Origin of tetracycline efflux proteins: conclusions from nucleotide sequence analysis

The sequences of six tetracycline efflux proteins and three transport proteins which have some resemblance to them were compared. The tetracycline efflux proteins fall into three families: (i) those encoded by pBR322, RP1, and Tn10 (Escherichia coli); (ii) pT181 (Staphylococcus aureus) and pTHT15 (Bacillus subtilis); and (iii) tet347 (Streptomyces rimosus). There is global sequence homology within each of the first two families, but there is none between the families. The pT181/pTHT15 family shares close homology with the N-terminal half of the methylenomycin A efflux protein (Streptomyces coelicor), while tet347 resembles the C-terminal half. Portions of the N-terminal half of the Tn10-encoded protein show significant resemblance to portions in the N-terminal half of the pT181/pTHT15 family, but this sometimes occurs among transport proteins which do not have a common substrate. Tetracycline efflux proteins, therefore, appear to have arisen on at least two, or possibly three, separate occasions, probably from other transport proteins.     

Effects of prostaglandins on fetal breathing do not involve peripheral chemoreceptors

In sheep, prostaglandin (PG) E2 inhibits fetal breathing movements and meclofenamate, a PG synthetase inhibitor, causes a marked stimulation of fetal breathing movements; the site of action of these agents is not known. To determine whether these effects are mediated through the peripheral chemoreceptors, we studied 13 fetal sheep at gestational ages of 127 to 138 days. Seven fetuses had bilateral section of the carotid sinus and vagus nerves (denervated); six had sham operations. Beginning at least 6 days after the operation, we infused PGE2 (0.6 microgram X kg-1 X min-1) into five denervated and five sham-operated fetuses and meclofenamate (0.4 mg X kg-1 X h-1) into six denervated and four sham-operated fetuses. Infusions averaged 20 h in duration. During preinfusion control periods, the incidence of fetal breathing movements (% of time) was lower in denervated than in sham-operated fetuses (18.9% vs. 31.5%; P less than 0.005). In both groups, the incidence of fetal breathing movements was decreased by PGE2 and was increased by meclofenamate; when expressed as absolute values, the magnitude of the changes with both agents was greater in sham-operated fetuses than denervated fetuses. However, the effects were similar in both groups when the changes were expressed as a percent of the respective control values. The incidence of fetal breathing movements (% of control) was decreased by PGE2 to 25.4% in denervated and to 28.2% in sham-operated fetuses and was increased by meclofenamate to 297.3% in denervated and to 304.0% in sham-operated fetuses.(ABSTRACT TRUNCATED AT 250 WORDS)     

A tetracycline efflux gene on Bacteroides transposon Tn4400 does not contribute to tetracycline resistance.

Previously, we demonstrated that the Bacteroides transposon Tn4351, which confers tetracycline resistance only on aerobically grown Escherichia coli, carries a gene that codes for a tetracycline-inactivating enzyme (B. S. Speer and A. A. Salyers, J. Bacteriol. 170:1423-1429, 1988). However, Park et al. (B. H. Park, M. Hendricks, M. H. Malamy, F. P. Tally, and S. B. Levy, Antimicrob. Agents Chemother. 31:1739-1743, 1987) showed that E. coli carrying a closely related transposon, Tn4400, exhibits energy-dependent efflux of tetracycline as well as tetracycline-inactivating activity (B. H. Park and S. B. Levy, Antimicrob. Agents Chemother. 32:1797-1800, 1988). This result raised the question of whether efflux or inactivation or a combination of the two was necessary for resistance conferred by both transposons. We showed that cells carrying Tn4351 did not exhibit the clear-cut efflux activity seen with cells carrying Tn4400 but rather exhibited a tetracycline accumulation profile which could be explained solely on the basis of inactivation of tetracycline in the cytoplasm and rapid diffusion of altered tetracycline out of the cell. Additionally, we were able to clone the efflux and tetracycline-modifying genes of Tn4400 separately. The region carrying the efflux gene spanned one of the two regions in which Tn4400 differs from Tn4351. A clone containing the corresponding region of Tn4351 did not exhibit efflux. Thus, it appears that Tn4351 does not have the efflux gene and that efflux makes no contribution to the resistance conferred by Tn4351. The MIC for cells carrying the subclone from Tn4400 that contained only the gene for tetracycline inactivation was the same that for cells carrying both the inactivation and efflux genes. Cells carrying only the gene for tetracycline efflux were tetracycline sensitive. This was true even when the efflux gene was on a high-copy-number plasmid which increased the level of efflux to that associated with the Tcr gene on pBR328. These results indicate that efflux activity does not contribute significantly to the tetracycline resistance conferred by Tn4400.     

The pleiotropic effects of his overexpression in Salmonella typhimurium do not involve AICAR-induced mutagenesis

Inhibition of cell division associated with overexpression of hisH and hisF in Salmonella typhimurium is strongly reminiscent of a cellular response to DNA damage. On these grounds, we investigated the involvement of a metabolite which appeared to represent a possible candidate for an endogenous mutagen: the base analog 5-amino-4-carboxamide imidazole riboside 5′-phosphate (AICAR), a by-product of HisH and HisF activity. However, we showed that AICAR is not an endogenous mutagen in S. typhimurium. Other types of DNA damage induced by his overexpression seem also unlikely, since similar mutation rates are found in hisO ⁺ and hisO ^c strains. We also show that AICAR production is not involved in the pleiotropic effects of his overexpression, since these are still observed in strains devoid of AICAR. Thus inhibition of cell division resulting from HisH and HisF overexpression must operate through a mechanism unrelated to the role of these proteins in histidine biosynthesis.     

The plasmid-encoded chloramphenicol-resistance protein of Rhodococcus fascians is homologous to the transmembrane tetracycline efflux proteins

The nucleotide sequence of the chloramphenicol-resistance gene (cmr) of Rhodococcus fascians NCPPB 1675 (located on the conjugative plasmid pRF2) allowed the identification of two possible open reading frames (ORFs), of which ORF1 was consistent with the mutational analysis. Biochemical analysis of cmr revealed that it does not encode an antibiotic-modifying enzyme. The amino acid sequence of ORF1 predicted a hydrophobic protein, with 12 putative membrane-spanning domains, homologous to proteins involved in the efflux of tetracycline across the plasma membrane. Expression of the cmr gene was induced by addition of chloramphenicol to the growth media. The promoter of this gene was restricted to 50 bp upstream from a 200 bp 5'-untranslated mRNA region, the latter containing two inverted repeats. At the amino acid level, the cmr gene is 52% identical to a previously identified chloramphenicol-resistance determinant in Streptomyces lividans, indicating a wider dispersion of this type of cmr gene among the actinomycetes.     

Dynamic alpha-helices: conformations that do not conform

Structural transitions are important for the stability and function of proteins, but these phenomena are poorly understood. An extensive analysis of Protein Data Bank entries reveals 103 regions in proteins with a tendency to transform from helical to nonhelical conformation and vice versa. We find that these dynamic helices, unlike other helices, are depleted in hydrophobic residues. Furthermore, the dynamic helices have higher surface accessibility and conformational mobility (P-value = 3.35e-07) than the rigid helices. Contact analyses show that these transitions result from protein-ligand, protein-nucleic acid, and crystal-contacts. The immediate structural environment differs quantitatively (P-value = 0.003) as well as qualitatively in the two alternate conformations. Often, dynamic helix experiences more contacts in its helical conformation than in the nonhelical counterpart (P-value = 0.001). There is differential preference for the type of short contacts observed in two conformational states. We also demonstrate that the regions in protein that can undergo such large conformational transitions can be predicted with a reasonable accuracy using logistic regression model of supervised learning. Our findings have implications in understanding the molecular basis of structural transitions that are coupled with binding and are important for the function and stability of the protein. Based on our observations, we propose that several functionally relevant regions on the protein surface can switch over their conformation from coil to helix and vice-versa, to regulate the recognition and binding of their partner and hence these may work as "molecular switches" in the proteins to regulate certain biological process. Our results supports the idea that protein structure-function paradigm should transform from static to a highly dynamic one.     

Volume-sensitive chloride channels do not mediate activation-induced chloride efflux in human neutrophils

Many agents that activate neutrophils, enabling them to adhere to venular walls at sites of inflammation, cause a rapid Cl(-) efflux. This Cl(-) efflux and the increase in the number and affinity of beta(2) integrin surface adhesion molecules (up-regulation) are all inhibited by ethacrynic acid and certain aminomethyl phenols. The effectiveness of the latter compounds correlates with their inhibition of swelling-activated Cl(-) channels (I(Clvol)), suggesting that I(Clvol) mediates the activator-induced Cl(-) efflux. To test this hypothesis, we used whole-cell patch clamp in hypotonic media to examine the effects of inhibitors of up-regulation on I(Clvol) in neutrophils and promyelocytic leukemic HL-60 cells. Both the channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid and [3-methyl-1-p-sulfophenyl-5-pyrazolone-(4)]-[1,3-dibutylbarbituric acid]-pentamethine oxonol (WW781), a nonpenetrating oxonol, inhibited I(Clvol) at concentrations similar to those that inhibit beta(2) integrin up-regulation. However, ethacrynic acid, at the same concentration that inhibits activator-induced Cl(-) efflux and up-regulation, had no effect on I(Clvol) and swelling-activated Cl(-) efflux, providing evidence against the involvement of I(Clvol) in the activator-induced Cl(-) efflux.     

Gene duplication in the evolution of the two complementing domains of gram-negative bacterial tetracycline efflux proteins

The resistance of Gram- bacteria to the broad-spectrum antibiotic tetracycline (Tc) results from energy-dependent drug efflux mediated by the tet gene product, the cytoplasmic membrane Tet protein. Amino acid (aa) sequences deduced from total tet nucleotide sequences of three different resistance determinants (classes A, B and C) indicate that the protein products [Tet(A), Tet(B), and Tet(C)] share a common ancestor. Hydropathic analysis of Tet sequences predicts twelve transmembrane segments in each protein, with six occurring in each half of the molecule. More importantly, the linear distributions of these segments in the N- and C-terminal halves are nearly identical, suggesting that the two halves of each Tet protein are related by a process of tandem gene duplication and divergence. Indeed, a variable but significant conservation of sequence was detected among the N- and C-terminal halves for all possible comparisons of the three proteins. Such conservation was not observed within other prokaryotic integral membrane proteins or when other prokaryotic proteins were compared to Tet halves. Similarity, both in sequence and in predicted transmembrane structural organization, strongly suggests that a common ancestor of Tet(A), Tet(B), and Tet(C) arose by duplication of a gene reading frame specifying a transmembrane protein of approximately 200 aa residues. The two halves of Tet proteins correspond to the two domains, alpha and beta, which have distinct, complementary roles in Tc efflux. Nevertheless, selective constraints to function in the cytoplasmic membrane have apparently led to maintenance of similar patterns of secondary structural organization in these complementary domains.     

Calcium binding to a human factor IXa derivative lacking gamma-carboxyglutamic acid: evidence for two high-affinity sites that do not involve beta-hydroxyaspartic acid

A derivative of human blood clotting factor IXa beta lacking gamma-carboxyglutamic acid (Gla) residues was prepared by limited proteolysis with chymotrypsin, and subsequently examined for its ability to bind calcium ions. By amino acid analysis, Gla-domainless human factor IXa beta contained 0.3-0.4 moles of beta-hydroxyaspartic acid per mole of protein. Equilibrium dialysis experiments demonstrated that Gla-domainless human factor IXa beta retained two high-affinity calcium binding sites (Kd=52 microM), a finding essentially identical to that observed for Gla-domainless bovine factor IX that contains 0.8-0.9 moles of beta-hydroxyaspartic acid per mole of protein. These data strongly suggest that the beta-hydroxyaspartic acid residue in these proteins does not participate in their high affinity calcium sites.     

Mitochondria from ejaculated human spermatozoa do not synthesize proteins

Sperm motility is dependent on mitochondrial ATP production that relies on the coordinated expression of the mitochondrial and nuclear genomes. It is generally accepted that mammalian ejaculated spermatozoa retain the ability to synthesize mtDNA-encoded proteins but not most of the nuclear ones. This implies an asynchronous regulation of the oxidative phosphorylation-related genes encoded by each genome. Trying to investigate this issue, we unexpectedly found that ejaculated human spermatozoa do not synthesize mtDNA-encoded proteins. Moreover, we estimated that the discrepancy between our observations and those published elsewhere was due to a chloramphenicol-sensitive protein synthesis attributed to mitochondria that instead corresponds to contaminating bacteria.     

Systems approaches reveal that ABCB and PIN proteins mediate co-dependent auxin efflux

Members of the B family of membrane-bound ATP-binding cassette (ABC) transporters represent key components of the auxin efflux machinery in plants. Over the last two decades, experimental studies have shown that modifying ATP-binding cassette sub-family B (ABCB) expression affects auxin distribution and plant phenotypes. However, precisely how ABCB proteins transport auxin in conjunction with the more widely studied family of PIN-formed (PIN) auxin efflux transporters is unclear, and studies using heterologous systems have produced conflicting results. Here, we integrate ABCB localization data into a multicellular model of auxin transport in the Arabidopsis thaliana root tip to predict how ABCB-mediated auxin transport impacts organ-scale auxin distribution. We use our model to test five potential ABCB–PIN regulatory interactions, simulating the auxin dynamics for each interaction and quantitatively comparing the predictions with experimental images of the DII-VENUS auxin reporter in wild-type and abcb single and double loss-of-function mutants. Only specific ABCB–PIN regulatory interactions result in predictions that recreate the experimentally observed DII-VENUS distributions and long-distance auxin transport. Our results suggest that ABCBs enable auxin efflux independently of PINs; however, PIN-mediated auxin efflux is predominantly through a co-dependent efflux where co-localized with ABCBs.

Predicting the experimentally observed root-tip auxin distribution requires ABCBs to efflux auxin independently, whereas PINs predominantly mediate auxin efflux where co-localized with ABCBs.     

Evidence that reactive oxygen species do not mediate NF-kappaB activation

It has been postulated that reactive oxygen species (ROS) may act as second messengers leading to nuclear factor (NF)-kappaB activation. This hypothesis is mainly based on the findings that N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), compounds recognized as potential antioxidants, can inhibit NF-kappaB activation in a wide variety of cell types. Here we reveal that both NAC and PDTC inhibit NF-kappaB activation independently of antioxidative function. NAC selectively blocks tumor necrosis factor (TNF)-induced signaling by lowering the affinity of receptor to TNF. PDTC inhibits the IkappaB-ubiquitin ligase activity in the cell-free system where extracellular stimuli-regulated ROS production does not occur. Furthermore, we present evidence that endogenous ROS produced through Rac/NADPH oxidase do not mediate NF-kappaB signaling, but instead lower the magnitude of its activation.