ABSTRACT
The near-minimal bacterium Mesoplasma florum is an interesting model for synthetic genomics and systems biology due to its small genome (~ 800 kb), fast growth rate, and lack of pathogenic potential. However, fundamental aspects of its biology remain largely unexplored. Here, we report a broad yet remarkably detailed characterization of M. florum by combining a wide variety of experimental approaches. We investigated several physical and physiological parameters of this bacterium, including cell size, growth kinetics, and biomass composition of the cell. We also performed the first genome-wide analysis of its transcriptome and proteome, notably revealing a conserved promoter motif, the organization of transcription units, and the transcription and protein expression levels of all protein-coding sequences. We converted gene transcription and expression levels into absolute molecular abundances using biomass quantification results, generating an unprecedented view of the M. florum cellular composition and functions. These characterization efforts provide a strong experimental foundation for the development of a genome-scale model for M. florum and will guide future genome engineering endeavors in this simple organism.
Subject(s)
Entomoplasmataceae/physiology , Base Sequence , Biomass , Entomoplasmataceae/genetics , Entomoplasmataceae/growth & development , Entomoplasmataceae/ultrastructure , Gene Expression Regulation, Bacterial , Genome, Bacterial , Intracellular Space/metabolism , Kinetics , Macromolecular Substances/metabolism , Nucleic Acids/metabolism , Open Reading Frames/genetics , Promoter Regions, Genetic/genetics , Ribosomes/metabolism , Temperature , Transcription Initiation Site , Transcription, GeneticABSTRACT
The mobilizable resistance island Salmonella genomic island 1 (SGI1) is specifically mobilized by IncA and IncC conjugative plasmids. SGI1, its variants and IncC plasmids propagate multidrug resistance in pathogenic enterobacteria such as Salmonella enterica serovars and Proteus mirabilis. SGI1 modifies and uses the conjugation apparatus encoded by the helper IncC plasmid, thus enhancing its own propagation. Remarkably, although SGI1 needs a coresident IncC plasmid to excise from the chromosome and transfer to a new host, these elements have been reported to be incompatible. Here, the stability of SGI1 and its helper IncC plasmid, each expressing a different fluorescent reporter protein, was monitored using fluorescence-activated cell sorting (FACS). Without selective pressure, 95% of the cells segregated into two subpopulations containing either SGI1 or the helper plasmid. Furthermore, FACS analysis revealed a high level of SGI1-specific fluorescence in IncC+ cells, suggesting that SGI1 undergoes active replication in the presence of the helper plasmid. SGI1 replication was confirmed by quantitative PCR assays, and extraction and restriction of its plasmid form. Deletion of genes involved in SGI1 excision from the chromosome allowed a stable coexistence of SGI1 with its helper plasmid without selective pressure. In addition, deletion of S003 (rep) or of a downstream putative iteron-based origin of replication, while allowing SGI1 excision, abolished its replication, alleviated the incompatibility with the helper plasmid and enabled its cotransfer to a new host. Like SGI1 excision functions, rep expression was found to be controlled by AcaCD, the master activator of IncC plasmid transfer. Transient SGI1 replication seems to be a key feature of the life cycle of this family of genomic islands. Sequence database analysis revealed that SGI1 variants encode either a replication initiator protein with a RepA_C domain, or an alternative replication protein with N-terminal replicase and primase C terminal 1 domains.
Subject(s)
Bacterial Proteins/genetics , Conjugation, Genetic/genetics , Genomic Islands/genetics , Phosphoproteins/genetics , Plasmids/genetics , Anti-Bacterial Agents/pharmacology , Chromosomes/drug effects , Chromosomes/genetics , DNA Helicases/genetics , Drug Resistance, Multiple, Bacterial/drug effects , Drug Resistance, Multiple, Bacterial/genetics , Plasmids/drug effects , Proteus mirabilis/genetics , Salmonella enterica/genetics , Trans-Activators/geneticsABSTRACT
Details of the "Trojan Horse" mechanism by which Zika virus (ZIKV) crosses the blood-brain barrier (BBB) remain unclear. However, the migration of ZIKV-infected monocytes to the brain is thought to be dependent on both pattern-recognition and chemokine receptors. In this study, we investigated whether the migration of ZIKV-infected MonoMac-1 (MM-1) cells through the BBB is dependent on chemokine receptor 7 (CCR7) and receptor for advanced glycation end (RAGE); we also determined whether high mobility group box protein 1 (HMGB1) could facilitate the permeabilization of endothelial cells. We demonstrated that ZIKV infects MM-1 cells, leading to milieu accumulation of HMGB1. Our results suggest that HMGB1 is involved in the dysregulation of primary human brain microvascular endothelial cell junction markers. Our results also indicate that the migration of ZIKV-infected monocytes is dependent on chemokine ligand 19 (CCL19), the natural ligand of CCR7, in conditions recapitulating inflammation. RAGE-dependent migration of ZIKV-infected cells declined during transmigration assays in the presence of RAGE receptor antagonist FPS-ZM1. Understanding the molecular role of monocyte trafficking during ZIKV infections could facilitate the development of new therapeutic strategies to prevent the deleterious consequences of ZIKV neuroinfection.
Subject(s)
Antigens, Neoplasm/physiology , Blood-Brain Barrier/physiology , Chemokine CCL19/physiology , HMGB1 Protein/physiology , Mitogen-Activated Protein Kinases/physiology , Monocytes/physiology , Receptors, CCR7/physiology , Zika Virus Infection , Animals , Cell Line , Cell Movement , Chlorocebus aethiops , Endothelial Cells/physiology , Humans , Monocytes/virology , Zika VirusABSTRACT
The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (γ-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.
Subject(s)
DNA Damage/radiation effects , DNA/radiation effects , Fluorescent Antibody Technique/methods , Low-Level Light Therapy/methods , Microscopy, Confocal/methods , HumansABSTRACT
Colonization of plant roots by Bacillus subtilis is mutually beneficial to plants and bacteria. Plants can secrete up to 30% of their fixed carbon via root exudates, thereby feeding the bacteria, and in return the associated B. subtilis bacteria provide the plant with many growth-promoting traits. Formation of a biofilm on the root by matrix-producing B. subtilis is a well-established requirement for long-term colonization. However, we observed that cells start forming a biofilm only several hours after motile cells first settle on the plant. We also found that intact chemotaxis machinery is required for early root colonization by B. subtilis and for plant protection. Arabidopsis thaliana root exudates attract B. subtilis in vitro, an activity mediated by the two characterized chemoreceptors, McpB and McpC, as well as by the orphan receptor TlpC. Nonetheless, bacteria lacking these chemoreceptors are still able to colonize the root, suggesting that other chemoreceptors might also play a role in this process. These observations suggest that A. thaliana actively recruits B. subtilis through root-secreted molecules, and our results stress the important roles of B. subtilis chemoreceptors for efficient colonization of plants in natural environments. These results demonstrate a remarkable strategy adapted by beneficial rhizobacteria to utilize carbon-rich root exudates, which may facilitate rhizobacterial colonization and a mutualistic association with the host. IMPORTANCE: Bacillus subtilis is a plant growth-promoting rhizobacterium that establishes robust interactions with roots. Many studies have now demonstrated that biofilm formation is required for long-term colonization. However, we observed that motile B. subtilis mediates the first contact with the roots. These cells differentiate into biofilm-producing cells only several hours after the bacteria first contact the root. Our study reveals that intact chemotaxis machinery is required for the bacteria to reach the root. Many, if not all, of the B. subtilis 10 chemoreceptors are involved in the interaction with the plant. These observations stress the importance of root-bacterium interactions in the B. subtilis lifestyle.
Subject(s)
Arabidopsis/microbiology , Bacillus subtilis/drug effects , Bacillus subtilis/physiology , Chemotaxis , Plant Roots/microbiology , Bacillus subtilis/growth & development , Bacterial Proteins/metabolism , Biofilms/growth & development , Exudates and Transudates/metabolism , Locomotion , Receptors, Cell Surface/metabolismABSTRACT
Considering the medical, biotechnological, and economical importance of actinobacteria, there is a continuous need to improve the tools for genetic engineering of a broad range of these microorganisms. Intergeneric conjugation has proven to be a valuable yet imperfect tool for this purpose. The natural resistance of many actinomycetes to nalidixic acid (Nal) is generally exploited to eliminate the sensitive Escherichia coli donor strain following conjugation. Nevertheless, Nal can delay growth and have other unexpected effects on the recipient strain. To provide an improved alternative to antibiotics, we propose a postconjugational counterselection using a diaminopimelic acid (DAP) auxotrophic donor strain. The DAP-negative phenotype was obtained by introducing a dapA deletion into the popular methylase-negative donor strain E. coli ET12567/pUZ8002. The viability of ET12567 and its ΔdapA mutant exposed to DAP deprivation or Nal selection were compared in liquid pure culture and after mating with Streptomyces coelicolor. Results showed that death of the E. coli ΔdapA Nal-sensitive donor strain occurred more efficiently when subjected to DAP deprivation than when exposed to Nal. Our study shows that postconjugational counterselection based on DAP deprivation circumvents the use of antibiotics and will facilitate the transfer of plasmids into actinomycetes with high biotechnological potential, yet currently not accessible to conjugative techniques.
Subject(s)
Actinobacteria/genetics , Conjugation, Genetic , Diaminopimelic Acid/metabolism , Escherichia coli/genetics , Anti-Bacterial Agents/metabolism , Escherichia coli/metabolism , Nalidixic Acid/metabolismABSTRACT
Small nucleolar RNAs (snoRNAs) are among the first discovered and most extensively studied group of small non-coding RNA. However, most studies focused on a small subset of snoRNAs that guide the modification of ribosomal RNA. In this study, we annotated the expression pattern of all box C/D snoRNAs in normal and cancer cell lines independent of their functions. The results indicate that C/D snoRNAs are expressed as two distinct forms differing in their ends with respect to boxes C and D and in their terminal stem length. Both forms are overexpressed in cancer cell lines but display a conserved end distribution. Surprisingly, the long forms are more dependent than the short forms on the expression of the core snoRNP protein NOP58, thought to be essential for C/D snoRNA production. In contrast, a subset of short forms are dependent on the splicing factor RBFOX2. Analysis of the potential secondary structure of both forms indicates that the k-turn motif required for binding of NOP58 is less stable in short forms which are thus less likely to mature into a canonical snoRNP. Taken together the data suggest that C/D snoRNAs are divided into at least two groups with distinct maturation and functional preferences.
Subject(s)
Nuclear Proteins/physiology , RNA, Small Nucleolar/metabolism , RNA-Binding Proteins/physiology , Repressor Proteins/physiology , Ribonucleoproteins, Small Nucleolar/physiology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Line , Cell Line, Tumor , Female , Humans , MCF-7 Cells , Nuclear Proteins/antagonists & inhibitors , Nucleic Acid Conformation , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , RNA Splicing Factors , RNA, Small Nucleolar/chemistry , RNA, Small Nucleolar/classification , Ribonucleoproteins, Small Nucleolar/antagonists & inhibitorsABSTRACT
Pre-mRNA alternative splicing is modified in cancer, but the origin and specificity of these changes remain unclear. Here, we probed ovarian tumors to identify cancer-associated splicing isoforms and define the mechanism by which splicing is modified in cancer cells. Using high-throughput quantitative PCR, we monitored the expression of splice variants in laser-dissected tissues from ovarian tumors. Surprisingly, changes in alternative splicing were not limited to the tumor tissues but were also found in the tumor microenvironment. Changes in the tumor-associated splicing events were found to be regulated by splicing factors that are differentially expressed in cancer tissues. Overall, â¼20% of the alternative splicing events affected by the down-regulation of the splicing factors QKI and RBFOX2 were altered in the microenvironment of ovarian tumors. Together, our results indicate that the tumor microenvironment undergoes specific changes in alternative splicing orchestrated by a limited number of splicing factors.
Subject(s)
Alternative Splicing , Ovarian Neoplasms/metabolism , RNA, Messenger/genetics , Cell Line, Tumor , Epithelial Cells/metabolism , Female , Gene Expression , Humans , Laser Capture Microdissection , Organ Specificity , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA Splice Sites , RNA Splicing Factors , RNA, Messenger/metabolism , RNA-Binding Proteins/physiology , Repressor Proteins/physiology , Stromal Cells/metabolism , Tumor MicroenvironmentABSTRACT
Several apoptotic regulators, including Bcl-x, are alternatively spliced to produce isoforms with opposite functions. We have used an RNA interference strategy to map the regulatory landscape controlling the expression of the Bcl-x splice variants in human cells. Depleting proteins known as core (Y14 and eIF4A3) or auxiliary (RNPS1, Acinus, and SAP18) components of the exon junction complex (EJC) improved the production of the proapoptotic Bcl-x(S) splice variant. This effect was not seen when we depleted EJC proteins that typically participate in mRNA export (UAP56, Aly/Ref, and TAP) or that associate with the EJC to enforce nonsense-mediated RNA decay (MNL51, Upf1, Upf2, and Upf3b). Core and auxiliary EJC components modulated Bcl-x splicing through different cis-acting elements, further suggesting that this activity is distinct from the established EJC function. In support of a direct role in splicing control, recombinant eIF4A3, Y14, and Magoh proteins associated preferentially with the endogenous Bcl-x pre-mRNA, interacted with a model Bcl-x pre-mRNA in early splicing complexes, and specifically shifted Bcl-x alternative splicing in nuclear extracts. Finally, the depletion of Y14, eIF4A3, RNPS1, SAP18, and Acinus also encouraged the production of other proapoptotic splice variants, suggesting that EJC-associated components are important regulators of apoptosis acting at the alternative splicing level.
Subject(s)
Alternative Splicing , Apoptosis/genetics , Exons , bcl-X Protein/genetics , Apoptosis Regulatory Proteins/genetics , Carrier Proteins/genetics , Co-Repressor Proteins , DEAD-box RNA Helicases/genetics , Eukaryotic Initiation Factor-4A , HEK293 Cells , HeLa Cells , Humans , Nuclear Proteins/genetics , RNA Interference , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , Ribonucleoproteins/genetics , SpliceosomesABSTRACT
Most human genes produce multiple mRNA isoforms through alternative splicing. However, the biological relevance of most splice variants remains unclear. In this study, we evaluated the functional impact of alternative splicing in cancer cells. We modulated the splicing pattern of 41 cancer-associated splicing events and scored the effects on cell growth, viability and apoptosis, identifying three isoforms essential for cell survival. Specifically, changing the splicing pattern of the spleen tyrosine kinase gene (SYK) impaired cell-cycle progression and anchorage-independent growth. Notably, exposure of cancer cells to epithelial growth factor modulated the SYK splicing pattern to promote the pro-survival isoform that is associated with cancer tissues in vivo. The data suggest that splicing of selected genes is specifically modified during tumor development to allow the expression of isoforms that promote cancer cell survival.
Subject(s)
Alternative Splicing , Cell Survival , Gene Expression Regulation , Intracellular Signaling Peptides and Proteins/genetics , Mitosis , Protein-Tyrosine Kinases/biosynthesis , Protein-Tyrosine Kinases/genetics , Apoptosis , Cell Line, Tumor , Epidermal Growth Factor/metabolism , Humans , Syk KinaseABSTRACT
OBJECTIVES: Obtaining stable fixation in cases of long bone non-union with segmental bone defects can be challenging. Bone quality is often sub-optimal. Locking plates and structural allografts have both been used clinically in these cases. The objective of this study was to determine the biomechanical characteristics of three constructs that have been employed in this context. METHODS: A biomechanical study was performed using 3rd Generation Composite Femurs as specimens. A diaphyseal segmental defect was created and fixed with one of three constructs: (1) lateral locking plate (LP); (2) lateral non-locking plate and medial allograft strut (S); (3) lateral non-locking plate and intramedullary fibula allograft (F). The "allografts" were fashioned from 3rd generation composite bones. Axial, torsional and bending stiffness as well as load to failure were determined using a materials testing machine. RESULTS: Overall, construct S was the stiffest and construct LP was the least stiff. Construct F had intermediate characteristics. Axial load to failure for construct S (6108N) and for construct F (5344N) was significantly greater than for construct LP (2855N). CONCLUSION: When maximal stiffness is desired, a construct with a structural allograft should be chosen over a locking plate. However, biological and anatomic factors must also be taken into account when using these constructs clinically.
Subject(s)
Bone Transplantation , Femoral Fractures/surgery , Fracture Fixation, Internal/instrumentation , Fractures, Ununited/surgery , Analysis of Variance , Biomechanical Phenomena , Bone Plates , Diaphyses/physiology , Femoral Fractures/physiopathology , Fracture Fixation, Internal/methods , Fractures, Ununited/physiopathology , HumansABSTRACT
The alternative splicing of Bcl-x generates the proapoptotic Bcl-x(S) protein and the antiapoptotic isoform Bcl-x(L). Bcl-x splicing is coupled to signal transduction, since ceramide, hormones, and growth factors alter the ratio of the Bcl-x isoforms in different cell lines. Here we report that the protein kinase C (PKC) inhibitor and apoptotic inducer staurosporine switches the production of Bcl-x towards the x(S) mRNA isoform in 293 cells. The increase in Bcl-x(S) elicited by staurosporine likely involves signaling events that affect splicing decisions, because it requires active transcription and no new protein synthesis and is independent of caspase activation. Moreover, the increase in Bcl-x(S) is reproduced with more specific inhibitors of PKC. Alternative splicing of the receptor tyrosine kinase gene Axl is similarly affected by staurosporine in 293 cells. In contrast to the case for 293 cells, PKC inhibitors do not influence the alternative splicing of Bcl-x and Axl in cancer cell lines, suggesting that these cells have sustained alterations that uncouple splicing decisions from PKC-dependent signaling. Using minigenes, we show that an exonic region located upstream of the Bcl-x(S) 5' splice site is important to mediate the staurosporine shift in Bcl-x splicing. When transplanted to other alternative splicing units, portions of this region confer splicing modulation and responsiveness to staurosporine, suggesting the existence of factors that couple splicing decisions with PKC signaling.
Subject(s)
Alternative Splicing/genetics , Protein Kinase C/metabolism , bcl-X Protein/metabolism , Alternative Splicing/drug effects , Caspases/metabolism , Cell Line, Tumor , Exons/genetics , Humans , Oncogene Proteins/metabolism , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins , RNA Precursors/genetics , RNA Splice Sites , Receptor Protein-Tyrosine Kinases/metabolism , Regulatory Sequences, Nucleic Acid/genetics , Staurosporine/pharmacology , Axl Receptor Tyrosine KinaseABSTRACT
BACKGROUND: We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins. In the present study, we have tested the inhibitory activity of RNA oligonucleotides carrying different tail structures. RESULTS: We show that an oligonucleotide with a 5' tail containing the human beta-globin branch site sequence inhibits the use of the 5' splice site of Bcl-xL, albeit less efficiently than a tail containing binding sites for the hnRNP A1/A2 proteins. A branch site-containing tail positioned at the 3' end of the oligonucleotide also elicited splicing inhibition but not as efficiently as a 5' tail. The interfering activity of a 3' tail was improved by adding a 5' splice site sequence next to the branch site sequence. A 3' tail carrying a Y-shaped branch structure promoted similar splicing interference. The inclusion of branch site or 5' splice site sequences in the Y-shaped 3' tail further improved splicing inhibition. CONCLUSION: Our in vitro results indicate that a variety of tail architectures can be used to elicit splicing interference at low nanomolar concentrations, thereby broadening the scope and the potential impact of this antisense technology.
Subject(s)
Alternative Splicing/genetics , Gene Silencing , Genetic Engineering/methods , Oligonucleotides, Antisense/genetics , RNA Splice Sites/genetics , Gene Expression Regulation/genetics , Genetic Variation/geneticsABSTRACT
Bcl-x is a member of the Bcl-2 family of proteins that are key regulators of apoptosis. The Bcl-x pre-mRNA is alternatively spliced to yield Bcl-x(S) and Bcl-x(L), two isoforms that have been associated, respectively, with the promotion and the prevention of apoptosis. We have investigated some of the elements and factors involved in the production of these two splice variants. Deletion mutagenesis using a human Bcl-x minigene identifies two regions in exon 2 that modulate Bcl-x 5'-splice site selection in human HeLa cells. One region (B3) is located upstream of the Bcl-x(L) 5'-splice site and enforces Bcl-x(L) production in cells and splicing extracts. The other region (B2) is located immediately downstream of the 5'-splice site of Bcl-x(S) and favors Bcl-x(S) production in vivo and in vitro. A 30-nucleotide G-rich element (B2G) is responsible for the activity of the B2 element. We show that recombinant heterogeneous nuclear ribonucleoprotein (hnRNP) F and H proteins bind to B2G, and mutating the G stretches abolishes binding. Moreover, the addition of hnRNP F to a HeLa extract improved the production of the Bcl-x(S) variant in a manner that was dependent on the integrity of the G stretches in B2G. Consistent with the in vitro results, small interfering RNA-mediated RNA interference targeting hnRNP F and H decreased the Bcl-x(S)/Bcl-x(L) ratio of plasmid-derived and endogenously produced Bcl-x transcripts. Our results document a positive role for the hnRNP F/H proteins in the production of the proapoptotic regulator Bcl-x(S.).