A Rhizobiales-Specific Unipolar Polysaccharide Adhesin Contributes to Rhodopseudomonas palustris Biofilm Formation across Diverse Photoheterotrophic Conditions.

Bacteria predominantly exist as members of surfaced-attached communities known as biofilms. Many bacterial species initiate biofilms and adhere to each other using cell surface adhesins. This is the case for numerous ecologically diverse Alphaprotebacteria, which use polar exopolysaccharide adhesins for cell-cell adhesion and surface attachment. Here, we show that Rhodopseudomonas palustris, a metabolically versatile member of the alphaproteobacterial order Rhizobiales, contains a functional unipolar polysaccharide (UPP) biosynthesis gene cluster. Deletion of genes predicted to be critical for UPP biosynthesis and export abolished UPP production. We also found that R. palustris uses UPP to mediate biofilm formation across diverse photoheterotrophic growth conditions, wherein light and organic substrates are used to support growth. However, UPP was less important for biofilm formation during photoautotrophy, where light and CO2 support growth, and during aerobic respiration with organic compounds. Expanding our analysis beyond R. palustris, we examined the phylogenetic distribution and genomic organization of UPP gene clusters among Rhizobiales species that inhabit diverse niches. Our analysis suggests that UPP is a conserved ancestral trait of the Rhizobiales but that it has been independently lost multiple times during the evolution of this clade, twice coinciding with adaptation to intracellular lifestyles within animal hosts. IMPORTANCE: Bacteria are ubiquitously found as surface-attached communities and cellular aggregates in nature. Here, we address how bacterial adhesion is coordinated in response to diverse environments using two complementary approaches. First, we examined how Rhodopseudomonas palustris, one of the most metabolically versatile organisms ever described, varies its adhesion to surfaces in response to different environmental conditions. We identified critical genes for the production of a unipolar polysaccharide (UPP) and showed that UPP is important for adhesion when light and organic substrates are used for growth. Looking beyond R. palustris, we performed the most comprehensive survey to date on the conservation of UPP biosynthesis genes among a group of closely related bacteria that occupy diverse niches. Our findings suggest that UPP is important for free-living and plant-associated lifestyles but dispensable for animal pathogens. Additionally, we propose guidelines for classifying the adhesins produced by various Alphaprotebacteria, facilitating future functional and comparative studies.

Genome-wide patterns of regulatory divergence revealed by introgression lines.

Understanding the genetic basis for changes in transcriptional regulation is an important aspect of understanding phenotypic evolution. Using interspecific introgression lines, we infer the mechanisms of divergence in genome-wide patterns of gene expression between the nightshades Solanum pennellii and S. lycopersicum (domesticated tomato). We find that cis- and trans-regulatory changes have had qualitatively similar contributions to divergence in this clade, unlike results from other systems. Additionally, expression data from four tissues (shoot apex, ripe fruit, pollen, and seed) suggest that introgressed regions in these hybrid lines tend to be downregulated, while background (nonintrogressed) genes tend to be upregulated. Finally, we find no evidence for an association between the magnitude of differential expression in NILs and previously determined sterility phenotypes. Our results contradict previous predictions of the predominant role of cis- over trans-regulatory divergence between species, and do not support a major role for gross genome-wide misregulation in reproductive isolation between these species.

N2 gas is an effective fertilizer for bioethanol production by Zymomonas mobilis.

A nascent cellulosic ethanol industry is struggling to become cost-competitive against corn ethanol and gasoline. Millions of dollars are spent on nitrogen supplements to make up for the low nitrogen content of the cellulosic feedstock. Here we show for the first time to our knowledge that the ethanol-producing bacterium, Zymomonas mobilis, can use N2 gas in lieu of traditional nitrogen supplements. Despite being an electron-intensive process, N2 fixation by Z. mobilis did not divert electrons away from ethanol production, as the ethanol yield was greater than 97% of the theoretical maximum. In a defined medium, Z. mobilis produced ethanol 50% faster per cell and generated half the unwanted biomass when supplied N2 instead of ammonium. In a cellulosic feedstock-derived medium, Z. mobilis achieved a similar cell density and a slightly higher ethanol yield when supplied N2 instead of the industrial nitrogen supplement, corn steep liquor. We estimate that N2-utilizing Z. mobilis could save a cellulosic ethanol production facility more than $1 million/y.

Variation in preen oil composition pertaining to season, sex, and genotype in the polymorphic white-throated sparrow.

Evidence for the the ability of birds to detect olfactory signals is now well documented, yet it remains unclear whether birds secrete chemicals that can be used as social cues. A potential source of chemical cues in birds is the secretion from the uropygial gland, or preen gland, which is thought to waterproof, maintain, and protect feathers from ectoparasites. However, it is possible that preen oil also may be used for individual recognition, mate choice, and signalling social/sexual status. If preen oil secretions can be used as socio-olfactory signals, we should be able to identify the volatile components that could make the secretions more detectable, determine the seasonality of these secretions, and determine whether olfactory signals differ among relevant social groups. We examined the seasonal differences in volatile compounds of the preen oil of captive white-throated sparrows, Zonotrichia albicollis. This species is polymorphic and has genetically determined morphs that occur in both sexes. Mating is almost exclusively disassortative with respect to morph, suggesting strong mate choice. By sampling the preen oil from captive birds in breeding and non-breeding conditions, we identified candidate chemical signals that varied according to season, sex, morph, and species. Linear alcohols with a 10-18 carbon chains, as well as methyl ketones and carboxylic acids, were the most abundant volatile compounds. Both the variety and abundances of some of these compounds were different between the sexes and morphs, with one morph secreting more volatile compounds in the non-breeding season than the other. In addition, 12 compounds were seasonally elevated in amount, and were secreted in high amounts in males. Finally, we found that preen oil signatures tended to be species-specific, with white-throated sparrows differing from the closely related Junco in the abundances and/or prevalence of at least three compounds. Our data suggest roles for preen oil secretions and avian olfaction in both non-social as well as social interactions.

A specialist herbivore uses chemical camouflage to overcome the defenses of an ant-plant mutualism.

Many plants and ants engage in mutualisms where plants provide food and shelter to the ants in exchange for protection against herbivores and competitors. Although several species of herbivores thwart ant defenses and extract resources from the plants, the mechanisms that allow these herbivores to avoid attack are poorly understood. The specialist insect herbivore, Piezogaster reclusus (Hemiptera: Coreidae), feeds on Neotropical bull-horn acacias (Vachellia collinsii) despite the presence of Pseudomyrmex spinicola ants that nest in and aggressively defend the trees. We tested three hypotheses for how P. reclusus feeds on V. collinsii while avoiding ant attack: (1) chemical camouflage via cuticular surface compounds, (2) chemical deterrence via metathoracic defense glands, and (3) behavioral traits that reduce ant detection or attack. Our results showed that compounds from both P. reclusus cuticles and metathoracic glands reduce the number of ant attacks, but only cuticular compounds appear to be essential in allowing P. reclusus to feed on bull-horn acacia trees undisturbed. In addition, we found that ant attack rates to P. reclusus increased significantly when individuals were transferred between P. spinicola ant colonies. These results are consistent with the hypothesis that chemical mimicry of colony-specific ant or host plant odors plays a key role in allowing P. reclusus to circumvent ant defenses and gain access to important resources, including food and possibly enemy-free space. This interaction between ants, acacias, and their herbivores provides an excellent example of the ability of herbivores to adapt to ant defenses of plants and suggests that herbivores may play an important role in the evolution and maintenance of mutualisms.

Non-growing Rhodopseudomonas palustris increases the hydrogen gas yield from acetate by shifting from the glyoxylate shunt to the tricarboxylic acid cycle.

When starved for nitrogen, non-growing cells of the photosynthetic bacterium Rhodopseudomonas palustris continue to metabolize acetate and produce H2, an important industrial chemical and potential biofuel. The enzyme nitrogenase catalyzes H2 formation. The highest H2 yields are obtained when cells are deprived of N2 and thus use available electrons to synthesize H2 as the exclusive product of nitrogenase. To understand how R. palustris responds metabolically to increase H2 yields when it is starved for N2, and thus not growing, we tracked changes in biomass composition and global transcript levels. In addition to a 3.5-fold higher H2 yield by non-growing cells we also observed an accumulation of polyhydroxybutyrate to over 30% of the dry cell weight. The transcriptome of R. palustris showed down-regulation of biosynthetic processes and up-regulation of nitrogen scavenging mechanisms in response to N2 starvation but gene expression changes did not point to metabolic activities that could generate the reductant necessary to explain the high H2 yield. We therefore tracked (13)C-labeled acetate through central metabolic pathways. We found that non-growing cells shifted their metabolism to use the tricarboxylic acid cycle to metabolize acetate in contrast to growing cells, which used the glyoxylate cycle exclusively. This shift enabled cells to more fully oxidize acetate, providing the necessary reducing power to explain the high H2 yield.

Investigation of scents on cheeks and foreheads of large felines in connection to the facial marking behavior.

We investigated head- and cheek-rubbing behavior in four species of large felines, lions (Leo panther), leopards (Panthera pardus), tigers (Panthera tigris), and cougars (Puma concolor), in captivity. Preliminary behavioral observations found that lions and tigers, but not leopards and cougars, showed behavioral responses to cardboard rubbing samples from head and cheek areas from conspecific felines, compared to the blank cardboard controls. In this context, surface samples on the facial areas of each species were collected to analyze volatile organic compounds that could be involved in the facial marking of felines. Previously developed stir bar surface sampling methodology was used. From all cheek and forehead samples, 100 volatile organic compounds were identified or tentatively identified. Among these, 41 have been previously reported to be present in feline urine and marking secretions. Several new compounds were identified on facial surfaces. Some of the compounds showed substantial quantitative differences among the species. One compound, that has not been reported previously in mammals, 3-acetamidofuran, was found in all investigated species. It was synthesized and tested for behavioral responses. No responses were elicited in a preliminary test. Future research will test other potential signaling compounds and their mixtures for ability to elicit behavioral responses.

Role of testosterone in stimulating seasonal changes in a potential avian chemosignal.

Songbird preen oil contains volatile and semivolatile compounds that may contain information about species, sex, individual identity, and season. We examined the relationship between testosterone (T) and the amounts of preen oil volatile and semivolatile compounds in wild and captive dark-eyed juncos (Junco hyemalis). In wild males and females, we observed an increase in volatile compound relative concentration early in the breeding season. This increase mirrored previously described seasonal elevation in T levels in wild males and females, suggesting a positive relationship between hormone levels and preen gland secretions, and a possible role for these secretions in signaling receptivity. In females, the greatest relative concentrations of most compounds were observed close to egg laying, a time when steroid hormones are high and also the only time that females respond to an injection of gonadotropin-releasing hormone with a short-term increase in T. In a study of captive juncos held on short days, we asked whether the seasonal increases observed in the wild could be induced with experimental elevation of T alone. We found that exogenous T stimulated the production of some volatile compounds in non-breeding individuals of both sexes. However, of the 15 compounds known to increase during the breeding season, only four showed an increase in relative concentration in birds that received T implants. Our results suggest that testosterone levels likely interact with other seasonally induced physiological changes to affect volatile compound amounts in preen oil.

Nickel2+-mediated assembly of an RNA-amino acid complex.

The RNA species SHR1 reacts with biocytin (epsilon-biotinoyl-L-lysine) in the presence of Ni(2+) or Pt(2+) to produce a metal-bridged complex that migrates more slowly than unreacted RNA in the presence of streptavidin (StrAv) on denaturing polyacrylamide gels. Mapping of reverse transcription pause sites identified G79 as a reactive nucleotide. G79 is near the 3' end of a 37 nucleotide core motif that is nearly as reactive as SHR1. SHR1 reacts with biocytin in the presence of Pt(2+) to yield a product that comigrates with the Ni(2+) product but that is much more stable, suggesting that the metal ion used in the reaction is present in the product, possibly linking the RNA to the amino acid. In support of this model, SHR1 shows a strong affinity for Ni(2+) in immobilized metal ion chromatography.