Thermoresponsive MALDI probe surfaces as a tool for protein on-probe purification.

Thin film depositions of rf plasma polymerized N-isopropylacrylamide (ppNIPAM) show a phase transition temperature below which the polymer surface is hydrophilic, and protein nonadsorptive, and above which the polymer surface is hydrophobic, and protein-retentive. Results presented here demonstrate that this thermoresponsive plasma polymer can be coated on the surface of a MALDI probe and subsequently used for on-probe biomolecule cleanup. Specifically, a contaminated biomolecule can be applied to the ppNIPAM coated MALDI probe surface at a temperature above the phase transition temperature, washed using solvent also held above the phase transition temperature, and then analyzed by reducing the probe temperature to room temperature before adding the MALDI matrix. With the use of this approach, it is demonstrated that cytochrome c contaminated with 0.3% SDS, which yields only a very weak MALDI ion signal as directly deposited, can be purified on-probe using the thermoresponsive plasma polymer to improve significantly the ion signal. It is further shown that the decontamination of whole cell protein extracts from cyanobacteria is augmented through the use of the ppNIPAM coated MALDI probe.

Antimicrobial properties of highly fluorinated tris(pyrazolyl)borates.

Highly fluorinated tris(pyrazolyl)borates were tested for their antimicrobial activity against various bacterial species. Both the silver(I) tris(pyrazolyl)borate [HB(3,5-(CF(3))(2)Pz)(3)]Ag(THF) (THF=tetrahydrofuran) and the sodium analog [HB(3,5-(CF(3))(2)Pz)(3)]Na(THF) appeared highly effective at inhibiting the growth of two different species of Gram-positive bacteria (i.e. being 12 and 21 fold more effective, respectively, (on a molar basis, based on the minimum inhibitory concentrations) against Staphylococcus aureus than silver sulfadiazine, a currently used silver antimicrobial). This suggests that the ligand portion of these molecules is responsible for the observed high effectiveness against the Gram-positive species. Furthermore, it appeared that the fluorinated substituents on the tris(pyrazolyl)borate were important for this high level of growth inhibition. Against two species of Gram-negative bacteria, including Pseudomonas aeruginosa, the fluorinated silver(I) tris(pyrazolyl)borate exhibited a moderate level of growth inhibition (similar to that of silver sulfadiazine), while the sodium analog showed very little ability to inhibit growth, indicating that for the Gram-negative species, the apparent responsible antimicrobial portion is the silver ion.

The response regulator RpaB binds the high light regulatory 1 sequence upstream of the high-light-inducible hliB gene from the cyanobacterium Synechocystis PCC 6803.

Cyanobacteria, like other photosynthetic organisms, respond to the potentially damaging effects of high-intensity light by regulating the expression of a variety of stress-responsive genes through regulatory mechanisms that remain poorly understood. The high light regulatory 1 (HLR1) sequence can be found upstream of many genes regulated by high-light (HL) stress in cyanobacteria. In this study, we identify the factor that binds the HLR1 upstream of the HL-inducible hliB gene in the cyanobacterium Synechocystis PCC 6803 as the RpaB (Slr0947) response regulator.

Negative control of the high light-inducible hliA gene and implications for the activities of the NblS sensor kinase in the cyanobacterium Synechococcus elongatus strain PCC 7942.

The hliA gene of the cyanobacterium Synechococcus elongatus PCC 7942 is known to be upregulated by high-intensity light through the activity of the NblS sensor kinase. In this work it was found that, within the hliA upstream region, changes to the sequence around -30 to -25 (relative to the transcriptional start site) resulted in elevated hliA expression, implicating this region in negative regulation of the gene. Electrophoretic mobility shift assays performed were consistent with a protein binding this region that acts to keep the gene off in lower light. A reduction in gene dosage of nblS in vivo resulted in enhanced hliA expression, suggesting that negative control of hliA is mediated through NblS. An extended version of the high light regulatory 1 (HLR1) motif (previously described in Synechocystis PCC 6803) was identified within the sequence surrounding -30 to -25 of hliA. The extended HLR1 sequence was found upstream of other NblS-controlled genes from S. elongatus and Synechocystis PCC 6803 and upstream of hli genes from a variety of cyanobacterial and related genomes. These results point to the evolutionary conservation of the HLR1 element and its importance in NblS-mediated signaling and yield new insight into NblS-mediated control of gene expression.

Antimicrobial properties of highly fluorinated silver(I) tris(pyrazolyl)borates.

Highly fluorinated tris(pyrazolyl)borate ligand [HB(3,5-(CF3)2Pz)3]- has been used in the isolation of air- and light-stable silver complex, [HB(3,5-(CF3)2Pz)3]Ag(OSMe2). It is a monomeric tetrahedral silver complex with an O-bonded dimethylsulfoxide ligand. The silver adduct [HB(3,5-(CF3)2Pz)3]Ag(OSMe2) and the related [HB(3,5-(CF3)2Pz)3] Ag(THF) (where OSMe2 = dimethyl sulfoxide; THF = tetrahydrofuran) show good antibacterial activity, and their antimicrobial efficacy against Staphylococcus aureus is greater than those of AgNO3 and silver sulfadiazine.

Control of photosynthetic and high-light-responsive genes by the histidine kinase DspA: negative and positive regulation and interactions between signal transduction pathways.

We have deleted a gene for a sensor histidine kinase, dspA (or hik33), in the cyanobacterium Synechocystis sp. strain PCC6803. In low and moderate light, the mutant grew slowly under photoautotrophic conditions, with a doubling time of approximately 40 h, and had severely reduced photosynthetic oxygen evolution. When the mutant was maintained in low or moderate light in the presence of glucose, its growth rate was only somewhat lower than that of wild-type cells. However, the mutant was light sensitive and rapidly died in high light. Furthermore, levels of many transcripts encoding genes associated with photosynthesis were altered in the mutant relative to wild-type Synechocystis sp. strain PCC6803 both in low light and following exposure to high light. There was constitutive expression of several high-light-inducible genes, including hli, psbAIII, and gpx2; there was little increased accumulation of sodB mRNA in high light; and the cells failed to accumulate cpcBA and psaAB mRNAs in low light in the presence of glucose, although a normal decline in the levels of these mRNAs was observed during exposure to high light. These results suggest that DspA is involved in controlling sets of photosynthetic and high-light-responsive genes, either directly or indirectly. These and other results, some of which are presented in a companion paper (C.-J. Tu, J. Shrager, R. Burnap, B. L. Postier, and A. R. Grossman, J. Bacteriol. 186:3889-3902, 2004), suggest that DspA acts as a global regulator that helps coordinate cellular metabolism with growth limitations imposed by environmental conditions.

Photosynthetic electron transport controls expression of the high light inducible gene in the cyanobacterium Synechococcus elongatus strain PCC 7942.

The hliA gene of Synechococcus, encoding a photoprotective high light inducible polypeptide, is up-regulated by high light (HL) or low intensity blue/UV-A light (BL). hliA expression was found to be up-regulated by KCN in low light (LL) (but not in the dark), and up-regulation in HL, BL, and LL (with KCN) was inhibited by 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone. A working hypothesis is proposed whereby up-regulation is in response to the reduced state of cytochrome b(6)f or a carrier beyond in photosynthesis. Modest up-regulation occurs in LL by treatment with 3-(3,4-dichlorophenyl)-1,1-dimethylurea, but this is related to effects on hliA mRNA stability rather than on transcription.

Light control of hliA transcription and transcript stability in the cyanobacterium Synechococcus elongatus strain PCC 7942.

The high-light-inducible proteins (HLIPs) of cyanobacteria are polypeptides involved in protecting the cells from high-intensity light (HL). The hliA gene encoding the HLIP from Synechococcus elongatus strain PCC 7942 is expressed in response to HL or low-intensity blue or UV-A light. In this study, we explore via Northern analysis details of the transcriptional regulation and transcript stability of the hliA gene under various light conditions. Transcript levels of the hliA gene increased dramatically upon a shift to HL or UV-A light to similar levels, followed by a rapid decrease in UV-A light, but not in HL, consistent with blue/UV-A light involvement in early stages of HL-mediated expression. A 3-min pulse of low-intensity UV-A light was enough to trigger hliA mRNA accumulation, indicating that a blue/UV-A photoreceptor is involved in upregulation of the gene. Low-intensity red light was found to cause a slight, transient increase in transcript levels (raising the possibility of red-light photoreceptor involvement), while light of other qualities had no apparent effect. No evidence was found for wavelength-specific attenuation of hliA transcript levels induced by HL or UV-A light. Transcript decay was slowed somewhat in darkness, and when photosynthetic electron transport was inhibited by darkness or treatment with DCMU, there appeared a smaller mRNA species that may represent a decay intermediate that accumulates when mRNA decay is slowed. Evidence suggests that upregulation of hliA by light is primarily a transcriptional response but conditions that cause ribosomes to stall on the transcript (e.g., a shift to darkness) can help stabilize hliA mRNA and affect expression levels.

nblS, a gene involved in controlling photosynthesis-related gene expression during high light and nutrient stress in Synechococcus elongatus PCC 7942.

The HliA protein of the cyanobacterium Synechococcus elongatus PCC 7942 is a small, thylakoid-associated protein that appears to play a role in photoprotection; its transcript rapidly accumulates in response to high-intensity light (HL) and the hli gene family is required for survival of cells in high light. In order to discover regulatory factors involved in HL acclimation in cyanobacteria, a screen was performed for chemically generated mutants unable to properly control expression of the hliA gene in response to HL. One such mutant was identified, and complementation analysis led to the identification of the affected gene, designated nblS. Based on its deduced protein sequence, NblS appears to be a membrane-bound, PAS-domain-bearing, sensor histidine kinase of two-component regulatory systems in bacteria. The nblS mutant was unable to properly control light intensity-mediated expression of several other photosynthesis-related genes, including all three psbA genes and the cpcBA genes. The mutant was also unable to control expression of the hliA and psbA genes in response to low-intensity blue/UV-A light, a response that may be related to the HL-mediated regulation of the genes. Additionally, in response to nutrient deprivation, the nblS mutant was unable to properly control accumulation of the nblA transcript and associated degradation of the light-harvesting phycobilisomes. The nblS mutant dies more rapidly than wild-type cells following exposure to HL or nutrient deprivation, likely due to its inability to properly acclimate to these stress conditions. Thus, the NblS protein is involved in the control of a number of processes critical for altering the photosynthetic apparatus in response to both HL and nutrient stress conditions.