EVOLUTIONARY ADAPTATION TO TEMPERATURE. VI. PHENOTYPIC ACCLIMATION AND ITS EVOLUTION IN ESCHERICHIA COLI

Acclimation refers to reversible, nongenetic changes in phenotype that are induced by specific environmental conditions. Acclimation is generally assumed to improve function in the environment that induces it (the beneficial acclimation hypothesis). In this study, we experimentally tested this assumption by measuring relative fitness of the bacterium Escherichia coli acclimated to different thermal environments. The beneficial acclimation hypothesis predicts that bacteria acclimated to the temperature of competition should have greater fitness than do bacteria acclimated to any other temperature. The benefit predicted by the hypothesis was found in only seven of 12 comparisons; in the other comparisons, either no statistically demonstrable benefit was observed or a detrimental effect of acclimation was demonstrated. For example, in a lineage evolutionarily adapted to 37°C, bacteria acclimated to 37°C have a higher fitness at 32°C than do bacteria acclimated to 32°C, a result exactly contrary to prediction; acclimation to 27°C or 40°C prior to competition at those temperatures confers no benefit over 37°C acclimated forms. Consequently, the beneficial acclimation hypothesis must be rejected as a general prediction of the inevitable result of phenotypic adjustments associated with new environments. However, the hypothesis is supported in many instances when the acclimation and competition temperatures coincide with the historical temperature at which the bacterial populations have evolved. For example, when the evolutionary temperature of the population was 37°C, bacteria acclimated to 37°C had superior fitness at 37°C to those acclimated to 32°C; similarly, bacteria evolutionarily adapted to 32°C had a higher fitness during competition at 32°C than they did when acclimated to 37°C. The more surprising results are that when the bacteria are acclimated to their historical evolutionary temperature, they are frequently competitively superior even at other temperatures. For example, bacteria that have evolved at either 20°C or 32°C and are acclimated to their respective evolutionary temperatures have a greater fitness at 37°C than when they are acclimated to 37°C. Thus, acclimation to evolutionary temperature may, as a correlated consequence, enhance performance not only in the evolutionary environment, but also in a variety of other thermal environments.     

Acclimation of arsenic-resistant Fe(II)-oxidizing bacteria in aqueous environment

This study investigated the potential of the Fe(II)-oxidizing bacteria in removing arsenic in aqueous environment. The bacteria were isolated from the batch of tap water and rusty iron wires, and were acclimated to culture media amended with arsenic concentrations, gradually increasing from 100 μg L⁻¹ to 100 mg L⁻¹. Acclimated bacteria with enhanced arsenic tolerance were used to remove arsenic from the aqueous solution. These bacteria belonged to Pseudomonas species according to 16S rRNA gene sequences. Extracellular enzymes produced by these bacteria played important roles in microbial Fe(II) oxidization and Fe oxide precipitation. Moreover, these bacteria survived and propagated in high arsenic condition (100 mg L⁻¹ As). However, after As(III/V) acclimation, morphological characteristics of the bacteria showed some changes, e.g., shrinking of long bacillus. XRD (X-ray diffraction) patterns indicated that Fe oxide precipitations by Fe(II)-oxidizing bacteria in Fe-rich culture medium were poorly-crystallized ferrihydrites. Adsorption on the biogenic ferrihydrites greatly contributed to high arsenic removal efficiency of Fe(II)-oxidizing bacteria.     

Singlet oxygen-mediated and EXECUTER-dependent signalling and acclimation of Arabidopsis thaliana exposed to light stress

Plants respond to environmental changes by acclimation that activates defence mechanisms and enhances the plant''s resistance against a subsequent more severe stress. Chloroplasts play an important role as a sensor of environmental stress factors that interfere with the photosynthetic electron transport and enhance the production of reactive oxygen species (ROS). One of these ROS, singlet oxygen (¹O₂), activates a signalling pathway within chloroplasts that depends on the two plastid-localized proteins EXECUTER 1 and 2. Moderate light stress induces acclimation protecting photosynthetic membranes against a subsequent more severe high light stress and at the same time activates ¹O₂-mediated and EXECUTER-dependent signalling. Pre-treatment of Arabidopsis seedlings with moderate light stress confers cross-protection against a virulent Pseudomonas syringae strain. While non-pre-acclimated seedlings are highly susceptible to the pathogen regardless of whether ¹O₂- and EXECUTER-dependent signalling is active or not, pre-stressed acclimated seedlings without this signalling pathway lose part of their pathogen resistance. These results implicate ¹O₂- and EXECUTER-dependent signalling in inducing acclimation but suggest also a contribution by other yet unknown signalling pathways during this response of plants to light stress.     

三种高盐苯胺废水对细菌群落结构的影响

采用PCR-DGGE技术分析了含有不同CODCr、盐、总磷、氨态氮、氯离子、苯胺浓度的3种高盐苯胺废水(1#、2#、3#)驯化的细菌群落结构变化,从分子水平阐明高盐苯胺废水对细菌群落结构的影响.结果表明,不同浓度高盐苯胺废水驯化过程中,2#废水随着浓度的升高,细菌群落多样性逐渐下降;1#废水驯化的细菌群落多样性指数以5%的废水最高,20%与40%的废水最低;3#废水驯化的细菌群落多样性指数以0%和5%的废水最高,20%的废水最低.不同废水对细菌群落多样性有不同的影响,表明适应和突变是驯化过程中细菌适应环境的一个重要作用力.3种不同高盐苯胺废水驯化的细菌群落多样性指数与CODCr、盐含量之间均存在显著负相关,但与氯离子和苯胺含量之间相关不显著.3种废水驯化的细菌的遗传多样性有较大差异,多态位点百分率、Shannon's信息指数和Nei's指数均以3#废水最高,1#废水次之,2#废水最低.但遗传多样性指标与CODCr、盐含量、氯离子和苯胺含量之间均相关不显著.可知细菌群落多样性的变化是由于废水中所含的污染物质的综合作用结果,而非单一的因素.3种废水的5种不同浓度驯化细菌之间的平均遗传距离分别为0.4724、0.4350和0.4902,其中3#废水驯化的细菌遗传变异最大.聚类分析表明,5种不同浓度高盐苯胺废水驯化细菌均可明显地分为两组.1#和2#废水均为0%、5%和10%废水驯化细菌聚成一组,而20%和40%废水驯化细菌聚成另一组; 3#废水为0%与5%废水驯化细菌聚成一组,而10%、20%和40%废水驯化细菌聚成另一组.不同的废水由于水质背景值不同,选择压力不同,导致细菌突变的阈值不同,使得诱导细菌变异程度不同,从而最终引起细菌群落结构的不同.驯化过程中微生物种群的变化使得整个微生物群落能够快速适应外部环境变化.     

The stringent response plays a key role in Bacillus subtilis survival of fatty acid starvation

The stringent response is a universal adaptive mechanism to protect bacteria from nutritional and environmental stresses. The role of the stringent response during lipid starvation has been studied only in Gram‐negative bacteria. Here, we report that the stringent response also plays a crucial role in the adaptation of the model Gram‐positive Bacillus subtilis to fatty acid starvation. B. subtilis lacking all three (p)ppGpp‐synthetases (Rel_Bs, RelP and RelQ) or bearing a Rel_Bs variant that no longer synthesizes (p)ppGpp suffer extreme loss of viability on lipid starvation. Loss of viability is paralleled by perturbation of membrane integrity and function, with collapse of membrane potential as the likely cause of death. Although no increment of (p)ppGpp could be detected in lipid starved B. subtilis, we observed a substantial increase in the GTP/ATP ratio of strains incapable of synthesizing (p)ppGpp. Artificially lowering GTP with decoyinine rescued viability of such strains, confirming observations that low intracellular GTP is important for survival of nutritional stresses. Altogether, our results show that activation of the stringent response by lipid starvation is a broadly conserved response of bacteria and that a key role of (p)ppGpp is to couple biosynthetic processes that become detrimental if uncoordinated.     

Molecular characterization of acutely and gradually heavy metal acclimated aquatic bacteria by FTIR spectroscopy

In the environment, bacteria can be exposed to the concentration gradient of toxic heavy metals (gradual) or sudden high concentration of them (acute). In both situations, bacteria get acclimated to toxic heavy metal concentrations. Acclimation causes metabolic and molecular changes in bacteria. In this study, we aimed to understand whether there are differences between molecular profiles of the bacteria (Brevundimonas, Gordonia and Microbacterium) which are under acute or gradual exposure to cadmium or lead by using ATR‐FTIR spectroscopy. Our results revealed the differences between the acclimation groups in membrane dynamics including changes in the structure and composition of the membrane lipids and proteins. Furthermore, protein concentrations decreased in acclimated bacterial groups. Also, a remarkable increase in exopolymer production occurred in acclimated groups. Interestingly, bacteria under acute cadmium exposure produced the significantly higher amount of exopolymer than they did under gradual exposure. On the contrary, under lead exposure gradually acclimate strains produced significantly higher amounts of exopolymer than those of acutely acclimated ones. This information can be used in bioremediation studies to obtain bacterial strains producing a higher amount of exopolymer.      

Perchlorate reduction by hydrogen autotrophic bacteria and microbial community analysis using high-throughput sequencing

Hydrogen autotrophic reduction of perchlorate have advantages of high removal efficiency and harmless to drinking water. But so far the reported information about the microbial community structure was comparatively limited, changes in the biodiversity and the dominant bacteria during acclimation process required detailed study. In this study, perchlorate-reducing hydrogen autotrophic bacteria were acclimated by hydrogen aeration from activated sludge. For the first time, high-throughput sequencing was applied to analyze changes in biodiversity and the dominant bacteria during acclimation process. The Michaelis–Menten model described the perchlorate reduction kinetics well. Model parameters q _max and K _s were 2.521–3.245 (mg ClO₄ ^?/gVSS h) and 5.44–8.23 (mg/l), respectively. Microbial perchlorate reduction occurred across at pH range 5.0–11.0; removal was highest at pH 9.0. The enriched mixed bacteria could use perchlorate, nitrate and sulfate as electron accepter, and the sequence of preference was: NO₃ ^? > ClO₄ ^? > SO₄ ^2?. Compared to the feed culture, biodiversity decreased greatly during acclimation process, the microbial community structure gradually stabilized after 9 acclimation cycles. The Thauera genus related to Rhodocyclales was the dominated perchlorate reducing bacteria (PRB) in the mixed culture.     

Bacterial community compositions of tomato (Lycopersicum esculentum Mill.) seeds and plant growth promoting activity of ACC deaminase producing Bacillus subtilis (HYT-12-1) on tomato seedlings

Study of endophytic bacteria within plant seeds is very essential and meaningful on account of their heritability and versatility. This study investigated Bacillus bacterial communities within the seeds of four commercial tomato varieties, by 16S rRNA gene PCR-RFLP (restriction fragment length polymorphism). Phylogenetic analysis of 16S rRNA gene sequences indicated that the 22 representative isolates belonged to five species of genus Bacillus and the bacterial compositions showed remarkable differences among tomato varieties. Isolates exhibited multiple plant growth promoting (PGP) traits: 37 % of indole-3-acetic acid production; 37 % of phosphate solubilization; 24 % of siderophores production; 85 % of potential nitrogen fixation and 6 % of 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. Isolate HYT-12-1 was shown to have highest ACC deaminase activity (112.02 nmol α-ketobutyrate mg^?1 protein h^?1) among the five ACC deamiase producing strains. 16S rRNA gene sequencing indicated that the isolate HYT-12-1 shared the highest sequence similarity (100 %) with B. subtilis. PGP experiments under gnotobiotic and greenhouse conditions revealed the ability of strain HYT-12-1 to enhance the growth of tomato seedlings. This is the first study to describe endophytic Bacillus communities within tomato seeds, and the results suggest that B. subtilis strain HYT-12-1 would have a great potential for industrial application as biofertilizer in the future.     

Survival of bacillus subtilis NB22, an antifungal-antibiotic iturin producer,and its transformant in soil-systems

Bacillus subtilis NB22 is an antifungal-antibiotic iturin producer that expresses broad suppressibility against phytopathogenic microorganisms. The survival of B. subtilis NB22-1, which is a spontaneous streptomycin-resistance mutant of NB22 was investigated in four different soils. After a gradual decline, the bacterial viable cell number stabilized at a level of 10⁴–10⁵ colony forming units/g-dry soil irrespective of soil differences. A similar decline and stabilization pattern was observed in the case of the transformant of B. subtilis NB22-1 with the plasmid pC194 in nonsterile soil. The transformant reached a much higher stabilized level in sterile soil than in nonsterile soil. However, significant loss of the plasmid was observed in both the soil systems after 10 to 20 d incubation. Plasmid pC194 was stable over a hundred generations in the strain when cultivated in a liquid complex medium, but unstable in minimal medium, indicating that the plasmid stability in soil does not necessarily reflect that in liquid culture.     

Bacterial Infection and Immune Responses in Lutzomyia longipalpis Sand Fly Larvae Midgut

The midgut microbial community in insect vectors of disease is crucial for an effective immune response against infection with various human and animal pathogens. Depending on the aspects of their development, insects can acquire microbes present in soil, water, and plants. Sand flies are major vectors of leishmaniasis, and shown to harbor a wide variety of Gram-negative and Gram-positive bacteria. Sand fly larval stages acquire microorganisms from the soil, and the abundance and distribution of these microorganisms may vary depending on the sand fly species or the breeding site. Here, we assess the distribution of two bacteria commonly found within the gut of sand flies, Pantoea agglomerans and Bacillus subtilis. We demonstrate that these bacteria are able to differentially infect the larval digestive tract, and regulate the immune response in sand fly larvae. Moreover, bacterial distribution, and likely the ability to colonize the gut, is driven, at least in part, by a gradient of pH present in the gut.     

The differential stress response of adapted chromite mine isolates Bacillus subtilis and Escherichia coli and its impact on bioremediation potential

In the current study, indigenous bacterial isolates Bacillus subtilis VITSUKMW1 and Escherichia coli VITSUKMW3 from a chromite mine were adapted to 100 mg L^?1 of Cr(VI). The phase contrast and scanning electron microscopic images showed increase in the length of adapted E. coli cells and chain formation in case of adapted B. subtilis. The presence of chromium on the surface of the bacteria was confirmed by energy dispersive X-ray spectroscopy (EDX), which was also supported by the conspicuous Cr–O peaks in FTIR spectra. The transmission electron microscopic (TEM) images of adapted E. coli and B. subtilis showed the presence of intact cells with Cr accumulated inside the bacteria. The TEM–EDX confirmed the internalization of Cr(VI) in the adapted cells. The specific growth rate and Cr(VI) reduction capacity was significantly higher in adapted B. subtilis compared to that of adapted E. coli. To study the possible role of Cr(VI) toxicity affecting the Cr(VI) reduction capacity, the definite assays for the released reactive oxygen species (ROS) and ROS scavenging enzymes (SOD and GSH) were carried out. The decreased ROS production as well as SOD and GSH release observed in adapted B. subtilis compared to the adapted E. coli corroborated well with its higher specific growth rate and increased Cr(VI) reduction capacity.     

Design of a PCR test based on the gyrA gene sequence for the identification of closely related species of the Bacillus subtilis group

A method for the taxonomic identification of seven closely related bacterial species of the Bacillus subtilis group (B. subtilis, B. amyloliquefaciens, B. licheniformis, B. vallismortis, B. atrophaeus, B. sonorensis, and B. mojavensis) using specific primers selected on the basis of the gyrA gene sequences was developed. The effectiveness of this method both for the identification of pure cultures of type strains of this group and for the precise species identification of collection and industrial bacterial strains was demonstrated. The principal possibility of using this method for detecting B. subtilis group bacteria in mixed cultures was shown.     

High-light-like photosynthetic responses of Cucumis sativus leaves acclimated to fluorescent illumination with a high red:far-red ratio: interaction between light quality and quantity

This study evaluated the photosynthetic responses of Cucumis sativus leaves acclimated to illumination from three-band white fluorescent lamps with a high red:far-red (R:FR) ratio (R:FR = 10.5) and the photosynthetic responses of leaves acclimated to metal-halide lamps that provided a spectrum similar to that of natural light (R:FR = 1.2) at acclimation photosynthetic photon flux density (PPFD) of 100 to 700 μmol m^?2 s^?1. The maximum gross photosynthetic rate (P _G) of the fluorescent-acclimated leaves was approximately 1.4 times that of the metal-halide-acclimated leaves at all acclimation PPFDs. The ratio of quantum efficiency of photosystem II (Φ_PSII) of the fluorescent-acclimated leaves to that of the metal-halide-acclimated leaves tended to increase with increasing acclimation PPFD, whereas the corresponding ratios for the leaf mass per unit area tended to decrease with increasing acclimation PPFD. These results suggest that the greater maximum P _G of the fluorescent-acclimated leaves resulted from an interaction between the acclimation light quality and quantity, which was mainly caused by the greater leaf biomass for photosynthesis per area at low acclimation PPFDs and by the higher Φ_PSII as a result of changes in characteristics and distribution of chloroplasts, or a combination of these factors at high acclimation PPFDs.     

A test for plasticity in sperm motility activation in response to osmotic environment in an anuran amphibian

Evolutionary theory predicts that selection will favor phenotypic plasticity in sperm traits that maximize fertilization success in dynamic fertilization environments. In species with external fertilization, osmolality of the fertilization medium is known to play a critical role in activating sperm motility, but evidence for osmotic‐induced sperm plasticity is limited to euryhaline fish and marine invertebrates. Whether this capacity extends to freshwater taxa remains unknown. Here, we provide the first test for plasticity in sperm‐motility activation in response to osmotic environment in an anuran amphibian. Male common eastern froglets (Crinia signifera) were acclimated to either low (0 mOsmol kg⁻¹) or high (50 mOsmol kg⁻¹) environmental osmolality, and using a split‐sample experimental design, sperm were activated across a range of osmolality treatments (0, 25, 50, 75, 100, and 200 ± 2 mOsmol kg⁻¹). Unexpectedly, there was no detectable shift in the optimal osmolality for sperm‐motility activation after approximately 13 weeks of acclimation (a period reflecting the duration of the winter breeding season). However, in both the low and high acclimation treatments, the optimal osmolality for sperm‐motility activation mirrored the osmolality at the natural breeding site, indicating a phenotypic match to the local environment. Previously it has been shown that C. signifera display among‐population covariation between environmental osmolality and sperm performance. Coupled with this finding, the results of the present study suggest that inter‐population differences reflect genetic divergence and local adaptation. We discuss the need for experimental tests of osmotic‐induced sperm plasticity in more freshwater taxa to better understand the environmental and evolutionary contexts favoring adaptive plasticity in sperm‐motility activation.     

GENITIC VARIABILITY IN TOXIN POTENCIES AMONG SEVENTEEN CLONES OF GAMBIERDISCUS TOXICUS (DINOPHYCEAE)1

Seventeen clones of the ciguatera-causing dinoflagellate Gambierdiscus toxicus Adachi and Fukuyo were acclimated to the same environments over several months. Significant variance components were detected between non-acclimated and acclimated cultures for cell potencies, yields and reproduction rates. The resultant variance in acclimated potencies among clones was statistically significant (P < 0.0001), indicating that potency can be used for genetic comparisons. However, cell potency differences for a clone of G. toxicus in the acclimated vs. non-acclimated phases can exceed genetic differences between clones. This stresses the need for a rigorous acclimation process. Caribbean isolates of G. toxicus were inherently more toxic than isolates from other areas. One Caribbean clone yielded 55 × 10^?4 mu (mouse units)·cell^?1 whereas clones Bermuda, the Bahamas, and Florida ranged from only 1.8 × 10^?4 mu·cell^?1 to a maximum of 19.8 × 10^?4 mu·cell^?1. Toxicity decreased with increasing latitude (r =–0.819, P < 0.01), indicating that environmental differences probably influenced the potencies. A comparison of acclimated reproduction rates at four light intensities also indicated that genetic differences among clones existed. The resulting reproduction rate/light slopes overlapped, indicating that the clones may be adapted to specific light regimes.     

Isolation and Characterization of Atrazine Mineralizing Bacillus subtilis Strain HB-6

Atrazine is a widely used herbicide with great environmental concern due to its high potential to contaminate soil and waters. An atrazine-degrading bacterial strain HB-6 was isolated from industrial wastewater and the 16S rRNA gene sequencing identified HB-6 as a Bacillus subtilis. PCR assays indicated that HB-6 contained atrazine-degrading genes trzN, atzB and atzC. The strain HB-6 was capable of utilizing atrazine and cyanuric acid as a sole nitrogen source for growth and even cleaved the s-triazine ring and mineralized atrazine. The strain demonstrated a very high efficiency of atrazine biodegradation with a broad optimum pH and temperature ranges and could be enhanced by cooperating with other bacteria, suggesting its huge potential for remediation of atrazine-contaminated sites. To our knowledge, there are few Bacillus subtilis strains reported that can mineralize atrazine, therefore, the present work might provide some new insights on atrazine remediation.     

Growth-phase dependence of bacterial membrane lipid profile and labeling for in-cell solid-state NMR applications

Cell labeling is a preliminary step in multiple biophysical approaches, including the solid-state nuclear magnetic resonance (NMR) study of bacteria in vivo. Deuterium solid-state NMR has been used in the past years to probe bacterial membranes and their interactions with antimicrobial peptides, following a standard labeling protocol. Recent results from our laboratory on a slow-growing bacterium has shown the need to optimize this protocol, especially the bacterial growth time before harvest and the concentration of exogenous labeled fatty acids to be used for both Escherichia coli and Bacillus subtilis. It is also essential for the protocol to remain harmless to cells while providing optimal labeling. We have therefore developed a fast and facile approach to monitor the lipid composition of bacterial membranes under various growth conditions, combining solution ³¹P NMR and GCMS. Using this approach, the optimized labeling conditions of Escherichia coli and Bacillus subtilis with deuterated palmitic acid were determined. Our results show a modification of B. subtilis phospholipid profile as a function of the growth stage, as opposed to E. coli. Our protocol recommends low concentrations of exogenous palmitic acid in the growth medium, and bacteria harvest after the exponential phase.     

Behavioral study of selected microorganisms in an aqueous electrohydrodynamic liquid bridge

An aqueous electrohydrodynamic (EHD) floating liquid bridge is a unique environment for studying the influence of protonic currents (mA cm^?2) in strong DC electric fields (kV cm^?1) on the behavior of microorganisms. It forms in between two beakers filled with water when high-voltage is applied to these beakers. We recently discovered that exposure to this bridge has a stimulating effect on Escherichia coli.. In this work we show that the survival is due to a natural Faraday cage effect of the cell wall of these microorganisms using a simple 2D model. We further confirm this hypothesis by measuring and simulating the behavior of Bacillus subtilis subtilis, Neochloris oleoabundans, Saccharomyces cerevisiae and THP-1 monocytes. Their behavior matches the predictions of the model: cells without a natural Faraday cage like algae and monocytes are mostly killed and weakened, whereas yeast and Bacillus subtilis subtilis survive. The effect of the natural Faraday cage is twofold: First, it diverts the current from passing through the cell (and thereby killing it); secondly, because it is protonic it maintains the osmotic pressure in the cell wall, thereby mitigating cytolysis which would normally occur due to the low osmotic pressure of the surrounding medium. The method presented provides the basis for selective disinfection of solutions containing different microorganisms.     

Novel Method for Selection of Antimicrobial Peptides from a Phage Display Library by Use of Bacterial Magnetic Particles

Antimicrobial peptides were isolated from a phage display peptide library using bacterial magnetic particles (BacMPs) as a solid support. The BacMPs obtained from “Magnetospirillum magneticum” strain AMB-1 consist of pure magnetite (50 to 100 nm in size) and are covered with a lipid bilayer membrane derived from the invagination of the inner membrane. BacMPs are easily purified from a culture of magnetotactic bacteria by magnetic separation. Approximately 4 × 10¹⁰ PFU of the library phage (complexity, 2.7 × 10⁹) was reacted with BacMPs. The elution of bound phages from BacMPs was performed by disrupting its membrane with phospholipase D treatment. Six candidate peptides, which were highly cationic and could bind onto the BacMP membrane, were obtained. They exhibited antimicrobial activity against Bacillus subtilis but not against Escherichia coli and Saccharomyces cerevisiae. The amino acid substitution of the selected peptide, KPQQHNRPLRHK (peptide 6-7), to enhance the hydrophobicity resulted in obvious antimicrobial activity against all test microorganisms. The present study shows for the first time that a magnetic selection of antimicrobial peptides from the phage display peptide library was successfully achieved by targeting the actual bacterial inner membrane. This BacMP-based method could be a promising approach for a high-throughput screening of antimicrobial peptides targeting a wide range of species.