Mismatch repair and the regulation of phase variation in Neisseria meningitidis

Neisseria meningitidis controls the expression of several genes involved in host adaptation by a process known as phase variation. The phase variation frequency of haemoglobin (Hb) receptors among clinical isolates of serogroups A, B and C differed drastically, ranging from approximately 10(-6) to 10(-2) cfu-1. Frequencies of phase variation are a genetic trait of a particular strain, as two unlinked Hb receptors, hpuAB and hmbR, phase varied with similar frequencies within a given isolate. Based on these frequencies, six Neisserial clinical isolates could be grouped into three distinct classes; slow, medium and fast. An increase in phase variation frequency was accompanied by high rates of spontaneous mutation to rifampicin and nalidixic acid resistance in one medium and one fast strain. The remaining three medium strains displayed elevated levels of phase variation without increases in overall mutability, as they possessed low rates of spontaneous mutation to drug resistance. The mismatch repair system of N. meningitidis was found to play an important role in determining the overall mutability of the clinical isolates. Inactivation of mismatch repair in any strain, regardless of its original phenotype, increased mutability to a level seen in the fast strain. Insertional inactivation of mutS and mutL in the slow strain led to 500- and 250-fold increases in hmbR switching frequency respectively. Concurrently, the frequency of spontaneous point mutations of mutS and mutL mutants from the slow strain was increased 20- to 30-fold to the level seen in the high strain. The status of Dam methylation did not correlate with either the phase variation frequency of Hb receptors or the general mutability of Neisserial strains. Analysis of an expanded set of isolates identified defects in mismatch repair as the genetic basis for strains displaying both the fast Hb switching and high mutation rate phenotypes. In conclusion, elevated frequencies of phase variation were accompanied by increased overall mutability in some N. meningitidis isolates including strains shown to be mismatch repair defective. Other isolates have evolved mechanisms that seem to affect only the switching frequency of phase-variable genes without an accompanied increased accumulation of spontaneous mutations.     

Pathways of ultraviolet mutability in Saccharomyces Cerevisiae. IV. The relation between canavanine toxicity and ultraviolet mutability to canavanine resistance.

Seven umr mutants of Saccharomyces cerevisiae which had reduced capacity for ultraviolet light (UV)-induced forward mutation from CAN1 to can1 were tested for sensitivity to L-canavanine relative to one wild-type UMR strain and one slightly UV-sensitive but phenotypically umr⁺ strain (mutant 306). Relative UV mutation resistance was estimated by dividing the UV fluence needed to yeild a particular induced mutation frequency by that needed to reach the same frequency in the genotypic wild-type strain. The umr5 and umr6 strains were especially sensitive to canavanine growth inhibition, while umr1 was no more sensitive than either wild type; umr2, umr3, umr4, a umr7, and α umr7 were equally sensitive to an intermediate degree. Incubation at 30°C of wildtype cells plated on canavanine-selective agar for increasingly longer times before UV irradiation resulted in decreasing UV mutation frequencies (reduced to 50% in 1.6 h). All umr strains tested in this way lost UV mutability faster than wild type, including mutant 306, umr1 (not sensitive to growth inhibition), and umr6 (very sensitive to growth inhibition). Cells were grown to stationary phase in YEDP growth medium and assayed for arginine and tryptophan transport into the cell. The umr6 strain, which had weak UV mutation resistance but high sensitivity to canavanine growth inhibition, transported arginine and tryptophan at essentially wild-type levels. The umr1 strain, however, which had moderate UV mutation resistance and normal canavanine toxicity, transported both amino acids at rates tenfold higher than wild type. The data suggest that increased canavanine toxicity does not necessarily lead to defective mutability at CAN1, and that mutational deficiency cannot result solely from increased canavanine toxicity. Although exposure to canavanine was shown to block mutation fixation and/or expression, it is suggested that the degree of growth inhibition is not strictly correlated with the degree of mutation resistance.     

Sterility and Hypermutability in the P-M System of Hybrid Dysgenesis in DROSOPHILA MELANOGASTER

Inbred wild strains of Drosophila melanogaster derived from the central and eastern United States were used to make dysgenic hybrids in the P-M system. These strains possessed P elements and the P cytotype, the condition that represses P element transposition. Their hybrids were studied for the mutability of the P element insertion mutation, snw, and for the incidence of gonadal dysgenesis (GD) sterility. All the strains tested were able to induce hybrid dysgenesis by one or both of these assays; however, high levels of dysgenesis were rare. Sets of X chromosomes and autosomes from the inbred wild strains were more effective at inducing GD sterility than were sets of Y chromosomes and autosomes. In two separate analyses, GD sterility was positively correlated with snw mutability, suggesting a linear relationship. However, one strain appeared to induce too much GD sterility for its level of snw destabilization, indicating an uncoupling of these two manifestations of hybrid dysgenesis.     

In vitro replication highlights the mutability of prions

Prions exist as strains, which are thought to reflect PrP^Sc conformational variants. Prion strains can mutate and it has been proposed that prion mutability depends on an intrinsic heterogeneity of prion populations that would behave as quasispecies. We investigated in vitro prion mutability of 2 strains, by following PrP^Sc variations of populations serially propagated in PMCA under constant environmental pressure. Each strain was propagated either at low dilution of the seed, i.e., by large population passages, or at limiting dilution, mimicking bottleneck events. In both strains, PrP^Sc conformational variants were identified only after large population passages, while repeated bottleneck events caused a rapid decline in amplification rates. These findings support the view that mutability is an intrinsic property of prions.     

Influence of iron depletion on growth and production of catechol siderophores by different Frankia strains

Nine strains of Frankia isolated from six Casuarinaceae (including four Casuarina sp., one Allocasuarina and one Gymnostoma) and one Elaeagnaceae (Hippophae¨ rhamnoides) were screened for growth and production of siderophores in an iron-deficient liquid medium. Siderophore production was detected only in four strains (Cj, G2, CH and G82) using the CAS and Arnow assays. Salicylates formed more than 90% and dihydroxybenzoates formed less than 10% of all catechol-type siderophores produced. Growth of the former strains was less affected by iron deficiency than that of strains Rif, Thr, URU, BR and RT which do not produce siderophores. Optimal siderophore production by strain Cj was noted when iron concentration reached 0.5m and was completely inhibited at an iron concentration of 10m. The kinetics of siderophore production by strain Cj showed that siderophore synthesis was detectable during the growth stationary phase. Growth of Cj (a siderophore-producing strain) and of RT (a non-siderophore-producing strain) differed when 2,2-dipyridyl or ethylene di(o-hydroxyphenyl) acetic acid (EDDHA) was added to the iron-deficient growth medium. Frankia strain RT was the most sensitive to the detrimental effect of both iron chelators.     

Characteristics of morphology and ultrastructure of bacteria of the genus Dethiosulfovibrio

Cell morphology and fine structure were studied in two strains of rod-shaped, strictly anaerobic, gram-negative sulfidogenic bacteria: strain SR12T (DSM 12538) and strain WS100 (DSM 12537) belonging to "Dethiosulfovibrio starorussensis." Cells of both strains, as well as cells of the type species of the genus Dethiosulfovibrio, D. peptidovorans, were found to possess multiple intracellular incomplete cross septa in the stationary growth phase.     

Surface structures, haemagglutination and cell surface hydrophobicity of Bacteroides fragilis strains.

Nineteen strains of Bacteroides fragilis were examined by negative staining for surface structures. One strain (ATCC 23745) possessed peritrichous fibrils, 16 strains carried peritrichous fimbriae and two strains carried no surface structures. The fimbriae had a diameter of 2.1 +/- 0.25 nm and appeared to be 'curly'. Only a small proportion (4 to 41%, depending on the strain) of cells in a population carried fimbriae or fibrils. Strain A312 Showed phase variation of fimbriae as expression of fimbriae was repressed at 20 degrees C and in early exponential phase at 37 degrees C. The fibrils on strain ATCC 23745 did not exhibit phase variation in response to changes in incubation temperature, growth phase or growth in two different media. Capsules were demonstrated by the Indian ink method on 18 of the 19 strains, varying in size from strain to strain and within the same population. Cultures often contained both capsulate and noncapsulate cells. All strains possessed an electron dense ruthenium red staining layer between 7.9 and 23.9 nm in width attached to the outer membrane. Cell surface hydrophobicity quantified by the hexadecane partition assay gave low values ranging from 6.6 to 52.1%. Only a few strains were able to haemagglutinate and these were only weakly active. There was no correlation between cell surface hydrophobicity, haemagglutinating activity and surface structures.     

Changes in bacterial cells induced by high pressure at subzero temperature

The aim of this study was to examine the effect of pressure treatment at 193 MPa and −20 °C on membrane damage, changes in activity of membrane-bound ATPases and degradation of nucleic acids. The experiments were carried out with three Escherichia coli strains, in the exponential and stationary phases of growth, and differing in sensitivity to pressure. All E. coli strains subjected to pressure in the exponential phase of growth were inactivated by 6 log cycles, independently of the strain, which was accompanied by a total loss of ability to plasmolyse, an increase in irreversible membrane permeability to PI, and a reduction of cellular ATP by more than 80%. After pressure treatment of stationary phase cells, the relationship between the inactivation level and the ability to plasmolyse was not as evident as in the case of exponential phase cells. Pressure treatment of two strains of E. coli K-12 and Ec160/59 in the stationary phase that decreased viability by no more than one log cycle led only to reversible permeabilization of bacterial membranes, while irreversible permeabilization was observed in the pressure sensitive E. coli IBA72 strain phase that was inactivated by 4.6 log cycles. The reduction of ATP and changes in ATPase activity after pressure treatment of tested E. coli strains in the stationary phase of growth depended on the stage of inactivation of the particular strain. Electrophoretic analysis showed degradation of RNA isolated after pressure treatment from cells of all E. coli strains tested in the exponential phase of growth. The changes of RNA induced by pressure were not visible in the case of cells in the stationary phase. The degradation of DNA isolated from pressure treated E. coli strains from the exponential as well as from the stationary phase of growth was not observed.     

Immunogenic protein variations of Clostridium chauvoei cellular antigens associated with the culture growth phase

The immunoprotective capacity of four Clostridium chauvoei strains at different growth stages is reported. In all the strains tested, the cells coming from the stationary phase were those with the highest immunoprotective capacity and, depending on the strain, this protective capacity diminished or even disappeared in other phases. Protein profiles were similar in all the strains and few proteins were differentially expressed during growth as shown by SDS-PAGE. For strain 17, a local strain, a clear relationship was observed between the diminution of immunogenicity and the total loss of protective capacity of sonicated cells at late stationary phase.     

Alteration of tyrosine isoaccepting transfer ribonucleic acid species in wild-type and asporogenous strains of Bacillus subtilis.

The relative amounts of two isoacceping species of tyrosine transfer ribonucleic acid, tRNATyrI and tRNATyrII, determined from reversed phase 5 profiles of tyrosyl-tRNA, prepared from Bacillus subtilis strain W168, were growth phase and medium dependent. The growth phase-dependent alterations in the relative amounts of tRNATyr species were also demonstrated in 11 asporogenous strains of B. subtilis. The proportion of tRNA-Tyr species and the extent of the alteration in their relative amounts during the transition from the exponential to the stationary phase of growth of these strains was not directly correlated with the formation of spores by strain W168 grown in various media or the stage at which the asporogenous strains are blocked in the process of sporulation.     

Response of Escherichia coli to ethyl methanesulfonate: Influence of growth phase and repair ability on survival and mutagenesis

The effects of altering the cell growth rate (physiological state) and DNA repair capacity (genetic state) on susceptibility to inactivation and mutagenesis by ethyl methanesulfonate (EMS) were studied in 4 strains of E. coli. Logarithmic and stationary phase cells of the polymerase I deficient mutant, P₃₄₇8 polA, a recombination deficient mutant, DZ₄₁₇ recA, and the respective parental strains, W₃₁₁₀pol⁺ and AB₂₅₃ rec⁺, were exposed to EMS and the surviving fraction and mutant frequency determined. At the same EMS concentration both mutants were more susceptible to inactivation than the parental strains. In all 4 strains, log phase cells were more sensitive to inactivation than stationary cells. The surviving fraction of stationary cells exceeded log cells by a factor of 18 for polA, 6 for recA, and about 2 for the parental strains. In all strains, except recA, log phase cells exhibited higher spontaneous mutant frequencies than stationary phase cells. At the same concentration of EMS, survivors of both polA and recA showed more than 10-fold higher induced frequencies than the wild types. However, at the same survival levels the repair deficient mutants exhibited induced mutant frequencies comparable to the repair proficient strains. There was no significant effect of growth phase on EMS induced mutability in recA or the parental strains. In marked contrast, the polymerase I deficient mutant shows both a higher spontaneous frequency and a greater than 10-fold higher EMS induced mutant frequency in log phase cultures compared to stationary phase cultures. Our results support the hypothesis that cellular susceptibility to alkylating agents is influenced by both the genetic capability for repair and the particular physiological state of the cell.     

Radiation resistance of lactobacilli isolated from radurized meat relative to growth and environment.

Of 113 lactobacilli isolated from radurized (5 kGy) minced meat, 7 Lactobacillus sake strains, 1 L. curvatus strain, and 1 L. farciminis strain were used for radiation resistance studies in a semisynthetic substrate (i.e., modified MRS broth). Five reference Lactobacillus spp., one Staphylococcus aureus strain, and one Salmonella typhimurium strain were used for comparative purposes. All L. sake isolates exhibited the phenomenon of being more resistant to gamma-irradiation in the exponential (log) phase than in the stationary phase of their growth cycles by a factor of 28%. Four references strains also exhibited this phenomenon, with L. sake (DSM 20017) showing a 68% increase in resistance in the log phase over the stationary phase. This phenomenon was not common to all bacteria tested and is not common to all strains with high radiation resistance. Four L. sake isolates and three reference strains were used in radiation sensitivity testing in a natural food system (i.e., meat). The bacteria were irradiated in minced meat and packaged under four different conditions (air, vacuum, CO2, and N2). Organisms exhibited the highest death rate (lowest D10 values [doses required to reduce the logarithm of the bacterial population by 1] ) under CO2 packaging conditions, but resistance to irradiation was increased under N2. The D10 values of the isolates were generally greater than those of the reference strains. The D10 values were also higher (approximately two times) in meat than in semisynthetic growth medium.     

Influence of iron depletion on growth and production of catechol siderophores by different Frankia strains

Nine strains of Frankia isolated from six Casuarinaceae (including four Casuarina sp., one Allocasuarina and one Gymnostoma) and one Elaeagnaceae (Hippophae¨ rhamnoides) were screened for growth and production of siderophores in an iron-deficient liquid medium. Siderophore production was detected only in four strains (Cj, G2, CH and G82) using the CAS and Arnow assays. Salicylates formed more than 90% and dihydroxybenzoates formed less than 10% of all catechol-type siderophores produced. Growth of the former strains was less affected by iron deficiency than that of strains Rif, Thr, URU, BR and RT which do not produce siderophores. Optimal siderophore production by strain Cj was noted when iron concentration reached 0.5μm and was completely inhibited at an iron concentration of 10μm. The kinetics of siderophore production by strain Cj showed that siderophore synthesis was detectable during the growth stationary phase. Growth of Cj (a siderophore-producing strain) and of RT (a non-siderophore-producing strain) differed when 2,2-dipyridyl or ethylene di(o-hydroxyphenyl) acetic acid (EDDHA) was added to the iron-deficient growth medium. Frankia strain RT was the most sensitive to the detrimental effect of both iron chelators.     

苏云金芽孢杆菌工程菌伴孢晶体的形态发生

苏云金芽孢杆菌在有帮助蛋白存在的情况下杀虫晶体蛋白获得了超量表达。通过透射电镜观察了Cry1Ac超量表达工程菌伴孢晶体的形态发生以及不同芽孢发育时期的晶体形态变化。结果表明,该工程菌的伴孢晶体在细胞不对称分裂的隔膜形成前就已出现,而且晶体发生的部位与芽孢无关。但晶体在形成初期往往靠近母细胞膜。观察结果还表明,大量表达的晶体蛋白不能马上参与到晶体合成,晶体形成的最佳时期是芽孢皮层形成期。母细胞大量液泡的产生与消失可能与晶体形成有关。此外,在超量表达工程菌中,Cry1Ac蛋白能在一个细胞内形成多个伴孢晶体,这在天然菌株中是罕见的。     

Survival of parental and genetically modified derivatives of a soil isolated Pseudomonas fluorescens under nutrient-limiting conditions

Wild type, a rifampicin-resistant mutant and three genetically modified derivatives of the soil isolate Pseudomonas fluorescens R2f were starved in pure cultures for periods of up to 70 d. Cells were starved after harvesting at a point early in the stationary phase of the growth curve and all five strains demonstrated the ability to survive nutrient deprivation and resuscitate rapidly when growth nutrients became available. No difference in total counts and metabolic activity was detected between the strains. Plate counts were similar for all strains up to day 35. Wild type and the rifampicin-resistant mutant strain showed greater recovery than the genetically modified strains on day 70. During the starvation period there was a significant decrease in cell lengths of all five strains, however, there was no significant difference between the strains. The shape of the starved cells varied with the growth phase at which they had been harvested. Cells taken from early stationary phase and starved produced predominantly rod-shaped cells whereas those taken from early log phase and starved produced small round cells. In experiments when the rifampicin-resistant mutant and the genetically modified strain Art-3 were starved at early log phase the cells were significantly smaller than respective cultures not exposed to the nutrient limiting conditions, and there was no significant difference in the response of the two strains. None of the cultures produced ultramicrobacteria, and none of the cultures entered a non-culturable state. Starvation at different cell densities did not affect the recoverability of the cells. The results of this study demonstrate that responses to starvation conditions by the genetically modified and parental strains are similar.     

Biosynthesis of endochitinase of Serratia marcescens.

The growth of a mutant strain of Serratia marcescens with high chitinase activity and the biosynthesis of endochitinase by this strain were investigated. The study was carried out using semisynthetic culture medium without inducers and culture medium containing colloidal chitin as a sole nitrogen and carbon source, with and without mitomycin C. The mutant strain, unlike the native one, was shown to produce endochitinase and to secrete the enzyme into the medium during the growth on culture medium without the inducers, chitin and mitomycin C. During growth on the medium with chitin the mutant strain differed from the native one with a short lag-phase of growth, the early appearance of endochitinase in the culture liquid and a high level of endochitinase activity. The difference between the strains disappeared after the addition of mitomycin C, an inducer of the cell SOS-response, to the culture medium containing chitin. Specific endochitinase activity of S. marcescens mutant strain grown on various culture media had two maxima, namely at the beginning and at the end of the stationary phase. Mitomycin C increased the specific activity in a second peak of endochitinase activity during the growth of the mutant strain.     

Autolysis in strains of viridans streptococci.

Seven strains of viridans streptococci of the species Streptococcus sanguis, S. mutans and S. mitis were investigated for autolysis. The effect of pH, salt concentration and temperature on the autolytic process was studied in Na2HPO4/NaH2PO4 buffer. Whole cells and walls of all strains autolysed most rapidly at pH values above 7. Autolysis of whole cells of S. sanguis and one strain of S. mitis (ATCC15909) was maximal in 0-05 TO 0-2 M buffer, while the two S. mutans strains and S. mitis ATCC15912 showed maximal autolysis in 0-5 and 1-0 M buffers. Cultures harvested in the stationary phase of growth possessed only slightly decreased autolytic activity compared with those from the exponential phase. Whole cells autolysed more rapidly at 37 degrees C Than at 45 degrees C and 10 degrees C. Autolysis of isolated walls of three strains of S. mitis (ATCC903, ATCC15909 and ATCC15912) was maximal at pH 7-0 AND 7-5 and in 1-0 M buffers. Streptococcus mitis ATCC15909 also showed maximal lysis in 0-01 M and 0-5 M buffers. An endopeptidase action of the autolytic system of S. mitis ATCC15912 was indicated by the progressive release of soluble amino groups during autolysis of the walls. No release of reducing groups was observed. Several free amino acids were released during autolysis of these walls, alanine, lysine and glutamic acid being in greatest quanitity.     

Transient self-inhibition of the growth of Lactobacillus delbrueckii subsp. bulgaricus in a pH-regulated fermentor

An industrial strain of Lactobacillus delbrueckii subsp. bulgaricus was grown in a synthetic medium on lactose as carbon substrate, in a pH-regulated fermentor. Growth proceeded in two distinct phases separated by a transient stationary phase. Various experimental approaches were used to identify the cause of this growth arrest. Growth experiments in L. bulgaricus culture supernatant fluids collected at different cultivation times in fermentor, and supplemented or not with various nutritional solutions, enabled us to discard the possibility of a nutritional limitation. Tube cultures of L. bulgaricus in medium supplemented with various lactic acid concentrations showed a potential inhibition by this metabolic end product but confirmed that this inhibition was not responsible for the cessation of growth. It was concluded that at least one inhibitory compound was produced during the growth phase of the strain, and this compound disappeared from the medium in the transient stationary phase, enabling the growth to start again later in the culture. Indeed, the stoichiometric analysis of the culture showed, firstly, that unidentified carbon compounds were produced from lactose during growth, which were probably converted in lactic acid during the transient stationary phase and, secondly, that part of the amino acids consumed gave catabolic end products. Finally, bacteriocin-like compounds were not considered to be responsible for this growth arrest.     

Experiences with serology of Plesiomonas shigelloides. 1. O-antigenic structure

With a set of 30 O-antisera, O-antigens were identified in 80% of 158 Plesiomonas shigelloides strains. Only strains of one serovar (018) regularly contained capsular antigen that caused their inagglutinability in the live state. Two groups of serovars displayed some O-antigenic relationship: 03 and 029; 08, 011 and 012. Each serovar in either group possessed a specific O-antigen and "group-common" minor antigens, which were designated I, II and III. Serovar 017 possessed O-antigen identical with that of Shigella sonnei phase I; this serovar was the most frequent one. Some serovars seemed to be ubiquitous; this was indicated by their wide geographic distribution and findings in man, domestic and feral animals, and water.