eDNA-stimulated cell dispersion from Caulobacter crescentus biofilms upon oxygen limitation is dependent on a toxin-antitoxin system.

In their natural environment, most bacteria preferentially live as complex surface-attached multicellular colonies called biofilms. Biofilms begin with a few cells adhering to a surface, where they multiply to form a mature colony. When conditions deteriorate, cells can leave the biofilm. This dispersion is thought to be an important process that modifies the overall biofilm architecture and that promotes colonization of new environments. In Caulobacter crescentus biofilms, extracellular DNA (eDNA) is released upon cell death and prevents newborn cells from joining the established biofilm. Thus, eDNA promotes the dispersal of newborn cells and the subsequent colonization of new environments. These observations suggest that eDNA is a cue for sensing detrimental environmental conditions in the biofilm. Here, we show that the toxin-antitoxin system (TAS) ParDE4 stimulates cell death in areas of a biofilm with decreased O2 availability. In conditions where O2 availability is low, eDNA concentration is correlated with cell death. Cell dispersal away from biofilms is decreased when parDE4 is deleted, probably due to the lower local eDNA concentration. Expression of parDE4 is positively regulated by O2 and the expression of this operon is decreased in biofilms where O2 availability is low. Thus, a programmed cell death mechanism using an O2-regulated TAS stimulates dispersal away from areas of a biofilm with decreased O2 availability and favors colonization of a new, more hospitable environment.

Bacteria are more social than what had long been expected. While they can swim around on their own, most of them in fact settle down as part of a surface-bound community. The plaque on our teeth and the gooey deposit in our bathroom pipes are the visible results of this communal lifestyle. Inside this slimy 'biofilm', cells share resources and are protected from toxic substances such as antibiotics. However, being tied to one spot is not always a good thing: it may be advantageous for a cell in a biofilm to strike out on its own and resume 'single life' if local conditions deteriorate. Caulobacter crescentus bacteria do not always have this choice, as adult cells in this species become permanently glued into place upon joining a biofilm. When these divide, however, their daughters have a choice: swim away, or stick with the group. Previous research has shown that this decision is influenced by the health of the community. Dying cells release DNA fragments which disable the structures allowing newborn cells to adhere to the environment, and a high mortality rate in the biofilm therefore forces unattached cells to leave the colony. Berne et al. wanted to build on these results and examine how exactly cells die in the biofilm. In particular, the deaths could be sudden and random, with the bacteria succumbing to injury; or they could result from cells activating one of their built-in self-destruct programs. To investigate this question, genetically modified C. crescentus bacteria were grown in the laboratory and exposed to different environments. Combining genetic and microscopic approaches revealed that as a biofilm becomes too crowded, certain individuals self-destruct via a cell death program known as the toxin-antitoxin system. Further experiments showed that low oxygen availability was the signal that triggered self-destruction. Drops in oxygen levels can happen when the environment becomes hostile or when a colony is overpopulated. The results by Berne et al. therefore suggest that by triggering self-destruction in certain members of the community, reduced oxygen access leads to newborn cells swimming away, which in turn prevents further overcrowding and allows new, more hospitable locations to be colonized. Biofilms are of growing interest in a wide range of human industries, but they also present formidable challenges. This is particularly the case in healthcare, as they tend to infest medical devices and help disease-causing species to resist treatments. Understanding how bacteria are encouraged to join or leave their colony is necessary to better control biofilms to our advantage.

The HmrABCX pathway regulates the transition between motile and sessile lifestyles in Caulobacter crescentus by a HfiA-independent mechanism.

Through its cell cycle, the bacterium Caulobacter crescentus switches from a motile, free-living state, to a sessile surface-attached cell. During this coordinated process, cells undergo irreversible morphological changes, such as shedding of their polar flagellum and synthesis of an adhesive holdfast at the same pole. In this work, we used genetic screens to identify genes involved in the regulation of the motile to sessile lifestyle transition. We identified a predicted hybrid histidine kinase that inhibits biofilm formation and activates the motile lifestyle: HmrA (Holdfast and motility regulator A). Genetic screens and genomic localization led to the identification of additional genes that regulate the proportion of cells harboring an active flagellum or a holdfast and that form a putative phosphorelay pathway with HmrA. Further genetic analysis indicates that the Hmr pathway is independent of the holdfast synthesis regulator HfiA and may impact c-di-GMP synthesis through the diguanylate cyclase DgcB pathway. Finally, we provide evidence that the Hmr pathway is involved in the regulation of sessile-to-motile lifestyle as a function of environmental stresses, namely excess copper and non-optimal temperatures.

The Two Chemotaxis Clusters in Caulobacter crescentus Play Different Roles in Chemotaxis and Biofilm Regulation.

The holdfast polysaccharide adhesin is crucial for irreversible cell adhesion and biofilm formation in Caulobacter crescentus Holdfast production is tightly controlled via developmental regulators, as well as via environmental and physical signals. Here, we identify a novel mode of regulation of holdfast synthesis that involves chemotaxis proteins. We characterized the two identified chemotaxis clusters of C. crescentus and showed that only the previously characterized major cluster is involved in the chemotactic response toward different carbon sources. However, both chemotaxis clusters encoded in the C. crescentus genome play a role in biofilm formation and holdfast production by regulating the expression of hfiA, the gene encoding the holdfast inhibitor HfiA. We show that CheA and CheB proteins act in an antagonistic manner, as follows: while the two CheA proteins negatively regulate hfiA expression, the CheB proteins are positive regulators, thus providing a modulation of holdfast synthesis and surface attachment.IMPORTANCE Chemosensory systems constitute major signal transduction pathways in bacteria. These systems are involved in chemotaxis and other cell responses to environment conditions, such as the production of adhesins to enable irreversible adhesion to a surface and surface colonization. The C. crescentus genome encodes two complete chemotaxis clusters. Here, we characterized the second novel chemotaxis-like cluster. While only the major chemotaxis cluster is involved in chemotaxis, both chemotaxis systems modulate C. crescentus adhesion by controlling expression of the holdfast synthesis inhibitor HfiA. Here, we identify a new level in holdfast regulation, providing new insights into the control of adhesin production that leads to the formation of biofilms in response to the environment.

Comparative Analysis of Ionic Strength Tolerance between Freshwater and Marine Caulobacterales Adhesins.

Bacterial adhesion is affected by environmental factors, such as ionic strength, pH, temperature, and shear forces. Therefore, marine bacteria must have developed adhesins with different compositions and structures than those of their freshwater counterparts to adapt to their natural environment. The dimorphic alphaproteobacterium Hirschia baltica is a marine budding bacterium in the clade CaulobacteralesH. baltica uses a polar adhesin, the holdfast, located at the cell pole opposite the reproductive stalk, for surface attachment and cell-cell adhesion. The holdfast adhesin has been best characterized in Caulobacter crescentus, a freshwater member of the Caulobacterales, and little is known about holdfast compositions and properties in marine Caulobacterales Here, we use H. baltica as a model to characterize holdfast properties in marine Caulobacterales We show that freshwater and marine Caulobacterales use similar genes in holdfast biogenesis and that these genes are highly conserved among the species in the two genera. We determine that H. baltica produces a larger holdfast than C. crescentus and that the holdfasts have different chemical compositions, as they contain N-acetylglucosamine and galactose monosaccharide residues and proteins but lack DNA. Finally, we show that H. baltica holdfasts tolerate higher ionic strength than those of C. crescentus We conclude that marine Caulobacterales holdfasts have physicochemical properties that maximize binding in high-ionic-strength environments.IMPORTANCE Most bacteria spend a large part of their life spans attached to surfaces, forming complex multicellular communities called biofilms. Bacteria can colonize virtually any surface, and therefore, they have adapted to bind efficiently in very different environments. In this study, we compare the adhesive holdfasts produced by the freshwater bacterium C. crescentus and a relative, the marine bacterium H. baltica We show that H. baltica holdfasts have a different morphology and chemical composition and tolerate high ionic strength. Our results show that the H. baltica holdfast is an excellent model to study the effect of ionic strength on adhesion and provides insights into the physicochemical properties required for adhesion in the marine environment.

Feedback regulation of Caulobacter crescentus holdfast synthesis by flagellum assembly via the holdfast inhibitor HfiA.

To permanently attach to surfaces, Caulobacter crescentusproduces a strong adhesive, the holdfast. The timing of holdfast synthesis is developmentally regulated by cell cycle cues. When C. crescentusis grown in a complex medium, holdfast synthesis can also be stimulated by surface sensing, in which swarmer cells rapidly synthesize holdfast in direct response to surface contact. In contrast to growth in complex medium, here we show that when cells are grown in a defined medium, surface contact does not trigger holdfast synthesis. Moreover, we show that in a defined medium, flagellum synthesis and regulation of holdfast production are linked. In these conditions, mutants lacking a flagellum attach to surfaces over time more efficiently than either wild-type strains or strains harboring a paralyzed flagellum. Enhanced adhesion in mutants lacking flagellar components is due to premature holdfast synthesis during the cell cycle and is regulated by the holdfast synthesis inhibitor HfiA. hfiA transcription is reduced in flagellar mutants and this reduction is modulated by the diguanylate cyclase developmental regulator PleD. We also show that, in contrast to previous predictions, flagella are not necessarily required for C. crescentus surface sensing in the absence of flow, and that arrest of flagellar rotation does not stimulate holdfast synthesis. Rather, our data support a model in which flagellum assembly feeds back to control holdfast synthesis via HfiA expression in a c-di-GMP-dependent manner under defined nutrient conditions.

Bacterial adhesion at the single-cell level.

The formation of multicellular microbial communities, called biofilms, starts from the adhesion of a few planktonic cells to the surface. The transition from a free-living planktonic lifestyle to a sessile, attached state is a multifactorial process that is determined by biological, chemical and physical properties of the environment, the surface and the bacterial cell. The initial weak, reversible interactions between a bacterium and a surface strengthen to yield irreversible adhesion. In this Review, we summarize our understanding of the mechanisms governing bacterial adhesion at the single-cell level, including the physical forces experienced by a cell before reaching the surface, the first contact with a surface and the transition from reversible to permanent adhesion.

Layered Structure and Complex Mechanochemistry Underlie Strength and Versatility in a Bacterial Adhesive.

While designing synthetic adhesives that perform in aqueous environments has proven challenging, microorganisms commonly produce bioadhesives that efficiently attach to a variety of substrates, including wet surfaces. The aquatic bacterium Caulobacter crescentus uses a discrete polysaccharide complex, the holdfast, to strongly attach to surfaces and resist flow. The holdfast is extremely versatile and has impressive adhesive strength. Here, we used atomic force microscopy in conjunction with superresolution microscopy and enzymatic assays to unravel the complex structure of the holdfast and to characterize its chemical constituents and their role in adhesion. Our data support a model whereby the holdfast is a heterogeneous material organized as two layers: a stiffer nanoscopic core layer wrapped into a sparse, far-reaching, flexible brush layer. Moreover, we found that the elastic response of the holdfast evolves after surface contact from initially heterogeneous to more homogeneous. From a composition point of view, besides N-acetyl-d-glucosamine (NAG), the only component that had been identified to date, our data show that the holdfast contains peptides and DNA. We hypothesize that, while polypeptides are the most important components for adhesive force, the presence of DNA mainly impacts the brush layer and the strength of initial adhesion, with NAG playing a primarily structural role within the core. The unanticipated complexity of both the structure and composition of the holdfast likely underlies its versatility as a wet adhesive and its distinctive strength. Continued improvements in understanding of the mechanochemistry of this bioadhesive could provide new insights into how bacteria attach to surfaces and could inform the development of new adhesives.IMPORTANCE There is an urgent need for strong, biocompatible bioadhesives that perform underwater. To strongly adhere to surfaces and resist flow underwater, the bacterium Caulobacter crescentus produces an adhesive called the holdfast, the mechanochemistry of which remains undefined. We show that the holdfast is a layered structure with a stiff core layer and a polymeric brush layer and consists of polysaccharides, polypeptides, and DNA. The DNA appears to play a role in the structure of the brush layer and initial adhesion, the peptides in adhesive strength, and the polysaccharides in the structure of the core. The complex, multilayer organization and diverse chemistry described here underlie the distinctive adhesive properties of the holdfast and will provide important insights into the mechanisms of bacterial adhesion and bioadhesive applications.

Evolutionary determinants of genome-wide nucleotide composition.

One of the long-standing mysteries of evolutionary genomics is the source of the wide phylogenetic diversity in genome nucleotide composition (G + C versus A + T), which must be a consequence of interspecific differences in mutation bias, the efficiency of selection for different nucleotides or a combination of the two. We demonstrate that although genomic G + C composition is strongly driven by mutation bias, it is also substantially modified by direct selection and/or as a by-product of biased gene conversion. Moreover, G + C composition at fourfold redundant sites is consistently elevated above the neutral expectation-more so than for any other class of sites.

Mutations in Sugar-Nucleotide Synthesis Genes Restore Holdfast Polysaccharide Anchoring to Caulobacter crescentus Holdfast Anchor Mutants.

Attachment is essential for microorganisms to establish interactions with both biotic and abiotic surfaces. Stable attachment of Caulobacter crescentus to surfaces requires an adhesive polysaccharide holdfast, but the exact composition of the holdfast is unknown. The holdfast is anchored to the cell envelope by outer membrane proteins HfaA, HfaB, and HfaD. Holdfast anchor gene mutations result in holdfast shedding and reduced cell adherence. Translocation of HfaA and HfaD to the cell surface requires HfaB. The Wzx homolog HfsF is predicted to be a bacterial polysaccharide flippase. An hfsF deletion significantly reduced the amount of holdfast produced per cell and slightly reduced adherence. A ΔhfsF ΔhfaD double mutant was completely deficient in adherence. A suppressor screen that restored adhesion in the ΔhfsF ΔhfaD mutant identified mutations in three genes: wbqV, rfbB, and rmlA Both WbqV and RfbB belong to a family of nucleoside-diphosphate epimerases, and RmlA has similarity to nucleotidyltransferases. The loss of wbqV or rfbB in the ΔhfsF ΔhfaD mutant reduced holdfast shedding but did not restore holdfast synthesis to parental levels. Loss of wbqV or rfbB did not restore adherence to a ΔhfsF mutant but did restore adherence and holdfast anchoring to a ΔhfaD mutant, confirming that suppression occurs through restoration of holdfast anchoring. The adherence and holdfast anchoring of a ΔhfaA ΔhfaD mutant could be restored by wbqV or rfbB mutation, but such mutations could not suppress these phenotypes in the ΔhfaB mutant. We hypothesize that HfaB plays an additional role in holdfast anchoring or helps to translocate an unknown factor that is important for holdfast anchoring.IMPORTANCE Biofilm formation results in increased resistance to both environmental stresses and antibiotics. Caulobacter crescentus requires an adhesive holdfast for permanent attachment and biofilm formation, but the exact mechanism of polysaccharide anchoring to the cell and the holdfast composition are unknown. Here we identify novel polysaccharide genes that affect holdfast anchoring to the cell. We identify a new role for the holdfast anchor protein HfaB. This work increases our specific knowledge of the polysaccharide adhesin involved in Caulobacter attachment and the general knowledge regarding production and anchoring of polysaccharide adhesins by bacteria. This work also explores the interactions between different polysaccharide biosynthesis and secretion systems in bacteria.

Obstruction of pilus retraction stimulates bacterial surface sensing.

It is critical for bacteria to recognize surface contact and initiate physiological changes required for surface-associated lifestyles. Ubiquitous microbial appendages called pili are involved in sensing surfaces and facilitating downstream behaviors, but the mechanism by which pili mediate surface sensing has been unclear. We visualized Caulobacter crescentus pili undergoing dynamic cycles of extension and retraction. Within seconds of surface contact, these cycles ceased, which coincided with synthesis of the adhesive holdfast required for attachment. Physically blocking pili imposed resistance to pilus retraction, which was sufficient to stimulate holdfast synthesis without surface contact. Thus, to sense surfaces, bacteria use the resistance on retracting, surface-bound pili that occurs upon surface contact.

Adhesins Involved in Attachment to Abiotic Surfaces by Gram-Negative Bacteria.

During the first step of biofilm formation, initial attachment is dictated by physicochemical and electrostatic interactions between the surface and the bacterial envelope. Depending on the nature of these interactions, attachment can be transient or permanent. To achieve irreversible attachment, bacterial cells have developed a series of surface adhesins promoting specific or nonspecific adhesion under various environmental conditions. This article reviews the recent advances in our understanding of the secretion, assembly, and regulation of the bacterial adhesins during biofilm formation, with a particular emphasis on the fimbrial, nonfimbrial, and discrete polysaccharide adhesins in Gram-negative bacteria.

Physiochemical properties of Caulobacter crescentus holdfast: a localized bacterial adhesive.

To colonize surfaces, the bacterium Caulobacter crescentus employs a polar polysaccharide, the holdfast, located at the end of a thin, long stalk protruding from the cell body. Unlike many other bacteria which adhere through an extended extracellular polymeric network, the holdfast footprint area is tens of thousands times smaller than that of the total bacterium cross-sectional surface, making for some very demanding adhesion requirements. At present, the mechanism of holdfast adhesion remains poorly understood. We explore it here along three lines of investigation: (a) the impact of environmental conditions on holdfast binding affinity, (b) adhesion kinetics by dynamic force spectroscopy, and (c) kinetic modeling of the attachment process to interpret the observed time-dependence of the adhesion force at short and long time scales. A picture emerged in which discrete molecular units called adhesins are responsible for initial holdfast adhesion, by acting in a cooperative manner.

Microchannel-nanopore device for bacterial chemotaxis assays.

Motile bacteria bias the random walk of their motion in response to chemical gradients by the process termed chemotaxis, which allows cells to accumulate in favorable environments and disperse from less favorable ones. In this work, we describe a simple microchannel-nanopore device that establishes a stable chemical gradient for chemotaxis assays in ≤1 min. Chemoattractant is dispensed by diffusion through 10 nm diameter pores at the intersection of two microchannels. This design requires no external pump and minimizes the effect of transmembrane pressure, resulting in a stable, reproducible gradient. The microfluidic platform facilitates microscopic observation of individual cell trajectories, and chemotaxis is quantified by monitoring changes in cell swimming behavior in the vicinity of the intersection. We validate this system by measuring the chemotactic response of an aquatic bacterium, Caulobacter crescentus, to xylose concentrations from 1.3 µM to 1.3 M. Additionally, we make an unanticipated observation of increased turn frequency in a chemotaxis-impaired mutant which provides new insight into the chemotaxis pathway in C. crescentus.

A bacterial extracellular DNA inhibits settling of motile progeny cells within a biofilm.

In natural systems, bacteria form complex, surface-attached communities known as biofilms. This lifestyle presents numerous advantages compared with unattached or planktonic life, such as exchange of nutrients, protection from environmental stresses and increased tolerance to biocides. Despite such benefits, dispersal also plays an important role in escaping deteriorating environments and in successfully colonizing favourable, unoccupied habitat patches. The α-proteobacterium Caulobacter crescentus produces a motile swarmer cell and a sessile stalked cell at each cell division. We show here that C. crescentus extracellular DNA (eDNA) inhibits the ability of its motile cell type to settle in a biofilm. eDNA binds to the polar holdfast, an adhesive structure required for permanent surface attachment and biofilm formation, thereby inhibiting cell attachment. Because stalked cells associate tightly with the biofilm through their holdfast, we hypothesize that this novel mechanism acts on swarmer cells born in a biofilm, where eDNA can accumulate to a sufficient concentration to inhibit their ability to settle. By targeting a specific cell type in a biofilm, this mechanism modulates biofilm development and promotes dispersal without causing a potentially undesirable dissolution of the existing biofilm.

CYP201A2, a cytochrome P450 from Rhodopseudomonas palustris, plays a key role in the biodegradation of tributyl phosphate.

Tributyl phosphate (TBP) is a toxic organophosphorous compound widely used in nuclear fuel processing and chemical industries. Rhodopseudomonas palustris, one of the most metabolically versatile photosynthetic bacteria, is shown here to degrade TBP efficiently under photosynthetic conditions. This study shows that this O(2)- and NADPH/FMNH(2)-dependent process was also catalyzed when TBP was incubated with membrane-associated proteins extracted from this strain. The effects of several regulators of cytochrome P450 activity on the TBP consumption suggest a key role for a cytochrome P450 in this process. Disruption of the rpa0241 gene encoding a putative cytochrome P450 led to a 60% decrease of the TBP catabolism, whereas reintroducing the gene in the mutant restored the wild-type phenotype. The rpa0241 gene was expressed and purified in Escherichia coli. Characterization by UV-visible spectroscopy of the purified recombinant membrane-bound protein (CYP201A2) encoded by the rpa0241 gene revealed typical spectral characteristics of cytochrome P450 with a large spin state change of the heme iron associated with binding of TBP (K (d) approximately 65 microM). It is proposed that CYP201A2 catalyzes the initial step of the biodegradation process of TBP.

Coimmobilization of a redox enzyme and a cofactor regeneration system.

The coimmobilization of nitrobenzene nitroreductase and glucose-6-phosphate dehydrogenase in silica particles enables the continuous conversion of nitrobenzene to hydroxylaminobenzene with NADPH recycling.

Application of a microfluidic reactor for screening cancer prodrug activation using silica-immobilized nitrobenzene nitroreductase.

The nitroreductase-catalyzed conversion of a strong electron-withdrawing nitro group to the corresponding electron-donating hydroxylamine is useful in a variety of biotechnological applications. Activation of prodrugs for cancer treatments or antibiotic therapy are the most common applications. Here, we show that a bacterial nitrobenzene nitroreductase (NbzA) from Pseudomonas pseudoalcaligenes JS45 activates the dinitrobenzamide cancer prodrug CB1954 and the proantibiotic nitrofurazone. NbzA was purified by affinity chromatography and screened for substrate specificity with respect to prodrug activation. To facilitate screening of alternate potential prodrugs, polyethyleneimine-mediated silica formation was used to immobilize NbzA with high immobilization yields and high loading capacities. Greater than 80% of the NbzA was immobilized, and enzyme activity was significantly more stable than NbzA in solution. The resulting silica-encapsulated NbzA was packed into a microfluidic microreactor that proved suitable for continuous operation using nitrobenzene, CB1954, and the proantibiotic nitrofurazone. The flow-through system provides a rapid and reproducible screening method for determining the NbzA-catalyzed activation of prodrugs and proantibiotics.

Tributyl phosphate degradation by Rhodopseudomonas palustris and other photosynthetic bacteria.

Tributyl phosphate (TBP) is widely used in nuclear fuel processing and other waste generating chemical industries. Although TBP is bacteriostatic, some microbes are resistant to it and may degrade it. Under dark aerobiosis, purple non-sulfur photosynthetic bacteria degraded up to 0.6 mM TBP, initially present at 2 mM, within 3 weeks and under photosynthetic conditions, Rhodopseudomonas palustris degraded 1.6 mM TBP within 3 weeks. The curing of the Rhodopseudomonas palustris endogenous plasmid demonstrated that the genes involved in the TBP degradation are chromosomal.