Bacillus subtilis tolerance of moderate concentrations of rifampin involves the sigma(B)-dependent general and multiple stress response.

Low concentrations of the RNA polymerase inhibitor rifampin added to an exponentially growing culture of Bacillus subtilis led to an instant inhibition of growth. Survival experiments revealed that during the growth arrest the cells became tolerant to the antibiotic and the culture was able to resume growth some time after rifampin treatment. L-[(35)S]methionine pulse-labeled protein extracts were separated by two-dimensional polyacrylamide gel electrophoresis to investigate the change in the protein synthesis pattern in response to rifampin. The sigma(B)-dependent general stress proteins were found to be induced after treatment with the antibiotic. Part of the oxidative stress signature was induced as indicated by the catalase KatA and MrgA. The target protein of rifampin, the beta subunit (RpoB) of the DNA-dependent RNA polymerase, and the flagellin protein Hag belonging to the sigma(D) regulon were also induced. The rifampin-triggered growth arrest was extended in a sigB mutant in comparison to the wild-type strain, and the higher the concentration, the more pronounced this effect was. Activity of the RsbP energy-signaling phosphatase in the sigma(B) signal transduction network was also important for this protection against rifampin, but the RsbU environmental signaling phosphatase was not required. The sigB mutant strain was less capable of growing on rifampin-containing agar plates. When plated from a culture that had already reached stationary phase without previous exposure to the antibiotic during growth, the survival rate of the wild type exceeded that of the sigB mutant by a factor of 100. We conclude that the general stress response of B. subtilis is induced by rifampin depending on RsbP activity and that loss of SigB function causes increased sensitivity to the antibiotic.     

Bacterial species and evolution: Theoretical and practical perspectives

A discussion of the species problem in modern evolutionary biology serves as the point of departure for an exploration of how the basic science aspects of this problem relate to efforts to map bacterial diversity for practical pursuits—for prospecting among the bacteria for useful genes and gene-products. Out of a confusing array of species concepts, the Cohesion Species Concept seems the most appropriate and useful for analyzing bacterial diversity. Techniques of allozyme analysis and DNA fingerprinting can be used to put this concept into practice to map bacterial genetic diversity, though the concept requires minor modification to encompass cases of complete asexuality. Examples from studies of phenetically definedBacillus species provide very partial maps of genetic population structure. A major conclusion is that such maps frequently reveal deep genetic subdivision within the phenetically defined specles; divisions that in some cases are clearly distinct genetic species. Knowledge of such subdivisions is bound to make prospecting within bacterial diversity more effective. Under the general concept of genetic cohesion a hypothetical framework for thinking about the full range of species conditions that might exist among bacteria is developed and the consequences of each such model for species delineation, and species identification are discussed. Modes of bacterial evolution, and a theory of bacterial speciation with and without genetic recombination, are examined. The essay concludes with thoughts about prospects for very extensive mapping of bacterial diversity in the service of future efforts to find useful products. In this context, evolutionary biology becomes the handmaiden of important industrial activities. A few examples of past success in commercializing bacterial gene-products from species ofBacillus and a few other bacteria are reviewed.     

Characteristics of substrates and inhibitors in binding to rat liver L-tryptophan 2,3-dioxygenase: a Fourier transform infrared and kinetic study.

Infrared spectroscopy and steady-state kinetics were applied to rat liver L-tryptophan 2,3-dioxygenase, in order to find relations between the structure and binding characteristics of its substrates and inhibitors. The binding characteristics were reflected by changes in the infrared CO stretch band(s) of an Fe(II)-CO complex of the enzyme upon addition of L-tryptophan and 12 analogs. The CO stretch band around 1961 cm-1 of the complex was not much affected by 1-methyl-D,L-tryptophan, a noncompetitive inhibitor, implying a binding at a site distant from the Fe(II)-CO vicinity. The spectral pattern was significantly changed by any of the other compounds which conserved an indole NH, indicative of its binding to the catalytic site. All substrates, which contained a complete CH(NH2)COOH group in addition to the NH, gave spectra similar to that of an L-tryptophan-bound complex. Spectral changes caused by six inhibitors, which lacked the complete CH(NH2)COOH, were different from one another and from those by the substrates. Hence, for an analog, the indole NH is indispensable to bind to the catalytic site, and the CH(NH2)COOH is important to take a correct configuration appropriate to the catalytic reaction. The reason why L- and D-isomers of 5-hydroxytryptohan are not substrates, in spite of their conservation of the required functional groups and correct binding to the catalytic site, has been ascribed to a possible distortion of the protein structure in the heme pocket due to a strong hydrogen bond from the hydroxyl group to an amino acid side chain.     

A technique for measurement of retrograde coronary blood flow in intact anesthetized dogs.

A technique for measurement of retrograde coronary blood flow in intact anesthetized dogs is described. Occlusion of the coronary artery is produced by the inflation of a small rubber balloon at the tip of a no. 9 cardiac catheter placed under fluorescopy in a branch of the left coronary artery. Blood which bleeds back from the occluded coronary artery through the no. 9 catheter is diverted into a small reservoir of 1-ml capacity. The time to fill this reservoir is recorded electrically. Retrograde coronary blood flow is calculated from the time required to fill this reservoir. Results indicate good repeatability of meadurements. The technique seems to be a simple, adequate, and convenient means for assessing agents for possible vasodilator action on the collateral circulation in intact animals.     

A comparison of methanobactins from Methylosinus trichosporium OB3b and Methylocystis strain Sb2 predicts methanobactins are synthesized from diverse peptide precursors modified to create a common core for binding and reducing copper ions

Methanobactins (mb) are low-molecular mass, copper-binding molecules secreted by most methanotrophic bacteria. These molecules have been identified for a number of methanotrophs, but only the one produced by Methylosinus trichosporium OB3b (mb-OB3b) has to date been chemically characterized. Here we report the chemical characterization and copper binding properties of a second methanobactin, which is produced by Methylocystis strain SB2 (mb-SB2). mb-SB2 shows some significant similarities to mb-OB3b, including its spectral and metal binding properties, and its ability to bind and reduce Cu(II) to Cu(I). Like mb-OB3b, mb-SB2 contains two five-member heterocyclic rings with associated enethiol groups, which together form the copper ion binding site. mb-SB2 also displays some significant differences compared to mb-OB3b, including the number and types of amino acids used to complete the structure of the molecule, the presence of an imidazolone ring in place of one of the oxazolone rings found in mb-OB3b, and the presence of a sulfate group not found in mb-OB3b. The sulfate is bonded to a threonine-like side chain that is associated with one of the heterocyclic rings and may represent the first example of this type of sulfate group found in a bacterially derived peptide. Acid-catalyzed hydrolysis and decarboxylation of the oxazolone rings found in mb-OB3b and mb-SB2 produce pairs of amino acid residues and suggest that both mb-OB3b and mb-SB2 are derived from peptides. In support of this, the gene for a ribosomally produced peptide precursor for mb-OB3b has been identified in the genome of M. trichosporium OB3b. The gene sequence indicates that the oxazolone rings in mb-OB3b are derived from the combination of a cysteine residue and the carbonyl from the preceding residue in the peptide sequence. Taken together, the results suggest methanobactins make up a structurally diverse group of ribosomally produced, peptide-derived molecules, which share a common pair of five-member rings with associated enethiol groups that are able to bind, reduce, and stabilize copper ions in an aqueous environment.     

LPS-induced chemokine expression in both MyD88-dependent and -independent manners is regulated by Cot/Tpl2-ERK axis in macrophages

LPS signaling is mediated through MyD88-dependent and -independent pathways, activating NF-κB, MAP kinases and IRF3. Cot/Tpl2 is an essential upstream kinase in LPS-mediated activation of ERKs. Here we explore the roles of MyD88 and Cot/Tpl2 in LPS-induced chemokine expression by studying myd88^?/? and cot/tpl2^?/? macrophages. Among the nine LPS-responsive chemokines examined, mRNA induction of ccl5, cxcl10, and cxcl13 is mediated through the MyD88-independent pathway. Notably, Cot/Tpl2-ERK signaling axis exerts negative effects on the expression of these three chemokines. In contrast, LPS-induced gene expression of ccl2, ccl7, cxcl2, cxcl3, ccl8, and cxcl9 is mediated in the MyD88-dependent manner. The Cot/Tpl2-ERK axis promotes the expression of the first four and inhibits the expression of the latter two. Thus, LPS induces expression of multiple chemokines through various signaling pathways in macrophages.     

Comparison of protein enrichment strategies for proteome analysis of plasma

Efforts to discover protein biomarkers in plasma are hampered by the high abundance of few proteins, which interfere with the detection of low‐abundant proteins. Different commercially available protein‐partitioning products were tested for their ability to lower the detection limit of proteins in 2‐D gels. Immuno‐depletion using polyclonal antibodies raised against the proteins of highest abundance (Seppro IgY14 System) was compared with a two‐step immuno‐depletion strategy, where depletion with the Seppro IgY14 column was followed by depletion with the Seppro IgY‐SuperMix system. The third strategy tested was protein pre‐fractionation using the ProteoMiner kit, where proteins compete for binding sites on bead‐bound peptide hexamers with different binding properties. The pre‐fractionated protein samples were analyzed using 2‐DE, which revealed stunning differences in protein patterns. However, detectable protein spots in the different plasma fractions contained exclusively high‐abundant proteins normally present in plasma at concentrations between 1 μg and 40 mg/mL.