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Cellular RNA polymerase in association with plasmid DNA segregates into the minicells of minicell-producing strains. In general, one "plasmid equivalent" of RNA polymerase, reflecting the size of the segregating plasmid DNA and its efficiency of segregation, entered the minicell with the plasmid. The amount of RNA polymerase (measured as the amount of enzyme activity purified from minicells and the rate of RNA synthesis in plasmid-containing minicells), and not the DNA content, appeared to be rate-limiting in plasmid-mediated transcription in minicells. The purified minicell and cellular RNA polymerases showed the same sensitivity to rifampin and streptolydigin; both were associated with sigma factor, although the minicell enzyme appeared to have slightly less than the cellular enzyme. These studies demonstrate that transcription of plasmid DNA in minicells is a function of the efficiency of segregation and the amount of RNA polymerase which enters with the plasmid DNA. Because RNA polymerase is limiting, plasmids with relatively weak promoters for the vector genes should be used when attempting to identify products from inserted foreign DNA.

Tetracycline resistance encoded by four genetically different determinants residing on plasmids in Escherichia coli was shown to be associated in each case with an energy-dependent decrease in accumulation of the antibiotic in whole cells in which resistance had been induced. The different class determinants examined were those on plasmids RP1 (class A), R222 (class B), R144 (class C), and RA1 (class D). This decrease in accumulation was attributable to an active efflux, because everted (inside-out) membrane vesicles made from tetracycline-induced E. coli cells containing any one of the four plasmids were shown to concentrate tetracycline by an active influx. This active uptake was not seen in inside-out vesicles from sensitive cells or uninduced R222-containing cells. In vesicles from induced R222-containing cells, the efflux appeared to be carrier-mediated with a Km of about 6 microM. These results demonstrate that active export of tetracycline is a common component of the mechanism for tetracycline resistance encoded by different plasmid-borne determinants in bacteria.

Escherichia coli sensitivity to tetracycline involves transport and accumulation of the antibiotic within the cell by two different uptake systems: an initial rapid uptake, which occurs over the initial 6 min of contact of the cell with tetracycline, and a slower uptake system, which continues indefinitely and whose rate of uptake is 1/10 that of the rapid system. Only the slow uptake system is blocked by inhibitors of energy-driven systems; it appears to be particularly dependent upon energy from oxidative phosphorylation. Although both uptake systems lead to accumulation of intracellular tetracycline and contribute to the cell's sensitivity, the rapid uptake system appears to be the more important. While these studies confirm active transport of tetracycline into the cell, they demonstrate that a critical uptake system which appears insensitive to metabolic inhibitors occurs initially.

An Enterobacter cloacae strain isolated from the faeces of a child with diarrhoea in Indonesia contained a transferable 216 MDa plasmid, pIN32, exhibiting IncHI2 phenotypic characters, including temperature sensitivity of transfer and the expression of H serotype pili at a repressed level. A derivative plasmid (pIN32-1), which had lost the IncHI2 phenotype, and contained only 60 MDa of the original replicon, was obtained after mating at 37 degrees C. It was IncFII, showed regions of homology with plasmid R100, determined IncFII serotype conjugative pili constitutively and was transfer-derepressed. After overnight growth at 37 degrees C in non-selective medium, pIN32 gave rise to another derivative, pIN32-2 (size 184.3 MDa), which retained the IncHI2 phenotype and several other pIN32 characters.

A unique form of congenital ichthyosis in two unrelated patients is described and characterized histologically by separation of the epidermis between the stratum corneum and the stratum granulosum. The clinical history, genetics, serially performed skin biopsies, and biochemical studies are reviewed. This form of ichthyosis is different from previously described entities. Lifelong peeling of the general body epidermis, pruritus, short stature, easily removed anagen hairs, and the ability to easily mechanically separate stratum corneum from the rest of the epidermis characterize the syndrome. In two families with this disorder, autosomal recessive inheritance is suggested. A low plasma tryptophan level as present in two patients with this disease. This inherited disorder of the epidermis was first described in 1924 before the genetics and histology of ichthyosis were extensively studied and is a distinct genetic and clinical entity to be considered in unusual cases of ichthyosis.

We previously found that murine leukemia cells of T cell, B cell, and erythroid ontogeny express a cell membrane antigen that cross-reacts with an idiotype of an anti-retroviral antibody. In the present study, the expression of this antigen (termed AVID, for anti-viral idiotype) by murine erythroleukemia (MEL) cells was examined during chemically induced differentiation. AVID expression by MEL cells was found to be lost when they were treated with either dimethyl sulfoxide or hexamethylene bisacetamide, two chemicals that induce MEL cells to terminally differentiate. The kinetics of disappearance of AVID during inducer treatment reflected the kinetics with which the inducers caused MEL cell commitment to terminal differentiation. Loss of AVID expression by inducer-treated cells was inhibited by dexamethasone, which inhibits commitment and MEL cell differentiation. The subset of inducer-treated cells that expressed the least amount of AVID contained the greatest number of cells committed to differentiate. These results indicate that AVID identifies a novel differentiation antigen of MEL cells.

A Tn5 insertional mutation on the Escherichia coli chromosome which caused a severalfold increase in susceptibility to structurally and functionally diverse antibiotics was found to map within the gene for polynucleotide phosphorylase (pnp) and to inactivate this enzyme, which is involved in RNA breakdown. The mutation also decreased the growth rate 10 to 25% and increased the rate of tetracycline uptake about 30%. The hypersensitivity due to the insertion was only partially complemented by a cloned pnp gene.

The fate of spontaneous chromosomal rifampin-resistant (Rifr) mutants of Pseudomonas putida and Pseudomonas fluorescens in sterile and live organic soil from which they were isolated was studied. In sterile native-soil assays, a Rifr mutant of P. putida showed no decrease in competitive fitness when compared with the wild-type parent. However, mutants of P. fluorescens were of two general categories. Group 1 showed no difference from the wild type in terms of growth rate, competitive fitness, and membrane protein composition. Group 2 showed a slower growth rate in both minimal and enriched media and an altered membrane protein profile. These mutants also demonstrated decreased competitive fitness compared with the wild-type strain. In live soil, the Rifr P. putida strain persisted throughout the 38-day test period with a decay rate of 0.7 log10 CFU/g of soil per 10 days. A group 1 Rifr P. fluorescens mutant maintained its inoculated titer for 7 to 10 days and then decayed at a rate of 0.2 to 0.4 log10 CFU/g of soil per 10 days. A group 2 Rifr P. fluorescens mutant remained at its titer for 1 to 5 days before decaying at a two- to threefold-faster rate. These findings indicate that rifampin resistance may not be an innocuous mutation in some pseudomonads and that marked strains should be compared with wild-type parents before being used as monitors of parental strain survival. Colonization of sterile soil with either the wild-type or mutant strain precluded normal colonization of the second added strain.(ABSTRACT TRUNCATED AT 250 WORDS)

We have detected Ca2+, Mg2+-dependent endonuclease activity in spleen cells of normal, Friend erythroleukemic, and phenylhydrazine-treated mice. When nuclei were isolated and incubated in the presence of Ca2+ and Mg2+ ions, the activity resulted in the production of 3'-OH termini in the cellular DNA and the release of chromatin due to internucleosomal DNA fragmentation. This enzyme activity was chromatin-bound and could be extracted from chromatin in an active form in 0.35 M KCl. The majority of endonuclease activity from erythroleukemic spleens was present in nuclei of precursor erythroid cells of low buoyant density (1.025-1.05 g/ml). Uninfected normal splenic tissue contained an endonuclease activity which was almost entirely confined to a B-lymphocyte population of high buoyant density (greater than 1.07 g/ml). Erythroid cell-enriched spleens from phenylhydrazine-treated mice exhibited a distribution of endonuclease activity in cells at low and high densities reflecting a mixture of erythroid and lymphoid cells. Cloned erythroleukemic cell lines propagated in vitro lacked cells of low density and showed no detectable endonuclease activity. However, nuclei from these cell lines were susceptible to exogenously added endonuclease extracted from erythroleukemic spleen cells. These same cell lines propagated as subcutaneous tumors contained endonuclease activity and a morphologically-similar low-density cell population which accounted for the endonuclease activity in these tumors. Nuclei from cloned lymphoid cell lines, representing different B-lymphocyte phenotypes, showed differences in the presence of endonuclease activity. Among the cell lines tested, only those expressing late B-cell markers showed detectable endonuclease activity.

Derivatives of bacteriophages lambda and M13 are in common use as vectors in recombinant DNA RESEARCH. These laboratory-derived phages have been designed to allow cloning of DNA fragments, but to be unable to survive outside a defined laboratory and/or host-cell environment. To assess the availability of wild-type lambda or M13 phages in the environment which might potentially rescue debilitated derivative phages, we have now examined the frequency of these and other bacteriophages in human and animal faeces. We detected coliphage in over two-thirds of the faecal samples. Of these, 1.2% of the samples contained lambda-like phage and 3.5% had phage indistinguishable from M13.

The class L (TetL) tetracycline resistance determinant from streptococci specified resistance and an energy-dependent decreased accumulation of tetracycline in both Streptococcus faecalis and Escherichia coli. Using E. coli, we showed that the reduced uptake resulted from active efflux. The streptococcal class M determinant, known to render the protein synthesis machinery of S. faecalis resistant to tetracycline inhibition, did not alter tetracycline transport in either host.

The radiologic finding of innumerable fine ridges in the outer soft tissues of a patient with severe generalized epidermolytic hyperkeratosis is presented as a sign of the disease. The radiodensity results from the density of keratin and low water content in the scales of epidermolytic hyperkeratosis.

Escherichia coli bearing a cryptic tetracycline resistance determinant from Bacteroides fragilis expressed low-level constitutive resistance to tetracycline under aerobic, but not anaerobic, growth conditions and accumulated less tetracycline aerobically than did isogenic susceptible cells. This decreased uptake was energy dependent and reversible by increased concentrations of tetracycline, suggesting a saturable carrier-mediated active efflux mechanism. Decreased uptake was not seen when the cells were grown and assayed anaerobically. Other tetracycline resistance determinants (classes A to E) isolated from gram-negative enteric bacteria expressed resistance and generated active efflux of tetracycline under anaerobic as well as aerobic conditions. When the Bacteroides determinant was placed in the same cell with any of the class A to E tetracycline resistance determinants, there was an increase in resistance under aerobic conditions of as much as 48% more than was projected by adding the resistances expressed by the determinants individually. In cells bearing the class A determinant together with the Bacteroides determinant, saturation of the active efflux system required over twofold more exogenous tetracycline than did cells bearing the class A determinant alone. We have designated this new tetracycline resistance determinant class F.

The structural gene region for tetracycline resistance on Tn10 consists of two complementation groups, tetA and tetB (M. S. Curiale and S. B. Levy, J. Bacteriol. 151:209-215, 1982). Using a series of deletion mutants, we have determined that the tetA region is 450 to 600 base pairs long and that the tetB region, which is adjacent to tetA, is 600 to 750 base pairs long. Point mutations in either tetA or tetB affected the amount and size of the inducible inner-membrane Tet protein synthesized in Escherichia coli maxicells. Moreover, deletions in these regions led to the synthesis of an appropriately smaller Tet protein. A single tetracycline-inducible RNA of about 1,200 bases was detected that was homologous with the tetracycline resistance structural gene region. These results indicate that the tetA and tetB complementation regions represent two parts of a single gene encoding two domains of the tetracycline resistance protein Tet.

Serial examination of five newly derived Friend murine tumors during early subcutaneous passages showed continuing changes in chromatin composition and structure over the first 10 to 20 passages followed by a period of stabilization over the subsequent 20 passages. These changes were reflected in a decrease in two major histone variants, H2A.1 and H2B.2, with a coordinate increase in histone variants, H2A.2 and H2B.1, and a changing nucleosome repeat length (NRL). The absolute values differed among the five tumors, but all five showed the same general direction of change. There was no obvious relationship among the NRL, H2A, and H2B histone variant values. A low H2A.1/H2A.2 ratio was found in Friend tumors of high malignant potential. Cell lines derived in vitro also showed directional changes in the H2A and H2B variants similar to those of their tumor cell parents, but with different kinetics. Our findings suggest that Friend tumor establishment is accompanied by an early period of chromatin reorganization marked by changes in several parameters of chromatin structure.

Tetracycline resistance (Tcr) determinants from three different strains of Haemophilus parainfluenzae expressed 10-fold higher levels of resistance when mated into Escherichia coli. No plasmid was found in any of the E. coli recipients, even in matings in which a plasmid was identified in the donor Haemophilus sp. The Tcr determinant from Haemophilus sp. caused instability of resident plasmids in the recipient E. coli: all plasmids were lost within 30 generations in antibiotic-free media. However, by serial subculture in antibiotics, stable resident plasmids were obtained which carried the Tcr determinant from Haemophilus sp. and were transferable by conjugation and transformation among E. coli strains. All Haemophilus determinants hybridized with a probe for the Tcr determinant on Tn10, which bears inducible Tcr. However, Haemophilus determinants were constitutively resistant to tetracycline in the Haemophilus donors and in the E. coli recipients. This constitutive expression was recessive to wild-type Tn10 in the same cell, indicating that the constitutive phenotype resulted from the absence of an active repressor. Restrictive enzyme analysis of various E. coli plasmid derivatives bearing a Tcr determinant from Haemophilus sp. demonstrated that the inserted DNA was of similar size (8.95 to 9.35 kilobases), close to that of Tn10. Heteroduplex analysis and DNA:DNA hybridization confirmed that the Tcr determinant from Haemophilus sp. had greater than 90% homology with the Tn10 determinant, including the DNA sequence for the repressor.

Studies of F plasmids in the same cell as a transfer-repressed IncFII R plasmid showed a 100 to 1,000-fold decrease in transfer and a 75-fold decrease in surface exclusion, but no detectable change was shown in the amount of TraTp synthesized. Moreover, a mutation in traJ on F which caused a 10(4)-fold reduction in transfer caused only a 3.6-fold decrease in TraTp. These two findings suggest that a significant amount of traT expression on F is independent of traJ. Furthermore, we showed, using immunoprecipitation of TraTp, that normal amounts of this protein could be present in the cell without producing normal levels of surface exclusion.

A prospective study was undertaken to determine whether feeding farm animals antibiotics in feed caused changes in the intestinal bacterial flora of farm dwellers and their neighbors. Chickens were fed tetracycline-supplemented feed (tet-feed), and, as expected, within one week their intestinal flora contained almost entirely tetracycline-resistant organisms. Increased numbers of resistant intestinal bacteria also appeared, but more slowly, in farm members, but not their neighbors. Within five and six months, 31.3 per cent of weekly fecal samples from farm dwellers contained greater than 80 per cent tetracycline-resistant bacteria as compared to 6.8 per cent of the samples from the neighbors (P less than 0.001). Seven of the 11 farm members, but only three of the 24 neighbors, had two or more fecal samples containing greater than 80 per cent tetracycline-resistant coliforms (P less than 0.01). These resistant bacteria contained transferable plasmids conferring multiple antibiotic resistances. Selective pressure by tet-feed for antibiotic-resistant bacteria in chickens extends to human beings in contact with chickens and the feed.

Specific gene transcription from chromatin has been examined in two different systems by using direct measurement of specific sequences by hybridization with excess DNA probe. An improved procedure has been used for hybridization of DNA immobilized on filters. This procedure is sensitive to less than one part in 10(5). Polyoma RNA sequences were detected a level of 0.004% in the overall transcript from chromatin of ts-a-transformed 3T3 fibroblasts. By the use of constructed plasmid DNA carrying rabbit globin sequences, globin RNA was titrated in the RNA transcribed from chromatin of bone marrow cells. Its relative frequency was 0.01%. Results obtained with this new approach confirm that DNA in chromatin is not randomly transcribed. They further illustrate that a reproducible assay system is now available for studying control elements active on gene expression at the transcriptional level, taking advantage of the constructed plasmids containing eukaryotic gene DNA fragments.

We investigated the relationship of the kallikrein-kinin system and the renin-angiotensin system in the regulation of blood pressure, salt and water excretion, and renal blood flow. Normotensive and hypertensive black and white men were studied during unresticted sodium intake as well as on a 10-meq/day sodium intake; potassium intake was held constant throughout the study (80 meq/day). During unrestricted sodium intake, urinary kallikrein activity was greater in white normotensives than white hypertensives or black normotensives. There was no difference (P greater than 0.05) between white and black hypertensives or between black normotensives and black hypertensives. All groups had greater urinary kallikrein activity on low sodium vs. unrestricted sodium intake, but the increase in black hypertensives was small, and they excreted significantly less kallikrein than the ogher groups on the low sodium diet. Plasma renin activity showed similar increments after sodium restriction in all groups. Urinary kallikrein activity correlated with renal blood flow in all groups except the black normotensives on low sodium intake. Renal blood flow could be correlated uniformly with log (urinary kallikrein activity/supine plasma renin activity) in all groups on either diet. Urinary sodium and potassium excretion and urine volume were not different among the groups. We conclude: (a) important racial differences exist in urinary kallikrein activity that are unrelated to sodium or potassium excretion or urine volume; (b) dietary sodium restriction further delineates racial differences and suggests alternative pathophysiologic mechanisms for huma hypertension; (c) urinary kallikrein activity correlates with renal blood flow; and (d) our data support the concept that the kallikrein-kinin system and the renin-angiotensin system contribute to the regulation of renal blood flow and may account for racial differences in renal vascular resistance.

Numerous tubuloreticular structures were seen on electron microscopic examination of involved and uninvolved skin of a neonate with lupus erythematosus. The patient had characteristic lupus skin lesions at birth that worsened for three months, and then gradually subsided. Their course was associated with systemic illness. Skin biopsy specimen was diagnostic. Direct immunogluorescence of the skin showed no abnormalities. Abnormal serological findings were noted in the mother, who had no overt clinical disease. The significance of these virus-like structures in lupus erythematosus is emphasized by the present case.

We investigated the possibility that variability in classification of patients with essential hypertension into low, normal, or high renin activity subgroups depends on the subject preparation that precedes the measurement of plasma renin activity (PRA). In 47 essential hypertensives, PRA was measured with patients supine and ambulatory who were receiving both an unrestricted dietray sodium intake and a low sodium diet. Additionally, PRA was determined following salt restriction, oral furosemide therapy, and ambulation. These results were compared, using several analytical techniques, to those of a group of age-, race-, and sex-matched normotensive controls. Extreme variability in classification was the rule, with only 28% of patients consistently classified as having normal PRA. No single approach provided maximal detection of both the low and high renin states. We conclude that renin classification of hypertensive patients requires a matched control population and that subtyping appears to be variable depending on diet, posture, and analytical approach.

Resistance to tetracycline (Tcr) mediated by Tn10 and related Tcr determinants involves an inner membrane protein, TET (similar but not identical for different determinants), and a proton motive force-dependent efflux of tetracycline which keeps the drug away from its intracellular target, the ribosome (L. M. McMurry, R. E. Petrucci, Jr., and S. B. Levy, Proc. Natl. Acad. Sci. USA 77:3974-3977, 1980). However, the amount of tetracycline accumulated by bacteria does not always correlate with their resistance levels, suggesting that an additional resistance mechanism may be present. When we permeabilized susceptible and resistant Tn10-bearing cells with toluene, we found that protein synthesis in the two strains became equally sensitive to tetracycline. Therefore, the protein synthesis machinery was not a source of resistance, and an intact membrane was required for resistance. To determine whether resistance was entirely dependent on energy, we measured susceptibility to tetracycline after inhibition of proton motive force by starvation and specific inhibitors. An 80 to 90% loss of Tcr (measured by protein synthesis) resulted from partial deenergization of resistant cells. A remaining resistance (10- to 20-fold greater than that of susceptible cells) could not be eliminated by further deenergization. These findings indicated that, to a major extent, expression of Tn10 resistance required energy, presumably for tetracycline efflux. They also suggested the existence of a small component of Tcr having little or no energy dependence. Whether this component depends on tetracycline efflux or some other mechanism is not known, but presumably both high- and low-energy components of resistance reflect activity of TET protein.

Mar (multiple antibiotic resistant) mutants of Escherichia coli express chromosomally mediated resistance to a variety of structurally unrelated hydrophilic and hydrophobic antibiotics. Insertion of transposon Tn5 into the marA locus at min 34.05 on the chromosome completely reverses the Mar phenotype (A. M. George and S. B. Levy, J. Bacteriol. 155:531-540, 1983). We found that among changes in the outer membrane of Mar mutants, porin OmpF was greatly reduced, although Mar mutants were more resistant than cells lacking only OmpF. Transduction of the marA region from a Mar strain, but not a wild-type strain, led to loss of OmpF. P1 transduction of marA::Tn5 into a Mar mutant partially restored OmpF levels. Therefore, OmpF reduction required a mutation in the marA region. Mar mutants of an ompF-lacZ operon fusion strain expressed 50 to 75% of the beta-galactosidase activity of the isogenic non-Mar parental strain, while Mar mutants of a protein fusion strain expressed less than 10% of the enzyme activity in the non-Mar strain. These changes were completely reversed by insertion of marA::Tn5. The responsiveness of OmpF-LacZ to osmolarity and temperature changes was similar in Mar and wild-type strains. Although some transcriptional control may have been present, OmpF reduction appeared to occur primarily by a posttranscriptional mechanism. The steady-state levels of ompF mRNA were twofold lower and the mRNA was five times less stable in the Mar mutant than in the wild-type strain. Expression of micF, which lowers ompF mRNA levels, was elevated in Mar strains, as revealed by a micF-lacZ fusion. Studies with strains deleted for the micF locus showed that the marA-dependent reduction of OmpF required an intact micF locus. Our findings suggest that the marA locus directly or indirectly increases micF expression, causing a posttranscriptional decrease in ompF mRNA and reduced amounts of OmpF.

The chromatin structure of morphologically-similar, but increasingly-malignant erythroleukemia cells was investigated using milk micrococcal nuclease digestion of isolated nuclei. The maximum solubilization of chromatin was unique for each of the three cell types: the least malignant (our Stage II) released 61% of its chromatin DNA, the most malignant (Stage IV), 46%, and the intermediate (Stage III) released 36%. An analysis of the nucleosome oligomers liberated by digestion also demonstrated differences. After 15 minutes of digestion when release was reaching its maximum, a greater proportion of large nucleosomal oligomers (sizes > trinucleosome) was released from Stage II nuclei than from Stage III or IV nuclei. The cell types also differed in the relative amount of H1-depleted mononucleosomes released. Analysis of the size of the double-stranded DNA associated with mononucleosomal particles showed that Stage III mononucleosomes were smaller (148 bp) than Stage IV (167 bp) or Stage II (190 bp). In addition, while the DNA of mononucleosomes depleted in H1 was smaller than that in the H1-containing species, relative size differences among the different cell types were retained. These data suggested that the difference in the mononuocleosome particle size resistant to nuclease digestion was independent of histone H1. Differences in nucleosome repeat length were also noted among the cell types. These studies have demonstrated dramatic differences in chromatin structure associated with malignant potential of an otherwise morphologically identical cell type. These findings may reflect changes in the relative amounts of H2a variants which we have previously described among the different malignant cell types.

Changes in the relative amount of two histone H2A subfractions have been observed in cells at different proliferative stages of Friend leukemia. Biochemical analyses of the purified H2A subfractions reveal them to be different in primary structure, and not the result of postsynthetic modifications of the same parent protein. Antibodies raised against the purified H2A.2 subfraction cross react with H2A.1 and H2A.2, but show high specificity for the immunizing subfraction at higher sera dilutions. Only H2A.2 contains a methionine which appears critical to an antigenic difference that immunologically distinguishes H2A.2 from H2A.1. The observed change in the relative amounts of two nonallelic variants of a histone coincident with changes in the physiologic states of the cell may indicate a correlation between genome structure and function.

The histone variant composition of normal and leukaemic lymphocytes was analysed using a method which circumvented endogenous cellular proteolysis. Whole cells were solubilized in buffer containing protamine which released the histones from the DNA and allowed their immediate analysis by Triton X-100-acetic acid-urea gel electrophoresis. The results were comparable to those obtained with histones which had been acid-extracted from cells, nuclei or chromatin; however, the new method was more reproducible since proteolysis was controlled. By histone variant analysis, chronic lymphocytic leukaemia patients could be separated into two groups. One group had lymphocytes with a histone H2a variant ratio of about 1.0; this finding resembled lymphocytes (75-85% T cells) from normal individuals. The larger group had lymphocytes with a reproducibly lower histone H2a variant ratio; these cells had a relative increase in the second variant, H2a.2. The groups of patients could not be distinguished by clinical disease state. No other differences were noted in the histone variant composition of lymphocytes from normal and leukaemic individuals.

E. coli minicells lack DNA, yet they make protein, the synthesis of which is sensitive to chloramphenicol but insensitive to rifamycin. This protein is coded for by very stable cellular mRNA with an estimated half-life of 40-80 min. In an R factor-containing minicell, two very different species of mRNA are observed: (i) R factor-specific mRNA with a short half-life whose synthesis is rifamycin-sensitive and (ii) cellular mRNA with a long half-life whose synthesis is rifamycin-insensitive. These findings indicate that minicells contain normal degradative mechanisms for mRNA and point out the existence of a unique class of very stable cellular mRNA. Greater than 80% of the rifamycin-insensitive protein synthesized goes into the outer minicell membrane. Relatively stable mRNA, half-life 5.5-11.5 min, for outer membrane protein in whole cells has been reported [Hirashima et al. (1973) J. Mol. Biol. 79, 373-389]. The stability of minicell mRNA is significantly greater. This and other observations suggest that there are two functional species of mRNA for outer membrane protein perhaps in different sites in the cell. Furthermore, these studies suggest that a class of cellular proteins is synthesized in bacteria without concomitant transcription and in the absence of association with chromosomal DNA.

The frequency of resistance to seven different antimicrobial agents was examined in the aerobic gram-negative gut flora of over 600 individuals from hospitals, from laboratories where antibiotics were used, and from urban and rural communities. In a majority (62.5%) of fecal samples from people without a recent history of taking antibiotics, 10% or more of the total organisms were resistant to at least one of the antibiotics. In about 40% of the samples, resistance to more than one drug was present at this level. More than one-third of the samples contained resistant organisms comprising 50% or more of the total flora examined. Organisms with coresistance to multiple drugs were found frequently. Individuals taking antibiotics produced more samples with a higher proportion (greater than 50%) of resistant bacteria, and these samples also had a significantly greater number of different resistance determinants. This extensive study revealed a high prevalence of resistant bacteria in the gut flora of ambulatory and hospitalized individuals whether or not they were taking antibiotics.

Clumping of bacteria containing R factors derepressed for pilus synthesis interfered with the separation of cells and minicells. Mucoid derivatives of these bacteria provided the means of preventing clumping and allowing a good separation. This method may be generally useful in dealing with autoagglutination of bacterial cultures.

The ability of Escherichia coli to colonize the large bowels of animals is related to many factors inherent to the intestinal environment and the bacterium. The use of germfree mice eliminates the competition between E. coli and the other microflora and allows most E. coli strains to colonize. We found that E. coli K-12 strains differing in chromosomal antibiotic resistance could monoassociate in germfree mice in large numbers. However, when two or more strains were in competition with each other, we detected quantitative differences in the abilities of the strains to colonize. The order of colonizing ability was as follows: nalidixic acid resistance greater than streptomycin resistance greater than rifampin resistance. We also found that a nalidixic acid-resistant strain bearing plasmid pBR322 colonized less efficiently and at lower levels when in competition with the nalidixic acid-resistant strain. Studies of the membrane proteins of the various strains indicated that changes in membrane proteins occurred concomitantly with altered resistance to antimicrobial agents. These results suggest that chromosomally linked alterations in antimicrobial sensitivity may also reflect changes in membrane proteins and a decreased ability to colonize mammalian intestines in otherwise isogenic bacterial strains.

These studies have demonstrated the poor survival of E. coli K-12 in the human and mouse gut and the absence of detectable transfer of pBR322 to endogenous intestinal flora. Even in the presence of mobilizing plasmids, no mobilization of pBR322 was detected, and transfer of the transferable plasmids was greatly reduced in the gut. Colonization of the mouse intestine by E. coli was shown to be affected by spontaneous chromosomal mutations to Sm or Rif resistance; these derivatives were weak competitors for colonization in the presence of the original E. coli K-12 organism. Moreover, E. coli K-12 appeared to be at a selective disadvantage in the human and mouse gut since even antibiotic selection failed to increase its survival. A background of data has been accumulated on the frequency of lambda, and M13 phages, and amber-suppressible bacteria in natural human and animal populations. These data should be useful to present and future risk-assessment evaluations. The frequency of antibiotic resistant coliforms in fecal samples from different human and animal populations was found to high. In the present study, antibiotic resistance was very common in both hospitalized and non-hospitalized populations. The percentage of resistant coliforms within individual specimens, however, was higher in patient population.

Using colony hybridization techniques and DNA probes derived from four distinct tetracycline resistance determinants, we have examined the frequency of these determinants among 225 lactose-fermenting coliforms isolated from fecal samples of both humans and animals. The class B, or Tn10-type determinant, occurred most frequently at 73.3%, followed by class A (on RP1) at 21.7%, and class C (on pSC101) at 8%; 3.5% of isolates harbored two of these determinants. Hybridization to class D, carried by plasmid RA1, was not found among any of the isolates. One isolate failed to hybridize to any of the probes and represents a fifth class of determinant. No dramatic differences were observed in the frequencies of these determinants among four populations examined: hospital, urban, rural, and laboratory. At low stringency conditions of hybridization we were able to demonstrate cross-hybridization of determinant A with class C DNA and limited reaction with class B DNA, but no reaction with class D DNA.

Increasing levels of resistance to tetracycline and to a number of other unrelated antibiotics, including chloramphenicol, beta-lactams, puromycin, and nalidixic acid, occurred in Escherichia coli after 50 to 200 generations of growth in the presence of subinhibitory concentrations of tetracycline or chloramphenicol. In the absence of selective pressure, resistances fell to low levels within 100 generations of growth. This amplification of resistance was observed in laboratory and naturally occurring E. coli strains as well as in polA and recA strains. With the exception of previously identified cmlA and cmlB mutations, tetracycline or chloramphenicol resistances were not P1 transducible. Coincident with the emergence of resistance was the appearance of a previously cryptic energy-dependent efflux system for tetracycline. The expression of resistance phenotypes and the tetracycline efflux system were temperature sensitive at 42 degrees C.

The Tn10-like constitutively expressed tetracycline resistance determinant from a Haemophilus parainfluenzae strain was cloned in Escherichia coli. Toxicity resulting from expression on multicopy plasmids necessitated its being cloned on a low-copy plasmid vector or in cells containing the Tn10-encoded repressor. Constitutive expression of tetracycline resistance was found to result from the synthesis of a truncated inactive repressor molecule. Instead of the 23-kilodalton repressor found in other Tn10-containing strains, this determinant encoded a 14.5-kilodalton molecule. The DNA sequence of the 700-base-pair region spanning the repressor gene and promoter-operator regions of the Haemophilus determinant was identical to that of the same region of Tn10, except for the absence of a single T X A base pair in the repressor gene. This deletion leads to premature termination of the protein. Antisera to the repressor suggested that the repressor was also absent in a second independently isolated H. parainfluenzae strain bearing a Tn10-like constitutive tetracycline resistance determinant.

Tetracycline resistance determinants are widespread and distinguishable genetically and biochemically. The nomenclature for this increasing number of determinants has been varied and inconsistent. This communication suggests ways of naming these determinants and their genes and gene products consistent with current genetic terminology.