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Dynamic metabolic efficiency of substrate utilization by copiotrophic marine bacteria

ABSTRACT: Under well-defined laboratory conditions, we grew R. pomeroyi DSS-3 and A. macleodii MIT1002 in batch cultures on a monosaccharide (glucose) and organic acid (acetate), provided either individually or in combination, and all at the same carbon equivalent. This batch culturing approach mimicked bacterial DOC assimilation in short-lived substrate ‘hot spots’, such as those formed by high phytoplankton extracellular release at peak photon availability. Measurements were made of bacterial metabolite uptake, respiration, and biomass accumulation through a growth cycle. Insights into bacterial core metabolism came from gene and protein expression measured at intervals during growth. Curated genome-scale models (flux balance analysis; FBA) were used to explore the metabolic foundation of CO2 production for insights into determinants of BGE and bCUE.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Bacteria Ruegeria Pomeroyi Dss-3 Alteromonas Macleodii

DISEASE(S): Disease Free

SUBMITTER: Matthew McIlvin

LAB HEAD: Mak Saito

PROVIDER: PXD045824 | Pride | 2023-10-27

REPOSITORIES: Pride

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			Action	DRS
	221018_ccomp_10_MIT_1002_Gluc_4_Ace_8_B_T8_30_mL.mzid.gz	Mzid
	221018_ccomp_10_MIT_1002_Gluc_4_Ace_8_B_T8_30_mL.mzid_221018_ccomp_10_MIT_1002_Gluc_4_Ace_8_B_T8_30_mL.MGF	Mzid
	221018_ccomp_10_MIT_1002_Gluc_4_Ace_8_B_T8_30_mL.raw	Raw
	221018_ccomp_11_DSS3_Ace12_b_30ml.mzid.gz	Mzid
	221018_ccomp_11_DSS3_Ace12_b_30ml.mzid_221018_ccomp_11_DSS3_Ace12_b_30ml.MGF	Mzid

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Project description:C. jejuni, a spiral-shaped gram-negative bacterium, is a leading bacterial cause of human foodborne illness. Acute disease is associated with C. jejuni invasion of the intestinal epithelium. Further, maximal host cell invasion requires the secretion of proteins termed Campylobacter invasion antigens (Cia). As bile acids are known to alter the pathogenic behavior of other gastrointestinal pathogens, we hypothesized that the virulence potential of Campylobacter may be triggered by the bile acid deoxycholate (DOC). In support of this hypothesis, culturing C. jejuni with a physiologically relevant concentration of DOC significantly altered the kinetics of cell invasion as evidenced by gentamicin-protection assays. In contrast to C. jejuni harvested from Mueller-Hinton (MH) agar plates, C. jejuni harvested from MH agar plates supplemented with DOC demonstrated Cia secretion as judged by metabolic labeling experiments. DOC was also found to induce the expression of the ciaB gene as judged by B-galactosidase reporter assays and real-time RT-PCR. Microarray analysis revealed that DOC induced the expression of virulence genes (i.e., ciaB, cmeABC, dccR, and tlyA). In summary, we demonstrate that it is possible to enhance the pathogenic behavior of C. jejuni by modifying the culture conditions. These results provide a foundation to identify genes expressed by C. jejuni in response to in vivo-like culture conditions. Keywords: Stress response For the expression profiling arrays, an indirect comparison of gene expression was performed, where the expression profile of the C. jejuni F38011 cultured in the presence and absence of DOC was measured separately on different slides as described previously (26). Briefly, Cy5 labeled reference DNA from the C. jejuni F38011 strain was mixed with Cy3 labeled test cDNA (C. jejuni F38011 cultured in the presence or absence of DOC) and hybridized to the Campylobacter cDNA array (26) on separate slides. DNA microarrays were scanned using an Axon GenePix 4000B microarray laser scanner (Axon Instruments, Union City, CA) and the data for spot and background intensities were processed using the GenePix 4.0 software. To compensate for any effect of the amount of template and uneven Cy-dye incorporation, data normalization was performed as previously described (26). For the comparison of genes differentially expressed in the presence and absence of DOC,six hybridization measurements were generated per biological experiment (three technical replicate arrays and two replicate features per array).

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Dynamic metabolic efficiency of substrate utilization by copiotrophic marine bacteria

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