Use of RNA Immunoprecipitation Method for Determining <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> RNA<Emphasis Type="Italic">-</Emphasis>Hfq Protein Associations In Vivo

Background

Soil bacterium Sinorhizobium meliloti (S. meliloti) forms an endosymbiotic partnership with Medicago truncatula (M. truncatula) roots which results in root nodules. The bacteria live within root nodules where they function to fix atmospheric N₂ and supply the host plant with reduced nitrogen. The bacterial RNA-binding protein Hfq (Hfq) is an important regulator for the effectiveness of the nitrogen fixation. RNA immunoprecipitation (RIP) method is a powerful method for detecting the association of Hfq protein with specific RNA in cultured bacteria, yet a RIP method for bacteria living in root nodules remains to be described.

Results

A modified S. meliloti gene encoding a His-tagged Hfq protein (Hfq^His) was placed under the regulation of the native Hfq gene promoter (P_hfqsm). The trans produced Hfq^His protein was accumulated at its nature levels during all stages of the symbiosis, allowing RNAs that associated with the given protein to be immunoprecipitated with the anti-His antibody against the protein from root nodule lysates. RNAs that associated with the protein were selectively enriched in the immunoprecipitated sample. The RNAs were recovered by a simple method using heat and subsequently analyzed by RT-PCR. The nature of PCR products was determined by DNA sequencing. Hfq association with specific RNAs can be analyzed at different conditions (e. g. young or older root nodules) and/or in wild-type versus mutant strains.

Conclusions

This article describes the RIP method for determining Sinorhizobium meliloti RNA-Hfq associations in vivo. It is also applicable to other rhizobia living in planta, although some tissue-specific modification related to sample disruption and homogenization may be needed.

     

Enhancing Functional Expression of Heterologous <Emphasis Type="Italic">Burkholderia</Emphasis> Lipase in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Functional expression of lipase from Burkholderia sp. C20 (Lip) in various cellular compartments of Escherichia coli was explored. The poor expression in the cytoplasm of E. coli was improved by several strategies, including coexpression of the cytoplasmic chaperone GroEL/ES, using a mutant E. coli host strain with an oxidative cytoplasm, and protein fusion technology. Fusing Lip with the N-terminal peptide tags of T7PK, DsbA, and DsbC was effective in enhancing the solubility and biological activity. Non-fused Lip or Lip fusions heterologously expressed in the periplasm of E. coli formed insoluble aggregates with a minimum activity. Biologically active and intact Lip was obtained upon the secretion into the extracellular medium using the native signal peptide and the expression performance was further improved by coexpression of the periplasmic chaperon Skp. The extracellular expression was even more effective when Lip was secreted as a Lip–HlyA fusion via the α-hemolysin transporter. Finally, Lip could be functionally displayed on the E. coli cell surface when fused with the carrier EstA.     

Hfq regulates the expression of the thermostable direct hemolysin gene in <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis>

Background  

The hfq gene is conserved in a wide variety of bacteria and Hfq is involved in many cellular functions such as stress responses and the regulation of gene expression. It has also been reported that Hfq is involved in bacterial pathogenicity. However, it is not clear whether Hfq regulates virulence in Vibrio parahaemolyticus. To evaluate this, we investigated the effect of Hfq on the expression of virulence-associated genes including thermostable direct hemolysin (TDH), which is considered to be an important virulence factor in V. parahaemolyticus, using an hfq deletion mutant.     

Expression of <Emphasis Type="Italic">recA</Emphasis> gene of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

Plasmids pKS5 and pKSrec30 carrying normal and mutant alleles of the Deinococcus recA gene controlled by the lactose promoter slightly increase radioresistance of Escherichia coli cells with mutations in genes recA and ssb. The RecA protein of D. radiodurans is expressed in E. coli cells, and its synthesis can be supplementary induced. The radioprotective effect of the xenologic protein does not exceed 1.5 fold and yields essentially to the contribution of plasmid pUC19-recA1.1 harboring the E. coli recA ⁺ gene in the recovery of resistance of the ΔrecA deletion mutant. These data suggest that the expression of D. radiodurans recA gene in E. coli cells does not complement mutations at gene recA in the chromosome possibly due to structural and functional peculiarities of the D. radiodurans RecA protein.     

Molecular cloning,expression in <Emphasis Type="Italic">Escherichia</Emphasis><Emphasis Type="Italic">coli</Emphasis> of Attacin A gene from <Emphasis Type="Italic">Drosophila</Emphasis> and detection of biological activity

Attacin, a 20 kDa antibacterial peptide, plays an important role in immunity. To understand this gene better, gene cloning, expression and biological activity detection of Attacin A was carried out in present study. The full-length open reading frame (ORF) coding for Attacin A gene was generated using RT-PCR which takes total RNA extracted from Drosophila as the template. The gene was inserted directionally into the prokaryotic expression vector pET-32a (+). The resulting recombinant plasmid was transformed into E. coli Rosetta. SDS–PAGE was carried out to detect the expression product which was induced by IPTG. The antimicrobial activity and hemolysis activity were tested in vitro after purification. Agarose gel electrophoresis indicated that the complete ORF of Attacin A gene has been cloned successfully from Drosophila stimulated by E. coli which includes 666 bp and encodes 221 AA. The gene encoding mature Attacin A protein was amplified by PCR from the recombinant plasmid containing Attacin A, which includes 570 bp in all. SDS–PAGE analysis demonstrated that the fusion protein expressed was approximately 39.2 kDa. Biological activities detection showed that this peptide exhibited certain antibacterial activity to several G− bacteria, as well as minor hemolysis activity for porcine red blood cells. In conclusion, Attacin A gene was cloned and expressed successfully. It was the basis for further study of Attacin.     

<Emphasis Type="Italic">Thermus thermophilus</Emphasis> L4 ribosomal protein: purification and sensitivity alteration against erythromycin of <Emphasis Type="Italic">E. coli</Emphasis> cells harboring a single amino acid mutant of TthL4 within its extended loop

Summary. Protein L4 from the thermophilic bacterium Thermus thermophilus (TthL4) was heterologously overproduced in Escherichia coli cells and purified under native conditions by using ion exchange chromatography. Although it’s known strong binding to RNA (23S rRNA as well as mRNA) the yield of the purified protein was 6 mg per 10 g of cells and it is similar to that referred for Thermotoga maritima L4 ribosomal protein. In addition, E. coli cells harboring the wild type Thermus thermophilus L4 (wtTthL4) ribosomal protein as well as its mutant having changed the highly conserved glutamic acid 56 by alanine (TthL4-Ala 56) were incorporated into E. coli ribosomes after transformation of the host cells with the recombined vector. The cells having incorporated the mutant TthL4-Ala56 are more sensitive against erythromycin related to that containing the wtTthL4 protein. The resistance to the drug indicates that the mutated amino acid Glu56 is probably critical for the local ribosomal conformation and that its mutation induces conformational disturbances that are “transferred” to the entrance of the major exit tunnel, the place where the drug does bind.     

A Simple Method for In Vivo Expression Studies of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">citri</Emphasis>

A major problem in studying bacterial plant pathogens is obtaining the microorganism directly from the plant tissue to perform in vivo expression (protein or mRNA) analyses. Here we report an easy and fast protocol to isolate Xanthomonas axonopodis pv. citri directly from the host plant, in sufficient amounts to perform protein fingerprinting by 2-D gel electrophoresis as well as RNA expression assays. The protein profile obtained was very similar to that of X. axonopodis pv. citri grown in the presence of a leaf extract of Citrus sinensis; however, some differential proteins expressed in vivo were observed. Total RNA extraction revealed typical 16S and 23S bands in the agarose gel, and RT-PCR reactions using primers specific for genes of the bacterium confirmed the quality of the RNA preparation. Also, RT-PCR reactions using plant ribosomal primers were employed, and no amplification product was obtained, indicating that plant RNA is not present in the bacterium RNA sample.     

Translation enhancement by optimized downstream box sequences in <Emphasis Type="Italic">Escherichia coli </Emphasis>and<Emphasis Type="Italic"> Mycobacterium smegmatis</Emphasis>

The effect on translation of downstream box sequences optimized for binding to Mycobacterium smegmatis and Escherichia coli 16S rRNA in the absence of a Shine--Dalgarno (SD) region was investigated. The relative translational efficiency of each construct in either M. smegmatis or E. coli was determined. Eradication of the SD region in the absence of a downstream box abolished the translation activity. In contrast, optimized downstream box constructs resulted in a 13- and 18-fold increase in protein synthesis, relative to non-optimized DB controls in E. coli and M. smegmatis, respectively.     

Challenges Associated with Heterologous Expression of <Emphasis Type="Italic">Flavobacterium psychrophilum</Emphasis> Proteins in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A two-parameter statistical model was used to predict the solubility of 96 putative virulence-associated proteins of Flavobacterium psychrophilum (CSF259-93) upon over expression in Escherichia coli. This analysis indicated that 88.5% of the F. psychrophilum proteins would be expressed as insoluble aggregates (inclusion bodies). These solubility predictions were verified experimentally by colony filtration blot for six different F. psychrophilum proteins. A comprehensive analysis of codon usage identified over a dozen codons that are used frequently in F. psychrophilum, but that are rarely used in E. coli. Expression of F. psychrophilum proteins in E. coli was often associated with production of minor molecular weight products, presumably because of the codon usage bias between these two organisms. Expression of recombinant protein in the presence of rare tRNA genes resulted in marginal improvements in the expressed products. Consequently, Vibrio parahaemolyticus was developed as an alternative expression host because its codon usage is similar to F. psychrophilum. A full-length recombinant F. psychrophilum hemolysin was successfully expressed and purified from V. parahaemolyticus in soluble form, whereas this protein was insoluble upon expression in E. coli. We show that V. parahaemolyticus can be used as an alternate heterologous expression system that can remedy challenges associated with expression and production of F. psychrophilum recombinant proteins.     

High-level expression of the xylanase from <Emphasis Type="Italic">Thermomyces lanuginosus</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

According to the amino acid sequence, a codon-optimized xylanase gene (xynA1) from Thermomyces lanuginosus DSM 5826 was synthesized to construct the expression vector pHsh-xynA1. After optimization of the mRNA secondary structure in the translational initiation region of pHsh-xynA1, free energy of the 70 nt was changed from −6.56 to −4.96 cal/mol, and the spacing between AUG and the Shine-Dalgarno sequence was decreased from 15 to 8 nt. The expression level was increased from 1.3 to 13% of total cell protein. A maximum xylanase activity of 47.1 U/mL was obtained from cellular extract. The recombinant enzyme was purified 21.5-fold from the cellular extract of Escherichia coli by heat treatment, DEAE-Sepharose FF column and t-Butyl-HIC column. The optimal temperature and pH were 65 °C and pH 6.0, respectively. The purified enzyme was stable for 30 min over the pH range of 5.0–8.0 at 60 °C, and had a half-life of 3 h at 65 °C.     

Expression of the bifunctional <Emphasis Type="Italic">Bacillus subtilis</Emphasis> TatAd protein in <Emphasis Type="Italic">Escherichia</Emphasis><Emphasis Type="Italic">coli</Emphasis> reveals distinct TatA/B-family and TatB-specific domains

In the Tat protein export pathway of Gram-negative bacteria, TatA and TatB are homologous proteins that carry out distinct and essential functions in separate sub-complexes. In contrast, Gram-positive Tat systems usually lack TatB and the TatA protein is bifunctional. We have used a mutagenesis approach to delineate TatA/B-type domains in the bifunctional TatAd protein from Bacillus subtilis. This involved expression of mutated TatAd variants in Escherichia coli and tests to determine whether the variants could function as TatA or TatB by complementing E. coli tatA and/or tatB mutants. We show that mutations in the C-terminal half of the transmembrane span and the subsequent FGP ‘hinge’ motif are critical for TatAd function with its partner TatCd subunit, and the same determinants are required for complementation of either tatA or tatB mutants in Escherichia coli. This is thus a critical domain in both TatA and TatB proteins. In contrast, substitution of a series of residues at the N-terminus specifically blocks the ability of TatAd to substitute for E. coli TatB. The results point to the presence of a universally conserved domain in the TatA/B-family, together with a separate N-terminal domain that is linked to the TatB-type function in Gram-negative bacteria.     

Effects of <Emphasis Type="Italic">Escherichia coli</Emphasis> RraA Orthologs of <Emphasis Type="Italic">Vibrio vulnificus</Emphasis> on the Ribonucleolytic Activity of RNase E In Vivo

RraA is a recently discovered protein inhibitor of RNase E that catalyzes the initial step in the decay and processing of numerous RNAs in Escherichia coli. In the genome of Vibrio vulnificus, two open reading frames that potentially encode proteins homologous to E. coli, RraA-designated RraAV1 and RraAV2, have respectively 80.1% and 59.0% amino acid identity to RraA. The authors report that coexpression of RraAV1 protein in E. coli cells overproducing RNase E rescued these cells from growth arrest and restored their normal growth, whereas coexpression of RraAV2 protein further inhibited the growth of E. coli cells, whose growth was already impaired by overproduction of RNase E. Analyses of the steady-state level of various RNase E substrates indicated that the coexpression of RraAV1 more efficiently inhibited RNase E action than coexpression of RraA, and consequently resulted in the more increased abundance of each RNA species tested in vivo. The inhibitory effect by RraAV2 coexpression on RNase E was observed only in the case of trpA mRNA, indicating the possibility of RNA substrate-dependent inhibition of RraAV2 on RNase E. The findings suggest that these regulators of ribonuclease activity have both a conserved inhibitory function and a differential inhibitory activity on RNase E-like enzymes across the species.     

Expression and single-step purification of mercury transporter (merT) from <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis> in <Emphasis Type="Italic">E. coli</Emphasis>

The mercury transporter, merT, from Cupriavidus metallidurans was cloned into pRSET-C and expressed in various E. coli hosts. Expression of merT gene failed in common expression hosts like E. coli BL21(DE3), E. coli BL21(DE3)pLysS and E. coli GJ1158 due to expression induced toxicity. The protein was successfully expressed in E. coli C43(DE3) as inclusion bodies. The inclusion bodies were solubilized with Triton X-100 detergent. The detergent solubilized protein with N-terminal His-tag was purified in a single-step by immobilized metal affinity chromatography with a yield of 8 mg l⁻¹.     

Heterologous expression of the <Emphasis Type="Italic">msp2 gene</Emphasis> from <Emphasis Type="Italic">Marasmius scorodonius</Emphasis>

For the heterologous expression of the msp2 gene from the edible mushroom Marasmius scorodonius in Escherichia coli the cDNA encoding the extracellular Msp2 peroxidase was cloned into the pBAD III expression plasmid. Expression of the protein with or without signal peptide was investigated in E. coli strains TOP10 and LMG194. Different PCR products were amplified for expression of the native target protein or a protein with a signal peptide. Omitting the native stop codon and adding six His-residues resulted in a fusion protein amenable to immune detection and purification by immobilised metal affinity chromatography. In E. coli the recombinant protein was produced in high yield as insoluble inclusion bodies. The influence of different parameters on MsP2 refolding was investigated. Active enzyme was obtained by glutathione-mediated oxidation in a medium containing urea, Ca²⁺, and hemin.     

Regioselective glucosidation of <Emphasis Type="Italic">trans</Emphasis>-resveratrol in <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing glucosyltransferase from <Emphasis Type="Italic">Phytolacca americana</Emphasis>

A glucosyltransferase (GT) of Phytolacca americana (PaGT3) was expressed in Escherichia coli and purified for the synthesis of two O-β-glucoside products of trans-resveratrol. The reaction was moderately regioselective with a ratio of 4′-O-β-glucoside: 3-O-β-glucoside at 10:3. We used not only the purified enzyme but also the E. coli cells containing the PaGT3 gene for the synthesis of glycoconjugates. E. coli cell cultures also have other advantages, such as a shorter incubation time compared with cultured plant cells, no need for the addition of exogenous glucosyl donor compounds such as UDP-glucose, and almost complete conversion of the aglycone to the glucoside products. Furthermore, a homology model of PaGT3 and mutagenesis studies suggested that His-20 would be a catalytically important residue.     

Combinatorial modulation of <Emphasis Type="Italic">galP</Emphasis> and <Emphasis Type="Italic">glk</Emphasis> gene expression for improved alternative glucose utilization

Phosphoenolpyruvate (PEP) is an important precursor for anaerobic production of succinate and malate. Although inactivating PEP/carbohydrate phosphotransferase systems (PTS) could increase PEP supply, the resulting strain had a low glucose utilization rate. In order to improve anaerobic glucose utilization rate for efficient production of succinate and malate, combinatorial modulation of galactose permease (galP) and glucokinase (glk) gene expression was carried out in chromosome of an Escherichia coli strain with inactivated PTS. Libraries of artificial regulatory parts, including promoter and messenger RNA stabilizing region (mRS), were firstly constructed in front of β-galactosidase gene (lacZ) in E. coli chromosome through λ-Red recombination. Most regulatory parts selected from mRS library had constitutive strengths under different cultivation conditions. A convenient one-step recombination method was then used to modulate galP and glk gene expression with different regulatory parts. Glucose utilization rates of strains modulated with either galP or glk all increased, and the rates had a positive relation with expression strength of both genes. Combinatorial modulation had a synergistic effect on glucose utilization rate. The highest rate (1.64 g/L h) was tenfold higher than PTS⁻ strain and 39% higher than the wild-type E. coli. These modulated strains could be used for efficient anaerobic production of succinate and malate.