BACKGROUND
the putative pathogen streptococcus infantarius subsp.
infantarius  is a lactic acid bacterium  commonly associated with the gastrointestinal tract of animals and humans  <cit> .
additionally, sii has been isolated from dairy products, feces , human blood  and human endocarditis   <cit> .
recently, it was identified as the predominant species in several spontaneously fermented african dairy products such as suusac, gariss and fènè <cit>  and in the mexican fermented maize beverage pozol <cit> .
sii belongs to the lancefield group d streptococcus bovis/streptococcus equinus complex  which comprises the species s. bovis, s. equinus, streptococcus lutetiensis , streptococcus gallolyticus subsp.
gallolyticus , streptococcus gallolyticus subsp.
macedonicus, streptococcus gallolyticus subsp.
pasteurianus and streptococcus alactolyticus <cit> .
the sbsec is commonly associated with many infectious diseases such as bacteremia, endocarditis and bloat  <cit> .
moreover, some members of the group, especially s. gallolyticus subsp.
gallolyticus, are suspected to play a role in colonic cancer development  <cit> , partly associated to increasing mrna levels of il- <dig>  il- <dig> and cox- <dig> in colorectal tissue, which contribute to inflammation caused tumor development  <cit> .
because of the high risk association of mainly s. gallolyticus subsp.
gallolyticus with infectious diseases and cancer, research on virulence within the sbsec group has largely focused on this species  <cit> .
virulence factors such as fibrinogen binding factor fimb, glucosyltransferase gtf and pilus subunit b pilb have been identified in several sbsec members  <cit> .
additionally, potential virulence factors such as adhesion proteins have been shown e.g.
the surface protein histone-like protein a , the “adhesion to collagen of the s. bovis group”  and “s.
bovis group surface protein”   <cit> .
however, many of these factors seem to be necessary for survival of sbsec in the gastrointestinal tract and should therefore be considered as niche factors  <cit> .
the pathogenicity of sii is less elucidated.
potential pro-inflammatory proteins were detected in sii and the species is also associated with non-colonic cancer  <cit> .
in parallel to s. gallolyticus subsp.
gallolyticus, a sii strain isolated from feces of an infected baby was able to translocate across a polarized epithelial monolayer of caco- <dig> cells, a property which potentially facilitates infection  <cit> .
this ability was so far only demonstrated for a single sii strain of clinical and not of food origin.
in a recent and broad clinical study on 58 s. bovis strains, only the subspecies s. infantarius subsp.
coli , but not sii, was isolated from blood of infected patients among 29 s. gallolyticus subsp.
gallolyticus and 12 s. gallolyticus subsp.
pasteurianus <cit> .
this suggests only a minor role of sii in infectious diseases.
nevertheless, the predominance of sii in african food fermentations  <cit>  and, as a consequence, the ingestion of high amounts of viable cells of this species by the consumer demands further research to elucidate any potential pathogenic traits of this sbsec member and possibly diverge dairy from clinical isolates.
streptococcus thermophilus is the only streptococcal species recommended by the qualified presumption of safety  for use in fermented food products  <cit> .
it displays an adaptation to the milk environment that is characterized by genome reduction, gene decay and loss of function, which is reflected by the high abundance of pseudogenes in all sequenced s. thermophilus genomes  <cit> .
genome reduction through loss or inactivation of virulence factors and long history of use contributed to the recognition of s. thermophilus by qps, despite its close genetic relationship to the sbsec  <cit> .
interestingly, streptococcus macedonicus aca-dc  <dig> , a greek cheese isolate, displayed comparable genome decay to s. thermophilus and could indicate parallel evolutionary adaptation to the dairy environment in other members of the sbsec and important contributions of certain members of the sbsec to dairy fermentations in europe  <cit> .
the predominance and probably exclusive habitat of the african sii variants in dairy fermentations suggests adaptation to the dairy environment similar to s. thermophilus <cit> .
this predominance seems directly related to the presence of a gal-lac operon in the african variant of sii <cit> , a feature that is absent in other members of the sbsec.
furthermore, african strains display a lactose fermentation pattern paralleling that of s. thermophilus <cit> .
the high prevalence of bacteriocin producers among african sii isolates likely contributes to the predominance of sii in african dairy fermentations  <cit> .
in this work, we present the complete genome sequence of sii cj <dig> isolated as representative predominant strain from spontaneously fermented camel milk suusac from kenya at over 108 cfu ml- <dig>  cj <dig> does not produce bacteriocin-like inhibitory substances  <cit> .
it was selected for genome sequencing due to genetic and metabolic evidence of a lactose fermentation pattern similar to s. thermophilus after studying of  <dig> different african sii isolates  <cit> .
a genomic comparison of strain cj <dig> to other pathogenic and non-pathogenic streptococci was performed in order to identify dairy adaptations and potential virulence factors in cj <dig>  our study provides new insight into streptococcal evolution in the previously untouched ecosystem of dairy fermentations in africa and provides new insight on safety and occurrence of horizontal gene transfer  of streptococci in food fermentations.
RESULTS
general genome properties
the genome of sii cj <dig> consists of a  <dig> ,420-bp circular molecule encoding  <dig> genes of which  <dig> encode for proteins    <cit> .
comparison of genes with their homologues in other streptococcal genomes, resulted in detection of  <dig>  genes that carry a deletion, insertion or premature stop, and that were therefore assigned as pseudogenes.
additionally,  <dig>  bp of plasmid related dna, designated psicj18- <dig>  providing  <dig> coding dna sequences  with only limited similarity to sbsec sequences were detected.
the nucleotide sequence  and g + c mol%-content of  <dig> out of  <dig> cds suggest a lactococcal origin .
a) n/a: not available; b) calculated according to: #pseudogenes/*100; c) the genome of atcc baa-102t was only aligned to that of cj <dig> but not completely assembled.
trna and rrna genes are possibly underestimated in the type strain as genome gaps were not closed.
hmp: human microbiome project http://www.hmpdacc.org.
the origin of the genome of cj <dig> was determined upstream of the dnaa gene and corresponds to the switch in gc-skew .
however, a shift towards the  <dig> o’clock position was detected for the terminus position, as is reflected by a switch in the gc-skew and in the cds-density on the forward and reverse strand , a feature also observed in s. gallolyticus subsp.
gallolyticus atcc  <dig> and s. gallolyticus subsp.
pasteurianus atcc  <dig>  <cit> .
the complete genome sequence was used to confirm the taxonomy of cj <dig> through alignment and subsequent phylogenetic analysis using 16s rrna and eight typical streptococcal genes .
all genes clearly positioned cj <dig> within the sbsec on the same branch as its closest relative sii atcc-baa-102t .
the highest bootstrap percentages were obtained for trees based on groel, recn and secy sequences .
comparison of cj <dig> to atcc baa-102t and other sbsec strains
the draft genome sequence of the sii atcc baa-102t type strain was used for a comparison to the african isolate cj <dig>  an in silico hybridization revealed that the organization of loci was highly conserved between cj <dig> and atcc baa-102t  and to the closely related species s. gallolyticus subsp.
macedonicus and s. gallolyticus subsp.
gallolyticus, albeit at a lesser degree .
the genome of cj <dig> is 37 kb larger than that of atcc baa-102t and harbors a number of variable regions and insertions compared to other streptococci, designated r1-r <dig> .
the major variable regions comprise phage-related proteins , proteins with high sequence identity to s. thermophilus  and a cluster of metabolic and hypothetical proteins specific for cj <dig> .
interestingly, r <dig> comprises many hypothetical proteins shared to the largest extent with the greek cheese isolate s. gallolyticus subsp.
macedonicus aca-dc  <dig>  and second to atcc baa-102t.
this suggests a closer relationship among these sbsec strains compared to the other strains used in genome analysis and might possibly even be related to the dairy origin.
remarkably, variable regions often possess a distinct base-deviation index in cj <dig>  indicating recent evolutionary origin due to little advanced amelioration .
for  <dig> cdss in cj <dig>  no homologous cds were detected in atcc baa-102t.
however, homologous cds were detected in other streptococci for  <dig> of them, whereas for the other  <dig> cds no significant hits were found in related strains  .
the reverse comparison revealed  <dig> cds from atcc baa-102t without orthologous cds in cj <dig>   <dig> of which encoded for hypothetical proteins .
this comparison of the african dairy isolate cj <dig> to the type strain reveals a high similarity in gene content and organisation.
however, there are some remarkable differences in gene content suggesting a distinct evolution of the two strains.
a)hmp human microbiome project http://www.hmpdacc.org.
carbohydrate metabolism
carbohydrate transport in bacteria is frequently mediated via phosphotransferase systems .
pts encoding operons were detected in both sii strains for the uptake of β-glucosides, lactose, fructose/mannose, fructose, sucrose, maltose/glucose and cellobiose.
such a wide variety of transport systems is often observed in gi-tract associated microbes  <cit> .
remarkably, the lactose pts gene locus in cj <dig>  is interrupted by three transposases, two truncating the β-glucoside bgl operon antiterminator upstream of the pts genes and one within the 6-phospho-β-galactosidase downstream of the pts genes, suggesting that the lactose pts in cj <dig> is not involved anymore in lactose utilization.
genes involved in galactose utilization in cj <dig> are organized in the operon galrkte <dig> operon .
however, compared to atcc baa-102t, cj <dig> harbors an additional gal-lac operon comprising genes galt/gale1m/lacsz  with high sequence identity  to s. thermophilus <cit>  and localized in region r <dig> .
also genes in the proximity of this gal-lac operon display high sequence identity to s. thermophilus, comprising among others the putative virulence gene encoding exfoliative toxin b , an acyl-coa dehydrogenase  and a macrophage infectivity potentiator  .
although the high sequence conservation indicates an s. thermophilus origin, the sequential order of genes is only conserved in the gal-lac operon.
mainly non-conserved dna sequences were localized downstream of the gal-lac operon and the truncated galt.
surprisingly, a second lacs  was detected in both sii strains not adjacent to either the gal or gal-lac operon.
this second lacs displays  <dig> % amino acid sequence identity between the two s. infantarius strains and lower identity  to the s. thermophilus-like lacs .
the physiological role of this second lacs is unknown.
to elucidate the role of two lactose transport systems in lactose metabolism of cj <dig>  knock-out  strains were constructed in the lactose translocater lacs , the β-galactosidase lacz  and the permease unit of the lactose pts encoding gene laciic  using a single-cross-over strategy .
phenotypes of ko strains were confirmed on bhi/x-gal/iptg agar media yielding blue colonies for cj18wt , cj18Δlaciic, cj18Δlacs and white colonies for cj18Δlacz.
this indicates no polar effects of lacs disruption on the expression of the lacz gene downstream of lacs .
the wild type cj <dig> and its mutant derivatives cj18Δlaciic, cj18Δlacs and cj18Δlacz grew similarly in control medium containing glucose as sole carbon source .
when grown with lactose as sole carbon source, cj18Δlaciic displayed a similar growth pattern as the wild type cj <dig> , indicating that lactose uptake in cj <dig> is not mediated by the lactose pts.
strains disrupted in genes of the gal-lac operon, cj18Δlacs and cj18Δlacz had clearly an impaired growth rate on lactose .
the growth characteristics of the mutant strains cj18Δlacs and cj18Δlacz on lactose show that lactose is utilized in cj <dig> via uptake by lacs and subsequently cleaved by lacz with a similar mechanism to the lactose metabolism of s. thermophilus.
acmr chloramphenicol resistant; emr erythromycin resistant.
additional features related to dairy environment
oligopeptide transporters are important during growth in milk for the uptake of peptides and amino acids  <cit> .
similar to atcc baa-102t, cj <dig> possesses an oppabcdf peptide transport system  but the genome of cj <dig> encodes two additional oppa  and, remarkably, a second oppabcdf encoding operon  with high sequence identity to streptococcus equi, streptococcus pyogenes or streptococcus gordonii .
single amino acid transport systems are conserved in both strains and in contrast to s. thermophilus strains, no reduction in amino acid biosynthesis pathways was observed for cj <dig>  both s. infantarius strains encode apparent complete pathways, such as histidine and glutamate biosynthesis or arginine catabolism .
capsular polysaccharides  and exopolysaccharides  are involved in the adhesion properties of bacteria through biofilm formation and serve as a defense mechanism against immune responses  <cit> .
furthermore, eps may contribute to the texture of many dairy products.
cj <dig> and atcc baa-102t both possess a conserved 5-kb operon for eps biosynthesis.
the genetic organization downstream of this cluster differs between the two s. infantarius strains.
cj <dig> harbors a number of additional eps and cps biosynthesis genes  that share highest protein sequence identities with proteins of species outside of the sbsec.
remarkably, the same region in cj <dig> contains wefc encoding a receptor polysaccharide phosphotransferase, also termed stealth protein.
this gene is absent in atcc baa-102t and displays high sequence homology to cpsj of s. thermophilus .
based on in silico analysis it was hypothesized to be involved in protection from the host immune system  <cit> .
the presence of a high variety of eps genes could be caused by selection during suusac manufacturing, but could also imply an additional virulence risk if a strain displays further virulence factors for e.g.
invasion, infection or toxin production.
adhesion and other virulence factors
adhesion of bacteria to surfaces is influenced by many factors such as eps or cps production as mentioned above, but also certain specific proteins.
a fibronectin binding protein fpb involved with adhesion to fibronectin and fibrinogen is present in both atcc baa-102t and cj <dig>  streptococcus bovis group surface proteins  are also involved in adhesion and found in both cj <dig>  and in atcc baa-102t .
five of these sbs are organized in a  <dig> -kb region  in cj <dig> comprising a truncated sbs  <dig> , an lpxtg-specific a/c-type sortase , sbs <dig>  and sbs <dig> .
this region upstream of sbs  <dig> is conserved in cj <dig> and atcc baa-102t, the dairy isolate s. gallolyticus subsp.
macedonicus aca-dc  <dig> and other s. gallolyticus strains.
the presence of sbs <dig> and sbs <dig> suggests that certain adhesion factors are shared among sbsec as commensal inhabitants of gastrointestinal tracts and detected also in the dairy strain s. thermophilus lmg <dig>  these factors might only contribute to virulence if further factors for invasion or toxin production are present as well.
other adhesion factors like s. bovis adhesion proteins  or others from non-sbsec origin, such as fima and fimb, are not present in both sii strains.
a hemolysin iii protein highly identical to that of the s. gallolyticus group including s. gallolyticus subsp.
macedonicus aca-dc  <dig>  as well as that of s. thermophilus lmd- <dig>  is encoded in both sii strains.
no defibrinated sheep blood hemolysing activity was detected for both strains.
a direct implication of virulence from the presence of a hemolysin gene except streptolysin o is not yet established for streptococci  <cit> .
typical virulence factors of non-sbsec-members s. pyogenes, s. agalactiae and s. pneumoniae had been used for the safety evaluation of s. thermophilus <cit> .
some of these virulence factors were previously found in s. gallolyticus ucn <dig> such as ssab/scaa/psaa , pilb , gtfbc , atla   <cit>  and used to screen strains in this study.
ssab/scaa/psaa was not detected in sii strains whereas atla displayed a lower protein sequence identity in cj <dig>  compared to the cheese isolate s. gallolyticus subsp.
macedonicus aca-dc  <dig> .
pro-inflammatory proteins  <cit>  were detected in both sii strains but also in s. thermophilus since they encode basic metabolic functions.
finally, comparison with an in silico genome containing antimicrobial resistance and virulence factor genes  <cit>  did not result in significant hits with any typical or concerning streptococcal virulence factors for both cj <dig> and atcc baa-102t.
natural competence
several regions potentially involved in natural competence were detected in both s. infantarius strains.
these include a competence operon , separate competence genes and a coia encoding gene involved in dna uptake.
furthermore, a cj18-unique restriction endonuclease and methylase were detected in region r <dig> .
in addition, both strains contain recombination proteins like reca, the rossman fold nucleotide-binding protein smf/dpra and the single-strand dna binding protein ssbb  <cit> .
the organization and mechanism of the competence-related genes  seems to be conserved in both atcc baa-102t and cj <dig> as well as most other streptococci  <cit> .
however, cj <dig> harbors an additional conjugal transfer protein  with high protein sequence identity  to s. thermophilus variant, suggesting a potentially increased capability for dna uptake compared to atcc baa-102t.
this is further supported by the apparent reduced activity of clustered regularly interspaced short palindromic repeats  and crispr associated genes  forming the crispr/cas system for defense against foreign dna  <cit> .
both cj <dig> and atcc baa-102t harbor single copies of csn <dig>  cas <dig> and cas <dig> in region r <dig> .
but remarkably, the cj <dig> proteins csn <dig>  cas <dig> and cas <dig> had higher identity  with the corresponding proteins in s. gallolyticus, s. bovis and s. equinus than with atcc baa-102t.
a crispr array comprises a leader sequence followed by identical repeated dna sequences intersected by highly variable spacer sequences.
cj <dig> comprises a crispr/cas section with  <dig> spacers whereas atcc baa-102t harbors  <dig> spacers.
the relative low number of crispr spacers predicts a lower crispr activity in cj <dig> and thus a decreased protection against foreign dna.
no dna sequence identity was detected between any of the spacers.
this indicates strain dependent cas/crispr activity in s. infantarius also reported for s. thermophilus strains  <cit> .
other features in the cj <dig> genome
production of bacteriocins is widely distributed among streptococci  <cit> .
s. infantarius cj <dig>  atcc baa-102t and also lp <dig>  possess a highly conserved bacteriocin abc-transporter accessory protein infae-acc, shared also with s. gallolyticus strains  and the bacteriocin abc-transporter infae-abc  located in region r <dig>  putative bacteriocin encoding genes were detected in atcc baa-102t, but none in strain cj <dig>  which confirms previous findings on its inability to produce bacteriocin-like inhibitory substances  <cit> .
unique phage-related genes are located in cj <dig> in regions r <dig> and r <dig> .
cj <dig> and atcc baa102t harbor both four and five phage integrase genes, respectively.
however, only one of them  has a homologous gene in atcc baa-102t , indicating possible distant relationship between these strains.
remarkable differences between both s. infantarius strains and their closest related species within the sbsec s. gallolyticus ucn <dig>  were a reduction in carbohydrate transport systems, e.g.
the absence of trehalose and mannitol transporting and degrading enzymes which play a role in maintenance in the bovine rumen.
this indicates a generally lower adaptation of s. infantarius to the bovine rumen as a habitat compared to s. gallolyticus and provides additional evidence to separate both species from each other.
discussion
fermented dairy products are important in africa as source of nutrients and as weaning food.
fermentation is an essential preservation method in the absence of refrigeration  <cit> .
analyses of dairy adaptations and potential virulence factors of bacteria leading spontaneous fermentation processes is therefore important to identify consumers’ health risk potential and unravel novel fermentative lactic acid bacteria strains.
in this study, we report the complete genome sequence of the african dairy isolate sii cj <dig>  the first complete assembled genome of a s. infantarius species.
whole genome comparison of sii cj <dig> to sii atcc baa-102t and related streptococci revealed substantial adaptations to the dairy environment in cj <dig>  paralleling that of s. thermophilus.
however, our data indicates that genome decay of sii cj <dig> is in a less advanced state compared to s. thermophilus, since most biosynthesis pathways seem to be intact and the number of pseudogenes  is smaller than for s. thermophilus .
this suggests that establishment of cj <dig> in the dairy environment is more recent than s. thermophilus strains or s. gallolyticus subsp.
macedonicus aca-dc  <dig>  based on genome decay, the most recent common ancestor for s. thermophilus strains was estimated to have lived  <dig> - <dig>  years ago, which is approximately the duration of human dairy activity  <cit> .
camels, however, were introduced in east africa only around  <dig>  years ago  <cit> , and the less advanced state of genome decay in cj <dig> may be related to the later start of african camel milk fermentation.
adaptation to the dairy environment in s. thermophilus consists of enhanced uptake of lactose and peptides and loss of other metabolic pathways.
cj <dig> displays a similar adaptation in the lactose metabolism through the transporter lacs and β-galactosidase lacz.
truncation of either lacs or lacz resulted in significant impaired growth on lactose, confirming the functionality of this acquired lactose utilization path.
neither the second lacs , present in both cj <dig> and atcc baa-102t, nor the lactose pts could take over lactose transport in the lacs ko strain.
the integration of transposases in the corresponding lactose pts gene cluster seems therefore a result of loss of essentiality after the acquirement of lacs and lacz.
moreover, a concurrent activity of both transporters potentially leads to misbalance in redox or phosphorylation status of the cell, and hence positive selection on truncation of the lactose pts gene cluster might have even occurred after acquirement of lacsz.
the release of galactose into the growth medium shows that lacs in cj <dig> functions as a highly efficient antiporter and the competitiveness of cj <dig> in the dairy environment seems therefore based on the acquired lacsz.
this facilitates efficient transport of lactose and as a consequence an increased lactose consumption and lactate production compared to atcc baa-102t   <cit> .
the role of other adaptations to the dairy environment, such as the presence of a second oppabcdf operon and an extended eps biosynthesis cluster is less clear.
enhanced uptake of casein derived peptides by the second peptide transporter could contribute to increased competiveness in milk.
the enlarged cluster of eps/cps-related proteins could contribute to survival during the suusac back-slopping process, via improved biofilm formation capabilities.
furthermore, eps contribute to texture of the fermented dairy product and the selection of strains for these textural properties might have occurred in the past  <cit> .
the more recent adaptation to the dairy environment of c <dig> is reflected by the lower number of pseudogenes and crispr spacers in cj <dig> compared to s. thermophilus or s. gallolyticus subsp.
macedonicus aca-dc  <dig>  cj <dig> harbors nine crispr spacers whereas typical widespread dairy starter strains of s. thermophilus such as cnrz  <dig> and lmg  <dig> harbor  <dig> and  <dig> spacers, respectively  <cit> .
phage infection and phage-related fermentation losses are major problems in dairy technology.
the number of crispr spacer in a bacterial genome is directly linked to phage contact history and presumptive resistance against phages of that particular strain  <cit> .
the african strain cj <dig> was apparently not continuously exposed to phage infections over prolonged periods.
this could be a result of the spontaneous nature of the traditional fermentation, which in contrast to industrial starter culture fermentations, does not rely on selected starter strains.
the absence of crispr spacer identity between cj <dig> and atcc baa-102t further shows that the african cj <dig> is only a distant relative of atcc baa-102t as previously observed in microevolution of crispr spacers in other genera  <cit> .
additionally, the presence of  <dig> cds in cj <dig> shared only with other streptococci but not with atcc baa-102t as well as the absence of  <dig> cds in cj <dig> present in atcc baa-102t indicates an ancestral streptococcal origin of these cdss and again only distant relation between the two sii strains.
another interesting feature of cj <dig> is its natural competence and dna uptake capability, paralleling that of other streptococci and lactic acid bacteria   <cit> .
as a possible result of this, the genome displays traces of hgt events from commensal bacteria encountered in milk such as lactococcus spp.
and s. thermophilus but also pathogens like s. agalactiae.
furthermore, the natural competence could potentially contribute to the uptake of mobile genetic elements and to spread of antibiotic resistance genes  <cit> .
therefore the apparent intact competence machinery is probably of high importance for persistence of the strain in the african dairy environment.
cj <dig> harbors none of the concerning typical streptococcal virulence factors  <cit>  and less sbsec-related virulence factors compared to e.g.
s. gallolyticus and s. bovis.
moreover, most of these potential virulence factors are related to adhesion and not directly to infection, cytotoxicity or toxin production and are therefore of less concern.
many factors found in cj <dig> are also present in the proclaimed safe strain s. gallolyticus subsp.
macedonicus aca-dc  <dig>  a species without qps-approval  <cit> .
some potential virulence factors or artifacts thereof were even found in s. thermophilus.
consequently, relying on genomic information alone, ingestion and digestion of large amounts of sii via suusac does not seem to be a direct health risk for adults.
however, the sbsec-associated health risks for immune-deprived people, a major concern in africa, and for children are less understood as epidemiological data on these diseases are not available.
furthermore, the uncertain association of sii with human diseases necessitates further elucidation of presumptive sii-specific virulence factors or the absence thereof in sii.
CONCLUSIONS
we assembled and analyzed the first complete genome sequence of the species s. infantarius.
the african dairy strain sii cj <dig> revealed many genetic adaptations to the dairy environment through acquired carbohydrate utilization pathways resulting in a lactose metabolism paralleling that of s. thermophilus.
potential mutations and insertions resulting in pseudogenes or truncated gene clusters indicate further evolution paralleling s. thermophilus.
however, gene decay is not as advanced as in the dairy isolates s. thermophilus or s. gallolyticus subsp.
macedonicus aca-dc  <dig> and the establishment in the dairy environment is therefore likely from a younger evolutionary period.
the species s. infantarius harbors less virulence factors compared to the s. gallolyticus group.
however, specific virulence factors for s. infantarius are not yet identified and epidemiological studies are necessary to prove the innocuity of african dairy sii strains and milks predominantly fermented with these strains.
this could prove traditional dairy fermentation in africa as ideal process to enhance food safety and shelf life as well as the later application of sii in an enhanced traditional fermentation technology paralleling the western dairy industry, but specific for africa.
conclusively, this study provides insight into the evolution of a novel dairy species and dairy environment in parallel to the western counterpart.

 the putative pathogen streptococcus infantarius subsp. infantarius is a lactic acid bacterium commonly associated with the gastrointestinal tract of animals and humans. additionally, sii has been isolated from dairy products, feces, human blood and human endocarditis. recently, it was identified as the predominant species in several spontaneously fermented african dairy products such as suusac, gariss and fènè and in the mexican fermented maize beverage pozol. sii belongs to the lancefield group d streptococcus bovis/streptococcus equinus complex which comprises the species s. bovis, s. equinus, streptococcus lutetiensis, streptococcus gallolyticus subsp. gallolyticus, streptococcus gallolyticus subsp. macedonicus, streptococcus gallolyticus subsp. pasteurianus and streptococcus alactolyticus. the sbsec is commonly associated with many infectious diseases such as bacteremia, endocarditis and bloat. moreover, some members of the group, especially s. gallolyticus subsp. gallolyticus, are suspected to play a role in colonic cancer development, partly associated to increasing mrna levels of il- il- and cox- in colorectal tissue, which contribute to inflammation caused tumor development. because of the high risk association of mainly s. gallolyticus subsp. gallolyticus with infectious diseases and cancer, research on virulence within the sbsec group has largely focused on this species. virulence factors such as fibrinogen binding factor fimb, glucosyltransferase gtf and pilus subunit b pilb have been identified in several sbsec members. additionally, potential virulence factors such as adhesion proteins have been shown e.g. the surface protein histone-like protein a, the “adhesion to collagen of the s. bovis group” and “s. bovis group surface protein”. however, many of these factors seem to be necessary for survival of sbsec in the gastrointestinal tract and should therefore be considered as niche factors. the pathogenicity of sii is less elucidated. potential pro-inflammatory proteins were detected in sii and the species is also associated with non-colonic cancer. in parallel to s. gallolyticus subsp. gallolyticus, a sii strain isolated from feces of an infected baby was able to translocate across a polarized epithelial monolayer of caco- cells, a property which potentially facilitates infection. this ability was so far only demonstrated for a single sii strain of clinical and not of food origin. in a recent and broad clinical study on 58 s. bovis strains, only the subspecies s. infantarius subsp. coli, but not sii, was isolated from blood of infected patients among 29 s. gallolyticus subsp. gallolyticus and 12 s. gallolyticus subsp. pasteurianus. this suggests only a minor role of sii in infectious diseases. nevertheless, the predominance of sii in african food fermentations and, as a consequence, the ingestion of high amounts of viable cells of this species by the consumer demands further research to elucidate any potential pathogenic traits of this sbsec member and possibly diverge dairy from clinical isolates. streptococcus thermophilus is the only streptococcal species recommended by the qualified presumption of safety for use in fermented food products. it displays an adaptation to the milk environment that is characterized by genome reduction, gene decay and loss of function, which is reflected by the high abundance of pseudogenes in all sequenced s. thermophilus genomes. genome reduction through loss or inactivation of virulence factors and long history of use contributed to the recognition of s. thermophilus by qps, despite its close genetic relationship to the sbsec. interestingly, streptococcus macedonicus aca-dc, a greek cheese isolate, displayed comparable genome decay to s. thermophilus and could indicate parallel evolutionary adaptation to the dairy environment in other members of the sbsec and important contributions of certain members of the sbsec to dairy fermentations in europe. the predominance and probably exclusive habitat of the african sii variants in dairy fermentations suggests adaptation to the dairy environment similar to s. thermophilus. this predominance seems directly related to the presence of a gal-lac operon in the african variant of sii, a feature that is absent in other members of the sbsec. furthermore, african strains display a lactose fermentation pattern paralleling that of s. thermophilus. the high prevalence of bacteriocin producers among african sii isolates likely contributes to the predominance of sii in african dairy fermentations. in this work, we present the complete genome sequence of sii cj isolated as representative predominant strain from spontaneously fermented camel milk suusac from kenya at over 108 cfu ml- cj does not produce bacteriocin-like inhibitory substances. it was selected for genome sequencing due to genetic and metabolic evidence of a lactose fermentation pattern similar to s. thermophilus after studying of different african sii isolates. a genomic comparison of strain cj to other pathogenic and non-pathogenic streptococci was performed in order to identify dairy adaptations and potential virulence factors in cj our study provides new insight into streptococcal evolution in the previously untouched ecosystem of dairy fermentations in africa and provides new insight on safety and occurrence of horizontal gene transfer of streptococci in food fermentations.  general genome properties the genome of sii cj consists of a,420-bp circular molecule encoding genes of which encode for proteins. comparison of genes with their homologues in other streptococcal genomes, resulted in detection of genes that carry a deletion, insertion or premature stop, and that were therefore assigned as pseudogenes. additionally,  bp of plasmid related dna, designated psicj18- providing coding dna sequences with only limited similarity to sbsec sequences were detected. the nucleotide sequence and g + c mol%-content of out of cds suggest a lactococcal origin. a) n/a: not available; b) calculated according to: #pseudogenes/*100; c) the genome of atcc baa-102t was only aligned to that of cj but not completely assembled. trna and rrna genes are possibly underestimated in the type strain as genome gaps were not closed. hmp: human microbiome project http://www.hmpdacc.org. the origin of the genome of cj was determined upstream of the dnaa gene and corresponds to the switch in gc-skew. however, a shift towards the o’clock position was detected for the terminus position, as is reflected by a switch in the gc-skew and in the cds-density on the forward and reverse strand, a feature also observed in s. gallolyticus subsp. gallolyticus atcc and s. gallolyticus subsp. pasteurianus atcc. the complete genome sequence was used to confirm the taxonomy of cj through alignment and subsequent phylogenetic analysis using 16s rrna and eight typical streptococcal genes. all genes clearly positioned cj within the sbsec on the same branch as its closest relative sii atcc-baa-102t. the highest bootstrap percentages were obtained for trees based on groel, recn and secy sequences. comparison of cj to atcc baa-102t and other sbsec strains the draft genome sequence of the sii atcc baa-102t type strain was used for a comparison to the african isolate cj an in silico hybridization revealed that the organization of loci was highly conserved between cj and atcc baa-102t and to the closely related species s. gallolyticus subsp. macedonicus and s. gallolyticus subsp. gallolyticus, albeit at a lesser degree. the genome of cj is 37 kb larger than that of atcc baa-102t and harbors a number of variable regions and insertions compared to other streptococci, designated r1-r. the major variable regions comprise phage-related proteins, proteins with high sequence identity to s. thermophilus and a cluster of metabolic and hypothetical proteins specific for cj. interestingly, r comprises many hypothetical proteins shared to the largest extent with the greek cheese isolate s. gallolyticus subsp. macedonicus aca-dc and second to atcc baa-102t. this suggests a closer relationship among these sbsec strains compared to the other strains used in genome analysis and might possibly even be related to the dairy origin. remarkably, variable regions often possess a distinct base-deviation index in cj indicating recent evolutionary origin due to little advanced amelioration. for cdss in cj no homologous cds were detected in atcc baa-102t. however, homologous cds were detected in other streptococci for of them, whereas for the other cds no significant hits were found in related strains. the reverse comparison revealed cds from atcc baa-102t without orthologous cds in cj of which encoded for hypothetical proteins. this comparison of the african dairy isolate cj to the type strain reveals a high similarity in gene content and organisation. however, there are some remarkable differences in gene content suggesting a distinct evolution of the two strains. a)hmp human microbiome project http://www.hmpdacc.org. carbohydrate metabolism carbohydrate transport in bacteria is frequently mediated via phosphotransferase systems. pts encoding operons were detected in both sii strains for the uptake of β-glucosides, lactose, fructose/mannose, fructose, sucrose, maltose/glucose and cellobiose. such a wide variety of transport systems is often observed in gi-tract associated microbes. remarkably, the lactose pts gene locus in cj is interrupted by three transposases, two truncating the β-glucoside bgl operon antiterminator upstream of the pts genes and one within the 6-phospho-β-galactosidase downstream of the pts genes, suggesting that the lactose pts in cj is not involved anymore in lactose utilization. genes involved in galactose utilization in cj are organized in the operon galrkte operon. however, compared to atcc baa-102t, cj harbors an additional gal-lac operon comprising genes galt/gale1m/lacsz with high sequence identity to s. thermophilus and localized in region r. also genes in the proximity of this gal-lac operon display high sequence identity to s. thermophilus, comprising among others the putative virulence gene encoding exfoliative toxin b, an acyl-coa dehydrogenase and a macrophage infectivity potentiator. although the high sequence conservation indicates an s. thermophilus origin, the sequential order of genes is only conserved in the gal-lac operon. mainly non-conserved dna sequences were localized downstream of the gal-lac operon and the truncated galt. surprisingly, a second lacs was detected in both sii strains not adjacent to either the gal or gal-lac operon. this second lacs displays % amino acid sequence identity between the two s. infantarius strains and lower identity to the s. thermophilus-like lacs. the physiological role of this second lacs is unknown. to elucidate the role of two lactose transport systems in lactose metabolism of cj knock-out strains were constructed in the lactose translocater lacs, the β-galactosidase lacz and the permease unit of the lactose pts encoding gene laciic using a single-cross-over strategy. phenotypes of ko strains were confirmed on bhi/x-gal/iptg agar media yielding blue colonies for cj18wt, cj18Δlaciic, cj18Δlacs and white colonies for cj18Δlacz. this indicates no polar effects of lacs disruption on the expression of the lacz gene downstream of lacs. the wild type cj and its mutant derivatives cj18Δlaciic, cj18Δlacs and cj18Δlacz grew similarly in control medium containing glucose as sole carbon source. when grown with lactose as sole carbon source, cj18Δlaciic displayed a similar growth pattern as the wild type cj, indicating that lactose uptake in cj is not mediated by the lactose pts. strains disrupted in genes of the gal-lac operon, cj18Δlacs and cj18Δlacz had clearly an impaired growth rate on lactose. the growth characteristics of the mutant strains cj18Δlacs and cj18Δlacz on lactose show that lactose is utilized in cj via uptake by lacs and subsequently cleaved by lacz with a similar mechanism to the lactose metabolism of s. thermophilus. acmr chloramphenicol resistant; emr erythromycin resistant. additional features related to dairy environment oligopeptide transporters are important during growth in milk for the uptake of peptides and amino acids. similar to atcc baa-102t, cj possesses an oppabcdf peptide transport system but the genome of cj encodes two additional oppa and, remarkably, a second oppabcdf encoding operon with high sequence identity to streptococcus equi, streptococcus pyogenes or streptococcus gordonii. single amino acid transport systems are conserved in both strains and in contrast to s. thermophilus strains, no reduction in amino acid biosynthesis pathways was observed for cj both s. infantarius strains encode apparent complete pathways, such as histidine and glutamate biosynthesis or arginine catabolism. capsular polysaccharides and exopolysaccharides are involved in the adhesion properties of bacteria through biofilm formation and serve as a defense mechanism against immune responses. furthermore, eps may contribute to the texture of many dairy products. cj and atcc baa-102t both possess a conserved 5-kb operon for eps biosynthesis. the genetic organization downstream of this cluster differs between the two s. infantarius strains. cj harbors a number of additional eps and cps biosynthesis genes that share highest protein sequence identities with proteins of species outside of the sbsec. remarkably, the same region in cj contains wefc encoding a receptor polysaccharide phosphotransferase, also termed stealth protein. this gene is absent in atcc baa-102t and displays high sequence homology to cpsj of s. thermophilus. based on in silico analysis it was hypothesized to be involved in protection from the host immune system. the presence of a high variety of eps genes could be caused by selection during suusac manufacturing, but could also imply an additional virulence risk if a strain displays further virulence factors for e.g. invasion, infection or toxin production. adhesion and other virulence factors adhesion of bacteria to surfaces is influenced by many factors such as eps or cps production as mentioned above, but also certain specific proteins. a fibronectin binding protein fpb involved with adhesion to fibronectin and fibrinogen is present in both atcc baa-102t and cj streptococcus bovis group surface proteins are also involved in adhesion and found in both cj and in atcc baa-102t. five of these sbs are organized in a -kb region in cj comprising a truncated sbs, an lpxtg-specific a/c-type sortase, sbs and sbs. this region upstream of sbs is conserved in cj and atcc baa-102t, the dairy isolate s. gallolyticus subsp. macedonicus aca-dc and other s. gallolyticus strains. the presence of sbs and sbs suggests that certain adhesion factors are shared among sbsec as commensal inhabitants of gastrointestinal tracts and detected also in the dairy strain s. thermophilus lmg these factors might only contribute to virulence if further factors for invasion or toxin production are present as well. other adhesion factors like s. bovis adhesion proteins or others from non-sbsec origin, such as fima and fimb, are not present in both sii strains. a hemolysin iii protein highly identical to that of the s. gallolyticus group including s. gallolyticus subsp. macedonicus aca-dc as well as that of s. thermophilus lmd- is encoded in both sii strains. no defibrinated sheep blood hemolysing activity was detected for both strains. a direct implication of virulence from the presence of a hemolysin gene except streptolysin o is not yet established for streptococci. typical virulence factors of non-sbsec-members s. pyogenes, s. agalactiae and s. pneumoniae had been used for the safety evaluation of s. thermophilus. some of these virulence factors were previously found in s. gallolyticus ucn such as ssab/scaa/psaa, pilb, gtfbc, atla and used to screen strains in this study. ssab/scaa/psaa was not detected in sii strains whereas atla displayed a lower protein sequence identity in cj compared to the cheese isolate s. gallolyticus subsp. macedonicus aca-dc. pro-inflammatory proteins were detected in both sii strains but also in s. thermophilus since they encode basic metabolic functions. finally, comparison with an in silico genome containing antimicrobial resistance and virulence factor genes did not result in significant hits with any typical or concerning streptococcal virulence factors for both cj and atcc baa-102t. natural competence several regions potentially involved in natural competence were detected in both s. infantarius strains. these include a competence operon, separate competence genes and a coia encoding gene involved in dna uptake. furthermore, a cj18-unique restriction endonuclease and methylase were detected in region r. in addition, both strains contain recombination proteins like reca, the rossman fold nucleotide-binding protein smf/dpra and the single-strand dna binding protein ssbb. the organization and mechanism of the competence-related genes seems to be conserved in both atcc baa-102t and cj as well as most other streptococci. however, cj harbors an additional conjugal transfer protein with high protein sequence identity to s. thermophilus variant, suggesting a potentially increased capability for dna uptake compared to atcc baa-102t. this is further supported by the apparent reduced activity of clustered regularly interspaced short palindromic repeats and crispr associated genes forming the crispr/cas system for defense against foreign dna. both cj and atcc baa-102t harbor single copies of csn cas and cas in region r. but remarkably, the cj proteins csn cas and cas had higher identity with the corresponding proteins in s. gallolyticus, s. bovis and s. equinus than with atcc baa-102t. a crispr array comprises a leader sequence followed by identical repeated dna sequences intersected by highly variable spacer sequences. cj comprises a crispr/cas section with spacers whereas atcc baa-102t harbors spacers. the relative low number of crispr spacers predicts a lower crispr activity in cj and thus a decreased protection against foreign dna. no dna sequence identity was detected between any of the spacers. this indicates strain dependent cas/crispr activity in s. infantarius also reported for s. thermophilus strains. other features in the cj genome production of bacteriocins is widely distributed among streptococci. s. infantarius cj atcc baa-102t and also lp possess a highly conserved bacteriocin abc-transporter accessory protein infae-acc, shared also with s. gallolyticus strains and the bacteriocin abc-transporter infae-abc located in region r putative bacteriocin encoding genes were detected in atcc baa-102t, but none in strain cj which confirms previous findings on its inability to produce bacteriocin-like inhibitory substances. unique phage-related genes are located in cj in regions r and r. cj and atcc baa102t harbor both four and five phage integrase genes, respectively. however, only one of them has a homologous gene in atcc baa-102t, indicating possible distant relationship between these strains. remarkable differences between both s. infantarius strains and their closest related species within the sbsec s. gallolyticus ucn were a reduction in carbohydrate transport systems, e.g. the absence of trehalose and mannitol transporting and degrading enzymes which play a role in maintenance in the bovine rumen. this indicates a generally lower adaptation of s. infantarius to the bovine rumen as a habitat compared to s. gallolyticus and provides additional evidence to separate both species from each other. discussion fermented dairy products are important in africa as source of nutrients and as weaning food. fermentation is an essential preservation method in the absence of refrigeration. analyses of dairy adaptations and potential virulence factors of bacteria leading spontaneous fermentation processes is therefore important to identify consumers’ health risk potential and unravel novel fermentative lactic acid bacteria strains. in this study, we report the complete genome sequence of the african dairy isolate sii cj the first complete assembled genome of a s. infantarius species. whole genome comparison of sii cj to sii atcc baa-102t and related streptococci revealed substantial adaptations to the dairy environment in cj paralleling that of s. thermophilus. however, our data indicates that genome decay of sii cj is in a less advanced state compared to s. thermophilus, since most biosynthesis pathways seem to be intact and the number of pseudogenes is smaller than for s. thermophilus. this suggests that establishment of cj in the dairy environment is more recent than s. thermophilus strains or s. gallolyticus subsp. macedonicus aca-dc based on genome decay, the most recent common ancestor for s. thermophilus strains was estimated to have lived -  years ago, which is approximately the duration of human dairy activity. camels, however, were introduced in east africa only around  years ago, and the less advanced state of genome decay in cj may be related to the later start of african camel milk fermentation. adaptation to the dairy environment in s. thermophilus consists of enhanced uptake of lactose and peptides and loss of other metabolic pathways. cj displays a similar adaptation in the lactose metabolism through the transporter lacs and β-galactosidase lacz. truncation of either lacs or lacz resulted in significant impaired growth on lactose, confirming the functionality of this acquired lactose utilization path. neither the second lacs, present in both cj and atcc baa-102t, nor the lactose pts could take over lactose transport in the lacs ko strain. the integration of transposases in the corresponding lactose pts gene cluster seems therefore a result of loss of essentiality after the acquirement of lacs and lacz. moreover, a concurrent activity of both transporters potentially leads to misbalance in redox or phosphorylation status of the cell, and hence positive selection on truncation of the lactose pts gene cluster might have even occurred after acquirement of lacsz. the release of galactose into the growth medium shows that lacs in cj functions as a highly efficient antiporter and the competitiveness of cj in the dairy environment seems therefore based on the acquired lacsz. this facilitates efficient transport of lactose and as a consequence an increased lactose consumption and lactate production compared to atcc baa-102t. the role of other adaptations to the dairy environment, such as the presence of a second oppabcdf operon and an extended eps biosynthesis cluster is less clear. enhanced uptake of casein derived peptides by the second peptide transporter could contribute to increased competiveness in milk. the enlarged cluster of eps/cps-related proteins could contribute to survival during the suusac back-slopping process, via improved biofilm formation capabilities. furthermore, eps contribute to texture of the fermented dairy product and the selection of strains for these textural properties might have occurred in the past. the more recent adaptation to the dairy environment of c is reflected by the lower number of pseudogenes and crispr spacers in cj compared to s. thermophilus or s. gallolyticus subsp. macedonicus aca-dc cj harbors nine crispr spacers whereas typical widespread dairy starter strains of s. thermophilus such as cnrz and lmg harbor and spacers, respectively. phage infection and phage-related fermentation losses are major problems in dairy technology. the number of crispr spacer in a bacterial genome is directly linked to phage contact history and presumptive resistance against phages of that particular strain. the african strain cj was apparently not continuously exposed to phage infections over prolonged periods. this could be a result of the spontaneous nature of the traditional fermentation, which in contrast to industrial starter culture fermentations, does not rely on selected starter strains. the absence of crispr spacer identity between cj and atcc baa-102t further shows that the african cj is only a distant relative of atcc baa-102t as previously observed in microevolution of crispr spacers in other genera. additionally, the presence of cds in cj shared only with other streptococci but not with atcc baa-102t as well as the absence of cds in cj present in atcc baa-102t indicates an ancestral streptococcal origin of these cdss and again only distant relation between the two sii strains. another interesting feature of cj is its natural competence and dna uptake capability, paralleling that of other streptococci and lactic acid bacteria. as a possible result of this, the genome displays traces of hgt events from commensal bacteria encountered in milk such as lactococcus spp. and s. thermophilus but also pathogens like s. agalactiae. furthermore, the natural competence could potentially contribute to the uptake of mobile genetic elements and to spread of antibiotic resistance genes. therefore the apparent intact competence machinery is probably of high importance for persistence of the strain in the african dairy environment. cj harbors none of the concerning typical streptococcal virulence factors and less sbsec-related virulence factors compared to e.g. s. gallolyticus and s. bovis. moreover, most of these potential virulence factors are related to adhesion and not directly to infection, cytotoxicity or toxin production and are therefore of less concern. many factors found in cj are also present in the proclaimed safe strain s. gallolyticus subsp. macedonicus aca-dc a species without qps-approval. some potential virulence factors or artifacts thereof were even found in s. thermophilus. consequently, relying on genomic information alone, ingestion and digestion of large amounts of sii via suusac does not seem to be a direct health risk for adults. however, the sbsec-associated health risks for immune-deprived people, a major concern in africa, and for children are less understood as epidemiological data on these diseases are not available. furthermore, the uncertain association of sii with human diseases necessitates further elucidation of presumptive sii-specific virulence factors or the absence thereof in sii.  we assembled and analyzed the first complete genome sequence of the species s. infantarius. the african dairy strain sii cj revealed many genetic adaptations to the dairy environment through acquired carbohydrate utilization pathways resulting in a lactose metabolism paralleling that of s. thermophilus. potential mutations and insertions resulting in pseudogenes or truncated gene clusters indicate further evolution paralleling s. thermophilus. however, gene decay is not as advanced as in the dairy isolates s. thermophilus or s. gallolyticus subsp. macedonicus aca-dc and the establishment in the dairy environment is therefore likely from a younger evolutionary period. the species s. infantarius harbors less virulence factors compared to the s. gallolyticus group. however, specific virulence factors for s. infantarius are not yet identified and epidemiological studies are necessary to prove the innocuity of african dairy sii strains and milks predominantly fermented with these strains. this could prove traditional dairy fermentation in africa as ideal process to enhance food safety and shelf life as well as the later application of sii in an enhanced traditional fermentation technology paralleling the western dairy industry, but specific for africa. conclusively, this study provides insight into the evolution of a novel dairy species and dairy environment in parallel to the western counterpart. 
the genome sequence of sii strain cj was compared with that of other streptococcus species to identify dairy adaptations including genome decay such as in streptococcus thermophilus, traits for its competitiveness in spontaneous milk fermentation and to assess potential health risks for consumers. dairy adaptations in cj are reflected by a high percentage of pseudogenes representing genome decay which includes the inactivation of the lactose phosphotransferase system by multiple transposases integration. natural competence genes are conserved in both sii strains, but cj contains a lower number of crispr spacers which indicates a reduced defense capability against alien dna. no classical streptococcal virulence factors were detected in both sii strains apart from those involved in adhesion which should be considered niche factors. metabolic adaptations to the dairy environment are evident and especially lactose uptake corresponds to s. thermophilus. genome decay is not as advanced as in s. thermophilus possibly due to a shorter history in dairy fermentations. the presence of lacs and lacz genes is the major dairy adaptation affecting lactose metabolism pathways also due to the disruption of laciiabc. we constructed mutant strains of lacs, lacz and laciiabc and analyzed the resulting strains of cj to confirm the redirection of lactose metabolism via lacs and lacz. macedonicusdairy fermentationlactose metabolismafricacamelhealth riskstreptococcus virulence factors the genome of sii cj harbors several unique regions in comparison to sii atcc baa-102t, among others an enlarged exo- and capsular polysaccharide operon; streptococcus thermophilus-associated genes; a region containing metabolic and hypothetical genes mostly unique to cj and the dairy isolate streptococcus gallolyticus subsp. streptococcus infantariusstreptococcus bovis/streptococcus equinus complexstreptococcus thermophilusstreptococcus gallolyticus subsp.
 streptococcus infantarius subsp. infantarius belongs to the streptococcus bovis/streptococcus equinus complex associated with several human and animal infections. sii is a predominant bacterium in spontaneously fermented milk products in africa. the genome sequence of sii strain cj was compared with that of other streptococcus species to identify dairy adaptations including genome decay such as in streptococcus thermophilus, traits for its competitiveness in spontaneous milk fermentation and to assess potential health risks for consumers.  the genome of sii cj harbors several unique regions in comparison to sii atcc baa-102t, among others an enlarged exo- and capsular polysaccharide operon; streptococcus thermophilus-associated genes; a region containing metabolic and hypothetical genes mostly unique to cj and the dairy isolate streptococcus gallolyticus subsp. macedonicus; and a second oligopeptide transport operon. dairy adaptations in cj are reflected by a high percentage of pseudogenes representing genome decay which includes the inactivation of the lactose phosphotransferase system by multiple transposases integration. the presence of lacs and lacz genes is the major dairy adaptation affecting lactose metabolism pathways also due to the disruption of laciiabc. we constructed mutant strains of lacs, lacz and laciiabc and analyzed the resulting strains of cj to confirm the redirection of lactose metabolism via lacs and lacz. natural competence genes are conserved in both sii strains, but cj contains a lower number of crispr spacers which indicates a reduced defense capability against alien dna. no classical streptococcal virulence factors were detected in both sii strains apart from those involved in adhesion which should be considered niche factors. sii-specific virulence factors are not described. several sii-specific regions encoding uncharacterized proteins provide new leads for virulence analyses and investigation of the unclear association of dairy and clinical sii with human diseases.  the genome of the african dairy isolate sii cj clearly differs from the human isolate atcc baa-102t. cj possesses a high natural competence predisposition likely explaining the enlarged genome. metabolic adaptations to the dairy environment are evident and especially lactose uptake corresponds to s. thermophilus. genome decay is not as advanced as in s. thermophilus possibly due to a shorter history in dairy fermentations. streptococcus infantariusstreptococcus bovis/streptococcus equinus complexstreptococcus thermophilusstreptococcus gallolyticus subsp. macedonicusdairy fermentationlactose metabolismafricacamelhealth riskstreptococcus virulence factors 