scholarly journals Functional Analysis of Alkane Hydroxylases from Gram-Negative and Gram-Positive Bacteria

2002 ◽  
Vol 184 (6) ◽  
pp. 1733-1742 ◽  
Author(s):  
Theo H. M. Smits ◽  
Stefanie B. Balada ◽  
Bernard Witholt ◽  
Jan B. van Beilen
Keyword(s):  
Functional Analysis ◽  
The Novel ◽  
Alkane Hydroxylase ◽  
Sequence Comparisons ◽  
Gram Positive Bacteria ◽  
Sequence Identity ◽  
Medium Chain Length ◽  
Protein Sequence Identity ◽  
Alkane Hydroxylases ◽  
Host Strains

ABSTRACT We have cloned homologs of the Pseudomonas putida GPo1 alkane hydroxylase from Pseudomonas aeruginosa PAO1, Pseudomonas fluorescens CHA0, Alcanivorax borkumensis AP1, Mycobacterium tuberculosis H37Rv, and Prauserella rugosa NRRL B-2295. Sequence comparisons show that the level of protein sequence identity between the homologs is as low as 35%, and that the Pseudomonas alkane hydroxylases are as distantly related to each other as to the remaining alkane hydroxylases. Based on the observation that rubredoxin, an electron transfer component of the GPo1 alkane hydroxylase system, can be replaced by rubredoxins from other alkane hydroxylase systems, we have developed three recombinant host strains for the functional analysis of the novel alkane hydroxylase genes. Two hosts, Escherichia coli GEc137 and P. putida GPo12, were equipped with pGEc47ΔB, which encodes all proteins necessary for growth on medium-chain-length alkanes (C6 to C12), except a functional alkane hydroxylase. The third host was an alkB knockout derivative of P. fluorescens CHA0, which is no longer able to grow on C12 to C16 alkanes. All alkane hydroxylase homologs, except the Acinetobacter sp. ADP1 AlkM, allowed at least one of the three hosts to grow on n-alkanes.

scholarly journals In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6

2008 ◽  
Vol 75 (2) ◽  
pp. 337-344 ◽  
Author(s):  
Daniel J. Koch ◽  
Mike M. Chen ◽  
Jan B. van Beilen ◽  
Frances H. Arnold
Keyword(s):  
Directed Evolution ◽  
Chain Length ◽  
Carbon Sources ◽  
Evolution System ◽  
Alkane Hydroxylase ◽  
Medium Chain ◽  
Medium Chain Length ◽  
Selection For ◽  
Alkane Hydroxylases

ABSTRACT Enzymes of the AlkB and CYP153 families catalyze the first step in the catabolism of medium-chain-length alkanes, selective oxidation of the alkane to the 1-alkanol, and enable their host organisms to utilize alkanes as carbon sources. Small, gaseous alkanes, however, are converted to alkanols by evolutionarily unrelated methane monooxygenases. Propane and butane can be oxidized by CYP enzymes engineered in the laboratory, but these produce predominantly the 2-alkanols. Here we report the in vivo-directed evolution of two medium-chain-length terminal alkane hydroxylases, the integral membrane di-iron enzyme AlkB from Pseudomonas putida GPo1 and the class II-type soluble CYP153A6 from Mycobacterium sp. strain HXN-1500, to enhance their activity on small alkanes. We established a P. putida evolution system that enables selection for terminal alkane hydroxylase activity and used it to select propane- and butane-oxidizing enzymes based on enhanced growth complementation of an adapted P. putida GPo12(pGEc47ΔB) strain. The resulting enzymes exhibited higher rates of 1-butanol production from butane and maintained their preference for terminal hydroxylation. This in vivo evolution system could be useful for directed evolution of enzymes that function efficiently to hydroxylate small alkanes in engineered hosts.

scholarly journals Synthesis and Bioassay of Novel Quinozolins

2019 ◽  
Vol 17 (72) ◽  
pp. 129-138
Author(s):  
Yasmine Kadom. Al-Majedy
Keyword(s):  
Twist Angle ◽  
Antimicrobial Properties ◽  
Heat Of Formation ◽  
Gram Negative Bacteria ◽  
The Novel ◽  
Gram Positive Bacteria ◽  
Visible Spectra ◽  
Ft Ir ◽  
Uv Visible

Novel Quinozolins were synthesized in a good yield through convert lacton to lactam and study the biological activity of the synthesized compounds. Quinozolins were characterized by elemental analysis, FT-IR and UV/visible spectra. The novel Quinozolins have been tested in vitro against (gram positive bacteria Staphylococcus aureus and against other gram negative bacteria, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Proteus vulgaris; in order to assess their antimicrobial properties. Moreover, charge, bond length, bond angle, twist angle, heat of formation and steric energy were calculated by using of the ChemOffice program. The study indicates that these Quinozolins have high activity against tested bacteria. Based on the reported results, it may be concluded that the coumarin act as synthons for synthesis of new Quinozolins derivatives through the replacement of oxygen atom by nitrogen atom.

scholarly journals First Report of Cucurbit aphid-borne yellows virus in Spain

Plant Disease ◽  
2004 ◽  
Vol 88 (8) ◽  
pp. 907-907 ◽  
Author(s):  
M. Juarez ◽  
V. Truniger ◽  
M. A. Aranda
Keyword(s):  
Nucleotide Sequence ◽  
Nucleotide Sequence Identity ◽  
Rt Pcr ◽  
Sequence Comparisons ◽  
E Coli ◽  
Sequence Identity ◽  
Tissue Print ◽  
Sigma Chemical ◽  
Beet Pseudo Yellows Virus ◽  
Cucumis Melo L

In late spring 2003, field-grown melon plants (Cucumis melo L.) showing bright yellowing of older leaves were observed near Valladolises in Campo de Cartagena, Murcia, Spain. Symptoms resembled those caused by viruses of the genus Crinivirus (family Closteroviridae), but absence or very low populations of whiteflies were observed. However, diseased foci showed clear indications of heavy aphid infestations. Later, during the fall of 2003, squash plants (Cucurbita pepo L.) grown in open fields in the same area showed similar symptoms. Tissue print hybridizations to detect Cucurbit yellow stunting disorder virus (CYSDV) and Beet pseudo yellows virus (BPYV) in symptomatic samples were negative. CYSDV and BPYV are two yellowing-inducing criniviruses previously described in Spain. In contrast, standard double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) with antiserum against Cucurbit aphid-borne yellows virus (CABYV; genus Polerovirus, family Luteoviridae) that was kindly provided by H. Lecoq (INRA-Montfavet Cedex, France) were consistently positive. Definitive confirmation of CABYV associated with symptomatic samples was obtained by performing reverse-transcription polymerase chain reaction (RT-PCR) analyses for the CABYV coat protein gene. Total RNA extracts (TRI reagent; Sigma Chemical, St. Louis, MO) were obtained from symptomatic and asymptomatic leaf samples and RT-PCR reactions were carried out using the primers 5′-GAATACGGTCGCGGCTAGAAATC-3′ (CE9) and 5′-CTATTTCGGGTTCTGGACCTGGC-3′ (CE10) based on the CABYV sequence published by Guilley et al. (2). A single DNA product of approximately 600 bp was obtained only from symptomatic samples. Amplified DNA fragments from two independent samples (samples 36-2 and 37-5) were cloned in E. coli and sequenced (GenBank Accession Nos. AY529653 and AY529654). Sequence comparisons showed a 95% nucleotide sequence identity between the two sequences. A 97% and 94% nucleotide sequence identity was found among 36-2 and 37-5, respectively and the CABYV sequence published by Guilley et al. (2). CABYV seems to be widespread throughout the Mediterranean Basin (1,3) but to our knowledge, it has not previously been described in Spain. Additionally, our data suggest that significant genetic variability might be present in the Spanish CABYV populations. References: (1) Y. Abou-Jawdah et al. Crop Prot. 19:217, 2000. (2) H. Guilley et al. Virology 202:1012, 1994. (3) H. Lecoq et al. Plant Pathol. 41:749, 1992.

scholarly journals Genome-based classification of Calidifontibacillus erzurumensis gen. nov., sp. nov., isolated from a hot spring in Turkey, with reclassification of Bacillus azotoformans as Calidifontibacillus azotoformans comb. nov. and Bacillus oryziterrae as Calidifontibacillus oryziterrae comb. nov.

2020 ◽  
Vol 70 (12) ◽  
pp. 6418-6427
Author(s):  
Ahmet Adiguzel ◽  
Hilal Ay ◽  
Mustafa Ozkan Baltaci ◽  
Sumeyya Akbulut ◽  
Seyda Albayrak ◽  
...  
Keyword(s):  
16S Rrna ◽  
Type Species ◽  
Hot Spring ◽  
Rrna Genes ◽  
Sequence Comparisons ◽  
Novel Genus ◽  
Content Type ◽  
Sequence Identity ◽  
Link Type ◽  
Bacillus Azotoformans

A novel Gram-stain-positive, rod-shaped, endospore-forming, motile, aerobic bacterium, designated as P2T, was isolated from a hot spring water sample collected from Ilica-Erzurum, Turkey. Phylogenetic analyses based on 16S rRNA gene sequence comparisons affiliated strain P2T with the genus Bacillus , and the strain showed the highest sequence identity to Bacillus azotoformans NBRC 15712T (96.7 %). However, the pairwise sequence comparisons of the 16S rRNA genes revealed that strain P2T shared only 94.7 % sequence identity with Bacillus subtilis subsp. subtilis NCIB 3610T, indicating that strain P2T might not be a member of the genus Bacillus . The digital DNA–DNA hybridization and average nucleotide identity values between strain P2T and B. azotoformans NBRC 15712T were 19.8 and 74.2 %, respectively. The cell-wall peptidoglycan of strain P2T contained meso-diaminopimelic acid. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an aminophospholipid, five unidentified phospholipids and two unidentified lipids while the predominant isoprenoid quinone was MK-7. The major fatty acids were iso-C15 : 0 and iso-C16 : 0. The draft genome of strain P2T was composed of 82 contigs and found to be 3.5 Mb with 36.1 mol% G+C content. The results of phylogenomic and phenotypic analyses revealed that strain P2T represents a novel genus in the family Bacillaceae , for which the name Calidifontibacillus erzurumensis gen. nov., sp. nov. is proposed. The type strain of Calidifontibacillus erzurumensis is P2T (=CECT 9886T=DSM 107530T=NCCB 100675T). Based on the results of the present study, it is also suggested that Bacillus azotoformans and Bacillus oryziterrae should be transferred to this novel genus as Calidifontibacillus azotoformans comb. nov. and Calidifontibacillus oryziterrae comb. nov., respectively.

scholarly journals Lysis Genes of the Bacillus subtilisDefective Prophage PBSX

1998 ◽  
Vol 180 (8) ◽  
pp. 2110-2117 ◽  
Author(s):  
Susanne Krogh ◽  
Steen T. Jørgensen ◽  
Kevin M. Devine
Keyword(s):  
Functional Analysis ◽  
Host Cell ◽  
Expression System ◽  
Cell Lysis ◽  
Lethal Gene ◽  
Gram Negative Bacteria ◽  
Gene Products ◽  
Gram Negative ◽  
Gram Positive Bacteria ◽  
Exported Protein

ABSTRACT Four genes identified within the late operon of PBSX show characteristics expected of a host cell lysis system; they arexepA, encoding an exported protein; xhlA, encoding a putative membrane-associated protein; xhlB, encoding a putative holin; and xlyA, encoding a putative endolysin. In this work, we have assessed the contribution of each gene to host cell lysis by expressing the four genes in different combinations under the control of their natural promoter located on the chromosome of Bacillus subtilis 168. The results show thatxepA is unlikely to be involved in host cell lysis. Expression of both xhlA and xhlB is necessary to effect host cell lysis of B. subtilis. Expression ofxhlB (encoding the putative holin) together withxlyA (encoding the endolysin) cannot effect cell lysis, indicating that the PBSX lysis system differs from those identified in the phages of gram-negative bacteria. Since host cell lysis can be achieved when xlyA is inactivated, it is probable that PBSX encodes a second endolysin activity which also uses XhlA and XhlB for export from the cell. The chromosome-based expression system developed in this study to investigate the functions of the PBSX lysis genes should be a valuable tool for the analysis of other host cell lysis systems and for expression and functional analysis of other lethal gene products in gram-positive bacteria.

scholarly journals Intra- and Interspecific Comparisons of Bacterial Diversity and Community Structure Support Coevolution of Gut Microbiota and Termite Host

2005 ◽  
Vol 71 (11) ◽  
pp. 6590-6599 ◽  
Author(s):  
Yuichi Hongoh ◽  
Pinsurang Deevong ◽  
Tetsushi Inoue ◽  
Shigeharu Moriya ◽  
Savitr Trakulnaleamsai ◽  
...  
Keyword(s):  
Community Structure ◽  
Gut Microbiota ◽  
Fragment Length Polymorphism ◽  
Clonal Analysis ◽  
Polymorphism Analysis ◽  
The Novel ◽  
Termite Species ◽  
Bacterial Phyla ◽  
Sequence Identity ◽  
Interspecific Comparisons

ABSTRACT We investigated the bacterial gut microbiota from 32 colonies of wood-feeding termites, comprising four Microcerotermes species (Termitidae) and four Reticulitermes species (Rhinotermitidae), using terminal restriction fragment length polymorphism analysis and clonal analysis of 16S rRNA. The obtained molecular community profiles were compared statistically between individuals, colonies, locations, and species of termites. Both analyses revealed that the bacterial community structure was remarkably similar within each termite genus, with small but significant differences between sampling sites and/or termite species. In contrast, considerable differences were found between the two termite genera. Only one bacterial phylotype (defined with 97% sequence identity) was shared between the two termite genera, while 18% and 50% of the phylotypes were shared between two congeneric species in the genera Microcerotermes and Reticulitermes, respectively. Nevertheless, a phylogenetic analysis of 228 phylotypes from Microcerotermes spp. and 367 phylotypes from Reticulitermes spp. with other termite gut clones available in public databases demonstrated the monophyly of many phylotypes from distantly related termites. The monophyletic “termite clusters” comprised of phylotypes from more than one termite species were distributed among 15 bacterial phyla, including the novel candidate phyla TG2 and TG3. These termite clusters accounted for 95% of the 960 clones analyzed in this study. Moreover, the clusters in 12 phyla comprised phylotypes from more than one termite (sub)family, accounting for 75% of the analyzed clones. Our results suggest that the majority of gut bacteria are not allochthonous but are specific symbionts that have coevolved with termites and that their community structure is basically consistent within a genus of termites.

Relationships of tailed phages: a survey of protein sequence identity

1995 ◽  
Vol 140 (10) ◽  
pp. 1871-1884 ◽  
Author(s):  
H. -W. Ackermann ◽  
A. Elzanowski ◽  
G. Fobo ◽  
G. Stewart
Keyword(s):  
Protein Sequence ◽  
Sequence Identity ◽  
Protein Sequence Identity
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