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In ascomycetous fungi, sexual mate recognition requires interaction of the Ste2 receptor protein produced by one partner with the α-factor peptide pheromone produced by the other partner. In some fungi, Ste2 is further needed for chemotropism towards plant roots to allow for subsequent infection and colonization. Here, we investigated whether this is also true for the pine pitch canker fungus, Fusarium circinatum, which is a devastating pathogen of pine globally. Ste2 knockout mutants were generated for two opposite mating-type isolates, after which all strains were subjected to chemotropism assays involving exudates from pine seedling roots and synthetic α-factor pheromone, as well as a range of other compounds for comparison. Our data show that Ste2 is not required for chemotropism towards any of these other compounds, but, in both wild-type strains, Ste2 deletion resulted in the loss of chemotropism towards pine root exudate. Also, irrespective of mating type, both wild-type strains displayed positive chemotropism towards α-factor pheromone, which was substantially reduced in the deletion mutants and not the complementation mutants. Taken together, these findings suggest that Ste2 likely has a key role during the infection of pine roots in production nurseries. Our study also provides a strong foundation for exploring the role of self-produced and mate-produced α-factor pheromone in the growth and overall biology of the pitch canker pathogen. [ABSTRACT FROM AUTHOR]

In ascomycetous fungi, sexual mate recognition requires interaction of the Ste2 receptor protein produced by one partner with the α-factor peptide pheromone produced by the other partner. In some fungi, Ste2 is further needed for chemotropism towards plant roots to allow for subsequent infection and colonization. Here, we investigated whether this is also true for the pine pitch canker fungus, Fusarium circinatum, which is a devastating pathogen of pine globally. Ste2 knockout mutants were generated for two opposite mating-type isolates, after which all strains were subjected to chemotropism assays involving exudates from pine seedling roots and synthetic α-factor pheromone, as well as a range of other compounds for comparison. Our data show that Ste2 is not required for chemotropism towards any of these other compounds, but, in both wild-type strains, Ste2 deletion resulted in the loss of chemotropism towards pine root exudate. Also, irrespective of mating type, both wild-type strains displayed positive chemotropism towards α-factor pheromone, which was substantially reduced in the deletion mutants and not the complementation mutants. Taken together, these findings suggest that Ste2 likely has a key role during the infection of pine roots in production nurseries. Our study also provides a strong foundation for exploring the role of self-produced and mate-produced α-factor pheromone in the growth and overall biology of the pitch canker pathogen.

ABSTRACT In Firmicutes, important processes such as competence development, sporulation, virulence, and biofilm formation are regulated by cytoplasmic quorum sensing (QS) receptors of the RRNPPA family using peptide-based communication. Although these systems regulate important processes in a variety of bacteria, their origin and diversification are poorly understood. Here, we integrate structural, genomic, and phylogenetic evidence to shed light on RRNPPA protein origin and diversification. The family is constituted by seven different subfamilies with different domain architectures and functions. Among these, three were found in Lactobacillales (Rgg, ComR, and PrgX) and four in Bacillales (AimR, NprR, PlcR, and Rap). The patterns of presence and the phylogeny of these proteins show that subfamilies diversified a long time ago, resulting in key structural and functional differences. The concordance between the distribution of subfamilies and the bacterial phylogeny was somewhat unexpected, since many of the subfamilies are very abundant in mobile genetic elements, such as phages, plasmids, and phage-plasmids. The existence of diverse propeptide architectures raises intriguing questions about their export and maturation. It also suggests the existence of diverse roles for the RRNPPA systems. Some systems encode multiple pheromones on the same propeptide or multiple similar propeptides, suggesting that they act as “chatterers.” Many others lack pheromone genes and may be “eavesdroppers.” Interestingly, AimR systems without associated propeptide genes were particularly abundant in chromosomal regions not classed as prophages, suggesting that either the bacterium or other mobile elements are eavesdropping on phage activity. IMPORTANCE Quorum sensing (QS) is a mechanism of bacterial communication, coordinating important decisions depending on bacterial population. QS regulates important processes not only in bacterial behavior but also in genetic mobile elements and host-guest interactions. In Firmicutes, the most important family of QS receptors is the RRNPPA family. Despite the importance of such systems in microbiology, we know little about RRNPPA origin and diversification. In this work, the combination of sequence analysis and structural biology allowed us to identify a very large number of novel systems but also to class of them in functional families and thereby study of their origin and functional diversification. Moreover, peptide pheromone analysis revealed new and intriguing mechanisms of communication, such as “eavesdropper” systems which only listen for the pheromone and “chatterers” that take control of the communication in their microenvironment.

In fungi, sexual reproduction primarily depends on the interaction between peptide pheromones and their receptors. Most ascomycete fungi produce two classes of peptide mating pheromones, a simple peptide and a modified peptide. These peptides are recognized by their corresponding receptors on the surface of cells of the opposite mating type to induce the mating reaction. Pheromone diversification may be associated with reproductive isolation, which restricts gene flow among populations; thus, it remains unclear how pheromones diversify without loss of successful mating. Here, I provide a brief review of recent findings on the 'asymmetric' diversification of peptide pheromones in the fission yeast Schizosaccharomyces pombe, and discuss evolution of the mating pheromones in fission yeast. [ABSTRACT FROM AUTHOR]

Work from our laboratories over the last 35 years that has focused on Ste2p, a G protein-coupled receptor (GPCR), and its tridecapeptide ligand α-factor is reviewed. Our work utilized the yeast Saccharomyces cerevisiae as a model system for understanding peptide-GPCR interactions. It explored the structure and function of synthetic α-factor analogs and biosynthetic receptor domains, as well as designed mutations of Ste2p. The results and conclusions are described using the nuclear magnetic resonance interrogation of synthetic Ste2p transmembrane domains (TMs), the fluorescence interrogation of agonist and antagonist binding, the biochemical crosslinking of peptide analogs to Ste2p, and the phenotypes of receptor mutants. We identified the ligand-binding domain in Ste2p, the functional assemblies of TMs, unexpected and interesting ligand analogs; gained insights into the bound α-factor structure; and unraveled the function and structures of various Ste2p domains, including the N-terminus, TMs, loops connecting the TMs, and the C-terminus. Our studies showed interactions between specific residues of Ste2p in an active state, but not resting state, and the effect of ligand activation on the dimerization of Ste2p. We show that, using a battery of different biochemical and genetic approaches, deep insight can be gained into the structure and conformational dynamics of GPCR-peptide interactions in the absence of a crystal structure.

Abstract: We are developing a rapid, time‐resolved method using laser‐activated cross‐linking to capture protein‐peptide interactions as a means to interrogate the interaction of serum proteins as delivery systems for peptides and other molecules. A model system was established to investigate the interactions between bovine serum albumin (BSA) and 2 peptides, the tridecapeptide budding‐yeast mating pheromone (α‐factor) and the decapeptide human gonadotropin‐releasing hormone (GnRH). Cross‐linking of α‐factor, using a biotinylated, photoactivatable p‐benzoyl‐L‐phenylalanine (Bpa)–modified analog, was energy‐dependent and achieved within seconds of laser irradiation. Protein blotting with an avidin probe was used to detect biotinylated species in the BSA‐peptide complex. The cross‐linked complex was trypsinized and then interrogated with nano‐LC–MS/MS to identify the peptide cross‐links. Cross‐linking was greatly facilitated by Bpa in the peptide, but some cross‐linking occurred at higher laser powers and high concentrations of a non‐Bpa–modified α‐factor. This was supported by experiments using GnRH, a peptide with sequence homology to α‐factor, which was likewise found to be cross‐linked to BSA by laser irradiation. Analysis of peptides in the mass spectra showed that the binding site for both α‐factor and GnRH was in the BSA pocket defined previously as the site for fatty acid binding. This model system validates the use of laser‐activation to facilitate cross‐linking of Bpa‐containing molecules to proteins. The rapid cross‐linking procedure and high performance of MS/MS to identify cross‐links provides a method to interrogate protein‐peptide interactions in a living cell in a time‐resolved manner. [ABSTRACT FROM AUTHOR]

Bacteria use diffusible chemical messengers, termed pheromones, to coordinate gene expression and behavior among cells in a community by a process known as quorum sensing. Pheromones of many gram-positive bacteria, such as Bacillus and Streptococcus, are small, linear peptides secreted from cells and subsequently detected by sensory receptors such as those belonging to the large family of RRNPP proteins. These proteins are cytoplasmic pheromone receptors sharing a structurally similar pheromone-binding domain that functions allosterically to regulate receptor activity. X-ray crystal structures of prototypical RRNPP members have provided atomic-level insights into their mechanism and regulation by pheromones. This review provides an overview of RRNPP prototype signaling; describes the structure-function of this protein family, which is spread widely among gram-positive bacteria; and suggests approaches to target RRNPP systems in order to manipulate beneficial and harmful bacterial behaviors. [ABSTRACT FROM AUTHOR]

Histidine kinase (HK) NisK is well known to sense lantibiotic nisin for regulating the biosynthesis of nisin. NisK possesses two trans-membrane segments and a large extracellular region and nisin contains 34 amino acids with five lanthionine rings. Unlike most peptide sensing HK with multi trans-membrane segments, NisK is a representative of a group of rarely reported HK that sense peptide as ligand. To reveal how NisK senses nisin molecule to regulate nisin biosynthesis, we constructed a reporter Lactococcus lactis strain with nisRK constitutively expressed and a reporter gene lacZ expressed under the control of promoter PnisA. We showed that the extracellular region of NisK was involved in recognizing nisin. Conserved residues in this group of HK were found in the extracellular region of NisK and mutagenesis of these residues in the reporter strain revealed that several hydrophobic residues including two aromatic residues are crucial for NisK sensing nisin and regulating nisin biosynthesis. Substitutions of hydrophobic regions in NisK extracellular domain showed that the first strand that was rich of hydrophobic amino acids was involved in regulating nisin biosynthesis. A negatively charged residue in the first bstrand also contributed to nisin biosynthesis. Protein binding analyses demonstrated that nisin could not interact with key NisK mutants, indicating these site in the extracellular region of NisK was involved in recognizing nisin. [ABSTRACT FROM AUTHOR]

Bacteria are considered "social" organisms able to communicate with one another using small hormone-like molecules (pheromones) in a process called quorum-sensing (QS). These signaling molecules increase in concentration as a function of bacterial cell density. For most human pathogens, QS is critical for virulence and biofilm formation, and the opportunity to interfere with bacterial QS could provide a sophisticated means for manipulating the composition of pathogenic biofilms, and possibly eradicating the infection. Streptococcus mutans is a well-characterized resident of the dental plaque biofilm, and is the major pathogen of dental caries (cavities). In S. mutans, its CSP QS signaling peptide does not act as a classical QS signal by accumulating passively in proportion to cell density. In fact, particular stresses such as those encountered in the oral cavity, induce the production of the CSP pheromone, suggesting that the pheromone most probably functions as a stress-inducible alarmone by triggering the signaling to the bacterial population to initiate an adaptive response that results in different phenotypic outcomes. This mini-review discusses two different CSP-induced phenotypes, bacterial "suicide" and dormancy, and the underlying mechanisms by which S. mutans utilizes the same QS signaling peptide to regulate two opposite phenotypes. [ABSTRACT FROM AUTHOR]

Mammals use pheromones to communicate with other animals of the same species. In mice, the VNO (vomeronasal organ) has a pivotal role in pheromone detection. We discovered a 7 kDa peptide, ESP1 (exocrine-gland-secreting peptide 1), in tear fluids from male mice that enhances the sexual behaviour of female mice via the VNO. NMR studies demonstrate that ESP1 adopts a compact structure with a helical fold stabilized by an intramolecular disulfide bridge. Functional analysis in combination with docking simulation indicates that ESP1 is recognized by a specific G-protein-coupled vomeronasal receptor, V2Rp5, via charge- charge interactions in the large extracellular region of the receptor. ESP1 is a member of the ESP family, which comprises 38 homologous genes in mice, and some of these genes are expressed in a sex- or agedependent manner. Most recently, ESP22 was found to be released specifically in juvenile tear fluids and to inhibit the sexual behaviour of adult male mice. These studies demonstrate that peptide pheromones are used for chemical communication in mice, and they indicate a structural basis for the narrowly tuned perception of mammalian peptide pheromones by vomeronasal receptors. [ABSTRACT FROM AUTHOR]

Highlights: [•] Sodefrin, a male newt pheromone elevates Ca2+ in the female vomeronasal (VN) cells. [•] Sodefrin increases the VN cell population that exhibits the elevation of [Ca2+]i. [•] Responsiveness of VN cells to sodefrin is sex- and hormone-dependent. [•] Dual pathways, namely PLC-IP3 and PLC-DAG-PKC are involved in the VN cell response. [•] This is the first to report the signal pathway for a vertebrate peptide pheromone. [Copyright &y& Elsevier]

Abstract: In ruminants, Streptococcus bovis is considered to be associated with acute rumen acidosis. To elucidate the regulatory mechanisms of S. bovis growth, we investigated the function of the two components of the peptide pheromone-signaling system, ComD and ComE, which are encoded by comD and comE, respectively, via the competence-stimulating peptide ComC, which is encoded by comC. Deletion of entire comC and two-thirds of comD resulted in decreased growth rate, which may be related to the change in the expression of several proteins, as shown by two-dimensional gel electrophoresis. The transcript level of comED was decreased by the disruption of comCD, suggesting that the transcription of comED might be stimulated by ComC. The transformation frequency was decreased by the disruption of comCD. Addition of recombinant ComC to S. bovis cultures increased the growth rate and transformation frequency. In the cultures of mixed ruminal microbes, addition of mature ComC peptide increased the number of S. bovis per total bacterial counts as estimated by the cDNA amounts of 16SrRNA. Thus, the peptide pheromone-signaling system via ComC, D, and E might be involved in the control of S. bovis growth in addition to competence development. This is the first report suggesting that an autoinducing peptide functions in the ruminal ecosystem. [Copyright &y& Elsevier]

Streptococcal competence-stimulating peptides (CSPs) were once thought to passively communicate population density in a process known classically as quorum sensing. However, recent evidence has shown that these peptides may also be inducible ‘alarmones,’ capable of conveying sophisticated messages in a population including the induction of altruistic cellular suicide under stressful conditions. We have previously characterized the alarmone response in Streptococcus mutans, a cariogenic resident of the oral flora, in which a novel bacteriocin-like peptide causes cell death in a subset of the population. Our objective in this work was to characterize the mechanism of immunity to cell death in S. mutans. Toward this goal, we have identified the conditions under which immunity is induced, and identified the regulatory system responsible for differential (and protective) expression of immunity. We also showed that CSP-induced death contributes to S. mutans biofilm formation through the release of chromosomal DNA into the extracellular matrix, providing a long sought-after mechanistic explanation for the role of CSP in S. mutans biofilm formation. [ABSTRACT FROM AUTHOR]

A locus encoding proteins with high homology to the pneumococcal BlpABCHR quorum-sensing system was identified in Streptococcus thermophilus LMG 18311. The BlpABCHR system regulates bacteriocin production in Streptococcus pneumoniae by monitoring the extracellular concentration of a peptide-pheromone encoded by blpC. The homologous system in S. thermophilus, termed StbABCHR, contains a corresponding gene ( stbC) encoding a possible peptide-pheromone (STP) that presumably controls bacteriocin production in S. thermophilus. We synthesized this peptide and found that it activates transcription of a gusA reporter gene placed behind the promoter of the bacteriocin-like gene stbD. Furthermore, deletion mapping and mutational analysis of the stbD promoter region were used to identify a degenerated direct repeat motif required for STP induced GusA expression. Our findings provide strong evidence that STP regulates bacteriocin production in S. thermophilus LMG 18311, and show that the StbABCHR quorum-sensing system can be exploited for inducible expression of recombinant proteins in this bacterial species. [ABSTRACT FROM AUTHOR]

Bacterial peptide pheromone has a high affinity to its membrane receptor. Fusion of these peptides to pore-forming antimicrobial peptide might enhance its bactericidal activity against pheromone-sensing bacteria. We constructed two chimeric peptides by fusing the pore-forming fungicidal peptide dhvar4 to the C-terminus of enterococcal peptide pheromones cCF10 and cOB1 individually. Comparison on the bactericidal activities against pheromone-sensing bacteria Enterococcus faecalis demonstrates that the chimeric peptides cCF10–dhvar4 and cOB1–dhvar4 are more potent than the parent peptide dhvar4. The LD50s of both chimeric peptides (1.0 μm) are 10 times lower than that of dhvar4 (10.8 μm). Free peptide pheromone could inhibit E. faecalis killing mediated by both chimeric peptides. As same as that of the parent peptide, both chimeric peptides kill bacteria by disrupting its cell membrane. These results indicate that fused enterococcal peptide pheromone increases the bactericidal activity of fungicidal peptide against E. faecalis by improving its ability to reach the cell membrane. [ABSTRACT FROM AUTHOR]

Bacteriocin biosynthesis inLactobacillus plantarumis an inducible process, triggered by the secreted inducer peptide pheromone IP-C11. The environmental concentration of IP-C11 is monitored by the membrane-bound histidine protein kinase PlnB, which is part of a two-component signal transduction pathway. Upon interaction with IP-C11, PlnB phosphorylates the cognate response regulator PlnC. This regulator subsequently activates transcription of the bacteriocin genes. PlnB belongs to the HPK10 subfamily of peptide-pheromone-activated histidine kinases. All members of this subfamily have an unusual polytopic membrane domain that previously has been shown to contain the peptide pheromone receptor. Employing an in vivo reporter assay, the present work investigated the receptor functionality of various mutagenized PlnB membrane domains. The results indicated that important determinants for receptor function locate to the most N-terminal extracytoplasmatic loop of the membrane domain. In addition, this region appears to be involved in the peptide pheromone interaction of ComD, another member of the HPK10 subfamily. [ABSTRACT FROM AUTHOR]

Pheromones have been implicated in the control of a number of behaviors in molluscs, but few peptide pheromones have been characterized in these animals. Peptide pheromones include: (1) a family of water-borne peptide pheromonal attractants (attractins) in the gastropod Aplysia that are released during egg laying and attract other Aplysia to form egg-laying and mating aggregations; (2) a tetrapeptide (ILME) in the cephalopod Sepia that elutes from egg masses and is thought to be involved in the transport of oocytes in the genital tract during egg laying; and (3) a Sepia sperm-attracting peptide (SepSAP; PIDPGVamide) that is released from oocytes during egg laying to facilitate external fertilization. [Copyright &y& Elsevier]

Lantibiotics are produced by a variety of Gram-positive bacteria. The production of these peptides appears to be regulated at the transcriptional level in a cell-density-dependent manner in various bacteria. This phenomenon has been studied in detail for the production of nisin by Lactococcus lactis, and the production of the structurally similar subtilin by Bacillus subtilis. In this paper, the molecular mechanism underlying regulation of nisin and subtilin production is reviewed. This quorum sensing, autoregulatory module includes the lantibiotics themselves as peptide pheromones, the signal transduction by the corresponding two-component regulatory systems, and the lantibiotic-responsive promoter elements in the biosynthesis gene clusters. Finally, the exploitation of these regulatory characteristics for the development of highly effective controlled gene expression systems in Gram-positive bacteria is discussed. [Copyright &y& Elsevier]

The production of the type I antimicrobial peptide (AMP) subtilin by Bacillus subtilis is regulated in a cell-density-dependent manner [Kleerebezem M, de Vos WM, Kuipers OP. The lantibiotics nisin and subtilin act as extracellular regulators of their own biosynthesis. In: Dunny GM, Winans SC, editors. Cell–cell signaling in bacteria. Washington, D.C., USA: ASM Press; 1999. p. 159–74; Stein T, Borchert S, Kiesau P, Heinzmann S, Kloss S, Klein C, Helfrich M, Entian KD. Dual control of subtilin biosynthesis and immunity in Bacillus subtilis. Mol Microbiol 2002;44:403–16; Stein T, Heinzmann S, Kiesau P, Himmel B, Entian KD. The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis. Mol Microbiol 2003;47:1627–36]. Three subtilin-responsive promoter elements within the spaBTCSIFEGRK are controlled by the specific cis-acting sequence element called the spa-box, which represents the binding site of the subtilin regulator SpaR [Stein T, Heinzmann S, Kiesau P, Himmel B, Entian KD. The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis. Mol Microbiol 2003;47:1627–36]. Here, we describe the functional characterization of the spaB, spaS and spaI promoters by transcriptional fusion with a promoterless β-glucuronidase encoding gusA gene. Within these gusA fusion constructs, transcription initiation start sites of the spaS and spaI promoters were mapped to be located downstream of the spa-box, which is in contrast to previous reports [Banerjee S, Hansen JN. Structure and expression of a gene encoding the precursor of subtilin, a small protein antibiotic. J Biol Chem 1988;263:9508–14; Stein T, Heinzmann S, Kiesau P, Himmel B, Entian KD. The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis. Mol Microbiol 2003;47:1627–36]. Nevertheless, all spa-promoters displayed typical cell-density-dependent activity in a subtilin-producing strain B. subtilis ATCC6633. Moreover, analysis of β-glucuronidase activities in a spaB mutant of B. subtilis ATCC6633 and a derivative of strain 168 that harbors the spaRK genes integrated in the chromosomal amyE locus, confirmed that these promoters are activated by subtilin-triggered, SpaRK-mediated, quorum-sensing control. Quantitative analysis showed that the spaS promoter strength at a given subtilin concentration appeared to be approximately five-fold higher than the spaB promoter, which in turn is approximately two-fold higher than the spaI promoter. Finally, it is shown that the elementary components involved in subtilin-mediated regulation are the two-component system, SpaRK, and a spa-box containing promoter. [Copyright &y& Elsevier]

Quorum sensing (QS) in Gram-negative bacteria is generally assumed to be mediated by N-acyl-homoserine lactone molecules while Gram-positive bacteria make use of signaling peptides. We analyzed the occurrence in Gram-negative bacteria of peptides and transporters that are involved in quorum sensing in Gram-positive bacteria. Many class II bacteriocins and inducing factors produced by lactic acid bacteria (LAB) and competence stimulating peptides (CSPs) synthesized by streptococci are processed by their cognate ABC-transporters during their secretion. During transport, a conserved leader sequence, termed the double-glycine motif (GG-motif), is cleaved off by the N-terminal domain of the transporter, which belongs to the Peptidase C39 protein family. Several peptides containing a GG-motif were recently described in Gram-negative bacteria (Trends Microbiol 2001;9:164–8). To screen for additional putative GG-motif containing peptides, an in silico strategy based on MEME, HMMER2.2 and Wise2 was designed. Using a curated training set, a motif model of the leader peptide was built and used to screen over 120 fully sequenced bacterial genomes. The screening methodology was applied at the nucleotide level as probably many small peptide genes have not been annotated and may be absent from the non-redundant databases. It was found that 33% of the screened genomes of Gram-negative bacteria contained one or more transporters carrying a Peptidase C39 domain, compared to 44% of the genomes of Gram-positive bacteria. The transporters can be subdivided into four classes on the basis of their domain organization. Genes coding for putative peptides containing 23–142 amino acids and a GG-motif were found in close association with genes coding for Peptidase C39 domain containing proteins. These peptides show structural similarity to bacteriocins and peptide pheromones of Gram-positive bacteria. The possibility of signal transduction based on peptide signaling in Gram-negative bacteria is discussed. [Copyright &y& Elsevier]

Work from our laboratories over the last 35 years that has focused on Ste2p, a G protein-coupled receptor (GPCR), and its tridecapeptide ligand α-factor is reviewed. Our work utilized the yeast Saccharomyces cerevisiae as a model system for understanding peptide-GPCR interactions. It explored the structure and function of synthetic α-factor analogs and biosynthetic receptor domains, as well as designed mutations of Ste2p. The results and conclusions are described using the nuclear magnetic resonance interrogation of synthetic Ste2p transmembrane domains (TMs), the fluorescence interrogation of agonist and antagonist binding, the biochemical crosslinking of peptide analogs to Ste2p, and the phenotypes of receptor mutants. We identified the ligand-binding domain in Ste2p, the functional assemblies of TMs, unexpected and interesting ligand analogs; gained insights into the bound α-factor structure; and unraveled the function and structures of various Ste2p domains, including the N-terminus, TMs, loops connecting the TMs, and the C-terminus. Our studies showed interactions between specific residues of Ste2p in an active state, but not resting state, and the effect of ligand activation on the dimerization of Ste2p. We show that, using a battery of different biochemical and genetic approaches, deep insight can be gained into the structure and conformational dynamics of GPCR-peptide interactions in the absence of a crystal structure. [ABSTRACT FROM AUTHOR]

Histidine kinase (HK) NisK is well known to sense lantibiotic nisin for regulating the biosynthesis of nisin. NisK possesses two trans-membrane segments and a large extracellular region and nisin contains 34 amino acids with five lanthionine rings. Unlike most peptide sensing HK with multi trans-membrane segments, NisK is a representative of a group of rarely reported HK that sense peptide as ligand. To reveal how NisK senses nisin molecule to regulate nisin biosynthesis, we constructed a reporter Lactococcus lactis strain with nisRK constitutively expressed and a reporter gene lacZ expressed under the control of promoter P nisA . We showed that the extracellular region of NisK was involved in recognizing nisin. Conserved residues in this group of HK were found in the extracellular region of NisK and mutagenesis of these residues in the reporter strain revealed that several hydrophobic residues including two aromatic residues are crucial for NisK sensing nisin and regulating nisin biosynthesis. Substitutions of hydrophobic regions in NisK extracellular domain showed that the first strand that was rich of hydrophobic amino acids was involved in regulating nisin biosynthesis. A negatively charged residue in the first βstrand also contributed to nisin biosynthesis. Protein binding analyses demonstrated that nisin could not interact with key NisK mutants, indicating these site in the extracellular region of NisK was involved in recognizing nisin.