RESUMO
In the search for the origin of Amyotrophic Lateral Sclerosis disease (ALS), we hypothesized earlier (Monselise, 2019) that D-amino acids produced by stressed microbiome may serve as inducers of the disease development. Many examples of D-amino acid accumulation under various stress conditions were demonstrated in prokaryotic and eukaryotic cells. In this work, wild-type Escherichia coli, members of the digestive system, were subjected to carbon and nitrogen starvation stress. Using NMR and LC-MS techniques, we found for the first time that D-glutamate accumulated in the stressed bacteria but not in control cells. These results together with the existing knowledge, allow us to suggest a new insight into the pathway of ALS development: D-glutamate, produced by the stressed microbiome, induces neurobiochemical miscommunication setting on C1q of the complement system. Proving this insight may have great importance in preventive medicine of such MND modern-age diseases as ALS, Alzheimer, and Parkinson.
Assuntos
Esclerose Lateral Amiotrófica , Escherichia coli , Ácido Glutâmico , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/microbiologia , Escherichia coli/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Estresse Fisiológico , Complemento C1q/metabolismo , Nitrogênio/metabolismo , Carbono/metabolismoRESUMO
Segregation of the replicating chromosome from a single to two nucleoid bodies is one of the major processes in growing bacterial cells. The segregation dynamics is tuned by intricate interactions with other cellular processes such as growth and division, ensuring flexibility in a changing environment. We hypothesize that the internal stochasticity of the segregation process may be the source of cell-to-cell phenotypic variability, in addition to the well-established gene expression noise and uneven partitioning of low copy number components. We compare dividing cell lineages with filamentous cells, where the lack of the diffusion barriers is expected to reduce the impact of other factors on the variability of nucleoid segregation dynamics. The nucleoid segregation was monitored using time-lapse microscopy in live E. coli cells grown in linear grooves. The main characteristics of the segregation process, namely, the synchrony of partitioning, rates of separation, and final positions, as well as the variability of these characteristics, were determined for dividing and filamentous lineages growing under the same conditions. Indeed, the gene expression noise was considerably homogenized along filaments as determined from the distribution of CFP and YFP stochastically expressed from the chromosome. We find that 1) the synchrony of nucleoid partitioning is progressively decreasing during consecutive cell cycles, but to a significantly lesser degree in filamentous than in dividing cells; 2) the mean partitioning rate of nucleoids is essentially the same in dividing and filamentous cells, displaying a substantial variability in both; and 3) nucleoids segregate to the same distances in dividing and filamentous cells. Variability in distances is increasing during successive cell cycles, but to a much lesser extent in filamentous cells. Our findings indicate that the variability of the chromosome segregation dynamics is reduced upon removal of boundaries between nucleoids, whereas the remaining variability is essentially inherent to the nucleoid itself.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Proteínas de Bactérias/genética , Variação Biológica da População , Segregação de Cromossomos , Cromossomos Bacterianos/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genéticaRESUMO
Duplication of the bacterial nucleoid is necessary for cell division hence specific arrest of DNA replication inhibits divisions culminating in filamentation, nucleoid dispersion and appearance of a-nucleated cells. It is demonstrated here that during the first 10 min however, Escherichia coli enhanced residual divisions: the proportion of constricted cells doubled (to 40%), nucleoids contracted and cells remodelled dimensions: length decreased and width increased. The preliminary data provides further support to the existence of temporal and spatial couplings between the nucleoid/replisome and the sacculus/divisome, and is consistent with the idea that bacillary bacteria modulate width during the division process exclusively.
Assuntos
Replicação do DNA , Escherichia coli/citologia , Escherichia coli/genética , Divisão Celular , Nucléolo Celular/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMO
AIM: Mycobacterium tuberculosis possesses an intracellular tagging and degradation system, which has emerged as a target for development of anti-tuberculosis agents. In this system, PafA is the ligase that marks proteins for degradation by their covalent modification with a protein modifier. Here, we studied pafA transcriptional regulation, which remained elusive despite its importance for M. tuberculosis virulence. MATERIALS & METHODS: Working with Mycobacterium smegmatis, a mycobacterial model organism, we examined the involvement of the global regulators PafB and PafC in pafA regulation. RESULTS: PafBC activated pafA transcription following DNA damage, resulting in efficient cellular recovery. CONCLUSION: The results unraveled the involvement of PafBC in pafA transcription, and revealed the importance of proper PafA regulation in mycobacterial physiology.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Mycobacterium smegmatis/genética , Transcrição Gênica , Ubiquitina-Proteína Ligases/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Dano ao DNA , Primers do DNA , DNA Bacteriano , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/metabolismo , Deleção de Sequência , Ubiquitina-Proteína Ligases/metabolismo , Virulência/genéticaRESUMO
The Z-ring plays a central role in bacterial division. It consists of FtsZ filaments, but the way these reorganize in the ring-like structure during septation remains largely unknown. Here, we measure the effective constriction dynamics of the ring. Using an oscillating optical trap, we can switch individual rod-shaped E. coli cells between horizontal and vertical orientations. In the vertical orientation, the fluorescent Z-ring image appears as a symmetric circular structure that renders itself to quantitative analysis. In the horizontal orientation, we use phase-contrast imaging to determine the extent of the cell constriction and obtain the effective time of division. We find evidence that the Z-ring constricts at a faster rate than the cell envelope such that its radial width (inwards from the cytoplasmic membrane) grows during septation. In this respect, our results differ from those recently obtained using photoactivated localization microscopy (PALM) where the radial width of the Z-ring was found to be approximately constant as the ring constricts. A possible reason for the different behavior of the constricting Z-rings could be the significant difference in the corresponding cell growth rates.
RESUMO
DNA-binding proteins play an important role in maintaining bacterial chromosome structure and functions. Heat-unstable (HU) histone-like protein is one of the most abundant of these proteins and participates in all major chromosome-related activities. Owing to its low sequence specificity, HU fusions with fluorescent proteins were used for general staining of the nucleoid, aiming to reveal its morphology and dynamics. We have exploited a single chromosomal copy of hupA-egfp fusion under the native promoter and used quantitative microscopy imaging to investigate the amount and dynamics of HUα in Escherichia coli cells. We found that in steady-state growing populations the cellular HUα content is proportional to the cell size, whereas its concentration is size independent. Single-cell live microscopy imaging confirmed that the amount of HUα exponentially increases during the cell cycle, but its concentration is maintained constant. This supports the existence of an auto-regulatory mechanism underlying the HUα cellular level, in addition to reflecting the gene copy number. Both the HUα amount and concentration strongly increase with the cell growth rate in different culture media. Unexpectedly, the HU/DNA stoichiometry also remarkably increases with the growth rate. This last finding may be attributed to a higher requirement for maintaining the chromosome structure in nucleoids with higher complexity.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/citologia , Escherichia coli/metabolismo , Proteínas de Transporte/genética , Ciclo Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , CinéticaRESUMO
DnaA, the initiator of chromosome replication in most known eubacteria species, is activated once per cell division cycle. Its overall activity cycle is driven by ATP hydrolysis and ADP-ATP exchange. The latter can be promoted by binding to specific sequences on the chromosome and/or to acidic phospholipids in the membrane. We have previously shown that the transition into an active form (rejuvenation) is strongly co-operative with respect to DnaA membrane occupancy. Only at low membrane occupancy is DnaA reactivation efficiently catalysed by the acidic phospholipids. The present study was aimed at unravelling the molecular mechanism underlying the occupancy-dependent DnaA rejuvenation. We found that truncation of the DnaA N-terminal completely abolishes the co-operative transformation between the high and low occupancy states (I and II respectively) without affecting the membrane binding. The environmentally sensitive fluorophore specifically attached to the N-terminal cysteines of DnaA reported on occupancy-correlated changes in its vicinity. Cross-linking of DnaA with a short homobifunctional reagent revealed that state II of the protein on the membrane corresponds to a distinct oligomeric form of DnaA. The kinetic transition of DnaA on the membrane surface is described in the present study by a generalized 2D condensation phase transition model, confirming the existence of two states of DnaA on the membrane and pointing to the possibility that membrane protein density serves as an on-off switch in vivo. We conclude that the DnaA conformation attained at low surface density drives its N-terminal-mediated oligomerization, which is presumably a pre-requisite for facilitated nt exchange.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Replicação do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Escherichia coli/química , Escherichia coli/genética , Cinética , Mutação , Transição de Fase , Conformação Proteica , Multimerização ProteicaRESUMO
The bacterial membrane exhibits a significantly heterogeneous distribution of lipids and proteins. This heterogeneity results mainly from lipid-lipid, protein-protein, and lipid-protein associations which are orchestrated by the coupled transcription, translation and insertion of nascent proteins into and through membrane (transertion). Transertion is central not only to the individual assembly and disassembly of large physically linked groups of macromolecules (alias hyperstructures) but also to the interactions between these hyperstructures. We review here these interactions in the context of the processes in Bacillus subtilis and Escherichia coli of nutrient sensing, membrane synthesis, cytoskeletal dynamics, DNA replication, chromosome segregation, and cell division.
RESUMO
The spatial organization of the Z-ring, the central element of the bacterial division machinery, is not yet fully understood. Using optical tweezers and subpixel image analysis, we have recently shown that the radial width of the Z-ring in unconstricted Escherichia coli is about 100 nm. The relatively large width is consistent with the observations of others. Moreover, simulation of the experimental FtsZ distribution using the theoretical three-dimensional (3D) point spread function was strongly in favour of a toroidal rather than a thin cylindrical model of the Z-ring. Here, we show that the low density of FtsZ filaments in the ring coincides within experimental uncertainty with the critical density of a 3D random network of cylindrical sticks. This suggests that the Z-ring may consist of a percolating network of FtsZ filaments. Several factors that are expected to affect the polymerization state and the extent of self-interaction of FtsZ within the Z-ring, as well as the functional implications of its sparse toroidal structure, are discussed in terms of percolation theory.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/ultraestrutura , Escherichia coli/química , Escherichia coli/ultraestrutura , Divisão Celular , Simulação por Computador , Modelos Biológicos , Pinças ÓpticasRESUMO
The bacterial membrane is characterized by a heterogeneous distribution of lipids and proteins and of higher level structures termed hyperstructures. The causes of this heterogeneity include lipid-lipid, protein-protein and protein-lipid interactions. The coupling of transcription, translation and insertion of nascent proteins into membrane, transertion, creates large membrane domains that are proposed to be important in the regulation and execution of the cell cycle and in other functions. In describing membrane heterogeneity, we suggest here that transertion is a global regulator coupling metabolism to the cell cycle.
Assuntos
Fenômenos Fisiológicos Bacterianos , Ciclo Celular , Membrana Celular/metabolismo , Regulação Bacteriana da Expressão Gênica , Metabolismo dos Lipídeos , Proteínas de Membrana/metabolismo , Biossíntese de Proteínas , Transcrição GênicaRESUMO
DnaA initiates chromosome replication in most known bacteria and its activity is controlled so that this event occurs only once every cell division cycle. ATP in the active ATP-DnaA is hydrolyzed after initiation and the resulting ADP is replaced with ATP on the verge of the next initiation. Two putative recycling mechanisms depend on the binding of DnaA either to the membrane or to specific chromosomal sites, promoting nucleotide dissociation. While there is no doubt that DnaA interacts with artificial membranes in vitro, it is still controversial as to whether it binds the cytoplasmic membrane in vivo. In this work we looked for DnaA-membrane interaction in E. coli cells by employing cell fractionation with both native and fluorescent DnaA hybrids. We show that about 10% of cellular DnaA is reproducibly membrane-associated. This small fraction might be physiologically significant and represent the free DnaA available for initiation, rather than the vast majority bound to the datA reservoir. Using the combination of mCherry with a variety of DnaA fragments, we demonstrate that the membrane binding function is delocalized on the surface of the protein's domain III, rather than confined to a particular sequence. We propose a new binding-bending mechanism to explain the membrane-induced nucleotide release from DnaA. This mechanism would be fundamental to the initiation of replication.
Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Cromossomos Bacterianos/genética , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/citologia , Escherichia coli/genética , Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Escherichia coli/metabolismo , Espaço Intracelular/metabolismo , Proteínas Luminescentes/metabolismo , Modelos Moleculares , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteína Vermelha FluorescenteRESUMO
We take the advantage of pyrene's unique spectral properties as a reliable polarity indicator to monitor pyrene localizations in the membrane depth by using wavelength selective fluorescence approach. We show that fine structure of pyrene fluorescence emission spectra and excimerization rate in model and native phospholipid membranes depend on the excitation wavelength. This phenomenon is not observed in neat solvents. In membranes, the dependence on the excitation wavelength reflects selective excitation of pyrene molecules located close to the membrane-water polar interface, or deep in the hydrophobic core of the membrane, verified with the aid of pyrene derivatives of fatty acids of various lengths.
Assuntos
Lipídeos de Membrana/química , Pirenos/química , Ácidos Graxos/química , Bicamadas Lipídicas/química , Fluidez de Membrana , Espectrometria de FluorescênciaRESUMO
In Escherichia coli and other bacteria, MinD, along with MinE and MinC, rapidly oscillates from one pole of the cell to the other controlling the correct placement of the division septum. MinD binds to the membrane through its amphipathic C-terminal alpha-helix. This binding, promoted by ATP-induced dimerization, may be further enhanced by a consequent attraction of acidic phospholipids and formation of a stable proteolipid domain. In the context of this hypothesis we studied changes in dynamics of a model membrane caused by MinD binding using membrane-embedded fluorescent probes as reporters. A remarkable increase in membrane viscosity and order upon MinD binding to acidic phospholipids was evident from the pyrene and DPH fluorescence changes. This viscosity increase is cooperative with regards to the concentration of MinD-ATP, but not of the ADP form, indicative of dimerization. Moreover, similar changes in the membrane dynamics were demonstrated in the native inverted cytoplasmic membranes of E. coli, with a different depth effect. The mobility of pyrene-labeled phosphatidylglycerol indicated formation of acidic phospholipid-enriched domains in a mixed acidic-zwitterionic membrane at specific MinD/phospholipid ratios. A comparison between MinD from E. coli and Neisseria gonorrhea is also presented.
Assuntos
Adenosina Trifosfatases/metabolismo , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Adenosina Trifosfatases/farmacologia , Trifosfato de Adenosina/metabolismo , Membrana Celular/efeitos dos fármacos , Difusão/efeitos dos fármacos , Escherichia coli/citologia , Proteínas de Escherichia coli/farmacologia , Polarização de Fluorescência , Lipossomos/metabolismo , Fosfolipídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Pirenos/farmacologiaRESUMO
MinD, a well-conserved bacterial amphitropic protein involved in spatial regulation of cell division, has a typical feature of reversible binding to the membrane. MinD shows a clear preference for acidic phospholipids organized into lipid domains in bacterial membrane. We have shown that binding of MinD may change the dynamics of model and native membranes (see accompanying paper [1]). On the other hand, MinD dimerization and anchoring could be enhanced on pre-existing anionic phospholipid domains. We have tested MinD binding to model membranes in which acidic and zwitterionic phospholipids are either well-mixed or segregated to phase domains. The phase separation was achieved in binary mixtures of 1-Stearoyl-2-Oleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol] (SOPG) with 1,2-Distearoyl-sn-Glycero-3-Phosphocholine (DSPC) or 1,2-Distearoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DSPG) and binding to these membranes was compared with that to a fluid mixture of SOPG with 1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (SOPC). The results demonstrate that MinD binding to the membrane is enhanced by segregation of anionic phospholipids to fluid domains in a gel-phase environment and, moreover, the protein stabilizes such domains. This suggests that an uneven binding of MinD to the heterogeneous native membrane is possible, leading to formation of a lipid-specific distribution pattern of MinD and/or modulation of its temporal behavior.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Varredura Diferencial de Calorimetria , Polarização de Fluorescência , Fluidez de Membrana , Transição de Fase , Fosfatidilcolinas/metabolismo , Ligação ProteicaRESUMO
Bacteria are the simplest living organisms. In particular, Escherichia coli has been extensively studied and it has become one of the standard model systems in microbiology. However, optical microscopy studies of single E. coli have been limited by its small size, approximately 1 x 3 microm, not much larger than the optical resolution, approximately 0.25 microm. As a result, not enough quantitative dynamical information on the life cycle of single E. coli is presently available. We suggest that, by careful analysis of images from phase contrast and fluorescence time-lapse microscopy, this limitation can be bypassed. For example, we show that applying this approach to monitoring morphogenesis in individual E. coli leads to a simple, quantitative description of this process. First, we find the time when the formation of the septum starts, tau(c). It occurs much earlier than the time when the constriction can be directly observed by phase contrast. Second, we find that the growth law of single cells is more likely bilinear/trilinear than exponential. This is further supported by the relations that hold between the corresponding growth rates. These methods could be further extended to study the dynamics of cell components, e.g., the nucleoid and the Z-ring.
Assuntos
Escherichia coli/citologia , Escherichia coli/crescimento & desenvolvimento , Interpretação de Imagem Assistida por Computador/métodos , Modelos Biológicos , Crescimento Celular , Simulação por ComputadorRESUMO
DnaA(L366K), in concert with a wild-type DnaA (wtDnaA) protein, restores the growth of Escherichia coli cells arrested in the absence of adequate levels of cellular acidic phospholipids. In vitro and in vivo studies showed that DnaA(L366K) alone does not induce the initiation of replication, and wtDnaA must also be present. Hitherto the different behavior of wt and mutant DnaA were not understood. We now demonstrate that this mutant may be activated at significantly lower concentrations of acidic phospholipids than the wild-type protein, and this may explain the observed growth restoration in vivo.
Assuntos
Cromossomos Bacterianos/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Fosfolipídeos/metabolismo , Ácidos/química , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Cinética , Lipossomos/metabolismo , Mutação , Fosfolipídeos/química , Ligação ProteicaRESUMO
The morphology and dynamics of DNA in a bacterial nucleoid affects the kinetics of such major processes as DNA replication, gene expression. and chromosome segregation. In this work, we have applied fluorescence correlation spectroscopy to assess the structure and internal dynamics of isolated Escherichia coli nucleoids. We show that structural information can be extracted from the amplitude of fluorescence correlation spectroscopy correlation functions of randomly labeled nucleoids. Based on the developed formalism we estimate the characteristic size of nucleoid structural units for native, relaxed, and positively supercoiled nucleoids. The degree of supercoiling was varied using the intercalating agent chloroquine and evaluated from fluorescence microscopy images. The relaxation of superhelicity was accompanied by 15-fold decrease in the length of nucleoid units (from approximately 50 kbp to approximately 3 kbp).
Assuntos
DNA Bacteriano/metabolismo , DNA Bacteriano/ultraestrutura , Escherichia coli/genética , Escherichia coli/ultraestrutura , Microscopia de Fluorescência/métodos , Espectrometria de Fluorescência/métodosRESUMO
DnaA is the initiator protein for chromosomal replication in bacteria; its activity plays a central role in the timing of the primary initiations within the Escherichia coli cell cycle. A controlled, reversible conversion between the active ATP-DnaA and the inactive ADP forms modulates this activity. In a DNA-dependent manner, bound ATP is hydrolyzed to ADP. Acidic phospholipids with unsaturated fatty acids are capable of reactivating ADP-DnaA by promoting the release of the tightly bound ADP. The nucleotide dissociation kinetics, measured in the present study with the fluorescent derivative 3'-O-(N-methylantraniloyl)-5'-adenosine triphosphate, was dependent on the density of DnaA on the membrane in a cooperative manner: it increased 5-fold with decreased protein density. At all surface densities the nucleotide was completely released, presumably due to protein exchange on the membrane. Distinct temperature dependences and the effect of the crowding agent Ficoll suggest that two functional states of DnaA exist at high and low membrane occupancy, ascribed to local macromolecular crowding on the membrane surface. These novel phenomena are thought to play a major role in the mechanism regulating the initiation of chromosomal replication in bacteria.
Assuntos
Proteínas de Bactérias/fisiologia , Membrana Celular/metabolismo , Proteínas de Ligação a DNA/fisiologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Trifosfato de Adenosina/farmacologia , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Corantes Fluorescentes/farmacologia , Hidrólise , Cinética , Modelos Químicos , Conformação Molecular , Nucleotídeos/química , Fosfolipídeos/química , Ligação Proteica , Temperatura , ortoaminobenzoatos/farmacologiaRESUMO
Phase variation in the colonial opacity of Streptococcus pneumoniae has been implicated as a factor in the pathogenesis of pneumococcal disease. This study examined the relationship between membrane characteristics and colony morphology in a few selected opaque-transparent couples of S. pneumoniae strains carrying different capsular types. Membrane fluidity was determined on the basis of intermolecular excimerization of pyrene and fluorescence polarization of 1,6-diphenyl 1,3,5-hexatriene (DPH). A significant decrease, 16 to 26% (P < or = 0.05), in the excimerization rate constant of the opaque variants compared with that of the transparent variants was observed, indicating higher microviscosity of the membrane of bacterial cells in the opaque variants. Liposomes prepared from phospholipids of the opaque phenotype showed an even greater decrease, 27 to 38% (P < or = 0.05), in the pyrene excimerization rate constant compared with that of liposomes prepared from phospholipids of bacteria with the transparent phenotype. These findings agree with the results obtained with DPH fluorescence anisotropy, which showed a 9 to 21% increase (P < or = 0.001) in the opaque variants compared with the transparent variants. Membrane fatty acid composition, determined by gas chromatography, revealed that the two variants carry the same types of fatty acids but in different proportions. The trend of modification points to the presence of a lower degree of unsaturated fatty acids in the opaque variants compared with their transparent counterparts. The data presented here show a distinct correlation between phase variation and membrane fluidity in S. pneumoniae. The changes in membrane fluidity most probably stem from the observed differences in fatty acid composition.