17Bacterial adhesion is affected by environmental factors, such as ionic strength, pH, 18 temperature, and shear forces, and therefore marine bacteria must have developed holdfasts 19 with different composition and structures than their freshwater counterparts to adapt to their 20 natural environment. The dimorphic a-proteobacterium Hirschia baltica is a marine budding 21 bacterium in the Caulobacterales clade. H. baltica uses a polar adhesin, the holdfast, located at 22 the cell pole opposite the reproductive stalk for surface attachment and cell-cell adhesion. The 23 holdfast adhesin has been best characterized in Caulobacter crescentus, a freshwater member 24 of the Caulobacterales, and little is known about holdfast composition and properties in marine 25 Caulobacterales. Here we use H. baltica as a model to characterize holdfast properties in 26 marine Caulobacterales. We show that freshwater and marine Caulobacterales use similar 27 genes in holdfast biogenesis and that these genes are highly conserved among the two genera. 28We also determine that H. baltica produces larger holdfast than C. crescentus and that those 29 holdfasts have a different chemical composition, as they contain N-acetylglucosamine and 30 galactose monosaccharide residues and proteins, but lack DNA. Finally, we show that H. baltica 31 holdfasts tolerate higher ionic strength than those of C. crescentus. We conclude that marine 32 Caulobacterales holdfasts have physicochemical properties that maximize binding in high ionic 33 strength environments. 34 35 IMPORTANCE 36 Most bacteria spend a large amount of their lifespan attached to surfaces, forming 37 complex multicellular communities called biofilms. Bacteria can colonize virtually any surface, 38 therefore they have adapted to bind efficiently in very different environments. In this study, we 39 compare the adhesive holdfasts produced by the freshwater bacterium C. crescentus and a 40 relative, the marine bacterium H. baltica. We show that H. baltica holdfasts have a different 41 3 morphology and chemical composition, and tolerate high ionic strength. Our results show that H. 42 baltica holdfast is an excellent model to study the effect of ionic strength on adhesion and 43 providing insights on the physicochemical properties required for adhesion in the marine 44 environment. 45 46 48 communities, known as biofilms (1). To irreversibly adhere to surfaces and form these complex 49 mutil-cellular communities, bacteria produce strong adhesins, mainly composed of proteins or 50 polysaccharides (2, 3). Bacterial adhesion is affected by different environmental conditions such 51 as pH, temperature, shear forces, and ionic strength (2,(4)(5)(6). In marine environments, bacteria 52 face 500 times higher ionic strength than in freshwater (7), therefore, marine bacteria have 53 evolved ways to overcome the effect of ionic strength and bind permanently to surfaces in high 54 salt environments such as seas and oceans. 55Caulobacterales are Alphaproteobacteria found in various habitats, from olig...

Differences in ionic strength, pH, temperature, shear forces, and other environmental factors impact adhesion, and organisms have evolved various strategies to optimize their adhesins for their specific environmental conditions. Many species of Alphaproteobacteria, including members of the order Caulobacterales, use a polar adhesin, called holdfast, for surface attachment and subsequent biofilm formation in both freshwater and marine environments. Hirschia baltica, a marine member of Caulobacterales, produces a holdfast adhesin that tolerates a drastically higher ionic strength than the holdfast produced by its freshwater relative, Caulobacter crescentus. In this work, we show that the holdfast polysaccharide deacetylase HfsH plays an important role in adherence in high-ionic-strength environments. We show that increasing expression of HfsH improves holdfast binding in high-ionic-strength environments. We conclude that HfsH plays a role in modulating holdfast binding at high ionic strength and hypothesize that this modulation occurs through varied deacetylation of holdfast polysaccharides.

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 Caulobacterales. H. 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.

The adhesion of organisms to surfaces in aquatic environments provides a diversity of benefits such as better access to nutrients or protection from the elements or from predation. Differences in ionic strength, pH, temperature, shear forces, and other environmental factors impact adhesion and organisms have evolved various strategies to optimize their adhesins for their specific environmental conditions. We know essentially nothing about how bacteria evolved their adhesive mechanisms to attach efficiently in environments with different physico-chemical conditions. Many species of Alphaproteobacteria, including members of the order Caulobacterales, use a polar adhesin, called holdfast, for surface attachment and subsequent biofilm formation in both freshwater and marine environments. Hirschia baltica, a marine member of Caulobacterales, produces a holdfast adhesin that tolerates a drastically higher ionic strength than the holdfast produced by its freshwater relative, Caulobacter crescentus. In this work, we show that the holdfast polysaccharide deacetylase HfsH plays an important role in adherence in high ionic strength environments. We show that deletion of hfsH in H. baltica disrupts holdfast binding properties and structure. Increasing expression of HfsH in C. crescentus improved holdfast binding in high salinity, whereas lowering HfsH expression in H. baltica reduced holdfast binding at high ionic strength. We conclude that HfsH plays a role in modulating holdfast binding at high ionic strength and hypothesize that this modulation occurs through varied deacetylation of holdfast polysaccharides.

A kernel screening assay (KSA) was used to assess the genetic and environmental effects on the vulnerability of maize to aflatoxin accumulation. Kernels of 26 inbred lines that had been grown in seven environments, and 190 lines of the Intermated B73xMo17 (IBM) population grown in one location in the United States, were inoculated with a toxigenic strain of A. flavus and incubated in the dark at 30°C for 6 days. Percent kernel colonization (PKC), sporulation and aflatoxin were influenced by the maize genotypes (G), the location (“ear environment” or E) and the GxE interactions. Overall, low broad‐sense heritabilities were observed for PKC, sporulation and aflatoxin. PKC was significantly correlated with sporulation in all environments. Aflatoxin was positively correlated with colonization for two and with sporulation for all ear environments. Higher grain sulphur or magnesium in IBM was associated with less colonization or aflatoxin. Postharvest susceptibility of maize to aflatoxin is thus influenced by factors that are modulated by the ear environment. In a KSA, sporulation could be a proxy test for aflatoxin accumulation.

For surface attachment and biofilm formation, bacteria produce adhesins that are composed of polysaccharides, proteins, and DNA. Species of the Caulobacterales produce a specialized polar adhesin, holdfast, which is required for permanent attachment to surfaces.

Bacteria use adhesins to colonize different surfaces and form biofilms. The species of the Caulobacterales order use a polar adhesin called holdfast, composed of polysaccharides, proteins, and DNA to irreversibly adhere to surfaces. In C. crescentus, a freshwater Caulobacterales, the holdfast is anchored at the cell pole via the holdfast anchor (Hfa) proteins HfaA, HfaB, and HfaD. HfaA and HfaD co-localize with holdfast and are thought to form amyloid-like fibers that anchor holdfast to the cell envelope. HfaB, a lipoprotein, is required for translocation of HfaA and HfaD to the cell surface. Deletion of the anchor proteins leads to a severe defect in adherence resulting from holdfast not properly attached to the cell and shed into the medium. This phenotype is greater in a ΔhfaB than a double ΔhfaA ΔhfaD mutant, suggesting that HfaB has other functions besides the translocation of HfaA and HfaD. Here, we identify an additional HfaB-dependent holdfast anchoring protein, HfaE, which is predicted to be a secreted protein. HfaE is highly conserved among Caulobacterales species with no predicted function. In planktonic culture, hfaE mutants produce holdfasts and rosettes similar to wild type. However, holdfasts from hfaE mutants bind to the surface but are unable to anchor cells, similar to other anchor mutants. We showed that fluorescently-tagged HfaE co-localizes with holdfast, and HfaE forms an SDS-resistant high molecular weight species consistent with amyloid fiber formation. We propose that HfaE is a novel holdfast anchor protein, and that HfaE functions to link holdfast material to the cell envelope.