X-gal® inhibits the swarming of Vibrio species

Journal of Microbiological Methods 66 (2006) 552 – 555 www.elsevier.com/locate/jmicmeth

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X-gal® inhibits the swarming of Vibrio species Moon-Young Kim a , Ra-Young Park a , Young-Hoon Bai b , Yoon-Young Chung c , Choon-Mee Kim a , Soo-Young Kim d , Joon-Haeng Rhee d , Sung-Heui Shin a,⁎ a

d

Research Center for Resistant Cells, Chosun University Medical School, 375 Seosuk-Dong, Dong-Gu, Gwangju 501-759, Republic of Korea b Department of Biology, Chosun University Medical School, Gwangju 501-759, Republic of Korea c Department of Anatomy, Chosun University Medical School, Gwangju 501-759, Republic of Korea Clinical Vaccine R&D Center, National Research Laboratory of Molecular Microbial Pathogenesis, Research Institute of Vibrio Infection and Genome Research Center for Enteropathogenic Bacteria, and Department of Microbiology, Chonnam National University Medical School, Gwangju 501-746, Republic of Korea Received 10 January 2006; accepted 12 January 2006 Available online 21 February 2006

Abstract The expressional levels of genes in swarmer cells can be determined by a simple method using X-gal-containing semisolid agars and lacZ-fusion transcription reporter strains of the genes concerned. However, X-gal alone inhibited the swarming of Vibrio, regardless of their ability to digest X-gal. Moreover, X-gal inhibited the growth of V. vulnificus containing functional lacZ. These effects of X-gal itself should be carefully considered when trying to determine the expression levels of genes in swarming cells using X-gal-containing semisolid agar. © 2006 Elsevier B.V. All rights reserved. Keywords: Vibrio; X-gal; β-galactosidase; Swarming

Swarming is a specialized form of surface motility displayed by several flagellated bacteria, and shares features with other surface phenomenon such as biofilm formation and host invasion. During swarming, the expressions of many virulence-related genes are up- or down-regulated. This surface-dependent regulation of virulence-related genes suggests that swarming can serve as a model for gene expression during the initial stages of host infections (Wang et al., 2004; Harshey, 2003). Therefore, it is important that simple or reliable methods are developed that enable the expressions of genes to be determined during swarming. ⁎ Corresponding author. Tel.: +82 62 230 6352; fax: +82 62 233 6052. E-mail address: [email protected] (S.-H. Shin). 0167-7012/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2006.01.004

One such method that can determine the expressions of certain virulence-related genes during swarming was introduced by Walker et al. (1999). This method consists of two steps: (1) the construction of lacZ-fusion transcriptional reporter strains for putative virulencerelated genes, and (2) the cultivation of these reporter strains on semisolid agars containing X-gal (5-bromo-4chloro-3-indolyl-β-D-galactopyranoside) as a chromogenic substrate for LacZ (β-galactosidase). The main advantage of this method is that it allows the gross discrimination of gene expression levels during swarming. In order to make it easier to discriminate gene expression levels, resulting swarming haloes should be as large as possible; that is to say, X-gal alone should not inhibit swarming. Therefore, here, we investigated the effect of X-gal on swarming.

M.-Y. Kim et al. / Journal of Microbiological Methods 66 (2006) 552–555

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2% NaCI-HI + DMF

2% NaCI-HI + PBS

2% NaCI-HI + X-gal

V. vulnificus

V. alginolyticus

V. cholerae

V. mimicus

V. parahaemolyticus

Fig. 1. Inhibition of Vibrio species swarming by X-gal. V. vulnificus MO6-24/O, V. alginolyticus, V. cholerae Non-O1, V. mimicus, or V. parahaemolyticus grown in 2% NaCl–HI agars were inoculated onto the surfaces of HI semisolid agars containing PBS, dimethylformamide (DMF) or 96 μg/ml of X-gal using the end of toothpick, and the plates were incubated at 37 °C for 8 h.

The following bacteria were used: Vibrio cholerae Non-O1 (clinical isolate), Vibrio parahaemolyticus (ATCC 27519), Vibrio alginolyticus (ATCC 17749), Vibrio mimicus (ATCC 33653), Vibrio vulnificus MO624/O (lacZ+), Proteus mirabilis (ATCC 2566), Serratia marcescens (ATCC 2216), and Escherichia coli DH5αcontaining the TOPO TA cloning vector (Invitrogen; lacZ+). A chromosomal lacZ-deletion mutant of V. vulnificus MO6-24/O (named CMM2101) was constructed by using a suicide vector pKAS32 as described previously (Kim et al., 2002). A lacZ-insertional mutant of E. coli DH5α was constructed by inserting a DNA fragment into the lacZ gene on TOPO TA cloning vector. A 355-bp DNA fragment from the V. vulnificus desA open reading frame (Tanabe et al., 2005) was amplified using the primer set (5′-atcccacgatgataagacaagtacagcg3′ and 5′-accttgagacgttacttcgcccaattgg-3′) and cloned 8

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into the lacZ of TOPO TA cloning vector, and then the plasmid was transformed into E. coli DH5α. E. coli showing white colony on LB agar containing ampicillin and X-gal was selected, and then insertional disruption of lacZ was confirmed by analyzing restriction fragments on agarose gel. Heart Infusion (HI, BD) agar (1.5% Bacto-agar) containing an additional 2% of NaCl was used to cultivate Vibrio species, and HI agar without added NaCl was used to cultivate the other bacteria. HI semisolid agar (0.3% Bacto-agar) with or without additional 2% NaCl was used to observe swarming. In order to observe the effect of X-gal (Bioneer and Duchefa Biochemie) on bacterial swarming, HI semisolid agars containing PBS, dimethylformamide (DMF; 50 μl/ml) or 96 μg/ml of X-gal dissolved with DMF were used. Bacteria grown on HI agars with/without 8

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HI + PBS

HI + X-gal

P. mirabilis

S. marcescens

Fig. 2. The facilitation of Proteus mirabilis and Serratia marcescens swarming by X-gal. Bacteria grown in HI agars were inoculated onto the surface of HI semisolid agars with PBS or 96 μg/ml of X-gal using the end of a toothpick, and the plates were incubated at 37 °C for 24 h.

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M.-Y. Kim et al. / Journal of Microbiological Methods 66 (2006) 552–555

A

2% NaCI-HI + PBS

B MO6-24/O (lacZ+)

E. coli (lacZ+)

MO6-24/O (lacZ-)

E. coli (lacZ-)

HI + PBS

2% NaCI-HI + X-gal

HI + X-gal

Fig. 3. (A) The lacZ mutation had no effect on the inhibition of Vibrio vulnificus swarming by X-gal. V. vulnificus MO6-24/O (lacZ+) and its lacZdeletion mutant (lacZ−) grown in 2% NaCl–HI agars were inoculated onto the surfaces of 2% NaCl–HI semisolid agars with PBS or 96 μg/ml of Xgal using the end of toothpick, and the plates were incubated at 37 °C for 8 h. (B) The lacZ mutation had no effect on the inhibition of E. coli swarming by X-gal. E. coli DH5α (lacZ+) and its lacZ-insertion mutant (lacZ−) grown in HI agars were inoculated onto the surfaces of HI semisolid agars with PBS or 96 μg/ml of X-gal using the end of a toothpick, and the plates were incubated at 37 °C for 24 h.

NaCl at 37 °C overnight were inoculated onto the surfaces of HI semisolid agars with/without NaCl and/ or X-gal using the end of toothpick, and incubated at 37 °C. To determine the effect of X-gal on bacterial growth, about 1 × 103 cfu/ml of bacteria were inoculated into HI broths with/without NaCl containing PBS, DMF or 96 μg/ml of X-gal, and cultured with vigorous shaking (220 rpm) at 37 °C for 8 h. Culture aliquots (100 μl) were serially diluted and spread on the HI agar surfaces, the agar plates were incubated at 37 °C for

24 h, and viable cells were counted. Bacterial growths were expressed as the means and standard errors of triplicate experiments. X-gal obviously inhibited the swarming of all Vibrio species used in this study (Fig. 1). In contrast, X-gal facilitated the swarming of P. mirabilis and S. marcescens (Fig. 2). E. coli did not show swarming (Fig. 3), as observed in a previous study (Niu et al., 2005). The swarming of all bacteria was not affected by DMF alone, which is used as the solvent for X-gal (Fig. 1). No difference was observed when X-gals

Viable cells (x 108 cfu/ml)

25 PBS DMF X-gal

20

15

10

5

0 VA

VC

VM

VP

Z+

lac

( VV

)

-)

cZ

VV

(la

Strains

Fig. 4. Effect of X-gal on the growth of Vibrio species during broth culture. V. alginolyticus (VA), V. cholerae Non-O1 (VC), V. mimicus (VM), V. parahaemolyticus (VP), V. vulnificus MO6-24/O (VV; lacZ+), or V. vulnificus MO6-24/O (VV; lacZ−) grown in 2% NaCl-HI agars were inoculated into 2% NaCl-HI broths containing PBS, dimethylformamide (DMF) or 96 μg/ml of X-gal to concentrations of 1 × 103 cfu/ml, and cultured with vigorous shaking (220 rpm) at 37 °C for 8 h. Culture aliquots (100 μl) were serially ten-fold diluted and spread on the HI agar surfaces, the agar plates were incubated at 37 °C for 24 h, and viable cells were counted. Bacterial growths were expressed as the means and standard errors of triplicate experiments.

M.-Y. Kim et al. / Journal of Microbiological Methods 66 (2006) 552–555

obtained from two different companies were used (data not shown). These inhibitory and stimulatory effects of X-gal on bacterial swarming did not appear to be affected by the presence or absence of the functional lacZ gene, which encodes β-galactosidase that digests X-gal. V. vulnificus, V. cholerae, and V. mimicus digested X-gal and exhibited blue colonies, but V. alginolyticus, V. parahaemolyticus, P. mirabilis, and S. marcescens did not exhibit blue colonies. Despite these different abilities to utilize X-gal, the swarming of all the Vibrio species was inhibited by X-gal, but the swarming of P. mirabilis and S. marcescens were rather stimulated by X-gal. Moreover, X-gal inhibited the swarming of a lacZ-deletion mutant (CMM2101) of V. vulnificus to the same degree as the swarming of wild type MO6-24/O strain containing functional lacZ. However, because E. coli DH5α did not show swarming until 24 h regardless of the presence or absence of functional lacZ, we did not determine the effect of X-gal on E. coli swarming (Fig. 3). Moreover, the inhibitory effect of X-gal on the swarming of Vibrio species did not appear to be due to the antibacterial activity of X-gal against them. X-gal did not inhibit the growth of the other Vibrio species and V. vulnificus containing mutated lacZ (Fig. 4), and the growth of P. mirabilis, S. marcescens and E. coli (data not shown). Exceptionally the growth of V. vulnificus containing functional lacZ was significantly inhibited by X-gal. Overall, our results indicate that the inhibitory effect of X-gal on the swarming of Vibrio species is neither related to their ability to digest X-gal nor consistent with the antibacterial activity of X-gal against them. We think that X-gal inhibits only the swarming of Vibrio species without growth inhibition because physiology of swarming cells is different from that of non-swarming cells (Harshey, 2003). To the best of our knowledge, the inhibitory effect of X-gal on the growth of V. vulnificus containing functional lacZ and the inhibitory effect of X-gal on the swarming of Vibrio species have not been reported previously and the underlying mechanisms are wholly unknown. The use of semisolid agars containing X-gal presents a simple and useful means of determining the expressions of genes in swarming cells by using the lacZfusion transcriptional reporter strains of the genes

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(Walker et al., 1999). In order to clearly discriminate swarming-associated gene expression levels on X-gal semisolid agars, the resulting swarming haloes should be as large as possible. However, our results showed that X-gal alone can inhibit the expression of the swarming phenotype of Vibrio species and the growth of V. vulnificus containing functional lacZ, and these inhibitory effects of X-gal may interfere with the discrimination of the expressional levels of genes regulated during swarming in Vibrio species. Therefore, when attempts are made to observe the expression levels of genes regulated during swarming in Vibrio species, or in other bacteria not tested in the present study, using X-gal semisolid agar, the inhibitory effects of X-gal alone on swarming and growth should be carefully considered. Acknowledgement This study was supported by a grant (R13-2003-009) from the Korean Ministry of Science and Technology and from the Korea Science and Engineering Foundation through the Research Center for Resistant Cells, and by a research fund from Chosun University (2005). References Harshey, R.M., 2003. Bacterial motility on a surface: many ways to a common goal. Annu. Rev. Microbiol. 57, 249–273. Kim, S.Y., Lee, S.E., Kim, Y.R., Kim, C.M., Ryu, P.Y., Choi, H.E., Chung, S.S., Rhee, J.H., 2002. Regulation of Vibrio vulnificus virulence by the LuxS quorum-sensing system. Mol. Microbiol. 48, 1647–1664. Niu, C., Graves, J.D., Mokuolu, F.O., Gilbert, S.E., Gilbert, E.S., 2005. Enhanced swarming of bacteria on agar plates containing the surfactant Tween-80. J. Microbiol. Methods 62, 129–132. Tanabe, T., Takata, N., Naka, A., Moon, Y.H., Nakao, H., Inoue, Y., Narimatsu, S., Yamamoto, S., 2005. Identification of an AraC-like regulator gene required for induction of the 78-kDa ferrioxamine B receptor in Vibrio vulnificus. FEMS Microbiol. Lett. 249, 309–314. Walker, K.E., Moghaddame-Jafari, S., Lockatell, C.V., Johnson, D., Belas, R., 1999. ZapA, the IgA-degrading metalloprotease of Proteus mirabilis, is a virulence factor expressed specifically in swarmer cells. Mol. Microbiol. 32, 825–836. Wang, Q., Frye, J.G., McClelland, M., Harshey, R.M., 2004. Gene expression patterns during swarming in Salmonella typhimurium: genes specific to surface growth and putative new motility and pathogenicity genes. Mol. Microbiol. 52, 169–187.