- Comparative genomic analysis of the secondary flagellar (flag-2) system in the order Enterobacterales. - Conclusions: The flag-2 flagellar system is a fairly common, but highly variable feature among members of the Enterobacterales. - Recent phylogenomic re-evaluation of the sole family in this order, the Enterobacteriaceae, has resulted in its division into eight distinct families [1].. - Cell Biology, University of the Witwatersrand, 2050 Wits, Johannesburg, South Africa. - Full list of author information is available at the end of the article. - The flag-2 locus of E. - Knock-out mu- tagenesis of the Y. - Here, by means of compara- tive genomic analyses, we have further analysed the flag- 2 locus and show it to present in a substantial number of taxa across a broad spectrum of the genera and fam- ilies in the order Enterobacterales. - The enterobacterial flag-2 locus comprises a large set of conserved genes for the synthesis and functioning of the secondary flagellar system, but also incorporates variable regions that may contribute to both structural and functional versatility of this system. - The finished and draft genomes of 4028 bacterial strains encompassing the taxonomic diversity of the order. - of the analysed taxa) strains were observed to possess an orthologous locus (Fig. - Additional file 1: Table S1) and these are distributed across a wide taxonomic breadth of the order. - As such, flag-2 loci occur in five of the eight families and 31/76 genera included in this study. - The highest prevalence can be observed in the family Budvicaceae (7/9 studied taxa) and Yersiniaceae (225/605 strains), while only taxa) of the family Enterobacteriaceae contained orthologous loci (Fig. - Notably, flag-2 loci are uni- versally present in several genera, including Citrobacter Clade D (30/30 strains) and Plesiomonas (8/8 strains), while in the two genera with the highest number of flag- 2 loci present, Yersinia (222/394 strains) and Escherichia (124/522 strains), 56 and 24% of the evaluated strains encode flag-2 systems, respectively. - For example, all 100 of the evaluated Y. - Molecular architecture of the flag-2 loci. - The flag-2 loci comprise of a set of co-localised genes within the genomes of the enterobacteria that harbour them (Fig. - 3), where gene loci responsible for the synthesis and functioning of the primary flagellar system are gen- erally dispersed across the enterobacterial chromosome [12]. - Of the 592 strains with flag-2 loci of strains with flag-2 loci) encode an orthologue complement of. - One of these conserved proteins is LafA, the flagellin counterpart of the flag-2 system, which is present in multiple copies in strains, with up to five copies (Y. - Multiple copies of the flagellin gene have also been observed in the flag-1 loci of many enterobacteria and have been suggested to contribute to the. - 1 Distribution of the flag-2 locus across the order Enterobacterales. - A circularized, topology-only ML phylogeny was constructed on the basis of the concatenated alignments of the house-keeping proteins GyrB, InfB, RecA and RpoB. - Cluster 1, comprised of fourteen genes lfhAB-lfiRQPNM-lafK-lfiEFGHIJ, encodes the proteins involved in regulation and assembly of the basal body components and is analogous to the flhAB-fliRQPNMEF- GHIJ genes in the flag-1 locus (Fig. - Cluster 3 comprises of the genes lafWZABCDEFSTU, which code for eleven proteins with substantially lower. - 2 Schematic comparison of the flag-1 and flag-2 loci of Escherichia sp. - A scale bar (4 kilobases) indicates the size of the loci. - 3 Schematic comparison of the flag-2 loci of representatives of each family within the order Enterobacterales. - Gene and en bloc deletion may have resulted in non- functionality of the flag-2 system in some. - While a substantial fraction of the flag-2 loci contain a complement of 39 conserved genes coding for proteins involved in flagellar biosynthesis and functioning, 22.13% of enterobacterial strains are missing at least one of these genes. - Transpos- ition appears to be a major driver of the observed en bloc gene deletions. - Previous analyses showed that in many Escherichia and Shigella strains, a deletion has occurred within the reading frames of the lfhA and lafU genes which occur at the 5′ and 3′ ends of the flag-2 locus, respectively, resulting in loss of the remaining locus between the lfhA and lafU pseudogene fragments. - Blast analyses of the lfhA and lafU genes and proteins against the 4028 Enterobacterales strains showed that this occurs in the genomes of of the strains (Fig. - For example, of the 100 E. - Similarly of the 66 Citrobacter Clade A strains lacking flag-2 loci show evidence of its deletion. - 1123/4028 analysed strains) prior to en bloc deletion of the locus in. - Alignment of the enterobacterial flag-2 loci and com- parative analysis of their encoded protein complements revealed that, although extensive synteny and a substan- tial set of conserved proteins occur among these loci (Fig. - 2), there are 349 distinct protein coding genes, which are not conserved among all enterobacterial flag-2 loci and which do not form part of the core set involved in flagellar biosynthesis and functioning. - VR1 occurs in the flag-2 loci of enterobacte- rial strains and is particularly prevalent in members of the family Budviciaceae (7/7 strains), Enterobacteriaceae (310/. - A total of 154 distinct proteins are encoded by the VR1 regions of the enterobacterial flag-2 loci. - This includes the flag-2 loci of all Budviciaceae and Hafniaceae and and 37.5% of the Yersiniaceae, Enterobacteri- aceae and Erwiniaceae, respectively. - By contrast, many of the proteins in VR1 share orthology with proteins in- volved in glycosylation and modification of the flagellar filament.. - The flag-2 variable region 1 (VR1) encodes the machinery for glycosylation, methylation and modification of the flagellum. - Here of the flag-2 loci contain genes cod- ing for glycosyltransferases which are predicted to be involved in flagellin glycosylation. - A further 70 strains encode five distinct glycosyltransferase orthologues (GT9–1 to GT9–5) of the GT9 family, which incorporates lipopoly- saccharide N-acetylglucosaminyltransferases and heptosyl- transferases [22]. - Given that distinct sugars can be incorporated by gly- cosyltransferases belonging to the different GT families, the type of sugars incorporated in the flagellin glycan cannot solely be determined on the basis of the type of glycosyltransferase present. - However, the VR1 region of 35 Citrobacter Clade A strains en- codes orthologues of the sialyltransferase PM0188 (pfam11477. - E-value: 2.54E-16) of Pasteurella multocida, suggesting that the flagellin glycan of the latter strains may incorporate neuraminic acid [23]. - These proteins are central to the synthesis of teichoic acid, phosphodiester-linked polyol glycopoly- mers that form a major part of the cell wall of most Gram-positive bacteria [24]. - The presence of ortholo- gues of genes involved in this function in VR1 suggest that these glycopolymers form part of the flagellin glycan of the enterobacterial flag-2 system.. - While the exact glycan sugar moieties of the flagellin glycans are difficult to determine, the presence of ortho- logues of diverse proteins which may modify or substi- tute the glycan chains suggest the flag-2 flagellin glycan is heavily decorated as has been observed in the primary flagellar system of many enterobacteria as well as Gram- positive bacteria [14, 18]. - Among the VR1-encoded pro- teins are orthologues of the acetyltransferases NeuD. - those of the Enterobacteriaceae Citrobacter Clade A and C and Escherichia, while that of Chania multitudinisentens RB-25 clusters with Pluralibacter (Enterobacteriaceae).. - and not with the other taxa, Erwinia and Izhakiella, of the Erwiniaceae. - Genomic regions derived through recent HGT events are often typified by G + C contents that vary substantially from the rest of the genome [26]. - 4.0 to + 5.0%) that of the remainder of the genome (Additional file 1: Table S2). - This may be attributed in part to the distinct G + C content of the VR1 and VR2 regions. - This is particularly evi- dent in the Enterobacteriaceae, where the flag-2 loci have an average G + C content 2.1% (range − 3.1 to + 5.8%) above that of the rest of the genome. - In one of the Enterobacteriaceae clades in the flag-2 tree, G + C de- viations are pronounced in Escherichia (G + C devi- ation. - By contrast, little is known about the function(s) of the secondary flagellar system among the Enterobacterales. - Here we have observed flag-2 loci in the genomes of members of the family Budviciaceae (two strains of Budvicia aquatica and four strains of P. - The flag-2 locus of the latter strain is, however, missing the lafZ gene.. - While the flag-2 loci of many of the enterobacterial taxa contain deletions or genes with disrupted reading frames, a substantial number (77.87% of strains contain- ing flag-2 loci) appear to encode the full complement of proteins for the synthesis and functioning of this flagel- lar system. - Ana- lysis of the proteins encoded in the VR1 and VR2 regions of the flag-2 loci revealed a number of putative secretion targets among the cargo proteins. - The VR1 re- gion of the Escherichia fergusonii YH17130 flag-2 locus incorporates a gene coding for a Type VI secretion sys- tem (T6SS) effector protein VgrG (COG3501. - Furthermore, within the VR1 regions of the Escherichia sp. - E-value: 1.88E-73), a phage lysozyme-like bacteriocin from Y, pestis and an ortholo- gue of the RNase toxin Ntox44 (pfam15607. - AAI) of the autotransporter MisL (PRK15313. - The en bloc deletions and gene disruptions can be envisaged to have a detrimental effect on functionality of the flag-2 system, as has been ob- served in Escherichia sp. - Further- more, pervasive deletion of the locus suggests that it may have been a far more common genomic feature and hints at a complex evolutionary history for the locus. - A recent study has identified orthologous flag-2 loci among members of the alpha-proteobacterial order Rhizobiales and furthermore, they are similar to the lateral flagellar system of Vibrio and Aeromonas spp. - comprehensive analysis across a broader taxonomic scope may shed further light on the evolution of the flag-2 system.. - Phylogenetic analysis on the basis of the conserved proteins encoded on the flag-2 loci suggest disparate evolutionary histories for these loci among the entero- bacterial taxa, with the flag-2 loci some taxa likely being derived through HGT events, while some lineages shows evidence of retention of this locus through vertical trans- mission. - Analysis of the flag-1 protein complement of forty-one motile species across eleven bacterial phyla showed extensive sequence similarity between the twenty-four core proteins conserved among all taxa [38].. - A communal feature of the flag-2 loci of most entero- bacterial taxa is the presence of cargo genes, integrated mainly in two variable regions, VR1 and VR2, which mainly encode the machinery for flagellin glycosylation, which as observed in the flag-1 locus, likely represents a common posttranslational feature of this flagellar sys- tem. - Also among the cargo genes are those which provide hints on the potential functions of the secondary flagellar system. - While earlier evidence largely advocated for the non-functionality of the flag-2 system, the eluci- dation of a functional flag-2 system in P. - As many of the taxa with intact flag-2 loci represent both clinical and plant pathogens, knock-out mutagenesis and further characterization are imperative for a deeper understand- ing of this intriguing flagellar system.. - Highly conserved and syntenous secondary flagellar (flag-2) loci occur in ~ 15% all screened members of the order Enterobacterales and likely represents important functional features in the taxa that incorporate them.. - Variable regions within the flag-2 loci code for proteins with roles in posttranslational modification of the flagel- lar system as well as those that provide hints into the functions of the flag-2 system. - determining the biological role(s) of the secondary fla- gellar system in members of the order Enterobacterales.. - Identification of flag-2 loci among the Enterobacterales The genomes of 4028 members of the order Enterobacter- ales were selected for analysis. - These complete flag-2 loci were structural annotated using the Prokaryotic GeneMark.hmm v.2 server [42] and the G + C% contents of each of the loci were calculated using BioEdit v . - Comparative genomic analyses of the enterobacterial flag-2 loci. - The presence/absence/deletion of the flag-2 locus was mapped onto this phylogeny. - The isolation source as well as habitat/lifestyles of the different strains are given. - Molecular characteristics of the flag-2 loci among 592 taxa in the Enterobacterales. - The sizes of the flag-2 loci, variable regions VR1 and VR2, their G + C contents. - Characteristics of the cargo genes encoded in the variable regions VR1 and VR2 and elsewhere in the enterobacterial flag-2 loci. - Cell Biology, University of the Witwatersrand. - TP was a post-graduate student who completed her MSc degree in Microbiology and Biotechnology at the University of the Witwatersrand. - Genome-based phylogeny and taxonomy of the ‘ Enterobacteriales. - The role of the bacterial flagellum in adhesion and virulence. - Structure of the bacterial flagellar hook and implication for the molecular universal joint mechanism. - Novel components of the flagellar system in Epsilonproteobacteria. - Flagellar glycosylation – a new component of the motility repertoire? Microbiol. - Comparative genomic analysis of the flagellin glycosylation island of the gram-positive thermophile Geobacillus. - Phylogenomic analyses of Bradyrhizobium reveal uneven distribution of the lateral and subpolar flagellar systems, which extends to Rhizobiales. - Stepwise formation of the bacterial flagellar system
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