SummaryCaulobacter crescentus attachment is mediated by the holdfast, a complex of polysaccharide anchored to the cell by HfaA, HfaB and HfaD. We show that all three proteins are surface exposed outer membrane (OM) proteins. HfaA is similar to fimbrial proteins and assembles into a high molecular weight (HMW) form requiring HfaD, but not holdfast polysaccharide. The HfaD HMW form is dependent on HfaA but not on holdfast polysaccharide. We show that HfaA and HfaD form homomultimers and that they require HfaB for stability and OM translocation. All three proteins localize to the late pre-divisional flagellar pole, remain at this pole in swarmer cells, and localize at the stalk tip after the stalk is synthesized at the same pole. Hfa protein localization requires the holdfast polysaccharide secretion proteins and the polar localization factor PodJ. An hfaB mutant is much more severely deficient in adherence and holdfast attachment than hfaA and hfaD mutants. An hfaA, hfaD double mutant phenocopies either single mutant, suggesting that HfaB is involved in holdfast attachment beyond secretion of HfaA and HfaD. We hypothesize that HfaB secretes HfaA and HfaD across the outer membrane, and the three proteins form a complex anchoring the holdfast to the stalk.

The field of cyanobacterial biofuel production is advancing rapidly, yet we know little of the basic biology of these organisms outside of their photosynthetic pathways. We aimed to gain a greater understanding of how the cyanobacterium Synechocystis PCC 6803 (Synechocystis, hereafter) modulates its cell surface. Such understanding will allow for the creation of mutants that autoflocculate in a regulated way, thus avoiding energy intensive centrifugation in the creation of biofuels. We constructed mutant strains lacking genes predicted to function in carbohydrate transport or synthesis. Strains with gene deletions of slr0977 (predicted to encode a permease component of an ABC transporter), slr0982 (predicted to encode an ATP binding component of an ABC transporter) and slr1610 (predicted to encode a methyltransferase) demonstrated flocculent phenotypes and increased adherence to glass. Upon bioinformatic inspection, the gene products of slr0977, slr0982, and slr1610 appear to function in O-antigen (OAg) transport and synthesis. However, the analysis provided here demonstrated no differences between OAg purified from wild-type and mutants. However, exopolysaccharides (EPS) purified from mutants were altered in composition when compared to wild-type. Our data suggest that there are multiple means to modulate the cell surface of Synechocystis by disrupting different combinations of ABC transporters and/or glycosyl transferases. Further understanding of these mechanisms may allow for the development of industrially and ecologically useful strains of cyanobacteria. Additionally, these data imply that many cyanobacterial gene products may possess as-yet undiscovered functions, and are meritorious of further study.

Polar development and cell division in Caulobacter crescentus are controlled and coordinated by multiple signal transduction proteins. divJ encodes a histidine kinase. A null mutation in divJ results in a reduced growth rate, cell filamentation, and mislocalized stalks. Suppressor analysis of divJ identified mutations in genes encoding the tyrosine kinase (divL) and the histidine kinase (cckA). The divL and cckA suppressor alleles all have single amino acid substitutions, some of which confer a temperature-sensitive phenotype, particularly in a wild-type background. Analysis of transcription levels from several positively regulated CtrA-dependent promoters reveals high expression in the divJ mutant, suggesting that DivJ normally serves to reduce CtrA activity. The divL and cckA suppressors reduce the amount of transcription from promoters positively regulated by CtrA, indicating that the mutations in divL and cckA are suppressing the defects of the divJ mutant by reducing the abnormally high level of CtrA activity. Immunoblotting showed no major perturbations in the CtrA protein level in any of these strains, suggesting that the high amount of CtrA activity seen in the divJ mutant and the reduced amount of activity in the suppressors are regulated at the level of activation and not transcription, translation, or degradation. In vivo phosphorylation assays confirmed that divJ mutants have elevated levels of CtrA phosphorylation and that this level is reduced in the suppressors with mutations in divL.The aquatic gram-negative bacterium Caulobacter crescentus has a dimorphic life cycle, beginning as a motile, piliated swarmer cell incapable of DNA replication. The swarmer cell differentiates into a stalked cell by ejecting its flagellum, retracting the pili, and synthesizing a stalk with an adhesive holdfast at the same pole that contained the flagellum. The stalked cell initiates DNA replication and cell division and synthesizes a new flagellum at the pole opposite the stalk, producing a new motile swarmer cell during each cell cycle. Multiple signal transduction proteins are involved in coordinating polar development and cell division in C. crescentus. The global response regulator CtrA controls the expression of at least 144 genes (25) involved in cell division, DNA methylation, holdfast synthesis, flagellum biogenesis, and pilus biogenesis (22,25,34,43). CtrA prevents the initiation of DNA replication in swarmer cells by binding to the origin of replication (35) and inhibits cell division by repressing transcription of ftsZ, which encodes the first cell division protein to localize to the site of division (22). Late in the cell cycle, CtrA activates transcription from the P QA promoter, which cotranscribes ftsQ and ftsA, ensuring ordered expression of ftsZ, ftsQ, and ftsA (22,38,50

Degradation of purines to uric acid is generally conserved among organisms, however, the end product of uric acid degradation varies from species to species depending on the presence of active catabolic enzymes. In humans, most higher primates and birds, the urate oxidase gene is non-functional and hence uric acid is not further broken down. Uric acid in human blood plasma serves as an antioxidant and an immune enhancer; conversely, excessive amounts cause the common affliction gout. In contrast, uric acid is completely degraded to ammonia in most fungi. Currently, relatively little is known about uric acid catabolism in the fungal pathogen Cryptococcus neoformans even though this yeast is commonly isolated from uric acid-rich pigeon guano. In addition, uric acid utilization enhances the production of the cryptococcal virulence factors capsule and urease, and may potentially modulate the host immune response during infection. Based on these important observations, we employed both Agrobacterium-mediated insertional mutagenesis and bioinformatics to predict all the uric acid catabolic enzyme-encoding genes in the H99 genome. The candidate C. neoformans uric acid catabolic genes identified were named: URO1 (urate oxidase), URO2 (HIU hydrolase), URO3 (OHCU decarboxylase), DAL1 (allantoinase), DAL2,3,3 (allantoicase-ureidoglycolate hydrolase fusion protein), and URE1 (urease). All six ORFs were then deleted via homologous recombination; assaying of the deletion mutants' ability to assimilate uric acid and its pathway intermediates as the sole nitrogen source validated their enzymatic functions. While Uro1, Uro2, Uro3, Dal1 and Dal2,3,3 were demonstrated to be dispensable for virulence, the significance of using a modified animal model system of cryptococcosis for improved mimicking of human pathogenicity is discussed.

Social Disruption (SDR), a murine social stressor, results in an increased reactivity of splenic CD11b+ macrophages to inflammatory stimuli. For example, SDR leads to increased cytokine production and glucocorticoid insensitivity upon lipopolysaccharide stimulation. Moreover, SDR enhances the ability of these macrophages to kill Escherichia coli partly through TLR4-induced expression of inducible nitric oxide synthase (iNOS). The MAPK p38 has extensive known relationships to a number of these SDR characteristics and previous work has also shown that levels of phosphorylated p38 are increased in CD11b+ splenocytes following SDR. As such, p38 is a likely common link among these various stress-induced immunological changes in this model. These experiments were designed to test the hypothesis that inhibiting p38 would significantly reduce the stress-induced increase in killing of E. coli. SDR normally significantly (p < 0.05) increases the percentage of bacteria killed; however, when p38 is inhibited, we found that the percentage killed is reduced to baseline. Additionally, iNOS expression in stressed animals stimulated with E.coli is significantly enhanced in SDR-exposed splenocytes but, p38 inhibition eliminated this increase. Taken together, the negation of the enhanced killing effect and significant decrease in iNOS expression following p38 inhibition supports the hypothesis that the MAPK p38 has an important role in the stress-induced increase in bacterial killing.