The Lifestyle of Vibrio cholerae Fosters Gene Transfers
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FEATURE ARTICLE
The Lifestyle of Vibrio cholerae
Fosters Gene Transfers
Growing on chitinous surfaces helps these bacteria to initiate horizontal gene
transfer and, perhaps, to swap pathogenic traits
Melanie Blokesch
Waterborne Vibrio cholerae bacteria cause chol- hotspots for transformation that foster the evolu-
era, a pandemic during the last 50 years across tion of V. cholerae and other vibrios. In a broader
Asia, Africa, and Latin America. Although most sense, by learning more about the environmental
infected individuals do not develop severe symp- lifestyle of V. cholerae, we can better understand
toms, some become violently ill with severe diar- the mechanisms by which this seemingly innoc-
rhea, vomiting, and cramps, and the loss of body uous microbe becomes a harmful human patho-
fluids, if untreated, can lead to shock and death. gen of great importance. This knowledge, in turn,
V. cholerae, which is primarily encountered in may help us in predicting the onset of cholera
estuaries, rivers, and coastal waters in its environ- outbreaks and facilitate efforts to control them.
mental reservoir, is found not only in a free-living
state but also associated with phytoplankton and
zooplankton (Fig. 1). Because factors such as in- V. cholerae in the Environment
creasing temperatures, the El Niño phenomenon, The epicenter of past cholera pandemics is the
and heavy rainfalls and floods can raise the abun- delta area where the Ganges and Brahmaputra
dance of phytoplankton and zooplankton, and, Rivers flow into the Bay of Bengal. In this region
along with them, cholera bacteria, changing cli- and elsewhere, V. cholerae is often found attached
matic conditions could well drive the re-emer- to small crustaceans and their molted exoskele-
gence of cholera, according to Rita Colwell from tons, and it emerges periodically as a human
the University of Maryland and her collaborators. pathogen. Such exoskeletons are made primarily
Moreover, V. cholerae’s strong association of chitin, a homopolymer of branched chains of
with the chitinous exoskeletons of zooplankton, -1,4-linked N-acetyl-D-glucosamine (GlcNAc).
which serves as a source of nutrition but also During the last decade, researchers realized
contributes to the onset of natural competence in that the link between this microbe and chitin
these bacteria, could mean that chitin surfaces are surfaces is of relevance to public health. As V.
cholerae forms biofılms on such chitin surfaces,
the bacteria concentrate, reaching levels of up to
SUMMARY 104 to 106 bacteria on single copepods (a domi-
➤ The human pathogen Vibrio cholerae and other Vibrio species are chitino- nant member of the zooplankton). Ingesting a
lytic bacteria that contribute to the recycling of carbon and nitrogen in few of those small crustaceans can be suffıcient to
aquatic environments. cause disease in humans. Notably, chitin also in-
➤ V. cholerae bacteria form biofilms on chitinous surfaces, enabling the creases the bacterium’s resistance to acid, mean-
bacteria to initiate a developmental program known as natural competence ing fewer bacteria are killed while passing the
for genetic transformation, a mode of horizontal gene transfer. stomach, thus increasing the chances of establish-
➤ Chitin-induced DNA exchange and transformations along such surfaces ing infections. Hence, drinking a single glass of
might contribute to the spread of pathogenic traits among different types contaminated water during a zooplankton bloom
of V. cholerae. in cholera-endemic regions can lead to the onset
➤ V. cholerae can grow on crab shells or on other chitin-containing materials of disease.
in the lab, making it possible to determine what signals regulate these Knowledge of the environmental lifestyle of
natural gene-exchange processes. this pathogen and how it is transmitted to hu-
64 • Microbe—Volume 9, Number 2, 2014
FEATURE ARTICLE
FIGURE 1
V. cholerae in the environment and the chain of transmission (based on work by Rita Colwell and collaborators). In
aquatic environments, V. cholerae is encountered as a free-living bacterium or in association with phyto- and
zooplankton. Changing factors like hours of sunlight, water temperatures, climate conditions, and human behavior
contribute to plankton blooms and, consequently, to elevated numbers of V. cholerae. Transmission to humans
most likely occurs through the ingestion of small crustaceans or their molts along with their accumulated bacterial
passengers. Scale bars: 1 m (left image), 3 m (right image).
mans can help in decreasing cholera cases. For in marine environments— equal to cellulose on
example, Rita Colwell and her collaborators in- land. The recycling of chitin by V. cholerae and
troduced a simple fıltration method, using folded other bacteria helps to maintain the global carbon
sari cloth to remove particles larger than 20 m, and nitrogen cycle, according to the late Saul
including zooplankton, from drinking water. Roseman of Johns Hopkins University in Balti-
Trial use of this method during 3 years in 65 more, Md., who pioneered research on chitin
Bangladeshi villages with a total population of degradation by the Vibrionaceae.
133,000 reduced cholera cases by 48% compared Colonization of chitinous surfaces by V. chol-
to a control group. erae is a consequence of (i) sensing and che-
motaxis; (ii) attachment and biofılm formation;
and can lead to (iii) chitin-induced natural com-
V. cholerae Associates with
petence, leading to DNA uptake and transforma-
Chitinous Surfaces
tion; and (iv) evolution, if new genes are acquired
Marine zooplankton produce more than 1011 (Fig. 2). The bacteria can secrete chitinases that
tons of chitin annually, providing a huge sub- degrade chitin throughout all these steps.
strate on which V. cholerae can attach and form There are two classes of chitinases: exochiti-
biofılms, protecting them against external stress- nases, which release diacetylchitobiose [GlcNAc)2]
es. V. cholerae not only binds to chitinous exo- units from the nonreducing end of the poly-
skeletons; it also plays an important role in saccharide chain, and endochitinases, which pro-
remineralizing chitin, preventing much of this duce multimers of GlcNAc by randomly cleaving
material from ever reaching the ocean floor even the glycosidic bonds within the chitin polymer,
though chitin is the most abundant polymer eventually releasing free oligosaccharides.
Microbe—Volume 9, Number 2, 2014 • 65
FEATURE ARTICLE
FIGURE 2
Chitin-dependent processes that V. cholerae undergoes. Marine vibrios, including V. cholerae, are chitinolytic
bacteria that can produce and sense diacetylchitobiose [(GlcNAc)2] gradients surrounding chitinous surfaces. After
chemotaxis towards the chitin surface, the bacteria attach, form microcolonies, and then develop into three-
dimensional biofilms. Under those chitin-dependent, high cell-density conditions, V. cholerae initiates natural
competence for genetic transformation, a mode of horizontal gene transfer, enabling the bacteria to take up free
DNA from the environment and to incorporate this material into its own chromosome by homologous
recombination. If the transforming DNA material carries a different O-antigen gene cluster (shown in green as part
of the transforming DNA), V. cholerae can exchange this region, thereby undergoing serogroup conversion (e.g.,
change of the O-antigen, illustrated on the right).
Once attached to the chitin surface by a type the V. cholerae genome and is transferred to
IV pilus (mannose-sensitive hemagglutinin pi- new strains via phage transduction, according
lus) and the GlcNAc-binding protein GbpA, the to Matthew Waldor and John Mekalanos at
bacteria form microcolonies that develop into Harvard Medical School in Cambridge, Mass. In
biofılms, according to Ronald Taylor and col- addition, its two pathogenicity islands as well
leagues at Dartmouth College in Hanover, N.H. as the Vibrio seventh pandemic islands, the
Maturation of biofılms involves both protein gene cluster encoding the O-antigen region of
components and exopolysaccharides, both of the lipopolysaccharide (LPS), an integrative and
which help to form the matrix that holds bacterial conjugative element, and other regions of the
cells within these biofılms, according to Fitnat two chromosomes bear hallmarks of recent gene
Yildiz at the University of California, Santa Cruz, acquisitions.
and Paula Watnick at Boston Children’s Hospi- Several mechanisms enable bacteria such as V.
tal, and their collaborators. Biofılm-forming V. cholerae to obtain DNA from external sources.
cholerae attach to chitinous crab shell fragments For example, chitin induces natural competence
via assorted fıbers and fılaments, according to our for genetic transformation in V. cholerae, accord-
scanning electron micrographs (Fig. 3). ing to work done by Gary Schoolnik and his col-
laborators at Stanford University in Stanford,
Calif., in which I also participated. Natural com-
Horizontal Gene Transfers among
petence, a mode of HGT, refers to the physiolog-
V. cholerae in Aquatic Environments
ical state in which a bacterium can take up free
How did V. cholerae gain pathogenic traits in the DNA from the environment and incorporate that
fırst place? The genome sequence of V. cholerae DNA into its genome via homologous recombi-
and genomic hybridization experiments suggest nation. Chitin also induces natural competence
horizontal gene transfers. For example, the chol- in other species of the genus Vibrio such as V.
era toxin phage CTX resides as a prophage in fıscheri, V. parahaemolyticus, and V. vulnifıcus.
66 • Microbe—Volume 9, Number 2, 2014
FEATURE ARTICLE
AUTHOR PROFILE
Blokesch: from Dolphins and Scuba to Vibrios and HGT
Melanie Blokesch grew up in a village near Munich, Germany. technology—talked with their daughters about science
She remembers always being fascinated by marine animals “whether it was about supernovae, lightning and faraday
and especially by dolphins and sharks, while being on holi- cages, steam engines, or hurricanes,” although her mother
days, an interest she indulged by swimming, snorkeling, and stopped short of encouraging her daughter’s first biological
scuba diving. Later, she found a way to integrate this passion experiments of collecting frogs. “She made me bring them
with her current microbiology research. “What I do now when back,” Blokesch recalls.
I am on vacation is take back some water samples and see She also credits a high school math teacher for fostering
what kind of Vibrios I can isolate” she says. her interest in science. “[The teacher] recommended me to
Blokesch, 37, is a tenure-track assistant professor at the attend an information day on girls in science,” Blokesch says.
Global Health Institute of the Swiss Federal Institute of “I wanted to study math after high school but wasn’t sure
Technology in Lausanne (EPFL), where she focuses on bacteria what I could do with it afterwards. So I ended up with biology
that are facultative human pathogens. “Time will tell whether and got so fascinated by bacteria and by what they are able
the knowledge on how and when new pathogens emerge will to do that I stayed in microbiology.” An important mentor was
help fight infectious diseases,” she says. “However, for sure, her thesis advisor August Böck, who always appreciated and
horizontal gene transfer (HGT)—the main topic of my research trusted her work.
group— does play a major role in the spread of antibiotic She earned her biology diploma in 2000 and her Ph.D. in
resistances. Thus, knowing what initiates HGT and the mech- microbiology in 2004, both from Ludwig-Maximilians-Univer-
anistic aspects of it is of prime importance.” sity in Munich. Between 2005 and 2009, she was a postdoc-
Blokesch studies Vibrio cholerae, the aquatic bacterium toral fellow doing research in the Microbiology and Immunol-
that causes cholera. “The goal of our research is to link the ogy department at Stanford University, after which she moved
ecology of the organism with the epidemiology of the disease to her current post in Switzerland. “I very much enjoyed my
in order to learn more about the pathogen’s evolution,” she time in California,” she says “but returned to Europe to be
says. “I often refer to the ‘three E’s’ when I talk about our closer to my family.”
research: ecology, evolution, and epidemiology.” Blokesch drew even closer to her older sister and father
Blokesch, the younger of two girls, believes her scientific following her mother’s recent death. Now that she is living in
curiosity comes from her mother, who worked as a chemistry Switzerland, she has rediscovered skiing and also plays bad-
lab technician before starting a family, but who never had the minton every week, enjoys squash, and continues to go scuba
opportunity to pursue her own scientific interests. “Unfortu- diving when plans allow, she says.
nately, she wasn’t encouraged by her own parents to follow
her dream and enter medical school,” Blokesch says of her Marlene Cimons
mother. Both parents— her father worked in information Marlene Cimons lives and writes in Bethesda, Md.
V. cholerae Associates with and Responds Although V. cholerae encompasses more than
to Chitin under Laboratory Conditions 200 different serogroups specifıed by the O-anti-
The chitin polymer’s insolubility complicates ef- gen region of the LPS, only strains belonging to
forts to study how V. cholerae associates with it. the O1 serogroup were thought to cause severe
As one approach, we use Dungeness crab shells, cholera. In the 1990s, a V. cholerae that converted
which we clean, break into small pieces, and ster- from the El Tor O1 serogroup to the new O139
ilize by autoclaving. V. cholerae readily colonize Bengal serogroup caused cholera outbreaks in
such surfaces when they are submerged in an Bangladesh and India. This unprecedented event
artifıcial sea water medium within 12-well plates was followed by spread of the O139 serogroup
(Fig. 4). We also use commercially available strain to other countries and fear of a new eighth
coarse chitin flakes aliquoted into tubes to better cholera pandemic.
standardize experimental procedure and to lower To test whether V. cholerae O1 can acquire
the risk of cross-contamination. Moreover, to the O139 gene cluster by chitin-induced natural
visualize bacterial attachment to chitin surfaces, transformation under laboratory conditions, we
we use chitin beads, which are amenable to light propagated a V. cholerae O1 El Tor strain as a
microscopy (Fig. 4). biofılm on crab shell fragments and then added
Microbe—Volume 9, Number 2, 2014 • 67FEATURE ARTICLE
FIGURE 3
V. cholerae forms biofilms on chitinous surfaces. Biofilm-forming V. cholerae cells bound to chitinous surfaces are
illustrated in these scanning electron micrographs. In the lower row bacteria that are attached to chitinous spikes
(porous looking surface) are shown at higher magnification, which allows the visualization of detailed structures
such as the polar flagellum, unidentified pili/fibers, and biofilm matrix components (as indicated by the white
arrows). Scale bars: 3 m (upper image), 0.3 m (lower images).
genomic DNA from the O139 Bengal strain as crobes. Indeed, because the O-antigen clusters
transforming material. We then selected for O1- of different serogroups of V. cholerae display a
to-O139 transformants by using a virulent bacte- GC-content that deviates from the rest of the
riophage specifıc for the O1 serogroup to carry chromosome, chitin-induced natural transfor-
out serogroup-specifıc, phage-mediated killing. mation could well be responsible for the diversity
The resulting phage-resistant transformants of natural V. cholerae serogroups. Moreover,
were genotypically identical to the O1 recipient because serogroup changes in toxigenic V. chol-
except for the acquired O139 serogroup gene erae strains, as observed for the O139 sero-
cluster (42 kbp) and the lost O1-specifıc gene group, might reoccur at any time, diagnostic tools
cluster (32 kbp). Testing with immunological and should take this scenario into consideration.
electron microscopy-based methods showed that
the serogroup convertants produce the new O-
Regulatory Network Drives Chitin-Induced
antigen.
Transformation in V. cholerae
These results provide a mechanistic explana-
tion for the appearance of the O139 Bengal vari- Our recent research focuses on the regulatory
ant of V. cholerae in 1992. More generally, they network that drives natural competence and
illustrate how genetic and ecological factors can transformation in V. cholerae. Although V. chol-
lead to the emergence of new pathogenic mi- erae cells respond uniformly to soluble chitin oli-
68 • Microbe—Volume 9, Number 2, 2014FEATURE ARTICLE
FIGURE 4
Colonization of chitin surfaces by V. cholerae under laboratory conditions. We and other researchers grow V.
cholerae on crab shell fragments (upper row), which first are cleaned to remove residual crabmeat, fragmented into
smaller size pieces, and autoclaved. Those sterilized crab shell fragments typically are incubated with the pathogen
in artificial seawater medium for several hours to days. Alternatively, we use sterilized chitin coarse flakes, which
provide an increased surface space, allowing growth to higher cell densities and more standardized experimental
approaches (middle row). Finally, chitin beads are useful for visualizing (fluorescently-labeled) V. cholerae cells that
associate with the chitin surface (lower row). With such chitin beads, we visualized both transcriptionally (e.g. a
competence gene fused to gfp; left image) and translationally (e.g. a competence protein fused to mCherry; right
image) fused reporter constructs.
gomers, competence induction around insoluble nal for intraspecies communication. System 2 is
biotic chitinous surfaces is nonsynchronized used for interspecies communication, which de-
and heterogeneous. Because the chitin surface pends on autoinducer 2 (AI-2), a furanosyl borate
can serve as the sole carbon source for V. chol- diester.
erae, we investigated the link between carbon How do these environmental signals foster
catabolite repression and chitin-induced natural natural competence and transformation? First,
transformation. Indeed, both colonization of the we learned that QS regulates only a small subset
chitin surface and induction of natural compe- of competence genes and that this regulation oc-
tence depend on the secondary messenger cyclic curs via a transcription factor that links chitin-
adenosine monophosphate (cAMP) in V. chol- induction and QS. More precisely, QS mediates a
erae. Moreover, quorum sensing (QS) also con- switch from DNA degradation around V. chol-
tributes to the onset of natural transformation in erae cells towards competence-mediated DNA
V. cholerae. uptake. Further, chitin-induced natural transfor-
Through QS, bacteria synthesize autoinducer mation depends on the species-specifıc signal,
(AI) molecules and secrete them into the envi- CAI-1, whereas the interspecies autoinducer
ronment to measure and respond to population AI-2 plays a negligible role. Importantly, when
density. Briefly, V. cholerae depends on two QS both autoinducers are absent, natural transfor-
circuits, according to Bonnie Bassler at Princeton mation is abolished.
University and her collaborators. The system 1 These results illustrate how V. cholerae can
cholera autoinducer 1 (CAI-1) serves as the sig- enhance species-specifıc DNA uptake, namely
Microbe—Volume 9, Number 2, 2014 • 69FEATURE ARTICLE
by coupling gene expression required for natural conversion of Vibrio cholerae in aquatic reservoirs.
transformation to the species-specifıc QS system PLoS Pathogens 3:e81.
1. This linkage could prove helpful in repairing Colwell, R. R., A. Huq, M. S. Islam, K. M. Aziz, M.
Yunus, N. H. Khan, A. Mahmud, R. B. Sack, G. B.
damaged genes. To follow up on this assumption
Nair, J. Chakraborty, D. A. Sack, and E. Russek-
we are now also studying the mechanistic aspects
Cohen. 2003. Reduction of cholera in Bangladeshi
of the DNA uptake process in V. cholerae. villages by simple fıltration. Proc. Natl. Acad. Sci USA
Melanie Blokesch is an Assistant Professor at the Swiss Federal 100:1051–1055.
Institute of Technology Lausanne (EPFL), Switzerland, Keyhani, N. O., and S. Roseman. 1999. Physiological
melanie.blokesch@epfl.ch aspects of chitin catabolism in marine bacteria.
Biochim. Biophys. Acta 1473:108 –122.
Lipp, E. K., A. Huq, and R. R. Colwell. 2002. Effects of
Acknowledgments global climate on infectious disease: the cholera mod-
el. Clin. Microbiol. Rev. 15:757–770.
I thank Graham Knott (BioEM facility of EPFL) for his
Lo Scrudato, M., and M. Blokesch. 2012. The regulatory
help and enthusiasm with the electron microscopy. I also
network of natural competence and transformation of
acknowledge all current and former members of the
Vibrio cholerae. PLoS Genetics 8: e1002778.
Blokesch lab for their scientifıc contributions. Finally, I
Meibom*, K. L., M. Blokesch*, N. A. Dolganov, C. Y.
thank Gary Schoolnik at Stanford University for intro-
Wu, and G. K. Schoolnik. 2005. Chitin induces natu-
ducing me into this fascinating topic. Research in the
ral competence in Vibrio cholerae. Science 310:1824 –
Blokesch laboratory is supported by grants from the Swiss 1827. (* contributed equally)
National Science Foundation (31003A_127029 and Ng, W. L., and B. L. Bassler. 2009. Bacterial quorum-
31003A_143356) and by a Starting Grant from the Euro- sensing network architectures. Annu. Rev. Genet. 43:
pean Research Council (VIR4ENV). 197–222.
Seitz, P., and M. Blokesch. 2013. Cues and regulatory
Suggested Reading pathways involved in natural competence and trans-
formation in pathogenic and environmental Gram-
Blokesch, M. 2012. Chitin colonization, chitin degrada- negative bacteria. FEMS Microbiol. Rev. 37:336 –363.
tion and chitin-induced natural competence of Vibrio (Epub, September 2012)
cholerae are subject to catabolite repression. Environ. Seitz, P., and M. Blokesch. 2013. DNA-uptake machin-
Microbiol. 14: 1898 –1912. ery of naturally competent Vibrio cholerae. Proc. Natl.
Blokesch, M., and G. K. Schoolnik. 2007. Serogroup Acad. Sci. USA, 110:17987–92.
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