ProP is a member of the major facilitator superfamily, a proton-osmolyte symporter, and an osmosensing transporter. ProP proteins share extended cytoplasmic carboxyl terminal domains (CTDs) implicated in osmosensing. The CTDs of the best characterized, group A ProP orthologs, terminate in sequences that form intermolecular, antiparallel a-helical coiled coils (e.g., ProPEc, from Escherichia coli). Group B orthologs lack that feature (e.g., ProPXc, from Xanthomonas campestris). ProPXc was expressed and characterized in E. coli to further elucidate the role of the coiled coil in osmosensing. The activity of ProPXc was a sigmoid function of the osmolality in cells and proteoliposomes. ProPEc and ProPXc attained similar activities at the same expression level in E. coli. ProPEc transports proline and glycine betaine with comparable high affinities at low osmolality. In contrast, proline weakly inhibited high-affinity glycine-betaine uptake via ProPXc. The K M for proline uptake via ProPEc increases dramatically with the osmolality. The K M for glycine-betaine uptake via ProPXc did not. Thus, ProPXc is an osmosensing transporter, and the C-terminal coiled coil is not essential for osmosensing. The role of CTD-membrane interaction in osmosensing was examined further. As for ProPEc, the ProPXc CTD co-sedimented with liposomes comprising E. coli phospholipid. Molecular dynamics simulations illustrated association of the monomeric ProPEc CTD with the membrane surface. Comparison with the available NMR structure for the homodimeric coiled coil formed by the ProPEc-CTD suggested that membrane association and homodimeric coiled-coil formation by that peptide are mutually exclusive. The membrane fluidity in liposomes comprising E. coli phospholipid decreased with increasing osmolality in the range relevant for ProP activation. These data support the proposal that ProP activates as cellular dehydration increases cytoplasmic cation concentration, releasing the CTD from the membrane surface. For group A orthologs, this also favors a-helical coiled-coil formation that stabilizes the transporter in an active form.

S ARS-CoV-2 originated in horseshoe bats and probably reached humans through an unidentified intermediary host (1). The virus is aerosolized and highly transmissible among humans; new variants have arisen and spread in successive waves across the world since late 2019. Since a report of SARS-CoV-2 infection in a dog in March 2020 (2), an ever-increasing range of species has been shown to be susceptible to infection, including household cats, dogs, ferrets, and hamsters (3-10).Companion animals have closest contact with humans, creating ample opportunity for exposure. Experimental infections have suggested that most companion animals are infected only transiently, as indicated by PCR positivity or virus isolation (11,12). Conversely, detection of antibodies by ELISA or neutralizing antibody assay suggests infection rates of 0.2%-43.9% related to factors such as the likelihood and frequency of interaction with infected humans (13-16). Infections in animals are typically subclinical or associated with transient respiratory or gastrointestinal disease (17,18). In rare cases, death has been attributed to SARS-CoV-2 infection; however, defining the contribution of SARS-CoV-2 to death in animals with underlying conditions such as cancer, bacterial pneumonia, or obesity is challenging. On the other hand, minks are highly susceptible to infection and pneumonia, and mortality rates of 35%-55% caused by SARS-CoV-2 infection were reported from outbreaks among farmed mink in Utah ( 19). Captive minks also contracted viruses with a unique amino acid substitution in the spike (S) protein that were subsequently retransmitted to humans and to community cats and dogs, around mink farms in the Netherlands (5,20). Similarly, infected pet Syrian hamsters may also retransmit SARS-CoV-2 to humans (21). More than 30% of free-ranging white-tailed deer tested in Ohio were SARS-CoV-2 positive by PCR, and a similarly high proportion of white-tailed deer in Texas and other North America locations had neutralizing antibodies (22,23). Experimentally, white-tailed deer transmitted SARS-CoV-2 to other deer vertically and horizontally by direct contact (24). It has not yet been determined if infected deer experience illness or have increased illness and death rates or if transmission is sustained among wild deer populations. However, such high

ProQ is a cytoplasmic protein with RNA chaperone activities that reside in FinO-and Hfq-like domains. Lesions at proQ decrease the level of the osmoregulatory glycine betaine transporter ProP. Lesions at proQ eliminated ProQ and Prc, the periplasmic protease encoded by the downstream gene prc. They dramatically slowed the growth of Escherichia coli populations and altered the morphologies of E. coli cells in high-salinity medium. ProQ and Prc deficiencies were associated with different phenotypes. ProQ-deficient bacteria were elongated unless glycine betaine was provided. High-salinity cultures of Prcdeficient bacteria included spherical cells with an enlarged periplasm and an eccentric nucleoid. The nucleoid-containing compartment was bounded by the cytoplasmic membrane and peptidoglycan. This phenotype was not evident in bacteria cultivated at low or moderate salinity, nor was it associated with murein lipoprotein (Lpp) deficiency, and it differed from those elicited by the MreB inhibitor A-22 or the FtsI inhibitor aztreonam at low or high salinity. It was suppressed by deletion of spr, which encodes one of three murein hydrolases that are redundantly essential for enlargement of the murein sacculus. Prc deficiency may alter bacterial morphology by impairing control of Spr activity at high salinity. ProQ and Prc deficiencies lowered the ProP activity of bacteria cultivated at moderate salinity by approximately 70% and 30%, respectively, but did not affect other osmoregulatory functions. The effects of ProQ and Prc deficiencies on ProP activity are indirect, reflecting their roles in the maintenance of cell structure.O smotic stress perturbs cell structure, composition, and function (1). Despite retaining their rod-like shape, Escherichia coli cells cultivated in high-salinity minimal medium maintain lower hydration, turgor pressure, and growth rate than those cultivated at a lower salinity that is optimal for growth (2). The elastic murein sacculus is believed to buffer effects of osmotically induced water fluxes on cell structure (3). E. coli can attenuate osmotically induced dehydration by accumulating small, uncharged, or zwitterionic organic solutes called osmolytes (1, 4, 5). For example, transporter ProP mediates the accumulation of diverse solutes, including proline and glycine betaine, thereby restoring cellular hydration and stimulating bacterial growth in high-salinity media (6, 7).ProQ is a cytoplasmic protein that binds RNA, facilitating RNA duplexing and strand exchange (8). Previous work showed that proQ lesions decreased ProP levels and attenuated ProP activity (the proQ transport phenotype). These effects occurred when bacteria expressed proP from the chromosome or a plasmid-based P BAD promoter during growth in low-to moderate-salinity media and were reversed by plasmid-based proQ expression (8, 9). Here, we show that proQ lesions dramatically slow the growth of E. coli populations in high-salinity medium and alter the morphologies of E. coli cells (the proQ growth and morphological phenotypes...

Here we describe the crystal structure of the N-terminal domain of the FK506-binding protein (FKBP) from wheat (wFKBP73), which is the first structure presenting three FK domains (wFK73_1, wFK73_2 and wFK73_3). The crystal model includes wFK73_2 and wFK73_3 domains and only part of the wFK73_1 domain. The wFK73_1 domain is responsible for binding FK506 and for peptidyl prolyl cis/trans isomerase (PPIase) activity, while the wFK73_2 and wFK73_3 domains lack these activities. A structure-based sequence comparison demonstrated that the absence of a large enough hydrophobic pocket important for PPIase activity, and of the conserved residues necessary for drug binding in the wFK73_2 and wFK73_3 domains explains the lack of these activities in these domains. Sequence and structural comparison between the three wFKBP73 domains suggest that the wFK73_2 domain is the most divergent. A structural comparison of the FK domains of wFKBP73 with other FKBPs containing more than one FK domain, revealed that while the overall architecture of each of the three FK domains displays a typical FKBP fold, their relative arrangement in space is unique and may have important functional implications. We suggest that the existence of FKBPs with three FK domains offers additional interactive options for these plant proteins enlarging the overall regulatory functions of these proteins.

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