High-Resolution Comparison of Bacterial Conjugation Frequencies

Summary

With an aim to understand the behaviors of various bacterial conjugative DNA elements under different conditions, we describe a protocol for detecting differences in conjugation frequency, with high resolution, to estimate how efficiently the donor bacterium initiates conjugation.

Abstract

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons of conjugation frequency under different conditions are required to understand how the conjugative element spreads in nature. However, conventional methods for comparing conjugation frequency are not appropriate for in-depth comparisons because of the high background caused by the occurrence of additional conjugation events on the selective plate. We successfully reduced the background by introducing a most probable number (MPN) method and a higher concentration of antibiotics to prevent further conjugation in selective liquid medium. In addition, we developed a protocol for estimating the probability of how often donor cells initiate conjugation by sorting single donor cells into recipient pools by fluorescence-activated cell sorting (FACS). Using two plasmids, pBP136 and pCAR1, the differences in conjugation frequency in Pseudomonas putida cells could be detected in liquid medium at different stirring rates. The frequencies of conjugation initiation were higher for pBP136 than for pCAR1. Using these results, we can better understand the conjugation features in these two plasmids.

Introduction

Bacterial conjugation of mobile genetic elements, conjugative plasmids, and integrative and conjugative elements (ICEs) is important for the horizontal spread of genetic information. It can promote rapid bacterial evolution and adaptation and transmit multidrug resistance genes^1,2. The conjugation frequency can be affected by proteins encoded on the conjugative elements for mobilization of DNA (MOB) and mating pair formation (MPF), including sex pili, which are classified according to MOB and MPF type^3,4,5. It can als....

Protocol

1. Preparation of a Donor with Green Fluorescent Protein (GFP)- and Kanamycin Resistance Gene-Tagged Plasmids

1. Introduction of marker genes into the target plasmid pBP136
   Note: The goal of this protocol is to generate pBP136::gfp. The bacterial strains and plasmids used in this study are listed in Table 1.
   1. Grow cultures of Escherichia coli DH10B harboring pBP136¹⁷ in 5 mL of sterile Luria broth (LB) and E. coli S17-1λpir¹⁸ harboring pJBA28¹⁹ [containing a kanamycin (Km....

Results

Comparison of conjugation frequency by the MPN method

In our previous report, we compared the conjugation frequencies of pBP136::gfp and pCAR1::gfp in three-fold diluted LB (1/3 LB) liquid medium with different stirring rates after a 45 min mating using 125 mL spinner flasks¹⁰. We compared the conjugation frequencies of pBP136::gfp and pCAR1::gfp with 10⁶ CFU/mL.......

Discussion

Here, we present a high-resolution protocol for detecting differences in conjugation frequency under different conditions, using a MPN method to estimate the number of transconjugants. One important step in the protocol is diluting the mixture of donor and recipient after mating until no transconjugants grow. Another step is adding high concentrations of antibiotics to the selective liquid medium to prevent further conjugation. These procedures can reduce the background caused by further conjugation in the selective medi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MoFlo XDP	Beckman-Coulter	ML99030	FACS
IsoFlow	Beckman-Coulter	8599600	Sheath solution
Fluorospheres (10 μm)	Beckman-Coulter	6605359	beads to set up the FACS
Incubator	Yamato Scientific Co. Ltd	211197-IC802
UV-VIS Spectrophotometer UV-1800	SIMADZU Corporation	UV-1800
96-well plates	NIPPON Genetics Co, Ltd	TR5003
microplate type Petri dish	AXEL	1-9668-01	for validation of sorting
membrane filter	ADVANTEC	C045A025A	for filter mating
pippettes	Nichiryo CO. Ltd	00-NPX2-20, 00-NPX2-200, 00-NPX2-1000	0.5-10 μL, 20-200 μL, 100-1000 μL
multi-channel pippetes	Nichiryo CO. Ltd	00-NPM-8VP, 00-NPM-8LP	0.5-10 μL, 20-200 μL
Tryptone	BD Difco	211705
Yeast extract	BD Difco	212750
NaCl	Sigma	S-5886
Agar	Nakarai tesque	01162-15
rifampicin	Wako	185-01003
gentamicin	Wako	077-02974
kanamycin	Wako	115-00342
Petri dish	AXEL	3-1491-51	JPND90-15
microtubes	Fukaekasei	131-815C
500 mL disposable spinner flask	Corning	CLS3578