Tracing Ancestors and Relatives of Escherichia coli B, and the Derivation of B Strains REL606 and BL21(DE3)

Antecedents of Escherichia coli B have been traced through publications, inferences, and personal communication to a strain from the Institut Pasteur in Paris used by d'Herelle in his studies of bacteriophages as early as 1918 (a strain not in the current collection). This strain appears to have passed from d'Herelle to Bordet in 1920, and from Bordet to at least three other laboratories by 1925. The strain that Gratia received from Bordet was apparently passed to Bronfenbrenner by 1924 and from him to Luria around 1941. Delbrück and Luria published the first paper calling this strain B in 1942. Its choice as the common host for phages T1-T7 by the phage group that developed around Delbrück, Luria, and Hershey in the 1940s led to widespread use of B along with E. coli K-12, chosen about the same time for biochemical and genetic studies by Tatum and Lederberg. Not all currently available strains related to B are descended from the B of Delbrück and Luria; at least three strains with somewhat different characteristics were derived independently by Hershey directly from the Bronfenbrenner strain, and a strain that appears to have passed from Bordet to Wollman is in the current Collection of the Institut Pasteur. The succession of manipulations and strains that led from the B of Delbrück and Luria to REL606 and BL21(DE3) is given, established in part through evidence from their recently determined complete genome sequences.     

Taxonomic characterisation of ceftazidime-resistant Brevundimonas isolates and description of Brevundimonas faecalis sp. nov

Three ceftazidime-resistant strains isolated from the sewage water of a municipal hospital in Palma de Mallorca, Spain, were analysed phenotypically and genotypically to clarify their taxonomic positions. Sequence determinations and phylogenetic analyses of the 16S rRNA genes indicated that strains CS20.3^T, CS39 and CS41 were affiliated with the species of the alphaproteobacterial genus Brevundimonas, most closely related to B. bullata, B. diminuta, B. naejangsanensis and B. terrae. Additional sequences analyses of the ITS1 region of the rRNA operon and the genes for the housekeeping enzymes DNA gyrase β-subunit and RNA polymerase β-subunit, genomic DNA–DNA hybridisation similarities, cell fatty acid profiles and physiological and biochemical characterizations supported the recognition of CS20.3^T (CCUG 58127^T = CECT 7729^T) as a distinct and novel species, for which the name Brevundimonas faecalis sp. nov. is proposed. Strains CS39 and CS41 were ascribed to the species B. diminuta.     

Identification and characterization of a 20β-HSDH from the anaerobic gut bacterium Butyricicoccus desmolans ATCC 43058

Members of the gastrointestinal microbiota are known to convert glucocorticoids to androstanes, which are subsequently converted to potent androgens by other members of the gut microbiota or host tissues. Butyricicoccus desmolans and Clostridium cadaveris have previously been reported for steroid-17,20-desmolase and 20β-hydroxysteroid dehydrogenase (HSDH) activities that are responsible for androstane formation from cortisol; however, the genes encoding these enzymes have yet to be reported. In this work, we identified and located a gene encoding 20β-HSDH in both B. desmolans and C. cadaveris. The 20β-HSDH of B. desmolans was heterologously overexpressed and purified from Escherichia coli. The enzyme was determined to be a homotetramer with subunit molecular mass of 33.8 ± 3.7 kDa. The r20β-HSDH displayed pH optimum in the reductive direction at pH 9.0 and in the oxidative direction at pH 7.0–7.5 with (20β-dihydro)cortisol and NAD(H) as substrates. Cortisol is the preferred substrate with K_m, 0.80 ± 0.06 μM; V_max, 30.36 ± 1.97 μmol·min⁻¹; K_cat, 607 ± 39 μmol·μM⁻¹·min⁻¹; K_cat/K_m, 760 ± 7.67. Phylogenetic analysis of the 20β-HSDH from B. desmolans suggested that the 20β-HSDH is found in several Bifidobacterium spp., one of which was shown to express 20β-HSDH activity. Notably, we also identified a novel steroid-17,20-desmolase-elaborating bacterium, Propionimicrobium lymphophilum, a normal inhabitant of the urinary tract.     

Methylobacterium bullatum sp. nov., a methylotrophic bacterium isolated from Funaria hygrometrica

A novel, pink-pigmented aerobic, facultatively methylotrophic bacterial strain (F3.2^T) isolated from the phyllosphere of Funaria hygrometrica, was analyzed using a polyphasic approach. Cells were Gram-negative, motile rods, strictly aerobic and non-spore-forming and exhibited surface structures varying in quantity, distribution and morphology. The isolate grew at 10–33 °C over a pH range of 5.5–8.0 and in the presence of less than 1.0% NaCl. Strain F3.2^T shared less than 70% DNA–DNA binding to the next type strain of the genus Methylobacterium (M. adhaesivum DSM 17169^T). In addition to the major cellular fatty acid C_18:1ω7c (81.7%), present in all Methylobacterium species (and also members of the genus Alphaproteobacteria), a high value (11.7%) of the fatty acids (summed feature) C_16:1ω7c and/or iso-C_15:02OH was determined. Phylogenetic analyses based on 16S rDNA and methanol dehydrogenase gene sequences, DNA–DNA hybridization values, chemotaxonomic and phenotypic characteristics indicate that the strain F3.2^T represents a novel species within the genus Methylobacterium. We propose the name Methylobacterium bullatum sp. nov. for this species. The type strain is the strain F3.2^T (DSM 21893^T = LMG 24788^T).     

Hyphomonas atlanticus sp. nov., isolated from the Atlantic Ocean and emended description of the genus Hyphomonas

A taxonomic study was carried out on strains 22II1-22F38^T and 22II-S13e, which were isolated from sea water and sediment from the Atlantic Ocean, respectively. The two strains were Gram-negative, oxidase and catalase positive, oval to pear shaped, and motile by a single polar flagellum. Phylogenetic analysis based on 16S rRNA gene sequences indicated that both strains belonged to the genus Hyphomonas, with highest sequence similarity (98.2%) to the type strains H. jannaschiana DSM 5153^T and H. johnsonii ATCC 43964^T. The genomic ANIm values and DNA-DNA hybridization estimate values between strain 22II1-22F38^T and seven type strains ranged from 82.84% to 84.10% and from 18.0% to 19.1%, respectively. Isolate 22II1-22F38^T had a G + C content of 58.3% and used Q-11 as the predominant respiratory quinone. The combined phenotypic and genotypic data showed that both strains represented a novel species of the genus Hyphomonas, for which the name Hyphomonas atlanticus sp. nov. is proposed, with the type strain being 22II1-22F38^T (=LMG 27916^T = MCCC 1A09418^T). In addition, we conclude that Hyphomonas hirschiana is a later synonym of Hyphomonas neptunium.     

Unconventional and effective methods for the regeneration of NAD(P) H in microorganisms or crude extracts of cells

Several yeasts, as well as aerobic and anaerobic bacteria catalyze the reduction of NAD and NADP in the presence of reduced methylviologen. The rates are usually much higher than those of reductions of unsaturated substrates by the organisms in cofermentations with carbohydrates. Since methylviologen can be continuously reduced at the cathode of an electrochemical cell it acts in catalytic amounts as a regenerable electron donor. Such systems may be superior to that with glucose as electron donor, because the NAD(P)H can be used exclusively for the reduction of the unsaturated substrate. The rate of the NAD(P)H formation depends very much on the organism and for the same organism on the growth procedure, the growth medium, the pretreatment of the cells, the pH, the buffer as well as on the ionic strength. Cells of Candida utilis which were frozen and thawed several times were superior to cells freshly harvested. Crude extracts revealed the best activities.Clostridia show the highest activities (up to 15 U per mg protein in the crude extract) and are suitable catalysts for the preparation of [4S-²H]NADH and [4S-²H]NADPH using ²H₂O-buffer in an electrochemical cell.The combinations of Alcaligenes eutrophus or Clostridium kluyveri and Candida utilis extracts in the presence of methylviologen are effective systems to reduce hydroxyacetone with hydrogen gas as electron donor or in an electrochemical cell. In this combination of microorganisms NADH is formed mainly by A. eutrophus or C. kluyveri and consumed for the reduction of hydroxyacetone by a reductase present in Candida utilis. The productivity numbers of such combinations are 10–30 times higher than those of yeasts alone.NAD(P)H regeneration, methylviologen-dependent NAD(P)H formation, deuterated NAD(P)H, Clostridia, yeast, bioreduction     

Degraded λ-carrageenan activates NF-κB and AP-1 pathways in macrophages and enhances LPS-induced TNF-α secretion through AP-1

Background

Carrageenan (CGN), a high molecular weight sulfated polysaccharide, is a traditional ingredient used in food industry. Its degraded forms have been identified as potential carcinogens, although the mechanism remains unclear.

Methods

The effects of degraded λ-carrageenan (λ-dCGN) on murine RAW264.7 cells and human THP-1-derived macrophage cells were investigated by studying its actions on tumor necrosis factor alpha (TNF-α) secretion, Toll-like receptor 4 (TLR4) expression, and activation of nuclear factor-κb (NF-κB) and activation protein-1 (AP-1) pathways.

Results

We found that λ-dCGN was much stronger than native λ-CGN in the activation of macrophages to secrete TNF-α. Treatment of RAW264.7 cells with λ-dCGN resulted in the upregulation of TLR4, CD14 and MD-2 expressions, but it did not increase the binding of lipopolysacchride (LPS) with macrophages. Meanwhile, λ-dCGN treatment activated NF-κB via B-cell lymphoma/leukemia 10 (Bcl10) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) phosphorylation. In addition, λ-dCGN induced extracellular signal-regulated kinases/1/2/mitogen-activated protein kinases (ERK1/2/MAPK) and AP-1 activation. Interestingly, pretreatment of RAW264.7 cells with λ-dCGN markedly enhanced LPS-stimulated TNF-α secretion. This pretreatment resulted in the enhanced phosphorylation of ERK1/2 and c-Jun N-terminal kinase (JNK) and intensified activation of AP-1.

Conclusions

λ-dCGN induced an inflammatory reaction via both NF-κB and AP-1, and enhanced the inflammatory effect of LPS through AP-1 activation.

General significance

The study demonstrated the role of λ-dCGN to induce the inflammatory reaction and to aggravate the effect of LPS on macrophages, suggesting that λ-dCGN produced during food processing and gastric digestion may be a safety concern.     

miR-202 suppresses cell proliferation in human hepatocellular carcinoma by downregulating LRP6 post-transcriptionally

MicroRNAs have emerged as important regulators of carcinogenesis. In the current study, we observed that microRNA-202 (miR-202) is downregulated in hepatocellular carcinoma (HCC) cells and tissues, indicating a significant correlation between miR-202 expression and HCC progression. Overexpression of miR-202 in HCC cells suppressed cell proliferation and tumorigenicity, while downregulation of miR-202 enhanced the cells’ proliferative capacity. Furthermore, we identified low-density lipoprotein receptor-related protein 6 (LRP6) as a direct target of miR-202. miR-202 suppresses the expression of LRP6 by binding to the 3′-untranslated region (UTR) of its mRNA. Finally, we found that silencing the expression of LRP6 is the essential biological function of miR-202 during HCC cell proliferation. Collectively, our findings reveal that miR-202 is a potential tumor suppressive miRNA that participates in carcinogenesis of human HCC by suppressing LRP6 expression.     

N-Acetylglucosamine Recognition by a Family 32 Carbohydrate-Binding Module from Clostridium perfringens NagH

Many carbohydrate-active enzymes have complex architectures comprising multiple modules that may be involved in catalysis, carbohydrate binding, or protein-protein interactions. Carbohydrate-binding modules (CBMs) are a common ancillary module whose function is to promote the adherence of the complete enzyme to carbohydrate substrates. CBM family 32 has been proposed to be one of the most diverse CBM families classified to date, yet all of the structurally characterized CBM32s thus far recognize galactose-based ligands. Here, we report a unique binding specificity and mode of ligand binding for a family 32 CBM. NagHCBM32-2 is one of four CBM32 modules in NagH, a family 84 glycoside hydrolase secreted by Clostridium perfringens. NagHCBM32-2 has the β-sandwich scaffold common to members of the family; however, its specificity for N-acetylglucosamine is unusual among CBMs. X-ray crystallographic analysis of the module at resolutions from 1.45 to 2.0 Å and in complex with disaccharides reveals that its mode of sugar recognition is quite different from that observed for galactose-specific CBM32s. This study continues to unravel the diversity of CBMs found in family 32 and how these CBMs might impart the carbohydrate-binding specificity to the extracellular glycoside hydrolases in C. perfringens.     

Luteolin as a glycolysis inhibitor offers superior efficacy and lesser toxicity of doxorubicin in breast cancer cells

Luteolin (Lu) exhibits a wide spectrum of anti-tumor activities, the present study was to observe whether Lu can sensitize breast cancer cells to doxorubicin (Dox) and to explain the basis underlying this phenomenon. In vitro, Lu at dose less than 100 μM had only slight effect on cells growth and cytotoxicity of Dox in 4T1 and MCF-7 cells under normoxia, but it could reverse tumor resistance to Dox and promote death of tumor cells under hypoxia. In vivo, Lu alone had also no effect on tumor growth delay, however, it could offer superior efficacy and lesser toxicity of Dox in 4T1 and MCF-7 bearing mice. Further study showed that Lu was able to suppress glycolytic flux but did not affect glucose uptake, the P-glycoprotein, anti-oxidative enzymes under hypoxia in vitro, and had not also effect on the intratumor Dox level in vivo. In addition, the activity of SOD and CAT was increased in serum and was decreased in tumor by Lu in vivo. These results suggest that luteolin as a glycolytic inhibitor might be a new adjuvant agent for chemotherapy.     

PI3 kinase/Akt signaling mediates epithelial-mesenchymal transition in hypoxic hepatocellular carcinoma cells

Hypoxia activates genetic programs that facilitate cell survival; however, in cancer, it may promote invasion and metastasis. Although the exact mechanisms driving hypoxia-induced invasion and metastasis remain elusive, we hypothesized that epithelial-mesenchymal transition (EMT) may play a major role. We investigated this in vitro by treating hepatocellular carcinoma cells under 1.0% O₂. After the hypoxia treatment, the cells exhibited some morphological changes including cell elongation, cytoskeletal rearrangement, and junctional disruption. Moreover, expression of the epithelia-specific marker E-cadherin was decreased and expression of the myofibroblast-specific marker vimentin was detected in the treated cells. Cell migration and ECM gel invasion were increased. These findings were consistent with events observed during EMT. Hypoxia-induced EMT is accompanied by increased phosphorylation, activation of Akt and the downstream signaling. Hypoxia-induced EMT was blocked by PI3K inhibitor LY294002. The results suggest that the PI3K/Akt-dependent signaling pathways serve to regulate hypoxia-induced EMT of hepatocellular carcinoma cells.     

我国海洋微生物菌种资源保藏与共享服务现状

海洋微生物多样性丰富。我国近年来在海洋微生物多样性调查、资源获取与开发利用等多方面取得重要进展。为促进资源的可持续高效利用,建立了专业的海洋微生物菌种保藏中心,目前库藏海洋微生物菌种2. 2万株。本文介绍了目前中国海洋微生物菌种保藏管理中心库藏资源概况与共享利用情况,以共享记录为基础,从已共享菌株的多样性、库藏资源共享利用以及共享用户等方面开展了统计分析。统计结果显示,共享菌株中超过80%为细菌,其次依次为丝状真菌、酵母、古菌及噬菌体。在菌株水平,已共享细菌覆盖了库藏细菌的28%;在种的水平,已共享细菌覆盖了库藏细菌的47%,包括变形菌纲3 178株,芽胞杆菌纲951株,放线菌纲817株。目前,库藏资源总共享比例约为30%,覆盖了库藏57%的属和49%的种。共享用户目前达266家,其中国内用户244家,国外用户22家。为大洋课题、国家973课题、863课题、自然基金、行业公益性项目、科技支撑及企业项目等各种科技计划,提供了重要的菌种资源保障。据不完全统计,共支撑发表SCI论文达500余篇。中国海洋微生物菌种保藏管理中心在菌株资源的收集、整理和共享等方面,为我国的科研事业提供了大量的资源支撑,取得了较好的社会效益。     

Regenerating islet-derived 1α (Reg-1α) protein is new neuronal secreted factor that stimulates neurite outgrowth via exostosin Tumor-like 3 (EXTL3) receptor

Regenerating islet-derived 1α (Reg-1α)/lithostathine, a member of a family of secreted proteins containing a C-type lectin domain, is expressed in various organs and plays a role in proliferation, differentiation, inflammation, and carcinogenesis of cells of the digestive system. We previously reported that Reg-1α is overexpressed during the very early stages of Alzheimer disease, and Reg-1α deposits were detected in the brain of patients with Alzheimer disease. However, the physiological function of Reg-1α in neural cells remains unknown. Here, we show that Reg-1α is expressed in neuronal cell lines (PC12 and Neuro-2a) and in rat primary hippocampal neurons (E17.5). Reg-1α is mainly localized around the nucleus and at the membrane of cell bodies and neurites. Transient overexpression of Reg-1α or addition of recombinant Reg-1α significantly increases the number of cells with longer neurites by stimulating neurite outgrowth. These effects are abolished upon down-regulation of Reg-1α by siRNA and following inhibition of secreted Reg-1α by antibodies. Moreover, Reg-1α colocalizes with exostosin tumor-like 3 (EXTL3), its putative receptor, at the membrane of these cells. Overexpression of EXTL3 increases the effect of recombinant Reg-1α on neurite outgrowth, and Reg-1α is not effective when EXTL3 overexpression is down-regulated by shRNA. Our findings indicate that Reg-1α regulates neurite outgrowth and suggest that this effect is mediated by its receptor EXTL3.     

HLA polymorphism in type 1 diabetes Tunisians

Several studies of the association between HLA and type 1 diabetes have been carried out revealing differences between ethnic groups. Our study, as part of the studies that should be performed about this association in the rest of the word, aims at elucidating the HLA DRB1, DQB1 polymorphism in Tunisian type 1 diabetes. This study includes 43 unrelated type 1 diabetes patients, and their mean age at onset is less than 15 years. Analysis of the frequency of alleles and haplotypes in these subjects, compared to a reference group (n = 101) led to the following results. 1) The Tunisian insulin-dependent diabetics present similarities as well as differences with other ethnic groups (Caucasians, North Africans). 2) The haplotype DRB1*04 DQ*0302 and DRB1*03 DQB1*0201 is positively associated to type 1 diabetes. 3) The heterozygotic genotype DRB1*04 DQB1*0302 / DRB1*03 DQB1*0201 is strongly associated to type 1 diabetes. 4) The haplotypes DRB1*01501 DQB1*0602 and DRB1*11 DQB1*0301 proved to be protective. In addition, the study of the subtypes DRB1*04 showed that alleles DRB1*0405 predispose to type 1 diabetes, whereas the allele DRB1*0403, which is in linkage disequilibrium with the DQB1*0402 in the Tunisian population, has a protective effect.     

Characterization of Transport Proteins for Aromatic Compounds Derived from Lignin: Benzoate Derivative Binding Proteins

In vitro growth experiments have demonstrated that aromatic compounds derived from lignin can be metabolized and represent a major carbon resource for many soil bacteria. However, the proteins that mediate the movement of these metabolites across the cell membrane have not been thoroughly characterized. To address this deficiency, we used a library representative of lignin degradation products and a thermal stability screen to determine ligand specificity for a set of solute-binding proteins (SBPs) from ATP-binding cassette (ABC) transporters. The ligand mapping process identified a set of proteins from Alphaproteobacteria that recognize various benzoate derivatives. Seven high-resolution crystal structures of these proteins in complex with four different aromatic compounds were obtained. The protein-ligand complexes provide details of molecular recognition that can be used to infer binding specificity. This structure-function characterization provides new insight for the biological roles of these ABC transporters and their SBPs, which had been previously annotated as branched-chain amino‐acid-binding proteins. The knowledge derived from the crystal structures provides a foundation for development of sequence-based methods to predict the ligand specificity of other uncharacterized transporters. These results also demonstrate that Alphaproteobacteria possess a diverse set of transport capabilities for lignin-derived compounds. Characterization of this new class of transporters improves genomic annotation projects and provides insight into the metabolic potential of soil bacteria.     

Isolation of laccase gene from Bacillus subtilis and analysis of its physicochemical properties

The present study reports the cloning and sequencing of lac2 from Bacillus subtilis. The gene is composed of 1542 bp and encodes a 514-amino acid protein. The gene has 86% homology with a published laccase with GeneID 936023. The lac2 gene was deposited in GenBank as a new nucleotide sequence. This new sequence was cloned into the multiple cloning site of pPIC9K to generate pPIC9K-lac2, which was then transformed into Pichia pastoris GS115 via electroporation. The recombinant GS115 (pPIC9K-lac2) was grown initially in BMGY medium and transferred to BMMY to induce gene expression for 48 h. The recombinant Lac2 protein shows laccase activity with α-naphthol and guaiacol as substrates. The optimal pH is between 3.2 and 4.7, and the optimal temperature is 25 °C for enzyme reaction.     

Efficiency of PCR-based methods in discriminating Bifidobacterium longum ssp. longum and Bifidobacterium longum ssp. infantis strains of human origin

Bifidobacterium longum is considered to play an important role in health maintenance of the human gastrointestinal tract. Probiotic properties of bifidobacterial isolates are strictly strain-dependent and reliable methods for the identification and discrimination of this species at both subspecies and strain levels are thus required. Differentiation between B. longum ssp. longum and B. longum ssp. infantis is difficult due to high genomic similarities. In this study, four molecular-biological methods (species- and subspecies-specific PCRs, random amplified polymorphic DNA (RAPD) method using 5 primers, repetitive sequence-based (rep)-PCR with BOXA1R and (GTG)₅ primers and amplified ribosomal DNA restriction analysis (ARDRA)) and biochemical analysis, were compared for the classification of 30 B. longum strains (28 isolates and 2 collection strains) on subspecies level. Strains originally isolated from the faeces of breast-fed healthy infants (25) and healthy adults (3) showed a high degree of genetic homogeneity by PCR with subspecies-specific primers and rep-PCR. When analysed by RAPD, the strains formed many separate clusters without any potential for subspecies discrimination. These methods together with arabionose/melezitose fermentation analysis clearly differentiated only the collection strains into B. longum ssp. longum and B. longum ssp. infantis at the subspecies level. On the other hand, ARDRA analysis differentiated the strains into the B. longum/infantis subspecies using the cleavage analysis of genus-specific amplicon with just one enzyme, Sau3AI. According to our results the majority of the strains belong to the B. longum ssp. infantis (75%). Therefore we suggest ARDRA using Sau3AI restriction enzyme as the first method of choice for distinguishing between B. longum ssp. longum and B. longum ssp. infantis.     

The deiodinases and the control of intracellular thyroid hormone signaling during cellular differentiation

Background

Thyroid hormone influences gene expression in virtually all vertebrates. Its action is initiated by the activation of T4 to T3, an outer ring deiodination reaction that is catalyzed by the type 1 or the type 2 iodothyronine selenodeiodinases (D1 or D2). Inactivation of T4 and T3 occurs via inner ring deiodination catalyzed by the type 3 iodothyronine selenodeiodinases (D3). The T4 concentration is generally quite stable in human plasma, with T3 levels also remaining constant. Deiodinase actions are tightly regulated in both pre- and post-natal life when they are required to make local adjustments of intracellular T3 concentrations in a precise spatio- and temporal manner. Although all the signals governing the dynamic expression of deiodinases in specific cell types are not known, many important regulatory factors have been deciphered.

Scope of review

This review provides striking examples from the recent literature illustrating how the expression of D2 and D3 is finely tuned during maturation of different organs, and how their action play a critical role in different settings to control intracellular T3 availability.

Major conclusions

Emerging evidence indicates that in various cell contexts, D2 and D3 are expressed in a dynamic balance, in which the expression of one enzyme is coordinately regulated with that of the other to tightly control intracellular T3 levels commensurate with cell requirements at that time.

General significance

Deiodinases control TH action in a precise spatio-temporal fashion thereby providing a novel mechanism for the local paracrine and autocrine regulation of TH action. This remarkable tissue-specific regulation of intracellular thyroid status remains hidden due to the maintenance of constant circulating TH concentrations by the hypothalamic–pituitary–thyroid axis. This article is part of a Special Issue entitled Thyroid hormone signalling.