rpoS基因在肠杆菌CGMCC 5087响应环境胁迫中的功能

[背景]苯乙醇(2-Phenylethanol,2-PE)是一种具有玫瑰香气味的高级香料添加剂,被广泛应用于香水、化妆品、食品和医药等领域.目前,利用工程菌合成苯乙醇有很好的应用前景.我们分离到一株肠杆菌(Enterobactersp.)CGMCC 5087,其可以通过苯丙酮酸途径合成2-PE.然而该菌的生长受到不同环...     

Divergent roles of RpoS in Escherichia coli under aerobic and anaerobic conditions

Escherichia coli exhibited different levels of rpoS expression and general stress resistance under aerobiosis and anaerobiosis. Expression measured using reporter gene fusions and protein levels was lower under anaerobic conditions. Consistent with earlier findings, rpoS mutants were selected in aerobic nutrient-limited cultures but rpoS mutants were not enriched under anaerobiosis. This result suggested that, despite its decreased level, RpoS had a function under anaerobic conditions not essential under aerobiosis. Competition experiments between rpoS(+) and rpoS bacteria confirmed the advantage conferred by RpoS under anaerobiosis. In contrast, stress resistance assays suggested RpoS made a greater contribution to general stress resistance under aerobiosis than anaerobiosis. These results indicate a significant, but different role of RpoS in aerobic and anaerobic environments.     

The in vitro fitness cost of antimicrobial resistance in Escherichia coli varies with the growth conditions

The objective of this study was to investigate the influence of stressful growth conditions on the fitness cost of antimicrobial resistance in Escherichia coli BJ4 caused by chromosomal mutations and plasmid acquisition. The fitness cost of chromosomal streptomycin resistance increased significantly when the bacteria were grown under all stress conditions tested, while the cost in 1/3 Luria–Bertani was not significantly changed in a streptomycin+rifampicin mutant. The increase in the fitness cost depended in a nonregular manner on the strain/stress combination. The fitness cost of plasmid-encoded resistance on R751 did not differ significantly, and was generally less under stressful growth conditions than in rich media. The fitness cost associated with R751 with the multiple drug resistance cassette from Salmonella Typhimurium DT104 increased significantly only under stressful conditions at low pH and at high-salt concentrations. Strains with an impaired rpoS demonstrated a reduced fitness only during growth in a high-salt concentration. In conclusion, it was demonstrated that bacterial fitness cost in association with antimicrobial resistance generally increases under stressful growth conditions. However, the growth potential of bacteria with antimicrobial resistances did not increase in a straightforward manner in these in vitro experiments and is therefore probably even more difficult to predict in vivo .     

Mutation of rpoS gene decreased resistance to environmental stresses, synthesis of extracellular products and virulence of Vibrio anguillarum

Vibrio anguillarum is a gram-negative halophilic bacterium that causes vibriosis in marine fish, freshwater fish and other aquatic animals. Bacteria have developed strategies to survive in harsh environments. The alternative σ factor, RpoS (σ^S), plays a key role in surviving under stress conditions in some gram-negative bacteria. An rpoS mutant of pathogenic V. anguillarum W-1 was constructed by homologous recombination. The sensitivity of the rpoS mutant to osmotic stress [2.4 M NaCl in artificial seawater (ASW)] did not change obviously, but the sensitivity of the rpoS mutant to high temperature (45 °C in ASW), UV-irradiation and oxidative stress (5 mM H₂O₂ in ASW) increased 33-fold, sixfold and 10-fold, respectively. The production of extracellular phospholipase, diastase, lipase, caseinase, hemolysin, catalase and protease of the rpoS mutant decreased markedly compared with those of the wild-type strain. Virulence of the rpoS mutant strain was also decreased when it was inoculated intraperitoneally into zebra fish; the lethal dose 50% of the wild type and the mutant was 8.66 × 10⁴ and 2.55 × 10⁶ CFU per fish, respectively. These results indicated that the RpoS of V. anguillarum plays important roles in bacterial adaptation to environmental stresses and its pathogenicity.     

Functional heterogeneity of RpoS in stress tolerance of enterohemorrhagic Escherichia coli strains

The stationary-phase sigma factor (RpoS) regulates many cellular responses to environmental stress conditions such as heat, acid, and alkali shocks. On the other hand, mutations at the rpoS locus have frequently been detected among pathogenic as well as commensal strains of Escherichia coli. The objective of this study was to perform a functional analysis of the RpoS-mediated stress responses of enterohemorrhagic E. coli strains from food-borne outbreaks. E. coli strains belonging to serotypes O157:H7, O111:H11, and O26:H11 exhibited polymorphisms for two phenotypes widely used to monitor rpoS mutations, heat tolerance and glycogen synthesis, as well as for two others, alkali tolerance and adherence to Caco-2 cells. However, these strains synthesized the oxidative acid resistance system through an rpoS-dependent pathway. During the transition from mildly acidic growth conditions (pH 5.5) to alkaline stress (pH 10.2), cell survival was dependent on rpoS functionality. Some strains were able to overcome negative regulation by RpoS and induced higher beta-galactosidase activity without compromising their acid resistance. There were no major differences in the DNA sequences in the rpoS coding regions among the tested strains. The heterogeneity of rpoS-dependent phenotypes observed for stress-related phenotypes was also evident in the Caco-2 cell adherence assay. Wild-type O157:H7 strains with native rpoS were less adherent than rpoS-complemented counterpart strains, suggesting that rpoS functionality is needed. These results show that some pathogenic E. coli strains can maintain their acid tolerance capability while compromising other RpoS-dependent stress responses. Such adaptation processes may have significant impact on a pathogen's survival in food processing environments, as well in the host's stomach and intestine.     

Plasticity and acclimation to light reflected in temporal and spatial changes of small-scale macroalgal distribution in a stream

The small-scale distribution pattern of macroalgae in the river Ilm, in Germany was monitored. These patterns were then related to abiotic factors and tested to discover whether the distribution of the common macroalgae, Cladophora glomerata (L.) Kütz. and Vaucheria sp., was linked to differences in their photosynthetic plasticity. Cladophora glomerata revealed higher maximum photosynthetic electron transport rates after acclimation to high light (HL) compared with low light (LL) acclimated samples. By contrast, Vaucheria sp. did not acclimate to different growth light conditions. The photosynthetic performance of both algae also varied according to diurnal conditions. High light caused a reversible decrease of the dark-adapted quantum yield (F(v)/F(m)) in C. glomerata and a concomitant reversible decrease of the light-adapted quantum yield (DeltaF/F'(m)). In Vaucheria sp., F(v)/F(m) remained mostly unchanged over the day, whereas DeltaF/F'(m) decreased during the morning at low light. Photosynthetic pigments confirmed acclimational differences between the species. HL C. glomerata showed increased chlorophyll a:chlorophyll b ratios, and higher amounts of xanthophyll-cycle pigments compared with LL samples, whereas Vaucheria sp. did not reveal differences between the light treatments. While preferences for substrate size, water velocity, and depth are similar for C. glomerata and Vaucheria sp., the physiological responses to light conditions are different. It is concluded that light conditions significantly affect the small-scale spatial distribution of macroalgae and that fitness is enhanced in species with a higher plasticity in photosynthetic acclimation in unstable environments.     

Fitness costs associated with low genetic variation are reduced in a harsher environment in amphibian island populations

A basic premise of conservation geneticists is that low levels of genetic variation are associated with fitness costs in terms of reduced survival and fecundity. These fitness costs may frequently vary with environmental factors and should increase under more stressful conditions. However, there is no consensus on how fitness costs associated with low genetic variation change under natural conditions in relation to the stressfulness of the environment. On the Swedish west coast, natterjack toad Bufo calamita populations show a strong population genetic structure and large variation in the amount of within-population genetic variation. We experimentally examined the survival of natterjack larvae from six populations with different genetic variation in three thermal environments corresponding to (a) the mean temperature of natural ponds (stable, laboratory), (b) a high temperature environment occurring in desiccating ponds (stable, laboratory) and (c) an outdoor treatment mimicking the natural, variable thermal conditions (fluctuating, semi-natural). We found that larvae in the outdoor treatment had poorer survival than larvae in the stable environments suggesting that the outdoor treatment was more stressful. Overall, populations with higher genetic variation had higher larval survival. However, a significant interaction between treatments and genetic variation indicated that fitness costs associated with low genetic variation were less severe in the outdoor treatment. Thus, we found no support for the hypothesis that fitness costs associated with low genetic variation increase under more stressful conditions. Our results suggest that natural thermal stress may mask fitness losses associated with low genetic variation in these populations.     

MiR-34a inhibits lymphatic metastasis potential of mouse hepatoma cells

Genetic adaptation is one of the key features of Escherichia coli (E. coli) that ensure its survival in different hostile environments. E. coli seems to initiate biofilm development in response to specific environmental cues. A number of properties inherent within bacterial biofilms indicate that their gene expression is different from that of planktonic bacteria. Two of the possible important genes are rpoS and bolA. The rpoS gene has been known as the alternative sigma (σ) factor, which controls the expression of a large number of genes, which are involved in responses to a varied number of stresses, as well as transition to stationary phase from exponential form of growth. Morphogene bolA response to stress environment leads to round morphology of E. coli cells, but little is known about its involvement in biofilms and its development or maintenance. The purpose of this study was to understand and analyse the responses of rpoS and bolA gene to sudden change in the environment. In this study, E. coli K-12 MG1655, rpoS, and bolA mutant strains were used and gene expression was studied. Results show that both genes contribute to the ability to respond and adapt in response to various types of stresses. RpoS response to various stress environments was somehow constant in both the planktonic and biofilm phases, whereas bolA responded well under various stress conditions, in both planktonic and biofilm mode, up to 5-6-fold change in the expression was noticed in the case of pH variation and hydrogen peroxide stress (H(2)O(2)) as compared with rpoS.     

Crl binds to domain 2 of σ(S) and confers a competitive advantage on a natural rpoS mutant of Salmonella enterica serovar Typhi

The RpoS sigma factor (σ(S)) is the master regulator of the bacterial response to a variety of stresses. Mutants in rpoS arise in bacterial populations in the absence of stress, probably as a consequence of a subtle balance between self-preservation and nutritional competence. We characterized here one natural rpoS mutant of Salmonella enterica serovar Typhi (Ty19). We show that the rpoS allele of Ty19 (rpoS(Ty19)) led to the synthesis of a σ(S)(Ty19) protein carrying a single glycine-to-valine substitution at position 282 in σ(S) domain 4, which was much more dependent than the wild-type σ(S) protein on activation by Crl, a chaperone-like protein that increases the affinity of σ(S) for the RNA polymerase core enzyme (E). We used the bacterial adenylate cyclase two-hybrid system to demonstrate that Crl bound to residues 72 to 167 of σ(S) domain 2 and that G282V substitution did not directly affect Crl binding. However, this substitution drastically reduced the ability of σ(S)(Ty19) to bind E in a surface plasmon resonance assay, a defect partially rescued by Crl. The modeled structure of the Eσ(S) holoenzyme suggested that substitution G282V could directly disrupt a favorable interaction between σ(S) and E. The rpoS(Ty19) allele conferred a competitive fitness when the bacterial population was wild type for crl but was outcompeted in Δcrl populations. Thus, these results indicate that the competitive advantage of the rpoS(Ty19) mutant is dependent on Crl and suggest that crl plays a role in the appearance of rpoS mutants in bacterial populations.     

Polymorphisms in rpoS and stress tolerance heterogeneity in natural isolates of Cronobacter sakazakii

Significant phenotypic diversity was observed when we examined the abilities of a number of Cronobacter sakazakii natural isolates to cope with various sublethal stress conditions (acid, alkaline, osmotic, oxidative, or heat stress). Levels of catalase activity and use of acetate as a carbon source, phenotypes commonly used as indirect assays to predict RpoS function, revealed a high correlation between predicted RpoS activity and tolerance to acid, alkaline, osmotic, and oxidative treatments. The rpoS genes were sequenced and analyzed for polymorphisms. Loss-of-function mutations were found in two strains; C. sakazakii DPC 6523 and the genome-sequenced strain C. sakazakii ATCC BAA-894. The complementation of these strains with a functional rpoS gene resulted in an increase in bacterial tolerance to acid, osmotic, and oxidative stresses. The pigmentation status of strains was also assessed, and a high variability in carotenoid content was observed, with a functional rpoS gene being essential for the production of the characteristic yellow pigment. In conclusion, the evidence presented in this study demonstrates that rpoS is a highly polymorphic gene in C. sakazakii, and it supports the importance of RpoS for the tolerance under stress conditions that C. sakazakii may encounter in the food chain and in the host during infection.     

Equivalent inbreeding depression under laboratory and field conditions in a tree-hole-breeding mosquito

Understanding the consequences of inbreeding has important implications for a wide variety of topics in population biology. However, most studies quantifying the effects of inbreeding are performed under artificial farm, greenhouse, laboratory or zoo conditions. Although several authors have argued that the deleterious effects of inbreeding (inbreeding depression) are likely to be more severe under natural field conditions than in artificial experimental environments, these arguments are usually speculative or based on indirect comparisons. We quantified the effects of inbreeding on fitness traits in a tree-hole-breeding mosquito Aedes geniculatus) under near-optimal laboratory conditions and in three natural tree holes. Our index of fitness (Ro) was lower in the field than in the laboratory and declined due to inbreeding in both However, environments, we found no significant interactions between inbreeding depression and environmental conditions. In both the field and laboratory a 10% increase in the inbreeding coefflicient (F) led to a 12-15) decline in fitness (Ro) These results suggest that inbreeding depression will not necessarily be more extreme under natural field conditions than in the laboratory.     

rpoS Mutations and Loss of General Stress Resistance in Escherichia coli Populations as a Consequence of Conflict between Competing Stress Responses

The general stress resistance of Escherichia coli is controlled by the RpoS sigma factor (phi(S)), but mutations in rpoS are surprisingly common in natural and laboratory populations. Evidence for the selective advantage of losing rpoS was obtained from experiments with nutrient-limited bacteria at different growth rates. Wild-type bacteria were rapidly displaced by rpoS mutants in both glucose- and nitrogen-limited chemostat populations. Nutrient limitation led to selection and sweeps of rpoS null mutations and loss of general stress resistance. The rate of takeover by rpoS mutants was most rapid (within 10 generations of culture) in slower-growing populations that initially express higher phi(S) levels. Competition for core RNA polymerase is the likeliest explanation for reduced expression from distinct promoters dependent on phi(70) and involved in the hunger response to nutrient limitation. Indeed, the mutation of rpoS led to significantly higher expression of genes contributing to the high-affinity glucose scavenging system required for the hunger response. Hence, rpoS polymorphism in E. coli populations may be viewed as the result of competition between the hunger response, which requires sigma factors other than phi(S) for expression, and the maintenance of the ability to withstand external stresses. The extent of external stress significantly influences the spread of rpoS mutations. When acid stress was simultaneously applied to glucose-limited cultures, both the phenotype and frequency of rpoS mutations were attenuated in line with the level of stress. The conflict between the hunger response and maintenance of stress resistance is a potential weakness in bacterial regulation.     

A Polyhydroxybutyrate-Producing <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. Isolated from Antarctic Environments with High Stress Resistance

Pseudomonas sp. 14-3, a strain that accumulates large quantities of polyhydroxybutyrate (PHB) when grown on octanoate, was isolated from Antarctic environments. This isolate was characterized on the basis of phenotypic features and partial sequencing of its 16S ribosomal RNA gene. Pseudomonas sp. 14-3 showed increased tolerance to both thermal and oxidative stress compared with three other Pseudomonas species. Stress tolerance of Pseudomonas sp. 14-3 was analyzed in polyhydroxyalkanoate accumulating and non-accumulating conditions, and increased levels of stress resistance were observed when PHB was produced. Pseudomonas sp. 14-3 was isolated from Antarctic regions, a habitat normally exposed to extreme conditions. An association between high PHB accumulation and high stress resistance in bacteria adapted to extreme environments is suggested.     

Quantitative genetic analysis of life-history traits of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> in stressful environments

Background  

Organisms live in environments that vary. For life-history traits that vary across environments, fitness will be maximised when the phenotype is appropriately matched to the environmental conditions. For the free-living nematode Caenorhabditis elegans, we have investigated how two major life-history traits, (i) the development of environmentally resistant dauer larvae and (ii) reproduction, respond to environmental stress (high population density and low food availability), and how these traits vary between lines and the genetic basis of this variation.     

Environmental dependence of inbreeding depression and purging in Drosophila melanogaster

Elimination or reduction of inbreeding depression by natural selection at the contributing loci (purging) has been hypothesized to effectively mitigate the negative effects of inbreeding in small isolated populations. This may, however, only be valid when the environmental conditions are relatively constant. We tested this assumption using Drosophila melanogaster as a model organism. By means of chromosome balancers, chromosomes were sampled from a wild population and their viability was estimated in both homozygous and heterozygous conditions in a favourable environment. Around 50% of the chromosomes were found to carry a lethal or sublethal mutation, which upon inbreeding would cause a considerable amount of inbreeding depression. These detrimentals were artificially purged by selecting only chromosomes that in homozygous condition had a viability comparable to that of the heterozygotes (quasi-normals), thereby removing most deleterious recessive alleles. Next, these quasi-normals were tested both for egg-to-adult viability and for total fitness under different environmental stress conditions: high-temperature stress, DDT stress, ethanol stress, and crowding. Under these altered stressful conditions, particularly for high temperature and DDT, novel recessive deleterious effects were expressed that were not apparent under control conditions. Some of these chromosomes were even found to carry lethal or near-lethal mutations under stress. Compared with heterozygotes, homozygotes showed on average 25% additional reduction in total fitness. Our results show that, except for mutations that affect fitness under all environmental conditions, inbreeding depression may be due to different loci in different environments. Hence purging of deleterious recessive alleles can be effective only for the particular environment in which the purging occurred, because additional load will become expressed under changing environmental conditions. These results not only indicate that inbreeding depression is environment dependent, but also that inbreeding depression may become more severe under changing stressful conditions. These observations have significant consequences for conservation biology.     

Lifetime fitness in two generations of Ipomopsis hybrids

Various models purporting to explain natural hybrid zones make different assumptions about the fitness of hybrids. One class of models assumes that hybrids have intrinsically low fitness due to genetic incompatibilities, whereas other models allow hybrid fitness to vary across natural environments. We used the intrinsic rate of increase to assess lifetime fitness of hybrids between two species of montane plants Ipomopsis aggregata and Ipomopsis tenuituba planted as seed into multiple field environments. Because fitness is predicted to depend upon genetic composition of the hybrids, we included F1 hybrids, F2 hybrids, and backcrosses in our field tests. The F2 hybrids had female fitness as high, or higher, than expected under an additive model of fitness. These results run counter to any model of hybrid zone dynamics that relies solely on intrinsic nuclear genetic incompatibilities. Instead, we found that selection was environmentally dependent. In this hybrid zone, cytoplasmic effects and genotype-by-environment interactions appear more important in lowering hybrid fitness than do intrinsic genomic incompatibilities between nuclear genes.