Nicotinamidase enzymes play a critical role in maintaining NAD
+ homeostasis and have been found to contribute to important biological processes in both prokaryotes and eukaryotes, including bacterial pathogenesis (
Anderson et al., 2003;
Balan et al., 2008;
Evans et al., 2010;
Frothingham et al., 1996;
Ghislain et al., 2002;
Hunt et al., 2007;
Kim et al., 2004;
Minard & McAlister-Henn, 2010;
Purser et al., 2003;
Raynaud et al., 1998;
Silva et al., 2009;
van der Horst et al., 2007;
Vrablik et al., 2009;
Wang & Pichersky, 2007). Because of the absence of nicotinamidases in mammals, these enzymes may be potential drug targets for mammalian pathogens (
Sauve, 2008), including the Lyme disease spirochaete
B. burgdorferi.
B. burgdorferi lacks the enzymes for
de novo biosynthesis of NAD
+ and must therefore rely on salvage of nicotinamide from the host for survival (
Casjens et al., 2000;
Fraser et al., 1997;
Purser et al., 2003). The
B. burgdorferi nicotinamidase, PncA, serves an essential physiological function required for survival of
B. burgdorferi throughout its infectious cycle (
Grimm et al., 2005;
Purser et al., 2003). However, in conflict with the essential
in vivo role of PncA, the current annotation of the
pncA ORF suggests that the encoded protein may lack catalytic activity due to the absence of an N-terminal aspartic acid that is conserved in the catalytic triad of nicotinamidase enzymes from other species (
French et al., 2010;
Purser et al., 2003). Using a genetic approach involving functional complementation of a
Salmonella mutant lacking nicotinamidase activity, we have found that addition of 48 nt of upstream flanking sequence to the annotated
pncA ORF results in production of an active nicotinamidase enzyme when expressed under the control of the
flaB promoter (). In contrast, the
pncA ORF as currently annotated, similarly expressed alone under the control of the
flaB promoter, did not yield nicotinamidase activity (). Furthermore, the plasmid carrying the
pncA ORF along with 48 nt of additional upstream sequence under the control of the
flaB promoter was capable of restoring mouse infectivity to spirochaetes lacking the entire lp25 plasmid. Recently, recombinant
B. burgdorferi PncA, expressed in and purified from
E. coli, was found to have poor catalytic activity (
French et al., 2010). Although the authors acknowledged that the current annotation of the
pncA ORF may be inaccurate and stated that additional upstream sequence was added to the
pncA gene in their expression plasmid, it remains unclear whether sufficient sequence was used to express enzymically active PncA (
French et al., 2010). We have identified a
pncA sequence that is sufficient to encode an active nicotinamidase enzyme, which supports
B. burgdorferi survival in the mouse. These data, as well as the
in vivo requirement for
pncA, raise the possibility of using exogenous expression of
pncA in spirochaetes lacking lp25 as an
in vivo genetic selection mechanism for shuttle-vector maintenance without the need for antibiotic selection. The requirement for
pncA in vivo is absolute, rather than the conditional requirement for antibiotic resistance, making
flaBp-
pncA a novel selection cassette with application in genetic studies
in vivo.
Purification of native PncA from
B. burgdorferi using a C-terminal FLAG-epitope tag followed by N-terminal sequencing determined that translation of
pncA initiates from the rare, non-canonical initiation codon AUU, which is 72 nt upstream of the current annotation for the start codon of the PncA protein (). The identified PncA start codon is 5 nt downstream of the putative ribosome-binding site and produces a 203 aa protein that harbours the N-terminal aspartic acid residue conserved in the catalytic triad of PncA homologues (). This is the first report of a
B. burgdorferi gene using AUU as the initiation codon. Bioinformatic analysis suggests that AUU is the initiation codon for the
pncA ORFs of all Lyme disease spirochaetes and that these ORFs are currently annotated incorrectly (). In contrast, the relapsing fever spirochaetes
Borrelia recurrentis and
Borrelia duttonii appear to initiate translation of
pncA using a canonical AUG initiation codon and are predicted to produce 204 aa PncA proteins, which include the conserved N-terminal aspartic acid residue (
Lescot et al., 2008) (). The few genes demonstrated to use an AUU initiation codon to initiate protein synthesis are bacterial
infC, which encodes initiation factor 3 (IF-3) (
Hu et al., 1993;
Liveris et al., 1993;
Pon & Gualerzi, 1986;
Sacerdot et al., 1996),
E. coli pcnB, which encodes poly(A) polymerase (PAPI) (
Binns & Masters, 2002),
orfB of bacterial transposable IS
3 element family member IS
911 (
Prère et al., 2011), gp26* of bacteriophage T4 (
Nivinskas et al., 1992) and a small number of eukaryotic genes (
Ivanov et al., 2008). Genes
infC and
pcnB are the only two examples in which AUU is the definitive initiation codon; the other examples use AUU as an alternative initiation codon in addition to a more conventional initiation site (
Binns & Masters, 2002).
Given the few characterized examples of AUU-initiated proteins, the biological significance of having an AUU start codon is unclear. Characterization of AUU-initiated translation of the
E. coli pcnB-encoded PAPI suggests a possible role for the non-canonical start codon in the control of protein production (
Binns & Masters, 2002). PAPI catalyses the template-independent addition of adenosine monophosphate to the 3′ ends of RNA species. Overproduction of
E. coli PAPI is deleterious for cell growth (
Binns & Masters, 2002;
Cao & Sarkar, 1992a,
b). Using experimentally modified genes, the efficiency of translation with AUU as an initiation codon has been found to be 1–13
% of that of AUG-initiated translation due to IF-3 initiation codon-mismatch discrimination and destabilization (
Sacerdot et al., 1996;
Sussman et al., 1996). Because of the reduced level of translation resulting from AUU initiation, the AUU initiation codon of
pcnB is believed to be the mechanism that limits PAPI synthesis (
Binns & Masters, 2002). We have not found overproduction of
B. burgdorferi PncA to be cytotoxic, nor has this been reported in the literature. No increase in
B. burgdorferi PncA production or change in spirochaete growth rate was observed when the AUU sequence was mutated to a canonical AUG (A. K. Linowski & M. W. Jewett, unpublished data). Elucidation of the role, if any, of the AUU initiation codon in translational control of
pncA is the focus of future studies.
B. burgdorferi is an A+T-rich organism with a G+C content of only 30 mol% (
Casjens et al., 2000;
Fraser et al., 1997). The codon-usage frequency of AUU in the
B. burgdorferi B31 genome is reported to be 55.7
%, compared with 18.1
% for AUG (
http://www.kazusa.or.jp/codon/). Further bioinformatics analysis is required to determine the frequency of AUU codons as putative initiation codons, rather than encoding an isoleucine residue. Our findings stress the importance of critical evaluation of genome annotation of ORFs of interest and highlight the possibility that additional AUU-initiated ORFs in the
B. burgdorferi genome may remain to be identified.
Together, our data have defined experimentally the complete amino acid sequence of the B. burgdorferi PncA protein. These findings provide a foundation for determination of the structure and catalytic function of this in vivo-essential protein that may contribute to rational design of nicotinamidase enzyme inhibitors that could serve as novel antimicrobials for the treatment of Lyme disease.
1,2 Sunny Jain,2 Angelika K. Linowski,2 Amit Sarkar,1 and Patricia A. Rosa1
This article has been 
