Supplemental Text 1 1

We examined colony-formation on various agar plates and auxotrophy for these isolates. 2

These isolates formed small non-β-hemolytic colonies on agar plates containing 5% 3

defibrinated sheep blood and various broths, including Todd-Hewitt broth (BD), 4

Müller-Hinton broth (Difco), Heart Infusion broth (BD), Brain Heart Infusion broth (BD), 5

Brucella broth (BD), Tryptic-Soy broth (BD), and Nutrient agar (Eiken Chemical). 6

These isolates formed small colonies on the following GBS selection media that are 7

commercially available in Japan: chromID™ Strepto B (bioMérieux), CHROMagar™ Strep 8

B (Kanto Chemistry), Pourmedia GBS semisolid agar medium (Eiken Chemical), and 9

Pourmedia GBS agar medium (Eiken Chemical). In addition, the three isolates formed small 10

colonies and did not produce a carotenoid pigment on a Pourmedia GBS semisolid agar 11

medium and a Pourmedia GBS agar medium. 12

Small colony variants (SCVs) are often linked to a deficiency in electron transport or 13

thymidine biosynthesis, which result in auxotrophy for hemin, menadione, or thymidine (1-3). 14

However, these isolates formed small non-β-hemolytic colonies on Müller-Hinton agar (BD) 15

containing 5% defibrinated sheep blood and supplemented with 0.1 µg/ml, 1 µg/ml, 10 µg/ml, 16

or 100 µg/ml of hemin, menadione, or thymidine (data not shown). The underlying reasons of 17

small non-β-hemolytic colony-formation by these isolates are presently unknown, and a 18

specific substance that can restore small colony formation to normal size colony formation 19

has not been identified. Therefore, we have not referred to these isolates as SCV in this 20

manuscript. 21

In addition, the Kirby-Bauer disk-diffusion method using oxacillin, ceftizoxime, and 22

ceftibuten disks for detecting PRGBS isolates, which was developed by our research group 23

(4), was used to evaluate whether this method could be used to successfully detect the three 24

isolates. Because the diameters of the growth inhibition zones around the disks in the three 25

isolates were notably smaller than those in the control strains 2603 V/R and NEM316, this 26

disk-diffusion method was found to be applicable for the identification of PRGBS isolates 27

despite their unusual phenotypes (Table S2). 28

29

Supplemental Text 2 30

The cyl operon in GBS has been reported to be essential for the production of hemolysin and 31

pigment. It contains 12 genes, and the deduced proteins of most of the genes are similar to 32

fatty acid biosynthesis enzymes (cylD, cylG, acpC, cylZ, and cylI). CylF shows similarities to 33

an aminomethyltransferase, and CylJ carries the conserved domain of a glycosyltransferase. 34

Two of the genes (cylX and cylK) display no significant similarities to genes of known 35

function in the GenBank database. cylE, which encodes a 78-kDa deduced protein, has been 36

proposed to encode the hemolysin molecule (5). The genes cylA and cylB display homology 37

to typical ABC transporters; CylA contains the ATP binding domain and CylB is a putative 38

transmembrane protein. The genes cylE, cylA, cylB, cylJ, and cylK were reported to be 39

responsible for the observed β-hemolysis activity on sheep blood agar plates (5-7). It was 40

previously reported that the ΔcylK mutant of GBS showed less β-hemolytic activity on a 41

horse blood agar plate (7). Similarly, in the present study, the three isolates formed small 42

non-β-hemolytic colonies on Todd-Hewitt broth supplemented with agar and 5% defibrinated 43

horse blood. 44

The Stp1/Stk1 (serine threonine phosphatase/serine threonine kinase) and CovS/CovR 45

(sensor histidine kinase/response regulator), which are two-component systems, have been 46

reported to be responsible for the β-hemolytic activity on blood agar plates. CovR (or CsrS) 47

and CovR (or CsrR) repress the transcription of cylE, which encodes the protein responsible 48

for hemolytic activity on the sheep blood agar plate (8). In addition, Stk1 regulates cylE 49

transcription through phosphorylation of CovR. The Δstp1 mutant showed no hemolytic 50

activity on the sheep blood agar plate in spite of not changing the transcriptional regulation of 51

cylE (9). 52

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