Cross-regulation of Aps-promoters in Lacticaseibacillus paracasei by the PsdR response regulator in response to lantibiotics.

The PsdRSAB and ApsRSAB detoxification modules, together with the antimicrobial peptides (AMPs)-resistance determinants Dlt system and MprF protein, play major roles in the response to AMPs in Lacticaseibacillus paracasei BL23. Sensitivity assays with a collection of mutants showed that the PsdAB ABC transporter and the Dlt system are the main subtilin resistance determinants. Quantification of the transcriptional response to subtilin indicate that this response is exclusively regulated by the two paralogous systems PsdRSAB and ApsRSAB. Remarkably, a cross-regulation of the derAB, mprF and dlt-operon genes-usually under control of ApsR-by PsdR in response to subtilin was unveiled. The high similarity of the predicted structures of both response regulators (RR), and of the RR-binding sites support this possibility, which we experimentally verified by protein-DNA binding studies. ApsR-P shows a preferential binding in the order PderA > Pdlt > PmprF > PpsdA. However, PsdR-P bound with similar apparent affinity constants to the four promoters. This supports the cross-regulation of derAB, mprF and the dlt-operon by PsdR. The possibility of cross-regulation at the level of RR-promoter interaction allows some regulatory overlap with two RRs controlling the expression of systems involved in maintenance of critical cell membrane functions in response to lantibiotics.

Antibiotic Potential of the Ambigol Cyanobacterial Natural Product Class and Simplified Synthetic Analogs.

The ambigols are cyanobacterial natural products characterized by three polychlorinated aromatic building blocks connected by biaryl and biaryl ether bridges. All ambigols known to date possess promising biological activities. Most significantly, ambigol A was reported to have antibacterial activity against Gram-positive bacteria, such as Bacillus megaterium and B. subtilis. We established a diverse compound library for in-depth biological evaluation building on our previous bio- and total synthetic research on this natural product family. To explore the antimicrobial potential in detail and to determine initial structure-activity relationships of this product class, a large set of dimeric and trimeric compounds were screened against selected bacterial and Candida target strains. Our results reveal exceptional antibiotic activity of the ambigols, especially against challenging clinical isolates.

3-Phenyllactic Acid and Polyphenols Are Substances Enhancing the Antibacterial Effect of Methylglyoxal in Manuka Honey.

Manuka honey is known for its unique antibacterial activity, which is due to methylglyoxal (MGO). After establishing a suitable assay for measuring the bacteriostatic effect in a liquid culture with a time dependent and continuous measurement of the optical density, we were able to show that honey differs in its growth retardingeffect on Bacillus subtilis despite the same content of MGO, indicating the presence of potentially synergistic compounds. In model studies using artificial honey with varying amounts of MGO and 3-phenyllactic acid (3-PLA), it was shown that 3-PLA in concentrations above 500 mg/kg enhances the bacteriostatic effect of the model honeys containing 250 mg/kg MGO or more. It has been shown that the effect correlates with the contents of 3-PLA and polyphenols in commercial manuka honey samples. Additionally, yet unknown substances further enhance the antibacterial effect of MGO in manuka honey. The results contribute to the understanding of the antibacterial effect of MGO in honey.

Phyletic Distribution and Diversification of the Phage Shock Protein Stress Response System in Bacteria and Archaea.

Maintaining cell envelope integrity is of vital importance for all microorganisms. Not surprisingly, evolution has shaped conserved protein protection networks that connect stress perception, transmembrane signal transduction, and mediation of cellular responses upon cell envelope stress. The phage shock protein (Psp) stress response is one such conserved protection network. Most knowledge about the Psp response derives from studies in the Gram-negative model bacterium Escherichia coli, where the Psp system consists of several well-defined protein components. Homologous systems were identified in representatives of the Proteobacteria, Actinobacteria, and Firmicutes. However, the Psp system distribution in the microbial world remains largely unknown. By carrying out a large-scale, unbiased comparative genomics analysis, we found components of the Psp system in many bacterial and archaeal phyla and describe that the predicted Psp systems deviate dramatically from the known prototypes. The core proteins PspA and PspC have been integrated into various (often phylum-specifically) conserved protein networks during evolution. Based on protein domain-based and gene neighborhood analyses of pspA and pspC homologs, we built a natural classification system for Psp networks in bacteria and archaea. We validate our approach by performing a comprehensive in vivo protein interaction study of Psp domains identified in the Gram-positive model organism Bacillus subtilis and found a strong interconnected protein network. Our study highlights the diversity of Psp domain organizations and potentially diverse functions across the plethora of the microbial landscape, thus laying the ground for studies beyond known Psp functions in underrepresented organisms. IMPORTANCE The PspA protein domain is found in all domains of life, highlighting its central role in Psp networks. To date, all insights into the core functions of Psp responses derive mainly from protein network blueprints representing only three bacterial phyla. Despite large overlaps in function and regulation, the evolutionary diversity of Psp networks remains largely elusive. Here, we present an unbiased protein domain- and genomic context-centered approach that describes and classifies Psp systems. Our results suggest so-far-unknown Psp-associated roles with other protein networks giving rise to new functions. We demonstrate the applicability of our approach by dissecting the Psp protein network present in Bacillus subtilis and demonstrate Psp domains working in concert with other cell envelope stress response systems. We find that the Psp-like protein universe reflects a surprising diversity within the bacterial and archaeal microbial world.

Regulation of heterologous subtilin production in Bacillus subtilis W168.

BACKGROUND: Subtilin is a peptide antibiotic (lantibiotic) natively produced by Bacillus subtilis ATCC6633. It is encoded in a gene cluster spaBTCSIFEGRK (spa-locus) consisting of four transcriptional units: spaS (subtilin pre-peptide), spaBTC (modification and export), spaIFEG (immunity) and spaRK (regulation). Despite the pioneer understanding on subtilin biosynthesis, a robust platform to facilitate subtilin research and improve subtilin production is still a poorly explored spot. RESULTS: In this work, the intact spa-locus was successfully integrated into the chromosome of Bacillus subtilis W168, which is the by far best-characterized Gram-positive model organism with powerful genetics and many advantages in industrial use. Through systematic analysis of spa-promoter activities in B. subtilis W168 wild type and mutant strains, our work demonstrates that subtilin is basally expressed in B. subtilis W168, and the transition state regulator AbrB strongly represses subtilin biosynthesis in a growth phase-dependent manner. The deletion of AbrB remarkably enhanced subtilin gene expression, resulting in comparable yield of bioactive subtilin production as for B. subtilis ATCC6633. However, while in B. subtilis ATCC6633 AbrB regulates subtilin gene expression via SigH, which in turn activates spaRK, AbrB of B. subtilis W168 controls subtilin gene expression in SigH-independent manner, except for the regulation of spaBTC. Furthermore, the work shows that subtilin biosynthesis in B. subtilis W168 is regulated by the two-component regulatory system SpaRK and strictly relies on subtilin itself as inducer to fulfill the autoregulatory circuit. In addition, by incorporating the subtilin-producing system (spa-locus) and subtilin-reporting system (PpsdA-lux) together, we developed "online" reporter strains to efficiently monitor the dynamics of subtilin biosynthesis. CONCLUSIONS: Within this study, the model organism B. subtilis W168 was successfully established as a novel platform for subtilin biosynthesis and the underlying regulatory mechanism was comprehensively characterized. This work will not only facilitate genetic (engineering) studies on subtilin, but also pave the way for its industrial production. More broadly, this work will shed new light on the heterologous production of other lantibiotics.

An Stomatin, Prohibitin, Flotillin, and HflK/C-Domain Protein Required to Link the Phage-Shock Protein to the Membrane in Bacillus subtilis.

Membrane surveillance and repair is of utmost importance to maintain cellular integrity and allow cellular life. Several systems detect cell envelope stress caused by antimicrobial compounds and abiotic stresses such as solvents, pH-changes and temperature in bacteria. Proteins containing an Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH)-domain, including bacterial flotillins have been shown to be involved in membrane protection and membrane fluidity regulation. Here, we characterize a bacterial SPFH-domain protein, YdjI that is part of a stress induced complex in Bacillus subtilis. We show that YdjI is required to localize the ESCRT-III homolog PspA to the membrane with the help of two membrane integral proteins, YdjG/H. In contrast to classical flotillins, YdjI resides in fluid membrane regions and does not enrich in detergent resistant membrane fractions. However, similarly to FloA and FloT from B. subtilis, deletion of YdjI decreases membrane fluidity. Our data reveal a hardwired connection between phage shock response and SPFH proteins.

Functional association of the stress-responsive LiaH protein and the minimal TatAyCy protein translocase in Bacillus subtilis.

The bacterial twin-arginine (Tat) pathway serves in the exclusive secretion of folded proteins with bound cofactors. While Tat pathways in Gram-negative bacteria and chloroplast thylakoids consist of conserved TatA, TatB and TatC subunits, the Tat pathways of Bacillus species and many other Gram-positive bacteria stand out for their minimalist nature with the core translocase being composed of essential TatA and TatC subunits only. Here we addressed the question whether the minimal TatAyCy translocase of Bacillus subtilis recruits additional cellular components that modulate its activity. To this end, TatAyCy was purified by affinity- and size exclusion chromatography, and interacting co-purified proteins were identified by mass spectrometry. This uncovered the cell envelope stress responsive LiaH protein as an accessory subunit of the TatAyCy complex. Importantly, our functional studies show that Tat expression is tightly trailed by LiaH induction, and that LiaH itself determines the capacity and quality of TatAyCy-dependent protein translocation. In contrast, LiaH has no role in high-level protein secretion via the general secretion (Sec) pathway. Altogether, our observations show that protein translocation by the minimal Tat translocase TatAyCy is tightly intertwined with an adequate bacterial response to cell envelope stress. This is consistent with a critical need to maintain cellular homeostasis, especially when the membrane is widely opened to permit passage of large fully-folded proteins via Tat.

Cold tolerance in the genus Arabidopsis.

PREMISE: Cold tolerance is an important factor limiting the geographic distribution and growing season for many plant species, yet few studies have examined variation in cold tolerance extensively within and among closely related species and compared that to their geographic distribution. METHODS: This study examines cold tolerance within and among species in the genus Arabidopsis. We assessed cold tolerance by measuring electrolyte leakage from detached leaves in multiple populations of five Arabidopsis taxa. The temperature at which 50% of cells were lysed was considered the lethal temperature (LT50 ). RESULTS: We found variability within and among taxa in cold tolerance. There was no significant within-species relationship between latitude and cold tolerance. However, the northern taxa, A. kamchatica, A. lyrata subsp. petraea, and A. lyrata subsp. lyrata, were more cold tolerant than A. thaliana and A. halleri subsp. gemmifera both before and after cold acclimation. Cold tolerance increased after cold acclimation (exposure to low, but nonfreezing temperatures) for all taxa, although the difference was not significant for A. halleri subsp. gemmifera. For all taxa except A. lyrata subsp. lyrata, the LT50 values for cold-acclimated plants were higher than the January mean daily minimum temperature (Tmin ), indicating that if plants were not insulated by snow cover, they would not likely survive winter at the northern edge of their range. CONCLUSIONS: Arabidopsis lyrata and A. kamchatica were far more cold tolerant than A. thaliana. These extremely cold-tolerant taxa are excellent candidates for studying both the molecular and ecological aspects of cold tolerance.

Identification of New Compounds from Sage Flowers (Salvia officinalis L.) as Markers for Quality Control and the Influence of the Manufacturing Technology on the Chemical Composition and Antibacterial Activity of Sage Flower Extracts.

Parts of Salvia species such as its flowers and leaves are currently used as a culinary herb and for some medicinal applications. To distinguish the different sage extracts it is necessary to analyze their individual chemical compositions. Their characteristic compounds might be established as markers to differentiate between sage flowers and leaf extracts or to determine the manufacturing technology and storage conditions. Tri-p-coumaroylspermidine can be detected only in flowers and has been described here for Salvia and Lavandula species for the first time. Markers for oxidation processes are the novel compounds salviquinone A and B, which were generated from carnosol by exposure to oxygen. Caffeic acid ethyl ester was established as an indirect marker for the usage of ethanol as extraction solvent. The compounds were identified by LC-QTOF-HRESIMS, LC-MS, NMR, IR, and single-crystal X-ray diffraction after isolation by semipreparative HPLC. Furthermore, sage flower resin showed interesting antibacterial in vitro activities against Gram-positive and Gram-negative bacteria.

One Ring, Two Membranes: IM30 Ring Complex and the Thylakoid Membrane Fusion.

Direct membrane fusion in chloroplasts and cyanobacteria is triggered by IM30 protein oligomers, but so far the exact mechanism of membrane binding has remained obscure. In this issue of Structure, Saur et al. (2017) describe the detailed structure of Janus-faced IM30 rings crucial for thylakoid membrane fusion and membrane layer architecture.

The black soldier fly, Hermetia illucens - a promising source for sustainable production of proteins, lipids and bioactive substances.

The growing demand worldwide for proteins and lipids cannot be met by the intensive use of agricultural land currently available. Insect mass cultures as a source for proteins and lipids have been in focus for various reasons. An insect with many positive properties is the black soldier fly, Hermetia illucens, whose larvae could be used for the sustainable production of proteins and lipids. Furthermore, the larvae produce bioactive substances which could potentially be used for human and animal welfare.

The applied side of antimicrobial peptide-inducible promoters from Firmicutes bacteria: expression systems and whole-cell biosensors.

The cell envelope is an essential bacterial structure that consists of the cytoplasmic membrane, the cell wall, and-in Gram-negative bacteria-the outer membrane. Because of its crucial functions, it represents a prime antibiotic target. Monitoring and maintaining its integrity are therefore keys to survival, especially in competitive environments where antibiotics represent one means of suppressing the growth of competitors. Resistance against external antibiotic threat, as well as auto-immunity against self-produced antibiotics, is often mediated by two-component systems (2CSs). They respond to antibiotic threat by inducing gene expression that results in the production of specific resistance determinants. The underlying transcriptional control is exhibited at the level of specific target promoters, which usually share a number of relevant features: They are tightly controlled and only induced in the presence of specific (sets of) antibiotics. This induction is dose dependent and often very sensitive, that is, it occurs well below inhibitory antibiotic concentrations. Because of these characteristics, a number of well-characterized cell envelope stress-inducible promoters have been developed for two different applied purposes: first, as whole-cell biosensors for antibiotic detection and mechanism-of-action studies, and second, as antibiotic-inducible expression systems for biotechnological purposes. The current state of research in both fields will be discussed in this review, focusing on 2CS-regulated promoters from Firmicutes bacteria that are induced to mediate resistance against antimicrobial peptides (AMPs) targeting the cell envelope.

The quorum-sensing regulator ComA from Bacillus subtilis activates transcription using topologically distinct DNA motifs.

ComA-like transcription factors regulate the quorum response in numerous Gram-positive bacteria. ComA proteins belong to the tetrahelical helix-turn-helix superfamily of transcriptional activators, which bind as homodimers to inverted sequence repeats in the DNA. Here, we report that ComA from Bacillus subtilis recognizes a topologically distinct motif, in which the binding elements form a direct repeat. We provide in vitro and in vivo evidence that the canonical and non-canonical site play an important role in facilitating type I and type II promoter activation, respectively, by interacting with different subunits of RNA polymerase. We furthermore show that there is a variety of contexts in which the non-canonical site can occur and identify new direct target genes that are located within the integrative and conjugative element ICEBs1. We therefore suggest that ComA acts as a multifunctional transcriptional activator and provides a striking example for complexity in protein-DNA interactions that evolved in the context of quorum sensing.

Contrasting Effects of the Cytotoxic Anticancer Drug Gemcitabine and the EGFR Tyrosine Kinase Inhibitor Gefitinib on NK Cell-Mediated Cytotoxicity via Regulation of NKG2D Ligand in Non-Small-Cell Lung Cancer Cells.

INTRODUCTION: Several cytotoxic anticancer drugs inhibit DNA replication and/or mitosis, while EGFR tyrosine kinase inhibitors inactivate EGFR signalling in cancer cell. Both types of anticancer drugs improve the overall survival of the patients with non-small-cell lung cancer (NSCLC), although tumors often become refractory to this treatment. Despite several mechanisms by which the tumors become resistant having been described the effect of these compounds on anti-tumor immunity remains largely unknown. METHODS: This study examines the effect of the cytotoxic drug Gemcitabine and the EGFR tyrosine kinase inhibitor Gefitinib on the expression of NK group 2 member D (NKG2D) ligands as well as the sensitivity of NSCLC cells to the NK-mediated lysis. RESULTS: We demonstrate that Gemcitabine treatment leads to an enhanced expression, while Gefitinib downregulated the expression of molecules that act as key ligands for the activating receptor NKG2D and promote NK cell-mediated recognition and cytolysis. Gemcitabine activated ATM and ATM- and Rad-3-related protein kinase (ATR) pathways. The Gemcitabine-induced phosphorylation of ATM as well as the upregulation of the NKG2D ligand expression could be blocked by an ATM-ATR inhibitor. In contrast, Gefitinib attenuated NKG2D ligand expression. Silencing EGFR using siRNA or addition of the PI3K inhibitor resulted in downregulation of NKG2D ligands. The observations suggest that the EGFR/PI3K pathway also regulates the expression of NKG2D ligands. Additionally, we showed that both ATM-ATR and EGFR regulate MICA/B via miR20a. CONCLUSION: In keeping with the effect on NKG2D expression, Gemcitabine enhanced NK cell-mediated cytotoxicity while Gefitinib attenuated NK cell killing in NSCLC cells.

Cold tolerance in Arabidopsis kamchatica.

UNLABELLED: • PREMISE OF THE STUDY: Cold tolerance is a critically important factor determining how plants will be influenced by climate change, including changes in snowcover and extreme weather events. Although a great deal is known about cold tolerance in Arabidopsis thaliana, it is not highly cold tolerant. This study examined cold tolerance and its genetic diversity in an herbaceous subarctic relative, Arabidopsis kamchatica, which generally occurs in much colder climates.• METHODS: Thermal analysis and electrolyte leakage were used to estimate supercooling points and lethal temperatures (LT50) in cold-acclimated and nonacclimated families from three populations of A. kamchatica.• KEY RESULTS: Arabidopsis kamchatica was highly cold tolerant, with a mean LT50 of -10.8°C when actively growing, and -21.8°C when cold acclimated. It also was able to supercool to very low temperatures. Surprisingly, actively growing plants supercooled more than acclimated plants (-14.7 vs. -12.7°C). There was significant genetic variation for cold tolerance both within and among populations. However, both cold tolerance and genetic diversity were highest in the midlatitude population rather than in the far north, indicating that adaptations to climate change are most likely to arise in the center of the species range rather than at the edges.• CONCLUSIONS: Arabidopsis kamchatica is highly cold tolerant throughout its range. It is far more freeze tolerant than A. thaliana, and supercooled to lower temperatures, suggesting that A. kamchatica provides a valuable complement to A. thaliana for cold tolerance research.

Genome size variation and evolution in allotetraploid Arabidopsis kamchatica and its parents, Arabidopsis lyrata and Arabidopsis halleri.

Polyploidization and subsequent changes in genome size are fundamental processes in evolution and diversification. Little is currently known about the extent of genome size variation within taxa and the evolutionary forces acting on this variation. Arabidopsis kamchatica has been reported to contain both diploid and tetraploid individuals. The aim of this study was to determine the genome size of A. kamchatica, whether there is variation in ploidy and/or genome size in A. kamchatica and to study how genome size has evolved. We used propidium iodide flow cytometry to measure 2C DNA content of 73 plants from 25 geographically diverse populations of the putative allotetraploid A. kamchatica and its parents, Arabidopsis lyrata and Arabidopsis halleri. All A. kamchatica plants appear to be tetraploids. The mean 2C DNA content of A. kamchatica was 1.034 pg (1011 Mbp), which is slightly smaller than the sum of its diploid parents (A. lyrata: 0.502 pg; A. halleri: 0.571 pg). Arabidopsis kamchatica appears to have lost ∼37.594 Mbp (3.6 %) of DNA from its 2C genome. Tetraploid A. lyrata from Germany and Austria appears to have lost ∼70.366 Mbp (7.2 %) of DNA from the 2C genome, possibly due to hybridization with A. arenosa, which has a smaller genome than A. lyrata. We did find genome size differences among A. kamchatica populations, which varied up to 7 %. Arabidopsis kamchatica ssp. kawasakiana from Japan appears to have a slightly larger genome than A. kamchatica ssp. kamchatica from North America, perhaps due to multiple allopolyploid origins or hybridization with A. halleri. However, the among-population coefficient of variation in 2C DNA content is lower in A. kamchatica than in other Arabidopsis taxa. Due to its close relationship to A. thaliana, A. kamchatica has the potential to be very useful in the study of polyploidy and genome evolution.

Subcellular localization, interactions and dynamics of the phage-shock protein-like Lia response in Bacillus subtilis.

The liaIH operon of Bacillus subtilis is the main target of the envelope stress-inducible two-component system LiaRS. Here, we studied the localization, interaction and cellular dynamics of Lia proteins to gain insights into the physiological role of the Lia response. We demonstrate that LiaI serves as the membrane anchor for the phage-shock protein A homologue LiaH. Under non-inducing conditions, LiaI locates in highly motile membrane-associated foci, while LiaH is dispersed throughout the cytoplasm. Under stress conditions, both proteins are strongly induced and colocalize in numerous distinct static spots at the cytoplasmic membrane. This behaviour is independent of MreB and does also not correlate with the stalling of the cell wall biosynthesis machinery upon antibiotic inhibition. It can be induced by antibiotics that interfere with the membrane-anchored steps of cell wall biosynthesis, while compounds that inhibit the cytoplasmic or extracytoplasmic steps do not trigger this response. Taken together, our data are consistent with a model in which the Lia system scans the cytoplasmic membrane for envelope perturbations. Upon their detection, LiaS activates the cognate response regulator LiaR, which in turn strongly induces the liaIH operon. Simultaneously, LiaI recruits LiaH to the membrane, presumably to protect the envelope and counteract the antibiotic-induced damage.

Cell envelope stress response in cell wall-deficient L-forms of Bacillus subtilis.

L-forms are cell wall-deficient bacteria that can grow and proliferate in osmotically stabilizing media. Recently, a strain of the Gram-positive model bacterium Bacillus subtilis was constructed that allowed controlled switching between rod-shaped wild-type cells and corresponding L-forms. Both states can be stably maintained under suitable culture conditions. Because of the absence of a cell wall, L-forms are known to be insensitive to ß-lactam antibiotics, but reports on the susceptibility of L-forms to other antibiotics that interfere with membrane-anchored steps of cell wall biosynthesis are sparse, conflicting, and strongly influenced by strain background and method of L-form generation. Here we investigated the response of B. subtilis to the presence of cell envelope antibiotics, with regard to both antibiotic resistance and the induction of the known LiaRS- and BceRS-dependent cell envelope stress biosensors. Our results show that B. subtilis L-forms are resistant to antibiotics that interfere with the bactoprenol cycle, such as bacitracin, vancomycin, and mersacidin, but are hypersensitive to nisin and daptomycin, which both affect membrane integrity. Moreover, we established a lacZ-based reporter gene assay for L-forms and provide evidence that LiaRS senses its inducers indirectly (damage sensing), while the Bce module detects its inducers directly (drug sensing).

Evolutionary genetics of an S-like polymorphism in Papaveraceae with putative function in self-incompatibility.

BACKGROUND: Papaver rhoeas possesses a gametophytic self-incompatibility (SI) system not homologous to any other SI mechanism characterized at the molecular level. Four previously published full length stigmatic S-alleles from the genus Papaver exhibited remarkable sequence divergence, but these studies failed to amplify additional S-alleles despite crossing evidence for more than 60 S-alleles in Papaver rhoeas alone. METHODOLOGY/PRINCIPAL FINDINGS: Using RT-PCR we identified 87 unique putative stigmatic S-allele sequences from the Papaveraceae Argemone munita, Papaver mcconnellii, P. nudicuale, Platystemon californicus and Romneya coulteri. Hand pollinations among two full-sib families of both A. munita and P. californicus indicate a strong correlation between the putative S-genotype and observed incompatibility phenotype. However, we also found more than two S-like sequences in some individuals of A. munita and P. californicus, with two products co-segregating in both full-sib families of P. californicus. Pairwise sequence divergence estimates within and among taxa show Papaver stigmatic S-alleles to be the most variable with lower divergence among putative S-alleles from other Papaveraceae. Genealogical analysis indicates little shared ancestral polymorphism among S-like sequences from different genera. Lack of shared ancestral polymorphism could be due to long divergence times among genera studied, reduced levels of balancing selection if some or all S-like sequences do not function in incompatibility, population bottlenecks, or different levels of recombination among taxa. Preliminary estimates of positive selection find many sites under selective constraint with a few undergoing positive selection, suggesting that self-recognition may depend on amino acid substitutions at only a few sites. CONCLUSIONS/SIGNIFICANCE: Because of the strong correlation between genotype and SI phenotype, sequences reported here represent either functional stylar S-alleles, tightly linked paralogs of the S-locus or a combination of both. The considerable complexity revealed in this study shows we have much to learn about the evolutionary dynamics of self-incompatibility systems.

In-depth profiling of the LiaR response of Bacillus subtilis.

The Lia system, a cell envelope stress response module of Bacillus subtilis, is comprised of the LiaRS two-component system and a membrane-anchored inhibitor protein, LiaF. It is highly conserved in the Firmicutes bacteria, and all orthologs investigated so far are activated by cell wall antibiotics. In response to envelope stress, the systems in Firmicutes cocci induce the expression of a number of genes that are involved in conferring resistance against its inducers. In contrast, a complete picture of the LiaR regulon of B. subtilis is still missing and no phenotypes could be associated with mutants lacking LiaRS. Here, we performed genome-wide transcriptomic, proteomic, and in-depth phenotypic profiling of constitutive "Lia ON" and "Lia OFF" mutants to obtain a comprehensive picture of the Lia response of Bacillus subtilis. In addition to the known targets liaIH and yhcYZ-yhdA, we identified ydhE as a novel gene affected by LiaR-dependent regulation. The results of detailed follow-up gene expression studies, together with proteomic analysis, demonstrate that the liaIH operon represents the only relevant LiaR target locus in vivo. It encodes a small membrane protein (LiaI) and a phage shock protein homolog (LiaH). LiaH forms large oligomeric rings reminiscent of those described for Escherichia coli PspA or Arabidopsis thaliana Vipp1. The results of comprehensive phenotype studies demonstrated that the gene products of the liaIH operon are involved in protecting the cell against oxidative stress and some cell wall antibiotics. Our data suggest that the LiaFSR system of B. subtilis and, presumably, other Firmicutes bacilli coordinates a phage shock protein-like response.