A comparative analysis of South African Life Sciences and Biology textbooks for inclusion of the nature of science

Umesh Ramnarain firstname.lastname@example.com
Department of Science and Technology Education
Faculty of Education
University of Johannesburg
South Africa

Keshni Padayachee
Department of Science and Technology Education
Faculty of Education
University of Johannesburg
South Africa

A comparative analysis of South African Life Sciences and Biology textbooks for inclusion of the nature of science
4B694CECAF2F363FEA5C204434E2ABDCBiology textbooksLife Sciences textbooksnature of scienceschool science curriculumscience textbook analysis
This study reports on the analysis of South African Life Sciences and Biology textbooks for the inclusion of the nature of science using a conceptual framework developed byChiappetta, Fillman and Sethna (1991).In particular, we investigated the differences between the representation of the nature of science in Biology textbooks that were written for a previous curriculum and the new Life Sciences textbooks that are in accord with the National Curriculum Statement.The analysis reflects that both Life Sciences and Biology textbooks still overwhelmingly represent the theme "Science as a body of knowledge" according to this framework.Despite significant curriculum reform that underlines a more balanced perspective of science encompassing the acquisition of knowledge through inquiry, the limited coverage given to the themes "The investigative nature of science", "Science as a way of thinking" and "The interaction of science, technology and society" does not reflect this reform.

Introduction

The value of textbooks in driving the teaching of science has been well-documented.The importance given to the textbook is encapsulated by Abd-El-Khalick, Waters and Le (2008:836), who remark that in the large majority of classrooms, textbooks become "the classroom, and determine what is taught and learned about science in these classrooms".Textbooks help translate the intentions of the curriculum into classroom practice by reflecting the goals of science learning, such as understanding the nature of science (NOS) and science content; developing inquiry skills; and understanding the interrelationship of science, technology, the environment and society (Albach & Kelly, 1998).The quality of textbooks, therefore, has a great impact on the quality of instruction (Lemmer, Edwards & Rapule, 2008).Furthermore, the availability of high quality textbooks is one of the critical factors in the successful implementation of curriculum reform (Swanepoel, 2010).

In South Africa, curriculum reform in school science has affirmed the prominence that ought to be given to the NOS in the teaching of school science subjects.Due to the value of the textbook in driving curriculum reform, this article reports on the analysis of South African Life Sciences and Biology textbooks for the inclusion of the NOS.In particular, we investigated the differences between the coverage given to the NOS in Biology textbooks that were written for the previous National Assembly Training and Education Department (NATED) 550 curriculum, and the new Life Sciences textbooks that are in accord with the National Curriculum Statement (NCS).

The construct "nature of science" has been advocated as an important educational outcome by various school science curricula worldwide.Lederman (2007:831), in fact, states that one would be hard pressed to find rhetoric arguing against its importance as a "prized educational outcome".Driver, Leach, Millar and Scott (1996) maintain that the NOS is a critical component of scientific literacy.While support for the NOS has been overwhelming in the science education community, there is some dissonance in the literature as to what it means (Laugksch, 2000).However, a review of the literature by Schwartz and Lederman (2002) shows that there is an acceptable level of agreement on what it entails.Lederman (2007:833) identifies the basic tenets of the nature of scientific knowledge as follows:

It is tentative (subject to change), empirically based (based on and/or derived from observations of the natural world), and subjective (involves personal background, biases and/or is theory laden); necessarily involves human inferences, imagination, and creativity (involves the invention of explanations; and is socially and culturally embedded).Two additional important aspects are the distinction between observations and inferences, and the functions of, and relationships between, scientific theories and laws.

In South Africa, the publication of the NCS for Life Sciences, a subject taught to students in the Further Education and Training phase (Grades 10-12) (Department of Education, 2003), marked a significant departure from the previous apartheid-era curriculum (NATED 550).NATED 550 had a narrow conception of scientific literacy that depicted science as a static body of knowledge.The subject Life Sciences was previously known as Biology, and the Biology syllabus required students to "learn chunks of biological facts that they had to regurgitate in tests and examinations" (Le Grange, 2008:94).Furthermore, Le Grange (2008) maintains that the school subject, Biology focused mainly on the study of plant and animal life, with the artificial separation of fact and value.This focus led to the privileging of factual knowledge over values under the influence of positivism.Mnguni (2013:2) cites Cotti and Schiro (2004) in describing this emphasis as "the scholar academic ideology that deals with disciplining students by transmitting discipline specific knowledge".

The view of science depicted by the previous NATED 550 curriculum was, therefore, incompatible with the tenets of the NOS as underlined by Lederman (2007).The NCS for Life Sciences states that "through the study of the Life Sciences, learners can develop an understanding of the nature of science" (Department of Education, 2003:9).This curriculum goal is in line with the tenets of the NOS encapsulated by Lederman (2007).

For an emerging economy such as South Africa to grow, higher-order intellectual skills should be strongly emphasised.Whereas skills in set tasks were valued in the past, today each worker is expected to think critically, solve abstract problems and generate new ideas for improvement (Castells, 2005).To enhance the transfer of these learnt skills to other environments, learners should be exposed to multiple contextual situations in which they engage with socio-scientific challenges such as Human Immunodeficiency Virus (HIV)/AIDS (Acquired immune deficiency syndrome), and environmental management (Mnguni, 2013).Lederman (2007) maintains that creativity and imagination play an important role in science, and regards this as a tenet of the NOS.This suggests that a science curriculum that places a high imperative on the NOS will inculcate in learners some of the higher-order skills required by the workforce of an emerging economy.

Despite the strong NCS focus on the NOS, teachers received little support when it comes to how to develop materials that would facilitate a legitimate view of the NOS.This lack of guidance on how to design curriculum materials opened the door for a plethora of textbooks entering the market.Owing to their limited capacity to design curriculum material, teachers relied heavily on the textbook as a vehicle for implementing the intended curriculum (Malcolm & Alant, 2004).This scenario accompanying curriculum reform in South Africa was not unexpected, because in order "to achieve large-scale reform, you cannot depend on people's capacity to bring about substantial change in the short run, so you need to propel the process with high quality teaching and training materials" (Fullan, 2001:79).It therefore becomes pivotal for teachers and curriculum planners to be aware of the quality of textbooks.

Despite the curriculum emphasis on learners acquiring an understanding of the NOS, and the key role of the textbook in supporting teachers to address this emphasis, analyses in other countries of science textbooks show that certain aspects of the NOS are not sufficiently addressed (Abd-El-Khalick et al., 2008;Chiappetta & Fillman, 2007;Lumpe & Beck, 1996;McComas, 2003).Abd-El-Khalick et al. (2008) in their analysis of high school Chemistry books used over four decades in the United States (US), rated them poorly in their representations of the NOS.More specifically, McComas (2003) found, in his analysis of US Biology textbooks, that the distinction between laws and theories (aspects of NOS) was not evident.He recommended that authors take more care to provide accurate and complete definitions coupled with useful examples.In South Africa, limited analysis of science textbooks has been undertaken, specifically analysis of the textbooks that have accompanied recent curriculum reform for their inclusion of the NOS.

The role of the textbook as a resource in advancing the tenets of the NOS is especially critical in the South African context.Studies on teacher understanding of the NOS in this country have revealed that teachers have an inadequate understanding of the NOS.A study conducted by Dekkers and Mnisi (2003) in the Limpopo Province found that the majority of teachers surveyed believed common myths about the NOS, as identified by McComas (1998).For example, teachers believed that "hypotheses become theories that in turn become laws" and that "scientific laws and other such ideas are absolute."A study by Linneman, Lynch, Kurup and Bantwini (2003) with teachers in the Eastern Cape arrived at a similar finding.Given this scenario, a shift in the practice of teachers can be supported through the development of textbooks that reflect a more balanced coverage of the NOS.

It is against this background that we investigated the way in which South African Life Sciences and Biology textbooks depict the NOS.Furthermore, we wanted to know how the coverage of the NOS in the current Life Sciences textbooks differed from that in the Biology textbooks that were used in the previous NATED 550 curriculum.Accordingly, the following research questions were formulated:

1)

To what extent do South African Life Sciences and Biology textbooks depict the nature of science? 2)

How do Life Sciences and Biology textbooks compare in the extent to which they cover the nature of science?

Conceptual Framework for Textbook Analysis

A critical aspect of any analysis of information is the conceptual framework used to guide the inquiry.We adopted a conceptual framework developed by Chiappetta et al. (1991) for analysing themes in the NOS.This framework has been employed extensively by researchers in their analysis of textbooks (Abd-El-Khalick, 2002;Chiappetta & Fillman, 2007;Chiappetta et al., 1991).This framework addresses the four themes described below.

1)

Science as a body of knowledge: This theme reflects science as a body of knowledge such as the facts, concepts, principles, laws, theories and models.

2)

The investigative nature of science: This theme reflects the active aspect of inquiry and learning, which involves the student in the methods and processes of science such as observing, measuring, classifying, inferring, recording data and making calculations.

3)

Science as a way of thinking: This theme represents thinking, reasoning and reflection, where the student is told how the specific enterprise operates.

4)

Interaction of science, technology and society: This theme pertains to the application of science and how technology helps or hinders humankind.

Each of the above themes in the NOS is elaborated on in greater detail through categories; this formed the basis for an analytical framework.The framework is presented in Table 1.

Science as a body of knowledge a) Knowledge presented as facts, concepts, laws, and principles b) Hypotheses, theories, and models c) Factual recall of information 2.

Methodology

A content analysis of three Grade Ten Life Sciences textbooks and three Biology textbooks was undertaken.Krippendorff (2004:18) defines this approach as "a research technique for making replicable and valid inferences from texts (or other meaningful matter) to the contexts of their use".Books that were extensively used in classrooms throughout South Africa were targeted for analysis.The selection of these books was based on information about schoolbook orders provided by the Department of Basic Education.The three books chosen for Life Sciences had the highest number of orders, and they collectively constituted close to 70% of all book orders for Grade Ten.Similarly, the Biology books chosen were the three most pop-ular in the NATED 550 curriculum.

We analysed the textbooks by applying the validated framework of Chiappetta et al. (1991).We first calculated 10% of pages for each textbook, as recommended by Chiappetta and Fillman (2007).We then applied a sliding scale, based on the proportion of pages allocated per content area, to calculate the number of pages to be analysed for each content area (Chiappetta & Fillman, 2007).The pages were then selected randomly by number generation from each content area.The four core content areas examined were: tissues, cells and molecular studies; structures and control processes; environmental studies; and diversity, change and continuity.The composition of each content area is shown in Table 2.In the analysis we first highlighted the units of analysis.The units of analysis included complete paragraphs, activities, worked examples, figures with captions, tables with captions, charts with captions, and marginal comments.The units were coded independently according to the NOS categories set out in Table 1.This entailed studying a unit, and identifying the theme and category to which the unit could best be related.A deductive process was therefore followed in coding the units from the textbooks according to the Chiappetta et al. (1991) framework.Examples of units that corresponded to the themes and categories are presented in the results section.After the coders completed assigning each unit of analysis to a category in one of the four themes of the NOS, the reliability in the coding of the units was determined by the use of Cohen's kappa statistic.Cohen's kappa is calculated as follows: k = (po − pc) / (1 − pc), where po is the proportion of ratings in which the two judges agree, and pc is the proportion of ratings for which agreement is expected by chance.Ethical clearance for this research was granted by the University of Johannesburg at which the first author is employed.

Results

The following pseudonyms are used for the three Life Sciences textbooks that were analysed: Approaching Life Sciences, The Basics of Life Sciences, and Curiosity in Life Sciences; and for the Biology textbooks, Diverse Biology: An Exploration of Biology and Classroom Biology.In total, 724 textbook units were coded to a particular category presented in Table 1.Between the coders there was agreement in coding 556 of these units, and this meant there was a 77% agreement in this process.The calculated kappa value was 0.7.According to Landis and Koch (1977), kappa values from 0.61 to 0.80 indicate a substantial level of agreement in coding.Coding differences were resolved through discussion.Consensus was eventually reached.

Table 3 shows the results on the coding of units for each of the six analysed textbooks.For example, in the book Approaching Life Sciences, 54% of units were considered to be related to the NOS theme "Science as a body of knowledge".The calculation of percentage coverage of the four themes was regarded as a valid means of comparison, as it addressed the implications of textbooks having different physical features such as page orientation, page size, and font type and size.

Figure 1 shows a visual comparison of the Life Sciences and Biology textbooks for the representation of the NOS themes.

We now discuss these results in terms of the four themes of the NOS.

Theme I: Science as a Body of Knowledge

The results show that the Life Sciences textbooks strongly manifest the theme "Science as a body of Knowledge", with 54% of the units of measurement devoted to this theme.The coverage in all three Biology textbooks is even more heavily dominated by this theme, with 84% of all units being coded to this theme.A deeper analysis showed that for the Life Sciences textbooks, 194 of the 227 units of analysis that were classified according to this theme corresponded to the category "Knowledge presented as facts, concepts, laws and principles".A similar observation was made from the coding of the Biology textbooks, with 92% of all units coded to this theme being categorised as "Knowledge presented as facts, concepts, laws and principles".The following two units extracted from Approaching Life Sciences and Classroom Biology exemplify the coverage in this category:

Living organisms are found in oceans, lakes and rivers (hydrosphere), as well as in the air or atmosphere.There is life on the surface of the Earth as well as in the soil, under the surface of the Earth (lithosphere).All these parts of the Earth where organisms live are part of the Biosphere.(Approaching Life Sciences) These organelles (ribosomes) are extremely small granules scattered in the cytoplasm of all cells.They occur singly or on the external surface of the endoplasmic reticulum or in groups in the cytoplasm where they are known as polyribosomes.

They contain ribonucleic acid (RNA) hence their name, and enzymes connected with protein synthesis.Ribosomes are the chief manufacturers of all cell proteins.(Classroom Biology)

The above unit from Approaching Life Sciences describes the biosphere of Earth.This unit corresponds to the category "Knowledge presented as facts, concepts, laws and principles", because the paragraph presents facts on the habitat of living organisms.A similar focus is noted in the unit from Classroom Biology, where facts are presented on the ribosome.

In promoting the theme "Science as a body of knowledge", there appears to be a belief that scientific knowledge can be acquired and understood by learners when presented through clear and coherent explanations (Chiappetta & Fillman, 2007).The visual representation of concepts is often used to enhance this knowledge acquisition.This was evident in diagrams from Curiosity in Life Sciences on the formation of proteins from amino acids, which showed the linking together of amino acids in long chains to form proteins.

It would appear that a strong goal in the teaching of science from the perspective of both Life Sciences and Biology textbook authors, is for learners to acquire scientific knowledge.With regard to curriculum reform, it is clear from the extensive coverage given to this theme that a strong goal in the learning of science remains the acquisition of knowledge.This focus coheres well with the curriculum imperative in the more recent Curriculum and Assessment Performance Statement (CAPS) for Life Sciences expressed through Specific Aim 1, which specifies the construction of ideas and concepts.The investigative approach to the teaching and learning of science is portrayed moderately by all three Life Sciences textbooks as evidenced by the 28% coding of units to the theme "The investigative nature of science".This coverage is even more limited in the three Biology textbooks, where only 12% of units were devoted to this theme.In terms of the CAPS, it is clear that this theme aligns closely with Specific Aim 2 on planning and carrying out of scientific investigations.A comparison of the textbooks of the two subjects suggests that the increased percentage coverage of this theme in the Life Sciences textbooks reflects attempts by textbook authors to address the investigative approach to learning.However, the scope given to this theme in the Life Sciences textbooks suggests that this learning outcome is not being adequately addressed.

For the Life Sciences textbooks, the units coded to the theme "The investigative nature of science" were overwhelmingly situated in the category "Learns through the use of materials" (107 of 120 units).An activity in The basics of Life Sciences on the use of yeast to show alcoholic fermentation falls within this category.In this coded unit, learners are presented with a diagram of the apparatus and then instructed to set up the apparatus.They are then given a procedure for conducting the investigation.The intention of this activity is to engage the learners in a hands-on learning situation in the manipulation of objects.In this and other units coded to this category, emphasis is placed on the development of procedural skills in handling the apparatus and making observations.However, the textbooks do not facilitate the development of other process skills such as hypothesising, planning an investigation, making an inference, analysing data, and reflecting on findings.This would suggest that the textbooks support learners in having only limited autonomy in scientific investigations.In other units, where this theme is addressed, the focus is on learners conducting investigations, with little or no scope being given to the design of investigations.

The analysis suggests an emphasis on learners doing practical work in confirming a given concept, law or principle that had been previously learned, rather than learners being given the opportunity to investigate their own ideas.

Theme III: Science as a Way of Thinking

The theme "Science as a way of thinking" is poorly represented in all six textbooks.For the Life Sciences textbooks, an average of 7% of units was coded to this theme.This theme was not addressed in any of the three Biology textbooks.Both sets of textbook authors do not convey, to any great extent, how the scientific enterprise operates.In the Life Sciences textbooks, where text was devoted to this theme, the focus was given to the category "Describes how a scientist discovered or experimented".The following example of such a unit is taken from Approaching Life Sciences:

In 1771, Joseph Priestly (1733-1804) placed a green plant into air in which a candle had burned out and found that ten days later another candle could be burned in the same air.He reported that he had 'accidently hit upon a method of restoring air that has been injured by the burning of candles'.He then tried the same experiment with a mouse and found that the mouse lived in the same air.

(Approaching Life Sciences)

The objective of this unit is to show how Priestley experimented on the purification of air using green plants.There was little or no evidence of the other categories in this theme.For example, little material highlighted the empirical NOS, the inductive and deductive NOS, and the relationship between evidence and proof.A few units related to the empirical NOS.The following is an example of a unit from Approaching Life Sciences that addresses the empirical NOS:

Measuring pH Collect a water sample.Add a few drops of universal indicator.Compare the colour of the water with the code on the indicator bottle.The pH for healthy, unpolluted water will be between 6.5 and 8.5.Interpret your results using the table on your right.(Approaching Life Sciences)

The average percentage coverage of units for all three Life Sciences books given to this theme was only 12%.The average coverage of this theme in the Biology textbooks was four percent.Despite the curriculum imperative as specified in Learning Outcome Three of the NCS for learners to understand the interrelationship between science, society and technology, it would appear that the Life Sciences textbooks have not, in any significant measure, increased the coverage given to this theme.

All six textbooks represented this theme to approximately the same degree.The great majority of units coded for this theme were located in the category "Usefulness of science and technology".Units coded to this category exemplified the applications of science and technology in society.Examples of such units from Curiosity in Life Sciences and An exploration of Biology are: Biological washing powders contain enzymes that can act on protein-based stains.The enzyme found in these washing powders is a protease that breaks down proteins in the stain, producing smaller molecules that can then be removed by the detergent in the washing powder.(Curiosity in Life Sciences) Farmers can practice good farming methods to control soil erosion by contour ploughing and planting.They can also leave a strip of natural bush or grassland on each side of a stream or river and not planting crops right up to the river banks.(An exploration of Biology)

The first excerpt explains the role of enzymes in washing powders and indicates its potential use in society, while the second describes good farming practices that can guide farmers in overcoming soil erosion.

Discussion

The analysis of the textbooks for the four themes reflects that both Life Sciences and Biology textbooks still overwhelmingly represent the theme "Science as a body of knowledge".Significant curriculum reform underlines a more balanced perspective of science encompassing the acquisition of knowledge through inquiry, the development of scientific thinking and the interaction between science, society and technology.The cover-age in the Life Sciences textbooks does not reflect this.A comparison of the Biology textbooks of the previous curriculum and the Life Sciences textbooks of the reformed curriculum revealed only minimal shifts towards addressing the other themes in the NOS.This finding correlates well with studies in other countries on the analysis of science textbooks against the background of curriculum reform.A study by Lumpe and Beck (1996) examined seven Biology textbooks using the same framework by Chiappetta et al. (1991).They concluded that the textbooks did not reflect the intended science education reform.Similarly, a study by Abd-El-Khalick (2002) reported that four popular science textbooks used in the US were devoid of important elements that define the NOS.

In view of the critical role played by the textbook in driving the teaching of science, the findings of this study raise concerns about the extent to which curriculum imperatives are being translated into practice.The findings suggest that the use of the textbook as a resource is undermining the advancement of the tenets of the NOS.A direct implication of this finding is that learners are not given the opportunity to acquire skills associated with activities that underlie the NOS.As pointed out earlier, growth in an emerging economy such as South Africa depends to a large extent on higher order skills in science and technology such as critical thinking, creativity, and problem solving.These skills could be acquired through learning that is aligned with the tenets of the NOS.

The reality of the classroom is that teachers give priority to assessments.The textbook industry in South Africa is huge, and publishers mandate writers to produce books that support teachers in preparing learners for these assessments to ensure a sizeable share of the market.Despite curriculum reform that strongly focuses on learner understanding of NOS, there is a strong focus on highstakes summative assessment in the form of tests and examinations in South Africa's education system.This is significant as Blanchard, Southerland, Osborne, Sampson, Annetta and Granger (2010) allude to studies (e.g.Saka, Southerland & Brooks, 2009;Shaver, Cuevas, Lee & Avalos, 2007) that indicate the high-stakes influence of standardised summative assessments on the classroom practice of science teachers.These assessments are heavily weighted towards testing science content knowledge, and hence the textbook coverage on the NOS is strongly skewed towards the theme "Science as a body of knowledge".Substantial change in the emphasis of textbook coverage can be achieved if assessments place more attention on the themes "Science as a way of investigating", "Science as a way of thinking" and "Science, technology and society".Furthermore, Abd-El-Khalick (2012) posits that NOS cannot be learned implicitly through engagement in doing science but "should rather be planned for instead of being anticipated as a side effect or secondary product" (Akindehin, 1988:73).We contend that a textbook that depicts a balanced perspective on the NOS can be employed as a resource that teachers can use in consciously addressing the goal of enhancing learners' conceptions of NOS.

We maintain that for there to be a radical shift towards a more balanced representation of the NOS in textbooks, publishers need to revisit the mandate given to textbook writers.In South Africa, new textbooks are first screened by a panel chosen by the Department of Basic Education before being approved for the list of books that schools can order from.In the review process, the panel evaluates the books against criteria drawn up by the department.To improve the chances of approval, publishers instruct writers to ensure that books adhere to these criteria.Given this scenario, it is likely that a change in the textbooks being produced can be effected should the department reflect in these selection criteria a more balanced perspective on the NOS.

investigative nature of science a) Learns through the use of materials b) Learns through the use of tables and charts c) Makes calculations d) Reasons out an answer e) Participates in thought experiments f) Gets information from the internet g) Uses scientific observation and inference h) Analyses and interprets data 3. Science as a way of thinking a) Description of how a scientist discovered or experimented b) Historical development of an idea c) Empirical basis of science d) Use of assumptions e) Inductive or deductive reasoning f) Cause and effect relationship g) Evidence and/or proof h) Presentation of scientific method(s) or problem solving i) Scepticism and criticism j) Human imagination and creativity k) Characteristics of scientists (subjectivity and bias) l) Various ways of understanding the natural world 4. Interaction of science, technology and society a) Usefulness of science and technology b) Negative effects of science and technology c) Discussion of social issues related to science and technology d) Careers in science and technology e) Contribution of diversity f) Societal or cultural influences g) Public or peer collaboration h) Limitations of science i) Ethics in science Source: Adapted fromChiappetta & Fillman (2007)

Table 2
2
Core content areas in textbooks
Core content areaTopicsTissues, cells and molecular studies Cell structureCell division and mitosisTissuesRelated diseases, e.g. cancerStructures and control processesEnergy releaseFood productionHuman nutrition and related diseases and allergiesGaseous exchange and related diseases and allergiesEnvironmental studiesBiosphere, biomes and ecosystemsLiving and non-living resourcesDiversity, change and continuityBiodiversity of plants and animals and conservationSignificance and value of biodiversity to ecosystem function and human survivalThreats to biodiversityParasitism and diseases, e.g. bilharzia

Table 3
3
The percentage of coverage of the four themes of the NOS in three Life Sciences and three Biology textbooks
NOS ThemesApproachingThe basics ofCuriosity inDiverseAn explorationClassroomLife SciencesLife SciencesLife SciencesBiologyof BiologyBiologyScience as a body of546344769481knowledgeThe investigative27223312619nature of scienceScience as a way of858000thinkingInteraction of1110151200science, technologyand societyFigure 1 A comparison of the Life Sciences and Biology textbooks used in this studyTheme II: The Investigative Nature of Science

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Representations of nature of science in high school chemistry textbooks over the past four decades. F Abd-El-Khalick, M Waters, A P Le, Journal of Research in Science Teaching. 4572008

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A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level

Stephan Binder
Georg Schendzielorz
Norma Stäbler
Karin Krumbach
Kristina Hoffmann
Michael Bott
Lothar Eggeling
A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level
M E T H O D Open Access
We present a novel method for visualizing intracellular metabolite concentrations within single cells of Escherichia coli and Corynebacterium glutamicum that expedites the screening process of producers. It is based on transcription factors and we used it to isolate new L-lysine producing mutants of C. glutamicum from a large library of mutagenized cells using fluorescence-activated cell sorting (FACS). This high-throughput method fills the gap between existing high-throughput methods for mutant generation and genome analysis. The technology has diverse applications in the analysis of producer populations and screening of mutant libraries that carry mutations in plasmids or genomes.

Background

Since the first demonstration of microbial product formation more than a century ago [1], vitamins, antibiotics, nucleotides, amino acids and organic acids have been produced in ever increasing quantities. For example, about three million tonnes of sodium glutamate are produced each year as a small microbial molecule. Bacterial synthesis is increasingly also used for the production of small molecules not naturally made by bacteria, such as pharmaceutical intermediates [2,3] or biofuels [4]. The combination of the successful application of microbial synthesis, progress in synthetic biology and changes in the global economy that necessitate intensified use of renewable raw materials indicates that microbial metabolite production will continue to expand.

Microorganisms are not naturally designed for profitable metabolite formation, however, and there is an unrelenting need to optimize strains and pathways. Current strain improvement strategies make use of a variety of methods for engineering and isolating microbial variants with the desired traits. These techniques fall into two major categories: 'rational' methods, which involve the targeted alteration of known genetic information; and 'random' approaches, which are typically based on the creation of mutant libraries containing nondirected changes in genotype with subsequent screening for phenotypes of interest. Both approaches have been successful but the use of mutant libraries has proven to have distinct advantages. The reason is that the exact genomic mutations necessary to adapt the cellular metabolism for increased product synthesis are often difficult to predict, and that 'rational' methods are restricted to known targets. Random approaches with subsequent screening for the phenotype of interest enable us to overcome these difficulties. They have made possible the commercialscale production of a variety of compounds, such as the unrivaled formation of succinate by Escherichia coli [5] or riboflavin by Bacillus subtilis [6]. Random and combinatorial approaches were also profitably used for the development of plasmid-encoded targets for the optimization of pathway flux in E. coli. This has been demonstrated with amorpha-4,11-diene production [2], which is an artemisinin precursor that is effective for the treatment of malaria, or with taxadiene production [3], an intermediate of the anticancer compound taxol.

However, with few exceptions, the evaluation of methods that utilize random approaches currently requires the cultivation of individual clones to determine production properties. This presents an obstacle. While high-throughput (HT) techniques for introducing genetic diversity and for product analysis or sequencing are well developed [7], comparable strategies for the identification and isolation of high-producer bacterial cells are still lacking. The opportunity to directly monitor product formation within single cells in vivo would add a new dimension to the characterization and development of microbial producers.

Here, we present examples of the monitoring of intracellular metabolite concentrations in single bacterial cells and demonstrate in an HT screen the isolation of new bacterial producer cells, as well as the identification of novel mutations based on whole-genome sequencing. The sensors we use are based on transcription factors (TFs) that regulate the transcriptional output of a target promoter in response to a cytosolic metabolite. Whereas the use of TFs to construct whole-culture biosensors for the detection of environmental small-molecule pollutants has long been established [8], this same approach has remained largely untranslated with respect to single-cell analysis and library screening. TFs are naturally targeted to a variety of small ligands, ranging from amino acids to sugars, sugar phosphates, vitamins, antibiotics, oxoacids and lipids [9]. They can also be engineered to obtain altered specificity [10,11], as recently summarized in a comprehensive review [12]. Coupling transcription of the target gene to a reporter protein provides a molecular device for recognition. This has already been successfully applied for screening in plate-based assays using colony color or colony size [10,13], for instance. Here we make full use of intracellular recognition of a specific metabolite in single cells by applying an autofluorescent protein as reporter and also fluorescence-activated cell sorting (FACS). This enables the isolation in HT screens of new bacterial small-molecule producers with random mutations introduced into the genome that enhance production of the molecule of interest, and we present an example of this.

Results

Schematic of approach

The workflow for HT selection of genomic variants of metabolite producers consists of the following steps: a) design of a suitable metabolite sensor, b) generation of genetic diversity in genomes of cells carrying the sensor, c) screening of the mutant library and selection of single producer cells via FACS, d) verification and characterization of mutants, and e) sequencing for target identification. We developed sensors for intracellular detection of basic amino acids, as well as of L-serine and Oacetyl-serine, and demonstrated the feasibility of the approach by isolating bacteria producing L-lysine from a library of randomly mutagenized wild-type (WT) cells, culminating in the identification of new useful mutations by whole-genome sequencing.

Design of L-lysine sensor

To develop a sensor suitable for intracellular L-lysine detection, we focused on the LysR-type transcriptional regulator (LTTR) LysG of Corynebacterium glutamicum. This protein senses elevated concentrations of basic amino acids, causing transcription of its target gene lysE, which encodes a basic amino acid exporter [14]. To explore the application of this native regulatory device to the conversion of an intracellular metabolite concentration into an optical output, we characterized the interaction of LysG with its target region upstream of lysE in a series of electrophoretic mobility shift assays ( Figure S1 in Additional file 1). These data were used to construct the metabolite sensor pSenLys shown in Figure S2 in Additional file 1. It contained lysG, together with the LysG-binding site in front of the lysE promoter driving transcription of eyfp coding for enhanced yellow fluorescent protein (EYFP). In addition, P tac -driven crimson was incorporated as a second fluorescence protein in the vector backbone. All strains and plasmids are listed in Table  S1 in Additional file 1.

Characterization of L-lysine sensor

The WT of C. glutamicum does not excrete L-lysine but there is a genealogy of defined mutants that exhibit increased L-lysine productivity [15]. We determined the cytosolic L-lysine concentration by silicone oil centrifugation and the response of pSenLys in these strains. The WT had a cytosolic L-lysine concentration below 5 mM, while the defined producers had steady-state concentrations ranging from roughly 8 to 25 mM (Figure 1a). A clear increase in specific EYFP fluorescence in cultures is seen with increasing cytosolic L-lysine concentration. As evident from microscopic inspection (insets in Figure 1a), pSenLys is a tool for visualizing cytosolic L-lysine concentrations also within single cells. The range of the cytosolic L-lysine concentration covered translates into a dynamic range of signal output of 8.3-fold.

The fluorescence signal from the pSenLys sensor also correlates with the extracellular L-lysine concentration that accumulates after glucose is consumed (Figure 1b). One instructive exception to this is strain DM1920, which accumulates extracellular L-lysine at concentrations comparable to that of DM1919 despite displaying lower fluorescence due to lower cytosolic concentrations of L-lysine. This is due to altered L-lysine export: Strain DM1919 has one copy of the exporter gene lysE and an export rate of 10.1 ± 0.4 nmol minute -1 mg(dry weight) -1 (Figure 1c), whereas strain DM1920 has two copies of lysE (Table S1 in Additional file 1), which results in an increased rate of export of L-lysine of 12.1 ± 0.6 nmol minute -1 mg(dry weight) -1 , and thus reduced cytosolic concentration. This observation provides an opportunity of influencing the read-out properties of a sensor that had not previously been taken into consideration. As demonstrated for the production of antibiotics [16], amino acids [17], and biofuels [4], small-molecule production depends on export proteins. The ability to manipulate export activities permits cytosolic concentration of the substrate to be increased or decreased, which may be helpful, for example, when using strains that display high productivity and high cytosolic concentrations of substrate.

To isolate single cell producers by HT flow cytometry, it is essential that the cells can be separated according to their fluorescent properties. To demonstrate that this is the case with pSenLys, sensor-carrying cells of WT, DM1728, and DM1919 grown in glucose minimal medium  )). (d) Differentiation of an equal mixture of cells of ATCC13032, DM1728 and DM1919 each carrying pSenLys by flow cytometry. The success of strain-specific sorting using gates P1 to P3 was over 90%. (e) Influence of dipeptide addition on specific fluorescence of C. glutamicum WT carrying pSenLys. The peptides Lys-Ala (circle), Arg-Ala (triangle) and His-Ala (square) were added to the cultures at the indicated concentrations. Additionally, Ala-Ala was added to give a total dipeptide concentration of 3 mM. The specific fluorescence was measured 1.5 h after dipeptide addition. FSC, forward scatter; RU, relative units.

were mixed in a 1:1:1 ratio to give a total of 4 × 10 7 cells ml -1 . This cell population was analyzed via FACS at a rate of 10 3 events per second. Clear differentiation of the population was achieved on the basis of intensity of the EYFP signal ( Figure 1d). Three further qualities were assessed (Table S2 in Additional file 1): 1) the sorting specificity achieved using gates P1 to P3 resulted in the selection of ≥ 89% of the L-lysine producer expected within the respective gate; 2) the recovery of viable single cells for each gate was ≥ 84%; 3) when DM1728 was mixed with a 10,000fold excess of WT cells and then re-isolated, 92% of the prepared cells were DM1728.

LysG recognizes L-arginine and L-histidine in addition to L-lysine [14]. Peptide addition is a proven method to increase the cytosolic pool of a specific amino acid in C. glutamicum or E. coli [18,19]. The fluorescence response of C. glutamicum carrying pSenLys to exogenously applied Lys-Ala is similar to endogenously synthesized L-lysine ( Figure 1e). The fluorescence response due to Arg-Ala supply was substantially greater, and that to His-Ala was greater still, which may indicate a higher affinity of these ligands for LysG.

Further metabolite sensors

To assess the general utility of TFs for reporting on smallmolecules at the single-cell level, we constructed pSenArg based on ArgP of E. coli controlling argO transcription as a function of cytosolic L-arginine [20]. When pSenArg was assayed for dipeptide responsiveness, the addition of Arg-Ala resulted in fluorescent E. coli cells (Figure 2, left), with fluorescence increasing in proportion to the dose of dipeptide ( Figure S3 in Additional file 1). As additional metabolite sensors, pSenSer and pSenOAS were constructed (Table S1 in Additional file 1), suitable for L-serine ( Figure 2, middle), and O-acetyl-L-serine detection in C. glutamicum (Figure 2, right), respectively.

Generation of genetic diversity and library screening

To demonstrate the feasibility of metabolite sensors for HT screening of mutant libraries, we introduced chromosomal mutations into the WT of C. glutamicum carrying pSenLys by treatment with MNNG (N-methyl-N'-nitro-Nnitrosoguanidine), one of the most effective chemical mutagens for creating genetic diversity [21]. While the separation of mixtures of defined producers could be achieved using FACS (Figure 1d), direct processing of the mutagenized library with FACS was not successful. We used a liquid culture recovery and enrichment step taking into account that metabolically active cells require 2 h for Crimson synthesis ( Figure S4 in Additional file 1), and that cells derived from the glycerol stock have to be incubated for 6 h on minimal medium to establish host-specific EYFP fluorescence. The suspension of mutagenized cells from glycerol stock (200 μl) was diluted into minimal medium containing IPTG (isopropyl-β-D-thiogalactopyranoside and, after 2 h, 6.5 × 10 6 Figure S3 of Additional file 1. The serine-producing strain C. glutamicum-Ser4 (middle) [45] with pSenSer is fluorescent (top), but the WT carrying pSenSer is not (bottom). (c) Fluorescence is seen with the L-cysteine producer C. glutamicum-Cys3 carrying pSenOAS (top right), but not with the control strain (bottom right). Epifluorescence microscopic analysis was done at λ ex = 490 to 510 nm and λ em = 520 to 550 nm.

Producer verification

The 270 colonies were used to inoculate microtiter plates, to enable HT screening of cultures using 0.75 ml minimal medium. L-lysine was detected in culture supernatants of 185 clones. Re-cultivation of 120 clones (Figure 3a) revealed that the L-lysine concentration ranged from 0.2 to 37 mM. Four clones accumulated 3.6 to 5 mM L-lysine plus 0.6 to 0.8 mM L-arginine and one clone 24.9 mM L-lysine plus 0.6 mM L-arginine. None of the mutants accumulated L-histidine, possibly due to the length of this pathway and its tight regulation.

Of the 120 mutants, 40 were selected randomly, and their culture fluorescence and growth recorded ( Figure S5 in Additional file 1). Using an expectation maximization algorithm [22], two clusters relating specific fluorescence to L-lysine accumulation were apparent (Figure 3b). The cluster represented by the lower curve showed characteristics similar to the defined recombinant strains used in Figure 1a, which are included in Figure 3b as gray dots and numbered in parentheses. This cluster includes the mutants of main interest. Mutants in the other cluster show in part extreme fluorescence at a comparatively low extracellular L-lysine accumulation. Since we screened for high fluorescence, it is possible that mutants with reduced L-lysine export activity -and therefore increased intracellular L-lysine concentration -accumulated. This finding warrants further exploration. Cellular export activity is influenced by a number of parameters, including the lipid environment of carriers and the composition of the outer membrane [4,16], which may cause mutants to excrete metabolites at different rates than the WT does.

Gene analysis in 40 mutants

We sequenced lysC, which encodes aspartate kinase in the 40 mutants described above (Figures 4 and 5a). To date, all L-lysine producers described have a mutation in lysC, preventing feedback inhibition of aspartate kinase activity by the concerted action of L-lysine plus L-threonine [23]. In 15 of the mutants that we found, lysC was mutated, including seven cases of the known mutation lysC-T308I, which is located in the regulatory β-subunit of the aspartate kinase [23]. New mutations -lysC-H357Y, lysC-T313I, lysC-G277D, and lysC-G277S -that also affect the regulatory subunit ( Figure S6  The spectrum of L-lysine accumulation in the culture supernatant of 120 mutants obtained using FACS selection. Mutants were grown in minimal medium with 4% (w/v) glucose and lysine concentrations determined after 48 h. (b) Specific culture fluorescence was determined for 40 arbitrarily chosen mutants. Two clusters are apparent after applying an expectation maximization (EM) algorithm to construct a distribution containing maximum likelihood estimates of the parameters in a Gaussian mixture model with two components for data in the 40-by-2 data matrix [22]. The heat bar on the right gives the probability of clones belonging to cluster one, which corresponds to that with the flat curve. The probability of clones belonging to cluster two, corresponding to that with the steep curve, uses the same heat bar with the highest probability in blue and lowest in red. The four gray circles marked (1) to (4) give lysine accumulation and specific fluorescence for the defined recombinants DM1728 (1), DM1800 (2), DM1730 (3), and DM1919 (4) used in Figure 1a. RU, relative units. found. We also sequenced hom in all 40 mutants, since reduced homoserine dehydrogenase activity reduces Lthreonine availability in cells and thus also reduces kinase activity (Figure 5a). Current approaches to engineering Llysine synthesis rely on just a single hom mutation [15]. Seven of the mutants isolated in this study carry novel mutations in hom (Figure 4).

In the remaining 18 mutants, neither lysC nor hom was mutated. We therefore sequenced thrB and thrC as further genes of L-threonine synthesis (Figure 5a). In strain K016 the mutation thrB-S102F was identified and in strain K074 the mutation thrC-A342V was found (Figure 4). The introduction of four selected mutations into the WT chromosome resulted in significant L-lysine concentrations ( Table S3 in Additional file 1), demonstrating that these new mutations cause increased L-lysine formation.

Genome sequence of mutant K051

We performed whole-genome sequencing on strain K051, which has no mutation in lysC, hom, thrB, or thrC yet accumulates L-lysine up to a concentration of 15.9 mM. Paired-end sequencing on an Illumina HiSeq 2000 provided more than 20 million reads. Trimming and mapping to the WT genome (NC_000913) [15] resulted in a 260-fold coverage (Table S4 in Additional file 1). The genome sequence of strain K051 has been deposited at the European Nucleotide Archive under accession number HE802067. Within K051, 268 SNPs are manifest. They are unevenly distributed in the genome (Figure 5b). The number of SNPs is within the range observed for E. coli treated with MNNG [24]. All of the SNPs identified are transitions, as expected with this mutagen, the majority of them resulting in amino acid exchanges (Figure 5b; Table S1 in Additional file 2). In addition, NCgl0863, which carries the amino acid exchange G54D, was partially duplicated, with the variant copy placed 6,108 bp distant from NCgl0863 in an intergenic region.

We searched the mutations in K051 for genes known to increase L-lysine production and to participate in the pathway from glucose uptake up to L-lysine excretion  ( Figure 5a). Specific mutations in zwf and gnd in the pentose phosphate pathway are known to increase L-lysine formation due to an increased supply of NADPH [25]; K051 has mutations in devB and tal that could also be effective. K051 also has mutations in pck and gltA, genes encoding phosphoenolpyruvate carboxykinase and citrate synthase, where reduced activities are known to increase the supply of pyruvate and oxaloacetate for L-lysine synthesis [26,27]. Also, mutations of branched-chain amino acid metabolism have been demonstrated to increase lysine formation, and K051 carries a mutation in ilvE, as well as in the Leu-tRNA synthetase LeuS. Of particular interest was the murE mutation (murE-G81E) in K051. This gene encodes UDP-N-acetylmuramyl-tripeptide synthetase, an enzyme that utilizes D, L-diaminopimelate as a substrate, as does the D, L-diaminopimelate decarboxylase, in L-lysine synthesis.

Influence of murE mutations on L-lysine synthesis

To determine whether the murE-G81E mutation identified could generate increased L-lysine formation, we introduced it by allelic replacement into DM1132, DM1728, DM1730, DM1800, and DM1933. The new strains were cultivated in parallel to their ancestor strains in shake flask cultivations and final L-lysine concentrations were determined after 48 h. As shown in Figure 6, the mutation caused strong L-lysine accumulation when introduced into the WT DM1132 and also DM1728, the strains that have few mutations and which form comparatively little L-lysine. Yet even with the best producer available, strain DM1933, a significant increase in L-lysine accumulation was determined. Given this finding, we sequenced murE in the remaining mutants isolated by our HT technology that had no identified mutation (Figure 4), and found murE-L121F in strain K055. Introduction of this specific mutation into the five defined L-lysine producers yielded increased L-lysine accumulation, too ( Figure 6). Whether the increases with the two murE mutations identified were due to increased availability of D, L-diaminopimelate for L-lysine synthesis, or whether a global regulatory effect pushes synthesis of D, L-diaminopimelate remains to be studied.

Discussion

The key requirement for visualization of single cells with elevated concentrations of a small molecule of interest is the availability of suitable in vivo sensor systems with sufficient sensitivity and specificity. There are a large number of options for developing customized reporters sensing intracellular metabolites. They are based on natural molecular recognition, allosteric switching, and gene regulation behavior of proteins and RNA. Every system has its own specific advantages and disadvantages, and the reader is referred to recent reviews on the numerous ideas and ongoing developments in the field [12,[28][29][30][31][32][33]. Whereas protein sensors based on periplasmic binding proteins and Förster resonance energy transfer (FRET) in principle enable concentration determinations in real time, use of TFs relies on expression of the reporter gene. This delay between ligand binding and the corresponding phenotypic change is not a disadvantage in developing or characterizing recombinant cells since stable genetically encoded genotypes are sought. With respect to the use of TFs in metabolite sensing for screening purposes, the present work based on LysR of C. glutamicum is the first example where the responsiveness of the optical output to an existing intracellular metabolite concentration is given, and where a TF-based sensor is used in an HT screen applying FACS for the isolation of new bacterial small-molecule producers. The responsiveness of TFs previously characterized is deduced from the external addition of the effector molecule and whole culture response. Although this may only be of limited significance for screening, it is disadvantageous for precise characterization since various processes such as active uptake, active export, diffusion and degradation of effector might result in a different cytosolic concentration than that present extracellularly. In the case of LysG-based pSenLys, we determined a detection range of 4 to 25 mM intracellular L-lysine. Sensor responsiveness is characterized by an analog-like response that, when fitted to the Hill equation, is described by n app of 3.19 ± 1.45. It enables the differentiation of WT from medium-and high-level producer cells (Table S2 in Additional file 1). As our intracellular determinations and the comparison of the isogenic strains with one copy and two copies of lysE revealed, the effective range of detection may be extended by altering export activity. This could be of relevance for further improvement of good producers. Sensor response and its usefulness will depend on the interplay between the cytosolic concentration of the small-molecule and export activity, as well as on the affinity of the sensor to the effector and target promoter site.

Three of the small-molecule sensors described in the present work are based on a LysR-type TF, and one on a ROK-type TF. Fortunately, the range of small molecules detectable by TFs is large. E. coli has more than 230 TFs, with many of them detecting small molecules. In bacteria, TFs have been found to sense sugars, sugar phosphates, vitamins, 2-oxoacids, ions, antibiotics, and acyl-CoA derivatives [9]. Moreover, TFs with new specificities can be generated [11]. An example is AraC, which has been switched from a natural L-arabinose sensor to a sensor detecting D-arabinose [34] or mevalonate [10], and the latter effector specificity has been used in a plate-based assay to screen for improved mevalonate producers. Other sensors that were given new specificities were developed from NahR or XylR for the detection of benzoic acid-related compounds [35], or TetR for structural derivatives of tetracycline [36]. Advances in the design of microbial-based molecular reporters and customizing ligand dependence derived from natural TFs have recently been reviewed [12].

Thus, sensors for a significant number of small molecules of biotechnological or pharmaceutical importance are within reach.

Whereas the WT of C. glutamicum does not excrete Llysine, cytosolic sensing and FACS as an efficient screen enabled the rapid isolation of 185 new mutants accumulating L-lysine in the culture supernatant. The current number of genes where mutations cause increased L-lysine synthesis is about 12 [37,38]. These mutations serve to increase flux through the L-lysine pathway itself, or to increase the pyruvate and oxaloacetate pool, or the NADPH supply. However, there are still unknown mutations to be discovered, since it is known that in an L-lysine-producing mutant developed over decades in classical screenings, many genes of biosynthesis pathways exhibit increased expression [39], and in a similarly derived L-arginine producer, arginine biosynthesis genes are highly expressed in a manner not achievable by plasmid-encoded expression [40]. Our approach provided alleles of known genes, and this is very useful for genomic reconstruction of producers where advantageous mutations are combined, and alleles may result in different productivity [2,41]. The number of 268 SNPs present in K051 is too great to study their individual impact on product formation, but new possibilities might be offered when more genome sequences become available. Striking was the murE mutation present in K051. We suggest that the catalytic activity of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-diaminopimelate ligase in MurE-G81E is reduced, with the consequence that more D, L-diaminopimelate is available for L-lysine synthesis. MurE of C. glutamicum is similar to MurE of Mycobacterium tuberculosis and E. coli, the crystal structures of which are known [42]. From these, it can be deduced that G81E is close to the nucleoside part of UDP-MurNAc-L-Ala-D-Glu, and L121F in the second mutant identified is close to the ATP-binding site. Thus, a reduced activity is meaningful, and in line with the increased L-lysine formation obtained with all strains when the murE mutations were introduced in their genomes. It is also in line with the reduced growth rates of these new recombinants (Table S5 in Additional file 1), since less D, L-diaminopimelate is channeled towards cell wall synthesis. An alternative to simple mass balance effects is that a lack of cell wall building blocks initiates a global response that has a positive effect on biosynthesis. We applied one of our transcriptional sensors for HT screening of a mutant library with chromosomal mutations, but the same principle may also be explored for HT screening of cells carrying plasmid libraries. This is attractive, since many pharmaceuticals currently produced microbially, such as amorpha-4,11-diene, taxadiene and lycopene, use plasmid-encoded biosynthesis pathways, for example, in E. coli [2,3,13]. Use of an appropriate sensor combined with FACS-assisted screening may significantly accelerate the development of producers for such small molecules, too. The HT selection routine for mutant isolation closes the gap between HT generation of mutant libraries and HT sequencing technologies, and further applications of sensing small molecules in single cells are in progress, such as the verification of producer population homogeneity and time-lapse microscopy of C. glutamicum in microfluidic chips [43].

Conclusions

This work examines visualization of the intracellular concentration of small molecules at the single cell level by the use of specific TFs. It opens up various possibilities to characterize and analyze single cells in populations with respect to their cytosolic small molecule concentration. We have demonstrated that the visualization of Llysine combined with HT sorting of genomic mutant libraries via FACS enables the isolation of new mutants. Together with whole-genome sequencing, this therefore establishes rapid access to new mutations to achieve more efficient product formation. In addition to the screening of cells with genomic mutations, the system is also suitable for screening cells with plasmid libraries to identify more efficient product accumulation.

Materials and methods

Sensor plasmid construction

The regulatory units were synthesized (LifeTechnologies GmbH, 64293 Darmstadt, Germany) and cloned into pJC1 using the restriction sites BamHI and SalI. An overview of the sensor plasmids is shown in Figure S2 in Additional file 1. The entire plasmid sequences were deposited at EMBL under the accession numbers HE583184 (pSenLys), HE583185 (pSenArg), HE583186 (pSenSer), and HE583187 (pSenOAS1).

FACS analysis and cell sorting

Cells were diluted to an optical density below 0.1 and immediately analyzed by a FACS ARIA II high-speed cell sorter (BD Biosciences, Franklin Lakes, NJ USA 07417) using excitation lines at 488 and 633 nm and detecting fluorescence at 530 ± 15 nm and 660 ± 10 nm at a sample pressure of 70 psi. Data were analyzed using BD DIVA 6.1.3 software. The sheath fluid was sterile filtered phosphate-buffered saline. Electronic gating was set to exclude non-bacterial particles on the basis of forward versus side scatter area. For sorting of Crimson-or EYFP-positive cells the next level of electronic gating was set to exclude non-fluorescent cells. Background was estimated using non-induced C. glutamicum for sorting of Crimson-positive cells. When sorting EYFPpositive cells, non-producing C. glutamicum cells were used.

Mutagenesis and library screening

C. glutamicum ATCC13032 carrying pSenLys was grown in 5 ml BHI complex medium (Difco Laboratories Inc., Detroit, MI 48201, USA) containing 25 μg ml -1 kanamycin to an optical density of 5 to ensure exponential growth. Whole-cell mutagenesis was done by the addition of MNNG dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 0.1 mg ml -1 and incubation for 15 mintes at 30°C. The treated cells were washed twice with 45 ml NaCl, 0.9% (w/v), resuspended in 10 ml BHI and regenerated for 3 h at 30°C and 180 rpm. Mutant cells were stored at -30°C as cryostocks in BHI containing 40% glycerol (w/v). Of the initial cells, 46.2% survived the MNNG treatment and among the surviving cells approximately 16% were auxotrophs. For FACS screening, the mutant stock population containing 7.5 × 10 8 viable cells per ml was diluted 1:100 in 20 ml minimal medium containing 0.1 mM IPTG to induce expression of the far-red fluorescent protein Crimson, which was taken as an indicator of metabolically active cells. After 2 h of cultivation, 6.5 × 10 6 cells were analyzed by FACS and 2 × 10 6 Crimson-positive cells collected in fresh 20 ml minimal medium without IPTG. After cultivation for a further 22 h, 1.8 × 10 7 cells were screened and 350 EYFP-positive cells spotted on Petri dishes containing minimal medium. Colonies grown after 48 h at 30°C were further analyzed.

HT cultivation and culture fluorescence analysis HT cultivation was done in 48-well Flowerplates (FPs; m2p-labs GmbH, 52499 Baesweiler, Germany) at 30°C, 990 rpm and a throw of ø 3 mm. The specific geometry of the FPs ensures high mass transfer performance and can be used together with the microcultivation system BioLector [44], allowing online monitoring of growth and fluorescence. The medium used for FP cultivations was the MOPS-buffered salt medium CGXII [45], with 4% glucose as substrate and 25 μg ml -1 kanamycin to select for maintenance of pSenLys. For offline cultivations, FPs were cultivated on a Microtron high-capacity microplate incubator operating at a shaker speed of 990 rpm, throw ø 3 mm (Infors AG, CH-4103 Bottmingen, Switzerland). Shake flask cultivations were used to compare the consequences of the murE mutations for L-lysine accumulation ( Figure 4b); these were done in 500 ml baffled Erlenmeyer flasks with 50 ml medium. The medium was the same as used in FP cultivations, except that the phosphate concentration was reduced by half. Cells pregrown in CGXII medium were used as inocula for all cultivations.

Amino acid quantification

Amino acids were quantified as their o-phthaldialdehyde derivatives via high-pressure liquid chromatography using a uHPLC 1290 Infinity system (Agilent, Santa Clara, CA 95051, USA) equipped with a Zorbax Eclipse AAA C18 3.5 micron 4.6 × 75 mm and a fluorescence detector. As eluent, a gradient of 0.01 M Na-borate buffer pH 8.2 with increasing concentrations of methanol was used, and detection of the fluorescent isoindole derivatives was at λ ex = 230 nm and λ em = 450 nm.

Determination of cytosolic amino acid concentrations and amino acid export rates Cells were pregrown as for FP cultivations for 24 h. They were washed once with fresh CGXII medium at room temperature and transferred into new medium in FPs to give an initial optical density of 10, which corresponds to 3.0 mg (dry weight) ml -1 . Cultures were incubated at 30°C on the Microtron high-capacity microplate incubator as above. Samples were processed at regular intervals to separate extra-and intracellular fluid by silicone oil centrifugation [46]. For the resulting fractions, amino acids were quantified as described above. The intracellular volume used to calculate the internal amino acid concentration was 1.6 μl mg (dry weight) -1 . When peptides were added (Figure 1e; Figure S3 in Additional file 1) mixtures of dipeptides at a final concentration of 3 mM were used, such as 1 mM Arg-Ala plus 2 mM Ala-Ala, to ensure that a constant supply of Arg-Ala-derived Arg is present over time in the cytosol at the lower Arg-Ala concentrations.

Epifluorescence microscopic analysis

Fluorescence imaging was performed using a fully motorized inverted microscope (Nikon Eclipse Ti) equipped with a focus assistant (Nikon PFS), Apo TIRF 100× Oil DIC N objective, NIKON DS-Vi1 color camera, ANDOR LUCA R DL604 camera, Xenon fluorescence light source and standard filters for EYFP detection (λ ex = 490 to 510 nm; λ em = 520 to 550 nm). Differential interference contrast (DIC) microscopy images as well as fluorescence images were captured and analyzed using the Nikon NIS Elements AR software package. Prior to analysis, cells were fixed on soft agarose-covered glass slides.

Additional material

Additional file 1: Supplementary Tables S1 to S4 and Figures S1 to S6. Table S1: strains used. Table S2: quality assessment of sorting cells carrying pSenLys. Table S3: L-lysine formation with mutations introduced by reverse engineering. Table S4: statistics on whole-genome sequencing of strain K051. Table S5: growth rates of murE mutants. Figure S1: isolation of LysG and characterization of the LysG binding site. Figure S2: the vector pSenLys and general configuration of sensor plasmids. Figure  S3: peptide-dose response curves with sensor-carrying E. coli and C. glutamicum. Figure S4: development of Crimson and EYFP signals in mixtures of ATCC13032 with DM1728 over time. Figure S5: growth curves and fluorescence of 40 mutant cultures. Figure S6: structural presentation of LysC and localization of mutations identified.

Figure 1
1Characterization of lysine sensor and lysine-producing recombinant cells. (a) Cytosolic L-lysine concentration in C. glutamicum WT and five defined lysine producer strains, all carrying pSenLys, and specific fluorescence (Sp. fluorescence) of the cultures. Error bars give the means of three independent cultures for each strain. Fitting the data to the Hill equation describes the signal transfer function by an n app of 3.19 ± 1.45 and this is shown as the red curve. (b) Extracellular accumulation of lysine after 48 h by the strains used in (a). (c) Lysine excretion rates of the same strains showing that strains with increased final lysine accumulation have increased excretion rates. Strain DM1920 has two copies of the lysine exporter gene lysE present in its chromosome and shows the highest excretion rate, but an intermediate cytosolic lysine concentration (color code as in (b

Figure 2
2Crimson-positive cells were sorted in minimal medium. After cultivation for a further 22 h, 350 EYFP positive cells were spotted onto minimal medium plates. Of these, 270 grew into colonies within 48 h. pSenSer E. coli C. glutamicum pSenArg pSenOAS Metabolite sensors reporting on cytosolic arginine, serine, and O-acetyl-serine. Cells of E. coli DH5α (left) carrying pSenArg fluoresce when Arg-Ala was added (top), but not following supplementation with Ala-Ala (bottom). The peptide-dose response curves for Arg-Ala, Lys-Ala and His-Ala are shown in

Figure 3
3Characterization of L-lysine-producing mutants isolated by FACS from a library of chemically mutagenized wild type cells. (a)

Figure 4
4Genetic characterization of mutants. In 40 mutants, targeted sequencing revealed mutations in lysC, hom, thrC and thrB, with some of the mutations already known from prior work (see text). Mutations that resulted in amino acid exchanges are indicated, along with growth rates and final external lysine titers. The mutation murE-G81E in strain K051 was identified by whole-genome sequencing. The second murE mutation, murE-L121F, was identified by subsequent targeted sequencing. In 15 mutants (plus K051), it was not possible to identify any mutation by site-directed mutagenesis.

Figure 5
5Sketch of the central metabolism and localization of mutations in C. glutamicum together with whole-genome sequencing results of the mutant K051. (a) Pathways and reactions required for and related to lysine synthesis. Genes that carry mutations leading to amino acid exchanges are indicated as follows: blue box, mutations known previously; yellow box, new mutations identified by targeted sequencing in 40 mutants; orange box, new mutations derived from the K051 genome. (b) Localization of the 268 SNPs in the genome of strain K051 determined by whole-genome sequencing. The size of the genome is 3.28 Mb, scale on the left. The mutations are classified into those causing an amino acid exchange, those that are silent, those leading to a stop codon, and those located in non-coding regions. The genome sequence of strain K051 is deposited at the European Nucleotide Archive under the accession number HE802067.

Figure 6
6Effect of murE mutations on lysine accumulation. Lysine production by different strains modified to carry a chromosomal murE mutation. Color code: gray, ancestor strains; orange, strains carrying the amino acid exchange L121F in MurE; green, strains carrying the amino acid exchange G81E in MurE.

Additional file 2 :
2All mutations of C. glutamicum strain K051. Abbreviations bp: base pair; EYFP: enhanced yellow fluorescent protein; FACS: fluorescence-activated cell sorting; FP: Flowerplate; HT: high throughput; IPTG: isopropyl-β-D-thiogalactopyranoside; MNNG: N-methyl-N'-nitro-Nnitrosoguanidine; SNP: single nucleotide polymorphism; TF: transcription factor; WT: wild type.
AcknowledgementsThis work was funded by the German Federal Ministry of Education and Research, FlexFit project, support code 0315589A. We thank Jessica Schneider, Thomas Bekel and Stephan Hans for support in genome analysis, Peter Droste for his introduction to the Omix software, Katharina Nöh for statistical analyses, Jan Marienhagen for useful discussions, Alexander Grünberger for preparation of microscopic pictures, and Tino Polen for help with data analysis.Authors' contributions SB performed experimental studies and the FACS analyses. GS constructed sensors and did the graphic work. NS and KH contributed to sensor construction. The determination of pool concentrations and export rates was done by KK, MB contributed to manuscript writing, and LE designed the project and wrote the paper. All authors have read and approved the manuscript for publication.Competing interestsThe authors declare that they have no competing interests.
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. Binder, Genome Biology. 13Binder et al. Genome Biology 2012, 13:R40 http://genomebiology.com/2012/13/5/R40