Transposition of IS 4 Family Insertion Sequences IS Teha3, IS Teha4, and IS Teha5 into the arc Operon Disrupts Arginine Deiminase System in Tetragenococcus halophilus

Abstract

Tetragenococcus halophilus, a halophilic lactic acid bacterium, is often used as a starter culture in the manufacturing of soy sauce. T. halophilus possesses an arginine deiminase system, which is responsible for the accumulation of citrulline, the main precursor of the potential carcinogen ethyl carbamate. In this study, we generated five derivatives lacking arginine deiminase activity from T. halophilus NBRC 12172 by UV irradiation. Using these derivatives as a fermentation starter prevented arginine deimination in soy sauce. DNA sequence analysis of the derivatives revealed that novel IS4 family insertion sequences, designated ISTeha3, ISTeha4, and ISTeha5, were transposed into the region around the arginine deiminase (arc) operon in the mutants. These insertion sequences contain a single open reading frame encoding a putative transposase and 13- to 15-bp inverted repeats at both termini, which are adjacent to 7- to 9-bp duplications of the target sequence. Investigation of wild strains isolated from soy sauce mash incapable of arginine deimination also indicated that insertion sequences are involved in the disruption of the arginine deiminase system in T. halophilusIMPORTANCE Insertion sequences play important roles in bacterial evolution and are frequently utilized in mutagenesis systems. However, the intrinsic insertion sequences of tetragenococci are not well characterized. Here, we identified three active insertion sequences of T. halophilus by transposition into the region around the arc operon. This report provides an example of insertion sequence-mediated generation and evolution of T. halophilus and primary information about their characteristics.

Keywords: Tetragenococcus halophilus; arginine deiminase system; ethyl carbamate; insertion sequence.

FIG 1

Arginine deimination pathway in T. halophilus and generation of EC (A) and schematic representation of the locus of the genes around the arc operon in the genome of T. halophilus NBRC 12172 and E. faecalis SD10 (B). (A) The broken line represents the cell membrane. Citrulline leakage out of the cell is caused by external factors, such as bacteriophage infection. (B) *MarR represents a putative MarR family transcriptional regulator gene. The arrows under lacD and MarR indicate the position on which the lacD and marR primers were designed. Percentages show the amino acid identities between T. halophilus and E. faecalis.

FIG 2

Location and orientation of ISs transposed around the arc operon in each derivative (A) and genetic structures (B) and nucleotide sequences of the 5′ and 3′ termini (C) of ISTeha3, ISTeha4, and ISTeha5. (A) Black arrows indicate the direction of the putative transposase transcription. The derivative names and designated IS names are indicated on the left side and right side of the arrows, respectively. (B) The black arrows indicate ORFs that encode a putative transposase. The triangles indicate IRs at the 5′ and 3′ termini. The nucleotide numbers refer to the start and stop positions of the ORFs. (C) Vertical lines between the sequences denote homologous nucleotides.

FIG 3

Relative expression levels of arcA in the strains determined by quantitative real-time PCR. The 16S rRNA gene was used as the reference. Data are expressed as means with error bars representing ±SD (n = 3). WT, wild-type strain.

FIG 4

pH transition of soy sauce mash treated with the strains (A) and arginine and ornithine content (B) and lactic acid content (C) in the soy sauce after 8 weeks of fermentation. (A) Data are expressed as the means with error bars representing +1 SD (n = 3). WT, wild-type strain. NS, without starter cultures. (B) Gray bar and black bar indicate arginine and ornithine, respectively. Data are expressed as the means with error bars representing ±SD. Ornithine contents were below the detection limit, except for those of the samples treated with the WT. (C) Data are expressed as the means with error bars representing ±SD.

FIG 5

Schematic representation of the locus of the genes between lacD and putative MarR family transcriptional regulator gene (A) and Northern blot analysis for arcA (B). (A) Broken lines represent the insertion or the replacement. *MarR represents a putative MarR family transcriptional regulator gene. (B) The result of Northern blot analysis is shown in the upper panel, designated arcA, and the total RNA stained with ethidium bromide is shown in the lower panel, designated EtBr. The RNA size on the membrane was estimated by the 16S rRNA (1.6 kb) and 23S rRNA (2.9 kb) patterns.