Chapter One - Physiological Role of Two-Component Signal Transduction Systems in Food-Associated Lactic Acid Bacteria

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Abstract

Two-component systems (TCSs) are widespread signal transduction pathways mainly found in bacteria where they play a major role in adaptation to changing environmental conditions. TCSs generally consist of sensor histidine kinases that autophosphorylate in response to a specific stimulus and subsequently transfer the phosphate group to their cognate response regulators thus modulating their activity, usually as transcriptional regulators. In this review we present the current knowledge on the physiological role of TCSs in species of the families Lactobacillaceae and Leuconostocaceae of the group of lactic acid bacteria (LAB). LAB are microorganisms of great relevance for health and food production as the group spans from starter organisms to pathogens. Whereas the role of TCSs in pathogenic LAB (most of them belonging to the family Streptococcaceae) has focused the attention, the roles of TCSs in commensal LAB, such as most species of Lactobacillaceae and Leuconostocaceae, have been somewhat neglected. However, evidence available indicates that TCSs are key players in the regulation of the physiology of these bacteria. The first studies in food-associated LAB showed the involvement of some TCSs in quorum sensing and production of bacteriocins, but subsequent studies have shown that TCSs participate in other physiological processes, such as stress response, regulation of nitrogen metabolism, regulation of malate metabolism, and resistance to antimicrobial peptides, among others.

Introduction

Regulation of cell physiology in response to changing conditions is a must for survival in the competitive environments that bacteria often face. Signal transduction systems are key players in the regulatory circuits that modulate bacterial physiology. Among them, two-component systems (TCSs) have been subjected to intensive research since their discovery (Nixon, Ronson, & Ausubel, 1986). TCSs are signal transduction pathways typically consisting of a sensor histidine kinase (HK), usually membrane bound, and a cytoplasmic response regulator (RR). Both proteins present a modular structure (Fig. 1). The HK has an N-terminal sensory domain that monitors the environmental signals and two modules involved in the phosphorylation reaction. The first domain holds the phosphorylatable His (histidine-phosphotransfer domain, Dhp). The second domain (CA domain) holds the ATP binding site and catalyzes the phosphorylation of the Dhp domain. The RR presents a conserved receiver domain (REC), where the phosphorylatable Asp residue is located, and a C-terminal effector domain. The domains involved in the phosphorylation reaction are homologous in all TCSs while the sensory and output domains of the HK and RR, respectively, are characteristic for each TCS and determine its specificity.

In general, detection of a specific stimulus triggers the HK autophosphorylation in the conserved His residue at the DHp domain and the subsequent transference of the phosphate group to the conserved Asp residue at the REC domain of its cognate RR (Fig. 1). Phosphorylation of the RR modulates its activity, which usually involves transcriptional regulation mediated by its C-terminal effector domain (Stock, Robinson, & Goudreau, 2000). HKs often also act as phosphatases for their cognate response regulators (Huynh & Stewart, 2011); in other cases, dephosphorylation of RRs is carried out by auxiliary phosphatases (Silversmith, 2010) or spontaneous hydrolysis. The final output response results from the balance of kinase and phosphatase activities. The mechanisms of signal transfer and regulation operated by TCSs will not be covered here as they have been extensively reviewed elsewhere (Casino et al., 2010, Galperin, 2010, Gao and Stock, 2009, Groisman, 2016, Huynh and Stewart, 2011, Jung et al., 2012, Krell et al., 2010, Mascher et al., 2006, Podgornaia and Laub, 2013, Salazar and Laub, 2015, Stock et al., 2000, Zschiedrich et al., 2016). These studies have shown that in many cases TCSs are integrated in complex regulatory networks that often involve a number of TCSs as well as other sensors.

The analyses of the HK and RR coding sequences and genetic organization have shown that TCS proteins belong to a limited number of families, which share common ancestry and domain structure (Whitworth & Cock, 2009). This has led to the proposal of a number of classification schemes based on phylogenetic reconstructions of conserved domains (Fabret et al., 1999, Grebe and Stock, 1999) or on the domain composition of TCS proteins (Galperin, 2006). Furthermore, TCSs are usually encoded by adjacent genes (although orphan genes, that is, unpaired HK or RR encoding genes, can also be found) and are arranged in the same order and orientation (Koretke, Lupas, Warren, Rosenberg, & Brown, 2000). The evolution of TCS has been the subject of a number of studies that have evidenced that coevolution of HK and RR pairs has been prevalent although examples of recruitment, i.e., duplication of one component and association with a nonorthologous partner, could also be observed (Alm et al., 2006, Koretke et al., 2000).

Beyond the basic scheme of signal transfer outlined earlier, more complex phosphotransfer relays also exist, which involve multiple phosphotransfer reactions among domains that can be found on separate polypeptides or as part of multidomain proteins (Appleby et al., 1996, Zhang, 2005). Besides, other auxiliary proteins can modulate the activities of TCSs (Buelow and Raivio, 2010, Gao and Stock, 2009). Furthermore, TCS can also integrate other signals through additional cytoplasmic sensory domains or through metabolites that can affect the phosphorylation state of the TCS proteins (Gao and Stock, 2009, Krell et al., 2010, Szurmant et al., 2007). Among the cytoplasmic sensory domains identified in HKs, PAS (PER, ARNT, SIM) and GAF (c-GMP–specific and c-GMP–stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA) are the most common (Galperin et al., 2001, Szurmant et al., 2007). PAS domains can receive signals by several mechanisms including signal binding to the PAS domain cavity, signal perception by cofactor-containing PAS domains, signal binding at the PAS domain–membrane interface, and signal-mediated modulation of inter-PAS domain disulfide bonds (reviewed in Krell et al., 2010). The role of GAF domains in TCS remains undetermined in many cases. It has been shown that GAF domains bind heme in some redox or oxygen-sensing HKs (Kumar, Toledo, Patel, Lancaster, & Steyn, 2007) and cyanobacterial photoreceptor HKs involved in phototaxis covalently bind tetrapyrrole pigments through their GAF domains (Ikeuchi & Ishizuka, 2008). A recent study has shown that the GAF domain of the Synechocystis sp. PCC 6803 cytoplasmic HK Hik2 possibly functions as a chloride sensor (Kotajima, Shiraiwa, & Suzuki, 2014). Some metabolites may also affect the phosphorylation state of TCSs. Several RRs have been shown to be phosphorylated by acetyl phosphate (acetyl-P) thus providing a possible link between TCS activity and metabolic state (Wolfe, 2010). Although some studies have noted that HK phosphatase activity may prevent this HK-independent phosphorylation, recent studies support the view of acetyl-P-dependent RR phosphorylation as a mechanism of regulation of TCS activity (Lima et al., 2012, Schrecke et al., 2013). Polyphosphate can also act as a phosphoryl donor for the MprB sensor protein of Mycobacterium tuberculosis (Sureka et al., 2007). Finally, other signal transducing pathways, such as those operated by Ser/Thr kinases, can modulate the activity of TCSs via posttranslational modification (Burnside & Rajagopal, 2012).

TCSs participate in most aspects of bacterial physiology, including motility, sporulation, competence, nutrient uptake, stress response, central metabolism, and virulence. TCSs are found in varying numbers in bacteria although, generally, bacteria with larger genomes encode more TCSs (Galperin, 2005, Sheng et al., 2012, Ulrich et al., 2005, Wuichet et al., 2010). In addition, the number of TCSs correlates with ecological niches. Free-living bacteria that inhabit changing or diverse environments usually harbor more TCSs than bacteria that live in constant environments, such as pathogenic bacteria, suggesting a correlation between metabolic versatility and number of TCSs (Capra and Laub, 2012, Galperin, 2005). In contrast to bacteria, TCSs are far less common in eukaryotes and completely absent in mammals. This, together with the role of some TCSs in pathogenesis, has driven their interest as potential targets for antimicrobial drugs. This situation also reflects on lactic acid bacteria (LAB) where a number of TCSs of pathogenic streptococci have been thoroughly characterized, whereas far less information is available about TCSs of commensal LAB.

The term lactic acid bacteria comprises a broad group of microorganisms characterized by their ability to degrade sugars mainly into lactic acid (Orla-Jensen, 1919). Originally classified on the basis of phenotypic traits that led to protracted controversies, the use of phylogenetic techniques based on DNA sequencing has shown that the major group of genera of LAB diverged from a common ancestor (Schleifer & Ludwig, 1995). It is becoming increasingly accepted by the scientific community that LAB species constitute the order Lactobacillales (phylum Firmicutes) and other species that have been traditionally considered as LAB must not be included in this group (Vandamme, De Bruyne, & Pot, 2014). The order Lactobacillales currently consists of six families: Aerococcaceae, Carnobacteriaceae, Enterococcaceae, Lactobacillaceae, Leuconostocaceae, and Streptococcaceae. However, even this family classification is questionable. Phylogenomic analyses have shown that, within the genus Lactobacillus, other genera belonging to families Lactobacillaceae and Leuconostocaceae, such as Fructobacillus, Leuconostoc, Oenococcus, Pediococcus and Weissella, are grouped within the lactobacilli as subclades (Claesson and van Sinderen, 2008, Makarova et al., 2006, Sun et al., 2015, Zhang et al., 2011). For the purposes of this review, we will therefore consider the members of families Lactobacillaceae and Leuconostocaceae as a single phylogenetic unit, and we will refer to them as lactobacilli throughout the text.

LAB have long been used for the transformation of raw foodstuffs into a variety of fermented products as their growth is associated with the acidification and production of antimicrobial substances that prevent the proliferation of pathogenic or spoilage organisms. Furthermore, the enzymatic processes associated to their growth contribute to the characteristic flavor and texture of these products. The continued use of LAB in these processes led to the adaptation of some strains (domestication) to specific food systems through repeated inoculation and selection. This process implied major genomic changes in food LAB strains, mainly loss of gene functions as a consequence of the adaptation to a nutrient-rich environment, but also gene gains associated to relevant technological properties (Douglas and Klaenhammer, 2010, Makarova et al., 2006). Other LAB, naturally associated to mucosal surfaces of humans and animals, are also used as probiotics since they are attributed health benefits (Tannock, 2004). Lactobacilli, in contrast to streptococci, have been rarely associated to disease (Cannon et al., 2005, Kamboj et al., 2015), but there is increasing concern about their possible role as reservoirs of potentially transmissible antimicrobial resistance genes (Devirgiliis et al., 2013, Jaimee and Halami, 2016). This highlights the need to understand not only the antibiotic resistance mechanisms of pathogenic bacteria, but also those present in commensal bacteria that are usually recognized as GRAS/QPS organisms such as most lactobacilli.

Due to its involvement in food production and health, leuconostoc, and specially lactobacilli, have been the subject of intensive research. Despite this, the role of TCSs in their physiology has been somehow neglected and our current knowledge on these systems is rather limited compared to other LAB such as streptococci. The aim of this review is to summarize the available evidence on the physiological role of TCSs in Lactobacillaceae and Leuconostocaceae as they comprise most food-associated LAB as well as many commensal species associated to plants and animals.

Section snippets

Number, Distribution, and Classification of TCSs in Lactobacilli

To our knowledge, only one study has dealt with the number and classification of TCSs in lactobacilli in an evolutionary setting using 19 genomic sequences of lactobacilli available at the time (Zúñiga, Gómez-Escoín, & González-Candelas, 2011). In this review, we have updated this previous work and used 98 complete genome sequences available at the Microbial Genome Database for Comparative Analysis (MBGD; http://mbgd.genome.ad.jp) (Uchiyama, Mihara, Nishide, & Chiba, 2015). The number of

Physiological Roles of TCSs in Lactobacilli

TCSs regulate essential physiological processes in many bacteria, and evidence obtained so far indicates that this is also the case in lactobacilli. The first indication on a functional role of TCSs of lactobacilli came from studies of bacteriocin production by lactobacilli. The genetic analyses of bacteriocin gene clusters revealed the presence of TCS-encoding genes in these clusters (Axelsson and Holck, 1995, Diep et al., 1994, Hühne et al., 1996). In Lactobacillus sakei, it was shown that

Concluding Remarks

Physiology and genetics of lactobacilli have been an important research area in the past decades due to their relevance in food production and health. However, the study of signal transduction pathways in these organisms has received relatively little attention. The progress made in the study of TCSs in lactobacilli has evidenced that these systems play important roles in the cell physiology of lactobacilli. The role of TCSs in bacteriocin production is relatively well known nowadays, but the

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