3.1 key nutrients serine, aspartate, asparagine, glutamate, glutamine,

3.1      ConclusionWGS, cgMLST and rMLSTgenomic data showed that all 30 RG-1 Campylobacter strains clustertogether in the phylogeny. This distinct group was far from the generalistST-21 and ST-45 clonal complexes of Campylobacter spp. The 16S rRNAphylogenic analysis confirmed that the RG-1 strains cluster close to C.

coli,but far from other species. The presence of the hipO and napAgene markers excludes the possibility that the RG-1 group is C. coli or C.jejuni subsp. doylei. These genomic properties and cell morphologyhave confirmed that this group are C.

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jejuni species. Further analysismight be necessary to confirm they are either C. jejuni subsp. jejunior that they belong to a new subspecies of C.

jejuni.  RG-1 strains were treated with themotility MHA medium in order to assist adaptation to the laboratory conditions.This treatment enhanced the bacterial growth by ~5-7 times and doubledmotility. Even though the RG-1 cells had adapted to the laboratory conditions,the growth and motility properties were still 3-8 and 2-6 times less incomparison to the reference strains, respectively. Nevertheless, RG-1 cellsgrew as well as reference strains when they were grown in the MHB rich mediumsupplemented with lab rat mucin.

This is probably due to a particularnutrient/growth factor within the rat mucin. Some other in vitro studies confirmed that the RG-1 strainsutilised the key nutrients serine, aspartate, asparagine, glutamate, glutamine,proline, succinic acid, formic acid, and pyruvic acid, as does NCTC11168. Theserine was used first followed by aspartate, glutamate and proline (Velayudhan et al.

, 2004). This order of amino acidpreference was similar to the well-studied NCTC11168. One of the cleardifferences between RG-1 and the control strain was that RG-1 did not utiliseL-fucose due to a missing genomic island (cj0480c-cj0480) neededto metabolise the carbon source. It is known that this pathway is absent inapproximately 50% of the sequenced C. jejuni and C. coli strains,including both human gastroenteritis causing C. jejuni 81-176 and 81116strains (Dwivedi et al.

, 2016).  RG-1 strains share the majority oftheir genome content (1540 core genes, 84.1% identity?). Meanwhile, 38.8% ofauxiliary genes (112/288) were from two large uncharacterised phage insertions.

The rest of the auxiliary genes were related to motility, chemotaxis, and membraneand transport proteins. These genes could be important for evolution of theRG-1 strains to a specific host environment. Significantly, cgMLST Cj0145 gene encoding putative TAT (Twin-ArginineTranslocation) was absent in all RG-1 strains. This pathway is involved in thestress response and aids C.

jejuni adaptation to different hosts (Hitchcock et al., 2010;Rajashekara et al., 2009).The loss of this cgMLST could restrict RG-1 to a specific niche.   Only 2.

2% of the RG-1 core genes(35 genes) might be rat specific genes. These were not cgMLST and were not inthe different selected sequence types of C. jejuni strains.

A group of 9genes are related to the uptake of iron. This pathway was not identical to the identified 7class of pumping iron in CampylobacterAD1 .However, based on percentage identity, this group of genes might have beenacquired from C. jejuni subsp. doylei269.

97 (92% identity) or C. helveticus (82% identity). On the other hand, RG-1 strains havelost three genes of the Ferric enterochelin uptake system including the cfrAgene and five genes of the Ferri-transferins uptake system, including the Cj0178gene. Different in vivo studies have demonstrated that C.

jejunirequires both CfrA and Cj0178 to be able to survive and colonise the intestinesof animals (Palyada et al., 2004; Zeng et al., 2009). In addition to the ironuptake system, RG-1 strains have a new operon of cdtABC genes. Theoperon might originate from the C. lari RM16701 strain(86% identity), which has a couple of deletions in the protein sequence of CdtA.It is well known that Campylobacter spp. are commensal bacteria topoultry.

Even though many of C. jejuni strains from broiler chickenshave cdt genes, they do not cause disease in the birds (Bang et al., 2001). On the other hand, RG-1 has lost some key systems. VitaminB5 biosynthesis has been identified as a host specificity factor. Interestingly,panBCD genes were absent in all RG-1 and RG-2 strains, but present in theother ST-21, ST-22 and ST-45 clonal complexes found in farm associated rats.

Sheppard et al. (2013)found that Campylobacter isolates from cattle have the required pathway,but the operon was not found in chicken associated strains. They proposed thatchickens could obtain the nutrients from rich vitamin B5 cereals and grain, andtherefore they do not need to synthesise it themselves. This may be true forthe RG-1 strains.

They could get vitamin B5 from the diet of the rats. Lastly,another host specific L-fucose utilisation operon was not found in the RG-1genomes. It has been studied that designing a mutation in the putative fucosepermease Cj0486 gene inhibited the uptake of fucose and reduced colonisationlevels in a piglet model, but without significant difference in a chicken model(Stahl et al., 2011).

Supplementing the chickens’ diet with L-fucose increased the level ofcolonisation by the wild strain. The fuc-positive NCTC1168 also formed amore sessile structure of biofilm than fuc-negative 81116 (Dwivedi et al., 2016).

The ability to use L-fucosemight allow the bacterial isolates to survive better in pigs over other hosts.Similarly, RG-1 strains might not be able to colonise pigs, suggesting thatthey have adapted to specific hosts.  To conclude, draft genomes, massspectrometry, and 16S rRNA have confirmed that the RG-1 strains are C.jejuni and presumably subsp.

jejuni. Approximately 15% of the RG-1core genes (148) were not cgMLST, and included a putative iron uptake systemand cdt genes. The strains have lost some virulence or host specific operonssuch as cgMLST Cj0145 putative TAT gene, Ferric enterochelin and Ferri-transferrinuptake systems, fuc pathway, and vitamin B5 biosynthesis. All RG-1strains were motile, did not have the ability to form biofilm on the glassmembrane filter, and grew poorly in rich medium compared to other differingsequence types. Growth of poorly growing RG-1 strains was encouraged throughthe addition of rat mucins to the rich medium. The loss of some host specificoperons and the improved growth of these RG-1 isolates in the rat mucinsuggests that this group of C.

jejuni strains may not be able to survivein different hosts and are likely restricted to specific hosts. To furtherinvestigate this, selected RG-1 strains were studied in both Galleriamelonella and Ross chicken in vivo models, as shown in chapter 5. The next chapter will be onthe ability of RG-2 strains to utilise glucose.     Appendices  Appendix 1: Growth of C. jejuni in small intestinal and caecal lab rat mucins.  The small intestine (S) and caecum (C) mucins were scraped from 4 white lab rats (~250g/rat) and the mucin was homogenised using a MP shaker.

Under my supervision, the growth experiment was done by Nancy, F. and Sarah, J. undergraduate students. MHB was supplemented with 5% mucin, 1% antibiotic and inoculated with 0.

002OD600 of bacterial cells, and incubated for 45h. Cells were then grown under microtiter plate growth assay conditions. CFU values are the mean of triplicate drops of three wells. Pattern filled histograms are growth in MHB without mucin.      Appendix 2: Concentration of amino acids in MEM-? medium. The medium was supplemented with 10% FBS and 20mM (A) proline and (B) serine.

Amino acid concentration was measured at 48h and 72h of incubation. The proline in A and serine in B data has been shown in Figure ?3.15, B.

  A B      Appendix 3: cgMLST 29 genes are absent (A) or incomplete (I) in at least one of the RG-1 strains.   Cj number Product Sequence length Genome position Absent (A) or Incomplete (I) gene among RG-1 Cj0118 conserved hypothetical protein 756 122366 I in Dg197 Cj0145* putative TAT (Twin-Arginine Translocation) pathway signal sequence domain protein 1782 148819 A in all Cj0174c|cfbpB* putative iron-uptake ABC transport system permease protein 1617 169946 A in all Cj0243c hypothetical protein 1167 224794 I in Dg150 Cj0430 putative integral membrane protein 1227 391711 I in Dg61 Cj0484 putative MFS (Major Facilitator Superfamily) transport protein 1233 451046 A in 36.6% Cj0561c putative periplasmic protein 930 524034 A in 36.

7% Cj0763c|cysE serine acetyltransferase 639 714138 I in Dg156 Cj0777 putative ATP-dependent DNA helicase 2031 728500 I in Dg189 Cj0799c|ruvA putative Holliday junction ATP-dependent DNA helicase 552 749307 I in Dg78 Cj0800c putative ATPase 1860 749834 I in Dg289 Cj0801 putative integral membrane protein (MviN homolog) 1452 751797 I in Dg189 Cj0810|nadE NH(3)-dependent NAD(+) synthetase 741 761404 I in Dg276 Cj0841c|mobB putative molybdopterin-guanine dinucleotide biosynthesis protein 492 789049 I in Dg189 Cj0850c putative MFS (Major Facilitator Superfamily) transport protein 1188 797653 I in Dg347 Cj1004 putative periplasmic protein 417 934201 I in Dg189 Cj1014c|livF branched-chain amino-acid ABC transport system ATP-binding protein 696 947343 I in Dg347 Cj1041c putative periplasmic ATP/GTP-binding protein 852 975230 A in 23.4% Cj1042c putative transcriptional regulatory protein 891 976144 A in 23.4% Cj1198|luxS S-ribosylhomocysteine lyase (autoinducer-2 production protein LuxS) 495 1127437 A in 36.7% Cj1064 pseudogene (nitroreductase) 620 1001218 A in 23.4% Cj1199 putative iron/ascorbate-dependent oxidoreductase 993 1128243 A in 36.7% Cj1201|metE 5-methyltetrahydropteroyltriglutamate– homocysteine methyltransferase 2265 1130028 A in 36.

7% Cj1202|metF 5,10-methylenetetrahydrofolate reductase 849 1132302 A in 36.7% Cj1295 conserved hypothetical protein 1308 1226978 I in Dg147 Cj1296 hypothetical protein 360 1228282 A in 23.4% Cj1346c 1-deoxy-D-xylulose 5-phosphate reductoisomerase 1071 1278851 I in Dg150 Cj1411c putative cytochrome P450 1362 1342550 I in Dg150 Cj1721c* putative outer membrane protein 645 1632901 A in all * absent in RG-1 including Dg147   Appendix 4: 148 non-cgMLST, but core genes in RG-1. RG-1 strains were Blasted in Genome Comparator (95% threshold) (Cody et al.

, 2017).   Cj NCTC11168 gene number Product Cj NCTC11168 gene number Product Cj0011c putative non-specific DNA binding protein. Cj0864 putative periplasmic protein Cj0014c putative integral membrane protein Cj0866 pseudogene (arylsulfatase) Cj0019c putative MCP-domain signal transduction protein Cj0874c putative cytochrome C Cj0020c cytochrome C551 peroxidase Cj0876c putative periplasmic protein Cj0030 hypothetical protein Cj0900c small hydrophobic protein Cj0037c putative cytochrome C Cj0973 hypothetical protein Cj0041|fliK putative flagellar hook-length control protein Cj0974 very hypothetical protein Cj0045c putative iron-binding protein Cj0983 putative lipoprotein Cj0057 putative periplasmic protein Cj0985c|hipO hippurate hydrolase Cj0058 putative peptidase C39 family protein Cj0986c putative integral membrane protein Cj0090 putative lipoprotein Cj0986c putative integral membrane protein Cj0092 putative periplasmic protein Cj0987c putative MFS (Major Facilitator Superfamily) transport protein Cj0093 putative periplasmic protein Cj0988c very hypothetical protein Cj0140 hypothetical protein Cj0990c hypothetical protein Cj0168c putative periplasmic protein Cj1018c|livK ABC transport system, periplasmic binding protein Cj0186c putative TerC family integral membrane protein Cj1019c|livJ  ABC transport system periplasmic binding protein Cj0199c putative periplasmic protein Cj1021c putative periplasmic protein Cj0201c putative integral membrane protein Cj1036c conserved hypothetical protein Cj0202c hypothetical protein Cj1055c putative sulfatase family protein Cj0203 putative citrate transporter Cj1060c putative membrane protein Cj0246c putative MCP-domain signal transduction protein Cj1077|ctsT putative periplasmic protein Cj0247c hypothetical protein Cj1079 putative periplasmic protein Cj0251c highly acidic protein Cj1110c putative MCP-type signal transduction protein Cj0263|zupT zinc transporter Cj1119c|pglG putative integral membrane protein Cj0299 putative periplasmic beta-lactamase Cj1135 putative two-domain glucosyltransferase Cj0327 putative endoribonuclease L-PSP family protein Cj1136 putative glycosyltransferase Cj0339 putative MFS (Major Facilitator Superfamily) transport protein Cj1149c|gmhA sedoheptulose 7-phosphate isomerase Cj0340 putative nucleoside hydrolase Cj1159c small hydrophobic protein Cj0380c hypothetical protein Cj1160c putative membrane protein Cj0414 putative oxidoreductase subunit Cj1189c|cetB bipartate energy taxis response protein cetB Cj0415 putative GMC oxidoreductase subunit Cj1191c putative PAS domain containing signal-transduction sensor protein Cj0416 hypothetical protein Cj1224 putative iron-binding protein Cj0417 hypothetical protein Cj1241 putative MFS (Major Facilitator Superfamily) transporter protein Cj0437|sdhA succinate dehydrogenase flavoprotein subunit Cj1255 putative isomerase Cj0438|sdhB putative succinate dehydrogenase iron-sulfur protein Cj1300 putative SAM domain containing methyltransferase Cj0439|sdhC putative succinate dehydrogenase subunit C Cj1305c hypothetical protein (617 family) Cj0455c putative membrane protein Cj1309c hypothetical protein Cj0494 putative exporting protein Cj1310c hypothetical protein (617 family) Cj0501 pseudogene (ammonium transporter) Cj1314c|hisF imidazole glycerol phosphate synthase subunit Cj0552 putative membrane protein Cj1315c|hisH imidazole glycerol phosphate synthase subunit Cj0553 putative integral membrane protein Cj1316c|pseA pseudaminic acid biosynthesis PseA protein Cj0554 hypothetical protein Cj1319 putative nucleotide sugar dehydratase Cj0555 putative dicarboxylate carrier protein MatC Cj1320 putative aminotransferase (degT family) Cj0556 putative amidohydrolase family protein Cj1327|neuB2 N-acetylneuraminic acid synthetase Cj0563 hypothetical protein Cj1328|neuC2 putative UDP-N-acetylglucosamine 2-epimerase Cj0564 putative integral membrane protein Cj1329 putative sugar-phosphate nucleotide transferase Cj0592c putative periplasmic protein Cj1330 hypothetical protein Cj0618 hypothetical protein  (617 family) Cj1331|ptmB acylneuraminate cytidylyltransferase (flagellin modification) Cj0659c putative periplasmic protein Cj1332|ptmA putative oxidoreductase (flagellin modification) Cj0660c putative transmembrane protein Cj1337|pseE PseE protein Cj0672 putative periplasmic protein Cj1360c putative proteolysis tag for 10Sa_RNA Cj0676|kdpA pseudogene (potassium-transporting ATPase A chain) Cj1406c putative periplasmic protein Cj0677|kdpB potassium-transporting ATPase B chain Cj1415c|cysC putative adenylylsulfate kinase Cj0678|kdpC pseudogene (potassium-transporting ATPase C chain) Cj1416c putative sugar nucleotidyltransferase Cj0679|kdpD truncated KdpD protein Cj1417c putative amidotransferase Cj0679|kdpD truncated KdpD protein Cj1418c putative transferase Cj0685c|cipA Invasion protein CipA Cj1419c putative methyltransferase Cj0685c|cipA Invasion protein CipA Cj1423c|hddC putative D-glycero-D-manno-heptose 1-phosphate guanosyltransferase Cj0692c putative membrane protein Cj1424c|gmhA2 phosphoheptose isomerase Cj0742 pseudogene (putative outer membrane protein) Cj1425c|hddA putative D-glycero-D-manno-heptose 7-phosphate kinase Cj0770c putative NLPA family lipoprotein Cj1456c putative periplasmic protein Cj0771c putative NLPA family lipoprotein Cj1521c putative CRISPR-associated protein Cj0772c putative NLPA family lipoprotein Cj1522c putative CRISPR-associated protein Cj0786 small hydrophobic protein Cj1523c putative CRISPR-associated protein Cj0814 hypothetical protein. Functional classification-Unknown Cj1547 Blc protein homolog Cj0815 hypothetical protein Cj1585c putative oxidoreductase Cj0816 hypothetical protein Cj1589 conserved hypothetical protein Cj0825 putative processing peptidase Cj1602 conserved hypothetical protein Cj0829c putative CoA binding domain containing protein Cj1625c|sdaC amino acid transporter Cj0830 putative integral membrane protein Cj1630|tonB2 putative TonB transport protein Cj0849c conserved hypothetical protein Cj1666c putative periplasmic protein Cj0851c putative integral membrane protein Cj1668c putative periplasmic protein Cj0859c hypothetical protein Cj1714 small hydrophobic protein Cj0860 putative integral membrane protein Cj1729c|flgE2 flagellar hook subunit protein    Appendix 5: Iron uptake groups of genes in Campylobacter. Genes that are missing in RG-1 indicated by underlining in red. Image taken from (Miller, C.

E. et al., 2009).

   AD1Thisneeds to be clearer. Class of 7 genes that are involved in pumping iron? Notsure what you mean


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