Persistence of
in the aquatic environment contributes to the fatal diarrheal disease cholera, which remains a global health burden. In the environment, bacteria face predation pressure by ...heterotrophic protists such as the free-living amoeba
. This study explores how a mutant of
adapts to acquire essential nutrients and survive predation. Here, we observed that up-regulation of iron acquisition genes and genes regulating resistance to oxidative stress enhances pathogen fitness. Our data show that
can defend predation to overcome nutrient limitation and oxidative stress, resulting in an enhanced survival inside the protozoan hosts.
Vibrio cholerae, the bacterial pathogen responsible for the diarrheal disease cholera, resides in the aquatic environment between outbreaks. For bacteria, genetic variation by lateral gene transfer ...(LGT) is important for survival and adaptation. In the aquatic environment, V. cholerae is predominantly found in biofilms associated with chitinous organisms or with chitin "rain". Chitin induces competency in V. cholerae, which can lead to LGT. In the environment, V. cholerae is also subjected to predation pressure by protist. Here we investigated whether protozoal predation affected LGT using the integron as a model. Integrons facilitate the integration of mobile DNA (gene cassettes) into the bacterial chromosome. We report that protozoal predation enhances transformation of a gene cassette by as much as 405-fold. We show that oxidative radicals produced in the protozoal phagosome induces the universal SOS response, which in turn upregulates the integron-integrase, the recombinase that facilitates cassette integration. Additionally, we show that during predation, V. cholerae requires the type VI secretion system to acquire the gene cassette from Escherichia coli. These results show that protozoal predation enhances LGT thus producing genetic variants that may have increased capacity to survive grazing. Additionally, the conditions in the food vacuole may make it a "hot spot" for LGT by accumulating diverse bacteria and inducing the SOS response helping drive genetic diversification and evolution.
Protozoan predation is one of the main environmental factors constraining bacterial growth in aquatic environments, and thus has led to the evolution of a number of defence mechanisms that protect ...bacteria from predation. These mechanisms may also function as virulence factors in infection of animal and human hosts. Whole transcriptome shotgun sequencing of Vibrio cholerae biofilms during predation by the amoebae, Acanthamoeba castellanii, revealed that 131 transcripts were significantly differentially regulated when compared to the non-grazed control. Differentially regulated transcripts included those involved in biosynthetic and metabolic pathways. The transcripts of genes involved in tyrosine metabolism were down-regulated in the grazed population, which indicates that the tyrosine metabolic regulon may have a role in the response of V. cholerae biofilms to A. castellanii predation. Homogentisate 1, 2-dioxygenase (HGA) is the main intermediate of the normal L-tyrosine catabolic pathway which is known to auto-oxidize, leading to the formation of the pigment, pyomelanin. Indeed, a pigmented mutant, disrupted in hmgA, was more resistant to amoebae predation than the wild type. Increased grazing resistance was correlated with increased production of pyomelanin and thus reactive oxygen species (ROS), suggesting that ROS production is a defensive mechanism used by bacterial biofilms against predation by amoebae A. castellanii.
The opportunistic pathogen Pseudomonas aeruginosa is ubiquitous in the environment, and in humans, it is capable of causing acute or chronic infections. In the natural environment, predation by ...bacterivorous protozoa represents a primary threat to bacteria. Here, we determined the impact of long-term exposure of P. aeruginosa to predation pressure. P. aeruginosa persisted when coincubated with the bacterivorous Acanthamoeba castellanii for extended periods and produced genetic and phenotypic variants. Sequencing of late-stage amoeba-adapted P. aeruginosa isolates demonstrated single nucleotide polymorphisms within genes that encode known virulence factors, and this correlated with a reduction in expression of virulence traits. Virulence for the nematode Caenorhabditis elegans was attenuated in late-stage amoeba-adapted P. aeruginosa compared to early-stage amoeba-adapted and nonadapted counterparts. Further, late-stage amoeba-adapted P. aeruginosa showed increased competitive fitness and enhanced survival in amoebae as well as in macrophage and neutrophils. Interestingly, our findings indicate that the selection imposed by amoebae resulted in P. aeruginosa isolates with reduced virulence and enhanced fitness, similar to those recovered from chronic cystic fibrosis infections. Thus, predation by protozoa and long-term colonization of the human host may represent similar environments that select for similar losses of gene function.
Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute infections in plants and animals, including humans, and chronic infections in immunocompromised and cystic fibrosis patients. This bacterium is commonly found in soils and water, where bacteria are constantly under threat of being consumed by bacterial predators, e.g., protozoa. To escape being killed, bacteria have evolved a suite of mechanisms that protect them from being consumed or digested. Here, we examined the effect of long-term predation on the genotypes and phenotypes expressed by P. aeruginosa. We show that long-term coincubation with protozoa gave rise to mutations that resulted in P. aeruginosa becoming less pathogenic. This is particularly interesting as similar mutations arise in bacteria associated with chronic infections. Importantly, the genetic and phenotypic traits possessed by late-stage amoeba-adapted P. aeruginosa are similar to those observed in isolates obtained from chronic cystic fibrosis infections. This notable overlap in adaptation to different host types suggests similar selection pressures among host cell types as well as similar adaptation strategies.
Corals host abundant microbial communities, or microbiomes, that play essential roles in the function of the coral holobiont. We examined the mucus microbiome in corals within the port of Singapore, ...where corals persist despite intense anthropogenic impacts. The coral mucus microbiomes of
Pectinia paeonia
and
Platygyra sinensis
at three reef sites were tracked by 16S rRNA gene amplicon sequencing from January 2019 to January 2020. Both coral species displayed spatial and temporal differences in microbiome composition, suggesting site specificity and seasonality in microbiome composition consistent with the monsoons. The temporal shifts in relative abundance of dominant taxa were different between the two coral species. Nonetheless, Proteobacteria was the most abundant phylum in both coral species and was reduced during the southwest (SW) monsoon, while Cyanobacteria and Crenarchaeota increased. The presumptive beneficial endosymbiont
Endozoicomonas
was only associated with corals at the reef site located the farthest from the Singapore mainland. The coral microbiomes reflected seasonal changes, while the seawater displayed distinct temporal microbial compositions and site-specific differentiation within all sampling dates. The persistence of coral reefs within the port of Singapore highlights the adaptive ability of corals to respond to stressful environments, and this study provides further evidence that a flexible microbiome could be an important part of the strategy employed by corals to remain resilient.
Quantitative real-time polymerase chain reaction (qRT-PCR) is a reliable technique for quantifying mRNA levels when normalised by a stable reference gene/s. Many putative reference genes are known to ...be affected by physiological stresses, such as nutrient limitation and hence may not be suitable for normalisation. In this study of Pseudomonas aeruginosa, the expression of 13 commonly used reference genes, rpoS, proC, recA, rpsL, rho, oprL, anr, tipA, nadB, fabD, ampC, algD and gyrA, were analysed for changes in expression under carbon starvation and nutrient replete conditions. The results showed that rpoS was the only stably expressed housekeeping gene during carbon starvation. In contrast, other commonly used housekeeping genes were shown to vary by as much as 10–100 fold under starvation conditions. This study has identified a suitable reference gene for qRT-PCR in P. aeruginosa during carbon starvation. The results presented here highlight the need to validate housekeeping genes under the chosen experimental conditions.
•Of thirteen putative housekeeping genes used previously for the normalisation of mRNA by qRT-PCR in Pseudomonas aeruginosa, only one gene was shown to be stable under carbon starvation conditions•The remaining putative housekeeping genes changed by 10–100 fold under starvation conditions•Housekeeping genes should be tested for stable expression under the particular conditions used, especially where physiological changes are being studied.
Vibrio vulnificus is an opportunistic pathogen that exhibits numerous virulence factors, including the secretion of a zinc metalloprotease and the production of a capsule. We have cloned and ...sequenced a gene from
V. vulnificus that is a homologue of the positive transcriptional regulator,
luxR, of the
lux operon in
Vibrio harveyi. This gene encodes a putative, single complete open reading frame designated
smcR, which shares greater than 75% nucleotide identity with
luxR of
V. harveyi. The deduced amino acid sequence of the putative SmcR protein is more than 90% identical and 95% similar to that of LuxR of
V. harveyi, suggesting that
V. vulnificus possesses a member of the family of signal–response genes recently described in
Vibrio cholerae and in
Vibrio parahaemolyticus. Our data also demonstrate that, in addition to
V. vulnificus, all six
Vibrio spp. tested contained genes that hybridized with the
luxR probe. We also present evidence that this regulatory protein was inherited from a common ancestor, and that the gene is ancient and widespread in marine
Vibrio spp.
Vibrio vulnificus is an opportunistic human pathogen and autochthonous inhabitant of coastal marine environments, where the bacterium is under constant predation by heterotrophic protists or ...protozoans. As a result of this selection pressure, genetic variants with antipredation mechanisms are selected for and persist in the environment. Such natural variants may also be pathogenic to animal or human hosts, making it important to understand these defense mechanisms. To identify antipredator strategies, 13 V. vulnificus strains of different genotypes isolated from diverse environments were exposed to predation by the ciliated protozoan Tetrahymena pyriformis, and only strain ENV1 was resistant to predation. Further investigation of the cell-free supernatant showed that ENV1 acidifies the environment by the excretion of organic acids, which are toxic to T. pyriformis. As this predation resistance was dependent on the availability of iron, transcriptomes of V. vulnificus in iron-replete and iron-deplete conditions were compared. This analysis revealed that ENV1 ferments pyruvate and the resultant acetyl-CoA leads to acetate synthesis under aerobic conditions, a hallmark of overflow metabolism. The anaerobic respiration global regulator
was upregulated when iron was available. An Δ
deletion mutant of ENV1 accumulated less acetate and, importantly, was sensitive to grazing by
. Based on the transcriptome response and quantification of metabolites, we conclude that ENV1 has adapted to overflow metabolism and has lost a control switch that shifts metabolism from acetate excretion to acetate assimilation, enabling it to excrete acetate continuously. We show that overflow metabolism and the acetate switch contribute to prey-predator interactions.
Bacteria in the environment, including
spp., interact with protozoan predators. To defend against predation, bacteria evolve antipredator mechanisms ranging from changing morphology, biofilm formation, and secretion of toxins or virulence factors. Some of these adaptations may result in strains that are pathogenic to humans. Therefore, it is important to study predator defense strategies of environmental bacteria. V. vulnificus thrives in coastal waters and infects humans. Very little is known about the defense mechanisms V. vulnificus expresses against predation. Here, we show that a V. vulnificus strain (ENV1) has rewired the central carbon metabolism, enabling the production of excess organic acid that is toxic to the protozoan predator
. This is a previously unknown mechanism of predation defense that protects against protozoan predators.
Mixed species biofilms exhibit increased tolerance to numerous stresses compared to single species biofilms. The aim of this study was to examine the effect of grazing by the heterotrophic protist, ...Tetrahymena pyriformis, on a mixed species biofilm consisting of Pseudomonas aeruginosa, Pseudomonas protegens, and Klebsiella pneumoniae. Protozoan grazing significantly reduced the single species K. pneumoniae biofilm, and the single species P. protegens biofilm was also sensitive to grazing. In contrast, P. aeruginosa biofilms were resistant to predation. This resistance protected the otherwise sensitive members of the mixed species biofilm consortium. Rhamnolipids produced by P. aeruginosa were shown to be the primary toxic factor for
However, a rhamnolipid-deficient mutant of P. aeruginosa (P. aeruginosa Δ
) maintained grazing resistance in the biofilm, suggesting the presence of at least one additional protective mechanism. P. aeruginosa with a deleted gene encoding the type III secretion system also resisted grazing. A transposon library was generated in the Δ
mutant to identify the additional factor involved in community biofilm protection. Results indicated that the Pseudomonas Quinolone Signal (PQS), a quorum sensing signaling molecule, was likely responsible for this effect. We confirmed this observation by showing that double mutants of Δ
and genes in the PQS biosynthetic operon lost grazing protection. We also showed that PQS was directly toxic to
. This study demonstrates that residing in a mixed species biofilm can be an advantageous strategy for grazing sensitive bacterial species, as P. aeruginosa confers community protection from protozoan grazing through multiple mechanisms.
Biofilms have been shown to protect bacterial cells from predation by protists. Biofilm studies have traditionally used single species systems, which have provided information on the mechanisms and regulation of biofilm formation and dispersal, and the effects of predation on these biofilms. However, biofilms in nature are comprised of multiple species. To better understand how multispecies biofilms are impacted by predation, a model mixed-species biofilm was here exposed to protozoan predation. We show that the grazing sensitive strains
and
gained associational resistance from the grazing resistant P. aeruginosa. Resistance was due to the secretion of rhamnolipids and quorum sensing molecule PQS. This work highlights the importance of using mixed species systems.
Predation by heterotrophic protists drives the emergence of adaptive traits in bacteria, and often these traits lead to altered interactions with hosts and persistence in the environment. Here we ...studied adaptation of the cholera pathogen, Vibrio cholerae during long-term co-incubation with the protist host, Acanthamoeba castellanii. We determined phenotypic and genotypic changes associated with long-term intra-amoebal host adaptation and how this impacts pathogen survival and fitness. We showed that adaptation to the amoeba host leads to temporal changes in multiple phenotypic traits in V. cholerae that facilitate increased survival and competitive fitness in amoeba. Genome sequencing and mutational analysis revealed that these altered lifestyles were linked to non-synonymous mutations in conserved regions of the flagellar transcriptional regulator, flrA. Additionally, the mutations resulted in enhanced colonisation in zebrafish, establishing a link between adaptation of V. cholerae to amoeba predation and enhanced environmental persistence. Our results show that pressure imposed by amoeba on V. cholerae selects for flrA mutations that serves as a key driver for adaptation. Importantly, this study provides evidence that adaptive traits that evolve in pathogens in response to environmental predatory pressure impact the colonisation of eukaryotic organisms by these pathogens.
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