Open Access

Comparative genomics of the dairy isolate Streptococcus macedonicus ACA-DC 198 against related members of the Streptococcus bovis/Streptococcus equinus complex

  • Konstantinos Papadimitriou1Email author,
  • Rania Anastasiou1,
  • Eleni Mavrogonatou2,
  • Jochen Blom3,
  • Nikos C Papandreou4,
  • Stavros J Hamodrakas4,
  • Stéphanie Ferreira5,
  • Pierre Renault6, 7,
  • Philip Supply5, 8, 9, 10, 11,
  • Bruno Pot8, 9, 10, 11 and
  • Effie Tsakalidou1
BMC Genomics201415:272

DOI: 10.1186/1471-2164-15-272

Received: 29 September 2013

Accepted: 1 April 2014

Published: 8 April 2014

Abstract

Background

Within the genus Streptococcus, only Streptococcus thermophilus is used as a starter culture in food fermentations. Streptococcus macedonicus though, which belongs to the Streptococcus bovis/Streptococcus equinus complex (SBSEC), is also frequently isolated from fermented foods mainly of dairy origin. Members of the SBSEC have been implicated in human endocarditis and colon cancer. Here we compare the genome sequence of the dairy isolate S. macedonicus ACA-DC 198 to the other SBSEC genomes in order to assess in silico its potential adaptation to milk and its pathogenicity status.

Results

Despite the fact that the SBSEC species were found tightly related based on whole genome phylogeny of streptococci, two distinct patterns of evolution were identified among them. Streptococcus macedonicus, Streptococcus infantarius CJ18 and Streptococcus pasteurianus ATCC 43144 seem to have undergone reductive evolution resulting in significantly diminished genome sizes and increased percentages of potential pseudogenes when compared to Streptococcus gallolyticus subsp. gallolyticus. In addition, the three species seem to have lost genes for catabolizing complex plant carbohydrates and for detoxifying toxic substances previously linked to the ability of S. gallolyticus to survive in the rumen. Analysis of the S. macedonicus genome revealed features that could support adaptation to milk, including an extra gene cluster for lactose and galactose metabolism, a proteolytic system for casein hydrolysis, auxotrophy for several vitamins, an increased ability to resist bacteriophages and horizontal gene transfer events with the dairy Lactococcus lactis and S. thermophilus as potential donors. In addition, S. macedonicus lacks several pathogenicity-related genes found in S. gallolyticus. For example, S. macedonicus has retained only one (i.e. the pil3) of the three pilus gene clusters which may mediate the binding of S. gallolyticus to the extracellular matrix. Unexpectedly, similar findings were obtained not only for the dairy S. infantarius CJ18, but also for the blood isolate S. pasteurianus ATCC 43144.

Conclusions

Our whole genome analyses suggest traits of adaptation of S. macedonicus to the nutrient-rich dairy environment. During this process the bacterium gained genes presumably important for this new ecological niche. Finally, S. macedonicus carries a reduced number of putative SBSEC virulence factors, which suggests a diminished pathogenic potential.

Keywords

Streptococcus Genome Adaptation Gene decay Pseudogene Horizontal gene transfer Pathogenicity Virulence factor Milk Niche

Background

Lactic acid bacteria (LAB) constitute a very important group of microorganisms for the food industry, as well as the health of humans and animals [1, 2]. Several species in this group have a long history of safe use in fermented foods and thus belong to the very few bacteria that may qualify for the "generally regarded as safe" (GRAS) or the "qualified presumption of safety" (QPS) status according to FDA and EFSA, respectively [3]. Other LAB species are commensals of the skin, the oral cavity, the respiratory system, the gastrointestinal tract (GIT) and the genitals of mammals or other organisms. Furthermore, the presence of specific LAB strains, called "probiotics", in certain niches of the body is considered to promote the health of the host [2]. This benign nature of LAB, as well as their economic value, often obscure the existence of notorious LAB pathogens that are among the leading causes of human morbidity and mortality worldwide [4].

This oxymoron about the vast differences in the pathogenic potential within the LAB group is probably best exemplified by streptococci. The genus basically consists of commensals that include several severe pathogens, like group A streptococci (GAS), group B streptococci (GBS) and Streptococcus pneumoniae [5]. Streptococcal pathogens are implicated in a plethora of diseases, ranging from mild (e.g. pharyngitis) to invasive and life-threatening (e.g. necrotizing fasciitis) infections [6]. In contrast, Streptococcus thermophilus is one of the most frequent starter LAB consumed by humans in yogurt and cheese [7]. It is believed that this is the only streptococcal species that, during its adaptation to the nutrient-rich milk environment, underwent extensive genome decay, resulting in the loss of pathogenicity-related genes present in members of the genus [7, 8].

Apart from S. thermophilus, other streptococci can grow in milk and milk products. Such streptococci mainly belong to the Streptococcus bovis/Streptococcus equinus complex (SBSEC) [9]. The exact route that would explain their presence in milk is yet unidentified. In theory, since some of them can naturally occur in the GIT or on the teat skin of lactating animals, they could be passively transmitted to raw milk. In addition, species of the SBSEC are known to be involved in human cases of endocarditis, meningitis, bacteremia and colon cancer [1012]. However, Streptococcus macedonicus, which is a member of this specific complex, has been suggested to be adapted to milk and it has been hypothesized that it could be non pathogenic. These assumptions were based on the fact that the primary ecological niche of S. macedonicus appears to be naturally fermented foods, mostly of dairy origin similarly to S. thermophilus [13]. Initial in vitro and in vivo evaluation did not support virulence of S. macedonicus ACA-DC 198 [14]. PCR and Southern blotting analyses indicated the absence of several Streptococcus pyogenes pathogenicity genes. In addition, oral administration of the organism at high dosages (8.9 log cfu daily) for an extended period of time (12 weeks) to mice did not result in any observable adverse effects including inflammation in the stomach or translocation from the GIT to the organs of the animals [14]. Moreover, strains of S. macedonicus have been shown to present important technological properties of industrial cultures like the production of texturizing exopolysaccharides and anti-clostridial bacteriocins [13].

Streptococcus macedonicus was originally isolated from traditional Greek Kasseri cheese [15] and it is phylogenetically related to Streptococcus gallolyticus subsp. gallolyticus and Streptococcus pasteurianus (formerly known as S. bovis biotypes I and II.2, respectively), as well as to Streptococcus infantarius (formerly known as S. bovis biotype II.1). The inclusion of S. macedonicus and S. pasteurianus as subspecies of S. gallolyticus subsp. gallolyticus (from this point on S. gallolyticus) has been previously suggested [16], but this taxonomic reappraisal has not been formally accepted so far [17]. Streptococcus gallolyticus and S. pasteurianus are considered pathogenic. Preliminary investigations concerning the mechanisms by which S. gallolyticus causes endocarditis indicated that S. macedonicus may lack at least some of the pathogenic determinants implicated in this disease [18, 19]. Furthermore, the recent study of the genome of S. infantarius subsp. infantarius CJ18 (from this point on S. infantarius) isolated from spontaneously fermented camel milk in Africa has indicated strain-dependent traits of adaptation to the dairy environment despite the fact that the species is considered as a putative pathogen [20]. Overall, the presence in fermented foods of SBSEC species with a currently unresolved pathogenicity status, such as S. macedonicus and S. infantarius, may represent an underestimated cause of concern in terms of food safety and public health, which needs to be addressed.

Here we present the first complete genome sequence of S. macedonicus in order to shed light on the biology of the species. We are particularly interested in assessing niche adaptation and in investigating the pathogenic potential of the strain analyzed based on comparative genomics against other complete genomes within the SBSEC. This is an important step to rationally deduce whether the bacterium is safe to be used as a starter or if extra technological measures are needed to avoid its presence in food fermentations.

Results and discussion

General features of Streptococcus macedonicusACA-DC 198 genome

The circular chromosome of S. macedonicus ACA-DC 198 consists of 2,130,034 bp (Figure 1) with a G + C content of 37.6%, which is among the lowest values within the available complete streptococcal genomes (39.3% ± 1.7%, n = 95 by May 2013). A total of 2,192 protein coding DNA sequences (CDSs) were annotated, covering 87.3% of the S. macedonicus chromosome. Of these, 192 were identified as putative pseudogenes according to GenePRIMP [21] analysis followed by manual curation. The bacterium also carries 18 rRNA genes organized in 5 clusters co-localized with most of the 70 tRNA genes. The S. macedonicus genome was found to be 220–232 kb smaller and only 30 kb larger than the genomes of S. gallolyticus and S. pasteurianus, respectively. Streptococcus infantarius has one of the smallest genome sizes within the SBSEC reported up to now (i.e. 141 kb smaller than that of S. macedonicus). The percentage of potential pseudogenes in S. macedonicus was 8.7%, in S. pasteurianus 7.7% and in S. infantarius 4.9%. In contrast, the percentage of pseudogenes in at least two S. gallolyticus strains (i.e. strains UCN34 and ATCC 43143) has been found to be 2.1% or less. This analysis is in accordance with previous findings [9, 22]. Based on the close phylogenetic relationship among the four species, these observations suggest that the genome of S. macedonicus, as well as those of S. pasteurianus and S. infantarius may be evolving under selective pressures that allow gene loss events and genome decay processes when compared to the S. gallolyticus genomes.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-15-272/MediaObjects/12864_2013_Article_5939_Fig1_HTML.jpg
Figure 1

The circular map of the genome of Streptococcus macedonicus ACA-DC 198. Genomic features appearing from the periphery to the centre of the map: 1. Forward CDSs (red); 2. Reverse CDSs (blue); 3. Putative pseudogenes (cyan); 4. rRNA genes (orange); 5. tRNA genes (green); 6.% GC plot; 7. GC skew.

Whole genome phylogeny, comparative genomics, and core genome analysis

A phylogenetic tree based on the currently available complete streptococcal genome sequences was constructed using the EDGAR software [23]. On this tree, S. gallolyticus, S. macedonicus, S. pasteurianus, as well as S. infantarius formed a single, monophyletic branch, providing strong evidence for the taxonomic integrity of the SBSEC (Additional file 1: Figure S1).

Subsequently, full chromosome alignments were performed using progressiveMAUVE [24]. The analysis revealed a mosaic pattern of homology organized in local collinear blocks (LCBs) among S. gallolyticus, S. macedonicus and S. pasteurianus (Figure 2A). Evidently, a significant portion of the genetic information has been overall conserved, as the majority of the LCBs are shared by all species. In addition, chromosomal rearrangements seem to have been rather minimal, as the number of LCBs showing a change in relative genomic position among the strains was low and their length short. Nevertheless, numerous differences were also detected. Some LCBs were common only among some of the strains, while some regions were identified as strain-specific (and hence not included within an LCB). The presence of such strain-specific regions suggests that, in addition to gene loss mentioned earlier, gene acquisition events mediated by horizontal gene transfer (HGT) may have played a role during the evolution of the three species (see below). Interestingly, the inclusion of the S. infantarius genome in the MAUVE analysis resulted in an increased number of LCBs with a decreased average length. As the level of sequence conservation of individual LCBs among the four species remains relatively high, this observation suggests that specific genome structure reorganization events occurred specifically in S. infantarius (Figure 2B). Analysis with the EDGAR software revealed a core genome of only 1,372 orthologous genes based on the sequence and the current annotation of S. gallolyticus, S. pasteurianus and S. macedonicus (Figure 3A, Additional file 2: Table S1) [23]. Once more, inclusion of S. infantarius increased the diversity, resulting in reduction of the core genome by more than 100 genes among the four species (Figure 3B, Additional file 3: Table S2). The significant percentage of variable genes within the four SBSEC species may underpin their adaptation to specific environments.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-15-272/MediaObjects/12864_2013_Article_5939_Fig2_HTML.jpg
Figure 2

Chromosome alignments of the Streptococcus bovis / Streptococcus equinus complex members as calculated by progressiveMauve. Chromosome alignments among Streptococcus gallolyticus, Streptococcus macedonicus and Streptococcus pasteurianus (A) and all the aforementioned streptococci and Streptococcus infantarius (B). Local collinear blocks (LCBs) of conserved sequences among the strains are represented by rectangles of the same colour. Connecting lines can be used to visualize synteny or rearrangement. LCBs positioned above or under the chromosome (black line) correspond to the forward and reverse orientation, respectively. The level of conservation is equivalent to the level of vertical colour filling within the LCBs (e.g. white regions are strain-specific). Sequences not placed within an LCB are unique for the particular strain.

https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-15-272/MediaObjects/12864_2013_Article_5939_Fig3_HTML.jpg
Figure 3

Core genome analysis of members of the Streptococcus bovis / Streptococcus equinus complex. Whole CDS Venn diagrams of Streptococcus gallolyticus, Streptococcus macedonicus and Streptococcus pasteurianus (A) or Streptococcus gallolyticus, Streptococcus infantarius, Streptococcus macedonicus and Streptococcus pasteurianus (B). In (B) Streptococcus gallolyticus ATCC 43143 was selected as a representative of the S. gallolyticus species, since it has the longest genome size among the three sequenced strains.

Genes involved in the survival in the GIT

It has been established that S. gallolyticus displays the notable ability to accumulate and metabolize a broad range of complex carbohydrates from plants when compared to other streptococci [25]. The necessity for this repertoire of carbohydrate-degrading activities has been considered to reflect the adaptation of S. gallolyticus to the rumen of herbivores [22, 25]. Preliminary analysis indicated that at least some of the relevant genes are either entirely absent or they have been converted into pseudogenes in the genomes of S. macedonicus, S. pasteurianus and S. infantarius (Table 1). The presence of pseudogenes related to carbohydrate metabolism reinforces the notion that S. macedonicus, S. pasteurianus and S. infantarius have undergone genome decay processes during adaptation to their ecological niches. The entire glycobiome of the SBSEC members was further analyzed based on the data available in the CAZy database (Additional file 4: Table S3) [26]. Important differences in the distribution of enzymes among the SBSEC members were observed for all CAZy categories including glycoside hydrolases (GHs), glycosyl tranferases, polysaccharide lyases, carbohydrate esterases and carbohydrate-binding modules (CBMs). Streptococcus macedonicus and Streptococcus infantarius had the smallest glycobiome within the SBSEC. The two strains had only 24 and 23 GHs, while the rest SBSEC members had more than 40. Given that most of these GHs are potentially involved in plant and dietary carbohydrate catabolism (e.g. GH1, GH3, GH13, GH36 etc.) [27], it could be hypothesized that S. macedonicus and S. infantarius have a diminished necessity for such enzymes in their ecological niche. Streptococcus pasteurianus had the highest number of GHs, some of which were unique among SBSEC (i.e. GH35, GH78, GH79, GH85, GH92, GH125). This observation indicates differences in the range of carbohydrates the strain is able to catabolize in comparison to the other members of the complex. Interestingly, none of the SBSEC members were found to carry GHs that are implicated in the degradation of host derived oligosaccharides (e.g. GH33 and GH98) [27]. In contrast, Streptococcus gallolyticus strains, S. macedonicus and S. infantarius had hits in the CBM41 family, while S. pasteurianus in the CBM32 family, both of which have been associated with the recognition of host glycans [27, 28].
Table 1

Genes in the Streptococcus bovis / Streptococcus equinus complex potentially involved in adaptation to the rumen

Function

Gene

S. gallolyticus UCN34

S. gallolyticus ATCC 43143

S. gallolyticus ATCC BAA-2069

S. pasteurianus ATCC 43144

S. macedonicus ACA-DC 198

S. infantarius CJ18

Pullulanase

- (a)

GALLO_1462

SGGB_1458

SGGBAA2069_c14850

SGPB_1362 (t)

SMA_1464 (s)

Sinf_1270

      

SMA_1465 (s)

 

Pullulanase

-

GALLO_0781

SGGB_0764

SGGBAA2069_c07530

-

SMA_0719 (p)

-

      

SMA_0720 (r)

 
      

SMA_0721 (p)

 

α-amylase, neopullulanase

-

GALLO_0753

SGGB_0736

SGGBAA2069_c07260

-

-

-

Fructan hydrolase

fruA

GALLO_0112

SGGB_0110

SGGBAA2069_c01280

-

-

-

Beta-1,4-endoglucanase V (cellulase)

-

GALLO_0330

SGGB_0358

SGGBAA2069_c03180

-

-

-

Cinnamoyl ester hydrolase

cinA

GALLO_0140

SGGB_0137

SGGBAA2069_c01580

-

-

-

Mannanase

-

GALLO_0162

SGGB_0206

SGGBAA2069_c01800

-

-

Sinf_0174 (p)

Endo-beta-1,4-galactanase

-

GALLO_0189

SGGB_0233

SGGBAA2069_c02070

SGPB_0176

SMA_0214 (p)

Sinf_0197 (p)

Pectate lyase

-

GALLO_1577

SGGB_1576

SGGBAA2069_c16050

-

-

Sinf_1418

Pectate lyase

-

GALLO_1578

SGGB_1577

SGGBAA2069_c16060

SGPB_1461 (p)

SMA_1582 (p)

-

      

SMA_1583 (s)

 
      

SMA_1584 (s)

 

Malate transporter

mleP

GALLO_2048

SGGB_2031

SGGBAA2069_c20060

SGPB_1855

SMA_1945

Sinf_1750

Malate dehydrogenase

mleS

GALLO_2049

SGGB_2032

SGGBAA2069_c20070

SGPB_1856

SMA_1946

Sinf_1751

PTS system, mannitol-specific IIBC component

mtlA

GALLO_0993

SGGB_0982

SGGBAA2069_c09680

-

SMA_0905 (p)

-

Mannitol operon transcriptional antiterminator

mtlR

GALLO_0994

SGGB_0983

SGGBAA2069_c09690

-

SMA_0906 (p)

-

      

SMA_0907

 
      

SMA_0908

 
      

SMA_0909

 
      

SMA_0910

 
      

SMA_0911

 
      

SMA_0912

 
      

SMA_0913

 
      

SMA_0914

 
      

SMA_0915

 
      

SMA_0916

 
      

SMA_0917

 

PTS system, mannitol-specific IIA component

mtlF

GALLO_0995

SGGB_0984

SGGBAA2069_c09700

-

-

-

Mannitol-1-phosphate 5-dehydrogenase

mtlD

GALLO_0996

SGGB_0985

SGGBAA2069_c09710

-

-

-

α-amylase

-

GALLO_0757

SGGB_0740

SGGBAA2069_c07300

-

-

-

α-amylase

amyE

GALLO_1632

SGGB_1646

SGGBAA2069_c16600

SGPB_1505 (p)

SMA_1612 (t)

Sinf_1443

α-amylase

-

GALLO_1043

SGGB_1033

SGGBAA2069_c10200

SGPB_0905

SMA_0972

Sinf_0846

tannase

tanA

GALLO_0933

SGGB_0917

SGGBAA2069_c09070 (s)

-

-

-

    

SGGBAA2069_c09080 (s)

   

Tannase (similar to tanA)

-

GALLO_1609

SGGB_1624

SGGBAA2069_c16370

-

-

-

Phenolic acid decarboxylase

padC

GALLO_2106

SGGB_2089

SGGBAA2069_c21040

SGPB_1899

SMA_2074

-

Carboxymuconolactone decarboxylase

-

GALLO_0906

SGGB_0891

SGGBAA2069_c08850

SGPB_0775

-

-

Bile salt hydrolase

bsh

GALLO_0818

SGGB_0803

SGGBAA2069_c07920

SGPB_0678

SMA_0753 (p)

Sinf_0639

(a) Not found; (t) Truncated; (s) Split CDSs corresponding to fragments of the original gene not yet characterized as pseudogenes; (p) Pseudogenes; (r) Transposase genes in italics.

Furthermore, S. gallolyticus can detoxify toxic compounds met in the rumen and other environments. Again, S. macedonicus, S. pasteurianus and S. infantarius miss some of the genes involved in detoxification (Table 1). None of them carry genes for tannin hydrolysis similar to GALLO_0933 or GALLO_1609. The potential to degrade additional phenolic compounds like gallic acid seems to be comparable between S. gallolyticus and S. pasteurianus. In contrast, S. infantarius has no orthologs of either PadC (GALLO_2106) or GALLO_0906, i.e. the two gallic acid decarboxylases found in S. gallolyticus UCN34, while S. macedonicus has retained only PadC. Furthermore, the bsh gene (GALLO_0818), coding for a bile salt hydrolase, is present in all four species with the exception of S. macedonicus, in which it appears as a pseudogene. Thus, our findings clearly suggest that not only S. macedonicus, but also S. pasteurianus and S. infantarius have deviated from S. gallolyticus in their potential to cope with the harsh environment of the GIT of herbivores.

Genes involved in the growth in milk or dairy products

Dairy LAB are considered fastidious microorganisms due to their adaptation to growth in milk that is particularly nutritious by nature. Lactose and milk proteins (both caseins and whey proteins) are characteristic of the dairy environment. LAB are able to ferment lactose to lactic acid and they have evolved a proteolytic system for the degradation of milk proteins down to amino acids [1, 29].

All SBSEC species are able to utilize lactose and to catabolize galactose. Sequence similarity searches revealed a gene cluster (SMA_0197 – SMA_0211) dedicated to lactose metabolism with a unique organization in SBSEC when compared to those previously reported for other LAB (Table 2). The typical sequence of lac genes is interrupted in the majority of SBSEC strains by genes coding for the IIA, IIB and IIC components of a PEP-PTS (SMA_0202 – SMA _0204). Annotation of this PEP-PTS varies among the SBSEC species/strains and for this reason functional analysis is required to properly determine its exact function. In contrast to other SBSEC species, these three PTS genes are absent from S. infantarius. The lactose-specific PTS found at the end of the lac gene cluster (SMA_0206 – SMA _0210) is also inactivated in S. infantarius through disruption of the lacT antiterminator gene by transposases [20]. Interestingly, the lac gene cluster in S. macedonicus contains two 6-phospho-beta-galactosidase (lacG) genes that may be indicative of adaptation of this particular species to milk. Galactose can also be catabolized through the Leloir pathway and a galRKTE operon coding for the relevant enzymes was previously determined in S. infantarius [30]. The gal operon is conserved in all SBSEC species analyzed here (Table 2).
Table 2

Genes in the Streptococcus bovis / Streptococcus equinus complex potentially involved in lactose and galactose metabolism

Function

Gene

S. gallolyticus UCN34

S. gallolyticus ATCC 43143

S. gallolyticus ATCC BAA-2069

S. pasteurianus ATCC 43144

S. macedonicus ACA-DC 198

S. infantarius CJ18

Lactose-specific PTS system repressor

lacR

GALLO_0176

SGGB_0220

SGGBAA2069_c01940

SGPB_0163

SMA_0197

Sinf_0181

Galactose-6-phosphate isomerase, LacA subunit

lacA

GALLO_0177

SGGB_0221

SGGBAA2069_c01950

SGPB_0164

SMA_0198

Sinf_0182

Galactose-6-phosphate isomerase, LacB subunit

lacB1

GALLO_0178

SGGB_0222

SGGBAA2069_c01960

SGPB_0165

SMA_0199

Sinf_0183

Tagatose-6-phosphate kinase

lacC

GALLO_0179

SGGB_0223

SGGBAA2069_c01970

SGPB_0166

SMA_0200

Sinf_0184

Tagatose 1,6-diphosphate aldolase

lacD2

GALLO_0180

SGGB_0224

SGGBAA2069_c01980

SGPB_0167

SMA_0201

Sinf_0185

Putative PTS system, IIA component

- (a)

GALLO_0181

SGGB_0225

SGGBAA2069_c01990

SGPB_0168

SMA_0202

-

Putative PTS system, IIB component

-

GALLO_0182

SGGB_0226

SGGBAA2069_c02000

SGPB_0169

SMA_0203

-

Putative PTS system, IIC component

-

GALLO_0183

SGGB_0227

SGGBAA2069_c02010

SGPB_0170

SMA_0204

-

Aldose 1-epimerase

lacX

GALLO_0184

SGGB_0228

SGGBAA2069_c02020

SGPB_0171

SMA_0205

Sinf_0186

Transcriptional antiterminator

lacT

GALLO_0185

SGGB_0229

SGGBAA2069_c02030

SGPB_0172

SMA_0206

Sinf_0187 (p)

       

Sinf_0188 (r)

       

Sinf_0189

       

Sinf_0190 (p)

6-phospho-beta-galactosidase

lacG

GALLO_0186

SGGB_0230

SGGBAA2069_c02040

SGPB_0173

SMA_0207

-

Transcriptional antiterminator

lacT

-

-

-

-

SMA_0208 (p)

-

Lactose-specific PTS system, IIA component

lacF

GALLO_0187

SGGB_0231

SGGBAA2069_c02050

SGPB_0174

SMA_0209

Sinf_0191

Lactose-specific PTS system, IIBC component

lacE

GALLO_0188

SGGB_0232

SGGBAA2069_c02060

SGPB_0175

SMA_0210

Sinf_0192

6-phospho-beta-galactosidase

lacG2

-

-

-

-

SMA_0211

Sinf_0193 (p)

       

Sinf_0194

       

Sinf_0195 (p)

Galactose repressor

galR

GALLO_0197

SGGB_0241

SGGBAA2069_c02150

SGPB_0184

SMA_0222

Sinf_0205

Galactokinase

galK

GALLO_0198

SGGB_0242

SGGBAA2069_c02160

SGPB_0185

SMA_0223

Sinf_0206

Galactose-1-P-uridyl transferase

galT

GALLO_0199

SGGB_0243

SGGBAA2069_c02170

SGPB_0186

SMA_0224

Sinf_0207

UDP-glucose 4-epimerase

galE

GALLO_0200

SGGB_0244

SGGBAA2069_c02180

SGPB_0187

SMA_0225

Sinf_0208

Beta-galactosidase

lacZ

-

-

-

SGPB_0344

-

-

Glucokinase

glcK

GALLO_0594

SGGB_0562

SGGBAA2069_c05300

SGPB_0467

SMA_0546

Sinf_0470

Beta-galactosidase

lacZ

-

-

-

-

-

Sinf_0935

Lactose and galactose permease

lacS

-

-

-

-

-

Sinf_0936

Aldose 1-epimerase

galM

-

-

-

-

-

Sinf_0937

UDP-glucose 4-epimerase

galE1

-

-

-

-

-

Sinf_0938

Galactose-1-P-uridyl transferase

galT

-

-

-

-

-

Sinf_0939 (p)

UDP-glucose 4-epimerase

lacS

-

-

-

-

-

Sinf_1514

Aldose 1-epimerase

lacX

-

-

-

-

SMA_1156

-

6-phospho-beta-galactosidase

lacG2

-

-

-

-

SMA_1157

-

Lactose-specific PTS system, IIBC component

lacE

-

-

-

-

SMA_1158

-

Lactose-specific PTS system, IIA component

lacF

-

-

-

-

SMA_1159

-

Tagatose 1,6-diphosphate aldolase

lacD

-

-

-

-

SMA_1160

-

Tagatose-6-phosphate kinase

lacC

-

-

-

-

SMA_1161

-

Galactose-6-phosphate isomerase, LacB subunit

lacB

-

-

-

-

SMA_1162

-

Galactose-6-phosphate isomerase, LacA subunit

lacA1

-

-

-

-

SMA_1163

-

Glucokinase

glcK

-

-

-

-

SMA_1164

-

Lactose phosphotransferase system repressor

lacR

-

-

-

-

SMA_1165

-

Transcription antiterminator

lacT

GALLO_1046

SGGB_1036

SGGBAA2069_c10230

SGPB_0907

-

-

Lactose-specific PTS system, IIA component

lacF

GALLO_1047

SGGB_1037

SGGBAA2069_c10240

SGPB_0908

-

-

Lactose-specific PTS system, IIBC component

lacE

GALLO_1048

SGGB_1038

SGGBAA2069_c10250

SGPB_0909

-

-

Phospho-beta-galactosidase

lacG

GALLO_1049

SGGB_1039

SGGBAA2069_c10260

SGPB_0910

-

-

Aldose 1-epimerase

galM

GALLO_0137

SGGB_0134

SGGBAA2069_c01550

SGPB_0130

-

-

UDP-glucose 4-epimerase

galE1

GALLO_0728

SGGB_0709

SGGBAA2069_c06910

SGPB_0601

-

-

(a) Not found; (p) Pseudogenes; (r) Transposase genes in italics.

A partial gal-lac operon galT(truncated)/galE1M/lacSZ with high sequence identity to S. thermophilus is also present in the genome of S. infantarius [30]. It has been demonstrated that the lactose and galactose permease (lacS) and the β-galactosidase (lacZ) are responsible for the uptake and initial hydrolysis of lactose in S. infantarius in a manner similar to that employed by S. thermophilus [20]. This gal-lac operon of S. infantarius is missing from the other SBSEC strains as a whole. A LacZ ortholog (SGPB_0344) is only present in S. pasteurianus and dispersed galE and galM genes can be found in the S. gallolyticus and S. pasteurianus genomes. Similarly to the presence of the extra gal-lac operon in S. infantarius, we detected a second lac gene cluster in S. macedonicus (SMA_1156 – SMA_1165), also suggesting adaptation to the milk environment. This second gene cluster is solely present in S. macedonicus and not in any other SBSEC member. Surprisingly, an additional lacTFEG region coding for a complete lactose PEP-PTS and a 6-phospho-beta-galactosidase is present in the genomes of S. gallolyticus and S. pasteurianus. This is an unexpected finding since S. gallolyticus and S. pasteurianus have hardly ever been related to milk up to now [9].

We then investigated the proteolytic system of S. macedonicus and the rest of the SBSEC members adapting the scheme previously described by Liu and co-workers (i.e. excluding housekeeping proteases or proteases involved in specific cellular processes other than the acquisition of amino acids) [29]. In milk, casein utilization by LAB is initiated after hydrolysis by a cell-envelope associated proteinase (CEP) releasing oligopeptides. The oligopeptides are then transferred intracellularly via specialized peptide transport systems where they are systematically degraded into amino acids by an array of intracellular peptidases. The four species have essentially the same proteolytic system, albeit showing some differences (Table 3). None of them has a typical PrtP CEP, but S. gallolyticus and S. infantarius carry a lactocepin coding gene. The lactocepin of the SBSEC shows ≥ 63% sequence similarity to the PrtS CEP involved in the degradation of milk proteins in S. thermophilus [31, 32]. The exact role of lactocepin in SBSEC species needs to be experimentally examined. SBSEC strains like S. macedonicus may require CEP activity to be provided by other bacteria when growing in milk. This is a common strategy of nonstarter LAB that rely on starter CEP-producing strains for casein hydrolysis [33]. Streptococcus infantarius carries two oligopeptide transport systems (Opp) [20], but all the other SBSEC species have only one such system. All SBSEC strains own a proton motive force (PMF)-driven DtpT transporter for the transport of di- and tri-peptides and they all possess an entire repertoire of proteolytic enzymes including endopeptidases, general aminopeptidases and specialized peptidases (Table 3). They only lack enzymes of the PepE/PepG (endopeptidases) and the PepI/PepR/PepL (proline peptidases) superfamilies in accordance to previous observations for streptococci and lactococci [29]. The conservation of this proteolytic system among streptococci in the SBSEC despite their presumed adaptation to different ecological niches [20, 22, 25] indicates that it may somehow be essential. Furthermore, S. macedonicus and the other SBSEC members are autotrophs for several amino acids (data not shown) and only S. pasteurianus has been reported to be unable to synthesize tryptophan [22]. Thus, the preservation of an entire proteolytic system by SBSEC members while retaining the ability to synthesize most, if not all, amino acids is puzzling, especially when considering that some of them have obviously undergone extensive genome decay processes. It could be hypothesized that this property of SBSEC species may provide a competitive advantage in poor environments, but this needs to be further investigated.
Table 3

Genes in the Streptococcus bovis / Streptococcus equinus complex potentially involved in proteolysis of milk proteins

Function

Gene

S. gallolyticus UCN34

S. gallolyticus ATCC 43143

S. gallolyticus ATCC BAA-2069

S. pasteurianus ATCC 43144

S. macedonicus ACA-DC 198

S. infantarius CJ18

Lactocepin

prtS

GALLO_0748

SGGB_0730

SGGBAA2069_c07210

-

-

Sinf_0588

Oligopeptide ABC transporter, substrate-binding protein

oppA

GALLO_0324

SGGB_0352

SGGBAA2069_c03120

SGPB_0276

SMA_0353

Sinf_0305

  

GALLO_1412

SGGB_1406

SGGBAA2069_c14340

SGPB_1328

SMA_1347

Sinf_1225

  

GALLO_1413

SGGB_1407

SGGBAA2069_c14350

  

Sinf_1226

       

Sinf_1825

Oligopeptide ABC transporter, permease protein

oppB

GALLO_0325

SGGB_0353

SGGBAA2069_c03130

SGPB_0277

SMA_0354

Sinf_0306

       

Sinf_1824

Oligopeptide ABC transporter, permease protein

oppC

GALLO_0326

SGGB_0354

SGGBAA2069_c03140

SGPB_0278

SMA_0355

Sinf_0307

       

Sinf_1823

Oligopeptide ABC transporter, ATP-binding protein

oppD

GALLO_0327

SGGB_0355

SGGBAA2069_c03150

SGPB_0279

SMA_0356

Sinf_0308

       

Sinf_1822

Oligopeptide ABC transporter, ATP-binding protein

oppF

GALLO_0328

SGGB_0356

SGGBAA2069_c03160

SGPB_0280

SMA_0357

Sinf_0309

       

Sinf_1821

Dipeptide/tripeptide permease

dtpT

GALLO_0638

SGGB_0613

SGGBAA2069_c05810

SGPB_0507

SMA_0600

Sinf_0519

Cysteine aminopeptidase C

pepC

GALLO_0478

SGGB_0452

SGGBAA2069_c04140

SGPB_0379

SMA_0442

Sinf_0388

Aminopeptidase N

pepN

GALLO_1143

SGGB_1134

SGGBAA2069_c11310

SGPB_1002

SMA_1066

Sinf_0984

Methionine aminopeptidase

pepM

GALLO_0775

SGGB_0758

SGGBAA2069_c07470

SGPB_0642

SMA_0713

Sinf_0604

Glutamyl aminopeptidase

pepA

GALLO_0101

SGGB_0101

SGGBAA2069_c01190

SGPB_0100

SMA_0113

Sinf_0111

  

GALLO_0151

SGGB_0195

SGGBAA2069_c01680

SGPB_0141

  

Endopeptidase

pepO

GALLO_2172

SGGB_2204

SGGBAA2069_c21680

SGPB_1933

SMA_2096

Sinf_1874

Oligoendopeptidase

pepF

GALLO_0669

SGGB_0651

SGGBAA2069_c06210

SGPB_0551

SMA_0630

Sinf_0554

  

GALLO_1516

SGGB_1511

SGGBAA2069_c15390

SGPB_1410

SMA_1526

Sinf_1335

Dipeptidase

pepD

GALLO_0732

SGGB_0713

SGGBAA2069_c06950

SGPB_0605

SMA_0668

Sinf_1301

Xaa-His dipeptidase

pepV

GALLO_0931

SGGB_0915

SGGBAA2069_c09050

SGPB_0797

SMA_0836

Sinf_0699

Peptidase T

pepT

GALLO_1366

SGGB_1360

SGGBAA2069_c13560

SGPB_1287

SMA_1297

Sinf_1183

X-prolyl-dipeptidyl aminopeptidase

pepX

GALLO_1959

SGGB_1942

SGGBAA2069_c19090

SGPB_1791

SMA_1862

Sinf_1676

Aminopeptidase P

pepP

GALLO_1901

SGGB_1885

SGGBAA2069_c18550

SGPB_1732

SMA_1811

Sinf_1626

Xaa-proline dipeptidase

pepQ

GALLO_1583

SGGB_1582

SGGBAA2069_c16110

SGPB_1466

SMA_1589

Sinf_1424

Apart from amino acids, S. gallolyticus UCN34 also carries complete pathways for the synthesis of a number of vitamins including riboflavin, nicotine amide, pantothenate, pyridoxine, and folic acid, while the biosynthetic pathways for biotin and thiamine are partial [25]. The genes potentially involved in the de novo biosynthesis of pyridoxine in the SBSEC strains were determined based on the respective pathway of S. pneumoniae D39 [34]. The corresponding loci are conserved among S. gallolyticus strains but once more S. macedonicus, S. pasteurianus and S. infantarius appear to have undergone a heterogeneous gene loss process, indicating the necessity for exogenous supply of some of these vitamins (Table 4). For example, S. macedonicus misses the bioBDY, panBCD and ribDEAH loci involved in the biosynthesis of biotin, pantothenate and riboflavin, respectively. In addition, the presence of pseudogenes or truncated/split genes may have disrupted the biosynthesis of pyridoxine, nicotine amide and thiamine through the routes analyzed here. It is not uncommon for LAB to be auxotrophic for several vitamins [35], though milk and other dairy products may contain all essential vitamins to sustain the growth of these microorgansims.
Table 4

Genes in the Streptococcus bovis / Streptococcus equinus complex potentially involved in the biosynthesis of vitamins

Vitamin

Gene

S. gallolyticus UCN34

S. gallolyticus ATCC 43143

S. gallolyticus ATCC BAA-2069

S. pasteurianus ATCC 43144

S. macedonicus ACA-DC 198

S. infantarius CJ18

Biotin (B8, partial)

bioB

GALLO_1916

SGGB_1900

SGGBAA2069_c18670

SGPB_1745

- (a)

-

 

bioD

GALLO_1915

SGGB_1899

SGGBAA2069_c18660

SGPB_1744

-

-

 

bioY

GALLO_1914

SGGB_1898

SGGBAA2069_c18650

SGPB_1743

-

-

 

pdxS

GALLO_1189

SGGB_1183

SGGBAA2069_c11790

-

SMA_1105 (s)

Sinf_1022

      

SMA_1106 (p)

 
 

pdxT

GALLO_1188

SGGB_1182

SGGBAA2069_c11780

-

SMA_1104

Sinf_1021

 

pdxR

GALLO_1111

SGGB_1101

SGGBAA2069_c10980

SGPB_0968

SMA_1031

Sinf_0955

Folic acid (B9)

folC

GALLO_1233

SGGB_1227

SGGBAA2069_c12240

SGPB_1087

SMA_1137

Sinf_1067

 

folE

GALLO_1232

SGGB_1226

SGGBAA2069_c12230

SGPB_1086

SMA_1136

Sinf_1066

 

folP

GALLO_1231

SGGB_1225

SGGBAA2069_c12220

SGPB_1085

SMA_1135

Sinf_1065

 

folB

GALLO_1230

SGGB_1224

SGGBAA2069_c12210

SGPB_1084

SMA_1134

Sinf_1064

 

folK

GALLO_1229

SGGB_1223

SGGBAA2069_c12200

SGPB_1083

SMA_1133

Sinf_1063

 

folD

GALLO_0622

SGGB_0594

SGGBAA2069_c05620

SGPB_0494

SMA_0581

Sinf_0503

Nicotine amide (NAD, B3)

nadA

GALLO_1937

SGGB_1920

SGGBAA2069_c18890

SGPB_1769

SMA_1844 (p)

Sinf_1655

 

nadB

GALLO_1936

SGGB_1919

SGGBAA2069_c18880

SGPB_1768

SMA_1840 (s)

Sinf_1654

      

SMA_1841 (s)

 
      

SMA_1842 (s)

 
      

SMA_1843 (p)

 
 

nadC

GALLO_1935

SGGB_1918

SGGBAA2069_c18870

SGPB_1767

SMA_1839

Sinf_1653

 

nadE

GALLO_0477

SGGB_0451

SGGBAA2069_c04130

SGPB_0377 (p)

SMA_0441

Sinf_0387

     

SGPB_0378 (p)

  

Pantothenate (B5)

panB

GALLO_0161

SGGB_0205

SGGBAA2069_c01790

-

-

-

 

panC

GALLO_0160

SGGB_0204

SGGBAA2069_c01780

-

-

Sinf_0173 (t)

 

panD

GALLO_0159

SGGB_0203

SGGBAA2069_c01770

-

-

Sinf_0172

 

panE

GALLO_0232

SGGB_0274

SGGBAA2069_c02470

SGPB_0217

SMA_0254

Sinf_0233 (p)

Riboflavin (B2)

ribD

GALLO_0692

SGGB_0673

SGGBAA2069_c06490

SGPB_0567

-

Sinf_0572

 

ribE

GALLO_0693

SGGB_0674

SGGBAA2069_c06500

SGPB_0568

-

Sinf_0573

 

ribA

GALLO_0694

SGGB_0675

SGGBAA2069_c06510

SGPB_0569

-

Sinf_0574

 

ribH

GALLO_0695

SGGB_0676

SGGBAA2069_c06520

SGPB_0570

-

Sinf_0575

 

ribF

GALLO_1160

SGGB_1152

SGGBAA2069_c11480

SGPB_1019

SMA_1086

Sinf_0999

Thiamine (B1, partial)

tenA

GALLO_1181

SGGB_1175

SGGBAA2069_c11710

SGPB_1039

-

Sinf_1014

 

thiE

GALLO_1178

SGGB_1172

SGGBAA2069_c11680

SGPB_1036

SMA_1100 (t)

Sinf_1011

 

thiM

GALLO_1179

SGGB_1173

SGGBAA2069_c11690

SGPB_1037

-

Sinf_1012

 

thiD

GALLO_1180

SGGB_1174

SGGBAA2069_c11700

SGPB_1038

-

Sinf_1013

 

thiI

GALLO_1346

SGGB_1341

SGGBAA2069_c13350

SGPB_1268

SMA_1273

Sinf_1163

 

thiN

GALLO_2003

SGGB_1987

SGGBAA2069_c19580

SGPB_1830

SMA_1899

Sinf_1712

(a) Not found; (s) Split CDSs corresponding to fragments of the original gene not yet characterized as pseudogenes; (p) Pseudogenes; (t) Truncated.

Genomic islands (GIs) and unique genes of Streptococcus macedonicus

GIs are sites of HGT that can uncover important features of the plasticity of a bacterial genome and they are primarily linked to gene gain processes. We used the IslandViewer application [36] to identify GIs of the SBSEC members in parallel. Streptococcus macedonicus had 14 predicted GIs with an average length of 18,109 bp corresponding to a total sequence of 253,523 bp or 11.9% the size of the bacterium’s genome (Additional file 5: Figure S2). This percentage of externally acquired DNA is higher compared to the other SBSEC members, in which it ranged from 8.8% in S. gallolyticus ATCC BAA-2069 down to 5.9% in S. gallolyticus UCN34.

As could be expected, the highest degree of sequence conservation among GIs was observed in the S. gallolyticus strains (e.g. S. gallolyticus UCN34 GIs 2, 6, 7, 8 and 9). When different SBSEC species were compared, a number of GIs were only partially conserved (e.g. S. gallolyticus UCN34 GIs 1, 3, 6, 7, 8 and 9). Unique GIs were also present in most genomes analyzed (e.g. S. pasteurianus GIs 2, 4, 6 and 8). Partially conserved GIs may be remnants of GIs acquired before speciation events in the SBSEC and their subsequent gene decay may be the result of adaptation to diverged ecological niches. The existence of unique GIs among the SBSEC species, whose acquisition must have been more recent (i.e. most probably after speciation), also points to the same direction. Furthermore, our analysis suggests that S. macedonicus shares stretches of GI sequences exclusively with S. infantarius among the SBSEC members (e.g. in S. macedonicus GIs 1, 4, 5, 6, 7, 8 and 14) in accordance with previous findings [20]. Potential donors of GI sequences were identified from best BLASTN hits showing sequence identity > 90%. In several instances sequence segments within S. macedonicus GIs may have derived from more than one donor (Additional file 6: Figure S3). Potential donors of the S. macedonicus GIs were Streptococcus agalactiae, Streptococcus intermedius, Streptococcus suis, Streptococcus uberis, Enterococcus faecium, Lactococcus garvieae and Pediococcus pentosaceus. Most importantly, Lactococcus lactis or S. thermophilus were found among these donors in 9 out of 14 S. macedonicus GIs and the same applies for S. infantarius in 6 out of 12 GIs. None of the GI sequences of the other SBSEC members could be linked to L. lactis or S. thermophilus apart from the S. gallolyticus ATCC BAA-2069 GI 6 that exhibited a 96% identity over an approximately 3 kb genomic region of S. thermophilus JIM 8232 (data not shown). These observations constitute additional evidence that S. macedonicus and S. infantarius are the only members of the complex that have extensively interacted with the dairy L. lactis and S. thermophilus.

We then calculated the unique genes (also referred here as singleton genes) of S. macedonicus against the other SBSEC species twice, taking or not into account the genome of S. infantarius. Results from singleton gene analysis using EDGAR [23] were manually curated to relieve the set from the high numbers of transposable elements. There was an important overlap between the list of genes found in GIs of S. macedonicus and the singleton genes (Additional file 7: Table S4 and Additional file 8: Table S5). Again, S. macedonicus and S. infantarius were found to share a number of genes that are absent from the other SBSEC genomes (Additional file 8: Table S5).

According to the aforementioned analysis S. macedonicus carries the complete biosynthetic pathways for two lantibiotic bacteriocins, i.e. the macedocin and the macedovicin peptides [37, 38]. The presence of both antimicrobials can provide an additional link between S. macedonicus and the milk environment. Production of macedocin has been observed only in milk up to now and proteolytic fragments of casein may trigger biosynthesis of this peptide [39]. In addition, the entire macedovicin gene cluster is practically identical (99% sequence identity over the entire length of the ~9.8 kb cluster) to the respective clusters of thermophilin 1277 and bovicin HJ50 found in the dairy isolates S. thermophilus SBT1277 and S. bovis HJ50, respectively [37]. The locus seems to have spread among the three strains by HGT and their common dairy origin increases the possibility that this exchange of genetic material has taken place in milk [37].

Another evident characteristic of the S. macedonicus genome was the presence of multiple restriction modification (RM) systems among the singleton genes (Additional file 9: Figure S4). Streptococcus macedonicus possesses the highest number of RM systems within the SBSEC and it is the only member of the group with all three types of RM systems. A yet unresolved difference in the number and the type of RM systems between commensal and dairy LAB has been previously observed [40, 41]. As mentioned earlier, phages are present in milk and dairy products often in high numbers [42] and traditional practices (e.g. backslopping) may promote the selection of phage resistant strains [40, 41]. In S. thermophilus RM systems are considered as important technological traits [8] and it has been previously suggested that genes of the type III RM system may provide a signature for milk adaptation [40]. Streptococcus macedonicus has two type III RM systems, one of which is inactive since it consists of pseudogenes. The increased number of RM systems of S. macedonicus compared to the other SBSEC members suggests that it should be particularly competent in resisting invading DNA. These findings coincide with the fact that S. macedonicus carries the highest number of spacers in its CRISPR (clustered regularly interspaced short palindromic repeats) locus within the SBSEC (Additional file 10: Table S6). Furthermore, BLASTN analysis of the spacers in the S. macedonicus CRISPR revealed that four of them, namely spacers 3, 5, 17 and 18, had hits in S. thermophilus phages (e.g. phages O1205, 7201, Abc2, etc.), S. thermophilus plasmids (e.g. pER36) or S. thermophilus CRISPR spacer sequences (data not shown). In contrast, among the 140 spacers of the different CRISPR found in the other SBSEC species, only one had a hit in L. lactis phage 1706 (spacer 35 in the CRISPR of S. pasteurianus). According to these findings the occurrence of S. macedonicus in the same habitat as that of S. thermophilus can be supported.

In addition, S. macedonicus contains singleton genes – several copies in some instances – coding for proteins involved in the transport and homeostasis of metal ions (Additional file 7: Table S4 and Additional file 8: Table S5). Some of these genes are also shared by S. infantarius, but not all. These genes may play a role in the transport of copper (e.g. copA and copB), cadmium (e.g. cadA and cadC), manganese (e.g. mntH) and magnesium (e.g. SMA_2044). Copper and cadmium are of no evident biological role for Lactobacillales [43] and thus transport systems for such metals in S. macedonicus should be perceived as a protective mechanism towards their deleterious effects (e.g. through oxidative stress). The presence of metal transport genes has been previously reported in several LAB including L. lactis and S. thermophilus strains [4348]. In our opinion the high number of metal transport associated genes in S. macedonicus was an unexpected observation and further investigation is required regarding their physiological relevance.

Distribution of virulence factors (VFs) within species of the SBSEC

One of the main goals behind the genome sequencing of S. macedonicus was to clarify its pathogenic potential. Unfortunately, despite the well-known association of S. bovis with human disease, especially endocarditis and colon cancer, there is very little knowledge about the pathogenicity mechanisms employed by members of the SBSEC. In Table 5 we have gathered genes previously assigned as potential VFs in SBSEC. The available studies have shed some light on the ability of S. gallolyticus to colonize host tissues, a step that is considered as a prerequisite for the initiation of the infection by this bacterium. Streptococcus gallolyticus UCN34 contains three pilus gene clusters which may mediate binding to the extracellular matrix (ECM), similarly to the clinical isolate TX20005 whose genome is partially characterized [25, 49]. The pil1 and pil3 of strain UCN34 have been found identical to the acb-sbs7-srtC1 and sbs15-sbs14-srtC3 loci of strain TX20005, respectively, but their additional predicted pilus gene cluster (i.e. pil2 vs. sbs12-sbs11-srtC2) was only distantly related [25]. While all three strains of S. gallolyticus carry the three pilus loci (as found in strain UCN34), S. macedonicus, S. pasteurianus and S. infantarius carry only the pil3 locus. Functional analysis indicated that pil1 is a crucial factor of S. gallolyticus UCN34 for binding to ECM, especially to collagen [18]. The preference of S. gallolyticus to bind to collagen is of particular importance, since it may allow the adherence of the bacterium to the collagen-rich surfaces of damaged heart valves and (pre)cancerous sites [50]. Besides the pilus loci, additional MSCRAMM (microbial surface recognizing adhesive matrix molecules) proteins have been predicted in S. gallolyticus, most of which are either absent or preudogenes in S. macedonicus, S. pasteurianus and S. infantarius (Table 5) [49]. The cell surface protein antigen c (PAc) also appears exclusively in the S. gallolyticus genomes, sometimes in more than one copy. Only the surface-exposed histone-like protein A (HlpA) and the autolysin (AtlA) are universally conserved in the SBSEC. HlpA has been shown to be a major heparin-binding protein regulating the ability of S. gallolyticus adherence to the heparan sulfate proteoglycans at the colon tumor cell surface [51]. AtlA is a fibronectin-binding protein which is a VF of S. mutans associated with infective endocarditis [52]. Furthermore, S. gallolyticus UCN34 carries loci for the biosynthesis of insoluble glucan polymers from sucrose and the synthesis of hemicellulose [25]. Insoluble glucan polymers may contribute to feedlot bloat in cattle [25], while hemicellulose could play a role in biofilm formation [53]. It is possible that the production of these polymers may vary among strains of S. gallolyticus (Table 5). Streptococcus macedonicus is devoid of the biosynthetic gene cluster of glucan, while the hemicellulose synthesis operon seems to be comprised of pseudogenes. Similarly, S. pasteurianus and S. infantarius seem to be also unable to synthesize both sugar polymers, either due to full or partial absence of the genetic loci.
Table 5

Genes in the Streptococcus bovis / Streptococcus equinus complex identified as putative virulence factors

Virulence factor

Gene

S. gallolyticus UCN34

S. gallolyticus ATCC 43143

S. gallolyticus ATCC BAA-2069

S. pasteurianus ATCC 43144

S. macedonicus ACA-DC 198

S. infantarius CJ18

Pilus 1 (pil1)

acb

GALLO_2179

SGGB_2211

SGGBAA2069_c21760

- (a)

-

-

 

sbs7

GALLO_2178

SGGB_2210

SGGBAA2069_c21750

SGPB_1938 (p)

-

-

 

srtC1

GALLO_2177

SGGB_2209

SGGBAA2069_c21740

-

-

Sinf_1876

Pilus 2 (pil2)

-

GALLO_1570

SGGB_1568

SGGBAA2069_c15960

-

-

-

 

-

GALLO_1569

SGGB_1567

SGGBAA2069_c15950

-

-

-

 

-

GALLO_1568

SGGB_1566

SGGBAA2069_c15940

-

-

-

Pilus 3 (pil3)

sbs15

GALLO_2040

SGGB_2022

SGGBAA2069_c19980

SGPB_1847

SMA_1939

Sinf_1744

 

sbs14

GALLO_2039

SGGB_2021

SGGBAA2069_c19970

SGPB_1846

SMA_1938

Sinf_1743

 

srtC3

GALLO_2038

SGGB_2020

SGGBAA2069_c19960

SGPB_1845

SMA_1937

Sinf_1742

Cell envelope proteinase (lactocepin)

sbs6

GALLO_0748

SGGB_0730

SGGBAA2069_c07210

-

-

Sinf_0588

Fructan hydrolase

sbs10

GALLO_0112

SGGB_0110

SGGBAA2069_c01280

-

-

-

Collagen adhesin

sbs13

GALLO_2032

SGGB_2016

SGGBAA2069_c19910

SGPB_1839 (p)

SMA_1932 (s)

Sinf_1737 (p)

     

SGPB_1840 (p)

SMA_1933 (p)

 
      

SMA_1934 (s)

 

Collagen adhesin

sbs16

GALLO_0577

SGGB_0544

SGGBAA2069_c05110

-

-

-

Cell surface protein antigen C (PAc)

-

GALLO_1675

SGGB_0154

SGGBAA2069_c13880

-

-

-

   

SGGB_1687

SGGBAA2069_c20560

   

Surrface-exposed histone-like protein A

hlpA

GALLO_0636

SGGB_0611

SGGBAA2069_c05790

SGPB_0505

SMA_0597

Sinf_0517

Autolysin

atlA

GALLO_1368

SGGB_1362

SGGBAA2069_c13580

SGPB_1289

SMA_1299

Sinf_1186 (t)

Glucan biosynthesis gene cluster

-

GALLO_1052

-

SGGBAA2069_c10370

-

-

-

 

-

GALLO_1053

SGGB_1042

SGGBAA2069_c10380

-

-

-

 

rggA

GALLO_1054

SGGB_1043

SGGBAA2069_c10390

-

-

Sinf_0876

 

gtfA

GALLO_1055

SGGB_1044

SGGBAA2069_c10400

-

-

Sinf_0877

 

rggB

GALLO_1056

SGGB_1045

SGGBAA2069_c10410

-

-

-

 

gtfB

GALLO_1057

SGGB_1046

SGGBAA2069_c10420

-

-

-

 

sbs2/gbpC

GALLO_1058

SGGB_1047

SGGBAA2069_c10430

-

SMA_0989 (p)

-

      

SMA_0990 (s)

 
      

SMA_0991 (s)

 

Hemicellulose biosynthesis gene cluster

-

GALLO_0364

SGGB_0392

SGGBAA2069_c03530

-

SMA_0392 (p)

Sinf_0344

 

-

GALLO_0365

SGGB_0393 (p)

SGGBAA2069_c03540 (s)

-

SMA_0393 (p)

-

   

SGGB_0394 (p)

SGGBAA2069_c03550 (s)

   
 

-

GALLO_0366

SGGB_0395

SGGBAA2069_c03560

-

SMA_0394 (p)

Sinf_0345

       

Sinf_0346 (s)

 

-

GALLO_0367

SGGB_0396

SGGBAA2069_c03570

-

-

-

Hemolysin TLY

-

GALLO_0630

SGGB_0605

SGGBAA2069_c05730

SGPB_0499

SMA_0591

Sinf_0511

Hemolysin III

-

GALLO_1262

SGGB_1256

SGGBAA2069_c12530

SGPB_1172

SMA_1191

Sinf_1093

Hemolysin A family protein

-

GALLO_1799

SGGB_1786

SGGBAA2069_c17570

SGPB_1603

SMA_1706

Sinf_1530

Exfoliative toxin B

-

-

-

-

-

-

Sinf_0933

Macrophage infectivity potentiator protein

-

-

-

-

-

-

Sinf_0931

(a) Not found; (p) Pseudogenes; (s) Split CDSs corresponding to fragments of the original gene not yet characterized as pseudogenes; (t) Truncated.

More genes whose products may be implicated in other interactions with the host cells beyond adherence could be identified. Despite the fact that the SBSEC members are considered non-hemolytic (as members of the group D streptococci), S. gallolyticus ATCC BAA-2069 has been reported to cause alpha-hemolysis on Schaedler Agar with 5% sheep blood [54]. Three hemolysins are conserved among the SBSEC members (Table 5). Sequence analysis of Sinf_1513 and Sinf_1683, also annotated as hemolysin genes, was not supportive of a hemolysin protein product (data not shown). Apart from hemolysins, a putative exfoliative toxin B (Sinf_0933) and a macrophage infectivity potentiator protein (Sinf_0931) are present in the S. infantarius genome [20]. Similar genes can be found in S. thermophilus strains but not in the other SBSEC species and in our opinion functional analysis is required to verify these annotations.

In order to expand our investigation for putative pathogenicity traits, we screened the genomes of S. macedonicus and its related SBSEC species using the VFDB (virulence factors database) [55] and the genes determined to encode putative VFs during this analysis are presented in Additional file 11: Table S7. Current results of comparative pathogenomics have allowed the classification of available streptococcal VFs in nine categories, i.e. adhesion factors, DNases, exoenzymes, immune evasion factors, immunoreactive antigens, factors involved in metal transport, proteases, superantigens and toxins [56]. The general profile of VFs for the six streptococci under investigation was rather similar and we determined a number of previously unidentified potential VFs dispersed among all or some of the SBSEC members. Several of these genes coding for putative VFs like the agglutinin receptor, the fibronectin/fibrinogen-binding protein (fbp54/pavA), the lipoprotein rotamase A (slrA), the plasmin receptor/GAPDH multifunctional protein, the streptococcal enolase exoenzyme, the pneumococcal surface antigen A and specific proteases (i.e. cppA, htrA/degP and tig/ropA) have been experimentally correlated with the virulence of pathogenic streptococci beyond SBSEC members [5767]. Some genes were also involved in the production of a capsule that enables bacterial cells to evade phagocytosis (Additional file 11: Table S7) [68]. According to our analysis, all SBSEC streptococci carry a main gene cluster spanning practically the same position in the chromosome that could be involved in the biosynthesis of a capsule (Additional file 12: Figure S5). Even though the cps clusters are identical between S. gallolyticus UCN34 and ATCC BAA-2069 [54], multiple sequence alignment indicates significant structural diversity in the rest of the strains. The existence of dispersed pseudogenes in the gene clusters of S. infantarius and S. macedonicus (e.g. SMA_0865 and SMA_0866) may prohibit the production of capsule substances. It should be emphasized that the strains of the SBSEC missed hits in several major categories of streptococcal VFs (e.g. DNases, immunoreactive antigens, superantigens and toxins) supporting a reduced pathogenic potential for the SBSEC in general.

Conclusions

In this study we presented the analysis of the first complete genome sequence of a dairy isolate of S. macedonicus. While comparative analysis among specific subgroups of the SBSEC species has been previously presented [20, 22, 25, 54], comparative genomics of the six complete genome sequences was missing. Most importantly, the inclusion of S. macedonicus into this analysis provided a better opportunity to assess niche adaptation of the SBSEC species that was so far limited by the presence of only one dairy isolate (i.e. S. infantarius CJ18) among four clinical strains.

Our findings clearly support two distinct evolutionary patterns within the SBSEC. On the one hand, S. gallolyticus is a species without apparent genome decay and the available genomes suggest that it is a robust bacterium able to thrive in the rumen of herbivores. On the other hand, the remaining SBSEC species, i.e. S. macedonicus, S. pasteurianus and S. infantarius exhibit decreased genome sizes accompanied by increased percentages of potential pseudogenes due to extensive genome decay, suggesting adaptation to nutrient-rich environments. This does not necessarily mean that the environment to which the three species have been adapted is the same. The three species appear with a reduced ability to catabolize complex plant carbohydrates and to detoxify substances met in the rumen, which indicates that they must have deviated from this niche. It has been proposed that S. pasteurianus may now reside in the human gut [22], while S. infantarius presents adaptations to milk [20]. Streptococcus macedonicus also possesses traits that may contribute to growth in the dairy environment, like the extra lactose gene cluster and its proteolytic system. However, all SBSEC strains, including clinical isolates, seem to be competent in the metabolism of lactose and galactose or the degradation of milk proteins. Taking into account these shared characteristics of all SBSEC species, it is tempting to speculate that their common ancestor may have been able to grow in milk.

In our opinion, several genome traits per se suggest adaptation of S. macedonicus to milk. This hypothesis is also supported by the predicted interspecies interactions of S. macedonicus with other bacteria. As it has been recently reported for S. infantarius [20], the S. macedonicus genome may have acquired genes originating from L. lactis and S. thermophilus through HGT. The predicted exposure of S. macedonicus to S. thermophilus phages, based on our CRISPR sequence analysis, is also in favor of this theory. No such evidence was found for the rest of the SBSEC members apart from S. infantarius. These findings are in accordance with the frequent isolation of S. macedonicus from dairy products [13] and the prevalence of S. infantarius in certain African fermented milks [20]. One additional question that arises is whether S. macedonicus and S. infantarius are specialized dairy microbes like S. thermophilus. We believe that the available data does not support this idea. Traits of milk adaptation have been shown to be strain-specific in S. infantarius [20]. In addition, the genome size of S. macedonicus is significantly larger, containing a higher number of functional genes in comparison to S. thermophilus. Streptococcus macedonicus and S. infantarius may thus represent intermediate evolutionary stages analogous to those followed by the ancestors of S. thermophilus before it became today’s starter culture.

Thus, the safety concerns raised from the presence of SBSEC members in foods remain, even if reports implicating S. macedonicus with disease are rather scarce [69, 70]. Our comparative genomic analysis showed that both S. macedonicus and S. infantarius miss several VFs that are highly conserved in S. gallolyticus. However, the interpretation of these findings becomes complicated as the available genome of the human blood isolate S. pasteurianus ATCC 43144 also exhibited diminished traits of pathogenicity similarly to the two dairy SBSEC members. Overall, our analysis provides evidence in agreement with the clinical perception that the members of the SBSEC are lower grade streptococcal pathogens [10]. In terms of food safety, the dairy SBSEC could thus constitute a risk factor similar to the presence of enterococci that are widely found in fermented products, but cause no major problem for the average healthy and adult consumer. Nevertheless, it is the correlation of the SBSEC microorganisms with human endocarditis and colon cancer in particular that may require special considerations. For example, it has been proposed that members of the SBSEC like S. gallolyticus may be part of the etiology of colon cancer by causing chronic inflammation [10]. In order to assess the pathogenicity of this group of streptococci, more research is needed on the specific mechanisms employed by SBSEC members to cause disease. More comparative and functional genomics studies comprising SBSEC genomes are necessary that will cover additional species of the complex, like the recently sequenced Streptococcus lutetiensis [71]. New clinico-epidemiological studies should also be undertaken in view of the most recent changes in the taxonomy of the SBSEC complex [72]. In the meantime, assuming the worse case scenario, we propose that the presence of SBSEC members including S. macedonicus and S. infantarius in foods should be avoided until their pathogenicity status is resolved.

Methods

Sequencing and annotation of the genome of Streptococcus macedonicusACA-DC 198

The genome of S. macedonicus ACA-DC 198 was sequenced and annotated as described previously [19]. In brief, we employed a sequencing strategy involving shotgun/paired-end pyrosequencing and shotgun Illumina sequencing with the 454 GS-FLX (Roche Diagnostics, Basel, Switzerland) and the Hiseq 2000 (Illumina, San Diego, CA), respectively. Sequences were assembled in two contigs corresponding to the complete genome sequence and the pSMA198 plasmid of S. macedonicus. The hybrid assembly was validated against an NheI optical map of the S. macedonicus genome generated at OpGen Technologies, Inc. (Madison, WI). The genome was annotated using the RAST [73] and the Basys [74] pipelines. Predictions of the two pipelines were compiled into a single annotation file after manual curation in the Kodon software environment (Applied Maths N.V., Sint-Martens-Latem, Belgium). Final corrections and quality assessment of the annotation were performed with the GenePRIMP pipeline [21]. GenePRIMP was also used for the identification of putative pseudogenes. The circular map of the S. macedonicus genome was generated by the DNAPlotter software [75].

Comparative genomics of Streptococcus macedonicusACA-DC 198 against related members of the SBSEC

The complete genome sequence of S. macedonicus was compared to those of S. gallolyticus strains UCN34, ATCC 43143 and ATCC BAA-2069, S. pasteurianus ATCC 43144 and S. infantarius CJ18 using a variety of tools. In order to visualize conserved genomic regions or chromosomal rearrangements, whole genome sequence alignments were performed by progressiveMAUVE [24]. Estimation of the differential gene content of the genomes, as well as whole genome phylogeny of streptococci was carried out within the EDGAR software framework [23]. Venn diagrams were designed with the VennDiagram package in R [76]. The glycobiome of the SBSEC members was determined based on the pre-computed data available in the CAZy database [26].

Additional analysis

Sequence similarity searches were performed with the BLAST suite [77]. Whenever necessary, protein sequences were analyzed in the CDD [78]. Figures showing similarity of gene clusters were constructed with the Easyfig comparison visualizer [79]. Potential VFs included in the VFDB [55] were identified in the SBSEC genomes with mpiBLAST, as implemented in the mGenomeSubtractor website [80]. In brief, the entire VFDB was uploaded as the reference sequence in the mGenomeSubtractor website and each genome was used as the query sequence. Only hits with H-value homology score > 0.6 were considered significant. CRISPRs were analyzed by the tools available in the CRISPRcompar web-service [81]. A general bit score cutoff value of 42.0 was applied during BLASTN of CRISPR spacers. GIs were identified and visualized by the IslandViewer application that utilizes three different prediction tools (i.e. IslandPick, SIGI-HMM and IslandPath-DIMOB) relying on either sequence composition or comparative genomics [36]. Genomic regions of RM systems were determined in the REBASE genomes database [82].

Availability of supporting data

The data set supporting the phylogenetic tree presented in Additional file 1: Figure S1 of this article is available in the [Dryad] repository, [unique persistent identifier doi:10.5061/dryad.7d039 and hyperlink to datasets in http://datadryad.org/]. Additional data sets supporting the results of this article are included within the article and its additional files.

Abbreviations

SBSEC: 

Streptococcus bovis/Streptococcus equinus complex

LAB: 

Lactic acid bacteria

GRAS: 

Generally regarded as safe

QPS: 

Qualified presumption of safety

GIT: 

Gastrointestinal tract

GAS: 

Group A streptococci

GBS: 

Group B streptococci

CDS: 

Coding DNA sequence

LCB: 

Local collinear block

HGT: 

Horizontal gene transfer

PEP-PTS: 

Phosphoenolpyruvate-dependent phosphotransferase system

CEP: 

Cell-envelope associated proteinase

PMF: 

Proton motive force

CRISPR: 

Clustered regularly interspaced short palindromic repeats

GI: 

Genomic island

RM: 

Restriction modification

VF: 

Virulence factor

ECM: 

Extracellular matrix

MSCRAMM: 

Microbial surface recognizing adhesive matrix molecule

CDD: 

Conserved domain database

VFDB: 

Virulence factors database.

Declarations

Acknowledgements

The present work was cofinanced by the European Social Fund and the National resources EPEAEK and YPEPTH through the Thales project.

Authors’ Affiliations

(1)
Laboratory of Dairy Research, Department of Food Science and Human Nutrition, Agricultural University of Athens
(2)
Laboratory of Cell Proliferation and Ageing, Institute of Biosciences and Applications, National Centre for Scientific Research "Demokritos"
(3)
Computational Genomics, Center for Biotechnology, Bielefeld University
(4)
Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens
(5)
Genoscreen, Genomic Platform and R&D, Campus de l’Institut Pasteur
(6)
INRA, UMR1319 Micalis
(7)
AgroParisTech, UMR Micalis
(8)
Institut Pasteur de Lille, Center for Infection and Immunity of Lille (CIIL)
(9)
Inserm U1019
(10)
CNRS UMR8204
(11)
Univ Lille de Nord France

References

  1. Konings WN, Kok J, Kuipers OP, Poolman B: Lactic acid bacteria: the bugs of the new millennium. Curr Opin Microbiol. 2000, 3 (3): 276-282. 10.1016/S1369-5274(00)00089-8.PubMedView ArticleGoogle Scholar
  2. Masood MI, Qadir MI, Shirazi JH, Khan IU: Beneficial effects of lactic acid bacteria on human beings. Crit Rev Microbiol. 2011, 37 (1): 91-98. 10.3109/1040841X.2010.536522.PubMedView ArticleGoogle Scholar
  3. Donohue DC, Gueimonde M: Some Considerations for the Safety of Novel Probiotic Bacteria. Lactic Acid Bacteria: Microbiological and Functional Aspects. Edited by: Lahtinen S, Salminen S, von Wright A, Ouwehand AC. 2012, Boca Raton: CRC Press Taylor & Francis Group, 4Google Scholar
  4. Woodford N, Livermore DM: Infections caused by Gram-positive bacteria: a review of the global challenge. J Infect. 2009, 59 (Suppl 1): S4-S16.PubMedView ArticleGoogle Scholar
  5. Facklam R: What happened to the streptococci: overview of taxonomic and nomenclature changes. Clin Microbiol Rev. 2002, 15 (4): 613-630. 10.1128/CMR.15.4.613-630.2002.PubMed CentralPubMedView ArticleGoogle Scholar
  6. Nobbs AH, Lamont RJ, Jenkinson HF: Streptococcus adherence and colonization. Microbiol Mol Biol Rev. 2009, 73 (3): 407-450. 10.1128/MMBR.00014-09.PubMed CentralPubMedView ArticleGoogle Scholar
  7. Bolotin A, Quinquis B, Renault P, Sorokin A, Ehrlich SD, Kulakauskas S, Lapidus A, Goltsman E, Mazur M, Pusch GD, Fonstein M, Overbeek R, Kyprides N, Purnelle B, Prozzi D, Ngui K, Masuy D, Hancy F, Burteau S, Boutry M, Delcour J, Goffeau A, Hols P: Complete sequence and comparative genome analysis of the dairy bacterium Streptococcus thermophilus. Nat Biotechnol. 2004, 22 (12): 1554-1558. 10.1038/nbt1034.PubMedView ArticleGoogle Scholar
  8. Hols P, Hancy F, Fontaine L, Grossiord B, Prozzi D, Leblond-Bourget N, Decaris B, Bolotin A, Delorme C, Dusko Ehrlich S, Guedon E, Monnet V, Renault P, Kleerebezem M: New insights in the molecular biology and physiology of Streptococcus thermophilus revealed by comparative genomics. FEMS Microbiol Rev. 2005, 29 (3): 435-463.PubMedGoogle Scholar
  9. Jans C, Lacroix C, Meile L: A novel multiplex PCR/RFLP assay for the identification of Streptococcus bovis/Streptococcus equinus complex members from dairy microbial communities based on the 16S rRNA gene. FEMS Microbiol Lett. 2012, 326 (2): 144-150. 10.1111/j.1574-6968.2011.02443.x.PubMedView ArticleGoogle Scholar
  10. Abdulamir AS, Hafidh RR, Abu Bakar F: The association of Streptococcus bovis/gallolyticus with colorectal tumors: the nature and the underlying mechanisms of its etiological role. J Exp Clin Cancer Res. 2011, 30: 11-10.1186/1756-9966-30-11.PubMed CentralPubMedView ArticleGoogle Scholar
  11. Galdy S, Nastasi G: Streptococcus bovis endocarditis and colon cancer: myth or reality? A case report and literature review. BMJ Case Rep. 2012, 2012: 1-2.Google Scholar
  12. Herrera P, Kwon YM, Ricke SC: Ecology and pathogenicity of gastrointestinal Streptococcus bovis. Anaerobe. 2009, 15 (1–2): 44-54.PubMedView ArticleGoogle Scholar
  13. De Vuyst L, Tsakalidou E: Streptococcus macedonicus, a multi-functional and promising species for dairy fermentations. Int Dairy J. 2008, 18 (5): 476-485. 10.1016/j.idairyj.2007.10.006.View ArticleGoogle Scholar
  14. Maragkoudakis PA, Papadelli M, Georgalaki M, Panayotopoulou EG, Martinez-Gonzalez B, Mentis AF, Petraki K, Sgouras DN, Tsakalidou E: In vitroand in vivosafety evaluation of the bacteriocin producer Streptococcus macedonicusACA-DC 198.Int J Food Microbiol. 2009, 133 (1–2): 141-147.PubMedView ArticleGoogle Scholar
  15. Tsakalidou E, Zoidou E, Pot B, Wassill L, Ludwig W, Devriese LA, Kalantzopoulos G, Schleifer KH, Kersters K: Identification of streptococci from Greek Kasseri cheese and description of Streptococcus macedonicus sp. nov. Int J Syst Bacteriol. 1998, 48 (Pt 2): 519-527.PubMedView ArticleGoogle Scholar
  16. Schlegel L, Grimont F, Ageron E, Grimont PA, Bouvet A: Reappraisal of the taxonomy of the Streptococcus bovis/Streptococcus equinus complex and related species: description of Streptococcus gallolyticus subsp. gallolyticus subsp. nov., S. gallolyticus subsp. macedonicus subsp. nov. and S. gallolyticus subsp. pasteurianus subsp. nov. Int J Syst Evol Microbiol. 2003, 53 (Pt 3): 631-645.PubMedView ArticleGoogle Scholar
  17. Whiley RA, Kilian M: International committee on systematics of prokaryotes subcommittee on the taxonomy of staphylococci and streptococci: minutes of the closed meeting, 31 July 2002, paris France. Int J Syst Evol Microbiol. 2003, 53 (3): 915-917. 10.1099/ijs.0.02589-0.View ArticleGoogle Scholar
  18. Danne C, Entenza JM, Mallet A, Briandet R, Debarbouille M, Nato F, Glaser P, Jouvion G, Moreillon P, Trieu-Cuot P, Dramsi S: Molecular characterization of a Streptococcus gallolyticus genomic island encoding a pilus involved in endocarditis. J Infect Dis. 2011, 204 (12): 1960-1970. 10.1093/infdis/jir666.PubMedView ArticleGoogle Scholar
  19. Papadimitriou K, Ferreira S, Papandreou NC, Mavrogonatou E, Supply P, Pot B, Tsakalidou E: Complete genome sequence of the dairy isolate Streptococcus macedonicus ACA-DC 198. J Bacteriol. 2012, 194 (7): 1838-1839. 10.1128/JB.06804-11.PubMed CentralPubMedView ArticleGoogle Scholar
  20. Jans C, Follador R, Hochstrasser M, Lacroix C, Meile L, Stevens MJ: Comparative genome analysis of Streptococcus infantariussubsp. infantariusCJ18, an African fermented camel milk isolate with adaptations to dairy environment.BMC Genomics. 2013, 14: 200-10.1186/1471-2164-14-200.PubMed CentralPubMedView ArticleGoogle Scholar
  21. Pati A, Ivanova NN, Mikhailova N, Ovchinnikova G, Hooper SD, Lykidis A, Kyrpides NC: GenePRIMP: a gene prediction improvement pipeline for prokaryotic genomes. Nat Methods. 2010, 7 (6): 455-457. 10.1038/nmeth.1457.PubMedView ArticleGoogle Scholar
  22. Lin IH, Liu TT, Teng YT, Wu HL, Liu YM, Wu KM, Chang CH, Hsu MT: Sequencing and comparative genome analysis of two pathogenic Streptococcus gallolyticus subspecies: genome plasticity, adaptation and virulence. PLoS One. 2011, 6 (5): e20519-10.1371/journal.pone.0020519.PubMed CentralPubMedView ArticleGoogle Scholar
  23. Blom J, Albaum SP, Doppmeier D, Puhler A, Vorholter FJ, Zakrzewski M, Goesmann A: EDGAR: a software framework for the comparative analysis of prokaryotic genomes. BMC Bioinforma. 2009, 10: 154-10.1186/1471-2105-10-154.View ArticleGoogle Scholar
  24. Darling AE, Mau B, Perna NT: ProgressiveMauve: multiple genome alignment with gene gain, loss and rearrangement. PLoS One. 2010, 5 (6): e11147-10.1371/journal.pone.0011147.PubMed CentralPubMedView ArticleGoogle Scholar
  25. Rusniok C, Couve E, Da Cunha V, El Gana R, Zidane N, Bouchier C, Poyart C, Leclercq R, Trieu-Cuot P, Glaser P: Genome sequence of Streptococcus gallolyticus: insights into its adaptation to the bovine rumen and its ability to cause endocarditis. J Bacteriol. 2010, 192 (8): 2266-2276. 10.1128/JB.01659-09.PubMed CentralPubMedView ArticleGoogle Scholar
  26. Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B: The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 2014, 42 (1): D490-D495.PubMed CentralPubMedView ArticleGoogle Scholar
  27. Crost EH, Tailford LE, Le Gall G, Fons M, Henrissat B, Juge N: Utilisation of mucin glycans by the human gut symbiont Ruminococcus gnavus is strain-dependent. PLoS One. 2013, 8 (10): e76341-10.1371/journal.pone.0076341.PubMed CentralPubMedView ArticleGoogle Scholar
  28. Ficko-Blean E, Boraston AB: Insights into the recognition of the human glycome by microbial carbohydrate-binding modules. Curr Opin Struct Biol. 2012, 22 (5): 570-577. 10.1016/j.sbi.2012.07.009.PubMedView ArticleGoogle Scholar
  29. Liu M, Bayjanov JR, Renckens B, Nauta A, Siezen RJ: The proteolytic system of lactic acid bacteria revisited: a genomic comparison. BMC Genomics. 2010, 11: 36-10.1186/1471-2164-11-36.PubMed CentralPubMedView ArticleGoogle Scholar
  30. Jans C, Gerber A, Bugnard J, Njage PM, Lacroix C, Meile L: Novel Streptococcus infantariussubsp. infantariusvariants harboring lactose metabolism genes homologous to Streptococcus thermophilus.Food Microbiol. 2012, 31 (1): 33-42. 10.1016/j.fm.2012.02.001.PubMedView ArticleGoogle Scholar
  31. Fernandez-Espla MD, Garault P, Monnet V, Rul F: Streptococcus thermophilus cell wall-anchored proteinase: release, purification, and biochemical and genetic characterization. Appl Environ Microbiol. 2000, 66 (11): 4772-4778. 10.1128/AEM.66.11.4772-4778.2000.PubMed CentralPubMedView ArticleGoogle Scholar
  32. Dandoy D, Fremaux C, de Frahan MH, Horvath P, Boyaval P, Hols P, Fontaine L: The fast milk acidifying phenotype of Streptococcus thermophilus can be acquired by natural transformation of the genomic island encoding the cell-envelope proteinase PrtS. Microb Cell Fact. 2011, 10 (Suppl 1): S21-10.1186/1475-2859-10-S1-S21.PubMed CentralPubMedView ArticleGoogle Scholar
  33. Calasso M, Gobbetti M: Lactic Acid Bacteria | Lactobacillus spp.: Other Species. Encyclopedia of Dairy Sciences. Edited by: Fuquay JW. 2011, San Diego: cademic Press, 125-131. 2View ArticleGoogle Scholar
  34. El Qaidi S, Yang J, Zhang JR, Metzger DW, Bai G: The vitamin B(6) biosynthesis pathway in Streptococcus pneumoniae is controlled by pyridoxal 5′-phosphate and the transcription factor PdxR and has an impact on ear infection. J Bacteriol. 2013, 195 (10): 2187-2196. 10.1128/JB.00041-13.PubMed CentralPubMedView ArticleGoogle Scholar
  35. LeBlanc JG, Laino JE, del Valle MJ, Vannini V, van Sinderen D, Taranto MP, de Valdez GF, de Giori GS, Sesma F: B-group vitamin production by lactic acid bacteria–current knowledge and potential applications. J Appl Microbiol. 2011, 111 (6): 1297-1309. 10.1111/j.1365-2672.2011.05157.x.PubMedView ArticleGoogle Scholar
  36. Dhillon BK, Chiu TA, Laird MR, Langille MG, Brinkman FS: IslandViewer update: improved genomic island discovery and visualization. Nucleic Acids Res. 2013, 41 (Web Server issue): W129-W132.PubMed CentralPubMedView ArticleGoogle Scholar
  37. Georgalaki M, Papadimitriou K, Anastasiou R, Pot B, Van Driessche G, Devreese B, Tsakalidou E: Macedovicin, the second food-grade lantibiotic produced by Streptococcus macedonicus ACA-DC 198. Food Microbiol. 2013, 33 (1): 124-130. 10.1016/j.fm.2012.09.008.PubMedView ArticleGoogle Scholar
  38. Georgalaki MD, Van Den Berghe E, Kritikos D, Devreese B, Van Beeumen J, Kalantzopoulos G, De Vuyst L, Tsakalidou E: Macedocin, a food-grade lantibiotic produced by Streptococcus macedonicus ACA-DC 198. Appl Environ Microbiol. 2002, 68 (12): 5891-5903. 10.1128/AEM.68.12.5891-5903.2002.PubMed CentralPubMedView ArticleGoogle Scholar
  39. Georgalaki M, Papadelli M, Chassioti E, Anastasiou R, Aktypis A, De Vuyst L, Van Driessche G, Devreese B, Tsakalidou E: Milk protein fragments induce the biosynthesis of macedocin, the lantibiotic produced by Streptococcus macedonicus ACA-DC 198. Appl Environ Microbiol. 2010, 76 (4): 1143-1151. 10.1128/AEM.00151-09.PubMed CentralPubMedView ArticleGoogle Scholar
  40. O’Sullivan O, O’Callaghan J, Sangrador-Vegas A, McAuliffe O, Slattery L, Kaleta P, Callanan M, Fitzgerald GF, Ross RP, Beresford T: Comparative genomics of lactic acid bacteria reveals a niche-specific gene set. BMC Microbiol. 2009, 9: 50-10.1186/1471-2180-9-50.PubMed CentralPubMedView ArticleGoogle Scholar
  41. Szczepankowska AK, Górecki RK, Kołakowski P, Bardowski JK: Lactic Acid Bacteria Resistance to Bacteriophage and Prevention Techniques to Lower Phage Contamination. Dairy Fermentation in R & D for Food, Health and Livestock Purposes. Edited by: Kongo JM. 2013, Croatia: InTechGoogle Scholar
  42. Marco MB, Moineau S, Quiberoni A: Bacteriophages and dairy fermentations. Bacteriophage. 2012, 2 (3): 149-158. 10.4161/bact.21868.PubMed CentralPubMedView ArticleGoogle Scholar
  43. Solioz M, Mermod M, Abicht HKM, Mancini S: Responses of Lactic Acid Bacteria to Heavy Metal Stress. Stress Responses of Lactic Acid Bacteria. Edited by: Tsakalidou E, Papadimitriou K. 2011, New York: Springer, 1Google Scholar
  44. Liu CQ, Khunajakr N, Chia LG, Deng YM, Charoenchai P, Dunn NW: Genetic analysis of regions involved in replication and cadmium resistance of the plasmid pND302 from Lactococcus lactis. Plasmid. 1997, 38 (2): 79-90. 10.1006/plas.1997.1301.PubMedView ArticleGoogle Scholar
  45. Magnani D, Barre O, Gerber SD, Solioz M: Characterization of the CopR regulon of Lactococcus lactis IL1403. J Bacteriol. 2008, 190 (2): 536-545. 10.1128/JB.01481-07.PubMed CentralPubMedView ArticleGoogle Scholar
  46. Schirawski J, Hagens W, Fitzgerald GF, Van Sinderen D: Molecular characterization of cadmium resistance in Streptococcus thermophilus strain 4134: an example of lateral gene transfer. Appl Environ Microbiol. 2002, 68 (11): 5508-5516. 10.1128/AEM.68.11.5508-5516.2002.PubMed CentralPubMedView ArticleGoogle Scholar
  47. Siezen RJ, Renckens B, van Swam I, Peters S, van Kranenburg R, Kleerebezem M, de Vos WM: Complete sequences of four plasmids of Lactococcus lactis subsp. cremoris SK11 reveal extensive adaptation to the dairy environment. Appl Environ Microbiol. 2005, 71 (12): 8371-8382. 10.1128/AEM.71.12.8371-8382.2005.PubMed CentralPubMedView ArticleGoogle Scholar
  48. Fallico V, McAuliffe O, Fitzgerald GF, Ross RP: Plasmids of raw milk cheese isolate Lactococcus lactissubsp. lactisbiovar diacetylactis DPC3901 suggest a plant-based origin for the strain.Appl Environ Microbiol. 2011, 77 (18): 6451-6462. 10.1128/AEM.00661-11.PubMed CentralPubMedView ArticleGoogle Scholar
  49. Sillanpaa J, Nallapareddy SR, Qin X, Singh KV, Muzny DM, Kovar CL, Nazareth LV, Gibbs RA, Ferraro MJ, Steckelberg JM, Weinstock GM, Murray BE: A collagen-binding adhesin, Acb, and ten other putative MSCRAMM and pilus family proteins of Streptococcus gallolyticus subsp. gallolyticus (Streptococcus bovis Group, biotype I). J Bacteriol. 2009, 191 (21): 6643-6653. 10.1128/JB.00909-09.PubMed CentralPubMedView ArticleGoogle Scholar
  50. Boleij A, Muytjens CM, Bukhari SI, Cayet N, Glaser P, Hermans PW, Swinkels DW, Bolhuis A, Tjalsma H: Novel clues on the specific association of Streptococcus gallolyticus subsp gallolyticus with colorectal cancer. J Infect Dis. 2011, 203 (8): 1101-1109. 10.1093/infdis/jiq169.PubMedView ArticleGoogle Scholar
  51. Boleij A, Schaeps RM, de Kleijn S, Hermans PW, Glaser P, Pancholi V, Swinkels DW, Tjalsma H: Surface-exposed histone-like protein a modulates adherence of Streptococcus gallolyticus to colon adenocarcinoma cells. Infect Immun. 2009, 77 (12): 5519-5527. 10.1128/IAI.00384-09.PubMed CentralPubMedView ArticleGoogle Scholar
  52. Jung CJ, Zheng QH, Shieh YH, Lin CS, Chia JS: Streptococcus mutans autolysin AtlA is a fibronectin-binding protein and contributes to bacterial survival in the bloodstream and virulence for infective endocarditis. Mol Microbiol. 2009, 74 (4): 888-902. 10.1111/j.1365-2958.2009.06903.x.PubMedView ArticleGoogle Scholar
  53. Garcia B, Latasa C, Solano C, Garcia-del Portillo F, Gamazo C, Lasa I: Role of the GGDEF protein family in Salmonella cellulose biosynthesis and biofilm formation. Mol Microbiol. 2004, 54 (1): 264-277. 10.1111/j.1365-2958.2004.04269.x.PubMedView ArticleGoogle Scholar
  54. Hinse D, Vollmer T, Ruckert C, Blom J, Kalinowski J, Knabbe C, Dreier J: Complete genome and comparative analysis of Streptococcus gallolyticussubsp. gallolyticus, an emerging pathogen of infective endocarditis.BMC Genomics. 2011, 12: 400-10.1186/1471-2164-12-400.PubMed CentralPubMedView ArticleGoogle Scholar
  55. Chen L, Xiong Z, Sun L, Yang J, Jin Q: VFDB 2012 update: toward the genetic diversity and molecular evolution of bacterial virulence factors. Nucleic Acids Res. 2012, 40 (Database issue): D641-D645.PubMed CentralPubMedView ArticleGoogle Scholar
  56. VFDB: Comparison of pathogenomic composition of Streptococcus. [http://www.mgc.ac.cn/cgi-bin/VFs/compvfs.cgi?Genus=Streptococcus],
  57. Demuth DR, Lammey MS, Huck M, Lally ET, Malamud D: Comparison of Streptococcus mutans and Streptococcus sanguis receptors for human salivary agglutinin. Microb Pathog. 1990, 9 (3): 199-211. 10.1016/0882-4010(90)90022-I.PubMedView ArticleGoogle Scholar
  58. Courtney HS, Li Y, Dale JB, Hasty DL: Cloning, sequencing, and expression of a fibronectin/fibrinogen-binding protein from group A streptococci. Infect Immun. 1994, 62 (9): 3937-3946.PubMed CentralPubMedGoogle Scholar
  59. Holmes AR, McNab R, Millsap KW, Rohde M, Hammerschmidt S, Mawdsley JL, Jenkinson HF: The pavA gene of Streptococcus pneumoniae encodes a fibronectin-binding protein that is essential for virulence. Mol Microbiol. 2001, 41 (6): 1395-1408. 10.1046/j.1365-2958.2001.02610.x.PubMedView ArticleGoogle Scholar
  60. Hermans PW, Adrian PV, Albert C, Estevao S, Hoogenboezem T, Luijendijk IH, Kamphausen T, Hammerschmidt S: The streptococcal lipoprotein rotamase A (SlrA) is a functional peptidyl-prolyl isomerase involved in pneumococcal colonization. J Biol Chem. 2006, 281 (2): 968-976. 10.1074/jbc.M510014200.PubMedView ArticleGoogle Scholar
  61. Terao Y, Yamaguchi M, Hamada S, Kawabata S: Multifunctional glyceraldehyde-3-phosphate dehydrogenase of Streptococcus pyogenes is essential for evasion from neutrophils. J Biol Chem. 2006, 281 (20): 14215-14223. 10.1074/jbc.M513408200.PubMedView ArticleGoogle Scholar
  62. Winram SB, Lottenberg R: The plasmin-binding protein Plr of group a streptococci is identified as glyceraldehyde-3-phosphate dehydrogenase. Microbiology. 1996, 142 (Pt 8): 2311-2320.PubMedView ArticleGoogle Scholar
  63. Pancholi V, Fischetti VA: alpha-enolase, a novel strong plasmin(ogen) binding protein on the surface of pathogenic streptococci. J Biol Chem. 1998, 273 (23): 14503-14515. 10.1074/jbc.273.23.14503.PubMedView ArticleGoogle Scholar
  64. Tseng HJ, McEwan AG, Paton JC, Jennings MP: Virulence of Streptococcus pneumoniae: PsaA mutants are hypersensitive to oxidative stress. Infect Immun. 2002, 70 (3): 1635-1639. 10.1128/IAI.70.3.1635-1639.2002.PubMed CentralPubMedView ArticleGoogle Scholar
  65. Angel CS, Ruzek M, Hostetter MK: Degradation of C3 by Streptococcus pneumoniae. J Infect Dis. 1994, 170 (3): 600-608. 10.1093/infdis/170.3.600.PubMedView ArticleGoogle Scholar
  66. Ibrahim YM, Kerr AR, McCluskey J, Mitchell TJ: Role of HtrA in the virulence and competence of Streptococcus pneumoniae. Infect Immun. 2004, 72 (6): 3584-3591. 10.1128/IAI.72.6.3584-3591.2004.PubMed CentralPubMedView ArticleGoogle Scholar
  67. Lyon WR, Caparon MG: Trigger factor-mediated prolyl isomerization influences maturation of the Streptococcus pyogenes cysteine protease. J Bacteriol. 2003, 185 (12): 3661-3667. 10.1128/JB.185.12.3661-3667.2003.PubMed CentralPubMedView ArticleGoogle Scholar
  68. Kadioglu A, Taylor S, Iannelli F, Pozzi G, Mitchell TJ, Andrew PW: Upper and lower respiratory tract infection by Streptococcus pneumoniae is affected by pneumolysin deficiency and differences in capsule type. Infect Immun. 2002, 70 (6): 2886-2890. 10.1128/IAI.70.6.2886-2890.2002.PubMed CentralPubMedView ArticleGoogle Scholar
  69. Herrero IA, Rouse MS, Piper KE, Alyaseen SA, Steckelberg JM, Patel R: Reevaluation of Streptococcus bovis endocarditis cases from 1975 to 1985 by 16S ribosomal DNA sequence analysis. J Clin Microbiol. 2002, 40 (10): 3848-3850. 10.1128/JCM.40.10.3848-3850.2002.PubMed CentralPubMedView ArticleGoogle Scholar
  70. Malkin J, Kimmitt PT, Ou HY, Bhasker PS, Khare M, Deng Z, Stephenson I, Sosnowski AW, Perera N, Rajakumar K: Identification of Streptococcus gallolyticus subsp. macedonicus as the etiological agent in a case of culture-negative multivalve infective endocarditis by 16S rDNA PCR analysis of resected valvular tissue. J Heart Valve Dis. 2008, 17 (5): 589-592.PubMedGoogle Scholar
  71. Jin D, Chen C, Li L, Lu S, Li Z, Zhou Z, Jing H, Xu Y, Du P, Wang H, Xiong Y, Zheng H, Bai X, Sun H, Wang L, Ye C, Gottschalk M, Xu J: Dynamics of fecal microbial communities in children with diarrhea of unknown etiology and genomic analysis of associated Streptococcus lutetiensis. BMC Microbiol. 2013, 13: 141-10.1186/1471-2180-13-141.PubMed CentralPubMedView ArticleGoogle Scholar
  72. Romero B, Morosini MI, Loza E, Rodriguez-Banos M, Navas E, Canton R, Campo RD: Reidentification of Streptococcus bovis isolates causing bacteremia according to the new taxonomy criteria: still an issue?. J Clin Microbiol. 2011, 49 (9): 3228-3233. 10.1128/JCM.00524-11.PubMed CentralPubMedView ArticleGoogle Scholar
  73. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O: The RAST Server: rapid annotations using subsystems technology. BMC Genomics. 2008, 9: 75-10.1186/1471-2164-9-75.PubMed CentralPubMedView ArticleGoogle Scholar
  74. Van Domselaar GH, Stothard P, Shrivastava S, Cruz JA, Guo A, Dong X, Lu P, Szafron D, Greiner R, Wishart DS: BASys: a web server for automated bacterial genome annotation. Nucleic Acids Res. 2005, 33 (Web Server issue): W455-W459.PubMed CentralPubMedView ArticleGoogle Scholar
  75. Carver T, Thomson N, Bleasby A, Berriman M, Parkhill J: DNAPlotter: circular and linear interactive genome visualization. Bioinformatics. 2009, 25 (1): 119-120. 10.1093/bioinformatics/btn578.PubMed CentralPubMedView ArticleGoogle Scholar
  76. Chen H, Boutros PC: VennDiagram: a package for the generation of highly-customizable Venn and Euler diagrams in R. BMC Bioinforma. 2011, 12: 35-10.1186/1471-2105-12-35.View ArticleGoogle Scholar
  77. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol. 1990, 215 (3): 403-410.PubMedView ArticleGoogle Scholar
  78. Marchler-Bauer A, Zheng C, Chitsaz F, Derbyshire MK, Geer LY, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Lanczycki CJ, Lu F, Lu S, Marchler GH, Song JS, Thanki N, Yamashita RA, Zhang D, Bryant SH: CDD: conserved domains and protein three-dimensional structure. Nucleic Acids Res. 2013, 41 (Database issue): D348-D352.PubMed CentralPubMedView ArticleGoogle Scholar
  79. Sullivan MJ, Petty NK, Beatson SA: Easyfig: a genome comparison visualizer. Bioinformatics. 2011, 27 (7): 1009-1010. 10.1093/bioinformatics/btr039.PubMed CentralPubMedView ArticleGoogle Scholar
  80. Shao Y, He X, Harrison EM, Tai C, Ou HY, Rajakumar K, Deng Z: mGenomeSubtractor: a web-based tool for parallel in silicosubtractive hybridization analysis of multiple bacterial genomes.Nucleic Acids Res. 2010, 38 (Web Server issue): W194-W200.PubMed CentralPubMedView ArticleGoogle Scholar
  81. Grissa I, Vergnaud G, Pourcel C: CRISPRcompar: a website to compare clustered regularly interspaced short palindromic repeats. Nucleic Acids Res. 2008, 36 (Web Server issue): W145-W148.PubMed CentralPubMedView ArticleGoogle Scholar
  82. Roberts RJ, Vincze T, Posfai J, Macelis D: REBASE–a database for DNA restriction and modification: enzymes, genes and genomes. Nucleic Acids Res. 2010, 38 (Database issue): D234-D236.PubMed CentralPubMedView ArticleGoogle Scholar

Copyright

© Papadimitriou et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.