Chromosomal instability in Streptomyces avermitilis: major deletion in the central region and stable circularized chromosome

<p>Abstract</p> <p>Background</p> <p>The chromosome of <it>Streptomyces </it>has been shown to be unstable, frequently undergoing gross chromosomal rearrangements. However, the mechanisms underlying this phenomenon remain unclear, with previous studies focused on two chromosomal ends as targets for rearrangements. Here we investigated chromosomal instability of <it>Streptomyces avermitilis</it>, an important producer of avermectins, and characterized four gross chromosomal rearrangement events, including a major deletion in the central region. The present findings provide a valuable contribution to the mechanistic study of genetic instability in <it>Streptomyces</it>.</p> <p>Results</p> <p>Thirty randomly-selected "bald" mutants derived from the wild-type strain all contained gross chromosomal rearrangements of various types. One of the bald mutants, SA1-8, had the same linear chromosomal structure as the high avermectin-producing mutant 76-9. Chromosomes of both strains displayed at least three independent chromosomal rearrangements, including chromosomal arm replacement to form new 88-kb terminal inverted repeats (TIRs), and two major deletions. One of the deletions eliminated the 36-kb central region of the chromosome, but surprisingly did not affect viability of the cells. The other deletion (74-kb) was internal to the right chromosomal arm. The chromosome of another bald mutant, SA1-6, was circularized with deletions at both ends. No obvious homology was found in all fusion sequences. Generational stability analysis showed that the chromosomal structure of SA1-8 and SA1-6 was stable.</p> <p>Conclusions</p> <p>Various chromosomal rearrangements, including chromosomal arm replacement, interstitial deletions and chromosomal circularization, occurred in <it>S. avermitilis </it>by non-homologous recombination. The finding of an inner deletion involving in the central region of <it>S. avermitilis </it>chromosome suggests that the entire <it>Streptomyces </it>chromosome may be the target for rearrangements, which are not limited, as previously reported, to the two chromosomal ends.</p

α-Lys424 Participates in Insertion of FeMoco to MoFe Protein and Maintains Nitrogenase Activity in Klebsiella oxytoca M5al

Our previous investigation of substrates reduction catalyzed by nitrogenase suggested that α-Ile423 of MoFe protein possibly functions as an electron transfer gate to Mo site of active center-“FeMoco”. Amino acid residue α-Lys424 connects directly to α-Ile423, and they are located in the same α-helix (α423-431). In the present study, function of α-Lys424 was investigated by replacing it with Arg (alkaline, like Lys), Gln (neutral), Glu (acidic), and Ala (neutral) through site-directed mutagenesis and homologous recombination. The mutants were, respectively, termed 424R, 424Q, 424E, and 424A. Studies of diazotrophic cell growth, cytological, and enzymatic properties indicated that none of the substitutions altered the secondary structure of MoFe protein, or normal expression of nifA, nifL, and nifD. Substitution of alkaline amino acid (i.e., 424R) maintained acetylene (C2H2) and proton (H+) reduction activities at normal levels similar to that of wild-type (WT), because its FeMoco content did not reduce. In contrast, substitution of acidic or neutral amino acid (i.e., 424Q, 424E, 424A) impaired the catalytic activity of nitrogenase to varying degrees. Combination of MoFe protein structural simulation and the results of a series of experiments, the function of α-Lys424 in ensuring insertion of FeMoco to MoFe protein was further confirmed, and the contribution of α-Lys424 in maintaining low potential of the microenvironment causing efficient catalytic activity of nitrogenase was demonstrated

Population response of intestinal microbiota to acute Vibrio alginolyticus infection in half-smooth tongue sole (Cynoglossus semilaevis)

IntroductionVibriosis causes enormous economic losses of marine fish. The present study investigated the intestinal microbial response to acute infection of half-smooth tongue sole with different-dose Vibrio alginolyticus within 72 h by metagenomic sequencing.MethodsThe inoculation amount of V. alginolyticus for the control, low-dose, moderate-dose, and high-dose groups were 0, 8.5 × 101, 8.5 × 104, and 8.5 × 107 cells/g respectively, the infected fish were farmed in an automatic seawater circulation system under a relatively stable temperature, dissolved oxygen and photoperiod, and 3 ~ 6 intestinal samples per group with high-quality DNA assay were used for metagenomics analysis.ResultsThe acute infections with V. alginolyticus at high, medium, and low doses caused the change of different-type leukocytes at 24 h, whereas the joint action of monocytes and neutrophils to cope with the pathogen infection only occurred in the high-dose group at 72 h. The metagenomic results suggest that a high-dose V. alginolyticus infection can significantly alter the intestinal microbiota, decrease the microbial α-diversity, and increase the bacteria from Vibrio and Shewanella, including various potential pathogens at 24 h. High-abundance species of potential pathogens such as V. harveyii, V. parahaemolyticus, V. cholerae, V. vulnificus, and V. scophthalmi exhibited significant positive correlations with V. alginolyticus. The function analysis revealed that the high-dose inflection group could increase the genes closely related to pathogen infection, involved in cell motility, cell wall/ membrane/envelope biogenesis, material transport and metabolism, and the pathways of quorum sensing, biofilm formation, flagellar assembly, bacterial chemotaxis, virulence factors and antibiotic resistances mainly from Vibrios within 72 h.DiscussionIt indicates that the half-smooth tongue sole is highly likely to be a secondary infection with intestinal potential pathogens, especially species from Vibrio and that the disease could become even more complicated because of the accumulation and transfer of antibiotic-resistance genes in intestinal bacteria during the process of V. alginolyticus intensified infection

Experimental Study on Zn-Doped Al-Rich Alloys for Fast on-Board Hydrogen Production

For the purpose of investigating the effect of Zn replacement of In3Sn on the hydrogen production performance of Al-rich alloy ingots, Al-Ga-In3Sn alloys with various Zn dosages (0&ndash;5 wt.%) were prepared by a traditional melting and casting technique. The phase compositions and microstructures were characterized using X-ray diffractometer (XRD) and scanning electron microscope (SEM) with an Energy Dispersed X-ray system (EDS). The SEM results indicate that, with a small amount of Zn instead of In3Sn, the number and total area of grain boundary (GB) phases will decrease gradually, and the average single GB area will eventually stabilize. The distribution of Zn in the alloy is similar to that of Ga, and an area with high Zn content appeared in the high-Zn-doped sample. The melting behaviors of Al with other metals were measured by DSC. The reaction of these alloys and water were investigated at different temperatures. Compared with Al-Ga-In3Sn alloy, low addition of Zn changed the composition of GB phase and increased the maximum hydrogen production rate. The reason for the changes in the hydrolysis reaction of Al with the addition of Zn was discussed

Degradation of Tetracycline in Water by Fe-Modified Sterculia Foetida Biochar Activated Peroxodisulfate

Tetracycline (TC) is a broad-spectrum antibiotic commonly, made use of in aquaculture and animal husbandry. After entering water bodies, it will represent a major threat to human health. In this study, sterculia foetida biochar (SFC) was readied by the combined hydrothermal pyrolysis (co-HTP) method with sterculia foetida as raw materials. Fen-SFC (Fe2-SFC, Fe3-SFC, and Fe4-SFC) was obtained by doping SFH with different concentrations of FeCl3. Finally, activation of peroxodisulfate (PDS) was achieved, using Fe3-SFC to degrade TC. The degradation of TC obeyed pseudo-second-order kinetics, and the constant of the reaction rate was 0.491 L mg&minus;1 min&minus;1. Radical trapping experiments, EPR test and electrochemical tests evidenced that the high catalytic performance of the Fe3-SFC/PDS system was ascribed to free radical pathway (&bull;OH and SO4&bull;&minus;) and non-radical pathway (1O2 and electron transfer), in which the latter plays a dominant role. This research not only demonstrates a new kind of biochar as an effective catalyst for PS activation, but also offers an avenue for the value-added reuse of sterculia foetida

Surface expression of protein A on magnetosomes and capture of pathogenic bacteria by magnetosome/antibody complexes

Magnetosomes are membrane-enclosed magnetite nanocrystals synthesized by magnetotactic bacteria (MTB). They display chemical purity, narrow size ranges, and species-specific crystal morphologies. Specific transmembrane proteins are sorted to the magnetosome membrane (MM). MamC is the most abundant MM protein of Magnetospirillum gryphiswaldense strain MSR-1. MamF is the second most abundant MM protein of MSR-1 and forms stable oligomers. We expressed staphylococcal protein A (SPA), an immunoglobulin-binding protein from the cell wall of Staphylococcus aureus, on MSR-1 magnetosomes by fusion with MamC or MamF. The resulting recombinant magnetosomes were capable of self-assembly with the Fc region of mammalian antibodies (Abs) and were therefore useful for functionalization of magnetosomes. Recombinant plasmids pBBR-mamC-spa and pBBR-mamF-spa were constructed by fusing spa (the gene that encodes SPA) with mamC and mamF, respectively. Recombinant magnetosomes with surface expression of SPA were generated by introduction of these fusion genes into wild-type MSR-1 or a mamF mutant strain. Studies with a Zeta Potential Analyzer showed that the recombinant magnetosomes had hydrated radii significantly smaller than those of WT magnetosomes and zeta potentials less than -30 mV, indicating that the magnetosome colloids were relatively stable. Observed conjugation efficiencies were as high as 71.24 µg Ab per mg recombinant magnetosomes, and the conjugated Abs retained most of their activity. Numbers of Vibrio parahaemolyticus (a common pathogenic bacterium in seafood) captured by recombinant magnetosome/ Ab complexes were measured by real-time fluorescence-based quantitative PCR. One mg of complex was capable of capturing as many as 1.74×107 Vibrio cells. The surface expression system described here will be useful for design of functionalized magnetosomes from MSR-1 and other MTB

Degradation of Tetracycline in Water by Fe-Modified Sterculia Foetida Biochar Activated Peroxodisulfate

Tetracycline (TC) is a broad-spectrum antibiotic commonly, made use of in aquaculture and animal husbandry. After entering water bodies, it will represent a major threat to human health. In this study, sterculia foetida biochar (SFC) was readied by the combined hydrothermal pyrolysis (co-HTP) method with sterculia foetida as raw materials. Fen-SFC (Fe2-SFC, Fe3-SFC, and Fe4-SFC) was obtained by doping SFH with different concentrations of FeCl3. Finally, activation of peroxodisulfate (PDS) was achieved, using Fe3-SFC to degrade TC. The degradation of TC obeyed pseudo-second-order kinetics, and the constant of the reaction rate was 0.491 L mg−1 min−1. Radical trapping experiments, EPR test and electrochemical tests evidenced that the high catalytic performance of the Fe3-SFC/PDS system was ascribed to free radical pathway (•OH and SO4•−) and non-radical pathway (1O2 and electron transfer), in which the latter plays a dominant role. This research not only demonstrates a new kind of biochar as an effective catalyst for PS activation, but also offers an avenue for the value-added reuse of sterculia foetida

Epsilon-Fe2O3 is a novel intermediate for magnetite biosynthesis in magnetotactic bacteria

Abstract Background Natural biological magnetite nanoparticles are widely distributed from microorganisms to humans. It is found to be very important in organisms, especially in navigation. Moreover, purified magnetite nanoparticles also have potential applications in bioengineering and biomedicine. Magnetotactic bacteria (MTB) is considered one of the most abundant species around the world which can form intracellular membrane enveloped magnetic nanoparticles, referred to as magnetosomes. To our knowledge, the biomineralization of magnetosome in MTB involves a serious of genes located on a large unstable genomic region named magnetosome island, which specially exists in MTB. The magnetite core of magnetosome formed via a Fe (III) ion intermediates, for instance, α-Fe2O3 and ferrihydrite. Though the biosynthesis of magnetosome represents a general biomineralization mechanism of biogenic magnetite, knowledge of magnetosome biosynthesis and biomineralization remains very limited. Method Cells used in this study were cultured in a 7.5-L bioreactor, samples for intermediate capture were taken each certain time interval after the generation of magnetosome biosynthesis condition. High-resolution transmission electron microscopy were used to analyze the detailed structure of magnetosomes. The parameters of the crystal structures were obtained by Fast Fourier Transform analyses. Results In this study, we identified a novel intermediate phase, ε-Fe2O3, during the magnetite maturation process in MTB via kinetic analysis. Unlike α-Fe2O3, which has been reported as a precursor during magnetosome biosynthesis in MTB before, ε-Fe2O3, due to its thermal instability, is a rare phase with scarce natural abundance. This finding confirmed that ε-Fe2O3 is an important novel intermediate during the biomineralization of magnetosome in MTB, and shed new light on the magnetosome biosynthesis pathway