Enhanced heterologous protein display on bacterial magnetic particles using a lon protease gene deletion mutant in Magnetospirillum magneticum AMB-1

ABSTRACT Previously we have shown that the African swine fever virus (ASFV) NL gene deletion mutant E70ΔNL is attenuated in pigs. Our recent observations that NL gene deletion mutants of two additional pathogenic ASFV isolates, Malawi Lil-20/1 and Pr4, remained highly virulent in swine (100% mortality) suggested that these isolates encoded an additional virulence determinant(s) that was absent from E70. To map this putative virulence determinant, in vivo marker rescue experiments were performed by inoculating swine with infection-transfection lysates containing E70 NL deletion mutant virus (E70ΔNL) and cosmid DNA clones from the Malawi NL gene deletion mutant (MalΔNL). A cosmid clone representing the left-hand 38-kb region (map units 0.05 to 0.26) of the MalΔNL genome was capable of restoring full virulence to E70ΔNL. Southern blot analysis of recovered virulent viruses confirmed that they were recombinant E70ΔNL genomes containing a 23- to 28-kb DNA fragment of the Malawi genome. These recombinants exhibited an unaltered MalΔNL disease and virulence phenotype when inoculated into swine. Additional in vivo marker rescue experiments identified a 20-kb fragment, encoding members of multigene families (MGF) 360 and 530, as being capable of fully restoring virulence to E70ΔNL. Comparative nucleotide sequence analysis of the left variable region of the E70ΔNL and Malawi Lil-20/1 genomes identified an 8-kb deletion in the E70ΔNL isolate which resulted in the deletion and/or truncation of three MGF 360 genes and four MGF 530 genes. A recombinant MalΔNL deletion mutant lacking three members of each MGF gene family was constructed and evaluated for virulence in swine. The mutant virus replicated normally in macrophage cell culture but was avirulent in swine. Together, these results indicate that a region within the left variable region of the ASFV genome containing the MGF 360 and 530 genes represents a previously unrecognized virulence determinant for domestic swine.

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Assembly of G Protein-Coupled Receptors onto Nanosized Bacterial Magnetic Particles Using Mms16 as an Anchor Molecule

Applied and Environmental Microbiology ◽

10.1128/aem.70.5.2880-2885.2004 ◽

2004 ◽

Vol 70 (5) ◽

pp. 2880-2885 ◽

Cited By ~ 49

Author(s):

Tomoko Yoshino ◽

Masayoshi Takahashi ◽

Haruko Takeyama ◽

Yoshiko Okamura ◽

Fukuichi Kato ◽

...

Keyword(s):

G Protein ◽

Magnetic Particles ◽

Lipid Membrane ◽

Specific Protein ◽

G Protein Coupled Receptors ◽

Native Conformation ◽

Wide Range ◽

Bacterial Magnetic Particles ◽

Magnetospirillum Magneticum ◽

G Protein Coupled

ABSTRACT G protein-coupled receptors (GPCRs) play a central role in a wide range of biological processes and are prime targets for drug discovery. GPCRs have large hydrophobic domains, and therefore purification of GPCRs from cells is frequently time-consuming and typically results in loss of native conformation. In this work, GPCRs have been successfully assembled into the lipid membrane of nanosized bacterial magnetic particles (BMPs) produced by the magnetic bacterium Magnetospirillum magneticum AMB-1. A BMP-specific protein, Mms16, was used as an anchor molecule, and localization of heterologous Mms16 on BMPs was confirmed by luciferase fusion studies. Stable luminescence was obtained from BMPs bearing Mms16 fused with luciferase at the C-terminal region. D1 dopamine receptor (D1R), a GPCR, was also efficiently assembled onto BMPs by using Mms16 as an anchor molecule. D1R-BMP complexes were simply extracted by magnetic separation from ruptured AMB-1 transformants. After washing, the complexes were ready to use for analysis. This system conveniently refines the native conformation of GPCRs without the need for detergent solubilization, purification, and reconstitution after cell disruption.

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The Expression Levels of Outer Membrane Proteins STM1530 and OmpD, Which Are Influenced by the CpxAR and BaeSR Two-Component Systems, Play Important Roles in the Ceftriaxone Resistance ofSalmonella entericaSerovar Typhimurium

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00216-11 ◽

2011 ◽

Vol 55 (8) ◽

pp. 3829-3837 ◽

Cited By ~ 31

Author(s):

Wensi S. Hu ◽

Hung-Wen Chen ◽

Rui-Yang Zhang ◽

Chung-Yi Huang ◽

Chi-Fan Shen

Keyword(s):

Outer Membrane ◽

Deletion Mutant ◽

Gene Deletion ◽

Mrna Levels ◽

Protein Levels ◽

Component Systems ◽

Two Component ◽

Serovar Typhimurium ◽

Two Component Systems ◽

Gene Deletion Mutant

ABSTRACTSignificant increases in STM3031, STM1530, and AcrD protein levels and significant decreases in OmpC and OmpD protein levels are present when the ceftriaxone-resistantSalmonella entericaserovar Typhimurium R200 strain is compared with the ceftriaxone-susceptible strain 01-4. AcrD is known to be involved in drug export, and STM3031 seems to play a key role in ceftriaxone resistance. Here, we examine the roles of STM1530, OmpC, and OmpD in ceftriaxone resistance. AnompDgene deletion mutant showed 4-fold higher ceftriaxone resistance than 01-4. AnompCgene deletion mutant showed 4-fold higher cephalothin and erythromycin resistance than 01-4, but there was no effect on ceftriaxone resistance. However, astm1530deletion mutant did show >64-fold lower ceftriaxone resistance than R200. Moreover, the STM3031 protein was significantly decreased in R200(Δstm1530) compared to R200. STM3031 expression has been shown to be influenced by the two-component system regulator genebaeR. CpxR seems to modulate BaeR. AcpxA-cpxRgene deletion mutant showed >2,048-fold lower ceftriaxone resistance than R200. The outer membrane protein profile of R200(ΔcpxAR) showed significant decreases in STM3031 and STM1530 compared to R200, while OmpD had returned to the level found in 01-4. Furthermore, thestm3031,stm1530, andompDmRNA levels were correlated with their protein expression levels in these strains, while decreases in the mRNA levels of the efflux pumpacrB,acrD, andacrFgenes were found in R200(ΔcpxAR). Findings similar to those for R200(ΔcpxAR) were found for R200(ΔbaeSR). These results, together with those for STM3031 and the fact that STM1530 is an outer membrane protein, suggest that STM1530 and OmpD are influenced by the CpxAR and BaeSR two-component systems and that this contributes toS. entericaserovar Typhimurium ceftriaxone resistance.

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6S-1 RNA Contributes to Sporulation and Parasporal Crystal Formation in Bacillus thuringiensis

Frontiers in Microbiology ◽

10.3389/fmicb.2020.604458 ◽

2020 ◽

Vol 11 ◽

Author(s):

Zhou Li ◽

Li Zhu ◽

Zhaoqing Yu ◽

Lu Liu ◽

Shan-Ho Chou ◽

...

Keyword(s):

Bacillus Thuringiensis ◽

Deletion Mutant ◽

Gene Deletion ◽

Gene Knockout ◽

Single Gene ◽

Crystal Formation ◽

Bacterial Cells ◽

Parasporal Crystal ◽

6S Rna ◽

Gene Deletion Mutant

6S RNA is a kind of high-abundance non-coding RNA that globally regulates bacterial transcription by interacting with RNA polymerase holoenzyme. Through bioinformatics analysis, we found that there are two tandem 6S RNA-encoding genes in the genomes of Bacillus cereus group bacteria. Using Bacillus thuringiensis BMB171 as the starting strain, we have explored the physiological functions of 6S RNAs, and found that the genes ssrSA and ssrSB encoding 6S-1 and 6S-2 RNAs were located in the same operon and are co-transcribed as a precursor that might be processed by specific ribonucleases to form mature 6S-1 and 6S-2 RNAs. We also constructed two single-gene deletion mutant strains ΔssrSA and ΔssrSB and a double-gene deletion mutant strain ΔssrSAB by means of the markerless gene knockout method. Our data show that deletion of 6S-1 RNA inhibited the growth of B. thuringiensis in the stationary phase, leading to lysis of some bacterial cells. Furthermore, deletion of 6S-1 RNA also significantly reduced the spore number and parasporal crystal content. Our work reveals that B. thuringiensis 6S RNA played an important regulatory role in ensuring the sporulation and parasporal crystal formation.

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