Anaerobic choline metabolism in microcompartments promotes growth and swarming of Proteus mirabilis - PubMed (original) (raw)
Anaerobic choline metabolism in microcompartments promotes growth and swarming of Proteus mirabilis
Eleanor Jameson et al. Environ Microbiol. 2016 Sep.
Abstract
Gammaproteobacteria are important gut microbes but only persist at low levels in the healthy gut. The ecology of Gammaproteobacteria in the gut environment is poorly understood. Here, we demonstrate that choline is an important growth substrate for representatives of Gammaproteobacteria. Using Proteus mirabilis as a model, we investigate the role of choline metabolism and demonstrate that the cutC gene, encoding a choline-trimethylamine lyase, is essential for choline degradation to trimethylamine by targeted mutagenesis of cutC and subsequent complementation experiments. Proteus mirabilis can rapidly utilize choline to enhance growth rate and cell yield in broth culture. Importantly, choline also enhances swarming-associated colony expansion of P. mirabilis under anaerobic conditions on a solid surface. Comparative transcriptomics demonstrated that choline not only induces choline-trimethylamine lyase but also genes encoding shell proteins for the formation of bacterial microcompartments. Subsequent analyses by transmission electron microscopy confirmed the presence of such novel microcompartments in cells cultivated in liquid broth and hyper-flagellated swarmer cells from solid medium. Together, our study reveals choline metabolism as an adaptation strategy for P. mirabilis and contributes to better understand the ecology of this bacterium in health and disease.
© 2015 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.
Figures
Figure 1
Neighbour‐joining phylogenetic tree, constructed from amino acid sequences of glycyl radical enzymes. The choline trimethylamine‐lyase,
CutC
, has been further split into two distinct sub‐clusters, type I and type II as previously described by Martínez‐del
C
ampo and colleagues (2015). Association of the
CutC
clusters with different glycyl radical enzyme‐containing microcompartment (
GRM
) loci is given in bracketed bales, as defined by Axen and colleagues (2014). The cut
C
genes corresponding to type I and II.b sub‐clusters are approximately 2.5 kb and include the functionally characterized cut
C
of
D
. desulfuricans. Type II.a genes include the cut
C
of
P
. mirabilis and are approximately 3.4 kb in length. The other sequence clusters represent characterized glycyl radical enzymes, except the cluster labelled ‘Unknown
CPFL
’ which has an unknown function (Lehtiö and Goldman, 2004). Bootstrap values > 70% are indicated on the nodes (1000 replicates). The scale bar indicates evolutionary distance in mutations per residue.
Figure 2
A. Quantification of trimethylamine (
TMA
) produced from recombinant
E
. coli carrying the codon‐optimized
P
. mirabilis cut
D
gene and either wild‐type cut
C
(first two bars) or a glycyl radical site
CutC
mutant (
G
1126
A
, third bar) during incubations in the defined medium supplemented with 1 mM choline chloride. Error bars represent standard deviations from three replicate cultures. B–D. Choline degradation and
TMA
production overlaid with growth in anaerobic P. mirabilis culture. B.
P
roteus mirabilis wild‐type, C.
P
roteus mirabilis cut
C
::kan mutant and D.
P
roteus mirabilis cut
C
::kan mutant complemented with native cut
CD
.
Figure 3
A. Anaerobic growth of
P
. mirabilis in liquid broth cultures in a defined medium at 37°C. The available carbon sources for
P
. mirabilis are indicated in the legend: glucose and choline (dashed 
), glucose only (solid 
), glycerol and choline (dashed 
), glycerol only (solid 
) and choline only (solid 
). Error bars represent standard deviation for three replicate cultures. B. Cumulative anaerobic swarm radiuses of
P
. mirabilis, incubated at 30°C, inoculated from an aerobic stationary phase culture. Error bars show standard deviation for three replicate plates. The carbon sources on the swarming agar plates are glucose and choline (dashed 
), glucose only (solid 
), glycerol and choline (dashed 
) or glycerol only (solid 
). Maximum swarm radius is 42 mm on petri dishes. C–F. Anaerobic swarming pattern of
P
. mirabilis on agar plates supplemented with (C) glycerol and choline (10 days growth), (D) glycerol (10 days growth), (E) glucose and choline (6 days growth) and (F) glucose (10 days growth).
Figure 4
Cumulative anaerobic swarming radiuses of
P
. mirabilis. Error bars show standard deviation for three replicate plates, grown on glycerol or glycerol plus choline for (A) wild‐type, (B) cut
C
::kan mutant and (C) cut
C
::kan mutant complemented with native cut
CD
.
Figure 5
A. Transmission electron microscopy (
TEM
) micrographs of
P
. mirabilis wild‐type cultivated anaerobically in a defined medium supplemented with (a) glucose, (b) choline or (c) glucose plus choline as the sole carbon sources. B. Transmission electron microscopy micrographs of cut
C
::kan mutant (a, d) and the complemented mutant (b, c, e, f) cultivated anaerobically on choline or glucose plus choline as the sole carbon sources. Microcompartments are indicated by white arrows.
EPS
, extracellular polysaccharides; *indicates protein aggregation; as indicates aberrant microcompartment structures.
Figure 6
Transmission electron microscopy micrographs of
P
. mirabilis swarming cells. The swarming agar was supplemented with (A) glycerol, (B) choline or (C) glycerol plus choline as the sole carbon sources. Microcompartments are indicated by white arrows.
References
- Armitage, J.P. (1981) Changes in metabolic activity of _Proteus mirabili_s during swarming. J Gen Microbiol 125: 445–450. - PubMed
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