Advances in Microbial Food Safety - ACS Publications - American

Chapter 1

Genomic and Proteomic Approaches for Studying Bacterial Stress Responses

Downloaded by 80.82.77.83 on November 9, 2017 | http://pubs.acs.org Publication Date: April 6, 2006 | doi: 10.1021/bk-2006-0931.ch001

Shivanthi Anandan Department of Bioscience and Biotechnology, Drexel University, Philadelphia, PA 19104

In the past the detection and analysis of pathogenic microorganisms in food has required the cultivation, isolation and identification of these organisms. This has been a time consuming endeavor. This approach has also not resolved the problem of those organisms that are "viable but nonculturable", that could not be identified by classical culture techniques. With the advent of genome sequencing, more molecular and global strategies for the identification of pathogenic organisms have become available. This presentation will describe and discuss molecular methods based on genomic and proteomic approaches to microbial identification. In addition, tools for analysis of gene expression in a community setting will be presented. Finally, strategies for the discovery of genes expressed during infection and pathogenesis will be considered.

© 2006 American Chemical Society

Juneja et al.; Advances in Microbial Food Safety ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

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2 Bacteria respond to varying environmental conditions by changing the expression of their genes. Most often this change in gene expression is coordinately controlled by a sigma factor that regulates the induction of a subset of genes in response to the change (8, 11, 12). This regulation is aimed at maintaining cellular homeostasis in the face of the changed environment. Although at first glance the study of bacterial stress responses does not seem to be of great utility, the information of how bacteria respond to stress is applicable to such diverse disciplines as medicine, pharmaceuticals and the food industry. In the food industry significant losses due to problems with food spoilage or pathogenic food-bome organisms are a reality. In order to minimize the risk of food contamination there is an urgent need to be able to detect the presence of spoilage and pathogenic organisms quickly and accurately. In addition, insights into bacterial responses to commonly used stress conditions used as food preservatives such as high salt and weak acids, can evaluate how spoilage and pathogenic organism will behave in these environments. In the field of infectious disease, study of bacterial responses to stress conditions within the host's body such as the acidic environment of the stomach and intestinal tract, will further our understanding of how pathogens evade these host defense systems. In the past, such analyses were hard to perform due to the lack of fast and specific methods that targeted cellular responses to stress. With the advent of the genomics era, novel techniques have been innovated that expedite the analysis of global changes in gene expression in relatively short periods of time. These techniques have impacted scientific research and have allowed a wealth of information to be gained in such divergent fields as food safety and medicine. The insights gained will drive the development of improved methods for food preservation and food safety and will catalyze the discovery of new vaccine and antimicrobial technologies.

Why Study Bacterial Stress Responses? Early studies on the physiology of bacterial species under stress conditions were carried out using exponentially growing laboratory cultures. Since then, many researchers have demonstrated that in natural habitats bacteria do not exhibit riotous, exponential growth., due partly to nutrient limitation and the build up of toxic metabolic by-products. In addition, the responses of bacterial species to stress often induces non-exponential growing phases such as stationary-phase in the bacterial population (15). Therefore, the responses shown by bacteria in exponential-phase are not generally the responses observed in stress induced situations, or in stationary-phase. In addition, one of the global responses to stress is the induction of cells that are either resistant to the stress or cells that enter a genetic program to form structures that protect them from the stress environment (15). Examples are Escherichia coli cells that show acidresistance in low p H environments, or Clostridium and Bacillus species that



3 form resistant spores in response to stress conditions. Therefore, the study of exponentially growing cells cannot begin to define and characterize the responses that occur in bacterial species when these organisms are confronted with a stressful, harsh environment. Very often the generalized stress response that is seen at the onset of exposure to stress in bacterial populations, is similar to responses observed in bacteria entering into stationary-phase (8, 12). The study of bacterial stress responses is very useful in a practical sense in food microbiology, infectious disease and in the study of the dynamics of natural populations. A detailed analysis of stress responses can be used to predict the behavior of a microorganism when faced with a particular environmental stress. These data can then be used to evaluate strategies for the preservation of food, evaluate the efficacy of specific drug therapies and for the analysis of bacterial populations in natural habitats.

The Rationale for Using Genomic Tools With the advent of genome sequencing, more global strategies for the identification of microorganisms at the molecular level have become available. These strategies also lend themselves to analyzing changes in gene expression at the global, rather than local level. Best of all, genomic tools bypass the need for culturing organisms, since all that is required to perform these analyses is either genomic D N A or total mRNA isolated from the organisms under investigation (26). Several excellent commercial kits are available that cheaply, consistently and with high efficiency can be used for the routine extraction of genomic D N A or total m R N A from microbes and even microbial populations. These extraction methods coupled with genomic and proteomic-based tools have revolutionized the analysis of bacterial stress responses in environmental microbiology as well as in food microbiology, and in the study of microbial infection and pathogenesis. Three common problems associated with classic culture-based methods of detection and analysis are: the lack of suitable culture media for fastidious strains or species in low abiundance, the presence of viable but nonculturable organisms and the difficulty of analyzing global gene expression under stress conditions.

The Lack of Appropriate Culture Media In the past, the detection and analysis of pathogenic microorganisms in food has required the cultivation, isolation and identification of these organisms. Identification of the microorganisms usually requires the culturing of these organisms on selective media combined with several metabolic tests. This is a


4 tedious and time consuming endeavor, that sometimes requires several weeks for a definitive answer. Culture-based methods can also require multiple enrichment steps to enable the isolation of those organisms present in small numbers or with fastidious growth requirements. A problem with culture-based methods is the lack of suitable growth media to support the growth of all but a few species. The number of existing microbial species is roughly estimated at 10 -10 . Kaeberlein et al. (73) argued that only a few thousand species have been isolated in pure culture because very few microbes isolated from the natural environment grow on nutrient media in the laboratory. Culture-based techniques therefore, have many drawbacks, and do not quickly and efficiently aid in bacterial isolation and characterization. Moreover, culture-based methods do not easily allow the growth of fastisious organisms. Downloaded by 80.82.77.83 on November 9, 2017 | http://pubs.acs.org Publication Date: April 6, 2006 | doi: 10.1021/bk-2006-0931.ch001

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Viable but Non-Culturable Organisms Viable but non-culturable ( V B N C ) organisms do not grow on the usual media used for the selection of most microbes, and thus can be missed during culture-based detection processes (5). Rice et al. (23) have defined the viable but non-culturable state as a physiological state having a specific block that prevents V B N C organisms from dividing and growing on media which normally supports their growth. Food-bome pathogenic microorganisms like Vibrio vulnificus can show the V B N C response, while still maintaining an infectious state (23). Since normal culture techniques will not be successful in isolating organisms in a V B N C state, these organisms still possess the ability to infect and cause disease in the host. V B N C organisms are a serious threat to human health and safety. In particular, those V B N C organisms that are carried in food (e.g. V. vulnificus) pose a serious threat to food safety and the health of the public.

Analysis of Global Gene Expression A third problem arises when trying to detect genes expressed in a particular organism under different growth or environmental conditions, or under normal and stress conditions. These experiments have usually involved culturing the organism under normal versus stress conditions, and then employing either Northern blot analysis or reverse-transcription polymerase chain reaction (RTPCR) to detect changes in gene expression. Under these experimental conditions, only a relatively few genes can be analyzed by either Northern blots or RT-PCR. A n extensive review on PCR-based techniques is presented in another chapter of this book by L i u and Fratamico, and therefore, PCR-based methods will not be discussed in this section. The situation is more complex when one is trying to


5 elucidate patterns of gene expression in a mixed microbial population. Again, culture-based techniques will not represent all the organisms present with a high degree of fidelity. A more accurate assessment of the organisms present and their gene expression profiles can be obtained using techniques that use molecular tools to target genetic loci.


Genome-Based Methods Genome-based methods encompass the techniques of D N A microarrays and proteomics (26, 27). D N A microarrays rely on the hybridization of D N A or R N A to selected oligonucleotides that represent the genome of the organism. These oligonucleotides are chosen by analysis of published genome sequence data. Proteomics uses two dimesional sodium dodecyl sulphate polyacrylamide gel electrophoresis (2-D SDS-PAGE) to separate the total proteins from a microbial species or population, followed by mass spectrometry of the isolated proteins for identification. Genomic and proteomic techniques do not require intact and viable microorganisms, bypassing the need for culturing the organisms under analysis.

DNA Micro-Array Based Detection D N A microarrays are fast becoming a very accurate technique for analysis of global gene expression, and for the detection of species in a microbial population. Essentially a glass surface, usually a slide, is spotted with a defined set of oligonucleotides that represent an entire genome or a subset of genes in a genome. The oligonucleotides are synthesized after careful analysis of the genome sequences of the organsism(s) and should be representative of the genome of the organisms under study. Several methods are available for the preparation of the microarray slide and these have been excellently reviewed by van Hal et al. (26). For analysis of global gene expression at the transcriptional level, total m R N A is extracted from the organism(s), labeled with a fluorescent tag, and the m R N A hybridized to the oligonucleotide containing slide or "chip." Detection of binding of specific m R N A to specific oligonucleotides is carried out by a microarray reader, which can be a charge-coupled device (CCD) camera, non-confocal laser scanner or confocal laser scanner. Commercial data acquisition and handling software are available for the analysis of the data generated by a microarray. In studying the response of genes to stress conditions, it is usual for two sets of fluorescently labeled m R N A to be hybridized separately to two D N A microarrays. One set of m R N A is isolated from organisms grown under standard conditions, and the second set is total m R N A



6 isolated from the same organism that has been subjected to the stress condition under study (e.g. heat shock, osmotic stress, cold shock). A comparison of the intensity of the fluorescent signals generated from both microarray sets reveals genes that are up-regulated or down-regulated in response to the stress condition. For detection of microorganisms, the microarray is hybridized with labeled genomic D N A fragments isolated from the sample. Analysis of the signals obtained with genomic D N A is similar to that described for mRNA. D N A microarray technology has been used in numerous examples to analyze gene expression. It has been used to detect the expression of "foreign" genes in genetically modified plants, to study genes expressed in response to hydrogen peroxide (oxidative stress) in the cyanobacterium Synechocystis sp. strain P C C 6803 (16), and to analyze genes expressed in the global stress response of the gram positive bacterium B. subtilis (21) to name but a few. The potential of this technique in the fields of infectious disease and pathogenesis are enormous, since genes that are specifically expressed during infection and disease can be identified by side-by-side comparisons with genes that are expressed by the pathogen in the free-living state. A limitation of this technique is that it is expensive and requires that the genome sequences of the organisms under study should be available for designing the oligonucleotides for the microarray.

Proteomics Proteomics-based techniques are used to determine the protein expression profile of an organism under given conditions (70, 20, 22). This technique is empirically more challenging than that of D N A microarrays, since it requires the extraction of total proteins in the cell. The profile of the extracted proteins should represent all protein classes present in the cell both qualitatively and in abundance. Proteins are then separated by two dimensional SDS-polyacrylamide gel electrophoresis, and the separated proteins identified by mass spectroscopy coupled with N-terminal sequencing of the mass spectroscopy generated peptides. The use of this technique is not as widespread as that of D N A microarrays due to the challenges associated with the purification and separation of the complex mixtures of proteins found in cell extracts. This technique has been used to study the cold adaptation of E. coli (19) and as a tool to improve the "substantial equivalence" of genetically modified organisms (6). Substantial equivalence refers to whether a food from a genetically modified organism corresponds totally from a digestive point of view, to the traditional one, and is a major issue in the controversy plaguing the use of transgenic organisms as sources of food.



7 Proteomics has also been used to analyze the proteins released during the ripening of Emmentaler cheese. In an innovative study, Gagnaire et al. (7), used proteomics to prepare a reference map of the different groups of proteins found within cheese. These authors were able to categorize the proteins found in the cheese into five classes: those involved in proteolysis, glycolysis, stress response, nucleotide repair and oxidation-reduction. In addition, information was obtained regarding the peptidases released into the cheese during the ripening process. This study enabled Gaganire et al. to differentiate between the various casein degradation mechanisms present, and to sugest that the streptococci within the cheese matrix are involved in peptide degradation and together with the indigenous lactobacilli contribute to the ripening process. Using proteomics these authors were able to derive a greater understanding of the microbial succession involved in the ripening of Emmentaler cheese, which information could not have been obtained using other protein separation technique. This example illustrates the power of proteomics as a tool for analyzing the composition of a complex mixture of proteins and peptides. The strength of genome-based technology relies on the accuracy and validity of the genome sequence information available (4). Very often, however, the information obtained from genomics and proteomics does not assign a putative function to the genes and proteins identified. If the genes/proteins identified by the genomics-based approaches have been previously well characterized, then it gives the researcher a starting point with which to set up future investigations. But, i f the gene or protein has only been annotated as a putative open reading frame without a function attributed to it, then this information does not yield any clues to the possible function of the gene/protein. The correlation between a gene/protein sequence and function in the organism has to be carried out by basic empirical research.

Techniques for Determining the Function of Identified Genes The identification of genes and proteins that are regulated by a particular stress response using genomic methods has to be correlated or, at least, associated with a particular function for the genomic information to have value. Techniques of classic microbial genetics are used to identify and characterize the function o f selected genes. In microbial genetics, gene function is usually identified by creating, isolating and identifying mutants in the signaling pathway or cellular process under study that correlates to a specific phenotype. Phenotypes that are selected for can be acid-resistance, high-salt resistance or avirulent mutants of pathogenic organisms. A n in-depth study of the aberrant mutant phenotype is then carried out to discover where in the process the precise malfunction occurs. The malfunctioning gene is then identified and the correct


8 function attributed to it. The power of microbial genetics is not only in the ability to create mutations by genetic or chemical means (mutagenesis), but also in the ability to identify the mutants (selection) and to recover the genetic site of the disruption. Transposon mutagenesis is a commonly used genetic technique for the in vitro or in vivo creation of mutant phenotypes.


Transposon mutagenesis In this type of mutagenesis, a transposon is delivered by electroporation, mating or conjugation to the wild-type cells of the organism of interest and allowed to randomly "hop" (or transpose) into any locus in the organism's genome. The transposition event is catalyzed by the enzyme transposase. B y transposing into a gene locus, the transposon creates a mutation in that gene by inserting into it. The insertion of the transposon generally inactivates the gene, such that the mutant created in this way has a loss of that particular gene's function. If the transposon locates into the regulatory regions of the gene, it can also cause up-regulation of the gene and create a situation where there is excess of that gene product in the cell. In either case, there is imbalance in the amount of the gene product in the cell that consequently causes a mutant phenotype. In the best case scenario, the mutant phenotype is an easily detectable and visible one, allowing for the easy isolation of these mutants. Most often, a clear, visible phenotype is not available. In these instances, many strategies have been described for the identification and isolation of the desired mutant phenotype. Discussed below are two approaches (signature-tagged mutagenesis and the negative selection method) that allow the identification and retrieval of aberrant genes in a pathway. Both methods employ negative selection strategies, that are so named because the identified cells are mutant in nature, allowing for easy retrieval of the mutant cells. Insertion of the transposon into genomic D N A can be done either in vitro or in vivo. Epicenter Technologies fwww.epicentre.com/transposome.asp) has developed a commercially available transposon mutagenesis system that can be used with extracted genomic D N A or with intact, viable cells. If extracted genomic D N A is used as the substrate for transposon activity, the transposon inserted D N A can be amplified in E. coli before it is introduced into viable cells with selection for the antibiotic marker on the transposon. If viable cells are used, the transposon is introduced into the cells by electroporation and after insertion the transposase enzyme is inactivated by salt. This method can only be used with those bacterial cells that allow electroporation for the introduction of D N A fragments. In systems where the introduction of D N A by electroporation is not an option, transposons can be introduced into cells via conjugation. Historically, a number of transposon mutagenesis schemes have been developed


9 for both gram positive and negative bacteria prior to the advent of the commercial kit, and can be used with high rates of success.


Signature-Tagged Mutagenesis This method of mutagenesis and selection (/ 7) has been used successfully to identify genes involved in the virulence process in Salmonella serovar Typhimurium, V. cholerae and Klebsiella pneumoniae (9). This technique combines insertional mutagenesis with the negative selection in vivo of avirulent or attenuated pathogenic strains. Transposon mutagenesis is used to generate a bank of mutant bacteria. This pool of mutant bacteria is then introduced into the host animal model. After incubation in the host, the bacteria are isolated and the signatures are amplified (Figure 1) to identify those tags that were lost due to death of the avirulent bacterial cells within the host. These dead bacteria represent those cells that were unable to infect the host successfully, due to the transposon insertion into a genetic locus essential for virulence. The "lost" tags can be identified by hybridization of the recovered tags to the master collection of bacteria containing all of the initially generated transposon tagged loci. Those bacteria that do not hybridize to the recovered tags and that represent the "lost" tags, contain transposon insertions in genes required for virulence. The transposon insertion site can be easily identified by locating the transposon itself, the D N A region containing the transposon isolated and the inactivated gene identified by D N A sequencing around the insertion site. The genes thus identified are required for the virulence process.

Negative Selection Strategy Using the codA Gene This genetic selection scheme was originated, developed and tested in our laboratory (2). The scheme is based on the fusion of a inducible promoter to the cytosine deaminase (codA) gene of E. coli (Figure 2). The promoter of choice used was the high-light regulated psbDII promoter (7) from the free-living cyanobacterium Synechococcus elongates. The psbDII promoter was fused to the codA gene such that all regulatory information (promoter sequences and the ribosomal binding site) were from the psbDII gene. The construct was then introduced by transformation into Synechococcus cells, and homologously recombined into a neutral site in the Synechococcus chromosome. Neutral sites are regions of the Synechococcus chromosome where genetic constructs can be recombined without any i l l effects on the growth and viability of the organism (3). The resulting strain was then mutagenized using iV-methyl-N -nitro-iVnitrosoguanidine ( M M N G ) to generate random, point mutations i n the


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Tagged genomic DNA carrying transposon

Amplify in suitable bacterial cells with selection for antibiotic marker


Transfer transposon containing isolates into host animal

After incubation in host, isolate microbial cells from host animal

Select for remaining tagged cells, and identify "lost" tags by hybridization to master dot blots of total tag-containing fragments

Figure 1. Diagrammatic representation of genes that are expressed in vivo using signature tagged mutagenesis.

WILD-TYPE CELLS Input Signal Intact pathway

ι

Activates promoter

\

1

Synthesize cytosine deaminase

Die when grown on on 5-FC

MUTANT CELLS Input Signal Defective pathway

CANNOT Activate promoter

ι

NO cytosine deaminase

Grow on on 5-FC

Figure 2. Cytosine deaminase-based negative selection scheme for the isolation of mutants.



11 chromosome, and the mutagenized cells grown on medium containing the selective agent 5-fluorocytosine. The enzyme cytosine deaminase, protein product of the codA gene, converts 5-fluorocytosine into the toxic product 5-fluorouracil (24). Synechococcus cells do not contain an intrinsic codA gene, and this gene is an excellent selective tool in this scheme. The scheme functions conceptually as follows. Cells that contain an intact high-light signaling pathway that regulates the psbDII promoter will express the codA gene in high light. Expression of this gene results in the synthesis of the enzyme cytosine deaminase which will convert 5-fluorocytosine to the toxic metabolite 5-fluorouracil, and cells growing on this substrate will die. However, cells in which the psbDII high-light pathway is defective due to a mutation in the pathway, will NOT express the codA gene and will survive when grown on 5-fluorocytosine due to their inability to convert this chemical into its toxic product. These cells will carry the desired mutations in the psbDII pathway. To identify the site of mutation, the resulting mutants can be individually "rescued" with genomic D N A fragment from wild-type Synechococcus. Rescued cells will display the wild-type ability to express the codA gene and will die when challenged with 5-fluorocytosine. This challenge can be used as cofirmation that the mutant phenotype has been rescued by the wild-type genomic D N A fragment. This negative selection scheme can be used where a clear, visible mutant phenotype is not available for the easy selection of mutants. The codA marker can be used in mammalian cells (14, 25) and in bacterial cells that lack a codA gene, or where the native codA gene has been disabled prior to use in this scheme. We tested this scheme with mutants that had been created by chemical mutagenesis. Other mutagenesis methods also lend themselves for use with this selection scheme. If transposon mutagenesis is used, the site of mutation can easily be located by isolating the region of transposon insertion in the genome.

In vivo Expression Technology (IVET) This method has been used to isolate genes that are expressed in the animal host during infection, but are not expressed in the free-living pathogenic organism. Mahan et al. (18) used this technique to isolate genes expressed during the infection of mice with the bacterium Salmonella serovar Typhimurium. Randomly generated Salmonella genomic D N A fragments were fused upstream of promoterless, tandomly arranged purA and lacZ genes, and the construct transformed into E. colt. The pur A gene is required for purine biosynthesis in the bacterium and the lacZ gene (when activated) serves as a reporter gene. The constructs were then transferred by conjugal mating from E. coli to a purA defective Salmonella strain, and integrated into the Salmonella chromosome by


12 homologous recombination. The resulting Salmonella cells were used to infect the animal host, the mouse. Salmonella cells were incubated in the mouse host for 2-3 days, to allow for selection of all Salmonella cells that had a purA phenotype. The purA phenotype would only have occurred i f the genomic D N A fragment cloned upstream o f the purA gene in the construct, contained a promoter that was activated upon infection of the mouse host. Any Salmonella cells that were purA defective would not be viable in the mouse host. Bacterial cells were recovered from the spleen of the host animals, and plated on indicator medium for lacZ gene expression. Mahan et al. (18) were interested in those Salmonella genes that were expressed in the host, but not in the free-living state on a laboratory medium. Thus, they isolated bacterial colonies that were Lac" and were white not blue in coloration on the indicator plate, since cells that were Lac on laboratory medium will contain D N A fragments that activated lacZ (and purA) expression in the free-living state. These authors were able to successfully identify genes associated with Salmonella virulence in the mouse host. The advent of genomics-hased techniques has revolutionized the analysis of bacterial gene expression in response to stress. For maximum impact and information, these techniques have to be coupled with classical microbial genetics methods to yield critical insights on bacterial stress responses. These data will greatly impact the fields of food safety, infectious disease and the design of antimicrobial technologies. +

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