Automated food microbiology: potential for the hydrophobic grid-membrane filter.
Bacterial counts obtained on hydrophobic grid-membrane filters were comparable to conventional plate counts for Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus in homogenates from a range of foods. The wide numerical operating range of the hydrophobic grid-membrane filters allowed sequential diluting to be reduced or even eliminated, making them attractive as components in automated systems of analysis. Food debris could be rinsed completely from the unincubated hydrophobic grid-membrane filter surface without affecting the subsequent count, thus eliminating the possibility of counting food particles, a common source of error in electronic counting systems. (+info
Effects of dispersed recreational activities on the microbiological quality of forest surface water.
The microbiological quality of forest surface waters in the Greenwater River watershed was examined to investigate the influence of heavy motorized camping in an area with no sanitary facilities. Indicator densities increased during weekend human-use periods when compared to weekdays. Increases in indicator densities were also noted downstream from heavily used camping areas when compared to upstream sites. Seasonal, weekly, and diurnal fluctuations in indicator densities were observed. This study suggests that potential health hazards exist in this watershed during periods of human use. (+info
Fecal coliform elevated-temperature test: a physiological basis.
The physiological basis of the Eijkman elevated-temperature test for differentiating fecal from nonfecal coliforms was investigated. Manometric studies indicated that the inhibitory effect upon growth and metabolism in a nonfecal coliform at 44.5 degrees C involved cellular components common to both aerobic and fermentative metabolism of lactose. Radioactive substrate incorporation experiments implicated cell membrane function as a principal focus for temperature sensitivity at 44.5 degrees C. A temperature increase from 35 to 44.5 degrees C drastically reduced the rates of [14C]glucose uptake in nonfecal coliforms, whereas those of fecal coliforms were essentially unchanged. In addition, relatively low levels of nonfecal coliform beta-galactosidase activity coupled with thermal inactivation of this enzyme at a comparatively low temperature may also inhibit growth and metabolism of nonfecal coliforms at the elevated temperature. (+info
Mechanism and specificity of the terminal thioesterase domain from the erythromycin polyketide synthase.
BACKGROUND: Polyketides are important compounds with antibiotic and anticancer activities. Several modular polyketide synthases (PKSs) contain a terminal thioesterase (TE) domain probably responsible for the release and concomitant cyclization of the fully processed polyketide chain. Because the TE domain influences qualitative aspects of product formation by engineered PKSs, its mechanism and specificity are of considerable interest. RESULTS: The TE domain of the 6-deoxyerythronolide B synthase was overexpressed in Escherichia coli. When tested against a set of N-acetyl cysteamine thioesters the TE domain did not act as a cyclase, but showed significant hydrolytic specificity towards substrates that mimic important features of its natural substrate. Also the overall rate of polyketide chain release was strongly enhanced by a covalent connection between the TE domain and the terminal PKS module (by as much as 100-fold compared with separate TE and PKS 'domains'). CONCLUSIONS: The inability of the TE domain alone to catalyze cyclization suggests that macrocycle formation results from the combined action of the TE domain and a PKS module. The chain-length and stereochemical preferences of the TE domain might be relevant in the design and engineered biosynthesis of certain novel polyketides. Our results also suggest that the TE domain might loop back to catalyze the release of polyketide chains from both terminal and pre-terminal modules, which may explain the ability of certain naturally occurring PKSs, such as the picromycin synthase, to generate both 12-membered and 14-membered macrolide antibiotics. (+info
Cytochrome P450 monooxygenases and insecticide resistance in insects.
Cytochrome P450 monooxygenases are involved in many cases of resistance of insects to insecticides. Resistance has long been associated with an increase in monooxygenase activities and with an increase in cytochrome P450 content. However, this increase does not always account for all of the resistance. In Drosophila melanogaster, we have shown that the overproduction of cytochrome P450 can be lost by the fly without a corresponding complete loss of resistance. These results prompted the sequencing of a cytochrome P450 candidate for resistance in resistant and susceptible flies. Several mutations leading to amino-acid substitutions have been detected in the P450 gene CYP6A2 of a resistant strain. The location of these mutations in a model of the 3D structure of the CYP6A2 protein suggested that some of them may be important for enzyme activity of this molecule. This has been verified by heterologous expression of wild-type and mutated cDNA in Escherichia coli. When other resistance mechanisms are considered, relatively few genetic mutations are involved in insecticide resistance, and this has led to an optimistic view of the management of resistance. Our observations compel us to survey in more detail the genetic diversity of cytochrome P450 genes and alleles involved in resistance. (+info
A single membrane-embedded negative charge is critical for recognizing positively charged drugs by the Escherichia coli multidrug resistance protein MdfA.
The nature of the broad substrate specificity phenomenon, as manifested by multidrug resistance proteins, is not yet understood. In the Escherichia coli multidrug transporter, MdfA, the hydrophobicity profile and PhoA fusion analysis have so far identified only one membrane-embedded charged amino acid residue (E26). In order to determine whether this negatively charged residue may play a role in multidrug recognition, we evaluated the expression and function of MdfA constructs mutated at this position. Replacing E26 with the positively charged residue lysine abolished the multidrug resistance activity against positively charged drugs, but retained chloramphenicol efflux and resistance. In contrast, when the negative charge was preserved in a mutant with aspartate instead of E26, chloramphenicol recognition and transport were drastically inhibited; however, the mutant exhibited almost wild-type multidrug resistance activity against lipophilic cations. These results suggest that although the negative charge at position 26 is not essential for active transport, it dictates the multidrug resistance character of MdfA. We show that such a negative charge is also found in other drug resistance transporters, and its possible significance regarding multidrug resistance is discussed. (+info
Membrane deinsertion of SecA underlying proton motive force-dependent stimulation of protein translocation.
The proton motive force (PMF) renders protein translocation across the Escherichia coli membrane highly efficient, although the underlying mechanism has not been clarified. The membrane insertion and deinsertion of SecA coupled to ATP binding and hydrolysis, respectively, are thought to drive the translocation. We report here that PMF significantly decreases the level of membrane-inserted SecA. The prlA4 mutation of SecY, which causes efficient protein translocation in the absence of PMF, was found to reduce the membrane-inserted SecA irrespective of the presence or absence of PMF. The PMF-dependent decrease in the membrane-inserted SecA caused an increase in the amount of SecA released into the extra-membrane milieu, indicating that PMF deinserts SecA from the membrane. The PMF-dependent deinsertion reduced the amount of SecA required for maximal translocation activity. Neither ATP hydrolysis nor exchange with external SecA was required for the PMF-dependent deinsertion of SecA. These results indicate that the SecA deinsertion is a limiting step of protein translocation and is accelerated by PMF, efficient protein translocation thereby being caused in the presence of PMF. (+info
Hsp60 is targeted to a cryptic mitochondrion-derived organelle ("crypton") in the microaerophilic protozoan parasite Entamoeba histolytica.
Entamoeba histolytica is a microaerophilic protozoan parasite in which neither mitochondria nor mitochondrion-derived organelles have been previously observed. Recently, a segment of an E. histolytica gene was identified that encoded a protein similar to the mitochondrial 60-kDa heat shock protein (Hsp60 or chaperonin 60), which refolds nuclear-encoded proteins after passage through organellar membranes. The possible function and localization of the amebic Hsp60 were explored here. Like Hsp60 of mitochondria, amebic Hsp60 RNA and protein were both strongly induced by incubating parasites at 42 degreesC. 5' and 3' rapid amplifications of cDNA ends were used to obtain the entire E. histolytica hsp60 coding region, which predicted a 536-amino-acid Hsp60. The E. histolytica hsp60 gene protected from heat shock Escherichia coli groEL mutants, demonstrating the chaperonin function of the amebic Hsp60. The E. histolytica Hsp60, which lacked characteristic carboxy-terminal Gly-Met repeats, had a 21-amino-acid amino-terminal, organelle-targeting presequence that was cleaved in vivo. This presequence was necessary to target Hsp60 to one (and occasionally two or three) short, cylindrical organelle(s). In contrast, amebic alcohol dehydrogenase 1 and ferredoxin, which are bacteria-like enzymes, were diffusely distributed throughout the cytosol. We suggest that the Hsp60-associated, mitochondrion-derived organelle identified here be named "crypton," as its structure was previously hidden and its function is still cryptic. (+info