Genetic instability of industrial strains of Penicillium chrysogenum

Summary It has shown that several characteristics of high-producing industrial strains of Penicillium chrysogenum tend to segregate in the course of cultivation (slant-to-slant transfer). Segregation includes a decrease in the yield of penicillin, mean conidial size, mean size of the nuclei, and an increase in the proportion of morphologically wild-type colonies. These lower-producing segregants also have a higher sensitivity against ultraviolet radiation and, as shown by cytofluorometric methods, a lower DNA content in the condia, a decrease in phosphate uptkae and in the activity of extracellular alkaline phosphatases compared to high-producing strains. Obviously, during mutagenesis/selection programmes ploidy mutants have been selected, which entails an increase in the number of genes coding enzymes responsible for penicillin biosynthesis. In the absence of selection pressure these high-producing strains segregate to lower-producing strains by chromosome losses in the course of slant-to-slant transfers. Offprint requests to: W. Künkel     

Heterozygous diploids ofPenicillium Chrysogenum and their segregation patterns

Several heterozygous diploids were made between genetically labelled derivatives of two strains ofPenicillium chrysogenum which produced relatively large amounts of penicillin and were of divergent lineage. The derivatives were labelled with spore colour and nutritional mutations. The diploids, although uniform in having wild type spore colour and being prototrophic, ranged from types having penicillin yields close to that of the original parents to types having less than a quarter of this titre level. Intermediate types had titre levels of about half to threequarters that of the high yielding diploids. Segregants were selected which had arisen naturally and also after nitrogen mustard treatment; most had the spore colour and auxotrophic phenotype of one or other immediate parent. From diploids of low and intermediate titre only haploid segregants with the genetical markers of one parent could be recovered with intact penicillin yield; haploids with the genetic markers of the other showed a marked reduction in yield. However, from diploids of high yield, both parental types could be recovered showing no loss of their original penicillin yield. The bearing of these results is discussed on the suggestion that different degrees of homozygosity between diploids may account for the titre variation observed. An alternative suggestion that mutations suppressive to penicillin titre might cause such variation is also considered.     

肺炎链球菌pbp2b和pbp1a基因突变与青霉素耐药的相关性研究

目的探讨耐青霉素肺炎链球菌pbp2b和pbp1 a基因的突变与青霉素耐药的关系,为明了肺炎链球菌的耐药性变异机制,防治其感染提供实验依据。方法从呼吸道感染患儿痰标本中分离肺炎链球菌163株,液体培养基连续稀释法测定其对青霉素的最小抑菌浓度(M IC),套式聚合酶链反应(nPCR)扩增pbp2b和pbp1 a基因,扩增产物直接DNA测序,所测序列与青霉素敏感株(SPN R6)的基因序列进行比较,并分析其氨基酸结构的改变。结果 163株肺炎链球菌中检出青霉素敏感菌75株,中度敏感17株,青霉素耐药菌71株(44%)。耐药菌中58株存在pbp2b突变(81.7%),其中,56株为点突变,2株为CCT插入突变;在27株有pbp2b基因突变的B型和C型耐药菌中,21株出现了不同程度的pbp1 a基因突变。PBP2B氨基酸结构改变以苏氨酸变为丙氨酸、精氨酸变为赖氨酸为主,PBP1A以丙氨酸变为苏氨酸、谷氨酸变为天门冬氨酸为主。结论肺炎链球菌的pbp2b和pbp1 a基因突变与对青霉素的耐药性密切相关,PBP2b突变导致低水平耐药;PBP2b和PBP1A突变导致高水平耐药。     

Prospects for increasing yield in macadamia using component traits and genomics

Selection of candidate cultivars in macadamia requires extensive phenotypic measurements over many years and trials. In particular, yield traits such as nut-in-shell yield and kernel yield are economically vital characteristics and therefore guide the selection process for new cultivars. However, these traits can only be measured in mature trees, resulting in long generation intervals and slow rates of genetic gain. In addition, these traits are expensive to measure. Strategies to reduce the generation interval and increase the intensity of selection include using yield component traits, identification of markers associated with component traits, and genomic selection for yield. Yield component traits that contribute to resource availability for fruit formation include floral and nut characteristics. In this review, these traits will be investigated to estimate their relative importance in macadamia breeding and their heritability and correlations with yield. Furthermore, the usefulness of genome-wide association studies regarding yield component traits will be reviewed. Genetic-based breeding techniques could exploit this information to increase yield gains per breeding cycle and estimate the quantitative nature of yield traits. Genomic selection uses genome-wide molecular markers to predict the phenotype of individuals at an early age before maturity, thereby reducing the cycle time and increasing gain per unit time in plant breeding programmes. This review evaluates the potential for measurement of yield component traits, genome-wide association studies, and genomic selection to be employed in the Australian macadamia breeding programme to accelerate gains for nut yield.     

alpha-Aminoadipate pool concentration and penicillin biosynthesis in strains of Penicillium chrysogenum

Intracellular amino acid pools in four Penicillium chrysogenum strains, which differed in their ability to produce penicillin, were determined under conditions supporting growth without penicillin production and under conditions supporting penicillin production. A significant correlation between the rate of penicillin production and the intracellular concentration of alpha-aminoadipate was observed, which was not shown with any other amino acid in the pool. In replacement cultivation, penicillin production was stimulated by alpha-aminoadipate, but not by valine or cysteine. Exogenously added alpha-aminoadipate (2 or 3 mM) maximally stimulated penicillin synthesis in two strains of different productivity. Under these conditions intracellular concentrations of alpha-aminoadipate were comparable in the two strains in spite of the higher rate of penicillin production in the more productive strain. Results suggest that the lower penicillin titre of strain Q 176 is due to at least two factors: (i) the intracellular concentration of alpha-aminoadipate is insufficient to allow saturation of any enzyme which is rate limiting in the conversion of alpha-aminoadipate to penicillin and (ii) the level of an enzyme, which is rate limiting in the conversion of alpha-aminoadipate to penicillin, is lower in Q 176 (relative to strain D6/1014/A). Results suggest that the intracellular concentration of alpha-aminoadipate in strain D6/1014/A is sufficiently high to allow saturation of the rate-limiting penicillin biosynthetic enzyme in that strain. The basis of further correlation of intracellular alpha-aminoadipate concentration and penicillin titre among strains D6/1014/A, P2, and 389/3, the three highest penicillin producers studied here, remains to be established.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of a forward and a reverse mutation assay in Salmonella typhimurium measuring L-arabinose resistance and histidine prototrophy

A forward and a reverse mutation assay designed to detect environmental mutagens have been compared in Salmonella typhimurium. The forward mutation assay scored resistance to L-arabinose and the reverse assay, reversion of histidine auxotrophy. Eighteen chemicals of different structural groups, all known to be mutagenic in the histidine reverse assay, were applied to strains carrying the genetic markers needed to perform both mutation assays. The mutagenicity of each chemical was determined by both plate and liquid tests. The plate test counted absolute numbers of surviving mutants and the liquid test separately measured survival and frequency of mutants among the survivors. All the chemicals used were found to be mutagenic in both mutation assays. The response of the L-arabinose assay was equal to or larger than the response of the histidine assay in the case of 16 chemicals. The two other compounds, 2-nitrofluorene and sodium azide, were detected more efficiently by the histidine assay. Sodium azide, a non-carcinogenic compound, is a potent mutagen in the histidine assay, but very weak in the L-arabinose assay.     

Comparison of N-ethyl-N-nitrosourea Effects in three strains of chlorococcal algae

Three strains of three different species of chlorococcal algae(Chlorella kesslerii Scenedesmus quadricauda andScenedesmus obliquus) were compared in their physiological and genetical responses to N-ethyl-N-nitrosourea (NEU). The mutagen was applied in the basic doses corresponding to their specific tolerance to it. Exponentially-graded doses derived from the basic ones acted for the same period of time and single basic doses acted at linearly-increased periods of time in parallel experiments. The influence of the mutagen on the cell cycle just in progress was characterized by the increase in the frequency of cell divisions which yielded a lower number of autospores. The conditions of the cells during the first three days after treatment were determined according to the average increase of their lag phase. The relative toxicity of the mutagen was expressed as the survival frequencies. The mutation effects were evaluated according to the frequency of permanent changes observed in the growing cell colonies : lethal, morphological and pigmentation. NEU was found to be a relatively suitable mutagen for the chlorococcal algae studied.     

Ecdysteroid titres during larval life and metamorphosis of Galleria mellonella

The ecdysteroid titre in Galleria larvae normally follows either a short larval cycle, which is characterized by a single peak on day 2, or a long metamorphic cycle, which includes a small peak on day 6 and a large one on day 7. Intermediate cycles and moult induction with reduced ecdysteroid concentrations occur under experimental conditions. The cycles and peaks are initiated and, until the brain critical period, driven by prothoracicotropic hormone (PTTH), while juvenile hormone (JH) titre determines which type of cycle will be activated. In larval development JH stimulates PTTH release but the metamorphic programme of neuroendocrine regulations involves blocking PTTH production by JH. This hormone then causes an interruption of the ecdysteroid cycle and extension of the last larval instar.     

Induction of Freeze-sensitive Mutants from a Freeze-tolerant Yeast,Torulaspora delbrueckii

Freeze-sensitive strains of yeast were induced from a freeze-tolerant yeast Torulaspora delbrueckii by incubation with ethyl-methane sulfonate as a mutagen. A maximum ratio of mutation was attained by the incubation at 30°C for 75min. One-hundred and fifty strains of freeze-sensitive yeast were selected by plating-culture for the first screening. The freeze-tolerance ratio of each strain was examined based on the fermentative activity before and after freezing in liquid medium and dough. Strain 60B3 showed the highest freeze-sensitivity in a pre-fermented frozen dough (pre-fermented at 30°C for 2h, and frozen at ?20°C for 7 days) among eight strains finally selected.     

Long-term endurance and selection studies in hydrogen and oxygen photoproduction by Chlamydomonas reinhardtii

Two wild-type strains of Chlamydomonas reinhardtii have been subjected to repeated cycles of anaerobiosis, carbon dioxide deprivation, and irradiation as a means of testing the long-term stability of hydrogen and oxygen photoproduction and the effectiveness of these conditions as selection or adaptation pressures for increasing hydrogen and/or oxygen yields. Simultaneous hydrogen and oxygen photoproduction yields were monitored in each culture for 160 h. The cells were then removed from the reaction chamber and used to inoculate fresh growth medium to produce the culture for the next experiment. This cycle was repeated five times. Yields of hydrogen and oxygen improved after three cycles and declined in the fourth and fifth; unlike the second and third cycles, extended periods of aerobic growth were used for the fourth and fifth cycles. The stability of hydrogen and oxygen photoproduction was greater in the fifth cycle than in any of the previous cycles. These subpopulations had hydrogen and oxygen production rates, at 160 h, which were nearly equal to the rates at the beginning of the fifth-cycle experiments. Time profiles of the relative hydrogen yields from each of the five cycles, prepared at 32, 80, and 120 h, show that the relative yield in each varies with the point in time at which the profile was taken. Chlorophyll retention increased with each successive cycle, indicating selection or adaptation for a more durable population of cells with respect to the light-harvesting component of the photosynthetic apparatus.     

Relationship between hormone titres and RNA and protein syntheses when the change to the pupal programme occurs in the silkworm, Bombyx mori

The information required for pupal programming was received early in the larval-pupal stage when the juvenile hormone level had fallen and the ecdysone titre was low. However, ecdysone titre at a threshold level was required to activate the pupal programme.RNA synthesis in the integument did not increase until the ecdysone titre reached the threshold level, whereas protein synthesis did not increase throughout the experimental period.     

Structural Variation among Wild and Industrial Strains of Penicillium chrysogenum

Strain selection and strain improvement are the first, and arguably most important, steps in the industrial production of biological compounds by microorganisms. While traditional methods of mutagenesis and selection have been effective in improving production of compounds at a commercial scale, the genetic changes underpinning the altered phenotypes have remained largely unclear. We utilized high-throughput Illumina short read sequencing of a wild Penicillium chrysogenum strain in order to make whole genome comparisons to a sequenced improved strain (WIS 54–1255). We developed an assembly-free method of identifying chromosomal rearrangements and validated the in silico predictions with a PCR-based assay and Sanger sequencing. Despite many rounds of mutagen treatment and artificial selection, WIS 54–1255 differs from its wild progenitor at only one of the identified rearrangements. We suggest that natural variants predisposed for high penicillin production were instrumental in the success of WIS 54–1255 as an industrial strain. In addition to finding a previously published inversion in the penicillin biosynthesis cluster, we located several genes related to penicillin production associated with these rearrangements. By comparing the configuration of rearrangement events among several historically important strains known to be high penicillin producers to a collection of recently isolated wild strains, we suggest that wild strains with rearrangements similar to those in known high penicillin producers may be viable candidates for further improvement efforts.     

Effect of corn-steep liquor on growth and mosquito larvicidal activity of Bacillus thuringiensis var israelensis de Barjac 1978 and B. sphaericus Neide 1904.

Influence of corn steep liquor on the cell yield and toxicity of three strains of B. thuringiensis var israelensis and two strains of B. sphaericus was studied and compared with peptone-yeast extract using a laboratory fermentor. Large increase in the cell yield of all the three strains of B. thuringiensis var israelensis was observed when cornsteep liquor was used as the sole nitrogen source. Significant increase in toxicity was also observed in B. thuringiensis var israelensis strains B17 and B113. Among the two B. sphaericus strains tested, the strain 1593 showed no significant change in cell yield and toxicity, whereas the strain VCRC B42 showed increased cell yield and toxicity in this medium. The results indicate that cornsteep liquor can effectively replace both peptone and yeast extract in the media presently used for large scale multiplication of the two larvicidal bacilli.     

Immobilization of permeabilized whole cell penicillin G acylase from Alcaligenes faecalis using pore matrix crosslinked with glutaraldehyde

The activity of penicillin G acylase from Alcaligenes faecalis increased 7.5-fold when cells were permeabilized with 0.3% (w/v) CTAB. The treated cells were entrapped by polyvinyl alcohol crosslinked with boric acid, and crosslinked with 2% (v/v) glutaraldehyde to increase the stability. The conversion yield of penicillin G to 6-aminopenicillanic acid was 75% by immobilized system in batch reaction. No activity was lost after 15 cycles and about 65% enzyme activity was retained at the end of the 31th cycle.     

Allozyme frequencies, heterozygosity and genetic distances following S1 recurrent selection in two synthetic maize populations

 Three cycles of S₁ recurrent selection for yield were carried out in two synthetic maize populations, EPS6 from humid Spain and EPS7 from arid Spain. One-hundred S₁ lines were evaluated from each cycle of selection and the ten highest-yielding S₁ lines were recombined to produce the next cycle. Changes in variability and genetic distances in two synthetic maize populations, following three cycles of recurrent selection, recombining ten S₁ lines in each cycle, were determined. Isozyme analysis was performed on 125 seedlings per cycle of selection (four cycles in each of two populations). Regressions of each allozyme frequency on cycles of selection were performed, genetic distances between populations were determined, and simple correlations between genetic distances and heterosis were calculated. The average heterozygosity per locus was also calculated for each population. Regression analysis did not reveal any common trend between EPS6 and EPS7 for changes in allele frequencies presumably due to selection. The number of polymorphic loci, the mean alleles per locus, and the mean heterozygosity did not show any reduction in variability. Finally, selection did not affect genetic distances among cycles of selection. The agronomic evaluation of the selection program, after three cycles of selection, revealed that the genetic variance was not significantly reduced for most traits, and that the heterosis among cycles of selection of both populations had not changed significantly. The conclusions based on isozyme data supported the deductions made from agronomic data. Three cycles of selection neither caused relevant changes on variability nor on genetic distance among cycles of selection of both maize synthetic populations. These data did not indicate any basis for increasing the number of S₁ lines recombined for recurrent selection. Received: 28 April 1997 / Accepted: 23 June 1997     

Improvement of A21978C production in Streptomyces roseosporus by reporter-guided rpsL mutation selection

Aims:  Daptomycin, one of the A21978C factors produced by Streptomyces roseosporus, is an acidic cyclic lipopeptide antibiotic with potent activity against a variety of Gram‐positive pathogens. To increase the titre of this extensively used and clinically important antibiotic, we applied a reported‐guided rpsL mutation selection system to generate strains producing high levels of A21978C. Methods and Results:  In the reporter design, dptE was chosen as the overexpressing target, and neo‐encoding neomycin phosphotransferase as the reporter. Using this reporter‐guided selection system, 20% of the selected, streptomycin‐resistant mutants produced greater amounts of A21978C than the starting strain. The selection system increased the screening efficiency about 10‐fold with a frequency of 1·7% A21978C overproducing strains among str^r mutants. A21978C production was increased approximately 2·2‐fold in the rpsL K43N mutant. Conclusions:  The combination of ribosome engineering and reporter‐guided mutant selection generated an A21978C overproducing strain that produced about twice as much A21978C as the parental strain. Significance and Impact of the Study:  The strategies presented here, which integrated the advantages of both ribosome engineering and reporter‐guided mutation selection, could be applied to other bacteria to improve their yield of secondary metabolites.     

Modified method of application of ethyl methanesulfonate for induction ofEscherichia coli Mutants

An efficient modification of ethyl methanesulfonate mutagenic action is based on mutagenization of small volumes of cell suspensions in micro-sample tubes. This provides for a rapid and safe handling of solutions of cancerogenic mutagens. A 3-4-h exposition to 30-40 mmol/L of mutagen appeared optimal, inducing more than 20% auxotrophs or, after a simultaneous application of the penicillin method, 60% of auxotrophs. Moreover, the modification proved its value in repeated applications of ethyl methanesulfonate, resulting in an accumulation of various mutation types. Consecutive mutations were accompanied by an increase in sensitivity. Based on the distribution of nucleoids in the mutagenized population, the penicillin method was modified to allow detection of mutants segregated from cells with several nucleoids.     

Methods for Mutation and Selection of the Ergot Fungus

A new method is described in which the Salkowski reaction is used for the rapid selection of alkaloid-producing mutants of the ergot fungus. This method was used to investigate the influence of a second mutation with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) on various mutants selected by a preliminary NTG mutation of Claviceps sp. strain SD 58. Three groups of mutants were used: high alkaloid producers, low alkaloid producers, and auxotrophs. Results indicated that a second mutation of all three types of mutants could improve alkaloid yield and vegetative vigor. In addition, a second mutation increased the frequency of auxotroph production. The difficulty of producing stable mutants from ergot strains that have multinucleated cells and that do not readily produce conidia in culture, such as an ergotamine-producing strain, was overcome by first forming protoplasts of the fungus and then subjecting them to the mutagen. Stable auxotrophs were obtained in this manner.     

Inferring somatic mutation rates using the stop-enhanced green fluorescent protein mouse

A new method is developed for estimating rates of somatic mutation in vivo. The stop-enhanced green fluorescent protein (EGFP) transgenic mouse carries multiple copies of an EGFP gene with a premature stop codon. The gene can revert to a functional form via point mutations. Mice treated with a potent mutagen, N-ethyl-N-nitrosourea (ENU), and mice treated with a vehicle alone are assayed for mutations in liver cells. A stochastic model is developed to model the mutation and gene expression processes and maximum-likelihood estimators of the model parameters are derived. A likelihood-ratio test (LRT) is developed for detecting mutagenicity. Parametric bootstrap simulations are used to obtain confidence intervals of the parameter estimates and to estimate the significance of the LRT. The LRT is highly significant (alpha < 0.01) and the 95% confidence interval for the relative effect of the mutagen (the ratio of the rate of mutation during the interval of mutagen exposure to the rate of background mutation) ranges from a minimum 200-fold effect of the mutagen to a maximum 2000-fold effect.     

Predicted and realized grain yield responses to full-sib family selection in CIMMYT maize (Zea mays L.) populations

Summary The maize (Zea mays L.) improvement program of the International Maize and Wheat Improvement Center (CIMMYT) develops broad-based maize populations and, until recently, improved all of them through full-sib family selection with international testing. The purpose of this study was to estimate the genetic and genetic × environment variance components for ten of those populations and to measure expected yield improvement from full-sib selection. Mean yield ranged from 3.35–6.81 t ha^–1. For five populations the average yield in the last cycle was higher than in the initial cycles. Several populations showed no improvement or yielded less in the final cycle of selection, either because selection intensity was low or because strong selection pressure was applied simultaneously for several traits. Variation resulting from differences among family means within cycles and from interaction between families and locations within cycles were significant in all populations and cycles. Results indicate that variability among full-sib families was maintained throughout the cycles for all populations. The large _ge ² / _g ² ratio shown by most populations suggests that yield response per cycle could be maximized if the environments in which progenies are tested were subdivided and classified into similar subsets. The proportion of the predicted response realized in improved yield varied for each population.Journal Paper No. 8640, Nebraska Agric. Res. Div. Project No. 12-159. Research was supported in part by USAID/USDA/ CSRS Research Grant No. 86-CRSR-2-2789