Analysis of gluconeogenic and anaplerotic enzymes in Campylobacter jejuni: an essential role for phosphoenolpyruvate carboxykinase

Abstract

Campylobacter jejuni is unable to utilize glucose as a carbon source due to the absence of the key glycolytic enzyme 6-phosphofructokinase. The genome sequence of C. jejuni NCTC 11168 indicates that homologues of all the appropriate enzymes for gluconeogenesis from phosphoenolpyruvate (PEP) are present, in addition to the anaplerotic enzymes pyruvate carboxylase (PYC), phosphoenolpyruvate carboxykinase (PCK) and malic enzyme (MEZ). Surprisingly, a pyruvate kinase (PYK) homologue is also present. To ascertain the role of these enzymes, insertion mutants in pycA, pycB, pyk and mez were generated. However, this could not be acheived for pckA, indicating that PCK is an essential enzyme in C. jejuni. The lack of PEP synthase and pyruvate orthophosphate dikinase activities confirmed a unique role for PCK in PEP synthesis. The pycA mutant was unable to grow in defined medium with pyruvate or lactate as the major carbon source, thus indicating an important role for PYC in anaplerosis. Sequence and biochemical data indicate that the PYC of C. jejuni is a member of the α 4 β 4 , acetyl-CoA-independent class of PYCs, with a 65 8 kDa subunit containing the biotin moiety. Whereas growth of the mez mutant was comparable to that of the wild-type, the pyk mutant displayed a decreased growth rate in complex medium. Nevertheless, the mez and pyk mutants were able to grow with pyruvate, lactate or malate as carbon sources in defined medium. PYK was present in cell extracts at a much higher specific activity [S 800 nmol min N1 (mg protein) N1 ] than PYC or PCK [T 65 nmol min N1 (mg protein) N1 ], was activated by fructose 1,6-bisphosphate and displayed other regulatory properties strongly indicative of a catabolic role. It is concluded that PYK may function in the catabolism of unidentified substrates which are metabolized through PEP. In view of the high K m of MEZ for malate (" " " 9 mM) and the lack of a growth phenotype of the mez mutant, MEZ seems to have only a minor anaplerotic role in C. jejuni.

Campylobacter jejuni Gene Expression in the Chick Cecum: Evidence for Adaptation to a Low-Oxygen Environment

“…The carbon sources that C. jejuni can utilize in the chicken cecum are unknown, although the most likely source of carbon and nitrogen in vivo is via deamination of amino acids, given that C. jejuni is asaccharolytic (41). In previous studies, in vitro experiments have shown that C. jejuni can grow in minimal media by using serine, aspartate, glutamate, and proline as carbon sources (20).…”

Sulphite : cytochrome c oxidoreductase deficiency in Campylobacter jejuni reduces motility, host cell adherence and invasion

“…Within those natural habitats, C. jejuni is able to metabolize products like free amino and keto acids generated by these accompanying bacterial species or from the host itself (Lee & Newell, 2006). Since C. jejuni lacks the glycolytic enzyme phosphofructokinase, glucose cannot be catabolized, and other products serve as electron donors for respiration, for instance succinate, malate, formate, Dlactate, hydrogen and NAD(P)H (Hoffman & Goodman, 1982;Parkhill et al, 2000;Kelly, 2001;Sellars et al, 2002;Velayudhan & Kelly, 2002). It has also been shown that sulphite can be exploited as a respiratory electron donor.…”

Metabolite and transcriptome analysis of Campylobacter jejuni in vitro growth reveals a stationary-phase physiological switch

“…The microaerophilic nature, complex nutritional requirements and relative difficulty of culturing C. jejuni have all contributed to the limited understanding of metabolism (Kelly, 2001(Kelly, , 2005Sellars et al, 2002). C. jejuni is unable to catabolize glucose and other hexose sugars due to the absence of the key glycolytic enzyme 6-phosphofructokinase (Parkhill et al, 2000;Velayudhan & Kelly, 2002 The importance of each amino acid in C. jejuni metabolism is yet to be fully established, however, and information on the carbon and nitrogen flow in this organism remains limited.…”

“…The microaerophilic nature, complex nutritional requirements and relative difficulty of culturing C. jejuni have all contributed to the limited understanding of metabolism (Kelly, 2001(Kelly, , 2005Sellars et al, 2002). C. jejuni is unable to catabolize glucose and other hexose sugars due to the absence of the key glycolytic enzyme 6-phosphofructokinase (Parkhill et al, 2000;Velayudhan & Kelly, 2002). Amino acids serve as major substrates for C. jejuni, and molecular detail concerning the utilization of L-serine, L-aspartate, L-proline and L-glutamate has begun to emerge (Guccione et al, 2008;Joshua et al, 2006;Kelly, 2005;Leach et al, 1997;Del Recio Leon-Kempis et al, 2006;Velayudhan et al, 2004).…”

“…The microaerophilic nature, complex nutritional requirements and relative difficulty of culturing C. jejuni have all contributed to the limited understanding of metabolism (Kelly, 2001(Kelly, , 2005Sellars et al, 2002). C. jejuni is unable to catabolize glucose and other hexose sugars due to the absence of the key glycolytic enzyme 6-phosphofructokinase (Parkhill et al, 2000;Velayudhan & Kelly, 2002 The importance of each amino acid in C. jejuni metabolism is yet to be fully established, however, and information on the carbon and nitrogen flow in this organism remains limited.Detailed understanding of the growth dynamics of C. jejuni during in vitro culture is lacking, and methods for growing this species are diverse and occasionally ill-considered. Batch culture remains an extremely common method of growing C. jejuni in vitro and is itself a common experimental system (Corcoran & Moran, 2007;Mohammed et al, 2005).…”