IMMUNOLOGY & MEDICAL MICROBIOLOGY

RESEARCH ARTICLE Comparative analysis of bio¢lm formation by main and nonmain subspecies Yersinia pestis strains Galina A. Eroshenko, Nadezhda A. Vid...
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RESEARCH ARTICLE

Comparative analysis of bio¢lm formation by main and nonmain subspecies Yersinia pestis strains Galina A. Eroshenko, Nadezhda A. Vidyaeva & Vladimir V. Kutyrev

IMMUNOLOGY & MEDICAL MICROBIOLOGY

Russian Anti-Plague Research Institute ‘Microbe’, Universitetskaya, Saratov, Russian Federation

Correspondence: Present address: Galina A. Eroshenko, Russian Anti-Plague Research Institute ‘Microbe’, Universitetskaya, 46, Saratov, 410005, Russian Federation. Tel.: 184 52 262 131; fax: 184 52 515 211; e-mail: [email protected] Received 23 November 2009; revised 8 June 2010; accepted 10 June 2010. Final version published online 5 July 2010. DOI:10.1111/j.1574-695X.2010.00719.x Editor: Gianfranco Donelli Keywords Yersinia pestis; biofilm formation; abiotic and biotic surfaces.

Abstract The biofilm-forming phenotype of 14 isolates from four ‘nonmain’ subspecies of Yersinia pestis was compared with eight isolates from the more commonly studied ‘main’ or epidemic subspecies of Y. pestis in this study. The four nonmain subspecies are more geographically limited, and are associated with certain mammalian hosts and regions of the Caucasus and Central Asia, whereas the main subspecies spread worldwide during the historic plague pandemics. With the main subspecies pestis, pigmentation on Congo red medium (CR1) correlated with biofilm formation on both abiotic and biotic surfaces. Main subspecies pestis strains that do not produce pigmentation on Congo red medium (CR ) have a deletion that includes the hmsF and hmsS genes known to be required for biofilm formation. CR strains of the nonmain subspecies, altaica and ulegeica, differed however from pestis and, while defective for biofilms on the two surfaces, both had intact hmsF and hmsS genes. The presence of rcsA was also investigated and results showed that it occurred with a 30-bp insertion in all forms of the subspecies. These findings suggest that biofilms are regulated differently in altaica and ulegeica than they are in pestis and also indicate that the rcsA pseudogene arose early in Y. pestis evolution, increasing the ability of the strain to form biofilm and thereby increasing its effective transmission.

Introduction The plague is a particularly dangerous vector-borne disease that still poses a significant threat to human health due to its circulation in numerous natural foci including those in the Russian Federation and its neighbouring countries. The causal agent of the plague is Yersinia pestis, which has a complex life cycle, involving survival in the mammal (37 1C) and flea vector (28 1C). Under these two different conditions, the plague agent expresses different pathogenic and housekeeping factors, most of which are shared with another pathogenic yersinia, Yersinia pseudotuberculosis. Genome sequencing of several Y. pestis and Y. pseudotuberculosis strains has indicated that the plague microorganism has recently evolved from the pseudotuberculosis agent (Parkhill et al., 2001; Chain et al., 2004). In a relatively short period of time, Y. pestis transformed from a saprophytic enteropathogenic bacterium to an obligate parasite, and systemic disease agent with new ways of transmission, via flea bite or droplet airway (Wren, 2003). During this FEMS Immunol Med Microbiol 59 (2010) 513–520

process, many characteristics in Y. pseudotuberculosis changed, including the ability to form biofilms on various surfaces. A bacterial biofilm is a complex, compact community of cells enclosed in an extracellular matrix, often attached to a surface (Costerton et al., 1995). Yersinia pseudotuberculosis uses a biofilm mode of growth to survive in the environment and to avoid ingestion by invertebrates (Darby et al., 2002; Joshua et al., 2003; Erickson et al., 2006). Its descendant, Y. pestis, further transformed this characteristic into the ability to form a bacterial block, a massive biofilm in the foregut of the flea vector. A blocked flea ensures effective transmission of the plague agent, as the flea regurgitates pieces of the bacterial biofilm into the bite site, injecting the bacteria into or under animal skin, thereby causing development of the bubonic plague and subsequent bacteremia (Hinnebusch et al., 1996; Jarrett et al., 2004). Although it has been demonstrated that early-phase transmission of Y. pestis by unblocked fleas could play an important role in the rapid spread of plague epizootics, transmission by blocked fleas 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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nevertheless remains one of the basic mechanisms for epizootic development as well as for the long-term maintenance of the plague agent in animal reservoirs (Voronova, 1989; Bazanova et al., 1991; Eisen et al., 2006). Ancient forms of the plague microorganism have survived in nature and are known as the Y. pestis nonmain subspecies. They occupy an intermediate position between Y. pseudotuberculosis and the highly virulent Y. pestis ssp. pestis. The nonmain subspecies – caucasica, altaica, hissarica and ulegeica – circulate in different natural plague foci in Russia and its neighbouring countries (Anisimov et al., 2004). They are selectively virulent for laboratory animals and have a low epidemic potential. The coexistence in nature of bacteria related to the evolutionary ancestor, intermediate virulence subspecies and highly virulent Y. pestis ssp. pestis gives an opportunity to retrace the steps of genome reorganization that led to the rapid evolution of a particularly dangerous infectious disease agent. Until now, biofilm formation has been studied in a limited number of strains, mainly in Y. pestis KIM strain and its derivatives (Darby et al., 2002; Kirillina et al., 2004; Bobrov et al., 2005, 2008; Erickson et al., 2006; Forman et al., 2006), and has not been explored in strains of the different subspecies, isolated from various natural plague foci. The aim of this work was to study biofilm formation in main and nonmain subspecies of Y. pestis strains from different origins and compare it to that in Y. pseudotuberculosis strains.

Materials and methods Bacterial strains, growth conditions, pigmentation analysis Twenty-two Y. pestis strains and four Y. pseudotuberculosis strains were used in this study (Table 1). Of the 22 Y. pestis strains used, eight were from the main subspecies pestis and 14 from the nonmain subspecies (caucasica, altaica, hissarica and ulegeica). Most of the strains were originally isolated from natural plague hotspots in the Russian Federation and surrounding areas, and two main subspecies Y. pestis strains, EV76 and 55-801, were isolated from Madagascar and Vietnam, respectively. All strains were received in lyophilized form from the state collection of pathogenic bacteria (Russian Anti-Plague Research Institute, Saratov). Bacteria were grown in Luria–Bertani (LB) broth and agar (pH 7.2) at 28 1C for 24–48 h. The ability of the strains to form pigmented colonies was studied by growing them for 4 days at 28 1C on a solid synthetic medium designed for pigmentation analysis [g L 1: 21.64 agar base, 2.16 D ( ) galactose, 0.06 DL-b-phenyl-a-alanine, 0.02 DL-valine, 0.12 L-arginine HCl, 0.03 DL-methionine, 0.05 L-cysteine HCl, 0.03 DLisoleucine, 0.0 glycine, 0.75 nonhydrated sodium sulphite, 0.03 Congo red, (pH 7.1  0.2)], produced by the Russian 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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Anti-Plague Research Institute ‘Microbe’. To ensure reliability of the results, all experiments were repeated three times.

Biofilm formation on abiotic surfaces Quantification of biofilms was performed by the crystal violet assay, with minor modifications of a previously reported method (O’Toole et al., 1999). Strains of Y. pestis and Y. pseudotuberculosis were grown overnight at 28 1C, then diluted 1 : 100 up to OD600 nm of 0.1 and 3 mL put in sterile polystyrol dishes. These were incubated at 28 1C for 48 h; then the broth was decanted. Bacteria were taken from dish walls for analysis by electron microscopy. Cells attached to dish walls were stained with 0.01% crystal violet in 96% ethanol at room temperature for 45 min. Dishes were washed three times with water, and biofilms were solubilized with a mixture of 80% ethanol and 20% acetone. A570 nm was measured on a Spectronic 5000 spectrophotometer.

Biofilm formation on nematode cuticles Studies of biofilm formation by Y. pestis strains on biotic surfaces were performed on the cuticle of the Caenorhabditis elegans nematode, wild-type strain N2 Bristol, obtained from Caenorhabditis Genetics Center (University of Minnesota). Twenty C. elegans adult worms were placed on a bacterial lawn (grown on NGM agar at room temperature for 24 h) for egg laying and then removed (Joshua et al., 2003). Dishes were incubated at 20 1C for 48 h. The ability of Y. pestis strains to form biofilms on biotic surfaces was quantified by calculating the percentage of blocked adult nematodes on a dish.

Electron microscopy Samples for electron microscopy were prepared by standard procedure using 1.5% OsO4 fixation followed by staining with 1% Rutenium red solution. The samples were then examined using transmission electron microscope Hitachi HU-12A (Japan) at accelerated voltage 50 kV.

PCR analysis Specific primers were used for PCR detection of the following genes: gmhA, primer design by Darby et al. (2005); hmsS, design by Tong et al. (2005); hmsP, design by Bobrov et al. (2005); speA and speC genes, design by Patel et al. (2006). For detection of hmsF, hmsT and rcsA, new primer pairs were constructed: hmsF-S – AAGACAGCACAGGGCGGAC and hmsF-R – TCCGTGGCCCACAGGTAA; hmsT-S – TCTACT GACAGCACGATATT and hmsT-As – TATCCAGGCCTAAA ACAC; and rcsA-S – TATTGTCGCTATGGTGGT and rcsAAs – TAGGCATCTCTGTCATCC. FEMS Immunol Med Microbiol 59 (2010) 513–520

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Table 1. Characteristics of Yersinia pestis and Yersinia pseudotuberculosis strains used in this study

Species, subspecies, strain, source, date of isolation

Formation of pigmented colonies

Y. pestis ssp. pestis CR strains EV76 (human, 1926), A`-161 (fleas of Rhanbomys opimus, 1962), 55–801 (human, 1967) CR1 strains 231 (corpse of Marmota baibacina, 1955), I-I996 100% (Citellus daurica, 1970), I-3223 (fleas of Citellus undulatus, 1987), A`-1836 (M. baibacina, 1983), A`-1793 (Citellus pigmaeus, 1978) Y. pestis ssp. caucasica CR1 strains 1146 (Microtus arvalis, 1962), 818 (fleas of M. arvalis, 100% 1968), 3544A`rm (M. arvalis, 1979) Y. pestis ssp. altaica CR strains I-2998 (Ochotona pricei, 1982) CR1 strains I-2359 (O. pricei, 1973), 30% I-2183 (Marmota, 1965) 100% Y. pestis ssp. hissarica CR1 strains A`-1249 (Microtus carruthersi, 1970), A`-1725 (Marmota 50% caudata, 1972), A`-1723 (M. carruthersi, 1970), A`-1728 (M. carruthersi, 1972) 100% Y. pestis ssp. ulegeica CR strains I-3069 (Microtus brandti, 1982) CR1 strains I-3131 (Ochotona pricei, 1984), 50% I-3130 (O. pricei, 1984), I-2422 100% (fleas of O. pricei, 1974) Y. pseudotuberculosis 2600 (Rhombomys opimus, 1976), 417 (Meriones erythrourus, 1976), 312 (human, 1973) 50–73 (human, 1973)

Presence of hmsP, hmsT; gmhA; speA, speC

Presence of 30-bp insertion in rcsA

1

1

1

1

1

1

23.3  1.25

1

1

1

1

5.3  1.15

1

1

1

1 1

1 1

1 1

1 1

5.3  1.15 8.0  1.04

1

1

1

1

5.3  1.15

1

1

1

1

8.0  1.04

1

1

1

1 1

1 1

1 1

1 1

5.3  1.15 8.0  1.04

1

1

Presence of genes hmsF, hms S

Biofilm Biofilm formation on formation on cuticle of abiotic surface nematode

1

Values are averages of three or more independent experiments, confidence intervals are indicated.

Results and discussion Pigmentation and biofilm formation by the main subspecies Y. pestis strains We explored biofilm formation by eight main subspecies Y. pestis strains. Six of them, namely A-161, I-1996, 231, I-3223, A-1836 and A-1793 were isolated from different natural plague foci (high-mountain, mountain and steppe) in the Russian Federation and neighbouring countries. One strain, vaccine EV76, was isolated in Madagascar and another, 55-801, in Vietnam (Table 1). Jackson & Burrows (1956) first discovered and defined the pigmentation phenotype (due to dye adsorption on the outer membrane of cells) in Y. pestis, and Surgalla & Beesley (1969) first correlated hemin binding to Congo red binding. More FEMS Immunol Med Microbiol 59 (2010) 513–520

recently, it has been established that biofilm production by Y. pestis KIM correlates with its ability to form pigmented colonies on a solid medium with hemin or Congo red (Darby et al., 2002; Kirillina et al., 2004; Bobrov et al., 2005, 2008; Erickson et al., 2006; Forman et al., 2006). To confirm this correlation with the main subspecies Y. pestis strains under investigation, we explored their ability to form pigmented colonies (Table 1). Bacteria were grown for 4 days at 28 1C on solid medium with Congo red. Of the eight main subspecies strains, five (231, I-1996, I-3223, A-1836, A-1793) were capable of forming pigmented (red) colonies (CR1 phenotype, 100%). The three other strains (EV76, A-161 and 55-801) did not form any pigmented colonies (CR phenotype). Biofilm formation by these strains on abiotic surfaces was subsequently investigated. The bacteria were grown in 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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polystyrol dishes in LB broth, and then cells attached to dish walls were stained with Crystal violet. A570 nm of the main subspecies CR1 strains (I-1996, I-3223, A-1836, A-1793) ranged from 1.0 to 1.2 OD, while that of the CR strains (EV76, A-161 and 55-801) ranged from 0.1 to 0.26 OD. The absorbance of the CR strains was therefore considered to be insignificant, while that of the CR1 strains was significantly higher due to a large number of cells in biofilms, attached to the dish walls. Electron microscopy analysis of the biofilms from the CR1 strains, showed that they consisted of cell aggregates embedded in an extracellular matrix (Fig. 1a). The CR isolates did not form biofilms under these conditions (Fig. 1b). Thus a distinct correlation between pigmentation and biofilm formation on abiotic surfaces was observed in natural main subspecies Y. pestis strains. In Y. pestis, products of the chromosomal hmsHFRS operon play a key role in pigmentation (Perry & Fetherston, 1997; Bobrov et al., 2005). These genes have also been shown to be necessary for biofilm formation (Joshua et al., 2003; Bobrov et al., 2008). The hms operon is part of the chromosomal pgm region that is often lost in main subspecies strains due to a large deletion (Kutyrev et al., 1992; Fetherston & Perry, 1994). To determine the reason for the absence of pigmentation and biofilm production in CR strains, we looked for their hms genes by PCR with primers

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specific for hmsF and hmsS. The results showed that these strains indeed lacked hmsF and hmsS (and probably the whole hms operon). The presence of these genes was confirmed in the five main subspecies (CR1) strains (Table 1). Furthermore, we looked for the presence of other genes known to take part in biofilm formation in the genomes of the main subspecies strains. Using PCR, we were able to detect the regulatory genes hmsT and hmsP, along with the heptose biosynthesis gene, gmhA, and the polyamine putrescine biosynthesis genes, speA and speC (Table 1) (Kirillina et al., 2004; Bobrov et al., 2005; Darby et al., 2005; Patel et al., 2006) in all eight Y. pestis strains. The correlation between pigmentation and biofilm production on a biotic surface was then investigated using C. elegans as a model system. The three CR strains did not produce biofilms on nematode cuticles and worms moved freely on the lawns of these strains (Fig. 2a). Conversely, a large number (23.3  1.25%) of C. elegans adult nematodes with well-formed biofilm fragments on their head and neck sections were seen on the lawns of the main subspecies CR1 strains. The worms were blocked by cohesive bacterial aggregates and made aberrant movements to escape (Fig. 2b). These results show that most (five out of eight) of the main subspecies Y. pestis strains produced biofilms on both

Fig. 1. Electron micrograph. Biofilm formation by CR1 Yersinia pestis 231 (a) and lack of biofilm formation by CR Y. pestis EV76 (b). Strains were grown at 28 1C for 48 h in LB broth in polystyrol dishes, then broth was decanted and bacteria were taken from dish walls for electron microscopy analysis. Samples for electron microscopy were prepared by standard procedure with 1.5% OsO4 fixation and following staining with 1% Rutenium red solution. Magnification,  1800.

Fig. 2. Biofilm formation by Yersinia pestis strains of the main subspecies on cuticle of nematode Caenorhabditis elegans: biofilm formation by CR1 strain 231 (b) and lack of biofilm formation by CR strain EV76 (a). Adult C. elegans were placed on the bacterial lawns for egg laying. Worms were then removed and cultures were incubated at 20 1C for 48 h. Biofilm-forming strains blocked nematodes, while those not forming a biofilm did not.

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abiotic and biotic surfaces and that these characteristics distinctly correlate with their ability to form pigmented colonies and with the presence of the hmsHFRS operon (Table 1).

Pigmentation and biofilm production by the nonmain subspecies Y. pestis strains Studying biofilm production by the nonmain subspecies Y. pestis strains was of significant interest, as this property had not been previously explored. Three strains from subspecies caucasica (1146, 818 and 3544 Arm, isolated in the Caucasus); three strains from subspecies altaica (I-2998, I-2359 and I-2183 from Altai); four strains from subspecies hissarica (A-1249, A-1725, A-1723 and A-1728 from Tadjikistan) and four strains from subspecies ulegeica (I-3069, I-3131, I-3130 and I-2422 from Mongolia) were used (Table 1). In these strains, a close correlation between pigmentation and biofilm production on abiotic surfaces was also observed. Three of the caucasica ssp. strains (818, 1146 and 3544 Arm), one altaica ssp. strain (I-2183), one hissarica ssp. strain (A1728) and two ulegeica ssp. strains (I-3130, I-2422) formed pigmented colonies (100%) on Congo red medium. These nonmain CR1 subspecies strains also produced visible biofilms on the walls of polystyrol dishes. Absorbance of the stained cells attached to dishes and solubilized in ethanol/ acetone mix was high, ranging from 1.0 to1.3 OD, almost equal to that observed in the main subspecies CR1 strains. In the case of the other nonmain subspecies strains, not all colonies formed on Congo red medium were pigmented. For the subspecies altaica strain I-2359, only 30% of the colonies were pigmented and for subspecies ulegeica strain I3131 and subspecies hissarica strains A-1723, A-1725, A1249) 50% of the colonies were pigmented. This percentage was preserved in the subcultured populations of CR1 as well as of CR colonies. This result indicates the necessity to further investigate the regulatory mechanism responsible for the lack of pigmented colonies formed in the altaica, ulegeica and hissarica ssp. Interestingly, the differences in percentage of pigmented colonies between strains of the nonmain subspecies had no significant effect on biofilm production on abiotic surfaces. The strains forming 30% and 50% pigmented colonies also produced visible biofilms in polystyrol dishes. Absorbance of their solubilized biofilms was 0.9–1.0 OD, only a little lower than that observed in the strains demonstrating 100% pigmentation. In our experiments, two strains of the nonmain subspecies, ulegeica (I-3069) and altaica (I-2998), reproducibly demonstrated the CR phenotype. These strains failed to produce biofilms on abiotic surfaces (absorbance o 0.1 OD). In all nonmain subspecies strains studied, positive signals for the hmsF and hmsS genes were observed by PCR FEMS Immunol Med Microbiol 59 (2010) 513–520

independent from their CR phenotypes, confirming the presence of these genes in these strains. Earlier, in our laboratory, whole hmsHFRS operons of some of the nonmain subspecies strains including strains I-3069 (CR , ulegeica) and I-2998 (CR , altaica) were sequenced. The nucleotide sequences of these nonmain subspecies CR strains and of the main subspecies CR1 strains, were identical with the exception of the caucasica strains. The caucasica strains contained two single nucleotide substitutions, one in hmsF and the other in hmsH (Sukhonosov & Krasnov, 2007; Sukhonosov et al., 2007). These results showed that the CR phenotype of the two altaica and ulegeica strains was not caused by deletions of or point mutations within the hms genes. PCR analysis also demonstrated the presence of the heptose biosynthesis gene (gmhA), the polyamine putrescine biosynthesis genes (speA and speC) as well as the regulatory genes (hmsP and hmsT) in genomes of all nonmain subspecies strains studied (Table 1). Thus the inability to form biofilms on abiotic surfaces by some of the nonmain subspecies Y. pestis strains is not related to the absence of the genes associated with these properties, the hmsHFRS operon and the regulatory genes hmsP, hmsT, as well as the gmhA, speA, speC genes. Preliminary data obtained in our laboratory show that the nucleotide sequences of hmsT and hmsP in the two CR strains of the altaica and ulegeica ssp. do not differ from those in other strains of these subspecies (not shown). It therefore seemed interesting for us to study the structure of another gene, rcsA, in the nonmain subspecies strains as rcsA has recently been shown to be a negative regulator of biofilm formation in fleas and a pseudogene in Y. pestis (Sun et al., 2008). We performed comparative sequence analysis of the rcsA gene in Y. pestis KIM, CO92, Pestoides F and Y. pseudotuberculosis IP32953 and YPIII present in the NCBI GenBank database. This analysis revealed that the Y. pestis rcsA gene contained a 30-bp insertion when compared with Y. pseudotuberculosis. We then examined the gene structure in natural Y. pestis strains using primers constructed for a variable gene locus. Results showed that the 30-bp insertion sequence was not only present in the rcsA gene in the main subspecies strains, but in all strains of caucasica, altaica, hissarica and ulegeica ssp. (Fig. 3). These data indicate the importance of switching off the rcsA gene function during the early stages of Y. pestis evolution (in the nonmain subspecies) and show that destruction of rcsA was a result of negative selection. Close correlation between pigmentation and biofilm production in the nonmain subspecies strains was also confirmed using the C. elegans model. CR strains of the nonmain subspecies Y. pestis strains (ulegeica I-3069, altaica I-2998) did not form biofilms on nematode cuticles and did not block the worms (Fig. 4a). In the strains that showed a reproducible difference in the percentage of colony 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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Fig. 3. PCR amplification of the rcsA gene in Yersinia pestis strains of the main and nonmain subspecies and in Yersinia pseudotuberculosis strains. Yersinia pestis strains of all subspecies contained an insertion of 30 bp in rcsA, while Y. pseudotuberculosis strains did not. Yersinia pestis strains of subspecies pestis: 1, EV76; 2, A-161; 3, 231; 4, I-1996; 5, A-1836; caucasica: 6, 1146; 7, 818; altaica: 8, I-2998; 9, I-2359; 10, I-2183; hissarica: 11, A1725; 12, A-1249; 13, A-1728; ulegeica: 14, I-3069; 15, I-3131; 16, I-3130. Yersinia pseudotuberculosis strains: 17, 312; 18, 417; 19, 50–73; 20, negative control. Electrophoresis was performed in 2% agarose gel. Sizes of PCR amplified rcsA gene fragments (313 and 283 bp) are indicated by arrows.

Fig. 4. Biofilm formation by Yersinia pestis strains of the nonmain subspecies on cuticle of Caenorhabditis elegans. Biofilm formation by CR1 strain A-1728 of hissarica ssp. (b) and lack of biofilm formation by CR strain I-2998 of altaica ssp. (a).

pigmentation (30% observed in altaica I-2359 and 50% in ulegeica I-3131 and hissarica A-1723, A-1725, A-1249), biofilm formation was still observed, but blocked fewer nematodes (5.3  1.15%), although most of the worms retained their motility. The same amount of blocked nematodes was seen on the lawns seeded with the 100% CR1 caucasica strains (818, 1146 and 3544 Arm). On the lawns of 100% CR1 (ulegeica I-2422, I-3130, altaica I-2183, hissarica A-1728) strains, blocked C. elegans nematodes were also seen among freely moving nematodes (Fig. 4b). The number of blocked nematodes was higher (8  1.04%) than that observed for the strains forming 30% and 50% pigmented colonies, but smaller than that for the main subspecies CR1 strains. In summary, most of the nonmain subspecies Y. pestis strains were able to produce biofilms on both abiotic and biotic surfaces and this ability correlated with the formation of pigmented colonies on solid Congo red medium. Evi2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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dently, these strains have another regulatory mechanism responsible for the formation of CR colonies, differing from that of the main subspecies strains. It is important to note that the difference in ability to form pigmented colonies has no influence on the virulence of the nonmain subspecies strains, as they are reportedly all virulent in mice, but avirulent in guinea pigs. Selective virulence for laboratory animals is a characteristic feature of the nonmain subspecies strains (Martinevskii, 1969).

Pigmentation and biofilm formation by Y. pseudotuberculosis strains To compare biofilm production in Y. pestis and its precursor, Y. pseudotuberculosis, we examined four strains of the latter. In this case, we failed to find a distinct correlation between pigmentation and biofilm production on abiotic surfaces. The strains used were Y. pseudotuberculosis 2600 and 417 FEMS Immunol Med Microbiol 59 (2010) 513–520

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(from Turkmenia) and 50–73, 312 (from the Far East of the Russian Federation). These strains did not produce pigmented colonies on Congo red medium, but they did produce biofilms on polystyrol dishes. A570 nm of solubilized biofilms was equal to 1.1–1.3 OD and comparable to that of the CR1 Y. pestis strains. Meanwhile, a correlation between pigmentation and biofilm production was observed on biotic surfaces. All four CR Y. pseudotuberculosis strains failed to produce biofilms on C. elegans nematode cuticles. Worms freely moved on their lawns in the usual sinusoidal way. These results are in accordance with the literature, which reports that most Y. pseudotuberculosis strains are not able to block the nematode C. elegans under laboratory conditions. Joshua et al. (2003) showed that of 41 Y. pseudotuberculosis strains belonging to 21 serovars, 76% did not produce biofilms on nematode cuticles. Evidently, the four isolates used in our study belong to the group of Y. pseudotuberculosis strains that do not produce biofilms on biotic surfaces under laboratory conditions. Genes of the hms operon, the regulatory genes hmsP and hmsT, as well as the genes gmhA, speA and speC, were detected in the four Y. pseudotuberculosis strains by PCR; however, the rcsA gene of these strains did not contain a 30-bp insertion (Fig. 3). Thus, in the Y. pseudotuberculosis strains studied, the CR phenotype was neither caused by deletion of the hmsHFRS operon nor by mutation of any of the other genes examined. It is likely that mechanisms for biofilm formation on abiotic and biotic surfaces in Y. pseudotuberculosis are different. They also differ from those of Y. pestis strains. To conclude, biofilm formation on abiotic and biotic surfaces was studied in natural main and nonmain subspecies Y. pestis strains. Strains of the highly virulent main subspecies pestis (plague agent) formed biofilms on polystyrol dishes and on C. elegans nematode cuticles. The number of blocked nematodes on the lawns of the main subspecies strains was higher than in the nonmain subspecies strains. Biofilm formation in the main subspecies strains strictly correlated with the presence of the hms operon and the CR1 phenotype. Deletion of the hms genes was the reason for the lack of biofilm production in the main subspecies CR strains. The majority of the nonmain subspecies strains also produced biofilms on both abiotic and biotic surfaces and these properties also correlated with the CR1 phenotype. Two CR strains failed to produce biofilms on abiotic surfaces and on nematode cuticles, although these strains contained genes from the hms operon as well as the hmsT, hmsP and other related genes. This indicates that the CR phenotype of these strains was caused not by a deletion of the hms operon but rather by other, as yet unknown, regulatory mechanisms. The nonmain subspecies Y. pestis strains capable of forming 30%, 50% and 100% pigmented FEMS Immunol Med Microbiol 59 (2010) 513–520

colonies, produced biofilms on nematode cuticles, but in smaller amounts than those produced by the main subspecies CR1 strains. It is likely that the evolution of Y. pestis towards the highly virulent main subspecies pestis required the enhanced ability to form biofilms on biotic surfaces, which was necessary to ensure effective transmission of the plague agent by the flea vector.

Acknowledgement This work was supported by the grant of RFFR N 08-0400731.

Authors’ contribution G.A.E, N.A.V. and V.V.K. contributed equally to this work.

References Anisimov AP, Lindler EL & Pier GB (2004) Intraspecific diversity of Yersinia pestis. Clin Microbiol Rev 17: 434–464. Bazanova LP, Zhovtyi I, Maevskii MP, Klimov VT & Popkov AF (1991) The seasonal dynamics of blocking in the flea Citellophorus tesquorum altaicus from the Tuva natural plague focus. Med Parazitol (Mosk) 1: 24–26 (in Russian). Bobrov AG, Kirillina O & Perry RD (2005) The phosphodiesterase activity of the HmsP EAL domain is required for negative regulation of biofilm formation in Yersinia pestis. FEMS Microbiol Lett 247: 123–130. Bobrov AG, Kirillina O, Forman S, Mack D & Perry RD (2008) Insights into Yersinia pestis biofilm development: topology and co-interaction of Hms inner membrane proteins involved in exopolysaccharide production. Environ Microbiol 10: 1419–1432. Chain PS, Carniel E, Larimer FW et al. (2004) Insights into evolution of Yersinia pestis through whole-genome comparison with Yersinia pseudotuberculosis. P Natl Acad Sci USA 191: 13826–13831. Costerton JW, Lewandowski Z, Caldwell DE, Korber DR & Lappin-Scott HM (1995) Microbial biofilms. Annu Rev Microbiol 49: 713–745. Darby C, Hsu JW, Ghori N & Falkow S (2002) Caenorhabditis elegans: plague bacteria biofilm blocks food intake. Nature 417: 243–244. Darby C, Ananth SL, Tan L & Hinnebusch BJ (2005) Identification of gmhA, a Yersinia pestis gene required for flea blockage, by using a Caenorhabditis elegans biofilm system. Infect Immun 73: 7236–7242. Eisen RJ, Bearden SW, Wilder AP, Montenieri JA, Antolin MF & Gage KL (2006) Early-phase transmission of Yersinia pestis by unblocked fleas as a mechanism explaining rapidly spreading plague epizootics. P Natl Acad Sci USA 103: 15380–15385. Erickson DL, Jarett CO, Wren BW & Hinnebusch BJ (2006) Serotype differences and lack of biofilm formation characterize

2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

c

520

Yersinia pseudotuberculosis infection of the Xenopsylla cheopis flea vector of Yersinia pestis. J Bacteriol 188: 1113–1119. Fetherston JD & Perry RD (1994) The pigmentation locus of Yersinia pestis KIM 61 is flanked by an insertion sequence and includes the structural genes for pesticin sensitivity and HMWP2. Mol Microbiol 13: 697–708. Forman SA, Bobrov G, Kirillina O, Craig SK, Abney J, Fetherston JD & Perry RD (2006) Identification of critical amino acid residues in the plague biofilm Hms proteins. Microbiology 152: 3399–3410. Hinnebusch BJ, Perry RD & Schwan TG (1996) Role of the Yersinia pestis hemin storage (hms) locus in the transmission of plague by fleas. Science 273: 367–370. Jackson S & Burrows TW (1956) The pigmentation of Pasteurella pestis on a defined medium containing haemin. Brit J Exp Pathol 37: 570–576. Jarrett CO, Deak E, Isherwood KE, Oyston PC, Fischer ER, Whitney AR, Kobayashi SD, DeLeo FR & Hinnebusch BJ (2004) Transmission of Yersinia pestis from an infectious biofilm in the flea vector. J Infect Dis 190: 783–792. Joshua GWP, Karlyshev AV, Smith MP, Isherwood KE, Titball RW & Wren BW (2003) A Caenorhabditis elegans model of Yersinia infection: biofilm formation on a biotic surface. Microbiology 149: 3221–3229. Kirillina O, Fetherston JD, Bobrov AG, Abney J & Perry RD (2004) HmsP, a putative phosphodiesterase, and HmsT, a putative diguanylate cyclase, control Hms-dependent biofilm formation in Yersinia pestis. Mol Microbiol 54: 75–88. Kutyrev VV, Filippov AA, Oparina OS & Protsenko OA (1992) Analysis of Yersinia pestis chromosomal determinants Pgm1 and Psts associated with virulence. Microb Pathogenesis 12: 177–186. Martinevskii IL (1969) Biology and Genetic Features of Plague and Plague-Related Microbes. Meditsina Press, Moscow, USSR (in Russian).

2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

c

G.A. Eroshenko et al.

O’Toole GA, Pratt LA, Watnik PI, Newman DK, Weaver VB & Kolter R (1999) Genetic approaches to study of biofilms. Method Enzymol 310: 91–109. Parkhill J, Wren BW, Thomson NR et al. (2001) Genome sequence of Yersinia pestis, the causative agent of plague. Nature 413: 523–527. Patel CN, Wortham BW, Lines JL, Fetherston JD, Perry RD & Oliveira MA (2006) Polyamines are essential for the formation of plague biofilm. J Bacteriol 188: 2355–2363. Perry RD & Fetherston JD (1997) Yersinia pestis – etiologic agent of plague. Clin Microbiol Rev 10: 35–66. Sukhonosov Iyu & Krasnov YaM (2007) Nucleotide sequence of genes of hms operon of Yersinia pestis strains of different origin. Fundamental and Clinical Medicine 440–441. Spb, St Petersburg. (in Russian). Sukhonosov Iyu, Boolgakova EG, Krasnov YaM, Guseva NP, Anisimova LV, Novichkova LA & Kutyrev VV (2007) Organization of hms operon of Yersinia pestis and Yersinia pseudotuberculosis strains of different origin. Genodiagnostics of Infectious Diseases, Vol. 1 (Pokrovskii VI, ed.), pp. 400–401. Universitetskaya kniga, Moscow (in Russian). Sun YC, Hinnebusch BJ & Darby C (2008) Experimental evidence for negative selection in the evolution of a Yersinia pestis pseudogene. P Natl Acad Sci USA 105: 8097–8101. Surgalla MJ & Beesley ED (1969) Congo red-agar plating medium for detecting pigmentation in Pasteurella pestis. Appl Microbiol 18: 834–837. Tong Z, Zhou D, Song Y et al. (2005) Genetic variations in pgm locus among natural isolates of Yersinia pestis. J Gen Appl Microbiol 51: 11–19. Voronova GA (1989) Enhanced blocking capacity of the plague microbe in the body of the flea. Parazitologiya 23: 427–429 (in Russian). Wren BW (2003) The Yersiniae – a model genus to study the rapid evolution of bacterial pathogens. Nat Rev Microbiol 1: 55–64.

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