A site-specific recombinase is required for competitive root colonization by Pseudomonas fluorescens WCS365

A colonization mutant of the efficient root-colonizing biocontrol strain Pseudomonas fluorescens WCS365 is described that is impaired in competitive root-tip colonization of gnotobiotically grown potato, radish, wheat, and tomato, indicating a broad host range mutation. The colonization of the mutant is also impaired when studied in potting soil, suggesting that the defective gene also plays a role under more natural conditions. A DNA fragment that is able to complement the mutation for colonization revealed a multicistronic transcription unit composed of at least six ORFs with similarity to lppL, lysA, dapF, orf235/233, xerC/sss, and the largely incomplete orf238. The transposon insertion in PCL1233 appeared to be present in the orf235/233 homologue, designated orf240. Introduction of a mutation in the xerC/sss homologue revealed that the xerC/sss gene homologue rather than orf240 is crucial for colonization. xerC in Escherichia coli and sss in Pseudomonas aeruginosa encode proteins that belong to the λ integrase family of site-specific recombinases, which play a role in phase variation caused by DNA rearrangements. The function of the xerC/sss homologue in colonization is discussed in terms of genetic rearrangements involved in the generation of different phenotypes, thereby allowing a bacterial population to occupy various habitats. Mutant PCL1233 is assumed to be locked in a phenotype that is not well suited to compete for colonization in the rhizosphere. Thus we show the importance of phase variation in microbe–plant interactions.

Human milk lactoferrin inactivates two putative colonization factors expressed by Haemophilus influenzae

Haemophilus influenzae is a major cause of otitis media and other respiratory tract disease in children. The pathogenesis of disease begins with colonization of the upper respiratory mucosa, a process that involves evasion of local immune mechanisms and adherence to epithelial cells. Several studies have demonstrated that human milk is protective against H. influenzae colonization and disease. In the present study, we examined the effect of human milk on the H. influenzae IgA1 protease and Hap adhesin, two autotransported proteins that are presumed to facilitate colonization. Our results demonstrated that human milk lactoferrin efficiently extracted the IgA1 protease preprotein from the bacterial outer membrane. In addition, lactoferrin specifically degraded the Hap adhesin and abolished Hap-mediated adherence. Extraction of IgA1 protease and degradation of Hap were localized to the N-lobe of the bilobed lactoferrin molecule and were inhibited by serine protease inhibitors, suggesting that the lactoferrin N-lobe may contain serine protease activity. Additional experiments revealed no effect of lactoferrin on the H. influenzae P2, P5, and P6 outer-membrane proteins, which are distinguished from IgA1 protease and Hap by the lack of an N-terminal passenger domain or an extracellular linker region. These results suggest that human milk lactoferrin may attenuate the pathogenic potential of H. influenzae by selectively inactivating IgA1 protease and Hap, thereby interfering with colonization. Future studies should examine the therapeutic potential of lactoferrin, perhaps as a supplement in infant formulas.

Selective maintenance of allozyme differences among sympatric host races of the apple maggot fly

Whether phytophagous insects can speciate in sympatry when they shift and adapt to new host plants is a controversial question. One essential requirement for sympatric speciation is that disruptive selection outweighs gene flow between insect populations using different host plants. Empirical support for host-related selection (i.e., fitness trade-offs) is scant, however. Here, we test for host-dependent selection acting on apple (Malus pumila)- and hawthorn (Crataegus spp.)-infesting races of Rhagoletis pomonella (Diptera: Tephritidae). In particular, we examine whether the earlier fruiting phenology of apple trees favors pupae in deeper states of diapause (or with slower metabolisms/development rates) in the apple fly race. By experimentally lengthening the time period preceding winter, we exposed hawthorn race pupae to environmental conditions typically faced by apple flies. This exposure induced a significant genetic response at six allozyme loci in surviving hawthorn fly adults toward allele frequencies found in the apple race. The sensitivity of hawthorn fly pupae to extended periods of warm weather therefore selects against hawthorn flies that infest apples and helps to maintain the genetic integrity of the apple race by counteracting gene flow from sympatric hawthorn populations. Our findings confirm that postzygotic reproductive isolation can evolve as a pleiotropic consequence of host-associated adaptation, a central tenet of nonallopatric speciation. They also suggest that one reason for the paucity of reported fitness trade-offs is a failure to consider adequately costs associated with coordinating an insect’s life cycle with the phenology of its host plant.

Overexpressed human CD44s promotes lung colonization during micrometastasis of murine fibrosarcoma cells: Facilitated retention in the lung vasculature

Normally nonmetastatic murine sis-transformed BALB/c 3T3 cells, transfected with human CD44s gene (hCD44s), acquire spontaneous metastatic capacity to the lung. The mechanism(s) of this facilitated micrometastasis was analyzed in an experimental metastasis model. Human CD44s overexpression promoted the earliest stages severalfold (initial implantation and subsequent stabilization of tumor cells) but was irrelevant for later stages (subsequent outgrowth) of lung experimental micrometastasis. By injecting mixed populations of parental (nonmetastatic) and CD44s-transfected cells, it was shown that cell–cell adhesion between tumor and parental cells was not promoted by hCD44s but that promotion of cell–cell adhesion to lung endothelium or specifically between transfected cells (via hyaluronan) are likely mechanisms. Results obtained with hCD44s-negative primary tumor cells and hCD44s-positive or -negative variants of lung micrometastatic cells (after s.c. injection of transfectants) confirmed the importance of CD44s overexpression for early but not late stages of experimental lung metastasis. Therefore, CD44s represents a metastasis-facilitating molecule that is irrelevant for primary tumor outgrowth but that promotes micrometastasis to the lungs at the very earliest stages.

Toward an understanding of the biochemical and pharmacological complexity of the saliva of a hematophagous sand fly Lutzomyia longipalpis

The saliva of blood-sucking arthropods contains powerful pharmacologically active substances and may be a vaccine target against some vector-borne diseases. Subtractive cloning combined with biochemical approaches was used to discover activities in the salivary glands of the hematophagous fly Lutzomyia longipalpis. Sequences of nine full-length cDNA clones were obtained, five of which are possibly associated with blood-meal acquisition, each having cDNA similarity to: (i) the bed bug Cimex lectularius apyrase, (ii) a 5′-nucleotidase/phosphodiesterase, (iii) a hyaluronidase, (iv) a protein containing a carbohydrate-recognition domain (CRD), and (v) a RGD-containing peptide with no significant matches to known proteins in the blast databases. Following these findings, we observed that the salivary apyrase activity of L. longipalpis is indeed similar to that of Cimex apyrase in its metal requirements. The predicted isoelectric point of the putative apyrase matches the value found for Lutzomyia salivary apyrase. A 5′-nucleotidase, as well as hyaluronidase activity, was found in the salivary glands, and the CRD-containing cDNA matches the N-terminal sequence of the HPLC-purified salivary anticlotting protein. A cDNA similar to α-amylase was discovered and salivary enzymatic activity demonstrated for the first time in a blood-sucking arthropod. Full-length clones were also found coding for three proteins of unknown function matching, respectively, the N-terminal sequence of an abundant salivary protein, having similarity to the CAP superfamily of proteins and the Drosophila yellow protein. Finally, two partial sequences are reported that match possible housekeeping genes. Subtractive cloning will considerably enhance efforts to unravel the salivary pharmacopeia of blood-sucking arthropods.

Is sexual selection and species recognition a continuum? Mating behavior of the stalk-eyed fly Drosophila heteroneura

If behavioral isolation between species can evolve as a consequence of sexual selection within a species, then traits that are both sexually selected and used as a criterion of species recognition by females should be identifiable. The broad male head of the Hawaiian picture-winged fly Drosophila heteroneura is a novel sexual dimorphism that may be sexually selected and involved in behavioral isolation from D. silvestris. We found that males with broad heads are more successful in sexual selection, both through female mate choice and through aggressive interactions. However, female D. heteroneura do not discriminate against hybrids on the basis of their head width. Thus, this novel trait is sexually selected but is not a major contributor to species recognition. Our methods should be applicable to other species in which behavioral isolation is a factor.

The role of phosphoglycans in Leishmania–sand fly interactions

Leishmania promastigotes synthesize an abundance of phosphoglycans, either attached to the cell surface through phosphatidylinositol anchors (lipophosphoglycan, LPG) or secreted as protein-containing glycoconjugates. These phosphoglycans are thought to promote the survival of the parasite within both its vertebrate and invertebrate hosts. The relative contributions of different phosphoglycan-containing molecules in Leishmania–sand fly interactions were tested by using mutants specifically deficient in either total phosphoglycans or LPG alone. Leishmania donovani promastigotes deficient in both LPG and protein-linked phosphoglycans because of loss of LPG2 (encoding the Golgi GDP-Man transporter) failed to survive the hydrolytic environment within the early blood-fed midgut. In contrast, L. donovani and Leishmania major mutants deficient solely in LPG expression because of loss of LPG1 (involved in biosynthesis of the core oligosaccharide LPG domain) had only a slight reduction in the survival and growth of promastigotes within the early blood-fed midgut. The ability of the LPG1-deficient promastigotes to persist in the midgut after blood meal excretion was completely lost, and this defect was correlated with their inability to bind to midgut epithelial cells in vitro. For both mutants, when phosphoglycan expression was restored to wild-type levels by reintroduction of LPG1 or LPG2 (as appropriate), then the wild-type phenotype was also restored. We conclude, first, that LPG is not essential for survival in the early blood-fed midgut but, along with other secreted phosphoglycan-containing glycoconjugates, can protect promastigotes from the digestive enzymes in the gut and, second, that LPG is required to mediate midgut attachment and to maintain infection in the fly during excretion of the digested blood meal.

Calcium imaging demonstrates colocalization of calcium influx and extrusion in fly photoreceptors

During illumination, Ca2+ enters fly photoreceptor cells through light-activated channels that are located in the rhabdomere, the compartment specialized for phototransduction. From the rhabdomere, Ca2+ diffuses into the cell body. We visualize this process by rapidly imaging the fluorescence in a cross section of a photoreceptor cell injected with a fluorescent Ca2+ indicator in vivo. The free Ca2+ concentration in the rhabdomere shows a very fast and large transient shortly after light onset. The free Ca2+ concentration in the cell body rises more slowly and displays a much smaller transient. After ≈400 ms of light stimulation, the Ca2+ concentration in both compartments reaches a steady state, indicating that thereafter an amount of Ca2+, equivalent to the amount of Ca2+ flowing into the cell, is extruded. Quantitative analysis demonstrates that during the steady state, the free Ca2+ concentration in the rhabdomere and throughout the cell body is the same. This shows that Ca2+ extrusion takes place very close to the location of Ca2+ influx, the rhabdomere, because otherwise gradients in the steady-state distribution of Ca2+ should be measured. The close colocalization of Ca2+ influx and Ca2+ extrusion ensures that, after turning off the light, Ca2+ removal from the rhabdomere is faster than from the cell body. This is functionally significant because it ensures rapid dark adaptation.

Function of bicoid and hunchback homologs in the basal cyclorrhaphan fly Megaselia (Phoridae)

The Drosophila gene bicoid functions at the beginning of a gene cascade that specifies anterior structures in the embryo. Its transcripts are localized at the anterior pole of the oocyte, giving rise to a Bicoid protein gradient, which regulates the spatially restricted expression of target genes along the anterior–posterior axis of the embryo in a concentration-dependent manner. The morphogen function of Bicoid requires the coactivity of the zinc finger transcription factor Hunchback, which is expressed in a Bicoid-dependent fashion in the anterior half of the embryo. Whereas hunchback is conserved throughout insects, bicoid homologs are known only from cyclorrhaphan flies. Thus far, identification of hunchback and bicoid homologs rests only on sequence comparison. In this study, we used double-stranded RNA interference (RNAi) to address the function of bicoid and hunchback homologs in embryos of the lower cyclorrhaphan fly Megaselia abdita (Phoridae). Megaselia-hunchback RNAi causes hunchback-like phenotypes as observed in Drosophila, but Megaselia-bicoid RNAi causes phenotypes different from corresponding RNAi experiments in Drosophila and bicoid mutant embryos. Megaselia-bicoid is required not only for the head and thorax but also for the development of four abdominal segments. This difference between Megaselia and Drosophila suggests that the range of functional bicoid activity has been reduced in higher flies.

Altered expression of the ToxR-regulated porins OmpU and OmpT diminishes Vibrio cholerae bile resistance, virulence factor expression, and intestinal colonization

The transmembrane transcriptional activators ToxR and TcpP modulate expression of Vibrio cholerae virulence factors by exerting control over toxT, which encodes the cytoplasmic transcriptional activator of the ctx, tcp, and acf virulence genes. However, ToxR, independently of TcpP and ToxT, activates and represses transcription of the genes encoding two outer-membrane porins, OmpU and OmpT. To determine the role of ToxR-dependent porin regulation in V. cholerae pathogenesis, the ToxR-activated ompU promoter was used to drive ompT transcription in a strain lacking OmpU. Likewise, the ToxR-repressed ompT promoter was used to drive ompU transcription in a strain lacking both ToxR and OmpT. This strategy allowed the generation of a toxR+ strain that expresses OmpT in place of OmpU, and a toxR− strain that expresses OmpU in place of OmpT. Growth rates in the presence of bile salts and other anionic detergents were retarded for the toxR+ V. cholerae expressing OmpT in place of OmpU, but increased in toxR− V. cholerae expressing OmpU in place of OmpT. Additionally, the toxR+ V. cholerae expressing OmpT in place of OmpU expressed less cholera toxin and toxin-coregulated pilus, and this effect was shown to be caused by reduced toxT transcription in this strain. Finally, the toxR+ V. cholerae expressing OmpT in place of OmpU was ≈100-fold reduced in its ability to colonize the infant-mouse intestine. Our results indicate that ToxR-dependent modulation of the outer membrane porins OmpU and OmpT is critical for V. cholerae bile resistance, virulence factor expression, and intestinal colonization.

The surface coat of procyclic Trypanosoma brucei: Programmed expression and proteolytic cleavage of procyclin in the tsetse fly

Trypanosoma brucei, the protozoan parasite causing sleeping sickness, is transmitted by a tsetse fly vector. When the tsetse takes a blood meal from an infected human, it ingests bloodstream form trypanosomes that quickly differentiate into procyclic forms within the fly's midgut. During this process, the parasite loses the 107 molecules of variant surface glycoprotein that formed its surface coat, and it develops a new coat composed of several million procyclin molecules. Procyclins, the products of a small multigene family, are glycosyl phosphatidylinositol-anchored proteins containing characteristic amino acid repeats at the C terminus [either EP (EP procyclin, a form of procyclin rich in Glu-Pro repeats) or GPEET (GPEET procyclin, a form of procyclin rich in Glu-Pro-Glu-Glu-Thr repeats)]. We have used a sensitive and accurate mass spectrometry method to analyze the appearance of different procyclins during the establishment of midgut infections in tsetse flies. We found that different procyclin gene products are expressed in an orderly manner. Early in the infection (day 3), GPEET2 is the only procyclin detected. By day 7, however, GPEET2 disappears and is replaced by several isoforms of glycosylated EP, but not the unglycosylated isoform EP2. Unexpectedly, we discovered that the N-terminal domains of all procyclins are quantitatively removed by proteolysis in the fly, but not in culture. These findings suggest that one function of the protease-resistant C-terminal domain, containing the amino acid repeats, is to protect the parasite surface from digestive enzymes in the tsetse fly gut.

Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme

Bacteriophage lytic enzymes quickly destroy the cell wall of the host bacterium to release progeny phage. Because such lytic enzymes specifically kill the species in which they were produced, they may represent an effective way to control pathogenic bacteria without disturbing normal microflora. In this report, we studied a murein hydrolase from the streptococcal bacteriophage C1 termed lysin. This enzyme is specific for groups A, C, and E streptococci, with little or no activity toward several oral streptococci or other commensal organisms tested. Using purified lysin in vitro, we show that 1,000 units (10 ng) of enzyme is sufficient to sterilize a culture of ≈107 group A streptococci within 5 seconds. When a single dose of lysin (250 units) is first added to the oral cavity of mice, followed by 107 live group A streptococci, it provides protection from colonization (28.5% infected, n = 21) compared with controls without lysin (70.5% infected, n = 17) (P < 0.03). Furthermore, when lysin (500 units) was given orally to 9 heavily colonized mice, no detectable streptococci were observed 2 h after lysin treatment. In all, these studies show that lysin represents a unique murein hydrolase that has a rapid lethal effect both in vitro and in vivo on group A streptococci, without affecting other indigenous microorganisms analyzed. This general approach may be used to either eliminate or reduce streptococci from the upper respiratory mucosal epithelium of either carriers or infected individuals, thus reducing associated disease.

Does recombination improve selection on codon usage? Lessons from nematode and fly complete genomes

Understanding the factors responsible for variations in mutation patterns and selection efficacy along chromosomes is a prerequisite for deciphering genome sequences. Population genetics models predict a positive correlation between the efficacy of selection at a given locus and the local rate of recombination because of Hill–Robertson effects. Codon usage is considered one of the most striking examples that support this prediction at the molecular level. In a wide range of species including Caenorhabditis elegans and Drosophila melanogaster, codon usage is essentially shaped by selection acting for translational efficiency. Codon usage bias correlates positively with recombination rate in Drosophila, apparently supporting the hypothesis that selection on codon usage is improved by recombination. Here we present an exhaustive analysis of codon usage in C. elegans and D. melanogaster complete genomes. We show that in both genomes there is a positive correlation between recombination rate and the frequency of optimal codons. However, we demonstrate that in both species, this effect is due to a mutational bias toward G and C bases in regions of high recombination rate, possibly as a direct consequence of the recombination process. The correlation between codon usage bias and recombination rate in these species appears to be essentially determined by recombination-dependent mutational patterns, rather than selective effects. This result highlights that it is necessary to take into account the mutagenic effect of recombination to understand the evolutionary role and impact of recombination.