Mary Lynne Perille Collins

Direct PCR detection of Escherichia coli O157:H7

Aims: This paper reports a simple, rapid approach for the detection of Shiga toxin (Stx)‐producing Escherichia coli (STEC).

Comparison of EPR-Visible Cu2+ Sites in pMMO from Methylococcus capsulatus (Bath) and Methylomicrobium album BG8

X-band (9.1 GHz) and S-band (3.4 GHz) electron paramagnetic resonance (EPR) spectra for particulate methane monooxygenase (pMMO) in whole cells from Methylococcus capsulatus (Bath) grown on (63)Cu and (15)N were obtained and compared with previously reported spectra for pMMO from Methylomicrobium album BG8. For both M. capsulatus (Bath) and M. album BG8, two nearly identical Cu(2+) EPR signals with resolved hyperfine coupling to four nitrogens are observed. The EPR parameters for pMMO from M. capsulatus (Bath) (g( parallel) = 2.244, A( parallel) = 185 G, and A(N) = 19 G for signal one; g( parallel) = 2.246, A( parallel) = 180 G, and A(N) = 19 G for signal two) and for pMMO from M. album BG8 (g( parallel) = 2.243, A( parallel) = 180 G, and A(N) = 18 G for signal one; g( parallel) = 2. 251, A( parallel) = 180 G, and A(N) = 18 G for signal two) are very similar and are characteristic of type 2 Cu(2+) in a square planar or square pyramidal geometry. In three-pulse electron spin echo envelope modulation (ESEEM) data for natural-abundance samples, nitrogen quadrupolar frequencies due to the distant nitrogens of coordinated histidine imidazoles were observed. The intensities of the quadrupolar combination bands indicate that there are three or four coordinated imidazoles, which implies that most, if not all, of the coordinated nitrogens detected in the continuous wave spectra are from histidine imidazoles.

Detection of Helicobacter pylori in water by direct PCR

Aims:  This paper demonstrates a rapid, simple method for the detection of Helicobacter pylori in water that eliminates the need for recovery of cells or DNA extraction prior to PCR.

Intracellular localization of the particulate methane monooxygenase and methanol dehydrogenase in Methylomicrobium album BG8

Abstract. The methanotrophic bacterium Methylomicrobium album BG8 uses methane as a sole source of carbon and energy. This bacterium forms an extensive intracytoplasmic membrane. The first enzymes of the methane oxidation pathway are the membrane-bound particulate methane monooxygenase and the periplasmic methanol dehydrogenase. Immunoelectron microscopy with specific antibodies was used to localize these enzymes to the intracytoplasmic membrane.

Role of the H Protein in Assembly of the Photochemical Reaction Center and Intracytoplasmic Membrane in Rhodospirillum rubrum

Rhodospirillum rubrum is a model for the study of membrane formation. Under conditions of oxygen limitation, this facultatively phototrophic bacterium forms an intracytoplasmic membrane that houses the photochemical apparatus. This apparatus consists of two pigment-protein complexes, the light-harvesting antenna (LH) and photochemical reaction center (RC). The proteins of the photochemical components are encoded by the puf operon (LHalpha, LHbeta, RC-L, and RC-M) and by puhA (RC-H). R. rubrum puf interposon mutants do not form intracytoplasmic membranes and are phototrophically incompetent. The puh region was cloned, and DNA sequence determination identified open reading frames bchL and bchM and part of bchH; bchHLM encode enzymes of bacteriochlorophyll biosynthesis. A puhA/G115 interposon mutant was constructed and found to be incapable of phototrophic growth and impaired in intracytoplasmic membrane formation. Comparison of properties of the wild-type and the mutated and complemented strains suggests a model for membrane protein assembly. This model proposes that RC-H is required as a foundation protein for assembly of the RC and highly developed intracytoplasmic membrane. In complemented strains, expression of puh occurred under semiaerobic conditions, thus providing the basis for the development of an expression vector. The puhA gene alone was sufficient to restore phototrophic growth provided that recombination occurred.

Type 2 Cu2+ in pMMO from Methylomicrobium album BG8

EPR spectra were obtained for the type 2 Cu2+ site in particulate methane monooxygenase (pMMO) from Methylomicrobium album BG8 grown on K15NO3 and 63Cu(NO3)2. The concentration of the type 2 Cu2+ signal was approximately 200 microM per 25 mg/ml protein in packed cells and membrane fractions, a concentration that is consistent with its attribution to pMMO, and the EPR parameters were consistent with electron paramagnetic resonance (EPR) parameters previously assigned to pMMO. The superhyperfine structure due to nitrogen is better resolved because I = 1/2 for 15N whereas I = 1 for 14N and A(15N)/A(14N) = 1.4. Under these conditions, superhyperfine structure is resolved in the g region of the X-band spectrum. At low microwave frequency (S-band) the resolution of the nitrogen superhyperfine structure improves. Signals are attributed to type 2 Cu2+ in which cupric ion is bound to four (less likely three) nitrogen donor atoms.

Concentration of Cu, EPR-detectable Cu, and formation of cupric-ferrocyanide in membranes with pMMO

EPR spectra were obtained for the type 2 Cu(2+) site in particulate methane monooxygenase, pMMO, from membrane fractions of Methylomicrobium album BG8. In addition to the EPR signal with g parallel = 2.24 and A parallel = 185 G found in both cells and membrane fractions, a second EPR signal with g parallel = 2.29 and A parallel = 146 G was found in membrane fractions and attributed to oxidation of cuprous sites. Comparison of EPR-detectable Cu(2+) with total copper determined by atomic absorption suggests that there are two or three EPR-silent coppers for every EPR-detectable copper and that there are approximately four coppers per enzyme composed of the 47, 27, and 25 kDa subunits. Treatment of membrane fractions loaded with pMMO with Fe(CN)6(3-) results in a new EPR signal that is attributed to CuFe(CN)6(2-), not to an intrinsic trimeric copper cluster as previously reported in studies with a related bacterium.

A new system for heterologous expression of membrane proteins: Rhodospirillum rubrum

Heterologous expression of membrane proteins has met with only limited success. This work presents a new host/vector system for the production of heterologous membrane proteins based on a mutant of the facultatively phototrophic bacterium Rhodospirillum rubrum. Under certain growth conditions, R. rubrum forms an intracytoplasmic membrane (ICM) that houses the photosynthetic apparatus, the structural proteins of which are encoded by puhA and pufBALM. The mutant R. rubrum H2, which was constructed by allelic exchange deleting puhA and pufBALM, does not form ICM. This strain was used as a host for a plasmid expressing the Pseudomonas aeruginosa membrane protein MscL from the Rhodobacter capsulatus puc promoter. ICM was formed in the H2 strain producing MscL but not in the vector control strain. These results suggest that a heterologous membrane protein stimulates ICM formation in R. rubrum and indicate that the capacity to form an ICM that can accommodate heterologous proteins makes R. rubrum a host that will be useful for membrane protein production. P. aeruginosa MscL, which forms inclusion bodies when produced in Escherichia coli, was expressed in R. rubrum H2 and purified from membranes with a yield of 22.8-23.4 mg/L culture (5.53-5.60 mg/g cell paste). Additionally Streptomyces lividans KcsA and P. aeruginosa CycB were produced and purified from R. rubrum H2 with yields of 13.7-14.4 mg/L culture (2.19-2.55 mg/g cell paste) and 6.6-7.4 mg/L culture (1.1-1.2mg/g cell paste), respectively.

Identity of succinate dehydrogenase in chemotrophically and phototrophically grown Rhodospirillum rubrum

The specific succinate dehydrogenase (EC 1.3.99.1 succinate: phenazine methosulfate oxidoreductase) activity of membranes of aerobically grown Rhodospirillum rubrum was found to be 4–7-fold greater than that of membranes from phototrophically grown cells. The enzymes obtained from cells grown under both conditions were compared in crossed immunoelectrophoresis and were shown to have the same electrophoretic mobility and immunological identity. As succinate dehydrogenase activity is associated with chromatophore membrane in phototrophically grown R. rubrum, this study demonstrates the presence of a chromatophore-specific component in aerobic cells. The succinate dehydrogenase activity of Triton extracts of membranes from phototrophic cells was subject to photoinactivation. The succinate dehydrogenase extracted from the membranes from aerobic cells was photoinactivated only if a bacteriochlorophyll a extract was added to the enzyme preparation. As this photopigment-dependent photoinactivation was inhibited by NaN3 or by a N2 atmosphere, it is attributed to the effects of singlet oxygen sensitized by bacteriochlorophyll a.

Isolation of intracytoplasmic membrane from the Methanotrophic bacterium Methylomicrobium album BG8

Abstract. Methane-oxidizing bacteria, including Methylomicrobium album BG8, form an intracytoplasmic membrane in addition to the cytoplasmic and outer membranes of the cell envelope. Techniques to isolate the intracytoplasmic membrane of M. album BG8 were developed. An intracytoplasmic membrane fraction was separated from a cell envelope fraction on the basis of sedimentation velocity in sucrose density gradients. Proteins associated with the particulate methane monooxygenase were found in both membrane fractions.