Histone-like Nucleoid Structuring Protein Research Articles

MucR protein: Three decades of studies have led to the identification of a new H-NS-like protein.

MucR belongs to a large protein family whose members regulate the expression of virulence and symbiosis genes in α-proteobacteria species. This protein and its homologs were initially studied as classical transcriptional regulators mostly involved in repression of target genes by binding their promoters. Very recent studies have led to the classification of MucR as a new type of Histone-like Nucleoid Structuring (H-NS) protein. Thus this review is an effort to put together a complete and unifying story demonstrating how genetic and biochemical findings on MucR suggested that this protein is not a classical transcriptional regulator, but functions as a novel type of H-NS-like protein, which binds AT-rich regions of genomic DNA and regulates gene expression.

Acetylation of xenogeneic silencer H-NS regulates biofilm development through the nitrogen homeostasis regulator in Shewanella.

Adjusting intracellular metabolic pathways and adopting suitable live state such as biofilms, are crucial for bacteria to survive environmental changes. Although substantial progress has been made in understanding how the histone-like nucleoid-structuring (H-NS) protein modulates the expression of the genes involved in biofilm formation, the precise modification that the H-NS protein undergoes to alter its DNA binding activity is still largely uncharacterized. This study revealed that acetylation of H-NS at Lys19 inhibits biofilm development in Shewanella oneidensis MR-1 by downregulating the expression of glutamine synthetase, a critical enzyme in glutamine synthesis. We further found that nitrogen starvation, a likely condition in biofilm development, induces deacetylation of H-NS and the trimerization of nitrogen assimilation regulator GlnB. The acetylated H-NS strain exhibits significantly lower cellular glutamine concentration, emphasizing the requirement of H-NS deacetylation in Shewanella biofilm development. Moreover, we discovered in vivo that the activation of glutamine biosynthesis pathway and the concurrent suppression of the arginine synthesis pathway during both pellicle and attached biofilms development, further suggesting the importance of fine tune nitrogen assimilation by H-NS acetylation in Shewanella. In summary, posttranslational modification of H-NS endows Shewanella with the ability to respond to environmental needs by adjusting the intracellular metabolism pathways.

Lysine acetylation regulates the AT-rich DNA possession ability of H-NS.

H-NS, the histone-like nucleoid-structuring protein in bacteria, regulates the stability of the bacterial genome by inhibiting the transcription of horizontally transferred genes, such as the type III and type VI secretion systems (T3/T6SS). While eukaryotic histone posttranslational modifications (PTMs) have been extensively studied, little is known about prokaryotic H-NS PTMs. Here, we report that the acetylation of H-NS attenuates its ability to silence horizontally transferred genes in response to amino acid nutrition and immune metabolites. Moreover, LC-MS/MS profiling showed that the acetyllysine sites of H-NS and K120 are indispensable for its DNA-binding ability. Acetylation of K120 leads to a low binding affinity for DNA and enhances T3/T6SS expression. Furthermore, acetylation of K120 impairs the AT-rich DNA recognition ability of H-NS. In addition, lysine acetylation in H-NS modulates in vivo bacterial virulence. These findings reveal the mechanism underlying H-NS PTMs and propose a novel mechanism by which bacteria counteract the xenogeneic silencing of H-NS.

The environmentally-regulated interplay between local three-dimensional chromatin organisation and transcription of proVWX in E. coli

Nucleoid associated proteins (NAPs) maintain the architecture of bacterial chromosomes and regulate gene expression. Thus, their role as transcription factors may involve three-dimensional chromosome re-organisation. While this model is supported by in vitro studies, direct in vivo evidence is lacking. Here, we use RT-qPCR and 3C-qPCR to study the transcriptional and architectural profiles of the H-NS (histone-like nucleoid structuring protein)-regulated, osmoresponsive proVWX operon of Escherichia coli at different osmolarities and provide in vivo evidence for transcription regulation by NAP-mediated chromosome re-modelling in bacteria. By consolidating our in vivo investigations with earlier in vitro and in silico studies that provide mechanistic details of how H-NS re-models DNA in response to osmolarity, we report that activation of proVWX in response to a hyperosmotic shock involves the destabilization of H-NS-mediated bridges anchored between the proVWX downstream and upstream regulatory elements (DRE and URE), and between the DRE and ygaY that lies immediately downstream of proVWX. The re-establishment of these bridges upon adaptation to hyperosmolarity represses the operon. Our results also reveal additional structural features associated with changes in proVWX transcript levels such as the decompaction of local chromatin upstream of the operon, highlighting that further complexity underlies the regulation of this model operon. H-NS and H-NS-like proteins are wide-spread amongst bacteria, suggesting that chromosome re-modelling may be a typical feature of transcriptional control in bacteria.

Decoding dissociation of sequence-specific protein-DNA complexes with non-equilibrium simulations.

Sequence-specific protein-DNA interactions are crucial in processes such as DNA organization, gene regulationand DNA replication. Obtaining detailed insights into the recognition mechanisms of protein-DNA complexes through experiments is hampered by a lack of resolution in both space and time. Here, we present a molecular simulation approach to quantify the sequence specificity of protein-DNA complexes, that yields results fast, and is generally applicable to any protein-DNA complex. The approach is based on molecular dynamics simulations in combination with a sophisticated steering potential and results in an estimate of the free energy difference of dissociation. We provide predictions of the nucleotide specific binding affinity of the minor groove binding Histone-like Nucleoid Structuring (H-NS) protein, that are in agreement with experimental data. Furthermore, our approach offers mechanistic insight into the process of dissociation. Applying our approach to the major groove binding ETS domain in complex with three different nucleotide sequences identified the high affinity consensus sequence, quantitatively in agreement with experiments. Our protocol facilitates quantitative prediction of protein-DNA complex stability, while also providing high resolution insights into recognition mechanisms. As such, our simulation approach has the potential to yield detailed and quantitative insights into biological processes involving sequence-specific protein-DNA interactions.

MucR from Sinorhizobium meliloti: New Insights into Its DNA Targets and Its Ability to Oligomerize.

Proteins of the MucR/Ros family play a crucial role in bacterial infection or symbiosis with eukaryotic hosts. MucR from Sinorhizobium meliloti plays a regulatory role in establishing symbiosis with the host plant, both dependent and independent of Quorum Sensing. Here, we report the first characterization of MucR isolated from Sinorhizobium meliloti by mass spectrometry and demonstrate that this protein forms higher-order oligomers in its native condition of expression by SEC-MALS. We show that MucR purified from Sinorhizobium meliloti can bind DNA and recognize the region upstream of the ndvA gene in EMSA, revealing that this gene is a direct target of MucR. Although MucR DNA binding activity was already described, a detailed characterization of Sinorhizobium meliloti DNA targets has never been reported. We, thus, analyze sequences recognized by MucR in the rem gene promoter, showing that this protein recognizes AT-rich sequences and does not require a consensus sequence to bind DNA. Furthermore, we investigate the dependence of MucR DNA binding on the length of DNA targets. Taken together, our studies establish MucR from Sinorhizobium meliloti as a member of a new family of Histone-like Nucleoid Structuring (H-NS) proteins, thus explaining the multifaceted role of this protein in many species of alpha-proteobacteria.

XrvB regulates the type III secretion system by directly repressing hrpG transcription in Xanthomonas oryzae pv. oryzicola

Xrv proteins are a group of regulators in Xanthomonas spp., belonging to the histone-like nucleoid-structuring (H-NS) proteins of Gram-negative bacteria. The rice bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc) harbors three Xrv proteins, the XrvA, XrvB, and XrvC. Here, we report that in Xoc, the XrvB but not XrvA and XrvC is involved in negative regulation of the type III secretion system (T3SS) encoded by hrp genes. As with other Xanthomonas spp., the T3SS is an essential virulence determinant of Xoc and the expression of the hrp genes in Xoc is controlled by the HrpG/HrpX regulatory cascade. HrpG positively regulates the expression of HrpX, which in turn activates the transcription of the hrp genes. We provide evidences to demonstrate that the XrvB binds to the promoter region of hrpG and represses its transcription. Furthermore, we found that XrvB production was induced in the Xoc cells cultured in a nutrient-rich medium compared to a hrp-inducing minimal medium. We also found that in Xoc, the hrpG expression level is inversely correlated with the content of XrvB, and XrvB occupancy at hrpG promoter region is positively correlated with XrvB levels. Our data suggest that XrvB is a determinative factor controlling the expression levels of HrpG. In addition, mutation analysis revealed that the Xoc XrvB also plays positive roles in regulating bacterial growth, cell motility, and stress tolerance. Our findings provide important insights into the molecular mechanism of T3SS expression regulation in Xoc.

Temperature-dependent carrier state mediated by H-NS promotes the long-term coexistence of Y. pestis and a phage in soil.

The study of carrier state phages challenged the canonical lytic-lysogenic binary, and carrier state appears to be ubiquitous and ecologically important. However, the mechanisms of the carrier state are not well elucidated due to the limited phage models. Herein, we reported phage HQ103, similar to Escherichia coli phage P2. In contrast to the temperate P2 phage, the HQ103 phage does not insert its genome into the bacterial chromosome and displays a dual behavior depending on the temperature. At 37°C, HQ103 lyses the host and forms clear plaques due to the truncation of repressor CI and mutation of promoter Pc. In contrast, HQ103 maintains a carrier state lifestyle with Y. pestis at an environmental temperature (21°C). Mechanistically, we found that the host-encoded histone-like nucleoid-structuring protein H-NS, which is highly expressed at 21°C to silence the Cox promoter Pe and inhibits the phage lytic cycle. Subsequently, the HQ103 carrier state Y. pestis could grow and co-exist with the phage in the soil at 21°C for one month. Thus, this study reveals a novel carrier state lifestyle of phage HQ103 due to the H-NS mediated xenogeneic silencing and demonstrates that the carrier state lifestyle could promote long-term phage-host coexist in nature.

Three-dimensional chromosome re-modelling: The integral mechanism of transcription regulation in bacteria.

Nucleoid-associated proteins (NAPs) are architectural proteins of the bacterial chromosome and transcription factors that dynamically organise the chromosome and regulate gene expression in response to physicochemical environmental signals. While the architectural and regulatory functions of NAPs have been verified independently, the coupling between these functions in vivo has not been conclusively proven. Here we describe a model NAP - histone-like nucleoid structuring protein (H-NS) - as a coupled sensor-effector that directly regulates gene expression by chromatin re-modelling in response to physicochemical environmental signals. We outline how H-NS-binding partners and post-translational modifications modulate the role of H-NS as a transcription factor by influencing its DNA structuring properties. We consolidate our ideas in models of how H-NS may regulate the expression of the proVWX and hlyCABD operons by chromatin re-modelling. The interplay between chromosome structure and gene expression may be a common - but, at present, under-appreciated - concept of transcription regulation in bacteria.

Localized modulation of DNA supercoiling, triggered by the Shigella anti-silencer VirB, is sufficient to relieve H-NS-mediated silencing.

In Bacteria, nucleoid structuring proteins govern nucleoid dynamics and regulate transcription. In Shigella spp., at ≤30°C, the histone-like nucleoid structuring protein (H-NS) transcriptionally silences many genes on the large virulence plasmid. Upon a switch to 37°C, VirB, a DNA binding protein and key transcriptional regulator of Shigella virulence, is produced. VirB functions to counter H-NS-mediated silencing in a process called transcriptional anti-silencing. Here, we show that VirB mediates a loss of negative DNA supercoils from our plasmid-borne, VirB-regulated PicsP-lacZ reporterin vivo. The changes are not caused by a VirB-dependent increase in transcription, nor do they require the presence of H-NS. Instead, the VirB-dependent change in DNA supercoiling requires the interaction of VirB with its DNA binding site, a critical first step in VirB-dependent gene regulation. Using two complementary approaches, we show that VirB:DNA interactions in vitro introduce positive supercoils in plasmid DNA. Subsequently, by exploiting transcription-coupled DNA supercoiling, we reveal that a localized loss of negative supercoils is sufficient to alleviate H-NS-mediated transcriptional silencingindependently of VirB. Together, our findings provide novel insight into VirB,a central regulator of Shigella virulenceand, more broadly, a molecular mechanism that offsets H-NS-dependent silencing of transcription in bacteria.

Serotype conversion gene rfbT is directly regulated by histone-like nucleoid structuring protein (H-NS) in V. cholerae O1.

Vibrio cholerae serogroup O1 (V. cholerae O1) is closely associated with cholera epidemics and has two main immunologically distinguishable serotypes, Ogawa and Inaba. Isolates serotype as Ogawa if the O-antigen polysaccharide (O-PS) is methylated or as Inaba if the O-PS is not methylated. This methylation is mediated by a methyltransferase encoded by the rfbT gene, and the mutation and low expression of rfbT results in serotype switch from Ogawa to Inaba. Previously, we have shown that cAMP receptor protein (CRP) activates rfbT. In this study, we demonstrated that histone-like nucleoid structuring protein (H-NS) is directly involved in the transcriptional repression of rfbT. This finding is supported by the analyses of rfbT mRNA level, rfbT-lux reporter fusions, electrophoretic mobility shift assay (EMSA), and DNase I footprinting assay. The rfbT mRNA abundances were significantly increased by deleting hns rather than fis which also preferentially associates with AT-rich sequences. A single-copy chromosomal complement of hns partly restored the down-regulation of rfbT. Analysis of rfbT-lux reporter fusions validated the transcriptional repression of hns. Subsequent EMSA and DNase I footprinting assay confirmed the direct binding of H-NS to rfbT promoter and mapped the exact binding site which was further verified by site-directed mutagenesis and promoter functional analysis. Furthermore, we found that in hns deletion mutant, CRP is no longer required for transcriptionally activating rfbT, suggesting that CRP functions as a dedicated transcription factor to relieve H-NS repression at rfbT. Together, this study expanded our understanding of the genetic regulatory mechanism of serotype conversion by global regulators in V. cholerae O1.

Two Trk/Ktr/HKT-type potassium transporters, TrkG and TrkH, perform distinct functions in Escherichia coli K-12

Escherichia coli K-12 possesses two versions of Trk/Ktr/HKT-type potassium ion (K+) transporters, TrkG and TrkH. The current paradigm is that TrkG and TrkH have largely identical characteristics, and little information is available regarding their functional differences. Here, we show using cation uptake experiments with K+ transporter knockout mutants that TrkG and TrkH have distinct ion transport activities and physiological roles. K+-transport by TrkG required Na+, whereas TrkH-mediated K+ uptake was not affected by Na+. An aspartic acid located five residues away from a critical glycine in the third pore-forming region might be involved in regulation of Na+-dependent activation of TrkG. In addition, we found that TrkG but not TrkH had Na+ uptake activity. Our analysis of K+ transport mutants revealed that TrkH supported cell growth more than TrkG; however, TrkG was able to complement loss of TrkH-mediated K+ uptake in E.coli. Furthermore, we determined that transcription of trkG in E.coli was downregulated but not completely silenced by the xenogeneic silencing factor H-NS (histone-like nucleoid structuring protein or heat-stable nucleoid-structuring protein). Taken together, the transport function of TrkG is clearly distinct from that of TrkH, and TrkG seems to have been accepted by E.coli during evolution as a K+ uptake system that coexists with TrkH.

H-NS Represses Biofilm Formation and c-di-GMP Synthesis in Vibrio parahaemolyticus

ObjectiveThis study aimed to investigate the regulation of histone-like nucleoid structuring protein (H-NS) on biofilm formation and cyclic diguanylate (c-di-GMP) synthesis in Vibrio parahaemolyticus RIMD2210633. MethodsRegulatory mechanisms were analyzed by the combined utilization of crystal violet staining, quantification of c-di-GMP, quantitative real-time polymerase chain reaction, LacZ fusion, and electrophoretic-mobility shift assay. ResultsThe deletion of hns enhanced the biofilm formation and intracellular c-di-GMP levels in V. parahaemolyticus RIMD2210633. H-NS can bind the upstream promoter–proximal DNA regions of scrA, scrG, VP0117, VPA0198, VPA1176, VP0699, and VP2979 to repress their transcription. These genes encode a group of proteins with GGDEF and/or EAL domains associated with c-di-GMP metabolism. ConclusionOne of the mechanisms by which H-NS represses the biofilm formation by V. parahaemolyticus RIMD2210633 may be via repression of the production of intracellular c-di-GMP.

SlyA regulates virulence gene expressions through activation of pchA regulatory gene in enterohemorrhagic Escherichia coli.

SlyA is a DNA-binding protein that alters the nucleoid complex composed of histone-like nucleoid-structuring protein (H-NS) and activates gene expression. In enterohemorrhagic Escherichia coli (EHEC), the expression of virulence genes is repressed by H-NS but is up-regulated in response to environmental factors by releasing a nucleoid complex. This study examined the effect of slyA deletion mutation in EHEC and discovered that the production of the locus of enterocyte effacement (LEE)-encoded EspB and Tir, as well as the cell adherence ability, was reduced in the mutant compared with the wild type. The promoter activity of the LEE1 operon, including the regulatory gene, ler, was reduced by slyA mutation, but tac promoter-controlled expression of pchA, which is a regulatory gene of LEE1, abolished the effect. The promoter activity of pchA was down-regulated by the slyA mutation. Furthermore, the coding region was required for its regulation and was bound to SlyA, which indicates the direct regulation of pchA by SlyA. However, the slyA mutation did not affect the butyrate-induced increase in pchA promoter activity. Additionally, the pchA promoter activity was increased via induction of lrp, a regulatory gene for butyrate response, in the slyA mutant and, conversely, by introducing high copies of slyA into the lrp mutant. These results indicate that SlyA is a positive regulator of pchA and is independent of the Lrp regulatory system. SlyA may be involved in the virulence expression in EHEC, maintaining a certain level of expression in the absence of a butyrate response.

Mechanistic basis for environment-controlled gene silencing by the histone-like nucleoid-structuring (H-NS) protein

Mechanistic basis for environment-controlled gene silencing by the histone-like nucleoid-structuring (H-NS) protein

Revisiting the Temperature Dependence of Protein Diffusion inside Bacteria: Validity of the Stokes-Einstein Equation.

Although the transport and mixing of proteins and other molecules inside bacteria rely on the diffusion of molecules, many aspects of the molecular diffusion in bacterial cytoplasm remain unclear or controversial, including how the diffusion-temperature relation follows the Stokes-Einstein equation. In this study, we applied single-particle tracking photoactivated localization microscopy to investigate the diffusion of histonelike nucleoid structuring (HNS) proteins and free dyes in bacterial cytoplasm at different temperatures. Although the diffusion of HNS proteins in both live and dead bacteria increased at higher temperatures and appeared to follow the Arrhenius equation, the diffusion of free dyes decreased at higher temperatures, questioning the previously proposed theories based on superthermal fluctuations. To understand the measured diffusion-temperature relations, we developed an alternative model, in which the bacterial cytoplasm is considered as a polymeric network or mesh. In our model, the Stokes-Einstein equation remains valid, while the polymeric network contributes a significant term to the viscosity experienced by the molecules diffusing in bacterial cytoplasm. Our model was successful in predicting the diffusion-temperature relations for both HNS proteins and free dyes in bacteria. In addition, we systematically examined the predicted diffusion-temperature relations with different parameters in the model, and predicted the possible existence of phase transitions.

Pat- and Pta-mediated protein acetylation is required for horizontally-acquired virulence gene expression in Salmonella Typhimurium.

Salmonella Typhimurium is a Gram-negative facultative pathogen that causes a range of diseases, from mild gastroenteritis to severe systemic infection in a variety of animal hosts. S. Typhimurium regulates virulence gene expression by a silencing mechanism using nucleoid-associated proteins such as Histone-like Nucleoid Structuring protein (H-NS) silencing. We hypothesize that the posttranslational modification, specifically protein acetylation, of proteins in gene silencing systems could affect the pathogenic gene expression of S. Typhimurium. Therefore, we created acetylation-deficient mutant by deleting two genes, pat and pta, which are involved in the protein acetylation pathway. We observed that the pat and pta deletion attenuates mouse virulence and also decreases Salmonella's replication within macrophages. In addition, the Δpat Δpta strain showed a decreased expression of the horizontally-acquired virulence genes, mgtC, pagC, and ugtL, which are highly expressed in low Mg2+. The decreased virulence gene expression is possibly due to higher H-NS occupancy to those promoters because the pat and pta deletion increases H-NS occupancy whereas the same mutation decreases occupancy of RNA polymerase. Our results suggest that Pat- and Pta-mediated protein acetylation system promotes the expression of virulence genes by regulating the binding affinity of H-NS in S. Typhimurium.

YbdO Promotes the Pathogenicity of Escherichia coli K1 by Regulating Capsule Synthesis.

Escherichia coli K1 is the most popular neonatal meningitis-causing Gram-negative bacterium. As a key virulence determinant, the K1 capsule enhances the survival of E. coli K1 in human brain microvascular endothelial cells (HBMECs) upon crossing the blood–brain barrier; however, the regulatory mechanisms of capsule synthesis during E. coli K1 invasion of HBMECs remain unclear. Here, we identified YbdO as a transcriptional regulator that promotes E. coli K1 invasion of HBMECs by directly activating K1 capsule gene expression to increase K1 capsule synthesis. We found that ybdO deletion significantly reduced HBMEC invasion by E. coli K1 and meningitis occurrence in mice. Additionally, electrophoretic mobility shift assay and chromatin immunoprecipitation–quantitative polymerase chain reaction analysis indicated that YbdO directly activates kpsMT and neuDBACES expression, which encode products involved in K1 capsule transport and synthesis by directly binding to the kpsM promoter. Furthermore, ybdO transcription was directly repressed by histone-like nucleoid structuring protein (H-NS), and we observed that acidic pH similar to that of early and late endosomes relieves this transcriptional repression. These findings demonstrated the regulatory mechanism of YbdO on K1 capsule synthesis, providing further insights into the evolution of E. coli K1 pathogenesis and host–pathogen interaction.

Histone-like nucleoid structuring (H-NS) protein silences the beta-glucoside (bgl) utilization operon in Escherichia coli by forming a DNA loop

The bgl operon of Escherichia coli encodes proteins mediating the metabolism of aromatic beta-glucosides, but the operon is silent in wild type cells. Insertion of an insertion sequence (IS) element in the regulatory region upstream of the bgl promoter activates expression of the operon. The repression mechanism involves the histone-like nucleoid structuring (H-NS) protein with two DNA binding sites, one in the region upstream of the promoter, and the other within the first structural gene of the operon, bglG. The detailed mechanism of repression is not well understood. Here, we first show two terminators flanking bglG are not required for bgl operon silencing. Instead, several lines of experimental evidence clearly suggest that the silencing mechanism involves looping of the DNA between H-NS’s two DNA binding sites. H-NS is known to preferentially bind to AT-rich curved DNA, and such regions are found in the vicinity of both sites. We show that strong repression is abolished by (1) preventing H-NS self-oligomerization while retaining DNA binding, (2) preventing or reducing H-NS binding to the bgl operon regulatory region, and (3) preventing or reducing H-NS binding to the binding site in the bglG gene. We also show that the phase of the DNA between these two binding sites is not important, and that large insertions of DNA in the putative loop region do not diminish repression. These results imply that H-NS depends on DNA looping to exert strong repression.

Histone-Like Nucleoid Structuring Protein Modulates the Fitness of tet(X4)-Bearing IncX1 Plasmids in Gram-Negative Bacteria.

The emergence of plasmid-mediated tigecycline resistance gene tet(X4) poses a challenging threat to public health. Based on the analysis of tet(X4)-positive plasmids in the NCBI database, we found that the IncX1-type plasmid is one of the most common vectors for spreading tet(X4) gene, but the mechanisms by which these plasmids adapt to host bacteria and maintain the persistence of antibiotic resistance genes (ARGs) remain unclear. Herein, we investigated the underlying mechanisms of how host bacteria modulate the fitness cost of IncX1 plasmids carrying tet(X4) gene. Interestingly, we found that the tet(X4)-bearing IncX1 plasmids encoding H-NS protein imposed low or no fitness cost in Escherichia coli and Klebsiella pneumoniae; instead, they partially promoted the virulence and biofilm formation in host bacteria. Regression analysis revealed that the expression of hns gene in plasmids was positively linked to the relative fitness of host bacteria. Furthermore, when pCE2::hns was introduced, the fitness of tet(X4)-positive IncX1 plasmid pRF55-1 without hns gene was significantly improved, indicating that hns mediates the improvement of fitness. Finally, we showed that the expression of hns gene is negatively correlated with the expression of tet(X4) gene, suggesting that the regulatory effect of H-NS on adaptability may be attributed to its inhibitory effect on the expression of ARGs. Together, our findings suggest the important role of plasmid-encoded H-NS protein in modulating the fitness of tet(X4)-bearing IncX1 plasmids, which shed new insight into the dissemination of tet(X4) gene in a biological environment.