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Glucose metabolism via the entner-doudoroff pathway in campylobacter: A rare trait that enhances survival and promotes biofilm formation in some isolates

Frontiers in Microbiology (2016) 7(NOV)
DOI: 10.3389/fmicb.2016.01877
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Abstract

Isolates of the zoonotic pathogen Campylobacter are generally considered to be unable to metabolize glucose due to lack of key glycolytic enzymes. However, the Entner-Doudoroff (ED) pathway has been identified in Campylobacter jejuni subsp. doylei and a few C. coli isolates. A systematic search for ED pathway genes in a wide range of Campylobacter isolates and in the C. jejuni/coli PubMLST database revealed that 1.7% of > 6,000 genomes encoded a complete ED pathway, including both C. jejuni and C. coli from diverse clinical, environmental and animal sources. In rich media, glucose significantly enhanced stationary phase survival of a set of ED-positive C. coli isolates. Unexpectedly, glucose massively promoted floating biofilm formation in some of these ED-positive isolates. Metabolic profiling by gas chromatography-mass spectrometry revealed distinct responses to glucose in a low biofilm strain (CV1257) compared to a high biofilm strain (B13117), consistent with preferential diversion of hexose-6-phosphate to polysaccharide in B13117. We conclude that while the ED pathway is rare amongst Campylobacter isolates causing human disease (the majority of which would be of agricultural origin), some glucose-utilizing isolates exhibit specific fitness advantages, including stationary-phase survival and biofilm production, highlighting key physiological benefits of this pathway in addition to energy conservation.

Figures

  • FIGURE 1 | The Entner-Doudoroff pathway in Campylobacter isolates. (A) Scheme of the ED pathway to illustrate how the reactions effectively bypass the lack of phosphofructokinase, which prevents glycolysis by the EMP pathway in Campylobacter, but can also allow cycling of triose-phosphate back to hexose phosphate. The key enzymes 6-phosphogluconate dehydratase (Edd) and 2-keto-3-deoxy-6-phosphogluconate aldolase (Eda) catalyze the dehydration of
  • TABLE 1 | Sources of ED-positive isolates from the Campylobacter jejuni/coli PubMLST database.
  • TABLE 2 | Allelic diversity of components of the glc locus among 113 ED-positive Campylobacter isolates.
  • FIGURE 2 | Phylogenetic relationships among Campylobacter glc loci. Maximum likelihood tree based on concatenated nucleotide sequences of genes comprising the glc locus, encoding the Entner-Doudoroff pathway, from 113 Campylobacter isolates. Colored strips adjacent to the phylogeny indicate species and source as shown in the inset legends. Known C. jejuni subsp. doylei isolates are indicated with filled circles. All C. coli isolates were assigned to C. coli clade 1, with the exception of a single clade 3 isolate, which is marked with a star. Isolates included in experiments carried out in this study are labeled. Roman numerals indicate groups of glc sequences referred to in the text. For major nodes, bootstrap values generated from 500 replicates are shown as percentages. The scale bar represents the number of nucleotide substitutions per site.
  • FIGURE 3 | Relationships among ED-positive Campylobacter isolates. NeighborNet graphs were generated based on whole-genome multilocus sequence typing (wgMLST) comparisons of 68 C. jejuni (A) and 38 C. coli (B) isolates, using NCTC11168 and 15-537360 (black squares), respectively, as reference genomes. ED allele types are shown as numbers adjacent to each isolate. Isolates included in experiments carried out in this study are labeled in full with the ED type in parentheses. Dashed lines and Roman numerals indicate groups of isolates with related ED types referred to in the text. The color of the filled circles indicates the source of each isolate as shown in the key (inset). All C. coli were assigned to C. coli clade 1 except for a single clade 3 isolate, which is marked with a star.
  • FIGURE 4 | Glucose stimulates growth of ED-positive but not ED-negative C. coli isolates. Four ED-positive C. coli isolates (B13117, CV1257, Dg172, and Dg349) and an ED-negative control C. coli strain (OXC6725) were inoculated into modified MCLMAN minimal media either without or with glucose supplementation. Cultures were incubated for 20 h at 37◦C under microaerobic conditions and viable cell numbers measured by plate counts. White bars, colony forming units (CFU) ml−1 of inoculum at time 0. Light gray bars, final CFU ml−1 in modified MCLMAN without glucose. Black bars, final CFU ml−1 in modified MCLMAN supplemented with 100 mM glucose. The bars show the mean and the error bars show the standard deviations of three independent cultures. Statistical significance is indicated by (∗∗p < 0.01) or (∗∗∗p < 0.001) as determined by Student’s t-test.
  • FIGURE 5 | Extended stationary phase survival with glucose. Viable cell numbers of ED-positive C. coli isolates (B13117, CV1257, Dg172, and Dg349) and an ED-negative control C. coli strain (OXC7218) were determined in TSB with or without supplementation with 100 mM glucose. Cultures were incubated for 7 days at 37◦C in microaerobic conditions and the viability determined by enumerating CFU at various time intervals. Results are mean and standard deviations of three independent cultures. The error bars are too small to be seen in some cases. (A) C. coli B13117; (B) C. coli CV1257; (C) C. coli Dg172; (D) C. coli Dg349; (E) C. coli OXC7218 (ED-negative control). Open circles and dashed line, cell numbers without glucose; filled circles and solid line, cell numbers with glucose. The statistical significance of the difference between the control without glucose and with added glucose at 7 days was tested by Student’s t-test, as indicated by (∗∗∗p < 0.001) or NS (not significant).
  • FIGURE 6 | Glucose stimulates production of floating biofilm that increases cell biomass. (A) The isolates of C. coli shown were cultivated for 4 days at 37◦C under microaerobic conditions in glucose-free TSB with and without supplementation with 100 mM glucose. The glucose-supplemented cultures of B13117 (ED+) and Dg349 (ED+) formed extensive floating biofilm (flocs; arrowed). CV1257 (ED+) and Dg172 (ED+) displayed denser growth with glucose but no biofilm formation, while a low level of aggregation was seen in the cultures of ED-negative OXC7218 regardless of glucose-supplementation. Pictures are representative cultures from four independent growths. (B) The four ED-positive C. coli isolates (B13117, CV1257, Dg172, and Dg349) and the ED-negative C. coli strain OXC7218 were cultivated for 7 days at 37◦C under microaerobic conditions in glucose-free TSB with or without glucose supplementation. The bars show dry weight biomass of 5 ml aliquots from cultures without glucose (white bars) and with glucose supplementation (black bars). The mean and standard deviations of four independent cultures for each condition are shown. The statistical significance of the difference between the dry weight without glucose and with added glucose was evaluated by Student’s t-test, as indicated by (∗∗∗p < 0.001) or NS (not significant). The arrows refer to the biofilm producing strains B13117 and Dg349 as in (A), which clearly produce more biomass with glucose compared to the other non-biofilm producing strains tested.

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Vegge, C. S., Jansen van Rensburg, M. J., Rasmussen, J. J., Maiden, M. C. J., Johnsen, L. G., Danielsen, M., … Kelly, D. J. (2016). Glucose metabolism via the entner-doudoroff pathway in campylobacter: A rare trait that enhances survival and promotes biofilm formation in some isolates. Frontiers in Microbiology, 7(NOV). https://doi.org/10.3389/fmicb.2016.01877

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