Rapid small scale preparation of bacterial genomic DNA, suitable for cloning and hybridization analysis

The uptake of noxythiolin by a urinary isolate of Escherichia coli was examined initially at 37°C but the adsorption isotherm was complicated by the concomitant degradation of the compound. When drug adsorption was investigated at 4°C, to reduce the degradation rate of the compound, it was observed that noxythiolin was taken up by the urinary isolate in a linear fashion. The resulting adsorption patterns are discussed in relation to their possible classification. The implications of this uptake are considered with respect to the antimicrobial activity of noxythiolin.     

Effects of small scale fluid motion on bacterial growth and respiration

1. Laboratory experiments were conducted to investigate the effect of small‐scale turbulent motion on the growth and respiration of bacteria in an oscillating grid apparatus. The experiments were performed under a range of energy dissipation levels similar to those occurring in freshwater systems. 2. The results showed that small‐scale turbulent motion does have an effect on bacterial growth and respiration. A higher gradient in the dissolved oxygen time series, higher 5‐day biochemical oxygen demand values, increased bacterial abundance, increased bacterial specific respiration, higher bacterial growth rate and increased nutrient uptake were all observed when the energy dissipation rate in the water column was increased. 3. This has implications for traditional laboratory procedures that are used to characterise bacterial metabolic rates under stagnant fluid‐flow conditions, such as biochemical oxygen demand (BOD), which would be influenced by the effects of the small‐scale fluid motion inherent in aquatic environments. According to our results, BOD values in natural systems experiencing fluid motion would be higher than traditional bottle‐derived rates.     

Remote control of mRNA cleavage by a small RNA

Comment on: Prévost K, et al. Genes Dev 2011; 25:385-96.     

Recognition and cleavage of 5-methylcytosine DNA by bacterial SRA-HNH proteins

SET and RING-finger-associated (SRA) domain is involved in establishment and maintenance of DNA methylation in eukaryotes. Proteins containing SRA domains exist in mammals, plants, even microorganisms. It has been established that mammalian SRA domain recognizes 5-methylcytosine (5mC) through a base-flipping mechanism. Here, we identified and characterized two SRA domain-containing proteins with the common domain architecture of N-terminal SRA domain and C-terminal HNH nuclease domain, Sco5333 from Streptomyces coelicolor and Tbis1 from Thermobispora bispora. Both sco5333 and tbis1 cannot establish in methylated Escherichia coli hosts (dcm⁺), and this in vivo toxicity requires both SRA and HNH domain. Purified Sco5333 and Tbis1 displayed weak DNA cleavage activity in the presence of Mg²⁺, Mn²⁺ and Co²⁺ and the cleavage activity was suppressed by Zn²⁺. Both Sco5333 and Tbis1 bind to 5mC-containing DNA in all sequence contexts and have at least a preference of 100 folds in binding affinity for methylated DNA over non-methylated one. We suggest that linkage of methyl-specific SRA domain and weakly active HNH domain may represent a universal mechanism in competing alien methylated DNA but to maximum extent minimizing damage to its own chromosome.     

Crystal structures of bacterial peptidoglycan amidase AmpD and an unprecedented activation mechanism

AmpD is a cytoplasmic peptidoglycan (PG) amidase involved in bacterial cell-wall recycling and in induction of β-lactamase, a key enzyme of β-lactam antibiotic resistance. AmpD belongs to the amidase_2 family that includes zinc-dependent amidases and the peptidoglycan-recognition proteins (PGRPs), highly conserved pattern-recognition molecules of the immune system. Crystal structures of Citrobacter freundii AmpD were solved in this study for the apoenzyme, for the holoenzyme at two different pH values, and for the complex with the reaction products, providing insights into the PG recognition and the catalytic process. These structures are significantly different compared with the previously reported NMR structure for the same protein. The NMR structure does not possess an accessible active site and shows the protein in what is proposed herein as an inactive “closed” conformation. The transition of the protein from this inactive conformation to the active “open” conformation, as seen in the x-ray structures, was studied by targeted molecular dynamics simulations, which revealed large conformational rearrangements (as much as 17 Å) in four specific regions representing one-third of the entire protein. It is proposed that the large conformational change that would take the inactive NMR structure to the active x-ray structure represents an unprecedented mechanism for activation of AmpD. Analysis is presented to argue that this activation mechanism might be representative of a regulatory process for other intracellular members of the bacterial amidase_2 family of enzymes.     

DNA gyrase on the bacterial chromosome: DNA cleavage induced by oxolinic acid.

Treatments in vivo of Escherichia coli with oxolinic acid, a potent inhibitor of DNA gyrase and DNA synthesis, lead to DNA cleavage when extracted chromosomes are incubated with sodium dodecyl sulfate. This DNA breakage has properties similar to those obtained in vitro with DNA gyrase reaction mixtures designed to assay production of supertwists: it is oxolinic acid-dependent, sodium dodecyl sulfate-activated, and at saturating drug concentrations produces double-strand DNA cleavage with a concommitant tight association of protein and DNA. In addition, identical treatments performed on a nalA mutant strain exhibit no DNA cleavage. Thus the DNA cleavage sites probably correspond to chromosomal DNA gyrase sites. Sedimentation measurements of the DNA cleavage products indicate that there are approximately 45 DNA breaks per chromosome. This value is similar to the number of domains of supercoiling found in isolated Escherichia coli chromosomes, suggesting one gyrase site per domain. At low oxolinic acid concentrations single-strand cleavages predominate after sodium dodecyl sulfate treatment, and the inhibition of DNA synthesis parallels the number of sites that obtain a single-strand scission. Double-strand breaks arise from the accumulation of single-strand cleavages in accordance with a model where each cleavage site contains two independent drug targets, one on each DNA strand. Since the nicking-closing subunit of gyrase is the target of oxolinic acid in vitro, we suggest that each gyrase site contains two nicking-closing subunits, one on each DNA strand, and that DNA synthesis requires both to be functional.     

The inhibition of boar acrosin amidase activity by sulfated polysaccharides

Carbohydrate-protein interactions are known to be important in gamete interactions. We therefore investigated the inhibition of boar sperm acrosin amidase activity by carbohydrates. The sulfated polysaccharides fucoidan and dextran sulfate inhibited amide hydrolysis whereas dextran and various monosaccharides did not inhibit acrosin amidase activity. The kinetics of the inhibition corresponded to those characteristic when multiple forms of an enzyme are present. Such a kinetic result was consistent with the presence of the known autolytically produced forms of acrosin. It was previously shown that sulfated polysaccharides inhibit sperm-egg binding and that acrosin binds carbohydrate. We propose that the sulfated polysaccharide inhibition of acrosin amidase activity observed here is causally related to the previously observed sulfated polysaccharide inhibition of sperm binding to the zona pellucida.     

Large scale preparation of calmodulin

A rapid large scale purification procedure based on three conventional purification steps, ammonium sulfate fractionation, DEAE cellulose and hydroxylapaptite chromotography yields gram amounts of calmodulin. The protein is more than 98% pure by SDS gel electrophoresis and amino acid composition. It is free of contaminating EGTA or EDTA and the omission of heat treatment or denaturing solvents during the preparation avoids possible denaturation of the protein and minimizes partial proteolysis.     

Correlated cleavage of damaged DNA by bacterial and human 8-oxoguanine-DNA glycosylases

Many enzymes acting on specific rare lesions in DNA are suggested to search for their targets by facilitated one-dimensional diffusion. We have used a recently developed correlated cleavage assay to investigate whether this mechanism operates for Fpg and OGG1, two structurally unrelated DNA glycosylases that excise an important oxidative lesion, 7,8-dihydro-8-oxoguanine (8-oxoG), from DNA. Similar to a number of other DNA glycosylases or restriction endonucleases, Fpg and OGG1 processively excised 8-oxoG from pairs with cytosine at low salt concentrations, indicating that the lesion search likely proceeds by one-dimensional diffusion. At high salt concentrations, both enzymes switched to a distributive mode of lesion search. Correlated cleavage of abasic site-containing substrates proceeded in the same manner as cleavage of 8-oxoG. Interestingly, both Fpg and especially OGG1 demonstrated higher processivity if the substrate contained 8-oxoG.A pairs, against which these enzyme discriminate. Introduction of a nick into the substrate DNA did not decrease the extent of correlated cleavage, suggesting that the search probably involves hopping between adjacent positions on DNA rather than sliding along DNA. This was further supported by the observation that mutant forms of Fpg (Fpg-F110A and Fpg-F110W) with different sizes of the side chain of the amino acid residue inserted into DNA during scanning were both less processive than the wild-type enzyme. In conclusion, processive cleavage by Fpg and OGG1 does not correlate with their substrate specificity and under nearly physiological salt conditions may be replaced with the distributive mode of action.     

Functional determinants of gate-DNA selection and cleavage by bacterial type II topoisomerases

Antibacterial fluoroquinolones trap a cleavage complex of gyrase and topoisomerase (topo) IV inducing site-specific DNA breakage within a bent DNA gate engaged in DNA transport. Despite its importance for drug action and in revealing potential sites of topoisomerase catalysis, the mechanism of DNA selectivity is poorly understood. To explore its functional basis, we generated mutant versions of the strongly cleaved E-site and used a novel competitive assay to examine their gemifloxacin-mediated DNA breakage by Streptococcus pneumoniae topo IV and gyrase. Parallel studies of Ca²⁺-induced cleavage distinguished ‘intrinsic recognition’ of DNA cleavage sites by topo IV from drug-induced preferences. Analysis revealed strong enzyme-determined requirements for −4G, −2A and −1T bases preceding the breakage site (between −1 and +1) and enzyme-unique or degenerate determinants at −3, plus drug-specific preferences at +2/+3 and for +1 purines associated with drug intercalation. Similar cleavage rules were seen additionally at the novel V-site identified here in ColE1-derived plasmids. In concert with DNA binding data, our results provide functional evidence for DNA, enzyme and drug contributions to DNA cleavage at the gate, suggest a mechanism for DNA discrimination involving enzyme-induced DNA bending/helix distortion and cleavage complex stabilization and advance understanding of fluoroquinolones as important cleavage-enhancing therapeutics.     

Production and purification of the bacterial autolysin N-acetylmuramoyl-L-alanine amidase B from Pseudomonas aeruginosa

The bacterial cell wall heteropolymer peptidoglycan is not a static structure as it is constantly being made and recycled throughout the bacterium's life cycle. This turnover of peptidoglycan is a highly coordinated event involving a complement of autolytic enzymes that include those with specificity for either the carbohydrate or the peptide linkages of peptidoglycan. One major class of these autolysins are the N-acetylmuramoyl-L-alanine amidases which cleave the amide linkage between the stem peptides and the lactyl moiety of muramoyl residues. They are required in the periplasm for cell separation during division and in both the periplasm and cytoplasm to trim soluble released PG fragments during turnover for recycling. The gene encoding N-acetylmuramoyl-L-alanine amidase B in Pseudomonas aeruginosa was cloned and over-expressed in Escherichia coli. The recombinant protein with a C-terminal His-tag was purified to apparent homogeneity by a combination of affinity and cation-exchange chromatographies using Ni(2+)NTA-agarose and Source S, respectively. Four separate assays involving zymography, light scattering, HPLC and MALDI-TOF mass spectrometry were used to confirm the activity of the protein as an N-acetylmuramoyl-L-alanine amidase.     

Large-scale preparation of bacterial cell membranes by tangential flow filtration

The preparation of cell membranes by ultracentrifugation of bacterial cell lysates, a pre-requisite for the purification of over-expressed membrane proteins, is both time-consuming and difficult to perform on a large scale. To overcome this bottleneck in the structural investigation of such proteins in the UK Membrane Protein Structure Initiative, we have investigated the alternative use of tangential flow filtration for preparation of membranes from Escherichia coli. This method proved to be superior to the conventional use of ultracentrifuges both in speed and in yield of membrane protein. Moreover, it could more readily be scaled up to process larger quantities of bacterial cells. Comparison of the purity and monodispersity of an over-expressed membrane protein purified from conventionally-prepared membranes and from membranes prepared by filtration revealed no substantial differences. The approach described should therefore be of general use for membrane protein preparation for a wide range of applications, including both structural and functional studies.