flavanones and proanthocyanidins, among which flavonols accounted for 45.78%. The period of rapid accumulation of flavonoids was 40–70 d following anthesis. The high expression of phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL) and UDP-glucose:flavonoids 3-o-glucosyltransferase (UF3GT) promoted the accumulation of total flavonoids, while the high expression of flavonoids 3′-hydroxylase (F3′H) and flavonols synthase (FLS) made flavanols the main component. Transcription factors such as the MYB-bHLH-WDR (MBW) complex and Selenium-binding protein (SBP) directly regulated the structural genes of flavonoid metabolism, while C2H2-type zinc finger (C2H2), Zinc-finger transcription factor (GATA), Dehydration-responsive element binding (DREB), Global Transcription factor Group E protein (GTE), Trihelix DNA-binding factors (Trihelix) and Phytochrome-interacting factor (PIF) indirectly promoted the synthesis of flavonoids through hormones such as brassinoidsteroids (BRs) and abscisic acid (ABA). These results provided valuable resources for the application of related genes in genetics and breeding.

Transcriptomic Changes Induced by Deletion of Transcriptional Regulator GCR2 on Pentose Sugar Metabolism in Saccharomyces cerevisiae.

Being a microbial host for lignocellulosic biofuel production, Saccharomyces cerevisiae needs to be engineered to express a heterologous xylose pathway; however, it has been challenging to optimize the engineered strain for efficient and rapid fermentation of xylose. Deletion of PHO13 (Δpho13) has been reported to be a crucial genetic perturbation in improving xylose fermentation. A confirmed mechanism of the Δpho13 effect on xylose fermentation is that the Δpho13 transcriptionally activates the genes in the non-oxidative pentose phosphate pathway (PPP). In the current study, we found a couple of engineered strains, of which phenotypes were not affected by Δpho13 (Δpho13-negative), among many others we examined. Genome resequencing of the Δpho13-negative strains revealed that a loss-of-function mutation in GCR2 was responsible for the phenotype. Gcr2 is a global transcriptional factor involved in glucose metabolism. The results of RNA-seq confirmed that the deletion of GCR2 (Δgcr2) led to the upregulation of PPP genes as well as downregulation of glycolytic genes, and changes were more significant under xylose conditions than those under glucose conditions. Although there was no synergistic effect between Δpho13 and Δgcr2 in improving xylose fermentation, these results suggested that GCR2 is a novel knockout target in improving lignocellulosic ethanol production.

Analysis of the proteolytic system of Streptococcus thermophilus strains CS5, CS9, CS18 and CS20

Streptococcus thermophilus is a common milk starter, and the ability to hydrolyse proteins is an important property. The proteolytic systems of S. thermophilus strains CS5, CS9, CS18 and CS20 have been investigated according to their genomes. The growth and proteolytic abilities of these strains have been analysed in combination with gene expressions of both the proteolytic system and the two-component systems (TCSs) of the specified strains. When the strains are grown in milk, prtS is a key determinant in the fermentation rate of PrtS+ strains (CS5, CS18 and CS20), but other genes are activated in the PrtS− strain CS9. The proteolytic system is regulated by the global transcriptional regulatory factor CodY, especially in the strain CS9. Furthermore, the expression of most genes involved in the proteolytic system and the TCSs were positively correlated with proteolytic activity of strains. The relationship between TCSs and proteolytic gene expression should be investigated in the future.

Eliciting the silent lucensomycin biosynthetic pathway in Streptomyces cyanogenus S136 via manipulation of the global regulatory gene adpA

Actinobacteria are among the most prolific sources of medically and agriculturally important compounds, derived from their biosynthetic gene clusters (BGCs) for specialized (secondary) pathways of metabolism. Genomics witnesses that the majority of actinobacterial BGCs are silent, most likely due to their low or zero transcription. Much effort is put into the search for approaches towards activation of silent BGCs, as this is believed to revitalize the discovery of novel natural products. We hypothesized that the global transcriptional factor AdpA, due to its highly degenerate operator sequence, could be used to upregulate the expression of silent BGCs. Using Streptomyces cyanogenus S136 as a test case, we showed that plasmids expressing either full-length adpA or its DNA-binding domain led to significant changes in the metabolome. These were evident as changes in the accumulation of colored compounds, bioactivity, as well as the emergence of a new pattern of secondary metabolites as revealed by HPLC-ESI-mass spectrometry. We further focused on the most abundant secondary metabolite and identified it as the polyene antibiotic lucensomycin. Finally, we uncovered the entire gene cluster for lucensomycin biosynthesis (lcm), that remained elusive for five decades until now, and outlined an evidence-based scenario for its adpA-mediated activation.

Improving Biotransformation Efficiency of Arthrobacter simplex by Enhancement of Cell Stress Tolerance and Enzyme Activity.

Arthrobacter simplex exhibits excellent Δ1-dehydrogenation ability, but the acquisition of the desirable strain is limited due to lacking of comprehensive genetic manipulation. Herein, a promoter collection for fine-tuning gene expression was achieved. Next, the expression level was enhanced and directed evolution of the global transcriptional factor (IrrE) was applied to enhance cell viability in systems containing more substrate and ethanol, thus contributing to higher production. IrrE promotes a stronger antioxidant defense system, more energy generation, and changed signal transduction. Using a stronger promoter, the enzyme activities were boosted but their positive effects on biotransformation performance were inferior to cell stress tolerance when exposed to challenging systems. Finally, an optimal strain was created by collectively reinforcing cell stress tolerance and catalytic enzyme activity, achieving a yield 261.8% higher relative to the initial situation. Our study provided effective methods for steroid-transforming strains with high efficiency.

Patient Stratification of Clear Cell Renal Cell Carcinoma Using the Global Transcription Factor Activity Landscape Derived From RNA-Seq Data.

Clear cell renal cell carcinoma represents the most common type of kidney cancer. Precision medicine approach to ccRCC requires an accurate stratification of patients that can predict prognosis and guide therapeutic decision. Transcription factors are implicated in the initiation and progression of human carcinogenesis. However, no comprehensive analysis of transcription factor activity has been proposed so far to realize patient stratification. Here we propose a novel approach to determine the subtypes of ccRCC patients based on global transcription factor activity landscape. Using the TCGA cohort dataset, we identified different subtypes that have distinct up-regulated biomarkers and altered biological pathways. More important, this subtype information can be used to predict the overall survival of ccRCC patients. Our results suggest that transcription factor activity can be harnessed to perform patient stratification.

Variations of Indole Metabolites and NRPS-PKS Loci in Two Different Virulent Strains of Xenorhabdus hominickii.

Xenorhabdus hominickii ANU1 is known to be an entomopathogenic bacterium symbiotic to nematode Steinernema monticolum. Another bacterial strain X. hominickii DY1 was isolated from a local population of S. monticolum. This bacterial strain X. hominickii DY1 was found to exhibit high insecticidal activities against lepidopteran and coleopteran species after hemocoelic injection. However, these two X. hominickii strains exhibited significant variations in insecticidal activities, with ANU1 strain being more potent than DY1 strain. To clarify their virulence difference, bacterial culture broths of these two strains were compared for secondary metabolite compositions. GC-MS analysis revealed that these two strains had different compositions, including pyrrolopyrazines, piperazines, cyclopeptides, and indoles. Some of these compounds exhibited inhibitory activities against phospholipase A2 to block eicosanoid biosynthesis and induce significant immunosuppression. They also exhibited significant insecticidal activities after oral feeding, with indole derivatives being the most potent. More kinds of indole derivatives were detected in the culture broth of ANU1 strain. To investigate variations in regulation of secondary metabolite production, expression level of leucine-responsive regulatory protein (Lrp), a global transcription factor, was compared. ANU1 strain exhibited significantly lower Lrp expression level than DY1 strain. To assess genetic variations associated with secondary metabolite synthesis, bacterial loci encoding non-ribosomal protein synthase and polyketide synthase (NRPS-PKS) were compared. Three NRPS and four PKS loci were predicted from the genome of X. hominickii. The two bacterial strains exhibited genetic variations (0.12∼0.67%) in amino acid sequences of these NRPS-PKS. Most NRPS-PKS genes exhibited high expression peaks at stationary phase of bacterial growth. However, their expression levels were significantly different between the two strains. These results suggest that differential virulence of the two bacterial strains is caused by the difference in Lrp expression level, leading to difference in the production of indole compounds and other NRPS-PKS-associated secondary metabolites.

Elevating NagZ Improves Resistance to β-Lactam Antibiotics via Promoting AmpC β-Lactamase in Enterobacter cloacae.

Enterobacter cloacae complex (ECC), one of the most common opportunistic pathogens causing multiple infections in human, is resistant to β-lactam antibiotics mainly due to its highly expressed chromosomal AmpC β-lactamase. It seems that regulation of chromosomal AmpC β-lactamase is associated with peptidoglycan recycling. However, underlying mechanisms are still poorly understood. In this study, we confirmed that NagZ, a glycoside hydrolase participating in peptidoglycan recycling in Gram-negative bacteria, plays a crucial role in developing resistance of E. cloacae (EC) to β-lactam antibiotics by promoting expression of chromosomal AmpC β-lactamase. Our data shows that NagZ was significantly up-regulated in resistant EC (resistant to at least one type of the third or fourth generation cephalosporins) compared to susceptible EC (susceptible to all types of the third and fourth generation cephalosporins). Similarly, the expression and β-lactamase activity of ampC were markedly enhanced in resistant EC. Moreover, ectopic expression of nagZ enhanced ampC expression and resistance to β-lactam antibiotics in susceptible EC. To further understand functions of NagZ in β-lactam resistance, nagZ-knockout EC model (ΔnagZ EC) was constructed by homologous recombination. Conversely, ampC mRNA and protein levels were down-regulated, and resistance to β-lactam antibiotics was attenuated in ΔnagZ EC, while specific complementation of nagZ was able to rescue ampC expression and resistance in ΔnagZ EC. More interestingly, NagZ and its hydrolyzates 1,6-anhydromuropeptides (anhMurNAc) could induce the expression of other target genes of AmpR (a global transcriptional factor), which suggested that the promotion of AmpC by NagZ is mediated AmpR activated by anhMurNAc in EC. In conclusion, these findings provide new elements for a better understanding of resistance in EC, which is crucial for the identification of novel potential drug targets.

Ferric uptake regulator (Fur) reversibly binds a [2Fe-2S] cluster to sense intracellular iron homeostasis in Escherichia coli

The ferric uptake regulator (Fur) is a global transcription factor that regulates intracellular iron homeostasis in bacteria. The current hypothesis states that when the intracellular "free" iron concentration is elevated, Fur binds ferrous iron, and the iron-bound Fur represses the genes encoding for iron uptake systems and stimulates the genes encoding for iron storage proteins. However, the "iron-bound" Fur has never been isolated from any bacteria. Here we report that the Escherichia coli Fur has a bright red color when expressed in E. coli mutant cells containing an elevated intracellular free iron content because of deletion of the iron-sulfur cluster assembly proteins IscA and SufA. The acid-labile iron and sulfide content analyses in conjunction with the EPR and Mössbauer spectroscopy measurements and the site-directed mutagenesis studies show that the red Fur protein binds a [2Fe-2S] cluster via conserved cysteine residues. The occupancy of the [2Fe-2S] cluster in Fur protein is ∼31% in the E. coli iscA/sufA mutant cells and is decreased to ∼4% in WT E. coli cells. Depletion of the intracellular free iron content using the membrane-permeable iron chelator 2,2´-dipyridyl effectively removes the [2Fe-2S] cluster from Fur in E. coli cells, suggesting that Fur senses the intracellular free iron content via reversible binding of a [2Fe-2S] cluster. The binding of the [2Fe-2S] cluster in Fur appears to be highly conserved, because the Fur homolog from Hemophilus influenzae expressed in E. coli cells also reversibly binds a [2Fe-2S] cluster to sense intracellular iron homeostasis.

A composite filter for low FDR of protein-protein interactions detected by in vivo cross-linking

In vivo chemical cross-linking combined with LCMSMS of digested extracts (in vivo CX-MS) can reveal stable and dynamic protein-protein interactions at proteome-wide scale and at peptide level. In vivo CX-MS requires a membrane permeable and cleavable cross-linker and a fast and sensitive search engine to identify the linked peptides. Here we explore the use of the search engine pLink 2 to identify cross-links induced in exponentially growing Bacillus subtilis cells treated in culture with bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). Cross-linked peptide pairs were identified by pLink 2 in very short time at an overall FDR of <5%. To also obtain a FDR <5% for non-redundant inter-protein cross-linked peptide pairs additional threshold values were applied for matched fragment intensity and for the numbers of unambiguous y and b ions assigned to both composite peptides. Also the mass- and charge-dependent retention times of target peptides purified by diagonal strong cation exchange chromatography were used as a criterion to distinguish true from false positives. After application of the composite filter new protein-protein interactions were revealed among others between the global transcriptional repressor AbrB and elongation factor Tu and between the essential protein YlaN of unknown function and the ferric uptake repressor Fur. SignificanceImportant for reliable identification of PPIs by chemical cross-linking in vivo is a low FDR of non-redundant inter-protein peptide pairs. Here we describe how to recognize the presence of spurious interactions in a dataset of cross-linked peptide pairs enriched by 2D strong cation exchange chromatography and identified by LCMSMS by taking into account chromatographic behavior of cross-linked peptide pairs and protein abundance of corresponding peptides. Based on these criteria we assessed that the FDR of the fraction of non-redundant inter-protein cross-linked peptide pairs was approx. 20–25% by interrogating an entire species specific database at an overall FDR of 5% or 0.1% with a search engine that otherwise scores best in sensitivity among other search engines. We have defined a composite filter to decrease this high FDR of inter-protein cross-linked peptide pairs to only about 2%.