Characterization of the RokA and HexA Broad-Substrate-Specificity Hexokinases from Bacteroides fragilis and Their Role in Hexose and N -Acetylglucosamine Utilization

Abstract

Bacteroides fragilis, a human gastrointestinal commensal and an opportunistic pathogen, utilizes simple and complex sugars and polysaccharides for growth in the large intestine and at sites of infection. Because B. fragilis lacks transport-linked sugar phosphorylation systems, cytoplasmic kinase(s) was expected to be required for the phosphorylation of hexoses and hexosamines. We have now identified two hexose kinases that are important for growth of B. fragilis on glucose, mannose, and other sugars. One kinase (RokA), a member of the ROK family of proteins, was found to be the sole kinase for activation of N-acetyl-D-glucosamine (NAG). The other kinase (HexA) is responsible for the majority of the glucose kinase activity in the cell, although a hexA deletion mutant strain was not defective for growth on any substrate tested. Deletion of both the rokA and hexA kinase genes resulted in inability of the cell to use glucose, mannose, NAG, and many other sugars. We purified RokA and determined its approximate molecular mass to be 36.5 kDa. The purified RokA protein was shown to phosphorylate several substrates, including glucose, NAG, and mannose, but not N-acetylmannosamine or N-acetylneuraminic acid. Phylogenetic analysis of RokA showed that it is most similar to kinases from the Cytophaga-Flavibacterium-Bacteroides group, while HexA was most similar to other bacterial hexokinases and eukaryotic hexokinases.The "nanaerobic" gram-negative organism Bacteroides fragilis (4) is capable of utilizing a wide variety of simple sugars and complex oligosaccharides as sources of carbon and energy for growth. This is a useful property for a bacterium that is both a gastrointestinal tract commensal and an opportunistic pathogen. In the large intestine, undigested dietary polysaccharides and host-derived glycoproteins, such as colonic mucin, can serve as growth substrates for B. fragilis (22). At sites of infection, the availability of host cell surface glycoproteins and glycolipids provides nutrients during the establishment phase and after abscess formation. As an example, the cell surface Lewis antigen contains galactose, mannose, N-acetyl-D-glucosamine (NAG), and N-acetylneuraminic acid (34, 46), all of which can serve as carbon and energy sources for B. fragilis. Godoy et al. (17) demonstrated that the B. fragilis neuraminidase (the product of the nanH gene) catalyzes the removal of terminal sialic acids from surface polysaccharides of CHO cells in monolayer cultures and in the rat granuloma pouch model system, both of which are glucose-limited growth conditions. Accordingly, ⌬nanH mutant strains are compromised for growth in these model systems. N-acetyl-D-glucosamine is an excellent carbon and energy source for B. fragilis and appears to be utilized more efficiently than glucose (8). Release of NAG from host glycoproteins and its transport into the cell can also influence anabolic cellular processes. NAG is a major component of the murein sacculus (21), and the ability to acquire or produce NAG affects cell wall product...

Sialic Acid ( N -Acetyl Neuraminic Acid) Utilization by Bacteroides fragilis Requires a Novel N -Acetyl Mannosamine Epimerase

“…Indeed, B. fragilis extracts containing NanE catalyzed the formation of NAG from manNAc ( Fig. 4A) and only formed NAG6-P if the extracts also contained the RokA kinase, which can phosphorylate NAG (6). In ⌬rokA extracts, only NAG could be detected; there was no production of NAG6-P (data not shown).…”

“…The reaction mixture contained ATP (2 mM), NADH (0.6 mM), phosphoenolpyruvate (5 mM), 1 l of pyruvate kinase (from rabbit muscle [EC 2.7.1.40], 1,351 U of activity/ml), 5 l of lactate dehydrogenase (12,500 U/ml), and 1 l of purified RokA protein (0.75 g [6]) in an assay buffer consisting of 0.1 M Tris-HCl (pH 7.0) and 0.01 M MgCl 2 . Purified NanE His6 protein and manNAc (final concentrations ranged from 33.5 M to 13.4 mM from a 0.67 M stock solution) were then added, and the reaction was monitored by measuring the decrease in the A 340 as NAD ϩ was formed from NADH at 25°C.…”

An Infant-associated Bacterial Commensal Utilizes Breast Milk Sialyloligosaccharides

“…While the ATCC15697 chromosome does not encode an epimerase with significant identity, ROK kinases appear in the sialic acid utilization cluster (Blon_0644) and upstream of nagA and nagB (Blon_0880) clustering with N-acylglucosamine 2-epimerase (pfam07221; Blon_0875). These kinases, and putative epimerase, may function in a similar manner to the B. fragilis system, mitigating the lack of a clearly identifiable nanK gene within the ATCC15697 chromosome (50).…”

Bacteroides: the Good, the Bad, and the Nitty-Gritty

“…Thus, they must have alternate ways of transporting sugars into the cell and attaching an active phosphate moiety. Recently, two broad-specificity hexokinases from B. fragilis were characterized, and their roles in hexose and NAG utilization were studied (31). These enzymes allow utilization of nutrients found in the gut (undigested dietary polysaccharides and host-derived glycoproteins) and at sites of infection (host cell surface antigens [including the Lewis antigen] and glycolipids) (31).…”