Salmonella typhi contains identical intervening sequences in all seven rrl genes.

Salmonella typhi Ty2 rrl genes contain intervening sequences (IVSs) in helix-25 but not in helix-45 on the basis of observed 23S rRNA fragmentation caused by IVS excision. We have confirmed this and shown all seven IVSs to be identical by isolating genomic DNA fragments containing each of the seven rrl genes from S. typhi Ty2 by use of pulsed-field gel electrophoresis; each rrl gene was amplified by PCR in the helix-25 and helix-45 regions and cycle sequenced. Thirty independent wild-type S. typhi strains, tested by genomic PCR and DraI restriction, also have seven rrl genes with helix-25 IVSs and no helix-45 IVSs. We propose that IVS homogeneity in S. typhi occurs because gene conversion drives IVS sequence maintenance and because adaptation to human hosts results in limited clonal diversity.     

Structural characterization of the antimicrobial peptide pleurocidin from winter flounder

Pleurocidin is an antimicrobial peptide that was isolated from the mucus membranes of winter flounder (Pseudopleuronectes americanus) and contributes to the initial stages of defense against bacterial infection. From NMR structural studies with the uniformly (15)N-labeled peptide, a structure of pleurocidin was determined to be in a random coil conformation in aqueous solution whereas it assumes an alpha-helical structure in TFE and in dodecylphosphocholine (DPC) micelles. From (15)N relaxation studies, the helix is a rigid structure in the membrane-mimicking environment. Strong NOESY cross-peaks from the pleurocidin to the aliphatic chain on DPC confirm that pleurocidin is contained within the DPC micelle and not associated with the surface of the micelle. From diffusion studies it was determined that each micelle contains at least two pleurocidin molecules.     

Transient-state reduction and steady-state kinetic studies of menaquinol oxidase from Bacillus subtilis, cytochrome aa3-600 nm. Spectroscopic characterization of the steady-state species.

Cytochrome aa3-600 or menaquinol oxidase, from Bacillus subtilis, is a member of the heme-copper oxidase family. Cytochrome aa3-600 contains cytochrome a, cytochrome a3, and CuB, and each is coordinated via histidine residues to subunit I. Subunit II of cytochrome aa3-600 lacks CuA, which is a common feature of the cytochrome c oxidase family members. Anaerobic reduction of cytochrome aa3-600 by the substrate analogue 2,3-dimethyl-1,4-naphthoquinone (DMN) resolves two distinct kinetic phases by stopped-flow, single-wavelength spectrometry. Global analysis of time-resolved, multiwavelength spectra shows that during these distinct phases cytochromes a and a3 are both reduced. Cyanide binding to cytochrome a3 enhances the fast phase rate, which in the presence of cyanide can be assigned to cytochrome a reduction, whereas cytochrome a3-cyanide reduction is slow. The steady-state activity of cytochrome aa3-600 exhibits saturation kinetics as a function of DMN concentration with a Km of 300 microM and a maximal turnover of 63.5 s(-1). Global kinetic analysis of steady-state spectra reveals a species that is characteristic of a partially reduced oxygen adduct of cytochrome a3-CuB, whereas cytochrome a remains oxidized. Electron paramagnetic resonance (EPR) spectroscopy of the oxidase in the steady state shows the expected signal from ferricytochrome a, and a new EPR signal at g = 2.01. A model of the catalytic cycle for cytochrome aa3-600 proposes initial electron delivery from DMN to cytochrome a, followed by rapid heme to heme electron transfer, and suggests possible origins of the radical signal in the steady-state form of the enzyme.     

Expression, purification, and characterization of the CuA-cytochrome c domain from subunit II of the Bacillus subtilis cytochrome caa3 complex in Escherichia coli

Cytochrome caa3 from Bacillus subtilis is a member of the heme-copper oxidase family of integral membrane enzymes that includes mitochondrial cytochrome c oxidase. Subunit II of cytochrome caa3 has an extra 100 amino acids at its C-terminus, relative to its mitochondrial counterpart, and this extension encodes a heme C binding domain. Cytochrome caa3 has many of the properties of the complex formed between mitochondrial cytochrome c and mitochondrial cytochrome c oxidase. To examine more closely the interaction between cytochrome c and the oxidase we have cloned and expressed the Cu(A)-cytochrome c portion of subunit II from the cytochrome caa3 complex of B. subtilis. We are able to express about 2000 nmol, equivalent to 65 mg, of the Cu(A)-cytochrome c protein per litre of Escherichia coli culture. About 500 nmol is correctly targeted to the periplasmic space and we purify 50% of that by a combination of affinity chromatography and ammonium sulfate fractionation. The cytochrome c containing sub-domain is well-folded with a stable environment around the heme C center, as its mid-point potential and rates of reduction are indistinguishable from values for the cytochrome c domain of the holo-enzyme. However, the Cu(A) site lacks copper leading to an inherent instability in this sub-domain. Expression of B. subtilis cytochrome c, as exemplified by the Cu(A)-cytochrome c protein, can be achieved in E. coli, and we conclude that the cytochrome c and Cu(A) sub-domains behave independently despite their close physical and functional association.     

Regulation of phenylacetic acid degradation genes of Burkholderia cenocepacia K56-2

Background  

Metabolically versatile soil bacteria Burkholderia cepacia complex (Bcc) have emerged as opportunistic pathogens, especially of cystic fibrosis (CF). Previously, we initiated the characterization of the phenylacetic acid (PA) degradation pathway in B. cenocepacia, a member of the Bcc, and demonstrated the necessity of a functional PA catabolic pathway for full virulence in Caenorhabditis elegans. In this study, we aimed to characterize regulatory elements and nutritional requirements that control the PA catabolic genes in B. cenocepacia K56-2.