Brighter Fluorescent Derivatives of UTI89 Utilizing a Monomeric vGFP.

Fluorescent proteins, especially green fluorescent protein (GFP), have been instrumental in understanding urinary tract infection pathogenesis by uropathogenic Escherichia coli (UPEC). We have used a recently developed GFP variant, vsfGFP-9, to create new plasmid- and chromosome-based GFP derivatives of the UPEC strain UTI89. The vsfGFP-9 strains are nearly 10× brighter with no in vitro growth or in vivo virulence defects compared to previously reported GFP-expressing UTI89 strains. The chromosomal vsfGFP-9 strain is equivalent to the wild type UTI89 during in vivo UTI, while both plasmid GFP constructs have an equivalent virulence defect compared to non-plasmid carrying UTI89. These new vsfGFP-9 expressing strains should be useful for further studies of the pathogenesis of UTI89, and similar strategies can be used to create improved fluorescent derivatives of other UPEC strains.

Rational Structure-Based Design of Bright GFP-Based Complexes with Tunable Dimerization.

Fluorescent proteins are transformative tools; thus, any brightness increase is a welcome improvement. We invented the "vGFP strategy" based on structural analysis of GFP bound to a single-domain antibody, predicting tunable dimerization, enhanced brightness (ca. 50%), and improved pH resistance. We verified all of these predictions using biochemistry, crystallography, and single-molecule studies. We applied the vsfGFP proteins in three diverse scenarios: single-step immunofluorescence in vitro (3× brighter due to dimerization); expression in bacteria and human cells in vivo (1.5× brighter); and protein fusions showing better pH resistance in human cells in vivo. The vGFP strategy thus allows upgrading of existing applications, is applicable to other fluorescent proteins, and suggests a method for tuning dimerization of arbitrary proteins and optimizing protein properties in general.

The condition-dependent transcriptional landscape of Burkholderia pseudomallei.

Burkholderia pseudomallei (Bp), the causative agent of the often-deadly infectious disease melioidosis, contains one of the largest prokaryotic genomes sequenced to date, at 7.2 Mb with two large circular chromosomes (1 and 2). To comprehensively delineate the Bp transcriptome, we integrated whole-genome tiling array expression data of Bp exposed to >80 diverse physical, chemical, and biological conditions. Our results provide direct experimental support for the strand-specific expression of 5,467 Sanger protein-coding genes, 1,041 operons, and 766 non-coding RNAs. A large proportion of these transcripts displayed condition-dependent expression, consistent with them playing functional roles. The two Bp chromosomes exhibited dramatically different transcriptional landscapes--Chr 1 genes were highly and constitutively expressed, while Chr 2 genes exhibited mosaic expression where distinct subsets were expressed in a strongly condition-dependent manner. We identified dozens of cis-regulatory motifs associated with specific condition-dependent expression programs, and used the condition compendium to elucidate key biological processes associated with two complex pathogen phenotypes--quorum sensing and in vivo infection. Our results demonstrate the utility of a Bp condition-compendium as a community resource for biological discovery. Moreover, the observation that significant portions of the Bp virulence machinery can be activated by specific in vitro cues provides insights into Bp's capacity as an "accidental pathogen", where genetic pathways used by the bacterium to survive in environmental niches may have also facilitated its ability to colonize human hosts.

Deciphering transcription factor binding patterns from genome-wide high density ChIP-chip tiling array data.

BACKGROUND: The binding events of DNA-interacting proteins and their patterns can be extensively characterized by high density ChIP-chip tiling array data. The characteristics of the binding events could be different for different transcription factors. They may even vary for a given transcription factor among different interaction loci. The knowledge of binding sites and binding occupancy patterns are all very useful to understand the DNA-protein interaction and its role in the transcriptional regulation of genes. RESULTS: In the view of the complexity of the DNA-protein interaction and the opportunity offered by high density tiled ChIP-chip data, we present a statistical procedure which focuses on identifying the interaction signal regions instead of signal peaks using moving window binomial testing method and deconvolving the patterns of interaction using peakedness and skewness scores. We analyzed ChIP-chip data of 4 different DNA interacting proteins including transcription factors and RNA polymerase in fission yeast using our procedure. Our analysis revealed the variation of binding patterns within and across different DNA interacting proteins. We present their utility in understanding transcriptional regulation from ChIP-chip data. CONCLUSIONS: Our method can successfully detect the signal regions and characterize the binding patterns in ChIP-chip data which help appropriate analysis of the ChIP-chip data.

Deconvolution of chromatin immunoprecipitation-microarray (ChIP-chip) analysis of MBF occupancies reveals the temporal recruitment of Rep2 at the MBF target genes.

MBF (or DSC1) is known to regulate transcription of a set of G(1)/S-phase genes encoding proteins involved in regulation of DNA replication. Previous studies have shown that MBF binds not only the promoter of G(1)/S-phase genes, but also the constitutive genes; however, it was unclear if the MBF bindings at the G(1)/S-phase and constitutive genes were mechanistically distinguishable. Here, we report a chromatin immunoprecipitation-microarray (ChIP-chip) analysis of MBF binding in the Schizosaccharomyces pombe genome using high-resolution genome tiling microarrays. ChIP-chip analysis indicates that the majority of the MBF occupancies are located at the intragenic regions. Deconvolution analysis using Rpb1 ChIP-chip results distinguishes the Cdc10 bindings at the Rpb1-poor loci (promoters) from those at the Rpb1-rich loci (intragenic sequences). Importantly, Res1 binding at the Rpb1-poor loci, but not at the Rpb1-rich loci, is dependent on the Cdc10 function, suggesting a distinct binding mechanism. Most Cdc10 promoter bindings at the Rpb1-poor loci are associated with the G(1)/S-phase genes. While Res1 or Res2 is found at both the Cdc10 promoter and intragenic binding sites, Rep2 appears to be absent at the Cdc10 promoter binding sites but present at the intragenic sites. Time course ChIP-chip analysis demonstrates that Rep2 is temporally accumulated at the coding region of the MBF target genes, resembling the RNAP-II occupancies. Taken together, our results show that deconvolution analysis of Cdc10 occupancies refines the functional subset of genomic binding sites. We propose that the MBF activator Rep2 plays a role in mediating the cell cycle-specific transcription through the recruitment of RNAP-II to the MBF-bound G(1)/S-phase genes.

Genomic binding profiling of the fission yeast stress-activated MAPK Sty1 and the bZIP transcriptional activator Atf1 in response to H2O2.

BACKGROUND: The evolutionally conserved MAPK Sty1 and bZIP transcriptional activator Atf1 are known to play a pivotal role in response to the reactive oxygen species in S. pombe. However, it is unclear whether all of the H(2)O(2)-induced genes are directly regulated by the Sty1-Atf1 pathway and involved in growth fitness under H(2)O(2)-induced stress conditions. METHODOLOGY/PRINCIPAL FINDINGS: Here we present the study on ChIP-chip mapping of the genomic binding sites for Sty1, Atf1, and the Atf1's binding partner Pcr1; the genome-wide transcriptional profiling of the atf1 and pcr1 strains in response to H(2)O(2); and the phenotypic assessment of approximately 90 Atf1/Pcr1-bound or unbound genes for growth fitness under H(2)O(2) conditions. ChIP-chip analysis shows that Atf1 and Pcr1 binding sites are overlapped in the genome and constitutively present before H(2)O(2) stress. On the other hand, Sty1 recruitment primarily occurs at the Atf1/Pcr1 binding sites and is induced by H(2)O(2). We found that Atf1/Pcr1 is clearly responsible for the high-level transcriptional response to H(2)O(2). Furthermore, phenotypic assessment indicates that among the H(2)O(2)-induced genes, Atf1/Pcr1-bound genes exhibit a higher likelihood of functional requirement for growth fitness under the stress condition than the Atf1/Pcr1-unbound genes do. Notably, we found that the Atf1/Pcr1-bound genes regardless of their responsiveness to H(2)O(2) show a high probability of requirement for growth fitness. CONCLUSION/SIGNIFICANCE: Together, our analyses on global mapping of protein binding sites, genome-wide transcriptional profiling, and phenotypic assessment provide insight into mechanisms for global transcriptional regulation by the Sty1-Atf1 pathway in response to H(2)O(2)-induced reactive oxygen species.

Genome-wide estimation of firing efficiencies of origins of DNA replication from time-course copy number variation data.

BACKGROUND: DNA replication is a fundamental biological process during S phase of cell division. It is initiated from several hundreds of origins along whole chromosome with different firing efficiencies (or frequency of usage). Direct measurement of origin firing efficiency by techniques such as DNA combing are time-consuming and lack the ability to measure all origins. Recent genome-wide study of DNA replication approximated origin firing efficiency by indirectly measuring other quantities related to replication. However, these approximation methods do not reflect properties of origin firing and may lead to inappropriate estimations. RESULTS: In this paper, we develop a probabilistic model - Spanned Firing Time Model (SFTM) to characterize DNA replication process. The proposed model reflects current understandings about DNA replication. Origins in an individual cell may initiate replication randomly within a time window, but the population average exhibits a temporal program with some origins replicated early and the others late. By estimating DNA origin firing time and fork moving velocity from genome-wide time-course S-phase copy number variation data, we could estimate firing efficiency of all origins. The estimated firing efficiency is correlated well with the previous studies in fission and budding yeasts. CONCLUSIONS: The new probabilistic model enables sensitive identification of origins as well as genome-wide estimation of origin firing efficiency. We have successfully estimated firing efficiencies of all origins in S. cerevisiae, S. pombe and human chromosomes 21 and 22.

Transformation/transcription domain-associated protein (TRRAP)-mediated regulation of Wee1.

The G2 DNA damage checkpoint inhibits Cdc2 and mitotic entry through the dual regulation of Wee1 and Cdc25 by the Chk1 effector kinase. Upregulation of Chk1 by mutation or overexpression bypasses the requirement for upstream regulators or DNA damage to promote a G2 cell cycle arrest. We screened in fission yeast for mutations that rendered cells resistant to overexpressed chk1(+). We identified a mutation in tra1, which encodes one of two homologs of transformation/transcription domain-associated protein (TRRAP), an ATM/R-related pseudokinase that scaffolds several histone acetyltransferase (HAT) complexes. Inhibition of histone deacetylases reverts the resistance to overexpressed chk1(+), suggesting this phenotype is due to a HAT activity, although expression of checkpoint and cell cycle genes is not greatly affected. Cells with mutant or deleted tra1 activate Chk1 normally and are checkpoint proficient. However, these cells are semi-wee even when overexpressing chk1(+) and accumulate inactive Wee1 protein. The changed division response (Cdr) kinases Cdr1 and Cdr2 are negative regulators of Wee1, and we show that they are required for the Tra1-dependent alterations to Wee1 function. This identifies Tra1 as another component controlling the timing of entry into mitosis via Cdc2 activation.

A Cds1-mediated checkpoint protects the MBF activator Rep2 from ubiquitination by anaphase-promoting complex/cyclosome-Ste9 at S-phase arrest in fission yeast.

Transcription of the MluI cell cycle box (MCB) motif-containing genes at G(1) phase is regulated by the MCB-binding factors (MBF) (also called DSC1) in Schizosaccharomyces pombe. Upon S-phase arrest, the MBF transcriptional activity is induced through the accumulation of the MBF activator Rep2. In this study, we show that the turnover of Rep2 is attributable to ubiquitin-mediated proteolysis. Levels of Rep2 oscillate during the cell cycle, with a peak at G(1) phase, coincident with the MBF activity. Furthermore, we show that Rep2 ubiquitination requires the function of the E3 ligase anaphase-promoting complex/cyclosome (APC/C). Ste9 can be phosphorylated by the checkpoint kinase Cds1 in vitro, and its inhibition/phosphorylation at S-phase arrest is dependent on the function of Cds1. Our data indicate that the Cds1-dependent stabilization of Rep2 is achieved through the inhibition/phosphorylation of APC/C-Ste9 at the onset of S-phase arrest. Stabilization of Rep2 is important for stimulating transcription of the MBF-dependent genes to ensure a sufficient supply of proteins essential for cell recovery from S-phase arrest. We propose that oscillation of Rep2 plays a role in regulation of periodic transcription of the MBF-dependent genes during cell cycle progression.

Localization of the antigenic sites of newcastle disease virus nucleocapsid using a panel of monoclonal antibodies.

A panel of six monoclonal antibodies (mAbs) against the nucleocapsid (NP) protein of Newcastle disease virus (NDV) was produced by immunization of Balb/c mice with purified recombinant NP protein. Western Blot analysis showed that all the mAbs recognized linearized NP epitopes. Three different NP antigenic sites were identified using deleted truncated NP mutants purified from Escherichia coli. One of the antigenic sites was located at the C-terminal end (residues 441 to 489) of the NP protein. Two other antigenic sites were located within the N-terminal end (residues 26-121 and 122-375). This study demonstrates that the N- and C-terminal ends of the NP proteins are responsible in eliciting immune response, thus it is most likely that these ends are exposed on the NP.

Adaptive expression responses in the Pol-gamma null strain of S. pombe depleted of mitochondrial genome.

BACKGROUND: DNA polymerase gamma(Pol-gamma) has been shown to be essential for maintenance of the mitochondrial genome (mtDNA) in the petite-positive budding yeast Saccharomyces cerevisiae. Budding yeast cells lacking mitochondria exhibit a slow-growing or petite-colony phenotype. Petite strains fail to grow on non-fermentable carbon sources. However, it is not clear whether the Pol-gamma is required for mtDNA maintenance in the petite-negative fission yeast Schizosaccharomyces pombe. RESULTS: We show that disruption of the nuclear gene pog1+ that encodes Pol-gamma is sufficient to deplete mtDNA in S. pombe. Cells bearing pog1Delta allele require substantial growth periods to form petite colonies. Mitotracker assays indicate that pog1Delta cells are defective in mitochondrial function and EM analyses suggest that pog1Delta cells lack normal mitochondrial structures. Depletion of mtDNA in pog1Delta cells is evident from quantitative real-time PCR assays. Genome-wide expression profiles of pog1Delta and other mtDNA-less cells reveal that many genes involved in response to stimulus, energy derivation by oxidation of organic compounds, cellular carbohydrate metabolism, and energy reserve metabolism are induced. Conversely, many genes encoding proteins involved in amino acid metabolism and oxidative phosphorylation are repressed. CONCLUSION: By showing that Pol-gamma is essential for mtDNA maintenance and disruption of pog1+ alters the genome-wide expression profiles, we demonstrated that cells lacking mtDNA exhibit adaptive nuclear gene expression responses in the petite-negative S. pombe.

Global profiling of DNA replication timing and efficiency reveals that efficient replication/firing occurs late during S-phase in S. pombe.

BACKGROUND: During S. pombe S-phase, initiation of DNA replication occurs at multiple sites (origins) that are enriched with AT-rich sequences, at various times. Current studies of genome-wide DNA replication profiles have focused on the DNA replication timing and origin location. However, the replication and/or firing efficiency of the individual origins on the genomic scale remain unclear. METHODOLOGY/PRINCIPAL FINDINGS: Using the genome-wide ORF-specific DNA microarray analysis, we show that in S. pombe, individual origins fire with varying efficiencies and at different times during S-phase. The increase in DNA copy number plotted as a function of time is approximated to the near-sigmoidal model, when considering the replication start and end timings at individual loci in cells released from HU-arrest. Replication efficiencies differ from origin to origin, depending on the origin's firing efficiency. We have found that DNA replication is inefficient early in S-phase, due to inefficient firing at origins. Efficient replication occurs later, attributed to efficient but late-firing origins. Furthermore, profiles of replication timing in cds1Delta cells are abnormal, due to the failure in resuming replication at the collapsed forks. The majority of the inefficient origins, but not the efficient ones, are found to fire in cds1Delta cells after HU removal, owing to the firing at the remaining unused (inefficient) origins during HU treatment. CONCLUSIONS/SIGNIFICANCE: Taken together, our results indicate that efficient DNA replication/firing occurs late in S-phase progression in cells after HU removal, due to efficient late-firing origins. Additionally, checkpoint kinase Cds1p is required for maintaining the efficient replication/firing late in S-phase. We further propose that efficient late-firing origins are essential for ensuring completion of DNA duplication by the end of S-phase.

Modulation of cell cycle-specific gene expressions at the onset of S phase arrest contributes to the robust DNA replication checkpoint response in fission yeast.

Fission yeast replication checkpoint kinases Rad3p and Cds1p are essential for maintaining cell viability after transient treatment with hydroxyurea (HU), an agent that blocks DNA replication. Although current studies have focused on the cyclin-dependent protein kinase Cdc2p that is regulated by these checkpoint kinases, other aspects of their functions at the onset of S phase arrest have not been fully understood. In this study, we use genome-wide DNA microarray analyses to show that HU-induced change of expression profiles in synchronized G(2) cells occurs specifically at the onset of S phase arrest. Induction of many core environmental stress response genes and repression of ribosomal genes happen during S phase arrest. Significantly, peak expression level of the MluI-like cell cycle box (MCB)-cluster (G(1)) genes is maintained at the onset of S phase arrest in a Rad3p- and Cds1p-dependent manner. Expression level maintenance of the MCB-cluster is mediated through the accumulation of Rep2p, a putative transcriptional activator of the MBF complex. Conversely, the FKH-cluster (M) genes are repressed during the onset of S phase arrest in a Rad3p-dependent manner. Repression of the FKH-cluster genes is mediated through the decreased levels of one of the putative forkhead transcription factors, Sep1p, but not Fkh2p. Together, our results demonstrate that Rad3p and Cds1p modulate transcriptional response during the onset of S phase arrest.

Purification and characterization of Nipah virus nucleocapsid protein produced in insect cells.

The nucleocapsid (N) protein of Nipah virus (NiV) is a major constituent of the viral proteins which play a role in encapsidation, regulating the transcription and replication of the viral genome. To investigate the use of a fusion system to aid the purification of the recombinant N protein for structural studies and potential use as a diagnostic reagent, the NiV N gene was cloned into the pFastBacHT vector and the His-tagged fusion protein was expressed in Sf9 insect cells by recombinant baculovirus. Western blot analysis of the recombinant fusion protein with anti-NiV antibodies produced a band of approximately 62 kDa. A time course study showed that the highest level of expression was achieved after 3 days of incubation. Electron microscopic analysis of the NiV recombinant N fusion protein purified on a nickel-nitrilotriacetic acid resin column revealed different types of structures, including spherical, ring-like, and herringbone-like particles. The light-scattering measurements of the recombinant N protein also confirmed the polydispersity of the sample with hyrdrodynamic radii of small and large types. The optical density spectra of the purified recombinant fusion protein revealed a high A(260)/A(280) ratio, indicating the presence of nucleic acids. Western blotting and enzyme-linked immunosorbent assay results showed that the recombinant N protein exhibited the antigenic sites and conformation necessary for specific antigen-antibody recognition.

Purification of the extra-cellular domain of Nipah virus glycoprotein produced in Escherichia coli and possible application in diagnosis.

The glycoprotein (G) of Nipah virus (NiV) is important for virus infectivity and induction of the protective immunity. In this study, the extra-cellular domain of NiV G protein was fused with hexahistidine residues at its N-terminal end and expressed in Escherichia coli. The expression under transcriptional regulation of T7 promoter yielded insoluble protein aggregates in the form of inclusion bodies. The inclusion bodies were solubilized with 8 M urea and the protein was purified to homogeneity under denaturing conditions using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The denatured protein was renatured by gradual removal of the urea. Light scattering analysis of the purified protein showed primarily monodispersity. The purified protein showed significant reactivity with the antibodies present in the sera of NiV-infected swine, as demonstrated in Western blot analysis and enzyme-linked immunosorbent assay (ELISA). Taken together, the data indicate the potential usefulness of the purified G protein for structural or functional studies and the development of immunoassay for detection of the NiV antibodies.

Identification of epitopes in the nucleocapsid protein of Nipah virus using a linear phage-displayed random peptide library.

A random peptide library of heptamers displayed on the surface of M13 bacteriophage was used to identify specific epitopes of antibodies in pooled sera of swine naturally infected by Nipah virus. The selected heptapeptides were aligned with protein sequences of Nipah virus and several putative epitopes were identified within the nucleocapsid protein. A total of 41 of 60 (68%) selected phage clones had inserts resembling a region with the sequence SNRTQGE, located at the C-terminal end (amino acids 503-509) of the nucleocapsid protein. The binding of antibodies in the swine and human antisera to the phage clone was inhibited by a synthetic peptide corresponding to this region. Epitopes identified by phage display are consistent with the predicted antigenic sites for the Nipah virus nucleocapsid protein. The selected phage clone used as a coating antigen discriminated swine and human Nipah virus sera-positive from sera-negative samples exhibiting characteristics, which might be attractive for diagnostic tests.

Solubility, immunogenicity and physical properties of the nucleocapsid protein of Nipah virus produced in Escherichia coli.

The nucleocapsid (N) protein of Nipah virus (NiV) can be produced in three Escherichia coli strains [TOP10, BL21(DE3) and SG935] under the control of trc promoter. However, most of the product existed in the form of insoluble inclusion bodies. There was no improvement in the solubility of the product when this protein was placed under the control of T7 promoter. However, the solubility of the N protein was significantly improved by lowering the growth temperature of E. coli BL21(DE3) cell cultures. Solubility analysis of N- and C-terminally deleted mutants revealed that the full-length N protein has the highest solubility. The soluble N protein could be purified efficiently by sucrose gradient centrifugation and nickel affinity chromatography. Electron microscopic analysis of the purified product revealed that the N protein assembled into herringbone-like particles of different lengths. The C-terminal end of the N protein contains the major antigenic region when probed with antisera from humans and pigs infected naturally.

Existence of two emm-like "mrp" and "emm" genes in the mga regulon of the Streptococcus pyogenes strain ST4547.

Streptococcus pyogenes ST4547 is an opacity factor negative strain, which has been recently reported as a new emm type from Malaysia. Nucleotide sequencing of the mga regulon of this strain showed the existence of two emm-like genes. The emm gene located upstream of the scpA gene comprises 1305 nucleotides encoding the putative precursor M protein of 435 amino acids in length with an M(r) of 49 kDa. or a predicted mature protein of 394 amino acids with an M(r) of 44.8 kDa. Another gene mrpST4547 was located upstream of the emm gene and downstream of the mga gene. The sequence of this mrp gene comprises 1167 nucleotides encoding a predicted protein of 388 amino acids in length with an M(r) of 42.2 kDa. or a predicted mature protein of 347 amino acids with an M(r) of 37.9 kDa. The mga regulon of strain ST4547 has a mosaic structure comprising segments, which originated from different OF positive and OF negative strains. The sequences flanking the hyper-variable and C repeats of the emmST4547 gene showed high similarity to corresponding regions in the mga regulon of OF positive strains notably M15, M4, M22 and M50. In contrast, the sequence within the hyper-variable and C repeat regions of the emmST4547 gene revealed high similarity to equivalent regions in the OF negative strains. These data indicates that horizontal transfer of emm-like gene could have occurred between OF positive and OF negative strains resulting in architectural divergence in the mga regulon.