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Comparative microaerobic growth of the C. jejuni wild-type (solid circles), canB mutant (open circles) and complemented canB strain (open squares) under 5% v/v CO2 (A) and 1% v/v CO2 (B) in the gas atmosphere. C. jejuni starter cultures were grown overnight in MHS broth under microaerobic conditions with 5% v/v CO2, before being inoculated in fresh, pre-warmed MHS broth equilibrated overnight with the indicated gas atmospheres. The data points represent the mean ± SEM of at least three independent growth experiments.
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Campylobacter jejuni, the leading cause of human bacterial gastroenteritis, requires low environmental oxygen and high carbon dioxide for optimum growth, but the molecular basis for the carbon dioxide requirement is unclear. One factor may be inefficient conversion of gaseous CO2 to bicarbonate, the required substrate of various carboxylases. Two p...
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... effect of deleting canB on the growth of C. jejuni NCTC 11168 was monitored at low (1% v/v) and high (5% v/v) CO2 conditions. Figure 3 compares the growth of wild-type, canB and a complemented strain under these two gas atmospheres. In the higher CO2 atmosphere (Fig. 3A), although the final cell yields of all strains were similar with an OD600 of ∼1.0, the canB mutant showed a slower growth rate than the wild-type, with approximate Gene organization and expression analysis of cj0229 and canB. A. Gene organization and neighbourhood of the C. jejuni NCTC 11168 chromosome containing potential CA encoding genes cj0229 and canB (cj0237). ...
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... effect of deleting canB on the growth of C. jejuni NCTC 11168 was monitored at low (1% v/v) and high (5% v/v) CO2 conditions. Figure 3 compares the growth of wild-type, canB and a complemented strain under these two gas atmospheres. In the higher CO2 atmosphere (Fig. 3A), although the final cell yields of all strains were similar with an OD600 of ∼1.0, the canB mutant showed a slower growth rate than the wild-type, with approximate Gene organization and expression analysis of cj0229 and canB. A. Gene organization and neighbourhood of the C. jejuni NCTC 11168 chromosome containing potential CA encoding ...
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... times of 4 h and 2.5 h respectively. Complemen- tation with the wild-type gene restored the mutant to a similar growth rate to the wild-type (Fig. 3A). Under the low CO2 (1% v/v) conditions, the wild-type and comple- mented strain both showed a similar longer doubling time of about 3 h, whereas the canB mutant did not show any significant growth during the experiment (Fig. 3B). These results clearly indicate that CanB plays a vital role in CO2 homeostasis in C. jejuni, especially ...
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... h respectively. Complemen- tation with the wild-type gene restored the mutant to a similar growth rate to the wild-type (Fig. 3A). Under the low CO2 (1% v/v) conditions, the wild-type and comple- mented strain both showed a similar longer doubling time of about 3 h, whereas the canB mutant did not show any significant growth during the experiment (Fig. 3B). These results clearly indicate that CanB plays a vital role in CO2 homeostasis in C. jejuni, especially under low CO2 ...
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Citations
... Both naked and test-forming foraminifers have β-CA (of the five nonhomologous gene families, α to ζ) (Fig. 8). The physiological role of β-CA is poorly understood, although assays in bacteria indicate that it provides HCO 3 − for oxaloacetate synthesis (59). In eukaryotes, both in silico predictions and experiments in fungi and fruit flies have shown that β-CAs localize to mitochondria (55,60). ...
Foraminifera, the most ancient known calcium carbonate-producing eukaryotes, are crucial players in global biogeochemical cycles and well-used environmental indicators in biogeosciences. However, little is known about their calcification mechanisms. This impedes understanding the organismal responses to ocean acidifi-cation, which alters marine calcium carbonate production, potentially leading to biogeochemical cycle changes. We conducted comparative single-cell transcriptomics and fluorescent microscopy and identified calcium ion (Ca 2+) transport/secretion genes and α-carbonic anhydrases that control calcification in a foraminifer. They actively take up Ca 2+ to boost mitochondrial adenosine triphosphate synthesis during calcification but need to pump excess intracellular Ca 2+ to the calcification site to prevent cell death. Unique α-carbonic anhydrase genes induce the generation of bicarbonate and proton from multiple CO 2 sources. These control mechanisms have evolved independently since the Precambrian to enable the development of large cells and calcification despite decreasing Ca 2+ concentrations and pH in seawater. The present findings provide previously unknown insights into the calcification mechanisms and their subsequent function in enduring ocean acidification.
... Among the ASVs that were more abundant in the high-treatment samples at later time points were members of the order Campylobacterales. Most species of this order are capnophiles that thrive in high-CO 2 environments (Al-Haideri et al. 2016;Waite et al. 2017). ASVs in the genus Vibrio initially had low abundances in the high-CO 2 treatment, but at later time points they were significantly more abundant in the high-CO 2 treatment than in the control. ...
Host‐associated microbial communities are fundamental to host physiology, yet it is unclear how these communities will respond to environmental disturbances. Here, we disentangle the environment‐linked and host‐linked effects of ocean acidification on oyster‐associated microbial communities. We exposed adult oysters (Crassostrea virginica) to CO2‐induced ocean acidification (400 vs. 2800 ppm) for 80 d. We measured the oyster extrapallial fluid pH and sampled the gills for microbial analysis at six time points. We found that different subsets of microbes were linked to acidification (n = 34 amplicon sequence variants [ASVs]) and to host response (n = 20 ASVs) with little overlap (n = 8 ASVs), suggesting that some members of the oyster microbiome were more responsive to environmental conditions while others were more tightly linked to host condition. Our results provide insight into which members of the oyster microbiome may contribute to the health and resistance of their host, and which members are the most vulnerable to changing environmental conditions.
... mrsB (cj1112c) encoding a methionine sulphoxide reductase, was shown to protect C. jejuni against oxidative and nitrosative stress 35 . Furthermore, canB displayed C. jejuni sequences in the hybrid strains, encoding carbonic anhydrase, an enzyme important for growth at low CO 2 concentrations 36 . A further oxidoreductase (Cj0833c) and genes encoding for the Ni/Fe hydrogenase small subunit hydA (Cj1267c) and hydA2 (Cj1399c) as well as nadD (Cj1404) involved in the synthesis of the redox cofactor NAD + were found to harbour C. jejuni sequences. ...
Campylobacter is the major bacterial agent of human gastroenteritis worldwide and represents a crucial global public health burden. Species differentiation of C. jejuni and C. coli and phylogenetic analysis is challenged by inter-species horizontal gene transfer. Routine real-time PCR on more than 4000 C. jejuni and C. coli field strains identified isolates with ambiguous PCR results for species differentiation, in particular, from the isolation source eggs. K-mer analysis of whole genome sequencing data indicated the presence of C. coli hybrid strains with huge amounts of C. jejuni introgression. Recombination events were distributed over the whole chromosome. MLST typing was impaired, since C. jejuni sequences were also found in six of the seven housekeeping genes. cgMLST suggested that the strains were phylogenetically unrelated. Intriguingly, the strains shared a stress response set of C. jejuni variant genes, with proposed roles in oxidative, osmotic and general stress defence, chromosome maintenance and repair, membrane transport, cell wall and capsular biosynthesis and chemotaxis. The results have practical impact on routine typing and on the understanding of the functional adaption to harsh environments, enabling successful spreading and persistence of Campylobacter.
... For this, a CA gene seems to be essential since species that need high carbon dioxide partial pressures (capnophiles) often have no detectable CA activity and some have lost CA genes (Ueda et al. 2008;Ueda et al. 2012). For the capnophile Campylobacter jejuni, it was shown that it contains a CA that is only active at high pH, but not under normal physiological pH (Al-Haideri et al. 2016). Also, CA deletion mutants often can only grow under high carbon dioxide partial pressures (Kusian et al. 2002;Merlin et al. 2003;Kumar et al. 2013), making them functional capnophiles. ...
... Two putative CA genes, Caut-bCA and Caut-gCA, were identified in the genome of C. autoethanogenum as potential members of the βand γ-classes of CA. However, a low sequence similarity of Caut-bCA with other members of the β-CA and a reported lack of CA activity for several γ-CA homologs Al-Haideri et al. 2016;Kaur et al. 2010;Ferry 2010), did not allow to ascribe CA function to either of these genes based on gene sequence alone. Neither transcription profile nor genomic contexts of the identified genes revealed a specific function for these genes. ...
Carbonic anhydrase catalyses the interconversion of carbon dioxide and water to bicarbonate and protons. It was unknown if the industrial-relevant acetogen Clostridium autoethanogenum possesses these enzymes. We identified two putative carbonic anhydrase genes in its genome, one of the β class and one of the γ class. Carbonic anhydrase activity was found for the purified β class enzyme, but not the γ class candidate. Functional complementation of an Escherichia coli carbonic anhydrase knock-out mutant showed that the β class carbonic anhydrase could complement this activity, but not the γ class candidate gene. Phylogenetic analysis showed that the β class carbonic anhydrase of Clostridium autoethanogenum represents a novel sub-class of β class carbonic anhydrases that form the F-clade. The members of this clade have the shortest primary structure of any known carbonic anhydrase.
... Species that need high carbon dioxide partial pressures (capnophiles) often have no detectable CA activity and some have lost CA genes (246,247). In the capnophile Campylobacter jejuni a CA was found only active at high pH, but not active under normal physiological pH (248). ΔCA mutants often can only grow under high carbon dioxide partial pressures (220,249,250) making them functional capnophiles. ...
... The assay is based on previously published methods (253)(254)(255)(256)and provides an alternative to stop-flow devices that allows for reasonable accurate high-throughput measurements. We have validated the assay using bovine CA and C. jejuni β-CA CanB (248), kindly supplied by D.J. Kelly of the University of Sheffield. The assay buffer was 50mM HEPES, 50mM NaSO4, 50 mM MgSO4, 0.004 % (w/v) phenol red at several pH values. ...
... Caut-gCA seems to be a typical member of the γ-CA family based both on sequence alignment comparison (figure 3-5) and phylogenetic analysis. Like some other members of this family (241,248,259,260), it did not show CA activity and therefore no extensive further sequence and phylogenetic analysis was performed on this protein. ...
Clostridium autoethanogenum is an anaerobic, facultative autotrophic bacterium that was isolated from rabbit faces in the last decennium of the twentieth century. It is used to convert carbon monoxide rich waste gas in to compounds such as acetate, ethanol, 2,3-butanediol and lactate. Carbon dioxide reacts with water to form carbonic acid and bicarbonate. This reaction is catalysed by enzymes called carbonic anhydrases. It was unknown if these enzymes were present in C. autoethanogenum. Genes encoding putative carbonic anhydrases were cloned and heterologous expressed. One gene encoded an active enzyme of a novel sub-clade of β-carbonic anhydrases. This gene was disrupted in the genome of C. autoethanogenum. The mutant was unable to grow at low pH and low carbon dioxide concentrations. Production of ethanol and 2,3-butanediol by WT C. autoethanogenum in carbon monoxide fed chemostat cultures was improved by employing phosphate limitation. A pilot study on the effect of phosphate limitation on rhamnose based growth showed 1,2-propanol and 1-propanol as native products of C. autoethanogenum. Acetolactate is the metabolic branch point for both branched chain amino acid and 2,3-butanediol production. An acetolactate synthase gene was deleted. The resulting mutant shows a subtle growth difference in media containing amino acids. Finally the strength of a series of heterologous promoters was determined in C. autoethanogenum. The research presented in this thesis improves our knowledge on C. autoethanogenum’s metabolism and offers tools to optimise it for product formation. This will enable improved exploitation of this organism for a carbon neutral future.
... For example, low oxygen concentrations (e.g., 5% O 2 ) and high growth temperatures (e.g., 37∼42 • C) are needed for the optimal growth of C. jejuni (Davis and DiRita, 2008). As a capnophile, additionally, C. jejuni requires CO 2 , and carbonic anhydrase that is encoded by canB contributes to C. jejuni growth under low (such as 1%) CO 2 conditions (Al-Haideri et al., 2016). ...
Campylobacter is a leading foodborne pathogen worldwide. Biofilm formation is an important survival mechanism that sustains the viability of Campylobacter under harsh stress conditions. Iron affects biofilm formation in some other bacteria; however, the effect of iron on biofilm formation has not been investigated in Campylobacter. In this study, we discovered that ferrous (Fe²⁺) and ferric (Fe³⁺) iron stimulated biofilm formation in Campylobacter jejuni. The sequestration of iron with an iron chelator prevented the iron-mediated biofilm stimulation. The level of total reactive oxygen species (ROS) in biofilms was increased by iron. However, the supplementation with an antioxidant prevented the total ROS level from being increased in biofilms by iron and also inhibited iron-mediated biofilm stimulation in C. jejuni. This suggests that iron promotes biofilm formation through oxidative stress. Based on the results of fluorescence microscopic analysis, Fe²⁺ and Fe³⁺ enhanced both microcolony formation and biofilm maturation. The levels of extracellular DNA and polysaccharides in biofilms were increased by iron supplementation. The effect of iron on biofilm formation was also investigated with 70 C. jejuni isolates from raw chicken. Regardless of the inherent levels of biofilm formation, iron stimulated biofilm formation in all tested strains; however, there were strain variations in iron concentrations affecting biofilm formation. The biofilm formation of 92.9% (65 of 70) strains was enhanced by either 40 μM Fe²⁺ or 20 μM Fe³⁺ or both (the iron concentrations that enhanced biofilm formation in C. jejuni NCTC 11168), whereas different iron concentrations were required to promote biofilms in the rest of the strains. The findings in this study showed that Fe²⁺ and Fe³⁺ contributed to the stimulation of biofilm formation in C. jejuni through oxidative stress.
... The input CO 2 concentration varied from 2% v/v to 10% v/v over the range of oxygen inputs used. Based on our previous work (Al-Haideri et al., 2016), 2% v/v CO 2 is in excess of the cells growth requirements. The culture volume was 885 ml, the temperature was maintained at 378C by a thermostatic water jacket, the gas sparging rate was 0.5 l min 21 with a stirring rate of 350 rpm and the pH was maintained at 7 6 0.1 with automatic addition of 1 M NaOH or H 2 SO 4 . ...
... The same steady-state samples as used for the microarray analysis were also used for RT-PCR of five selected genes that showed up or downregulation. Procedures for RNA isolation, purification and analysis by RT-PCR using the gyrA gene for normalisation, followed the standard methods described in our previous work (Al-Haideri et al., 2016). The primers used are shown in Supporting Information Table S1. ...
Campylobacter jejuni, the most frequent cause of food-borne bacterial gastroenteritis worldwide, is a microaerophile that has to survive high environmental oxygen tensions, adapt to oxygen limitation in the intestine and resist host oxidative attack. Here, oxygen-dependent changes in C. jejuni physiology were studied at constant growth rate using carbon (serine)-limited continuous chemostat cultures. We show that a perceived aerobiosis scale can be calibrated by the acetate excretion flux, which becomes zero when metabolism is fully aerobic (100% aerobiosis). Transcriptome changes in a downshift experiment from 150% to 40% aerobiosis revealed many novel oxygen-regulated genes and highlighted re-modelling of the electron transport chains. A label-free proteomic analysis showed that at 40% aerobiosis, many proteins involved in host colonisation (e.g. PorA, CadF, FlpA, CjkT) became more abundant. PorA abundance increased steeply below 100% aerobiosis. In contrast, several citric-acid cycle enzymes, the peptide transporter CstA, PEB1 aspartate/glutamate transporter, LutABC lactate dehydrogenase and PutA proline dehydrogenase became more abundant with increasing aerobiosis. We also observed a co-ordinated response of oxidative stress protection enzymes and Fe-S cluster biogenesis proteins above 100% aerobiosis. Our approaches reveal key virulence factors that respond to restricted oxygen availability and specific transporters and catabolic pathways activated with increasing aerobiosis. This article is protected by copyright. All rights reserved.
... In the intestine, however, C. jejuni may be able to acquire the CO 2 generated as a catabolic end product from the fermentation activity of the intestinal microbiota or directly from the host [69]. Various CO 2 -incorporating metabolic reactions require hydrogen carbonate (HCO 3 -) instead of gaseous CO 2 ( Fig 6A), and the hydration of CO 2 to HCO 3 depends in C. jejuni on the carbonic anhydrase CanB (CJJ81176_0262) (Fig 6B), which promotes its growth in vitro [70]. Besides facilitating the carboxylation reactions, the generated HCO 3 might be required for maintaining the intracellular pH homeostasis [71]. ...
... Inactivation of pycA resulted in a slight but significant reduction in 13 C-labeling of the detected amino acids, mainly Asp (Fig 6D), demonstrating the redundancy in HCO 3 incorporation by the carboxylating enzymes in C. jejuni (Fig 6A). The inability of the C. jejuni pycA mutant to metabolize HCO 3 led to a reduction in its in vitro growth, especially when cultivated with Ser and lactate as sole energy source (Fig 6E), similar to what has been previously described for the canB mutant [70]. However, the pyc mutants showed no significant colonization defect in mice ( Fig 6A), indicating that the amount of OAA, generated through the catabolism of growth substrates that fuel the TCA cycle, is sufficient for its gluconeogenic requirements. ...
Campylobacter jejuni is one of the leading infectious causes of food-borne illness around the world. Its ability to persistently colonize the intestinal tract of a broad range of hosts, including food-producing animals, is central to its epidemiology since most infections are due to the consumption of contaminated food products. Using a highly saturated transposon insertion library combined with next-generation sequencing and a mouse model of infection, we have carried out a comprehensive genome-wide analysis of the fitness determinants for growth in vitro and in vivo of a highly pathogenic strain of C. jejuni. A comparison of the C. jejuni requirements to colonize the mouse intestine with those necessary to grow in different culture media in vitro, combined with isotopologue profiling and metabolic flow analysis, allowed us to identify its metabolic requirements to establish infection, including the ability to acquire certain nutrients, metabolize specific substrates, or maintain intracellular ion homeostasis. This comprehensive analysis has identified metabolic pathways that could provide the basis for the development of novel strategies to prevent C. jejuni colonization of food-producing animals or to treat human infections.
... Carbonic anhydrase enzymes catalyze the reversible hydration of CO 2 to bicarbonate and can be divided into at least six different phylogenetic groups, with varying expressions across kingdoms (Bury-Moné et al., 2008;Al-Haideri et al., 2015). These groups have similar function but differing structures, which are believed to have arisen by convergent evolution (Smith and Ferry, 2000). ...
Campylobacter contaminated poultry meat is a major source of human foodborne illness. Campylobacter coli strain OR12 is a robust colonizer of chickens that was previously shown to outcompete and displace other Campylobacter strains from the chicken’s gastrointestinal tract. This strain is capable of aerobic growth on blood agar. Serial aerobic passage increased this aerotolerance as assessed by quantitative assays for growth and survival on solid media. Aerotolerance was also associated with increased peroxide stress resistance. Aerobic passage did not alter cellular morphology or motility or hinder the microaerobic growth rate. Colonization of broiler chickens by aerotolerant C. coli OR12 was significantly lower than the wild-type strain at 3 days after challenge but not by 7 days, suggesting adaptation had occurred. Bacteria recovered from chickens had retained their aerotolerance, indicating this trait is stable. Whole genome sequencing enabled comparison with the wild-type sequence. Twenty-three point mutations were present, none of which were in genes known to affect oxidative stress resistance. Insertions or deletions caused frame shifts in several genes including, phosphoglycerate kinase and the b subunit of pyruvate carboxylase that suggest modification of central and carbohydrate metabolism in response to aerobic growth. Other genes affected include those encoding putative carbonic anhydrase, motility accessory factor, filamentous haemagglutinin, and aminoacyl dipeptidase proteins. Aerotolerance has the potential to affect environmental success and survival. Increased environmental survival outside of the host intestinal tract may allow opportunities for transmission between hosts. Resistance to oxidative stress may equate to increased virulence by virtue of reduced susceptibility to oxidative free radicals produced by host immune responses. Finally, resistance to ambient atmospheric oxygen may allow increased survival on chicken skin, and therefore constitutes an increased risk to public health.
... Supporting this, even epsilonproteobacterial genera that associate with terrestrial animals, such as Campylobacter, Helicobacter and Wolinella, retain physiological characteristics reflective of a hydrothermal environment where they most likely evolved. These include a need for high CO 2 (Al-Haideri et al., 2016), intolerance to high levels of O 2 (Kendall et al., 2014), and the ability to use hydrogen as an electron donor (Wolin et al., 1961). Below, the underlying geochemistry of deep-sea vents and its effect on the growth conditions of the microbes inhabiting this habitat are discussed. ...
... Another important and poorly understood phenomenon is the capnophilic (CO 2 -loving) nature of Epsilonproteobacteria. As mentioned above, heterotrophic Campylobacter strains require high levels of CO 2 (≈ 10% v/v in headspace gas), which may be related to the activity of carbonic anhydrases that convert CO 2 to bicarbonate for carboxylation reactions (Al-Haideri et al., 2016). Previous studies have shown that oxygen tolerance may be enhanced when CO 2 above atmospheric levels is provided (Bolton and Coates, 1983). ...
Chemoautotrophic ecosystems at deep-sea hydrothermal vents were discovered in 1977, but not until 1995 were free-living autotrophic Epsilonproteobacteria identified as important microbial community members. Because the deep-sea is food-starved, the autotrophic metabolism of hydrothermal vent Epsilonproteobacteria may be very important for deep-sea consumers. However, quantifying their metabolic activities in situ has remained difficult, and biochemical mechanisms underlying their autotrophic physiology are poorly described. To gain insight into environmental processes, an approach was developed for incubations of microbes at in situ pressure and temperature (25 MPa, 24°C) with various combinations of electron donors/acceptors (H₂ , O₂ and NO₃- and ¹³HCO₃-) as a tracer to track carbon fixation. During short (18-24 h) incubations of low-temperature vent fluids from Crab Spa (9°N East Pacific Rise), the concentration of electron donors/acceptors and cell numbers were monitored to quantify microbial processes. Measured rates were generally higher than previous studies, and the stoichiometry of microbially-catalyzed redox reactions revealed new insights into sulfur and nitrogen cycling. Single-cell, taxonomically-resolved tracer incorporation showed Epsilonproteobacteria dominated carbon fixation, and their growth efficiency was calculated based on electron acceptor consumption. Using these data, in situ primary productivity, microbial standing stock, and average biomass residence time of the deep-sea vent subseafloor biosphere were estimated. Finally, the population structures of the most abundant genera Sulfurimonas and Thioreductor were shown to be strongly influenced by pO₂ and temperature respectively, providing a mechanism for niche differentiation in situ. To gain insights into the core biochemical reactions underlying autotrophy in Epsilonprotebacteria, a theoretical metabolic model of Sulfurimonas denitrificans was developed. Validated iteratively by comparing in silico yields with data from chemostat experiments, the model generated hypotheses explaining critical, yet so far unresolved reactions supporting chemoautotrophy in Epsilonproteo bacteria. For example, it provides insight into how energy is conserved during sulfur oxidation coupled to denitrification, how reverse electron transport produces ferredoxin for carbon fixation, and why aerobic growth yields are only slightly higher compared to denitrification. As a whole, this thesis provides important contributions towards understanding core mechanisms of chemoautrophy, as well as the in situ productivity, physiology and ecology of autotrophic Epsilonproteobacteria.
