A positivity result in the theory of Macdonald polynomials

We outline here a proof that a certain rational function Cn(q, t), which has come to be known as the “q, t-Catalan,” is in fact a polynomial with positive integer coefficients. This has been an open problem since 1994. Because Cn(q, t) evaluates to the Catalan number at t = q = 1, it has also been an open problem to find a pair of statistics a, b on the collection

Similarities between the DNA replication initiators of Gram-negative bacteria plasmids (RepA) and eukaryotes (Orc4p)/archaea (Cdc6p)

The proteins responsible for the initiation of DNA replication are thought to be essentially unrelated in bacteria and archaea/eukaryotes. Here we show that RepA, the initiator from the Pseudomonas plasmid pPS10, and the C-terminal domain of ScOrc4p, a subunit of Saccharomyces cerevisiae (Sc) origin recognition complex (ORC), share sequence similarities. Based on biochemical and spectroscopic evidence, these similarities include common structural elements, such as a winged-helix domain and a leucine-zipper dimerization motif. We have also found that ScOrc4p, as previously described for RepA-type initiators, interacts with chaperones of the Hsp70 family both in vitro and in vivo, most probably to regulate the assembly of active ORC. In evolutionary terms, our results are compatible with the recruitment of the same protein module for initiation of DNA replication by the ancestors of present-day Gram-negative bacteria plasmids, archaea, and eukaryotes.

Purification and molecular cloning of an inducible gram-negative bacteria-binding protein from the silkworm, Bombyx mori.

A 50-kDa hemolymph protein, having strong affinity to the cell wall of Gram(-) bacteria, was purified from the hemolymph of the silkworm, Bombyx mori. The cDNA encoding this Gram(-) bacteria-binding protein (GNBP) was isolated from an immunized silkworm fat body cDNA library and sequenced. Comparison of the deduced amino acid sequence with known sequences revealed that GNBP contained a region displaying significant homology to the putative catalytic region of a group of bacterial beta-1,3 glucanases and beta-1,3-1,4 glucanases. Silkworm GNBP was also shown to have amino acid sequence similarity to the vertebrate lipopolysaccharide receptor CD14 and was recognized specifically by a polygonal anti-CD14 antibody. Northern blot analysis showed that GNBP was constitutively expressed in fat body, as well as in cuticular epithelial cells of naive silkworms. Intense transcription was, however, rapidly induced following a cuticular or hemoceolien bacterial challenge. An mRNA that hybridized with GNBP cDNA was also found in the l(2)mbn immunocompetent Drosophila cell line. These observations suggest that GNBP is an inducible acute phase protein implicated in the immune response of the silkworm and perhaps other insects.

Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites

The conserved two-component regulatory system GacS/GacA determines the expression of extracellular products and virulence factors in a variety of Gram-negative bacteria. In the biocontrol strain CHA0 of Pseudomonas fluorescens, the response regulator GacA is essential for the synthesis of extracellular protease (AprA) and secondary metabolites including hydrogen cyanide. GacA was found to exert its control on the hydrogen cyanide biosynthetic genes (hcnABC) and on the aprA gene indirectly via a posttranscriptional mechanism. Expression of a translational hcnA′-′lacZ fusion was GacA-dependent whereas a transcriptional hcnA-lacZ fusion was not. A distinct recognition site overlapping with the ribosome binding site appears to be primordial for GacA-steered regulation. GacA-dependence could be conferred to the Escherichia coli lacZ mRNA by a 3-bp substitution in the ribosome binding site. The gene coding for the global translational repressor RsmA of P. fluorescens was cloned. RsmA overexpression mimicked partial loss of GacA function and involved the same recognition site, suggesting that RsmA is a downstream regulatory element of the GacA control cascade. Mutational inactivation of the chromosomal rsmA gene partially suppressed a gacS defect. Thus, a central, GacA-dependent switch from primary to secondary metabolism may operate at the level of translation.

Inactivation of the α C protein antigen gene, bca, by a novel shuttle/suicide vector results in attenuation of virulence and immunity in group B Streptococcus

The α C protein of group B Streptococcus (GBS) is a major surface-associated antigen. Although its role in the biology and virulence of GBS has not been defined, it is opsonic and capable of eliciting protective immunity. The α C protein is widely distributed among clinical isolates and is a potential protein carrier and antigen in conjugate vaccines to prevent GBS infections. The structural gene for the α C protein, bca, has been cloned and sequenced. The protein encoded by bca is related to a class of surface-associated proteins of Gram-positive cocci involved in virulence and immunity. To investigate the potential roles of the α C protein, bca null mutants were generated in which the bca gene was replaced with a kanamycin resistance cassette via homologous recombination using a novel shuttle/suicide vector. Studies of lethality in neonatal mice showed that the virulence of the bca null mutants was attenuated 5- to 7-fold when compared with the isogenic wild-type strain A909. Significant differences in mortality occurred in the first 24 h, suggesting that the role of the α antigen is important in the initial stages of the infection. In contrast to A909, bca mutants were no longer killed by polymorphonuclear leukocytes in the presence of α-specific antibodies in an in vitro opsonophagocytic assay. In contrast to previous studies, α antigen expression does not appear to play a role in resistance to opsonophagocytosis in the absence of α-specific antibodies. In addition, antibodies to the α C protein did not passively protect neonatal mice from lethal challenge with bca mutants, suggesting that these epitopes are uniquely present within the α antigen as expressed from the bca gene. Therefore, the α C protein is important in the pathogenesis of GBS infection and is a target for protective immunity in the development of GBS vaccines.

Staphylococcus aureus sortase mutants defective in the display of surface proteins and in the pathogenesis of animal infections

Many Gram-positive bacteria covalently tether their surface adhesins to the cell wall peptidoglycan. We find that surface proteins of Staphylococcus aureus are linked to the cell wall by sortase, an enzyme that cleaves polypeptides at a conserved LPXTG motif. S. aureus mutants lacking sortase fail to process and display surface proteins and are defective in the establishment of infections. Thus, the cell wall envelope of Gram-positive bacteria represents a surface organelle responsible for interactions with the host environment during the pathogenesis of bacterial infections.

Structure of sortase, the transpeptidase that anchors proteins to the cell wall of Staphylococcus aureus

Surface proteins of Gram-positive bacteria play important roles during the pathogenesis of human infections and require sortase for anchoring to the cell-wall envelope. Sortase cleaves surface proteins at the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine (T) and the amino group of cell-wall crossbridges. The NMR structure of sortase reveals a unique β-barrel structure, in which the active-site sulfhydryl of cysteine-184 is poised for ionization by histidine-120, presumably enabling the resultant thiolate to attack the LPXTG peptide. Calcium binding near the active site stimulates catalysis, possibly by altering the conformation of a surface loop that recognizes newly translocated polypeptides. The structure suggests a mechanistic relationship to the papain/cathepsin proteases and should facilitate the design of new antiinfective agents.

Processing of the leader mRNA plays a major role in the induction of thrS expression following threonine starvation in Bacillus subtilis.

The threonyl-tRNA synthetase gene, thrS, is a member of a family of Gram-positive genes that are induced following starvation for the corresponding amino acid by a transcriptional antitermination mechanism involving the cognate uncharged tRNA. Here we show that an additional level of complexity exists in the control of the thrS gene with the mapping of an mRNA processing site just upstream of the transcription terminator in the thrS leader region. The processed RNA is significantly more stable than the full-length transcript. Under nonstarvation conditions, or following starvation for an amino acid other than threonine, the full-length thrS mRNA is more abundant than the processed transcript. However, following starvation for threonine, the thrS mRNA exists primarily in its cleaved form. This can partly be attributed to an increased processing efficiency following threonine starvation, and partly to a further, nonspecific increase in the stability of the processed transcript under starvation conditions. The increased stability of the processed RNA contributes significantly to the levels of functional RNA observed under threonine starvation conditions, previously attributed solely to antitermination. Finally, we show that processing is likely to occur upstream of the terminator in the leader regions of at least four other genes of this family, suggesting a widespread conservation of this phenomenon in their control.

Functional expression of mouse Mdr1 in an outer membrane permeability mutant of Escherichia coli.

Functional expression of the multidrug resistance protein P-glycoprotein (P-gp) in Escherichia coli is providing an appropriate system for structure/function studies and might provide an invaluable tool to screen potential P-gp substrates and inhibitors. The major problem encountered in such studies, however, is the impermeability of the outer membrane of Gram-negative bacteria, which protects microorganisms against the cytotoxic effects of many lipophilic cancer drugs and blocks accessibility of P-gp reversal agents. In the present study we have constructed, by mutagenesis, a "leaky" (containing a permeable outer membrane) strain of E. coli, which is significantly more susceptible to the toxic effect of known P-gp substrates and cytotoxic agents. Expression of mouse Mdr1 in the mutant confers cross-resistance to daunomycin, quinidine, chloroquine, rhodamine 6G, and puromycin. Most importantly, reserpine and doxorubicin completely abolish Mdr1-mediated rhodamine resistance. The results provide strong support for previous observations, suggesting that Mdr1 can be expressed functionally in E. coli and indicate that the leaky mutant will be useful for further structure/function studies of the heterologously expressed eukaryotic drug efflux protein.

Copy-up mutants of the plasmid RK2 replication initiation protein are defective in coupling RK2 replication origins.

The broad host range plasmid RK2 replicates and regulates its copy number in a wide range of Gram-negative bacteria. The plasmid-encoded trans-acting replication protein TrfA and the origin of replication oriV are sufficient for controlled replication of the plasmid in all Gram-negative bacteria tested. The TrfA protein binds specifically to direct repeat sequences (iterons) at the origin of replication. A replication control model, designated handcuffing or coupling, has been proposed whereby the formation of coupled TrfA-oriV complexes between plasmid molecules results in hindrance of origin activity and, consequently, a shut-down of plasmid replication under conditions of higher than normal copy number. Therefore, according to this model, the coupling activity of an initiation protein is essential for copy number control and a copy-up initiation protein mutant should have reduced ability to form coupled complexes. To test this model for plasmid RK2, two previously characterized copy-up TrfA mutations, trfA-254D and trfA-267L, were combined and the resulting copy-up double mutant TFrfA protein TrfA-254D/267L was characterized. Despite initiating runaway (uncontrolled) replication in vivo, the copy-up double-mutant TrfA protein exhibited replication kinetics similar to the wild-type protein in vitro. Purified TrfA-254D, TrfA-267L, and TrfA-254D/267L proteins were then examined for binding to the iterons and for coupling activity using an in vitro ligase-catalyzed multimerization assay. It was found that both single and double TrfA mutant proteins exhibited substantially reduced (single mutants) or barely detectable (double mutant) levels of coupling activity while not being diminished in their capacity to bind to the origin of replication. These observations provide direct evidence in support of the coupling model of replication control.

Mucosal and systemic immune responses to a recombinant protein expressed on the surface of the oral commensal bacterium Streptococcus gordonii after oral colonization.

To circumvent the need to engineer pathogenic microorganisms as live vaccine-delivery vehicles, a system was developed which allowed for the stable expression of a wide range of protein antigens on the surface of Gram-positive commensal bacteria. The human oral commensal Streptococcus gordonii was engineered to surface express a 204-amino acid allergen from hornet venom (Ag5.2) as a fusion with the anchor region of the M6 protein of Streptococcus pyogenes. The immunogenicity of the M6-Ag5.2 fusion protein was assessed in mice inoculated orally and intranasally with a single dose of recombinant bacteria, resulting in the colonization of the oral/pharyngeal mucosa for 10-11 weeks. A significant increase of Ag5.2-specific IgA with relation to the total IgA was detected in saliva and lung lavages when compared with mice colonized with wild-type S. gordonii. A systemic IgG response to Ag5.2 was also induced after oral colonization. Thus, recombinant Gram-positive commensal bacteria may be a safe and effective way of inducing a local and systemic immune response.

Pseudomonas syringae Hrp type III secretion system and effector proteins

Pseudomonas syringae is a member of an important group of Gram-negative bacterial pathogens of plants and animals that depend on a type III secretion system to inject virulence effector proteins into host cells. In P. syringae, hrp/hrc genes encode the Hrp (type III secretion) system, and avirulence (avr) and Hrp-dependent outer protein (hop) genes encode effector proteins. The hrp/hrc genes of P. syringae pv syringae 61, P. syringae pv syringae B728a, and P. syringae pv tomato DC3000 are flanked by an exchangeable effector locus and a conserved effector locus in a tripartite mosaic Hrp pathogenicity island (Pai) that is linked to a tRNALeu gene found also in Pseudomonas aeruginosa but without linkage to Hrp system genes. Cosmid pHIR11 carries a portion of the strain 61 Hrp pathogenicity island that is sufficient to direct Escherichia coli and Pseudomonas fluorescens to inject HopPsyA into tobacco cells, thereby eliciting a hypersensitive response normally triggered only by plant pathogens. Large deletions in strain DC3000 revealed that the conserved effector locus is essential for pathogenicity but the exchangeable effector locus has only a minor role in growth in tomato. P. syringae secretes HopPsyA and AvrPto in culture in a Hrp-dependent manner at pH and temperature conditions associated with pathogenesis. AvrPto is also secreted by Yersinia enterocolitica. The secretion of AvrPto depends on the first 15 codons, which are also sufficient to direct the secretion of an Npt reporter from Y. enterocolitica, indicating that a universal targeting signal is recognized by the type III secretion systems of both plant and animal pathogens.

Advances in random matrix theory, zeta functions, and sphere packing

Over four hundred years ago, Sir Walter Raleigh asked his mathematical assistant to find formulas for the number of cannonballs in regularly stacked piles. These investigations aroused the curiosity of the astronomer Johannes Kepler and led to a problem that has gone centuries without a solution: why is the familiar cannonball stack the most efficient arrangement possible? Here we discuss the solution that Hales found in 1998. Almost every part of the 282-page proof relies on long computer verifications. Random matrix theory was developed by physicists to describe the spectra of complex nuclei. In particular, the statistical fluctuations of the eigenvalues (“the energy levels”) follow certain universal laws based on symmetry types. We describe these and then discuss the remarkable appearance of these laws for zeros of the Riemann zeta function (which is the generating function for prime numbers and is the last special function from the last century that is not understood today.) Explaining this phenomenon is a central problem. These topics are distinct, so we present them separately with their own introductory remarks.

The cell wall components peptidoglycan and lipoteichoic acid from Staphylococcus aureus act in synergy to cause shock and multiple organ failure.

Although the incidence of Gram-positive sepsis has risen strongly, it is unclear how Gram-positive organisms (without endotoxin) initiate septic shock. We investigated whether two cell wall components from Staphylococcus aureus, peptidoglycan (PepG) and lipoteichoic acid (LTA), can induce the inflammatory response and multiple organ dysfunction syndrome (MODS) associated with septic shock caused by Gram-positive organisms. In cultured macrophages, LTA (10 micrograms/ml), but not PepG (100 micrograms/ml), induces the release of nitric oxide measured as nitrite. PepG, however, caused a 4-fold increase in the production of nitrite elicited by LTA. Furthermore, PepG antibodies inhibited the release of nitrite elicited by killed S. aureus. Administration of both PepG (10 mg/kg; i.v.) and LTA (3 mg/kg; i.v.) in anesthetized rats resulted in the release of tumor necrosis factor alpha and interferon gamma and MODS, as indicated by a decrease in arterial oxygen pressure (lung) and an increase in plasma concentrations of bilirubin and alanine aminotransferase (liver), creatinine and urea (kidney), lipase (pancreas), and creatine kinase (heart or skeletal muscle). There was also the expression of inducible nitric oxide synthase in these organs, circulatory failure, and 50% mortality. These effects were not observed after administration of PepG or LTA alone. Even a high dose of LTA (10 mg/kg) causes only circulatory failure but no MODS. Thus, our results demonstrate that the two bacterial wall components, PepG and LTA, work together to cause systemic inflammation and multiple systems failure associated with Gram-positive organisms.

Disparate responses to oxidative stress in saprophytic and pathogenic mycobacteria.

To persist in macrophages and in granulomatous caseous lesions, pathogenic mycobacteria must be equipped to withstand the action of toxic oxygen metabolites. In Gram-negative bacteria, the OxyR protein is a critical component of the oxidative stress response. OxyR is both a sensor of reactive oxygen species and a transcriptional activator, inducing expression of detoxifying enzymes such as catalase/hydroperoxidase and alkyl hydroperoxidase. We have characterized the responses of various mycobacteria to hydrogen peroxide both phenotypically and at the levels of gene and protein expression. Only the saprophytic Mycobacterium smegmatis induced a protective oxidative stress response analogous to the OxyR response of Gram-negative bacteria. Under similar conditions, the pathogenic mycobacteria exhibited a limited, nonprotective response, which in the case of Mycobacterium tuberculosis was restricted to induction of a single protein, KatG. We have also isolated DNA sequences homologous to oxyR and ahpC from M. tuberculosis and Mycobacterium avium. While the M. avium oxyR appears intact, the oxyR homologue of M. tuberculosis contains numerous deletions and frameshifts and is probably nonfunctional. Apparently the response of pathogenic mycobacteria to oxidative stress differs significantly from the inducible OxyR response of other bacteria.