Shumin Hu

Shandong University, Jinan, Shandong Sheng, China

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Publications (8)21.91 Total impact

  • Tingting Guo, Shumin Hu, Jian Kong
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    ABSTRACT: Three putative promoter regions were identified preceding the nisZ gene in Lactococcus lactis HSM-22. To investigate their function in the control of nisZ biosynthesis, green fluorescence protein (GFP) was adopted as probe to determine activities of the three promoters. The results showed that PnisZ-0 containing two sets of the -35 and -10 regions exhibited the same maximum activity as promoter PnisZ-2 containing the putative promoter region near the start codon. However, the GFP expression level directed by PnisZ-0 was twofold higher than that found with PnisZ-2 under low-dose nisin, indicating that promoter PnisZ-1 distant from the start codon could be important in response to the inducer nisin. Then, Pnis-2 was randomized to develop functional promoters through the degenerate oligonucleotide approach in L. lactis. 35 inducible promoters and 14 constitutive promoters were obtained, covering 3-5 logs of expression levels in small increments of activity. Sequence analysis revealed that base changes in both consensus sequence and spacing sequence resulted in remarkable decrease of promoter activity, while the sequence outside -35 and -10 regions would influence the promoter function radically. The functional promoters were evaluated for the efficiency and stability to control β-galactosidase (Gal) expression in L. lactis. High correlation was obtained between the Gal activity and promoter strength, suggesting that promoters developed here have the potential for fine tuning gene expression in L. lactis.
    Current Microbiology 01/2013; · 1.52 Impact Factor
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    ABSTRACT: It was previously shown that the surface (S)-layer proteins covering the cell surface of Lactobacillus crispatus K313 were involved in the adherence of this strain to human intestinal cell line HT-29. To further elucidate the structures and functions of S-layers, three putative S-layer protein genes (slpA, slpB, and slpC) of L. crispatus K313 were amplified by PCR, sequenced, and characterized in detail. Quantitative real-time PCR analysis reveals that slpA was silent under the tested conditions; whereas slpB and slpC, the putative amino acid sequences which exhibited minor similarities to the previously reported S-layer proteins in L. crispatus, were actively expressed. slpB, which was predominantly expressed in L. crispatus K313, was further investigated for its functional domains. Genetic truncation of the untranslated leader sequence (UTLS) of slpB results in a reduction in protein production, indicating that the UTLS contributed to the efficient S-layer protein expression. By producing a set of N- and C-terminally truncated recombinant SlpB proteins in Escherichia coli, the cell wall-binding region was mapped to the C terminus, where rSlpB(380-501) was sufficient for binding to isolated cell wall fragments. Moreover, the binding ability of the C terminus was variable among the Lactobacillus species (S-layer- and non-S-layer-producing strains), and teichoic acid may be acting as the receptor of SlpB. To determine the adhesion region of SlpB to extracellular matrix proteins, ELISA was performed. Binding to immobilized types I and IV collagen was observed with the His-SlpB(1-379) peptides, suggesting that the extracellular matrix protein-binding domain was located in the N terminus.
    Applied Microbiology and Biotechnology 04/2012; · 3.69 Impact Factor
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    ABSTRACT: Currently, there is an increasing interest in the use of probiotics as an alternative strategy to antimicrobial compounds. In this study, two high adhesive strains Lactobacillus crispatus K313 adhering to HT-29 cells as well as Lb. crispatus K243 adhering to collagen type IV were isolated from chicken intestines. SDS-PAGE analysis revealed the presence of the potential S-proteins SlpA and SlpB in Lb. crispatus K243 and K313. SlpA and SlpB, rich in hydrophobic amino acids, were proved to be involved in adhering to collagen type IV and HT-29 cells, respectively, based on the LiCl treatment assay. After removal of S-proteins, the viability and tolerance of the two Lb. crispatus strains to simulated gastric and small intestinal juice were reduced, indicating the protective role of S-proteins against the hostile environments. Lb. crispatus K313 exhibited the stronger autoaggregation ability and inhibitive activity against Salmonella braenderup H9812 adhesion to HT-29 cells than the strain K243. To elucidate the inhibitive mechanism, cultured epithelial cells were exposed with Lb. crispatus strains, and followed by a challenge with S. braenderup H9812. The pro-inflammatory signaling factors (IL-8, CXCL1 and CCL20) from HT-29 were detected by real-time PCR technology. The results showed that both of Lb. crispatus strains down-regulated the transcription level of those pro-inflammatory genes induced by S. braenderup H9812 by 36.2-58.8%. ELISA analysis was further confirmed that Lb. crispatus K243 and K313 inhibited the IL-8 secretion triggered by S. braenderup H9812 by 32.8% and 47.0%, indicating that the two isolates could attenuate the pro-inflammatory signaling induced by S. braenderup H9812, and have the potential application in clinical practice to prevent diarrhea.
    Veterinary Microbiology 04/2012; 159(1-2):212-20. · 3.13 Impact Factor
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    ABSTRACT: Lactococcus lactis is a well-studied bacterium widely used in dairy fermentation and capable of producing metabolites with organoleptic and nutritional characteristics. For fine tuning of the distribution of glycolytic flux at the pyruvate branch from lactate to diacetyl and balancing the production of the two metabolites under aerobic conditions, a constitutive promoter library was constructed by randomizing the promoter sequence of the H(2)O-forming NADH oxidase gene in L. lactis. The library consisted of 30 promoters covering a wide range of activities from 7,000 to 380,000 relative fluorescence units using a green fluorescent protein as reporter. Eleven typical promoters of the library were selected for the constitutive expression of the H(2)O-forming NADH oxidase gene in L. lactis, and the NADH oxidase activity increased from 9.43 to 58.17-fold of the wild-type strain in small steps of activity change under aerobic conditions. Meanwhile, the lactate yield decreased from 21.15 ± 0.08 mM to 9.94 ± 0.07 mM, and the corresponding diacetyl production increased from 1.07 ± 0.03 mM to 4.16 ± 0.06 mM with the intracellular NADH/NAD(+) ratios varying from 0.711 ± 0.005 to 0.383 ± 0.003. The results indicated that the reduced pyruvate to lactate flux was rerouted to the diacetyl with an almost linear flux variation via altered NADH/NAD(+) ratios. Therefore, we provided a novel strategy to precisely control the pyruvate distribution for fine tuning of the lactate and diacetyl production through promoter engineering in L. lactis. Interestingly, the increased H(2)O-forming NADH oxidase activity led to 76.95% lower H(2)O(2) concentration in the recombinant strain than that of the wild-type strain after 24 h of aerated cultivation. The viable cells were significantly elevated by four orders of magnitude within 28 days of storage at 4°C, suggesting that the increased enzyme activity could eliminate H(2)O(2) accumulation and prolong cell survival.
    PLoS ONE 01/2012; 7(4):e36296. · 3.53 Impact Factor
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    ABSTRACT: Previous studies have revealed that the C-terminal region of the S-layer protein from Lactobacillus is responsible for the cell wall anchoring, which provide an approach for targeting heterologous proteins to the cell wall of lactic acid bacteria (LAB). In this study, we developed a new surface display system in lactic acid bacteria with the C-terminal region of S-layer protein SlpB of Lactobacillus crispatus K2-4-3 isolated from chicken intestine. Multiple sequence alignment revealed that the C-terminal region (LcsB) of Lb. crispatus K2-4-3 SlpB had a high similarity with the cell wall binding domains SA and CbsA of Lactobacillus acidophilus and Lb. crispatus. To evaluate the potential application as an anchoring protein, the green fluorescent protein (GFP) or beta-galactosidase (Gal) was fused to the N-terminus of the LcsB region, and the fused proteins were successfully produced in Escherichia coli, respectively. After mixing them with the non-genetically modified lactic acid bacteria cells, the fused GFP-LcsB and Gal-LcsB were functionally associated with the cell surface of various lactic acid bacteria tested. In addition, the binding capacity could be improved by SDS pretreatment. Moreover, both of the fused proteins could simultaneously bind to the surface of a single cell. Furthermore, when the fused DNA fragment of gfp:lcsB was inserted into the Lactococcus lactis expression vector pSec:Leiss:Nuc, the GFP could not be secreted into the medium under the control of the nisA promoter. Western blot, in-gel fluorescence assay, immunofluorescence microscopy and SDS sensitivity analysis confirmed that the GFP was successfully expressed onto the cell surface of L. lactis with the aid of the LcsB anchor. The LcsB region can be used as a functional scaffold to target the heterologous proteins to the cell surfaces of lactic acid bacteria in vitro and in vivo, and has also the potential for biotechnological application.
    Microbial Cell Factories 01/2011; 10:86. · 3.31 Impact Factor
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    ABSTRACT: Capsid protein (Cap) of porcine circovirus type 2 (PCV2) encoded by orf2 is a main structural protein with strong immunoreactivity. However, capsid protein is expressed poorly in prokaryotic organisms because of differences in codon usage. In this study, we introduce 24 synonymous mutations into orf2 by mutagenic primers and overlap extension polymerase chain reaction (OE-PCR) technique. Fourteen rare codons of orf2 were replaced with preferable codons used in Escherichia coli cells. Moreover, the nuclear localization signal (NLS) region rich in rare codon clusters at the 5′ end was deleted. The codon-optimized genes demonstrated higher levels of expression compared with wild-type genes. The influence of rare codons on the gene expression was eliminated by mutation. Western blot analysis confirmed the immunoreactivity of the proteins expressed by mutated genes. Further testing demonstrated that the mutated genes were also expressed successfully in Lactococcus lactis NZ9000. The immunologically active Cap proteins produced by recombinant strains have the potential applications for serological diagnostic assays and vaccine development against PCV2-associated diseases. Keywords orf2 –Rare codons– Escherichia coli – Lactococcus lactis –Expression
    World Journal of Microbiology and Biotechnology 01/2011; 27(3):651-657. · 1.26 Impact Factor
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    ABSTRACT: The endolysin Lyb5, from Lactobacillus fermentum temperate bacteriophage phiPYB5, showed a broad lytic spectrum against Gram-positive as well as Gram-negative bacteria. Sequence analysis revealed that the C terminus of the endolysin Lyb5 (Ly5C) contained three putative lysin motif (LysM) repeat regions, implying that Ly5C was involved in bacterial cell wall binding. To investigate the potential of Ly5C for surface display, green fluorescent protein (GFP) was fused to Ly5C at its N or C terminus and the resulting fusion proteins were expressed in Escherichia coli. After being mixed with various cells in vitro, GFP was successfully displayed on the surfaces of Lactococcus lactis, Lactobacillus casei, Lb. brevis, Lb. plantarum, Lb. fermentum, Lb. delbrueckii, Lb. helveticus, and Streptococcus thermophilus cells. Increases in the fluorescence intensities of chemically pretreated L. lactis and Lb. casei cells compared to those of nonpretreated cells suggested that the peptidoglycan was the binding ligand for Ly5C. Moreover, the pH and concentration of sodium chloride were optimized to enhance the binding capacity of GFP-Ly5C, and high-intensity fluorescence of cells was observed under optimal conditions. All results suggested that Ly5C was a novel anchor for constructing a surface display system for lactic acid bacteria (LAB). To demonstrate the applicability of the Ly5C-mediated surface display system, beta-galactosidase (beta-Gal) from Paenibacillus sp. strain K1, replacing GFP, was functionally displayed on the surfaces of LAB cells via Ly5C. The success in surface display of GFP and beta-Gal opened up the feasibility of employing the cell wall anchor of bacteriophage endolysin for surface display in LAB.
    Applied and Environmental Microbiology 02/2010; 76(8):2410-8. · 3.95 Impact Factor
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    ABSTRACT: A specific method to identify nisin-producing strains was developed based on Nisin-Controlled gene Expression (NICE) vector pSec:Nuc. The plasmid pSec:Nuc was transformed into non-nisin-producing strain Lactococcus lactis NZ9000, a host commonly used for the NICE system. The generating strain L. lactis NZ9000/pSec:Nuc could sense extracellular inducer nisin and efficiently secrete a reporter protein Nuc, the staphylococcal nuclease (Nuc) into the medium. Instead of using purified nisin, the culture supernatants of nisin-producing strains were also used as inducers. Therefore, the NICE system could be used to identify nisin-producing strains. With this principle, 4 among 56 lactococci strains isolated from raw milk were identified as nisin producers. The results were further confirmed by polymerase chain reaction amplification with their genomic DNA as templates, and nucleotide sequencing revealed that three of them produced nisin A, and the others produced nisin Z. Those results made it possible to isolate and identify nisin-producing strains specifically and rapidly using NICE system.
    Current Microbiology 03/2009; 58(6):604-8. · 1.52 Impact Factor