Mutation in Escherichia coli K-12 Mediating Spherelike Envelopes and Changed Tolerance to Ultraviolet Irradiation and Some Antibiotics

In a mutation experiment with a rough, ampicillin-resistant strain of Escherichia coli K-12, two smooth ampicillin-sensitive mutants were isolated. One of the mutants (with the envA gene) was recently described. The second mutant (strain D23) with the envB gene which has been mapped to a position close to streptomycin resistance (strA) at 64 min is described. The envB gene gives rise to spherelike cells. Electron microscopy revealed an abnormal septum formation and a circular distribution of the nuclear material. Strain D23 (with envB) showed a changed resistance to several antibiotics as well as an increased tolerance to ultraviolet irradiation. The envB gene decreased the ampicillin resistance mediated by the ampA gene (at 82 min), but no effect was found on episomally mediated penicillin resistance.     

Leakage of periplasmic enzymes from envA1 strains of Escherichia coli

Previous work ascribed antibiotic hypersensitivity of the envA1 mutant to lowered lipopolysaccharide levels and exposure of the lipid bilayer. In the detailed characterization of the EnvA permeability phenotype presented here, the envA1 mutation was shown to confer leakage of the periplasmic enzymes beta-lactamase and RNase I. Leakage was observed in three different genetic backgrounds, including the original envA1 strain and its parent. In contrast, no detectable leakage of the cytoplasmic enzyme beta-galactosidase was observed. Sensitivity of envA1 strains to a range of antibiotics not previously reported was tested, and lipophilicity (partition coefficient) of a number of antibiotics was determined. On the basis of observations of periplasmic leakage and sensitivity to large hydrophilic antibiotics and lysozyme, part of the permeability phenotype of the envA1 mutant is proposed to be due to transient rupture and resealing of the EDTA-sensitive outer membrane layer. In this regard, the EnvA permeability phenotype falls into a general class of permeability/leaky mutants of both Escherichia coli and Salmonella typhimurium.     

Characterization of heat resistant mutant strains of Rhizobium sp. [Cajanus] for growth, survival and symbiotic properties

Fourteen heat resistant mutant strains were isolated from a wild-type strain (PP201, Nod⁺ Fix⁺) of Rhizobium sp. (Cajanus) by giving it a heat shock of 43°C. These mutant strains showed a greater increase in optical density (O.D.) and a higher viable cell count in both rhizospheric and non-rhizospheric soil at high temperature. Symbiotic studies showed that pigeon pea plants inoculated with a few mutant strains had ineffective nodules (Nod⁺ Fix⁻) under controlled temperature (43°C) conditions, but under natural high temperature (40–45°C) conditions, the host plants infected with all the mutant strains showed higher total shoot nitrogen than the plants inoculated with the parent strain. Four mutant strains (HR-3, HR-6, HR-10 and HR-12) were found to be highly efficient for all the symbiotic parameters, and thus have the potential to be used as bioinoculants in the North-Western regions of India during the summer season.     

Isolation and analysis of a mutant of Escherichia coli hyper-resistant to near-ultraviolet light plus 8-methoxypsoralen

A mutant of Escherichia coli K12 was isolated which shows enhanced resistance towards near-ultraviolet (NUV) light plus 8-methoxypsoralen (MPS) compared with its wild-type parent strain. The PUVA (NUV + MPS)-resistant strain remains as sensitive for far-ultraviolet (FUV) light as its parent strain. A recA- derivative of this mutant strain was as sensitive to PUVA as its reca- parental strain. A polyacrylamide gel electrophoresis study of total cell lysates from the mutant bacteria showed that a protein of approximately 55 kd was synthesised in higher concentrations compared with its synthesis in the wild-type parent strain. Furthermore, synthesis of this protein was reduced in the recA- derivative of the mutant strain suggesting that the recA gene product might be acting as a regulator of the synthesis of the 55-kd protein. It is suggested that in E. coli damage to DNA by PUVA can be repaired by a specific RecA LexA-inducible repair system and the repair efficiency is enhanced if the 55-kd protein is present in concentrations higher than that synthesised by the wild-type parent E. coli.     

Resistance of Escherichia coli to penicillins. 8. Physiology of a class II ampicillin-resistant mutant

Class II ampicillin-resistant mutants of Escherichia coli are defined as having a twofold increase in penicillinase-mediated ampicillin resistance when determined by colony formation tests on plates. In this paper, one class II mutant has been compared to its parent strain. In liquid medium, the mutant was less resistant than the parent strain both in the absence and in the presence of R1 and R-factor mediating penicillinase activity. The penicillinase activity was found to be almost completely bound to the cells in the parent strain, whereas it was excreted to a great extent in the class II mutant strain. In liquid medium, resistance was well correlated to the cell-bound penicillinase activity, whereas the excreted penicillinases were also of great importance for survival on ampicillin plates. The mutant also had a changed resistance to a great number of other antibacterial drugs. The mutant was found to be more sensitive than the parent strain to osmotic shock, especially when treated with ethylenediaminetetraacetic acid or washed with sodium ions. However, the osmotic stability was restored by the presence of 1 mm Mg(2+) ions. The class II mutant was more sensitive than the parent strain to sodium cholate, and it adsorbed the phages T4 and T3-1 at a slower rate than did the parent strain. The two strains adsorbed T6 at the same rate. The class II phenotype could be gradually reversed by increasing concentrations of divalent cations. The pleiotropic changes in the phenotype are apparently unrelated to the specific targets for the antibacterial agents tested. They are secondary consequences of a cell envelope mutation. The findings indicate that the class II mutation mediates a structural change in the lipopolysaccharide of the cell envelope.     

Cloning and characterization of the Aeromonas caviae recA gene and construction of an A. caviae recA mutant.

A recombinant plasmid carrying the recA gene of Aeromonas caviae was isolated from an A. caviae genomic library by complementation of an Escherichia coli recA mutant. The plasmid restored resistance to both UV irradiation and to the DNA-damaging agent methyl methanesulfonate in the E. coli recA mutant strain. The cloned gene also restored recombination proficiency as measured by the formation of lac+ recombinants from duplicated mutant lacZ genes and by the ability to propagate a strain of phage lambda (red gam) that requires host recombination functions for growth. The approximate location of the recA gene on the cloned DNA fragment was determined by constructing deletions and by the insertion of Tn5, both of which abolished the ability of the recombinant plasmid to complement the E. coli recA strains. A. caviae recA::Tn5 was introduced into A. caviae by P1 transduction. The resulting A. caviae recA mutant strain was considerably more sensitive to UV light than was its parent. Southern hybridization analysis indicated that the A. caviae recA gene has diverged from the recA genes from a variety of gram-negative bacteria, including A. hydrophila and A. sobria. Maxicell labeling experiments revealed that the RecA protein of A. caviae had an Mr of about 39,400.     

Phenethyl Alcohol as a Suppressor of the EnvA Phenotype Associated with the envA Gene in Escherichia coli K-12

In Escherichia coli K-12 the envA gene was previously shown to mediate chain formation and a decreased tolerance to several antibacterial agents. Phenethyl alcohol at low concentrations has now been found to increase the tolerance to actinomycin D, ampicillin, rifampin, and gentian violet in strains containing envA. The increased tolerance to gentian violet was correlated to a decreased uptake of the dye. A phenotype suppression of chain formation and colony morphology in envA mutants was also obtained. Except for an increase in palmitic acid, chemical analysis revealed no differences between an envA and its wild-type strain in the lipopolysaccharide part of the envelope. However, a decrease in the amount of phosphatidylglycerol and a C18: 1 fatty acid was observed in the extractable lipids of a strain containing envA. Growth in the presence of phenethyl alcohol reversed the changes in fatty acid and the phospholipid composition. Phenethyl alcohol was found to cause an immediate but transient inhibition of ribonucleic acid synthesis. It is suggested that this inhibition affects the penetrability barrier of the outer cell envelope layers in strains containing envA.     

Rapid Method for Isolation of Large Quantities of Outer Membrane from Escherichia coli K-12 and Its Application to the Study of Envelope Mutants

A rapid method for the isolation of large quantities of bacterial outer membrane is described. This cell envelope component was removed from plasmolyzed cells of Escherichia coli K-12 by lysozyme-ethylenediaminetetra-acetic acid treatment, aggregated by lowering the pH to 5.0, and recovered by centrifugation. Aggregates of membrane fragments were clearly identified in an electron microscope. A criterion of homogeneity of the preparation was obtained by isopycnic sucrose gradient centrifugation. A single band appeared at a density of 1.24 g/cc. The cytoplasmic membrane marker, succinate dehydrogenase activity, was 40 times lower in the outer membrane preparation than in complete cell envelope preparations. A rich activity was, however, found for the outer membrane marker, phospholipase A. The compositions of outer membranes from a transductant pair were compared. One transductant was a chain-forming, antibiotic-supersensitive envA strain, whereas the other contained the envA(+) allele. The envA strain showed a slightly modified protein pattern and a lower relative content of phosphatidylglycerol.     

Overproduction of outer membrane protein suppresses envA-induced hyperpermeability.

A quantitative study on outer membrane components was performed in a number of envelope mutants of Escherichia coli K-12 exhibition different permeability properties for antimicrobial agents. The envA1 allele causing an increased influx for both hydrophobic and hydrophilic drugs was found to be associated with a deficiency in the amount of lipopolysaccharides. The sefA1 envA1 double mutant was found to have a higher outer membrane buoyant density, apparently due to an increase in protein content. This double mutant was still low in lipopolysaccharide content.     

Sequence analysis of the Gluconobacter oxydans RecA protein and construction of a recA-deficient mutant.

The deduced amino acid sequence of Gluconobacter oxydans RecA protein shows 75.2, 69.4, and 66.2% homology with those from Aquaspirillum magnetotacticum, Escherichia coli, and Pseudomonas aeruginosa, respectively. The amino acid residues essential for function of the recombinase, protease, and ATPase in E. coli recA protein are conserved in G. oxydans. Of 24 amino acid residues believed to be the ATP binding domain of E. coli RecA, 17 are found to be identical in G. oxydans RecA. Interestingly, nucleotide sequence alignment between the SOS box of G. orphans recA gene and those from different microorganisms revealed that all the DNA sequences examined have dyad symmetry that can form a stem-loop structure. A G. oxydans recA-deficient mutant (LCC96) was created by allelic exchange using the cloned recA gene that had been insertionally inactivated by a kanamycin-resistance cassette. Such replacement of the wild-type recA with a kanamycin resistance gene in the chromosome was further verified by Southern hybridization. Phenotypically, the recA-deficient mutant is significantly more sensitive to UV irradiation than the wild-type strain, suggesting that the recA gene of G. oxydans ATCC9324 plays a role in repairing DNA damage caused by UV irradiation. Moreover, the mutant strain is much more plasmid transformable than its parent strain, illustrating that G. oxydans LCC96 could be used as a host to take up the recombinant plasmid for gene manipulation.     

Methionine sulphoxide reductase is an important antioxidant enzyme in the gastric pathogen Helicobacter pylori

The ability of Helicobacter pylori to colonize the stomach requires that it combat oxidative stress responses imposed by the host. The role of methionine sulfoxide reductase (Msr), a methionine repair enzyme, in H. pylori stress resistance was evaluated by a mutant analysis approach. An msr mutant strain lacked immunologically detectable sulphoxide reductase protein and also showed no enzyme activity when provided with oxidized methionines as substrates. The mutant strain showed diminished growth compared to the parent strain in the presence of chemical oxidants, and showed rapid viability loss when exposed to oxidizing conditions. The stress resistance and enzyme activity could be recovered by complementing the mutant with a functional copy of the msr gene. Upon fractionation of parent strain and the complemented mutant cells into membranes and cytoplasmic proteins, most of the immunologically detectable Msr was localized to the membrane, and this fraction contained all of the Msr activity. Qualitative detection of the whole cell protein pattern using 2,4-dinitro phenyl hydrazine (DNPH) showed a far greater number of oxidized protein species in the mutant than in the parent strain when the cells were subjected to oxygen, peroxide or s-nitrosoglutathione (GSNO) induced stress. Importantly, no oxidized proteins were discerned in either strain upon incubation in anaerobic conditions. A mutant strain that synthesized a truncated Msr (corresponding to the MsrA domain) was slightly more resistant to oxidative stress than the msr strain. Mouse colonization studies showed Msr is an important colonization factor, especially for effective longer-term (14 and 21 days) colonization. Complementation of the mutant msr strain by chromosomal insertion of a functional gene restored mouse colonization ability.     

Toxic fatty acid resistant mutant of Trichophyton rubrum.

A mutant of Trichophyton rubrum which could tolerate high concentration (110 microgram/ml) of undecanoic acid was isolated from undecanoic (27.5 microgram/ml) sensitive parent. The mutant showed cross resistance towards other fatty acids like propionic acid, caprylic acid and undecenoic acid which were toxic for the parent strain. Development of fatty acid resistance in the mutant strain was associated with increased pigment production. There was no difference in colony or cell morphology between fatty acid resistant mutant and fatty acid sensitive parent.     

Isolation, oxygen sensitivity, and virulence of NADH oxidase mutants of the anaerobic spirochete Brachyspira (Serpulina) hyodysenteriae, etiologic agent of swine dysentery.

Brachyspira (Serpulina) hyodysenteriae, the etiologic agent of swine dysentery, uses the enzyme NADH oxidase to consume oxygen. To investigate possible roles for NADH oxidase in the growth and virulence of this anaerobic spirochete, mutant strains deficient in oxidase activity were isolated and characterized. The cloned NADH oxidase gene (nox; GenBank accession no. U19610) on plasmid pER218 was inactivated by replacing 321 bp of coding sequence with either a gene for chloramphenicol resistance (cat) or a gene for kanamycin resistance (kan). The resulting plasmids, respectively, pCmDeltaNOX and pKmDeltaNOX, were used to transform wild-type B. hyodysenteriae B204 cells and generate the antibiotic-resistant strains Nox-Cm and Nox-Km. PCR and Southern hybridization analyses indicated that the chromosomal wild-type nox genes in these strains had been replaced, through allelic exchange, by the inactivated nox gene containing cat or kan. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis revealed that both nox mutant cell lysates were missing the 48-kDa Nox protein. Soluble NADH oxidase activity levels in cell lysates of Nox-Cm and Nox-Km were reduced 92 to 96% compared to the activity level in parent strain B204. In an aerotolerance test, cells of both nox mutants were at least 100-fold more sensitive to oxygen exposure than were cells of the wild-type parent strain B204. In swine experimental infections, both nox mutants were less virulent than strain B204 in that fewer animals were colonized by the mutant cells and infected animals displayed mild, transient signs of disease, with no deaths. These results provide evidence that NADH oxidase serves to protect B. hyodysenteriae cells against oxygen toxicity and that the enzyme, in that role, contributes to the pathogenic ability of the spirochete.     

Properties of a Novel Pleiotropic Bacteriophage-Resistant Mutant of Staphylococcus aureus H

A phage-resistant mutant of Staphylococcus aureus H (Sm(R)), S. aureus 52A5, was previously shown to lack polymeric teichoic acid. This paper characterizes other phenotypic differences between the strains. In broth cultures the mutant cells grew more slowly, were larger, and formed much larger clumps than the parent strain. The clumps of cells appeared to be covalently linked and could only be separated by mild sonic energy-a process which yielded viable cells. Mutant and parent cells autolyzed at equal rates, whereas isolated cell walls of the mutant strain autolyzed faster than the wild type. Nevertheless, the specific activity of the autolytic enzyme in the wild type soluble fraction was much higher than in the mutant. In contrast to the parent, strain 52A5 failed to accumulate nucleotide-bound murein precursors when treated with penicillin. Mutant strains with these characteristics were repeatedly isolated both spontaneously and by chemical mutagenesis. Strain 52A5 was shown to be fully revertible. Thus, it appears to be a pleiotropic mutation, and the possible nature of the defect which causes these varied effects is discussed.     

Mutation induction in Escherichia coli WP2 uvrA by Cerenkov emission associated with 137Cs gamma irradiation

Evidence is presented for the mutation of the tryptophan-requiring bacterial strain Escherichia coli WP2 uvrA from auxotrophy to prototrophy, and from streptomycin sensitivity to resistance, by Cerenkov emission associated with 137Cs gamma irradiation. Furthermore, the data strongly suggest a more than additive interaction between the gamma-induced damage and that induced by Cerenkov emission for both mutations scored. An additional observation is that mutant yields (expressed as mutants/10(7) survivors) show a dependence on the number of viable cells plated for both uv (254 nm) and Cerenkov-induced mutations, but not for those induced by gamma irradiation. This demonstrates another similarity between uv- and Cerenkov-induced damage.     

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.     

The contribution of bacteriocin to inhibition of Listeria monocytogenes by Carnobacterium piscicola strains in cold-smoked salmon systems

AIMS: To study the importance of bacteriocin production for the antilisterial effect of a bacteriocinogenic Carnobacterium piscicola strain A9b on growth of Listeria monocytogenes in broth and cold-smoked salmon systems. METHODS AND RESULTS: Acriflavin treatment of strain A9b resulted in loss of bacteriocin production and of immunity to carnobacteriocin B2. Two plasmids present in the wild-type were lost in the variant that was also more sensitive to bavaricin and leucocin A than the wild-type indicating cross-resistance to class IIa bacteriocins. The growth rate of the bac- mutant was higher than that of the wild-type at 5 and 37 degrees C but not at 25 or 30 degrees C. In salmon juice the maximum cell density of L. monocytogenes was suppressed 3 and 6 log by co-culture with C. piscicola A9b bac- and bac+, respectively, as compared with the control. Sterile filtered cultures of C. piscicola A9b bac- caused a limited suppression of the maximum cell density of L. monocytogenes similar to that observed when sterile buffer was added in equal amounts. Semi-purified carnobacteriocin B2 caused a 3.5 log decline in viable cell count after 6 day of incubation in cold-smoked salmon juice at 5 degrees C. High resistance level to carnobacteriocin B2 was observed for L. monocytogenes cells exposed to semi-purified and in situ produced carnobacteriocin B2. CONCLUSIONS: The presence of bacteriocin production in C. piscicola enhances its inhibition of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the emergence of resistance, a bacteriocin negative lactic acid bacteria may be more suited for practical use as a bioprotective agent against L. monocytogenes in ready-to-eat foods.     

猪传染性胸膜肺炎放线杆菌毒素apxH基因无药物抗性标记突变株HBC-/GFP+的构建及其生物学特性研究

通过使用枯草芽胞杆菌一个蔗糖敏感sacB基因发展了一种依靠蔗糖的负向筛选系统,这种方法允许没有标记突变的基因进入胸膜肺炎放线杆菌染色体。首先,构建了猪传染性胸膜肺炎放线杆菌毒素apxⅡ基因GFP插入失活型的重组质粒pOSAKCG,其中一个表达盒含有氨苄青霉素基因和以外膜蛋白omlA作为启动子表达sacB基因。重组质粒pOSAKCG通过电穿孔转化,它的突变apxⅡCA基因与野生型亲本菌株胸膜肺炎放线杆菌HB03染色体上野生型apxⅡCA基因发生同源交换,两步法筛选获得了apxⅡ基因突变株HBC^-／GFP^ ,PCR和Southern blot对突变株进行初步鉴定,进一步对突变株的一些生物学特性,包括它的溶血活性、免疫原性、生长特性及其对小鼠的安全性进行了研究。结果表明,无药物抗性标记突变株的构建是成功的。该突变株的构建为进一步研究突变株作为载体和疫苗奠定了坚实的基础。     

Escherichia coli resistance to chlorine and glutathione synthesis in response to oxygenation and starvation

Reduced glutathione (GSH) levels and resistance to chlorine were measured for two isogenic Escherichia coli strains stressed by oxygenation and/or starvation. The E. coli mutant deficient in GSH was not more sensitive to the oxidant than its parent strain when the bacteria were cultured with a low oxygenation rate. Starvation or oxygenation increased the resistance of the parent strain to chlorine, while the resistance of the deficient strain remained unchanged.