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Objective: To examine attitudes, conflicts, images, circumstances, and time-period effects associated with corporal punishment and other forms of adult-to-child violence during the early 20th century in the United States. Method: A sample of 147 letters, referencing corporal punishment and dating from 1924 to 1939, were analyzed using both qualitative and quantitative techniques. The letters were addressed to Angelo Patri (1876-1965), a popular child-rearing expert during the interwar years (also known as the Machine Age), and written primarily by middle class parents with everyday worries about child rearing and proper discipline. Results: People who sought advice emphasized the practical significance of corporal punishment over and above the idea that it violated children's rights to be protected against harm. One in four letters cited conflicts with significant others about corporal punishment. Generally, children were perceived as frail, defiant, or feral. Rarely, were they seen as devilish or, conversely, innocent. Children's disobedience and disrespect were cited more than other misbehaviors as reasons for corporal punishment. Age and gender of the focal child varied by time period when letters from the 1920s and 1930s were compared. Conclusions: A full understanding of parent-to-child violence cannot be achieved without a firm grasp of its genealogy. The growing popularity of child psychology during the Machine Age had a measurable impact on how children were viewed. A utilitarian frame of interpretation was an important part of the everyday ''work'' associated with child rearing during this time, foreshadowing the tendency today to emphasize efficacy more than rights when evaluating the legitimacy of corporal punishment.

The release of non-disinfected wastewater into the marine environment is a common practice in many countries; nevertheless, the molecular mechanisms involved in determining the survival of enteric bacteria in seawater are poorly understood, in spite of the obvious public health implications. In a methodological attempt to address this issue, a plasmid-based collection of 687 Escherichia coli distinct promoter::luxCDABE fusions was screened to identify promoters that are induced upon exposure to seawater. The luminescence driven by 22 out of these promoters reproducibly increased at least two-fold in an artificial seawater medium; only 9 of the corresponding genes have previously been assigned a function. The most prominent characteristic of the induced genes was that most (18 out of 22) were under rpoS control. The induction of these seawater-responsive promoters was evaluated in different media to identify the cause of the increased transcription. Salinity or osmolarity was instrumental in only four cases, and in three promoters, increased pH also seemed to play a role; however, the most significant environmental effector in inducing the majority of the seawater-induced promoters appeared to be nutrient limitation. [ABSTRACT FROM AUTHOR]

The genome-wide transcription profile of Escherichia coli cells treated with hydrogen peroxide was examined with a DNA microarray composed of 4,169 E. coli open reading frames. By measuring gene expression in isogenic wild-type and oxyR deletion strains, we confirmed that the peroxide response regulator OxyR activates most of the highly hydrogen peroxide-inducible genes. The DNA microarray measurements allowed the identification of several new OxyR-activated genes, including the hemH heme biosynthetic gene; the six-gene suf operon, which may participate in Fe-S cluster assembly or repair; and four genes of unknown function. We also identified several genes, including uxuA, encoding mannonate hydrolase, whose expression might be repressed by OxyR, since their expression was elevated in the DeltaoxyR mutant strain. In addition, the induction of some genes was found to be OxyR independent, indicating the existence of other peroxide sensors and regulators in E. coli. For example, the isc operon, which specifies Fe-S cluster formation and repair activities, was induced by hydrogen peroxide in strains lacking either OxyR or the superoxide response regulators SoxRS. These results expand our understanding of the oxidative stress response and raise interesting questions regarding the nature of other regulators that modulate gene expression in response to hydrogen peroxide.

Acivicin, a modified amino acid natural product, is a glutamine analog. Thus, it might interfere with metabolism by hindering glutamine transport, formation, or usage in processes such as transamidation and translation. This molecule prevented the growth of Escherichia coli in minimal medium unless the medium was supplemented with a purine or histidine, suggesting that the HisHF enzyme, a glutamine amidotransferase, was the target of acivicin action. This enzyme, purified from E. coli, was inhibited by low concentrations of acivicin. Acivicin inhibition was overcome by the presence of three distinct genetic regions when harbored on multicopy plasmids. Comprehensive transcript profiling using DNA microarrays indicated that histidine biosynthesis was the predominant process blocked by acivicin. The response to acivicin, however, was quite complex, suggesting that acivicin inhibition resonated through more than a single cellular process.

Mitomycin C (MMC), a DNA-damaging agent, is a potent inducer of the bacterial SOS response; surprisingly, it has not been used to select resistant mutants from wild-type Escherichia coli. MMC resistance is caused by the presence of any of four distinct E. coli genes (mdfA, gyrl, rob, and sdiA) on high-copy-number vectors. mdfA encodes a membrane efflux pump whose overexpression results in broad-spectrum chemical resistance. The gyrI (also called sbmC) gene product inhibits DNA gyrase activity in vitro, while the rob protein appears to function in transcriptional activation of efflux pumps. SdiA is a transcriptional activator of ftsQAZ genes involved in cell division.

In Escherichia coli the amplification of sdiA, a positive activator of ftsQAZ, genes that are essential for septation, results in mitomycin C resistance. To help us understand this resistance phenotype, genes whose expression was altered by increased sdiA dosage were identified using a DNA microarray-based, comprehensive transcript profiling method. The expression of 62 genes was reduced by more than threefold; of these, 41 are involved in motility and chemotaxis. Moreover, the expression of 75 genes, 36 of which had been previously characterized, was elevated at least threefold. As expected, increased sdiA dosage led to significantly elevated sdiA and 'ddlB-ftsQAZ-lpxC operon expression. Transcription of two genes, uvrY and uvrC, located downstream of sdiA and oriented in the same direction, was elevated about 10-fold, although the intervening gene, yecF, of opposite polarity was unaffected by increased sdiA dosage. Three genes (mioC and gidAB) flanking the replication origin, oriC, were transcribed more often when sdiA dosage was high, as were 12 genes within 1 min of a terminus of replication, terB. Transcription of the acrABDEF genes, mapping in three widely spaced loci, was elevated significantly, while several genes involved in DNA repair and replication (e.g., nei, recN, mioC, and mcrC) were moderately elevated in expression. Such global analysis provides a link between septation and the response to DNA-damaging agents.

Gene expression profiling provides powerful analyses of transcriptional responses to cellular perturbation. In contrast to DNA array-based methods, reporter gene technology has been underused for this application. Here we describe a genomewide, genome-registered collection of Escherichia coli bioluminescent reporter gene fusions. DNA sequences from plasmid-borne, random fusions of E. coli chromosomal DNA to a Photorhabdus luminescens luxCDABE reporter allowed precise mapping of each fusion. The utility of this collection covering about 30% of the transcriptional units was tested by analyzing individual fusions representative of heat shock, SOS, OxyR, SoxRS, and cya/crp stress-responsive regulons. Each fusion strain responded as anticipated to environmental conditions known to activate the corresponding regulatory circuit. Thus, the collection mirrors E. coli's transcriptional wiring diagram. This genomewide collection of gene fusions provides an independent test of results from other gene expression analyses. Accordingly, a DNA microarray-based analysis of mitomycin C-treated E. coli indicated elevated expression of expected and unanticipated genes. Selected luxCDABE fusions corresponding to these up-regulated genes were used to confirm or contradict the DNA microarray results. The power of partnering gene fusion and DNA microarray technology to discover promoters and define operons was demonstrated when data from both suggested that a cluster of 20 genes encoding production of type I extracellular polysaccharide in E. coli form a single operon.

A nearly complete collection of 4,290 Escherichia coli open reading frames was amplified and arrayed in high density on glass slides. To exploit this reagent, conditions for RNA isolation from E. coli cells, cDNA production with attendant fluorescent dye incorporation, DNA-DNA hybridization, and hybrid quantitation have been established. A brief isopropyl-beta-D-thiogalactopyranoside (IPTG) treatment elevated lacZ, lacY, and lacA transcript content about 30-fold; in contrast, most other transcript titers remained unchanged. Distinct RNA expression patterns between E. coli cultures in the exponential and transitional phases of growth were catalogued, as were differences associated with culturing in minimal and rich media. The relative abundance of each transcript was estimated by using hybridization of a genomic DNA-derived, fluorescently labeled probe as a correction factor. This inventory provided a quantitative view of the steady-state level of each mRNA species. Genes the expression of which was detected by this method were enumerated, and results were compared with the current understanding of E. coli physiology.

Escherichia coli strains containing plasmid-borne fusions of the recA promoter-operator region to the Vibrio fischeri lux genes were previously shown to increase their luminescence in the presence of DNA damage hazards, and thus to be useful for genotoxicant detection. The present study expands previous work by demonstrating and investigating the luminescent response of these strains to ultraviolet radiation. Several genetic variants of the basic recA'::lux design were examined, including a tolC modification of membrane efflux capacity, a chromosomal integration of the recA'::lux fusion, a different lux reporter (Photorhabdus luminescens instead of V. fischeri, allowing the assay to be run at 37 degrees C), and a different host bacterium (Salmonella typhimurium instead of E. coli). Generally, two modifications provided the fastest responses: the use of the S. typhimurium host or the P. luminescens lux reporter. Highest sensitivity, however, was demonstrated in an E. coli strain in which a single copy of the V. fischeri lux fusion was integrated into the bacterial chromosome.

Quaternary ammonium functionalized poly(propyleneimine) dendrimers were synthesized and their antibacterial properties were evaluated using a bioluminescence method. These quaternary ammonium dendrimers are very potent biocides. The antibacterial properties depend on the size of the dendrimer, the length of hydrophobic chains in the quaternary ammonium groups, and the counteranion. Since these dendrimers are well characterized and monodisperse, they also serve as an effective system to study the structure-activity relationship. The antimicrobial properties of these dendrimer biocides have a parabolic dependence on molecular weight, which is different from the bell-shaped molecular weight dependence of conventional polymer biocides. The dependence on the hydrophobic chain of the quaternary ammonium structure is similar to conventional polymer biocides, and shows a parabolic relationship with dendrimer biocides carrying C10 hydrophobes the most potent. The antimicrobial properties of these novel biocides with bromide anions are more potent than those with chloride anions. Biocides derived from hyperbranched polymers were also synthesized and found to possess somewhat lower effectiveness.

The expression pattern of 1,529 yeast genes in response to sulfometuron methyl (SM) was analyzed by DNA microarray technology. SM, a potent herbicide, inhibits acetolactate synthase, a branched-chain amino acid biosynthetic enzyme. Exposure of yeast cells to 0.2 microg/ml SM resulted in 40% growth inhibition, a Gcn4p-mediated induction of genes involved in amino acid and cofactor biosynthesis, and starvation response. The accumulation of intermediates led to the induction of stress response genes and the repression of genes involved in carbohydrate metabolism, nucleotide biosynthesis, and sulfur assimilation. Extended exposure to SM led to a relaxation of the initial response and induction of sugar transporter and ergosterol biosynthetic genes, as well as repression of histone and lipid metabolic genes. Exposure to 5 microg/ml SM resulted in >98% growth inhibition and stimulated a similar initial expression change, but with no relaxation after extended exposure. Instead, more stress response and DNA damage repair genes become induced, suggesting a serious cellular consequence. Other salient features of metabolic regulation, such as the coordinated expression of cofactor biosynthetic genes with amino acid biosynthetic ones, were evident from our data. A potential link between SM sensitivity and ergosterol metabolism was uncovered by expression profiling and confirmed by genetic analysis.

Escherichia coli strains containing plasmid-borne fusions of Vibrio fischeri lux to the recA promoter-operator region were previously shown to be potentially useful for detecting genotoxicants. In an attempt to improve past performance, the present study examines several modifications and variations of this design, singly or in various combinations: (1) modifying the host cell's toxicant efflux capacity via a tolC mutation; (2) incorporating the lux fusion onto the bacterial chromosome, rather then on a plasmid; (3) changing the reporter element to a different lux system (Photorhabdus luminescens), with a broader temperature range; (4) using Salmonella typhimurium instead of an E. coli host. A broad spectrum of responses to pure chemicals as well as to industrial wastewater samples was observed. Generally, fastest responses were exhibited by Sal94, a S. typhimurium strain harboring a plasmid-borne fusion of V. fischeri lux to the E. coli recA promoter. Highest sensitivity, however, was demonstrated by DPD3063, an E. coli strain in which the same fusion was integrated into the bacterial chromosome, and by DPD2797, a plasmid-bearing tolC mutant. Overall, the two latter strains appeared to perform better and seemed preferable over the others. The sensor strains retained their sensitivity following a 2-month incubation after alginate-embedding, but at the cost of a significantly delayed response.

The recombinant Escherichia coli strain DPD2794 containing a recA::luxCDABE fusion is used to detect genotoxicity of various chemicals. Genotoxic agents were previously categorized into two groups, Direct DNA Damaging (DDD) agents and Indirect DNA Damaging (IDD) agents; these two groups have been distinguished with this strain. Minimum detectable concentrations of the DDD agents were about one to five orders of magnitude lower than those of the IDD agents. The response patterns of this strain to DDD agents differed from those to IDD agents in terms of kinetics and the forms of the dose-dependent response., (Copyright 1999 Elsevier Science B.V.)

We report here the first quantitative study of the branched-chain amino acid biosynthetic pathway in Salmonella typhimurium LT2. The intracellular levels of the enzymes of the pathway and of the 2-keto acid intermediates were determined under various physiological conditions and used for estimation of several of the fluxes in the cells. The results led to a revision of previous ideas concerning the way in which multiple acetohydroxy acid synthase (AHAS) isozymes contribute to the fitness of enterobacteria. In wild-type LT2, AHAS isozyme I provides most of the flux to valine, leucine, and pantothenate, while isozyme II provides most of the flux to isoleucine. With acetate as a carbon source, a strain expressing AHAS II only is limited in growth because of the low enzyme activity in the presence of elevated levels of the inhibitor glyoxylate. A strain with AHAS I only is limited during growth on glucose by the low tendency of this enzyme to utilize 2-ketobutyrate as a substrate; isoleucine limitation then leads to elevated threonine deaminase activity and an increased 2-ketobutyrate/2-ketoisovalerate ratio, which in turn interferes with the synthesis of coenzyme A and methionine. The regulation of threonine deaminase is also crucial in this regard. It is conceivable that, because of fundamental limitations on the specificity of enzymes, no single AHAS could possibly be adequate for the varied conditions that enterobacteria successfully encounter.

The first common enzyme of isoleucine and valine biosynthesis, acetolactate synthase (ALS), is specifically inhibited by the herbicide sulfometuron methyl (SM). To further understand the physiological consequences of flux alterations at this point in metabolism, Escherichia coli genes whose expression was induced by partial inhibition of ALS were sought. Plasmid-based fusions of random E. coli DNA fragments to Photorhabdus luminescens luxCDABE were screened for bioluminescent increases in actively growing liquid cultures slowed 25% by the addition of SM. From more than 8,000 transformants, 12 unique SM-inducible promoter-lux fusions were identified. The lux reporter genes were joined to seven uncharacterized open reading frames, f253a, f415, frvX, o513, o521, yciG, and yohF, and five known genes, inaA, IdcC, osmY, poxB, and sohA. Inactivation of the rpoS-encoded sigma factor, sigmaS, reduced basal expression levels of six of these fusions 10- to 200-fold. These six genes defined four new members of the sigmaS regulon, f253a, IdcC, yciG, and yohF, and included two known members, osmY and poxB. Furthermore, the weak acid salicylate, which causes cytoplasmic acidification, also induced increased bioluminescence from seven SM-inducible promoter-lux fusion-containing strains, namely, those with fusions of the sigmaS-controlled genes and inaA. The pattern of gene expression changes suggested that restricted ALS activity may result in intracellular acidification and induction of the sigmaS-dependent stress response.

A miniature bioreactor was fabricated as a contactor between biosensing cells and toxic materials. This miniature bioreactor (58 mL working volume) showed performance similar to that of a conventional bioreactor, as well as the advantages of easy installation, facile operation, and small medium requirements during long-term continuous operation. A performance evaluation measured the response to ethanol in continuous operation by using a recombinant bioluminescent Escherichia coli strain. Continuous cultures were repeatedly induced by the ethanol challenge. Steady-state cell concentrations (OD) were found to be decreased, the induced specific bioluminescence (SBL) peak value was found to be increased, and the peak response time, which is the time constant of this continuous monitoring system, was found to be decreased with increasing dilution rate. Finally on- and off-line bioluminescence monitoring was shown to be reliable, suggesting that this system is suitable for applications such as monitoring the influent and effluent streams of waste water biotreatment plants.

The effects of temperature, growth stage, and inducer (ethanol) concentration on the kinetics and magnitude of the stress response were investigated by using an Escherichia coli strain with the grpE heat shock promoter fused to the Vibrio fischeri lux genes. When stressed, the cells responded by changing the level of specific light emission, which was measured both on- and off-line. These measurements were used to characterize and optimize the sensitivity of the construct by determining the conditions at which the culture exhibited maximum specific bioluminescence and minimum response time to ethanol induction in batch cultivation. The results of the batch study were then applied to continuous cultivation, and the effect of dilution rate was determined. These results are of considerable interest in the development of an on-line biological sensor system for the detection and toxicity assessment of chemical pollutants.

To investigate the interactions of heavy metals with cells, a minimal medium for the growth of enteric bacteria using glycerol-2-phosphate as the sole phosphorus source was developed that avoided precipitation of Pb2+ with inorganic phosphate. Using this medium, spontaneous mutants of Escherichia coli resistant to addition of Pb(NO3)2 were isolated. Thirty-five independent mutants all conferred a low level of resistance. Disk diffusion assays on solid medium were used to survey the response of E. coli and Salmonella typhimurium mutants altered in global regulatory networks to Pb(NO3)2 and CdCl2. Strains bearing mutations in oxyR and rpoH were the most hypersensitive to these compounds. Based upon the response of strains completely devoid of isozymes needed to inactivate reactive oxygen species, this hypersensitivity to lead and cadmium is attributable to alteration in superoxide dismutase rather than catalase levels. Similar analysis of chaperone-defective mutants suggests that these metals damage proteins in vivo.

The herbicide suphometuron methyl inhibits the utilization of pyruvate and 2-ketobutyrate by the branched-chain amino acid biosynthetic enzyme acetolactate synthase. Eighteen insertions of the transposon Tn10 into the genome of Salmonella typhimurium LT2 caused hypersensitivity to this herbicide. Five of these insertions conferred a partial auxotrophic requirement. Concurrent herbicide sensitivity and heat-labile pantothenate auxotrophy was due to panD::Tn10 mutations, while coincident sulphometuron methyl sensitivity and thiamin auxotrophy was attributable to thiA::Tn10 mutations. The phenotypes of these mutations suggested that coenzyme A and thiamin pyrophosphate availability modulated the cells' response to sulphometuron methyl. A model suggesting a key role for 2-ketobutyrate accumulation in herbicide action is supported by the function of thiamin pyrophosphate in 2-ketoacid metabolism and the known role of a 2-ketoacid in coenzyme A synthesis.

The way in which proteins attain and maintain their final form is of fundamental importance. Recent work has focused on the role of a set of ubiquitous proteins, termed chaperonins, in the assembly of phage and multisubunit proteins. The range of chaperonin action is unknown; they could interact with most cellular polypeptides or have a limited subset of protein partners. Included in the chaperonin family is the essential heat-shock regulated Escherichia coli groEL gene product. Over-expression of the groE operon in E. coli causes enhanced assembly of heterologously expressed ribulose bisphosphate carboxylase subunits and suppresses the heat-sensitive mutant phenotype of several dnaA alleles. It has been inferred that suppression of heat-sensitive mutations is confined to dnaA alleles and that this confinement could reflect an interaction between the groE operon products and a dnaA protein aggregate at the replication origin. We now report that multiple copies of the groE operon suppress mutations in genes encoding several diverse proteins. Our data indicate a general role for the groE operon products, the GroEL and GroES proteins, in the folding-assembly pathways of many proteins.