scholarly journals Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

2015 ◽  
Vol 81 (9) ◽  
pp. 3205-3217 ◽  
Author(s):  
Steven D. Smith ◽  
Romain Bridou ◽  
Alexander Johs ◽  
Jerry M. Parks ◽  
Dwayne A. Elias ◽  
...  
Keyword(s):  
Amino Acid ◽  
Inorganic Mercury ◽  
Desulfovibrio Desulfuricans ◽  
Site Directed Mutagenesis ◽  
Mercury Methylation ◽  
Amino Acid Residues ◽  
Content Type ◽  
Anaerobic Microorganisms ◽  
C Terminus ◽  
Methylation Capacity

ABSTRACTMethylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting ofhgcAandhgcB, encodes two of the proteins essential for this activity.hgcAencodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereashgcBencodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylatorDesulfovibrio desulfuricansND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. This study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.

scholarly journals Identification and Characterization of Porphyromonas gingivalis Client Proteins That Bind to Streptococcus oralis Glyceraldehyde-3-Phosphate Dehydrogenase

2012 ◽  
Vol 81 (3) ◽  
pp. 753-763 ◽  
Author(s):  
Kazuhiko Maeda ◽  
Hideki Nagata ◽  
Masae Kuboniwa ◽  
Miki Ojima ◽  
Tsukasa Osaki ◽  
...  
Keyword(s):  
Amino Acid ◽  
Porphyromonas Gingivalis ◽  
Interaction Analysis ◽  
Site Directed Mutagenesis ◽  
Receptor Protein ◽  
Oral Streptococci ◽  
Amino Acid Residues ◽  
Content Type ◽  
Streptococcus Oralis ◽  
Client Proteins

ABSTRACTCoaggregation ofPorphyromonas gingivalisand oral streptococci is thought to play an important role inP. gingivaliscolonization. Previously, we reported thatP. gingivalismajor fimbriae interacted withStreptococcus oralisglyceraldehyde-3-phosphate dehydrogenase (GAPDH), and that amino acid residues 166 to 183 of GAPDH exhibited strong binding activity towardP. gingivalisfimbriae (H. Nagata, M. Iwasaki, K. Maeda, M. Kuboniwa, E. Hashino, M. Toe, N. Minamino, H. Kuwahara, and S. Shizukuishi, Infect. Immun.77:5130–5138, 2009). The present study aimed to identify and characterizeP. gingivaliscomponents other than fimbriae that interact withS. oralisGAPDH. A pulldown assay was performed to detect potential interactions betweenP. gingivalisclient proteins andS. oralisrecombinant GAPDH with amino acid residues 166 to 183 deleted by site-directed mutagenesis. Seven proteins, namely,tonB-dependent receptor protein (RagA4), arginine-specific proteinase B, 4-hydroxybutyryl-coenzyme A dehydratase (AbfD), lysine-specific proteinase, GAPDH, NAD-dependent glutamate dehydrogenase (GDH), and malate dehydrogenase (MDH), were identified by two-dimensional gel electrophoresis followed by proteomic analysis using tandem mass spectrometry. Interactions between these client proteins andS. oralisGAPDH were analyzed with a biomolecular interaction analysis system.S. oralisGAPDH showed high affinity for five of the seven client proteins (RagA4, AbfD, GAPDH, GDH, and MDH). Interactions betweenP. gingivalisandS. oraliswere measured by a turbidimetric method and fluorescence microscopy. RagA4, AbfD, and GDH enhanced coaggregation, whereas GAPDH and MDH inhibited coaggregation. Furthermore, the expression ofluxSinP. gingivaliswas upregulated by RagA4, AbfD, and GDH but was downregulated by MDH. These results indicate that the fiveP. gingivalisclient proteins function as regulators inP. gingivalisbiofilm formation with oral streptococci.

scholarly journals A Cytoplasmic Antiholin Is Embedded In Frame with the Holin in a Lactobacillus fermentum Bacteriophage

2018 ◽  
Vol 84 (6) ◽  
Author(s):  
Tingting Guo ◽  
Yongping Xin ◽  
Chenchen Zhang ◽  
Jian Kong
Keyword(s):  
Amino Acid ◽  
Host Cell ◽  
Transmembrane Domain ◽  
Cell Lysis ◽  
Lactobacillus Fermentum ◽  
Host Cells ◽  
Amino Acid Residues ◽  
Content Type ◽  
C Terminus ◽  
Infection Cycles

ABSTRACT In double-stranded DNA bacteriophages, infection cycles are ended by host cell lysis through the action of phage-encoded endolysins and holins. The precise timing of lysis is regulated by the holin inhibitors, named antiholins. Sequence analysis has revealed that holins with a single transmembrane domain (TMD) are prevalent in Lactobacillus bacteriophages. A temperate bacteriophage of Lactobacillus fermentum , ϕPYB5, has a two-component lysis cassette containing endolysin Lyb5 and holin Hyb5. The hyb5 gene is 465 bp long, encoding 154 amino acid residues with an N-terminal TMD and a large cytoplasmic C-terminal domain. However, the N terminus contains no dual-start motif, suggesting that Hyb5 oligomerization could be inhibited by a specific antiholin. Two internal open reading frames in hyb5 , hyb5 157–465 and hyb5 209–328 , were identified as genes encoding putative antiholins for Hyb5 and were coexpressed in trans with lyb5-hyb5 in Escherichia coli . Surprisingly, host cell lysis was delayed by Hyb5 157–465 but accelerated by abolishment of the translation initiation site of this protein, indicating that Hyb5 157–465 acts as an antiholin to holin Hyb5. Moreover, deletion of 45 amino acid residues at the C terminus of Hyb5 resulted in early cell lysis, even in the presence of Hyb5 157–465 , implying that the interaction between Hyb5 157–465 and Hyb5 occurs at the C terminus of the holin. In vivo and in vitro , Hyb5 157–465 and Hyb5 were detected in the cytoplasmic and membrane fractions, respectively, and pulldown assays confirmed direct interaction between Hyb5 157–465 and Hyb5. All the results suggest that Hyb5 157–465 is an antiholin of Hyb5 that is involved in lysis timing. IMPORTANCE Phage-encoded holins are considered to be the “molecular clock” of phage infection cycles. The interaction between a holin and its inhibitor antiholin precisely regulates the timing of lysis of the host cells. As a prominent biological group in dairy processes, phages of lactic acid bacteria (LAB) have been extensively genome sequenced. However, little is known about the antiholins of LAB phage holins and the holin-antiholin interactions. In this work, we identified an in-frame antiholin against the class III holin of Lactobacillus fermentum phage ϕPYB5, Hyb5, and demonstrated its interaction with the cognate holin, which occurred in the bacterial cytoplasm.

scholarly journals Synergistic Effects of a Chalkophore, Methanobactin, on Microbial Methylation of Mercury

2020 ◽  
Vol 86 (11) ◽  
Author(s):  
Xixiang Yin ◽  
Lihong Wang ◽  
Lijie Zhang ◽  
Hongmei Chen ◽  
Xujun Liang ◽  
...  
Keyword(s):  
Food Web ◽  
Synergistic Effects ◽  
Inorganic Mercury ◽  
Desulfovibrio Desulfuricans ◽  
Geobacter Sulfurreducens ◽  
Mercury Methylation ◽  
Copper Binding ◽  
Thiol Compounds ◽  
Content Type ◽  
Net Production

ABSTRACT Microbial production of the neurotoxin methylmercury (MeHg) is a significant health and environmental concern, as it can bioaccumulate and biomagnify in the food web. A chalkophore or a copper-binding compound, termed methanobactin (MB), has been shown to form strong complexes with mercury [as Hg(II)] and also enables some methanotrophs to degrade MeHg. It is unknown, however, if Hg(II) binding with MB can also impede Hg(II) methylation by other microbes. Contrary to expectations, MB produced by the methanotroph Methylosinus trichosporium OB3b (OB3b-MB) enhanced the rate and efficiency of Hg(II) methylation more than that observed with thiol compounds (such as cysteine) by the mercury-methylating bacteria Desulfovibrio desulfuricans ND132 and Geobacter sulfurreducens PCA. Compared to no-MB controls, OB3b-MB decreased the rates of Hg(II) sorption and internalization, but increased methylation by 5- to 7-fold, suggesting that Hg(II) complexation with OB3b-MB facilitated exchange and internal transfer of Hg(II) to the HgcAB proteins required for methylation. Conversely, addition of excess amounts of OB3b-MB or a different form of MB from Methylocystis strain SB2 (SB2-MB) inhibited Hg(II) methylation, likely due to greater binding of Hg(II). Collectively, our results underscore the complex roles of microbial exogenous metal-scavenging compounds in controlling net production and bioaccumulation of MeHg in the environment. IMPORTANCE Some anaerobic microorganisms convert inorganic mercury (Hg) into the neurotoxin methylmercury, which can bioaccumulate and biomagnify in the food web. While the genetic basis of microbial mercury methylation is known, factors that control net methylmercury production in the environment are still poorly understood. Here, it is shown that mercury methylation can be substantially enhanced by one form of an exogenous copper-binding compound (methanobactin) produced by some methanotrophs, but not by another. This novel finding illustrates that complex interactions exist between microbes and that these interactions can potentially affect the net production of methylmercury in situ.

scholarly journals The C-Terminal Region of Bacteroides fragilis Toxin Is Essential to Its Biological Activity

2006 ◽  
Vol 74 (10) ◽  
pp. 5595-5601 ◽  
Author(s):  
Cynthia L. Sears ◽  
Simy L. Buckwold ◽  
Jai W. Shin ◽  
Augusto A. Franco
Keyword(s):  
Biological Activity ◽  
Amino Acid ◽  
Point Mutations ◽  
Bacteroides Fragilis ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
C Terminus

ABSTRACT To evaluate the role of the C-terminal region in Bacteroides fragilis toxin (BFT) activity, processing, and secretion, sequential C-terminal truncation and point mutations were created by site-directed mutagenesis. Determination of BFT activity on HT29/C1 cells, cleavage of E-cadherin, and the capacity to induce interleukin-8 secretion by wild-type BFT and C-terminal deletion mutants showed that deletion of only 2 amino acid residues at the C terminus significantly reduced BFT biological activity and deletion of eight or more amino acid residues obliterated BFT biologic activity. Western blot and reverse transcription-PCR analyses indicated that BFT mutants lacking seven or fewer amino acid residues in the C-terminal region are processed and expressed similar to wild-type BFT. However, BFT mutants lacking eight or more amino acids at the C terminus are expressed similar to wild-type BFT but are unstable. We concluded that the C terminus of BFT is not tolerant of modest amino acid deletions, suggesting that it is biologically important for BFT activity.

scholarly journals Novel Bacillus thuringiensis δ-Endotoxin Active against Locusta migratoria manilensis

2011 ◽  
Vol 77 (10) ◽  
pp. 3227-3233 ◽  
Author(s):  
Yan Wu ◽  
Cheng-Feng Lei ◽  
Dan Yi ◽  
Peng-Ming Liu ◽  
Mei-Ying Gao
Keyword(s):  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Locusta Migratoria ◽  
Significant Activity ◽  
Amino Acid Residues ◽  
Cry Protein ◽  
Content Type ◽  
Amino Terminal ◽  
C Terminus ◽  
Locusta Migratoria Manilensis

ABSTRACTA novel δ-endotoxin gene was cloned from aBacillus thuringiensisstrain with activity againstLocusta migratoria manilensisby PCR-based genome walking. The sequence of thecrygene was 3,432 bp long, and it encoded a Cry protein of 1,144 amino acid residues with a molecular mass of 129,196.5 kDa, which exhibited 62% homology with Cry7Ba1 in the amino acid sequence. The δ-endotoxin with five conserved sequence blocks in the amino-terminal region was designated Cry7Ca1 (GenBank accession no.EF486523). Protein structure analysis suggested that the activated toxin of Cry7Ca1 has three domains: 227 residues forming 7 α-helices (domain I); 213 residues forming three antiparallel β-sheets (domain II); and 134 residues forming a β-sandwich (domain III). The three domains, respectively, exhibited 47, 44, and 34% sequence identity with corresponding domains of known Cry toxins. SDS-PAGE and Western blot analysis showed that Cry7Ca1, encoded by the full-length open reading frame of thecrygene, the activated toxin 1, which included three domains but without the N-terminal 54 amino acid residues and the C terminus, and the activated toxin 2, which included three domains and N-terminal 54 amino acid residues but without the C terminus, could be expressed inEscherichia coli. Bioassay results indicated that the expressed proteins of Cry7Ca1 and the activated toxins (toxins 1 and 2) showed significant activity against 2nd instar locusts, and after 7 days of infection, the estimated 50% lethal concentrations (LC50s) were 8.98 μg/ml for the expressed Cry7Ca1, 0.87 μg/ml for the activated toxin 1, and 4.43 μg/ml for the activated toxin 2. The δ-endotoxin also induced histopathological changes in midgut epithelial cells of adultL. migratoria manilensis.

scholarly journals Primary- and secondary-structural analysis of a unique prokaryotic metallothionein from a Synechococcus sp. cyanobacterium

1988 ◽  
Vol 251 (3) ◽  
pp. 691-699 ◽  
Author(s):  
R W Olafson ◽  
W D McCubbin ◽  
C M Kay
Keyword(s):  
Amino Acid ◽  
Metal Binding ◽  
Helical Structure ◽  
Basic Amino Acid ◽  
Cysteine Residues ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Empirical Relations ◽  
Heavy Metal Binding ◽  
Synechococcus Sp

Biochemical and physiological studies of Synechococcus cyanobacteria have indicated the presence of a low-Mr heavy-metal-binding protein with marked similarity to eukaryotic metallothioneins (MTs). We report here the characterization of a Synechococcus prokaryotic MT isolated by gel-permeation and reverse-phase chromatography. The large number of variants of this molecule found during chromatographic separation could not be attributed to the presence of major isoproteins as assessed by amino acid analysis and amino acid sequencing of isoforms. Two of the latter were shown to have identical primary structures that differed substantially from the well-described eukaryotic MTs. In addition to six long-chain aliphatic residues, two aromatic residues were found adjacent to one another near the centre of the molecule, making this the most hydrophobic MT to be described. Other unusual features included a pair of histidine residues located in repeating Gly-His-Thr-Gly sequences near the C-terminus and a complete lack of association of hydroxylated residues with cysteine residues, as is commonly found in eukaryotes. Similarly, aside from a single lysine residue, no basic amino acid residues were found adjacent to cysteine residues in the sequence. Most importantly, sequence alignment analyses with mammalian, invertebrate and fungal MT sequences showed no statistically significant homology aside from the presence of Cys-Xaa-Cys structures common to all MTs. On the other hand, like other MTs, the prokaryotic molecule appears to be free of alpha-helical structure but has a considerable amount of beta-structure, as predicted by both c.d. measurements and the Chou & Fasman empirical relations. Considered together, these data suggested that some similarity between the metal-thiolate clusters of the prokaryote and eukaryote MTs may exist.

Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function

1997 ◽  
Vol 75 (6) ◽  
pp. 687-696 ◽  
Author(s):  
Tamo Fukamizo ◽  
Ryszard Brzezinski
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Binding ◽  
Structure And Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Streptomyces Sp ◽  
Binding Cleft ◽  
And Function

Novel information on the structure and function of chitosanase, which hydrolyzes the beta -1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation. Key words: chitosanase, amino acid sequence, overexpression system, reaction mechanism, site-directed mutagenesis.

scholarly journals Identification and characterization of amphibian SLC26A5 using RNA-Seq

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Zhongying Wang ◽  
Qixuan Wang ◽  
Hao Wu ◽  
Zhiwu Huang
Keyword(s):  
Amino Acid ◽  
Inner Ear ◽  
Hair Cells ◽  
Rana Catesbeiana ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Rna Seq ◽  
American Bullfrog ◽  
Frog Species

Abstract Background Prestin (SLC26A5) is responsible for acute sensitivity and frequency selectivity in the vertebrate auditory system. Limited knowledge of prestin is from experiments using site-directed mutagenesis or domain-swapping techniques after the amino acid residues were identified by comparing the sequence of prestin to those of its paralogs and orthologs. Frog prestin is the only representative in amphibian lineage and the studies of it were quite rare with only one species identified. Results Here we report a new coding sequence of SLC26A5 for a frog species, Rana catesbeiana (the American bullfrog). In our study, the SLC26A5 gene of Rana has been mapped, sequenced and cloned successively using RNA-Seq. We measured the nonlinear capacitance (NLC) of prestin both in the hair cells of Rana’s inner ear and HEK293T cells transfected with this new coding gene. HEK293T cells expressing Rana prestin showed electrophysiological features similar to that of hair cells from its inner ear. Comparative studies of zebrafish, chick, Rana and an ancient frog species showed that chick and zebrafish prestin lacked NLC. Ancient frog’s prestin was functionally different from Rana. Conclusions We mapped and sequenced the SLC26A5 of the Rana catesbeiana from its inner ear cDNA using RNA-Seq. The Rana SLC26A5 cDNA was 2292 bp long, encoding a polypeptide of 763 amino acid residues, with 40% identity to mammals. This new coding gene could encode a functionally active protein conferring NLC to both frog HCs and the mammalian cell line. While comparing to its orthologs, the amphibian prestin has been evolutionarily changing its function and becomes more advanced than avian and teleost prestin.

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