Adherence to epithelial cells and ultrastructure of fosfomycin-resistant mutants of group A streptococci

Adherence of three strains of group A streptococci and their fosfomycin-resistant mutants to HEp-2 tissue culture cells was compared with some cell-surface characteristics, i.e. ultrastructure and hydrophobicity. Among Fosr mutants, both well-adhering and weakly adhering mutants were found. Clonal analysis of the mutants proved their greater stability in the adherence. Well-adhering parent strains of streptococci and Fosr mutants exhibited surface fibrillae in contrast to weakly adhering Fosr mutants which were devoid of fibrillae or contined fibrillae of lower electron density. Decrease of adherence of Fosr mutants of two strains was accompanied by a decrease of their hydrophobicity.     

Isolation and Preliminary Characterization of Temperature-Sensitive Mutants of Influenza Virus

Isolation of temperature-sensitive (ts) mutants was attempted from the WSN strain of influenza A virus which was grown and assayed in MDBK cells. After growth of wild-type virus in the presence of 5-fluorouracil, 15 ts mutants were selected for which the ratio of plaquing efficiency at 39.5 C to that at 33 C was 10⁻³ or less. In pairwise crosses of ts mutants, recombination and complementation were either very efficient or undetectable. It is suggested, therefore, that the viral genome consists of physically discrete units and recombination occurs as an exchange of these units. All 15 mutants have been assigned with certainty into five recombination groups. Three mutants are suspected to be double mutants. Any two complementing mutants always recombined with each other, and noncomplementing mutants did not recombine. In physiological tests, mutants showed diverse patterns of functional defects at the nonpermissive temperature. However, it was not always possible to correlate these physiological defects with the results of genetic characterization.     

菲白竹组培苗白化、绿化突变体的超微结构及15个叶绿体编码基因的表达

以菲白竹（Pleioblastus fortunei）组培苗绿化突变体及经60Coγ射线辐射获得的白化突变体为实验材料,从生长特性、超微结构及15个叶绿体编码基因的拷贝数及转录水平方面进行了研究。结果显示,白化突变体具有分化不定芽和不定根的能力,其叶肉细胞中叶绿体缺失或结构不完整,基因拷贝数与野生型无明显差异,部分基因转录水平低于野生型,psbA基因的转录水平高于野生型。绿化突变体叶色一致且能稳定遗传,与野生型绿色组织细胞的超微结构相比,其叶绿体基粒片层数量少,结构松散,基因拷贝数低或相当于野生型,多数叶绿体编码基因转录水平较野生型呈下调趋势,atpI基因转录水平高于野生型。psaA基因在两种突变体中均不表达,仅在野生型中有微弱表达。     

Phenotypes of pleiotropic-negative sporulation mutants of Bacillus subtilis

The phenotypic properties of representatives of the five genetic classes of pleiotropic-negative sporulation mutants have been investigated. Protease production, alkaline and neutral proteases, was curtailed in spoA mutants, but the remainder of mutant classes produced both proteases, albeit at reduced levels. The spoA and spoB mutants plaqued phi2 and phi15 at high efficiency, but the efficiency of plating of these phages on spoE, spoF, and spoH mutants was drastically reduced. Antibiotic was produced by the spoH mutants and to a degree by some spoF mutants, but the other classes did not produce detectable activity. The spoA mutants were less responsive to catabolite repression of histidase synthesis by glucose than was the wild type. Severe catabolite repression could be induced in spoA mutants by amino acid limitation, suggesting that the relaxation of catabolite repression observed is not due to a defect in the mechanism of catabolite repression. Although others have shown a perturbation in cytochrome regulation in spoA and spoB mutants, the primary dehydrogenases, succinate dehydrogenase and reduced nicotinamide adenine dinucleotide dehydrogenase, leading to these cytochromes are unimpaired in all mutant classes. A comparison of the structural components of cell walls and membranes of spoA and the wild type is made. The pleiotropic phenotypes of these mutants are discussed.     

菲白竹组培苗白化、绿化突变体的超微结构及15个叶绿体编码基因的表达

以菲白竹(Pleioblastus fortunei)组培苗绿化突变体及经⁶⁰Co γ射线辐射获得的白化突变体为实验材料, 从生长特性、超微结构及15个叶绿体编码基因的拷贝数及转录水平方面进行了研究。结果显示, 白化突变体具有分化不定芽和不定根的能力, 其叶肉细胞中叶绿体缺失或结构不完整, 基因拷贝数与野生型无明显差异, 部分基因转录水平低于野生型, psbA基因的转录水平高于野生型。绿化突变体叶色一致且能稳定遗传, 与野生型绿色组织细胞的超微结构相比, 其叶绿体基粒片层数量少, 结构松散, 基因拷贝数低或相当于野生型, 多数叶绿体编码基因转录水平较野生型呈下调趋势, atpI基因转录水平高于野生型。psaA基因在两种突变体中均不表达, 仅在野生型中有微弱表达。     

The effect of surface charge balance on thermodynamic stability and kinetics of refolding of firefly luciferase

Thermodynamic stability and refolding kinetics of firefly luciferase and three representative mutants with depletion of negative charge on a flexible loop via substitution of Glu by Arg (ER mutant) or Lys (EK mutant) as well as insertion of another Arg in ER mutants (ERR mutant) was investigated. According to thermodynamic studies, structural stability of ERR and ER mutants are enhanced compared to WT protein, whereas, these mutants become prone to aggregation at higher temperatures. Accordingly, it was concluded that enhanced structural stability of mutants depends on more compactness of folded state, whereas aggregation at higher temperatures in mutants is due to weakening of intermolecular repulsive electrostatic interactions and increase of intermolecular hydrophobic interactions. Kinetic results indicate that early events of protein folding are accelerated in mutants.     

Biochemical Characterization of the ctr Mutants of Escherichia coli

A test procedure based on complementation in mixed extracts is described for the assay of heat-stable protein and enzyme I of the phosphoenolpyruvate-dependent phosphotransferase system. The test was used to assay a collection of pleiotropic carbohydrate mutants of Escherichia coli (ctr mutants) and revertants of these mutants. All mutants were found to lack enzyme I of the phosphoenolpyruvate-dependent transferase system. Revertants of these mutants to complete wild phenotype regained enzyme I-forming ability. Reversion to partial wild type was not accompanied by restoration of enzyme I-forming ability.     

Physiological characterization of temperature-sensitive mutants of mengovirus.

Twenty-four temperature-sensitive mutants of mengovirus were characterized physiologically with respect to phenotype. The mutants were separated into four classes on the basis of viral RNA synthesis. L-67-S cells infected with five of the mutants synthesized little viral RNA at 39.5 C. These mutants are designated RNA-. One mutant is designated RNA* since its RNA synthesis is altered at both 39.5 and 31.5 C. The other mutants were divided into two groups, RNA plus or minus (25 TO 49% of wild-type RNA synthesis) and RNA plus (50 to 100% of wild-type RNA synthesis). The time of expression of the mutation in the RNA- mutants was estimated from the results of reciprocal temperature-shift experiments. The mutatation in ts12 appears to be expressed at the time RNA synthesis normally begins. The defect in three of the mutants was expressed 1 to 2 h before RNA synthesis is normally detectable. Protein synthesis is required before RNA synthesis begins when the cells are shifted from 39.5 to 31.5 C. The RNA polymerase synthesized by cells infected with these RNA- mutants at 31.5 C was stable and fully active when assayed at 39.5 C in vitro. The sedimentation profiles of the viral RNA synthesized by cells infected with RNA plus and RNA plus or minus mutants are similar to wild-type profiles with the exception of ts148. Cells infected with this RNA plus or minus mutant synthesize RNA that sediments in a sucrose gradient like replicative-intermediate RNA, but little mature viral RNA is evident. The results of step-up experiments indicate that the temperature-sensitive period for the majority of the RNA plus and RNA plus and minus mutants extends through most of the replicative cycle. The temperature-sensitive defect of four of the mutants, however, was expressed in the first hour, suggesting that some undefined early function is required for the eventual maturation of mengovirus. The virions of three of the RNA- mutants were more thermolabile than wild-type virions. Five of the RNA plus and RNA plus or minus mutants were also thermolabile. Genetic complementation at a significant level was not detectable in mixed infections of the mutants described.     

Genetic and Physiological Properties of Temperature-Sensitive Mutants of Cocal Virus

Temperature-sensitive (ts) mutants of Cocal virus (VSV Cocal) were isolated after treatment with the base analogue mutagen, 5-fluorouracil. These mutants could be classified into four mutually complementing groups. Weak complementation was detected between certain pairs of VSV Cocal ts mutants and ts mutants of vesicular stomatitis virus (VSV) Indiana, but no complementation was observed with ts mutants of VSV New Jersey. Two complementing ts mutants of Chandipura virus, an unrelated rhabdovirus, did not complement any VSV mutant, Thus, ability to complement in the VSV group appears to be correlated with serological relationships.The RNA and protein-synthesizing capacities of these ts mutants have been determined, and it is possible to establish a correspondence between the VSV Cocal and the VSV Indiana complementation groups.     

The induction of mutants in a non-acid-fast strain ofMycobacterium phlei with nitrous acid

The inactivation and mutagenic effets of nitrous acid on a non-acid-fast strain ofMycobacterium phlei were studied. It was found that 0.017m NaNO₂ at pH 4.4 may be used for the induction of auxotrophic mutants, scotochromogenic and achromogenic mutants and STM-resistant mutants. Three doubly auxotrophic mutants, three mutants requiring amino acids and three mutants depending on vitamins were obtained. One mutant was not classified. Eighteen scotochromogenic mutants were isolated, seventeen of them were orange. Only ten achromogenic mutants were isolated. Twelve scotochromogenic and eight achromogenic mutants could be used in further genetic studies as they did not revert spontaneously to photochromogeny. Six auxotrophic mutants could be used due to their low frequency of spontaneous reversions. The frequency of STM-resistant mutants increased on an average seven-fold after the mutagenic treatment as compared with the spontaneous frequency.     

A complementation analysis of mobilization deficient mutants of the plasmid colE1

Summary Hydroxylamine was used to induce mutants of the ColE1 derived plamid pML2 that are inefficiently mobilized (Mob^-) during conjugation by an Hfr donor. The ability of those mutants to be complemented by deletion mutants and Tn3 insertion mutants of ColE1 was examined. Three complementation groups were identified and localized on the ColE1 genetic map (Mob1, Mob2, and Mob3). One hydroxylamine mutant was not complemented by any mobilization deficient mutant but was complemented by mobilizable ColE1 mutants. Two hydroxylamine mutants were not complemented by any ColE1 derivatives. A mutant that had its relaxation nick site deleted had a markedly reduced mobilizability. The relationship between DNA relaxation, replication and mobilization is considered.     

An improved method for isolation of mutator mutants from mouse FM3A cells and their characterization

An improved method to select mutator mutants was developed. By this new method, mutator mutants were isolated efficiently, and 7 mutants were obtained from cultured mouse FM3A cells. These mutator mutants have an elevated rate of spontaneous mutation at 3 genetic loci (resistance to ouabain, blasticidin S, and tunicamycin). The sensitivity of these mutants to aphidicolin and arabinofuranosylcytosine was the same as in the wild-type cells. Determination of the size of the cellular dNTP pool revealed that there was no large imbalance in the precursor pool in the mutator mutants. These results suggested that the mutator character may be due to alteration in some factor(s) correlated directly to DNA replication. Also, there was no change in the sensitivity of all these mutator mutants to DNA damaging agents.     

Characterization of nonattaching mutants of Agrobacterium tumefaciens.

The first step in tumor formation by Agrobacterium tumefaciens is the site-specific binding of the bacteria to plant host cells. Transposon mutants of the bacteria which fail to attach to carrot suspension culture cells were isolated. These mutants showed no significant attachment to carrot cells with either microscopic or viable cell count assays of bacterial binding. The nonattaching mutants were all avirulent. When revertants of the mutants were obtained by enriching for bacteria which do bind to carrot cells, the bacteria were found to have regained the ability to bind to carrot cells and virulence simultaneously. These results suggest that the ability of the bacteria to bind to plant cells is required for virulence. Like the parent strain, all of the nonattaching mutants synthesized cellulose, but unlike the parent strain, they failed to aggregate carrot suspension culture cells. The transposon Tn5, which was used to obtain the mutants, was located on a 12-kilobase EcoRI fragment of the bacterial chromosomal DNA in all of the nonattaching mutants from strain C58. That the mutant phenotype was due to the Tn5 insertion was shown by cloning the Tn5-containing DNA fragment from the mutant bacteria and using it to replace the wild-type fragment in the parent strain by marker exchange. The resulting bacteria had the same mutant phenotype as the original Tn5 mutants; they did not attach to carrot cells, they did not cause the aggregation of carrot cells, and they were avirulent. No difference was seen between the parent strain and the nonattaching mutants in hydrophobicity, motility, flagella, fimbriae, beta-2-glucan content, size of lipopolysaccharide, or ability of the lipopolysaccharide to inhibit bacterial attachment to tissue culture cells. Differences were seen between the parent strain and the nonattaching mutants in the polypeptides removed from the bacteria during the preparation of spheroplasts. Three of the mutants were lacking a polypeptide of about 34 kilodaltons (kDa). One mutant was lacking the 34-kDa polypeptide and another polypeptide of about 38 kDa. The fifth mutant was lacking a polypeptide slightly smaller than the 34-kDa polypeptide missing in the other four mutants. These missing polypeptides all reappeared in the revertants of the mutants. Thus, bacterial binding to plant cells appears to require the presence of these polypeptides.     

Pleiotropic effects of alkaline phosphatase regulatory mutations phoB and phoT on anaerobic growth of and polyphosphate synthesis in Escherichia coli.

The appearance during anaerobiosis of spontaneous phoT phoB double mutants in a phoT background is described. During both exponential growth and stationary phase, selection against the phoT mutants relative to the wild type was evident. This reduction in viability of phoT mutants was suppressed in phoT phoB double mutants. Sensitivity to anaerobiosis was shown to be correlated with polyphosphate overproduction. A possible pleiotropic function of phoT and phoB is suggested.     

Isolation and characteristics of rifampicin-resistant mutants of Streptomyces erythraeus strain

Erythromycin-producing strains of S. erythraeus were characterized with respect to formation of spontaneous and induced rifampicin-resistant mutants. It was shown that the frequency of spontaneous rifampicin-resistant mutants formed by various strains amounted to 0.9.10(-8) = 9.1.10(-7). In some events the exposure to nitrosoguanidine increased the frequency of such mutants by 2 orders of magnitude. The rifampicin-resistant mutants differed in antibiotic resistance. It was found that a significant part of the rifampicin-resistant mutants became sensitive to heating (19.1 per cent) and lost the ability to form aeromycelium (21.8 per cent).     

Isolation of the rec Mutants in Staphylococcus aureus

A histidine auxotroph (his-) of Staphylococcus aureus MS3937 and mutants sensitive to ultraviolet (UV) irradiation were obtained. The UV-sensitive mutants were found also to be sensitive to N-methyl-N'-nitro-N-nitrosoguanidine and mitomycin C, and their sensitivity was accounted for by a defect in deoxyribonucleic acid repair. Transduction of either chromosomal or plasmid markers to UV-sensitive mutants showed that these staphylococcus mutants are of the recA (reckless) type mutants as reported in Escherichia coli and Salmonella typhimurium; therefore the UV-sensitive mutants (uvr-) were renamed recombination-deficient mutants (rec-). The biochemical and genetic properties of these mutants are described, and their usefulness for studies of staphylococcal plasmids is discussed.     

Isolation of Conditionally Putrescine-Deficient Mutants of Escherichia coli

Mutants defective in the conversion of arginine to putrescine were found by screening clones from mutagenized cultures for inability to produce urea during growth in arginine-supplemented media. Two partially blocked mutants were isolated; one was deficient in arginine decarboxylase and the other was deficient in agmatine ureohydrolase. As predicted from the pattern of putrescine synthesis in Escherichia coli, these mutants were conditionally putrescine-deficient. When grown in either minimal or ornithine-supplemented media, conditions which lead to preferential utilization of the ornithine to putrescine pathway, the mutants had normal intracellular polyamine levels. However, when the mutants were placed in arginine-supplemented media, the level of intracellular putrescine was lowered markedly. Under conditions where intracellular putrescine was 1% of normal, the doubling time of the mutants was increased approximately 10%. The putrescine-deficient mutants had wild-type morphology, normal levels of protein and ribonucleic acid (RNA), and stringent amino acid control of RNA synthesis.