Doreen E. Culham

Cardiolipin promotes polar localization of osmosensory transporter ProP in Escherichia coli

The osmolality required to activate osmosensory transporter ProP and the proportion of cardiolipin (CL) among the phospholipids of Escherichia coli rise with growth medium osmolality. Most CL synthesis has been attributed to the cls gene product. Transcription of cls increased with osmolality. The proportion of CL was low and osmolality‐independent in cls– bacteria. It increased more dramatically on the transition to stationary phase in cls– than cls+ bacteria. Thus, Cls is responsible for osmoregulated CL synthesis and other enzymes may contribute to CL accumulation during stationary phase. The proportion of phosphatidylglycerol (PG) was elevated and it increased with medium osmolality in cls– bacteria. A cls defect impaired growth of E. coli on solid and in liquid media at low and, more strongly, at high osmolality. Bacteria cultured at high osmolality without osmoprotectant were shorter and rounder than those cultured at low osmolality or with glycine betaine. Fluorescence microscopy showed that CL and ProP colocalize at the poles and near the septa of dividing E. coli cells. The polar localization of ProP was independent of its expression level but correlated with the proportion and polar localization of CL. Association with CL (and not PG) may be required for polar ProP localization.

The ion coupling and organic substrate specificities of osmoregulatory transporter ProP in Escherichia coli.

Transporter ProP of Escherichia coli, a member of the major facilitator superfamily, mediates osmoprotective proline or glycine betaine accumulation by bacteria exposed to high osmolality environments. Morpholinopropane sulfonic acid, a common constituent of microbiological media, accumulates in osmoadapting E. coli cells but it is not osmoprotective and it did not influence proP transcription or ProP activity. The apparent K(m) for proline uptake via ProP increased with decreasing pH in the range 7.5-4. ProP-dependent proline uptake by de-energized bacteria was associated with alkalinization of the external medium. Thus ProP mediates cotransport of H(+) and zwitterionic proline and a transporter functional group with a pK(a) of 5-6 is implicated in catalysis. Exogenous proline or glycine betaine elicits K(+) release from osmoadapting E. coli cells and ProP activity is stimulated by exogenous K(+). However, uptake of proline or glycine betaine stimulated K(+) efflux from K(+)-loaded bacteria which expressed either ProP or alternative, osmoregulatory transporter ProU. This indicated that ProP was unlikely to mediate K(+) efflux. Zwitterions ectoine, pipecolate, proline betaine, N,N-dimethylglycine, carnitine and 1-carboxymethylpyridinium were identified as alternative ProP substrates. Choline, a cation and a structural analogue of glycine betaine, was a low affinity inhibitor but not a substrate of ProP.

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.

The osmotic stress response and virulence in pyelonephritis isolates of Escherichia coli: contributions of RpoS, ProP, ProU and other systems

Trehalose synthesis (RpoS-dependent) and betaine uptake mediated by transporters ProP and ProU contribute to the osmotolerance of Escherichia coli K-12. Pyelonephritis isolates CFT073 and HU734 were similar and diminished in osmotolerance, respectively, compared to E. coli K-12. The roles of RpoS, ProP and ProU in osmoregulation and urovirulence were assessed for these isolates. Strain HU734 expressed an RpoS variant which had low activity and a C-terminal extension. This bacterium accumulated very little trehalose and had poor stationary-phase thermotolerance. For E. coli CFT073, introduction of an rpoS deletion impaired trehalose accumulation, osmotolerance and stationary-phase thermotolerance. The rpoS defects accounted for the difference in osmotolerance between these strains in minimal medium of very high osmolality (1.4 mol kg(-1)) but not in medium of lower osmolality (0.4 mol kg(-1)). The slow growth of both pyelonephritis isolates in high-osmolality medium was stimulated by glycine betaine (GB) and deletion of proP and/or proU impaired GB uptake. An HU734 derivative lacking both proP and proU retained osmoprotective GB uptake activity that could be attributed to system BetU, which is not present in strain K-12 or CFT073. BetU transported GB (K(m), 22 microM) and proline betaine. High-osmolality human urine (0.92 mol kg(-1)) included membrane-permeant osmolyte urea (0.44 M) plus other constituents which contributed an osmolality of only approximately 0.4 mol kg(-1). Strains HU734 and CFT073 showed correspondingly low GB uptake activities after cultivation in this urine. Deletion of proP and proU slowed the growth of E. coli HU734 in this high-osmolality human urine (which contains betaines) but had little impact on its colonization of the murine urinary tract after transurethral inoculation. By contrast, deletion of rpoS, proP and proU had no effect on the very rapid growth of CFT073 in high-osmolality urine or on its experimental colonization of the murine urinary tract. RpoS-dependent gene expression is not essential for growth in human urine or colonization of the murine urinary tract. Additional osmoregulatory systems, some not present in E. coli K-12 (e.g. BetU), may facilitate growth of pyelonephritis isolates in human urine and colonization of mammalian urinary tracts. The contributions of systems ProP and ProU to urinary tract colonization cannot be definitively assessed until all such systems are identified.

Osmoregulatory transporter ProP influences colonization of the urinary tract by Escherichia coli.

Osmoregulatory transporters ProP and ProU mediate the use of betaines as osmoprotectants by Escherichia coli. Glycine betaine and proline betaine are present in mammalian urines. Betaine uptake may therefore facilitate the growth of E. coli in the urinary tract, an environment of fluctuating osmolality. ProP transporter activity was approximately threefold higher in a pyelonephritis isolate, E. coli HU734, than in E. coli K-12. The growth rate of E. coli HU734 in aerated minimal salts medium was reduced twofold by 0.2 M NaCl in the absence and by 0.55 M NaCl in the presence of glycine betaine. Maximal growth rate stimulation was achieved when glycine betaine was added at a concentration as low as 25 microM. Deletion of the proP locus impaired the growth rate of E. coli HU734 in human urine but not in minimal medium supplemented with NaCl (0.4 M), with or without glycine betaine (0.1 mM). The expression of pyelonephritis-associated (P) pili was reduced when E. coli HU734 was cultured in a rich culture medium (LB) of elevated salinity. The proP lesion had no influence on P pilus expression in vitro or on the recovery of bacteria from the kidneys of inoculated mice. However, it did reduce their recovery from the bladders of inoculated mice 100-fold. These data provide the first direct evidence that osmoprotective betaine accumulation and transporter ProP are pertinent to both growth in human urine and colonization of the murine urinary tract by uropathogenic E. coli.

ProQ is an RNA chaperone that controls ProP levels in Escherichia coli.

Transporter ProP mediates osmolyte accumulation in Escherichia coli cells exposed to high osmolality media. The cytoplasmic ProQ protein amplifies ProP activity by an unknown mechanism. The N- and C-terminal domains of ProQ are predicted to be structurally similar to known RNA chaperone proteins FinO and Hfq from E. coli. Here we demonstrate that ProQ is an RNA chaperone, binding RNA and facilitating both RNA strand exchange and RNA duplexing. Experiments performed with the isolated ProQ domains showed that the FinO-like domain serves as a high-affinity RNA-binding domain, whereas the Hfq-like domain is largely responsible for RNA strand exchange and duplexing. These data suggest that ProQ may regulate ProP production. Transcription of proP proceeds from RpoD- and RpoS-dependent promoters. Lesions at proQ affected ProP levels in an osmolality- and growth phase-dependent manner, decreasing ProP levels when proP was expressed from its own chromosomal promoters or from a heterologous plasmid-based promoter. Small RNA molecules are known to regulate cellular levels of sigma factor RpoS. ProQ did not act by changing RpoS levels since proQ lesions did not influence RpoS-dependent stationary phase thermotolerance and they affected ProP production and activity similarly in bacteria without and with an rpoS defect. Taken together, these results suggest that ProQ does not regulate proP transcription. It may act as an RNA-binding protein to regulate proP translation.

Osmoregulatory Systems of Escherichia coli: Identification of Betaine-Carnitine-Choline Transporter Family Member BetU and Distributions of betU and trkG among Pathogenic and Nonpathogenic Isolates

Multiple transporters mediate osmoregulatory solute accumulation in Escherichia coli K-12. The larger genomes of naturally occurring strains such as pyelonephritis isolates CFT073 and HU734 may encode additional osmoregulatory systems. CFT073 is more osmotolerant than HU734 in the absence of organic osmoprotectants, yet both strains grew in high osmolality medium at low K(+) (micromolar concentrations) and retained locus trkH, which encodes an osmoregulatory K(+) transporter. Both lacked the trkH homologue trkG. Transporters ProP and ProU account for all glycine-betaine uptake activity in E. coli K-12 and CFT073, but not in HU734, yet elimination of ProP and ProU impairs the growth of HU734, but not CFT073, in high osmolality human urine. No known osmoprotectant stimulated the growth of CFT073 in high osmolality minimal medium, but putative transporters YhjE, YiaMNO, and YehWXYZ may mediate uptake of additional osmoprotectants. Gene betU was isolated from HU734 by functional complementation and shown to encode a betaine uptake system that belongs to the betaine-choline-carnitine transporter family. The incidence of trkG and betU within the ECOR collection, representatives of the E. coli pathotypes (PATH), and additional strains associated with urinary tract infection (UTI) were determined. Gene trkG was present in 66% of the ECOR collection but only in 16% of the PATH and UTI collections. Gene betU was more frequently detected in ECOR groups B2 and D (50% of isolates) than in groups A, B1, and E (20%), but it was similar in overall incidence in the ECOR collection and in the combined UTI and PATH collections (32 and 34%, respectively). Genes trkG and betU may have been acquired by lateral gene transfer, since trkG is part of the rac prophage and betU is flanked by putative insertion sequences. Thus, BetU and TrkG contribute, with other systems, to the osmoregulatory capacity of the species E. coli, but they are not characteristic of a particular phylogenetic group or pathotype.

The role of the carboxyl terminal α‐helical coiled‐coil domain in osmosensing by transporter ProP of Escherichia coli

Concentrative uptake of osmoprotectants via transporter ProP contributes to the rehydration of Escherichia coli cells that encounter high osmolality media. A member of the major facilitator superfamily, ProP is activated by osmotic upshifts in whole bacteria, in cytoplasmic membrane vesicles and in proteoliposomes prepared with the purified protein. Soluble protein ProQ is also required for full osmotic activation of ProP in vivo. ProP is differentiated from structural and functional homologues by its osmotic activation and its C‐terminal extension, which is predicted to form an α‐helical coiled‐coil. A synthetic polypeptide corresponding to the C‐terminus of ProP (ProP‐p) formed a dimeric α‐helical coiled‐coil. A derivative of transporter ProP lacking 26 C‐terminal amino acids was expressed but inactive. A derivative harbouring amino acid changes K460I, Y467I and H495I (each at the core, coiled‐coil ‘a’ position) required a larger osmotic upshift for activation than did the wild type transporter. The same changes extended, stabilized and altered the oligomeric state of the coiled‐coil formed by ProP‐p. Amino acid change R488I (also at the ‘a’ position) further increased the magnitude of the osmotic upshift required to activate ProP, reduced the activity attained and rendered ProP activation transient. Unexpectedly, replacement R488I destabilized the coiled‐coil formed by ProP‐p. The activity and osmotic activation of ProP were even more strongly attenuated by helix‐destabilizing change I474P. These data demonstrate that the carboxyl terminal domain of ProP can form a homodimeric α‐helical coiled‐coil with unusual properties. They implicate the C‐terminal domain in the osmotic activation of ProP. Copyright

An Escherichia coli Reference Collection Group B2- and Uropathogen-Associated Polymorphism in the rpoS-mutS Region of the E. coli Chromosome

Chromosomal DNAs of enterohemorrhagic, uropathogenic, and laboratory attenuated Escherichia coli strains differ in the rpoS-mutS region. Many uropathogens lack a deletion and an insertion characteristic of enterohemorrhagic strains. At the same chromosomal position, they harbor a 2.1-kb insertion of unknown origin with a base composition suggestive of horizontal gene transfer. Unlike virulence determinants associated with urinary tract infection and/or neonatal meningitis (pap or prs, sfa, kps, and hly), the 2.1-kb insertion is shared by all group B2 strains of the E. coli Reference Collection.

Transmembrane Helix I and Periplasmic Loop 1 of Escherichia coli ProP Are Involved in Osmosensing and Osmoprotectant Transport

Osmoregulatory transporters stimulate bacterial growth by mediating osmoprotectant uptake in response to increasing osmotic pressure. The ProP protein of Escherichia coli transports proline and other osmoprotectants. Like LacY, ProP is a member of the major facilitator superfamily and a H(+)-solute symporter. ProP is regulated by osmotic pressure via a membrane potential-dependent mechanism. A homology model predicts that ionizable and polar residues, highly conserved among ProP homologues, cluster deep within the N-terminal helix bundle of ProP. Chemical labeling of introduced cysteine (Cys) residues supported the homology model by confirming the predicted positions of transmembrane helix I (TMI) and periplasmic loop 1. Replacements of residues in the putative polar cluster impaired or altered ProP function, suggesting that they are important for osmosensing and may interact with the transport substrates. Asn34, Glu37, Phe41, Tyr44, and Ala48 line the most polar face of TMI; Tyr44 is on the periplasmic side of the putative polar cluster, and Ala59 is in periplasmic loop 1. The N-ethylmaleimide reactivities of Cys introduced at positions 41, 44, 48, and 59 increased with osmotic pressure, whereas the reactivities of those at cytoplasm-proximal positions 34 and 37 did not. Replacements of polar cluster residues that blocked transport also affected the NEM reactivity of Cys44 and its osmolality dependence. This report and previous work suggest that conformational changes associated with osmosensing may shift the equilibria between outward- and inward-facing transport pathway intermediates.