Biomedical subjects

Gregory M Cook

Publications and source records attributed to Gregory M Cook.

26 records · Page 2Linked to original sources

PubMed2003

Bioenergetic properties of the thermoalkaliphilic Bacillus sp. strain TA2.A1.

The thermoalkaliphilic Bacillus sp. strain TA2.A1 was able to grow in pH-controlled batch culture containing a nonfermentable growth substrate from pH 7.5 to 10.0 with no significant change in its specific growth rate, demonstrating that this bacterium is a facultative alkaliphile. Growth at pH 10.0 was sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone, suggesting that a proton motive force (Deltap) generated via aerobic respiration was an obligate requirement for growth of strain TA2.A1. Strain TA2.A1 exhibited intracellular pH homeostasis as the external pH increased from 7.5 to 10.0; however, the maximum DeltapH generated over this pH range was only 1.1 units at an external pH of 9.5. The membrane potential (Deltapsi) was maintained between -114 mV and -150 mV, and little significant change was observed over the pH range for growth. In contrast, the Deltap declined from -164 mV at pH 7.5 to approximately -78 mV at pH 10.0. An inwardly directed sodium motive force (DeltapNa(+)) of -100 mV at pH 10.0 indicated that cellular processes (i.e., solute transport) dependent on a sodium gradient would not be affected by the adverse Deltap. The phosphorylation potential of strain TA2.A1 was maintained between -300 mV and -418 mV, and the calculated H(+)/ATP stoichiometry of the ATP synthase increased from 2.0 at pH 7.5 to 5.7 at pH 10.0. Based on these data, vigorous growth of strain TA2.A1 correlated well with the DeltapNa(+), phosphorylation potential, and the ATP/ADP ratio, but not with Deltap. This communication represents the first report on the bioenergetics of an extremely thermoalkaliphilic aerobic bacterium.

Adenosine Triphosphate↗

PubMed2003

Characterization of a vancomycin-resistant Enterococcus faecalis (VREF) isolate from a dog with mastitis: further evidence of a clonal lineage of VREF in New Zealand.

We report here on the characterization of a vancomycin-resistant Enterococcus faecalis (VREF) isolated from a dog with mastitis. The isolate was positive for the vanA, ermB, and tet(M) genes, with vanA and ermB carried on the same transferable plasmid. Comparison of this isolate with VREF from poultry and human sources in New Zealand demonstrated identical SmaI macrorestriction patterns and Tn1546-like elements. This is further evidence of a clonal lineage of VREF in New Zealand.

Animals↗

PubMed2002

A novel haem compound accumulated in Escherichia coli overexpressing the cydDC operon, encoding an ABC-type transporter required for cytochrome assembly.

cydDC genes encode a heterodimeric ABC transporter required for assembly of the membrane-bound cytochrome bd quinol oxidase and periplasmic cytochromes. Here, we demonstrate that overexpression of functional cydDC genes on a multicopy plasmid results in elevated levels of cytochromes b and d, but most notably formation in anaerobically grown cells of a novel haem-containing component P-574. The pigment has a distinctive absorbance at 574-579 nm and 448 nm in reduced minus oxidised spectra and renders over-producing cells reddish in colour. The highest levels of P-574 were observed in mutants (cydAB) in the structural genes for the polypeptides of cytochrome bd. P-574 is labile; its spectral signal is reduced in cells that are frozen-thawed or subjected to mechanical disruption. P-574 was not detected in cytoplasmic or periplasmic fractions and was predominantly associated with the cell membrane. P-574 did not bind CO or cyanide. Production of P-574 was dependent on haem biosynthesis indicating that it is a haem-containing molecule or derived from haem biosynthesis. These findings suggest that P-574 may result from association of a haem compound with overexpressed transporter subunits, but not with oxidase subunits, and are consistent with an intimate link between the transporter and haem processing during oxidase assembly.

ATP-Binding Cassette Transporters↗

PubMed2002

Phenotypic and molecular characterization of community occurring, Western Samoan phage pattern methicillin-resistant Staphylococcus aureus.

In New Zealand, it is estimated that greater than half of the methicillin-resistant Staphylococcus aureus (MRSA) strains recovered from patients belong to what has been termed Western Samoan phage pattern types 1 and 2 (WSPP1, WSPP2). These strains differ from classical MRSA isolates in terms of their lack of multiresistance and community occurrence, suggesting that such strains possess properties and/or characteristics different from those of other MRSA. To address this hypothesis, 10 WSPP1 and WSPP2 isolates from Western Samoa, New Zealand and Australia were compared with common hospital MRSA isolates. All WSPP isolates were identical with regard to pulsed-field gel electrophoretic pattern of SmaI-digested DNA, coagulase gene restriction fragment length polymorphism pattern and localization of mecA to a 194 kb SmaI digestion fragment. The WSPP strains were no more resistant/sensitive to various environmental stresses (e.g. skin fatty acids, UV light, desiccation) compared with hospital epidemic MRSA strains, except for their higher tolerance to salt. In terms of virulence, the WSPP MRSA were quantitatively better at attaching to the epithelial cell line HEp2, were uniformly egg-yolk opacity factor negative and produced higher levels of haemolytic toxins compared with non-WSPP MRSA isolates.

Australia↗

PubMed2001

Intracellular pH regulation by Mycobacterium smegmatis and Mycobacterium bovis BCG.

Mycobacteria are likely to encounter acidic pH in the environments they inhabit; however intracellular pH homeostasis has not been investigated in these bacteria. In this study, Mycobacterium smegmatis and Mycobacterium bovis [Bacille Calmette--Guérin (BCG)] were used as examples of fast- and slow-growing mycobacteria, respectively, to study biochemical and physiological responses to acidic pH. M. smegmatis and M. bovis BCG were able to grow at pH values of 4.5 and 5.0, respectively, suggesting the ability to regulate internal pH. Both species of mycobacteria maintained their internal pH between pH 6.1 and 7.2 when exposed to decreasing external pH and the maximum Delta pH observed was approximately 2.1 to 2.3 units for both bacteria. The Delta pH of M. smegmatis at external pH 5.0 was dissipated by protonophores (e.g. carbonyl cyanide m-chlorophenylhydrazone), ionophores (e.g. monensin and nigericin) and N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the proton-translocating F(1)F(0)-ATPase. These results demonstrate that permeability of the cytoplasmic membrane to protons and proton extrusion by the F(1)F(0)-ATPase plays a key role in maintaining internal pH near neutral. Correlations between measured internal pH and cell viability indicated that the lethal internal pH for both strains of mycobacteria was less than pH 6.0. Compounds that decreased internal pH caused a rapid decrease in cell survival at acidic pH, but not at neutral pH. These data indicate that both strains of mycobacteria exhibit intracellular pH homeostasis and this was crucial for the survival of these bacteria at acidic pH values.

Cell Membrane Permeability↗

PubMed2000

Oxidase and periplasmic cytochrome assembly in Escherichia coli K-12: CydDC and CcmAB are not required for haem-membrane association.

The mechanism(s) that bacteria use to transport haem into and across the cytoplasmic membrane to complete the assembly of periplasmic cytochromes is unknown. The authors have tested directly the role(s) of two ATP-binding cassette (ABC) transporters - the cydDC and ccmAB gene products - in Escherichia coli by measuring haem uptake in everted (inside-out) membrane vesicles. If haem is exported to the periplasm in vivo, the same process should result in active accumulation in such everted vesicles. [14C]Haemin (chloride) with bovine serum albumin (BSA) as a carrier protein was accumulated in intact everted membrane vesicles by an energy-independent mechanism. The kinetics of this process were biphasic: rapid uptake/binding was followed by a slower uptake of haem, which was inhibited by a large excess of unlabelled haemin-BSA, but not by BSA. However, accumulated haemin was not chased out of the vesicles by unlabelled haemin-BSA, suggesting specific binding of haemin with the membrane or transport into the lumen of the vesicle. Neither ATP nor a protonmotive force (delta(p)) generated by lactate oxidation was required for haemin binding or subsequent transport, and carbonyl cyanide m-chlorophenylhydrazone (CCCP), sodium vanadate and monensin had no effect on haemin transport. The rate of haemin uptake following the initial rapid binding was proportional to the external haemin concentration, suggesting that the uptake process was driven by the haemin concentration gradient across the cell membrane. The kinetics of [14C]haemin uptake were similar in wild-type and cydD1 or delta(ccmA) mutants, suggesting that the activity of neither the CydDC nor CcmAB transporters is essential for haem export to the periplasm. Cytochrome d levels were unaffected by mutations in trxB (encoding thioredoxin reductase), trxA (thioredoxin), or grx (glutaredoxin), suggesting that the CydDC transporter does not export these components of reducing pathways for cytochrome assembly.

ATP-Binding Cassette Transporters↗

PubMed1998

A factor produced by Escherichia coli K-12 inhibits the growth of E. coli mutants defective in the cytochrome bd quinol oxidase complex: enterochelin rediscovered.

Escherichia coli produces an extracellular factor that inhibits the aerobic growth of Cyd- mutants, defective in the synthesis or assembly of the cytochrome bd-type quinol oxidase. This paper shows that such a factor is the iron-chelating siderophore enterochelin. Mutants in entA or aroB, defective in the production of enterochelin, did not produce the factor that inhibits the growth of cydAB and cydDC mutants; purified enterochelin inhibited the growth of Cyd- mutants, but not that of wild-type cells. Other iron-chelating agents, particularly ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA), whose complex with Fe(III) has a large stability constant (log K = 33.9), also inhibited the growth of Cyd- mutants at micromolar concentrations, but not that of wild-type cells. Supplementation of agar plates with Fe(III) or boiled catalase prevented the inhibition of Cyd- mutants by the extracellular factor. Spontaneous mutants isolated by being able to grow in the presence of the extracellular factor on plates also showed increased resistance to iron chelators. The reducing agent ascorbate, ascorbate plus In(III), ascorbate plus Ga(III), or Ga(III) alone, also alleviated inhibition by the extracellular factor, presumably by reducing iron to Fe(II) and complexing of the siderophore with alternative trivalent metal cations. The preferential inhibition of Cyd- mutants by the extracellular factor and other iron chelators is not due to decrease in expression, activity or assembly of cytochrome bo', the major alternative oxidase mediating quinol oxidation. Cyd- mutants overproduce siderophores, presumably reflecting intracellular iron deprivation.

Ascorbic Acid↗

PubMed1996

Catabolite repression and inducer control in Gram-positive bacteria.

Results currently available clearly indicate that the metabolite-activated protein kinase-mediated phosphorylation of Ser-46 in HPr plays a key role in catabolite repression and the control of inducer levels in low-GC Gram-positive bacteria. This protein kinase is not found in enteric bacteria such as E. coli and Salmonella typhimurium where an entirely different PTS-mediated regulatory mechanism is responsible for catabolite repression and inducer concentration control. In Table 2 these two mechanistically dissimilar but functionally related processes are compared (Saier et al., 1995b). In Gram-negative enteric bacteria, an external sugar is sensed by the sugar-recognition constituent of an Enzyme II complex of the PTS (IIC), and a dephosphorylating signal is transmitted via the Enzyme IIB/HPr proteins to the central regulatory protein, IIAGlc. Targets regulated include (1) permeases specific for lactose, maltose, melibiose and raffinose, (2) catabolic enzymes such as glycerol kinase that generate cytoplasmic inducers, and (3) the cAMP biosynthetic enzyme, adenylate cyclase that mediates catabolite repression (Saier, 1989, 1993). In low-GC Gram-positive bacteria, cytoplasmic phosphorylated sugar metabolites are sensed by the HPr kinase which is allostericlaly activated. HPr becomes phosphorylated on Ser-46, and this phosphorylated derivative regulates the activities of its target proteins. These targets include (1) the PTS, (2) non-PTS permeases (both of which are inhibited) and (3) a cytoplasmic sugar-P phosphatase which is activated to reduce cytoplasmic inducer levels. Other important targets of HPr(ser-P) action are (4) the CcpA protein and probably (5) the CepB transcription factor. These two proteins together are believed to determine the intensity of catabolite repression. Their relative importance depends on physiological conditions. Both proteins may respond to the cytoplasmic concentration of HPr(ser-P) and appropriate metabolites. CepA possibly binds sugar metabolites such as FBP as well as HPr(ser-P). Because HPr(his-P, ser-P) does not bind to CepA, the regulatory cascade is also sensitive to the external PTS sugar concentration. Mutational analyses (unpublished results) suggest that CepA may bind to a site that includes His-15. Interestingly, both the CepA protein in the Gram-positive bacterium, B. subtilis, and glycerol kinase in the Gram-negative bacterium, E. coli, sense both a PTS protein and a cytoplasmic metabolic intermediate. The same may be true of target permeases and enzymes in both types of organisms, but this possibility has not yet been tested. The parallels between the Gram-negative and Gram-positive bacterial regulatory systems are superficial at the mechanistic level but fundamental at the functional level. Thus, the PTS participates in regulation in both cases, and phosphorylation of its protein constituents plays key roles. However, the stimuli sensed, the transmission mechanisms, the central PTS regulatory proteins that effect allosteric regulation, and some of the target proteins are completely different. It seems clear that these two transmission mechanisms evolved independently. They provide a prime example of functional convergence.

Adenosine Triphosphate↗