Cell division inhibitor minC from Lactobacillus Acidophilus VTCC-B-871 and detection of antimicrobial activities in functional study

The minC gene of Lactobacillus acidophilus VTCC-B-871 was identified based on the genome data of L. rhamnous strains in genebank, and antimicrobial activities other than cell division inhibitor was detected. After cloning and sequencing, there was 99.1% homology between minC gene of L. acidophilus VTCC-B-871 and L. rhamnous GG using Blast search. By analysis, the result also showed the close relation between these strains that was necessary in the identification of these probiotics. Besides, using pET21a(+) vector for the over-expression of minC protein in Escherichia coli BL21(DE3)plysS under 2 mM Isopropyl β-D-1-thiogalactopyranoside within 5 h, the filamentation occurred and showed that minC was functional across species. Especially, the study detected successfully, the antimcrobial activities on Pseudomonas aeruginosa ATCC 27853 in the cultures containing glucose, saccharose and manitol. The antimicrobial agent still showed the activities after treated with temperature at 100, 90, 80, 70, and 60°C in 15 min, but loss activity after protease K treatment at 10 mg/ml for 30 min. The characterization pointed the antimicrobial agent seemed as colicin in E. coli. The present work was the first reported in characterizing the Lactobacillus minC homolog that might be useful in probiotic classification and antibiotic production.


INTRODUCTION
MinC was one of cell division inhibitors in Escherichia coli (E.coli), Neiseiria gonorrhea (Szeto et al., 2001) and Bacillus subtilis (Stahlber et al., 2004).In E. coli, the Min system was composed of the minC, minD and minE proteins encoded by the minB operon (De Boer et al., 1988Boer et al., , 1989)).Most previous studies showed the effects of Min system in cell division, and only one study showed that minC was a nanorecorder (Bhomkar et al., 2011).However, there was no study on the oscillation of minC causing the alteration in secondary products in organisms that will produce and improve many products in the pharmaceutica l field.MinC is dynamically related to the FtsZ and other proteins (De Boer et al., 1989).Colicins are bacterial antibiotic toxins produced by E. coli cells and are active against E. coli and closely related strains.To penetrate the target cell, colicins bind to an outer membrane receptor at the cell surface and then translocate their N-terminal domain through the outer membrane and the periplasm.Once fully translocated, the N-terminal domain triggers entry of the catalytic Cterminal domain by an unknown process.Colicin K uses the Tsx nucleoside-specific receptor for binding at the cell surface, the OmpA protein for translocation through the outer membrane, and the TolABQR proteins for the transit through the periplasm (Benedetti et al., 1991;Aurélie et al., 2010).Colicins also interacted with FtsH (Mathieu et al., 2011).Here, we initiated studies to identify minC gene in L. acidophilus VTCC-B-871.That would be the way to identify L. acidophilus and rhamnosusm, probiotics commonly used in healthcare (Kaur et al., 2001).Besides, the functional study on secondary product producing of the host, the secondary product was antimicrobial agents on pathgens.Until now, the pathogens as Candida albicans, Salmonella typhi, Staphylococcus aureus, and Pseudomonas aeruginosa were the causes of infectious diseases.
For the above reasons, the study focused on the minC gene from L. acidophilus VTCC-B-871 to support the identification of Lactobacillus probiotics and to improve the secondary product in host.Firstly, minC gene in L. acidophilus VTCC-B-871 should be cloned and identified, followed by function study as cell division and antmicrobial activities.

Plasmids, bacterial strains, growth conditions
The pGEM-T vectors used for molecular cloning and E. coli JM109, BL21(DE3)pLysS were purchased by Promega.The pET21(a+) used for over-expression was purchased by Novagen.L. acidophilus VTCC-B-871 was purchased by Vietnam type culture collection (VTCC).E. coli JM109 was used as a host to clone Lactobacillus minC gene.E. coli BL21(DE3)pLysS was used as an expression strain.Lactobacillus strain were grown on deMan, Rogosa and Sharpe (MRS) for 72 to 96 h at 30°C.E. coli strains were grown in Luria-Bertani (LB) for 18 to 24 h at 37°C, with shaking at 200 rpm.When required, antibiotics were added to media in the following concentrations: 100 µg of ampicillin/ml, 5, 20, 34 µg of chloramphenicol/ml for E. coli.

DNA isolation
The L. acidophillus was incubated in MRS broth at 30°C in 48 to 72 h.The white colonies with the milky smell were picked up and transferred onto MRS agar.The incubation condition was at 37°C in 24 to 48 h aerobically.The genomic DNA was isolated according to Sambrook et al. (1989).

Sequencing and analysis of minC
Based on the similarity of minC of L. rhamnosus strains sequenced and deposited in DNA Data Bank of Japan (DDBJ), the minC of L. acidophilus was thought to have a high similarity at the 5 and 3 ends of minC of L. rhamnosus.In order to clone, the primers used to isolate minC were designated from the minC of L. rhamnosus.The primer set was as sense 5'-GTGGACAGTGTGGTCTTAAAAGGTC-3' and an antisense 5'-CTACATTGCCTCCATTTGCTTAAATAATC-3'.The genomic DNA from L. acidophilus VTCC-B-871 strain was used as template to perform Polymerase chain reaction (PCR).The PCR fragment was amplified and sequencing was performed with the ABI PRIZM 310 genetic analyzer using the BigDye terminator cycle sequencing ready reaction kit according to the manufacturer's protocols.The Lactobacillus minC gene was confirmed and analyzed using Fasta.The protein molecular mass, pI, were calculated on an ExPASy Proteomics Server.The sequence data obtained in this study has been submitted to the DDBJ.

Overexpression of minC and microscopy studies
The L. acidophilus minC gene was amplified by PCR with a sense primer CHE1 (5'-CATATGGACAGTGTGGTCTTAAAAGGTC-3') (the NdeI site is underlined) and an antisense CHE2 (5'-CTCGAGCATTGCCTCCATTTGCTTAAATAATC-3') (the XhoI site is underlined).The amplified minC was subcloned into pGEM-T vector and then checked again by DNA sequencing.The minC fragment was cut out from pGEM-T vector by NdeI and XhoI double-digestion and inserted into the same sites of pET-21(a+) to produce pET-21(a+)/minC.E. coli BL21(DE3)pLysS transformed with pET-21(a+)/minC was grown at 37°C in Luria-Bertani (LB) medium supplemented with appropriate antibiotics but chloramphenicol at 5, 20, and 34 µg/µl to OD 600 = 0.4 to 0.5, after which 2 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to culture to induce at 37°C for 5 h.Light microscopy was used to observe the morphological changes in E. coli.

Antimicrobial activity test
Antimicrobial effects were tested on S. aureus ATCC 25923, E. coli ATCC 9637, Salmonella typhi ATCC 19430, C. albicans ATCC 14053, P. aeruginosa ATCC 27853 by the agar diffusion method.The tested microorganisms were propagated twice and then grown for 18 to 24 h in 10 ml of appropriate growth media.Turbidity of the culture broth was compared with McFarland tubes to give an estimate of bacterial population (1 × 10 6 CFU/ml).Supernatant of the cell after expression were collected after centrifugation at 12,000 × g for 15 min, and the clear supernatant was sterilized by filtration (0.45 μm), thus yielding cell-free filtrates.
The wells (ø 6 mm) were then prepared and filled using 100 μl of cell-free filtrate.The inoculated plates were incubated for 18 to 24 h at appropriate temperatures, and the diameter of the inhibition zone was measured in millimeters with calipers.The measurements recorded were from the edge of the zone to the edge of the wall.The study was also optimized for the antimicrobial activities in cultures containing 1% glucose, 1% saccharose and 1% manitol.

Partial characterization of the antimicrobial agent
To test the thermostability and sensitivity to protease, both tests were performed according to Lewus et al. (1991).100 μl of the supernatant were incubated 15 min at 60, 70, 80, 90 and 100°C within 15 min, followed by the bacteriocin test.To test the sensitivity to protease, the supernatant was treated with proteinase K (10 mg/ml) and the mixture was incubated 30 min at 37°C, followed by the bacteriocin tests.

Analysis of minC gene of L. acidophilus VTCC-B-871
After PCR performance, a PCR product was amplified (Figure 1).After cloning, a minC gene of 657 bp was sequenced.This sequence showed 99.1% simlariry to L. rhamnosus GG and was deposited in the DDBJ database under accession no.AB755424.The minC protein of L. acidophillus VTCC-B-871 consisted of 218 amino acids with a calculated pI of 5.31 and Mw of 23509 Da.The protein exhibits 99% identity to minC from L. rhamnosus GG (ID: 8421052).Interestingly, there were similarities of 60 to 70 nucleotides at both 5' end and 3' end of L. rhamnosus GG and L. acidophillus VTCC-B-871.Figures 2 and 3 showed the similarity of the nucleotides in the sequence at both ends and the difference of the nucleotides of A to C at the position of 70 nt and G at the 590 nt of the sequence, respectively.Based on the similarity, the designated primer set would be used to identify these Lactobacillus strains.

Expression of minC gene in E. coli and its effect on cell division
To study the function of L. acidophillus minC protein, this work tried to test whether the L. acidophilus minC protein was functional in E. coli cells, the E. coli BL21(DE3)plysS was introduced with plasmid pET-21(a+)/minC.The recombinant E. coli cells were cultured in media containing without or with chloramphenicol at 5, 20 and 34 µg/µl.
The over-expression was done under Isopropyl β-D-1thiogalactopyranoside (IPTG) with or without combination of glucose, saccharose, manitol.Under the condition of 2 mM IPTG for expression, the cells transformed with the pET-21(a+) exhibited normal rod-shaphed morphology (Figure 4) and pET-21(a+)/minC exhibited the filament formation after 5 h of over-expression.This result indicated that Lactobacillus minC was functional across species.The morphology of E. coli harboring minC in different sugars also formed filamentation after 5 hoverexpression.
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Antimicrobial activities
The supernatants of the overexpressed cell cultures with and without glucose, saccharose, manitol were centrifuged and used to test the actvities on the above mentioned pathogens.The results showed the activities on P. aeruginosa (Figure 5).The activities were also obtained in the cultures without ampicillin and cloramphenicol surprisingly (Figure 5).The antimicrobial agent was tested on the other strains (data not shown).
The antimicrobial agent might be the colicin secreted from E. coli.This result revealed that minC might be related to bacteriocin production.The tests also used the samples from the E. coli without harborring Lactobacillus minC.Consequently, there was no inhibition zone formed by this strain (Figure 5).Presented in Figure 5 was the inhibition of the growth of P. aeruginosa ATCC 27853 by the action of the supernatants.The inhibition zone diameters were 17 to 19 mm and were not significant between conditions (P < 0.05).To characterize the agent, the supernatant was treated with high temperature and protease K which showed that the antimicrobial agent in the supernatant was heat-stable and damage in protease K (Figure 6).However, there was not significant in five levels of tested temperature (P < 0.05).The results presented the inhibition zone in Figure 6.

DISCUSSION
The PCR product was amplified succesfully in Figure 1 and the minC sequence was confirmed and analysed in Figure 2. It meant that L. acidophilus and rhamnosus had the similarity in cell division.Especially, the sequences had the similarity at both ends that might be conveniet to identify these strain or that they were closely related.The minC gene was overexpressed in cultures with and without sugars, but the filamentation fomation still developed.The sugars supplied the energy for cell growth as Adenosine-5'-triphosphate (ATP) or Guanosine-5'triphosphate (GTP).MinC activated with minD and FtsZ (De Boer et al., 1991).However, minD required ATP while FtsZ required GTP for their functions (Walker et al., 1982).The inhibition on Pseudomonas might reveal a colicin in the host E. coli.The study tried to test the activities in cultures without any antibiotic as well as in heat treatment and consequently, there were also the inhibition.In the Figure 5, the supernatants from E. coli without harboring minC did not show the antimicrobial acivities.Therefore, minC of Lactobacillus introduced into E. coli had oscillation and interaction with minCDE of E. coli and change the conformation of the cell mem rane and caused colicin to e secreted.In the lower concentration of chloramphenicol as 5 µg/ml, the activities was weaker than the concentration of chloramphenicol at 20 and 34 µg/ml (Figure 5).With the result without chloramphenol in the combination of the  control, the used antibiotics were lysed during culture (Benjamin et al., 2009).Clearly, minC play a role in antimicrobial agent or bacteriocin production.

CONCLUSION
The study was the first report that minC play a role in antimicrobial agent or suspected colicin.The study also detected minC in L. acidophilus that was benefit in identi-fication of L. rhamnosus and L. acidophilus.The minC homologue was also over-expressed in E. coli successfully.

Figure 1 .
Figure 1.The PCR product of minC gene from isolated Lactobacillus acidophilus.A: λ/Hind III marker, B: PCR product.The arrow showed the PCR product.

Figure 2 .
Figure 2. Comparision of the nucleotide segment at both 5 and 3 ends of L. acidophilus and L. rhamnosus using Blast search.The red letters indicate the similarity at 5 and 3 ends.The green letters indicate that the different nucleotides appeared first at both ends.The stars show the sequence remaining portion.

Figure 3 .Figure 4 .
Figure 3.The chromatogram portion of sequence showed the first different site and the last different sites at the both ends of nucleotide sequence.The upper: the black short line on the peaks of A and C indicate that the different nucleotides appeared first at 5 end.The lower: the black short line on the peaks of C indicate that the complementary nucleotide G appeared at 3 end.
Thanks to the grant from National Foundation of Science and Technology of Vietnam-NAFOSTED and the support of Hochiminh city International University for this work.