Genes Encoding Binding Proteins Research Articles

Expression of the Biofilm-Associated Genes in Methicillin-Resistant Staphylococcus aureus in Biofilm and Planktonic Conditions.

The role of genes that are essential for development of Staphylococcus aureus biofilm during infection is not fully known. mRNA from two methicillin-resistant S. aureus strains that formed weak and strong biofilm on polystyrene plates were isolated at five time points from cells grown in biofilm and planktonic culture. Quantitative real-time PCR analysis showed that the expression levels of investigated genes under biofilm conditions were significantly higher than under planktonic conditions. The expression levels of the gene encoding elastin binding protein (ebps) and laminin binding protein (eno) were significantly increased in biofilm at 3 h, both in strongly and weakly adhering strain. The peak expression of fib gene encoding fibrinogen binding protein was found at 6 and 8 h in the case of strongly and weakly adhering strain, respectively. The expression of icaA and icaD genes in both strains was significantly higher under biofilm conditions when comparing to planktonic cells during 12 h. The expression level of the genes encoding binding proteins and the glucosamine polymer polysaccharide intercellular adhesin (PIA) slowly decreased after 24 h. Finally, we found that the expression levels of genes encoding binding factors in weakly adhering strain were significantly lower than in strongly adhering strain.

Role of cleavage and polyadenylation specificity factor 100: anchoring poly(A) sites and modulating transcription termination.

CPSF100 is a core component of the cleavage and polyadenylation specificity factor (CPSF) complex for 3'-end formation of mRNA, but it still has no clear functional assignment. CPSF100 was reported to play a role in RNA silencing and promote flowering in Arabidopsis. However, the molecular mechanisms underlying these phenomena are not fully understood. Our genetics analyses indicate that plants with a hypomorphic mutant of CPSF100 (esp5) show defects in embryogenesis, reduced seed production or altered root morphology. To unravel this puzzle, we employed a poly(A) tag sequencing protocol and uncovered a different poly(A) profile in esp5. This transcriptome-wide analysis revealed alternative polyadenylation of thousands of genes, most of which result in transcriptional read-through in protein-coding genes. AtCPSF100 also affects poly(A) signal recognition on the far-upstream elements; in particular it prefers less U-rich sequences. Importantly, AtCPSF100 was found to exert its functions through the change of poly(A) sites on genes encoding binding proteins, such as nucleotide-binding, RNA-binding and poly(U)-binding proteins. In addition, through its interaction with RNA Polymerase II C-terminal domain (CTD) and affecting the expression level of CTD phosphatase-like 3 (CPL3), AtCPSF100 is shown to potentially ensure transcriptional termination by dephosphorylation of Ser2 on the CTD. These data suggest a key role for CPSF100 in locating poly(A) sites and affecting transcription termination.

HP-23: Co-ordinated Regulation of Gene Expression in H. Pylori in Response to Low pH and Iron Limiting Growth Conditions

AbstractEstablishing a successful infection within the gastric environment and its varied and changing niches requires adaptation achieved through regulation of bacterial gene expression. In the gastric environment in addition to the low pH Helicobacter pylori is required to adapt to conditions of iron limitation. In this study, we used cDNA microarrays to identify genes whose expression was altered when the bacterium was grown under differing conditions of pH (5 versus 7) and iron limitation. cDNA microarray analyses following exposure to different growth conditions were carried out for three H. pylori strains 26695 (gastritis-associated), J99 (ulcer-associated) and AG-1 (atrophic gastritis-associated). Genes were identified whose expression was acid pH-dependent in all the strains: 68 genes were up-regulated, these included urease-related genes (ureA, ureB, ureI, UreF), amidase (aimE) and genes involved in flagellar synthesis (flgB, fliM, flgE). Additionally, up-regulation of transport-related genes and genes encoding binding proteins (glnQ, glnH, exbD) were noted. Furthermore, 37 genes were down-regulated from different functional groups (e.g., outer membrane proteins and chaperones). At pH 7, iron limitation mainly induced motility-associated genes, whereas at pH5, iron depletion although inducing down-regulation of 12 genes, led to the increased expression of 21 genes including those coding for carbonic anhydrase and proteins involved in metal ion transport, instead of genes coding for urease and amidase enzymes. Genes encoding virulence factors (e.g., cagA and vacA) and proteins involved in motility were over-expressed under all the conditions of stress. Collectively, as effectors of the H. pylori stress response, this transcriptome analysis implicating up-regulation of various metal responsive regulators and metal transport-related genes, in addition to induction of ammonia-producing enzymes and virulence factors (CagA and VacA), emphasizes the link between the bacterial response to acidity, metal metabolism and virulence. Moreover, this response was similar in H. pylori strains associated with different pathologies.

Differential Gene Expression Profiling of Staphylococcus aureus Cultivated under Biofilm and Planktonic Conditions

It is well known that biofilm formation by pathogenic staphylococci on implanted medical devices leads to "chronic polymer-associated infections." Bacteria in these biofilms are more resistant to antibiotics and the immune defense system than their planktonic counterparts, which suggests that the cells in a biofilm have altered metabolic activity. To determine which genes are up-regulated in Staphylococcus aureus biofilm cells, we carried out a comparative transcriptome analysis. Biofilm growth was simulated on dialysis membranes laid on agar plates. Staphylococci were cultivated planktonically in Erlenmeyer flasks with shaking. mRNA was isolated at five time points from cells grown under both conditions and used for hybridization with DNA microarrays. The gene expression patterns of several gene groups differed under the two growth conditions. In biofilm cells, the cell envelope appeared to be a very active compartment since genes encoding binding proteins, proteins involved in the synthesis of murein and glucosaminoglycan polysaccharide intercellular adhesin, and other enzymes involved in cell envelope synthesis and function were significantly up-regulated. In addition, evidence was obtained that formate fermentation, urease activity, the response to oxidative stress, and, as a consequence thereof, acid and ammonium production are up-regulated in a biofilm. These factors might contribute to survival, persistence, and growth in a biofilm environment. Interestingly, toxins and proteases were up-regulated under planktonic growth conditions. Physiological and biochemical tests for the up-regulation of urease, formate dehydrogenase, proteases, and the synthesis of staphyloxanthin confirmed the microarray data.

A novel mode of sensory transduction in archaea: binding protein-mediated chemotaxis towards osmoprotectants and amino acids.

Directly upstream of the Halobacterium salinarum transducer genes basT and htpIV we identified two open reading frames (orfs) with significant homologies to genes encoding binding proteins for amino acids and compatible solutes, respectively. Behavioral testing of deletion mutants indicates that halobacterial chemotaxis towards branched-chain amino acids as well as compatible osmolytes of the betaine family requires both a binding and a transducer protein. We therefore named the binding/transducer proteins BasB/BasT for branched-chain and sulfur-containing amino acids and CosB/CosT for compatible solutes. Our data support a signaling mechanism with the binding proteins functioning as lipid-anchored receptors interacting with the extracellular domain of their cognate transducers. Inspection of the halobacterial genome suggests that BasB and CosB exclusively mediate chemotaxis responses without any additional role in transport, which is in contrast to bacterial binding proteins, which are always part of ABC transport systems. The CosB/CosT system is the first instance of a chemotaxis signaling pathway for organic osmolytes in the living world and natural abundance 13C-NMR analysis of cytoplasmic extracts suggests that H.salinarum utilizes these solutes for osmotic adaptation.

Insulin-like Growth Factor 1 Binding Protein 3 Synthesis by Aortic Endothelial Cells Is a Function of Cell Density

Proliferating bovine aortic endothelial cells in culture do not express the insulin-like growth factor 1 binding protein 3 gene, in contrast to the genes encoding binding proteins 2, 4, 5, and 6. The binding protein 3 gene is activated only at cell confluency with continual transcription thereafter, for at least an eight-day post-confluent period, both in the presence or absence of serum. Secretion of protein product into the medium parallels transcription. DNA synthesis is inversely related to the amount of total insulin-like growth factor 1 binding protein 3 in the culture medium. The addition of anti-binding protein 3 antibody to media significantly increases the rate of DNA synthesis by extensively post-confluent cells. These data suggest (1) aortic endothelial cells are an important source of circulating binding protein 3, (2) binding protein 3 has an inhibitory effect upon the growth of endothelial cells, (3) the triggering of binding protein 3 synthesis after confluency suggests a role in maintaining the growth-arrested monolayer state of the cellsin vivo.

Cloning and Nucleotide Sequences of livB and liv, the Structural Genes Encoding Binding Proteins of the High-Affinity Branched-Chain Amino Acid Transport in Salmonella typhimurium

The liv gene cluster responsible for encoding the high-affinity branched-chain amino acid transport proteins in Salmonella typhimurium was mapped in the 7.6-kilobase HindIII-SacI segment of plasmid pMN12 by utilizing the gene dosage effect. By subcloning and biochemical analysis, the livB and livC structural genes encoding the leucine-, isoleucine-, valine-, threonine-binding protein (LIVT-BP) and the leucine-specific binding protein (L-BP), respectively, were localized within the 3,617-base HindIII-BstEII segment. Upon determining the nucleotide sequence of the 3,617 bases, we found that the coding sequence of the livB gene (1,095 base pairs) starts at the position 355 and specifies the precursor LIVT-BP of 365 amino acid residues, and the livC gene (1,107 base pairs) starts at the position 2,452 and encodes the precursor L-BP of 369 amino acid residues. The two genes, separated by a 1-kilobase intergenic region, each possess potential promoters and rho-independent transcriptional terminators. The mature LIVT-BP and L-BP are produced by removing the putative 21 and 23 signal peptides from the respective precursors. In comparison with the analogous two binding proteins from Escherichia coli K-12, strong homologies are observed.