Solution structure of the C-terminal antiparallel coiled-coil domain from Escherichia coli osmosensor ProP

Bacteria respond to increasing medium osmolality by accumulating organic solutes that are compatible with cellular functions. Transporter ProP of Escherichia coli, a proton symporter and a member of the major facilitator superfamily, senses osmotic shifts and responds by importing osmolytes such as glycine betaine. ProP contains a cytoplasmic, C-terminal extension that is essential for its activity. A peptide corresponding to the C-terminal extension of ProP forms a homodimeric alpha-helical coiled-coil even though some of its heptad a positions are not occupied by hydrophobic amino acid residues. Unexpectedly, amino acid replacement R488I, occurring at a heptad a position, destabilized the coiled-coil formed by the ProP peptide and attenuated the response of the intact transporter to osmotic upshifts in vivo. Thus, ProP was proposed to dimerize via an antiparallel coiled-coil. We used nuclear magnetic resonance (NMR) spectroscopy to determine the structure of the synthetic peptide corresponding to residues 468-497 of ProP. This region did form an antiparallel coil-coil in which critical residue R488 specifies the antiparallel coiled-coil orientation by forming stabilizing salt-bridges. Charged residues (both acidic and basic) are clustered on the c/g surface of the coiled-coil whereas polar residues are distributed on the b/e surface. This causes the structure to be bent, in contrast to other known antiparallel coiled-coils (those from the hepatitis delta antigen (PDB ID code 1A92) and the bovine F(1) ATPase inhibitor protein (PDB ID code 1HF9)). The coiled-coil and its possible importance for osmosensing are discussed.     

A structural model for the osmosensor, transporter, and osmoregulator ProP of Escherichia coli

Transporter ProP of Escherichia coli, a member of the major facilitator superfamily (MFS), acts as an osmosensor and an osmoregulator in cells and after purification and reconstitution in proteoliposomes. H(+)-osmoprotectant symport via ProP is activated when medium osmolality is elevated with membrane impermeant osmolytes. The three-dimensional structure of ProP was modeled with the crystal structure of MFS member GlpT as a template. This GlpT structure represents the inward (or cytoplasm)-facing conformation predicted by the alternating access model for transport. LacZ-PhoA fusion analysis and site-directed fluorescence labeling substantiated the membrane topology and orientation predicted by this model and most hydropathy analyses. The model predicts the presence of a proton pathway within the N-terminal six-helix bundle of ProP (as opposed to the corresponding pathway found within the C-terminal helix bundle of its paralogue, LacY). Replacement of residues within the N-terminal helix bundle impaired the osmotic activation of ProP, providing the first indication that residues outside the C-terminal domain are involved in osmosensing. Some residues that were accessible from the periplasmic side, as predicted by the structural model, were more susceptible to covalent labeling in permeabilized membrane fractions than in intact bacteria. These residues may be accessible from the cytoplasmic side in structures not represented by our current model, or their limited exposure in vivo may reflect constraints on transporter structure that are related to its osmosensory mechanism.     

Purification and reconstitution of an osmosensor: transporter ProP of Escherichia coli senses and responds to osmotic shifts

The ProP protein of Escherichia coli is an osmoregulatory H+-compatible solute cotransporter. ProP is activated by an osmotic upshift in both whole cells and membrane vesicles. We are using biochemical and biophysical techniques to explore the osmosensory and catalytic mechanisms of ProP. We now report the purification and reconstitution of the active transporter. Protein purification was facilitated by the addition of six histidine (His) codons to the 3' end of proP. The recombinant gene was overexpressed from the E. coli galP promoter, and ProP-(His)6 was shown to be functionally equivalent to wild-type ProP by enzymatic assay of whole cells. ProP-(His)6, purified by Ni2+ (NTA) affinity chromatography, cross-reacted with antibodies raised against the ProP protein. ProP-(His)6 was reconstituted into Triton X-100 destabilized liposomes prepared with E. coli phospholipid. The reconstituted transporter mediated proline accumulation only if (1) a membrane potential was generated by valinomycin-mediated K+ efflux and (2) the proteoliposomes were subjected to an osmotic upshift (0.6 M sucrose). Activity was also stimulated by DeltapH. Pure ProP acts, in the proteoliposome environment, as sensor, transducer, and respondent to a hyperosmotic shift. It is the first such osmosensor to be isolated.     

Structure and function of transmembrane segment XII in osmosensor and osmoprotectant transporter ProP of Escherichia coli

Escherichia coli transporter ProP acts as both an osmosensor and an osmoregulator. As medium osmolality rises, ProP is activated and mediates H+-coupled uptake of osmolytes like proline. A homology model of ProP with 12-transmembrane (TM) helices and cytoplasmic termini was created, and the protein's topology was substantiated experimentally. Residues 468-497, at the end of the C-terminal domain and linked to TM XII, form an intermolecular, homodimeric alpha-helical coiled-coil that tunes the transporter's response to osmolality. We aim to further define the structure and function of ProP residues Q415-E440, predicted to include TM XII. Each residue was replaced with cysteine (Cys) in a histidine-tagged, Cys-less ProP variant (ProP*). Cys at positions 415-418 and 438-440 were most reactive with Oregon Green Maleimide (OGM), suggesting that residues 419 through 437 are in the membrane. Except for V429-I433, reactivity of those Cys varied with helical periodicity. Cys predicted to face the interior of ProP were more reactive than Cys predicted to face the lipid. The former may be exposed to hydrated polar residues in the protein interior, particularly on the periplasmic side. Intermolecular cross-links formed when ProP* variants with Cys at positions 419, 420, 422, and 439 were treated with DTME. Thus TM XII can participate, along its entire length, in the dimer interface of ProP. Cys substitution E440C rendered ProP* inactive. All other variants retained more than 30% of the proline uptake activity of ProP* at high osmolality. Most variants with Cys substitutions in the periplasmic half of TM XII activated at lower osmolalities than ProP*. Variants with Cys substitutions on one face of the cytoplasmic half of TM XII required a higher osmolality to activate. They included elements of a GXXXG motif that are predicted to form the interface of TM XII with TM VII. These studies define the position of ProP TM XII within the membrane, further support the predicted structure of ProP, reveal the dimerization interface, and show that the structure of TM XII influences the osmolality at which ProP activates.     

Redesigned purification yields a fully functional PutA protein dimer from Escherichia coli.

Proline utilization by Escherichia coli and Salmonella typhimurium requires expression of genes putP (encoding a proline transporter) and putA. Genetic data indicate that the PutA protein is both put repressor and a respiratory chain-linked dehydrogenase. We report a redesigned purification procedure as well as the physical characteristics and biological activities of the PutA protein purified from E. coli. The purified protein was homogeneous as determined by electrophoresis performed under denaturing and nondenaturing conditions. Its N-terminal sequence corresponded to that predicted by the DNA sequence. We showed copurification of proline and delta 1-pyrroline-5-carboxylate dehydrogenase activities. Purified PutA protein bound put DNA in vitro in an electrophoretic band-shift assay and it could be reconstituted to inverted membrane vesicles, yielding proline dehydrogenase activity. The Stokes radius and Svedberg coefficient of the protein were determined to be 7.1 nm and 9.9 S, respectively. These hydrodynamic data revealed that the protein in our preparation was dimeric with a molecular mass of 293 kDa and that it had an irregular shape indicated by the friction factor (f/f0) of 1.6.     

Requirements for osmosensing and osmotic activation of transporter ProP from Escherichia coli

Transporter ProP of Escherichia coli, a solute-H+ symporter, can sense and respond to osmotic upshifts imposed on cells, on membrane vesicles, or on proteoliposomes that incorporate purified ProP-(His)6. In this study, proline uptake catalyzed by ProP was used as a measure of its osmotic activation, and the requirements for osmosensing were defined using the proteoliposome system. The initial rate of proline uptake increased with decreasing external pH and increasing DeltaPsi, lumen negative. Osmotic upshifts increased DeltaPsi by concentrating lumenal K+, but osmotic activation of ProP could be distinguished from this effect. Osmotic activation of ProP resulted from changes in Vmax, though osmotic shifts also increased the KM for proline. Osmotic activation could be described as a reversible, osmotic upshift-dependent transition linking (at least) two transporter protein conformations. No correlation was observed between ProP activation and the position of the anions of activating sodium salts within the Hofmeister series of solutes. Both the magnitude of the osmotic upshift required to activate ProP and the ProP activity attained were similar for membrane-impermeant osmolytes, including NaCl, glucose, and PEG 600. The membrane-permeant osmolytes glycerol, urea, PEG 62, and PEG 106 failed to activate ProP. Two poly(ethylene glycol)s, PEG 150 and PEG 200, were membrane-permeant and did not cause liposome shrinkage, but they did partially activate ProP-(His)6.     

Formation of an antiparallel, intermolecular coiled coil is associated with in vivo dimerization of osmosensor and osmoprotectant transporter ProP in Escherichia coli

Membrane transporter ProP from Escherichia coli senses extracellular osmolality and responds by mediating the uptake of osmoprotectants such as glycine betaine when osmolality is high. Earlier EPR and NMR studies showed that a peptide replica of the cytoplasmic ProP carboxyl terminus (residues D468-R497) forms a homodimeric, antiparallel, alpha-helical coiled coil in vitro stabilized by electrostatic interactions involving R488. Amino acid replacement R488I disrupted coiled-coil formation by the ProP peptide, elevated the osmolality at which ProP became active, and rendered the osmolality response of ProP transient. In the present study, either E480 or K473 was replaced with cysteine (Cys) in ProP, a Cys-less, fully functional, histidine-tagged ProP variant, to use Cys-specific cross-linking approaches to determine if antiparallel coiled-coil formation and dimerization of the intact protein occur in vivo. The Cys at positions 480 would be closer in an antiparallel dimer than those at positions 473. These replacements did not disrupt coiled-coil formation by the ProP peptide. Partial homodimerization of variant ProP-E480C could be demonstrated in vivo and in membrane preparations via Cys-specific cross-linking with dithiobis(maleimidoethane) or by Cys oxidation to cystine by copper phenanthroline. In contrast, these reagents did not cross-link ProP with Cys at position 133 or 241. Cross-linking of ProP with Cys at position 473 was limited and occurred only if ProP was overexpressed, consistent with an antiparallel orientation of the coiled coil in the intact protein in vivo. Although replacement E480C did not alter transporter activity, replacement K473C reduced the extent and elevated the threshold for osmotic activation. K473 may play a role in ProP structure and function that is not reflected in altered coiled-coil formation by the corresponding peptide. Substitution R488I affected the activities of ProP-(His)(6), ProP-E480C, and ProP-K473C as it affected the activity of ProP. Surprisingly, it did not eliminate cross-linking of Cys at position 480, and it elevated cross-linking at position 473, even when ProP was expressed at physiological levels. This suggested that the R488I substitution may have changed the relative orientation of the C-termini within the dimeric protein from antiparallel to parallel, resulting in only transient osmotic activation. These results suggest that ProP is in monomer-dimer equilibrium in vivo. Dimerization may be mediated by C-terminal coiled-coil formation and/or by interactions between other structural domains, which in turn facilitate C-terminal coiled-coil formation. Antiparallel coiled-coil formation is required for activation of ProP at low osmolality.     

The sucrose permease of Escherichia coli: functional significance of cysteine residues and properties of a cysteine-less transporter

The sucrose (CscB) permease belongs to the oligosaccharide:H(+) symporter family of the Major Facilitator Superfamily and is homologous to the lactose permease from Escherichia coli. Sucrose transport in cells expressing sucrose permease is completely inhibited by N-ethylmaleimide (NEM), suggesting that one or more of the seven native Cys residues may be important for transport. In this paper, each Cys residue was individually replaced with Ser, and transport activity, membrane expression, and NEM sensitivity are documented. All seven single Cys-->Ser mutants are expressed normally in the membrane and catalyze sucrose transport with activities ranging from 80% to 180% of wild type. Six of the seven Ser mutants are completely inactivated by NEM, while Cys122-->Ser permease is insensitive to the sulfhydryl reagent, indicating that NEM inhibition results from alkylation of Cys122. Subsequently, a sucrose permease devoid of Cys residues (Cys-less) was constructed in which all Cys residues were replaced with Ser simultaneously by using a series of overlap-extension PCRs. Membrane expression and kinetic parameters for Cys-less [K(m) 4.8 mM, V(max) 192 nmol min(-1) (mg of protein)(-1)] are essentially identical to those of wild type [K(m) 5.4 mM, V(max) 196 nmol min(-1) (mg of protein)(-1)]. However, Cys-less permease catalyzes sucrose accumulation to steady-state levels that are approximately 2-fold higher than those of wild type. As anticipated, Cys-less permease is completely resistant to NEM inhibition. The observations demonstrate that Cys residues play no functional role in sucrose permease. Furthermore, the approach described to create the Cys-less transporter is generally applicable to other proteins. An application of Cys-less permease in the study of the substrate binding site is presented in the accompanying paper.     

Streamlined assessment of membrane permeability and its application to membrane engineering of Escherichia coli for octanoic acid tolerance

Journal of Industrial Microbiology & Biotechnology - The economic viability of bio-production processes is often limited by damage to the microbial cell membrane and thus there is a demand for...     

High salt-induced conversion of Escherichia coli GroEL into a fully functional thermophilic chaperonin

The GroE chaperonin system can adapt to and function at various environmental folding conditions. To examine chaperonin-assisted protein folding at high salt concentrations, we characterized Escherichia coli GroE chaperonin activity in 1.2 m ammonium sulfate. Our data are consistent with GroEL undergoing a conformational change at this salt concentration, characterized by elevated ATPase activity and increased exposure of hydrophobic surface, as indicated by increased binding of the fluorophore bis-(5, 5')-8-anilino-1-naphthalene sulfonic acid to the chaperonin. The presence of the salt results in increased substrate stringency and dependence on the full GroE system for release and productive folding of substrate proteins. Surprisingly, GroEL is fully functional as a thermophilic chaperonin in high concentrations of ammonium sulfate and is stable at temperatures up to 75 degrees C. At these extreme conditions, GroEL can suppress aggregation and mediate refolding of non-native proteins.     

ProP‐ProP and ProP‐phospholipid interactions determine the subcellular distribution of osmosensing transporter ProP in Escherichia coli

Osmosensing transporter ProP protects bacteria from osmotically induced dehydration by mediating the uptake of zwitterionic osmolytes. ProP activity is a sigmoidal function of the osmolality. ProP orthologues share an extended, cytoplasmic C‐terminal domain. Orthologues with and without a C‐terminal, α‐helical coiled‐coil domain respond similarly to the osmolality. ProP concentrates at the poles and septa of Escherichia coli cells in a cardiolipin (CL)‐dependent manner. The roles of phospholipids and the C‐terminal domain in subcellular localization of ProP were explored. Liposome association of peptides representing the C‐terminal domains of ProP orthologues and variants in vitro was compared with subcellular localization of the corresponding orthologues and variants in vivo. In the absence of coiled‐coil formation, the C‐terminal domain bound liposomes and ProP concentrated at the cell poles in a CL‐independent manner. The presence of the coiled‐coil replaced those phenomena with CL‐dependent binding and localization. The effects of amino acid replacements on lipid association of the C‐terminal peptide fully recapitulated their effects on the subcellular localization of ProP. These data suggest that polar localization of ProP results from association of its C‐terminal domain with the anionic lipid‐enriched membrane at the cell poles. The coiled‐coil domain present on only some orthologues renders that phenomenon CL‐dependent.     

The orientation of cytochromes in membrane multilayers prepared from aerobically grown Escherichia coli K12

Centrifugation of membrane vesicles, prepared from ultrasonically disrupted Escherichia coli K12, on to a planar surface followed by slow, partial dehydration results in a high degree of parallel orientation of the membrane planes with respect to each other and the supporting surface. Rotation of such membrane multilayers about a single axis parallel with the membrane planes within the magnetic field of an electron paramagnetic resonance (e.p.r.) spectrometer allows the orientation of anisotropic paramagnetic centres to be deduced. Computer simulations of the angular dependence of cytochrome e.p.r. spectra show two, or perhaps three, cytochromes, well-oriented with respect to the membrane plane. A low-spin cytochrome is oriented with the normal to its haem plane lying in the membrane plane. One (or perhaps two) high-spin cytochrome(s) lies with its haem plane making an angle of 45 degrees with the membrane plane. The orientation of the low-spin cytochrome haem is thus the same as that of haems in b-type cytochromes and cytochrome oxidases of the a type found in the mitochondria of higher animal and microbial cells and the bacterium Paracoccus denitrificans (Erecińska et al., 1979). The possible identity of this low-spin component as the terminal oxidase, cytochrome o, is discussed.     

Cardiolipin controls the osmotic stress response and the subcellular location of transporter ProP in Escherichia coli

The phospholipid composition of the membrane and transporter structure control the subcellular location and function of osmosensory transporter ProP in Escherichia coli. Growth in media of increasing osmolality increases, and entry to stationary phase decreases, the proportion of phosphatidate in anionic lipids (phosphatidylglycerol (PG) plus cardiolipin (CL)). Both treatments increase the CL:PG ratio. Transporters ProP and LacY are concentrated with CL (and not PG) near cell poles and septa. The polar concentration of ProP is CL-dependent. Here we show that the polar concentration of LacY is CL-independent. The osmotic activation threshold of ProP was directly proportional to the CL content of wild type bacteria, the PG content of CL-deficient bacteria, and the anionic lipid content of cells and proteoliposomes. CL was effective at a lower concentration in cells than in proteoliposomes, and at a much lower concentration than PG in either system. Thus, in wild type bacteria, osmotic induction of CL synthesis and concentration of ProP with CL at the cell poles adjust the osmotic activation threshold of ProP to match ambient conditions. ProP proteins linked by homodimeric, C-terminal coiled-coils are known to activate at lower osmolalities than those without such structures and coiled-coil disrupting mutations raise the osmotic activation threshold. Here we show that these mutations also prevent polar concentration of ProP. Stabilization of the C-terminal coiled-coil by covalent cross-linking of introduced Cys reverses the impact of increasing CL on the osmotic activation of ProP. Association of ProP C termini with the CL-rich membrane at cell poles may raise the osmotic activation threshold by blocking coiled-coil formation. Mutations that block coiled-coil formation may also block association of the C termini with the CL-rich membrane.     

Expression of soluble and fully functional ricin A chain in Escherichia coli is temperature-sensitive

Linkage of ricin A chain (RA) to a cell surface binding antibody or other ligand can result in a potent cytotoxic agent. We expressed the primary sequence for RA in Escherichia coli to facilitate production and to obtain protein free of naturally occurring contaminants, i.e. ricin B chain. Differences in the level of expression and in the characteristics of the expressed protein were noted when several different host/vector systems were tested. Recombinant RA (rRA) was expressed directly under control of the phage lambda major leftward promoter (PL) and the E. coli trp promoter. It was also expressed fused to E. coli alkaline phosphatase sequences, both in the same reading frame for secretion and out-of-reading frame for expression in a cistron-like arrangement. Expression in the PL promoter system, which is temperature-regulated, was achieved at 37 degrees C as well as at 42 degrees C. The protein expressed at these different temperatures had grossly different properties. Whereas rRA expressed at 37 degrees C was soluble and fully active, that produced at 42 degrees C was aggregated, insoluble, and reduced in activity. Soluble rRA could be converted to the insoluble form by incubation at 42 degrees C in vivo, but not in vitro. Hence, this difference in properties does not simply reflect an inherent thermal instability of the protein. Conditions present in vivo, including the possible association with other proteins, are apparently required for this effect on rRA.     

A positively charged region is a determinant of the orientation of cytoplasmic membrane proteins in Escherichia coli

Basic amino acid residues were introduced into an extracellular (periplasmic) domain, preceding a membrane-spanning hydrophobic domain, of SecY, an integral cytoplasmic membrane protein. The localization of the domain was monitored as to the alkaline phosphatase activity of TnPhoA fused adjacent to the domain. The alkaline phosphatase activity of such Escherichia coli cells drastically decreased when positive charges were introduced, indicating that on the introduction the SecY domain showed a change in localization from the periplasm to the cytoplasm. In another experiment, positive charges were introduced to the same periplasmic domain of another SecY-PhoA fusion protein, in which PhoA is fused to the cytoplasmic domain of SecY following the particular hydrophobic domain. The alkaline phosphatase activity increased drastically when positive charges were introduced, indicating that the SecY domain fused to PhoA showed a change in localization from the cytoplasm to the periplasm. In both experiments, the removal of a large amino-terminal portion of the SecY domain did not alter the effect of the positive charge introduction. Changes in localization of SecY domains thus demonstrated were also supported by a protease accessibility test on spheroplasts. It is proposed that a positively charged region adjacent to a membrane-embedded hydrophobic region tends to be stabilized on the cytoplasmic surface of the membrane, which in turn endows the hydrophobic region with the ability to act as a stop-transfer sequence or a signal sequence and consequently determines the orientation of the hydrophobic region in the membrane.     

Escherichia coli and its application in a mediated amperometric glucose sensor

Escherichia coli cells, which contain apo-glucose dehydrogenase, were used in constructing a mediated amperometric glucose sensor. The E. coli modified glucose sensor, which was prepared by immobilizing E. coli cells behind a dialysis membrane on a carbon paste electrode containing 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q(0)), produced a current for the electrocatalytic oxidation of glucose with Q(0) as an electron transfer mediator only after the addition of a trace amount of pyrroloquinoline quinone (PQQ), the cofactor of the enzyme. This allows a novel method of glucose measurements free from the interference of the redox active substances, if contained, in a sample solution. The glucose sensor was insensitive to dioxygen; the currents measured under anaerobic and aerobic conditions, and even under dioxygen saturated conditions were almost the same in magnitude at a given concentration of glucose over the range of 0.2-10 mM. Response time of the glucose sensor was 2 min to attain 90% level of the steady-state current. The E. coli modified glucose sensor was reusable when treated with ethylenediaminetetraacetic acid (EDTA). When E. coli cells were lyophilized, they could be stored at room temperature in a dry box for more than six months without loss of the catalytic activity.     

Studies of the Escherichia coli Trp repressor binding to its five operators and to variant operator sequences.

The Escherichia coli Trp repressor binds to promoters of very different sequence and intrinsic activity. Its mode of binding to trp operator DNA has been studied extensively yet remains highly controversial. In order to examine the selectivity of the protein for DNA, we have used electromobility shift assays (EMSAs) to study its binding to synthetic DNA containing the core sequences of each of its five operators and of operator variants. Our results for DNA containing sequences of two of the operators, trpEDCBA and aroH are similar to those of previous studies. Up to three bands of lower mobility than the free DNA are obtained which are assigned to complexes of stoichiometry 1 : 1, 2 : 1 and 3 : 1 Trp repressor dimer to DNA. The mtr and aroL operators have not been studied previously in vitro. For DNA containing these sequences, we observe predominantly one retarded band in EMSA with mobility corresponding to 2 : 1 complexes. We have also obtained retardation of DNA containing the trpR operator sequence, which has only been previously obtained with super-repressor Trp mutants. This gives bands with mobilities corresponding to 1 : 1 and 2 : 1 complexes. In contrast, DNA containing containing a symmetrized trpR operator sequence, trpRs, gives a single retarded band with mobility corresponding solely to a 1 : 1 protein dimer-DNA complex. Using trpR operator variants, we show that a change in a single base pair in the core 20 base pairs can alter the number of retarded DNA bands in EMSA and the length of the DNase I footprint observed. This shows that the binding of the second dimer is sequence selective. We propose that the broad selectivity of Trp repressor coupled to tandem 2 : 1 binding, which we have observed with all five operator sequences, enables the Trp repressor to bind to a limited number of sites with diverse sequences. This allows it to co-ordinately control promoters of different intrinsic strength. This mechanism may be of importance in a number of promoters that bind multiple effector molecules.     

Glycine and its N-methylated analogues cause pH-dependent membrane damage to enterotoxigenic Escherichia coli

The current study first investigates the emulsifying potential of glycine and its N-methylated derivatives N-methylglycine (sarcosine), N,N-dimethylglycine (DMG) and N,N,N-trimethylglycine (betaine) under varying pH conditions. Subsequently, the effect of these test compounds on the membrane integrity of enterotoxigenic Escherichia coli (ETEC) was evaluated. Oil in water emulsions containing each compound show that DMG is a more potent enhancer of emulsification than glycine, sarcosine and betaine under the conditions tested. Flow cytometry was used to investigate whether the emulsifying potential is associated with an effect on ETEC membrane integrity. The bacteria were exposed to each of the test compounds under varying pH conditions and membrane integrity was assessed using the LIVE/DEAD BacLight kit. Results show a membrane deteriorating effect caused by glycine, sarcosine and DMG, but not by betaine. This effect is pH- and time-dependent and has an apparent threshold at pH 9.0. Conventional plate counts confirmed concomitant changes in culturability of the membrane comprised bacteria.