A direct PCR approach with low-biomass insert opens new horizons for molecular sciences on cryptogam communities.

Molecular sequence data have transformed research on cryptogams (e.g., lichens, microalgae, fungi, and symbionts thereof) but methods are still strongly hampered by the small size and intermingled growth of the target organisms, poor cultivability and detrimental effects of their secondary metabolites. Here, we aim to showcase examples on which a modified direct PCR approach for diverse aspects of molecular work on environmental samples concerning biocrusts, biofilms, and cryptogams gives new options for the research community. Unlike traditional approaches, this methodology only requires biomass equivalent to colonies and fragments of 0.2 mm in diameter, which can be picked directly from the environmental sample, and includes a quick DNA lysis followed by a standardized PCR cycle that allows co-cycling of various organisms/target regions in the same run. We demonstrate that this modified method can (i) amplify the most widely used taxonomic gene regions and those used for applied and environmental sciences from single colonies and filaments of free-living cyanobacteria, bryophytes, fungi, and lichens, including their mycobionts, chlorobionts, and cyanobionts from both isolates and in situ material during co-cycling; (ii) act as a tool to confirm that the dominant lichen photobiont was isolated from the original sample; and (iii) optionally remove inhibitory secondary lichen substances. Our results represent examples which highlight the method's potential for future applications covering mycology, phycology, biocrusts, and lichenology, in particular.IMPORTANCECyanobacteria, green algae, lichens, and other cryptogams play crucial roles in complex microbial systems such as biological soil crusts of arid biomes or biofilms in caves. Molecular investigations on environmental samples or isolates of these microorganisms are often hampered by their dense aggregation, small size, or metabolism products which complicate DNA extraction and subsequent PCRs. Our work presents various examples of how a direct DNA extraction and PCR method relying on low biomass inserts can overcome these common problems and discusses additional applications of the workflow including adaptations.

Nanosecond time-resolved infrared spectroscopy for the study of electron transfer in photosystem I.

Microsecond time-resolved step-scan FTIR difference spectroscopy was used to study photosystem I (PSI) from Thermosynechococcus vestitus BP-1 (T. vestitus, formerly known as T. elongatus) at 77 K. In addition, photoaccumulated (P700+-P700) FTIR difference spectra were obtained at both 77 and 293 K. The FTIR difference spectra are presented here for the first time. To extend upon these FTIR studies nanosecond time-resolved infrared difference spectroscopy was also used to study PSI from T. vestitus at 296 K. Nanosecond infrared spectroscopy has never been used to study PSI samples at physiological temperatures, and here it is shown that such an approach has great value as it allows a direct probe of electron transfer down both branches in PSI. In PSI at 296 K, the infrared flash-induced absorption changes indicate electron transfer down the B- and A-branches is characterized by time constants of 33 and 364 ns, respectively, in good agreement with visible spectroscopy studies. These time constants are associated with forward electron transfer from A1- to FX on the B- and A-branches, respectively. At several infrared wavelengths flash-induced absorption changes at 296 K recover in tens to hundreds of milliseconds. The dominant decay phase is characterized by a lifetime of 128 ms. These millisecond changes are assigned to radical pair recombination reactions, with the changes being associated primarily with P700+ rereduction. This conclusion follows from the observation that the millisecond infrared spectrum is very similar to the photoaccumulated (P700+-P700) FTIR difference spectrum.

Common loss of far-red light photoacclimation in cyanobacteria from hot and cold deserts: a case study in the Chroococcidiopsidales.

Deserts represent an extreme challenge for photosynthetic life. Despite their aridity, they are often inhabited by diverse microscopic communities of cyanobacteria. These organisms are commonly found in lithic habitats, where they are partially sheltered from extremes of temperature and UV radiation. However, living under the rock surface imposes additional constraints, such as limited light availability, and enrichment of longer wavelengths than are typically usable for oxygenic photosynthesis. Some cyanobacteria from the genus Chroococcidiopsis can use this light to photosynthesize, in a process known as far-red light photoacclimation, or FaRLiP. This genus has commonly been reported from both hot and cold deserts. However, not all Chroococcidiopsis strains carry FaRLiP genes, thus motivating our study into the interplay between FaRLiP and extreme lithic environments. The abundance of sequence data and strains provided the necessary material for an in-depth phylogenetic study, involving spectroscopy, microscopy, and determination of pigment composition, as well as gene and genome analyses. Pigment analyses revealed the presence of red-shifted chlorophylls d and f in all FaRLiP strains tested. In addition, eight genus-level taxa were defined within the encompassing Chroococcidiopsidales, clarifying the phylogeny of this long-standing polyphyletic order. FaRLiP is near universally present in a generalist genus identified in a wide variety of environments, Chroococcidiopsis sensu stricto, while it is rare or absent in closely related, extremophile taxa, including those preferentially inhabiting deserts. This likely reflects the evolutionary process of gene loss in specialist lineages.

SepT, a novel protein specific to multicellular cyanobacteria, influences peptidoglycan growth and septal nanopore formation in Anabaena sp. PCC 7120.

IMPORTANCE: Multicellular organization is a requirement for the development of complex organisms, and filamentous cyanobacteria such as Anabaena represent a paradigmatic case of bacterial multicellularity. The Anabaena filament can include hundreds of communicated cells that exchange nutrients and regulators and, depending on environmental conditions, can include different cell types specialized in distinct biological functions. Hence, the specific features of the Anabaena filament and how they are propagated during cell division represent outstanding biological issues. Here, we studied SepT, a novel coiled-coil-rich protein of Anabaena that is located in the intercellular septa and influences the formation of the septal specialized structures that allow communication between neighboring cells along the filament, a fundamental trait for the performance of Anabaena as a multicellular organism.

Interlaboratory Reproducibility in Growth and Reporter Expression in the Cyanobacterium Synechocystis sp. PCC 6803.

In recent years, a plethora of new synthetic biology tools for use in cyanobacteria have been published; however, their reported characterizations often cannot be reproduced, greatly limiting the comparability of results and hindering their applicability. In this interlaboratory study, the reproducibility of a standard microbiological experiment for the cyanobacterial model organism Synechocystis sp. PCC 6803 was assessed. Participants from eight different laboratories quantified the fluorescence intensity of mVENUS as a proxy for the transcription activity of the three promoters PJ23100, PrhaBAD, and PpetE over time. In addition, growth rates were measured to compare growth conditions between laboratories. By establishing strict and standardized laboratory protocols, reflecting frequently reported methods, we aimed to identify issues with state-of-the-art procedures and assess their effect on reproducibility. Significant differences in spectrophotometer measurements across laboratories from identical samples were found, suggesting that commonly used reporting practices of optical density values need to be supplemented by cell count or biomass measurements. Further, despite standardized light intensity in the incubators, significantly different growth rates between incubators used in this study were observed, highlighting the need for additional reporting requirements of growth conditions for phototrophic organisms beyond the light intensity and CO2 supply. Despite the use of a regulatory system orthogonal to Synechocystis sp. PCC 6803, PrhaBAD, and a high level of protocol standardization, ∼32% variation in promoter activity under induced conditions was found across laboratories, suggesting that the reproducibility of other data in the field of cyanobacteria might be affected similarly.

Compensatory Transcriptional Response of Fischerella thermalis to Thermal Damage of the Photosynthetic Electron Transfer Chain.

Key organisms in the environment, such as oxygenic photosynthetic primary producers (photosynthetic eukaryotes and cyanobacteria), are responsible for fixing most of the carbon globally. However, they are affected by environmental conditions, such as temperature, which in turn affect their distribution. Globally, the cyanobacterium Fischerella thermalis is one of the main primary producers in terrestrial hot springs with thermal gradients up to 60 °C, but the mechanisms by which F. thermalis maintains its photosynthetic activity at these high temperatures are not known. In this study, we used molecular approaches and bioinformatics, in addition to photophysiological analyses, to determine the genetic activity associated with the energy metabolism of F. thermalis both in situ and in high-temperature (40 °C to 65 °C) cultures. Our results show that photosynthesis of F. thermalis decays with temperature, while increased transcriptional activity of genes encoding photosystem II reaction center proteins, such as PsbA (D1), could help overcome thermal damage at up to 60 °C. We observed that F. thermalis tends to lose copies of the standard G4 D1 isoform while maintaining the recently described D1INT isoform, suggesting a preference for photoresistant isoforms in response to the thermal gradient. The transcriptional activity and metabolic characteristics of F. thermalis, as measured by metatranscriptomics, further suggest that carbon metabolism occurs in parallel with photosynthesis, thereby assisting in energy acquisition under high temperatures at which other photosynthetic organisms cannot survive. This study reveals that, to cope with the harsh conditions of hot springs, F. thermalis has several compensatory adaptations, and provides emerging evidence for mixotrophic metabolism as being potentially relevant to the thermotolerance of this species. Ultimately, this work increases our knowledge about thermal adaptation strategies of cyanobacteria.

Identification of far-red light acclimation in an endolithic Chroococcidiopsis strain and associated genomic features: Implications for oxygenic photosynthesis on exoplanets.

Deserts represent extreme habitats where photosynthetic life is restricted to the lithic niche. The ability of rock-inhabiting cyanobacteria to modify their photosynthetic apparatus and harvest far-red light (near-infrared) was investigated in 10 strains of the genus Chroococcidiopsis, previously isolated from diverse endolithic and hypolithic desert communities. The analysis of their growth capacity, photosynthetic pigments, and apcE2-gene presence revealed that only Chroococcidiopsis sp. CCMEE 010 was capable of far-red light photoacclimation (FaRLiP). A total of 15 FaRLiP genes were identified, encoding paralogous subunits of photosystem I, photosystem II, and the phycobilisome, along with three regulatory elements. CCMEE 010 is unique among known FaRLiP strains by undergoing this acclimation process with a significantly reduced cluster, which lacks major photosystem I paralogs psaA and psaB. The identification of an endolithic, extremotolerant cyanobacterium capable of FaRLiP not only contributes to our appreciation of this phenotype's distribution in nature but also has implications for the possibility of oxygenic photosynthesis on exoplanets.

Generation of Synthetic Shuttle Vectors Enabling Modular Genetic Engineering of Cyanobacteria.

Cyanobacteria have raised great interest in biotechnology due to their potential for a sustainable, photosynthesis-driven production of fuels and value-added chemicals. This has led to a concomitant development of molecular tools to engineer the metabolism of those organisms. In this regard, however, even cyanobacterial model strains lag behind compared to their heterotrophic counterparts. For instance, replicative shuttle vectors that allow gene transfer independent of recombination into host DNA are still scarce. Here, we introduce the pSOMA shuttle vector series comprising 10 synthetic plasmids for comprehensive genetic engineering of Synechocystis sp. PCC 6803. The series is based on the small endogenous plasmids pCA2.4 and pCB2.4, each combined with a replicon from Escherichia coli, different selection markers as well as features facilitating molecular cloning and the insulated introduction of gene expression cassettes. We made use of genes encoding green fluorescent protein (GFP) and a Baeyer-Villiger monooxygenase (BVMO) to demonstrate functional gene expression from the pSOMA plasmids in vivo. Moreover, we demonstrate the expression of distinct heterologous genes from individual plasmids maintained in the same strain and thereby confirmed compatibility between the two pSOMA subseries as well as with derivatives of the broad-host-range plasmid RSF1010. We also show that gene transfer into the filamentous model strain Anabaena sp. PCC 7120 is generally possible, which is encouraging to further explore the range of cyanobacterial host species that could be engineered via pSOMA plasmids. Altogether, the pSOMA shuttle vector series displays an attractive alternative to existing plasmid series and thus meets the current demand for the introduction of complex genetic setups and to perform extensive metabolic engineering of cyanobacteria.

From manganese oxidation to water oxidation: assembly and evolution of the water-splitting complex in photosystem II.

The manganese cluster of photosystem II has been the focus of intense research aiming to understand the mechanism of H2O-oxidation. Great effort has also been applied to investigating its oxidative photoassembly process, termed photoactivation that involves the light-driven incorporation of metal ions into the active Mn4CaO5 cluster. The knowledge gained on these topics has fundamental scientific significance, but may also provide the blueprints for the development of biomimetic devices capable of splitting water for solar energy applications. Accordingly, synthetic chemical approaches inspired by the native Mn cluster are actively being explored, for which the native catalyst is a useful benchmark. For both the natural and artificial catalysts, the assembly process of incorporating Mn ions into catalytically active Mn oxide complexes is an oxidative process. In both cases this process appears to share certain chemical features, such as producing an optimal fraction of open coordination sites on the metals to facilitate the binding of substrate water, as well as the involvement of alkali metals (e.g., Ca2+) to facilitate assembly and activate water-splitting catalysis. This review discusses the structure and formation of the metal cluster of the PSII H2O-oxidizing complex in the context of what is known about the formation and chemical properties of different Mn oxides. Additionally, the evolutionary origin of the Mn4CaO5 is considered in light of hypotheses that soluble Mn2+ was an ancient source of reductant for some early photosynthetic reaction centers ('photomanganotrophy'), and recent evidence that PSII can form Mn oxides with structural resemblance to the geologically abundant birnessite class of minerals. A new functional role for Ca2+ to facilitate sustained Mn2+ oxidation during photomanganotrophy is proposed, which may explain proposed physiological intermediates during the likely evolutionary transition from anoxygenic to oxygenic photosynthesis.

Changes in supramolecular organization of cyanobacterial thylakoid membrane complexes in response to far-red light photoacclimation.

Cyanobacteria are ubiquitous in nature and have developed numerous strategies that allow them to live in a diverse range of environments. Certain cyanobacteria synthesize chlorophylls d and f to acclimate to niches enriched in far-red light (FRL) and incorporate paralogous photosynthetic proteins into their photosynthetic apparatus in a process called FRL-induced photoacclimation (FaRLiP). We characterized the macromolecular changes involved in FRL-driven photosynthesis and used atomic force microscopy to examine the supramolecular organization of photosystem I associated with FaRLiP in three cyanobacterial species. Mass spectrometry showed the changes in the proteome of Chroococcidiopsis thermalis PCC 7203 that accompany FaRLiP. Fluorescence lifetime imaging microscopy and electron microscopy reveal an altered cellular distribution of photosystem complexes and illustrate the cell-to-cell variability of the FaRLiP response.

Infrared nanoscopy and tomography of intracellular structures.

Although techniques such as fluorescence-based super-resolution imaging or confocal microscopy simultaneously gather both morphological and chemical data, these techniques often rely on the use of localized and chemically specific markers. To eliminate this flaw, we have developed a method of examining cellular cross sections using the imaging power of scattering-type scanning near-field optical microscopy and Fourier-transform infrared spectroscopy at a spatial resolution far beyond the diffraction limit. Herewith, nanoscale surface and volumetric chemical imaging is performed using the intrinsic contrast generated by the characteristic absorption of mid-infrared radiation by the covalent bonds. We employ infrared nanoscopy to study the subcellular structures of eukaryotic (Chlamydomonas reinhardtii) and prokaryotic (Escherichia coli) species, revealing chemically distinct regions within each cell such as the microtubular structure of the flagellum. Serial 100 nm-thick cellular cross-sections were compiled into a tomogram yielding a three-dimensional infrared image of subcellular structure distribution at 20 nm resolution. The presented methodology is able to image biological samples complementing current fluorescence nanoscopy but at less interference due to the low energy of infrared radiation and the absence of labeling.

Two novel heteropolymer-forming proteins maintain the multicellular shape of the cyanobacterium Anabaena sp. PCC 7120.

Polymerizing and filament-forming proteins are instrumental for numerous cellular processes such as cell division and growth. Their function in stabilization and localization of protein complexes and replicons is achieved by a filamentous structure. Known filamentous proteins assemble into homopolymers consisting of single subunits - for example, MreB and FtsZ in bacteria - or heteropolymers that are composed of two subunits, for example, keratin and α/ß tubulin in eukaryotes. Here, we describe two novel coiled-coil-rich proteins (CCRPs) in the filament-forming cyanobacterium Anabaena sp. PCC 7120 (hereafter Anabaena) that assemble into a heteropolymer and function in the maintenance of the Anabaena multicellular shape (termed trichome). The two CCRPs - Alr4504 and Alr4505 (named ZicK and ZacK) - are strictly interdependent for the assembly of protein filaments in vivo and polymerize nucleotide independently in vitro, similar to known intermediate filament (IF) proteins. A ΔzicKΔzacK double mutant is characterized by a zigzagged cell arrangement and hence a loss of the typical linear Anabaena trichome shape. ZicK and ZacK interact with themselves, with each other, with the elongasome protein MreB, the septal junction protein SepJ and the divisome associate septal protein SepI. Our results suggest that ZicK and ZacK function in cooperation with SepJ and MreB to stabilize the Anabaena trichome and are likely essential for the manifestation of the multicellular shape in Anabaena. Our study reveals the presence of filament-forming IF-like proteins whose function is achieved through the formation of heteropolymers in cyanobacteria.

Structural Determinants and Their Role in Cyanobacterial Morphogenesis.

Cells have to erect and sustain an organized and dynamically adaptable structure for an efficient mode of operation that allows drastic morphological changes during cell growth and cell division. These manifold tasks are complied by the so-called cytoskeleton and its associated proteins. In bacteria, FtsZ and MreB, the bacterial homologs to tubulin and actin, respectively, as well as coiled-coil-rich proteins of intermediate filament (IF)-like function to fulfil these tasks. Despite generally being characterized as Gram-negative, cyanobacteria have a remarkably thick peptidoglycan layer and possess Gram-positive-specific cell division proteins such as SepF and DivIVA-like proteins, besides Gram-negative and cyanobacterial-specific cell division proteins like MinE, SepI, ZipN (Ftn2) and ZipS (Ftn6). The diversity of cellular morphologies and cell growth strategies in cyanobacteria could therefore be the result of additional unidentified structural determinants such as cytoskeletal proteins. In this article, we review the current advances in the understanding of the cyanobacterial cell shape, cell division and cell growth.

Light-driven formation of manganese oxide by today's photosystem II supports evolutionarily ancient manganese-oxidizing photosynthesis.

Water oxidation and concomitant dioxygen formation by the manganese-calcium cluster of oxygenic photosynthesis has shaped the biosphere, atmosphere, and geosphere. It has been hypothesized that at an early stage of evolution, before photosynthetic water oxidation became prominent, light-driven formation of manganese oxides from dissolved Mn(2+) ions may have played a key role in bioenergetics and possibly facilitated early geological manganese deposits. Here we report the biochemical evidence for the ability of photosystems to form extended manganese oxide particles. The photochemical redox processes in spinach photosystem-II particles devoid of the manganese-calcium cluster are tracked by visible-light and X-ray spectroscopy. Oxidation of dissolved manganese ions results in high-valent Mn(III,IV)-oxide nanoparticles of the birnessite type bound to photosystem II, with 50-100 manganese ions per photosystem. Having shown that even today's photosystem II can form birnessite-type oxide particles efficiently, we propose an evolutionary scenario, which involves manganese-oxide production by ancestral photosystems, later followed by down-sizing of protein-bound manganese-oxide nanoparticles to finally yield today's catalyst of photosynthetic water oxidation.

Femtosecond visible transient absorption spectroscopy of chlorophyll-f-containing photosystem II.

The recently discovered, chlorophyll-f-containing, far-red photosystem II (FR-PSII) supports far-red light photosynthesis. Participation and kinetics of spectrally shifted far-red pigments are directly observable and separated from that of bulk chlorophyll-a We present an ultrafast transient absorption study of FR-PSII, investigating energy transfer and charge separation processes. Results show a rapid subpicosecond energy transfer from chlorophyll-a to the long-wavelength chlorophylls-f/d The data demonstrate the decay of an ∼720-nm negative feature on the picosecond-to-nanosecond timescales, coinciding with charge separation, secondary electron transfer, and stimulated emission decay. An ∼675-nm bleach attributed to the loss of chl-a absorption due to the formation of a cation radical, PD1+•, is only fully developed in the nanosecond spectra, indicating an unusually delayed formation. A major spectral feature on the nanosecond timescale at 725 nm is attributed to an electrochromic blue shift of a FR-chlorophyll among the reaction center pigments. These time-resolved observations provide direct experimental support for the model of Nürnberg et al. [D. J. Nürnberg et al., Science 360, 1210-1213 (2018)], in which the primary electron donor is a FR-chlorophyll and the secondary donor is chlorophyll-a (PD1 of the central chlorophyll pair). Efficient charge separation also occurs using selective excitation of long-wavelength chlorophylls-f/d, and the localization of the excited state on P720* points to a smaller (entropic) energy loss compared to conventional PSII, where the excited state is shared over all of the chlorin pigments. This has important repercussions on understanding the overall energetics of excitation energy transfer and charge separation reactions in FR-PSII.

Global distribution of a chlorophyll f cyanobacterial marker.

Some cyanobacteria use light outside the visible spectrum for oxygenic photosynthesis. The far-red light (FRL) region is made accessible through a complex acclimation process that involves the formation of new phycobilisomes and photosystems containing chlorophyll f. Diverse cyanobacteria ranging from unicellular to branched-filamentous forms show this response. These organisms have been isolated from shaded environments such as microbial mats, soil, rock, and stromatolites. However, the full spread of chlorophyll f-containing species in nature is still unknown. Currently, discovering new chlorophyll f cyanobacteria involves lengthy incubation times under selective far-red light. We have used a marker gene to detect chlorophyll f organisms in environmental samples and metagenomic data. This marker, apcE2, encodes a phycobilisome linker associated with FRL-photosynthesis. By focusing on a far-red motif within the sequence, degenerate PCR and BLAST searches can effectively discriminate against the normal chlorophyll a-associated apcE. Even short recovered sequences carry enough information for phylogenetic placement. Markers of chlorophyll f photosynthesis were found in metagenomic datasets from diverse environments around the globe, including cyanobacterial symbionts, hypersaline lakes, corals, and the Arctic/Antarctic regions. This additional information enabled higher phylogenetic resolution supporting the hypothesis that vertical descent, as opposed to horizontal gene transfer, is largely responsible for this phenotype's distribution.

Fischerella thermalis: a model organism to study thermophilic diazotrophy, photosynthesis and multicellularity in cyanobacteria.

The true-branching cyanobacterium Fischerella thermalis (also known as Mastigocladus laminosus) is widely distributed in hot springs around the world. Morphologically, it has been described as early as 1837. However, its taxonomic placement remains controversial. F. thermalis belongs to the same genus as mesophilic Fischerella species but forms a monophyletic clade of thermophilic Fischerella strains and sequences from hot springs. Their recent divergence from freshwater or soil true-branching species and the ongoing process of specialization inside the thermal gradient make them an interesting evolutionary model to study. F. thermalis is one of the most complex prokaryotes. It forms a cellular network in which the main trichome and branches exchange metabolites and regulators via septal junctions. This species can adapt to a variety of environmental conditions, with its photosynthetic apparatus remaining active in a temperature range from 15 to 58 °C. Together with its nitrogen-fixing ability, this allows it to dominate in hot spring microbial mats and contribute significantly to the de novo carbon and nitrogen input. Here, we review the current knowledge on the taxonomy and distribution of F. thermalis, its morphological complexity, and its physiological adaptations to an extreme environment.

The MarR-Type Repressor MhqR Confers Quinone and Antimicrobial Resistance in Staphylococcus aureus.

Aims: Quinone compounds are electron carriers and have antimicrobial and toxic properties due to their mode of actions as electrophiles and oxidants. However, the regulatory mechanism of quinone resistance is less well understood in the pathogen Staphylococcus aureus. Results: Methylhydroquinone (MHQ) caused a thiol-specific oxidative and electrophile stress response in the S. aureus transcriptome as revealed by the induction of the PerR, QsrR, CstR, CtsR, and HrcA regulons. The SACOL2531-29 operon was most strongly upregulated by MHQ and was renamed as mhqRED operon based on its homology to the Bacillus subtilis locus. Here, we characterized the MarR-type regulator MhqR (SACOL2531) as quinone-sensing repressor of the mhqRED operon, which confers quinone and antimicrobial resistance in S. aureus. The mhqRED operon responds specifically to MHQ and less pronounced to pyocyanin and ciprofloxacin, but not to reactive oxygen species (ROS), hypochlorous acid, or aldehydes. The MhqR repressor binds specifically to a 9-9 bp inverted repeat (MhqR operator) upstream of the mhqRED operon and is inactivated by MHQ in vitro, which does not involve a thiol-based mechanism. In phenotypic assays, the mhqR deletion mutant was resistant to MHQ and quinone-like antimicrobial compounds, including pyocyanin, ciprofloxacin, norfloxacin, and rifampicin. In addition, the mhqR mutant was sensitive to sublethal ROS and 24 h post-macrophage infections but acquired an improved survival under lethal ROS stress and after long-term infections. Innovation: Our results provide a link between quinone and antimicrobial resistance via the MhqR regulon of S. aureus. Conclusion: The MhqR regulon was identified as a novel resistance mechanism towards quinone-like antimicrobials and contributes to virulence of S. aureus under long-term infections.