Plasmids
Comparative RAST analysis of the draft assemblies with that of the virulence plasmid, pESA3 (131,196 bp in size [37]), shown in Additional file 4: Table S4, revealed the presence of coding sequences for the predicted alleles of the pESA3-like, RepFIB virulence plasmid originally described by Franco et al. [43]. pESA3-like plasmids contain a common backbone set of alleles represented by the plasmid origin of replication gene, repA, an ABC iron transporter gene cluster (identified by the presence of eitA) and a Cronobactin (an aerobactin-like siderophore) gene cluster (identified by the presence of iucC). Prototypical
C. sakazakii
strain BAA-894 also possesses plasmidborne gene sequences for a
Cronobacter
plasminogen activator gene (cpa), genes encoding an ~ 17-kbp type six secretion system (T6SS) and, in approx. 20% of
C. sakazakii
strains (however, not found in BAA-894), possess genes of the ~ 27-kbp gene filamentous hemagglutinin (FHA) gene cluster represented by the presence of fhaB [44, 43]. Interestingly, results of PCR analysis of the strains reported in the present study, shown in Table 6, revealed that all of the strains were PCR-positive for repA, cpa, eitA, and iucC. All of the strains were also PCR-positive for the T6SS’s IntLeft (IntL) gene locus, but only seven, 11, and three of the strains were PCR-positive for the other three T6SS alleles (vgrG, R end, IntR). These results suggest that the T6SS gene cluster is highly variable in these strains, similar to what Franco et al. [43] and Yan et al. [45, 46] had previously reported. In addition, six of the strains were PCR-positive for fhaB, signifying that these strains possess the FHA gene cluster. Only one of the strains was PCR-positive for pESA2-like plasmids, while five of the strains were PCR-positive for the
C. turicensis
-like pCTU3 plasmid which was identified by Stephan et al. [47]. RAST analysis was used to determine if any of the 26 plant-origin strains harbored the small cryptic CSK29544_2p-like plasmid which has been found in other
C. sakazakii
strains such as
C. sakazakii
strain SP291 (CSK29544_2p is homologous to pSP291–3), a highly persistent environmental strain found associated with an Irish PIF manufacturing facility [45, 46]. According to the
C. sakazakii
NCBI website (https://www.ncbi.nlm.nih.gov/genome/genomes/1170?), the species type strain,
C. sakazakii
29544T harbors three plasmids CSK29544_1p (pESA3-like virulence plasmid, 93,905 bp in size), CSK29544_2p (a small cryptic plasmid, 4938 bp in size), and CSK29544_3p (a pESA2- like conjugative plasmid, 53,457 bp in size). CSK29544_2p contains five genes encoding for a methyl-accepting chemotaxis protein, a hypothetical protein and a plasmid mobilization relaxosome protein cluster, MobCABD. Our analysis showed that none of the strains harbored this plasmid (data not shown).
Table 6 Prevalence and distribution of pESA3 alleles associated with the virulence plasmid and pESA2/pCTU3 plasmids harbored by 26 spice-associated C. sakazakii isolates Chromosomal traits
Next generation genome sequencing of the different
Cronobacter
species revealed a species-level bidirectional divergence which is hypothesized to be driven by niche adaptation [35]. Figure 2 illustrates this phylogenetic divergence, using the kSNP3 tool [48], of the strains reported in this study with representative strains of each species. The phylogeny among these strains followed similar sequence type evolutionary lineages which were reported by Chase et al. [36] and Gopinath et al. [18]. Furthermore,
Cronobacter
possess a diversity of remarkable features which support the organism’s capability to survive under severe environmental growth conditions such as xerotolerant econiches confined to the production of dried foods, such as PIF [35, 29, 30]. The physiological mechanisms of desiccation survival are thought to involve both primary and secondary desiccation responses; and involve the efflux of various sugars such as trehalose and other osmoprotectants [29, 30]. Genomically, several genes involved in osmotic responses were found within these spice-associated strains; furthermore, these genes were shown by Srikumar et al. [30] to be transcriptionally highly up-regulated in
C. sakazakii
cells grown under xerotolerant growth conditions. For example, DnaJ and DnaK, (Additional file 3: Table S3) in strain MOD1_O23mB, represented by locus tags: C5975_08705 and C5975_08710 are two co- expressed chaperone proteins which are classified in COG O and were found in all of the strains analyzed in this study. DnaJ participates actively in the response to hyperosmotic and heat shock by preventing the aggregation of stress-denatured proteins and acts in association with DnaK and GrpE (locus tag C5975_09365). DnaJ is considered to be the nucleotide exchange factor for DnaK and may function as a thermosensor. Unfolded proteins bind initially to DnaJ. It is also hypothesized that DnaJ, DnaK, and GrpE act together in the replication of plasmids through activation of initiation proteins. Another protein, Aquaporin Z (classified in COG M, represented here as an example in strain MOD1_O23mB (locus tag: C5975_14540) Additional file 3: Table S3), was found in all strains and is a porin-like channel protein that permits osmotically driven movement of water in both directions. It is thought to be involved in osmoregulation and in the maintenance of cell turgor pressure during volume expansion in rapidly growing cells. It is thought that Aquaporin Z opens in response to the stretch forces in the membrane lipid bilayer and that it may also participate in the regulation of osmotic pressure changes within the cell during osmotic stress. Thus, Aquaporin Z mediates rapid entry or exit of water in response to abrupt changes in osmolarity. Aquaporin Z is also a member of the major intrinsic protein (MIP) superfamily which functions primarily as water-selective membrane channels that transport water, small neutral molecules, and ions out of and between cells. Still another protein, ProQ (as example, locus C5975_18900 in strain MOD1_O23mB in Additional file 3: Table S3), is classified in COG T; and is a protein that is a structural element that influences the osmotic activation of the proline/betaine transporter ProP at a post-translational level. It also acts as a proton symporter that senses osmotic shifts and responds by importing osmolytes such as proline, glycine betaine, stachydrine, pipecolic acid, ectoine and taurine into the cell. ProP is thought to have a dual role in that it serves the cell as both an osmosensor and an osmoregulator which is available to participate in the bacterial osmoregulatory response [29, 30]. The channel opens in response to the stretch forces in the membrane lipid bilayer and may also participate in the regulation of osmotic pressure changes within the cell. Other proteins such a TreF (an alpha, alpha-trehalase, MOD1_O23mB locus C5975_10755, COG G, Additional file 3: Table S3) was found and is thought to provide cells with the ability to utilize trehalose under high osmolarity growth conditions by splitting it into glucose molecules that can subsequently be taken up by the phosphotransferase-mediated uptake system. Another set of proteins encoded by the mdoHGC operon (COG P, MOD1_O23mB locus C5975_17925, C5975_17930, C5975_17940 in Additional file 3: Table S3), which is involved in the biosynthesis of osmoregulated periplasmic glucans (OPGs), was found to be highly up-regulated in
C. sakazakii
grown under xerotolerant growth conditions [30]. The roles of the OPGs are complex and vary considerably among bacteria, but OPGs are thought to be a part of a signal transduction pathway(s) and are thought to indirectly regulate genes involved in virulence. The total number of OPGs increases when the osmolarity growth conditions decreases [49]. In general, EggNOG analysis identified 10 proteins per strain that were involved in the osmotolerance response. Another group of chaperone-like proteins which these
C. sakazakii
strains possessed are also annotated as heat shock proteins, and consist of IbpA (C5975_06750), DiaA (C5975_07735), and HtpX (C5975_18890), and Hsp15 (C5975_00700, COG M). There were in general between 11 and 17 heat shock-related proteins found by EggNOG analysis. Other sets of proteins found associated with these strains include 22–27 fimbriae proteins, however no curli proteins were found. There were 23–28 different efflux pump-associated proteins including proteins involved with the efflux or transport of threonine, homoserine lactone (locus tag C5975_00275), p-hydroxybenzoic acid (locus tag C5975_07280), glutathione-regulated potassium (locus tag C5975_00475, C5975_00480, C5975_08855, C5975_08860, KefGFCB), RND efflux (C5975_02520, Transporter), proteins associated with heavy metal efflux of nickel/cobalt (C5975_13445, RcnB), cobalt/magnesium (C5975_08880, ApaG), and manganese ions (C5975_18840, MntP), sugar efflux (C5975_13720, SetB), and multidrug resistance (MdtA, MdtH, MdtD). There were on average 5–13, 1–10, 15–20 proteins that were annotated as integrases, transposases, and recombinase-like proteins, respectively. All of these genes have been observed in other
C. sakazakii
genomes [16, 18, 19]. Interestingly, there was a large difference (11–63) in the number of phage-associated proteins among the strains. For example
C. sakazakii
strain Jor96 possessed phage proteins annotated for lambda, GP49-like, P2, Mu, and cp-933 k phages. Lastly there was also a wide difference in the number of both toxin-antitoxin type I and type II toxin-antitoxin family proteins found among the genomes; examples include type I toxin-antitoxin system hok family toxin and type II toxin- antitoxin systems such as RelE/ParE, RelE/DinJ, and HipA families.
Among the spice-associated
C. sakazakii
strains, 4 to 7 hemolysin- related proteins were identified. For example
C. sakazakii
strain MOD1_Jor93 possessed six alleles encoding for hemolysin-related proteins, such as four COG category U (intracellular trafficking and secretion) genes. A hemolysin secretion/activation protein homologous to the ShlB/FhaC/HecB family of alleles was found in MOD1_Jor93 (C5940_08565, Additional file 3: Table S3). This Pfam annotated allele shares homology with a group of sequences that are related to ShlB from
Serratia
marcescens
[50]. It is hypothesized that ShlB is an outer membrane protein possibly involved in either a Type V or a two-partner secretion system where it functions to secrete and activate a ShlA type hemolysin. The activation of ShlA is thought to occur during secretion when ShlB imposes a conformational change in the inactive hemolysin to form the active protein. Though ShlA was not found in MOD1_Jor93, this protein was found in MOD1_Jor20 (C5932_21600).
There were three proteins defined as COG category S (function unknown) which included a hemolysin expression modulating protein, a putative hemolysin, and COG1272, a predicted membrane hemolysin III which Cruz et al. previously described [51].
Other virulence-related proteins included MsgA (analogous with a DNA damage- inducible protein, DinI family protein). Every genome possessed genes for this protein. The same protein is found in Salmonella enterica subsp. enterica. It is thought that MsgA in
Salmonella
is required for intramacrophage survival and seems to be independent of the PhoP regulon [52]. Other virulence factor-like proteins found were ImpE and SrfB [46].
Xylose and arabinose account for more than 30% of the total sugars in agricultural residues and in fact, Xylose is the second most abundant sugar in nature besides glucose and primarily exists as D-xylose [53]. However, it is usually found as a polymeric component of plant cell wall matrix polysaccharides such as xylans, e.g., arabinoxylans, hemicellulose (xylan, glucuronoxylan), and xyloglucan [53]. Complex interactions are thought to exist between human pathogens and a plant’s indigenous microflora, including phytopathogens, which are associated with fresh produce [53].
Xanthomonas
pathogens such as
X. campestris
pathovars cause diseases of agronomic importance throughout the world; examples include black rot disease in crucifers such as cauliflower, cabbage, garden cress, bok choy, broccoli, and brussel sprouts; and in fact these pathovars can affect all cultivated brassicas. Also,
X. campestris
pv. vesicatoria (now reclassified as
X. euvesicatoria
), causes bacterial spot disease on pepper and tomato plants, and
X. campestris
pv. malvacearum (now
X. axonopodis
pv. malvacearum), causes angular leaf spot of cotton [54, 55]. These phytopathogens possess a number of plant cell wall-degrading enzymes (as part of the carbohydrate utilization with TonB-dependent outer membrane transporter system regulon, CUT), which are secreted by a type II secretion system (T2SS) and are required for virulence and pathogenesis. These pathogens also possess two major xylanase-related genes, xynA and xynB, which could influence biofilm formation and virulence by weakening the plant cell wall structure through degradation causing the release of nutrients during plant colonization [54]. A xylanolytic-like system, ubiquitous in lignocellulose-degrading bacteria, is also found in E. coli [56], and thought to play important roles in biofilm formation, nutrient uptake and adaptation of these
Proteobacteria
to the plant phyllosphere [56]. Functional metagenomic findings reported by Carter et al. [57] and transcriptional analyses suggest that E. coli O157:H7 competes with spinach indigenous microflora for essential macronutrients which is thought to lead to its ability to contaminate spinach [57, 58].
A xylose utilization operon (average size of ~ 16,771 bp; 11 genes) which possessed a G + C content of 54.9%, was found among the spice-associated
C. sakazakii
strains. A map of the operon for
C. sakazakii
strain MOD1_AS15 is shown in Fig. 3a. The operon consists of the following genes: xylA (xylose isomerase, locus tag C5965_02230), xylB (xylulose kinase, locus tag C5965_02235), xylF (D-xylose ABC transporter substrate binding protein, locus tag C5965_02225), xylG (xylose ABC transporter ATP binding protein, locus tag C5965_02220), xylH (a sugar ABC transporter permease, locus tag C5965_02215), which is part of the ABC transporter complex XylFGH. This latter complex is involved in D-xylose uptake, xylR (an AraC-like xylose operon transcription regulator, locus tag C5965_02210), bax (an ATP- ribonucleoside binding protein, locus tag C5965_02205), an α-amylase gene (amy1, locus tag C5965_02200), a valine-pyruvate transaminase gene (avtA, locus tag C5965_02195), xylS (an α- xylosidase gene, locus tag C5965_02190), and a proposed α-xynT (glycoside-pentoside- heuronide family transporter, locus tag C5965_02185). Outside of the xylose utilization operon are other xyloside uptake genes and genes encoding degradation enzymes, such as a second xynT (a proposed β-xynT, locus tag C5965_04340), xynB (a β-xylosidase, locus tag C5965_04335), and xylE (a proton-sugar symporter (locus tag C5965_09300). This shares significant homology with xylE of E.coil, which is a member of the major facilitator superfamily (MFS) of transporters) possessed by E. coli and other bacteria [56]. The genomic structure of the
Cronobacter
xylose utilization operon was similar to that found in E. coli strain K-12 (strain MG1655; GenBank assembly accession: GCA_000005845; RefSeq assembly accession:GCF_000005845) except that two genes present in the
Cronobacter
xylose operon, xylS and α- xynT are missing from within the operon in E. coli strain MG1655 which resulted in ~ 13,041 bp sized operon. Additionally, there was a size difference (ranging from 16,340 to 16,790 bp) observed among the operons possessed by the twenty-six
C. sakazakii
strains, and there were four strains which differed in that bax and the α-xynT were either truncated or duplicated.
Previously we reported the presence of a xylose utilization operon in
C. sakazakii
strain GP1999, which was isolated from a tomato’s rhizoplane/rhizosphere continuum [16]. Furthermore the xylose utilization operon was found in 29 other
C. sakazakii
strains [19] which were obtained from foods of plant origin and dried-food manufacturing environments, supporting the hypothesis that plants may be the ancestral econiche for
Cronobacter
spp., as posited by Schmid et al. [17] and Joseph et al. [32]. Among these strains, we also observed differences in size of the operon [19]. In comparison, the CUT-like xylose utilization operon possessed by
X. axonopodis
pv. citri strain AW12879 (NCBI GenBank assembly accession number: GCA_000349225; RefSeq assembly accession: GCF_000349225) comprises a total of 13 genes and was 25,382 bp in size. Noteworthy, within this operon, an IS3 family transposase was located next to an α- glucosidase gene. Additional differences found were the presence of a TonB-dependent receptor gene and a LacI family transcriptional regulator gene (data not shown).
In the current report, we show the G + C content of a 17, 970 bp region upstream and a 17,422 bp region downstream of the
C. sakazakii
xylose utilization operon possessed G + C contents of 58.1 and 59.6%, respectively (data not shown). This change in G + C content suggests that the
Cronobacter
xylose utilization operon may be a predicted genomic (GI) or metabolic island [59]. Because bacterial genomes evolve through re-combinational events such as mutations, rearrangements, or horizontal gene transfer, we looked for clusters of genes of known or predicted GIs. Genomic islands were historically classified into distinct subtypes depending on the functions they encoded: e.g., symbiotic islands, metabolic islands, fitness islands, pathogenicity islands, or antibiotic resistance islands [60]. However, such G + C content change was not seen in the genomes of the E. coli and the
X. axonopodis
pv. citri strains. As shown in Fig. 3b, and similar to the xylose operon of E. coli strain MG1655 a number of sequence repeats (two in the case of MG1655) were located throughout the
Cronobacter
xylose operon (up to six sequence repeat regions were observed in some strains) suggesting that these are binding sites for regulatory proteins or that they may be evidence of past transpositions. For any one strain, there were multiple sequence repeats found. Table 7 shows examples of the various inverted repeats, palindromes and direct repeats observed in two
C. sakazakii
strains MOD1_Jor151 and MOD1_Jor173. Inverted and direct repeats were sometimes found in two different genes within the same strain (MOD1_Jor151 amy1 and xylS or xylG and xynT); while palindromic sequence was found in bax of MOD1_Jor151. Occasionally, the size of the sequence repeat varied between 15 or 16 bases (which are the default parameters for the sequence repeats finder algorithm within Geneious). Finally, the location of the sequence repeats and type of sequence repeats found among the strains generally followed sequence type evolutionary lines with the exception of ST4 strains (MOD1_Jor148, MOD1_Jor154) and ST643 strain (MOD1_Jor103) which possessed different palindromic sequences which were associated with hypothetical protein or bax. Additional file 5: Table S5 shows the location of each the identical repeat regions within each strain’s xylose utilization operon. It should be noted that other palindromic inverted repeats (IR) of 10 to 13 nucleotides, separated by a 10-bp spacer, forming a stem-loop structure, are found on the virulence plasmids, pESA3 and pCTU1. Furthermore, Franco et al. [43] showed that a conserved pCTU1 region was located upstream of this IR, while the
Cronobacter
plasminogen activator locus on pESA3 was located downstream from this sequence repeat. Also, the upstream flanking gene seen in the
Cronobacter
xylose utilization operon was identified as a hydrolase and the downstream flanking gene was identified as DUF- 2778. These two genes and their locations were conserved throughout the 26 spice-associated
C
.
sakazakii
genomes. Figure 3c shows an alignment of a xylB gene that has the IR repeat region from strain MOD1_Jor22 compared to strain MOD1_AS15 which lacks this repeat region. Note that bax can contain two to three identical repeat regions suggesting that this is a highly regulated gene. Bax has been shown to induce cell apoptosis of
Arabidopsis
protoplast cells through reactive oxygen independent and dependent processes namely DNA fragmentation, increased vacuolation, and loss of plasma membrane integrity [61]. Together, these results suggest that there is a virulence factor function to Bax and that the
Cronobacter
xylose utilization operon may be a predicted metabolic island.
Table 7 Summary of inverted repeat, palindrome, and direct repeat present in C. sakazakii strains MOD1_Jor151 and MOD1_Jor173 genomesa Figure 4 illustrates the proposed molecular basis of how
C. sakazakii
(strain MOD1_Jor22 as an example) may utilize D-xylose, xylose-containing plant cell wall polymers (xylans, hemicellulose-like, and cellulose) or α- and β-xylosides. D-xylose enters the cytoplasm of a cell either by diffusion or by transport and binds to the AraC-like positive xylose operon transcription regulator, XylR. XylR is, identical to AraC which activates the transcription of the analogous arabinose utilization operon, araBAD, araE and araFGH operons, but represses the transcription of the araC operon. Once bound, XylR actuates the xylose regulon by activating the transcription of the xylFGH, xylR, xylAB, and xylE genes. In fact, in E. coli, the xylose transporters XylE and XylFGH can transport both arabinose and xylose; conversely the arabinose transporters
Ara
E and
Ara
FGH can take up xylose, even in the absence of arabinose [56]. As with arabinose, expression of the XylE and XylFGH transporters increases the rate of xylose uptake and further enhances activation of the regulon. Another set of genes, which are also outside the operon, may be triggered through the proposed activation of the xylose regulon: xynA encoding for Xylanase A (xynA, locus tag C5934_19110) which is an Endo-1,4-β-xylanase and may be secreted by a proposed type 2 secretion system. A third pathway of xylose utilization, also seen in E. coli, was found in these
Cronobacter
spice strain’s genomes and includes a xylulose reductase, an oxidoreductase (locus tag C5934_08370), and a NAD(P)-dependent alcohol dehydrogenase (locus tag C5934_08415) which are thought to be activated under anaerobic growth conditions [56]. D-xylose, or transported α/β-xylosides (via α/β-XynTs) are converted to D-xylose by α/β-xylosidases (XylS/XynB) within the cell. It is not certain, at this time, how xylans are converted to α-xylosides in the extracellular milieu. However, the fact
Cronobacter
possess an α-xylosidases (xylS) and an adjacent xynT gene, suggests that that α- xylosides may be transported into the cell and then converted to D-Xylose, which is then converted to D-xylulose by xylose isomerase (XylA) and then phosphorylated by Xylulose kinase (XylB). Then, xylulose 5-phosphate is metabolized by the enzymes of the pentose phosphate pathway [56]. Together these results support those reported by Srikumar et al. [30], which suggest that 5-carbon sugar physiological mechanisms utilized by
Cronobacter
plays important roles in its overall survival strategy.