Role of Bacillus Subtilis RNA Polymerase in Controlling Gene Expression

Objective

This study will address two significant questions regarding the general stress response in the bacterium BACILLUS SUBTILIS and related Gram positive pathogens such as LISTERIA MONOCYTOGENES: (1) What cellular functions are affected by the response; and (2) How are stress signals sensed and conveyed to the Sigma-B transcription factor that controls the response?

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NON-TECHNICAL SUMMARY: The general stress response allows bacteria to survive in the environment, in food, and in pathogenic interactions, thereby increasing the threat of human illness. We want to understand the general stress response in the bacterium B. subtilis, beginning with the sensors which detect the stress, extending through the signaling network which conveys these stress signals to the regulator activating the response, and ending with the physiological role of the newly made proteins. Study of this mechanism in B. subtilis will help explain the response in the related bacteria Staphylococcus aureus and Listeria monocytogenes, allowing control of these harmful food-borne pathogens.

APPROACH: Study will focus on the transcriptional regulation of genes induced in B. SUBTILIS and related Gram positive pathogens during the general stress response. This response is controlled by the Sigma-B transcription factor, whose activity is modulated by a complex signal transduction network. The short-term objectives are (1) how do genes under Sigma-B control bring about a stress-resistant state; and (2) what is the molecular mechanism by which Sigma-B is activated in response to diverse stress signals?

PROGRESS: 2007/01 TO 2007/12 
OUTPUTS: We study bacterial stress signaling networks that function via the partner switching mechanism, in which key protein interactions are controlled by serine phosphorylation. We use BACILLUS SUBTILIS as a model for related Gram positive pathogens, many of which cause food-borne illnesses by surviving harsh or stressful conditions. We addressed three questions in analyzing critical features of the Sigma-B regulatory network, which controls a general stress response: (i) How does the Sigma-B network integrate diverse stress signals? The network consists of two branches converging on common regulatory elements. Each branch is specific for one of the signal classes that activate the Sigma-B transcription factor: energy or environmental stress. We have found that the energy branch terminates with a serine phosphatase with three domains: an N-terminal PAS domain, a central coiled coil, and a C-terminal PP2C phosphatase. We are completing a genetic and biochemical analysis of the contribution of each domain to signal sensing and propagation (Brody et al., in preparation). (ii) What features of the mechanism allow it to adapt to different signaling tasks? We constructed mathematical models to compare the performance of two well-studied partner switching networks: the Sigma-B general stress network and the Sigma-F sporulation network, which controls a critical step of the developmental process in BACILLUS SUBTILIS and other bacteria. The two networks have clear topological differences, and we asked how their different arrangement of similar components affects network performance (Igoshin et al., 2007). (iii) How does expression of genes under Sigma-B control contribute to stress survival? Here we studied the five-gene ssrA operon that is controlled by Sigma-B, characterizing the transcriptional organization of the operon and the contribution of each gene product to bacterial growth at low and high temperatures (Shin and Price, 2007). PARTICIPANTS: Chester W. Price, PI; Margaret S. Brody, Staff Research Associate III; Ji-Hyun Shin, Postdoctoral Scientist (lab member, no support from agency); Oleg A. Igoshin, Postdoctoral Scientist (UC Davis collaborator, no support from agency); Michael A. Savageau, Professor (UC Davis collaborator, no support from agency). TARGET AUDIENCES: Target audience comprised other microbiologists who study bacteria important for food safety and spoilage. Efforts included publication of two papers in peer-reviewed international journals and informal discussions with target audience at an international meeting on the Molecular Genetics of Bacteria and Phages, held in Madison, Wisconsin on August 7-12, 2007.
 
IMPACT: 2007/01 TO 2007/12 
General stress responses controlled by the Sigma-B transcription factor and sporulation processes controlled by the Sigma-F factor allow bacteria to resist food processing treatments such as acid, cold, ethanol, heat, and salt. Underscoring the value of the BACILLUS SUBTILIS model we use, the signaling pathways that activate Sigma-B and Sigma-F in related pathogens are the same as the ones we study in the non-pathogenic BACILLUS SUBTILIS. For example, in these related pathogens Sigma-B contributes to human illness in three ways. (1) Direct contribution: In BACILLUS ANTHRACIS, LISTERIA MONOCYTOGENES and STAPHYLOCOCCUS AUREUS, loss of Sigma-B function leads to decreased virulence in test animals. (2) Interaction with virulence pathways: In LISTERIA MONOCYTOGENES and STAPHYLOCOCCUS AUREUS, interactions exist between Sigma-B and signaling pathways that control virulence determinants. The emerging picture is that pathogens have complex regulatory networks that integrate signals from the environment to produce graded responses, and Sigma-B figures prominently in such networks. (3) Persistence in the environment: Expression of genes under Sigma-B control contribute to pathogen survival in foods and in medical facilities, exposing victims to a larger infectious dose. This is particularly evident for LISTERIA MONOCYTOGENES. Sigma-B allows growth at low temperature or at high osmolarity, which constitutes the principle threat of this pathogen to the food supply. Understanding the Sigma-B network will help control LISTERIA MONOCYTOGENES by suggesting ways to defeat the potent stress response of the organism. Our experimental OUTPUTS have led to the following specific OUTCOMES: (i) A bioinformatics approach has shown that the energy branch of the Sigma-B network constitutes a signaling module that is found in diverse bacteria, with the ability to couple an energy stress input to many different signal output domains (Brody et al., in preparation). (ii) A mathematical modeling approach has shown that the different topological arrangement of otherwise similar components of a signaling network result in different system properties that correlate with regulatory demands (Igoshin et al., 2007). (iii) A genetic and physiological analysis has shown that the ssrA ribosome rescue system is under Sigma-B control and is important for growth at both unusually low and unusually high temperatures (Shin and Price, 2007).
 
PROGRESS: 2006/01/01 TO 2006/12/31 
We study bacterial signaling networks that function via the partner switching mechanism, in which key protein interactions are controlled by serine phosphorylation. We use BACILLUS SUBTILIS as a model for related Gram positive pathogens, many of which cause food-borne illnesses by surviving harsh conditions. We addressed three questions in analyzing critical features of the mechanism and its role in survival and pathogenesis. (i) How widespread is the mechanism? Genome analysis finds partner-switching components in diverse bacteria, suggesting the mechanism is ancient and can accomplish different signaling tasks. However, the mechanism had not been characterized outside of the Gram positive lineage in which it was discovered. We therefore performed a significant experimental test of the predicted regulators in the Gram negative, obligate intracellular pathogen CHLAMYDIA TRACHOMATIS, the leading cause of sexually transmitted disease in developed countries and of preventable blindness in the developing world. Our demonstration that the core of the mechanism is conserved in CHLAMYDIA indicates that its basic features are maintained over a large evolutionary span, and in a bacterium that occupies a very different niche from the free-living BACILLUS. Based on the properties of its input phosphatases we propose that the pathway controls an important part of the developmental cycle within the host, in response to signals external to the CHLAMYDIA membrane (Hua et al., 2006). (ii) What features of the mechanism are important for its different signaling tasks? We constructed mathematical models of well-studied networks, such as that for the Sigma-F transcription factor, which controls a critical step of the sporulation process in BACILLUS SUBTILIS and other bacteria. Our model predicts that the self-reinforcing formation of a long-lived complex transforms the network into an essentially irreversible hysteretic switch, explaining its sharp, robust and irreversible activation (Igoshin et al., 2006). A mathematical comparison of the Sigma-F network and the similar but functionally distinct Sigma-B network is in progress. Sigma-B controls the general stress response in BACILLUS SUBTILIS and related bacteria, and contributes to growth and survival under harsh conditions. (iii) How does the Sigma-B network integrate diverse stress signals? The network consists of two branches converging on common regulatory elements. Each branch is specific for one of the signal classes that activate Sigma-B: energy or environmental stress. The environmental branch contains five paralogous proteins that function as negative regulators. We have genetic and biochemical evidence that a sixth paralog, YtvA, acts as a positive regulator in the same branch. YtvA differs from the negative regulators by its FMN-containing Light-Oxygen-Voltage domain, which has the photochemistry of a blue-light sensor. These results are the first demonstration that blue light controls a stress response in a non-photosynthetic bacterium, and support a model in which a large signaling complex integrates multiple environmental signals to modulate the general stress response (Gaidenko et al., 2006).
 
IMPACT: 2006/01/01 TO 2006/12/31 
The sporulation process controlled by the Sigma-F regulator and the general stress response controlled by the Sigma-B regulator allow bacteria to resist food processing treatments such as acid, cold, ethanol, heat, and salt. Underscoring the value of the BACILLUS SUBTILIS model we use, the signaling pathways that activate Sigma-F and Sigma-B in related pathogens are the same as in BACILLUS SUBTILIS. In these bacteria Sigma-B affects pathogenesis three ways. (i) Direct contribution: In BACILLUS ANTHRACIS and STAPHYLOCOCCUS AUREUS, loss of Sigma-B leads to decreased virulence in test animals. (ii) Interaction with virulence pathways: In LISTERIA MONOCYTOGENES and STAPHYLOCOCCUS AUREUS, interactions exist between Sigma-B and signaling pathways that control virulence determinants. The emerging picture is that pathogens have complex regulatory networks that integrate signals from the environment to produce graded responses, and Sigma-B figures prominently in such networks. (iii) Persistence in the environment. Expression of genes under Sigma-B control contribute to pathogen survival in foods and in medical facilities, exposing victims to a larger infectious dose. This is particularly evident for LISTERIA MONOCYTOGENES. Sigma-B allows growth at low temperature or at high osmolarity, which constitute the main threat of this pathogen to the food supply. LISTERIA MONOCYTOGENES accounts for 25% of the fatalities due to food-borne illness. Understanding the Sigma-B network will help control the pathogen by suggesting compounds that target Sigma-B signaling without harming humans.

PROGRESS: 2005/01/01 TO 2005/12/31 
The general stress response contributes to bacterial survival in the environment, in foods, and in pathogenic interactions. We study this response using BACILLUS SUBTILIS as a model for related Gram positive pathogens. Among these organisms the response is controlled by the Sigma-B transcription factor. In the BACILLUS model, we want to understand the response beginning with the sensors that detect diverse stresses, extending through the signal transduction network that conveys these stresses to Sigma-B, and ending with the role of the genes under Sigma-B control. The signal transduction network is of particular interest because it functions via the partner switching mechanism, in which key protein interactions are controlled by serine phosphorylation. Genome analysis finds this mechanism in diverse bacterial groups, suggesting it is very ancient and can be configured to accomplish different signaling tasks. However, the mechanism had not been experimentally characterized outside of the Gram positive lineage in which it was originally discovered. This year we have undertaken three new lines of investigation to establish the critical features of partner switching mechanism and its role in both bacterial survival and pathogenesis. (i) We performed a significant experimental test of the predicted partner switching regulators encoded by the genome of the obligate intracellular pathogen CHLAMYDIA TRACHOMATIS, the leading cause of sexually transmitted disease in developed countries and of preventable blindness in the developing world. Our experimental demonstration that the core of the partner switching mechanism is conserved in CHLAMYDIA indicates that its basic features are maintained over a large evolutionary span, and in a bacterium that occupies a very different niche from the free-living BACILLUS. Based on the predicted properties of its input phosphatases we propose that the partner switching pathway controls an important aspect of the developmental cycle within the host, in response to signals external to the CHLAMYDIA cytoplasmic membrane. (ii) We have begun a molecular genetic investigation of the partner switching pathway in the food-borne pathogen LISTERIA MONOCYTOGENES, where it controls activation of the general stress transcription factor Sigma-B, much like in the closely related BACILLUS model. In LISTERIA, Sigma-B and the general stress response are important for growth in the cold and under osmotic stress, which constitute the principal threat of this pathogen in food. We are now establishing which environmental stress signals are conveyed by the different parts of the partner switching pathway in LISTERIA. (iii) We have begun to construct mathematical models of the best studied partner switching signaling pathways to establish which features are important for function. For example, the Sigma-F factor of BACILLUS SUBTILIS controls a critical step of the sporulation process. Our model predicts that the self-reinforcing formation of a long-lived complex transforms the Sigma-F partner switching pathway into an essentially irreversible hysteretic switch and explains its sharp, robust and irreversible activation.
 
IMPACT: 2005/01/01 TO 2005/12/31 
The general stress response that is activated by means of Sigma-B allows bacteria to resist food processing treatments such as acid, cold, ethanol, heat, and salt. Underscoring the value of the BACILLUS SUBTILIS model we use, the signaling pathways that activate Sigma-B in related pathogenic bacteria function in essentially the same way as in BACILLUS SUBTILIS. Sigma-B affects pathogenesis in three ways. (i) Direct contribution: In BACILLUS ANTHRACIS, MYCOBACTERIUM TUBERCULOSIS and STAPHYLOCOCCUS AUREUS, loss of Sigma-B function leads to decreased virulence in test animals. (ii) Interaction with virulence pathways: In LISTERIA MONOCYTOGENES and STAPHYLOCOCCUS AUREUS, intriguing interactions exist with signaling pathways that control expression of virulence determinants. The emerging picture is that human pathogens have complex regulatory networks that integrate signals from the external environment to produce graded responses, and that Sigma-B often figures prominently in such networks. (iii) Persistence in the environment. Expression of genes under Sigma-B control contribute to pathogen survival in foods and in medical facilities, exposing victims to a larger infectious dose. This is particularly evident for LISTERIA MONOCYTOGENES, wherein Sigma-B allows growth at low temperature or at high osmolarity, features that constitute the principle threat of this pathogen to the food supply. However, it is also true for STAPHYLOCOCCUS AUREUS and STAPHYLOCOCCUS EPIDERMIDIS, which can cause serious human infections.
 
PROGRESS: 2004/01/01 TO 2004/12/31 
The general stress response contributes to bacterial survival in the environment, in foods, and in pathogenic interactions. We study this response using BACILLUS SUBTILIS as a model for related Gram-positive pathogens. Among these organisms the response is controlled by the Sigma-B transcription factor. In the BACILLUS model, we want to understand the response beginning with the sensors, which detect diverse stresses, extending through the signal transduction network, which conveys these stresses to Sigma-B, and ending with the role of the genes under Sigma-B control. The signal transduction network functions via the 'partner switching' mechanism, in which protein interactions are controlled by serine phosphorylation. In outline, the network comprises two upstream pathways (environmental and energy), which converge on the direct regulators of Sigma-B. Here we ask (i) how is specificity maintained in parallel partner switching pathways; and (ii) how do the environmental regulators transmit diverse stress signals? One, we showed that a parallel switching pathway is insulated from the Sigma-B pathway by the combined action of its serine kinase and phosphatase. Two, we determined that four newly identified regulators act collectively as redundant co-antagonists in a large signaling complex, and that a threonine residue conserved among them is critical for stress transmission. In a complementary study, we tested key predictions of our signaling model by determining the in vivo phosphorylation states of environmental regulators, confirming the role of the critical threonine.
 
IMPACT: 2004/01/01 TO 2004/12/31 
The general stress response confers resistance to food processing treatments such as acid, cold, ethanol, heat, and salt. Our work in B. SUBTILIS will help control the related food pathogens STAPHYLOCOCCUS AUREUS and LISTERIA MONOCYTOGENES, in which loss of Sigma-B causes multiple stress sensitivity and decreased virulence. Our study will also explain the principles governing a broad array of signaling pathways that function by the partner switching mechanism.

PROGRESS: 2003/01/01 TO 2003/12/31 
The general stress response contributes to bacterial survival in the environment, in foods, and in pathogenic interactions. We study this response using BACILLUS SUBTILIS as a model for related Gram positive pathogens. Among these organisms the general stress response is controlled by the Sigma-B transcription factor. Two important questions are (i) how does this response confer multiple stress resistance; and (ii) how is Sigma-B activated by diverse stress signals? During the past year we addressed both questions. First, we developed a new method to analyze DNA array data called "correlation selection". This method allows statistical analysis of the combined DNA array data from our lab and others around the world, even those using significantly different experimental methodology. We identified new Sigma-B dependent genes not found in earlier analyses, and we used RACE-PCR to confirm that many of these genes are directly under Sigma-B control (manuscript in preparation). Second, Sigma-B activity is modulated by a "partner switching" signaling mechanism in which protein interactions are governed by serine phosphorylation. Energy or environmental stresses are conveyed to Sigma-B by two independent pathways, each terminating with a differentially regulated serine phosphatase whose activation triggers the response. We showed by genetic and biochemical analysis that the partner switching pathway controlling the RsbU environmental-signaling phosphatase is unusual in that it contains redundant co-antagonist proteins in a large, 700 kDa complex called a "signalsome" (submitted).
 IMPACT: 2003/01/01 TO 2003/12/31 
Bacterial stress response systems contribute to food-borne illness. Our work in B. SUBTILIS will help control the related human pathogens B. ANTHRACIS, STAPHYLOCOCCUS AUREUS and LISTERIA MONOCYTOGENES. Homologues of the B. SUBTILIS regulators are also found in diverse Gram negative pathogens, such as VIBRIO, XANTHAMONAS and CHLAMYDIA species, indicating that this signaling mechanism is widespread.
 
PROGRESS: 2002/01/01 TO 2002/12/31 
General stress response contributes to bacterial survival in the environment, foods, and pathogenic interactions. We study this response using BACILLUS SUBTILIS as a model for related Gram positive pathogens. In these organisms the general stress response is controlled by the Sigma-B transcription factor. During the past year we addressed two important questions: (i) how does this response confer multiple stress resistance; and (ii) how does Sigma-B activate the response? First, in a significant extension of our earlier genomic approach to find genes controlled by Sigma-B, we developed a powerful new method to analyze DNA array data called "correlation selection" (unpublished). This method allows statistically sound analysis of the combined array data from our lab and others around the world, even those using significantly different experimental methodology. Second, Sigma-B is controlled by a "partner switching" signaling mechanism in which protein interactions are governed by serine phosphorylation. Energy or environmental stresses are conveyed to Sigma-B by two independent pathways, each terminating with a differentially regulated serine phosphatase whose activation triggers the response. We showed that the related PrpC serine phosphatase is not part of the Sigma-B regulatory network but instead controls other major functions in stationary phase cells. We also showed by genetic and biochemical analysis that the partner switch controlling the RsbU environmental-signaling phosphatase is unusual in that it contains redundant co-antagonist proteins (unpublished).
 
IMPACT: 2002/01/01 TO 2002/12/31 
Bacterial stress response systems contribute to food-borne illness and infection. Our B. SUBTILIS study will help control related human pathogens B. ANTHRACIS, S. AUREUS, M. TUBERCULOSIS, and L. MONOCYTOGENES. Coantagonist proteins like those we found in B. SUBTILIS are also in L. MONOCYTOGENES, suggesting signals (e.g. cold shock) are similarly conveyed to Sigma-B in this cryotolerant pathogen.

PROGRESS: 2001/01/01 TO 2001/12/31 
The general stress response contributes to bacterial survival in the environment, in foods, and in pathogenic interactions. We study the general stress response of the bacterium BACILLUS SUBTILIS as a model for related Gram positive human pathogens. In B. SUBTILIS this response is controlled by the Sigma-B transcription factor. Two important questions are (i) how does the general stress response confer multiple stress resistance; and (ii) how does Sigma-B activate the response? I reviewed this field during the project period (Price, 2001) and published research papers that addressed both questions. (i) Using genomic DNA array studies we found that Sigma-B directs the expression of more than 200 general stress genes (Price et al., 2001). Our key findings were surprising and open new research avenues. For one, we now know the general stress response extensively alters cellular physiology, but gene products with a direct protective function are not common. Moreover, the B. SUBTILIS response resembles that of yeast, suggesting that both prokaryotic and eukaryotic cells counter stress in fundamentally similar ways. (ii) Sigma-B activity is itself controlled by a new mechanism of signal transduction which integrates both environmental and energy stresses, conveyed via independent signaling pathways. Our current goal is to identify sensor and regulatory proteins which comprise these pathways. We have discovered five new regulators in the environmental signaling pathway (Akbar et al., 2001) and one new regulator in the energy signaling pathway (Brody et al., 2001).
 
IMPACT: 2001/01/01 TO 2001/12/31 
The stress response systems of bacteria contribute strongly to their capacity to cause food-borne illness and human infection. Our study of the general stress response in BACILLUS SUBTILIS provides information useful to control the related human pathogens BACILLUS ANTHRACIS, LISTERIA MONOCYTOGENES, MYCOBACTERIUM TUBERCULOSIS, and STAPHYLOCOCCUS AUREUS.
 
PROGRESS: 2000/01/01 TO 2000/12/31 
Bacterial stress responses contribute to food-borne illness directly by influencing bacterial pathogenicity and indirectly by promoting bacterial survival in foods. The general stress response of the Gram positive bacterium BACILLUS SUBTILIS is a model for Gram positive pathogenic organisms that are more difficult to study because they lack a good genetic system. The Sigma-B transcription factor of BACILLUS SUBTILIS directs the expression of more than 200 general stress genes. Sigma-B activity is itself controlled by a new mechanism of signal transduction that integrates both environmental and energy stresses, which are conveyed to Sigma-B via independent signal transduction pathways. I have reviewed this field during the last project period (Price, 2000). We have now discovered five new regulators in the environmental signaling pathway (Akbar et al., 2001) and one new regulator in the energy signaling pathway (publication in preparation). Our goal is to understand the complete signal transduction pathway, from the sensors that detect the external and internal stress signals to the target genes whose expression is controlled by Sigma-B to confer a multiple stress resistance. In related bacteria this multiple stress resistance includes the formation of biofilms in a Sigma-B dependent manner. These biofilms are the cause of persistent contamination in food processing plants and of persistent infection via medical devices.
 
IMPACT: 2000/01/01 TO 2000/12/31
The stress response systems of bacteria contribute strongly to their capacity to cause food-borne illnesses. Study of the general stress response in BACILLUS SUBTILIS provides an excellent model for the Gram positive food-borne pathogens STAPHYLOCOCCUS AUREUS and LISTERIA MONOCYTOGENES, in which Sigma-B and its distinctive signaling pathway have recently been discovered.

PROGRESS: 1999/01/01 TO 1999/12/31 
Bacterial stress responses contribute directly to food-borne illness by influencing bacterial pathogenicity and indirectly by promoting survival in foods. The general stress response of the Gram positive bacterium Bacillus subtilis is a model for Gram positive pathogenic organisms that are more difficult to study because they lack a good genetic system. The Sigma-B transcription factor of B. subtilis controls the expression of more than 100 general stress genes. Sigma-B activity itself is controlled by a new mechanism of signal transduction that integrates both environmental and energy stresses. We have addressed the regulation of Sigma-B and the role of Sigma-B-dependent genes in three new ways. First, a biochemical and molecular genetic study found the mechanism by which a key environmental stress regulator activates Sigma-B (Gaidenko et al). Second, we identified and characterized the transcriptional control of two genes which are solely under Sigma-B control and which contribute to the general stress response (Akbar et al.). Third, we used a combined genetic and biochemical approach to identify an entirely new branch of the Sigma-B signaling pathway, one which activates Sigma-B only in response to energy stress (Vijay et al.). Based on this study, we developed a new model of Sigma-B regulation. This model provides the potential to interfere with Sigma-B signaling in related pathogenic bacteria and thereby control them without the use of conventional antibiotics.
 
IMPACT: 1999/01/01 TO 1999/12/31 
The stress response systems of bacteria contribute strongly to their capacity to cause food-borne illnesses. Study of the general stress response in Bacillus subtilis provides an excellent model for the Gram positive food-borne pathogens Staphylococcus aureus and Listeria monocytogenes, in which Sigma-B and its distinctive signaling pathway have recently been discovered.
 
PROGRESS: 1999/01/01 TO 1999/12/31 
Bacterial stress responses contribute directly to food-borne illness by influencing bacterial pathogenicity and indirectly by promoting survival in foods. The general stress response of the Gram positive bacterium Bacillus subtilis is a model for Gram positive pathogenic organisms that are more difficult to study because they lack a good genetic system. The Sigma-B transcription factor of B. subtilis controls the expression of more than 100 general stress genes. Sigma-B activity itself is controlled by a new mechanism of signal transduction that integrates both environmental and energy stresses. We have addressed the regulation of Sigma-B and the role of Sigma-B-dependent genes in three new ways. First, a biochemical and molecular genetic study found the mechanism by which a key environmental stress regulator activates Sigma-B (Gaidenko et al). Second, we identified and characterized the transcriptional control of two genes which are solely under Sigma-B control and which contribute to the general stress response (Akbar et al.). Third, we used a combined genetic and biochemical approach to identify an entirely new branch of the Sigma-B signaling pathway, one which activates Sigma-B only in response to energy stress (Vijay et al.). Based on this study, we developed a new model of Sigma-B regulation. This model provides the potential to interfere with Sigma-B signaling in related pathogenic bacteria and thereby control them without the use of conventional antibiotics.
 
IMPACT: 1999/01/01 TO 1999/12/31 
The stress response systems of bacteria contribute strongly to their capacity to cause food-borne illnesses. Study of the general stress response in Bacillus subtilis provides an excellent model for the Gram positive food-borne pathogens Staphylococcus aureus and Listeria monocytogenes, in which Sigma-B and its distinctive signaling pathway have recently been discovered.

PROGRESS: 1998/01/01 TO 1998/12/31 
The stress responses of Gram positive bacteria contribute strongly to serious food-borne illness. The general stress response of the Gram positive bacterium BACILLUS SUBTILIS serves as a model for similar responses in these pathogenic organisms. The sigma-B transcription factor controls the expression of more than 100 general stress genes. Sigma-B activity itself is controlled by a new mechanism of signal transduction which integrates both environmental and energy stresses (Kang, 1998). We have addressed the regulation of sigma-B and the role of sigma-B-dependent genes in three new ways. First, we characterized the sigB operon of the closely related BACILLUS LICHENIFORMIS in order to learn which motifs were conserved among sigma-B regulators and therefore were likely important for function (Brody & Price, 1998). Second, we found that sigma-B was important for survival under acid and alkaline stress conditions (Gaidenko & Price, 1998). Third, we used a genetic approach for a structure-function analysis of a key sigma-B regulator (Kang et al., 1998).

PROGRESS: 1997/01/01 TO 1997/12/01 
Stress responses are an important part of pathogeneses in Gram negative bacteria. Less is known regarding stress responses in Gram positive bacteria, a group containing many human pathogens which cause food-borne illness. The general stress response of the Gram positive bacterium BACILLUS SUBTILIS is controlled by the sigma-B tanscription factor, which associates with RNA polymerase to direct the expression of more than forty stress genes. sigma-B activity is in turn controlled by the newly discovered partner switching mechanism of signal transduction, which uses two coupled modules to integrate the two different classes of signals to which sigma-B responds: environmental stress and energy stress. Key questions we are now addressing include (1) what are the molecular details by which the partner switching mechanism functions; (2) what is the molecular machinery which senses energy stress and conveys these signals to the downstream partner switching module; and (3) what machinery senses environmental stress and conveys these signals to the upstream partner switching module (AKBAR ET AL., 1997). Answers to these questions will contribute to an understanding of how pathogens persist in the natural environment and in food systems to cause food-borne infections and intoxications.

PROGRESS: 1996/01 TO 1996/12 
The stress response is an important part of pathogeneses in Gram negative bacteria. Less is known regarding stress response in Gram positive bacteria, a group containing many human pathogens which cause food-borne illness. We have now established key features of the mechanism controlling the general stress response in the Gram positive bacterium BACILLUS SUBTILIS. This stress response is governed by the alternative transcription factor sigma-B, which associates with the catalytic core of RNA polymerase to direct transcription of over 40 stress genes. The activity of sigma-B is in turn controlled by a multi-component regulatory network, which we studied using a combined genetic and biochemical approach (KANG ET AL., 1966; REDFIELD and PRICE, 1966; YANG ET AL., 1966). Our work on this network defines a newly discovered means of signal transduction in bacteria, a "partner switching" mechanism in which key protein-protein interactions are controlled by reversible phosphorylation. We have also continued analyzing sigma-B-dependent genes of B. SUBTILIS in order to learn their protective role (AKBAR ET AL., 1996; VARON ET AL., 1996). Lastly, we (SUH ET AL., 1996) have the genes needed to study structure-function relationships in RNA polymerase core subunits using IN VITRO reconstitution methods.
 
PROGRESS: 1994/01 TO 1994/12 
We conduct a molecular genetic analysis of RNA polymerase to learn its role in controlling gene expression in response to environmental, morphological, and cell cycle signals in BACILLUS SUBTILIS, a gram-positive, spore-forming bacterium. Significant bacterial products such as antibiotics, insecticidal toxins, industrially useful enzymes, and food-borne toxins are made by B. SUBTILIS or similar organisms, and RNA polymerase is the enzyme of central importance in controlling bacterial gene expression. Recent progress includes: (1) The sigma-B transcription factor binds RNA polymerase and reprograms its promoter specificity to turn on new sets of genes of previously unknown function. We developed a genetic method to identify genes controlled by sigma-B; all of these newly identified genes are expressed most actively when cells are subjected to general stresses such as heat, ethanol, or osmotic shock (VARON, 1994). These results are generally significant because genes important for bacterial pathogenesis are often expressed as part of a general stress response, about which little is known in gram positive bacteria. (2) We isolated the region encoding the beta subunit of the RNA polymerase core enzyme and found its genetic and transcriptional organization differs materially from that in ESCHERICHIA COLI. We also found that a beta segment previously considered dispensable was in fact highly conserved among the second largest subunits of eukaryotic and prokaryotic RNA polymerases.
 
PROGRESS: 1993/01 TO 1993/12 
We conduct a molecular genetic analysis of RNA polymerase to learn its role in controlling gene expression in response to environmental, morphological, and cell cycle signals in BACILLUS SUBTILIS, a gram-positive, spore-forming prokaryote. This is important because significant bacterial products such as antibiotics, insecticidal toxins, industrially useful enzymes, and food-borne toxins are made by B. SUBTILIS or similar organisms, and because RNA polymerase is the enzyme of central importance in controlling bacterial gene expression. Recent progess includes: (1) The sigma-B transcription factor binds RNA polymerase and reprograms its promoter specificity to turn on new sets of genes of unknown function. We developed a new genetic method to identify genes transcribed by sigma-B and found that it controls a large regulon expressed in the stationary growth phase (BOYLAN ET AL., 1993a). The function of one new gene suggested that the sigma-B regulon counters environmental stresses (VARON ET AL., 1993). (2) We found that sigma-B activity is induced by salt, heat, or ethanol stress, and that these signals pass through the same regulatory network that controls sigma activity in response to stationary phase signals. We concluded that sigma-B controls a general stress regulon (BOYLAN ET AL., 1993b). (3) We characterized new regulatory elements that control sigma-B in response to nutritional signals (WISE, 1993). (4) Spore formation begins with an unusual, asymmetric cell division.
 
PROGRESS: 1992/01 TO 1992/12 
We conduct a molecular genetic analysis of RNA polymerase to learn its role in controlling gene expression during the exponential and stationary growth phases in BACILLUS SUBTILIS, a gram-positive, spore-forming prokaryote. This is important because significant bacterial products such as antibiotics, insecticidal toxins, industrially useful enzymes, and food-borne toxins which cause human illness are made during the stationary phase, and because RNA polymerase is the enzyme of central importance in controlling bacterial gene expression. Our recent progress is in three areas: (1) We completed a genetic analysis of the elaborate regulatory system that controls activity of sigma-B, a transcription factor which in turn controls stationary phase gene expression by directing RNA polymerase to a specific set of genes. The sigma-B gene lies third in a four gene operon; we showed that each of the other genes in the operon has a role in regulating sigma-B activity. The products of these three genes form a hierarchical control system responsive to stationary phase signals. Reported in BOYLAN ET AL., 1992. (2) The physiological role of sigma-B in stationary phase metabolism remains unknown. To learn this role we developed and used a genetic technique to identify six new genes that are controlled by sigma-B. The function of one of these genes suggests that sigma-B controls a regulon responsive to environmental stress in stationary phase. Two manuscripts in preparation.
 
PROGRESS: 1991/01 TO 1991/12 
We conduct a molecular genetic analysis of the role of RNA polymerase in controlling gene expression during the exponential and stationary phases of growth in BACILLUS SUBTILIS, a gram-positive, spore-forming prokaryote. This is important because many significant bacterial products such as antibiotics, insecticidal toxins, industrially useful enzymes, and food-borne toxins which cause human illness are made during the stationary phase, and because RNA polymerase is the enzyme of central importance in controlling bacterial gene expression. Our recent progress is in two areas: (1) We have completed a genetic analysis of an elaborate regulatory system that controls activity of sigma-B, a transcription factor which in turn controls stationary phase gene expression by directing RNA polymerase to transcribe a specific class of genes. The gene for sigma-B lies third in a four gene operon, and we have shown that each of the other genes in the operon has a role in regulating sigma-B activity. The products of these three genes form a hierarchical control system responsive to stationary phase signals. Manuscript submitted for publication. (2) The exact physiological role of sigma-B with regard to regulating and integrating stationary phase metabolism remains unknown. To learn this role we are identifying genes that require sigma-B for maximal expression, using a new genetic technique that we have developed. One gene newly discovered using our technique is CSBA, which we have isolated and thoroughly characterized.
 
PROGRESS: 1990/01 TO 1990/12 
We conduct a molecular genetic analysis of the role of RNA polymerase in controlling gene expression during the exponential and stationary phases of growth in BACILLUS SUBTILIS, a gram-positive, spore-forming prokaryote. This is essential because many significant bacterial products such as antibiotics, insecticidal toxins, industrially important enzymes, and toxins which cause human illness are made during the stationary phase, and because RNA polymerase is the enzyme of central importance in controlling bacterial gene expression. Our recent progress is in two areas: (1) We have done a genetic and biochemical analysis of gene products that control expression and activity of sigma-B, a transcription factor which in turn regulates stationary phase gene expression. Genetic organization of the sigma-B operon is similar to the SPOIIA operon, which encodes the sporulation-essential sigma-F. These two operons may therefore control alternate branches of stationary phase gene expression. (Reported in KALMAN ET AL., 1990.) (2) The exact physiological role of sigma-B with regard to regulating and integrating station phase metabolism remains unknown. To learn this role we are identifying genes that require sigma-B for maximal expression, and we have developed a new genetic technique to isolate such genes. One gene newly discovered using our technique is CSBA.

PROGRESS: 1989/01 TO 1989/12 
We conduct a molecular genetic analysis of the role of RNA polymerase in controlling gene expression during the exponential and stationary phases of growth in Bacillus subtilis, a gram-positive, spore-forming prokaryote. This is significant because many important bacterial products such as toxins, enzymes and antibiotics are made during the stationary phase. Our progress is in three areas: (1) We have done a genetic and biochemical analysis of three gene products that control expression and activity of sigma-B, a transcription factor which in turn regulates stationary phase gene expression. Genetic organization of the sigma-B operon is similar to the spoIIA operon, which encodes the sporulation-essential sigma-F. These two operons may therefore control alternate branches of stationary phase gene expression. (2) The exact physiological role of sigma-B remains unknown. We intend to learn this role by identifying genes that require sigma-B for maximal expression. By a novel genetic technique we isolated one such gene, csbA, which is maximally expressed in stationary phase under conditions inimical to sporulation, supporting the hypothesis that sigma-B regulates an alternate pathway. (3) We identified in the B. subtilis spc operon a homologue to E. coli secY, a central component of the secretory machinery. B. subtilis secY is highly expressed from a unique promoter early in stationary phase, the time of active protein secretion.

PROGRESS: 1988/01 TO 1988/12 
I am continuing a molecular genetic analysis of the function and regulation of RNA polymerase, a complex enzyme central to control of gene expression during growth and sporulation in the gram-positive prokaryote Bacillus subtilis. At least nine different sigma factors associate with the catalytic core of the polymerase (comprising the alpha, beta, and beta' core subunits) to alter promoter selectivity and thereby alter transcription of at least nine different classes of genes. Our progress falls into three areas. (1) We isolated the sigB gene encoding the alternative sigma-B of unknown function. Although sigma-B is clearly not essential for sporulation, we discovered that sigB lies in an operon with organization similar to the spo11A operon, which encodes the sporulation-essential alternative sigma-F. We suggest these two sigma factors mediate alternative pathways of stationary phase gene expression. (2) Our ongoing molecular analysis of the alpha gene defined a very large ribosomal protein operon with both striking similarities and differences compared to the alpha operon of the gram-negative bacterium E. coli. We are exploring how the organizational differences affect regulation of the region. (3) One gene within the alpha operon encodes the analog to E. coli SecY, an integral membrane protein central to the protein secretory pathway. And just downstream of the alpha operon is another operon with strong similarities to ATP-dependent periplamic permease systems of E. coli.
 
PROGRESS: 1987/01 TO 1987/12 
I am continuing a molecular genetic analysis of the function and regulation of the RNA polymerase complex, an enzyme central to control of gene expression during growth and sporulation of the gram-positive prokaryote Bacillus subtilis. At least seven different sigma factors can associate with the catalytic core of the polymerase (containing the alpha, beta, and beta' subunits) to direct the transcription of seven different classes of genes. Our progress this year falls in three areas. (1) We isolated the gene for one of the alternative sigma factors, sigma-B (previously called sigma-37) which had been widely assumed to regulate the sporulation process. However, a null sigma-B mutation (made in vitro) had no effect on either growth or sporulation. We have determined that the sigma-B gene (sigB) is the third in a four-gene operon, that the gene immediately downstream of sigB is a negative regulator of sigB expression, and that a sigB is strongly induced at the beginning of stationary phase. Thus the unknown function of sigma-B is likely manifest at this time. (2) Our ongoing molecular analysis of the alpha subunit gene suggests that B. subtilis and E. coli regulate genes central to growth rate control in fundamentally different ways. (3) Mutations that block sporulation early in the process (spoO mutations) are thought to affect transcription of developmental genes. We have found expression of enzymes subject to carbon catabolite control is altered in a spoO background.

PROGRESS: 1986/01 TO 1986/12 
I am continuing a molecular genetic analysis of the function and regulation of the RNA polymerase complex, an enzyme central to control of gene expression in growing and sporulating Bacillus subtilis. At least six different sigma factors can associate with the polymerase core (alpha, beta and beta' subunits) and direct the transcription of different classes of genes. We used recombinant DNA methods to isolate and characterize the gene for the alpha core subunit. By classical genetics and DNA sequence analysis, we mapped the alpha gene in the main ribosomal protein gene cluster. Our ongoing molecular analysis of alpha suggests that B. subtilis and E. coli regulate genes central to growth rate control in fundamentally different ways. We have also isolated the gene for a 37,000 dalton minor sigma factor (sigma-37) that was generally assumed to regulate the sporulation process. However, our null sigma-37 mutation (made in vitro) had no effect on growth or sporulation in rich media. Future research should elucidate sigma-37 function and regulation.

PROGRESS: 1985/01 TO 1985/12 
My research involves a molecular genetic analysis of the function and regulationof RNA polymerase, a multi-subunit enzyme which is central to regulation of gene expression in growing and sporulating B. subtilis. Five different sigma subunits can direct the polymerase to transcribe different classes of genes. We have used recombinant DNA methods to isolate and characterize the gene for the most abundant sigma, sigma-43 (molecular mass 43,000 daltons). Using similar methods, we have isolated the gene encoding alpha, another polymerase subunit, and the one for sigma-37, a factor which may control expression of sporulation genes. Molecular and genetic analyses of these genes are in progress. We have also completed a physiological analysis of early-blocked sporulation mutations (spo0) which may interact with the polymerase to regulate gene expression (see Price & Doi, MGG 201:88, 1985). All the spo0 mutants have substantially greater than wild-type derepression of carbon-sensitive genes when grown on a poor carbon source that induces sporulation, suggesting that spo0 gene products are involved in carbon control of gene expression. These results are being prepared for publication. Three graduate students participate in these projects.

Investigators

Price, Chester

Institution

University of California - Davis

Start date

2005

End date

2010

Funding Source

Nat'l. Inst. of Food and Agriculture

Project number

CA-D*-MIC-4559-H

Accession number

96431