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Campylobacter jejuni causes acute gastroenteritis worldwide and is transmitted primarily through poultry, in which it is often a commensal member of the intestinal microbiota. Previous transcriptome sequencing (RNA-Seq) experiment showed that transcripts from an operon encoding a high-affinity phosphate transporter (PstSCAB) of C. jejuni were among the most abundant when the bacterium was grown in chickens. Elevated levels of the pstSCAB mRNA were also identified in an RNA-Seq experiment from human infection studies. In this study, we explore the role of PstSCAB in the biology and colonization potential of C. jejuni. Our results demonstrate that cells lacking PstSCAB survive poorly in stationary phase, in nutrient-limiting media, and under osmotic conditions reflective of those in the chicken. Polyphosphate levels in the mutant cells were elevated at stationary phase, consistent with alterations in expression of polyphosphate metabolism genes. The mutant strain was highly attenuated for colonization of newly hatched chicks, with levels of bacteria at several orders of magnitude below wild-type levels. Mutant and wild type grew similarly in complex media, but the pstS::kan mutant exhibited a significant growth defect in minimal medium supplemented with l-lactate, postulated as a carbon source in vivo. Poor growth in lactate correlated with diminished expression of acetogenesis pathway genes previously demonstrated as important for colonizing chickens. The phosphate transport system is thus essential for diverse aspects of C. jejuni physiology and in vivo fitness and survival. IMPORTANCE Campylobacter jejuni causes millions of human gastrointestinal infections annually, with poultry a major source of infection. Due to the emergence of multidrug resistance in C. jejuni, there is need to identify alternative ways to control this pathogen. Genes encoding the high-affinity phosphate transporter PstSCAB are highly expressed by C. jejuni in chickens and humans. In this study, we address the role of PstSCAB on chicken colonization and other C. jejuni phenotypes. PstSCAB is required for colonization in chicken, metabolism and survival under different stress responses, and during growth on lactate, a potential growth substrate in chickens. Our study highlights that PstSCAB may be an effective target to develop mechanisms for controlling bacterial burden in both chicken and human.

Inorganic polyphosphate (polyP) is synthesized by bacteria under stressful environmental conditions and acts by a variety of mechanisms to promote cell survival. While the kinase that synthesizes polyP (PPK, encoded by the ppk gene) is well known, ppk transcription is not activated by environmental stress and little is understood about how environmental stress signals lead to polyP accumulation. Previous work has shown that the transcriptional regulators DksA, RpoN (σ54) and RpoE (σ24) positively regulate polyP production, but not ppk transcription, in Escherichia coli . In this work, we examine the role of the alternative sigma factor RpoN and nitrogen starvation stress response pathways in controlling polyP synthesis. We show that the RpoN enhancer binding proteins GlnG and GlrR impact polyP production, and uncover a new role for the nitrogen phosphotransferase regulator PtsN (EIIANtr) as a positive regulator of polyP production, acting upstream of DksA, downstream of RpoN and apparently independently of RpoE. However, neither these regulatory proteins nor common nitrogen metabolites appear to act directly on PPK, and the precise mechanism(s) by which polyP production is modulated after stress remain(s) unclear. Unexpectedly, we also found that the genes that impact polyP production vary depending on the composition of the rich media in which the cells were grown before exposure to polyP-inducing stress. These results constitute progress towards deciphering the regulatory networks driving polyP production under stress, and highlight the remarkable complexity of this regulation and its connections to a broad range of stress-sensing pathways.

Many bacterial species, including Escherichia coli (E. coli) utilize the enzyme polyphosphate kinase (PPK) to synthesize polyphosphate (polyP) in response to biological stress. Multiple studies have shown that impairing PPK activity impairs bacterial pathogenicity and survival, which has made it a target for therapeutic development. Unfortunately, PPK regulation is poorly understood. To address this, we previously described a series of mutations in E. coli ppk, termed ppk*, that result in high polyP accumulation in vivo. However, the specific activity of PPK* enzyme in vitro, when purified using a C‐term 6X His‐tag (PPK*‐HT), was comparable to purified WT enzyme (PPK‐HT). PPK activity is tied to its oligomeric state, with the dimeric form synthesizing polyP. To determine if the ppk* mutation altered oligomerization, we tested PPK‐HT and PPK*‐HT enzymes in an analytical ultracentrifuge. We found that the PPK‐HT enzyme was mostly dimeric but had a small monomeric population. Conversely, the PPK*‐HT enzyme was exclusively dimeric. This suggested that the ppk* mutation may affect oligomerization, but the His‐tag masked any effect on activity. We next hypothesized that the C‐term His tag alters the in vitro activity. To test this, we compared the specific activities of PPK‐HT and PPK*‐HT to that of PPK purified using a smaller C‐term C tag (PPK‐CT) and PPK enzyme purified with no tags (PPK‐NT). PPK*‐HT and PPK‐HT had comparable activities in vitro but were significantly more active than the untagged enzyme. PPK‐CT and PPK‐NT had comparable activities. This suggests that the C‐tag does not alter PPK activity while the His‐tag does. We next tested the purified enzymes’ sensitivity to substrate by testing PPK activity in 6mM and 20mM ATP. We found that the His‐tag increased the enzyme’s activity in the higher ATP concentrations. Comparing the 20mM and 6mM samples, the His‐tag enzymes showed an increase in activity of ~3.5 fold compared to their 6mM conditions. Conversely, the PPK‐CT and PPK‐UT only showed a 1.4‐ and 1.9‐ fold increase in activity, respectively. PolyP synthesis impairs bacterial growth and replication. We hypothesized that a more active PPK would impair recovery following a prolonged stationary phase. We developed strains carrying one of four expression vectors: an empty vector control (EV), PPK‐HT, PPK‐CT, or native PPK. Using these four strains, we developed a bacterial growth recovery curve assay. We found that while the PPK‐CT and untagged PPK strains had comparable recovery and growth rates, the PPK‐HT strain had a slower recovery rate resulting in a later shift into log phase. Taken together, our results have significant implications for polyP biology. We have shown that the standard purification method produces an enzyme with altered substrate sensitivities, making it a poorer candidate for drug development studies. C‐tag enzyme is comparable to untagged enzyme, making it a reliable candidate for future studies. Finally, the in vitro differences produce an in vivo phenotype, making it easy to test ...

Many bacterial species, including Escherichia coli (E. coli), utilize the enzyme polyphosphate kinase (PPK) to synthesize polyphosphate (polyP) in response to biological stresses. Multiple studies have shown that impairing PPK activity impairs bacterial pathogenicity and survival. Further, no PPK homolog has been identified in mammalian species, making it a promising therapeutic target. While the structure, kinetics, and mechanism of PPK is well‐characterized, its regulation is poorly understood. We previously described a series of mutations in E. coli ppk, termed ppk*, that result in high polyP accumulation in vivo. However, the specific activity of PPK* enzyme in vitro, when purified using a C‐term 6X His‐tag (PPK*‐HT), was comparable to purified WT enzyme (PPK‐HT). Previous reports have indicated that N‐term modifications impair PPK activity. We hypothesized that the C‐term His tag alters the in vitro activity. To test this, we compared the specific activities of PPK purified using a smaller C‐term C tag (PPK‐CT) and the C‐term His tag enzymes to a natively purified PPK enzyme with no tags and a detagged PPK purified using the N‐term Profinity tag (PPK*‐DT and PPK‐DT). We found that the PPK*‐HT and PPK‐HT maintained comparable activities, but were significantly more active than the other purified enzymes. PPK‐CT and native PPK had comparable activities, suggesting the smaller C‐tag does not alter PPK activity. Both PPK*‐DT and PPK‐DT had drastically reduced activity, likely due to two N‐term residues left behind by the Profinity elution process. PPK*‐DT had very low activity, while the PPK‐DT enzyme had no detectable activity, suggesting that the PPK* mutation does increase the specific activity in the absence of a C‐term His tag. As polyP synthesis is heavily involved in bacterial stress responses that reduce growth and replication under unfavorable conditions, we hypothesized that a more active PPK would impair recovery following a prolonged stationary phase. To determine if the C‐term modifications affect bacterial growth and recovery, we developed strains carrying one of four expression vectors: an empty vector control (EV), PPK‐HT, PPK‐CT, or native PPK. Using these strains, we measured the effect of tagged PPK on recovery from stationary phase over 24 hours. We found that while the PPK‐CT and native PPK strains had comparable recovery and and shifted into log phase at roughly the same time, the PPK‐CT strain had a modestly slower recovery resulting in a later shift into log phase. Importantly, the empty vector control had the fastest recovery rate, which is consistent with our understanding of polyP synthesis acting to impair growth. Taken together, our results have significant implications for the field of polyP biology. We have shown that the current field‐wide purification system produces a more active PPK, which results in both in vivo and in vitro differences compared to native PPK. In the future we will focus on understanding how these modifications affect PPK's activity, whether the termini repres...