Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance

The self‐assembling MexA‐MexB‐OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR‐wt as well as a selected set of MDR single mutants distant from the proposed DNA‐binding helix. Although DNA affinity and MexA‐MexB‐OprM repression were both drastically impaired in the selected MexR‐MDR mutants, MexR‐wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR‐MDR mutants, secondary structure content and oligomerization properties were very similar to MexR‐wt despite their lack of DNA binding. Despite this, the MexR‐MDR mutants showed highly varying stabilities compared with MexR‐wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA‐binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR‐wt in both free and DNA‐bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations—stability, domain interactions, and internal hydrophobic surfaces—are also critical for the regulation of MexR DNA binding.     

Identification of Cys385 in the isolated kinase insertion domain of heme-regulated eIF2 alpha kinase (HRI) as the heme axial ligand by site-directed mutagenesis and spectral characterization

Heme-regulated eIF2alpha kinase (HRI) is an important enzyme that modulates protein synthesis during cellular emergency/stress conditions, such as heme deficiency in red cells. It is essential to identify the heme axial ligand(s) and/or binding sites to establish the heme regulation mechanism of HRI. Previous reports suggest that a His residue in the N-terminal region and a Cys residue in the C-terminal region trans to the His are axial ligands of the heme. Moreover, mutational analyses indicate that a residue located in the kinase insertion (KI) domain between Kinase I and Kinase II domains in the C-terminal region is an axial ligand. In the present study, we isolate the KI domain of mouse HRI and employ site-directed mutagenesis to identify the heme axial ligand. The optical absorption spectrum of the Fe(III) hemin-bound wild-type KI displays a broad Soret band at around 373nm, while that of the Fe(II) heme-bound protein contains a band at 422nm. Spectral titration studies conducted for both the Fe(III) hemin and Fe(II) heme complexes with KI support a 1:1 stoichiometry of heme iron to protein. Resonance Raman spectra of Fe(III) hemin-bound KI suggest that thiol is the axial ligand in a 5-coordinate high-spin heme complex as a major form. Electron spin resonance (ESR) spectra of Fe(III) hemin-bound KI indicate that the axial ligands are OH(-) and Cys. Since Cys385 is the only cysteine in KI, the residue was mutated to Ser, and its spectral characteristics were analyzed. The Soret band position, heme spectral titration behavior and ESR parameters of the Cys385Ser mutant were markedly different from those of wild-type KI. Based on these spectroscopic findings, we conclude that Cys385 is an axial ligand of isolated KI.     

Proteomic profile changes in membranes of ethanol-tolerant <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Clostridium thermocellum, a cellulolytic, thermophilic anaerobe, has potential for commercial exploitation in converting fibrous biomass to ethanol. However, ethanol concentrations above 1% (w/v) are inhibitory to growth and fermentation, and this limits industrial application of the organism. Recent work with ethanol-adapted strains suggested that protein changes occurred during ethanol adaptation, particularly in the membrane proteome. A two-stage Bicine-doubled sodium dodecyl sulfate-polyacrylamide gel electrophoresis protocol was designed to separate membrane proteins and circumvent problems associated with membrane protein analysis using traditional gel-based proteomics approaches. Wild-type and ethanol-adapted C. thermocellum membranes displayed similar spot diversity and approximately 60% of proteins identified from purified membrane fractions were observed to be differentially expressed in the two strains. A majority (73%) of differentially expressed proteins were down-regulated in the ethanol-adapted strain. Based on putative identifications, a significant proportion of these down-regulated proteins were involved with carbohydrate transport and metabolism. Approximately one-third of the up-regulated proteins in the ethanol-adapted species were associated with chemotaxis and signal transduction. Overall, the results suggested that membrane-associated proteins in the ethanol-adapted strain are either being synthesized in lower quantities or not properly incorporated into the cell membrane.     

The brefeldin A-inhibited guanine nucleotide-exchange protein, BIG2, regulates the constitutive release of TNFR1 exosome-like vesicles

The type I, 55-kDa tumor necrosis factor receptor (TNFR1) is released from cells to the extracellular space where it can bind and modulate TNF bioactivity. Extracellular TNFR1 release occurs by two distinct pathways: the inducible proteolytic cleavage of TNFR1 ectodomains and the constitutive release of full-length TNFR1 in exosome-like vesicles. Regulation of both TNFR1 release pathways appears to involve the trafficking of cytoplasmic TNFR1 vesicles. Vesicular trafficking is controlled by ADP-ribosylation factors (ARFs), which are active in the GTP-bound state and inactive when bound to GDP. ARF activation is enhanced by guanine nucleotide-exchange factors that catalyze replacement of GDP by GTP. We investigated whether the brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins, BIG1 and/or BIG2, are required for TNFR1 release from human umbilical vein endothelial cells. Effects of specific RNA interference (RNAi) showed that BIG2, but not BIG1, regulated the release of TNFR1 exosome-like vesicles, whereas neither BIG2 nor BIG1 was required for the IL-1beta-induced proteolytic cleavage of TNFR1 ectodomains. BIG2 co-localized with TNFR1 in diffusely distributed cytoplasmic vesicles, and the association between BIG2 and TNFR1 was disrupted by BFA. Consistent with the preferential activation of class I ARFs by BIG2, ARF1 and ARF3 participated in the extracellular release of TNFR1 exosome-like vesicles in a nonredundant and additive fashion. We conclude that the association between BIG2 and TNFR1 selectively regulates the extracellular release of TNFR1 exosome-like vesicles from human vascular endothelial cells via an ARF1- and ARF3-dependent mechanism.     

Conformational stabilization at the active site of molluskan (Rapana thomasiana) hemocyanin by a cysteine-histidine thioether bridge A study by mass spectrometry and molecular modeling

In some type-3 copper proteins (molluskan hemocyanin, catechol oxidase and fungal tyrosinase) one of the histidine residues, liganding the Cu(A) atom of the dinuclear copper active site, is covalently linked to a cysteine residue by a thioether bridge. The purpose of this study was to disclose the function of this bridge. Mass spectral analysis of a peptide, isolated from Rapana thomasiana (gastropodan mollusk) hemocyanin, indicated a stabilization of the peptide structure in the region of the bridge. Molecular modeling of three thioether containing type-3 copper proteins using the dead-end elimination method showed that the concerned histidine would be very flexible if not linked to the cysteine. Also, the side chain orientation of the histidine is rather exceptional, as evidenced by statistical data from the protein databank. It is suggested that the role of the bridge is to fix the histidine in an orientation that is optimal for coordination of the Cu(A) atom.     

Evolutionary relationships and reproductive isolating mechanisms in the rice frog (Fejervarya limnocharis) species complex from Sri Lanka, Thailand, Taiwan and Japan, inferred from mtDNA gene sequences, allozymes, and crossing experiments

The rice frog (Fejervarya limnocharis) species complex is widely distributed, from India to Japan, and most prevalently in Southeast Asia. Conspicuous morphological variation has been reported for this species complex throughout its distribution range. In the present study, we used mtDNA gene sequence and allozyme analyses to infer evolutionary affinities within this species complex using eight populations (Sri Lanka; Bangkok and Ranong in Thailand; Taiwan; and Hiroshima, Okinawa, Ishigaki and Iriomote in Japan). We also conducted crossing experiments among four populations from Japan, Thailand, and Sri Lanka in order to find out more about the reproductive isolating mechanisms that might exist among the East, Southeast, and South Asian populations of this species complex. The crossing experiments revealed that the Sri Lanka population is reproductively isolated from the Hiroshima, Bangkok, and Ranong populations by complete hybrid inviability, and that the Bangkok population may be reproductively isolated from the Hiroshima population by partial hybrid inviability. Thus, it is not unreasonable to regard the Sri Lanka population as a species separated from F. limnocharis. The mtDNA and allozyme data showed that the Ranong population is most closely related to the Bangkok population in nuclear genome, but more similar to the Okinawa and Taiwan populations in mtDNA genome. The present, preliminary survey may raise questions about the species status of these particular populations and also about the nature of the biological species concept.     

Mechanism of reduced colonic contractility in experimental colitis: role of sarcoplasmic reticulum pump isoform-2

Inflammatory bowel diseases (IBD) such as Crohn’s disease and ulcerative colitis are inflammatory disorders associated with decreased colonic contractility. Here we show that, in experimental colitis in rat induced by trinitrobenzenesulfonic acid, there is a decrease in contraction in response to carbamoylcholine and the sarco/endoplasmic reticulum Ca⁺² (SERCA) pump inhibitor thapsigargin. However, the decrease in contractility may occur due to decrease in the SERCA pump levels or their inactivation. Therefore, we examined the protein and mRNA levels for SERCA2 isoform, which is predominant isoform in colonic smooth muscle. There was a decrease in the levels of SERCA2 protein and mRNA levels in inflamed colonic muscle. These findings suggest that decreased SERCA pump levels is responsible for a decrease in the Ca⁺² stores in the sarco/endoplasmic reticulum that causes a decrease in the contractility in colonic smooth muscle leading to poor bowel movements.     

The 32 kDa subunit of replication protein A (RPA) participates in the DNA replication of Mung bean yellow mosaic India virus (MYMIV) by interacting with the viral Rep protein

Mung bean yellow mosaic India virus (MYMIV) is a member of genus begomoviridae and its genome comprises of bipartite (two components, namely DNA-A and DNA-B), single-stranded, circular DNA of about 2.7 kb. During rolling circle replication (RCR) of the DNA, the stability of the genome and maintenance of the stem–loop structure of the replication origin is crucial. Hence the role of host single-stranded DNA-binding protein, Replication protein A (RPA), in the RCR of MYMIV was examined. Two RPA subunits, namely the RPA70 kDa and RPA32 kDa, were isolated from pea and their roles were validated in a yeast system in which MYMIV DNA replication has been modelled. Here, we present evidences that only the RPA32 kDa subunit directly interacted with the carboxy terminus of MYMIV-Rep both in vitro as well as in yeast two-hybrid system. RPA32 modulated the functions of Rep by enhancing its ATPase and down regulating its nicking and closing activities. The possible role of these modulations in the context of viral DNA replication has been discussed. Finally, we showed the positive involvement of RPA32 in transient replication of the plasmid DNA bearing MYMIV replication origin using an in planta based assay.     

Formation of Tellurium Nanocrystals during Anaerobic Growth of Bacteria That Use Te Oxyanions as Respiratory Electron Acceptors

Certain toxic elements support the metabolism of diverse prokaryotes by serving as respiratory electron acceptors for growth. Here, we demonstrate that two anaerobes previously shown to be capable of respiring oxyanions of selenium also achieve growth by reduction of either tellurate [Te(VI)] or tellurite [Te(IV)] to elemental tellurium [Te(0)]. This reduction achieves a sizeable stable-Te-isotopic fractionation (isotopic enrichment factor [] = −0.4 to −1.0 per ml per atomic mass unit) and results in the formation of unique crystalline Te(0) nanoarchitectures as end products. The Te(0) crystals occur internally within but mainly externally from the cells, and each microorganism forms a distinctly different structure. Those formed by Bacillus selenitireducens initially are nanorods (~10-nm diameter by 200-nm length), which cluster together, forming larger (~1,000-nm) rosettes composed of numerous individual shards (~100-nm width by 1,000-nm length). In contrast, Sulfurospirillum barnesii forms extremely small, irregularly shaped nanospheres (diameter < 50 nm) that coalesce into larger composite aggregates. Energy-dispersive X-ray spectroscopy and selected area electron diffraction indicate that both biominerals are composed entirely of Te and are crystalline, while Raman spectroscopy confirms that they are in the elemental state. These Te biominerals have specific spectral signatures (UV-visible light, Raman) that also provide clues to their internal structures. The use of microorganisms to generate Te nanomaterials may be an alternative for bench-scale syntheses. Additionally, they may also generate products with unique properties unattainable by conventional physical/chemical methods.